It has been suggested previously that chlorpropamide and other hypoglycemic sulfonylureas interfere with hepatic triglyceride breakdown. Since ketogenesis from endogenous hepatic lipid stores is a measure of hepatic triglyceride hydrolysis, ketogenesis derived from endogenous lipids as well as ketogenesis derived from exogenously added isotopic oleate was determined in isolated hepatocytes from fasted rats in an attempt to identify the nature of the direct effects of sulfonylureas on hepatic lipid metabolism. Ketogenesis from endogenous lipids was inhibited by 1 mM chlorpropamide, while ketone production from exogenous oleate did not change. The effect of chlorpropamide on hepatic triglyceride metabolism was further studied in the isolated perfused liver of normal rats in the presence of a continuous [3H]oleate infusion and in isolated liver cells incubated in the presence of [3H]oleate. In liver perfusion experiments, 1 mM chlorpropamide enhanced the incorporation of tritium into triglycerides (but not other lipid classes) and increased both liver triglyceride content and triglyceride secretion. Using isolated cells similar effects could be demonstrated at 0.5 mM chlorpropamide. Chlorpropamide, tolbutamide, and carbutamide, all of which inhibited endogenous ketogenesis in isolated liver cells, also inhibited lysosomal triglyceride lipase activity in rat liver homogenates. The drugs were not inhibitory towards alkaline lipase activity. Demethylglycodiazin (2-benzolsulfonamid--5-(beta-hydroxyethoxy)-pyrimidin), which did not inhibit endogenous ketogenesis in isolated liver cells, did not affect lysosomal lipase activity. The lysosomotropic drug chloroquine was markedly antiketogenic when tested in liver cells. The reduction in endogenous ketogenesis, the enhanced accumulation of liver triglycerides, and the stimulation of hepatic triglyceride output by chlorpropamide are ascribed to an interference of the drug with hepatic triglyceride breakdown. The present results also suggest that the lysosomes play a significant role in hepatic lipolysis.
L J Debeer, J Thomas, G Mannaerts, P J DeSchepper
To examine the origin of digitalis-induced ventricular tachycardia (VT), acetyl strophanthidin (AS) (25 mug/min) was perfused into a limited zone of myocardium in intact anesthetized dogs through a catheter placed fluoroscopically in the left anterior descending artery without ischemia. A second catheter in the great cardiac vein sampled venous effluent from this region. His and left bundle branch depolarizations were recorded and bipolar intramural electrograms from endocardial and epicardial sites within the anterior descending region were obtained. No conduction alterations preceded arrhythmia. Cardiac venous K+ rose from 3.3 +/- to 4.4 +/- 0.2 meq/liter (P less than 0.001), indicating egress from the perfused zone. 10 animals (Group 1) were sacrificed 2 min after onset of VT while 11 (Group 2) continued until fibrillation (4-14 min). All showed normal (endocardial leads to epicardial) transmural depolarization during sinus rhythm, but 10/21 demonstrated reversal, usually late during VT, including 8/11 in Group 2. Epicardial activation preceded fascicular activation and QRS. Recordings from the border and circumflex regions in 10 additional dogs (Group 3) demonstrated activation reversal only in the border zone. Myocardial K+ was reduced (mean 63 +/- 1 mueq/g) and Na+ increased (mean 41 +/- 2 mueq/g) in the perfused zone (nonperfused circumflex area K+ 72 +/- 1, Na+ 33 +/- 1 mueq/g, P less than 0.001 for both); changes were similar in inner and outer ventricular wall. In related experiments, subepicardial injections of AS induced activation reversal within the immediate area, similar to recordings during coronary infusion. Reversed transmural activation with early epicardial depolarization suggest VT arises within myocardium; electrolyte gradients between adjacent regions may be causative.
P O Ettinger, J Calabro, T J Regan, H A Oldewurtel
A multiple inert gas elimination method was used to study the mechanism of impaired gas exchange in 23 patients with advanced chronic obstructive pulmonary disease (COPD). Three patterns of ventilation-perfusion (Va/Q) inequality were found: (a) A pattern with considerable regions of high (greater than 3) VA/Q, none of low (less than 0.1) VA/Q, and essentially no shunt. Almost all patients with type A COPD showed this pattern, and it was also seen in some patients with type B. (b) A pattern with large amounts of low but almost none of high VA/Q, and essentially no shunt. This pattern was found in 4 of 12 type B patients and 1 of type A. (c) A pattern with both low and high VA/Q areas was found in the remaining 6 patients. Distributions with high VA/Q areas occurred mostly in patients with greatly increased compliance and may represent loss of blood-glow due to alveolar wall destruction. Similarly, well-defined modes of low VA/Q areas were seen mostly in patients with severe cough and sputum and may be due to reduced ventilation secondary to mechanical airways obstruction or distortion. There was little change in the VA/Q distributions on exercise or on breathing 100% O2. The observed patterns of VA/Q inequality and shunt accounted for all of the hypoxemia at rest and during exercise. There was therefore no evidence for hypoxemia caused by diffusion impairment. Patients with similar arterial blood gases often had dissimilar VA/Q patterns. As a consequence the pattern of VA/Q inequality could not necessarily be inferred from the arterial PO2 and PCO2.
P D Wagner, D R Dantzker, R Dueck, J L Clausen, J B West
Cell populations obtained by bronchial lavage from human subjects were examined for the presence of cells related to the mast cell-basophil series. Such bronchial lumen histamine-containing cells (BLHCC) were identified. The BLHCC stained with toluidine blue may be identified by bright field or dark field microscopy. The BLHCC are alive as evidenced by ability to release histamine (H) after exposure to anti-IgE or calcium ionophore. Although H release from peripheral blood leukocytes by these two agents is potentiated by the presence of D2O, H release from BLHCC of the same subjects by anti-IgE or calcium ionophore was not potentiated by D2O. In studies comparing bronchial cell populations of humans and rhesus monkeys with peripheral blood leukocyte populations of the same subjects, the histamine content of the bronchial cell population was much higher in rhesus monkeys. IgE/Alb ratios of respiratory secretions and serum of the same human subjects were of the same order of magnitude in contrast to previous comparisons done on these fluids in rhesus monkeys.
R Patterson, J M McKenna, I M Suszko, N H Solliday, J J Pruzansky, M Roberts, T J Kehoe
In advanced osteoarthritis, all of the cartilaginous components are lost from the joint surface. Although mechanisms exist for proteoglycan degradation, there is not known to be any system for removal of the collagen. This study suggests that the loss of the collagen components may be a function of articular cartilage collagenase. The enzyme in normal human cartilage is bound to an inhibitor and appears to be present in very small amounts. Attempts to demonstrate collagenase activity in ground human articular cartilage or in its lysosomal fraction were unsuccessful. 7-Day cartilage tissue cultures also failed to demonstrate the presence of the enzyme; but the same culture fluid, incubated with trypsin, showed significant degradation of collagen, suggesting that trypsin destroyed the inhibitor. 7-Day culture fluids were then chromatographed on a heparin-charged Sepharose 4B affinity column that had been activated with cyanogen bromide. This removed the inhibitor, and the chromatographed fluid from osteoarthritic cartilage released 42% of the incorporated counts of the collagen substrate, whereas normal cartilage released 10.1% and a trypsin control, 6.4%. Electrophoresis of the degradation products of the enzyme-collagen complex incubated at 37 degrees C revealed breakdown was complete to small dialyzable fragments, while at 25 degrees C larger fragments were split off.
M G Ehrlich, H J Mankin, H Jones, R Wright, C Crispen, G Vigliani
Recent models of the urinary concentrating mechanism have postulated that urea in the medullary interstitium creates a transtubular concentration gradient for sodium between fluid at the end of the descending limb of Henle's loop and the medullary interstitium, favoring the passive outward movement of sodium from Henle's thin ascending limb. These experiments were designed to determine whether such a gradient normally exists. Young nondiuretic Munich-Wistar rats were prepared for micropuncture of the exposed left renal papilla. Samples of loop of Henle fluid and vasa recta plasma (assumed to reflect the composition of interstitial fluid) were obtained from adjacent sites. Loop fluid values in 21 comparisons from 18 rats (mean +/- SE) were: sodium 344 +/- 12 meq/liter; potassium, 26 +/- 2 meq/liter; osmolality, 938 +/- 37 mosmol/kg H23. Vasa recta plasma values (in corresponding units of measurement) were: sodium, 284 +/- 11; potassium, 34 +/- 2; osmolality, 935 +/- 34. Mean values of paired differences (loop fluid minus vasa recta plasma) were: delta sodium, 60 +/- 11.1 (P less than 0.001); delta potassium, -8.0 +/- 2.1 (P less than 0.001); delta osmolality, 4 +/- 16 (NS). Corrected for plasma water, the loop fluid minus vasa recta differences (in milliequivalents per kilogram H2O) were: delta sodium, 40 +/- 11.4 (P less than 0.005); delta potassium, -9.7 +/- 1.9 (P less than 0.001). We interpret these findings to indicate that in the papilla of nondiuretic rats, a significant difference in sodium concentration exists across the thin loop of Henle favoring outward movement of sodium, which confirms a key requirement of the passive models. A concentration difference for potassium in the reverse direction was also observed.
P A Johnston, C A Battilana, F B Lacy, R L Jamison
Collagens in normal human liver and in alcoholic cirrhotic liver were investigated. Collagens were solubilized by limited proteolysis with pepsin under nondenaturing conditions, and after purification, were fractionated into types I and III by selective precipitation with NaCl. After carboxymethyl cellulose and agarose chromatography, the resulting alpha-chains from each of the collagen types were analyzed with respect to their amino acid and carbohydrate compositions. A comparison of the results obtained from normal liver with those from the diseases organ revealed no significant differences. The isolated human liver alpha1(I) and alpha1(III) chains were digested with CNBr and the generated peptides were separated and purified by a combination of ion-exchange and molecular sieve chromatography. The molecular weight and the amino acid and the carbohydrate compositions of each of the peptides were identical to those of the corresponding human skin peptides except for the slightly higher content of hydroxylysine in some of the peptides. The relative content of type III in relation to type I collagen in both normal anc cirrhotic liver was determined by digesting washed liver homogenates directly with CNBr and quantitating the resultant alpha1(I) and alpha 1(III) peptides after chromatographic separation. The relative quantities of these peptides indicated that normal human liver contained an average of 47% type III, with the remainder being type I. Cirrhotic liver, on the other hand, contained a significantly smaller proportion of type III, ranging from 18 to 34% in different samples, with a corresponding increase in type I. These findings indicate that although the amino acid and carbohydrate compositions of collagens deposited in cirrhotic liver are normal, the fibrotic process of alcoholic liver disease in humans is accompanied by an alteration in tissue collagen polymorphism, and suggest that the observed alterations may have pathogenetic implications.
J M Seyer, E T Hutcheson, A H Kang
Superoxide anion (O-2-) generation by human peripheral blood polymorphonuclear leukocytes is enhanced when these cells encounter appropriate soluble or particulate stimuli. O-2- generation requires intact, viable cells and proceeds independently of phagocytosis. To investigate the possibility that the O-2--generating system is associated with the outer surface of the polymorphonuclear leukocyte plasma membrane, we have examined the effects upon O-2- production of p-diazobenzenesulfonic acid, a reagent which can react predominantly with proteins of the external cell membrane. When normal human polymorphonuclear leukocytes were preincubated with cytochalasin B (to minimize endocytosis) and then exposed to the surface-active lectin, concanavalin A, the cells were stimulated to generate O-2- in a concentration- and time-dependent fashion and selectively to discharge the granule-associated enzyme, lysozyme, into the surrounding medium. These responses, as well as cellular binding of [H] concanavalin A, could be blocked by alpha-methyl-D-mannoside. Brief treatment (less than 5 min at 4 degrees C) of the cells with p-diazobenzenesulfonic acid (1.0-5.0 mM) significantly interfered with concanavalin A-mediated O-2- generation but had no influence upon lysozyme release or upon binding of [3H] concanavalin A. The diazonium salt did not alter cell viability or the specific activity of the cytoplasmic enzyme, lactate dehydrogenase (inhibitable under conditions which allowed entry of this reagent into the cytosol). p-Diazobenzenesulfonic acid, therefore, very likely exerted its effects at the cell surface of the intact polymorphonuclear leukocyte, selectively inhibiting O-2- production (either directly or indirectly) without influencing another response to lectin-cell contact: release of lysozyme. These results support the possibility that a polymorphonuclear leukocyte ectoenzyme is responsible for O-2- production.
I M Goldstein, M Cerqueira, S Lind, H B Kaplan
Gastric emptying and secretion, as well as intragastric volume and composition, were determined simultaneously in three patients with Zollinger-Ellison syndrome and in seven normal subjects. Gastric hypersecretion was observed in patients with Zollinger-Ellison syndrome and in normal subjects receiving pentagastrin. In contrast, the fraction of gastric contents emptied per minute (fractional rate of emptying) was increased in Zollinger-Ellison patients and unchanged or decreased in normal subjects receiving pentagastrin. The increased fractional rate of gastric emptying in patients with Zollinger-Ellison syndrome persisted despite abolition of gastric hypersecretion by metiamide. Thus, the increased fractional gastric emptying seen in patients with Zollinger-Ellison syndrome is not attributable to hypergastrinemia, or to gastric hypersecretion per se. Instead, it appears to be caused by an undefined nervous or humoral factor.
A Dubois, P V Eerdewegh, J D Gardner
A soluble nuclear antigen that reacts with sera obtained from patients with systemic lupus erythematosus and Sjögren's syndrome has been described. The antigen, tentatively named the Ha antigen after the prototype serum, was shown to react with specific antibodies by precipitin, complement fixation, and immunofluorescence techniques. The Ha antigen prepared from isolated nuclei of calf thymus glands, calf liver, and rat liver showed identical immunological reactivities; a wide distribution among different species and tissues is presumed. The Ha antigen was destroyed by trypsin and relatively mild heat or pH variation from neutrality, but was resistant to DNase or RNase. Many of these characteristics are similar to those of the "B" antigen to which antibodies have recently been described in Sjögren's syndrome. The nuclear origin of the Ha antigen was confirmed by the speckled nuclear immunofluorescence staining pattern given by purified antibody to Ha obtained from a specific immune precipitate. Preliminary results showed approximately 13% of patients with systemic lupus erythematosus and 30% of patients with Sjögren's syndrome had precipitating antibodies to the Ha antigen.
M Akizuki, R Powers, H R Holman
Approximately 4 h after an initial bronchoalveolar lavage (BAL) of a primate's lung, an appreciable number of polymorphonuclear leukocytes (PMNs) were noted to accumulate in respiratory fluids when lavage was repeated. Whereas, alveolar macrophages (90%) and lymphocytes (7%) were the principal respiratory cells recovered initially from lavage fluid, later samples contained 45-90% PMNs To explain the observed ingress of PMNs into lung fluids, concentrated BAL fluid was tested for chemoattractant activity. Such fluid obtained 4 and 24 h after an initial lavage contained material that produced directed migration (chemotaxis) for PMNs and mononuclear cells isolated from peripheral blood of normal donors. Gel filtration chromatography of BAL disclosed two peaks of chemotactic activity in the effluent fractions. Material from the column with an estimated molecular weight of 15,000 daltons was chemotactic for both PMNs and mononuclear cells. Because it was susceptible to inactivation with antiserum against the fifth component of complement, resistant to heating, and unaffected by antiserum against C3, this factor was considered analogous to the cleavage product of the fifth component of complement. C5a. In addition chemotactic activity for PMNs only was contained in an effluent peak having a molecular weight of about 5,000 daltons. This material was heat labile but unaffected by antisera to complement components. To locate the possible source of these factors in respiratory fluid, in vitro cultures of alveolar macrophages were established. These cells, whether stimulated by phagocytosis of opsonized bacteria or merely by attachment to a glass surface, produced chemotactic material which had physical characteristics similar to the small molecular weight material in BAL. Moreover, it induced preferential chemotaxis for PMNs. Thus, in primate lungs, at least two chemotactic substances may generate an inflammatory response; one which is a fragment of the complement component C5 and another small molecular weight factor which is released from alveolar macrophages.
J A Kazmierowski, J I Gallin, H Y Reynolds
A fluormetric method for the determination of pyridine nucleotides has been adapted for use in studying the reduced pyridine nucleotide oxidases in human polymorphonuclear leukocytes. In the presence of strong base the oxidized forms of the pyridine nucleotides form a highly fluorescent product. The small amounts of NAD(P) formed by the oxidase reactions can be determined with great sensitivity. This method has been compared to the radioisotopic assay for NADPH oxidation. Both methods gave essentially the same results in terms of nanomoles NADP produced by control, resting, and phagocytizing samples. Both NADPH and NADH oxidase activities were insensitive to cyanide. NADPH oxidation had a pH optimum of 5.5, while that for NADH appeared to be 6.0. Granules isolated from phagocytizing cells routinely showed more activity toward both substrates (two to threefold) than granules from resting cells. Both activities were located primarily in a granule fraction prepared by differential centrifugation. Oxidation of NADPH was routinely four to five times that of NADH at all except very high substrate levels. Measurable NADH oxidation was rarely seen below 0.80 mM NADH, while NADPH oxidation was easily measurable at 0.20 mM. One patient with chronic granulomatous disease was studied. At low substrate levels, there was no activity toward either substrate in granules isolated from either resting or phagocytizing cells of this patient, while granules isolated from normal control cells showed substantial activity at these substrate levels. Purification of the activities had been initiated with linear sucrose gradients. Both activities co-sediment to a very dense region of the gradient, a region different from that in which membrane or azurophil granules usually equilibrate. The peak gradient fractions show a 10-30-fold increase in specific activity over comparable granule fractions. These data suggest that the oxidase activities are associated with one enzyme that has different affinities for the two substrates ans support the contention that the oxidation of NADPH is responsible for the metabolic burst accompanying phagocytosis in human PMNL.
D Iverson, L R DeChatelet, J K Spitznagel, P Wang
Acid-base disturbances may develop secondary to the changes in renal tubular function and bone dynamics which attend phosphate depletion (PD). This work characterizes the acid-base status of rats fed a low phosphate diet. After 18 days, PD rats had marked calciuria (pair-fed controls: 0.3 +/- 0.2; PD 32.2 +/- 2.5 mueq/h; P less than 0.001), severe bicarbonaturia (controls: 0; PD 17.6 +/- 0.2 meq/h; P less than 0.001), and negative net acid excretion (controls: 44.5 +/- 2.9; PD: --6.6 +/- 2.5 meq/h; P less than 0.001), but plasma pH, HCO3, and PCO2 were equal in both groups. After 45 days, plasma HCO3 fell to 21.1 +/- 0.9 meq/liter in PD (controls: 23.6 +/- 0.5 meq/liter; P less than 0.05), while bicarbonaturia (controls: 0.4 +/- 0.2; PD: 3.8 +/- 1 mueq/h; P less than 0.02) and calciuria were present but diminished. These data suggested the coexistence of bone HCO3 mobilization and renal HCO3 wasting in PD. To test this thesis, bicarbonaturia was eliminated by nephrectomy. 24 h later plasma HCO3 was higher in PD rats (controls: 19.3 +/- 0.02; PD: 22.6 +/- 0.8 meq/liter; P less than 0.05), consistend with the presence of extrarenal HCO3 production. After inhibition of bone resorption with colchicine (1 mg/kg), plasma HCO3 decreased to 16.8 +/- 0.6 meq/liter in PD rats (controls): 26.4 +/- 1 meq/liter; P less than 0.001) while bicarbonaturia persisted. These data indicate that the plasma HCO3 in PD is the net result of renal HCO3 wasting and bone HCO3 mobilization. These combined effects maintain normal blood HCO3 initially (18 days) but with time (45 days), bone resorption diminishes and the acidifying renal tubular defect predominates.
M Emmett, S Goldfarb, Z S Agus, R G Narins
Cyclic somatostatin was administered intravenously (10 mug/min for 60 min) to 10 healthy overnight fasted (postabsorptive) subjects and to 5 healthy 60-h fasted subjects. In both groups, arterial insulin and glucagon fell 50% and splanchnic release of these hormones was inhibited. In the overnight fasted subjects splanchnic glucose output fell 70%, splanchnic uptake of lactate and pyruvate was unchanged, alanine uptake fell by 25%, and glycerol uptake rose more than twofold in parallel with an increase in arterial glycerol. In the 60-h fasted group splanchnic glucose output was less than 40% of that observed in the overnight fasted subjects. Somatostatin led to a further decrease (--70%) in glucose production. Splanchnic uptake of lactate and pyruvate fell by 30-40%, amino acid uptake was unchanged, while uptake of glycerol rose fivefold. Total uptake of glucose precursors thus exceeded the simultaneous glucose output by more than 200%. Splanchnic uptake of FFA rose fourfold during somatostatin while output of beta-hydroxybutyrate increased by 75%. Estimated hepatic blood flow fell 25-35% and returned to base line as soon as the somatostatin infusion ended. It is concluded that (a) somatostatin-induced hypoglucagonemia results in inhibition of splanchnic glucose output in glycogen-depleted, 60-h fasted subjects as well as in postabsorptive subjects, indicating an effect of glucagon on hepatic gluconeogenesis as well as glycogenolysis; (b) the glucagonsensitive step(s) in gluconeogenesis affected by somatostatin involves primarily intra-hepatic disposal rather than net hepatic uptake of glucose precursors; (c) splanchnic uptake of fatty acids and ketone output are increased in the face of combined insulin and glucagon deficiency; and (d) diminished splanchnic blood flow may contribute to some of the effects of somatostatin on splanchnic metabolism.
J Wahren, S Efendić, R Luft, L Hagenfeldt, O Björkman, P Felig
The present investigation was designed to quantitatively assess the possible influence of countercurrent exchange on passive absorption from the small intestine of the dog. Villus blood flow was measured with a modification of the microsphere method. Simultaneously, the absorption from the gut lumen of five diffusible gases (H2, He, CH4, 133Xe, and CO) was determined. Villus blood flow averaged 0.247 +/- 0.03 (SEM) ml/min per g. The observed absorption of H2, He, CH4, and 133Xe was only 16.2 +/- 1.8, 12.8 +/- 2.3, 12.0 +/- 1.8, and 15.8 +/- 1.4 %, respectively, of what this villus blood flow could carry away if it reached perfect equilibrium with the luminal gases. This low absorption rate could result from diffusion limitation to absorption or countercurrent exchange. The diffusive permeability of the barrier seperating the luminal gases and villus blood flow was assessed by measuring the absorption rate of CO. Because absorbed CO binds tightly to hemoglobin, it cannot exchange, and when present in low concentrations its uptake is entirely diffusion limited. Knowledge of the diffusion rate through tissue of the unbound gases relative to that of CO made it possible to calculate the degree to which each of the unbound gases should equilibrate with villus tip blood. The percentage equilibration between lumen and blood at the villus tip for H2, He, CH4, and 133Xe was 99.7, 99.9, 75.6, and 36.0% , respectively. Each of these values greatly exceeded the percentage equilibration of blood leaving the villus (calculated from the observed absorption rate and villus blood flow) and indicated an exchange of 83.8, 87.2, 84.1, and 56.1% of initially absorbed H2, He, CH4, and 133Xe. This result is in accord with theoretical calculations which suggest that countercurrent exchange should be exceedingly efficient in the dog. The striking effect of countercurrent exchange on passive absorption in the dog differs from our previous studies in the rabbit where no exchange was demonstrated. This marked species difference may result from anatomical differences in villus architecture. The dog has long, densely packed villi while the rabbit has broad, widely spaced villi. In the dog, only the villus tips may equilibrate with the lumen, hence a countercurrent gradient may be established in the villus. The entire villus of the rabbit may equilibrate with the lumen and no gradient for countercurrent exchange can therefore be established.
J H Bond, D G Levitt, M D Levitt
The molecular basis for the aberrant catecholamine responsiveness of the adenylate cyclase of adrenocortical carcinoma 494 was explored. The adenylate cyclase of this corticosteroid-producing, transplanted, adrenal cancer of the rat was stimulated not only by adrenocorticotropic hormone and fluoride, but also by the beta-adrenergic agonist, isoproterenol. The adenylate cyclase of normal adrenal tissue was unresponsive to isoproterenol. Direct binding studies with the specific high affinity B-adrenergic ligand, (-)[3H]dihydroalprenolol, demonstrated the pressure of 0.094 pmol of specific binding sites per milligram of tumor membrane protein. By contrast, normal adrenal membranes contained too few binding sites to accurately measure and study using these techniques. The tumor binding sites had high affinity for (-)[3H] dihydroalprenolol with an equilibrium dissociation constant of 2.1 nM. Adrenergic agonists competed for the binding sites in an order of potency, [(-) isoproterenol greater than (-) epinephrine (-) norepinephrine], paralleling their order of potency as beta-adrenergic agonists. The beta-adrenergic antagonist, (-) propranolol, competed for binding, causing half-mzximal inhibition of specific binding at a concentration of 6 nM. The alpha-adrenergic antagonist, phentolamine, and several catecholamine metabolites and precursors did not effectively compete for the binding sites at high concentrations. Binding was stereospecific, the (+) stereoisomers of beta-adrenergic agonists and antagonists requiring 40- to 300-fold higher concentrations than the corresponding (-) stereoisomers to half maximally inhibit (-) [3H] dihydroalprenolol binding. These results indicate that adrenocortical carcinoma 494 membranes contain beta-adrenergic receptor-binding sites which are not normally present in membranes of adrenal tissue. These ectopic beta-adrenergic receptors presumably confer on the neoplastic tissue the catecholamine sensitivity of its adenylate cyclase.
L T Williams, T B Gore, R J Lefkowitz
Five patients with glucagonomas had elevated plasma levels of total glucagon immunoreactivity. Gel filtrations of these plasma samples on Bio-Gel P30 columns showed that most of the immunoreactivity eluted in the 3,500-(true glucagon) and 9,000-dalton fractions. After the administration of alpha cell effectors, changes in total glucagon immunoreactivity were seen which were accounted for primarily by the 3,500-dalton species, but there were also changes in the 9,000-dalton moiety. Venous effluent plasma from tumors of two subjects contained elevated concentrations of glucagon immunoreactivity in both fractions. When material from both the 3,500- and 9,000-dalton peaks were serially diluted in a glucagon immunoassay, parallel displacement curves were found, suggesting that both have similar or identical antigenic determinants. Thus, with conversion to a neoplastic state, alpha cells of glucagonomas, much like beta cells of insulinomas, may secrete an increased amount of a larger, 9,000-mol wt glucagon species which may be a prohormone.
G C Weir, E S Horton, T T Aoki, D Slovik, J Jaspan, A H Rubenstein
Human diabetics on intermediate and long-acting insulin occasionaly become hypoglycemic during exercise. We have shown previously that during exercise, hypoglycemia did not occur in depancreatized insulin-infused dogs because the increments in glucose production and utilization were proportional and of the same magnitude as in normal dogs. Therefore, to elucidate the mechanism of the glucose-lowering effect of strenuous exercise, we measured glucose production and utilization, metabolic clearance of glucose, and serum immunoreactive insulin in postabsorptive depancreatized dogs 8 h after a subcutaneous injection of protamine zinc and crystalline insulin. During rest, plasma glucose was stable, but ranged between hypoglycemia and hyperglycemia. Hyperglycemia was associated with overproduction of glucose, indicating insulin deficiency despite normal or elevated serum immunoreactive insulin. Glucose clearance, as in normal dogs, increased threefold but glucose production increased only marginally (50%) and, consequently, glucose decreased in plasma. The decrease of plasma glucose was directly proportional to the preexercise concentration and production of glucose. The magnitude of inhibition glucose production was not correlated with the serum immunoreactive insulin indicating either that some released insluin was not active or that a moderate immunoreactive insulin increment induced a near-maximal inhibition. It is concluded that in depancreatized dogs injected with protamine zinc insulin, exercise accelerates mobilization of insulin from its injection site presumably because of increased blood and lymph flow. Glucose utilization did not exceed that in normal dogs, but hepatic glucose production failed to increase sufficiently to meet the needs of muscle in exercise.
R Kawamori, M Vranic
Insulin, as well as other ligands which increase intracellular guanosine 3',5'-cyclic monophosphate (cGMP), augments thymic-derived (T)- lymphocyte effector activity as revealed by alloimmune lymphocyte-mediated cytotoxicity. The observation that insulin binds only to monocytes among circulating nonimmune human mononuclear cells fosterd reexamination of the mechanism by which insulin augments T-lymphocyte function. This report concerns a test of the hypothesis that the T cell is directly affected by insulin and that an insulin receptor emerges upon T lymphocytes consequent to immune activation. Spleens were removed from rats skin grafted across a major histocompatibility barrier. Lymphocytes were harvested from Ficoll-Hypaque density gradients and subsequently enriched for T cells by passage over one or two nylon wool columns. This population was composed of more than 98% T cells as assessed by surface marker techniques (Ig staining, erythrocyte antibody, and erythrocyte antibody complement rosetting, anti-T staining). There was no loss of augmentation of lymphocyte-mediated cytotoxicity induced by insulin, carbamycholine, and 8-bromo-cGMP in the purified cells when compared to unfractionated cells 7 days after transplantation. 125I-insulin bound saturably to the allostimulated T-enriched lymphocytes with maximum binding at 12.8 +/- 0.2 pg and a dissociation constant at equilibrium of 1.3 nM. In contrast, insulin receptors were not present on nonimmune T-enriched cells or on T cells from animals that received syngeneic grafts. The affinity of the lymphocyte insulin receptor was similar to that of more conventional insulin-sensitive tissues e.g., liver, adipocyte. After 89% of T cells from spleens on day 7 were lysed with anti-thy 1.1 antibody and complement, the ability to measure specific insulin binding was lost. These data confirm a physiologic role for insulin in T-lymphocyte effector function and describe the emergence of insulin receptors concomitant with cell sensitivity to ligand. Such receptors may play a role in hormonal modulation of the immune response.
J H Helderman, T B Strom
Intact sheep antidigoxin antibodies and their Fab fragments have both been found to exert profound effects on digoxin pharmacokinetics in [3H] digoxin-treated dogs. Both classes of molecule remove digoxin from the extravascular space and sequester it in the circulation in protein-bound form, a form in which the digoxin is presumably inactive. These two classes of molecule differ, however, in that the intact antibody molecules interfere with digoxin excretion, thereby promoting the retention of the glycoside; this retained digoxin is eventually released in free, active form when the administered antibody is metabolically degraded. In contrast, urinary excretion of digoxin continues in Fab-treated dogs, with significant quantities of digoxin being excreted promptly in the urine in complex with Fab fragments. These differences in urinary excretion, together with the probable decreased immunogenicity of sheep antidigoxin Fab fragments, suggest that such fragments possess potential advantages over intact antibody molecules for use in the therapy of life-threatening digoxin intoxication in man.
V P Butler Jr, D H Schmidt, T W Smith, E Haber, B D Raynor, P Demartini
The paroxysmal nocturnal hemoglobinuria (PNH) platelet differs from the normal human platelet in its interaction with activated complement components: (a) when complement is activated by the alternative pathway, greater amounts of C3 are fixed to the PNH platelet than to the normal platelet; (b) the platelet-release reaction, as measured by serotonin release, occurs after C3 fixation to the PNH platelet. This reaction does not occur with normal platelets; (c) although serotonin release mediated by antibody alone was the same for normal and PNH platelets, antibody-initiated complement activation resulted in the fixation of greater amounts of C3 to PNH platelets and greater consequent serotonin release; and (d) nearly maximal serotonin release; and (d) nearly maximal serotonin release from PNH platelets occurs after the fixation of C3 (or perhaps C5) to the membrane without completion of the terminal sequence. In contrast, completion of the terminal complement sequence beyond C5 is required for maximal serotonin release from normal platelets. These abnormalities of interaction of complement components and PNH platelets may explain the occurrence of thromboses in this disease.
R H Dixon, W F Rosse
To investigate the role of glucagon in regulating hepatic glucose production in man, selective glucagon deficiency was produced in four normal men by infusing somatostatin (0.9 mg/h) and regular pork insulin (150-muU/kg per min) for 2 h. Exogenous glucose was infused to maintain euglycemia. Arterial plasma glucagon levels fell by greater than 50% whereas plasma insulin levels were maintained in the range of 10-14 muU/ml. In response to these hormonal changes, net splanchnic glucose production (NSGP) fell by 75% and remained suppressed for the duration of the study. In contrast, when somatostatin alone was administered to normal men, resulting in combined insulin and glucagon deficiency (euglycemia again maintained), NSGP fell markedly but only transiently, reaching its nadir at 15 min. Thereafter, NSGP rose progressively, reaching the basal rate at 105 min. These data indicate that the induction of selective glucagon deficiency in man (with basal insulin levels maintained) is associated with a marked and sustained fall in hepatic glucose production. We conclude, therefore, that basal glucagon plays an important role in the maintenance of basal hepatic glucose production in normal man.
J E Liljenquist, G L Mueller, A D Cherrington, U Keller, J-L Chiasson, J M Perry, W W Lacy, D Rabinowitz