The direct effects of an enkephalin analogue, (D-Ala2/MePhe4/Met/(O)-o1) enkephalin (DAMME), on insulin release from isolated islets of Langerhans of the rat have been investigated. DAMME had a dose-dependent effect on insulin secretion: low concentrations (10(-10) to 10(-8) mol/l) were stimulatory while high concentrations (10(-5) mol/l) were inhibitory in the presence of 8 mmol/l glucose. Similar effects were found with met-enkephalin, and with the longer acting alanine substituted met-enkephalin. Morphine sulphate (5 X 10(-7) mol/l) also stimulated insulin release. The effects of enkephalin and morphine were blocked by the specific opiate antagonist naloxone hydrochloride (1.2 X 10(-6) mol/l). The insulin secretory response of perifused islets to enkephalins and morphine was rapid, corresponding to the first phase of glucose induced insulin release. These observations suggest that there may be opiate receptors in islets, and that opioid peptides could modulate insulin release.

We have shown that nitric oxide treatment for 30-90 min causes inhibition of insulin secretion, DNA damage and disturbs sub-cellular organization in rat and human islets of Langerhans and HIT-T15 cells. Here rat islets and beta-cell lines were treated with various free radical generating systems S-nitrosoglutathione (nitric oxide), xanthine oxidase plus hypoxanthine (reactive oxygen species), 3-morpholinosydnonimine (nitric oxide, super-oxide, peroxynitrite, hydrogen peroxide) and peroxynitrite and their effects over 4 h to 3 days compared with those of the cytokine combination interleukin-1beta, tumour necrosis factor-alpha and interferon-gamma. End points examined were de novo protein synthesis, cellular reducing capacity, morphological changes and apoptosis by acridine orange cytochemistry, DNA gel electrophoresis and electron microscopy. Treatment (24-72 h) with nitric oxide, superoxide, peroxynitrite or combined cytokines differentially decreased redox function and inhibited protein synthesis in rat islets of Langerhans and in insulin-containing cell lines; cytokine effects were arginine and nitric oxide dependent. Peroxynitrite gave rare apoptosis in HIT-T15 cells and superoxide gave none in any cell type, but caused the most beta cell-specific damage in islets. S-nitroso-glutathione was the most effective agent at causing DNA laddering or chromatin margination characteristic of apoptotic cell death in insulin-containing cells. Cytokine-induced apoptosis was observed specifically in islet beta cells, combined cytokine effects on islet function and death most resembled those of the mixed radical donor SIN-1.

When islets were cultured with interleukin-1 beta (1 or 100 pmol/l) for 12 h in arginine-containing medium, cyclic GMP levels were increased 1.6- and 4.5-fold respectively. The arginine analogue, N-omega-nitro-L-arginine methyl ester, which blocks nitric oxide formation and partially reverses inhibition of insulin secretion by 100 pmol/l interleukin-1 beta, largely, but not completely, blocked generation of cyclic GMP. Treatment of islets with 100 pmol/l interleukin-1 beta for 12 h significantly decreased islet cyclic AMP generation in the absence of isobutylmethylxanthine (from 13.1 +/- 0.7 to 9.3 +/- 0.8 fmol/micrograms islet protein), this fall was arginine-dependent and may have resulted from an effect on a cyclic AMP phosphodiesterase, since it was masked if isobutylmethylxanthine was present. Isobutylmethylxanthine (0.4 mmol/l) reduced the inhibitory potency of interleukin-1 beta in 15 h slightly but significantly from 80.5 to 59.0%. The morpholinosydnonimine SIN-1, which is a nitric oxide donor, inhibited insulin secretion, raised islet cyclic GMP and lowered cyclic AMP; its effects were similar to those of interleukin-1 beta. However, 6-anilinoquinoline-5,8-quinone, [LY83583 (1-10 mumol/l)], inhibited insulin secretion, and significantly decreased cyclic GMP while 8-bromocyclic GMP stimulated insulin secretion. Both low- and high-dose interleukin-1 beta treatment give a large arginine-dependent and a small, yet significant, arginine-independent increase in cyclic GMP. The inhibitory effect of SIN-1 or interleukin-1 beta on insulin secretion seems to depend to a small extent on decreased islet cyclic AMP, though sustained increases in nitric oxide or depleted islet GTP may directly affect the secretory process.

1. The concentrations of cyclic AMP were compared in islets of Langerhans isolated from the pancreases of normal female and pregnant rats and were higher in islets in pregnancy. 2. There was also a significant increase in adenylate cyclase activity in homogenates of islets from pregnant rats compared with those from normal rats. 3. Increased cyclic AMP concentration in islets from pregnant rats was reflected in increased protein kinase activity. When the cyclic AMP-dependent protein kinase activity was increased by 3-isobutyl-1-methylxanthine this stimulated activity was significantly greater in pregnancy. 4. Insulin-secretion studies with islets from normal and pregnant rats showed that theophylline or 3-isobutyl-1-methylxanthine, which raise intracellular cyclic AMP concentrations, caused a significantly greater insulin secretion in pregnancy. 5. It was also found that in the presence of a glucose concentration too low to stimulate insulin secretion, the latter could be induced if the cyclic AMP concentrations were raised sufficiently with 3-isobutyl-1-methylxanthine. 6. It is suggested that the higher cyclic AMP concentrations observed in islets in pregnancy mediate the greater insulin-secretory capacity, as well as the greater sensitivity of these islets to low glucose concentrations.