Rapid non-genomic activation of cytosolic cyclic AMP-dependent protein kinase activity and [Ca2+]i by 17β-oestradiol in female rat distal colon

1 In this study, the effect of 17 beta-oestradiol on adenosine 3':5'-cyclic monophosphate (cyclic AMP)dependent protein kinase (PKA) activity was investigated. 2 Rapid (within 15 min) activation of basal PKA activity was observed in cytosolic fractions by 17 beta-oestradiol but not by 17 alpha-oestradiol, progesterone or testosterone. This stimulation was abolished by the specific PKA inhibitor PKI but not by the classical oestrogen receptor antagonist tamoxifen. 3 17 beta-Oestradiol did not stimulate basal PKA activity in membrane fractions or in cytosolic fractions from male rats. 4 The increase in cytosolic PKA activity was indirect as (i) it was inhibited by the adenylyl cyclase inhibitor SQ22536, (ii) it was mimicked by forskolin and (iii) 17 beta-oestradiol did not cause a stimulation of basal PKA activity in either type I or type II commercially available PKA holoenzymes. 5 Protein kinase C delta (PKC delta) was directly activated by 17 beta-oestradiol. The specific PKC inhibitor, bisindolylmaleimide I (GF 109203X), abolished the 17 beta-oestradiol-induced PKA activation. 6 17 beta-Oestradiol stimulated an increase in free intracellular calcium ion concentration ([Ca2+](i)) in isolated female but not male rat colonic crypts. This was inhibited by verapamil, nifedipine and zero extracellular [Ca2+] but unaffected by tamoxifen. 17 alpha-Oestradiol, testosterone and progesterone failed to increase [Ca2+](i). 7 PKC and PKA inhibitors abolished the 17 beta-oestradiol-induced increase in [Ca2+](i). 8 These results demonstrate the existence of a novel 17 beta-oestradiol-specific PKA and Ca2+ signalling pathway, which is both sex steroid- and gender-specific, in rat distal colonic epithelium.