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Biomedical subjects

G W Sharp

Publications and source records attributed to G W Sharp.

At least 127 records · Page 7Linked to original sources
PubMed1978

Effects of cholera toxin and guanosine 5'-[betagamma-imido]triphosphate on beta-adrenergic-receptor affinity.

A comparison was made of the effects of cholera toxin and p[NH]ppG on the binding affinity of beta-adrenergic receptors in toad erythrocyte membranes. This was determined by studying the ability of isoproterenol and propranolol to compete for the receptor with (-)-[3H]dihydroalprenolol. p[NH]ppG decreased the receptor affinity for the agonist isoproterenol (i.e. a 'right' shift in the displacement-concentration curve), but was without effect on the affinity for the antagonist propranolol. Toad erythrocyte membranes after treatment with cholera toxin exhibited increased receptor affinity for isoproterenol (i.e. a 'left' shift in the displacement curve), but did not affect the affinity for propranolol. p[NH[ppG was able to exert its right shift even in cholera-toxin treated membranes. The ability of cholera toxin to alter beta-adrenergic-receptor affinity is interpreted as further evidence that the toxin affects the nucleotide-regulatory component of adenylate cyclase. The regulatory component affected may be the catecholamine-sensitive guanosine triphosphatase.

Alprenolol↗
PubMed1978

Studies on the time course and rate-limiting steps in the activation of adenylate cyclase in rat liver by cholera toxin.

Cholera toxin stimulates adenylate cyclase in rat liver after intravenous injection. The stimulation follows a short latent period of 10min, and maximum stimulation was attained at 120min. Half-maximal stimulation was achieved at 35min. In contrast with this lengthy time course in the intact cell, adenylate cyclase in broken-cell preparations of rat liver in vitro were maximally stimulated by cholera toxin (in the presence of NAD+) in 20min with half-maximal stimulation in 8min. Binding of cholera toxin to cell membranes by the B subunits is followed by translocation of the A subunit into the cell or cell membrane, and separation of the A1 polypeptide chain from the A2 chain by disulphide-bond reduction, and finally activation of adenylate cyclase by the A1 chain and NAD+. As the binding of cholera toxin is rapid, two possible rate-limiting steps could be the determinants of the long time course of action. These are translocation of the A1 chain from the outside of the cell membrane to its site of action (this includes the time required for separation from the whole toxin) or the availability of NAD+ for activation. When NAD+ concentrations in rat liver were elevated 4-fold, by the administration of nicotinamide, no change in the rate of activation of adenylate cyclase by cholera toxin was observed. Thus the intracellular concentration of NAD+ is not rate-limiting and the major rate-limiting determinant in intact cells must be between the time of toxin binding to the cell membrane and the appearance of subunit A1 at the enzyme site.

Adenylyl Cyclases↗
PubMed1978

Glucose stimulated proinsulin biosynthesis rates of turn off after cessation of the stimulus.

Proinsulin biosynthesis was stimulated progressively to a plateau level at 45 min by 5.5 mmol/1 glucose. The rate of biosynthesis promptly decreased following cessation of either 15 or 60 min exposure to this concentration of glucose. Actinomycin D had no effect on the rate of decline. In contrast, exposure to 20 mmol/1 glucose caused a prolonged increase in proinsulin biosynthesis which was still apparent at 75 min. After removal of the stimulus there was an initial decrease in the rate of proinsulin biosynthesis followed by persistently elevated rates. When proinsulin biosynthesis was stimulated by 20 mmol/1 glucose in the presence of actinomycin D, there was a rapid switch off of biosynthesis and no persistent effect. Thus, two controls for regulation of proinsulin biosynthesis can be characterised: a prompt, rapidly reversible stimulation in response to low or high glucose concentrations and a persistent stimulation in response to high glucose concentrations. These two effects may represent the translational and transcriptional effects of glucose respectively.

Animals↗
PubMed1978

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.

Animals↗
PubMed1978

Studies on the dual effects of glucose on 45Ca++ efflux from isolated rat islets.

45Ca++ efflux studies were performed on rat islets of Langerhans which were loaded to isotopic equilibrium during 48 h in tissue cultures. 45Ca++ loading was 50% complete at 1 h, 80% at 4 h, and reached, at equilibrium, a content equal to 10-11 pmol/islet. The islets responded to glucose stimulation with a rapid and markedly biphase insulin release. Under normal conditions, glucose stimulated 45Ca++ efflux with an initial surge (simultaneous with the first peak of insulin release), which declined rapidly to 50% of the peak value and then slowly declined for the remainder of the glucose stimulation. Special conditions were required to uncover an early inhibition of 45Ca++ efflux; these were the lowering of the temperature of the perifusate from 37 C to 30 C or below, or reduction of the medium Ca++ concentration to 0.1 mM or less. Under zero calcium conditions the glucose inhibition of 45Ca++ efflux can be rigorously interpreted as an inhibition of calcium efflux. The studies at low temperature or low Ca++ concentrations revealed two effects of glucose on 45Ca++ efflux: an initial inhibition followed by a stimulation, the inhibitory effect was obscured by the rapidity of onset of the stimulatory effect under normal conditions. At low temperature it was also possible to inhibit glucose-stimulated insulin release, although the stimulated 45Ca++ efflux remained unchanged. At 30 C or in experiments with 0.3 mM Ca++, glucose-stimulated insulin release preceded the stimulation of 45Ca++ efflux. It therefore, is, concluded that the stimulated 45Ca++ efflux is a consequence, rather than a determinant, of stimulus-secretion coupling. The stimulated efflux is dependent on the presence of Ca++ in the medium and is independent of emiocytosis. This latter finding excludes the secretory granules as a significant source of glucose-stimulated 45Ca++ extrusion.

Animals↗
PubMed1977

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.

Animals↗
PubMed1977

Somatostatin inhibition of pancreatic glucagon release from monolayer cultures and interactions with calcium.

The effects of somatostatin (SRIF) on glucagon release have been studied in the monolayer culture of newborn rat pancreas. It was found that SRIF inhibited glucagon release rapidly and in a dose dependent manner at concentrations of 1-1000 ng/ml. SRIF inhibited glucagon release under basal conditions and after stimulation by arginine, 3-isobutyl-1-methylxanthine (IBMX), high Ca++ concentrations, ionophore A23187 and Ca++, and Ba++. SRIF inhibited ionophore-induced glucagon release over 60 min when a low concentration of A23187 was used (0.1 microgram/ml) but not when a high concentraion (10 microgram/ml) was used. The stimulant effect of 10 microgram/ml A23187 was, however, inhibited by SRIF during short periods of incubation. The per cent inhibition of arginine-stimulated glucagon release due to SRIF remained unchanged when the Ca++ concentration in the medium was varied from 1-10 mM. It is concluded that SRIF promptly inhibits glucagon release under basal conditions or when stimulated by a variety of agents. Thus, the action of SRIF appears to be basic to the granule release process and not specifically antagonisitc to any particular stimulants. Further, as SRIF inhibits release due to raised cytosol Ca++ (e.g., ionophore-Ca++ or high Ca++ experiments) the action is probably at a late point in the release mechanism.

Animals↗
PubMed1976

The activation of adenylate cyclase by cholera toxin: possible interaction with the nucleotide regulatory site.

The application of cholera toxin to intact cells causes a stimulation of adenylate cyclase activity. The effect is characterized by a lag period followed by a progressive rise in enzyme activity over several hours. Only a few minutes' exposure to the toxin is required to produce effects lasting over several days. Stimulation of adenylate cyclase by cholera toxin in broken cell preparations requires the presence of nicotinamide-adenine dinucleotide (NAD) and an unidentified component of the cytosol. Guanyl nucleotides and certain non-hydrolysable analogues of guanosine triphosphate also stimulate adenylate cyclase. Stimulation by the analogues results in a highly activated enzyme which has characterisitcs similar to those of adenylate cyclase after stimulation by cholera toxin. Thus the stimulation is irreversible, the enzyme may be "solubilized" by non-ionic detergents in the activated state, and responses to certain hormones are enhanced. Therefore the possibility exists that cholera toxin acts on the guanyl nucleotide regulatory protein of the adenylate cyclase complex. In exploring this possibility it was found pretreatment with cholera toxin not only blocked the stimulatory effect of subsequently added guanylylimidodi-phosphate (GppNHp) but that the latter reduced the stimulation by toxin. Similarly, pretreatment with GppNHp blocked the effect of cholera toxin. The similarities in the effects of cholera toxin and GppNHp, together with the mutual interference of their activities, suggests that cholera toxin acts at the same regulatory site at which guanyl nucleotides exert their effects on adenylate cyclase.

Adenylyl Cyclases↗
PubMed1976

Calcium induced glucagon release in monolayer culture of the endocrine pancreas. Studies with ionophore A23187.

The possible role of Ca2+ in glucagon release has been investigated by the use of ionophore A23187. This ionophore permits Ca2+ entry down a suitable concentration gradient by complexing and releasing Ca2+, thereby acting as a carrier in plasma membranes. Cultured cells obtained by enzymatic digestion of pancreases from newborn rats were studied on the third day of culture. As expected the effects of the ionophore were dependent upon the presence of Ca2+ in the medium. However, either stimulation or inhibition of glucagon release resulted when different concentrations of ionophore and Ca2+ were used. With 1.0 mM Ca2+ in the medium, glucagon release was stimulated in the presence of 0.01 and 0.1 mug/ml ionophore, but inhibited in the presence of 3.0 and 10.0 mug/ml. With 0.1 mug/ml ionophore, glucagon release was stimulated by 0.3 and 1.0 mC Ca2+ but not by 2.5 mM Ca2+. With 10 mug/ml ionophore glucagon release was stimulated by 0.03, 0.1 and 0.3 mM Ca2+, whereas at 1.0 mM, glucagon release was depressed. These findings suggest that by increasing Ca2+, glucagon is released from the A-cells, whereas too large an increase in Ca2+ is inhibitory. The effect to stimulate release was not completely specific for Ca2+ in that while the ionophore did not stimulate release in the presence of either Mg2+ or Sr2+ in the absence of Ca2+, it did stimulate release when Ba2+ was tested. Furthermore Ba2+ at 0.3 mM was stimulatory even in the absence of ionophore. Glucagon release in the absence of ionophore was also enhanced by addition of 30 mM Ca2+ or by omission of Ca2+ from the medium. It is concluded that Ca2+, which plays an essential role in the stimulus-secretion coupling in several different cell types, may be involved in the stimulation of glucagon release from the A-cells of the pancreas.

Animals↗
PubMed1976

Stimulatory and inhibitory effects of cyclic AMP on pancreatic glucagon release from monolayer cultures and the controlling role of calcium.

When glucagon release from monolayer cultures of newborn rat pancreas was measured over four hours in media containing 2.5 mM Ca++, a significant cyclic AMP-related inhibition of release was observed. This was noted whether intracellular cyclic AMP levels were raised by the addition of exogenous cyclic AMP or dibutyryl cyclic AMP, by phosphodiesterase inhibition with theophylline, or by the stimulation of adenylate cyclase with cholera toxin. The inhibition was concentration dependent for cyclic AMP and could not be reproduced by the addition of AMP, ADP or ATP. Adenosine also inhibited glucagon release while ATP was stimulatory. From time course studies it appeared that the inhibitory effects of cyclic AMP and cholera toxin were progressive after two hours of incubation. With cholera toxin an early stimulation of glucagon release was observed. The effects of cyclic AMP and cholera toxin on arginine-stimulated glucagon release were to stimulate further the glucagon release during the first hour of the incubation. Thus, the effects of raising intracellular cyclic AMP levels were biphasic in that both an early stimulation and a late inhibition of glucagon release were observed. In examining the nature of these responses a remarkable controlling role for Ca++ was uncovered: at Ca concentrations of 0.3 mM and lower no effect of cyclic AMP on glucagon release was found. With 1 mM Ca++ in the medium cyclic AMP stimulated glucagon release early (30 min) and thereafter had no further effect. In the presence of 2.5 mM Ca++ cyclic AMP did not stimulate early but did cause the delayed inhibition of release. It is concluded that the effect of cyclic AMP on glucagon release can be either stimulatory or inhibitory depending upon the Ca++ concentration of the medium and the duration of exposure to raised cyclic AMP levels.

Adenine Nucleotides↗
PubMed1976

Proinsulin synthesis in islets of Langerhans from spiny mice (Acomys cahirinus). Comparison with rats and mice.

Proinsulin synthesis in response to different concentrations of glucose has been studied in islets of Langerhans of acomys, rat and mouse. The response to increased glucose was small in acomys when compared with rat or mouse. The time course of the effect of glucose on proinsulin synthesis was also studied in sequential 15 min periods up to 60 min. With 5.5 mM glucose the rate of 3H-leucine incorporation for acomys islets were greater over the periods 15-30, 30-45 and 45-60 min after the addition of glucose to the islets than at 0-15 min. By contrast rat islets exhibited a greater glucose-induced stimulation between 15-30 and 30-45 min relative to the 0-15 min period and a further increase of the incorporation rate between 45 and 60 min. With 27.5 mM glucose acceleration of incorporation relative to the first 15 min period occurred at 15-30 min; it was then stable up to 60 min for islets both from acomys and from rat. Incorporation into total protein was much greater in acomys than in rat or mouse: thus in 2.75 mM glucose, acomys islets incorporated 5 and 19 times more 3H-leucine into total protein than did islets from rat or mouse respectively. In 27.5 mM glucose the incorporation into total protein of the islets from acomys was 4 and 13 times greater. Islet content of insulin was similar for all three species. No significant changes in ATP content were observed in response to changes in glucose concentration from 2.75 to 27.5 mM. The results demonstrate a decreased responsiveness of proinsulin synthesis to glucose in acomys and are discussed in terms of the known decreased sensitivity of insulin release in this animal.

Adenosine Triphosphate↗
PubMed1976

Effects of streptozotocin in vitro on proinsulin biosynthesis, insulin release and ATP content of isolated rat islets of Langerhans.

Proinsulin synthesis, insulin release and intracellular ATP concentrations were measured in isolated rat islets of Langerhans under control conditions in vitro incubation and after treatment with several concentrations of streptozotocin for different periods of time. It was found that streptozotocin inhibited proinsulin synthesis, as well as insulin release, in a time and concentration dependent manner. The characteristics of the inhibition of these two processes were similar in general terms, but one dissimilarity was noted, i.e. after 60 min exposure to a high concentration of streptozotocin, proinsulin synthesis was inhibited more than insulin release. ATP content was reduced by high concentrations of streptozotocin, but it was found that proinsulin synthesis and insulin release could be inhibited without any effect on ATP content by a low (6.22 mM) concentration of streptozotocin. The effect of streptozotocin on proinsulin synthesis was judged to be the result of a target specificity for the B-cell rather than a specific effect on proinsulin relative to total protein synthesis.

Adenosine Triphosphate↗