Geoffrey W. G. Sharp

The Roles of Intracellular and Extracellular Ca++ in Glucose-Stimulated Biphasic Insulin Release by Rat Islets

Verapamil, an agent known rapidly to block calcium uptake into islets of Langerhans, has been used to study the roles of intra- and extracellular calcium in the two phases of glucose-induced insulin release. Rates of calcium uptake and insulin release during the first phase were measured simultaneously over 5 min in rat islets after maintenance in tissue culture for 2 days. Rates of (45)Ca(++) efflux and insulin release during the first and second phases were also measured simultaneously under perifusion conditions. For this, islets were loaded with (45)Ca(++) during the entire maintenance period to complete isotopic equilibrium. Under static incubation conditions 5 muM Verapamil had no effect upon Ca(++) uptake or insulin release in the presence of 2.8 mM glucose. By contrast, glucose-stimulated calcium influx was totally abolished without there being any significant effect upon first phase insulin release. Thus first phase insulin release is independent of increased uptake of extracellular calcium. The lack of effect of 5 muM Verapamil blockade on first phase insulin release was confirmed, under perifusion conditions, and was in marked contrast to the observed 55% inhibition of second phase release. (45)Ca(++) efflux was inhibited during both phases of the insulin release response. The results show that increased calcium uptake in response to glucose is not involved in the mechanism of first phase insulin release but is required for the full development and maintenance of the second phase release. It seems possible that intracellular calcium is the major regulatory control for first phase insulin release and that intracellular calcium and increased uptake of extracellular calcium contribute almost equally to the second phase of glucose-induced release.

Somatostatin- and epinephrine-induced modifications of 45Ca++ fluxes and insulin release in rat pancreatic islets maintained in tissue culture.

The effects of somatostatin and epinephrine have been studied with regard to glucose-induced insulin release and (45)Ca(++) uptake by rat pancreatic islets after 2 days in tissue culture and with regard to (45)Ca(++) efflux from islets loaded with the radio-isotope during the 2 days of culture. (45)Ca(++) uptake, measured simultaneously with insulin release, was linear with time for 5 min. (45)Ca(++) efflux and insulin release were also measured simultaneously from perifused islets. Glucose (16.7 mM) markedly stimulated insulin release and (45)Ca(++) uptake. Somatostatin inhibited the stimulation of insulin release by glucose in a concentration-related manner (1-1,000 ng/ml) but was without effect on the glucose-induced stimulation of (45)Ca(++) uptake. Similarly, under perifusion conditions, both phases of insulin release were inhibited by somatostatin while no effect was observed on the pattern of (45)Ca(++) efflux after glucose.Epinephrine, in contrast to somatostatin, caused a concentration-dependent inhibition of the stimulation of both insulin release and (45)Ca(++) uptake by glucose. Both phases of insulin release were inhibited by epinephrine and marked inhibition could be observed with no change in the characteristic glucose-evoked pattern of (45)Ca(++) efflux (e.g., with 10 nM epinephrine). The inhibitory effect of epinephrine on (45)Ca(++) uptake and insulin release appeared to be mediated via an alpha-adrenergic mechanism, since is was abolished in the presence of phentolamine. Somatostatin inhibits insulin release without any detectable effect upon the handling of calcium by the islets. In contrast, inhibition of insulin release by epinephrine is accompanied by a partial inhibition of glucose-induced Ca(++) uptake.

Localization of the action of cholera toxin on adenyl cyclase in mucosal epithelial cells of rabbit intestine

Brush borders and plasma membranes have been purified from mucosal epithelial cells of rabbit ileum under control conditions and after treatment for 3 hr with cholera toxin in vivo. The activity of several enzymes in these preparations was measured. It was concluded that adenyl cyclase, like NaK-ATPase, seems not to be a normal constituent of brush borders. Both these enzymes are present in plasma membrane preparations derived largely from the basal and lateral margins of the epithelial cells, both may be phospholipid dependent enzymes and both are affected by cholera toxin. Adenyl cyclase activity is increased while NaK-ATPase is decreased. The activities of alkaline phosphatase, leucineaminopeptidase, 5-nucleotidase, glucose-6-phosphatase, and Mg-ATPase were not found to be affected by the toxin. Cholera toxin, which makes contact with the luminal side of the epithelial cells, in the natural disease and in the experimental model, would appear to exert its pathologic effect on adenyl cyclase at the opposite (basal and lateral) side of the cells.

Stimulation of Adenylate Cyclase by Ca2+ and Calmodulin in Rat Islets of Langerhans: Explanation for the Glucose-induced Increase in Cyclic AMP Levels

The effect of Ca2+ and calmodulin has been studied on adenylate cyclase activity in homogenates of rat islets of Langerhans. EGTA had a stimulatory effect on the enzyme in accord with the known inhibitory effect of Ca2+. In contrast, the addition of Ca2+ together with calmodulin is stimulatory and demonstrates the existence of a Ca2+-dependent adenylate cyclase in islets of Langerhans. It is suggested that the glucose-induced increase in cyclic AMP concentrations in intact islets is a secondary consequence of the glucose-induced increase in cytosol free-Ca2+ concentrations which, with calmodulin, causes an increase in the activity of adenylate cyclase.

Adenyl cyclase in the toad bladder

Abstract Preparation of hormone sensitive adenyl cyclase and some properties of this enzyme were investigated in mucosal cells of toad bladder. This tissue responds to antidiuretic hormone by an increase in the active transport of Na + , enhancement of permeability to water, urea and certain other small molecules, and by an increase in osmotic water flow across the tissue. These actions of the hormone are all thought to be mediated by adenyl cyclase. Antidiuretic hormones, NaF and Mg 2+ were all found to stimulate the enzyme although the stimulation by Mg 2+ of basal enzyme activity was slight. The hormonal activation was usually greater that of fluoride under optimal conditions. The apparent K m for ATP as derived by measurement of half maximal activity was approx. 1·10 −4 M. Optimal pH was 7·5. At high concentrations both theophylline and, to a lesser extent, cyclic AMP inhibited adenyl cyclase activity. The adenyl cyclase activity was highest in the whole homogenate and the sediment from a 1000 × g centrifugation, results that are in accord with a localization of the enzyme in the cell membrane. Barely detectable activity occurred in the 10 000 × g supernatant. When high concentrations of protein were present in the enzyme assay mixture, inhibition of enzyme activity was observed. The enzyme was labile and deterioration set in when homogenization was too vigorous or prolonged, or when the cells were disrupted by sonication. Ageing of the preparation, even when frozen at −80°, resulted in loss of activity, the loss being more marked for hormonal stimulation than for fluoride stimulation. Divalent cations had marked effects on the enzyme activity. Concentrations of Mg 2+ from 1 to 25 mM had a stimulatory effect upon the enzyme and upon stimulation by hormone and fluoride. Greater concentrations had an inhibitory effect. Ca 2+ was inhibitory at all concentrations above 1·10 −5 M . Thus optimal conditions for the activity of adenyl cyclase have been defined and the preparation should allow further elucidation of the action of hormones on active Na + transport and osmotic water flow in this tissue.

Dopamine Stimulation of Active Na and Cl Absorption in Rabbit Ileum: INTERACTION WITH α2-ADRENERGIC AND SPECIFIC DOPAMINE RECEPTORS

The effects of dopamine on active intestinal ion transport have been evaluated. An epithelial sheet preparation of rabbit ileum was used in vitro with the Ussing chamber-voltage clamp technique. Dopamine, in the presence of 1 mM ascorbic acid, added to the serosal bathing solution caused a dose-dependent decrease in short-circuit current, with a half-maximal effect at 1.2 muM and maximal effect of -50 muA/cm(2) at 50 muM; dopamine decreased the potential difference, and increased the conductance and net Na and net Cl absorption. There was no effect on the residual ion flux. Dopamine did not alter the change in short-circuit current caused by mucosal glucose (10 mM) or serosal theophylline (10 mM). Mucosal dopamine had no effect. The effect of dopamine on short-circuit current was inhibited by the dopamine antagonists haloperidol and domperidone and the alpha(2)-adrenergic antagonist yohimbine; there was no effect of the alpha(1)-antagonist prazosin and the beta-antagonist propranolol. In addition, the alpha(2)-adrenergic agonist clonidine, but not the alpha(1)-agonist methoxamine caused a dose-dependent decrease in short-circuit current. The ileal effects of dopamine did not occur via conversion into norepinephrine or release of norepinephrine from the peripheral nerves since peripheral sympathectomy with 6-hydroxydopamine did not alter the dopamine-induced change in ileal short-circuit current. The dopamine effects were not associated with a change in basal ileal cyclic AMP content but were associated with a decrease in total ileal calcium content as measured by atomic absorption spectrometry and as estimated by (45)Ca(++) uptake. The decrease in calcium content could be attributed to a dopamine-induced decrease in (45)Ca(++) influx from the serosal surface. Because of the presence of dopamine in ileal mucosa and these effects on ileal electrolyte transport, it is possible that dopamine may be involved in the physiologic regulation of active intestinal electrolyte absorption.

A Study of Intercellular Spaces in the Rabbit Jejunum during Acute Volume Expansion and after Treatment with Cholera Toxin

The effects of acute volume expansion and of intraluminal administration of cholera toxin have been examined in rabbit jejunum. Acute volume expansion was shown to reverse the normal reabsorptive flux of water and cause significant fluid secretion. Phase and electronmicroscopic examination of the jejunal epithelium showed that marked distension of the intercellular spaces had occurred. Examination of the jejunal epithelium after treatment with cholera toxin showed that, in association with high rates of fluid secretion, the intercellular spaces were extremely small and lateral membranes of adjacent cells were in close apposition to one another. Thus the mechanisms of fluid secretion in these two situations would appear to be quite different. The secretion associated with volume expansion, and accompanied by a rise in venous pressure and bullous deformations of terminal junctions, could well be due to hydrostatic pressure applied through intercellular channels. The secretion of cholera appears to be unrelated to hydrostatic pressure and is more likely due to body-to-lumen active ion transport.

Evidence for the Involvement of Na/Ca Exchange in Glucose-induced Insulin Release from Rat Pancreatic Islets

Glucose-induced inhibition of Ca(++) extrusion from the beta-cell may contribute to the rise in cytosol Ca(++) that leads to insulin release. To study whether interference with Na/Ca exchange is involved in this inhibition the effects of glucose were compared to those of ouabain. This substance inhibits Na/K ATPase, decreases the transmembrane Na(+) gradient in islets, and thus interferes with Na/Ca exchange. Collagenase isolated rat islets were maintained for 2 d in tissue culture with a trace amount of (45)Ca(++). Insulin release and (45)Ca(++) efflux were then measured during perifusion. In Ca(++)-deprived medium (to avoid changes in tissue specific radioactivity) 16.7 mM glucose inhibited (45)Ca(++) efflux. Initially 1 mM ouabain inhibited (45)Ca(++) efflux in a similar fashion, the onset being even faster than that of glucose. The effects of 16.7 mM glucose and ouabain were not additive, indicating that both substances may interfere with Na/Ca exchange. In the presence of Ca(++), 16.7 mM glucose induced biphasic insulin release. Ouabain alone caused a gradual increase of insulin release. Again, the effects of ouabain and 16.7 mM glucose were not additive. In contrast, at a submaximal glucose concentration (7 mM) ouabain enhanced both phases of release. An important role for Na/Ca exchange is suggested from experiments in which Ca(++) was removed at the time of glucose-stimulation (16.7 mM). The resulting marked inhibition of insulin release was completely overcome during first phase by ouabain added at the time of Ca(++) removal; second phase was restored to 60%. This could be due to the rapid inhibitory action of ouabain on Ca(++) efflux thereby preventing loss of cellular calcium critical for glucose to induce insulin release. It appears, therefore, that interference with Na/Ca exchange is an important event in the stimulation of insulin release by glucose.

Properties of Adenyl Cyclase from Human Jejunal Mucosa during Naturally Acquired Cholera and Convalescence

The enterotoxin of Vibrio cholerae causes copious fluid production throughout the lenght of the small intestine. As this is thought to be mediated by stimulation of adenyl cyclase, a study has been made of the activity and properties of this enzyme in jejunal biopsy tissue taken from patients during the diarrheal phase of cholera and after recovery. Adenyl cyclase activity during cholera was increased more than twofold relative to the enzyme in convalescence. Under both conditions stimulation by prostaglandin E(1) (PGE(1)) and by fluoride was observed. The responsiveness to PGE(1) was not altered in cholera; the total activity of the fluoride-stimulated enzyme was similar, a finding that suggests cholera toxin stimulates pre-existing enzyme in the intestinal cell. The enzymes during cholera and convalescence were similar in all other properties examined. Optimal Mg(++) concentration was 10 mM; Mn(++) at 5 mM stimulated the enzyme but could not replace Mg(++) except in the presence of 10 mM fluoride. Calcium was markedly inhibitory at concentrations greater than 10(-4) M. The pH optimum was 7.5 and the Michaelis constant (K(m)) for ATP concentration approximated 10(-4) M. Thus the interaction of cholera toxin with human intestinal adenyl cyclase does not alter the basic properties of the enzyme. When biopsy specimens were maintained intact in oxygenated Ringers solution at 0 degrees C, no loss of activity was observed at 1(1/2) and 3 hr. In contrast, when the cells were homogenized, rapid loss of activity, with a half-life of 90 min was seen even at 0 degrees C. Consequently for comparative assays of human jejunal adenyl cyclase, strict control of the experimental conditions is required. It was under such conditions that a twofold increase in basal adenyl cyclase activity during cholera was observed.

Macromolecular binding of aldosterone in the toad bladder

Abstract Protein-bound aldosterone has been extracted by sonication of nuclei from the mucosal cells of toad bladder. After incubation of hemibladders in 10 and 100 nM (+)-[ 3 H]aldosterone, 1.5·10 −14 and 7.7·10 −14 moles bound aldosterone per 100 μg DNA can be extracted, respectively. Most of the bound aldosterone dissociates rapidly, and 4 h after isolation only a stable component of 20% remains. Dissociation is accelerated at room temperature. The stability of the complex is unaffected by dithiothreitol but enhanced by 25% glycerol. An excess of deoxycorticosterone, hydrocortisone, spirolactone (SC 14266) and unlabeled aldosterone, when added to the incubation medium, displace bound aldosterone while testosterone has no effect on binding by purified nuclei. This suggests mineralocorticoid specificity for the binding protein. [ 3 H]Hydrocortisone (100 nM), a less active mineralocorticoid than aldosterone, binds primarily to the nonspecific, nondissociable binding sites at this concentration. One high-molecular-weight component and one component of molecular weight of approx. 100 000 were demonstrated by agarose column chromatography. Both components are stable and unaffected by deoxycorticosterone. The mineralocorticoid-specific complex can be equated with the rapidly dissociable fraction. When nuclei are sonicated and (+)-[ 3 H]aldosterone is then added a nonsaturable nonspecific binding occurs which is increased with time and unaffected by temperature. Thus three aldosterone binding complexes have been isolated from toad-bladder mucosal-cell nuclei one of which is rapidly dissociable and mineralocorticoid specific.