adenosine-3--5--cyclic-phosphorothioate and Insulinoma

It has previously been suggested that ACTH and ACTH-related peptides may act as paracrine modulators of insulin secretion in the islets of Langerhans. We have, therefore, examined the expression and function of the ACTH receptor (the melanocortin 2 receptor, MC2-R) in human and mouse primary islet tIssue and in the MIN6 mouse insulinoma cell line. Mouse MC2-R mRNA was detected in both MIN6 cells and mouse islet tIssue by PCR amplification of cDNA. In perifusion experiments with MIN6 pseudo-islets, a small, transient increase in insulin secretion was obtained when ACTH(1-24) (1 nM) was added to medium containing 2 mM glucose (control) but not when the medium glucose content was increased to 8 mM. Further investigations were performed using static incubations of MIN6 cell monolayers; ACTH(1-24) (1 pM-10 nM) provoked a concentration-dependent increase in insulin secretion from MIN6 monolayer cells that achieved statistical significance at concentrations of 1 and 10 nM (150 +/- 13.6% basal secretion; 187 +/- 14.9% basal secretion, P<0.01). Similar responses were obtained with ACTH(1-39). The phosphodiesterase inhibitor IBMX (100 microM) potentiated the responses to sub-maximal doses of ACTH(1-24). Two inhibitors of the protein kinase A (PKA) signaling pathway, Rp-cAMPS (500 microM) and H-89 (10 microM), abolished the insulin secretory response to ACTH(1-24) (0.5-10 nM). Treatment with 1 nM ACTH(1-24) caused a small, statistically significant increase in intracellular cAMP levels. Secretory responses of MIN6 cells to ACTH(1-24) were also influenced by changes in extracellular Ca2+ levels. Incubation in Ca2+-free buffer supplemented with 0.1 mM EGTA blocked the MIN6 cells' secretory response to 1 and 10 nM ACTH(1-24). Similar results were obtained when a Ca2+ channel blocker (nitrendipine, 10 microM) was added to the Ca2+-containing buffer. ACTH(1-24) also evoked an insulin secretory response from primary tIssues. The addition of ACTH(1-24) (0.5 nM) to perifusions of mouse islets induced a transient increase in insulin secretion at 8 mM glucose. Perifused human primary islets also showed a secretory response to ACTH(1-24) at basal glucose concentration (2 mM) with a rapid initial spike in insulin secretion followed by a decline to basal levels. Overall the results demonstrate that the MC2-R is expressed in beta-cells and suggest that activation of the receptor by ACTH initiates insulin secretion through the activation of PKA in association with Ca2+ influx into

Topics: 1-Methyl-3-isobutylxanthine; Adrenocorticotropic Hormone; Animals; Calcium Channel Blockers; Cell Line; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Humans; Insulin; Insulin Secretion; Insulinoma; Islets of Langerhans; Isoquinolines; Mice; Nitrendipine; Paracrine Communication; Phosphodiesterase Inhibitors; Receptor, Melanocortin, Type 2; RNA, Messenger; Stimulation, Chemical; Sulfonamides; Thionucleotides

The regulation of a calcium-activated nonselective cation (Ca-NS+) channel by analogues of cyclic AMP has been investigated in the rat insulinoma cell line, CRI-G1. The activity of the channel is modulated by cyclic AMP in a complex way. In the majority of patches (83%) tested concentrations of cyclic AMP of 10 microM and above cause an inhibition of channel activity which is immediately reversible on washing. In contrast, lower concentrations of cyclic AMP, between 0.1 and 1.0 microM, produce a transient activation of channel activity in most patches (63%) tested. One group of analogues, including N6-monobutyryl cyclic AMP and N6, 2'-O-dibutyryl cyclic AMP reduced the activity of the Ca-NS+ channel at all concentrations tested and 2'-O-Monobutyryl cyclic AMP produced inhibition in all patches tested except one, at all concentrations. A second group produced dual concentration-dependent effects on Ca-NS+, low concentrations stimulating and high concentrations inhibiting channel activity. 6-Chloropurine cyclic AMP and 8-bromo cyclic AMP produced effects similar to those of cyclic AMP itself. In contrast, 8-[4-chlorophenylthio] cyclic AMP also showed a dual action, but with a high level of activation at all concentrations tested up to 1 mM. Ca-NS+ channel activity was also predominantly activated by low concentrations of Sp-cAMPS. The activating effects of both Sp-cAMPS and cyclic AMP are antagonized by Rp-cAMPS, which by itself only produced a weak inhibition of Ca-NS+ channel activity even at concentrations of 10 microM and above. The results are discussed in terms of a model in which cyclic AMP, and other cyclic nucleotides, modulate the activity of the Ca-NS+ channel by binding to two separate sites.

Topics: Animals; Binding Sites; Bucladesine; Calcium; Cell Line; Cell Membrane; Cyclic AMP; Insulinoma; Ion Channels; Models, Biological; Pancreatic Neoplasms; Rats; Stereoisomerism; Structure-Activity Relationship; Thionucleotides; Tumor Cells, Cultured