The diaphragm: contractile properties and fatigue.

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Authors

D F Rochester

Hemodynamic versus adrenergic control of cat right ventricular hypertrophy.

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The purpose of this study was to determine whether cardiac hypertrophy in response to hemodynamic overloading is a primary result of the increased load or is instead a secondary result of such other factors as concurrent sympathetic activation. To make this distinction, four experiments were done; the major experimental result, cardiac hypertrophy, was assessed in terms of ventricular mass and cardiocyte cross-sectional area. In the first experiment, the cat right ventricle was loaded differentially by pressure overloading the ventricle, while unloading a constituent papillary muscle; this model was used to ask whether any endogenous or exogenous substance caused uniform hypertrophy, or whether locally appropriate load responses caused ventricular hypertrophy with papillary muscle atrophy. The latter result obtained, both when each aspect of differential loading was simultaneous and when a previously hypertrophied papillary muscle was unloaded in a pressure overloaded right ventricle. In the second experiment, epicardial denervation and then pressure overloading was used to assess the role of local neurogenic catecholamines in the genesis of hypertrophy. The degree of hypertrophy caused by these procedures was the same as that caused by pressure overloading alone. In the third and fourth experiments, beta-adrenoceptor or alpha-adrenoceptor blockade was produced before and maintained during pressure overloading. The hypertrophic response did not differ in either case from that caused by pressure overloading without adrenoceptor blockade. These experiments demonstrate the following: first, cardiac hypertrophy is a local response to increased load, so that any factor serving as a mediator of this response must be either locally generated or selectively active only in those cardiocytes in which stress and/or strain are increased; second, catecholamines are not that mediator, in that adrenergic activation is neither necessary for nor importantly modifies the cardiac hypertrophic response to an increased hemodynamic load.

Authors

G Cooper 4th, R L Kent, C E Uboh, E W Thompson, T A Marino

Polymorphism of theophylline metabolism in man.

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To determine whether genetic mechanisms control large interindividual variations in theophylline elimination in normal uninduced human subjects, and, if so, to test the possibility that these genetic factors are transmitted as a simple Mendelian trait, theophylline was administered to 79 unrelated adults, six sets of monozygotic twins, six sets of dizygotic twins, and six two-generation families. Thereafter, in urine collected from each subject at regular intervals for 48 h, concentrations of theophylline and its three principal metabolites were measured and rate constants of formation of these metabolites calculated. The twin study, designed to determine the relative contributions of genetic and environmental factors to large interindividual variation in theophylline elimination, revealed predominantly genetic control. Values for this genetic component, designated heritability (H1(2)), of interindividual variation in rate constants of metabolite formation were 0.61, 0.84, and 0.95 for 3-methylxanthine, 1-methyluric acid, and 1,3-dimethyluric acid, respectively. H1(2) for the overall theophylline elimination rate constant (kel) was lower (0.34). In the 79 unrelated adults, each distribution curve for rate constants of formation of each theophylline metabolite appeared to be trimodal. By contrast, the distribution curve for the overall theophylline elimination rate constant appeared to be either unimodal or bimodal. The extent of interindividual variation was fourfold for theophylline kel and 6-8-fold for the three principal metabolites. High correlations among the three rate constants in individual subjects suggested their regulation by a single shared factor. In six families carefully selected to be under near basal environmental conditions so that hepatic theophylline metabolism of each family member would be neither markedly induced nor inhibited, phenotypes for theophylline metabolite rate constants were assigned. This assignment of phenotype was made by the position of each family member's rate constant on the three distribution curves that were generated from the 79 unrelated subjects. In each family, pedigree analysis of the three phenotypes for each rate constant was consistent with their control by two alleles at a single genetic locus and with autosomal codominant transmission. Frequencies of the two alleles at each genetic locus controlling rate constants of formation of theophylline metabolites were similar (p = 0.49, 0.53, and 0.52). In the three families studied with antipyrine (AP) as well as with theophylline, AP k(el) correlated (r approximately 0.7) with each rate constant of theophylline metabolite formation, as well as with theophylline k(el). While these results are compatible with a common regulatory element in the AP and theophylline polymorphisms, other evidence suggests more than a single genetic polymorphism. This additional evidence includes different gene frequencies for the AP (p approximately 0.1) and theophylline (p approximately 0.5) polymorphisms, different genotype assignments in several families for some theophylline metabolites, different distribution curves for theophylline k(el) from those for the three theophylline metabolites in 79 unrelated subjects, and finally low correlations between AP metabolite rate constants and theophylline metabolite rate constants in the three families receiving both drugs.

Authors

C A Miller, L B Slusher, E S Vesell

Direct and indirect effects of calcium entry blocking agents on isovolumic left ventricular relaxation in conscious dogs.

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To assess the direct and indirect effects of the commonly used calcium entry blockers (CEB) upon the major determinants of isovolumic left ventricular relaxation, we administered equidepressant intracoronary (IC, n = 7) and equihypotensive intravenous (n = 12) dosages of diltiazem (16 +/- 3 SE micrograms/kg IC and 63 +/- 9 micrograms/kg i.v.), verapamil (10 +/- 2 and 57 +/- 5 micrograms/kg), and nifedipine (1 +/- 0.1 and 8 +/- 0.3 micrograms/kg) to preinstrumented awake dogs with normal ventricular function. The time constant of left ventricle (LV) relaxation, analyzed by two methods (T1, from the linear relation of the natural logarithm of LV pressure and time; T2, from the linear relation of LV pressure and negative high fidelity LV pressure), was significantly and equivalently prolonged by IC diltiazem (T1 + 48%, P less than 0.02), verapamil (T1 + 43%, P less than 0.001), and nifedipine (T1 + 30%, P less than 0.03). Lesser amounts of each CEB that did not affect rate of LV pressure development or extent of shortening produced no change in T1 or T2. By contrast, intravenous calcium entry blockade either produced no significant change (diltiazem and verapamil) or shortened (nifedipine T1 - 18%, P less than 0.01) LV isovolumic relaxation. However, after beta adrenergic blockade with propranolol (2 mg/kg i.v., n = 6) no change in ventricular relaxation was observed during nifedipine and the time constant was significantly prolonged by verapamil (T1 + 15%, P less than 0.05). We conclude that calcium entry blockade directly impairs normal left ventricular relaxation: This effect is closely linked to the negative inotropic properties of these drugs. The prolongation of isovolumic relaxation produced by calcium blockade is attenuated or even reversed by reflex sympathetic stimulation and favorably altered loading conditions during systemic administration.

Authors

R A Walsh, R A O'Rourke

Rabbits immunized with thyroid-stimulating hormone produce autoantiidiotypic thyroid-stimulating antibodies.

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We immunized rabbits with thyroid-stimulating hormone (TSH) to investigate the hypothesis that such immunization could result in production of thyroid-stimulating autoantiidiotypic antibodies to anti-TSH. Thyroid-stimulating immunoglobulin (TSI) appeared in the serum of several rabbits after immunization. At 160 d, TSI equivalent to 6-18 microU TSH/1.5 mg IgG was present in two of six human (h)TSH-, two of six hTSH beta chain-, and two of the four surviving bovine (b)TSH-immunized animals. Control (human serum albumin-immunized rabbits) serum TSI was 4.3 +/- 0.4 (mean +/- SD) at this time. Antiidiotypic antibodies that could bind to monoclonal anti-hTSH were found in the sera of the bTSH-immunized rabbits. The peak TSI activity occurred 3 mo after a TSH booster immunization and declined gradually during subsequent weeks. Evidence that antiidiotypic antibodies to anti-TSH can cause thyroid stimulation strengthens the notion that such antibodies may be the cause of Graves' hyperthyroidism.

Authors

G N Beall, B Rapoport, I J Chopra, S R Kruger

Aluminum deposition at the osteoid-bone interface. An epiphenomenon of the osteomalacic state in vitamin D-deficient dogs.

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Although aluminum excess is an apparent pathogenetic factor underlying osteomalacia in dialysis-treated patients with chronic renal failure, the mechanism by which aluminum impairs bone mineralization is unclear. However, the observation that aluminum is present at osteoid-bone interfaces in bone biopsies of affected patients suggests that its presence at calcification fronts disturbs the cellular and/or physiochemical processes underlying normal mineralization. Alternatively, aluminum at osteoid-bone interfaces may reflect deposition in preexistent osteomalacic bone without direct effects on the mineralization process. We investigated whether aluminum accumulates preferentially in osteomalacic bone and, if so, whether deposition of aluminum occurs at calcification fronts and specifically inhibits mineralization. Aluminum chloride (1 mg/kg) was administered intravenously three times per week for 3 wk to five normal and five vitamin D-deficient osteomalacic dogs. Before administration of aluminum the vitamin D-deficient dogs had biochemical and bone biopsy evidence of osteomalacia. Bone aluminum content in the osteomalacic dogs (15.1 +/- 2.2 micrograms/g) and the plasma aluminum concentration (10.4 +/- 2.1 micrograms/liter) were no different than those of normal dogs (10.5 +/- 3.5 micrograms/g and 11.9 +/- 1.2 microgram/liter, respectively). After the 3 wk of aluminum administration the plasma phosphorus, parathyroid hormone, and 25-hydroxyvitamin D concentrations were unchanged in normal and vitamin D-deficient dogs. Similarly, no alteration in bone histology occurred in either group. In contrast, bone aluminum content increased to a greater extent in the vitamin D-deficient dogs (390.3 +/- 24.3 micrograms/g) than in the normal dogs (73.6 +/- 10.6 micrograms/g). Moreover, aluminum localized at the osteoid-bone interfaces of the osteomalacic bone in the vitamin D-deficient dogs, covering 42.9 +/- 9.2% of the osteoid-bone surface. Further, in spite of continued aluminum chloride administration (1 mg/kg two times per week), vitamin D repletion of the vitamin D-deficient dogs for 11 wk resulted in normalization of their biochemistries. In addition, while normal dogs maintained normal bone histology during the period of continued aluminum administration, vitamin D repletion of the vitamin D-deficient dogs induced healing of their bones. Indeed, the appearance of aluminum in the cement lines of the healed bones indicated that mineralization had occurred at sites of prior aluminum deposition. These observations illustrate that aluminum deposition in osteomalacic bone may be a secondary event that does not influence bone mineralization. Thus, although aluminum may cause osteomalacia in chronic renal failure, its presence at mineralization fronts may not be the mechanism underlying this derangement.

Authors

L D Quarles, V W Dennis, H J Gitelman, J M Harrelson, M K Drezner

Carbohydrate fermentation in the human colon and its relation to acetate concentrations in venous blood.

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There is now substantial evidence that some dietary polysaccharides, notably dietary fiber, escape absorption in the small bowel and are then broken down in the large intestine of man. The main end products of this colonic digestive process, which is anerobic, are short chain fatty acids (SCFA), and acetic, propionic, and butyric acids. Although these acids are known to be absorbed from the colon, their subsequent fate and significance is unknown. We have measured venous blood SCFA levels in healthy subjects after a 16-h fast, and then following oral doses of either 50 mmol SCFA, 5, 10, or 20 g doses of the fermentable carbohydrate lactulose, or 20 g of pectin. Fasting venous blood acetate was 53.8 +/- 4.4 mumol/liter (SEM) (n = 14). Fasting arterial blood acetate, taken simultaneously with venous blood in six subjects, was higher; 125.6 +/- 13.5 mumol/liter (arterial) vs. 61.1 +/- 6.9 mumol/liter (venous). Significant levels of propionate or butyrate were not detected in any blood samples. Following an oral dose of 50 mmol mixed SCFA, venous blood acetate reached a peak of 194.1 +/- 57.9 mumol/liter at 45 min and returned to fasting levels at 2 h. Blood acetate also rose in response to lactulose, peak levels occurring 2-4 h after the dose: 5 g, 98.6 +/- 23.1 mumol/liter; 10 g, 127.3 +/- 18.2 mumol/liter; and 20 g, 181.3 +/- 23.9 mumol/liter. Pectin fermentation was much slower, with blood acetate levels starting to rise after 6 h and remaining elevated at about twice fasting levels for the subsequent 18 h. However, areas under the blood acetate curves were closely related (r = 0.97; n = 5), whatever the source of acetate. These studies show that the large intestine makes an important contribution to blood acetate levels in man and that fermentation may influence metabolic processes well beyond the wall of this organ.

Authors

E W Pomare, W J Branch, J H Cummings

Differing effects of antiinsulin serum and antiinsulin receptor serum on 123I-insulin metabolism in rats.

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Anesthetized rats were treated with saline, antiinsulin receptor serum, or antiinsulin serum, and the biodistribution of high pressure liquid chromatography-purified 123I-Tyr A14-insulin was studied by scintillation scanning. Time activity curves over organs of interest were calibrated by sacrificing the rats at the end of the experiment and directly determining the radioactivity in the blood, liver, and kidneys. Saline-treated rats exhibited normal insulin biodistribution. The highest concentration of 123I-insulin was found in the liver, and reached 30% of total injected dose between 3 and 5 min after injection. After this peak, activity rapidly decreased with a t1/2 of 6 min. Activity of 123I-insulin in kidney showed a more gradual rise and fall and was approximately 15% of injected dose at its maximum. In rats treated with antiinsulin antiserum, insulin biodistribution was markedly altered. Peak liver activity increased with increasing antibody concentration with up to 90% of injected dose appearing in the liver. In addition, there was no clearance of the liver 123I-insulin over 30 min. Autoradiographic studies demonstrated that in contrast to the normal rats in which radioactivity was associated with hepatocytes, in rats passively immunized with anti-insulin serum, 125I-insulin was associated primarily with the Kuppfer cells. In contrast, antibodies to the insulin receptor markedly inhibited 123I-insulin uptake by the liver. Kidney activity increased, reflecting the amount of free 123I-insulin that reached this organ. This is similar to the pattern observed when insulin receptors are saturated with a high concentration of unlabeled insulin. Thus, both insulin antibodies and anti-receptor antibodies alter the distribution of insulin, but with very different patterns. The use of 123I-insulin and scintillation scanning allows one to study specific alterations in insulin distribution in animal models of insulin-resistant states, and should also be useful in human disease states.

Authors

J C Sodoyez, F Sodoyez Goffaux, R von Frenckell, C J De Vos, S Treves, C R Kahn

Cleavage of blood coagulation factor XIII and fibrinogen by thrombin during in vitro clotting.

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Thrombin cleavage of blood coagulation Factor XIII (a2b2) and fibrinogen was studied during in vitro clotting to determine the physiologic sequence of these events. First, the time course of fibrin formation and cleavage of Factor XIII was measured in platelet-rich plasma. Cleavage of fibrinogen was measured by using a radioimmunoassay for fibrinopeptide A. Conversion of trace amounts of radioiodinated a-chains of 125I-Factor XIII to thrombin-modified a-chains was measured in unreduced 10% sodium dodecyl sulfate-polyacrylamide gels. During spontaneous clotting, a similar percentage of 125I-Factor XIII and fibrinogen was cleaved at each time point. Visible gelation of polymerized fibrin monomer occurred when 24 +/- 8% of fibrinogen was cleaved and 21 +/- 6% of Factor XIII was converted to Factor XIII'. Thrombin cleavage of Factor XIII and fibrinogen was also studied in platelet-poor plasma to which thrombin was added. In order to measure Factor XIIIa activity, fibrin polymerization was completely inhibited by the addition of Gly-Pro-Arg-Pro. Factor XIIIa formation was measured by the incorporation of [3H]putrescine into casein. The concentration of added thrombin required to cleave 50% of fibrinogen and Factor XIII was 0.65 U/ml and 0.35 U/ml, respectively. The rate of cleavage of fibrinogen by thrombin was 43-fold greater than cleavage of Factor XIII. Lower Gly-Pro-Arg-Pro concentrations were used to determine the effects of incompletely inhibiting fibrin polymerization on cleavage of Factor XIII and fibrinogen. Thrombin cleavage of Factor XIII but not fibrinogen was dependent on the extent of fibrin polymerization. The more marked the degree of inhibition of fibrin polymerization, the slower the rate of Factor XIIIa formation. Thus, in platelet-rich plasma, thrombin cleavage of Factor XIII and fibrinogen are closely related events during spontaneous clotting. Furthermore, cleavage of Factor XIII during clotting is enhanced by fibrin polymerization in platelet-poor plasma.

Authors

C S Greenberg, C C Miraglia, F R Rickles, M A Shuman

Pancreatic enzyme response to a liquid meal and to hormonal stimulation. Correlation with plasma secretin and cholecystokinin levels.

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Pancreatic trypsin output and plasma secretin and cholecystokinin (CCK) levels were measured in five healthy volunteers to investigate the mechanisms involved in regulating postprandial pancreatic secretion. The pancreas was stimulated by a liquid test meal or by either intravenous secretin (1-82 pmol/kg-1 per h-1) or caerulein, a CCK analogue (2.3-37 pmol/kg-1 per h-1), or by a combination of secretin and caerulein. Pancreatic secretion was assessed by a marker perfusion technique (polyethylene glycol [PEG 4000]), plasma secretin, and CCK by specific radioimmunoassays. Increasing doses of secretin produced increasing bicarbonate output (P less than 0.01), whereas trypsin was not stimulated over basal. Graded caerulein produced a stepwise increase in trypsin and bicarbonate output (P less than 0.01). Potentiation occurred for bicarbonate secretion between secretin and caerulein, but not for trypsin output. Postprandial trypsin secretion averaged 29.1 IU/min-1 over 150 min (equal to 55% of maximal response to caerulein). The peak trypsin response amounted to 90% of maximal caerulein. Significant increases of plasma secretion (P less than 0.05) and CCK (P less than 0.01) were observed after the meal. Comparison of enzyme and CCK responses to the testmeal or to exogenous caerulein suggested that the amount of CCK released after the meal could account for the postprandial trypsin secretion. We conclude that (a) the postprandial enzyme response in man is submaximal in comparison to maximal exogenous hormone stimulation; (b) CCK is a major stimulatory mechanism of postprandial trypsin secretion, whereas secretin is not involved; and (c) Potentiation of enzyme secretion is not a regulatory mechanism of the postprandial secretory response.

Authors

C Beglinger, M Fried, I Whitehouse, J B Jansen, C B Lamers, K Gyr

Mechanism of preservation of glomerular perfusion and filtration during acute extracellular fluid volume depletion. Importance of intrarenal vasopressin-prostaglandin interaction for protecting kidneys from constrictor action of vasopressin.

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Glomerular circulatory dynamics were assessed in 60 adult anesthetized rats, which were either deprived or not deprived of water for 24-48 h. Water-deprived rats (n = 21) were characterized by a depressed level of single nephron glomerular filtration rate (SNGFR) when compared with nonwater-deprived controls (n = 8) (23.2 +/- 1.3 vs. 44.8 +/- 4.1 nl/min). This was primarily due to decreased glomerular plasma flow rate (71 +/- 5 vs. 169 +/- 23 nl/min) and glomerular capillary ultrafiltration coefficient (0.028 +/- 0.003 vs. 0.087 +/- 0.011 nl/[s . mmHg]). Infusion of saralasin to these water-deprived rats resulted in significant increases in plasma flow rate and ultrafiltration coefficient, and decline in arteriolar resistances. Consequently, SNGFR increased by approximately 50% from pre-saralasin levels. When water-deprived saralasin-treated rats were given a specific antagonist to the vascular action of arginine vasopressin (AVP), d(CH2)5Tyr(Me)AVP, a fall in systemic blood pressure occurred, on average from 102 +/- 5 to 80 +/- 5 mmHg, unaccompanied by dilation of renal arterioles, so that both plasma flow rate (129 +/- 8 vs. 85 +/- 13 nl/min) and SNGFR (31.0 +/- 2.9 vs. 18.2 +/- 4.4 nl/min) decreased. This more selective extrarenal constrictor action of AVP was further documented in additional studies in which cardiac output and whole kidney blood flow rate were simultaneously measured. In water-diuretic rats, administration of a moderately pressor dose of AVP (4 mU/kg per min) resulted in a significant rise in kidney blood flow rate (from 8.8 +/- 1.2 to 9.6 +/- 1.3 ml/min). The higher kidney blood flow rate occurred despite a fall in cardiac output (from 111 +/- 7 to 98 +/- 9 ml/min), and was associated with a significant increase in the ratio of systemic vascular to renal vascular resistance (on average from 0.083 +/- 0.014 to 0.106 +/- 0.019). Furthermore, infusion of d(CH2)5Tyr(Me)AVP to water-deprived animals (n = 6) to antagonize endogenous AVP resulted in systemic but not renal vasodilation, so that kidney blood flow rate fell (by approximately 30%), as did systemic-to-renal resistance ratio (by approximately 30%). When the above two experiments were repeated in indomethacin-treated animals, exogenous AVP administration in water-diuretic rats (n = 6) and antagonism of endogenous AVP in water-deprived rats (n = 7) caused, respectively, parallel constriction and dilation in systemic and renal vasculatures. The net effect was unaltered systemic to renal vascular resistance ratio in both cases. These results indicate that (1) unlike angiotensin II, AVP maintains glomerular perfusion and filtration in acute extracellular fluid volume depletion by a more selective constriction of the extrarenal vasculature. (2) The relative renal insensitivity to the vasoconstrictor action of AVP appears to be due to an AVP-induced release of a potent renal vasodilator, sensitive to indomethacin, presumably prostaglandins.

Authors

A Yared, V Kon, I Ichikawa

Gamma interferon is spontaneously released by alveolar macrophages and lung T lymphocytes in patients with pulmonary sarcoidosis.

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Gamma interferon (IFN gamma) is a potent immune mediator that plays a central role in enhancing cellular immune processes. This study demonstrates that while lung mononuclear cells from normal individuals spontaneously release little or no interferon (less than 10 U/10(6) cells per 24 h), those from patients with pulmonary sarcoidosis spontaneously release considerable amounts (65 +/- 20 U/10(6) cells per 24 h, P less than 0.02 compared to normals). Furthermore, cells from patients with active disease release far more interferon than those from patients with inactive disease (101 +/- 36 compared to 24 +/- 8 U/10(6) cells per 24 h, P less than 0.02). Characterization of this interferon using acid sensitivity, specific antibody inhibition, and target cell specificity criteria demonstrated that it was almost entirely IFN gamma. This spontaneous release of IFN gamma appeared to be compartmentalized to the lung of these patients in that their blood mononuclear cells spontaneously released little or no IFN gamma (P less than 0.02, compared to sarcoidosis lung mononuclear cells) and no IFN gamma was detected in their serum. Both lung T lymphocytes and alveolar macrophages contributed to the spontaneous release of IFN gamma by lung mononuclear cells from sarcoid patients; purified preparations of T lymphocytes and alveolar macrophages from these patients spontaneously released similar amounts of IFN gamma (56 +/- 21 and 32 +/- 11 U/10(6) cells per 24 h, respectively, P greater than 0.3). At least one role for IFN gamma in the pathogenesis of sarcoidosis appeared to be related to activation of alveolar macrophages, as alveolar macrophages recovered from patients with active disease spontaneously killed [3H]uridine-labeled tumor cell targets (17.7 +/- 4.5% cytotoxicity compared with 2.8 +/- 0.9% in normals, P less than 0.02) and purified IFN gamma enhanced the ability of alveolar macrophages from sarcoidosis patients with inactive disease to kill similar targets (P less than 0.001, compared to alveolar macrophages cultured in medium alone). Treatment of sarcoid patients with corticosteroids, a therapy known to suppress the activity of the disease, caused a marked reduction in the level of spontaneous IFN gamma release by lung mononuclear cells compared with untreated patients (P less than 0.02), which suggests that the effectiveness of corticosteroid therapy in controlling active pulmonary sarcoidosis may, at least in part, be due to suppression of IFN gamma release.

Authors

B W Robinson, T L McLemore, R G Crystal

Suppression of normal human erythropoiesis by gamma interferon in vitro. Role of monocytes and T lymphocytes.

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Interferons (IFN) have been shown to suppress the proliferation of human erythroid progenitors (erythroid burst-forming units [BFU-E] and colony-forming units [CFU-E]) in vitro. To examine the mechanism(s) underlying this inhibitory activity, the effect of different doses (50-10,000 U) of a highly purified preparation of recombinant DNA produced human gamma-IFN on erythroid colony formation by normal human bone marrow BFU-E and CFU-E in the presence and absence of monocytes and/or T lymphocytes was studied. The addition of gamma-IFN to whole marrow caused suppression of BFU-E (6-65%) and CFU-E (31-79%) in a dose-dependent fashion. This inhibition occurred both with the direct addition of gamma-IFN to the culture plates as well as by the preincubation of marrow cells with gamma-IFN followed by the washing of the cells; at the highest concentration of gamma-IFN (10,000 U), near-maximal inhibition of colony formation occurred with as little as 15 min of preexposure (BFU-E, 50%; CFU-E, 81%). Removal of monocytes and/or T lymphocytes before the addition of gamma-IFN significantly reduced the inhibitory effects of this lymphokine (BFU-E, -1 to 38%; CFU-E, -8 to 67%). Co-culture of purified autologous monocytes or T cells preexposed to gamma-IFN with monocyte and T cell-depleted marrow cells resulted in highly significant inhibition of erythroid colony formation even when these treated cells comprised less than 1% of the total nucleated cell populations in culture. The inhibitory action of gamma-IFN was not prevented or reversed by erythropoietin. These results demonstrate that the inhibitory effects of gamma-IFN on erythropoiesis are mediated to a significant degree through accessory cell populations, and suggest that gamma-IFN may represent a useful tool in the study of the role of immunocompetent cells in the regulation of erythropoiesis in vitro.

Authors

S W Mamus, S Beck-Schroeder, E D Zanjani

Reduction of sympathetic inotropic response after ischemia in dogs. Contributor to stunned myocardium.

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Eight open chest dogs underwent 25 min of coronary occlusion to determine whether brief myocardial ischemia disrupts the normal myocardial inotropic response to sympathetic nervous stimulation. If so, this could represent a mechanism contributing to postischemic myocardial dysfunction. Myocardial segment shortening was measured using ultrasonic dimension crystals before and after coronary artery occlusion and reperfusion. Left ansa subclavia stimulation and systemic norepinephrine (NE) infusion were used to test the myocardial inotropic response to neural stimulation and direct exposure to the sympathetic mediator, respectively. Before coronary artery occlusion, base-line preischemic segment shortening (12.5 +/- 1.6%) (SEM) increased during both sympathetic stimulation (20.2 +/- 1.4%) and NE infusion (19.7 +/- 1.1%). The control segment responded similarly. After ischemia and reperfusion there was no significant change in heart rate, aortic or left ventricular pressures, nor changes in control segment shortening. In contrast, shortening in the postischemic segment was markedly reduced compared to baseline (4.1 +/- 2.4%), and no longer responded to sympathetic stimulation (2.4 +/- 2.8%), while responsiveness to systemic NE was maintained (12.9 +/- 2.0%), P less than 0.001, which suggested injury to the sympathetic-neural axis during the period of ischemia. This reduced response to neural stimulation was persistent for up to 2 h after reperfusion. Left atrial or intracoronary infusion of bretylium tosylate, which releases norepinephrine from nerve terminals, resulted in an immediate inotropic response in the postischemic segment, which indicated that total depletion of NE from nerve terminals during the ischemic period had not occurred. Disruption of sympathetic neural responsiveness is likely a component of the mechanism of postischemic myocardial dysfunction whenever there is appreciable sympathetic drive to the heart.

Authors

A A Ciuffo, P Ouyang, L C Becker, L Levin, M L Weisfeldt

A unique control mechanism in the regulation of insulin secretion. Secretagogue-induced somatostatin receptor recruitment.

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In this study, we have correlated the translocation of somatostatin (SRIF) receptors from the cell interior to the plasma membrane with the ability of SRIF to inhibit insulin release. Islets were perifused with glucose (30, 100, 165, 200, or 300 mg/dl) in the presence of sodium isethionate. Sodium isethionate inhibits insulin release, but not the recruitment of SRIF receptors. Thus, the recruitment of SRIF receptors to the surface membrane continued without the lysis of secretion vesicles. SRIF binding rose from 3.75 +/- 0.16 to 6.46 +/- 0.28 fmol/10 islets as glucose concentration increased. Sodium isethionate was then removed, islets perifused with low glucose (30 mg/dl), and challenged with 400 microM isobutylmethylxanthine (IBMX) with or without SRIF (5 micrograms/ml). In the islets perifused with high glucose concentration, IBMX lysed a greater number of vesicles and caused enhanced release of insulin. The greater the number of secretion vesicles marginated to the plasma membrane by glucose, the greater the response to IBMX. Colchicine (1 mM) prevented secretion vesicle migration and this potentiation effect of higher concentrations of glucose was eliminated. In experiments with IBMX and SRIF, the degree of inhibition of IBMX-induced insulin release by SRIF was proportional to the magnitude of SRIF binding to these islets. SRIF inhibited insulin release by 20 microU/100 islets initially perifused with low glucose (30 mg/dl) and by 875 microU/100 islets perifused with high glucose (300 mg/dl). The maximal effect of SRIF was observed when its binding reached a level of 5.4 fmol/10 islets. We conclude that inhibition of insulin release by SRIF is proportional to the SRIF receptor concentration, and that translocation of SRIF receptors during exocytosis plays an important role in paracrine regulation of insulin secretion by rendering the islets more sensitive to SRIF.

Authors

B Draznin, J W Leitner, K E Sussman

Relationship of urinary and blood carbon dioxide tension during hypercapnia in the rat. Its significance in the evaluation of collecting duct hydrogen ion secretion.

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This study was designed to establish the relationship between urinary pCO2 and systemic blood pCO2 during acute hypercapnia and to investigate the significance of this relationship to collecting duct hydrogen ion (H+) secretion when the urine is acid and when it is highly alkaline. In rats excreting a highly alkaline urine, an acute increase in blood pCO2 (from 42 +/- 0.8 to 87 +/- 0.8 mmHg) resulted in a significant fall in urine minus blood (U-B) pCO2 (from 31 +/- 2.0 to 16 +/- 4.2 mmHg, P less than 0.005), a finding which could be interpreted to indicate inhibition of collecting duct H+ secretion by hypercapnia. The urinary pCO2 of rats with hypercapnia, unlike that of normocapnic controls, was significantly lower than that of blood when the urine was acid (58 +/- 6.3 and 86 +/- 1.7 mmHg, P less than 0.001) and when it was alkalinized in the face of accelerated carbonic acid dehydration by infusion of carbonic anhydrase (78 +/- 2.7 and 87 +/- 1.8 mmHg, P less than 0.02). The finding of a urinary pCO2 lower than systemic blood pCO2 during hypercapnia suggested that the urine pCO2 prevailing before bicarbonate loading should be known and the blood pCO2 kept constant to evaluate collecting duct H+ secretion using the urinary pCO2 technique. In experiments performed under these conditions, sodium bicarbonate infusion resulted in an increment in urinary pCO2 (i.e., a delta pCO2) which was comparable in hypercapnic and normocapnic rats (40 +/- 7.2 and 42 +/- 4.6 mmHg, respectively) that were alkalemic (blood pH 7.53 +/- 0.02 and 7.69 +/- 0.01, respectively). The U-B pCO2, however, was again lower in hypercapnic than in normocapnic rats (15 +/- 4.0 and 39 +/- 2.5 mmHg, respectively, P less than 0.001). In hypercapnic rats in which blood pH during bicarbonate infusion was not allowed to become alkalemic (7.38 +/- 0.01), the delta pCO2 was higher than that of normocapnic rats which were alkalemic (70 +/- 5.6 and 42 +/- 4.6 mmHg, respectively, P less than 0.005) while the U-B pCO2 was about the same (39 +/- 3.7 and 39 +/- 2.5 mmHg). We further examined urine pCO2 generation by measuring the difference between the urine pCO2 of a highly alkaline urine not containing carbonic anhydrase and that of an equally alkaline urine containing this enzyme. Carbonic anhydrase infusion to hypercapnic rats that were not alkalemic resulted in a fall in urine pCO(2) (from 122+/-5.7 to 77+/-2.2 mmHg) which was greater (P <0.02) than that seen in alkalemic normocapnic controls (from 73+/- 1.9 to 43+/-1.3 mmHg) with a comparable urine bicarbonate concentration and urine nonbicarbonate buffer capacity. CO(2) generation, therefore, from collecting dust H(+) secretion and titration of bicarbonate, was higher in hypercapnic rats that in normocapnic controls. We conclude that in rats with actue hypercapnia, the U-B p(CO(2)) achieved during bicarbonate loading greatly underestimates collecting duct H(+) secretion because it is artificially influenced by systemic blood pCO(2). the deltapCO(2) is a better qualitative index of collecting duct H+ secretion that the U-B pCO(2), because it is not artificially influenced by systemic blood pCO(2) and it takes into account the urine PCO(2) prevailing before bicarbonate loading.

Authors

D C Batlle, M Downer, C Gutterman, N A Kurtzman

Abnormal redox status of membrane-protein thiols in sickle erythrocytes.

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Abstract

Although sickle erythrocytes (RBC) undergo excessive autooxidation, investigators have not found evidence for abnormal oxidation of protein thiols in sickle RBC membranes (e.g., protein aggregates linked by intermolecular disulfide bonds). However, the conventional techniques heretofore used cannot detect more subtle changes in thiol status such as abnormal intramolecular disulfide bonds. We examined RBC membranes using thiol-disulfide exchange chromatography which partitions sodium dodecyl sulfate-solubilized proteins on the basis of reactive thiols, yielding gel-bound (reduced-thiol) and filtrate (oxidized/blocked-thiol) fractions. Membranes from normal RBC partition so that only 13.6 +/- 1.4% of all membrane protein is found in the filtrate fraction. An abnormally increased amount of membrane protein from sickle RBC (21.5 +/- 4.3%) partitions into the filtrate fraction (P less than 0.001). Since sickle RBC do not have high molecular weight aggregates of membrane protein, this indicates abnormal intramolecular thiol oxidation in sickle RBC membranes. Treatment of normal RBC with thiol blockers and oxidants simulates this shift of membrane protein into the filtrate fraction. Analysis using polyacrylamide gel electrophoresis reveals that the filtrate fraction derived from normal RBC consists mostly of band 7 and glycophorins, with only trace amounts of other membrane proteins. Superimposed upon this normal background, sickle RBC filtrates are enriched with all proteins (including cytoskeletal protein bands 1, 2, 2.1, and 4.1), suggesting a generalized oxidative perturbation of sickle RBC membranes. These observations support the concept that excessive RBC autooxidation may play a role in sickle disease pathophysiology, and they perhaps help explain the development of those membrane abnormalities that may reflect cytoskeletal dysfunction in sickle erythrocytes.

Authors

B H Rank, J Carlsson, R P Hebbel

Partial restoration of impaired interleukin-2 production and Tac antigen (putative interleukin-2 receptor) expression in patients with acquired immune deficiency syndrome by isoprinosine treatment in vitro.

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Abstract

The in vitro effects of isoprinosine (ISO) on interleukin-2 (IL-2) production, the expression of Tac antigen (IL-2 receptor) on lymphocytes, and the ability of Leu 3(+) cells to absorb interleukin-1 (IL-1) were investigated in 10 patients with acquired immune deficiency syndrome (AIDS). In 9 of the 10 patients, production of IL-2 from mononuclear cells and Leu 3(+) cells was depressed; expression of Tac antigen on mononuclear cells and Leu 2(+) cells was found to be depressed in 9 of 10 patients. The ability of the Leu 3(+) lymphocytes to absorb IL-1 was depressed in all (four of four) patients studied. After ISO treatment, IL-2 production, Tac antigen expression and IL-1 absorption were restored to normal or near normal levels in most of the patients. These results suggest that ISO has an immunostimulating capacity in AIDS patients and that the potential of ISO in immune response restoration in AIDS patients deserves critical consideration.

Authors

K Y Tsang, H H Fudenberg, G M Galbraith, R P Donnelly, L R Bishop, W R Koopmann

Modulation of procollagen gene expression by retinoids. Inhibition of collagen production by retinoic acid accompanied by reduced type I procollagen messenger ribonucleic acid levels in human skin fibroblast cultures.

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Abstract

Recent clinical observations have suggested that retinoids, which are in frequent use in dermatology, can affect the connective tissue metabolism in skin and other tissues. In this study, we examined the effects of several retinoids on the metabolism of collagen by human skin fibroblasts in culture. Incubation of cultured fibroblasts with all-trans-retinoic acid or 13-cis-retinoic acid, in 10(-5) M or higher concentrations, markedly reduced the procollagen production, as measured by synthesis of radioactive hydroxyproline. The effect was selective in that little, if any, inhibition was noted in the incorporation of [3H]leucine into the noncollagenous proteins, when the cells were incubated with the retinoids in 10(-5) M concentration. Similar reduction in procollagen production was noted with retinol and retinal, whereas an aromatic analogue of retinoic acid ethyl ester (RO-10-9359) resulted in a slight increase in procollagen production in these cultures. The reduction in procollagen production by all-trans-retinoic acid was accompanied by a similar reduction in pro alpha 2(I) of type I procollagen specific messenger RNA (mRNA), as detected by dot blot and Northern blot hybridizations. Hybridizations with human fibronectin and beta-actin specific DNA probes indicated that the levels of the corresponding mRNAs were not affected by the retinoids, further suggesting selectivity in the inhibition of procollagen gene expression. Further control experiments indicated that all-trans-retinoic acid, under the culture conditions employed, did not affect the posttranslational hydroxylation of prolyl residues, the mannosylation of newly synthesized procollagen, the specific radioactivity of the intracellular prolyltransfer RNA pool, or DNA replication. All-trans-retinoic acid also elicited a reduction in trypsin-activatable collagenase, but not in the activity of prolyl hydroxylase or an elastaselike neutral protease in the fibroblast cultures. Incubation of three fibroblast lines established from human keloids with all-trans-retinoic acid or 13-cis-retinoic acid also resulted in a marked reduction in procollagen production. The results, therefore, suggest that further development of retinoids might provide a novel means of modulating collagen gene expression in patients with various diseases affecting the connective tissues.

Authors

H Oikarinen, A I Oikarinen, E M Tan, R P Abergel, C A Meeker, M L Chu, D J Prockop, J Uitto

Nifedipine increases cholesteryl ester hydrolytic activity in lipid-laden rabbit arterial smooth muscle cells. A possible mechanism for its antiatherogenic effect.

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Abstract

Calcium and cholesterol (CHOL) accumulation are characteristic features of human atherosclerotic plaques. Calcium channel blockers have been shown to increase calcium levels in myocardial cells and suppress free and esterified CHOL deposition in arteries of CHOL-fed animals. To test the hypothesis that Nifedipine alters CHOL metabolism, thereby decreasing free and esterified CHOL accumulation in smooth muscle cells (SMC), we cultured arterial SMC from rabbits fed a normal or egg-supplemented diet for 6 mo. Cultured cells were treated with 0.1 mg/liter Nifedipine every 3 d during a 1-wk experiment. Although Nifedipine significantly increased lysosomal and cytoplasmic cholesteryl ester (CE) hydrolase activity in normal SMC via increased levels of intracellular cyclic AMP, no change in total CHOL content was observed after 1 wk of Nifedipine treatment. Contrary to these observations, lipid-laden SMC demonstrated a significant 50% loss in CHOL and CE after treatment with Nifedipine, due in part to the observed increase in CE hydrolytic activities. These data support our hypothesis that Nifedipine decreases CHOL and CE accumulation in arterial SMC by increasing arterial CE hydrolysis.

Authors

O R Etingin, D P Hajjar

An evaluation of the significance of microscopic colitis in patients with chronic diarrhea.

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Abstract

Some patients with chronic idiopathic diarrhea have an apparent nonspecific inflammation of colonic mucosa, even though their colons appear normal by barium enema and colonoscopy. This has been referred to as microscopic colitis. However, the significance of this finding is unclear, because the ability of pathologists to accurately distinguish mild degrees of abnormality has not been established. Furthermore, even if the mucosa of these patients is nonspecifically inflamed, it is not known whether this is associated with deranged colonic function that could contribute to the development of chronic diarrhea. To assess these questions, we first examined colonic biopsy specimens in a blinded fashion, comparing biopsy results from patients with microscopic colitis with biopsy specimens from subjects in two control groups. This analysis revealed that colonic mucosa from six patients with microscopic colitis was in fact abnormal. For example, their mucosa contained an excess of both neutrophiles and round cells in the lamina propria, cryptitis, and reactive changes. These and other differences were statistically significant. Second, colonic absorption, measured by the steady state nonabsorbable marker perfusion method, was severely depressed in the patients. For example, mean water absorption rate was 159 ml/h in normal subjects and was reduced to only 26 ml/h in six patients with microscopic colitis. Results of net and unidirectional electrolyte fluxes and of electrical potential difference suggested that colonic fluid absorption was abnormal because of reduced active and passive sodium and chloride absorption and because of reduced Cl/HCO3 exchange. Small intestinal fluid and electrolyte absorption was abnormally reduced in two of the six patients, suggesting the possibility of coexistent small intestinal involvement in some of these patients. We conclude that nonspecific inflammation of colonic mucosa is associated with a severe reduction of colonic fluid absorption, and that the latter probably contributes to the development of chronic diarrhea.

Authors

G W Bo-Linn, D D Vendrell, E Lee, J S Fordtran

Recognition and characterization of stage-specific oocyst/sporozoite antigens of Toxoplasma gondii by human antisera.

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Abstract

Human infection with Toxoplasma gondii is presumed due to the ingestion of either tissue cysts containing bradyzoites or oocyst/sporozoites that are excreted in the feces of infected cats. The incidence of human infection in the general population by either of these routes is unknown. We have previously described unique stage-specific oocyst/sporozoite antigens identified by murine hybridoma monoclonal antibodies. We obtained acute and convalescent antitoxoplasma antisera from patients in an epidemiologically well-documented outbreak of oocyst-transmitted infection associated with the ingestion of contaminated water. An enzyme-linked immunosorbent assay comparing equal numbers of tachyzoites (invasive stage) and oocyst/sporozoite (excreted stage) indicated that these antisera recognized antigens from both life forms. Absorption of pooled antisera with purified oocyst/sporozoites reduced both the antioocyst immunoglobulin G (IgG) and immunoglobulin M (IgM) titer but had only minimal effect on the antitachyzoite titer. Absorption of the antisera with tachyzoites reduced the IgG and IgM antioocyst and antitachyzoite titer. A sodium dodecyl sulfate-polyacrylamide gel analysis of radioiodinated oocyst/sporozoites shows that the principal stage-specific surface proteins of the oocyst/sporozoite have approximate Mr of 67,000 and 25,000. Periodic acid and silver stain of purified oocyst/sporozoite identified bands of similar molecular weight not present in the tachyzoite preparation. Western blot analysis of purified parasites assayed with human antioocyst antisera identified specific oocyst/sporozoite antigens not present on the tachyzoites. At least two major stage-specific oocyst/sporozoite antigens of approximate Mr of 67,000 and 190,000 were identified by the infected patients' antisera and not by the normal controls. Reaction to these oocyst/sporozoite antigens was seen primarily in the IgM fraction of the acute phase and the IgG fraction of convalescent phase antisera. Neither absorption of the antisera with tachyzoites nor periodate treatment of the oocyst/sporozoites reduced the antibody recognition of these stage-specific antigens. These data suggest that individuals infected by a presumed oocyst-transmitted route develop antibodies against unique stage-specific oocyst/sporozoite antigens.

Authors

L H Kasper, P L Ware

Pharmacologic effects of autacoids on subsets of T cells. Regulation of expression/function of histamine-2 receptors by a subset of suppressor cells.

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Abstract

Autacoids (principally histamine, beta adrenergic catecholamines, and prostaglandins E and A) have only recently been recognized as substantive moderators of a number of immune functions. If autacoids are to be considered as potential therapeutic immunomodulators, it is necessary to understand their effects on subsets of T cells while they are and are not in contact with each other. This report demonstrates that autacoid receptors are nonrandomly distributed on phenotypically and functionally distinct subsets of human T cells. Each human T cell subset responded to both histamine and isoproterenol, but the dose response curve and maximal efficacy varied widely between the subsets. The suppressor T cells were more responsive to both histamine and isoproterenol than helper/inducer T cells (TH) or cytotoxic T cells (Tc). We found that after mitogenic stimulation the response to histamine, but not isoproterenol, was greatly increased only in TH (Leu 3+) and Tc (Leu 2+, 9.3+) subsets, and that this effect may be regulated by suppressor T cells (Leu 2+, 9.3-). The dramatic rise in cAMP accumulation in response to histamine in mitogen-treated TH and Tc was totally blocked by an H2 antagonist (cimetidine), but not by an H1 antagonist (mepyramine). These findings indicate interdependence of (a) immunologically uncommitted subsets in their response to selected drugs, and (b) control of basal- and autacoid-induced cAMP production, as well as (c) increased qualitative and quantitative selectivity, which is caused by mitogen. If we had performed these experiments only on unseparated cells we would not have observed the remarkable selectivity of autacoid effects on subsets of T cells.

Authors

M M Khan, P Sansoni, E G Engleman, K L Melmon

Pulsatile growth hormone secretion in normal man during a continuous 24-hour infusion of human growth hormone releasing factor (1-40). Evidence for intermittent somatostatin secretion.

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Abstract

Growth hormone (GH) secretory patterns were studied in a patient with ectopic growth hormone releasing factor (GRF) secretion and in normal men given continuous infusions of human growth hormone releasing factor (1-40)-OH (hGRF-40). In the patient with ectopic GRF secretion, GH secretion was pulsatile despite continuously elevated immunoreactive GRF levels. To determine if pulsatile GH secretion is maintained in normal subjects, we administered to six healthy young men vehicle or hGRF-40, 2 ng/kg per min, for 24 h and gave a supramaximal intravenous bolus dose of hGRF-40, 3.3 micrograms/kg, after 23.5 h of infusion. hGRF-40 infusion resulted in greater GH secretion than did vehicle infusion and pulsatile GH secretion was maintained throughout hGRF-40 infusion. During the 23.5 h of vehicle infusion, total GH secretion (microgram; mean +/- SEM) was 634 +/- 151 compared with 1,576 +/- 284 during hGRF-40 infusion (P = 0.042). The GH response to the intravenous bolus of hGRF-40 was greater after vehicle infusion than after hGRF-40 infusion; 877 +/- 170 and 386 +/- 125 micrograms of GH was secreted after the bolus on vehicle and hGRF-40 days, respectively (P = 0.015). The total amount of GH secreted during the 25.5 h of the two study days was not different; 1,504 +/- 260 and 1,952 +/- 383 micrograms were secreted during vehicle and hGRF-40 days, respectively (P = 0.36). Not only was pulsatile GH secretion maintained during hGRF-40 infusion, but there was augmentation of naturally occurring GH pulses, which is in contrast to the effect of gonadotropin-releasing hormone on gonadotropin secretion. We suggest that GH pulses are a result of GRF secretion that is associated with a diminution or withdrawal of somatostatin secretion.

Authors

M L Vance, D L Kaiser, W S Evans, R Furlanetto, W Vale, J Rivier, M O Thorner

Effects of platelet-modifying drugs on arterial thromboembolism in baboons. Aspirin potentiates the antithrombotic actions of dipyridamole and sulfinpyrazone by mechanism(s) independent of platelet cyclooxygenase inhibition.

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Abstract

To resolve questions of drug actions, efficacy, and interactions for platelet-modifying agents used clinically, we have compared the relative capacities and mechanisms of aspirin, dipyridamole, sulfinpyrazone, and dazoxiben to prevent arterial thromboembolism in a baboon model. In 136 studies the agents were given twice daily by oral administration both singly and in combination. The antithrombotic efficacy of a given therapy was determined by its capacity to interrupt steady-state platelet utilization induced by thrombogenic arteriovenous cannulae. When given alone, dipyridamole and sulfinpyrazone reduced the rate at which platelets were utilized by thrombus formation in a dose-dependent manner with essentially complete interruption by dipyridamole at 10 mg/kg per d. In contrast, neither aspirin (2-100 mg/kg per d) nor dazoxiben (20-100 mg/kg per d) decreased cannula platelet consumption detectably despite the striking reduction in the capacity of platelets to produce thromboxane B2. However, aspirin, but not dazoxiben, potentiated the antithrombotic effects of dipyridamole and sulfinpyrazone in a dose-dependent fashion without changing the pharmacokinetics for any of the agents. Complete potentiation required aspirin at 20 mg/kg per d to be given with each dose of dipyridamole. Because dazoxiben's blockade of platelet thromboxane A2 production was not associated with antithrombotic potentiation, and because complete potentiation by aspirin required a dose that fully inhibited vascular production of prostaglandin I2 (PGI2), we conclude that aspirin's potentiating effect on dipyridamole is independent of PGI2 production or inhibition of thromboxane A2 formation. In addition, because frequent repeated and synchronous dosing of aspirin was necessary, aspirin's potentiating effects appear to be produced by mechanism(s) unrelated to its potent, irreversible inhibition of platelet cyclooxygenase.

Authors

S R Hanson, L A Harker, T D Bjornsson

(-)-Adrenaline-induced, calcium-dependent phosphorylation of proteins in human platelets.

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Abstract

In human platelets, adrenaline stimulated, approximately four-fold, as compared with controls, the phosphorylation of primarily two proteins of apparent molecular weights of 20,000 and 40,000, respectively. Maximum phosphorylation occurred after incubation for 1 min and was inhibited by the addition of either yohimbine, prostaglandin E1, or EGTA. Phosphorylation of the two proteins was accompanied by diacylglycerol formation. The (-)-adrenaline-induced phosphorylation of proteins corresponds to the activation of a calcium-dependent protein kinase partially purified by DEAE-cellulose and Sephadex G150 column chromatography. The enzymatic activity was modulated by addition of (-)-adrenaline and CaCl2, by diolein, and in the presence of membranes or phosphatidylinositol but not phosphatidylethanolamine and phosphatidylcholine. A phospholipid-dependent reaction appears to be involved in the molecular mechanism of action of adrenaline.

Authors

L H Block, H Jaksche, P Erne, P Bolli, F R Bühler

Regulation of cation content and cell volume in hemoglobin erythrocytes from patients with homozygous hemoglobin C disease.

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Abstract

Erythrocytes from patients with homozygous hemoglobin C disease (CC cells) contain less K, Na, and water than do erythrocytes from normal subjects that contain only hemoglobin A (AA cells). In this paper, we provide evidence that the reduced K content and volume of CC cells are due to the activity in these but not in AA cells of a K transport system that is: (a) insensitive to ouabain and bumetanide, and (b) stimulated by increased cell volume, and dependent on internal pH (pHi). When the cation and water content of CC cells was increased (by making the membrane temporarily permeable to cations with nystatin) and the cells were then incubated in an isotonic medium containing 140 mM NaCl and 4 mM KCl, they lost K and shrunk back toward the original volume. This regulatory K and volume decrease was not inhibited by ouabain or bumetanide. When CC cells were incubated in a hypotonic medium, with ouabain and bumetanide, they also lost K and shrunk toward the original volume. This behavior was not observed in control AA cells. The ouabain- and bumetanide-resistant K efflux from CC cells was volume and pH dependent: K efflux from CC cells rose from 5-6 to 20-25 mmol/liter of cells X h, when cell volume was increased by increasing cell solute content (nystatin method) or by exposure to hypotonic media. In CC cells, the dependence of K efflux on pHo had a bell shape, with a maximal flux (20-25 mmol/liter of cells X h) at pHo 6.8-7.0. In contrast, the K efflux from control cells was minimal at pH 7.4 (1.2 mmol/liter of cells X h) and was slightly stimulated by both acid and alkaline pH. In order to study the effect of pHi and pHo on K efflux, CC cells were incubated with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (150 microM) and acetazolamide (1 mM) at different pHi (6.7, 7.3, and 7.8), and resuspended in media with different pHo (6.75, 7.4, and 8): K efflux was stimulated by reducing pHi but was independent of pHo. The ouabain- and bumetanide-resistant K efflux from CC cells was not inhibited by some inhibitors of the Ca2+-activated K permeability. It seems likely that the genetically determined change in the primary structure of hemoglobin C directly or indirectly causes this modification in K transport. One possible mechanism could involve an electrostatic interaction between C hemoglobin and components of the erythrocyte membrane.

Authors

C Brugnara, A S Kopin, H F Bunn, D C Tosteson

Generation of free radical intermediates from foreign compounds by neutrophil-derived oxidants.

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Abstract

A large number of foreign compounds, including many drugs, industrial pollutants, and environmental chemicals, can be oxidized under appropriate conditions to potentially toxic free radical intermediates. We evaluated the ability of the oxidants produced by the neutrophil myeloperoxidase system to generate free radical intermediates from several such compounds. Sodium hypochlorite or hypochlorous acid produced by human peripheral blood neutrophils and trapped in the form of taurine chloramine were both found to be capable of producing free radicals from chlorpromazine, aminopyrine, and phenylhydrazine. These radical intermediates were demonstrated by visible light spectroscopy and by direct electron spin resonance (for the chlorpromazine and aminopyrine radicals) or by spin-trapping (for the phenyl radical generated from phenylhydrazine). Stable oxidants produced by the neutrophils (i.e., those present in the supernatants of stimulated neutrophils in the absence of added taurine) also were found to be capable of generating free radical intermediates. The production of the oxidants and the ability of neutrophil supernatants to generate these radicals were almost completely eliminated by sodium azide, a myeloperoxidase inhibitor. We suggest that the oxidation by neutrophils of certain chemical compounds to potentially damaging electrophilic free radical forms may represent a new metabolic pathway for these substances and could be important in the processes of drug toxicity and chemical carcinogenesis.

Authors

B Kalyanaraman, P G Sohnle

Role of hepatic autoregulation in defense against hypoglycemia in humans.

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Abstract

To assess the role of hepatic autoregulation in defense against hypoglycemia, we compared the effects of complete blockade of glucose counterregulation with those of blockade of only neurohumoral counterregulation during moderate (approximately 50 mg/dl) and severe (approximately 30 mg/dl) hypoglycemia induced by physiologic hyperinsulinemia during subcutaneous infusion of insulin in normal volunteers. Compared with observations in control experiments, neurohumoral counterregulatory blockade (somatostatin, propranolol, phentolamine, and metyrapone), during which identical moderate hypoglycemia was achieved using the glucose clamp technique, resulted in suppressed glucose production (0.62 +/- 0.08 vs. 1.56 +/- 0.07 mg/kg per min at 12 h, P less than 0.01) and augmented glucose utilization (2.17 +/- 0.18 vs. 1.57 +/- 0.07 mg/kg per min at 12 h, P less than 0.01). Complete blockade of counterregulation (neurohumoral blockade plus prevention of hypoglycemia) did not further enhance the suppressive effects of insulin on glucose production. However, when severe hypoglycemia was induced during neurohumoral counterregulatory blockade, glucose production was nearly two times greater (1.05 +/- 0.05 mg/kg per min at 9 h) than that observed during complete counterregulatory blockade (0.58 +/- 0.08 mg/kg per min at 9 h, P less than 0.01) and that observed during mere neurohumoral blockade with moderate hypoglycemia (0.59 +/- 0.06 mg/kg per min at 9 h, P less than 0.01). These results demonstrate that glucose counterregulation involves both neurohumoral and hepatic autoregulatory components: neurohumoral factors, which require only moderate hypoglycemia for their activation, augment glucose production and reduce glucose utilization; hepatic autoregulation requires severe hypoglycemia for its activation and may thus serve as an emergency system to protect the brain when other counterregulatory factors fail to prevent threatening hypoglycemia.

Authors

G Bolli, P De Feo, G Perriello, S De Cosmo, M Ventura, P Campbell, P Brunetti, J E Gerich

Alpha thalassemia changes erythrocyte heterogeneity in sickle cell disease.

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Abstract

Homozygous alpha-thalassemia has the beneficial effect in sickle cell anemia of reducing the hemolytic severity while changing several other hematological parameters. We examined in detail the cellular basis of some of these hematologic alterations. We find that the broad distribution in erythrocyte density and the large proportion of dense cells associated with sickle cell anemia are both reduced with coexisting alpha-thalassemia. Measurements of glycosylated hemoglobin levels as a function of cell density indicate that the accelerated increase in cell density, beyond normal cell aging, in sickle cell anemia is also reduced with alpha-thalassemia. The patients with homozygous alpha-thalassemia and sickle cell disease have slightly lower levels of hemoglobin F than the nonthalassemic sickle cell patients. Examination of hemoglobin F production revealed that the proportion of hemoglobin F containing reticulocytes remained unchanged, as did the proportion of hemoglobin F in cells containing hemoglobin F (F cells). Preferential survival of F cells occurs in sickle cell anemia, with or without alpha-thalassemia, and the slight difference in hemoglobin F levels appear to reflect differences in numbers of circulating F cells. Thus, in sickle cell disease with coexisting alpha-thalassemia, the change in the erythrocyte density profile, possibly due to inhibition of polymerization-related increases in cell density, explains the hematological improvement.

Authors

C T Noguchi, G J Dover, G P Rodgers, G R Serjeant, S E Antonarakis, N P Anagnou, D R Higgs, D J Weatherall, A N Schechter

Carbon dioxide causes exocytosis of vesicles containing H+ pumps in isolated perfused proximal and collecting tubules.

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Abstract

In the turtle bladder it has recently been shown that CO2 stimulates H+ secretion, at least in part, by causing fusion of vesicles enriched in H+ pumps with the luminal plasma membrane. To test for the presence of this mechanism in the kidney we perfused collecting ducts and proximal straight tubules on the stage of an inverted epifluorescence microscope with fluorescein isothiocyanate dextran (70,000 mol wt) in CO2-free medium. After washout we noted punctate fluorescence in endocytic vesicles in some collecting ducts and in all proximal straight tubule cells. More cells took up fluorescent dextran in outer medullary than in cortical collecting ducts. Using the pH dependence of the excitation spectrum of fluorescein we found the pH of the vesicles to be acid (approximately pH 6). Addition of proton ionophores increased vesicular pH by 0.6 +/- 0.1 U, suggesting that the acidity of the vesicles was caused by H+ pumps. CO2 added to the medium (25 mmHg, pH 7.6 at 37 degrees C) reduced fluorescence intensity by 24 +/- 5% in cortical collecting ducts, 27 +/- 5% in medullary collecting ducts, and 25 +/- 5% in proximal straight tubules. Since this effect was prevented by the prior addition of colchicine to the bath, we believe that CO2 caused a decrease in cytoplasmic fluorescence by stimulating exocytotic fusion of the vesicles and thereby secretion of fluorescent dextran. This exocytotic fusion also occurred when tubules that were loaded with fluorescent dextran at a pCO2 of 37 mmHg were exposed isohydrically to a pCO2 of 114 mmHg; the mean decrease was 53 +/- 4%. We conclude that some cells in the collecting ducts and all cells in the proximal straight tubule incorporate fluorescent dextran into the apical cytoplasmic vesicles and acidify them with H+ pumps. CO2 causes fusion of these vesicles with the luminal membrane, but whether CO2 stimulates H+ secretion by increasing the number of functioning H+ pumps remains to be determined.

Authors

G J Schwartz, Q Al-Awqati

Characterization of antigens from nontypable Haemophilus influenzae recognized by human bactericidal antibodies. Role of Haemophilus outer membrane proteins.

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Abstract

Major outer membrane antigens, proteins, and lipopolysaccharides (LPSs), from nontypable Haemophilus influenzae were characterized and examined as targets for complement-dependent human bactericidal antibodies. Outer membranes from two nontypable H. influenzae isolates that caused otitis media and pneumonia (middle ear and transtracheal aspirates) were prepared by shearing organisms in EDTA. These membranes were compared with membranes prepared independently by spheroplasting and lysozyme treatment of whole cells and found to have: similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the proteins; identical densities (rho = 1.22 g/cm3); and minimal d-lactose dehydrogenase activity indicating purity from cytoplasmic membranes. Outer membranes were solubilized in an LPS-disaggregating buffer and proteins were separated from LPS by molecular sieve chromatography. The SDS-PAGE patterns of outer membrane proteins (OMPs) from the two strains differed in the major band although other prominent bands appeared similar in molecular weight. LPS prepared by hot phenol water extraction of each of the strains contained 45% (pneumonia isolate) and 60% (otitis isolate) lipid (wt/wt), 49% and 50% carbohydrate (wt/wt), respectively, and less than 1%, 3-deoxy-manno octulosonic acid. Immunoglobulin M (IgM) purified from normal human serum (NHS) plus complement was bactericidal for both strains. Purified immunoglobulin G (IgG) from NHS killed the middle ear isolate and immune convalescent IgM from the serum of the patient with pneumonia killed his isolate. NHS or convalescent serum were absorbed with OMPs and LPS (0.6-110 micrograms) from each of the strains and immune specific inhibition of bactericidal antibody activity by each antigen was determined. OMPs from the pulmonary isolate inhibited bactericidal antibody activity directed against the isolate in both NHS (1.5 microgram of antigen) and immune serum (0.75 microgram of antigen). OMPs (60 micrograms) from the ear isolate also inhibited bactericidal activity in the respective immune serum. LPSs exhibited minimal inhibition (greater than 110 micrograms). Three human sera (two normal, one immune) were selectively depleted of 80% of antibody activity against OMPs (measured by enzyme-linked immunosorbent assay) by affinity chromatography using OMPs from the pulmonary isolate coupled to a solid phase. These OMP antibody-depleted sera also showed an 88% reduction of bactericidal activity against this strain. Immunopurified antibody against OMPs eluted from the solid phase was bactericidal.

Authors

H E Gnehm, S I Pelton, S Gulati, P A Rice

Mechanism of insulin resistance in human liver cirrhosis. Evidence of a combined receptor and postreceptor defect.

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Abstract

Insulin resistance in liver cirrhosis may depend on either reduced sensitivity (receptor defect) and/or reduced response to insulin (postreceptor defect). To clarify the mechanism of such resistance, a [3H]glucose infusion (0.2 microCi/min) was performed for 120 min before and during a euglycemic clamp at approximately 100, 1,000, and 10,000 microU/ml steady state plasma insulin concentration in 18 compensated cirrhotics with portal hypertension and impaired glucose tolerance, and 18 healthy volunteers with no family history of diabetes, matched for sex, age, and weight. Mean fasting plasma insulin (29.2 +/- 3.4 SEM vs. 14.8 +/- 1.1 microU/ml) was significantly higher (P less than 0.001) in cirrhotics, while fasting plasma glucose was much the same in the two groups. Glucose use (milligrams per kilogram per minute) was significantly lower in cirrhotics at all three steady state plasma insulin levels: 3.04 +/- 0.34 vs. 7.72 +/- 0.61 (P less than 0.001) at approximately 100; 6.05 +/- 1.07 vs. 11.45 +/- 1.24 (P less than 0.001) at approximately 1,000; and 11.69 +/- 0.69 vs. 14.13 +/- 0.74 (P less than 0.05) at approximately 10,000 microU/ml. Mean plasma C-peptide was significantly higher in cirrhotics both basally and during the steady states (P less than 0.001); it was completely suppressed at approximately 10,000 microU/ml in controls and only 57.5% of the baseline in cirrhotics. Endogenous glucose production (milligrams per kilogram per minute) was much the same in the two groups in the fasting state and almost entirely suppressed in the controls (0.10 +/- 0.05 vs. 0.48 +/- 0.11, P less than 0.001) at approximately 100 microU/ml; at approximately 1,000 microU/ml a residual glucose production, 0.07 +/- 0.05, was observed in the cirrhotics only. In addition, insulin binding and 3-ortho-methyl-glucose transport were studied in vitro in six cirrhotics and six controls. Insulin binding to circulating monocytes and isolated adipocytes was significantly lower (P less than 0.025) in cirrhotics in all insulin concentration studies. Glucose transport values on isolated adipocytes were significantly lower in cirrhotics both basally (P less than 0.001) and at maximal insulin concentration (P less than 0.05). These results suggest that insulin resistance in human cirrhosis is more dependent on depressed peripheral glucose use than on increased endogenous glucose production, and that a combined receptor and postreceptor defect in insulin action on target cells seems to be present.

Authors

P Cavallo-Perin, M Cassader, C Bozzo, A Bruno, P Nuccio, A M Dall'Omo, M Marucci, G Pagano

Diarrhea in streptozocin-treated rats. Loss of adrenergic regulation of intestinal fluid and electrolyte transport.

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Abstract

Diarrhea was noted in rats with streptozocin-induced chronic diabetes. We have investigated the possibility that this diarrhea is a consequence of altered neuronal control of water and electrolyte absorption in the intestinal epithelium. In particular, we examined noradrenergic control because alpha-2-adrenergic agonists are known to stimulate intestinal fluid absorption. When compared with nondiabetic littermates, chronically diabetic rats exhibited significant impairment of fluid absorption by the ileum and colon, but not the jejunum. This impairment of intestinal fluid absorption was not found in either insulin-treated or untreated acutely diabetic (7 d) animals. Mucosal histology appeared normal in all of the above groups. Mucosal norepinephrine stores in the jejunum and ileum of chronically diabetic rats were estimated in vitro by the short-circuit current (Isc) response to tyramine, an agent that effectively releases stored norepinephrine. Pargyline was added to inhibit enzymatic destruction of the added tyramine. In chronically diabetic rats, the Isc response to tyramine was significantly decreased in ileum, but not in jejunum. However, when these responses were expressed as a fraction of the maximal Isc tissue response to exogenously added epinephrine, significant decreases were noted in both ileum and jejunum. In tissues from acutely diabetic rats, Isc responses to tyramine and epinephrine were no different from controls. When sympathetic denervation was produced in nondiabetic rats by treatment with 6-OH-dopamine, the pattern of impaired fluid absorption that developed was the same as that observed in chronically diabetic rats. We conclude that impaired intestinal mucosal absorption of fluid and electrolytes slowly develops in rats made diabetic with streptozocin and that this absorptive impairment is due to a loss of normally present noradrenergic innervation of enterocytes.

Authors

E B Chang, R M Bergenstal, M Field

Use of a murine monoclonal antibody for detection of circulating plasma DF3 antigen levels in breast cancer patients.

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Abstract

The murine monoclonal antibody (MAb), designated DF3, reacts with a 300,000-mol wt mammary epithelial antigen. A sequential double-determinant radioimmunoassay (RIA) has been developed to monitor circulating DF3 antigen. Using this assay, we have demonstrated that 33 of 36 normal women had plasma RIA antigen levels less than 150 U/ml. In contrast, 33 of 43 patients (76%) with metastatic breast cancer had RIA DF3 antigen levels greater than or equal to 150 U/ml. The difference between these two groups was statistically significant (P less than 0.001). Similar results have been obtained with a double-determinant enzyme-linked immunoassay (EIA). Only 6 of 111 age-matched normal subjects had EIA DF3 antigens levels greater than or equal to 30 U/ml, while 42 of 58 patients (72%) with breast cancer had levels equal to or above this value. Thus, similar patterns of specificity are obtained with the EIA or RIA. The elevation of circulating DF3 antigen levels in breast cancer patients has been confirmed by transfer blot assays. MAb DF3 reactivity occurred predominantly with circulating antigens of three different molecular weights ranging from 300,000 to approximately 400,000 mol wt. We also demonstrate that patients with both primary and metastatic breast cancer who were free of detectable disease at the time of sampling have DF3 antigen levels that are similar to those obtained from normal subjects. While patients with hepatoma (27%) and ovarian carcinoma (47%) also had elevated circulating DF3 antigen levels, the results suggest that DF3 antigen levels may be useful in distinguishing breast cancer patients from those with esophageal, gastric, colorectal, pancreatic, and lung carcinomas. Furthermore, the results of the RIA, EIA, and transblot analyses demonstrate that the measurement of circulating DF3 antigen levels provides a new and potentially useful marker to follow the clinical course of patients with metastatic breast cancer.

Authors

D F Hayes, H Sekine, T Ohno, M Abe, K Keefe, D W Kufe

Histamine modulates contraction and cyclic nucleotides in cultured rat mesangial cells. Differential effects mediated by histamine H1 and H2 receptors.

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Abstract

Histamine influences the glomerular microcirculation and modulates immune-inflammatory responses. In the rat kidney, histamine is synthesized by glomeruli and stimulates cyclic nucleotide production specifically in glomeruli. We investigated the in vitro effect of histamine on cyclic nucleotide accumulation in rat cultured glomerular mesangial and epithelial cells. Histamine stimulated cyclic AMP (cAMP) accumulation in cultured mesangial cells (64.0 +/- 22.1 to 511.4 +/- 86.6 pmol/mg protein, n = 9) but had no effect on cAMP accumulation in epithelial cells. This effect was dose-dependent and time-dependent. Stimulation of cAMP accumulation occurred in the range of 5 X 10(-6) M-10(-4) M histamine with a half maximal stimulatory effect of 2 X 10(-5) M. Initial stimulation was noted by 30 s, and maximum stimulation was observed at 5 min. The H2 antagonist cimetidine (10(-4) M) abolished the stimulatory effect of histamine (10(-4) M), while equimolar concentrations of the H1 antagonist diphenhydramine had no significant effect on cAMP accumulation. Moreover, the specific H2 agonist dimaprit, but not the H1 agonist 2-pyridylethylamine, stimulated cAMP accumulation. Histamine had no effect on cAMP accumulation in epithelial cells or on cyclic guanosine monophosphate accumulation in epithelial or mesangial cells. Since the in vivo infusion of histamine reduces ultrafiltration coefficient and since mesangial cell contraction is thought to be responsible for the reduction in the ultrafiltration coefficient, we examined the effect of histamine on the contractile property of mesangial cells. Histamine (5 X 10(-6)-10(-4) M) contracted mesangial cells, and the H1 antagonist diphenhydramine (10(-4) M) but not the H2 antagonist cimetidine (10(-4) M) prevented histamine (10(-4) M) induced contraction. In addition, the H1 agonist 2-pyridylethylamine, but not the H2 agonist dimaprit, contracted mesangial cells. Histamine and its specific agonists and antagonists induced contraction of isolated glomeruli as assessed by glomerular planar surface area in a manner parallel to their effect on mesangial cells. Cinnarizine (10(-5) M), a Ca++ channel blocker, or Ca++, Mg++-free medium prevented histamine (10(-4) M) induced mesangial cell and glomerular contraction. Thus, histamine enhances cAMP accumulation specifically in mesangial cells via an H2 receptor. In contrast, histamine contracts mesangial cells and glomeruli via an H1 receptor, an effect that is dependent on extracellular Ca++ entry. These findings show that histamine potentially influences intraglomerular hemodynamics via effects on mesangial cell contraction. Moreover, our findings considered with the in vivo observation that histamine reduces kf via and H1 receptor provide further support of the hypothesis that mesangial cell contraction regulates the glomerular capillary surface area available for filtration. Our studies also show that this contractile effect of histamine is dependent on extracellular calcium. The presence of a cAMP system sensitive to histamine may have major implications in the pathogenesis of inflammatory glomerulopathies. Mesangial cells possess characteristics similar to circulating and tissue immune effector cells, including lysosomal enzyme release, oxygen radical production, and release of a number of immunomodulatory factors. Histamine and cAMP have been shown to modulate such characteristics of inflammatory cells. It is therefore conceivable that histamine, via its interaction with H2 receptors and subsequent generation cAMP, may have profound effects on such properties of mesangial cells, suggesting that this autacoid may modulate not only glomerular hemodynamics but also immune, inflammatory responses within the glomerulus.

Authors

J R Sedor, H E Abboud

Use of SM-1 monoclonal antibody and human complement in selective killing of small cell carcinoma of the lung.

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Abstract

SM-1 is a murine monoclonal antibody strongly reactive with a cell membrane antigen of small cell carcinoma (SCC) of the lung but unreactive with the membrane of most other carcinomas and normal tissues including normal bone marrow. We have found that in the presence of human complement, SM-1 antibody is highly cytotoxic to SCC cells. Using three treatments with antibody and complement, more than 99% of SCC cells in culture were lysed, as determined by the chromium release and clonogenic assays. Similar efficiency of SCC cell lysis was observed when one SM-1 antibody treatment was followed by three treatments with human complement. In contrast, there was little antibody-dependent lysis of non-small cell lung cancer cells, other carcinomas, and leukemia cell lines. The amount of chromium released from normal bone marrow cells treated with SM-1 antibody and complement was minimal and was mainly due to the effect of complement alone. Clonogenic assays, including colony-forming unit-granulocytic/monocytic, erythroid burst-forming unit, and colony-forming unit-granulocytic/erythroid/monocytic/megakaryocytic, also showed no significant SM-1 antibody-dependent cytotoxicity on normal bone marrow precursors. Since SM-1 antibody is selectively cytotoxic to SCC cells in the presence of human complement, it is a potentially useful agent for the selective eradication of tumor cell contamination in marrows of patients with metastatic small cell lung cancer and possibly for in vivo serotherapy.

Authors

M Mabry, J A Speak, J D Griffin, R A Stahel, S D Bernal

Arginine vasotocin in ovine fetal blood, urine, and amniotic fluid.

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Abstract

Arginine vasotocin ([8-arginine]-oxytocin) (AVT), the primary antidiuretic principle in submammalian vertebrates, has been reported to be present in mammalian pituitary and pineal glands. Although the most phyletically ubiquitous of the known neurohypophysial peptides, AVT is still not recognized as a mammalian hormone. We examined plasma, urine, and amniotic fluid from fetal lambs by radioimmunoassay (RIA) for evidence of AVT to assess the possibility of its being such a hormone. Measureable quantities of AVT-like immunoreactivity (irAVT) were observed in fetal plasma (2.4 +/- 0.2 pg/ml), urine (1.4 +/- 0.2 pg/ml), and amniotic fluid (1.9 +/- 0.2 pg/ml). Since the AVT antiserum shows minimal cross-reactivity with arginine vasopressin (AVP) and oxytocin (OT), measurements of AVP and OT concentrations in the same biological fluids also were conducted with specific antisera. The results suggest that the irAVT was not accountable on the basis of cross-reaction. To further verify the identity of the irAVT, a high pressure liquid chromatography system using RIA as a means of detection was developed. This system is sufficiently sensitive to allow the separation and quantitation of picogram quantities of the synthetic peptides AVT, AVP, and OT. In this system, the irAVT in fetal plasma, urine, and amniotic fluid appeared as a single peak coeluting with synthetic AVT. These results indicate that AVT is present in ovine fetal plasma and support the view that the fetus secretes this peptide. The physiological significance of circulating AVT remains to be defined.

Authors

M G Ervin, R D Leake, M G Ross, G C Calvario, D A Fisher

Mechanism of action of gemfibrozil on lipoprotein metabolism.

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Abstract

Gemfibrozil is a potent lipid regulating drug whose major effects are to increase plasma high density lipoproteins (HDL) and to decrease plasma triglycerides (TG) in a wide variety of primary and secondary dyslipoproteinemias. Its mechanism of action is not clear. Six patients with primary familial endogenous hypertriglyceridemia with fasting chylomicronemia (type V lipoprotein phenotype) with concurrent subnormal HDL cholesterol levels (HDL deficiency) were treated initially by diet and once stabilized, were given gemfibrozil (1,200 mg/d). Each patient was admitted to the Clinical Research Center with metabolic kitchen facilities, for investigation of HDL and TG metabolism immediately before and after 8 wk of gemfibrozil treatment. Gemfibrozil significantly increased plasma HDL cholesterol, apolipoprotein (apo) AI, and apo AII by 36%, 29%, and 38% from base line, respectively. Plasma TG decreased by 54%. Kinetics of apo AI and apo AII metabolism were assessed by analysis of the specific radioactivity decay curves after injection of autologous HDL labeled with 125I. Gemfibrozil increased synthetic rates of apo AI and apo AII by 27% and 34%, respectively, without changing the fractional catabolic rates. Stimulation of apo AI and apo AII synthesis by gemfibrozil was associated with the appearance in plasma of smaller (and heavier) HDL particles as assessed by gradient gel electrophoresis and HDL composition. Postheparin extra-hepatic lipoprotein lipase activity increased significantly by 25% after gemfibrozil, and was associated with the appearance in plasma of smaller very low density lipoprotein particles whose apo CIII:CII ratio was decreased. These data suggest that gemfibrozil increases plasma HDL levels by stimulating their synthesis. Increased transport (turnover) of HDL induced by gemfibrozil may be significant in increasing tissue cholesterol removal in these patients.

Authors

K Saku, P S Gartside, B A Hynd, M L Kashyap

Abnormality of phospholipid transverse diffusion in sickle erythrocytes.

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Abstract

We have used spin-labeled analogues of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to compare the transverse diffusion rates of lipids in normal and sickle erythrocytes. The beta-chain of the spin-labeled lipids was a short chain (five carbons) providing the spin labels with a relative water solubility, and hence permitting their rapid incorporation into cell membranes. The orientation of the labeled lipids in the membranes was assayed by selective chemical reduction of the nitroxide labels embedded in the outer leaflet. We have found that all three spin-labeled phospholipids are initially incorporated in the outer leaflet. Upon incubation at 4 degrees C the aminophospholipids, not the phosphatidylcholine, diffuse toward the inner leaflet within 3 h. The transverse diffusion rate of aminophospholipids is reduced by 41% (phosphatidylserine) and 14% (phosphatidylethanolamine) in homozygote sickle cells (SS) when compared with normal cells (AA) or heterozygote cells (AS or SC). At equilibrium the asymmetric distribution of spin-labeled phospholipids resulting from this selective diffusion is also reduced in SS cells when compared with AA, SC, or AS cells. This reduced asymmetry was not found in a reticulocyte-rich blood sample (hemoglobin A), indicating that the age of the cell cannot be responsible for this phenomenon. Moreover, because at low temperatures the sickling process does not occur, the observed perturbations in phospholipid organization reflect preexisting membrane abnormalities in sickle cells. Ghosts loaded with ATP give the same results. Varying the concentration of intracellular calcium had no effect on lipid diffusion, except at very high free calcium concentrations (3 microM) when diffusion was practically abolished. We suggest that membrane protein alterations may be part of the explanation of the observed abnormalities.

Authors

A Zachowski, C T Craescu, F Galacteros, P F Devaux

Immunization with a Plasmodium falciparum merozoite surface antigen induces a partial immunity in monkeys.

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Abstract

Saimiri monkeys immunized with a Plasmodium falciparum merozoite polypeptide of 41 kD mol wt are resistant to a blood challenge infection that induces a fulminant infection in control monkeys. The sera of the immunized monkeys reacted, as shown by the indirect immunofluorescence technique, with the apical part of the merozoites from five isolates or clones of P. falciparum. Whether the immunogen was dissolved in nonionic detergent (NP-40) or in sodium dodecyl sulfate (SDS) had a marked influence on the level of protection in immunized monkeys. Thus, monkeys immunized with the antigen solubilized in a nonionic detergent developed much lower parasitemia than monkeys immunized with denatured antigen (antigen eluted from SDS polyacrylamide gel electrophoresis).

Authors

L H Perrin, B Merkli, M S Gabra, J W Stocker, C Chizzolini, R Richle

Cross-reactive idiotypes in immunoglobulin A-deficient sera.

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Abstract

Mice of inbred strains immunized with simple antigens can produce antibodies that share similar V regions, which result in serologic similarities called cross-reactive idiotypes (CRI). In this study, we considered the possibility that IgA-deficient humans, who are continuously immunized via the intestinal tract by dietary protein, might also produce antibodies sharing CRI. For this, anti-casein antibodies were isolated from the blood of 16 adult IgA-deficient donors (4 Finns and 12 North Americans) and an autologous anti-anti-casein from the blood of one of the Finnish donors. In addition, a heterologous anti-anti-casein was raised to the casein-anti-casein immune complexes of this donor. Comparing the activities of the two anti-idiotypes, it was found that both bind anti-casein in the region of the antigen binding site, but that each binds additional determinants not located within this region, with the heterologous reagent having more affinity for these latter determinants than the autologous anti-idiotype. Using both reagents in enzyme-linked immunosorbent assay inhibition assays, extensive cross-reactivities between anti-caseins were demonstrated. Using the autologous anti-idiotype, 5 of 16 anti-caseins were found to share CRI, and with the heterologous reagent 12 of 16 shared CRI. In both assays, the anti-caseins of Finnish donors displayed more cross-reactivity than those derived from Northern American donors. These studies show that specific, commonly shared CRI can be identified in this human system in which antibodies are raised as a result of natural immunization across the gastrointestinal mucosa.

Authors

M K Cheung, C Cunningham-Rundles, J Koistinen

Bovine milk lipoprotein lipase transfers tocopherol to human fibroblasts during triglyceride hydrolysis in vitro.

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Abstract

Lipoprotein lipase appears to function as the mechanism by which dietary vitamin E (tocopherol) is transferred from chylomicrons to tissues. In patients with lipoprotein lipase deficiency, more than 85% of both the circulating triglyceride and tocopherol is contained in the chylomicron fraction. The studies presented here show that the in vitro addition of bovine milk lipoprotein lipase (lipase) to chylomicrons in the presence of human erythrocytes or fibroblasts (and bovine serum albumin [BSA]) resulted in the hydrolysis of the triglyceride and the transfer of both fatty acids and tocopherol to the cells; in the absence of lipase, no increase in cellular tocopherol was detectable. The incubation system was simplified to include only fibroblasts, BSA, and Intralipid (an artificial lipid emulsion containing 10% soybean oil, which has gamma but not alpha tocopherol). The addition of lipase to this system also resulted in the transfer of tocopherol (gamma) to the fibroblasts. Addition of both lipase and its activator, apolipoprotein CII, resulted in a further increase in the cellular tocopherol content, but apolipoprotein CII alone had no effect. Heparin, which is known to prevent the binding of lipoprotein lipase to the cell surface membrane, abrogated the transfer of tocopherol to fibroblasts without altering the rate of triglyceride hydrolysis. Thus, in vitro tocopherol is transferred to cells during hydrolysis of triglyceride by the action of lipase, and for this transfer of tocopherol to occur, the lipase itself must bind to the cell membrane.

Authors

M G Traber, T Olivecrona, H J Kayden

Activation of the respiratory burst enzyme from human neutrophils in a cell-free system. Evidence for a soluble cofactor.

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Abstract

Activation of the respiratory burst in phagocytic cells, an important host defense process, is not yet well understood. We now report the development of a cell-free system for activation of NADPH oxidase, the respiratory burst enzyme, in human neutrophils. Activation was achieved by the addition of arachidonic acid to a postnuclear supernatant (500 g) from disrupted unstimulated cells (no arachidonate, 0.2; with arachidonate, 3.4 nmol superoxide anion/min per mg) and was dependent on both the concentration of arachidonate and on the amount of cellular material present. Activity stimulated by arachidonate appeared to be NADPH oxidase based on a Michaelis constant for NADPH of 32 microM and a pH optimum of 7.0-7.5. Separation of the 500-g supernatant by high speed centrifugation revealed a requirement for both soluble and particulate cofactors. Activation of NADPH oxidase by arachidonate did not occur in the high speed pellet fraction from unstimulated cells but could be restored by the addition of the high speed supernatant. In addition, priming of intact neutrophils with low concentrations of the chemoattractant N-formyl-methionyl-leucyl-phenylalanine or the tumor promoter phorbol myristate acetate replaced the soluble factor requirement for NADPH oxidase activation by arachidonate in the high speed pellet. This cell-free system can now be used to provide further insight into the biochemical basis of priming and the terminal mechanisms involved in the activation of NADPH oxidase.

Authors

L C McPhail, P S Shirley, C C Clayton, R Snyderman

Activation of human neutrophil nicotinamide adenine dinucleotide phosphate, reduced (triphosphopyridine nucleotide, reduced) oxidase by arachidonic acid in a cell-free system.

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Abstract

Sonicates from unstimulated human neutrophils produce no measurable superoxide since the superoxide-generating enzyme, NADPH oxidase, is inactive in these preparations. Previous attempts to activate the oxidase in disrupted cells with conventional neutrophil stimuli have been unsuccessful. This report describes a cell-free system in which arachidonic acid (82 microM) was able to activate superoxide generation that was dependent upon the presence of NADPH and the sonicate. For activation to occur, both the particulate and supernatant fractions of the sonicate must be present. Calcium ions, which are required for activation of intact neutrophils by arachidonate, were not necessary in the cell-free system. In quantitative terms, the superoxide-generating activity in the cell-free system could account for at least 20-50% of the superoxide rate observed in intact neutrophils stimulated with arachidonate. Sonicates from patients with chronic granulomatous disease (CGD) could not be activated by arachidonic acid in the cell-free system. In three patients representing both genetic forms of CGD, the defect appeared to reside in the particulate fraction. The soluble cofactor was normal in all three patients and could be used to activate normal neutrophil pellets in the presence of arachidonic acid. Thus, at least a portion of the activation mechanism in the neutrophil, that residing in the soluble phase, appeared to be normal in patients with CGD.

Authors

J T Curnutte