CORRECTION

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Abstract

Authors

Canine renal receptors for parathyroid hormone. Down-regulation in vivo by exogenous parathyroid hormone.

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Abstract

Chronic elevation of circulating parathyroid hormone (PTH) is associated with decreased target cell responsiveness to PTH. To study the subcellular mechanism of this phenomenon we evaluated PTH receptors and adenylate cyclase activity in renal cortical membranes prepared before and after infusion of bovine parathyroid gland extract (PTE) into thyroparathyroidectomized dogs. PTE infusion resulted in a 53% decrease in the number of high-affinity receptors (P less than 0.01) associated with a 66% decrease in PTH-stimulated adenylate cyclase (P less than 0.01) relative to paired base-line values. Both the equilibrium constant of dissociation (KD) for PTH binding and the concentration of PTH that caused half-maximal stimulation of adenylate cyclase were in the range of 1 to 4 nM, and were unaffected by the PTE infusion. Responsiveness of the renal adenylate cyclase to sodium fluoride was 88% of base-line values. Infusion of the PTE vehicle alone did not affect PTH receptor number or blunt the adenylate cyclase response to PTH. Pretreatment of the membranes made after PTE infusion with guanosine triphosphate (GTP), which is known to produce dissociation of receptor-bound PTH, failed to restore either receptor number or PTH-stimulated adenylate cyclase. This finding was not due to a lack of efficacy of the GTP pretreatment, because identical GTP pretreatment restored PTH binding to base-line values in membranes partially occupied by incubation with PTH in vitro. Thus, simple residual occupancy of PTH receptors by the infused hormone did not appear to account for the observed receptor loss. The results of this study suggest that target cell resistance to PTH in patients with hyperparathyroidism might occur, at least in part, due to down-regulation of PTH receptors by circulating hormone.

Authors

C A Mahoney, R A Nissenson

Effects of dietary-induced hyperparathyroidism on the parathyroid hormone-receptor-adenylate cyclase system of canine kidney. Evidence for postreceptor mechanism of desensitization.

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Abstract

The present studies were designed to examine the consequences of chronic mild elevations of endogenous parathyroid hormone (PTH) in vivo on the PTH receptor-adenylate cyclase system of canine kidney cortex. Hyperparathyroidism was induced in normal dogs by feeding a diet low in calcium, high in phosphorus to the animals for a period of 6-9 wk. This maneuver resulted in a two to threefold increase in the plasma levels of carboxy-terminal immunoreactive PTH. This degree of hyperparathyroidism is similar to that seen in patients with hyperparathyroidism and normal renal function. After 6-9 wk on the diet the animals were killed and basolateral renal cortical membranes prepared for the study of the PTH receptor-adenylate cyclase system in vitro. The dietary hyperparathyroidism resulted in desensitization of the PTH-responsive adenylate cyclase (Vmax 3,648 +/- 654 pmol cyclic (c)AMP/mg protein per 30 min in hyperparathyroid animals vs. 5,303 +/- 348 in normal controls). The Kact (concentration of PTH required for half-maximal enzyme activation) was unchanged. However, PTH receptor binding (125I-norleucyl8-norleucyl18-tyrosinyl34, 125I[Nle8, Nle18, Tyr34] bPTH (1-34) NH2 as radioligand) was not different in the two groups of animals. Thus, dietary hyperparathyroidism resulted in an uncoupling of the PTH receptor-adenylate cyclase system. This defect was not corrected by guanyl nucleotides in vitro, and the effects of guanyl nucleotides on PTH binding and enzyme activation appeared normal. NaF-stimulated enzyme activity was reduced in the hyperparathyroid animals (8,285 +/- 607 pmol cAMP/mg protein per 30 min vs. 10,851 +/- 247 in controls). These data indicate that desensitization of the PTH-responsive adenylate cyclase system of canine kidney as a result of mild chronic elevations of endogenous PTH is due to a postreceptor defect, demonstrable by NaF activation, not corrected by guanyl nucleotides, leading to abnormal PTH-receptor adenylate cyclase coupling.

Authors

J Tamayo, E Bellorin-Font, K J Martin

Chronic hyperglycemia with secondary hyperinsulinemia inhibits the maturational response of fetal lamb lungs to cortisol.

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Abstract

I tested the hypothesis that chronic hyperglycemia with secondary hyperinsulinemia inhibits the stimulation of fetal lung maturation by cortisol. Glucose was infused (16 +/- 2 mg/kg per min, mean +/- SE) from 112 through 130 d gestation into five chronically catheterised twin fetal lambs from which tracheal fluid could be collected. In addition, cortisol was infused (420 micrograms/h) from 128 through 130 d gestation into both the five glucose-treated twins and the five twin controls. Serum glucose (48 +/- 2 mg/dl) and insulin levels (45 +/- 3 microU/ml) were significantly higher in the glucose-treated fetuses than serum glucose (23 +/- 2 mg/dl, P less than 0.001) and insulin (15 +/- 3 microU/ml, P less than 0.001) in the controls. Serum cortisol levels were less than 2 micrograms/dl before 128 d gestation and rose to greater than 6 micrograms/dl, P less than 0.001 during cortisol infusion in both the glucose-treated and control fetuses. Cortisol treatment of control fetuses was associated with a 4.8-fold increase in surface active material (SAM) flux into tracheal fluid, and a 7.7-fold increase in total phospholipid content, a 9.5-fold increase in mixed lecithin content, a 10.5-fold increase in disaturated phosphatidylcholine content, and a 5.6-fold increase in phosphatidylglycerol content of the tracheal fluid (all P less than 0.001). In the glucose-treated fetuses there were no significant changes in the tracheal fluid SAM flux and phospholipid content following cortisol administration. In lung wash from the control fetuses treated with cortisol there was 8.9-fold more SAM, and on thin-layer chromatography there was 5.6-fold more total phospholipids, 3.9-fold more mixed lecithin, 6.2-fold more disaturated phosphatidylcholine, and 2.5-fold more phosphatidylglycerol when compared with lung wash from the glucose-treated fetuses treated with cortisol (all P less than 0.001). Lung volumes at maximal inflation pressure during air pressure-volume studies were 1.8-fold greater in the cortisol-treated control fetuses than in the glucose-treated fetuses, P less than 0.025. Chronic hyperglycemia with secondary hyperinsulinemia inhibits the maturational response of fetal lamb lungs to cortisol. A similar mechanism may operate in utero to increase the incidence of respiratory distress syndrome in infants of diabetic mothers with poor maternal glucose homeostasis. Moreover, on the basis of these data, prenatal treatment of infants of diabetic mothers with corticosteroids might not be expected to enhance fetal lung maturation.

Authors

D Warburton

Myeloperoxidase-dependent effect of amines on functions of isolated neutrophils.

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Abstract

Isolated neutrophilic leukocytes were incubated with primary amines and related nitrogenous compounds. Stimulation of neutrophil oxygen (O2) metabolism with phorbol myristate acetate or opsonized zymosan resulted in production of hydrogen peroxide (H2O2), myeloperoxidase-catalyzed oxidation of chloride (C1-) to hypochlorous acid (HOC1), and the reaction of HOC1 with the added compounds to yield nitrogen-chlorine (N-C1) derivatives. Formation of N-C1 derivatives of low lipid solubility resulted in accumulation of the derivatives in the extracellular medium. These oxidizing agents were identified and measured on the basis of their absorption spectra and their ability to oxidize 5-thio-2-nitrobenzoic acid to the disulfide form. The yield of N-Cl derivatives was in the order: taurine greater than Tris greater than spermidine greater than spermine greater than glucosamine greater than putrescine greater than guanidinoacetate. Accumulation of N-C1 derivatives was also observed in the absence of added amines, owing to the reaction of HOC1 with endogenous taurine and other amines that were released from the cells into the medium. In the presence of compounds that yield lipophilic N-C1 derivatives, little or no accumulation of oxidizing agents was observed. Instead, these compounds inhibited the accumulation of N-C1 derivatives that was obtained with taurine, and their effect was competitive with taurine. Inhibition was in the order: methylamine greater than ethanolamine greater than phenylethylamine greater than p-toluenesulfonamide greater than ammonia greater than guanidine. Formation of lipophilic N-C1 derivatives also resulted in inhibition of O2 uptake and glucose metabolism. Inhibition was prevented by adding catalase to eliminate H2O2, dapsone to inhibit myeloperoxidase, taurine to compete for reaction with HOC1, or compounds that are rapidly oxidized by HOC1 or N-C1 derivatives, to reduce these oxidizing agents. The results indicate that: (a) formation of N-C1 derivatives that do not penetrate biological membranes can protect leukocytes against the cytotoxicity of HOC1 and lipophilic N-C1 derivatives, and (b) formation of membrane-permeable N-C1 derivatives in the absence of target cells or readily oxidized substances results in oxidative attack by the N-C1 derivatives on leukocyte components and inhibition of leukocyte functions.

Authors

E L Thomas, M B Grisham, M M Jefferson

Regulation of vascular prostaglandin synthesis by metabolites of arachidonic acid in perfused rabbit aorta.

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To address the hypothesis that metabolites of arachidonic acid are important regulators of prostaglandin (PG) synthesis in intact vascular tissue, we studied arachidonate metabolism in rabbit aortas in response to a continuous infusion of arachidonic acid, 10 micrograms/ml. Prostacyclin (PGI2; measured as 6-keto-PGF1 alpha) production rate accelerated during the first 2 min, reached peak velocity at 2 min, and then progressively decelerated. The velocity profile of PGI2 production was similar to that previously reported for cyclooxygenase holoenzyme assayed in vitro, and was consistent with progressive inactivation of the enzymes leading to PGI2 synthesis. We determined the specific inhibition of cyclooxygenase and prostacyclin synthetase by measuring PGI2 and PGE2 production rates and by infusing cyclic endoperoxides. Our results indicate preferential inactivation of cyclooxygenase during arachidonate metabolism, most likely due to cyclooxygenase-derived oxidative intermediates. This was a dose-dependent response and resulted in a progressive decrease in the 6-keto-PGF1 alpha/PGE2 ratio. Exogenously added 15-hydroperoxy eicosatetraenoic acid, on the other hand, actually stimulated cyclooxygenase activity at low doses, while markedly inhibiting prostacyclin synthetase. This finding, along with the accelerating nature of arachidonate metabolism, is consistent with the concept of "peroxide tone" as a mediator of cyclooxygenase activity in this system. These results demonstrate that arachidonate metabolites regulate PG synthesis in intact blood vessels. The progressive enzymatic inhibition intrinsic to arachidonate metabolism may be a model for similar changes occurring in states of enhanced lipid peroxidation. These metabolic alterations might greatly influence the numerous vascular functions known to involve arachidonic acid metabolism.

Authors

R S Kent, S L Diedrich, A R Whorton

Angiotensin II-induced hypertension in the rat. Effects on the plasma concentration, renal excretion, and tissue release of prostaglandins.

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Abstract

We examined in rats the effects of intraperitoneal angiotensin II (AII) infusion for 12 d on urinary excretion, plasma concentration, and in vitro release of prostaglandin (PG) E2 and 6-keto-PGF1 alpha, a PGI2 metabolite. AII at 200 ng/min increased systolic blood pressure (SBP) progressively from 125 +/- 3 to 170 +/- 9 mmHg (P less than 0.01) and elevated fluid intake and urine volume. Urinary 6-keto-PGF1 alpha excretion increased from 38 +/- 6 to 55 +/- 5 and 51 +/- 7 ng/d (P less than 0.05) on days 8 and 11, respectively, of AII infusion, but urinary PGE2 excretion did not change. Relative to a control value of 129 +/- 12 pg/ml in vehicle-infused (V) rats, arterial plasma 6-keto-PGF1 alpha concentration increased by 133% (P less than 0.01) with AII infusion. Aortic rings from AII-infused rats released more 6-keto-PGF1 alpha (68 +/- 7 ng/mg) during 15-min incubation in Krebs solution than did rings from V rats (40 +/- 3 ng/mg); release of PGE2, which was less than 1% of that of 6-keto-PGF1 alpha, was also increased. Slices of inner renal medulla from AII-infused rats released more 6-keto-PGF1 alpha (14 +/- 1 ng/mg) during incubation than did slices from V rats (8 +/- 1 ng/mg, P less than 0.05), but PGE2 release was not altered. In contrast, AII infusion did not alter release of 6-keto-PGF1 alpha or PGE2 from inferior vena cava segments or from renal cortex slices. Infusion of AII at 125 ng/min also increased SBP, plasma 6-keto-PGF1 alpha concentration, and in vitro release of 6-keto-PGF1 alpha from rings of aorta and renal inner medulla slices; at 75 ng/min AII had no effect. SBP on AII infusion day 11 correlated positively with both 6-keto-PGF1 alpha plasma concentration (r = 0.54) and net aortic ring release (r = 0.70) when data from all rats were combined. We conclude that augmentation of PGI2 production is a feature of AII-induced hypertension. The enhancement of PGI2 production may be an expression of nonspecific alteration in vascular structure and metabolic functions during AII-induced hypertension, as well as the result of a specific effect of the peptide on the arachidonate-prostaglandin system.

Authors

D I Diz, P G Baer, A Nasjletti

Tolbutamide perifusion of rat islets. Sequential changes in calcium, phosphorus, sodium, potassium, and chlorine in single beta cells.

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Abstract

Fluctuations of calcium, phosphorus, sodium, potassium, and chlorine in beta cells were followed during rat islet perifusion with tolbutamide and related to insulin secretion. In 24 paired experiments two chambers containing 100 islets were perifused with buffered medium containing 4.2 mM glucose alone or with added tolbutamide (200 micrograms/ml). Effluent was collected frequently for insulin determinations. At eight different time intervals from 0 to 20 min islets were acutely fixed, prepared for scanning electron microscopy and beta cells in islet tissue were identified. Element content in 480 single cells was measured by energy dispersive x-ray analysis. Tolbutamide elicited typical monophasic insulin release that exceeded control islet secretory rates from 2 to 6 min with a peak value at 3 min. This pattern was preceded by monophasic calcium accumulation in beta cells that abruptly rose 150% above control cells at 1 min and declined to base line by 4 min. The rapid ascent of calcium was associated with significant depressions of sodium and potassium content without alterations of cell phosphorus. Chlorine fell at 2 min and then rose greater than 50% above control cells at 4 min. After 6 min insulin secretion and element content remained near control levels. We conclude that monophasic calcium accumulation in beta cells is the earliest, most predictive event of islet insulin secretion after a tolbutamide stimulus. Oscillations of beta cell sodium and potassium reciprocally relate to calcium, and an elevation of chlorine content is a relatively late phenomenon in the stimulus-secretion coupling process.

Authors

R K Kalkhoff, K A Siegesmund, R F Dragen

Adenosine deaminase deficiency with normal immune function. An acidic enzyme mutation.

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Abstract

In most instances, marked deficiency of the purine catabolic enzyme adenosine deaminase results in lymphopenia and severe combined immunodeficiency disease. Over a 2-yr period, we studied a white male child with markedly deficient erythrocyte and lymphocyte adenosine deaminase activity and normal immune function. We have documented that (a) adenosine deaminase activity and immunoreactive protein are undetectable in erythrocytes, 0.9% of normal in lymphocytes, 4% in cultured lymphoblasts, and 14% in skin fibroblasts; (b) plasma adenosine and deoxyadenosine levels are undetectable and deoxy ATP levels are only slightly elevated in lymphocytes and in erythrocytes; (c) no defect in deoxyadenosine metabolism is present in the proband's cultured lymphoblasts; (d) lymphoblast adenosine deaminase has normal enzyme kinetics, absolute specific activity, S20,w, pH optimum, and heat stability; and (e) the proband's adenosine deaminase exhibits a normal apparent subunit molecular weight but an abnormal isoelectric pH. In contrast to the three other adenosine deaminase-deficient healthy subjects who have been described, the proband is unique in demonstrating an acidic, heat-stable protein mutation of the enzyme that is associated with less than 1% lymphocyte adenosine deaminase activity. Residual adenosine deaminase activity in tissues other than lymphocytes may suffice to metabolize the otherwise lymphotoxic enzyme substrate(s) and account for the preservation of normal immune function.

Authors

P E Daddona, B S Mitchell, H J Meuwissen, B L Davidson, J M Wilson, C A Koller

Heterogeneity of human factor V deficiency. Evidence for the existence of antigen-positive variants

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Functional human Factor V has been purified using a rapid immunoaffinity method. Following barium citrate adsorption of plasma, Factor V was precipitated with polyethylene glycol at a concentration between 5 and 14%. The resulting preparation was applied to a column containing an immobilized immunoadsorbent consisting of an IgG fraction containing a naturally occurring human monoclonal (IgG4λ) antibody with inhibitory activity against human Factor V. The solid phase immunoglobulin quantitatively bound Factor V from human plasma. The bound Factor V was effectively eluted with a Tris buffer pH 7.2 containing 1.2 M NaCl and 1 M α-methyl-D-mannoside. The isolated native Factor V with high specific activity (92 U/mg) showed a single band (Mr, 350,000) on both reduced and nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Factor V was purified 5,100-fold over plasma with an overall yield of 77%. The purified Factor V when subjected to thrombin activation exhibited an 18-fold increase in coagulant activity.

Authors

Hui Chong Chiu, Eugene Whitaker, Robert W. Colman

Antibodies of patients with Lyme disease to components of the Ixodes dammini spirochete.

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Abstract

Lyme disease is an inflammatory disorder of skin, joints, nervous system, and heart. The disease is associated with a preceding tick bite and is ameliorated by penicillin treatment. A spirochete (IDS) isolated from Ixodes dammini ticks has been implicated as the etiologic agent of Lyme disease. We examined the antibody responses of Lyme disease patients to IDS lysate components in order to further understand the pathogenesis of this disease. The components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, reacted with patients' sera, and the bound IgG was detected with 125I-labeled protein A (western blot). We found that (a) Lyme disease patients had antibodies to IDS components (b) most patients studied had antibodies to two components with apparent subunit molecular weights of 41,000 and 60,000, and (c) the patients' antibody responses during illness and remission were specific, for the most part, for the IDS. In contrast to the findings with Lyme disease sera, sera from controls showed little reactivity with IDS components in either the western blots or a derivative solid-phase radioimmunoassay.

Authors

A G Barbour, W Burgdorfer, E Grunwaldt, A C Steere

Saccharides mediate the attachment of rat macrophages to bone in vitro.

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Macrophages (M phi) are multipotential cells capable of giving rise to osteoclasts and of resorbing bone. Since both of these processes are ultimately dependent upon the attachment of cells to a mineralized bone surface, we have examined in this study the mechanism by which such attachment is achieved. The data show that elicited rat peritoneal M phi bind to bone in a temperature-dependent and -saturable manner with half-maximal attachment occurring within 10 min at 37 degrees C and reaching a plateau by approximately 60 min. The kinetics of binding are essentially the same whether devitalized bone particles or viable calvaria are used as a substrate. The attachment of M phi to bone is inhibited by some sugars (e.g., N-acetyl-galactosamine, thiogalactoside, beta-lactose), fetuin and asialofetuin, and by pretreating the bone with periodate. Binding is also significantly reduced when M phi are preincubated with tunicamycin and swainsonine at nontoxic concentrations sufficient to inhibit or alter glycosylation. On the other hand, exposing the cells to neuraminidase increases the capacity of M phi to bind to bone. Collectively, our observations indicate that the attachment of M phi to bone is a highly regulated process and is mediated, at least in part, by saccharides located on both the cell and the bone surface.

Authors

Z Bar-Shavit, S L Teitelbaum, A J Kahn

Defective binding of macrophages to bone in rodent osteomalacia and vitamin D deficiency. In vitro evidence for a cellular defect and altered saccharides in the bone matrix.

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In the osteomalacic as well as normal skeleton, few osteoclasts are associated with osteoid-covered bone surfaces. The reason for this particular cellular deficit is not clear, but may relate to the inability of osteoclasts and/or osteoclast precursors (monocyte-macrophages) to attach to immature, unmineralized bone matrix, a step apparently essential for normal resorptive activity and osteoclast differentiation. In this study, we have examined cell-bone binding using macrophages (M phi) and bone isolated from vitamin D-deficient rats and hypophosphatemic, osteomalacic mice and from their normal counterparts. The data show that M phi-bone attachment is greatly reduced (P less than 0.001) in both vitamin D deficiency and hypophosphatemia, but that the mechanisms responsible for this reduction are apparently different in the two disorders. In hypophosphatemia, the reduction in binding appears solely attributable to the absence or inaccessibility of bone matrix oligosaccharides or glycoproteins essential to the attachment process. In vitamin D deficiency, on the other hand, not only is the bone matrix defective as a binding substrate, but the M phi, per se, is limited in its capacity to attach to normal, vitamin D-deficient, and hypophosphatemic bone.

Authors

Z Bar-Shavit, A J Kahn, S L Teitelbaum

Early membrane damage during coronary reperfusion in dogs. Detection by radiolabeled anticardiac myosin (Fab')2.

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Abstract

There is currently great interest in acute coronary reperfusion as a therapeutic modality for severe myocardial ischemia. While some studies have demonstrated a reduction in the overall extent of necrosis by early reperfusion, other studies have identified potentially deleterious effects produced by reflow. Because membrane disruption may be an important mechanism of irreversible cell injury, we measured changes in cell membrane integrity early during reperfusion using radiolabeled anticardiac myosin (Fab')2 antibody fragments in dogs. Our method involved brief periods of exposure to the (Fab')2 so that the levels of (Fab')2 binding indicated the degree of membrane disruption at discrete times during the progression of cell injury. In the first protocol (Fab')2 fragments labeled with either 125I and 131I were injected into the left circumflex coronary artery at the onset of reflow and at 45 min of reflow after a 1-h circumflex artery occlusion. Coronary sinus flow was diverted for 5 min following each injection to prevent recirculation. The (Fab')2 binding ratio (ischemic/control) increased during the first 45 min of reflow in each of eight experiments (mean increase 170%, P less than 0.01). No significant increase in (Fab')2 binding was observed in five additional experiments in which nonspecific (Fab')2 was injected. This indicates that the increase in binding seen with antimyosin-specific (Fab')2 was due to changes in specific binding rather than to alterations in (Fab')2 delivery produced by changes in blood flow distribution. The increase in membrane damage during reflow was confirmed by a second protocol in which each animal received only a single left atrial injection of (Fab')2 followed by rapid excision of the heart. The (Fab')2 binding ratio was 1.7 +/- 0.3 (SEM) in the group that received (Fab')2 at the onset of reflow and 3.7 +/- 0.6 (SEM) (P less than 0.05) in the group that received (Fab')2 after 45 min of reflow. In a third set of experiments in which hyperosmotic mannitol was infused during reflow the mean increase in (Fab')2 binding using the first protocol was only 80 +/- 40 vs. 170 +/- 30% without mannitol (P less than 0.05). Thus, membrane damage develops early during coronary reperfusion following 1 h of circumflex coronary artery occlusion, and part of this membrane damage can be prevented by altering the conditions of reflow. A method involving brief exposure of the myocardium to antimyosin (Fab')2 is promising for detecting changes in membrane integrity during evolving ischemic injury.

Authors

L H Frame, J A Lopez, B A Khaw, J T Fallon, E Haber, W J Powell Jr

Muscle phosphofructokinase deficiency. Biochemical and immunological studies of phosphofructokinase isozymes in muscle culture.

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Abstract

Muscle cultures from three unrelated patients with muscle phosphofructokinase (PFK; EC 2.7.1.11) deficiency (Glycogenosis type VII; Tarui disease) had normal PFK activity and normal morphology. Chromatographic and immunological studies showed that normal muscle cultures express all three PFK subunits, M (muscle-type), L (liver-type), and P (platelet-type) and contain multiple homotetrameric and heterotetrameric isozymes. Muscle cultures from patients lack catalytically active M subunit-containing isozymes, but this is compensated for by the presence of P- and L-containing isozymes. Despite the lack of muscle-type PFK activity, presence of immunoreactive M subunit was demonstrable by indirect immunofluorescence, suggesting a mutation of the structural gene coding for the M-subunit of PFK.

Authors

M Davidson, A F Miranda, A N Bender, S DiMauro, S Vora

Neural control of the sphincter of Oddi. A physiological role of 5-hydroxytryptamine in the regulation of basal sphincter of Oddi motor activity in the cat.

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Abstract

The effect of 5-hydroxytryptamine (5-HT) on the sphincter of Oddi (SO) was studied in the cat. The SO had two motor responses to 5-HT: the most common was an initial contraction followed by a more prolonged relaxation, and the other was an exclusive relaxation. Tetrodotoxin did not impair the magnitude of the net contraction induced by 5-HT, but it completely blocked the relaxation. Methysergide partially inhibited the SO contraction in response to submaximal doses of 5-HT (5-20 micrograms/kg). Atropine decreased the SO excitatory response to all doses of 5-HT. The combination of atropine and methysergide completely antagonized the 5-HT excitatory effect, which changed the SO biphasic response to an exclusive relaxation. After tetrodotoxin, the effect of 5-HT was almost completely antagonized by methysergide alone. The SO contraction and relaxation caused by 5-HT were almost completely blocked by 5-HT tachyphylaxis. In contrast, a 5-HT depletion with reserpine enhanced the sensitivity of the SO to 5-HT, responding to doses a thousand times smaller than in control animals. Hexamethonium, phentolamine, propranolol, and 5-methoxy-N,N-dimethyltryptamine did not antagonize the 5-HT-induced contraction or relaxation. These findings indicate that 5-HT caused SO contraction by stimulating postganglionic cholinergic neurons and the smooth muscle directly and caused relaxation by stimulating postganglionic, noncholinergic, nonadrenergic inhibitory neurons. 5-HT blockade or depletion resulted in a significant reduction in basal tonic pressures and in the amplitude of phasic contractions, which suggested that serotonergic neurons may play a physiologic role in the regulation of basal SO motor activity.

Authors

J Behar, P Biancani

Neoantigen of the polymerized ninth component of complement. Characterization of a monoclonal antibody and immunohistochemical localization in renal disease.

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Abstract

A monoclonal antibody to a neoantigen of the C9 portion of the membrane attack complex (MAC) of human complement has been developed and characterized. The distribution of this neoantigen was assessed by indirect immunofluorescence microscopy in nephritic and nonnephritic renal diseases. The antibody (Poly C9-MA) reacted on enzyme-linked immunosorbent assay (ELISA) with a determinant in complement-activated serum that was undetectable in normal human serum (NHS). Zymosan particles incubated in NHS had positive immunofluorescent staining with Poly C9-MA; however, binding of Poly C9-MA was not observed with zymosan particles incubated in sera deficient in individual complement components C3, C5, C6, C7, C8, or C9. Reconstitution of C9-deficient sera with purified C9 restored the fluorescence with Poly C9-MA. Poly C9-MA reacted positively by ELISA in a dose-dependent manner with purified MC5b-9 solubilized from membranes of antibody-coated sheep erythrocytes treated with NHS but not with intermediate complement complexes. Poly C9-MA also reacted in a dose-dependent manner on ELISA and in a radioimmunoassay with polymerized C9 (37 degrees C, 64 h) (poly C9) but not with monomeric C9. Increasing amounts of either unlabeled poly C9 or purified MC5b-9 inhibited the 125I-poly C9 RIA in an identical manner. These studies demonstrate that Poly C9-MA recognizes a neoantigen of C9 common to both the MAC and to poly C9. By immunofluorescence, Poly C9-MA reacted minimally with normal kidney tissue in juxtaglomerular loci, the mesangial stalk, and vessel walls. Poly C9-MA stained kidney tissue from patients with glomerulonephritis in a pattern similar to that seen with polyclonal anti-human C3. In tissue from patients with nonnephritic renal disease--diabetes, hypertension, and obstructive uropathy--Poly C9-MA was strongly reactive in the mesangial stalk and juxtaglomerular regions, tubular basement membranes, and vascular walls. Poly C9-MA binding was especially prominent in areas of advanced tissue injury. Poly C9-MA frequently stained loci where C3 was either minimally present or absent. These studies provide strong evidence for complement activation not only in nephritic but also in nonnephritic renal diseases.

Authors

R J Falk, A P Dalmasso, Y Kim, C H Tsai, J I Scheinman, H Gewurz, A F Michael

Anaphylactic release of a prekallikrein activator from human lung in vitro.

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Abstract

We have demonstrated the in vitro IgE-mediated release of a prekallikrein activator from human lung. The lung prekallikrein activator was partially purified by sequential chromatography on sulfopropyl-Sephadex, DEAE-Sephacel, and Sepharose 6B. Purified human prekallikrein was converted to its active form (kallikrein) by the lung protease. The generated kallikrein was shown to be biologically active; that is, it generates bradykinin from purified human high-molecular weight kininogen and also cleaves benzoyl-propyl-phenyl-arginyl-p-nitroanilide, a known synthetic substrate of kallikrein. The lung prekallikrein activator differs from the known physiologic activators of prekallikrein (the activated forms of Hageman factor) with respect to: (a) size (it has a mol wt of approximately 175,000); (b) synthetic substrate specificity (D-propyl/phenyl/arginyl-p-nitroanilide is a substrate for the activated forms of Hageman factor, but not the lung protease); (c) antigenic specificity (an anti-Hageman factor immunoadsorbent column did not remove significant amounts of the lung protease, while it removed most of the activity of activated Hageman factor fragments); and (d) inhibition profile (the lung proteases was not inhibited by corn trypsin inhibitor). This prekallikrein activator provides a physiologic mechanism by which prekallikrein can be directly activated during IgE-mediated reactions of the lung. While the role of this lung prekallikrein activator in immediate hypersensitivity reactions and in other inflammatory processes is not clear, it does represent a first and important interface between IgE-mediated reactions and the Hageman factor-dependent pathways of the inflammatory response.

Authors

H L Meier, A P Kaplan, L M Lichtenstein, S Revak, C G Cochrane, H H Newball

Defective adenosine triphosphate synthesis. An explanation for skeletal muscle dysfunction in phosphate-deficient mice.

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Abstract

The basis for skeletal muscle dysfunction in phosphate-deficient patients and animals is not known, but it is hypothesized that intracellular phosphate deficiency leads to a defect in ATP synthesis. To test this hypothesis, changes in muscle function and nucleotide metabolism were studied in an animal model of hypophosphatemia. Mice were made hypophosphatemic through restriction of dietary phosphate intake. Gastrocnemius function was assessed in situ by recording isometric tension developed after stimulation of the nerve innervating this muscle. Changes in purine nucleotide, nucleoside, and base content of the muscle were quantitated at several time points during stimulation and recovery. Serum concentration and skeletal muscle content of phosphorous are reduced by 55 and 45%, respectively, in the dietary restricted animals. The gastrocnemius muscle of the phosphate-deficient mice fatigues more rapidly compared with control mice. ATP and creatine phosphate content fall to a comparable extent during fatigue in the muscle from both groups of animals; AMP, inosine, and hypoxanthine (indices of ATP catabolism) appear in higher concentration in the muscle of phosphate-deficient animals. Since total ATP use in contracting muscle is closely linked to total developed tension, we conclude that the comparable drop in ATP content in association with a more rapid loss of tension is best explained by a slower rate of ATP synthesis in the muscle of phosphate-deficient animals. During the period of recovery after muscle stimulation, ATP use for contraction is minimal, since the muscle is at rest. In the recovery period, ATP content returns to resting levels more slowly in the phosphate-deficient than in the control animals. In association with the slower rate of ATP repletion, the precursors inosine monophosphate and AMP remain elevated for a longer period of time in the muscle of phosphate-deficient animals. The slower rate of ATP repletion correlates with delayed return of normal muscle contractility in the phosphate-deficient mice. These studies suggest that the slower rate of repletion of the ATP pool may be the consequence of a slower rate of ATP synthesis and this is in part responsible for the delayed recovery of normal muscle contractility.

Authors

B D Hettleman, R L Sabina, M K Drezner, E W Holmes, J L Swain

Differential effects of oral, peripheral intravenous, and intraportal glucose on hepatic glucose uptake and insulin and glucagon extraction in conscious dogs.

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Abstract

The effect of equal (1.1 +/- 0.1 g/kg body wt) amounts of glucose administered orally, or by peripheral intravenous or intraportal infusion on hepatic glucose uptake and fractional hepatic extraction of insulin and glucagon was studied in conscious dogs with chronically implanted Doppler flow probes on the portal vein and hepatic artery and catheters in the portal vein, hepatic vein, carotid artery, and superior mesenteric vein. Portal vein and hepatic vein plasma flow increased only after oral glucose administration. Arterial plasma glucose increased equally to 150-160 mg/100 ml after all three routes of glucose administration. Portal vein glucose was similar after oral (195 +/- 15 mg/100 ml) and intraportal glucose infusion (215 +/- 11 mg/100 ml) and significantly higher than after peripheral intravenous glucose. Hepatic glucose uptake after oral (68 +/- 4%) and intraportal glucose administration (65 +/- 7%) significantly exceeded that after peripheral intravenous glucose infusion (23 +/- 5%). The amount of insulin above basal presented to the liver during the 180 min after oral glucose was 7.6 +/- 1.3 U, 4.3 +/- 0.6 U after intraportal glucose, and 4.1 +/- 0.6 U after peripheral intravenous glucose. Hepatic extraction of insulin increased significantly after oral glucose (42 +/- 3 to 61 +/- 4%), but was unchanged after intraportal and peripheral intravenous glucose administration. When the portal vein glucose levels achieved during peripheral intravenous glucose infusion for 90 min were maintained by a subsequent 90-min intraportal glucose infusion, hepatic glucose uptake was significantly greater during the intraportal glucose infusion. Glucagon secretion was suppressed equally after oral glucose, intraportal glucose, and peripheral intravenous glucose administration; fractional hepatic extraction of that hormone, which was significantly less than that of insulin, was unchanged. These results indicate that hepatic glucose uptake is significantly greater after oral and intraportal glucose administration than after peripheral intravenous glucose infusion. This difference is not simply related to the amount of glucose or insulin presented to the liver and the increased hepatic glucose uptake did not depend solely upon the augmented fractional hepatic extraction of insulin. Hepatic extraction of insulin and hepatic glucose uptake appear to be regulated independently.

Authors

T Ishida, Z Chap, J Chou, R Lewis, C Hartley, M Entman, J B Field

Hemolytic disease of the newborn caused by a new deletion of the entire beta-globin cluster.

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Abstract

We describe a new type of gamma delta beta-thalassemia in four generations of a family of Scotch-Irish descent. The proposita presented with hemolytic disease of the newborn, which was characterized by a microcytic anemia. Initial restriction endonuclease analysis of the DNA showed no grossly abnormal patterns, but studies of polymorphic restriction sites and gene dosage revealed an extensive deletion that removed all the beta- and beta-like globin genes from the affected chromosome. In situ hybridization of chromosome preparations with radioactive beta-globin gene probes showed that only one 11p homolog contained the beta-globin gene cluster in the affected family members.

Authors

M Pirastu, Y W Kan, C C Lin, R M Baine, C T Holbrook

Intestinal assimilation of a proline-containing tetrapeptide. Role of a brush border membrane postproline dipeptidyl aminopeptidase IV.

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Abstract

The mechanism of hydrolysis and absorption of a proline-containing tetrapeptide, Leu-Pro-Gly-Gly (10 mM) by rat intestine was examined in vivo by using jejunal perfusion methods. The peptide substrate and hydrolysis products were analyzed by use of an automated amino acid analyzer. Leucine, proline, and glycine were absorbed by the intestine at a significantly higher rate from the tetrapeptide than from an equivalent amino acid mixture. The analysis of the hydrolytic products in the lumen during in vivo perfusion of the tetrapeptide showed that two dipeptides, Leu-Pro and Gly-Gly, were the major products. These two dipeptides were also the major hydrolytic products when a purified rat intestinal brush border membrane preparation was incubated with Leu-Pro-Gly-Gly. The rate of hydrolysis of the tetrapeptide was much higher than that for several other proline-containing peptides (Leu-Pro, Pro-Leu, and Pro-Gly-Gly) that were tested. Studies using Gly-Pro-beta-naphthylamide, a specific substrate for postproline dipeptidyl aminopeptidase IV, showed that this enzyme is mainly localized to the brush border membrane and is responsible for the hydrolysis of the tetrapeptide into the two dipeptides Leu-Pro and Gly-Gly. Thus, brush border membrane dipeptidyl aminopeptidase IV very likely plays an important role at the intestinal mucosal cell surface in the final stages of digestion of proline-containing peptides.

Authors

A Morita, Y C Chung, H J Freeman, R H Erickson, M H Sleisenger, Y S Kim

Specific plasma membrane receptors for reovirus on rat pituitary cells in culture.

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Abstract

Specific cellular and host tropism is a characteristic property of many viruses mediated by the interaction of viral attachment proteins with components of the plasma membrane of the cell. We have studied the binding of virus to cells quantitatively by using type 3 reovirus labeled with 125I and GH4C1 pituitary cells in culture. Binding was rapid at both 4 degrees and 15 degrees C and was stable over a 9-h period. Unlabeled virus inhibited binding of the labeled virus in a dose-dependent manner. Scatchard analysis revealed 4,200 viral binding sites/cell with an apparent affinity of 1.2 X 10(-11) M. Also, binding of type 3 reovirus was inhibited by antibodies directed against the viral hemagglutinin and partially inhibited by type 2 reovirus, but was unaffected by type 1 reovirus or a variety of other ligands that bind to receptors on GH4C1 cells. These data indicate that reovirus binds to a high affinity, specific receptor on target cells, which may control its tropism and ultimate disease expression.

Authors

E Maratos-Flier, C R Kahn, D R Spriggs, B N Fields

Effects of secretin on peritubular capillary physical factors and proximal fluid reabsorption in the rat.

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Abstract

The effects of secretin vasodilation on peritubular capillary Starling forces and absolute proximal reabsorption were examined in the rat. Secretin was infused at 75 mU/kg per min into the aorta above the left renal artery. Efferent plasma flow increased from 125 +/- 28 to 230 +/- 40 nl/min with secretin infusion. Single nephron filtration rate (44 +/- 6 vs. 44 +/- 7 nl/min) and absolute proximal reabsorption (21 +/- 5 vs. 21 +/- 4 nl/min) were not significantly changed. Peritubular capillary and interstitial hydrostatic pressures increased with secretin infusions (from 9 +/- 0.4 to 15 +/- 0.7 mmHg and from 3 +/- 0.2 to 4 +/- 0.2 mmHg, respectively). Both peritubular capillary and interstitial oncotic pressures decreased (from 25 +/- 2 to 20 +/- 2 mmHg and from 10 +/- 1 to 4 +/- 1 mmHg, respectively) during secretin infusion. The net reabsorption pressure for peritubular capillary uptake significantly decreased from 9 +/- 2 to 5 +/- 2 mmHg and the coefficient of reabsorption increased from 3 +/- 1 to 6 +/- 2 nl/min per mmHg. We conclude that although secretin causes a vasodilation and decreases net reabsorption pressure, absolute proximal reabsorption is unchanged. Peritubular capillary uptake is maintained, and since net reabsorption pressure is decreased, the coefficient of reabsorption is increased.

Authors

J I Mertz, J A Haas, T J Berndt, J C Burnett Jr, F G Knox

Receptors for bradykinin in intact cultured human fibroblasts. Identification and characterization by direct binding study.

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Abstract

Bradykinin receptors on cultured human fibroblasts were characterized using [2,3-prolyl-3,4-3H(N)]bradykinin as radioligand. During incubation with intact fibroblasts, intact [3H]bradykinin was lost much more rapidly at 37 degrees than at 4 degrees C as determined by bioassay, high-performance liquid chromatography, and ion-exchange chromatography, and is likely to be degraded. At 4 degrees, but not at 37 degrees C, bradykinin remained intact in the presence of 2 mM bacitracin, but not in the presence of soybean trypsin inhibitor or SQ-20881, an inhibitor of kininase II. Specific binding at 4 degrees C was saturable with a maximum number of binding sites of 230 +/- 18 fmol/mg protein (mean +/- SE, n = 4) and a dissociation constant of 4.6 +/- 0.5 nM (mean +/- SE, n = 4). Linear Scatchard plots, Hill coefficients close to unity (0.95-1.06), and the failure of excess bradykinin to influence dissociation kinetics are consistent with a single component binding system with no significant cooperativity. Na+ at physiological concentrations and Ca++ or Mg++ at 3-10 mM reduced binding by 25%. The relative potencies of bradykinin analogues and unrelated peptides in competing for [3H]bradykinin binding indicated a specificity of the binding sites consistent with that of a B2 type receptor. Potencies of the peptides in displacing [3H]bradykinin correlated with their abilities to release prostacyclin, determined as its metabolite 6-keto-PGF1 alpha. This system, the first in which bradykinin receptors on human cells have been characterized, should prove useful for investigation of the regulation of bradykinin-influenced biological processes.

Authors

A A Roscher, V C Manganiello, C L Jelsema, J Moss

Antigen-induced human T cell help. Precursor frequency, radiation sensitivity, and allogeneic effects.

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Abstract

We have recently noted marked differences between the in vitro responses of human B lymphocytes to stimulation with soluble antigens vs. stimulation with mitogens. In the present study, these differences were analyzed in terms of the precursor frequencies for the T cells and B cells involved and in terms of the radiation sensitivity of the T cells providing help in the two systems. Marked differences were found between antigen-induced and mitogen-induced systems with regard to T cell precursor frequencies and radiation sensitivity. In contrast, the precursor frequencies for the B cells involved in the two systems were approximately the same. In addition, having developed a system for the study of human antigen-specific B cell responses, we were interested in delineating the nature of the allogeneic effects that might be operative in this system. Marked allogeneic effects, both positive and negative, were noted in this system and will need to be taken into account in any studies that try to address the question of the genetic restriction, if any, that exists in human antigen-specific T cell-B cell collaboration. Appreciation of the marked differences between the antigen-specific and mitogen-induced activation and immunoregulation of human B cell responses will be of importance in understanding the relationship between specificity and nonspecificity of antibody production in normal and disease states.

Authors

H C Lane, G Whalen, A S Fauci

Metabolic compensation for profound erythrocyte adenylate kinase deficiency. A hereditary enzyme defect without hemolytic anemia.

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Abstract

A child with hemolytic anemia was found to have severe erythrocyte adenylate kinase (AK) deficiency, but an equally enzyme-deficient sibling had no evidence of hemolysis. No residual enzyme activity was found in erythrocytes by spectrophotometric methods that could easily have detected 0.1% of normal activity. However, concentrated hemolysates were shown to have the capacity to generate small amounts of ATP and AMP from ADP after prolonged incubation. Hemolysates could also catalyze the transfer of labeled gamma-phosphate from ATP to ADP. Intact erythrocytes were able to transfer phosphate from the gamma-position of ATP to the beta-position, albeit at a rate substantially slower than normal. They could also incorporate 14C-labeled adenine into ADP and ATP. Thus, a small amount of residual AK-like activity representing about 1/2,000 of the activity normally present could be documented in the deficient erythrocytes. The residual activity was not inhibited by N-ethylmaleimide, which completely abolishes the activity of the normal AK1 isozyme of erythrocytes. The minute amount of residual activity in erythrocytes could represent a small amount of the AK2 isozyme, which has not been thought to be present in erythrocytes, or the activity of erythrocyte guanylate kinase with AMP substituting as substrate for GMP. Peripheral blood leukocytes, cultured skin fibroblasts, and transformed lymphoblasts from the deficient subject manifested about 17, 24, and 74%, respectively, of the activity of the concurrent controls. This residual activity is consistent with the existence of genetically independent AK isozyme, AK2, which is known to exist in these tissues. The cause of hemolysis in the proband was not identified. Possibilities include an unrelated enzyme deficiency or other erythrocyte enzyme defect and intraction of another unidentified defect with AK deficiency.

Authors

E Beutler, D Carson, H Dannawi, L Forman, W Kuhl, C West, B Westwood

Elastase and alpha 1-proteinase inhibitor activity in tracheal aspirates during respiratory distress syndrome. Role of inflammation in the pathogenesis of bronchopulmonary dysplasia.

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Abstract

Pulmonary effluent samples were obtained from 26 preterm or term infants throughout the period of endotracheal intubation. Infants with respiratory distress syndrome, infants with this disorder developing bronchopulmonary dysplasia, and intubated infants without lung disease were compared daily in terms of lung effluent cellularity, albumin, elastase activity, alpha 1-proteinase content and activity, and elastase inhibitory capacity. The elastase activity was determined to be neutrophilic in origin. Polyacrylamide gel electrophoresis of pulmonary effluents from two infants with respiratory distress syndrome and exposed to FiO2 greater than or equal to 0.6 up to 6 d revealed cleavage of alpha 1-proteinase inhibitor to a 47,000-mol weight fragment suggestive of oxidation. Pulmonary effluent neutrophils, macrophages, and elastase activity were increased by day 3 of life in infants with respiratory distress syndrome eventually developing bronchopulmonary dysplasia. Elastase inhibitory capacity and alpha 1-proteinase inhibitor activity were reduced in infants developing chronic lung disease. Bronchopulmonary dysplasia developed in infants with enhanced inflammatory response, but with less or inhibited antiprotease activity.

Authors

T A Merritt, C G Cochrane, K Holcomb, B Bohl, M Hallman, D Strayer, D K Edwards 3rd, L Gluck

Urinary calcium excretion in familial hypocalciuric hypercalcemia. Persistence of relative hypocalciuria after induction of hypoparathyroidism

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Abstract

Familial hypocalciuric hypercalcemia (FHH) is an autosomal dominant trait comprising hypercalcemia, hypophosphatemia, parathyroid hyperplasia, and unusually low renal clearance of calcium. We evaluated the role of parathyroid hormone in the relative hypocalciuria of FHH and characterized the renal transport of calcium in this disorder using three previously hypercalcemic FHH patients with surgical hypoparathyroidism and three controls with surgical hypoparathyroidism. Intravenous infusion of calcium chloride in two patients with FHH and in three controls increased serum calcium from a mean basal of 5.0 to a mean peak of 6.8 meq/liter in two FHH patients and from 4.2 to 5.7 in three control subjects. Urinary calcium in a third FHH patient was studied without calcium infusion during recovery from hypercalcemia of vitamin D intoxication. At all serum concentrations of calcium, calcium clearance was lower in FHH than in controls; at base-line serum calcium, the ratio of calcium clearance to inulin clearance (CCa/CIN) in FHH subjects was 32% of that in controls and decreased to 19% during hypercalcemia. Calcium infusion increased the ratio of sodium clearance to inulin clearance in controls from a base line of 0.020 to 0.053 at peak concentrations of calcium in serum, but did not affect this parameter in FHH (0.017 at base-line serum calcium vs. 0.019 at peak).

Authors

M. F. Attie, J. R. Gill Jr., J. L. Stock, A. M. Spiegel, R. W. Downs Jr., M. A. Levine, S. J. Marx

Purification and comparison of outer membrane protein P2 from Haemophilus influenzae type b isolates.

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Abstract

Haemophilus influenzae type b isolates have been subdivided based on differences in major outer membrane protein (OMP) profiles resolved on gradient and modified Laemmli sodium dodecyl sulfate-polyacrylamide gel electrophoresis systems. Although 21 subtypes have been observed, 86% of invasive isolates have one of five common subtypes and 71% of isolates have one of three subtypes. In isolates with two of the most common outer membrane subtypes, one major OMP has an apparent molecular weight of 37,000. In isolates with another common OMP subtype, a cross-reactive protein with an apparent molecular weight of 36,500 is observed. All three proteins have been designated P2. Protein P2 from these prototype isolates was solubilized with Zwittergent 3-14 and purified to homogeneity. Based on amino acid compositions, cyanogen bromide cleavage products, staphylococcal V8 protease, and chymotryptic peptide maps, the P2 protein from the three isolates has been highly conserved. Rabbit antibody prepared against P2 from one strain was cross-reactive with P2 isolated from the other two heterologous strains by Western blot. This antibody passively protected infant rats against type b Haemophilus infection caused by the homologous organism, but not against challenge by a strain with the heterologous 36,500 mol wt P2 protein. Thus, although the P2 protein from isolates with different OMP subtypes are closely related, the protection experiments suggest that determinants on the cell surface interacting with protective antibody may be strain or subtype specific.

Authors

R S Munson Jr, J L Shenep, S J Barenkamp, D M Granoff

Genetic regulation of a structural polymorphism of human C3b receptor.

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Abstract

Two forms of the human C3b receptor (C3bR), which have relative molecular weights (Mr) of 250,000 and 260,000 and are designated F and S, respectively, have been identified in specific immunoprecipitates from erythrocytes and leukocytes by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both forms of the receptor were visualized on gels by autoradiography of 125I-labeled antigen and by silver nitrate staining. Individual donors expressed one of three possible patterns of C3bR, either the F or S form alone or both, and these patterns represented stable phenotypic characteristics of their erythrocytes and polymorphonuclear and mononuclear leukocytes. Removal of N-linked oligosaccharides by endoglycosidase-F treatment decreased the Mr of both forms but did not abolish the difference in their electrophoretic mobilities. That both forms of the receptor were functional was indicated by the capacity of all antigenic C3bR sites on erythrocytes from individuals having any of the three phenotypes to bind dimeric C3b with affinities ranging from 3 to 5 X 10(7) M-1. Analyses of the occurrence of the F and S forms of C3bR in 76 individuals from 15 families revealed that this polymorphism was regulated by two alleles transmitted in an autosomal codominant manner. Of 111 normal unrelated individuals, 64.9% were homozygous for the F form (FF), 1.8% were homozygous for the S form (SS), and 33.3% were heterozygotes (FS). This distribution did not differ from that calculated by the Hardy-Weinberg equilibrium based on two codominant alleles that regulate the expression of the F and S forms and that have frequencies of 81.5 and 18.5%, respectively. The locus regulating structural polymorphism of C3bR is designated C3BRM (M for mobility or Mr), and is distinct from the recently described locus regulating the quantitative expression of C3bR on erythrocytes.

Authors

W W Wong, J G Wilson, D T Fearon

Evidence for genetic restriction in the suppression of erythropoiesis by a unique subset of T lymphocytes in man.

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Abstract

The suppression of erythropoiesis by lymphocytes from patients with a T cell lymphoproliferative syndrome and pure erythrocyte aplasia has been previously demonstrated. To study the nature of the suppressor cell and possible genetic restriction of this suppression, we investigated a patient with severe anemia, splenomegaly, lymphocytosis, and erythroid aplasia. A 3-mo course of low-dose daily oral cyclophosphamide achieved a complete remission for over 12 mo. The surface phenotype of his lymphocytes was analyzed by means of antibodies to lineage, differentiation, and activation-specific surface antigens. The cells expressed mature T cell antigens T3, T8, and T11, while lacking T1. Immature T cell, B cell, and the monocyte-specific antigen Mo2 were absent, while Mo1, a monocyte-associated antigen not normally expressed on T cells, was present. T10 and Ia expressed as activation antigens were also present. The cells, cryopreserved at diagnosis, were thawed and co-cultured in plasma clot with patient remission marrow samples at T cell/bone marrow ratios of 1:1 and 2:1. There was nearly 90% suppression of erythroid colony-forming unit expression and 60% suppression of erythroid burst-forming unit expression at 2:1 T cell to bone marrow ratios and somewhat less suppression at 1:1. Granulocyte/macrophage progenitor expression was unaffected. Erythroid progenitor differentiation in the marrows of two HLA identical siblings was similarly suppressed. The cells were co-cultured with the marrows of nine nonrelated donors to investigate the potential genetic restriction of this suppression. Colony suppression equal to that observed in the marrow of the patient and his siblings was found in studies of two partially HLA identical individuals. No suppression was detected in marrow co-cultures of two entirely HLA dissimilar individuals. These results show that suppression of erythropoiesis by a unique subset of T8, Mo1, Ia-positive lymphocytes isolated from a patient with lymphocytosis and erythrocyte aplasia is genetically restricted.

Authors

J M Lipton, L M Nadler, G P Canellos, M Kudisch, C S Reiss, D G Nathan

Role in nude mice of interferon and natural killer cells in inhibiting the tumorigenicity of human hepatocellular carcinoma cells infected with hepatitis B virus

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Abstract

The human hepatoma cell line, PLC/PRF/5, which is persistently infected with hepatitis B virus (HBV), has integrated HBV-DNA, secretes HBV surface antigen (HBsAg), and does not grow readily in congenitally athymic (nu/nu) mice. The present investigation was undertaken to ascertain whether the low tumorigenicity of this cell line was governed by a host immune response and/or was related to expression of HBsAg. Subcutaneous injection of 4-5 X 106 cells into BALB/c nude mice produced localized encapsulated tumors with morphologic features of primary hepatocellular carcinoma in 25% of the animals within 29-40 d. No tumor growth was observed at lower cell inocula. In contrast, SK-HEP-1, an HBV-negative human hepatoma cell line, produced tumors at 1-5 X 106 cells inocula in 66% of the animals. Immunosuppression of mice with antilymphocyte serum (ALS) or irradiation increased tumor incidence in mice inoculated with 1 X 106 PLC/PRF/5 cells to almost 100% and produced local invasiveness. Immunosuppression also reduced the latency, i.e., time to tumor appearance, and increased mean tumor weight. These results suggest that tumorigenicity was limited by the host immune response.

Authors

Daniel Shouval, Bracha Rager-Zisman, Phuc Quan, David A. Shafritz, Barry R. Bloom, Lola M. Reid

Hepatic bilirubin uptake in the isolated perfused rat liver is not facilitated by albumin binding.

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Abstract

Bilirubin uptake by the liver is a rapid process of high specificity that has kinetic characteristics which suggest carrier-mediation. In the circulation, bilirubin is readily bound to albumin, from which it is extracted by the liver. Although several studies suggested that it is the small, unbound fraction of bilirubin which interacts with hepatocytes and is removed from the circulation, recent experiments have been interpreted as suggesting that binding to albumin facilitates ligand uptake. A liver cell surface receptor for albumin has been postulated. The present study was designed to examine directly whether albumin facilitates the hepatic uptake of bilirubin and whether uptake of bilirubin depends on binding to albumin. Rat liver was perfused with a protein-free fluorocarbon medium, and single-pass uptake of 1, 10, or 200 nmol of [3H]bilirubin was determined after injection as an equimolar complex with 125I-albumin, with 125I-ligandin, or free with only a [14C]sucrose reference. Uptake of 10 nmol of [3H]bilirubin was 67.5 +/- 3.7% of the dose when injected with 125I-albumin, 67.4 +/- 6.5% when injected with 125I-ligandin, and 74.9 +/- 2.4% when injected with [14C]sucrose (P greater than 0.1). At 200 nmol, uptake fell to 46.4 +/- 3.1% (125I-albumin) and 63.3 +/- 3.4% [( 14C]sucrose) of injected [3H]bilirubin (P less than 0.01), which suggests saturation of the uptake mechanism. When influx was quantitated by the model of Goresky, similar results were obtained. When [3H]bilirubin was injected simultaneously with equimolar 125I-albumin and a [14C]sucrose reference, there was no delay in 125I-albumin transit as compared with that of [14C]sucrose. This suggested that the off-rate of albumin from a putative hepatocyte receptor would have to be very rapid, which is unusual for high affinity receptor-ligand interaction. There was no evidence for facilitation of bilirubin uptake by binding to albumin or for interaction of albumin with a liver cell surface receptor. These results suggest that the hepatic bilirubin uptake mechanism is one of high affinity which can extract bilirubin from circulating carriers such as albumin, ligandin, or fluorocarbon.

Authors

Y R Stollman, U Gärtner, L Theilmann, N Ohmi, A W Wolkoff

Mitochondrial dysfunction induced by fatty acid ethyl esters, myocardial metabolites of ethanol.

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Abstract

Mechanisms responsible for alcohol-induced heart muscle disease have been difficult to elucidate partly because of previously obscure, demonstrable cardiac metabolism of ethanol. Recently, fatty acid ethyl esters were identified in our laboratory and found to be myocardial metabolites of ethanol. In the present study, they have been shown to induce mitochondrial dysfunction. Incubation of isolated myocardial mitochondria with fatty acid ethyl esters led to a concentration-dependent reduction of the respiratory control ratio index of coupling of oxidative phosphorylation and decrement of maximal rate of oxygen consumption. Furthermore, fatty acid ethyl esters were demonstrated to bind to mitochondria in vitro, and, importantly, 72% of intracellularly synthesized ethyl esters were found to bind to mitochondria isolated from intact tissue incubated with ethanol. Protein binding of fatty acid ethyl esters was markedly less than that of fatty acids. Because uncoupling of mitochondrial oxidative phosphorylation correlated with the cleavage of fatty acid ethyl ester shown to be initially bound to mitochondria, with resultant generation of fatty acid, a potent uncoupler, in a locus in or near the mitochondrial membrane, fatty acid ethyl esters may contribute to a potentially toxic shuttle for fatty acid with transport from physiological intracellular binding sites to the mitochondrial membrane; direct effects of fatty acid ethyl esters may also be deleterious. Operation of this shuttle as a result of ethanol ingestion and subsequent accumulation of fatty acid ethyl esters may account for the impaired mitochondrial function and inefficient energy production associated with toxic effects of ethanol on the heart.

Authors

L G Lange, B E Sobel

Norepinephrine-stimulated hypertrophy of cultured rat myocardial cells is an alpha 1 adrenergic response.

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Abstract

We have shown recently that norepinephrine stimulates muscle cell hypertrophy in primary cultures from the neonatal rat ventricle and that this stimulation is not blocked by the beta adrenergic antagonist propranolol. The present study was done to define the adrenergic specificity of the myocyte hypertrophic response to norepinephrine. 90% pure, single-cell cultures of nongrowing myocytes were maintained in serum-free medium 199 with transferin and insulin. Myocyte size was quantitated 48 h after addition of adrenergic agents, by measuring cell volume, cell surface area, and cell protein. L-norepinephrine increased myocyte size to a maximum 150% of control; half-maximum effect was obtained at a concentration of 0.2 microM. This increase in cell size was inhibited by the nonselective alpha adrenergic antagonist phentolamine and by the alpha 1 adrenergic antagonists prazosin and terazosin; it was not inhibited by propranolol or by the alpha 2 adrenergic antagonist yohimbine. The beta adrenergic agonist isoproterenol did not increase cell size. Thus, norepinephrine-stimulated hypertrophy of cultured rat myocardial cells is an alpha 1 adrenergic response.

Authors

P Simpson