Non-genomic actions of 17 beta-oestradiol in mouse pancreatic beta-cells are mediated by a cGMP-dependent protein kinase

1. Intracellular calcium concentration ([Ca2+](i)) was measured in mouse wh ole islets of Langerhans using the calcium-sensitive fluorescent dye Indo-1 . 2. Application of physiological concentrations of 17 beta-oestradiol in the presence of a stimulatory glucose concentration (8 mM) potentiated the [Ca 2+](i) signal in 83% of islets tested. Potentiation was manifested as eithe r an increase in the frequency or duration of [Ca2+](i) oscillations. 3. The effects caused by 17 beta-oestradiol were mimicked by the cyclic nuc leotide analogues 8-bromoguanosine-3',5'-cyclic monophosphate (8-Br-cGMP) a nd 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP). 4. Direct measurements of both cyclic nucleotides demonstrated that nanomol ar concentrations of 17 beta-oestradiol in the presence of 8 mM glucose inc reased cGMP levels, yet cAMP levels were unchanged. The increment in cGMP w as similar to that induced by 11 mM glucose. 5. Patch-clamp recording in intact cells showed that 8-Br-cGMP reproduced t he inhibitory action of 17 beta-oestradiol on ATP-sensitive K+ (K-ATP) chan nel activity This was not a membrane-bound effect since it could not be obs erved in excised patches. 6. The action of 17 beta-oestradiol on K-ATP channel activity was not modif ied by the specific inhibitor of soluble guanylate cyclase (sGC) LY 83583. This result indicates a likely involvement of a membrane guanylate cyclase (mGC). 7. The rapid decrease in K-ATP channel activity elicited by 17 beta-oestrad iol was greatly reduced using Rp-8-pCPT-cGMPS, a specific blocker of cGMP-d ependent protein kinase (PKG). Conversely, Rp-cAMPS, which inhibits cAMP-de pendent protein kinase (PKA), had little effect, 8. The results presented here indicate that rapid, non-genomic effects of 1 7 beta-oestradiol after interaction with its binding site at the plasma mem brane of pancreatic beta-cells is a cGMP-dependent phosphorylation process.