Non-genomic actions of 17beta-oestradiol in mouse pancreatic beta-cells are mediated by a cGMP-dependent protein kinase.
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 17beta-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 17beta-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 17beta-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 17beta-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 17beta-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 17beta-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 17beta-oestradiol after interaction with its binding site at the plasma membrane of pancreatic beta-cells is a cGMP-dependent phosphorylation process.
Ropero
A B
AB
Institute of Bioengineering and Department of Physiology, Miguel Hernández University, San Juan Campus, Alicante, Spain.
Fuentes
E
E
Rovira
J M
JM
Ripoll
C
C
Soria
B
B
Nadal
A
A
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology*; D005456:Fluorescent Dyes; D007211:Indoles; D007515:Islets of Langerhans / Q000187:drug effects* / Q000201:enzymology*; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000502:physiology; D013873:Thionucleotides / Q000494:pharmacology
C090860:8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphorothioate; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007211:Indoles; D015221:Potassium Channels; D013873:Thionucleotides; D015124:8-Bromo Cyclic Adenosine Monophosphate; D004958:Estradiol; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D006152:Cyclic GMP; C048960:indo-1; D002118:Calcium