Activation of phospholipase C-epsilon by heterotrimeric G protein betagamma-subunits.

PLC-epsilon was identified recently as a phosphoinositide-hydrolyzing phospholipase C (PLC) containing catalytic domains (X, Y, and C2) common to all PLC isozymes as well as unique CDC25- and Ras-associating domains. Novel regulation of this PLC isozyme by the Ras oncoprotein and alpha-subunits (Galpha(12)) of heterotrimeric G proteins was illustrated. Sequence analyses of PLC-epsilon revealed previously unrecognized PH and EF-hand domains in the amino terminus. The known interaction of Gbetagamma subunits with the PH domains of other proteins led us to examine the capacity of Gbetagamma to activate PLC-epsilon. Co-expression of Gbeta(1)gamma(2) with PLC-epsilon in COS-7 cells resulted in marked stimulation of phospholipase C activity. Gbeta(2) and Gbeta(4) in combination with Ggamma(1), Ggamma(2), Ggamma(3), or Ggamma(13) also activated PLC-epsilon to levels similar to those observed with Gbeta(1)-containing dimers of these Ggamma-subunits. Gbeta(3) in combination with the same Ggamma-subunits was less active, and Gbeta(5)-containing dimers were essentially inactive. Gbetagamma-promoted activation of PLC-epsilon was blocked by cotransfection with either of two Gbetagamma-interacting proteins, Galpha(i1) or the carboxyl terminus of G protein receptor kinase 2. Pharmacological inhibition of PI3-kinase-gamma had no effect on Gbeta(1)gamma(2)-promoted activation of PLC-epsilon. Similarly, activation of Ras in the action of Gbetagamma is unlikely, because a mutation in the second RA domain of PLC-epsilon that blocks Ras activation of PLC failed to alter the stimulatory activity of Gbeta(1)gamma(2). Taken together, these results reveal the presence of additional functional domains in PLC-epsilon and add a new level of complexity in the regulation of this novel enzyme by heterotrimeric G proteins.

Fuel and hormone regulation of phospholipase C beta 1 and delta 1 overexpressed in RINm5F pancreatic beta cells.

The mechanism by which glucose and other fuels stimulate phosphoinositide-specific phospholipase C (PLC) in pancreatic islet beta cells is not known. Previous studies have suggested that glucose may couple to PLC beta 1 and PLC delta 1. To determine directly if fuels activate these PLC isozymes, clones stably overexpressing PLC beta 1 or PLC delta 1 were generated in the fuel-sensitive beta cell line RINm5F, and secretagogue regulation of these PLC isoforms was determined. Overexpression of PLC beta 1 or PLC delta 1 significantly increased PLC activity in isolated cell fractions, consistent with overexpression of active PLC isoforms in these clones. In paired experiments, stimulation of inositol phosphate (IP) accumulation by the fuel glyceraldehyde was enhanced in clones overexpressing PLC beta 1, in parallel with the G-protein alpha subunit activator, AlF(4)(-), suggesting a coupling between glyceraldehyde and this PLC isoform. In contrast, overexpression of PLC delta 1 had no effect on glyceraldehyde- or AlF(4)(-)-stimulated IP accumulation. Similarly, IP accumulation stimulated by ionomycin was enhanced in PLC beta 1, but not PLC delta 1 clones, indicating that increases in intracellular free calcium [Ca(2+)](i) can regulate PLC beta 1 but not PLC delta 1 overexpressed in this cell line. Interestingly, [Arg(8)] vasopressin-stimulated, but not carbachol-stimulated, IP accumulation was significantly increased in clones overexpressing either PLC beta 1 or PLC delta 1. These studies illustrate unique pathways coupling diverse secretagogues to specific PLC isoforms in islet beta cells, and demonstrate that glyceraldehyde can activate PLC beta 1 but not PLC delta 1; whereas, vasopressin, but not carbachol, can stimulate either isoform.

Phospholipase C(epsilon): a novel Ras effector.

Three classes of mammalian phosphoinositide-specific phospholipase C (PLC) have been characterized, PLCbeta, PLCgamma and PLCdelta, that are differentially regulated by heterotrimeric G-proteins, tyrosine kinases and calcium. Here we describe a fourth class, PLCepsilon, that in addition to conserved PLC domains, contains a GTP exchange factor (GRF CDC25) domain and two C-terminal Ras-binding (RA) domains, RA1 and RA2. The RA2 domain binds H-Ras in a GTP-dependent manner, comparable with the Ras-binding domain of Raf-1; however, the RA1 domain binds H-Ras with a low affinity in a GTP-independent manner. While G(alpha)q, Gbetagamma or, surprisingly, H-Ras do not activate recombinant purified protein in vitro, constitutively active Q61L H-Ras stimulates PLC(epsilon) co-expressed in COS-7 cells in parallel with Ras binding. Deletion of either the RA1 or RA2 domain inhibits this activation. Site-directed mutagenesis of the RA2 domain or Ras demonstrates a conserved Ras-effector interaction and a unique profile of activation by Ras effector domain mutants. These studies identify a novel fourth class of mammalian PLC that is directly regulated by Ras and links two critical signaling pathways.

Cadmium disrupts the signal transduction pathway of both inhibitory and stimulatory receptors regulating chloride secretion in the shark rectal gland.

The heavy metal cadmium causes nephrotoxicity and alters the transport function of epithelial cells. In the shark rectal gland, chloride secretion is regulated by secretagogues and inhibitors acting through receptors coupled to G proteins and the cyclic AMP-protein kinase A pathway. We examined the effects of cadmium on the response to the inhibitory peptide somatostatin (SRIF), and to the stimulatory secretagogues forskolin and vasoactive intestinal peptide (VIP). In control experiments, SRIF (100 nM) entirely inhibited the chloride secretory response to 10 microM forskolin (maximum chloride secretion with forskolin 1984 +/- 176 microEq/h/g; with forskolin + SRIF 466 +/- 93 microEq/h/g, P < 0.001). Cadmium (25 microM) entirely reversed the inhibitory response to SRIF (chloride secretion 2143 +/- 222 microEq/h/g) and caused an overshoot (2917 +/- 293 microEq/h/g) that exceeded the response to forskolin (P < 0.01). Cadmium also enhanced forskolin-stimulated chloride secretion (2628 +/- 418 vs. 1673 +/- 340 microEq/h/g, P < 0.02) and reversed the declining phase of the forskolin response. Cadmium had a concentration-dependent, biphasic effect on the response to VIP. Cd (10-100 microM) increased both chloride secretion and tissue cyclic AMP content, whereas higher concentrations (1 mM) inhibited chloride secretion and cyclic AMP accumulation. Our findings provide evidence that Cd disrupts the signal transduction pathways of both inhibitory receptors and secretagogues regulating cAMP mediated transport in an intact epithelia. The results are consistent with direct effects of cadmium on adenylate cyclase and/or phosphodiesterase activity in this marine epithelial model.

Time-dependent effects of cholinergic stimulation on beta cell responsiveness.

The effects of cholinergic stimulation on beta cell insulin secretory and phosphoinositide (PI) responses were determined in freshly isolated rat islets. Increasing the glucose level perifusing the islet from 5.6 to 8mM was accompanied by a modest insulin secretory response. The further addition of 10 microM carbachol increased peak first- and second-phase responses by 2.6- and 6. 8-fold, respectively. In the presence of 5.6 mM glucose, this low level (10 microM) of carbachol increased insulin release two- to three-fold, a response that was maintained for at least 60 min. In contrast to these acute stimulatory actions in the presence of glucose, chronic 3.5-h exposure of islets to 10 microM carbachol abolished beta cell insulin secretory responses to stimulation, with the combination of 8 mM glucose plus 10 microM carbachol. However, the further addition of 200 microM tolbutamide to these islets increased insulin secretory rates significantly. To establish the role of islet cell PI hydrolysis in these secretory responses, additional studies were conducted with islets whose PI pools were labeled with [3H]inositol. Acute exposure to 10 microM carbachol alone significantly increased inositol phosphate accumulation and the efflux of [3H]inositol, even in the absence of glucose. Including 10 microM carbachol during the labeling period with [3H]inositol resulted in significant impairments in subsequently measured inositol phosphate accumulation and [3H]inositol efflux responses to 8 mM glucose plus carbachol stimulation. Prior long-term exposure to 10 microM carbachol also induced heterologous desensitization: 20 mM glucose-stimulated insulin release and inositol phosphate accumulation were impaired in a parallel fashion. Chronic carbachol exposure had no deleterious effect on the usage of 8 or 20 mM glucose or on the insulin content of the islet. The acute stimulatory effects of carbachol on inositol phosphate accumulation as well as its inhibitory effect on 20 mM glucose-stimulated insulin release after prolonged exposure to the muscarinic agonist were significantly reduced by atropine. These findings demonstrate that changes in PI hydrolysis parallel those observed with insulin secretion and suggest that alterations in phospholipase C activation may account, at least in part, for the insulin secretory responses observed.

Regulation of insulin release by phospholipase C activation in mouse islets: differential effects of glucose and neurohumoral stimulation.

Rat islets respond to glucose stimulation with a marked first and second phase increase in insulin secretion. In contrast, mouse islets have a similar first phase response but little second phase secretion. In these studies, we determined if activation of phospholipase C (PLC) accounts for these differences in second phase insulin secretion in these two species. Stimulation of freshly isolated mouse and rat islets with 15 mM glucose resulted in comparable first phase insulin secretion; however, the second phase response from mouse islets was only doubled from 28 +/- 6 to 60 +/- 7 pg/islet.min compared with an increase from 24 +/- 4 to 1064 +/- 93 pg/islet.min from rat islets. The addition of the muscarinic agonist carbachol (100 microM) in the presence of 15 mM glucose, however, markedly increased second phase insulin release from mouse islets to 801 +/- 80 pg/islet.min. Similar increases in second phase insulin release from mouse islets were obtained with the addition of 500 nM of the protein kinase C activator tetradecanoyl phorbol acetate in the presence of 15 mM glucose. However, the incretin factor glucagon-like peptide-1, which elevates islet cAMP levels, had little effect on second phase insulin release in the mouse. An analysis of PLC-mediated phosphoinositide (PI) hydrolysis revealed that 15 mM glucose increased inositol phosphate (IP) accumulation 0.5-fold above baseline in mouse islets compared with 3.7-fold in rat islets. In contrast, carbachol stimulated IP accumulation 3.5-fold in both mouse and rat islets. Analysis of PLC isozymes with isozyme specific monoclonal antibodies, demonstrated that mouse islets express 14 +/- 4% of PLC-delta 1 and 18 +/- 6% of PLC-beta 1 compared with rat islets but similar amounts of the PLC-gamma 1 (117 +/- 16%). These findings suggest that the decreased second phase insulin secretory response in mouse compared with rat islets results, at least in part, from an inability of high glucose to stimulate comparable increments in PI hydrolysis. This lack of glucose responsiveness may be due to the pronounced underexpression of specific PLC isozymes in the mouse.

Effects of short-term culturing on islet phosphoinositide and insulin secretory responses to glucose and carbachol.

The ability of glucose and carbachol, alone or in combination, to stimulate islet cell phosphoinositide (PI) hydrolysis and insulin secretory responses in freshly isolated or in 20-24 h cultured rat islets was assessed. In freshly isolated, 3H-inositol-prelabeled islets, 20 mM glucose alone or 1 mM carbachol alone stimulated significant increments in 3H-inositol efflux and inositol phosphate (IP) accumulation. When stimulated with both agonists, a dramatic and synergistic effect on IP accumulation was noted. Carbachol (1 mM) alone had no sustained stimulatory effect on insulin secretion. Glucose (20 mM) alone induced a biphasic insulin secretory response. When compared to prestimulatory secretory rates of 18 +/- 4 pg/islet/min, peak first and second phase responses now averaged 422 +/- 61 and 1016 +/- 156 pg/islet/min, respectively. In contrast to freshly studied islets, culturing islets for 20-24 h in CMRL-1066 medium attenuated all measured responses. The increases in 3H-inositol efflux rates in response to glucose, carbachol, or their combination were significantly less than those observed with fresh islets. The IP responses were also attenuated. Second phase insulin secretory responses to 20 mM glucose alone 68 +/- 9 pg/islet/min) or the combination of 20 mM glucose plus 1 mM carbachol (358 +/- 85 pg/islet/min) were also significantly decreased when compared with fresh islets. We conclude from these studies that the process of culturing islets for one day in CMRL-1066 significantly decreases islet cell PI hydrolysis and insulin secretory responsiveness. These observations may help to explain the discordant conclusions reached concerning the involvement of PI hydrolysis and protein kinase C activation in the regulation of insulin release from freshly isolated versus cultured islets.

Synergistic interaction of glucose and neurohumoral agonists to stimulate islet phosphoinositide hydrolysis.

The interaction between neurohumoral agonists and glucose to stimulate phosphoinositide (PI)-specific phospholipase C (PLC) and insulin release was examined. In freshly isolated rat islets, maximal glucose (40 mM), cholecystokinin (CCK; 300 nM), or carbachol (CCh; 1 mM) stimulated PI hydrolysis 6.5-, 9.8-, and 5.7-fold, respectively, above basal. The combination of glucose and CCK or of glucose and CCh, but not of CCK and CCh, synergistically increased PI hydrolysis 23.2- and 21.6-fold, respectively, indicating that these secretagogues activate PLC by distinct pathways and that there is an interaction between them. This synergy was maximal at physiological concentrations of stimulatory glucose (8-10 mM) and was paralleled by a marked synergistic stimulation of insulin secretion. The enhanced PI response was partially Ca2+ dependent and may involve the activation of distinct isozymes of PLC, which we identify in islets. These studies demonstrate for the first time a unique and highly sensitive synergistic interaction between glucose and neurohumoral agonists to stimulate PI hydrolysis, and they suggest that enhanced PI hydrolysis is important in the potentiation of glucose- and neurohumoral-stimulated insulin secretion.

Islet phosphoinositide hydrolysis and insulin secretory responses from prediabetic fa/fa ZDF rats.

The sequence of events that culminate in the development of diabetes in the fa/fa Zucker diabetic fatty (ZDF) rat is unclear. In the present series of experiments islets from 5 week old prediabetic fa/fa male rats were isolated and their phosphoinositide (PI) hydrolysis and insulin secretory responses compared to those obtained from lean nondiabetic age- and weight-matched control rats. Peak first and second phase insulin secretory responses to 20mM glucose averaged 77 +/- 10 (mean +/- SE, n = 7) and 491 +/- 47 pg/islet/min from lean, nondiabetic control islets. The comparable responses from fa/fa prediabetic rat islets were significantly greater, 264 +/- 51 and 810 +/- 78 pg/islet/min. In a parallel fashion 3H-inositol efflux and inositol phosphate responses from prediabetic rat islets were also greater than comparable control responses. These findings demonstrate that significant increases in the phospholipase C-mediated hydrolysis of islet PI pools and insulin release in response to hyperglycemic stimulation can be detected prior to the emergence of diabetes in the fa/fa ZDF rat. These early changes in beta cell responsiveness to glucose may contribute to the hyperinsulinemia and subsequent insulin resistance characteristic of this animal model of non-insulin dependent diabetes mellitus.

Calcium and a mitochondrial signal interact to stimulate phosphoinositide hydrolysis and insulin secretion in rat islets.

Fuel metabolism generates multiple signals that interact to stimulate insulin secretion. These studies explored the mechanism by which fuels activate phosphoinositide (PI) hydrolysis and the role of this signal transduction pathway in fuel-stimulated insulin secretion. High potassium (30 mM), which depolarizes the membrane and increases Ca2+ influx, caused only a transient monophasic release of insulin. In contrast, glucose (20 mM) or monomethylsuccinate (MMSucc; 10 mM) markedly stimulated a sustained insulin secretory response, indicating that fuel metabolism generates a signal(s) in addition to Ca2+ influx that is required for a sustained secretory response. On the other hand, diazoxide, an ATP-sensitive K+ channel activator that prevents membrane depolarization and Ca2+ influx in response to fuel metabolism, reduced the secretory responses to glucose and MMSucc to baseline levels, demonstrating that Ca2+ influx was essential to fuel-stimulated insulin secretion. The further addition of high K+ bypassed the diazoxide block and restored insulin secretory rates. The insulin secretory response to glucose or MMSucc in the presence of diazoxide and K+ was inhibited by the Ca2+ channel antagonist nitrendipine and the protein kinase-C inhibitor staurosporine. Changes in PI hydrolysis paralleled those in insulin secretion. High potassium alone induced only a modest 2.5-fold increase in inositol phosphate accumulation. This response was significantly less than that to glucose or MMSucc, which increased inositol phosphate accumulation by 6.8- or 5.2-fold, respectively. Like its effect on secretion, diazoxide markedly reduced glucose- or MMSucc-stimulated PI hydrolysis, and this inhibition was reversed with high K+. In contrast, diazoxide had no effect on receptor-activated PI hydrolysis stimulated by 100 nM cholecystokinin (CCK), and the effects of CCK were not dependent on added fuel, indicating that fuel and CCK activate PI hydrolysis by distinct pathways. These findings demonstrate that mitochondrial metabolism of glucose or MMSucc generates a signal(s) that interacts with Ca2+ influx to stimulate PI hydrolysis and sustained insulin secretion. This pathway of fuel-activated PI hydrolysis is distinct from that of CCK receptor-activated PI hydrolysis. These studies suggest that fuel-activated PI hydrolysis plays an important role in fuel-stimulated insulin secretion.

Endogenous adenosine is an autacoid feedback inhibitor of chloride transport in the shark rectal gland.

The present studies define the physiologic role of endogenous adenosine in the perfused shark rectal gland, a model epithelia for hormone-stimulated chloride transport. Chloride ion secretion, and venous adenosine and inosine concentrations increased in parallel in response to hormone stimulation. From a basal rate of 157 +/- 26 mu eq/h per g, chloride secretion increased to 836 +/- 96 and 2170 +/- 358 with 1 and 10 microM forskolin, venous adenosine increased from 5.0 +/- 1 to 126 +/- 29 and 896 +/- 181 nM, and inosine increased from 30 +/- 9 to 349 +/- 77 and 1719 +/- 454 nM (all P less than 0.01). Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, completely blocked the release of adenosine and inosine. Inhibition of chloride transport with bumetanide, an inhibitor of the Na+/K+/2Cl- cotransporter, or ouabain, an inhibitor of Na+/K+ ATPase activity, reduced venous adenosine and inosine to basal values. When the interaction of endogenous adenosine with extracellular receptors was prevented by adenosine deaminase, NBTI, or 8-phenyltheophylline, the chloride transport response to secretagogues increased by 1.7-2.3-fold. These studies demonstrate that endogenous adenosine is released in response to hormone-stimulated cellular work and acts at A1 adenosine receptors as a feedback inhibitor of chloride transport.

A1 adenosine receptors inhibit chloride transport in the shark rectal gland. Dissociation of inhibition and cyclic AMP.

In the in vitro perfused rectal gland of the dogfish shark (Squalus acanthias), the adenosine analogue 2-chloroadenosine (2Clado) completely and reversibly inhibited forskolin-stimulated chloride secretion with an IC50 of 5 nM. Other A1 receptor agonists including cyclohexyladenosine (CHA), N-ethylcarboxamideadenosine (NECA) and R-phenylisopropyl-adenosine (R-PIA) also completely inhibited forskolin stimulated chloride secretion. The "S" stereoisomer of PIA (S-PIA) was a less potent inhibitor of forskolin stimulated chloride secretion, consistent with the affinity profile of PIA stereoisomers for an A1 receptor. The adenosine receptor antagonists 8-phenyltheophylline and 8-cyclopentyltheophylline completely blocked the effect of 2Clado to inhibit forskolin-stimulated chloride secretion. When chloride secretion and tissue cyclic (c)AMP content were determined simultaneously in perfused glands, 2Clado completely inhibited secretion but only inhibited forskolin stimulated cAMP accumulation by 34-40%, indicating that the mechanism of inhibition of secretion by 2Clado is at least partially cAMP independent. Consistent with these results, A1 receptor agonists only modestly inhibited (9-15%) forskolin stimulated adenylate cyclase activity and 2Clado markedly inhibited chloride secretion stimulated by a permeant cAMP analogue, 8-chlorophenylthio cAMP (8CPT cAMP). These findings provide the first evidence for a high affinity A1 adenosine receptor that inhibits hormone stimulated ion transport in a model epithelia. A major portion of this inhibition occurs by a mechanism that is independent of the cAMP messenger system.