HIT T15 Research Articles

The effect of gliquidone on KATP channels in pancreatic β-cells, cardiomyocytes, and vascular smooth muscle cells

AimsSulfonylurea drugs exert an insulinotropic effect through ATP-sensitive potassium (KATP) channel inhibition in pancreatic islet cells. These channels are also expressed in cardiomyocytes and vascular smooth muscle cells (VSMCs), suggesting potential for adverse cardiovascular effects. We evaluated the effects of Gliquidone (Glq) on sulfonylurea receptors in HIT-T15 cells (SUR1), cardiomyocytes (SUR2A), and VSMCs (SUR2B). MethodsThe concentration-dependent effects of Glq (0.001–500μM) on KATP channels were assessed using whole-cell patch clamp in HIT-T15 cells, rat cardiomyocytes, and VSMCs. Parallel studies using Glibenclamide (Glb) (0.001–10μM) and Gliclazide (Glc) (0.01–500μM)were conducted as controls. ResultsIn HIT–T15 cells, Glb exhibited the lowest IC50 (0.03μM), as compared to Glq (0.45μM) and Glc (1.21μM). However, Glq had higher IC50 in cardiomyoctes and VSMCs, as compared to Glb (119.1 vs. 0.01 and 149.7 vs. 0.09μM, respectively), suggesting that Glq is more selective to β-cells than Glb. Thus, Glq may have fewer side effects in cardiomyoctes and VSMCs. ConclusionsGlq is a highly selective SUR secretagogue with moderate affinity to β-cells, but low affinity to cardiomyocytes and VSMCs. Our data also reveal the non-selective nature of Glb, as evidenced by high binding affinity to KATP channels in all the three cell types examined.

Phosphatase inhibitors induce defective hormone secretion in insulin-secreting cells and entry into apoptosis.

A long-term (> or =24 h) exposure of insulin-secreting HIT T15 cells to the phosphatase inhibitor, okadaic acid (OA), at concentrations inhibiting serine/threonine phosphatases 1 (PP1) and 2A (PP2A) reduced proliferation and insulin secretion. The reduced proliferation was related to the induction of apoptosis as evidenced by morphological criteria and the occurrence of internucleosomal DNA fragmentation after 15 h in 50 nM OA. The compromised insulin secretion was not simply a consequence of a lowered hormone content and cell growth, but comprised also a complete suppression of secretion stimulated by K+ depolarisation and forskolin. K+ depolarisation of HIT cells cultured for 24 h in 50 nM OA resulted in a nearly unimpaired influx of Ca2+, but did not induce secretion. These observations suggest that the secretory defect may be localised distal to Ca2+ influx in stimulus secretion coupling of insulin-secreting cells.

An Autocrine Insulin Feedback Loop Maintains Pancreatic β-Cell 3-Phosphorylated Inositol Lipids

Phosphatidylinositol 3-kinases (PI3Ks) have a central role in pancreatic beta-cell function. Downstream events include the regulation of K(ATP) channel activity, insulin secretion, gene transcription, and cell survival. Fewer data are available on the 3-phosphorylated inositol lipids (3-PIs) that are the primary products of these kinases. We characterized these PI3K products in insulin-secreting HIT T15 cells and were able to demonstrate, for the first time the presence of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)]. We then showed that glucose can significantly increase PtdIns(3,4,5)P(3), PtdIns(3,4)P(2), and notably PtdIns(3,5)P(2). We investigated the mechanism(s) whereby these molecules are generated under both basal and glucose-stimulated conditions. We postulated that insulin exocytosis could drive the rises in 3-PIs. In our experimental system, we could detect a rise in insulin secretion within 1 min of glucose stimulation, thus allowing the possibility that early rises in 3-PIs are regulated by secreted insulin. This was confirmed because blockade of the beta-cell insulin receptor completely abrogated the glucose-mediated increase of all three lipids, driving their concentrations below basal levels. Using primary pancreatic islets and either blockade of the insulin receptor or antibodies to insulin, we verified that basal insulin secretion is responsible for the maintenance of 3-PIs. Therefore, autocrine insulin signaling, a feature compromised in diabetes, is essential to up-regulate both basal and glucose-stimulated levels of a vital family of second messengers that preserve and drive pancreatic beta-cell function.

Susceptibility of insulinoma cells to cadmium and modulation by L-type calcium channels

Cadmium (Cd), a toxic metal that induces apoptosis and necrosis in a variety of cells, accumulates in pancreas and may be a cause of diabetes in humans. In the insulinoma cells line HIT-T15 (HIT), we measured internal calcium (Ca) and Cd levels by the fluorescent dye Fura-2 and confirm that L-type voltage-dependent calcium channels (VDCC) play a major role in glucose response and represent a pathway of Cd influx in these cells. Therefore we examined the role of VDCC in acute Cd poisoning by comparing its accumulation and cytotoxic effect in HIT cells and in epithelial-like VDCC-free HeLa cells. Cultures were incubated with 10-300 microM Cd for 15 min-6 h. While negligible at the end of the treatment, HIT cell death was evident after 18-24 h, and it was time-, dose- and serum-dependent. Short (< or = 60 min) Cd treatments with lower doses (< or = 100 microM in serum-free medium) induced delayed apoptotic cell death, as demonstrated by DNA fragmentation on agarose gels and segmentation of DAPI-stained nuclei. Longer incubations and/or higher concentrations caused mainly necrosis. The same treatments were largely harmless in HeLa cells, in which neither death nor DNA fragmentation was observed. The Ca antagonist nimodipine was capable to prevent HIT cell death at lower doses of Cd and to restore the apoptotic condition at higher doses, indicating that reduction of Cd flux through VDCC modulates Cd toxicity. These data demonstrate a specific sensitivity to Cd of insulinoma cells that can be significant for pancreatic beta-cell pathology.

β-Cell toxicity of ATP-sensitive K + channel-blocking insulin secretagogues

A prolonged exposure of isolated pancreatic islets to insulin secretagogues, the imidazolines phentolamine, alinidine and idazoxan (100 μM each), the sulfonylurea tolbutamide (500 μM), or the alkaloid quinine (100 μM) resulted in morphological damage of 4–18% of β-cells compared to less than 2% in controls. Thus, the question arose whether K ATP channel-blocking insulin secretagogues are β-cell toxic as has already been suggested for sulfonylureas. The concentration- and time-dependency of the secretagogue-associated toxicity was documented by viability assays in insulin-secreting HIT T15 cells. Treatment for 24 h with idazoxan reduced MTT conversion by 50% at 100 μM and by 98% at 1000 μM. Phentolamine and quinine reduced viability comparably at 1000 μM, but were less toxic at 100 μM. On the other hand, the imidazoline alinidine and the sulfonylurea tolbutamide were only moderately toxic (less than 40% viability loss at 1000 μM). The imidazoline efaroxan appeared even to be non-toxic. Apoptotic DNA fragmentation and DEVD-caspase activation was observed at 100 μM of idazoxan and phentolamine, whereas at 1000 μM signs of necrosis predominated. Alinidine, tolbutamide and quinine treatment did not increase markers of apoptotic cell death. Blocking Ca 2+ influx by D600 did not diminish secretagogue-associated toxicity. Electron microscopy confirmed the validity of these observations for β-cells in intact mouse islets. In summary, β-cell toxicity of the tested insulin secretagogues varied widely and did not depend on a prolonged Ca 2+ influx via L-type Ca 2+ channels. Thus, secretagogue-mediated closure of K ATP channels is apparently not per se β-cell toxic.

Hypoglycemic effect of S(−)-hydroxyhexamide, a major metabolite of acetohexamide, and its enantiomer R(+)-hydroxyhexamide

A short-lasting hypoglycemic effect was observed when S(−)-hydroxyhexamide ( S-HH), a major metabolite of acetohexamide, and its enantiomer R(+)-hydroxyhexamide ( R-HH), were administered orally to rats. Since the reductive metabolism of acetohexamide is known to be reversible in rats, oral administration of R-HH may exhibit the hypoglycemic effect through the generation of acetohexamide. However, oral administration of R-HH to rabbits, in spite of their inability to oxidize R-HH to acetohexamide, caused a significant decrease and increase, respectively, of plasma glucose and insulin levels. Furthermore, both S-HH and R-HH were found to stimulate the secretion of insulin from hamster HIT T15 cells (pancreatic β-cells). These results provide further evidence that both R-HH and S-HH exhibit a significant hypoglycemic effect.

Glibenclamide but not other sulphonylureas stimulates release of neuropeptide Y from perifused rat islets and hamster insulinoma cells.

We have studied the effects of first and second generation sulphonylureas on the release of insulin and neuropeptide tyrosine (NPY) from hamster insulinoma tumour (HIT T15) cells and isolated rat islets. In the presence of 5.5 mmol/l glucose all sulphonylureas stimulated insulin release from the HIT cells (P<0.01 ANOVA, n> or =4) but only glibenclamide (GLIB, 10 micromol/l) stimulated the release of NPY (mean+/-s.e.m. control 11.1+/-1.3 vs GLIB 28.4+/-4.1 fmol/h per 10(6) cells, P<0001, n=16). In isolated perifused rat islets both glibenclamide (10 micromol/l) (control 3.5+/-0.3 vs GLIB 6. 3+/-0.2 fmol/min per islet, P<0.01, n=6) and tolbutamide (50 micromol/l) (control 4.7+/-0.1 vs TOLB 6.7+/-0.3 fmol/min per islet, P<0.01, n=6) enhanced glucose (8 mmol/l)-stimulated insulin release. However, only glibenclamide stimulated the release of NPY from the islets (control 3.4+/-0.8 vs GLIB 24.5+/-5 attomol/min per islet, P<0.01, n=6). Similar results were obtained in islets isolated from dexamethasonetreated rats. Glibenclamide treatment of HIT cells showed a prompt insulin release (10 min) while NPY secretion was slower (60 min), suggesting that internalization of the sulphonylurea is required to stimulate NPY release. Glibenclamide, the most common oral therapeutic agent in type 2 diabetes mellitus, is associated with release of the autocrine insulin secretion inhibitor, NPY.

Expression of Thyrotropin-Releasing Hormone (TRH) receptor subtype 1 in mouse pancreatic islets and HIT-T15, an insulin-secreting clonal β cell line

Thyrotropin-releasing hormone (TRH), originally isolated as a hypothalamic hormone, has been reported to be present and released from the pancreatic β cells, affecting pancreatic functions. However, it still remains unclear whether TRH receptor is expressed in the pancreas. In the present study, we characterized TRH receptors (TRHR) in mouse pancreatic islets and HIT-T15 cells, a hamster clonal β cell line. RT-PCR study showed significant expression of TRHR subtype 1 (TRHR1) mRNA in both mouse pancreatic islets and HIT-T15 (HIT) cells. In contrast, there was no expression of TRHR2 mRNA, a novel subtype of TRHR which is expressed predominantly in the central nervous system. Sequencing analysis demonstrated that TRHR1 of the islets was identical to that in the pituitary, and cloned hamster TRHR1 shared 93.3% homology with that of the mouse at the nucleic acid level. Northern blot analysis of TRHR 1 mRNA in HIT-T15 cells showed a single strong hybridization signal approximately 3.7 kb in length. Furthermore, Scatchard plot analysis in HIT-T15 cells revealed that the Kd value for MeTRH was 0.63 nM. Significant elevation of intracellular calcium concentration was observed in response to as little as 10 nM TRH, and this was not affected by removal of extracellular calcium. This is the first description indicating the presence of functional TRH receptor subtype 1 in the pancreatic β cells, and our observations suggested the regulation of pancreatic function by TRH through autocrine or paracrine mechanisms.

( R)-ACX is a novel sufonylurea compound with potent, quick and short-lasting hypoglycemic activity

We investigated the mechanism of the hypoglycemic effect of ( R)-4-(1-acetoxyethyl)- N-(cyclohexylcarbamoyl)benzene-sulfonamide [( R)-acetoxyhexamide; ( R)-ACX], a new sulfonylurea compound. ( R)-ACX potently stimulated the release of insulin from cultured pancreatic β-cells (HIT T15 cells), established from hamster islet cells SV40-transformed. When ( R)-ACX was orally administered to fasted rats, it decreased the plasma glucose level in a dose-dependent manner. The hypoglycemic effect of ( R)-ACX was quick and short lasting, as compared to that of acetohexamide and glibenclamide. The quick and short-lasting hypoglycemic effect of ( R)-ACX was thought likely to result from rapid absorption of ( R)-ACX and rapid elimination of ( R)-ACX and its metabolite, ( R)-hydroxyhexamide. Furthermore, ( R)-ACX was found to suppress the increase of blood glucose level due to starch loading in fasted mice. ( R)-ACX may be useful in the control of postprandial hyperglycemia to patients with non-insulin-dependent diabetic mellitus.

An endogenous peptide isolated from the gut, NK-lysin, stimulates insulin secretion without changes in cytosolic free Ca 2+ concentration

We have recently isolated and cloned a novel endogenous peptide from pig intestine, NK-lysin (NKL). In the present study we show that NKL (1–100 nM) potently and reversibly stimulates insulin secretion in rat pancreatic islets and in the β-cell line HIT T15. This effect of NKL was not accompanied by changes in cytoplasmic free calcium concentration. The stimulatory activity of NKL on insulin release was also observed in permeabilized islets under Ca 2+-clamped conditions. Preincubation of HIT T15 cells with NKL for 1 h or 24 h did not influence cell viability. Possible mechanisms of insulinotropic activity of NKL are discussed.

Cloning of the Promoters for the β-Cell ATP-Sensitive K-Channel Subunits Kir6.2 and SUR1

The beta-cell ATP-sensitive potassium channel (K-ATP channel), which regulates insulin secretion, is composed of two types of subunits: 1) a sulfonylurea receptor (SUR1) and 2) an inwardly rectifying potassium channel (Kir6.2). We have isolated clones containing 5'-flanking DNA for both genes by hybridization screening of a human genomic library. Sequencing of over one kilobase of each upstream region has revealed that the putative promoters are G + C rich, with no TATA box. Several E-boxes and potential Sp1 sites are present in both promoters, and the Kir6.2 upstream region contains an Alu repeat. Using a luciferase reporter gene in transient transfection assays, we demonstrate that the upstream DNA contains promoters that are active in the beta-cell lines HIT T15 and MIN6. The promoters are completely inactive in the fibroblast cell line COS7 but show some activity in HepG2 (liver) and HEK293 (epithelial) cell lines. Deletion analysis suggests that a short (173-base pair [bp]) fragment of SUR1 5'-flanking sequence is sufficient for maximal promoter activity. In contrast, over 900 bp of Kir6.2 5' sequence are required for similar high level expression, and deletion of the Alu repeat results in an increase in promoter activity.

Citrulline-Argininosuccinate-Arginine Cycle Coupled to Ca2+-Signaling in Rat Pancreatic β-Cells

Pancreatic β-cells possess nitric oxide (NO) synthases (NOSs) which synthesize NO and L-citrulline from L-arginine. The present study was designed to explore the mechanism of citrulline and arginine metabolism in β-cells and its possible coupling to β-cell functions. The enzymes involved in citrulline-arginine metabolism, argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and NOS were expressed in rat islets and insulinoma HIT T15 cells. In the presence of stimulatory glucose, L-citrulline and L-argininosuccinate at physiological concentrations (0.1-1 mM) increased cytosolic Ca2+concentration ([Ca2+]i) in rat β-cells. The citrulline-induced [Ca2+]iincrease was inhibited by a NOS inhibitor, NG-monomethyl-L-arginine (NMMA). L-citrulline also stimulated NO production in HIT cells, which was inhibited by NMMA. In conclusion, L-citrulline is metabolized by ASS-ASL-NOS cycle to produce NO, which in turn increases [Ca2+]iin β-cells.

Modulation of tyrosine kinase activity has multiple actions on insulin release from the pancreatic beta-cell: studies with lavendustin A.

We investigated the role of tyrosine kinases in the regulation of insulin release from a hamster beta-cell line, HIT T15, using selective tyrosine kinase inhibitors. Genistein increased the insulin release induced by glucose, but herbimycin A, tyrphostins and the erbstatin analogue failed to change the release. Lavendustin A at 0.1 nM-1 microM caused a concave-shaped inhibition of the insulin release stimulated by 7 mM glucose. The inhibitory effect of lavendustin A was overcome by higher concentrations of glucose. Lavendustin B, the negative control analogue, had no effect on the release. Lavendustin A at a nanomolar range progressively inhibited insulin release by high K+ (50 mM)-depolarization, whereas the inhibitor did not change the insulin release by Ca2+ ionophore (A23187). On the contrary, lavendustin A at 10 nM significantly increased insulin release when glucose-induced insulin release was enhanced by either 5 microM forskolin or 162 nM 12-O-tetradecanoylphorbol 13-acetate. Lavendustin A failed to influence the Ca(2+)-induced insulin release from HIT cells permeabilized with streptolysin-O. These findings suggest that tyrosine kinases may play versatile roles in the control of insulin release from the pancreatic beta-cell.

Ca2+/calmodulin and cyclic 3,5' adenosine monophosphate control movement of secretory granules through protein phosphorylation/dephosphorylation in the pancreatic beta-cell.

We observed the movement of insulin granules in living transformed hamster pancreatic beta-cells (HIT T15) with a light microscope, where secretory granules are moving in the cytoplasmic space. Velocity of the typical granule movement was approximately 1.5 microns/sec. A stimulatory concentration of glucose activated the movement of the secretory granules. Forskolin, an activator of adenylate cyclase, increased the movement, resulting in changes in intracellular localization of the granules. Acetylcholine also activated the granule movement, whereas high K+ and tolbutamide, which cause Ca2+ influx through the voltage-dependent Ca2+ channel, had only little effect. The movement was abolished by BAPTA, the intracellular Ca2+-chelator. Activation of protein kinase C by 12-O-tetradecanoyl-phorbol 13-acetate failed to affect this movement. The motile events were inhibited by the calmodulin antagonist, W-7, and dramatically increased by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. These results suggest protein phosphorylation by Ca2+/calmodulin- and cAMP-dependent protein kinases play a positive role in the control of the insulin granule movements, which results in potentiation of insulin release from the pancreatic beta-cell.

Ca2+/calmodulin and cyclic 3,5' adenosine monophosphate control movement of secretory granules through protein phosphorylation/dephosphorylation in the pancreatic beta-cell

We observed the movement of insulin granules in living transformed hamster pancreatic beta-cells (HIT T15) with a light microscope, where secretory granules are moving in the cytoplasmic space. Velocity of the typical granule movement was approximately 1.5 microns/sec. A stimulatory concentration of glucose activated the movement of the secretory granules. Forskolin, an activator of adenylate cyclase, increased the movement, resulting in changes in intracellular localization of the granules. Acetylcholine also activated the granule movement, whereas high K+ and tolbutamide, which cause Ca2+ influx through the voltage-dependent Ca2+ channel, had only little effect. The movement was abolished by BAPTA, the intracellular Ca2+-chelator. Activation of protein kinase C by 12-O-tetradecanoyl-phorbol 13-acetate failed to affect this movement. The motile events were inhibited by the calmodulin antagonist, W-7, and dramatically increased by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. These results suggest protein phosphorylation by Ca2+/calmodulin- and cAMP-dependent protein kinases play a positive role in the control of the insulin granule movements, which results in potentiation of insulin release from the pancreatic beta-cell.

Imidazoline compounds stimulate insulin release by inhibition of K(ATP) channels and interaction with the exocytotic machinery.

A novel imidazoline compound, RX871024, was used to investigate the mechanisms by which imidazoline derivatives promote insulin secretion in rat pancreatic beta-cells and HIT T15 cells. RX871024 stimulated insulin release from rat pancreatic beta-cells and HIT T15 cells in a glucose-dependent way. This effect was not related to alpha2-adrenergic, I1-, and I2-imidazoline receptors. RX871024 promoted insulin release by at least two modes of action. One included an increase in cytoplasmic free Ca2+ concentration ([Ca2+]i), subsequent to blocking of ATP-dependent K+ channels, membrane depolarization, and activation of voltage-dependent Ca2+ channels. The other, a more distal effect of imidazoline, affected the exocytotic machinery and was unrelated to changes in membrane potential and [Ca2+]i. The mechanism of RX871024-induced insulin release was dependent on protein kinases A and C. The sensitizing effect of a low dose of RX871024 on glucose-induced insulin secretion suggests that imidazoline compounds of this kind may constitute the basis for development of a new class of oral hypoglycemic agents.

Identification of functional ionotropic glutamate receptor proteins in pancreatic β-cells and in islets of Langerhans

The presence of ionotropic glutamate receptor proteins in islets of Langerhans and pancreatic β-cell lines (MIN6, HIT T15, RINm5F) was investigated. For this purpose immunoblot analysis of β-cell membranes was performed with subunit-specific antibodies. We identified NMDAR1 subunits of the NMDA and KA-2 subunits of the kainate receptors, but did not detect GluR1 subunits of the AMPA receptor. The receptor subunits present were shown to be glycosylated. β-cell membranes contained specific binding sites for glutamate receptor ligands, and NMDA increased insulin secretion. These results demonstrate that ionotropic glutamate receptor proteins, similar to those in the central nervous system, are expressed in rat pancreatic β-cells.

3H]cirazoline as a tool for the characterization of imidazoline sites.

Recent studies have shown that cirazoline, an alpha 1-adrenoceptor agonist, has greater affinity than do other imidazoline or guanidinium compounds at imidazoline recognition sites. In this report we used [3H]cirazoline as a probe to characterize imidazoline recognition sites present in membrane homogenates of rat brain and kidney as well as pancreatic beta HIT T15 cells. Specific binding of [3H]cirazoline to these various homogenates was saturable and reversible and was resolved into two classes of high affinity binding sites. Competition inhibition studies of [3H]cirazoline binding to these different membrane preparations were performed with alkaloid, phenylethylamine, imidazoline, and guanidinium compounds. Catecholamines and non-imidazoline adrenoceptor ligands such as epinephrine, benextramine, prazosin, propranolol, rauwolscine, or adrenoceptor ligands such as epinephrine, benextramine, prazosin, propranolol, rauwolscine, or yohimbine did not compete with [3H]cirazoline (Ki > 10 microM). Under our experimental conditions, only guanidinium and imidazoline derivatives had high affinities for [3H]cirazoline binding sites. Unlabeled cirazoline, clonidine, bromoxidine, idazoxan, and amiloride had the highest affinities with this respective rank order. These results suggest that [3H]cirazoline is a novel high affinity radioligand that specifically labels nonadrenergic imidazoline-guanidinium sites in the brain, kidney, and beta cells. Furthermore, the obtained rank order of inhibition suggests that [3H]cirazoline binding does not distinguish between I1 and I2 sites. In addition, we compared the specific binding of [3H]cirazoline with that of the alpha 2-adrenoceptor antagonist [3H]rauwolscine in chinese hamster ovary (CHO) cell lines stably expressing human alpha 2C2-, alpha 2C4-, and alpha 2C10-adrenoceptor subtypes. Using [3H]rauwolscine as a probe, each of these transfected cell lines expressed high levels for the three different alpha 2-adrenoceptor subtypes (Bmax values were between 2 and 7 pmol.mg-1 protein). In contrast, none of these cell lines displayed measurable imidazoline recognition sites. In summary, [3H]cirazoline is a novel high affinity radioligand that specifically labels imidazoline recognition sites without significant alpha- or beta-adrenoceptor binding. Furthermore, our results using alpha 2-adrenoceptor transfected cells confirm that the imidazoline recognition sites and each of the cloned alpha 2-adrenoceptor subtypes represent distinct macromolecular entities.

Characterisation of endoplasmic reticulum and plasma membrane Ca 2+-ATPases in pancreatic β-cells and in islets of Langerhans

We have investigated the plasma membrane (PMCA) and endoplasmic reticulum (SERCA) Ca 2+-ATPases involved in active transport of Ca 2+ in pancreatic β-cell lines (MIN6, HIT T15, RINm5F) and in islets of Langerhans. Under selective membrane phosphorylation conditions (at low ATP concentration, in the presence of Ca 2+ and La 3+ and in the absence of Mg 2+ at 4°C) the only labelled proteins are the phosphoenzyme intermediates of the Ca 2+-ATPases. Under these conditions, β-cells membranes incorporated 32P from [γ- 32P]ATP into two proteins with molecular mass on acidic SDS-polyacrylamide gels of around 115 and 150 kDa. The 150 kDa band was identified as PMCA (i) by reaction with a monoclonal anti-human erythrocyte plasma membrane Ca 2+-ATPase antibody; (ii) by its typical tryptic cleavage pattern which generated an 80 kDa band; (iii) by lack of inhibition of its autophosphorylation by SERCA-specific inhibitors. The 115 kDa band was identified as SERCA (i) by reaction with a polyclonal anti-rat fast skeletal muscle Ca 2+-ATPase antibody; (ii) by the concentration-dependent inhibition of its autophosphorylation by thapsigargin and 2,5- di( t-butyl)-1,4-benzohydroquinone (tBHQ), which are specific inhibitors of SERCA. The 115 kDa band was further characterised as the SERCA-2b isoform by reaction with a polyclonal rabbit antibody against the 12 C-terminal amino acids of SERCA-2b.

P1-305 - Effects of lavendustin A. a tyrosine kinase inhibitor. on insulin release from a pancreatic B-cell line. HIT T15

P1-305 - Effects of lavendustin A. a tyrosine kinase inhibitor. on insulin release from a pancreatic B-cell line. HIT T15