Estradiol-stimulated nitric oxide release in human granulocytes is dependent on intracellular calcium transients: evidence of a cell surface estrogen receptor.
We tested the hypothesis that estrogen acutely stimulates constitutive nitric oxide synthase activity in human granulocytes by acting on a cell surface estrogen receptor (ER). The release of nitric oxide was measured in real time with an amperometric probe. Exposure of granulocytes to 17beta-estradiol stimulated NO release within seconds in a concentration-dependent manner. The NO release was also stimulated by 17beta-estradiol conjugated to bovine serum albumin (E(2)-BSA), which suggests mediation by a cell surface receptor. Tamoxifen, an ER inhibitor, antagonized the action of both 17beta-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 medium, the 17beta-estradiol-stimulated release of NO from granulocytes was shown to be dependent on intracellular calcium ([Ca(2+)]i) transients in a tamoxifen-sensitive process. Exposure to BAPTA-AM (1,2bis-(-aminophenoxy)ethans-N,N,N', N'-tetraacetic acid tetra(acetoxyymethyl) ester), a [Ca(2+)]i chelator, reduced [Ca(2+)]i in response to E(2)-BSA, and depleting [Ca(2+)]i stores abolished the effect of 17beta-estradiol on NO release. Confocal photomicrographs 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 tamoxifen-sensitive manner. Finally, using reverse transcriptase-polymerase chain reaction, human neutrophil granulocytes expressed ERalpha but not ERbeta, suggesting that ERalpha may be the membrane receptor for 17beta-estradiol. The study demonstrated that a physiological dose of estrogen down-regulates granulocyte activity by acutely stimulating NO release via the activation of a cell surface ER which is coupled to increases in [Ca(2+)]i. (Blood. 2000;95:3951-3958)
Stefano
G B
GB
Neuroscience Research Institute, State University of New York at Old Westbury, Old Westbury, NY 11568-0210, USA. stefanog@surg.som.sunysb.edu
Cadet
P
P
Breton
C
C
Goumon
Y
Y
Prevot
V
V
Dessaint
J P
JP
Beauvillain
J C
JC
Roumier
A S
AS
Welters
I
I
Salzet
M
M
D000818:Animals; D002118:Calcium / Q000097:blood; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002417:Cattle; D004958:Estradiol / Q000494:pharmacology*; D047628:Estrogen Receptor alpha; D004966:Estrogens, Conjugated (USP) / Q000494:pharmacology*; D006098:Granulocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D009569:Nitric Oxide / Q000097:blood*; D011960:Receptors, Estrogen / Q000097:blood* / Q000235:genetics; D012710:Serum Albumin, Bovine / Q000494:pharmacology*
D047628:Estrogen Receptor alpha; D004966:Estrogens, Conjugated (USP); D011960:Receptors, Estrogen; C043090:estradiol-bovine serum albumin; D012710:Serum Albumin, Bovine; D009569:Nitric Oxide; D004958:Estradiol; D002118:Calcium