Rapid effect of estrogen on ATP-induced [Ca2+]1 signaling in cultured DRG neurons from wild type and ERKO mice

Abstract

Background: There is an increasing awareness of sex-related differences in pain mechanisms underlying differences in clinical presentations of pain syndromes. One such mechanism may he the interaction of putative nociceptive stimuli and estrogen receptors (ER) on peripheral afferent nerve terminalS. We have previously demonstrated that estrogen (1713estradiol) rapidly attenuates the ATP-induced (P2X receptor mediated) [Ca2+], increase by inhibiting L-type voltage-gated calcium channels in cultured DRG neurons from male rats (Chaban et al, 2003). Aims: To study the rapid effect of 17[3-estradiol on ATP-induced lCa2§ transients in cultured DRG neurons from wild type and estrogen receptor-a 'knock out' (ERKO) mice. Methods: Primary cultures of mice lumbosacral DRG neurons were collected under sterile technique, loaded with Fura-2 and tested for ATP-induced changes in [Ca 2. ], by fluorescent ratio imaging. A polyclonal antibody was used for ER immunocytochemistry. Results: Cultured mice DRG neurons expressed both ERc~ and Er[3 by immunocytochemistry. ATP (20 p-M), an algesic agent, caused [Ca2+]~ transients in 48% of small to medium size DRG neurons (25-40 I~M) from wild type mice (273_ + 124 nM, n = 38). A 5 rain incubation with estradiol (50 nM1 p.M) inhibited ATP-induced [Ca2*]~ (137 -+ 11.3, P < .05) with [C5o = 17 nM. The effect of estradiol was reversible. Pretreatment with purinoreceptor antagonist PPADS (5 jzM) or chelating extracellular Ca 2+ with BAPTA (10 raM) eliminated ATP-induced [Ca2+]~ transients. Estradiol coupled to bovine serum albumin (E-6-BSA), which does not penetrate the plasma membrane had the same effect, suggesting that estrogen acts at membrane-associated estrogen receptor. This effect was steroidand stereo-specific since pure estrogen receptor antagonist ICI 182,780 (1 p-M) blocked the estrogen-induced inhibition and 17cx-estradiol had no effect. In DRG neurons from ER~x 'knock out' mice, estradiol did not block ATP-induced [Ca2+]~ flux indicating that the estrogen inhibition is dependent on ERa. Conclusions: 1) Estrogen presumably acts through a membrane receptor similar to ERa to modulate the P2X receptor mediated [Ca a§ ]~ response of DRG cells. 2) The present results demonstrate for the first time an important nonreproductive role of ERc~ in modulating Ca 2+ signaling at the level of primary afferent neuron, thereby modulating the sensitivity to painful stimuli in the periphery. Supported by NIH grants P50 DK64539 (EAM), DK58173 (EAM) and DA13185 (PEM)