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

1 Intracellular calcium concentration ([Ca 2+ ] 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 m m ) potentiated the [Ca 2+ ] i signal in 83 % of islets tested. Potentiation was manifested as either an increase in the frequency or duration of [Ca 2+ ] 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 m m glucose increased cGMP levels, yet cAMP levels were unchanged. The increment in cGMP was similar to that induced by 11 m m glucose. 5 Patch‐clamp recording in intact cells showed that 8‐Br‐cGMP reproduced the inhibitory action of 17β‐oestradiol on ATP‐sensitive K + (K ATP ) 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 K ATP 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 K ATP 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.