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G W Sharp

Publications and source records attributed to G W Sharp.

At least 91 records · Page 5Linked to original sources
PubMed1985

Studies on role of calmodulin in Ca2+ regulation of rabbit ileal Na and Cl transport.

To study the role of calmodulin in regulation of rabbit ileal active electrolyte transport by Ca2+, the effects of the Ca2+-calmodulin antagonist naphthalenesulfonamides W12 and W13 and the weak Ca2+-calmodulin antagonist promethazine, a phenothiazine, were studied on basal ileal Na and Cl transport and on secretion stimulated by Ca2+ and by cAMP. The naphthalenesulfonamides and promethazine all stimulated basal ileal Na and Cl absorption. The stimulation of Na absorption was dependent on Cl in the bathing solutions, and the stimulation of Cl absorption was Na dependent. This suggests that the transport process stimulated was the neutral, linked NaCl absorptive process. W13, which is a better Ca2+-calmodulin antagonist but has similar hydrophobic properties to W12, stimulated active ileal absorption at a lower concentration than W12, suggesting that the naphthalenesulfonamide-induced stimulation of active ileal absorption was not due to the hydrophobic properties of these drugs but could be due to their effects on the calcium-binding protein calmodulin. W12, W13, and promethazine did not alter paracellular transport, not affecting dilution potentials or structural features of the paracellular pathway. W12 and W13 did not decrease the changes in active ileal Na and Cl transport caused by increasing ileal cAMP content or intracellular Ca2+. This suggests that calmodulin is not directly involved in the active electrolyte secretion caused by increased intestinal Ca2+ or cAMP.

Animals↗
PubMed1985

Inhibition of glucose-induced insulin release by xylazine.

The effects of xylazine, a sedative that is extensively used in veterinary medicine, on glucose-stimulated insulin release have been studied using isolated perifused rat pancreatic islets. Xylazine had no effect on insulin release under basal conditions. Glucose-stimulated insulin release was inhibited in a concentration-dependent manner over the range of 10(-9)-10(-6) M. Maximum inhibition even with 10(-5) M xylazine was around 80%, which suggested that xylazine is only a partial agonist at its receptor. The inhibitory effect could be completely eliminated by yohimbine, an alpha 2-adrenergic antagonist, and was unaffected by prazosin, an alpha 1-adrenergic antagonist. That xylazine is a partial agonist was verified by the fact that when present in excess, it could stimulate insulin release which had already been inhibited to 100% by epinephrine. It is concluded that xylazine is a potent partial alpha 2-adrenergic agonist and that this mechanism is responsible, at least in part, for the hyperglycemia and insulinopenia associated with its use.

Animals↗
PubMed1985

Role of calcium in cholinergic stimulation of lacrimal gland protein secretion.

To characterize the role of Ca2+ in cholinergic stimulation of lacrimal gland protein secretion, the effects of inhibitors of cellular Ca2+ handling on protein secretion were investigated. Protein secretion was measured from rat exorbital glands using either pieces of gland in perifusion or acini isolated by collagenase digestion. Peroxidase was used as a measure of protein secretion. An inhibitor of Ca2+ influx via voltage sensitive Ca2+ channels (verapamil) at 10(-5) and 5 X 10(-5) M did not alter protein secretion stimulated by the cholinergic agonist carbachol at 10(-5) M. Inhibition of Ca2+ efflux via Na+/Ca2+ exchange by removal of extracellular Na+ or by inhibition of Na+-K+-ATPase activity using ouabain (10(-3) M) or extracellular K+ removal did not stimulate protein secretion. In contrast, inhibition of Ca2+ release from intracellular stores with TMB-8 at 100 micron completely blocked protein secretion stimulated by carbachol at 10(-5) M. Similarly, the Ca2+/calmodulin (CaM) antagonists W-13 and W-12 decreased carbachol-induced protein secretion with potencies similar to those which inhibit Ca2+/CaM dependent processes. We conclude that cholinergic agonists stimulate lacrimal gland protein secretion primarily by mobilizing Ca2+ from intracellular stores and that one mechanism by which this Ca2+ could activate secretion is in conjunction with calmodulin.

Animals↗
PubMed1984

Ca2+-calmodulin-, cyclic AMP- and cyclic GMP-induced phosphorylation of proteins in purified microvillus membranes of rabbit ileum.

Evidence is available to suggest that Ca2+-calmodulin and cyclic nucleotides are involved in the regulation of ion transport in rabbit ileum. Since both Ca2+-calmodulin and cyclic nucleotides exert many of their effects by phosphorylation, the effects of Ca2+-calmodulin and cyclic nucleotides on phosphorylation of purified microvillus membrane from rabbit ileal mucosa were evaluated. Ca2+-calmodulin increased phosphorylation of five microvillus-membrane peptides, with Mr values of 137000, 77000, 58000, 53000 and 50000. The increases in phosphorylation caused by Ca2+-calmodulin were: Mr-137000 peptide, 111 +/- 26%; Mr-77000 peptide, 71 +/- 17%; Mr-58000 peptide, 51 +/- 8%; Mr-53000 peptide, 113 +/- 20%. These increases were maximal at 1 microM-calmodulin and 0.3-0.9 microM free Ca2+; concentrations of Ca2+ causing half-maximal effects on phosphorylation for the different peptides were 0.06-0.12 microM. Cyclic AMP and cyclic GMP increased phosphorylation of two peptides, of Mr 137000 and 85000. The concentrations of cyclic nucleotides giving half-maximal phosphorylation of the Mr-137000 peptide were 0.3 microM-cyclic AMP and 4.6 microM-cyclic GMP, and for the Mr-85000 peptide, 3.9 microM-cyclic AMP and 0.05 microM-cyclic GMP. The maximal increase in phosphorylation of the Mr-137000 peptide was 200% for cyclic AMP and 95% for cyclic GMP, and that of the Mr-85000 peptide was 220% for cyclic AMP and 120% for cyclic GMP. These studies demonstrate the existence of Ca2+-calmodulin-, cyclic AMP- and cyclic GMP-dependent protein kinases and substrate proteins in purified rabbit ileal microvillus membranes and that Ca2+ can regulate phosphorylation of these proteins over the presumed physiological concentration range of cytosol free Ca2+.

Animals↗
PubMed1984

Cyclic nucleotide-dependent enzyme secretion in the rat lacrimal gland.

To characterize the role of cyclic nucleotides in secretion of enzymes by the lacrimal gland, pieces of rat exorbital glands were perfused with (1) 8-bromoadenosine-3',5'-cyclic monophosphate (8 Br cyclic AMP), (2) 8-bromoguanosine-3',5'-cyclic monophosphate (8 Br cyclic GMP), (3) forskolin, a stimulator of adenylate cyclase activity, (4) 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterase activity, or (5) carbachol, a cholinergic agonist. As a measure of enzyme secretion, timed collections of the perifusate effluent were analysed for peroxidase, an enzyme secreted by the lacrimal gland. Control peroxidase secretion was 0.3-0.9 (u./min per milligram protein). Peroxidase secretion was stimulated by 8 Br cyclic AMP (1 mM), but not by 8 Br cyclic GMP (1 mM). A 2-fold increase was detected. Peroxidase secretion was also stimulated by forskolin (60 microM), IBMX (1 mM), and the cholinergic agonist carbachol, which all stimulated peroxidase secretion 2-or 3-fold. The effect of maximally effective concentrations of IBMX (1 mM) and carbachol (0.1 mM) on secretion was additive. Finally, Ca2+ depletion in the presence of EGTA (1 mM) inhibited both IBMX-and carbachol-induced secretion by 45% and 60% respectively. We conclude that cyclic AMP, but not cyclic GMP, can stimulate lacrimal gland enzyme secretion. Cyclic AMP appears to utilize a pathway separate from but convergent with cholinergic agonists.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Mechanisms of synergism between glucose and cAMP on stimulation of insulin release.

The mechanism of synergism between glucose and adenosine 3',5'-cyclic monophosphate (cAMP) on insulin release has been studied. Synergism may result from 1) inhibition of Na+-Ca2+ exchange by glucose and 2) a cAMP-induced sensitization of the release machinery to Ca2+. To distinguish between these two possibilities, isolated rat pancreatic islets were perifused with agents that raise intracellular levels of cAMP [3-isobutyl-1-methylxanthine (IBMX) and forskolin] and others that increase intracellular concentrations of Ca2+ either by blocking Na2+-Ca2+ exchange (ouabain and choline-Ringer solution) or by causing increased Ca2+ influx (KCl, carbachol, and 10 mM Ca2+). The results indicate that both the combination of cAMP and increased Ca2+ influx or blocked Na2-Ca2+ exchange and increased Ca2+ influx potentiated insulin release. When the relative potentiating abilities of cAMP and blocked Na2+-Ca2+ exchange were compared by determining the individual effects of IBMX and 1 mM ouabain (a concentration that causes similar inhibition of 45C2+ efflux as 16.7 mM glucose) in the presence of carbachol, cAMP was only 1.4 times more potent as a potentiating agent than blocked Na+-Ca2+ exchange. The greatest potentiation of insulin release was observed when Na+-Ca2+ exchange was blocked in the presence of increased levels of intracellular cAMP.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Unexpected potentiation of insulin release by the calcium store blocker TMB-8.

Insulin release from rat pancreatic islets, stimulated by cAMP, 3-isobutyl-1-methylxanthine, or forskolin, is potentiated by TMB-8 [8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate], a drug that blocks the efflux of calcium from intracellular calcium stores without affecting influx. There is a short latent period before the potentiation occurs which can be reduced by preincubation with the drug. TMB-8 alone neither stimulates nor inhibits insulin release. The results suggest that an intracellular store fills with calcium because of the blocked efflux and unchanged influx, and loses its ability to regulate the cytosol Ca++ concentration. Under basal conditions, in the presence or absence of TMB-8, the plasma membrane is able to regulate the cytosol Ca++ concentration at low levels so that no change in insulin release occurs. However, when 3-isobutyl-1-methylxanthine, cAMP, or forskolin add an additional Ca++ load to the cytosol, the regulator capacity of the plasma membrane is overwhelmed, and cytosol Ca++ rises to higher levels than in the absence of TMB-8 because the filled store is no longer regulating. Thus, insulin release is potentiated.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Dantrolene and basal ileal sodium and chloride transport: involvement of calcium stores.

The effect of dantrolene on active ion transport in rabbit ileum was determined using the Ussing chamber short-circuiting technique. Dantrolene prevents the release of calcium from intracellular stores in skeletal muscle and was used to probe the role of intracellular calcium stores in intestinal ion transport. A saturated solution of dantrolene (approx 25 microM) decreased ileal short-circuit current and potential difference, increased conductance and mucosal-to-serosal and net Na and Cl fluxes, but did not alter serosal-to-mucosal Na and Cl fluxes. The dantrolene stimulation of active Na and Cl absorption was specific since it did not alter glucose-dependent Na absorption, transport changes caused by Ca2+ ionophore A23187, or the increase in short-circuit current caused by dibutyryl cAMP or theophylline. These effects were associated with an increase in total ileal calcium content and a decreased rate of 45Ca2+ efflux without any change in 45Ca2+ influx from the serosal or mucosal surfaces. These findings are consistent with an effect of dantrolene to stimulate active ileal Na and Cl absorption by a mechanism involving lowered cytosol Ca2+ levels and compatible with trapping calcium in intracellular stores. It thus appears as if intracellular calcium stores have an important role in the control of basal ion transport in the intestine.

Animals↗
PubMed1983

Effects of forskolin on insulin release and cyclic AMP content in rat pancreatic islets.

The effects of forskolin, a diterpene stimulator of adenylate cyclase, on cyclic AMP content and insulin release, have been studied in rat pancreatic islets. In the presence of 2.8 mM glucose, 0.3 to 30 microM forskolin gave concentration-dependent increases in cyclic AMP content to as much as seven-fold without causing any significant increase in insulin release. With 5.6 mM glucose, cyclic AMP contents were elevated by forskolin to the same extent as with 2.8 mM glucose but, under these conditions, insulin release was stimulated. 3 microM forskolin was a maximal concentration with respect to insulin release, but not for the elevation of cyclic AMP levels. Concentration-dependent increases in both insulin release and cyclic AMP content were detected over the range of 0.3 to 3 microM forskolin. The effect of forskolin to stimulate insulin release was rapid and was reversible within 15 minutes of removal of the drug. When tested with 16.7 mM glucose, forskolin potentiated both phases of stimulated release, with the greater effect upon the first phase.

Animals↗
PubMed1983

Involvement of Ca2+ in the impaired glucose-induced insulin release from islets cultured at low glucose.

Islet culture at a low glucose concentration results in a progressive impairment of glucose-induced insulin release. The role of Ca2+ in this defect was studied by comparing rat islets cultured for 6 days either at 8.3 mM (control) or 2.8 mM glucose. For measurement of 45Ca content and 45Ca2+ efflux, islets were kept in the presence of 45Ca2+ throughout. In islets cultured at 8.3 mM glucose, stimulation with 16.7 mM glucose during perifusion caused a typical biphasic pattern of insulin release paralleled by an increase in the rate of 45Ca2+ efflux. Both effects of glucose were markedly reduced in islets kept at 2.8 mM glucose, despite a similar insulin content. Islet 45Ca content was reduced. Both 45Ca content and insulin release were restored when islets were kept for an additional 24 h at 8.3 mM glucose. Insulin release induced by 3-isobutyl-1-methylxanthine (IBMX) or alpha-ketoisocaproic acid was not impaired, demonstrating that there is no generalized release defect. In contrast, glyceraldehyde- or K+-induced release was decreased. In islets maintained at 2.8 mM glucose, the stimulatory effect of glucose on Ca2+ uptake and the inhibitory effect on Ca2+ efflux (in the absence of Ca2+) were found to be operative. A defect may therefore lie distal to the Ca2+ uptake step involving either the mechanism by which glucose uses cellular Ca or another step yet to be identified.

Animals↗
PubMed1982

Ca2+- and calmodulin-dependent protein phosphorylation in rat lacrimal gland.

Ca2+, in homogenized lacrimal glands, enhanced phosphorylation of several peptides. Phosphorylation of two of these peptides was further stimulated by addition of the Ca2+-binding protein calmodulin and decreased by trifluoperazine, an inhibitor of Ca2+--calmodulin-dependent activity. Thus, Ca2+--calmodulin-dependent protein kinases and their substrates are present in lacrimal gland and could have an important role in lacrimal-gland function.

Animals↗
PubMed1982

Selective inhibition of glucose-stimulated insulin release by dantrolene.

Dantrolene sodium, which interferes with excitation-contraction coupling by inhibiting the Ca2+ release from sarcoplasmic reticulum in muscle, was used to investigate the role of stored calcium in the stimulation of insulin release by various secretagogues. Insulin release was measured simultaneously with 45Ca2+ uptake or 45Ca2+ efflux from isolated rat pancreatic islets. Glucose-stimulated insulin release was inhibited by dantrolene (10-100 microM) as was glyceraldehyde- or mannose-stimulated release. In contrast, dantrolene failed to inhibit insulin release stimulated by leucine, arginine, ouabain, potassium, or 3-isobutyl-1-methylxanthine. Although dantrolene lowered glucose-stimulated 45Ca2+ uptake, nonspecific blockade of voltage-dependent Ca2+ channels may not be a primary action of dantrolene because K+-stimulated 45Ca2+ uptake was not inhibited. Glucose utilization (3H2O formation) was unaffected by dantrolene, whereas glucose oxidation (14CO2 production) was decreased. In the absence of Ca2+, the glucose-inhibited 45Ca2+ efflux was unchanged. At normal Ca2+, dantrolene inhibited glucose-stimulated 45Ca2+ efflux and veratridine induced insulin release. This suggests an interference with mobilization of beta-cell calcium stores. The selective action of dantrolene on insulin release makes it an interesting tool for further studies on stimulus-secretion coupling.

Animals↗
PubMed1982

Chloroquine stimulates absorption and inhibits secretion of ileal water and electrolytes.

The effects of chloroquine diphosphate, a drug with "'membrane-stabilizing" properties, were studied on basal ileal absorption and on ileal secretion induced by increased intracellular cAMP levels and calcium (serotonin). The studies were performed on rat (in vivo) and rabbit ileum (in vitro). Intraluminal chloroquine (10(-4) M) reversed cholera toxin- and theophylline-induced secretion in rat ileum but did not alter the cholera toxin- and theophylline-induced increases in cAMP content. Addition of chloroquine (10(-4) M) to the mucosal surface of rabbit ileum did not alter basal active electrolyte transport or the serotonin-induced decreased Na and Cl absorption but inhibited the theophylline-induced C1 secretion. Addition of chloroquine (10(-4)) M) to the serosal surface stimulated net Na and Cl absorption. This effect may involve intracellular calcium. Chloroquine increased the rabbit ileal calcium content and decreased 45Ca2+ influx from the serosal surface. Both the mucosal and serosal effects of chloroquine described led to a net increase in absorptive function of the intestine and should prove useful in developing treatment of diarrheal diseases.

Absorption↗
PubMed1982

Dopamine stimulation of active Na and Cl absorption in rabbit ileum: interaction with alpha 2-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.

Animals↗