Estradiol-stimulated nitric oxide release in human granulocytes is dependent on intracellular calcium transients: evidence of a cell surface estrogenreceptor

We tested the hypothesis that estrogen acutely stimulates constitutive nitr ic oxide synthase activity in human granulocytes by acting on a cell surfac e estrogen receptor (ER), The release of nitric oxide was measured in real time with an amperometric probe, Exposure of granulocytes to 17 beta-estrad iol stimulated NO release within seconds in a concentration-dependent manne r. The NO release was also stimulated by 17 beta-estradiol conjugated to bo vine serum albumin (E-2-BSA), which suggests mediation by a cell surface re ceptor. Tamoxifen, an ER inhibitor, antagonized the action of both 17 beta- estradiol and E-2-BSA, whereas ICI 182,780, an inhibitor of the nuclear ER, had no effect. Using dual emission microfluorometry in a calcium-free medi um, the 17 beta-estradiol-stimulated release of NO from granulocytes was sh own to be dependent on intracellular calcium ([Ca2+]i) transients in a tamo xifen-sensitive process. Exposure to BAPTA-AM (1,2bis-(-aminophenoxy)ethans -N,N,N',N'-tetraacetic acid tetra(acetoxyymethyl) ester), a [Ca2+]i chelato r, reduced [Ca2+]i in response to E-2-BSA, and depleting [Ca2+]i stores abo lished the effect of 17 beta-estradiol on NO release, Confocal photomicrogr aphs using E-2-BSA-FITC (fluorescein isothiocyanate) revealed cell membrane reactivity, Estrogen-stimulated NO release had an immunosuppressive effect , and it initiated granulocyte rounding and loss of adherence in a tamoxife n-sensitive manner. Finally, using reverse transcriptase-polymerase chain r eaction, human neutrophil granulocytes expressed ER alpha but not ER beta, suggesting that ER alpha may be the membrane receptor for 17 beta-estradiol , The study demonstrated that a physiological dose of estrogen down-regulat es granulocyte activity by acutely stimulating NO release via the activatio n of a cell surface ER which is coupled to increases in [Ca2+]i, (C) 2000 b y The American Society of Hematology .