New insights concerning the glucose-dependent insulin secretagogue action of glucagon-like peptide-1 in pancreatic beta-cells.

The GLP-1 receptor is a Class B heptahelical G-protein-coupled receptor that stimulates cAMP production in pancreatic beta-cells. GLP-1 utilizes this receptor to activate two distinct classes of cAMP-binding proteins: protein kinase A (PKA) and the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs). Actions of GLP-1 mediated by PKA and Epac include the recruitment and priming of secretory granules, thereby increasing the number of granules available for Ca(2+)-dependent exocytosis. Simultaneously, GLP-1 promotes Ca(2+) influx and mobilizes an intracellular source of Ca(2+). GLP-1 sensitizes intracellular Ca(2+) release channels (ryanodine and IP (3) receptors) to stimulatory effects of Ca(2+), thereby promoting Ca(2+)-induced Ca(2+) release (CICR). In the model presented here, CICR activates mitochondrial dehydrogenases, thereby upregulating glucose-dependent production of ATP. The resultant increase in cytosolic [ATP]/[ADP] concentration ratio leads to closure of ATP-sensitive K(+) channels (K-ATP), membrane depolarization, and influx of Ca(2+) through voltage-dependent Ca(2+) channels (VDCCs). Ca(2+) influx stimulates exocytosis of secretory granules by promoting their fusion with the plasma membrane. Under conditions where Ca(2+) release channels are sensitized by GLP-1, Ca(2+) influx also stimulates CICR, generating an additional round of ATP production and K-ATP channel closure. In the absence of glucose, no "fuel" is available to support ATP production, and GLP-1 fails to stimulate insulin secretion. This new "feed-forward" hypothesis of beta-cell stimulus-secretion coupling may provide a mechanistic explanation as to how GLP-1 exerts a beneficial blood glucose-lowering effect in type 2 diabetic subjects.

Syntaxin-3 and syntaxin-1A inhibit L-type calcium channel activity, insulin biosynthesis and exocytosis in beta-cell lines.

AIMS/HYPOTHESIS: Syntaxin-1A (Syn-1A) is known to play a negative regulatory role in insulin secretion but the precise mechanisms for its action are not clear. Syn-2, -3 and -4 are also present in islet beta cells but their functions are not known. Here, we investigated the role of these syntaxins in the insulin secretory process. METHODS: We examined the following effects of Syn-1, -2, -3 and -4 expression in insulinoma beta-cell lines. Endogenous insulin secretion was measured by batch radioimmunoassay (RIA) and single cell patch clamp capacitance measurements. The L-type Ca(2+) channel activity was studied by patch clamp electrophysiology. Insulin gene transcription was examined by Northern blotting and measurement of insulin gene promoter activity by the co-expression of cyan fluorescent protein-labelled rat insulin promoter. RESULTS: Syn-1A or -3, but not Syn-2 or -4 overexpression, inhibited K(+)-induced insulin release as determined by RIA (49.7 +/- 5.5 % and 49.1 +/- 6.2 %, respectively) and electrophysiologic membrane capacitance measurements (68.0 +/- 21.0 % and 58.0 +/- 13.2 %, respectively). Overexpressed Syn-1A and -3, but not Syn-2, inhibited Ca(2+) channel current amplitude by 39.5 +/- 11.6 % and 52.7 +/- 6.0 %, respectively. Of note, overexpression of Syn-1A and -3 also reduced single cell (by confocal microscopy) and total cellular endogenous insulin content (by RIA) by 24.8 +/- 4.2 % and 31.8 +/- 3.9 %, respectively. This correlated to a reduction in endogenous insulin mRNA by 24.5 +/- 4.2 % and 25.7 +/- 4.2 %, respectively. This inhibition of insulin biosynthesis is mainly at the level of insulin gene transcription as demonstrated by an inhibition of insulin gene promoter activity (53.3 +/- 9.15 % and 39.0 +/- 6.8 %, respectively). CONCLUSIONS/INTERPRETATION: These results demonstrate that Syn-1A and -3 possess strong inhibitory actions on both insulin exocytosis and insulin biosynthesis whereas Syn-2 and -4 do not inhibit the insulin secretory process.

cAMP-regulated guanine nucleotide exchange factor II (Epac2) mediates Ca2+-induced Ca2+ release in INS-1 pancreatic beta-cells.

1. The signal transduction pathway responsible for cAMP-dependent Ca2+-induced Ca2+ release (CICR) from endoplasmic reticulum Ca2+ stores was assessed in the insulin-secreting cell line INS-1. 2. CICR was triggered by the GLP-1 receptor agonist exendin-4, an effect mimicked by caffeine, Sp-cAMPS or forskolin. CICR required influx of Ca2+ through L-type voltage-dependent Ca2+ channels, and was blocked by treatment with nimodipine, thapsigargin, or ryanodine, but not by the IP3 receptor antagonist xestospongin C. 3. Treatment with the cAMP antagonist 8-Br-Rp-cAMPS blocked CICR in response to exendin-4, whereas the PKA inhibitor H-89 was ineffective when tested at a concentration demonstrated to inhibit PKA-dependent gene expression. 4. RT-PCR of INS-1 cells demonstrated expression of mRNA coding for the type-II isoform of cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF-II, Epac2). 5. CICR in response to forskolin was blocked by transient transfection and expression of a dominant negative mutant isoform of cAMP-GEF-II in which inactivating mutations were introduced into the exchange factor's two cAMP-binding domains. 6. It is concluded that CICR in INS-1 cells results from GLP-1 receptor-mediated sensitization of the intracellular Ca2+ release mechanism, a signal transduction pathway independent of PKA, but which requires cAMP-GEF-II.

Germination and Establishment of Infection on Grape Berries by Single Airborne Conidia of Botrytis cinerea.

Table grapes (cv. Dauphine) at different phenological stages were dusted in a settling tower with dry conidia of Botrytis cinerea. The berries were incubated for periods of 3 to 96 h at high relative humidity (RH; ±93% RH, moist berries), or were covered with a film of water (wet berries). Germination of the solitary conidia, appressorium formation, stilbene and suberin induction by germlings, and germling viability were examined by fluorescence microscopy after each incubation period. Isolation and freezing studies were conducted to determine surface colonization (berries left unsterile) and penetration (surface-disinfested berries). Symptoms were determined on berries incubated at a specific wetness regime, kept dry for 10 days, and then incubated for 4 days at high RH. Microscopic observations indicated that germination was delayed on immature berries, but proceeded at a high rate on mature berries. Growth was invariably restricted on moist berries. Attempted penetration was always direct. Stilbene and suberin were generally induced early and were intense on berries at the pea-size and bunch closure stages. Dieback of conidia and germlings occurred at a significantly higher rate on wet than moist berries, and was more pronounced on immature than on mature berries. The segment isolation and freezing studies showed that infections in grape berry cheeks established by this infection mode were few. Extended incubation periods did not lead to substantially higher rates of surface colonization and skin penetration. Disease symptoms did not develop during the 14-day period on the berries transferred to dry perspex chambers, irrespective of phenological stage, incubation period, or wetness regime. According to these findings, this mode of infection should not contribute to a gradual build-up of secondary inoculum in the vineyard, and to B. cinerea epiphytotics.

Expression of cAMP-regulated guanine nucleotide exchange factors in pancreatic beta-cells.

The insulinotropic hormone glucagon-like peptide-1 (GLP-1) binds to a Gs-coupled receptor on pancreatic beta-cells and potentiates glucose-induced insulin secretion, insulin gene transcription, and beta-cell growth. These stimulatory effects have been attributed to the elevation of intracellular cAMP levels, though it is now apparent that some stimulatory effects of GLP-1 occur independently of the cAMP-mediated activation of protein kinase A (PKA). The nature of this alternative, PKA-independent signaling pathway remains unknown. Here we present evidence for the expression of type 1 and type 2 cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs) in beta-cells. GEFs are activated by their binding of cAMP. Because cAMP-GEFs activate Ras/MAPK proliferation signaling pathways, they may play an important role in PKA-independent, GLP-1-mediated, signaling pathways in the regulation of beta-cell growth and differentiation.

Insulinotropic toxins as molecular probes for analysis of glucagon-likepeptide-1 receptor-mediated signal transduction in pancreatic beta-cells.

Cholera toxin, pertussis toxin, mastoparan, maitotoxin, and alpha-latrotoxin are complex protein or polyether-based toxins of bacterial, insect, or phytoplankton origin that act with high potency at the endocrine pancreas to stimulate secretion of insulin from beta-cells located in the islets of Langerhans. The remarkable insulinotropic properties of these toxins have attracted considerable attention by virtue of their use as selective molecular probes for analyses of beta-cell stimulus-secretion coupling. Targets of the toxins include heptahelical cell surface receptors, GTP-binding proteins, ion channels, Ca(2+) stores, and the exocytotic secretory apparatus. Here we review the value of insulinotropic toxins from the perspective of their established use in the study of signal transduction pathways activated by the blood glucose-lowering hormone glucagon-like peptide-1 (GLP-1). Our analysis of one insulinotropic toxin (alpha-latrotoxin) leads us to conclude that there exists a process of molecular mimicry whereby the 'lock and key'analogy inherent to hormone-receptor interactions is reproduced by a toxin related in structure to GLP-1.

Glucagon-like peptide 1 stimulates insulin gene promoter activity by protein kinase A-independent activation of the rat insulin I gene cAMP response element.

Glucagon-like peptide 1 (GLP-1), a hormonal activator of adenyl cyclase, stimulates insulin gene transcription, an effect mediated by the cAMP response element (CRE) of the rat insulin I gene promoter (RIP1). Here we demonstrate that the signaling mechanism underlying stimulatory effects of GLP-1 on insulin gene transcription results from protein kinase A (PKA)-independent activation of the RIP1 CRE. Although GLP-1 stimulates cAMP production in rat INS-1 insulinoma cells, we find accompanying activation of a -410-bp RIP1 luciferase construct (-410RIP1-LUC) to exist independently of this second messenger. GLP-1 produced a dose-dependent stimulation of -410RIP1-LUC (EC50 0.43 nmol/l), an effect reproduced by the GLP-1 receptor agonist exendin-4 and abolished by the antagonist exendin(9-39). Activation of RIP1 by GLP-1 was not affected by cotransfection with dominant-negative Gs alpha, was not blocked by cAMP antagonist Rp-cAMPS, and was insensitive to PKA antagonist H-89. Truncation of -410RIP1-LUC to generate -307-, -206-, and -166-bp constructs revealed 2 segments of RIP1 targeted by GLP-1. The first segment, not regulated by forskolin, was located between -410 and -307 bp of the promoter. The second segment, regulated by both GLP-1 and forskolin, included the CRE and was located between -206 and -166 bp. Consistent with these observations, stimulatory effects of GLP-1 at RIP1 were reduced after introduction of delta-182 and delta-183/180 inactivating deletions at the CRE. The action of GLP-1 at -410RIP1-LUC was also reduced by cotransfection with A-CREB, a genetically engineered isoform of the CRE binding protein CREB, which dimerizes with and prevents binding of basic-region-leucine-zipper (bZIP) transcription factors to the CRE. In contrast, the action of GLP-1 at the CRE was not blocked by cotransfection with M1-CREB, an isoform that lacks a consensus serine residue serving as substrate for PKA-mediated phosphorylation. On the basis of these studies, it is proposed that PKA-independent stimulatory actions of GLP-1 at RIP1 are mediated by bZIP transcription factors related in structure but not identical to CREB.

Race Determination and Vegetative Compatibility Grouping of Fusarium oxysporum f. sp. melonis from South Africa.

Isolates of Fusarium oxysporum f. sp. melonis (72 total) obtained from 30 fields in 17 melonproducing regions in South Africa were race typed, using differential cvs. CM 17187, Doublon, Perlita, and Topmark, and grouped on the basis of vegetative compatibility. Fifty-four isolates were identified as race 0, eight as race 1, and ten as race 2. Race 0 occurred in 15 of 17 regions, whereas race 1 was sporadically recovered. Race 2 was obtained from only four fields located in one geographic region. Perlita plants (carrying the gene Fom3) inoculated with local isolates of races 0 and 2 and reference isolates of race 0 became stunted, and their leaves became yellow, thickened, and brittle. Using two inoculation methods, similar symptoms were induced by reference and local isolates of race 0 on Perlita seedlings. The results indicated that Fom3 in Perlita confers a tolerant reaction compared with the resistant reaction of gene Fom1 in Doublon and, therefore, should not be used alone in race determination tests. All isolates belonged to vegetative compatibility group 0134, indicating a high degree of genetic homogeneity among the South African F. oxysporum f. sp. melonis population.

cAMP-dependent mobilization of intracellular Ca2+ stores by activation of ryanodine receptors in pancreatic beta-cells. A Ca2+ signaling system stimulated by the insulinotropic hormone glucagon-like peptide-1-(7-37).

Glucagon-like peptide-1 (GLP-1) is an intestinally derived insulinotropic hormone currently under investigation for use as a novel therapeutic agent in the treatment of type 2 diabetes mellitus. In vitro studies of pancreatic islets of Langerhans demonstrated that GLP-1 interacts with specific beta-cell G protein-coupled receptors, thereby facilitating insulin exocytosis by raising intracellular levels of cAMP and Ca2+. Here we report that the stimulatory influence of GLP-1 on Ca2+ signaling results, in part, from cAMP-dependent mobilization of ryanodine-sensitive Ca2+ stores. Studies of human, rat, and mouse beta-cells demonstrate that the binding of a fluorescent derivative of ryanodine (BODIPY FL-X ryanodine) to its receptors is specific, reversible, and of high affinity. Rat islets and BTC3 insulinoma cells are shown by reverse transcriptase polymerase chain reaction analyses to express mRNA corresponding to the type 2 isoform of ryanodine receptor-intracellular Ca2+ release channel (RYR2). Single-cell measurements of [Ca2+]i using primary cultures of rat and human beta-cells indicate that GLP-1 facilitates Ca2+-induced Ca2+ release (CICR), whereby mobilization of Ca2+ stores is triggered by influx of Ca2+ through L-type Ca2+ channels. In these cells, GLP-1 is shown to interact with metabolism of D-glucose to produce a fast transient increase of [Ca2+]i. This effect is reproduced by 8-Br-cAMP, but is blocked by a GLP-1 receptor antagonist (exendin-(9-39)), a cAMP antagonist ((Rp)-cAMPS), an L-type Ca2+ channel antagonist (nimodipine), an antagonist of the sarco(endo)plasmic reticulum Ca2+ ATPase (thapsigargin), or by ryanodine. Characterization of the CICR mechanism by voltage clamp analysis also demonstrates a stimulation of Ca2+ release by caffeine. These findings provide new support for a model of beta-cell signal transduction whereby GLP-1 promotes CICR by sensitizing intracellular Ca2+ release channels to the stimulatory influence of cytosolic Ca2+.

Leptin suppression of insulin secretion and gene expression in human pancreatic islets: implications for the development of adipogenic diabetes mellitus.

Previously we demonstrated the expression of the long form of the leptin receptor in rodent pancreatic beta-cells and an inhibition of insulin secretion by leptin via activation of ATP-sensitive potassium channels. Here we examine pancreatic islets isolated from pancreata of human donors for their responses to leptin. The presence of leptin receptors on islet beta-cells was demonstrated by double fluorescence confocal microscopy after binding of a fluorescent derivative of human leptin (Cy3-leptin). Leptin (6.25 nM) suppressed insulin secretion of normal islets by 20% at 5.6 mM glucose. Intracellular calcium responses to 16.7 mM glucose were rapidly reduced by leptin. Proinsulin messenger ribonucleic acid expression in islets was inhibited by leptin at 11.1 mM, but not at 5.6 mM glucose. Leptin also reduced proinsulin messenger ribonucleic acid levels that were increased in islets by treatment with 10 nM glucagon-like peptide-1 in the presence of either 5.6 or 11.1 mM glucose. These findings demonstrate direct suppressive effects of leptin on insulin-producing beta-cells in human islets at the levels of both stimulus-secretion coupling and gene expression. The findings also further indicate the existence of an adipoinsular axis in humans in which insulin stimulates leptin production in adipocytes and leptin inhibits the production of insulin in beta-cells. We suggest that dysregulation of the adipoinsular axis in obese individuals due to defective leptin reception by beta-cells may result in chronic hyperinsulinemia and may contribute to the pathogenesis of adipogenic diabetes.

Pertussis toxin-sensitive GTP-binding proteins characterized in synaptosomal fractions of embryonic avian cerebral cortex.

Pertussis toxin (PTX)-sensitive GTP-binding proteins (G proteins) are essential intermediaries subserving neuronal signal transduction pathways that regulate excitation-secretion coupling. Despite this established role, relatively little is known regarding the identity, subcellular distribution, and relative abundance of this class of G proteins in synaptic nerve endings. Here, sucrose density gradient centrifugation was combined with 1- and 2-dimensional gel electrophoresis to characterize PTX-sensitive G protein alpha subunits in synaptosomal fractions of embryonic (day 12) chick cerebral cortical homogenates. These findings demonstrate multiple isoforms of M(r) 40-41 kDa Gi alpha and G(o) alpha subunits that can be identified on the basis of PTX-catalyzed ADP-ribosylation and immunoblot analysis.

Black widow spider alpha-latrotoxin: a presynaptic neurotoxin that shares structural homology with the glucagon-like peptide-1 family of insulin secretagogic hormones.

alpha-Latrotoxin is a presynaptic neurotoxin isolated from the venom of the black widow spider Latrodectus tredecimguttatus. It exerts toxic effects in the vertebrate central nervous system by depolarizing neurons, by increasing [Ca2+]i and by stimulating uncontrolled exocytosis of neurotransmitters from nerve terminals. The actions of alpha-latrotoxin are mediated, in part, by a GTP-binding protein-coupled receptor referred to as CIRL or latrophilin. Exendin-4 is also a venom toxin, and it is derived from the salivary gland of the Gila monster Heloderma suspectum. It acts as an agonist at the receptor for glucagon-like peptide-1(7-36)-amide (GLP-1), thereby stimulating secretion of insulin from pancreatic beta-cells of the islets of Langerhans. Here is reported a surprising structural homology between alpha-latrotoxin and exendin-4 that is also apparent amongst all members of the GLP-1-like family of secretagogic hormones (GLP-1, glucagon, vasoactive intestinal polypeptide, secretin, pituitary adenylyl cyclase activating polypeptide). On the basis of this homology, we report the synthesis and initial characterization of a chimeric peptide (Black Widow GLP-1) that stimulates Ca2+ signaling and insulin secretion in human beta-cells and MIN6 insulinoma cells. It is also reported here that the GTP-binding protein-coupled receptors for alpha-latrotoxin and exendin-4 share highly significant structural similarity in their extracellularly-oriented amino-termini. We propose that molecular mimicry has generated conserved structural motifs in secretagogic toxins and their receptors, thereby explaining the evolution of defense or predatory strategies that are shared in common amongst distantly related species including spiders, lizards, and snakes. Evidently, the toxic effects of alpha-latrotoxin and exendin-4 are explained by their ability to interact with GTP-binding protein-coupled receptors that normally mediate the actions of endogenous hormones or neuropeptides.

Effects of Fruit and Pollen Exudates on Growth of Botrytis cinerea and Infection of Plum and Nectarine Fruit.

Sugars in exudates from Harry Pickstone plum and Sunlite nectarine fruit and from pollen of weeds commonly found in orchards were determined by gas-liquid chromatography, and their effect on the development of Botrytis cinerea was determined in vitro and in vivo. Fructose, glucose, and sorbitol were the only sugars detected in exudates of immature fruit. They occurred at low concentrations, but their concentration generally increased as fruit ripened. Sucrose was first detected during maturation. In nectarine, an increase in sugar concentration, especially sucrose, was pronounced during the period of rapid cell enlargement, which occurred approximately 2 weeks before harvest. Absorbance readings of culture media amended with sugar indicated that the hexose sugars (fructose and glucose) and sucrose did not markedly influence growth of B. cinerea at concentrations below 0.22 and 0.12 mM, respectively. The hexose sugars caused a steady increase in growth when supplied at concentrations in excess of 0.44 mM, and sucrose caused a steady increase in growth at 0.23 mM. The stimulatory effect of fruit exudates on growth of B. cinerea on glass slides coincided with the period of rapid sugar release from the fruit and the shift in susceptibility to decay. Only fructose (1.72 mM) and glucose (0.72 mM) were detected in nectarine pollen exudates. Pollen exudates from weeds stimulated fungal growth and significantly increased the aggressiveness of the pathogen on plum and nectarine fruit when added to conidia during the last 4 weeks prior to the picking-ripe stage. The study showed that changes in the composition of nectarine and plum fruit exudates may contribute to the late-season susceptibility of these fruit to B. cinerea infection.

Leptin suppression of insulin secretion by the activation of ATP-sensitive K+ channels in pancreatic beta-cells.

In the genetic mutant mouse models ob/ob or db/db, leptin deficiency or resistance, respectively, results in severe obesity and the development of a syndrome resembling NIDDM. One of the earliest manifestations in these mutant mice is hyperinsulinemia, suggesting that leptin may normally directly suppress the secretion of insulin. Here, we show that pancreatic islets express a long (signal-transducing) form of leptin-receptor mRNA and that beta-cells bind a fluorescent derivative of leptin (Cy3-leptin). The expression of leptin receptors on insulin-secreting beta-cells was also visualized utilizing antisera generated against an extracellular epitope of the receptor. A functional role for the beta-cell leptin receptor is indicated by our observation that leptin (100 ng/ml) suppressed the secretion of insulin from islets isolated from ob/ob mice. Furthermore, leptin produced a marked lowering of [Ca2+]i in ob/ob beta-cells, which was accompanied by cellular hyperpolarization and increased membrane conductance. Cell-attached patch measurements of ob/ob beta-cells demonstrated that leptin activated ATP-sensitive potassium channels (K(ATP)) by increasing the open channel probability, while exerting no effect on mean open time. These effects were reversed by the sulfonylurea tolbutamide, a specific inhibitor of K(ATP). Taken together, these observations indicate an important physiological role for leptin as an inhibitor of insulin secretion and lead us to propose that the failure of leptin to inhibit insulin secretion from the beta-cells of ob/ob and db/db mice may explain, in part, the development of hyperinsulinemia, insulin resistance, and the progression to NIDDM.

Signal transduction of PACAP and GLP-1 in pancreatic beta cells.

PACAP and GLP-1 depolarize pancreatic beta cells and stimulate insulin secretion in the presence of glucose. Depolarization occurs through at least two distinct mechanisms: (1) closure of ATP-sensitive K+ channels, and (2) activation of nonselective cation channels (NSCCs). Under physiological conditions the NSCCs carry a predominantly Na(+)-dependent current. The current may also have a Ca2+ component, but this remains to be determined. Acting together, these two signaling systems reinforce each other and serve to promote membrane depolarization, a rise of [Ca2+]i, and exocytosis of insulin-containing secretory granules. The NSCCs in beta cells are dually regulated by intracellular cAMP and [Ca2+]i. In view of this dual regulation, it appears likely that NSCC channel activation results from signaling events occurring not only at the plasma membrane (gating of channels by cAMP; protein kinase A-mediated phosphorylation of channels) but also at intracellular sites (mobilization of calcium stores by an as yet to be determined process). It is noteworthy that activation of NSCCs has also been reported following stimulation of beta-cells with maitotoxin, or after depletion of intracellular Ca2+ stores. Therefore, the possibility arises that PACAP, GLP-1, and maitotoxin all act on the same types of ion channels in these cells, and that these channels are sensitive to alterations in the content of intracellular calcium. FIGURE 6 summarizes our current knowledge concerning the properties of the PACAP and GLP-1 signaling systems as they pertain to the regulation of NSCCs and intracellular calcium homeostasis in the beta cell. Given that PACAP and GLP-1 are proven to be exceptionally potent insulin secretagogues, it is of considerable interest to determine their usefulness as blood glucose-lowering agents. Initial evaluations of the therapeutic effectiveness of GLP-1 indicate a role for this peptide in the treatment of NIDDM, and also possibly insulin-dependent diabetes mellitus (IDDM). A very attractive feature of such a strategy is the demonstrated lack of hypoglycemic side effects attendant to administration of GLP-1 to diabetic subjects. These observations reinforce the notion that peptides of the PACAP/glucagon/VIP family represent important pharmacological tools for use in experimental therapeutics.

Activation of a cAMP-regulated Ca(2+)-signaling pathway in pancreatic beta-cells by the insulinotropic hormone glucagon-like peptide-1.

Glucagon-like peptide-1 (GLP-1) is an intestinally derived insulinotropic hormone that is currently under investigation for use in the treatment of diabetes mellitus. To investigate the Ca2+ signaling pathways by which GLP-1 may stimulate the secretion of insulin from pancreatic beta-cells, we examined its effects on the concentration of free intracellular Ca2+ ([Ca2+]i) while simultaneously determining what action it exerts on ion channel function. Measurements of [Ca2+]i were obtained from single rat beta-cells and from beta TC6 and HIT-T15 insulinoma cells loaded with the Ca2+ indicator fura-2, and changes in membrane potential and current were monitored using the perforated patch clamp technique. We report a previously undocumented action of GLP-1 and analogs of cAMP (8-bromo-cAMP, Sp- or Rp-adenosine 3',5'-cyclic monophosphothionate triethylamine) to raise [Ca2+]i that is attributable to the activation of a prolonged inward current designated here as IcAMP. Activation of IcAMP is associated with an increased membrane conductance, membrane depolarization, and triggers large increases of [Ca2+]i. IcAMP is primarily a Na+ current that is blocked by extracellularly applied La3+ or by intracellular administration of Ca2+ chelators (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl, EGTA) and which exhibits a reversal potential of about -26 mV. We propose that IcAMP results from the opening of nonselective cation channels that are activated by intracellular Ca2+ and cAMP and which might play an important role in the regulation of insulin secretion from pancreatic beta-cells.

Pituitary adenylate cyclase-activating polypeptide induces the voltage-independent activation of inward membrane currents and elevation of intracellular calcium in HIT-T15 insulinoma cells.

The secretion of insulin by pancreatic beta-cells is controlled by synergistic interactions of glucose and hormones of the glucagon-related peptide family, of which pituitary adenylate cyclase-activating polypeptide (PACAP) is a member. Here we show by simultaneous recording of intracellular calcium ion ([Ca2+]i) and membrane potential that both PACAP-27 and PACAP-38 depolarize HIT-T15 cells and raise [Ca2+]i. PACAP stimulation can result in membrane depolarization by two distinct mechanisms: 1) PACAP reduces the membrane conductance and increases membrane excitability; and 2) PACAP activates a pronounced inward current that is predominantly a Na+ current, blockade by La3+, and which exhibits a reversal potential of about -28 mV. Activation of this current does not require membrane depolarization, because the response is observed when cells are held under voltage clamp at -70 mV. This current may result from the cAMP-dependent activation of nonspecific cation channels because the current is also observed in response to forskolin or membrane-permeant analogs of cAMP. We also suggest that PACAP raises [Ca2+]i and stimulates insulin secretion by three distinct mechanisms: 1) depolarization activates Ca2+ influx through L-type voltage-dependent calcium channels, 2) mobilization of intracellular Ca2+ stores, and 3) entry of Ca2+ via voltage-independent Ca2+ channels. These effects of PACAP may play an important role in a neuro-entero-endocrine loop regulating insulin secretion from pancreatic beta-cells during the transition period from fasting to feeding.

Voltage-independent calcium channels mediate slow oscillations of cytosolic calcium that are glucose dependent in pancreatic beta-cells.

Pancreatic beta-cells and HIT-T15 cells exhibit oscillations of cytosolic calcium ([Ca2+]i) that are dependent on glucose metabolism and appear to trigger pulsatile insulin secretion. Significantly, differences in the pattern of this [Ca2+]i oscillatory activity may have important implications for our understanding of how glucose homeostasis is achieved during the feeding and fasting states. When single beta-cells are exposed to a stepwise increase in glucose concentration that mimics the transition from fasting to feeding states, fast irregular oscillations of [Ca2+]i are observed. Alternatively, when single beta-cells are equilibrated in a steady state concentration of glucose that mimics the fasting state, slow periodic oscillations of [Ca2+]i are noted. Here we report a fundamental difference in the mechanism by which glucose induces these two types of [Ca2+]i oscillatory activity. In agreement with previous studies, we substantiate a role for L-type voltage-dependent Ca2+ channels as mediators of the fast oscillations of [Ca2+]i observed after a stepwise increase in glucose concentration. In marked contrast, we report that voltage-independent calcium channels (VICCs) mediate slow oscillations of [Ca2+]i that occur when beta-cells are equilibrated in steady state concentrations of glucose. Slow [Ca2+]i oscillations are mediated by VICCs which are pharmacologically and biophysically distinguishable from voltage-dependent Ca2+ channels that mediate fast oscillations. Specifically, slow [Ca2+]i oscillations are blocked by extracellular La3+, but not by nifedipine, and are independent of changes in membrane potential. Measurement of membrane conductance also indicate an important role for VICCs, as demonstrated by a steady state inward Ca2+ current that is blocked by La3+. The steady state Ca2+ current appears to generate slow [Ca2+]i oscillations by triggering Ca(2+)-induced Ca2+ release from intracellular Ca2+ stores, a process that is mimicked by extracellular application of caffeine, a sensitizer of the ryanodine receptor/Ca2+ release channel. Depletion of intracellular Ca2+ stores with thapsigargin stimulated Mn2+ influx, suggesting the presence of Ca(2+)-release-activated Ca2+ channels. Taken together, these observations are consistent with a role for VICCs (possibly G-type channels) and/or Ca(2+)-release-activated Ca2+ channels as mediators of slow [Ca2+]i oscillations in beta-cells. We propose that slow oscillations of [Ca2+]i probably serve as important initiators of insulin secretion under conditions in which tight control of glucose homeostasis is necessary, as is the case during the fasting normoglycemic state.