Normal Rat Pancreatic Islets Research Articles

Somatostatin Receptor Expression and Biological Functions in Endocrine Pancreatic Cells

Type 1 diabetes is resulting from the selective destruction of insulin-producing betacells within the pancreatic islets. Somatostatin acts as an inhibitor of hormone secretion through specific receptors (sst1-5).All ssts were expressed in normal rat and mouse pancreatic islets, although the expression intensity and the co-expression pattern varied between ssts as well as between species. This may reflect a difference in response to somatostatin in islet cells of the two species.The Non-Obese Diabetic (NOD) mouse model is an experimental model of type 1 diabetes, with insulitis accompanied by spontaneous hyperglycaemia. Pancreatic specimens from NOD mice at different age and stage of disease were stained for ssts. The islet cells of diabetic NOD mice showed increased islet expression of sst2–5 compared to normoglycemic NOD mice. The increase in sst2-5 expression in the islets cells may suggest either a contributing factor in the process leading to diabetes, or a defense response against ongoing beta-cell destruction.Somatostatin analogues were tested on a human endocrine pancreatic tumour cell line and cultured pancreatic islets. Somatostatin analogues had an effect on cAMP accumulation, chromogranin A secretion and MAP kinase activity in the cell line. Treatment of rat pancreatic islets with somatostatin analogues with selective receptor affinity was not sufficient to induce an inhibition of insulin and glucagon secretion. However, a combination of selective analogues or non-selective analogues via costimulation of receptors can cause inhibition of hormone production. For insulin and glucagon, combinations of sst2 + sst5 and sst1 + sst2, respectively, showed a biological effect.In summary, knowledge of islet cell ssts expression and the effect of somatostatin analogues with high affinity to ssts may be valuable in the future attempts to influence beta-cell function in type 1 diabetes mellitus, since down-regulation of beta-cell function may promote survival of these cells during the autoimmune attack.

PACAP is expressed in secretory granules of insulin and glucagon cells in human and rodent pancreas: Evidence for generation of cAMP compartments uncoupled from hormone release in diabetic islets

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an islet neuropeptide with potent insulinotropic action. The current study investigates PACAP expression in normal human and rat pancreatic islets, and whether it is altered in diabetic state. To that end, PACAP immunoreactivity was studied by immunofluorescence methods enhanced by the catalyzed reporter deposition (CARD) technique. Insulin and cyclic adenosine monophosphate (cAMP) generation induced by PACAP were investigated in islets isolated from the spontaneously diabetic Goto–Kakizaki (GK) rat. PACAP immunoreactivity was observed in virtually all insulin and glucagon cells in both species, but not in somatostatin or pancreatic polypeptide (PP) cells; this co-localization pattern was unaltered in diabetic pancreata. In normal human pancreas, PACAP was further localized ultrastructurally to the secretory granules of insulin and glucagon cells. PACAP significantly potentiated glucose-stimulated insulin release in isolated islets of normal but not of GK rats. PACAP failed to enhance cAMP generation in normal islets, but induced ∼5-folds exaggeration in the diabetic islets. In conclusion, using improved immunocytochemistry techniques and electron microscopy (EM), PACAP was shown to be expressed both in normal and diabetic islet cells and localized to secretory granules of insulin and glucagon cells. Furthermore, the insulinotropic action of PACAP was markedly impaired in diabetic islets in spite of exaggerated cAMP response.

Expression and cellular distribution pattern of plasma membrane calcium pump isoforms in rat pancreatic islets.

This work is aimed at identifying the presence and cellular distribution pattern of plasma membrane calcium pump (PMCA) isoforms in normal rat pancreatic islet. Microsomal fractions of isolated islets and exocrine tissue were analyzed to detect different PMCA isoforms. The cellular distribution pattern of these PMCAs in the islets was also studied in fixed pancreas sections incubated with antibodies against PMCAs and insulin. Antibody 5F10, which reacts with all PMCA variants, showed multiple bands in the blots in the 127-134 kDa region, indicating the presence of several isoforms. Microsomes also reacted positively with specific antibodies for individual PMCA isoforms, generating a band of the expected size. Antibody 5F10 immunocytochemically labeled the plasma cell membrane of both b- and non-b-cells, but predominantly the former. All islet cells were also labeled with antibodies against isoforms 1 and 4, while the antibody reacting with isoform 3 labeled exclusively b-cells. A few b- and non-b-cells were positively labeled with the antibody reacting with PMCA b variant. Negative results were obtained with the antibody against isoform 2. Further studies, together with previous reports on the modulatory effect of insulin secretagogues and blockers upon PMCA activity, may provide evidence of the importance of this particular PMCA expression for islet function under normal and pathological conditions.

Expression, processing, and secretion of the neuroendocrine VGF peptides by INS-1 cells.

The neurotropin-inducible gene vgf is expressed in neuronal and endocrine tissues. It encodes a secretory protein that is proteolytically processed in neuronal cells to low molecular mass polypeptides. In the present report, we show that vgf is expressed in different insulinoma cell lines and in normal rat pancreatic islets. In the insulinoma-derived beta-cell line INS-1, vgf messenger RNA was transcriptionally up-regulated by increased levels ofintracellular cAMP, but not by the addition of glucose (20 mM) or phorbol 12-myristate 13-acetate (100 nM). Furthermore, nerve growth factor failed to stimulate vgf gene expression. In INS-1 cells, the VGF protein was shown to be processed in a post endoplasmic reticulum compartment to produce a peptide profile similar to that seen in neurons. The release of such VGF peptides occurred at a low rate in the absence of secretory stimuli (<2%/h). A 3-fold increase in the rate of release was seen after the addition of glucose (15 mM), a 4-fold increase was seen after (Bu)2cAMP (1 mM), and a 6-fold increase was seen after phorbol 12-myristate 13-acetate (100 nM). These results indicated that insulin-containing cells produce VGF-derived peptides that are released via a regulated pathway in response to insulin secretagogues.

RINm5f cells express inactivating BK channels whereas HIT cells express noninactivating BK channels.

Large-conductance Ca2+- and voltage-activated BK-type K+ channels are expressed abundantly in normal rat pancreatic islet cells and in the clonal rat insulinoma tumor (RINm5f) and hamster insulinoma tumor (HIT) beta cell lines. Previous work has suggested that the Ca2+ sensitivity of BK channels in RIN cells is substantially less than that in HIT cells, perhaps contributing to differences between the cell lines in responsiveness to glucose in mediating insulin secretion. In both RIN cells and normal pancreatic beta cells, BK channels are thought to play a limited role in responses of beta cells to secretagogues and in the electrical activity of beta cells. Here we examine in detail the properties of BK channels in RIN and HIT cells using inside-out patches and whole cell recordings. BK channels in RIN cells exhibit rapid inactivation that results in an anomalous steady-state Ca2+ dependence of activation. In contrast, BK channels in HIT cells exhibit the more usual noninactivating behavior. When BK inactivation is taken into account, the Ca2+ and voltage dependence of activation of BK channels in RIN and HIT cells is essentially indistinguishable. The properties of BK channel inactivation in RIN cells are similar to those of inactivating BK channels (termed BKi channels) previously identified in rat chromaffin cells. Inactivation involves multiple, trypsin-sensitive cytosolic domains and exhibits a dependence on Ca2+ and voltage that appears to arise from coupling to channel activation. In addition, the rates of inactivation onset and recovery are similar to that of BKi channels in chromaffin cells. The charybdotoxin (CTX) sensitivity of BKi currents is somewhat less than that of the noninactivating BK variant. Action potential voltage-clamp waveforms indicate that BK current is activated only weakly by Ca2+ influx in RIN cells but more strongly activated in HIT cells even when Ca2+ current magnitude is comparable. Concentrations of CTX sufficient to block BKi current in RIN cells have no effect on action potential activity initiated by glucose or DC injection. Despite its abundant expression in RIN cells, BKi current appears to play little role in action potential activity initiated by glucose or DC injection in RIN cells, but BK current may play an important role in action potential repolarization in HIT cells.

CCK receptor subtype in insulin-producing cells: a combined functional and in situ hybridization study in rat islets and a rat insulinoma cell line

Cholecystokinin (CCK) stimulates insulin secretion. It is, however, not established whether CCK receptors are expressed in insulin-producing cells. We therefore investigated, by in situ hybridization, whether CCK-A or CCK-B receptor mRNA could be detected in normal rat pancreatic islets and in the rat insulinoma cell line, RINm5F. The CCK-A, but not the CCK-B, receptor transcript was detected in both islets and RINm5F cells. Islet CCK-A receptors were mostly confined to the center of the islets corresponding to the distribution of the B cells. In RINm5F cells, insulin release was not significantly affected by cholecystokinin (CCK)-8-S (10 −13 to 10 −7 M), which is in contrast to the insulinotropic effect of CCK-8-S in normal rat islets. Similarly, in FURA-2AM-loaded cells, CCK-8-S (10 −11 to 10 −7 M) was without effect on the intracellular Ca 2+ concentration ([Ca 2+]ic) in RINm5F cells, whereas CCK-8-S (10 −7 M) markedly increased [Ca 2+]ic (by 366±2 nM ( P<0.001) in normal rat islet cells. We conclude that the CCK-A, but not the CCK-B, receptor subtype is expressed in both normal rat islets and in the rat insulinoma-derived cell line RINm5F. There is, however, a functional difference between normal islets and the RINm5F cells with respect to effects of CCK-8-S on insulin release and [Ca 2+]ic.

Inhibition of glucose-induced insulin secretion by long-term preexposure of pancreatic islets to leptin

Here we evaluated the effect of leptin on glucose-induced insulin secretion by normal rat pancreatic islets. We show in perifusion experiments that leptin had no acute effect on the secretory activity of β-cells. However, following preexposure to leptin a pronounced time- and dose-dependent inhibition of both first and second phases of secretion was observed. Maximum inhibition was obtained at 24 h and with 100 nM leptin. This inhibition did not involve a decrease in cellular insulin content. It was also not observed with islets from fa/fa rats. Leptin thus inhibits insulin secretion by a mechanism which requires long-term preexposure to the hormone and which may involve alteration in β-cell gene expression.

Inhibitory effect of sulfonylureas on protein phosphatase activity in rat pancreatic islets.

We have measured protein phosphatase (PP) activity in crude homogenates as well as in the total 105.000 x g supernatant and precipitate fractions from normal rat pancreatic islets. On the basis of the inhibition produced by either 1 nM or 1 microM okadaic acid, both PP1 and PP2A activity were present in crude islet homogenates in equivalent proportions (53% and 47%, respectively); PP1 was the main activity present in the precipitate, whereas in the supernatant it was PP2A. Tolbutamide, glybenclamide and glyclazide significantly decreased PP activity in islet homogenates in a dose-dependent manner, with a K10.5 value that in the case of glybenclamide correlated with its Kd for binding site, its EC50 on KATP channel, and its EC50 on insulin release. These data indicate that PPs play a role in the control of insulin secretion and suggest a further possible target for sulfonylureas within their overall action as insulin secretagogues.

Characterization of prenylcysteine methyltransferase in insulin-secreting cells

Prenylcysteine carboxymethyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, was characterized in insulin-secreting INS-1 cells and normal rat pancreatic islets. The activity of this enzyme was monitored by the methylation of an artificial substrate (a prenylated cysteine analogue) with S-adenosy1[methyl-3H]methionine as methyl donor. More than 95% of the methyltransferase activity was associated with the membranes, and high-salt treatment only partially extracted the enzyme from the membranes. The highest specific activity was in the insulin-granule-enriched 25000 g pellet obtained by differential centrifugation. However, a highly purified insulin-enriched fraction obtained by density centrifugation in Percoll did not exhibit methyltransferase activity. The analyses of marker enzymes for cellular organelles revealed that the methyltransferase was co-localized, with the plasma membrane and probably the endoplasmic reticulum, but not with the mitochondria or lysosomes. Guanosine 5'-[gamma-thio]-triphosphate failed to increase methyltransferase activity directly, although it promotes the methylation of GTP-binding proteins. Mastoparan, Ca2+, cAMP and the protein kinase C activator phorbol 12-myristate 13-acetate did not alter enzyme activity. In addition, methyltransferase activity was not stably modified by stimulation of intact cells using glucose or other agents. However, the carboxymethylation of certain low-molecular-mass G-proteins is increased by glucose stimulation; conversely, treatment of cells with N-acetyl-S-trans,trans-farnesyl-L-cysteine inhibited glucose- and forskolin-induced insulin secretion. These results suggest that the membrane-associated prenylcysteine carboxymethyltransferase may be constitutively active and that the methylation of target proteins in vivo is regulated by the access of these proteins to the methyltransferase, as well as by their active (GTP-liganded) configuration.

Cytosolic biosynthesis of GTP and ATP in normal rat pancreatic islets

GTP and ATP are necessary for glucose-induced insulin secretion; however, the biosynthetic pathways of purine nucleotides have not been studied in pancreatic islets. The present work examines the cytosolic pathways of purine nucleotide synthesis using intact rat islets cultured overnight in RPMI 1640 medium containing either [ 14C]glycine (to label the de novo pathway) or [ 3H]hypoxanthine (to mark the salvage pathway), with or without mycophenolic acid or l-alanosine (selective inhibitors of cytosolic GTP and ATP synthesis, respectively). Addition of mycophenolic acid decreased total GTP content (mass) by 73–81%; although the incorporation of labeled hypoxanthine into GTP also fell by 87%, the incorporation of glycine did not change. Similarly, l-alanosine decreased ATP mass by 26–33% in the presence of either label; whereas the incorporation of hypoxanthine into ATP fell 59%, the incorporation of glycine was again not significantly decreased. Thus, both the de novo and salvage purine nucleotide biosynthetic pathways are present in rat islets; however, the salvage pathway appears to be quantitatively the more important source of nucleotides. This conclusion was supported by additional studies of the effects on nucleotide content and insulin secretion of various site-specific inhibitors of purine synthesis. These findings have potential relevance to the processes of mitogenesis, cell proliferation and differentiation of islet cells, as well as for the control of insulin secretion.

Subcellular localization and kinetic characterization of guanine nucleotide binding proteins in normal rat and human pancreatic islets and transformed β cells

The subcellular localization and the kinetics of the GTPase activities of monomeric and heterotrimeric GTP-binding proteins were investigated in normal rat and human pancreatic islets and were compared to those obtained using a transformed hamster β cell line (HIT cells). The [α- 32P]GTP overlay technique revealed the presence of at least four low-molecular-mass proteins (approx. 20–27 kDa) in normal rat islets, which were enriched in the secretory granule fraction compared to the membrane fraction (with little abundance of these proteins in the cytosolic fraction). In contrast, in HIT cells, these proteins (at least six) were predominantly cytosolic. Three of these proteins were immunologically identified as rab3A, rac2, and CDC42Hs in islets as well as in HIT cells. In addition, pertussis toxin augmented the ribosylation of at least one heterotrimeric G-protein of about 39 kDa (probably G i and/or G o) in the membrane and secretory granule fractions of normal rat and human islets, whereas at least three such Ptx substrates (36–39 kDa) were found in HIT cell membranes. Kinetic analyses of the intrinsic specific GTPase activities revealed the presence of at least three such activities ( K m for GTP of 372 nM, 2.2 μM, and 724 μM) in islet homogenates which were differentially distributed in various subcellular fractions; similar activities were also demonstrable in HIT cell homogenates. Thus, these studies demonstrate the presence of both monomeric as well as trimeric G-proteins intrinsic to the secretory granules of normal rat islets which can be ascribed to beta cells; since these G-proteins are regulated by insulinotropic lipids (as described in the accompanying article), such proteins may couple the activation of phospholipases (endogenous to islets) to the exocytotic secretion of insulin. These findings also suggest that caution is necessary in extrapolating data concerning G-proteins from cultured, transformed β cell lines to the physiology of normal islets, in view of both qualitative and quantitative differences between the two preparations.

Stimulation by prostaglandin E2 of a high-affinity GTPase in the secretory granules of normal rat and human pancreatic islets

Recent reports of a pertussis-toxin (Ptx)-sensitive inhibition of glucose-induced insulin release by prostaglandin E2 (PGE2) in transformed beta-cells prompted us to look for the presence of prostaglandin-regulatable GTP-binding proteins (G-proteins) on the secretory granules of normal pancreatic islets. PGE2 (but not PGF2 alpha, PGA2, PGB2 or PGD2) stimulated in a concentration-dependent manner a high-affinity GTPase activity in the secretory-granule-enriched fractions of both normal rat and human islets. Similar results were found after sucrose-density-gradient-centrifugation-based isolation of secretory granules to those after a differential-centrifugation procedure. Half-maximal stimulation occurred at 800 nM PGE2, a concentration known to inhibit both phases of glucose-induced insulin secretion from pure beta-cell lines. The GTPase stimulatory effect of PGE2 was blocked virtually totally by Ptx pretreatment; it was not due to an effect on substrate binding since no measurable effect of PGE2 on binding of guanosine 5'-[gamma-[35S]thio]triphosphate was observed in cognate fractions. Other Ptx-sensitive inhibitors of insulin secretion (such as adrenaline or clonidine) also stimulated GTPase activity, suggesting that one (or more) inhibitory exocytotic G-proteins (i.e. a putative GEi) is located on the secretory granules. These studies demonstrate, for the first time in an endocrine gland, the presence of a regulatable G-protein, strategically located on the secretory granules where it might regulate the exocytotic cascade distal to both plasma-membrane events and the generation of soluble mediators of insulin secretion.

Amino-terminal parathyroid hormone-related protein: specific binding and cytosolic calcium responses in rat insulinoma cells.

PTH-related protein (PTHrP), originally identified through its causative role in human humoral hypercalcemia of malignancy, is now known to be a normal gene product expressed in a wide variety of neuroendocrine, epithelial, and mesoderm-derived tissues. PTHrP gene expression has recently been demonstrated in fetal and adult, benign and malignant, as well as human and rodent pancreatic islets. As in other tissues, the role of PTHrP expression in the normal islet is only beginning to be explored. In the current report, PTHrP expression in the normal rat pancreatic islet was confirmed using an affinity-purified antiserum directed against the N-terminal, biologically active region of the molecule. The effects of PTHrP on the islet were then explored using rat insulinoma (RIN m5F) cells. Synthetic PTHrP-(1-36) bound specifically, but with low affinity (Kd, approximately 10(-7) M) to RIN cell membranes. PTHrP-(1-36) failed to stimulate cAMP production in RIN cells, although RIN cells displayed a normal adenylate cyclase response to glucagon-like peptide-1-(7-36). In contrast, PTHrP-(1-36) induced a rapid dose-dependent rise in intracellular calcium in RIN cells in doses as low as 10(-12)-10(-10) M. These findings 1) confirm that PTHrP is expressed by islet cells, 2) demonstrate that the effects of PTHrP on the pancreatic islet are mediated, as in keratinocytes and lymphocytes, by a receptor related to but distinct from the PTH receptor, and 3) suggest that PTHrP functions in the islet as an autocrine or paracrine factor. Further studies are required to determine the physiological consequences of PTHrP expression by the pancreatic islet.

Effect of fusidic acid on interleukin-1 (IL-1)- and IL-6-induced pancreatic beta-cell functions in rats.

Fusidic acid and its sodium salt (fusidin) are anti-staphylococcal drugs with a steroidal primary structure. Both compounds have been shown to prevent the lymphocyte co-stimulatory activities of the cytokines, interleukin (IL)-1 and IL-6, in a manner similar to that of cyclosporin A. As shown in this paper, fusidin also prevents the inhibitory effect of human recombinant IL-1 beta (rIL-1 beta) and the stimulatory effect of human rIL-6, on glucose-induced insulin production in vitro by normal rat pancreatic islets. The drug also inhibited rIL-1 beta-induced IL-6 production by the islets. Fusidin showed a dose-related effect at pharmacologically relevant concentrations from 3 to 30 micrograms/ml, and the drug was progressively less active when added 1, 4 and 24 h after rIL-1 beta. Electron microscopical studies showed that beta cells cultured for 72 h with rIL-1 beta accumulated less lipid in the presence of fusidin, most probably reflecting the functionally protective effect of the drug. Other characteristic ultrastructural changes induced in beta cells by rIL-1 beta were, however, not altered. It is suggested that fusidin may prove clinically effective as a modulator of IL-1- and IL-6-induced changes in beta-cell functions.

Inhibition of Na/Ca exchange in pancreatic islet cells by 3′,4′-dichlorobenzamil

Na/Ca exchange may play a role in Ca 2+ extrusion from the pancreatic B cell. The role played by the exchanger was examined by characterizing the effects of 3′-4′-dichlorobenzamil on ionic fluxes and insulin release in normal rat pancreatic islet cells. 3′,4′-Dichlorobenzamil potently inhibited 45Ca uptake mediated by reverse Na/Ca exchange ( ic 50: 18 μm) in islet cells. The drug failed to decrease intracellular pH but reduced 86Rb outflow from perifused islets. The effects of glucose and 3′,4′-dichlorobenzamil on 86Rb outflow were not additive. The drug potently blocked 45Ca uptake through voltage-sensitive Ca 2+ channels (IC 50: 7.5μM). In the presence of extracellular Ca 2+ and 3′,4′-dichlorobenzamil, glucose lost part of its ability to reduce 45Ca outflow. The drug failed to affect the secondary rise in 45Ca outflow induced by the sugar. In the absence of extracellular Ca 2+,3′,4′-dichlorobenzamil induced a delayed inhibition of 45Ca outflow, the effect of the sugar and the drug being not additive. This effect of 3′,4′-dichlorobenzamil and its ability to impair the inhibitory effect of glucose were reproduced by the removal of extracellular Na + and disappeared under the latter experimental condition. 3′,4′-Dichlorobenzamil did not affect insulin release in the absence of glucose but significantly increased glucose-induced insulin release when used at a high concentration. It is concluded that 3′,4′-dichlorobenzamil is a potent inhibitor of the process of Na/Ca exchange in the pancreatic B cell. Unfortunately, the drug is of poor specificity and blocks, in the same range of concentrations, both K + channels and voltage-sensitive Ca 2+ channels. The data also indicate that glucose inhibits 45Ca outflow from pancreatic islets to a great extent (at least 75%) by inhibiting Na/Ca exchange. The type of Na/Ca exchange that is inhibited by glucose, remains to be elucidated

Characterization of the process of sodium-calcium exchange in pancreatic islet cells

Na(+)-Ca2+ exchange may play a role in Ca2+ extrusion from the pancreatic B-cell. The characteristics of the process working in its reverse mode were examined in normal rat pancreatic islet cells. Isosmotical replacement of extracellular Na+ by sucrose induced a concentration-dependent increase in 45Ca uptake, displaying a pharmacological sensitivity compatible with an uptake mediated by Na(+)-Ca2+ exchange. Glucose, up to 2.8 mM, stimulated reverse Na(+)-Ca2+ exchange. Likewise, membrane depolarization activated the process but only under raised intracellular Na+ activity. In conclusion, the B-cell Na(+)-Ca2+ exchange displays properties similar to those observed in other cells: reversibility and sensitivity to membrane potential. When working in its reverse mode the exchanger displays a quite large capacity. The role played by the exchanger in the process of insulin release warrants further investigation.

Protein phosphorylation in pancreatic islets induced by 3-phosphoglycerate and 2-phosphoglycerate.

We have shown previously that 3-phosphoglycerate, which is a glycolytic metabolite of glucose, induces protein phosphorylation in bovine and rat brain and in rat heart, kidney, liver, lung, and whole pancreas. Since glycolytic metabolism of glucose is of paramount importance in insulin release, we considered the possibility that 3-phosphoglycerate may act as a coupling factor, and we searched for evidence for the existence of 3-phosphoglycerate-dependent protein phosphorylation systems in freshly isolated normal rat pancreatic islets. Membrane and cytosol fractions were incubated with [gamma-32P]ATP and appropriate test substances and were subjected to NaDodSO4/PAGE and autoradiography. As little as 0.005 mM 3-phosphoglycerate or 2-phosphoglycerate stimulated the phosphorylation of a 65-kDa cytosol protein by as early as 0.25 min. The phosphate bond of the 65-kDa phosphoprotein was sufficiently stable to withstand dialysis; the radioactivity could not be chased out by subsequent exposure to ATP, ADP, 3-phosphoglycerate, or 2,3-bisphosphoglycerate. Moreover, cAMP, cGMP, phorbol 12-myristate 13-acetate, or calcium failed to stimulate the phosphorylation of the 65-kDa protein. Phosphoglycerate-dependent protein phosphorylation in islets may have relevance to stimulation of insulin secretion.

Effects of verapamil and nifedipine on gliclazide-induced increase in cytosolic free Ca2+ in pancreatic islet cells.

The changes in cytosolic free calcium [Ca2+]i induced by the sulfonylurea gliclazide and potassium in normal rat pancreatic islet cells were measured using the fluorescent Ca2+ indicator fura-2. Both in the absence or presence of 5.6 mM glucose, gliclazide caused a rapid and sustained increase in [Ca2+]i. The phenylalkylamine verapamil reduced these increases, but the Ca2+ channel blocker was more potent in the presence than in the absence of glucose. In contrast, nifedipine, a Ca2+ channel blocker of another chemical type, reduced to a similar extent the increase in [Ca2+]i evoked by gliclazide in the absence and presence of glucose. In the absence of glucose, a rise in extracellular K+ concentration from 5 to 20 or 30 mM also induced a rapid and sustained rise in [Ca2+]i. Verapamil more markedly reduced the rise in [Ca2+]i induced by 30 mM than by 20 mM K+. It is concluded that gliclazide increases Ca2+ inflow into normal islet cells primarily, if not exclusively, by opening voltage-sensitive Ca2+ channels. The differential sensitivity toward verapamil of gliclazide-induced rise in [Ca2+]i can be explained by the use-dependent block exerted by Ca2+ channel blockers of the phenylalkylamine type.

Glyburide increases cytosolic-free calcium concentrations in normal rat pancreatic islet cells

We have attempted to delineate the effect of glyburide on the regulation of cytosolic-free calcium concentrations, [Ca 2+]i, in normal rat pancreatic islet cells. In the presence of extracellular calcium (1 mmol/L), glyburide increased [Ca 2+]i from 70 nmol/L to 260 nmol/L in a dose-dependent manner. The maximal effect was seen at a concentration of 2 μmol/L with half-maximal stimulation observed at .25 μmol/L. The effect of glyburide (.25 μmol/L) was inhibited 90% by the calcium channel blocker, verapamil (30 μmol/L). At a maximally effective concentration of glyburide (2 μmol/L), the inhibitory effect of verapamil was only 17%. In the absence of extracellular calcium, glyburide increased [Ca 2+]i from 55 nmol/L to 107 nmol/L, indicating its ability to mobilize intracellular calcium stores. These results correlated well with the ability of glyburide (2 μmol/L) to stimulate insulin secretion both in the presence (from 38 ± 5 μmol/L/10 islets to 131 ± 28 μU/10 islets) and in the absence ( from 49 ± 4 μU 10 islets to 93 ± 7 μU 10 islets ) of extracellular Ca 2+. The present observations suggest that glyburide promotes calcium influx via voltage-dependent calcium channels and may mobilize intracellular calcium stores.

Hexose metabolism in pancreatic islets: Regulation of mitochondrial hexokinase binding

A major fraction of hexokinase was found to be bound, presumably to mitochondria, in both normal and tumoral rat pancreatic islet cells examined after either mechanical disruption or digitonin treatment. Spermidine enhanced the binding and glucose 6-phosphate caused the release of hexokinase to and from islet mitochondria, in a manner comparable to that seen in parotid or brain homogenates. In hepatocytes, some hexokinase, but no glucokinase, was found in the bound form. In islet cells, however, the pattern of glucokinase binding was similar to that of hexokinase. It is speculated that the preferential location of both hexokinase and glucokinase on mitochondria may favor the maintenance of a high cytosolic ATP content in islet cells.