Artificial rearing of neonatal rats on a high-carbohydrate (HC) milk formula resulted in the immediate onset of hyperinsulinemia. This study examines, in islets of 12-day-old HC rats, adaptive changes that support the hyperinsulinemic... more
Artificial rearing of neonatal rats on a high-carbohydrate (HC) milk formula resulted in the immediate onset of hyperinsulinemia. This study examines, in islets of 12-day-old HC rats, adaptive changes that support the hyperinsulinemic state. Increases in plasma glucagon-like peptide-1 (GLP-1) levels and islet GLP-1 receptor mRNA supported increased insulin secretion by HC islets. Isolated HC islets, but not mother-fed (MF) islets, secreted moderate amounts of insulin in a glucose- and Ca2+-independent manner. Under stringent Ca2+-free conditions and in the presence of glucose, GLP-1 plus acetylcholine augmented insulin release to a larger extent in HC islets. Levels of adenylyl cyclase type VI mRNA and activities of protein kinase A, protein kinase C, and calcium calmodulin kinase II were increased in HC islets. A tenfold increase in norepinephrine concentration was required to inhibit insulin secretion in HC islets compared with MF islets, indicating reduced sensitivity to adrenerg...
Research Interests:
OF PROSTAGLANDIN SYNTHESIS IN THE FELINE ADRENAL CORTEX by Suza nne Ga l e Laychock Depa rtm ent of Pharmacology Medical College of Virginia Schoo l of Bas ic Sciences Vi rg inia Commonwea lth Uni versit y August, 1976 Studies were ca rri... more
OF PROSTAGLANDIN SYNTHESIS IN THE FELINE ADRENAL CORTEX by Suza nne Ga l e Laychock Depa rtm ent of Pharmacology Medical College of Virginia Schoo l of Bas ic Sciences Vi rg inia Commonwea lth Uni versit y August, 1976 Studies were ca rri ed out on th e feline adrena l gland to ascerta in th e role of prostaglandins in the mechanism of a ct ion of ACTH. Using tritiat ed arachidonic acid as a prostag landin (PG) precursor , it was demonstrated by co lumn and thin layer chromatography techniques that i so lated trypsinized adre nocorti ca l ce ll s possess an ac tive PG synth etase capable of synthes izing ra d iolabe led PGE, PGF, and PGA/B-like su b s ta nces . Concentra tion s of ACTH (125 250 μU ) which s ti mu late s t e roidog e nesis enhanced the conver sion of radiolabeled arachidonic acid to PGE, PGF and th e PGA/B products extrac ted from corti ca l ce ll s and incubation media. PG biosyn thes i s by isolated cortical ce lls was studied by radio immunoassay (RIA) using a nti...
Research Interests:
The rate of glucose utilization in isolated pancreatic islets of the rat was inhibited by the alpha 2-adrenoceptor agonists clonidine and epinephrine. Yohimbine reversed the inhibition. alpha 1 or beta-adrenoceptor agonists had little or... more
The rate of glucose utilization in isolated pancreatic islets of the rat was inhibited by the alpha 2-adrenoceptor agonists clonidine and epinephrine. Yohimbine reversed the inhibition. alpha 1 or beta-adrenoceptor agonists had little or no effect on glucose utilization. Stimulation of muscarinic receptors by carbamylcholine reversed the effect of clonidine. Pertussis toxin blocked the effect of clonidine on glucose utilization, and potentiated the response to carbamylcholine. 8-Bromo-cAMP did not affect glucose utilization in the presence of clonidine. Thus, alpha 2-adrenoceptors negatively modulate glucose utilization, and the effect is mediated by an inhibitory guanine nucleotide regulatory protein, but not by cAMP.
Research Interests:
Glucose utilization in isolated islets of Langerhans of the rat was determined by measuring the conversion of [5-3H]glucose (10 mM) to 3H2O. The alpha 2-adrenoceptor agonists clonidine, epinephrine, and norepinephrine in the presence of... more
Glucose utilization in isolated islets of Langerhans of the rat was determined by measuring the conversion of [5-3H]glucose (10 mM) to 3H2O. The alpha 2-adrenoceptor agonists clonidine, epinephrine, and norepinephrine in the presence of the alpha 1-adrenoceptor antagonist prazosin and the beta-adrenoceptor antagonist propranolol inhibited glucose utilization by as much as 50%. Yohimbine, an alpha 2-adrenoceptor antagonist, reversed the reduction in glucose utilization evoked by alpha 2 receptor agonists. The cholinomimetics carbachol and muscarine, and 8-bromo-cyclic GMP, but not other cyclic nucleotides, reversed the clonidine-induced suppression of glucose utilization. 3-Isobutyl-1-methylxanthine potentiated the stimulation of glucose utilization by carbachol with clonidine. In contrast, the beta-adrenoceptor agonist isoproterenol did not affect glucose utilization. Forskolin, which activates adenylate cyclase, reduced glucose utilization and did not affect the inhibitory response to clonidine. The ester phorbol 12,13-dibutyrate induced a latent reversal of the effects of clonidine. Insulin release paralleled changes in glucose utilization with alpha 2-adrenoceptor agonists. Carbachol and 8-bromo-cyclic GMP antagonized the alpha 2-adrenoceptor-induced inhibition of insulin release. During sustained insulin release (60 min), 8-bromo-cyclic AMP became a more potent modulator of secretion than 8-bromo-cyclic GMP in the presence of clonidine, although glucose utilization was not enhanced by 8-bromo-cyclic AMP.
Research Interests:
Research Interests:
Research Interests:
Rat pancreatic islets methylate phosphatidylethanolamine (PE) lipids to form phosphatidylcholine (PC) with S-adenosyl-L-[methy-3H]methionine as the methyl donor. Islet PE-N-methyltransferase had activity optima at pH 6-7 and 8-9.... more
Rat pancreatic islets methylate phosphatidylethanolamine (PE) lipids to form phosphatidylcholine (PC) with S-adenosyl-L-[methy-3H]methionine as the methyl donor. Islet PE-N-methyltransferase had activity optima at pH 6-7 and 8-9. S-Adenosyl-L-homocysteine, sodium deoxycholate, and Triton X-100 inhibited methylation in islet homogenates. Addition of phosphatidyl-N-monomethylethanolamine and phosphatidyl-N,N-dimethylethanolamine (PDME) enhanced [3H]methyl incorporation into PDME and PC, respectively. Isoproterenol, but not glucose, stimulated phospholipid methylation in islet homogenates. Propranolol inhibited the isoproterenol effect. In intact islets, glucose or isoproterenol stimulated insulin release and incorporation of [3H]methyl groups from [methyl-3H]methionine into phospholipids. Isoproterenol enhanced to a similar extent glucose-stimulated methylation and hormone release. Neither 2-deoxyglucose, tolbutamide, nor 8-bromo-cyclic AMP stimulated islet phospholipid methylation. The methyl-transferase inhibitor 3-deazaadenosine inhibited both glucose and isoproterenol-stimulated methyltransferase activity and insulin release. Propranolol inhibited the beta-adrenergic potentiation of glucose-induced phospholipid methylation and insulin release. These data suggest that PE-N-methyltransferase plays a role in amplification of the islet cell stimulus-secretion coupling response to certain secretagogues.
Research Interests:
The pancreas is an exocrine and an endocrine organ. The acinar cells secrete proteolytic, lipolytic and amylytic enzymes in the pancreatic juice that flows through a ductal network to the duodenum. These enzymes are important for... more
The pancreas is an exocrine and an endocrine organ. The acinar cells secrete proteolytic, lipolytic and amylytic enzymes in the pancreatic juice that flows through a ductal network to the duodenum. These enzymes are important for digestion. The pancreatic islets of Langerhans in the pancreas secrete hormones into the blood that are essential for maintenance of normal blood glucose levels or euglycemia. Diabetes mellitus is a disease of the endocrine pancreas. Type 1 diabetes is characterized as an absolute lack of insulin in the B-cells of the pancreatic islet. Insulin must be replaced for optimal control of blood glucose levels. Type 2 diabetes is characterized by a relative insufficiency of insulin secretion and peripheral insulin resistance. Oral hypoglycemic agents, injectable incretins and insulin are used to control blood glucose levels in type 2 diabetes. Oral hypoglycemic agents can target insulin secretion, hepatic glucose release, kidney glucose transporters, and glucose uptake and metabolism in insulin target tissues. Carbohydrate metabolism in the GI tract is also a target for reducing prandial glucose levels in blood. Maintaining euglycemia often requires combination pharmacologic therapy.
Research Interests:
Research Interests:
ABSTRACT
Research Interests:
Research Interests:
There is now little doubt that muscle contraction and secretory activity involve fundamentally similar processes, with an increase in free Ca2+ within the cytosol providing the critical link between membrane activation by a stimulus and... more
There is now little doubt that muscle contraction and secretory activity involve fundamentally similar processes, with an increase in free Ca2+ within the cytosol providing the critical link between membrane activation by a stimulus and the specific tissue response (Rubin, 1974a; Douglas, 1975a). On the one hand, Ca2+ is instrumental in initiating muscle myofilament contraction while, on the other hand, Ca2+ facilitates the fusion of secretory granule membranes with the cell membrane of exocytotic tissues. The fact that Ca2+ is a mediator in these two basic biological processes has prompted speculation that they share a similar molecular basis involving a contractile event (Poisner, 1970; Kuo and Coffee, 1976; Trifaro and Ulpian, 1976).
Prostaglandins biosynthesized from 3H-arachidonic acid by trypsin-dispersed cat adrenocortical cells were isolated by silicic acid and thin layer chromatography. PGE, PGF, and a third component with mobility properties indistinguishable... more
Prostaglandins biosynthesized from 3H-arachidonic acid by trypsin-dispersed cat adrenocortical cells were isolated by silicic acid and thin layer chromatography. PGE, PGF, and a third component with mobility properties indistinguishable from either PGA or PGB were identified both in cortical cell homogenates and incubation medium. Concentrations of ACTH (125-250 muU) which stimulate steroidogenesis enhanced the conversion of labeled precursor to all three of these prostaglandins. These findings provide further evidence for the proposal that prostaglandins function as a critical link in ACTH-induced steroidogenesis.
Research Interests:
Neonatal rat pups were artificially reared on isocaloric diets high in carbohydrate (HC) or high in fat (HF) or were naturally reared on mother's milk (MF). The HC adult rats were hyperinsulinemic, normoglycemic, and obese. This study... more
Neonatal rat pups were artificially reared on isocaloric diets high in carbohydrate (HC) or high in fat (HF) or were naturally reared on mother's milk (MF). The HC adult rats were hyperinsulinemic, normoglycemic, and obese. This study investigates pancreatic islet insulin release (IR) of the adult first-generation (1-) diet-regulated animals and their second-generation (2-) progeny. Male rat 1-HC islets had higher basal IR than either 1-MF or 1-HF control groups. In addition, glucose (17 mM) failed to increase IR above basal values in 1-HC islets, whereas it stimulated IR in 1-MF and 1-HF islets. Similar secretory responses were evoked by 2-ketoisocaproic acid (2-KIC). Female rat 1-MF and 1-HF islets also had higher glucose-stimulated IR compared with 1-HC islets. Male rat 2-HC islets had higher basal IR and reduced sensitivity to glucose and 2-KIC compared with 2-MF islets, which coincided with hyperinsulinemia. Glyceraldehyde-3-phosphate dehydrogenase activity in 1-HC and 2-HC...
Research Interests:
Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [14C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid... more
Isolated rat pancreatic acini were employed to demonstrate that the exocrine pancreas can metabolize [14C]-arachidonic acid by way of the lipoxygenase pathway as well as the cyclooxygenase pathway. Analysis by high performance liquid chromatography delineated a monohydroxy acid, presumably 12-L-hydroxy-5,8-10,14-eicosatetraenoic acid (12-HETE) as the major lipoxygenase product. The formation of this hydroxy arachidonate derivative was stimulated by the calcium ionophore ionomycin. Stimulation of the lipoxygenase pathway by ionomycin was confirmed by thin layer chromatography. In addition, 6-keto-PGF1 alpha, PGF2 alpha, and PGE2 were identified; and ionomycin, carbamylcholine, and caerulein enhanced the formation of these metabolites of the cyclooxygenase pathway. Ionomycin induced stimulation of HETE formation was inhibited by ETYA and nordihydroguaiaretic acid, but spontaneous and evoked enzyme secretion was unaffected. Thus, although ionomycin, a pancreatic secretagogue, stimulate...
Research Interests:
Isolated pancreatic islets of Langerhans of the rat which were sonicated and incubated with radiolabeled arachidonic acid for 1 hr synthesized several species of prostaglandins (PGs). Both thin-layer and high-performance liquid (HPLC)... more
Isolated pancreatic islets of Langerhans of the rat which were sonicated and incubated with radiolabeled arachidonic acid for 1 hr synthesized several species of prostaglandins (PGs). Both thin-layer and high-performance liquid (HPLC) chromatographic techniques demonstrated the synthesis by islet sonicates of PGF2 alpha and PGE2 equivalents, in addition to the 15-keto-13, 14-dihydro metabolites of these primary PGs. In addition, HPLC allowed the identification of 6-keto-PGF1 alpha (the metabolite of prostacyclin) as a major PG synthesized from arachidonate by this tissue. Islet vascular elements, as well as endocrine cells, may contribute to the synthesis of the latter compound. Lesser amounts of arachidonate were incorporated into PG-like compounds eluting as thromboxane. The synthesis of PGs was sensitive to the protein concentration of islet sonicate, and a five-fold dilution of protein resulted in a comparable reduction in arachidonate incorporation into PGs. Labeled arachidonat...
Research Interests:
Isolated islets were incubated with [32P]P1 and radiolabelling of polyphosphoinositides were determined. Labelling equilibrium was approached after 45 min, with a half-time of 15 min. D-Glucose decreased the amount of [32P]PO4 in... more
Isolated islets were incubated with [32P]P1 and radiolabelling of polyphosphoinositides were determined. Labelling equilibrium was approached after 45 min, with a half-time of 15 min. D-Glucose decreased the amount of [32P]PO4 in phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 4-phosphate (PtdIns4P) within 0.5 min, and loss of radiolabel was still evident at 1 min. [32P]PO4 levels in polyphosphoinositides returned to basal levels within 5 min. Neither D-galactose nor D-glucose after pretreatment of islets with mannoheptulose elicited the polyphosphoinositide effect. The glucose-stimulated breakdown of polyphosphoinositides was inhibited by EGTA; re-addition of Ca2+ partially restored the glucose effect. Ionomycin and tolbutamide promoted the rapid breakdown of PtdIns(4,5)P2, whereas the breakdown of PtdIns4P was less rapid and of a lesser magnitude. The results suggest that the Ca2+-dependent breakdown of polyphosphoinositides in an early metabolic eve...
Research Interests:
A significant proportion of the steroidogenic response of isolated rat adrenocortical cells to dibutyryl cyclic AMP does not require extracellular calcium, and this component is profoundly depressed by low concentrations of the putative... more
A significant proportion of the steroidogenic response of isolated rat adrenocortical cells to dibutyryl cyclic AMP does not require extracellular calcium, and this component is profoundly depressed by low concentrations of the putative calcium antagonist, TMB-8. The inhibition is reversed by either the readdition of calcium or the calcium ionophore A23187. The steroidogenic response to pregnenolone, whose mode of action does not require calcium, was not depressed by TMB-8. Corticotropin (ACTH)-induced steroidogenesis, which requires extracellular calcium, was markedly depressed by TMB-8, although enhanced cyclic AMP formation is only slightly depressed by this drug. Adrenal cortical microsomes possess an ATP-dependent 45calcium (45Ca2+) uptake system which responded to EGTA with a rapid efflux of 45Ca2+; EGTA-induced calcium efflux from this microsomal fraction was markedly reduced by a concentration of TMB-8 that blocked dibutyryl cyclic AMP-evoked steroidogenesis. TMB-8 produced ...
Research Interests:
Biochemical evidence in support of a role for arachidonic acid 5-lipoxygenase activity in pancreatic islet insulin secretion has been obtained. Peptidyl leukotriene metabolism was studied in rat islets using a dual-labeling technique in... more
Biochemical evidence in support of a role for arachidonic acid 5-lipoxygenase activity in pancreatic islet insulin secretion has been obtained. Peptidyl leukotriene metabolism was studied in rat islets using a dual-labeling technique in extended culture, with analysis of arachidonic acid metabolites by reverse-phase high-performance liquid chromatography. The production of [3H]arachidonoyl/[35S]cysteinyl leukotrienes C4 and E4 by islets was compared with that by mouse resident peritoneal macrophages and with the lipoxygenase metabolism of rabbit polymorphonuclear leukocytes. The stimulus-specific nature of leukotriene biosynthesis was characterized by low basal biosynthesis in unstimulated islet cells with a calcium-mediated activation of 5-lipoxygenase product formation.
Research Interests:
It is hoped that his review enables the reader to appreciate the complexities implicit in the interactions among Ca2+, cyclic nucleotides, and phospholipid-metabolizing pathways in cell signal transduction. The interactions are varied and... more
It is hoped that his review enables the reader to appreciate the complexities implicit in the interactions among Ca2+, cyclic nucleotides, and phospholipid-metabolizing pathways in cell signal transduction. The interactions are varied and intricate, often involving several levels of cell amplification mechanisms. Upsetting the balance of fatty acids in membrane phospholipids can have detrimental effects on adenylate cyclase. Thus, n - 3 fatty acid enrichment of phospholipids suppresses adenylate cyclase activity. The effects of significant alterations in dietary fatty acids, such as might occur with the current vogue for n - 3 eicosapentaenoic acid and docosahexaenoic acid (fish oil) dietary enrichment regimens, will need to be assessed more fully with regard to stimulus-induced changes in cyclic nucleotide production in various tissues. Since the n - 3 fatty acids have not been demonstrated to affect guanylate cyclase activity, dietary changes in certain of these fatty acids would ...
Research Interests:
Phospholipase A2 and acyltransferase activities were identified in membranes associated with purified pancreatic zymogen granules. In homogenate and granule membranes, phospholipase activity was linearly related to protein concentration... more
Phospholipase A2 and acyltransferase activities were identified in membranes associated with purified pancreatic zymogen granules. In homogenate and granule membranes, phospholipase activity was linearly related to protein concentration and was Ca2(+)-dependent with an alkaline pH optimum. The Ca2+ sensitivity was observed over the range of concentrations through which intracellular ionic Ca2+ is elevated by physiological stimuli in intact cells. Intact zymogen granules and granule membranes also demonstrated reacylating activity in the presence and absence of an exogenous acceptor. Reacylating activity was related to the concentration of lyosphospholipid added and was optimally activated at alkaline pH. A more rapid rate of reacylation was observed when [14C]arachidonoyl CoA was employed as the donor molecule rather than [3H]arachidonate (plus coenzyme A); this suggests the absence of acyl-CoA synthetase in the purified granule membranes. We conclude that granule membrane phospholi...
Research Interests:
Isolated rat pancreatic islets prelabeled with myo-[3H]inositol respond to glucose and carbamylcholine with increased [3H] inositol phosphate (InsP) production. Prostaglandin E2 (PGE2) inhibits the effects of glucose and carbamylcholine... more
Isolated rat pancreatic islets prelabeled with myo-[3H]inositol respond to glucose and carbamylcholine with increased [3H] inositol phosphate (InsP) production. Prostaglandin E2 (PGE2) inhibits the effects of glucose and carbamylcholine on [3H]InsP production. Ionomycin reversed the effect of PGE2 on glucose-stimulated [3H]InsP production. The cyclooxygenase inhibitors indomethacin, ibuprofen, and eicosatetraynoic acid potentiated [3H]InsP production in response to 5 and 10 mM glucose but not to 17 mM glucose. Indomethacin did not affect the carbamylcholine response. Unsaturated fatty acids, including arachidonic acid, linolenic acid, eicosapentaenoic acid, oleic acid, and eicosatetraynoic acid, increased [3H]InsP production. Arachidonic acid potentiated [3H]InsP accumulation in response to low concentrations of glucose. Indomethacin potentiated the response to arachidonic acid. delta 9-Tetrahydrocannabinol, which mobilizes endogenous fatty acids, also potentiated glucose-stimulated...
Research Interests:
Trypsin-dispersed cat adrenocortical cells were incubated at 37 degrees C in modified Eagle's medium containing [14C]arachidonic acid of sodium [14C]-acetate and then in non-radioactive medium. Radioactive incorporation was obtained... more
Trypsin-dispersed cat adrenocortical cells were incubated at 37 degrees C in modified Eagle's medium containing [14C]arachidonic acid of sodium [14C]-acetate and then in non-radioactive medium. Radioactive incorporation was obtained in all phospholipids, with the greatest amount of radioactivity in phosphatidylcholine, followed by phosphatidylethanolamine, phosphatidyl-serine, and phosphatidylinositol. Concentrations of individual phospholipids generally paralleled the relative amounts of corresponding radiolabeled phospholipids, although the percentage of phosphatidylinositol was considerably lower than its radioactive counterpart, resulting in a high specific activity of this particular phospholipid. Although a potently steroidogenic concentration of corticotropin failed to enhance release of label from any particular phospholipid, analysis of specific activity showed that corticotropin stimulation was accompanied by an increased turnover of phosphatidylinositol and phosphatid...
Research Interests:
Research Interests:
Artificial rearing of 4-day-old rat pups on a high-carbohydrate (HC) milk formula results in the immediate onset of hyperinsulinemia. To evaluate these early changes, studies on pancreatic function were carried out on 12-day-old HC rats... more
Artificial rearing of 4-day-old rat pups on a high-carbohydrate (HC) milk formula results in the immediate onset of hyperinsulinemia. To evaluate these early changes, studies on pancreatic function were carried out on 12-day-old HC rats and compared with age-matched mother-fed (MF) pups. The plasma insulin and glucagon contents were increased sixfold and twofold, respectively, in HC rats compared with MF rats. There was a distinct leftward shift in the glucose-stimulated insulin secretory pattern for HC islets. HC islets secreted insulin in the absence of any added glucose and in the presence of Ca(2+) channel inhibitors. The activities of glucokinase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate dehydrogenase complex were significantly increased in HC islets compared with MF islets. The protein contents of GLUT-2 and hexokinase were significantly increased in HC islets. These findings indicate that a nutritional intervention in the form of a HC formula only du...
Research Interests:
Research Interests: Biology, Medicine, Fetal development, Pregnancy, Humans, and 4 moreMetabolic diseases, Female, Newborn Infant, and Fetus
Research Interests:
Obese individuals are more likely to suffer from diseases termed the "metabolic syndrome," which includes type 2 diabetes. It is now recognized that early life dietary experiences play an important role in the etiology of such... more
Obese individuals are more likely to suffer from diseases termed the "metabolic syndrome," which includes type 2 diabetes. It is now recognized that early life dietary experiences play an important role in the etiology of such diseases. In this context, the consequences of a high carbohydrate (HC) dietary intervention in neonatal rats is being studied in our laboratory. Artificial rearing of 4-day-old rat pups on a HC milk formula up to Day 24 results in the immediate onset of hyperinsulinemia, which persists throughout the period of dietary intervention. Several adaptations at the biochemical, cellular, and molecular levels in the islets of…
Research Interests:
Islets of Langerhans isolated from rat pancreata were incubated with (¹â´C)choline to determine the biosynthesis of PC by the CDP choline to determine the biosynthesis of PC by the CDPcholine pathway. Recovery of (¹â´C)PC in islet... more
Islets of Langerhans isolated from rat pancreata were incubated with (¹â´C)choline to determine the biosynthesis of PC by the CDP choline to determine the biosynthesis of PC by the CDPcholine pathway. Recovery of (¹â´C)PC in islet membranes was time-related, and stimulated by glucose (17mM) during 60 min. The rate of PC synthesis was constant during 60 min with glucose stimulation. In contrast, the sulfonylurea tolbutamide (2 mM) reduced the recovery of (¹â´C)choline in PC, and 8-bromo-cyclic AMP (5 mM) did not significantly affect (¹â´C)PC recovery. Incubation of islets in Ca/sup 2 +/-free medium enhanced glucose-stimulated recovery of (¹â´C)choline-labeled PC due to the inhibition of phospholipase and phospholipid hydrolysis. Inhibition of CTP:phosphocholine cytidylyltransferase with 5-deoxy-5'-isobutylthioadenosine (SIBA) reduced (¹â´C)PC levels and insulin release in a concentration dependent manner. Treatment with SIBA also reduced Mg/sup 2 +/-dependent Ca/sup ...
Research Interests:
ABSTRACT
Research Interests:
Research Interests:
The present study was undertaken to determine whether nitric oxide (NO) alters voltage-dependent changes in intracellular calcium levels ([Ca2+]i) using PC12 cells as a neuronal model. The addition to PC12 cells of sodium nitroprusside... more
The present study was undertaken to determine whether nitric oxide (NO) alters voltage-dependent changes in intracellular calcium levels ([Ca2+]i) using PC12 cells as a neuronal model. The addition to PC12 cells of sodium nitroprusside (SNP), which spontaneously releases NO in aqueous solution, significantly inhibited the KCl-stimulated increase in [Ca2+]i. The inhibitory action of SNP was concentration-dependent and was mimicked by hydroxylamine which also generates NO. Both L-type (nifedipine sensitive) and N-type (omega-conotoxin sensitive) voltage-dependent Ca2+ channels are present in PC12 cells and may be affected by NO-generating agents. In contrast, SNP did not alter [Ca2+]i in response to purinergic receptor stimulation. Preincubation of PC12 cells with 8-bromo-cyclic GMP also inhibited the KCl-stimulated increase in [Ca2+]i. In addition, inclusion of the guanylyl cyclase inhibitor, LY83583, blocked the inhibitory action of SNP on the voltage-sensitive changes in [Ca2+]i. The results suggest that NO selectively inhibits voltage-dependent calcium influx in neuronal cells through a cyclic GMP-dependent mechanism.
Research Interests:
Delta-9-tetrahydrocannabinol ((-)delta 9 THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in... more
Delta-9-tetrahydrocannabinol ((-)delta 9 THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. The bicyclic synthetic cannabinoid [3H]CP-55,940 has been used as a ligand to demonstrate the presence of a cannabinoid receptor in mammalian brain. We now report that [3H]CP-55,940 binds to live sea urchin (Strongylocentrotus purpuratus) sperm in a concentration, sperm density, and time-dependent manner. Specific binding of [3H]CP-55,940 to sperm, defined as total binding displaced by (-)delta 9THC, was saturable: KD 5.16 +/- 1.02 nM; Hill coefficient 0.98 +/- 0.004. This suggests a single class of receptor sites and the absence of significant cooperative interactions. Sea urchin sperm contain 712 +/- 122 cannabinoid receptors per cell. Binding of [3H]CP-55,940 to sperm was reduced in a dose-dependent manner by increasing concentrations of CP-55,940, (-)delta 9THC, and (+)delta 9THC. The rank order of potency to inhibit binding of [3H]CP-55,940 to sperm and to block the egg jelly stimulated acrosome reaction was: CP-55,940 > (-)delta 9THC > (+)delta 9THC. These findings show that sea urchin sperm contain a stereospecific cannabinoid receptor that may play a role in inhibition of the acrosome reaction. The radioligand binding data obtained with live sea urchin sperm are remarkably similar to those previously published by other investigators using [3H]CP-55,940 on mammalian brain and nonneural tissues. The cannabinoid binding properties of this receptor appear to have been highly conserved during evolution. We postulate that the cannabinoid receptor may modulate cellular responses to stimulation.
Research Interests:
Research Interests:
A role for prolactin (PRL) in the regulation of adenylyl cyclase (AC), cyclic AMP (cAMP) formation and insulin secretion was studied in isolated rat pancreatic islets cultured for 4 days at 5.5 mM glucose in the absence (control) or... more
A role for prolactin (PRL) in the regulation of adenylyl cyclase (AC), cyclic AMP (cAMP) formation and insulin secretion was studied in isolated rat pancreatic islets cultured for 4 days at 5.5 mM glucose in the absence (control) or presence of PRL (500 ng/ml). In PRL-treated islets, stimulation by glucose (8 mM), carbamylcholine chloride (CCh) and phorbol dibutyrate increased cAMP levels 40, 89, and 151%, respectively, above similarly stimulated control islets without PRL. Moreover, insulin secretion in PRL-treated islets was more than doubled in response to 8 mM glucose plus glucagon-like peptide 1 compared with control islets. PRL also increased protein kinase C (PKC) activity in cultured islets. When islets were cultured at an insulin secretion desensitizing concentration of glucose (11 mM) for 4 days, there was a decrease in forskolin-stimulated cAMP production. However, the presence of PRL with 11 mM glucose prevented the glucose-induced decrease in cAMP production. Insulin secretion in response to 17 mM glucose was also higher (P<0.02) in islets cultured with 11 mM glucose plus PRL compared with islets cultured with 11 mM glucose alone. Islet AC types -III, -V, and -VI mRNA levels increased relative to 18s rRNA following PRL treatment. In contrast, culture at 11 mM glucose decreased relative AC-III, -V and -VI mRNA levels by as much as 50%. Culture with PRL prevented the decrease in AC expression during islet culture with 11 mM glucose, and the mRNA levels remained similar to control islets cultured at 5.5 mM glucose. Thus, PRL not only increased islet AC expression and activity and insulin secretory responsiveness, but also protected islets from chronic glucose-induced inhibition of these beta-cell activation parameters.
Research Interests:
Research Interests: Biology, Medicine, Biological Sciences, Dexamethasone, Cell line, and 15 moreBlood Glucose, Insulin, Mice, Nitric oxide, Animals, Metallothionein, Insulinoma, Nitric Oxide Synthase, Glucocorticoids, islets of Langerhans, Interleukin, Pancreatic islets, Drug synergism, Medical and Health Sciences, and In Vitro Techniques
Insulin secretion from beta cells of the islets of Langerhans in the endocrine pancreas is regulated by glucose, glucose metabolites, metabolic intermediates such as ATP, acetyl CoA and reduced pyridine nucleotides, and classical second... more
Insulin secretion from beta cells of the islets of Langerhans in the endocrine pancreas is regulated by glucose, glucose metabolites, metabolic intermediates such as ATP, acetyl CoA and reduced pyridine nucleotides, and classical second messengers. Receptor responses transduced by guanine nucleotide binding proteins modulate metabolic activity, the generation of second messengers, and cell depolarization during stimulus-response coupling in the beta cell. This review will consider insulin secretion as regulated by glucose metabolic pathways and second messengers.
Research Interests:
Research Interests:
Functional and molecular biological evidence exists for the expression of ryanodine receptors in non-muscle cells. In the present study, RT-PCR and 5'-rapid amplification of cDNA 5'-end (5'-RACE analysis) provided... more
Functional and molecular biological evidence exists for the expression of ryanodine receptors in non-muscle cells. In the present study, RT-PCR and 5'-rapid amplification of cDNA 5'-end (5'-RACE analysis) provided evidence for the presence of a type 1 ryanodine receptor/Ca2+ channel (RyR1) in diverse cell types. In parotid gland-derived 3-9 (epithelial) cells, the 3'-end 1589 nucleotide sequence for a rat RyR shared 99% homology with rat brain RyR1. Expression of this RyR mRNA sequence in exocrine acinar cells, endocrine cells, and liver in addition to skeletal muscle and cardiac muscle, suggests wide tissue distribution of the RyR1. Positive identification of a 5'-end sequence was made for RyR1 mRNA in rat skeletal muscle and brain, but not in parotid cells, pancreatic islets, insulinoma cells, or liver. These data suggest that a modified RyR1 is present in exocrine and endocrine cells, and liver. Western blot analysis showed L-type Ca2+ channel-related proteins in parotid acinar cells, which were of comparable size to those identified in skeletal and cardiac muscle, and in brain. Immunocytochemistry carried out on intact parotid acini demonstrated that the dihydropyridine receptor was preferentially co-localized with the IP3 receptor in the apical membranes. From these data we conclude that certain non-muscle cells express a modified RyR1 and L-type Ca2+ channel proteins. These receptor/channels may play a role in Ca2+ signaling involving store-operated Ca2+ influx via receptor-mediated channels.
Research Interests: Microbiology, Chemistry, Medical Microbiology, Membrane Biology, Medicine, and 15 moreGene expression, Brain, Liver, Mice, Animals, Calcium channels, Insulinoma, Rats, Myocardium, Parotid Gland, Base Sequence, Biochemistry and cell biology, islets of Langerhans, Molecular Sequence Data, and Nucleotide sequence
Research Interests:
Although genes and dietary habits are generally implicated in the aetiology of the prevailing obesity epidemic, the steep increase in the incidence of obesity within a relatively short span of time suggests that other contributing factors... more
Although genes and dietary habits are generally implicated in the aetiology of the prevailing obesity epidemic, the steep increase in the incidence of obesity within a relatively short span of time suggests that other contributing factors may be at play. The role of nutritional experience during the very early periods of life is increasingly being recognized as contributing to growth and metabolic changes in later life. Epidemiological data and studies from animal models have established a strong correlation between an aberrant intrauterine environment and adult-onset disorders in offspring. The nutritional experience in the immediate postnatal life is another independent factor contributing to the development of metabolic diseases in adulthood. Although studies on the small-litter rat model have shown that overnourishment during the suckling period results in adult-onset metabolic disorders, our studies have shown that a change in the quality of calories-specifically, increased carbohydrate intake by newborn rat pups in the immediate postnatal period-results in chronic hyperinsulinaemia and adult-onset obesity. Several functional alterations in islets and in the hypothalamic energy homeostatic mechanism appear to support this phenotype. Remarkably, female rats that underwent the high-carbohydrate dietary modification as neonates spontaneously transmitted the obesity phenotype to their offspring, thus establishing a vicious generational effect. The high-carbohydrate diet-fed rat model has particular relevance in the context of the current human infant feeding practices: reduction in breast feeding and increase in formula feeding for infants, accompanied by early introduction of carbohydrate-enriched baby foods.
Research Interests: Genetics, Metabolism, Biology, Diet, Medicine, and 15 moreMetabolic Bone Disease, Humans, Female, Animals, Male, Animal Model, Infant feeding, Clinical Sciences, Breast feeding, Dietary Carbohydrates, Fetus, Metabolic Disorder, Dietary Habits, High Carbohydrate Diet, and Infant nutritional physiological phenomena
Cells from primary islets and beta-cell lines form pseudoislets (PIs) in static cultures. Interestingly, MIN6 beta-cells with aberrant regulation of proliferation form PIs which cease to grow after a week in culture. This growth arrest is... more
Cells from primary islets and beta-cell lines form pseudoislets (PIs) in static cultures. Interestingly, MIN6 beta-cells with aberrant regulation of proliferation form PIs which cease to grow after a week in culture. This growth arrest is attributed to a pro-apoptotic and anti-proliferative PI environment. We hypothesized that cell necrosis due to poor nutrient transport in dishes rather than apoptosis effects the observed PI size restriction. Formation of beta-cell PIs was explored in stirred-suspension bioreactors with enhanced mass transfer. Cells in stirred-suspension proliferated continuously and the PI size increased for two weeks. Bioreactor PIs displayed regulated basal insulin secretion and enhanced responsivity to glucose and incretins. Compared to dishes, cell viability in the bioreactor was higher with lower released lactate dehydrogenase activity. Similar expression of p21 and p27 in monolayers and PIs did not suggest an anti-proliferative PI milieu. Caspase-2, -8 and -9 activities were comparable in dish and bioreactor PIs, and the latter continued to grow after one week of culture. Thus, apoptosis is not sufficient to explain the differences in PI size between dishes and bioreactor. Moreover, the bioreactor method described here may be used to generate PIs with increased cell viability and function for research and clinical applications.
Research Interests:
The Journal of Lipid Research Skip to main page content. ...
Research Interests:
Research Interests:
ABSTRACT
Research Interests: Endocrinology, Computer Science, Biology, Calcium, Medicine, and 15 moreCats, Biological Sciences, Nicotine, Animals, Acetylcholine, Perfusion, Proteins, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Cells, Exocytosis, Adrenocorticotropic Hormone, Cycloheximide, Adrenal Medulla, Adrenal glands, and Medical and Health Sciences
L-Arginine (L-Arg) is metabolized by nitric oxide synthase to the reactive intermediate nitric oxide. Since nitric oxide stimulates guanylyl cyclase and cGMP synthesis, L-Arg effects on cGMP accumulation in isolated pancreatic islets of... more
L-Arginine (L-Arg) is metabolized by nitric oxide synthase to the reactive intermediate nitric oxide. Since nitric oxide stimulates guanylyl cyclase and cGMP synthesis, L-Arg effects on cGMP accumulation in isolated pancreatic islets of the rat and RINm5F insulinoma cells were determined. Both L-Arg and glucose stimulation increased islet cGMP levels, and glucose potentiated the response to L-Arg alone. A competitive inhibitor of L-Arg metabolism to nitric oxide, NG-monomethyl-L-arginine, reduced glucose- and L-Arg-stimulated insulin release and glucose-induced increases in cGMP; however, basal insulin release was slightly increased. D-Arg and L-ornithine did not affect islet cGMP levels, although insulin release was stimulated. RINm5F cell cGMP levels and insulin release increased in response to L-Arg in a concentration- and time-related manner, whereas glucose and L-histidine were without effect. 8-Bromo-cGMP also slightly increased RINm5F cell insulin release. Sodium nitroprusside as a source of nitric oxide increased RINm5F cell cGMP production. Methylene blue and LY83583, inhibitors of soluble guanylyl cyclase activation, reduced RINm5F cell cGMP levels in the presence and absence of L-Arg; LY83583 also reduced glucose-stimulated cGMP levels in islets. Insulin release by glucose and L-Arg was also inhibited by methylene blue and LY83583 in islets. We conclude that glucose and L-Arg stimulate guanylyl cyclase activity and cGMP formation in beta-cells at least in part through metabolism to the reactive intermediate nitric oxide. However, neither nitric oxide nor cGMP synthesis is obligatory for insulin secretion.