Gender, estrogen, and NOS: cautions about generalizations.

Evidence from observational studies as well as prospective randomized trials indicates that the incidence of coronary artery disease is less in premenopausal women compared with age-matched men and in postmenopausal women who are using estrogen replacement therapy.1,2 The mechanisms by which estrogen reduces development of cardiovascular disease are multifactorial and, in addition to alterations in lipid metabolism, include actions on all components of the vascular wall (endothelial, smooth muscle, and adventitial cells), neurons, and blood elements (platelets and leukocytes). Changes in production of nitric oxide (NO) have been implicated as one of the cellular biochemical-related pathways regulated by estrogen that may contribute to gender and hormonal differences in the progression of cardiovascular disease.3–9 NO is synthesized from L-arginine by the enzyme nitric oxide synthase (NOS). NOS consists of three isoforms: type I, neuronal; type II, inducible; and type III, endothelial/ constitutive. In this issue of Circulation Research, Garcı́aDurán et al10 add to the accumulating body of evidence suggesting that estrogen directly modulates expression of NOS, in particular the neuronal isoform (type I) in neutrophils. With use of Western blot analysis of protein isolated from neutrophils, levels of neuronal NOS were greater in neutrophils from premenopausal women during the ovulatory phase of the estrus cycle when estrogen is high compared with the follicular phase when circulating levels of estrogen fall. In addition, expression of neuronal NOS increased in neutrophils of postmenopausal women who were using transdermal estrogen replacement (50 mg/d) for 4 months. The range of circulating estrogen over which these changes in neuronal NOS occurred was physiological, between 5310 and 2310 mol/L. These observations suggest that changes in circulating levels of oxidized products of nitric oxide in blood of women during different stages of the estrus cycle as well as with estrogen replacement therapy may be derived from cells other than the those of the endothelium.11–14 Garcı́a-Durán et al10 examined the gender specificity of estrogen on expressional control of neuronal NOS using neutrophils from male subjects. In these cells, there was both a time and dose dependency of induction of neuronal NOS by estrogen. This induction was receptor mediated and showed biphasic stimulation with increases in NOS from 10 to 10 mol/L of estrogen and inhibition at higher concentrations. These concentration ranges are similar to those shown to stimulate neuronal NOS isolated from rabbit cerebellum.15 The observation that estrogen can modulate NOS isoform in male cells is consistent with observations of increases in reactive hyperemia, a vascular response mediated by endothelium-derived NOS, in male to female transsexuals.16 As provocative as these results might be, much remains to be learned regarding effects of sex steroid hormones on leukocyte function and how these effects relate to gender differences in expression of cardiovascular or other diseases. Although the kinetics of the response of induction of neuronal NOS was defined for neutrophils isolated from males, the question arises as to whether the kinetic relationship of induction of neuronal NOS might be altered on a background of concentrations of endogenous hormones such as estrogen itself, progesterone, or testosterone, as has been reported for macrophages.17 The physiological significance of modulation of NOS isoforms by estrogen remains to be tested definitively in different models of cardiovascular disease. Although mechanical vascular injury and high-cholesterol feeding are common manipulations in experimental animals to mimic the pathogenesis of cardiovascular disease in humans, few experimental studies are designed to examine relationships between cardiovascular disease and infections such as Chlamydia, cytomegalovirus, endocarditis, syphilis, or human immunodeficiency virus.18–24 Functionally, increased expression of neuronal NOS in male neutrophils correlated with increased production of NO, reduced expression of CD18 antigen, and reduced adhesion of neutrophils to a plastic surface. Decreased neutrophil adherence may not be “beneficial” when neutrophil infiltration is a first-line defense to limit infective processes. With immunological challenge, NO will be induced in cells other than neutrophils, and cytokines will increase expression of type II inducible NOS.25,26 Because NOS is regulated by NO itself, increased production of NO from other isoforms, especially the inducible isoform, may in fact decrease NO production in other cell types independent of the mechanism by which that isoform of NOS may be upregulated by estrogen. Therefore, effects of modulation of NOS isoforms by estrogen should be considered in the context of integrated physiological function. Expressional control of neuronal NOS in male neutrophils seems to be initiated by receptor activation. However, the specific subtype of the estrogen receptor, that is, estrogen receptor a or b, was not identified. Expression of estrogen The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Departments of Surgery and Physiology, The Mayo Clinic and Foundation, Rochester, Minn. Correspondence to Virginia M. Miller, PhD, The Mayo Clinic and Foundation, Departments of Surgery and Physiology, 200 First St SW, Rochester, MN 55905. E-mail [email protected] (Circ Res. 1999;85:979-981.) © 1999 American Heart Association, Inc.