cyclic-gmp and Insulinoma

Recent investigations have demonstrated an influence of melatonin on insulin secretion in pancreatic beta-cells. The effects are receptor-mediated via two parallel signaling pathways. The aim of this study was to examine the relevance of a second melatonin receptor (MT2) as well as the involvement of a third signaling cascade in mediating melatonin effects, i.e. the cyclic guanosine monophosphate (cGMP) pathway. Our results demonstrate that the insulin-inhibiting effect of melatonin could be partly reversed by preincubation with the unspecific melatonin receptor antagonist luzindole as well as by the MT2-receptor-specific antagonist 4P-PDOT (4-phenyl-2-propionamidotetraline). As melatonin is known to modulate cGMP concentration via the MT2 receptor, these data indicate transmission of the melatonin effects via the cGMP transduction cascade. Molecular investigations established the presence of different types of guanylate cyclases, cGMP-specific phosphodiesterases and cyclic nucleotide-gated channels in rat insulinoma beta-cells (INS1). Moreover, variations in mRNA expression were found when comparing day and night values as well as different states of glucose metabolism. Incubation experiments provided evidence that 3-isobutyl-1-methylxanthine (IBMX)-stimulated cGMP concentrations were significantly decreased in INS1 cells exposed to melatonin for 1 hr in a dose- and time-dependent manner. This effect could also be reversed by application of luzindole and 4P-PDOT. Stimulation with 8-Br-cGMP resulted in significantly increased insulin production. In conclusion, it could be demonstrated that the melatonin receptor subtype MT2 as well as the cGMP signaling pathway are involved in mediating the insulin-inhibiting effect of melatonin.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Brain; Cell Line, Tumor; Colforsin; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 2; Cyclic Nucleotide-Gated Cation Channels; Diabetes Mellitus, Type 2; Dose-Response Relationship, Drug; Glucose; Guanylate Cyclase; Insulin; Insulin Secretion; Insulin-Secreting Cells; Insulinoma; Melatonin; Pineal Gland; Rats; Rats, Wistar; Receptor, Melatonin, MT2; Signal Transduction; Tetrahydronaphthalenes; Tryptamines

Preceding the onset of type 1 diabetes mellitus, pancreatic islets are infiltrated by macrophages secreting interleukin-1beta (IL-1beta) which induces beta-cell apoptosis and exerts inhibitory actions on islet beta-cell insulin secretion. IL-1beta seems to act chiefly through induction of nitric oxide (NO) synthesis. Hence, IL-1beta and NO have been implicated as key effector molecules in type 1 diabetes mellitus. In this paper, the influence of endogenously produced and exogenously delivered NO on the regulation of cell proliferation, cell viability and discrete parts of the stimulus-secretion coupling in insulin-secreting RINm5F cells was investigated. Because vitamin E may delay diabetes onset in animal models, we also investigated whether tocopherols may protect beta-cells from the suppressive actions of IL-1 and NO in vitro. To this end, the impact of NO on insulin secretory responses to activation of phospholipase C (by carbamylcholine), protein kinase C (by phorbol ester), adenylyl cyclase (by forskolin), and Ca(2+) influx through voltage-activated Ca(2+) channels (by K(+)-induced depolarization) was monitored in culture after treatment with IL-1beta or by co-incubation with the NO donor spermine-NONOate. It was found that cell proliferation, viability, insulin production and the stimulation of insulin release evoked by carbamylcholine and phorbol ester were impeded by IL-1beta or spermine-NONOate, whereas the hormone output by the other secretagogues was not altered by NO. Pretreatment with gamma-tocopherol (but not alpha-tocopherol) afforded a partial protection against the inhibitory effects of NO, whereas specifically inhibiting inducible NO synthase with N-nitro-L-arginine completely reversed the IL-1beta effects. In contrast, inhibiting guanylyl cyclase with ODQ (1H-[1,2, 4]oxadiazolo[4,3-alpha]-quinoxaline-1-one) or blocking low voltage-activated Ca(2+) channels with NiCl(2) failed to influence the actions of NO. In conclusion, our data show that NO inhibits growth and insulin secretion in RINm5F cells, and that gamma-tocopherol may partially prevent this. The results suggest that phospholipase C or protein kinase C may be targeted by NO. In contrast, cGMP or low voltage-activated Ca(2+) channels appear not to mediate the toxicity of NO in these cells. These adverse effects of NO on the beta-cell, and the protection by gamma-tocopherol, may be of importance for the development of the impaired insulin secretion characterizing type 1 diabetes

Topics: Adenylyl Cyclases; Animals; Calcium; Calcium Channels; Carbachol; Cell Division; Cell Line; Cell Survival; Cholinergic Agonists; Colforsin; Cyclic GMP; Enzyme Inhibitors; Insulin; Insulin Secretion; Insulinoma; Interleukin-1; Islets of Langerhans; Models, Chemical; Nitric Oxide; Nitrogen Oxides; Oxadiazoles; Phorbol Esters; Protein Kinase C; Quinoxalines; Rats; Spermine; Type C Phospholipases; Vitamin E

The effect of nitric oxide (NO) donors on high-voltage-activated Ca2+ channels in insulin-secreting RINm5F cells was investigated using the patch-clamp technique in the whole-cell configuration. Sodium nitroprusside (SNP, 2-400 microM) induced a dose-dependent reduction in Ba2+ currents with maximal inhibition of 58%. The IC50 for SNP was 45 microM. A different NO donor, (+/-)S-nitroso-N-acetylpenicillamine (SNAP, 500 microM), also produced a 50% decrease in current amplitude. When 200 microM SNP was administered together with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidozoline-1-oxyl-3-oxide (carboxy-PTIO, 300 microM), the Ba2+ current inhibition was lowered to 7%. Administration of 500 microM 8-bromoguanosine 3':5'-cyclic monophosphate sodium salt (8-Br-cGMP) mimicked the effects of SNP, causing a comparable decrease (56%) in peak-current amplitude. When soluble guanylyl cyclase was blocked by 10 microM 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ), the inhibitory effect of 200 microM SNP was reduced from 39% to 15%. The SNP-induced current decrease was 36% of controls after the blockade of L-type Ca2+ channels and 30% in the presence of 2.5 microM omega-conotoxin-MVIIC. These data indicate that NO inhibits both L-type and P/Q-type Ca2+ channels in RINm5F cells, probably by an increase in the intracellular levels of cGMP. NO may then significantly influence the Ca2+-dependent release of hormones from secretory cells as well as that of neurotransmitters from nerve terminals.

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Cyclic GMP; Electrophysiology; Enzyme Inhibitors; Insulinoma; Membrane Potentials; Nitric Oxide; Pancreatic Neoplasms; Patch-Clamp Techniques; Penicillamine; Rats; S-Nitroso-N-Acetylpenicillamine; Sodium-Potassium-Exchanging ATPase; Tumor Cells, Cultured

Topics: Amino Acid Sequence; Animals; Bacterial Toxins; Cyclic GMP; Enterotoxins; Escherichia coli Proteins; Guanylate Cyclase; In Vitro Techniques; Insulinoma; Islets of Langerhans; Molecular Sequence Data; Nitroprusside; Pancreatic Neoplasms; Point Mutation; Rats; RNA, Messenger; Sequence Deletion; Signal Transduction; Transfection; Tumor Cells, Cultured

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Cyclic GMP; Diabetes Mellitus, Type 1; Free Radicals; Humans; Insulinoma; Interferon-gamma; Interleukin-1; Islets of Langerhans; Nitric Oxide; Nitric Oxide Synthase; omega-N-Methylarginine; Pancreatic Neoplasms; Rats; Tumor Cells, Cultured

L-Arginine (L-Arg) is metabolized by nitric oxide synthase to the reactive intermediate nitric oxide. Since nitric oxide stimulates guanylyl cyclase and cGMP synthesis, L-Arg effects on cGMP accumulation in isolated pancreatic islets of the rat and RINm5F insulinoma cells were determined. Both L-Arg and glucose stimulation increased islet cGMP levels, and glucose potentiated the response to L-Arg alone. A competitive inhibitor of L-Arg metabolism to nitric oxide, NG-monomethyl-L-arginine, reduced glucose- and L-Arg-stimulated insulin release and glucose-induced increases in cGMP; however, basal insulin release was slightly increased. D-Arg and L-ornithine did not affect islet cGMP levels, although insulin release was stimulated. RINm5F cell cGMP levels and insulin release increased in response to L-Arg in a concentration- and time-related manner, whereas glucose and L-histidine were without effect. 8-Bromo-cGMP also slightly increased RINm5F cell insulin release. Sodium nitroprusside as a source of nitric oxide increased RINm5F cell cGMP production. Methylene blue and LY83583, inhibitors of soluble guanylyl cyclase activation, reduced RINm5F cell cGMP levels in the presence and absence of L-Arg; LY83583 also reduced glucose-stimulated cGMP levels in islets. Insulin release by glucose and L-Arg was also inhibited by methylene blue and LY83583 in islets. We conclude that glucose and L-Arg stimulate guanylyl cyclase activity and cGMP formation in beta-cells at least in part through metabolism to the reactive intermediate nitric oxide. However, neither nitric oxide nor cGMP synthesis is obligatory for insulin secretion.

Topics: Aminoquinolines; Animals; Arginine; Cyclic GMP; Glucose; Guanylate Cyclase; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Male; Methylene Blue; Nitroprusside; Pancreatic Neoplasms; Rats; Rats, Inbred Strains; Tumor Cells, Cultured

Topics: Adenoma, Islet Cell; Animals; Atrial Natriuretic Factor; Cyclic GMP; Insulin; Insulin Secretion; Insulinoma; Pancreatic Neoplasms; Rats; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface; Tumor Cells, Cultured