Intestinal CART is a regulator of GIP and GLP-1 secretion and expression.

Impaired incretin effect is a culprit in Type 2 Diabetes. Cocaine- and amphetamine-regulated transcript (CART) is a regulatory peptide controlling pancreatic islet hormone secretion and beta-cell survival. Here we studied the potential expression of CART in enteroendocrine cells and examined the role of CART as a regulator of incretin secretion and expression. CART expression was found in glucose-dependent insulinotropic polypeptide (GIP)-producing K-cells and glucagon-like peptide-1 (GLP-1)-producing L-cells in human duodenum and jejunum and circulating CART levels were increased 60 min after a meal in humans. CART expression was increased by fatty acids and GIP, but unaffected by glucose in GLUTag and STC-1 cells. Exogenous CART had no effect on GIP and GLP-1 expression and secretion in GLUTag or STC-1 cells, but siRNA-mediated silencing of CART reduced GLP-1 expression and secretion. Furthermore, acute intravenous administration of CART increased GIP and GLP-1 secretion during an oral glucose-tolerance test in mice. We conclude that CART is a novel constituent of human K- and L-cells with stimulatory actions on incretin secretion and that interfering with the CART system may be a therapeutic avenue for T2D.

The human L-type calcium channel Cav1.3 regulates insulin release and polymorphisms in CACNA1D associate with type 2 diabetes.

AIMS/HYPOTHESIS: Voltage-gated calcium channels of the L-type have been shown to be essential for rodent pancreatic beta cell function, but data about their presence and regulation in humans are incomplete. We therefore sought to elucidate which L-type channel isoform is functionally important and its association with inherited diabetes-related phenotypes. METHODS: Beta cells of human islets from cadaver donors were enriched using FACS to study the expression of the genes encoding voltage-gated calcium channel (Cav)1.2 and Cav1.3 by absolute quantitative PCR in whole human and rat islets, as well as in clonal cells. Single-cell exocytosis was monitored as increases in cell capacitance after treatment with small interfering (si)RNA against CACNA1D (which encodes Cav1.3). Three single nucleotide polymorphisms (SNPs) were genotyped in 8,987 non-diabetic and 2,830 type 2 diabetic individuals from Finland and Sweden and analysed for associations with type 2 diabetes and insulin phenotypes. RESULTS: In FACS-enriched human beta cells, CACNA1D mRNA expression exceeded that of CACNA1C (which encodes Cav1.2) by approximately 60-fold and was decreased in islets from type 2 diabetes patients. The latter coincided with diminished secretion of insulin in vitro. CACNA1D siRNA reduced glucose-stimulated insulin release in INS-1 832/13 cells and exocytosis in human beta cells. Phenotype/genotype associations of three SNPs in the CACNA1D gene revealed an association between the C allele of the SNP rs312480 and reduced mRNA expression, as well as decreased insulin secretion in vivo, whereas both rs312486/G and rs9841978/G were associated with type 2 diabetes. CONCLUSION/INTERPRETATION: We conclude that the L-type calcium channel Cav1.3 is important in human glucose-induced insulin secretion, and common variants in CACNA1D might contribute to type 2 diabetes.

Impact of transcription factor 7-like 2 (TCF7L2) on pancreatic islet function and morphology in mice and men.

Common genetic variations in the gene encoding transcription factor 7-like 2 (TCF7L2) reveal the strongest association with type 2-diabetes known to date. These lead to impaired insulin production and output, but the mechanisms of disease remain incompletely known. In this issue of Diabetologia, two publications provide new insights into TCF7L2-dependent diabetes.

Role of TCF7L2 risk variant and dietary fibre intake on incident type 2 diabetes.

AIMS/HYPOTHESIS: The T allele of transcription factor 7-like 2 gene variant, TCF7L2 rs7903146, increases the risk of type 2 diabetes by 40-50%. As TCF7L2 rs7903146 has been associated with diminished incretin effect we investigated whether interaction between dietary intake of carbohydrate, fat, protein or fibre and this variant affects the risk of type 2 diabetes. METHODS: A cohort of 24,799 non-diabetic individuals from the Malmö Diet and Cancer Study (MDCS), with dietary data obtained by a modified diet history method, were followed up for 12 years, with 1,649 recordings of incident type 2 diabetes made. Risk of type 2 diabetes in strata of diet quintiles was analysed prospectively adjusting for potential confounders. Cross-sectional analyses were performed on baseline fasting glucose and HbA(1c) levels in a subset of 5,216 randomly selected individuals from the MDCS. RESULTS: The elevated risk of type 2 diabetes with rs7903146 (OR 1.44, 95% CI 1.33, 1.56, p = 4.6 × 10(-19)) increased with higher intake of dietary fibre (OR 1.24, 95% CI 1.04, 1.47 to OR 1.56, 95% CI 1.31, 1.86 from the lowest to highest quintile; p (interaction) = 0.049). High intake of dietary fibre was inversely associated with diabetes incidence only among CC genotype carriers (OR 0.74, 95% CI 0.58, 0.94 per quintile, p = 0.025). The T allele was associated with 0.027% elevated HbA(1c) (p = 0.02) and this effect increased with higher intake of fibre (from -0.021% to 0.079% for the lowest to the highest quintile, p (interaction) = 0.02). Each quintile of higher fibre intake was associated with lower HbA(1c) levels among CC and CT but not among TT genotype carriers (-0.036%, p = 6.5 × 10(-7); -0.023%, p = 0.009; and 0.012%, p = 0.52, respectively). CONCLUSIONS/INTERPRETATION: Our study suggests that dietary fibre intake may modify the association between TCF7L2 rs7903146 and incidence of type 2 diabetes, and that higher fibre intake may associate with protection from type 2 diabetes only among non-risk allele carriers.

γ-Aminobutyric acid (GABA) signalling in human pancreatic islets is altered in type 2 diabetes.

AIMS/HYPOTHESIS: γ-Aminobutyric acid (GABA) is a signalling molecule in the interstitial space in pancreatic islets. We examined the expression and function of the GABA signalling system components in human pancreatic islets from normoglycaemic and type 2 diabetic individuals. METHODS: Expression of GABA signalling system components was studied by microarray, quantitative PCR analysis, immunohistochemistry and patch-clamp experiments on cells in intact islets. Hormone release was measured from intact islets. RESULTS: The GABA signalling system was compromised in islets from type 2 diabetic individuals, where the expression of the genes encoding the α1, α2, ß2 and ß3 GABA(A) channel subunits was downregulated. GABA originating within the islets evoked tonic currents in the cells. The currents were enhanced by pentobarbital and inhibited by the GABA(A) receptor antagonist, SR95531. The effects of SR95531 on hormone release revealed that activation of GABA(A) channels (GABA(A) receptors) decreased both insulin and glucagon secretion. The GABA(B) receptor antagonist, CPG55845, increased insulin release in islets (16.7 mmol/l glucose) from normoglycaemic and type 2 diabetic individuals. CONCLUSIONS/INTERPRETATION: Interstitial GABA activates GABA(A) channels and GABA(B) receptors and effectively modulates hormone release in islets from type 2 diabetic and normoglycaemic individuals.

Enhancement of glucagon secretion in mouse and human pancreatic alpha cells by protein kinase C (PKC) involves intracellular trafficking of PKCalpha and PKCdelta.

AIMS/HYPOTHESIS: Protein kinase C (PKC) regulates exocytosis in various secretory cells. Here we studied intracellular translocation of the PKC isoenzymes PKCalpha and PKCdelta, and investigated how activation of PKC influences glucagon secretion in mouse and human pancreatic alpha cells. METHODS: Glucagon release from intact islets was measured in static incubations, and the amounts released were determined by RIA. Exocytosis was monitored as increases in membrane capacitance using the patch-clamp technique. The expression of genes encoding PKC isoforms was analysed by real-time PCR. Intracellular PKC distribution was assessed by confocal microscopy. RESULTS: The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated glucagon secretion from mouse and human islets about fivefold (p < 0.01). This stimulation was abolished by the PKC inhibitor bisindolylmaleimide (BIM). Whereas PMA potentiated exocytosis more than threefold (p < 0.001), BIM inhibited alpha cell exocytosis by 60% (p < 0.05). In mouse islets, the PKC isoenzymes, PKCalpha and PKCbeta1, were highly abundant, while in human islets PKCeta, PKCepsilon and PKCzeta were the dominant variants. PMA stimulation of human alpha cells correlated with the translocation of PKCalpha and PKCdelta from the cytosol to the cell periphery. In the mouse alpha cells, PKCdelta was similarly affected by PMA, whereas PKCalpha was already present at the cell membrane in the absence of PMA. This association of PKCalpha in alpha cells was principally dependent on Ca(2+) influx through the L-type Ca(2+) channel. CONCLUSIONS/INTERPRETATION: PKC activation augments glucagon secretion in mouse and human alpha cells. This effect involves translocation of PKCalpha and PKCdelta to the plasma membrane, culminating in increased Ca(2+)-dependent exocytosis. In addition, we demonstrated that PKCalpha translocation and exocytosis exhibit differential Ca(2+) channel dependence.

Unique splicing pattern of the TCF7L2 gene in human pancreatic islets.

AIMS/HYPOTHESIS: Intronic variation in the TCF7L2 gene exhibits the strongest association to type 2 diabetes observed to date, but the mechanism whereby this genetic variation translates into altered biological function is largely unknown. A possible explanation is a genotype-dependent difference in the complex splicing pattern; however, this has not previously been characterised in pancreatic or insulin target tissues. Here, the detailed TCF7L2 splicing pattern in five human tissues is described and dependence on risk genotype explored. METHODS: RT-PCR and quantitative real-time PCR were employed to characterise TCF7L2 splicing in pancreatic islets, blood lymphocytes, skeletal muscle and subcutaneous and visceral adipose tissue from non-diabetic individuals. RESULTS: The mapping of TCF7L2 splice variants shows a specific pattern in pancreatic islets, with four predominant transcripts and high usage of the variable exons 4 and 15. The overall concentration of TCF7L2 mRNA is highest in islets and fat and lower in blood and muscle. No significant difference in overall amount or splicing pattern was observed between carriers and non-carriers of the rs7903146 risk (T) allele. However, incorporation of exon 4 in islets correlates positively with plasma HbA(1c) levels (r = 0.758; p = 0.018). CONCLUSIONS/INTERPRETATION: There were pronounced tissue-specific differences in the splicing of TCF7L2 with forms containing exon 4 and 15 being most abundant in islets. The incorporation of exon 4 in islets correlated with HbA(1c) levels. Further experiments will be needed to determine the direction of this correlation, and larger cohorts needed to unequivocally resolve whether there is a relationship between genotype and splicing in islets.

Polymorphisms in the gene encoding the voltage-dependent Ca(2+) channel Ca (V)2.3 (CACNA1E) are associated with type 2 diabetes and impaired insulin secretion.

AIMS/HYPOTHESIS: Glucose-stimulated insulin secretion is dependent on the electrical activity of beta cells; hence, genes encoding beta cell ion channels are potential candidate genes for type 2 diabetes. The gene encoding the voltage-dependent Ca(2+) channel Ca(V)2.3 (CACNA1E), telomeric to a region that has shown suggestive linkage to type 2 diabetes (1q21-q25), has been ascribed a role for second-phase insulin secretion. METHODS: Based upon the genotyping of 52 haplotype tagging single nucleotide polymorphisms (SNPs) in a type 2 diabetes case-control sample (n = 1,467), we selected five SNPs that were nominally associated with type 2 diabetes and genotyped them in the following groups (1) a new case-control sample of 6,570 individuals from Sweden; (2) 2,293 individuals from the Botnia prospective cohort; and (3) 935 individuals with insulin secretion data from an IVGTT. RESULTS: The rs679931 TT genotype was associated with (1) an increased risk of type 2 diabetes in the Botnia case-control sample [odds ratio (OR) 1.4, 95% CI 1.0-2.0, p = 0.06] and in the replication sample (OR 1.2, 95% CI 1.0-1.5, p = 0.01 one-tailed), with a combined OR of 1.3 (95% CI 1.1-1.5, p = 0.004 two-tailed); (2) reduced insulin secretion [insulinogenic index at 30 min p = 0.02, disposition index (D (I)) p = 0.03] in control participants during an OGTT; (3) reduced second-phase insulin secretion at 30 min (p = 0.04) and 60 min (p = 0.02) during an IVGTT; and (4) reduced D (I) over time in the Botnia prospective cohort (p = 0.05). CONCLUSIONS/INTERPRETATION: We conclude that genetic variation in the CACNA1E gene contributes to an increased risk of the development of type 2 diabetes by reducing insulin secretion.

Anaplerosis via pyruvate carboxylase is required for the fuel-induced rise in the ATP:ADP ratio in rat pancreatic islets.

AIMS/HYPOTHESIS: The molecular mechanisms of insulin release are only partially known. Among putative factors for coupling glucose metabolism to insulin secretion, anaplerosis has lately received strong support. The anaplerotic enzyme pyruvate carboxylase is highly expressed in beta cells, and anaplerosis influences insulin secretion in beta cells. By inhibiting pyruvate carboxylase in rat islets, we aimed to clarify the hitherto unknown metabolic events underlying anaplerotic regulation of insulin secretion. METHODS: Phenylacetic acid (5 mmol/l) was used to inhibit pyruvate carboxylase in isolated rat islets, which were then assessed for insulin secretion, fuel oxidation, ATP:ADP ratio, respiration, mitochondrial membrane potential, exocytosis and ATP-sensitive K(+) channel (K(ATP)-channel) conductance. RESULTS: We found that the glucose-provoked rise in ATP:ADP ratio was suppressed by inhibition of pyruvate carboxylase. In contrast, fuel oxidation, respiration and mitochondrial membrane potential, as well as Ca(2+)-induced exocytosis and K(ATP)-channel conductance in single cells, were unaffected. Insulin secretion induced by alpha-ketoisocaproic acid was suppressed, whereas methyl-succinate-stimulated secretion remained unchanged. Perifusion of rat islets revealed that inhibition of anaplerosis decreased both the second phase of insulin secretion, during which K(ATP)-independent actions of fuel secretagogues are operational, as well as the first and K(ATP)-dependent phase. CONCLUSIONS/INTERPRETATION: Our results are consistent with the concept that anaplerosis via pyruvate carboxylase determines pyruvate cycling, which has previously been shown to correlate with glucose responsiveness in clonal beta cells. These processes, controlled by pyruvate carboxylase, seem crucial for generation of an appropriate ATP:ADP ratio, which may regulate both phases of fuel-induced insulin secretion.

Insulin granule dynamics in pancreatic beta cells.

Glucose-induced insulin secretion in response to a step increase in blood glucose concentrations follows a biphasic time course consisting of a rapid and transient first phase followed by a slowly developing and sustained second phase. Because Type 2 diabetes involves defects of insulin secretion, manifested as a loss of first phase and a reduction of second phase, it is important to understand the cellular mechanisms underlying biphasic insulin secretion. Insulin release involves the packaging of insulin in small (diameter approximately 0.3 micro m) secretory granules, the trafficking of these granules to the plasma membrane, the exocytotic fusion of the granules with the plasma membrane and eventually the retrieval of the secreted membranes by endocytosis. Until recently, studies on insulin secretion have been confined to the appearance of insulin in the extracellular space and the cellular events preceding exocytosis have been inaccessible to more detailed analysis. Evidence from a variety of secretory tissues, including pancreatic islet cells suggests, however, that the secretory granules can be functionally divided into distinct pools that are distinguished by their release competence and/or proximity to the plasma membrane. The introduction of fluorescent proteins that can be targeted to the secretory granules, in combination with the advent of new techniques that allow real-time imaging of granule trafficking in living cells (granule dynamics), has led to an explosion of our knowledge of the pre-exocytotic and post-exocytotic processes in the beta cell. Here we discuss these observations in relation to previous functional and ultra-structural data as well as the secretory defects of Type 2 diabetes.

Glucose dependence of insulinotropic actions of pituitary adenylate cyclase-activating polypeptide in insulin-secreting INS-1 cells.

The cAMP-elevating pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates insulin release in pancreatic B-cells. Here, we have investigated its potentiating action in rat insulinoma INS-1 cells. In intact cells, PACAP-27 (100 nM) stimulated glucose-induced insulin secretion by >60%. Using the patch-clamp technique with single-cell exocytosis monitored as increases in cell capacitance, we observed that at 10 mM and 20 mM extracellular glucose, PACAP-27 acted mainly by a >50% enhancement of depolarization-elicited Ca(2+) entry, whereas at low (3 mM) glucose, the predominant effect of the peptide was a twofold increase in Ca(2+) sensitivity of insulin exocytosis. The latter effect was mimicked by glucose itself in a dose-dependent fashion. PACAP-27 exerts a prolonged effect on insulin secretion that is dissociated from changes of cytoplasmic cAMP. Whereas an elevation of cellular cAMP content (135%) could be observed 2 min after addition of PACAP-27, after 30 min preincubation with the peptide, cAMP concentrations were not different from basal. Yet, such pretreatment with PACAP-27 stimulated subsequent insulin release by congruent with60%. This sustained action is likely to reflect an increased degree of protein-kinase-A-dependent phosphorylation, and inhibitors of the kinase largely prevented the PACAP-mediated effects.

Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells.

The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.

Priming of insulin granules for exocytosis by granular Cl(-) uptake and acidification.

ATP-dependent priming of the secretory granules precedes Ca(2+)-regulated neuroendocrine secretion, but the exact nature of this reaction is not fully established in all secretory cell types. We have further investigated this reaction in the insulin-secreting pancreatic B-cell and demonstrate that granular acidification driven by a V-type H(+)-ATPase in the granular membrane is a decisive step in priming. This requires simultaneous Cl(-) uptake through granular ClC-3 Cl(-) channels. Accordingly, granule acidification and priming are inhibited by agents that prevent transgranular Cl(-) fluxes, such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and an antibody against the ClC-3 channels, but accelerated by increases in the intracellular ATP:ADP ratio or addition of hypoglycemic sulfonylureas. We suggest that this might represent an important mechanism for metabolic regulation of Ca(2+)-dependent exocytosis that is also likely to be operational in other secretory cell types.

Perchlorate stimulates insulin secretion by shifting the gating of L-type Ca2+ currents in mouse pancreatic B-cells towards negative potentials.

The effects of the chaotrophic anion perchlorate (ClO4-) on glucose-induced electrical activity, exocytosis and ion channel activity in mouse pancreatic B-cells were investigated by patch-clamp recordings and capacitance measurements. ClO4- stimulated glucose-induced electrical activity and increased the action potential frequency by 70% whilst not affecting the membrane potential when applied in the presence of a subthreshold concentration of the sugar. ClO4- did not influence ATP-dependent K (KATP) channel activity and voltage-gated delayed K+ current. Similarly, ClO4- had no effect on Ca2+-dependent exocytosis. The stimulation of electrical activity and insulin secretion was instead attributable to an enhancement of the whole-cell Ca2+ current. This effect was particularly pronounced at voltages around the threshold for action potential initiation and a doubling of the current amplitude was observed at -30 mV. This was due to a 7-mV shift in the gating of the Ca2+ current towards negative voltages. The action of ClO4- was more pronounced when added in the presence of 0.1 mM BAY K8644, whereas no stimulation was observed when applied at a maximal concentration of the agonist (1 mM). Single-channel recordings revealed that the effect of ClO4- on whole-cell currents was principally due to a 60% increase in the mean duration of the long openings and the number of active channels. We propose that ClO4- stimulates insulin secretion and electrical activity by exerting a BAY K8644-like action on Ca2+ channel gating.

Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells.

We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.

CaM kinase II-dependent mobilization of secretory granules underlies acetylcholine-induced stimulation of exocytosis in mouse pancreatic B-cells.

1. Measurements of cell capacitance were used to investigate the mechanisms by which acetylcholine (ACh) stimulates Ca2+-induced exocytosis in single insulin-secreting mouse pancreatic B-cells. 2. ACh (250 microM) increased exocytotic responses elicited by voltage-clamp depolarizations 2.3-fold. This effect was mediated by activation of muscarinic receptors and dependent on elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) attributable to mobilization of Ca2+ from intracellular stores. The latter action involved interference with the buffering of [Ca2+]i and the time constant (tau) for the recovery of [Ca2+]i following a voltage-clamp depolarization increased 5-fold. As a result, Ca2+ was present at concentrations sufficient to promote the replenishment of the readily releasable pool of granules (RRP; > 0.2 microM) for much longer periods in the presence than in the absence of the agonist. 3. The effect of Ca2+ on exocytosis was mediated by activation of CaM kinase II, but not protein kinase C, and involved both an increased size of the RRP from 40 to 140 granules and a decrease in tau for the refilling of the RRP from 31 to 19 s. 4. Collectively, the effects of ACh on the RRP and tau result in a > 10-fold stimulation of the rate at which granules are supplied for release.

The stimulatory action of tolbutamide on Ca2+-dependent exocytosis in pancreatic beta cells is mediated by a 65-kDa mdr-like P-glycoprotein.

Intracellular application of the sulfonylurea tolbutamide during whole-cell patch-clamp recordings stimulated exocytosis >5-fold when applied at a cytoplasmic Ca2+ concentration of 0.17 microM. This effect was not detectable in the complete absence of cytoplasmic Ca2+ and when exocytosis was elicited by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The stimulatory action could be antagonized by the sulfonamide diazoxide, by the Cl--channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), by intracellular application of the antibody JSB1 [originally raised against a 170-kDa multidrug resistance (mdr) protein], and by tamoxifen (an inhibitor of the mdr- and volume-regulated Cl- channels). Immunocytochemistry and Western blot analyses revealed that JSB1 recognizes a 65-kDa protein in the secretory granules. This protein exhibited no detectable binding of sulfonylureas and is distinct from the 140-kDa sulfonylurea high-affinity sulfonylurea receptors also present in the granules. We conclude that (i) tolbutamide stimulates Ca2+-dependent exocytosis secondary to its binding to a 140-kDa high-affinity sulfonylurea receptor in the secretory granules; and (ii) a granular 65-kDa mdr-like protein mediates the action. The processes thus initiated culminate in the activation of a granular Cl- conductance. We speculate that the activation of granular Cl- fluxes promotes exocytosis (possibly by providing the energy required for membrane fusion) by inducing water uptake and an increased intragranular hydrostatic pressure.

The voltage sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery.

Although N- and P-type Ca2+ channels predominant in fast-secreting systems, Lc-type Ca2+ channels (C-class) can play a similar role in certain secretory cells and synapses. For example, in retinal bipolar cells, Ca2+ entry through the Lc channels triggers ultrafast exocytosis, and in pancreatic beta-cells, evoked secretion is highly sensitive to Ca2+. These findings suggest that a rapidly release pool of vesicles colocalizes with the Ca2+ channels to allow high Ca2+ concentration and a tight coupling of the Lc channels at the release site. In binding studies, we show that the Lc channel is physically associated with synaptotagmin (p65) and the soluble N-ethylmaleimide-sensitive attachment proteins receptors: syntaxin and synaptosomal-associated protein of 25 kDa. Soluble N-ethylmaleimide-sensitive attachent proteins receptors coexpressed in Xenopus oocytes along with the Lc channel modify the kinetic properties of the channel. The modulatory action of syntaxin can be overcome by coexpressing p65, where at a certain ratio of p65/syntaxin, the channel regains its unaltered kinetic parameters. The cytosolic region of the channel, Lc753-893, separating repeats II-III of its alpha1C subunit, interacts with p65 and "pulls" down native p65 from rat brain membranes. Lc753-893 injected into single insulin-secreting beta-cell, inhibits secretion in response to channel opening, but not in response to photolysis of caged Ca2+, nor does it affect Ca2+ current. These results suggest that Lc753-893 competes with the endogenous channel for the synaptic proteins and disrupts the spatial coupling with the secretory apparatus. The molecular organization of the Lc channel and the secretory machinery into a multiprotein complex (named excitosome) appears to be essential for an effective depolarization evoked exocytosis.

Rapid ATP-dependent priming of secretory granules precedes Ca(2+)-induced exocytosis in mouse pancreatic B-cells.

1. The glucose and ATP dependence of exocytosis were investigated in single mouse pancreatic B-cells by monitoring changes in cell capacitance evoked by voltage-clamp depolarizations, infusion of high [Ca2+]i buffers or photorelease of caged Ca2+ or ATP. 2. In intact B-cells, using the perforated patch whole-cell technique, glucose (5 mM) increased exocytotic responses evoked by membrane depolarization 5-fold over that observed in the absence of the sugar. Increasing the glucose concentration to 20 mM produced a further doubling of exocytosis. The stimulatory action of glucose was attributable to glucose metabolism and abolished by mannoheptulose, an inhibitor of glucose phosphorylation. 3. Exocytosis triggered by infusion of high [Ca2+]i and ATP was reduced by 80% when ATP was replaced by its non-hydrolysable analogue adenosine 5'-[beta, gamma-methylene]triphosphate (AMP-PCP) in standard whole-cell experiments. Exocytosis elicited by GTP gamma S was similarly affected by replacement of ATP with the stable analogue. 4. Photoreleasing ATP in the presence of 170 nM [Ca2+]i, following the complete wash-out of endogenous ATP produced a prompt (latency, < 400 ms) and biphasic stimulation of exocytosis. 5. Elevation of [Ca2+]i to exocytotic levels by photorelease from Ca(2+)-nitrophenyl EGTA preloaded into the cell evoked a biphasic stimulation in the presence of Mg-ATP. The response consisted of an initial rapid (completed in < 200 ms) phase followed by a slower (lasting > or = 10 s) sustained component. Replacement of ATP with AMP-PCP abolished the late component but did not affect the initial phase. The latency between elevation of [Ca2+]i and exocytosis was determined as < 45 ms. Inclusion of N-ethylmaleimide (NEM; 0.5 mM for 3 min) in the intracellular solution exerted effects similar to those obtained by substituting AMP-PCP for ATP. 6. We conclude that the B-cell contains a small pool (40 granules) of primed granules which are immediately available for release and which are capable of undergoing exocytosis in an ATP-independent fashion. We propose that this pool of granules is preferentially released during first phase glucose-stimulated insulin secretion. The short latency between the application of ATP and the onset of exocytosis finally suggests that priming takes place with sufficient speed to participate in the rapid adjustment of the secretory capacity of the B-cell.