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

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Abstract
1 Intracellular calcium concentration ([Ca2+]i) was measured in mouse whole islets of Langerhans using the calcium-sensitive fluorescent dye Indo-1. 2 Application of physiological concentrations of 17β-oestradiol in the presence of a stimulatory glucose concentration (8 mm) potentiated the [Ca2+]i signal in 83 % of islets tested. Potentiation was manifested as either an increase in the frequency or duration of [Ca2+]i oscillations. 3 The effects caused by 17β-oestradiol were mimicked by the cyclic nucleotide analogues 8-bromoguanosine-3′,5′-cyclic monophosphate (8-Br-cGMP) and 8-bromoadenosine-3′,5′-cyclic monophosphate (8-Br-cAMP). 4 Direct measurements of both cyclic nucleotides demonstrated that nanomolar concentrations of 17β-oestradiol in the presence of 8 mm glucose increased cGMP levels, yet cAMP levels were unchanged. The increment in cGMP was similar to that induced by 11 mm glucose. 5 Patch-clamp recording in intact cells showed that 8-Br-cGMP reproduced the inhibitory action of 17β-oestradiol on ATP-sensitive K+ (KATP) channel activity. This was not a membrane-bound effect since it could not be observed in excised patches. 6 The action of 17β-oestradiol on KATP channel activity was not modified 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 KATP channel activity elicited by 17β-oestradiol was greatly reduced using Rp-8-pCPT-cGMPS, a specific blocker of cGMP-dependent protein kinase (PKG). Conversely, Rp-cAMPS, which inhibits cAMP-dependent protein kinase (PKA), had little effect. 8 The results presented here indicate that rapid, non-genomic effects of 17β-oestradiol after interaction with its binding site at the plasma membrane of pancreatic β-cells is a cGMP-dependent phosphorylation process.

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