Proton transport and cell function.

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Abstract

The past five years have witnessed an explosion of information on the many and varied roles of H+ transport in cell function. H+ transport is involved in three broad areas of cell function: (a) maintenance and alteration of intracellular pH for initiation of specific cellular events, (b) generation of pH gradients in localized regions of the cell, including gradients involved in energy transduction, and (c) transepithelial ion transport. These processes each involve one or more of several H+ translocating mechanisms. The first section of this review will discuss these H+ translocating mechanisms and the second part will deal with the cellular functions controlled by H+ transport.

Authors

H E Ives, F C Rector Jr

An altered platelet granule glycoprotein in patients with essential thrombocythemia.

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Abstract

The protein profiles of washed platelets from nine patients with essential thrombocythemia were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In four patients, an additional protein band (reduced Mr of 170,000) was clearly identified in both unstimulated platelet preparations and thrombin-released supernatant fractions. This band was also evident, though to a lesser extent, in three more patients, but it could not be located in the two remaining patients nor in any of ten controls. Subsequent characterization of the 170,000 reduced protein in one patient indicated that (a) it was glycosylated, as judged by periodic acid-Schiff staining, and (b) that native protein was a disulfide-linked multimer (possibly trimeric), which (c) partially bound to the activated platelet plasma membrane in the presence of calcium, and (d) was immune precipitated by anti-glycoprotein G antisera. The combined evidence is consistent with the 170,000 reduced protein being a modified form of the normal subunit of the platelet alpha-granule constituent, glycoprotein G (also termed thrombospondin and thrombin-sensitive protein).

Authors

W J Booth, M C Berndt, P A Castaldi

Cloning of chromosomal DNA from Haemophilus influenzae. Its use for studying the expression of type b capsule and virulence.

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Abstract

Haemophilus influenzae may make any one of six chemically distinct capsular polysaccharides, but only strains of capsular serotype b commonly cause systemic infection (e.g., meningitis) in humans. Molecular cloning of DNA was used to investigate the expression of type b capsule and its association with H. influenzae virulence. A virulent H. influenzae type b strain was used to construct a lambda library of chromosomal DNA in Charon 4. Two independently isolated recombinant phage were isolated from the library and were found to possess DNA necessary for expression of type b capsule. Using a well-characterized rat model of H. influenzae systemic infection, we showed that type b transformants elicited by the cloned DNA were pathogenic, causing bacteremia and meningitis, whereas the untransformed capsule-deficient H. influenzae organisms were not. A 4.4-kb EcoRI fragment, common to both DNA clones, was used to characterize clinical isolates representing all six encapsulated serotypes as well as several capsule-deficient H. influenzae by Southern hybridization analysis. The probe hybridized to an identical sized (4.4 kb) fragment of EcoRI-digested chromosomal DNA from eight independently isolated type b strains. Single bands of homology to the probe were also found in EcoRI fragments of chromosomal DNA obtained from 33 encapsulated, nontype b H. influenzae. However, the size of these EcoRI fragments proved to be characteristic for each of the different capsular serotypes. These studies provide a basis for pursuing the molecular analysis of the epidemiology and virulence of pathogenic H. influenzae.

Authors

E R Moxon, R A Deich, C Connelly

Two distinct forms of Factor VIII coagulant protein in human plasma. Cleavage by thrombin, and differences in coagulant activity and association with von Willebrand factor.

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Abstract

We have characterized Factor VIII coagulant protein, present in normal human plasma, that reacts with a specific human 125I-labeled anti-human VIII:C antigen Fab antibody fragment. Two major Factor VIII coagulant antigen populations were present. The first, approximately 85% of the total antigen, was bound to von Willebrand factor and when tested in a standard one-stage assay had Factor VIII coagulant activity. The second antigenic population, eluting near fibrinogen when plasma was gel filtered, was not bound to von Willebrand protein, did not have Factor VIII coagulant activity unless activated, but did block anti-VIII:C Fab neutralization of clotting activity. The two antigenic populations were separable by cryoprecipitation and agarose gel electrophoresis. Although the two antigenic populations differed in their Factor VIII coagulant activity and in their binding to von Willebrand factor, the principal member of both populations is of mol wt 2.4 X 10(5). Both antigens, when proteolyzed by thrombin, were quickly converted to a 1 X 10(5)-mol wt form in association with the appearance of VIII:C activity. The 1 X 10(5)-mol wt antigen was further slowly degraded to an 8 X 10(4)-mol wt form while Factor VIII coagulant activity declined. These results demonstrate the presence of an inactive Factor VIII coagulant protein in plasma, not associated with von Willebrand factor, that can react with thrombin to yield Factor VIII coagulant activity.

Authors

M J Weinstein, L E Chute

Systemic circulatory adjustments to acute hypoxia and reoxygenation in unanesthetized sheep. Role of renin, angiotensin II, and catecholamine interactions.

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Abstract

The hemodynamic consequences of the hypoxic inhibition of angiotensin-converting enzyme activity were studied in chronically instrumented unanesthetized sheep (n = 8) breathing a hypoxic gas mixture for 60 min (PaO2 = 31 mm Hg) followed by reoxygenation with room air. Changes in cardiac output, vascular pressures, blood flow distribution, arterial pH, PaCO2, PaO2, and arterial levels of plasma renin activity, angiotensin II, bradykinin, and catecholamines were measured at selected time points. Seven additional sheep underwent the same protocol but received saralasin, an angiotensin II receptor blocker beginning at 55 min of hypoxia and extending into the reoxygenation period. During hypoxia, both groups developed identical hemodynamic patterns including a rise in cardiac output (25%), blood pressure (15%), and preferential blood flow distribution to the heart, brain, adrenals, diaphragm, and skeletal muscle, as well as a decrease in the fraction of cardiac output to the kidneys and most of the gut. This was associated with a decrease in angiotensin II concentrations (from 35 to 17 pg/ml) in spite of a doubling in plasma renin activity and catecholamines. Bradykinin levels did not change. Upon reoxygenation, bolus production of angiotensin II (from 17 to 1,819 pg/ml) occurred in spite of a constant level of plasma renin activity. Concurrently, different hemodynamic patterns between control and saralasin groups emerged upon reoxygenation, including an elevation from base line in blood pressure and systemic vascular resistance in the control group. Cardiac work (heart-rate systolic pressure product) in the control group remained elevated upon reoxygenation while coronary blood flow returned to base-line values. Saralasin reduced cardiac work upon reoxygenation and restored the match between coronary blood flow and work. We conclude that plasma renin activity and oxygen tension together govern angiotensin II levels for an optimal level of systemic vasomotor tone during hypoxia. However, upon reoxygenation, bolus production of angiotensin II may result in pathophysiologic circulatory patterns, such as impairment in oxygen delivery to the myocardium proportional to persistently elevated cardiac work in the immediate postresuscitation period.

Authors

D Davidson, S A Stalcup

Nature of the renal injury following total renal ischemia in man.

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The effects of total renal ischemia (TRI) of 15-87 min duration due to suprarenal clamping of the aorta were studied in 15 mannitol-treated patients undergoing abdominal aortic surgery. 15 patients undergoing similar surgery but requiring only infrarenal clamping served as controls. 1-2 h following TRI, GFR was reduced to only 39% of that in controls, 23 +/- 5 vs. 59 +/- 7 ml/min (P less than 0.001). This could not be ascribed to impaired renal plasma flow (RPF), which was mildly reduced to 331 +/- 71 and was not different from the value in controls, 407 +/- 66 ml/min. However, impaired PAH extraction (43 +/- 7%) and isosthenuria, not present in controls, suggest a primary role for tubular injury in lowering GFR at this time. 24 h following TRI, the GFR remained depressed below controls, 45 +/- 8 vs. 84 +/- 8 ml/min (P less than 0.005), while the transglomerular sieving of neutral dextrans was significantly enhanced (radius interval, 24-40 A). A theoretical analysis of transcapillary solute exchange revealed that these findings could be largely explained by a selective reduction of either RPF (-61%) or of transmembrane hydraulic pressure difference (-18%) below control values. Alternately, a combination of these two factors with changes of smaller magnitude could explain the findings. In contrast, a selective increase in oncotic pressure or decrease of the glomerular ultrafiltration coefficient could be excluded as a cause of hypofiltration 24 h after TRI. These observations lead us to suggest that the transient azotemia observed following TRI is due to a self-limited injury to the nephron that is identical to that seen in overt and sustained forms of acute renal failure.

Authors

B D Myers, D C Miller, J T Mehigan, C O Olcott 4th, H Golbetz, C R Robertson, G Derby, R Spencer, S Friedman

Human auto-antiidiotypes regulating T cell-mediated reactivity to tetanus toxoid.

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Abstract

While investigating the effect on B cells of repetitive in vivo immunization with tetanus toxoid (TT), we observed the subsequent development of specific anergy for T cell delayed hypersensitivity (DTH) to TT. This appeared approximately 35 d after a series of five booster immunizations. Concurrently, in vitro T cell blastogenic responses were preserved. Serum obtained when the skin tests were nonreactive demonstrated a profound inhibitory activity on T cell reactivity. This activity was shown to be anti-antibody activity that was both anti-F(ab)'2 and, specifically, anti-TT F(ab)'2. It blocked binding of TT to a pool of allogeneic antibodies and also inhibited allogeneic antigen-specific T cell blastogenesis. Thus, we could identify activity in the serum of hyperimmunized individuals that appeared auto-anti-idiotypic (anti-id) and represented a single or family of major crossreacting idiotypes (id) for TT. The expression of the auto-anti-id correlated with the loss of T cell reactivity in vivo and in vitro. Subsequent examinations revealed persistent, specific cutaneous anergy beyond six months, which was then associated with a failure of T cells to react with antigen in vitro. Mixing experiments with cells from these later times and cryopreserved autologous cells obtained prior to hyperimmunization revealed there had been the development of antigen-specific T suppressor cells. Thus, in vivo DTH tolerance following hyperimmunization was associated with an inhibitory serum activity that appeared to be anti-id. Persistence of tolerance (greater than 6 mo) occurred with the development of T suppressor cells.

Authors

A Saxon, E Barnett

Autoradiographic characterization of beta adrenergic receptors in coronary blood vessels and myocytes in normal and ischemic myocardium of the canine heart.

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Abstract

This light microscopic autoradiographic study was performed to test the hypotheses that (a) the density of beta adrenergic receptors (BAR) may differ in various components of the heart and (b) BAR in certain components of the heart may exhibit a selective response to pharmacologic and pathological stimuli. Blocks of canine left ventricle were frozen and tissue sections cut and incubated in (-)[3H]dihydroalprenolol (DHA) to label the BAR. For total and nonspecific binding, serial sections were incubated with and without 10(-5) M (+/-)propranolol. Scintillation spectrometry of sections demonstrated rapid binding, saturability, stereospecificity, a dissociation constant (KD) of 3.2 +/- 0.5 nM (SD) (n = 3), and a maximal binding of 31.3 +/- 3.1 fmol/mg of tissue protein. Isoproterenol was 12.5 times more effective than norepinephrine in displacing DHA. Sections incubated with 10(-5) - 10(-8) M metoprolol, a beta one selective antagonist, demonstrated a KD of 0.7 X 10(-6) M. For autoradiography, emulsion-coated coverslips were attached to the slides. After exposure, the slides were developed and stained, and grain density quantified. Specific BAR binding (n = 4 dogs) was 1,047 +/- 131 (SEM) grains/10(-2) mm2 for myocardial arterioles, 219 +/- 30 for myocardial arteries, 31 +/- 12 for the proximal left anterior descending coronary artery (LAD), and 231 +/- 34 for cardiac myocytes. Specific binding in the presence of 10(-5) M metoprolol was reduced approximately 75% for both arterioles and myocytes. However, at 10(-6) M metoprolol, the percent reduction in specific DHA binding was greater for myocytes (50%) than for arterioles (0%), and at 10(-7) M metoprolol, the percent reduction in specific DHA binding was 17% for myocytes with no reduction over arterioles. After 1 h of LAD occlusion, a selective increase (18%) in BAR density occurred over cardiac myocytes, but not over blood vessels in the ischemic myocardium. Thus, (a) specific BAR binding was five times greater in arterioles than in small arteries and myocardium and 34 times greater than in the proximal LAD; (b) BAR of myocytes were more sensitive than those of arterioles to displacement by the beta one selective antagonist, metoprolol; and (c) a selective increase in BAR occurs in cardiac myocytes but not in blood vessels after 1 h of ischemia in this experimental model.

Authors

K H Muntz, E G Olson, G R Lariviere, S D'Souza, A Mukherjee, J T Willerson, L M Buja

Control of cardiac output in thyrotoxic calves. Evaluation of changes in the systemic circulation.

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The contribution of peripheral vascular factors to the high output state in thyrotoxicosis was examined in 11 calves treated with daily intramuscular injections of L-thyroxine (200 micrograms/kg) for 12-14 d. Thyroxine treatment increased cardiac output from 14.1 +/- 1.4 to 24.7 +/- 1.4 liters/min (P less than 0.001) and decreased systemic vascular resistance from 562 +/- 65 to 386 +/- 30 dyn-s/cm5 (P less than 0.01). Blood volume was increased from 65 +/- 4 ml/kg in the euthyroid state to 81 +/- 6 ml/kg when the animals were thyrotoxic (P less than 0.05). The role of low peripheral vascular resistance in maintenance of the high output state was evaluated by infusion of phenylephrine at two dosages (2.5 and 4.0 micrograms/kg per min). In the euthyroid state, no significant decrease in cardiac output was observed at either level of pressor infusion. In the thyrotoxic state, the higher dosage of phenylephrine increased peripheral resistance to the euthyroid control level and caused a small (6%) decrease in cardiac output (P less than 0.05). This small decrease in cardiac output probably could be attributed to the marked increase in left ventricular afterload caused by the pressor infusion as assessed from measurements of intraventricular pressure and dimensions. Changes in the venous circulation were evaluated by measurement of mean circulatory filling pressure and the pressure gradient for venous return in six animals during cardiac arrest induced by injection of acetylcholine into the pulmonary artery. Mean circulatory filling pressure increased from 10 +/- 1 mmHg in the euthyroid state to 16 +/- 2 mmHg (P less than 0.01) during thyrotoxicosis, while pressure gradient for venous return increased from 10 +/- 1 to 14 +/- 2 mmHg (P less than 0.02). These changes in venous return curves were not affected significantly by ganglionic blockade with trimethapan (2.0 mg/kg per min) before cardiac arrest. Thus, the high output state associated with thyrotoxicosis is not dependent upon a low systemic vascular resistance, but is associated with increases in blood volume, mean circulatory filling pressure, and pressure gradient for venous return.

Authors

S Goldman, M Olajos, E Morkin

Myeloperoxidase modulates the phagocytic activity of polymorphonuclear neutrophil leukocytes. Studies with cells from a myeloperoxidase-deficient patient.

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Abstract

Patients lacking the primary granulae enzyme, myeloperoxidase (MPO), do not usually show any increased susceptibility to infection or altered inflammatory response, in contrast to several other biochemical defects in polymorphonuclear neutrophils. We have now evaluated the role of MPO on phagocyte function in a patient with complete MPO deficiency suffering from generalized pustular psoriasis. We found that the MPO-deficient neutrophils showed enhanced phagocytosis (greater than 200% of normal) of IgG- and C3b-opsonized yeast particles and prolonged N-formylmethionyl-leucyl-phenylaline-mediated stimulation of superoxide production. When purified human MPO was added to normal neutrophils during cell adhesion, their Fc- and C3b-mediated phagocytosis was reduced without affecting cell viability. 1 microgram/ml of MPO reduced the Fc and C3b phagocytosis to 47 and 65%, respectively, whereas 10 micrograms/ml reduced the activity to 20 and 54%. Both attachment and ingestion were reduced to a similar extent, indicating that MPO affected the receptor function per se. When MPO was added to the hyperactive MPO-deficient cells, phagocytosis was reduced more rapidly. Catalase, azide, and methionine eliminated the inhibitory effect, and catalase and methionine, in fact, enhanced the phagocytic activity of adherent neutrophils. These data indicate that, apart from being a potent antimicrobial system, the oxidizing activity of the MPO-H2O2-halide system may modulate the inflammatory response by impairing certain receptor-mediated recognition mechanisms of phagocytic cells, which otherwise could elicit inflammatory reactions and tissue injury.

Authors

O Stendahl, B I Coble, C Dahlgren, J Hed, L Molin

Lingual lipase in cystic fibrosis. Quantitation of enzyme activity in the upper small intestine of patients with exocrine pancreatic insufficiency.

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Abstract

We have measured the level of lingual lipase activity in gastric and duodenal aspirates of five patients with cystic fibrosis (CF) and pancreatic insufficiency. Lingual lipase activity (measured in vitro by the hydrolysis of long-chain triglyceride, tri-[3H]olein, at pH 4.2 and expressed in nanomoles FFA released per milliliter aspirate per minute) and pH in gastric and duodenal aspirates were measured at 10-min intervals during a a 30-min basal period and at 15-min intervals during a 2-h period after the ingestion of a test meal. In gastric aspirates, lingual lipase activity decreased from basal levels of 200 +/- 34 nmol FFA released per milliliter per minute (similar to values reported previously in normal subjects (Hamosh M., H. L. Klaeveman, R. O. Wolf, and R. O. Scow, 1975, J. Clin. Invest., 55:908-913) to 79 +/- 15 nmol FFA/ml per min during the first postprandial hour and returned to basal levels during the second postprandial hour, (206 +/- 39 nmol FFA/ml per min). Duodenal aspirates, obtained during basal conditions, had lingual lipase activity similar to that in the stomach, 178 +/- 63 nmol FFA/ml per min. Enzyme activity levels were 56 +/- 14 and 113 +/- 29 during the first and second postprandial hours. Measurements of total lipase activity delivered to the ligament of Treitz showed that lingual lipase amounted to 91.22 +/- 4.06% of the total lipase activity in the upper small intestine during the 150-min study period. The basal and postprandial gastric pH levels in the five CF patients studied (3.2 +/- 0.44, 4.0 +/- 0.16, and 4.4 +/- 0.4 for basal and first and second postprandial hours, respectively) did not differ from previously reported values for normal subjects. The pH of duodenal aspirates was however significantly lower (P less than 0.001) in CF patients, both under basal conditions (5.0 +/- 0.26) and during the first and second postprandial hours (4.9 +/- 0.13 and 4.4 +/- 0.36, respectively), than in normal subjects. The low postprandial duodenal pH enables lingual lipase to act not only in the stomach but to continue the hydrolysis of dietary fat in the upper small intestine of CF patients. The data presented show that lingual lipase remains fully active in CF and accounts for greater than 90% of total lipase activity in the upper small intestine. We suggest that, because of low intestinal pH in CF, enzyme replacement therapy containing lingual lipase could improve fat absorption in CF patients to a greater extent than the pancreatic preparations now in use.

Authors

C K Abrams, M Hamosh, V S Hubbard, S K Dutta, P Hamosh

Selective inhibition of acetaminophen oxidation and toxicity by cimetidine and other histamine H2-receptor antagonists in vivo and in vitro in the rat and in man.

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Abstract

Acetaminophen-induced hepatotoxicity results from hepatic enzymatic oxidation of acetaminophen to a toxic, electrophilic intermediate. Acetaminophen is ordinarily eliminated after conjugation with glucuronic acid and sulfate to nontoxic derivatives. Cimetidine has been shown to inhibit the hepatic oxidation of a number of drugs and to protect rats from acetaminophen-induced hepatic necrosis. The aim of this study was to define the mechanism by which cimetidine reduced acetaminophen-induced hepatic necrosis and to determine whether inhibition of formation of the reactive metabolite(s) of acetaminophen occurred also in man. In vivo cimetidine pretreatment decreased covalent binding of [3H]acetaminophen to the liver from 552 +/- 23.8 to 170 +/- 31.6 nmol/g protein 2 h after a toxic dose of acetaminophen in 3-methylcholanthrene pretreated rats (P less than 0.05). Cimetidine pretreatment also significantly reduced the rate of hepatic glutathione depletion. Both cimetidine and metiamide produced dose-dependent inhibition of acetaminophen oxidation in vitro, whereas inhibition by ranitidine and cimetidine sulfoxide was quantitatively less. Inhibition of acetaminophen oxidation by cimetidine and metiamide was primarily competitive with an inhibition constant (Ki) of 130 +/- 16 and 200 +/- 50 microM, respectively. By contrast, cimetidine inhibited acetaminophen glucuronidation minimally with a Ki of 1.39 +/- 0.23 mM. Similar results were obtained using human liver microsomes as a source of enzymes. In a dose-related fashion, cimetidine also reduced acetaminophen-induced toxicity to human lymphocytes when incubated with microsomes and NADPH. Pharmacokinetics of acetaminophen elimination were studied in normal volunteers with and without co-administration of cimetidine 300 mg every 6 h. In normal volunteers, cimetidine decreased the fractional clearance of the oxidized (potentially toxic) metabolites of acetaminophen more than the conjugated metabolites. This finding confirmed the hypothesis that cimetidine is a relatively selective inhibitor of the oxidation of acetaminophen to reactive metabolites in man as well as in animals. When considered together with the results of previous studies showing improved survival and decreased hepatoxicity in acetaminophen-poisoned animals, the present results provide a rational basis for assessing possible benefits of cimetidine treatment of acetaminophen overdoses in man.

Authors

M C Mitchell, S Schenker, K V Speeg Jr

Characterization and partial purification of an endothelial cell growth factor from human platelets.

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Abstract

Platelets have been shown to affect the growth of vascular endothelial cells. This report describes the characterization and partial purification from human platelets of a novel growth factor which can stimulate human endothelial cells to synthesize DNA and grow. Platelets were lysed by sonication and the particulate fraction removed by ultracentrifugation at 100,000 g. The supernatant of the platelet lysate stimulated the incorporation of [3H]thymidine into DNA of endothelial cells by 20-fold and caused a threefold increase of cell number in 2 d in culture. Gel filtration on Sephacryl S-200 and dialysis with exclusion membranes resulted in a 50-fold purification of this growth-promoting substance. Two peaks of endothelial-growth factor (ENDO-GF) were observed with apparent molecular weights of 65,000 and 135,000. Further characterization showed that ENDO-GF differed from platelet-derived growth factor since it was very heat labile and more potent in stimulating growth in endothelial cells than in fibroblasts. The isolation of an ENDO-GF from platelets suggests that platelets may have a role in the growth and healing processes of human endothelium.

Authors

G L King, S Buchwald

Influence of methimazole on murine thyroiditis. Evidence for immunosuppression in vivo.

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Abstract

Thionamide drugs are immunosuppressives in vitro. To examine this action in vivo, A/J mice were immunized with human thyroglobulin (hTg) (0.5 mg intraperitoneal injections for 5 d) beginning on days 6, 24, and 43 with or without methimazole (M) (0.05%) and l-thyroxine (T4) (0.1 micrograms/ml to prevent thyroid hypertrophy) in their water supply. Groups (n = 8) were killed on days 37, 42, and 59. Spontaneous splenic IgG-secreting cells determined by Staphylococcus protein A-linked sheep erythrocytes (SRBC) via indirect plaque-forming cell (PFC) assay indicated polyclonal stimulation induced by the hTg exposure (controls = 2,285 +/- 599, hTg-only = 5,570 +/- 470 PFC per 10(6) spleen cells), but this was significantly reduced in the M plus T4-treated group (3,640 +/- 415 PFC, P = 0.05). hTg antibody was measured by specific PFC assay using hTg-linked SRBC. Anti-hTg PFC were absent in controls and were 147 +/- 41, 25 +/- 8, and 173 +/- 58 PFC per 10(6) spleen cells in the hTg-only groups on days 37, 42, and 59, respectively. Anti-hTg PFC results in the M plus T4-treated animals were significantly reduced to 0, 15 +/- 5, and 63 +/- 30 anti-hTg PFC. Histological examination revealed a marked thyroiditis in hTg-only animals and a significantly reduced degree of mononuclear cell infiltration and follicular destruction in the M plus T4-treated groups (graded 1.9 compared with 3.6 in hTg-only P = less than 0.01). Examination of IgG deposition using fluorescent anti-mouse IgG revealed a similar granular pattern and degree of staining in both immunized groups. Control animals that received concurrent T4 administration alone showed similar hTg-induced murine thyroiditis to non-T4-treated animals and could not explain the apparent immunosuppression observed. In conclusion, these data demonstrated that M reduced both the splenic immune response and the degree of thyroiditis after heterologous Tg immunization, while a quantitative difference in the circulating and intrathyroidally deposited Tg antibody was not detected.

Authors

T F Davies, I Weiss, M A Gerber

Epinephrine supports the postabsorptive plasma glucose concentration and prevents hypoglycemia when glucagon secretion is deficient in man.

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We hypothesized that adrenergic mechanisms support the postabsorptive plasma glucose concentration, and prevent hypoglycemia when glucagon secretion is deficient. Accordingly, we assessed the impact of glucagon deficiency, produced by infusion of somatostatin with insulin, without and with pharmacologic alpha- and beta-adrenergic blockade on the postabsorptive plasma glucose concentration and glucose kinetics in normal human subjects. During somatostatin with insulin alone mean glucose production fell from 1.5 +/- 0.05 to 0.7 +/- 0.2 mg/kg per min and mean plasma glucose declined from 93 +/- 3 to 67 +/- 4 mg/dl over 1 h; glucose production then increased to base-line rates and plasma glucose plateaued at 64-67 mg/dl over 2 h. This plateau was associated with, and is best attributed to, an eightfold increase in mean plasma epinephrine. It did not occur when adrenergic blockade was added; glucose production remained low and mean plasma glucose declined progressively to a hypoglycemic level of 45 +/- 4 mg/dl, significantly (P less than 0.001) lower than the final value during somatostatin with insulin alone. These data provide further support for the concept that maintenance of the postabsorptive plasma glucose concentration is a function of insulin and glucagon, not of insulin alone, and that adrenergic mechanisms do not normally play a critical role. They indicate, however, that an endogenous adrenergic agonist, likely adrenomedullary epinephrine, compensates for deficient glucagon secretion and prevents hypoglycemia in the postabsorptive state in humans. Thus, postabsorptive hypoglycemia occurs when both glucagon and epinephrine are deficient, but not when either glucagon or epinephrine alone is deficient, and insulin is present.

Authors

S G Rosen, W E Clutter, M A Berk, S D Shah, P E Cryer

Increased proteolysis. An effect of increases in plasma cortisol within the physiologic range.

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Prolonged exposure to glucocorticoids in pharmacologic amounts results in muscle wasting, but whether changes in plasma cortisol within the physiologic range affect amino acid and protein metabolism in man has not been determined. To determine whether a physiologic increase in plasma cortisol increases proteolysis and the de novo synthesis of alanine, seven normal subjects were studied on two occasions during an 8-h infusion of either hydrocortisone sodium succinate (2 micrograms/kg X min) or saline. The rate of appearance (Ra) of leucine and alanine were estimated using [2H3]leucine and [2H3]alanine. In addition, the Ra of leucine nitrogen and the rate of transfer of leucine nitrogen to alanine were estimated using [15N]leucine. Plasma cortisol increased (10 +/- 1 to 42 +/- 4 micrograms/dl) during cortisol infusion and decreased (14 +/- 2 to 10 +/- 2 micrograms/dl) during saline infusion. No change was observed in plasma insulin, C-peptide, or glucagon during either saline or cortisol infusion. Plasma leucine concentration increased more (P less than 0.05) during cortisol infusion (120 +/- 1 to 203 +/- 21 microM) than saline (118 +/- 8 to 154 +/- 4 microM) as a result of a greater (P less than 0.01) increase in its Ra during cortisol infusion (1.47 +/- 0.08 to 1.81 +/- 0.08 mumol/kg X min for cortisol vs. 1.50 +/- 0.08 to 1.57 +/- 0.09 mumol/kg X min). Leucine nitrogen Ra increased (P less than 0.01) from 2.35 +/- 0.12 to 3.46 +/- 0.24 mumol/kg X min, but less so (P less than 0.05) during saline infusion (2.43 +/- 0.17 to 2.84 +/- 0.15 mumol/kg X min, P less than 0.01). Alanine Ra increased (P less than 0.05) during cortisol infusion but remained constant during saline infusion. During cortisol, but not during saline infusion, the rate and percentage of leucine nitrogen going to alanine increased (P less than 0.05). Thus, an increase in plasma cortisol within the physiologic range increases proteolysis and the de novo synthesis of alanine, a potential gluconeogenic substrate. Therefore, physiologic changes in plasma cortisol play a role in the regulation of whole body protein and amino acid metabolism in man.

Authors

P S Simmons, J M Miles, J E Gerich, M W Haymond

Interaction of von Willebrand factor with human platelets in the plasma milieu.

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The binding of von Willebrand factor (vWf) to stimulated platelets in the plasma milieu was performed using a radiolabeled monoclonal antibody to vWf. Plasma proteins specifically inhibited the thrombin- and ADP/epinephrine-induced vWf binding to activated platelets but did not inhibit the ristocetin-induced vWf binding. When normal plasma was heat defibrinated, monoclonal-labeled vWf was bound to platelets following thrombin or ADP/epinephrine stimulation. Furthermore, monoclonal-labeled vWf from afibrinogenemic plasma bound normally to platelets. The binding of vWf to stimulated platelets in either heat-defibrinated normal plasma or afibrinogenemic plasma was specifically inhibited by the addition of normal plasma fibrinogen in a concentration-dependent manner. At levels of fibrinogen less than 1 mg/ml, however, vWf binding could be demonstrated. The inhibition by fibrinogen of vWf binding to platelets was competitive and overcome by increased concentrations of vWf. These studies show that thrombin-induced and ADP/epinephrine-induced vWf binding to platelets does not occur in the plasma milieu, although at reduced levels of fibrinogen, vWf binding to stimulated platelets can be demonstrated.

Authors

J Schullek, J Jordan, R R Montgomery

Characterization of hepatic low density lipoprotein binding and cholesterol metabolism in normal and homozygous familial hypercholesterolemic subjects.

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Patients with familial hypercholesterolemia have elevated levels of plasma low density lipoproteins (LDL), increased hepatic synthesis of apolipoprotein B-containing lipoproteins, defective binding of low density lipoproteins to fibroblasts, and premature atherosclerosis. The role of a hepatic low density lipoprotein receptor in normal man and its importance in the pathogenesis of familial hypercholesterolemia have not been previously determined. In the present study, direct comparison was made of the binding of LDL to hepatic membranes from normal and receptor-negative homozygous familial hypercholesterolemic subjects. The effects of calcium, EDTA, and temperature on the binding of lipoproteins to the hepatic membranes were also evaluated. At 4 degrees C, no significant difference in specific binding of LDL to hepatic membranes from normal and familial hypercholesterolemic subjects was observed. At 37 degrees C, both total and specific binding of LDL were significantly reduced in patients with familial hypercholesterolemia. Hepatic membrane binding of LDL from the two patients homozygous for receptor-negative familial hypercholesterolemia was 53 and 59% of normal. The activity of the rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase was normal; however, the total hepatic cholesterol and cholesteryl ester content was significantly increased from 53 to 129%. These results indicate that patients with familial hypercholesterolemia have a defect in the interaction of hepatic membranes with low density lipoproteins. This defect may lead to accelerated atherosclerosis by decreasing the cellular catabolism of LDL and enhancing the production of LDL, which is characteristic of patients homozygous for familial hypercholesterolemia.

Authors

J M Hoeg, S J Demosky Jr, E J Schaefer, T E Starzl, H B Brewer Jr

Characterization of inactive renin ("prorenin") from renin-secreting tumors of nonrenal origin. Similarity to inactive renin from kidney and normal plasma.

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Abstract

Inactive renin comprises well over half the total renin in normal human plasma. There is a direct relationship between active and inactive renin levels in normal and hypertensive populations, but the proportion of inactive renin varies inversely with the active renin level; as much as 98% of plasma renin is inactive in patients with low renin, whereas the proportion is consistently lower (usually 20-60%) in high-renin states. Two hypertensive patients with proven renin-secreting carcinomas of non-renal origin (pancreas and ovary) had high plasma active renin (119 and 138 ng/h per ml) and the highest inactive renin levels we have ever observed (5,200 and 14,300 ng/h per ml; normal range 3-50). The proportion of inactive renin (98-99%) far exceeded that found in other patients with high active renin levels. A third hypertensive patient with a probable renin-secreting ovarian carcinoma exhibited a similar pattern. Inactive renins isolated from plasma and tumors of these patients were biochemically similar to semipurified inactive renins from normal plasma or cadaver kidney. All were bound by Cibacron Blue-agarose, were not retained by pepstatin-Sepharose, and had greater apparent molecular weights (Mr) than the corresponding active forms. Plasma and tumor inactive renins from the three patients were similar in size (Mr 52,000-54,000), whereas normal plasma inactive renin had a slightly larger Mr than that from kidney (56,000 vs. 50,000). Inactive renin from each source was activated irreversibly by trypsin and reversibly by dialysis to pH 3.3 at 4 degrees C; the reversal process followed the kinetics of a first-order reaction in each instance. The trypsin-activated inactive renins were all identical to semipurified active renal renin in terms of pH optimum (pH 5.5-6.0) and kinetics with homologous angiotensinogen (Michaelis constants, 0.8-1.3 microM) and inhibition by pepstatin or by serial dilutions of renin-specific antibody. These results indicate that a markedly elevated plasma inactive renin level distinguishes patients with ectopic renin production from other high-renin hypertensive states. The co-production of inactive and active renin by extrarenal neoplasms provides strong presumptive evidence that inactive renin is a biosynthetic precursor of active renin. The unusually high proportion of inactive renin in plasma and tumor extracts from such patients is consistent with ineffective precursor processing by neoplastic tissue, suggesting that if activation of "prorenin" is involved in the normal regulation of active renin levels it more likely occurs in the tissue of origin (e.g., kidney) than in the circulation.

Authors

S A Atlas, T E Hesson, J E Sealey, B Dharmgrongartama, J H Laragh, M C Ruddy, M Aurell

Possible mechanism of phorbol diester-induced maturation of human promyelocytic leukemia cells.

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Abstract

The phorbol diesters are the most potent inducers of differentiation of the promyelocytic leukemia cell line, HL-60. Soluble phorbol diester receptors from HL-60 cells were obtained from the cytosolic fraction and from the particulate fraction by either divalent ion chelation or detergent extraction. The partially purified soluble phorbol diester receptors required exogenous Ca2+ and phospholipid for maximal binding and displayed a dissociation constant (KD) of 8.1 nM for [3H]phorbol 12,13-dibutyrate. Phorbol diester analogues inhibited [3H]phorbol 12,13-dibutyrate binding in a stereospecific manner consistent with their biologic potency. The soluble phorbol diester receptors prepared by all three methods copurified in a constant ratio with the Ca2+/phospholipid-dependent protein kinase C through ammonium sulfate precipitation, DEAE ion exchange, and gel filtration chromatography. Partially purified protein kinase C was directly activated by the phorbol diesters even in the absence of exogenous Ca2+. The ability of a series of phorbol analogues to activate the kinase correlated with their known activity as inducers of cell differentiation. In addition, phorbol diester stimulation altered the phosphate acceptor substrate profile of protein kinase C, at least in part, by alteration of the Michaelis constant (Km). These data suggest that protein kinase C is the phorbol diester receptor and that phorbol diester-induced macrophage maturation of HL-60 cells may be mediated by activation of intracellular protein kinase C.

Authors

G R Vandenbark, L J Kuhn, J E Niedel

Biopterin synthesis defect. Treatment with L-dopa and 5-hydroxytryptophan compared with therapy with a tetrahydropterin.

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Abstract

We have identified a generalized deficiency of monoamine neurotransmitters in a patient with a defect in biopterin synthesis. Neurotransmitter precursors (L-3,4-dihydroxyphenylalanine [L-dopa]; 5-hydroxytryptophan [5-HTP] and a tetrahydropterin [6-methyltetrahydropterin (6MPH4)] were investigated for their ability to normalize monoamine neurotransmitter metabolism. Before treatment, the concentrations of dopamine (DA), norepinephrine, epinephrine, and six monoamine metabolites were very low or undetectable in plasma, cerebrospinal fluid, or urine. L-Dopa and 5-HTP replacement was begun at age 7 mo. This therapy generally corrected the deficiency of monoamines and their metabolites, and improved neurological development until the age of 25 mo. Despite these benefits, the intermittent administration of L-dopa could not produce a stable improvement of acute neurological function or DA metabolism. In the 3 h after L-dopa administration, plasma DA and the motor activity and alertness of the patient rose and fell in parallel. Doses of L-dopa that were clinically optimal produced normal plasma levels of norepinephrine and epinephrine, but excessive concentrations of DA and its metabolites. Furthermore, the clinical and biochemical effects of L-dopa were inhibited by phenylalanine and 5-HTP, respectively, demonstrating that these amino acids have antagonistic pharmacological effects. Physiological correction of the monoamine deficit and the hyperphenylalaninemia of this disorder was attempted at age 35 mo using high doses (8-38 mg/kg per d) of 6MPH4. 6MPH4, a synthetic analogue of tetrahydrobiopterin, controlled the hyperphenylalaninemia. Significant concentrations of 6MPH4 were obtained in the cerebrospinal fluid; no neurological improvement or stimulation of monoamine synthesis in the central nervous system was detected. These findings indicate the complexity in replacement therapy with L-dopa and 5-HTP, but suggest that this treatment may be partially effective in biopterin-deficient patients who are unresponsive to high doses of tetrahydropterins.

Authors

R R McInnes, S Kaufman, J J Warsh, G R Van Loon, S Milstien, G Kapatos, S Soldin, P Walsh, D MacGregor, W B Hanley

Role of iron in Trypanosoma cruzi infection of mice.

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Abstract

The role of iron in experimental infection of mice with Trypanosoma cruzi was investigated. B6 mice had a transient parasitemia and a transient anemia, both of maximal intensity 28 d after the inoculation of T. cruzi. There was a biphasic hypoferremic host response to infection with T. cruzi with the peak hypoferremia also occurring 28 d after inoculation of the parasite. The mortality rate from infection was increased from 23% in phosphate-buffered saline-treated B6 mice to 50% in a group of B6 mice receiving iron-dextran (P less than or equal to 0.025), whereas depletion of iron stores with the iron chelator desferrioxamine B and an iron-deficient diet provided complete protection of B6 mice (P less than or equal to 0.05). The mortality rate in the highly susceptible C3H strain was reduced from 100% in the control group to 45% (P less than or equal to 0.025) in the iron-depleted group. The tissue iron stores were altered in mice receiving either iron-dextran or desferrioxamine B and an iron-deficient diet. In vitro, T. cruzi was shown to require both a heme and a nonheme iron source for an optimal growth rate. The effects of iron excess or depletion on the outcome of infection with T. cruzi correlated both with the growth requirements of the parasite for iron and with the availability of intracellular iron. Thus, it was suggested that the hypoferremic response, by sequestering iron within intracellular stores, potentially enhanced the pathogenicity of the intracellular parasites. Furthermore, the in vivo effects of iron excess and depletion correlated with an effect of iron on the growth rate and pathogenicity of the parasite.

Authors

R G Lalonde, B E Holbein

Static and dynamic rigidities of normal and sickle erythrocytes. Major influence of cell hemoglobin concentration.

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Abstract

Static and dynamic deformabilities of erythrocytes are important determinants of microcirculatory blood flow. To determine the influence of increased cellular hemoglobin concentration on these properties, we quantitated static and dynamic deformabilities of isolated subpopulations of oxygenated normal and sickle erythrocytes with defined cell densities using micromechanical manipulations of individual cells. The rheological properties measured to characterize static deformability were membrane extensional rigidity and bending rigidity. To characterize dynamic deformability of the cells, we measured the time constants for rapid elastic recovery from extensional and bending deformations. The extensional rigidity of sickle cells increased with increasing cell hemoglobin concentration while that of normal cells was independent of the state of cell hydration. Moreover, sickle cells were found to exhibit inelastic behavior at much lower cell hemoglobin concentrations than normal cells. In contrast, the dynamic rigidity of both normal and sickle cells was increased to the same extent at elevated hemoglobin concentrations. Moreover, this increase in dynamic rigidity with increasing cellular dehydration was much more pronounced than that seen for static rigidity. Both the increased static and dynamic rigidities of the dehydrated sickle cells could be greatly improved by hydrating the cells. This suggests that increased bulk hemoglobin concentration, which is perhaps inordinately increased adjacent to the membrane, plays a major role in regulating the rigidity of sickle cells. In addition, irreversible membrane changes also appear to accompany cell dehydration in vivo, resulting in increased membrane shear rigidity and plastic flow. We expect that the marked increases in rigidity of dehydrated sickle cells observed here may have a major influence on the dynamics of their circulation in the microvasculature.

Authors

E Evans, N Mohandas, A Leung

Reactions of immunoglobulin G-binding ligands with platelets and platelet-associated immunoglobulin G.

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Abstract

Immunoglobulin G (IgG) bound to platelets is usually detected by one of two general methods: binding of labeled anti-IgG or consumption of anti-IgG. The latter method gives, in general, values 5-10-fold greater than the former under the same conditions. To investigate these discrepancies, we have compared the detection of platelet-bound IgG by a labeled anti-IgG binding assay and by a quantitative antiglobulin consumption test using the same antibodies. The interaction of 125I-labeled monoclonal anti-IgG or polyclonal anti-IgG with washed and IgG-coated platelets was studied. The binding of these ligands to washed normal platelets was largely (50-80%) nonspecific; the binding was not saturable and was only partially inhibitable by excess unlabeled anti-IgG. The binding of anti-IgG to platelets coated with anti-PIA1, a platelet-specific IgG antibody, appeared to be saturable and inhibitable; the dissociation constant (KD) of this IgG-anti-IgG reaction was 4.9 X 10(-9) for monoclonal and 1.4 X 10(-7) for polyclonal anti-IgG. The ratio of sites present on the membrane (determined by 131I-labeled anti-PIA1) to the number of binding sites for anti-IgG determined by Scatchard analysis was 0.53 for monoclonal anti-IgG and 1.3 for polyclonal anti-IgG. The binding of monoclonal anti-IgG to platelet-bound immune complexes or IgG aggregates appeared to be complex. 131I-Labeled IgG was affixed to platelets and was detected by three tests: direct binding of radiolabeled monoclonal anti-IgG and quantitative antiglobulin consumption (QAC) tests, which were quantitated either by measuring directly the amount of radiolabeled anti-IgG consumed from fluid phase (direct QAC), or indirectly by reference to a calibration curve relating the consumption of anti-IgG by known amounts of fluid-phase, non-immune IgG (indirect QAC). The amount of platelet-bound IgG detected by the direct binding of 125I-labeled monoclonal anti-IgG and by the direct QAC approximated that known to be bound to the platelet. The results of the indirect QAC test were 10-fold greater. The discrepancy appears to be due to the fact that there is a difference between the IgG-anti-IgG interaction when IgG is bound to a platelet and when it is in solution or bound to plastic nonspecifically or specifically. This difference results in a falsely high value for platelet-bound IgG when fluid-phase or plastic-bound IgG is used to calibrate the antiglobulin consumption test.

Authors

W F Rosse, D V Devine, R Ware

Correlation between ventilation and brain blood flow during sleep.

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Abstract

The relationships between brain blood flow (BBF) and ventilation (VI) were studied during sleep in 13 goats. Unilateral BBF was continuously measured with an electromagnetic flow probe; total and regional BBF were assessed by the radioactive microsphere technique in four animals. Interacting changes in VI and BBF occurred during both slow wave (SWS) and rapid eye movement (REM) sleep. During SWS, significant decreases in VI and increases in arterial PCO2 occurred compared to wakefulness. BBF during SWS correlated linearly with arterial CO2 tension (PaCO2); nd the relationship was similar to that for awake goats breathing CO2. During REM sleep, VI was significantly less than both the awake (W) and SWS states due principally to a decrease in tidal volume. BBF during REM sleep was significantly and substantially increased compared with both the W and SWS states; this increase was shared by all brain areas. The increase in BBF during REM sleep was greater than that predicted from changes in PaCO2. In five goats provided with chronic sagittal sinus fistulae, arteriovenous oxygen difference was measured in separate studies and found to be significantly lower during REM sleep compared with W; brain O2 consumption was similar in magnitude in the REM and W states. Thus, the high BBF of REM sleep was also unexplained by an increase of brain metabolic activity. We conclude that, during SWS, increases in BBF are explained by hypoventilation and hypercapnia. In contrast, during REM sleep, BBF is substantially in excess of that expected from PaCO2 or brain metabolism. It is postulated that this excess of BBF during REM sleep could reduce the central chemoreceptor pH relative to that present in SWS. The combination of reduction of sensitivity to CO2 and lower tissue PCO2 during REM sleep makes it likely that the output of the central chemoreceptors during this state is less than that during SWS and wakefulness. This may contribute to the low tidal volume and respiratory irregularities of this sleep period.

Authors

T V Santiago, E Guerra, J A Neubauer, N H Edelman

Angiotensin II directly stimulates sodium transport in rabbit proximal convoluted tubules.

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Abstract

Numerous previous studies have proposed a role for angiotensin II (AII) in the renal regulation of salt balance. At least one nephron site, the proximal convoluted segment, has been implicated in this role. We used in vitro microperfusion of rabbit proximal convoluted tubules to further examine this question. To insure use of appropriate in vivo concentrations as well as potency of the hormone in vitro, we measured plasma AII levels by radioimmunoassay in normal, sodium-depleted, and adrenalectomized rabbits, and measured AII activity by bioassay after incubation in various microperfusion baths. Plasma levels ranged from approximately 2 X 10(-11) to 5 X 10(-11) M. AII activity was stable in Ringer's solution plus albumin, but not in rabbit serum or Ringer's solution plus fetal calf serum. In Ringer's solution plus albumin, physiologic concentrations of AII stimulated volume reabsorption (Jv). 10(-11) M AII increased Jv by 16% (P less than 0.01). 10(-10) M AII produced a lesser increase, 7.5% (P less than 0.05). At a frequently studied, but probably pharmacologic dose, 10(-7) M AII inhibited Jv by 24% (P less than 0.001). AII at 10(-11) M did not stimulate Jv in the presence of 10(-7) M saralasin. Though previous studies have suggested agonistic effects of saralasin alone in epithelia, we found no significant effect of 10(-7) M saralasin on Jv. None of the AII doses measurably changed transepithelial voltage. We conclude that AII in physiologic doses directly stimulates Jv in proximal convoluted tubules and this effect is probably receptor mediated and, within the limits of detection, electroneutral.

Authors

V L Schuster, J P Kokko, H R Jacobson

Ontogeny of Fc receptors and complement receptor (CR3) during human myeloid differentiation.

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Abstract

Two different Fc receptors for IgG (Fc gamma R) have been identified on human leukocytes: a high avidity receptor (Fc gamma Rhi) present on monocytes but not on neutrophils, and a low avidity receptor (Fc gamma Rlo) present on neutrophils but not on monocytes. Fc gamma Rlo can be inhibited and the receptor precipitated by monoclonal antibody 3G8. We have used this monoclonal antibody to study the course of Fc gamma Rlo appearance on bone marrow cells, leukocytes of patients with chronic myelogenous leukemia (CML), and HL-60 and U937 cells induced to differentiate with agents such as dimethyl sulfoxide (DMSO), retinoic acid, phorbol myristate acetate, and lymphokine. We report that Fc gamma Rlo is a late differentiation antigen, first expressed at the metamyelocyte stage. Since precursors to metamyelocytes bear Fc gamma R, and the promyelocyte line HL-60 bears Fc gamma Rhi, there must be a progressive loss of Fc gamma Rhi during myeloid differentiation and the reciprocal expression of Fc gamma Rlo. Results of immunoprecipitation and polyacrylamide gel analysis of the proteins are consistent with these results. We have also studied the receptor for the C3bi complement component (CR3), which is blocked and immunoprecipitated by monoclonal antibody OKM10. During DMSO-driven differentiation of HL-60 cells, we find that CR3 is induced on all cells, whereas Fc gamma Rlo is induced on only 24% of cells, suggesting that CR3 appears earlier during differentiation than Fc gamma Rlo does.

Authors

H B Fleit, S D Wright, C J Durie, J E Valinsky, J C Unkeless

Role of endogenous secretin in acid-induced inhibition of human gastric function.

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Abstract

The role of secretin in the inhibition of gastric acid secretion that occurs during acidification of the gastric lumen was studied in nine healthy men. Gastric acid secretion was stimulated by 500-ml meals of 8% peptone solution, and the pH of the stomach was maintained at 5.5, 2.5, or 2.0 by intragastric titration. The increase in plasma secretin was measured, after extraction, by a new secretin radioimmunoassay. After determining the intravenous dose of secretin required to reproduce plasma secretin concentrations achieved during pH 2.5 and 2.0 meals, similar doses were given during administration of a pH 5.5 peptone meal. The doses of secretin led to plasma secretin concentrations that averaged 3.4 pM, not different from the 3.2 and 3.9 pM concentrations achieved during acidified meals. However, exogenous secretin infusion failed to inhibit acid secretion or gastrin response to peptone, although significant inhibitions occurred in both during peptone meals given at pH 2.5 or 2.0. When secretin infusions were given at fivefold higher rates, plasma gastrin responses again failed to demonstrate significant inhibition. Gastric emptying was inhibited significantly by both acidified peptone meals but only slightly (P = 0.053) during exogenous infusion of physiologic secretin doses. The decrease in acid secretion could be explained by decreased gastrin release, but neither of these findings could be explained by circulating secretin concentrations. These results cast strong doubt on a physiological role of secretin in inhibition of acid secretion in man.

Authors

J H Kleibeuker, V E Eysselein, V E Maxwell, J H Walsh

Arginine vasopressin potentiates adrenocorticotropin release induced by ovine corticotropin-releasing factor.

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Abstract

Arginine vasopressin (AVP) stimulates ACTH release in man and acts synergistically with synthetic ovine corticotropin-releasing factor (oCRF) in vitro. This study was designed to examine in man the combined effects of synthetic AVP (10 U intramuscularly) and oCRF (1 micrograms/kg intravenously) on ACTH release. Five normal male volunteers participated in five separate experiments: (a) AVP alone; (b) oCRF alone; (c) AVP followed by oCRF 15 min later; (d) simultaneous AVP and oCRF; and (e) insulin-induced hypoglycemia. Plasma immunoreactive ACTH (IR-ACTH) and IR-cortisol were measured for 4 h after injection of each hormone; basal levels for all subjects were less than or equal to 9 +/- 1.2 pg/ml and 4.9 +/- 0.4 micrograms/dl (mean +/- SE), respectively. AVP and oCRF, when given individually, caused rapid rises in IR-ACTH to similar peak levels of 25 +/- 6.6 and 33 +/- 4.6 pg/ml, respectively. AVP given 15 min before oCRF caused a 2.6-fold potentiation of the oCRF response, with a peak IR-ACTH of 85 +/- 4.6 pg/ml. AVP given at the same time as oCRF produced a fourfold potentiation of the peak IR-ACTH response to 132 +/- 11 pg/ml. These ACTH responses were far greater than those previously observed after 30-fold greater doses of oCRF alone. By way of comparison, insulin-induced hypoglycemia caused a peak IR-ACTH of 169 +/- 20 pg/ml. IR-ACTH returned to base line at 60-90 min after AVP alone, whereas the prolonged effect of oCRF was apparent whether it was given alone or in combination with AVP. The mean peak IR-cortisol responses to AVP, oCRF, and AVP given 15 min before oCRF were similar (16.5 +/- 0.9, 16.4 +/- 2.3, and 18.5 +/- 0.8 micrograms/dl, respectively), but the peak IR-cortisol responses to AVP and oCRF given simultaneously and to insulin-induced hypoglycemia were 1.5 and 1.7 times greater, respectively. IR-cortisol returned to base line within 2-3 h after AVP alone, but remained elevated for at least 4 h after oCRF alone or in combination with AVP. These results indicate that AVP acts synergistically with oCRF to release ACTH in man and suggest that AVP may play a physiologic role in modulating the ACTH response mediated by corticotropin-releasing factor.

Authors

C R DeBold, W R Sheldon, G S DeCherney, R V Jackson, A N Alexander, W Vale, J Rivier, D N Orth

Encainide and its metabolites. Comparative effects in man on ventricular arrhythmia and electrocardiographic intervals.

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Abstract

To assess the relative contributions of encainide and its putatively active metabolites, O-demethyl encainide (ODE) and 3 methoxy-O-demethyl encainide (3MODE), to the drug's pharmacologic effects, we compared intravenous infusions and sustained oral therapy in two phenotypically distinct groups of patients, extensive and poor metabolizers of encainide. Unlike poor metabolizers, extensive metabolizers had appreciable quantities of both metabolites detectable in plasma and had fourfold shorter elimination half-lives for encainide. By quantitating electrocardiogram intervals, arrhythmia frequency, and plasma concentrations, we found that, in poor metabolizers, arrhythmia suppression and ventricular complex (QRS) prolongation were correlated positively with encainide concentrations (r greater than or equal to 0.570, P less than 0.014). In these two subjects, antiarrhythmic concentrations of encainide (greater than 265 ng/ml) were at least fivefold higher than those sustained in the six extensive metabolizers during steady state oral therapy. In extensive metabolizers, encainide concentrations were uncorrelated with effects. Arrhythmia suppression and QRS prolongation in extensive metabolizers correlated best with ODE (r greater than or equal to 0.816, P less than 0.001); QTc change correlated positively with both 3MODE and ODE. Arrhythmia suppression paralleled QRS prolongation; the relationship between them appeared similar in both phenotypic groups. In most patients, extensive metabolizers, encainide effects during oral therapy are mediated by metabolites, probably ODE.

Authors

E L Carey Jr, H J Duff, D M Roden, R K Primm, G R Wilkinson, T Wang, J A Oates, R L Woosley

Inhibition of platelet-aggregating activity in thrombotic thrombocytopenic purpura plasma by normal adult immunoglobulin G.

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Abstract

Plasma from patients with thrombotic thrombocytopenic purpura (TTP) caused the aggregation of autologous and homologous platelets, and effect which was inhibited by normal plasma. IgG purified from seven normal adults at a concentration of 0.7 mg/ml completely inhibited the platelet aggregation induced by plasma obtained from two TTP patients with active disease. The inhibition of platelet aggregation by human adult IgG was concentration dependent, and the inhibitory activity of human IgG was neutralized by rabbit antihuman IgG. Fab fragments inhibited the TTP plasma-induced platelet aggregation as well as intact IgG, whereas Fc fragments had no effect. Platelet aggregation caused by ADP, collagen, epinephrine, or thrombin was not affected by purified human IgG. The prior incubation of IgG with TTP plasma caused a significantly greater reduction of platelet aggregation by TTP plasma than that of IgG and platelet suspension, suggesting that the IgG inhibits TTP plasma-induced platelet aggregation through direct interaction with platelet aggregating factor in TTP plasma. IgG obtained initially from five infants and young children under the age of 4 yr did not possess any inhibitory activity. When one of the children reached 3 yr of age, his IgG inhibited the aggregation induced by one TTP plasma, but not that caused by another plasma. The IgG procured from the same boy at 4 yr of age inhibited the aggregation induced by both TTP plasmas. The IgG purified from the TTP plasma during active disease failed to inhibit the aggregation caused by the same plasma. After recovery, however, the IgG effectively inhibited aggregation. These observations suggest that platelet-aggregating factors present in the TTP plasma are heterogeneous in nature and that the IgG present in the normal adult plasma, which inhibits the TTP plasma-induced platelet aggregation, may be partially responsible for the success of plasma infusion therapy in TTP.

Authors

E C Lian, P T Mui, F A Siddiqui, A Y Chiu, L L Chiu

Regulation of human peripheral blood monocyte DR antigen expression in vitro by lymphokines and recombinant interferons.

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Abstract

The in vitro regulation of adult human monocyte DR antigen expression was studied. Normally about 75% of freshly obtained human peripheral blood monocytes express DR antigens as determined by anti-DR and complement-mediated cytotoxicity assays. DR expression on monocytes in unfractionated peripheral blood mononuclear cell cultures persisted to variable degrees for up to 5 d of incubation. However, when the mononuclear cells were thoroughly depleted of nonadherent cells, cultured monocytes consistently exhibited progressively decreased DR expression over 2-5 d of incubation. Readdition of nonadherent cells to the adherent cell population prevented or delayed this decrease in monocyte DR antigen expression. Thus, monocyte DR expression diminished markedly during in vitro incubation; however, the presence of nonadherent cells somehow interfered with this process. In other experiments, peripheral adherent monocytes, which had been cultured for 2-3 d to reduce their DR expression, could be induced to reexpress DR antigens after 2 d of incubation with unpurified lymphokine-containing culture supernatants, recombinant human interferon-alpha, or recombinant human gamma interferon (IFN-gamma). The reinduction of DR expression on human monocytes by lymphokines was abrogated by an antiserum produced to the synthetic N-terminal amino acids of human IFN-gamma, indicating that IFN-gamma is the active mediator in the lymphokine-containing preparations. Monocytes cultured with lymphokines or recombinant interferons also could initiate a significantly greater mixed lymphocyte response than control monocytes. Thus, IFN-gamma-containing lymphokines and recombinant interferons are required to induce human monocyte DR expression and accessory cell capacity in vitro, since in their absence monocytes become DR antigen-deficient. Finally, incubation of unfractionated human mononuclear cells with anti-human IFN-gamma also promoted the loss of monocyte DR expression. These findings suggest that resting lymphocytes are probably capable of producing sufficient IFN-gamma in vitro to result in the maintenance of the monocyte DR phenotype.

Authors

M B Sztein, P S Steeg, H M Johnson, J J Oppenheim

Effect of chronic sham feeding on maximal gastric acid secretion in the dog.

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Abstract

The reason for increased maximal acid secretory capacity in some patients with duodenal ulcer is uncertain. We postulated that chronically increased cephalic-vagal stimulation may be a cause of increased maximal acid output. To study this, we prepared six male, mongrel dogs with a vagally innervated gastric fistula, a vagally denervated fundic (Heidenhain) pouch, and a cervical esophagostomy. Physiological cephalic-vagal stimulation was accomplished by sham feeding, which increased acid output from the vagally innervated stomach but not from the vagally denervated pouch. During an initial 6-wk control period, dogs were fed by mouth once daily at 3 p.m. Then, a 6-wk period of sham feeding was carried out, during which animals were sham fed with blenderized dog chow from 8 a.m. to 3 p.m. every day (a 7-h period of continuous cephalic-vagal stimulation), after which animals were fed by mouth. After 6 wk of daily sham feeding, maximal acid output in response to intravenous pentagastrin (16 micrograms/kg per h) increased by 27 +/- 4% in the vagally innervated stomach (P less than 0.01). Maximal acid output then returned to control levels after a final 6-wk recovery period with no sham feeding. No changes in maximal acid output occurred in the vagally denervated pouch during the 18-wk study. No changes in basal acid secretion or responsiveness of parietal cells to submaximal doses of pentagastrin occurred in the fistula or pouch during chronic sham feeding. We conclude that chronic physiological cephalic-vagal stimulation can increase maximal acid secretory capacity. Our studies also suggest that the effect of chronically increased vagal stimulation on maximal acid secretory capacity is reversible.

Authors

R C Thirlby, M Feldman

Peripheral tissue mechanism for maintenance of serum triiodothyronine values in a thyroxine-deficient state in man.

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Abstract

The present study was undertaken to define the source of endogenous triiodothyronine (T3) production responsible for maintaining serum T3 levels in euthyroid subjects with depressed serum thyroxine (T4) values. After withdrawal from 4 wk of exogenous T3 administration, a 22% decline in serum T3 values (from 129 +/- 6 to 99 +/- 4 ng/dl) was observed in six euthyroid subjects, despite a twofold reduction in serum T4 concentrations (from 7.5 +/- 0.5 to 3.2 +/- 0.5 micrograms/dl). This was accompanied by a nearly twofold increase in serum T3/T4 ratio values (17 +/- 1 to 29 +/- 6) but no significant alteration in reverse T3/T4 ratio values. This phenomenon did not appear to be thyroid stimulating hormone (TSH) dependent, since base-line serum TSH values were subnormal. Nor was it dependent on changes in thyroid gland function, since a blunted T3 response to exogenous bovine TSH occurred and pharmacologic doses of iodide did not influence the phenomenon. The finding in three athyreotic subjects that serum T3/T4 ratio values increased from 14 +/- 1 on T4 therapy (mean serum T4, 9.6 +/- 0.8 micrograms/dl and T3, 132 +/- 8 ng/dl) to 40 +/- 2 after withdrawal from 2 wk of T3 administration (serum T4 1.2 +/- 0.1 micrograms/dl and T3 46 +/- 3 ng/dl) provided direct evidence that an alteration in peripheral thyroid hormone metabolism was probably responsible for these findings previously observed in euthyroid subjects. The results of this study support the possible existence in euthyroid man of a peripheral tissue autoregulatory mechanism for maintaining serum T3 values in states of T4 deficiency. Whether this process involves an alteration in the efficiency of T4 to T3 conversion or the rate of T3 clearance is presently unknown.

Authors

S M Lum, J T Nicoloff, C A Spencer, E M Kaptein

Calcitriol but no other metabolite of vitamin D is essential for normal bone growth and development in the rat.

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Abstract

To determine the relative importance of different metabolites of vitamin D in bone growth and development, weanling male rat pups suckled by vitamin D-deficient mothers were given either calcitriol (1,25-dihydroxycholecalciferol) by continuous subcutaneous infusion, oral calcidiol (25-hydroxycholecalciferol), or oral 24,24-difluoro-25-hydroxycholecalciferol, a synthetic compound that can undergo 1-hydroxylation but not 24-hydroxylation, as their sole source of vitamin D for 40 d. Pups raised in the same manner, but given no vitamin D, served as controls. The three metabolites compared were given in doses that restored normal plasma calcium levels and normal increments in body weight. After in vivo double tetracycline labeling, bone histomorphometry by standard methods was performed on one femur and one tail vertebra. There were no significant differences between the three metabolite-treated groups in length, periosteal or endosteal diameter, cortical cross-sectional area, cortical porosity, osteoid thickness and volume, appositional rate and bone formation rate in the femur, or in qualitative and quantitative indices of endochondral ossification in the tail vertebra. All three groups differed markedly from the untreated controls with respect to all measurements. Collectively, the data indicate that neither calcidiol nor any 24-hydroxylated metabolite of calcidiol is needed in the rat (other than as a precursor) for longitudinal or transverse bone growth, for normal endochondral ossification, or for normal periosteal and endosteal formation, mineralization, and resorption of bone. Calcitriol was fully active with respect to each of the indices listed when given in a manner resembling its continuous endogenous production by the kidney, suggesting that previous reports of incomplete skeletal response to calcitriol result from its rapid clearance and infrequent oral administration. We demonstrated that calcitriol is the only metabolite that is both necessary and sufficient for normal bone growth and development in the rat, but our data do not indicate the extent to which its beneficial skeletal effects were mediated by direct action on bone, either of calcitriol itself or of some metabolite thereof, or by restoration of normal plasma levels of calcium and phosphate.

Authors

A M Parfitt, C H Mathews, R Brommage, K Jarnagin, H F DeLuca

In vitro enhancement of immunoglobulin gene expression in chronic lymphocytic leukemia.

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Abstract

B cell chronic lymphocytic leukemia (CLL) cells appear to be arrested in their differentiation so that little immunoglobulin is secreted in most cases. To determine their capacity for further differentiation we stimulated cells from a series of 10 cases of CLL with a phorbol ester and assayed for production of immunoglobulin protein, accumulation of immunoglobulin mRNA, and alterations in cell surface markers. We found that cells from all cases were induced to secret monoclonal immunoglobulin of the same heavy and light chain type as the surface membrane immunoglobulin type. Immunoglobulin secretion was preceded by a rapid increase in the levels of mRNA coding for IgM, predominantly the secretory form, mu s-mRNA, rather than the membrane form, mu m-mRNA. A similar selection of mu s- over mu m-mRNA is known to occur in plasma cells by a mechanism of differential processing of mRNA from a single mu-chain gene. Except for a decline in the expression of surface IgD, cell surface determinants remained unaffected both in terms of the percentage of positive cells and the relative number of sites per cell. In contrast to previous studies, these results indicate that CLL cells consistently retain the capacity to further differentiate toward plasma cells and secrete immunoglobulin. The immunoglobulin secretion is mediated, at least in part, by a developmentally regulated increment in mu s-mRNA.

Authors

J Cossman, L M Neckers, R M Braziel, J B Trepel, S J Korsmeyer, A Bakhshi

Normal testicular structure and reproductive function in deermice lacking retinol and alcohol dehydrogenase activity.

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Abstract

It was found that a strain of deermice (Peromyscus maniculatus), which genetically lacks liver alcohol dehydrogenase activity also displays no such activity in the testis and is devoid of the enzyme activity that converts retinol to retinal, both in liver and in the testis; nevertheless, these animals exhibit normal reproduction and testicular histology. Therefore, one must reconsider the theory that the testicular atrophy and aspermatogenesis commonly found in alcoholics is due, at least in part, to interaction of ethanol with these enzyme activities in the testis.

Authors

M A Leo, C S Lieber