Epac2-dependent mobilization of intracellular Ca²+ by glucagon-like peptide-1 receptor agonist exendin-4 is disrupted in ß-cells of phospholipase C-ε knockout mice.

Calcium can be mobilized in pancreatic ß-cells via a mechanism of Ca(2+)-induced Ca(2+) release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR in ß-cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide-specific phospholipase C- (PLC-) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC- gene expression in mice disrupts the action of exendin-4 to facilitate CICR in the ß-cells of these mice. Thus, in the present study, in which wild-type (WT) C57BL/6 mouse ß-cells were loaded with the photolabile Ca(2+) chelator NP-EGTA, the UV flash photolysis-catalysed uncaging of Ca(2+) generated CICR in only 9% of the ß-cells tested, whereas CICR was generated in 82% of the ß-cells pretreated with exendin-4. This action of exendin-4 to facilitate CICR was reproduced by cAMP analogues that activate protein kinase A (6-Bnz-cAMP-AM) or Epac2 (8-pCPT-2'-O-Me-cAMP-AM) selectively. However, in ß-cells of PLC- KO mice, and also Epac2 KO mice, these test substances exhibited differential efficacies in the CICR assay such that exendin-4 was partly effective, 6-Bnz-cAMP-AM was fully effective, and 8-pCPT-2'-O-Me-cAMP-AM was without significant effect. Importantly, transduction of PLC- KO ß-cells with recombinant PLC- rescued the action of 8-pCPT-2'-O-Me-cAMP-AM to facilitate CICR, whereas a K2150E PLC- with a mutated Ras association (RA) domain, or a H1640L PLC- that is catalytically dead, were both ineffective. Since 8-pCPT-2'-O-Me-cAMP-AM failed to facilitate CICR in WT ß-cells transduced with a GTPase activating protein (RapGAP) that downregulates Rap activity, the available evidence indicates that a signal transduction 'module' comprised of Epac2, Rap and PLC- exists in ß-cells, and that the activities of Epac2 and PLC- are key determinants of CICR in this cell type.

PKA-dependent potentiation of glucose-stimulated insulin secretion by Epac activator 8-pCPT-2'-O-Me-cAMP-AM in human islets of Langerhans.

Potential insulin secretagogue properties of an acetoxymethyl ester of a cAMP analog (8-pCPT-2'-O-Me-cAMP-AM) that activates the guanine nucleotide exchange factors Epac1 and Epac2 were assessed using isolated human islets of Langerhans. RT-QPCR demonstrated that the predominant variant of Epac expressed in human islets was Epac2, although Epac1 was detectable. Under conditions of islet perifusion, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) potentiated first- and second-phase 10 mM glucose-stimulated insulin secretion (GSIS) while failing to influence insulin secretion measured in the presence of 3 mM glucose. The insulin secretagogue action of 8-pCPT-2'-O-Me-cAMP-AM was associated with depolarization and an increase of [Ca(2+)](i) that reflected both Ca(2+) influx and intracellular Ca(2+) mobilization in islet beta-cells. As expected for an Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) failed to stimulate phosphorylation of PKA substrates CREB and Kemptide in human islets. Furthermore, 8-pCPT-2'-O-Me-cAMP-AM (10 microM) had no significant ability to activate AKAR3, a PKA-regulated biosensor expressed in human islet cells by viral transduction. Unexpectedly, treatment of human islets with an inhibitor of PKA activity (H-89) or treatment with a cAMP antagonist that blocks PKA activation (Rp-8-CPT-cAMPS) nearly abolished the action of 8-pCPT-2'-O-Me-cAMP-AM to potentiate GSIS. It is concluded that there exists a permissive role for PKA activity in support of human islet insulin secretion that is both glucose dependent and Epac regulated. This permissive action of PKA may be operative at the insulin secretory granule recruitment, priming, and/or postpriming steps of Ca(2+)-dependent exocytosis.

Phospholipase C-ε links Epac2 activation to the potentiation of glucose-stimulated insulin secretion from mouse islets of Langerhans.

Glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells is potentiated by cAMP-elevating agents, such as the incretin hormone glucagon-like peptide-1 (GLP-1), and cAMP exerts its insulin secretagogue action by activating both protein kinase A (PKA) and the cAMP-regulated guanine nucleotide exchange factor designated as Epac2. Although prior studies of mouse islets demonstrated that Epac2 acts via Rap1 GTPase to potentiate GSIS, it is not understood which downstream targets of Rap1 promote the exocytosis of insulin. Here, we measured insulin secretion stimulated by a cAMP analog that is a selective activator of Epac proteins in order to demonstrate that a Rap1-regulated phospholipase C-epsilon (PLC-ε) links Epac2 activation to the potentiation of GSIS. Our analysis demonstrates that the Epac activator 8-pCPT-2'-O-Me-cAMP-AM potentiates GSIS from the islets of wild-type (WT) mice, whereas it has a greatly reduced insulin secretagogue action in the islets of Epac2 (-/-) and PLC-ε (-/-) knockout (KO) mice. Importantly, the insulin secretagogue action of 8-pCPT-2'-O-Me-cAMP-AM in WT mouse islets cannot be explained by an unexpected action of this cAMP analog to activate PKA, as verified through the use of a FRET-based A-kinase activity reporter (AKAR3) that reports PKA activation. Since the KO of PLC-ε disrupts the ability of 8-pCPT-2'-O-Me-cAMP-AM to potentiate GSIS, while also disrupting its ability to stimulate an increase of ß-cell [Ca2+]i, the available evidence indicates that it is a Rap1-regulated PLC-ε that links Epac2 activation to Ca2+-dependent exocytosis of insulin.

Enhanced Rap1 activation and insulin secretagogue properties of an acetoxymethyl ester of an Epac-selective cyclic AMP analog in rat INS-1 cells: studies with 8-pCPT-2'-O-Me-cAMP-AM.

To ascertain the identities of cyclic nucleotide-binding proteins that mediate the insulin secretagogue action of cAMP, the possible contributions of the exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) were evaluated in a pancreatic beta cell line (rat INS-1 cells). Assays of Rap1 activation, CREB phosphorylation, and PKA-dependent gene expression were performed in combination with live cell imaging and high throughput screening of a fluorescence resonance energy transfer-based cAMP sensor (Epac1-camps) to validate the selectivity with which acetoxymethyl esters (AM-esters) of cAMP analogs preferentially activate Epac or PKA. Selective activation of Epac or PKA was achieved following exposure of INS-1 cells to 8-pCPT-2'-O-Me-cAMP-AM or Bt(2)cAMP-AM, respectively. Both cAMP analogs exerted dose-dependent and glucose metabolism-dependent actions to stimulate insulin secretion, and when each was co-administered with the other, a supra-additive effect was observed. Because 2.4-fold more insulin was secreted in response to a saturating concentration (10 microm) of Bt(2)cAMP-AM as compared with 8-pCPT-2'-O-Me-cAMP-AM, and because the action of Bt(2)cAMP-AM but not 8-pCPT-2'-O-Me-cAMP-AM was nearly abrogated by treatment with 3 microm of the PKA inhibitor H-89, it is concluded that for INS-1 cells, it is PKA that acts as the dominant cAMP-binding protein in support of insulin secretion. Unexpectedly, 10-100 microm of the non-AM-ester of 8-pCPT-2'-O-Me-cAMP failed to stimulate insulin secretion and was a weak activator of Rap1 in INS-1 cells. Moreover, 10 microm of the AM-ester of 8-pCPT-2'-O-Me-cAMP stimulated insulin secretion from mouse islets, whereas the non-AM-ester did not. Thus, the membrane permeability of 8-pCPT-2'-O-Me-cAMP in insulin-secreting cells is so low as to limit its biological activity. It is concluded that prior reports documenting the failure of 8-pCPT-2'-O-Me-cAMP to act in beta cells, or other cell types, need to be re-evaluated through the use of the AM-ester of this cAMP analog.

Glucose-dependent potentiation of mouse islet insulin secretion by Epac activator 8-pCPT-2'-O-Me-cAMP-AM.

Epac2 is a cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF) that is proposed to mediate stimulatory actions of the second messenger cAMP on mouse islet insulin secretion. Here we have used methods of islet perifusion to demonstrate that the acetoxymethyl ester (AM-ester) of an Epac-selective cAMP analog (ESCA) penetrates into mouse islets and is capable of potentiating both first and second phases of glucose-stimulated insulin secretion (GSIS). When used at low concentrations (1-10 µM), 8-pCPT-2'-O-Me-cAMP-AM activates Rap1 GTPase but exhibits little or no ability to activate protein kinase A (PKA), as validated in assays of in vitro PKA activity (phosphorylation of Kemptide), Ser (133) CREB phosphorylation status, RIP1-CRE-Luc reporter gene activity, and PKA-dependent AKAR3 biosensor activation. Since quantitative PCR demonstrates Epac2 mRNA to be expressed at levels ca. 5.3-fold greater than that of Epac1, available evidence indicates that Epac2 does in fact mediate stimulatory actions of cAMP on mouse islet GSIS.

Linopirdine modulates calcium signaling and stimulus-secretion coupling in adrenal chromaffin cells by targeting M-type K+ channels and nicotinic acetylcholine receptors.

Adrenal chromaffin cells synthesize and release catecholamines and several other transmitters that play important physiological roles in the coordinated response to stress or danger. The main trigger for secretion is acetylcholine (ACh) released from splanchnic nerve terminals that activates nicotinic ACh receptors (nAChRs) on the chromaffin cells, causing membrane depolarization and Ca2+ entry primarily through voltage-gated Ca2+ channels (Ca-channels). G protein-coupled receptors (GPCRs) can also trigger secretion, and it has been suggested that closure of M-type K+ channels might contribute to this process. However, GPCRs have multiple effects on calcium signaling and secretion, including release of intracellular Ca2+ stores, activation of second messenger pathways and kinases, and Ca2+ entry through store/receptor-operated channels. Hence, the effects of M-channel closure on [Ca2+]i signaling and transmitter release remain unclear. We have investigated the effects of linopirdine, a relatively selective blocker of M-channels, on stimulus-secretion coupling in chromaffin cells. Linopirdine produced a small increase in [Ca2+]i in approximately 63% of cells because of influx through Ca-channels. However, this was not sufficient to promote catecholamine release. We also show that linopirdine reduced cholinergic-stimulated increases in [Ca2+]i and secretion, primarily through potent block of nAChRs and a subtle effect on Ca2+ entry via Ca-channels. Hence, our data support the hypothesis that M-channels help control the excitability of chromaffin cells, but additional pathways need to be recruited by GPCRs to trigger catecholamine release. Furthermore, linopirdine potently targets nAChRs to modulate stimulus-secretion coupling in adrenal chromaffin cells.

Multilocus analysis of hypertension: a hierarchical approach.

While hypertension is a complex disease with a well-documented genetic component, genetic studies often fail to replicate findings. One possibility for such inconsistency is that the underlying genetics of hypertension is not based on single genes of major effect, but on interactions among genes. To test this hypothesis, we studied both single locus and multilocus effects, using a case-control design of subjects from Ghana. Thirteen polymorphisms in eight candidate genes were studied. Each candidate gene has been shown to play a physiological role in blood pressure regulation and affects one of four pathways that modulate blood pressure: vasoconstriction (angiotensinogen, angiotensin converting enzyme - ACE, angiotensin II receptor), nitric oxide (NO) dependent and NO independent vasodilation pathways and sodium balance (G protein-coupled receptor kinase, GRK4). We evaluated single site allelic and genotypic associations, multilocus genotype equilibrium and multilocus genotype associations, using multifactor dimensionality reduction (MDR). For MDR, we performed systematic reanalysis of the data to address the role of various physiological pathways. We found no significant single site associations, but the hypertensive class deviated significantly from genotype equilibrium in more than 25% of all multilocus comparisons (2,162 of 8,178), whereas the normotensive class rarely did (11 of 8,178). The MDR analysis identified a two-locus model including ACE and GRK4 that successfully predicted blood pressure phenotype 70.5% of the time. Thus, our data indicate epistatic interactions play a major role in hypertension susceptibility. Our data also support a model where multiple pathways need to be affected in order to predispose to hypertension.