This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Komesaroff, P. A. Articles by Westerman, R. A. Search for Related Content PubMed PubMed Citation Articles by Komesaroff, P. A. Articles by Westerman, R. A.

A Novel, Nongenomic Action of Estrogen on the Cardiovascular System

Baker Medical Research Institute (P.A.K., C.V.S.B.), PO Box 348, Prahran, Victoria 3181, Australia and International Diabetes Institute (R.A.W.), Kooyong Road, Caulfield, Victoria, Australia

Address all correspondence and requests for reprints to: P.A. Komesaroff, Baker Medical Research Institute, P.O. Box 348, Prahran, Victoria 3181, Australia; E-mail: Paul.Komesaroff{at}Baker.Edu.Au

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
To examine the time course and mechanisms of action of single doses of estrogen on the skin microvasculature, two double-blind placebo-controlled cross-over studies were conducted in healthy young men using the noninvasive technique of laser Doppler velocimetry with iontophoretic application of vasodilator substances. Estradiol (2 mg sublingually) produced a significant increase in the response to the endothelial vasodilator acetylcholine (ACh) after 15 min, but not after 20 or 30 min. The mean plasma estradiol concentration increased from 89.4 ± 9 pmol/L at baseline to 486.6 ± 218 pmol/L at 15 min. An iv bolus of 25 mg conjugated equine estrogens produced significant increases in the responses to ACh at 15 and 20 min but not at 30 min. There was no change in responses to the nonendothelial vasodilators sodium nitroprusside or nicotine, and administration of placebo produced no change in ACh responses at any time point. These experiments show that, at plasma estradiol concentrations within the physiological range for premenopausal women, estrogens act directly on the cutaneous microvasculature through a rapid onset, rapid offset, nongenomic mechanism that is specific to the endothelium; in addition, it supports the view that estrogens can act on the male cardiovascular system in a manner that is potentially clinically beneficial.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
ESTROGENS are widely considered to play a role in reducing the risk of coronary heart disease in women (1), primarily through actions on lipid levels and vascular reactivity in large resistance and capacitance vessels (2). Like other steroid hormones, estrogens act through genomic mechanisms as transcription factors, and there is also evidence for nongenomic mechanisms of action (2, 3). The studies reported here sought to test the hypothesis that a single dose of estrogen that produces physiological estradiol concentrations may, within a nongenomic time frame, lead to significant effects on the microvasculature.

The technique employed was that of laser Doppler velocimetry with iontophoretic application of vasodilator substances to the forearm skin. Direct current iontophoresis is a well-established, safe, and efficient method of effecting transdermal delivery of drugs that has been widely used in neurophysiological research (4). Laser Doppler velocimetry is a noninvasive technique for assessing blood flux in superficial cutaneous microvessels. It involves the direction onto the skin of a noninjurious beam of infrared light from a low power laser via a fibre optic probe and detection of a frequency shift produced by scatter of photons from moving red blood cells. The combination of laser Doppler velocimetry and iontophoresis has been used since the 1980s to study the physiology of neurovascular responses to various stimuli in skin (5) and recently to demonstrate a microvascular endothelial defect in Type II diabetics (6).

The studies examined the blood flux responses to iontophoretic application of acetylcholine (ACh), an endothelium-dependent dilator (7), and the nonendothelial vasodilator sodium nitroprusside (SNP) before and after sublingual and intravenous administration of estrogen and placebo. To clarify mechanisms, the neurogenic flare responses to iontophoretic application of nicotine (8) and to the endothelium-dependent response to ischemia were also assessed.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Laser Doppler velocimetry

Blood flow was measured with a dual channel Moor DT4 laser Doppler flowmeter (Moor Instruments, Devon, England), which employs an 810 nm probe to detect blood flow in the superficial 1–2 mm of skin (9), so that the blood vessels imaged were arterioles, capillaries, post-capillary venules, and venules of the superficial dermal plexus (10). A continuous tracing of blood flux was made using a chart recorder. The time constant applied to the output signal was 0.1 sec for both laser Doppler flowmeters.

Iontophoresis

Drugs were iontophoresed from specially-designed polyvinylchloride chambers containing a reservoir about 0.5 mL in capacity and a central well 6 mm in diameter (for the laser probe) applied to the forearm with adhesive tape (5). The solutions used were acetylcholine (BDH Chemicals, Dorset, UK), sodium nitroprusside BP (David Bull Laboratories, Melbourne, Victoria, Australia) and nicotine sulphate (Sigma, St. Louis, MO), each at a concentration of 10 mg/mL. A current of 0.04 mA was passed for 30 sec through a circular chamber 8 mm in diameter, giving a charge density of 0.08 mA/cm². SNP was administered with a cathodal charge and ACh with an anodal charge. Blood flux was measured continuously for 4 min after completion of stimulus for ACh and nicotine, and for 6 min for SNP.

Post-ischemic hyperemia

For assessment of the post-ischemic hyperemic response, a sphygmomanometer cuff was applied to the upper arm, and flux was recorded continuously before and after inflation to a pressure in excess of systolic pressure for 4.5 min; recording was continued until baseline levels were regained.

Design of experiments

Two randomized, placebo-controlled, double-blind, cross-over studies were conducted on nonsmoking, nonobese healthy males ages 20–45 yr, on no medications. The project was approved by the Alfred Hospital Ethics Committee, and all subjects gave informed consent. Testing was conducted with ACh until a stable baseline was obtained, and then SNP (and, in the intravenous experiment, nicotine) was iontophoresed. In the first experiment (n = 10; mean age 30.5 yr ± 2.9 SEM), a single dose of estradiol (2 mg) (Estrace, Bristol-Myer-Squibb, NJ) or placebo was administered sublingually; in the second (n = 6; mean age 30.3 yr ± 3.8), a single bolus of conjugated equine estrogens (25 mg) (Premarin USP, Ayerst Laboratories, Sydney, Australia) or diluent alone was injected into a right antecubital vein over 1 min. (One vial of Premarin contains lactose 200 mg, sodium citrate 12.5 mg, and dimethyl polysiloxane 0.2 mg; the diluent consists of 2% benzyl alcohol and water for injection USP.) ACh testing was repeated 5, 10, 15, 20 and 30 min after administration of drug, SNP testing after 25 min and, in experiment 2, nicotine after 25 min. Subjects were randomly assigned to estrogen or placebo, and testing was repeated at least a week later with the cross-over substance. For experiment 1, estradiol levels were measured at 0, 15 and 30 min after administration of estrogen by a standard radioimmunoassay. The experiments were carried out in a temperature controlled room (21.5–22.5 C).

Statistics and analysis

Total microvascular blood flux was calculated as the area under the flux curve for the period assessed, and results were quoted as blood flux (units volt.minutes) ± SEM. Data were analyzed for changes in response to ACh, SNP, and nicotine following administration of estrogen or placebo. Comparisons were made, using a paired t-test (with appropriate corrections), between baseline values and integrated flow achieved in the various settings and, using analysis of variance with repeat measures, between responses obtained with estrogen and placebo. Subject numbers were chosen to ensure a power of 70% to detect a change of 10% in response variables. Values of test statistics were considered statistically significant if P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Table 1 shows the effects on blood flux of sublingual and iv estradiol on the vasodilatory responses to ACh, and Figures 1 and 2 show the changes from baseline and the effects of SNP, nicotine, and ischemia. At 15 min after sublingual estrogen administration a significant (P < 0.02) increase in the ACh response was seen compared with baseline, and the total ACh response was significantly greater with estrogen than with placebo (P < 0.001). No significant differences were observed at 20 and 30 min or between the SNP responses after administration of estrogen and placebo. Marked increases in mean plasma estradiol levels were noted at 15 min after sublingual estrogen, and a further increase was seen after 30 min, presumably reflecting continuing gastric absorption (see Table 1).

View larger version (23K): [in this window] [in a new window]   Figure 1. Effects of a single sublingual dose of estrogen, given at t = 0, on forearm microvascular blood flux responses to ACh and SNP (n = 10). Left panel: Blood flux/baseline flux responses to iontophoretically applied ACh at times after sublingual estrogen or placebo; * P < 0.02. Right panel: Response to SNP after estrogen and placebo at t = 25. Bars are + SEM.

View larger version (20K): [in this window] [in a new window]   Figure 2. Effects of a single iv dose of estrogen, given at t = 0, on forearm microvascular blood flux responses to ACh, SNP, nicotine, and ischemia (n = 6). Left panel: blood flux/baseline flux responses to iontophoretically applied ACh at times after IV estrogen or diluent; * P < 0.02. Right panel: Responses to iontophoretic SNP and nicotine and to ischemia after estrogen and placebo. SNP and nicotine were applied at t = 25 and ischemia at t = 30 min.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is well established that estrogens can act on coronary arteries, forearm resistance vessels, and some other vascular beds to increase arterial blood flow and decrease vascular resistance through actions on the vascular endothelium and smooth muscle (2, 7, 11). Hitherto, however, the evidence for a microvascular action has only been indirect: for example, it has been shown that intracoronary estradiol infusion increases coronary flow and decreases coronary resistance (12) and that finger skin blood flow (13) varies with sex hormone status. Because the microvasculature is of importance in controlling local blood flow to end organs, a role for estrogens is likely to be of significant physiological interest.

Classically, estrogens, like other steroid hormones, have been considered to act through their binding to intracellular receptors which act as ligand-dependent transcription factors to stimulate new protein production (14). However, as with other steroid hormones, in recent years evidence has accumulated that they may also act rapidly, though nongenomic mechanisms (15, 16, 17). In particular, several studies have suggested that estrogens in varying doses may potentiate ACh responses in large vessels within a nongenomic time frame (3, 18, 19). In the present study, the rapidity of the response to estrogen administration strongly implies a nongenomic mechanism of action. The results suggest that in the forearm microvascular bed the effects of estrogen become apparent within 15 min of administration of a pulse of hormone, at a time when circulating hormone concentrations correspond to levels occurring in women during the follicular and luteal phases, but do not persist beyond 30 min, even where hormone levels are continuing to increase because of ongoing gastric absorption. This sudden onset, sudden offset, pattern of action has not been observed before and could play a role in effecting rapid, fine adjustments to regional blood flow in response to changing local requirements. Caution should be exercised in extending these results to cycling women, however, because the role of the changes during the menstrual cycle in the relative concentrations of progesterone and estrogen is unknown.

These studies provide some evidence regarding the mechanisms of action of estrogen on the microvasculature. ACh is known to cause vasodilation through increased endothelial release of nitric oxide in arteries and veins and other endothelium-dependent mechanisms (2, 20). It can act through muscarinic receptors, known to be located on endothelial tissues, and through nicotinic receptors, located in neural tissue (8). Sodium nitroprusside is a nitric oxide donor that acts on vascular smooth muscle independently of the endothelium (7). The post-ischemic hyperemic response involves both endothelium-dependent dilatation and direct actions of metabolites relaxing microvascular smooth muscle (21, 22). The lack of an effect of estrogen on the response to nicotine suggests that nicotinic receptors and, therefore, neuronal mechanisms, are unlikely to be involved. The lack of an effect on the sodium nitroprusside or ischemic responses also excludes nonendothelial nitric oxide pathways. Accordingly, it appears likely that the effect of estrogen is specific to the endothelial actions of ACh.

These experiments show that acute administration of estrogen in men produces rapid cardiovascular changes. In men, estrogens are produced in significant quantities by local tissue aromatization of androgenic precursors from the testes and adrenal glands. They appear to play an important role in bone metabolism, and there is preliminary evidence that they may have a role in controlling endothelial function (23). These experiments suggest that estrogens are active in the male cardiovascular system, although the physiological significance of this is unclear.

These studies also show that laser Doppler velocimetry with low voltage direct current iontophoresis of vasoactive substances provides a convenient, noninvasive method for studying the effects of hormones on the microvasculature and raises the possibility of clinical applications in this area. Despite its obvious attractions, however, the technique is subject to certain limitations. The most important of these arise from the extreme sensitivity of the laser Doppler technique and the high variability of cutaneous blood flow, which at the present time restrict its use in cross-sectional studies or as a clinical tool.

In conclusion, these studies show that 1) estrogens act on the cutaneous microvascular bed to enhance responses to the endothelial vasodilator acetylcholine at plasma estradiol concentrations within the physiological range for premenopausal women; 2) the effect is of rapid onset, commencing within 15 minutes after delivery of the estrogen dose, and of rapid offset, with no effect detectable at 30 min; 3) the mechanisms of the microvascular actions of estrogens do not appear to include modulation of nicotinic receptors on nerves or nitric oxide effects on vascular smooth muscle; and 4) estrogens can act on the male cardiovascular system in a manner that is potentially clinically beneficial.

	Acknowledgments

 
Paul Komesaroff is assisted by the Victorian Health Promotion Foundation.

Received August 26, 1997.

Accepted April 3, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Stampfer MJ, Colditz GA, Willett WC, et al. 1991 Postmenopausal estrogen therapy and cardiovascular disease. N Engl J Med. 325:756–762.[Abstract]
2. White MM, Zamudio S, Stevens T, et al. 1995 Estrogen, progesterone and vascular reactivity: potential cellular mechanisms. Endocr Rev. 16:739–751.[CrossRef][Medline]
3. Gilligan DM, Badar DM, Panza JA, Quyyumi AA, Cannon RA. 1994 Acute vascular effects of estrogen in postmenopasual women. Circulation. 90:786–791.[Medline]
4. Chien YW, Siddiqui O, Shi WM, Lelawpngs P, Liu JC. 1989 Direct current iontophoretic transdermal delivery of peptide and protein drugs. J Pharm Sci. 78:376–383.[Medline]
5. Westerman RA, Widdop RE, Hannford J, et al. 1988 Laser Doppler velocimetry in the measurement of neurovascular function. Aust Phys Eng Sci Med. 11:53–66.
6. Morris SJ, Shore AC, Tooke JE. 1995 Responses of the skin microcirculation to acetylcholine and sodium nitroprusside in patients with NIDDM. Diabetologia. 38:1337–1344.[Medline]
7. Sudhir K, Jennings GL, Funder JW, Komesaroff PA. 1996 Estrogen enhances basal nitric oxide release in the forearm vasculature in perimenopausal women. Hypertension. 28:330–335.[Abstract/Free Full Text]
8. Izumi H, Karita K. 1992 Axon flare evoked by nicotine in human skin. Jpn J Physiol. 42:721–730.[CrossRef][Medline]
9. Anderson RR, Parrish JA. 1981 Optical properties of human skin. In: Marks R, Payne PA, eds. Bioengineering and the skin. Lancaster, Boston; MTP Press: 147–194.
10. Ryan TJ. 1992 Cutaneous circulation. In: Goldsmith LA, ed. Biochemistry, physiology and molecular biology of the skin, 2nd ed. London: Oxford University Press; 1019–1083.
11. Sarrel PM, Poole-Wilson PA, Collins P. 1993 Beneficial effect of oestrogen on exercise-induced myocardial ischaemia in women with coronary artery disease. Lancet. 342:133–135.[CrossRef][Medline]
12. Gilligan DM. 1994 Effects of physiological levels of estrogen on coronary vasomotor function. Circulation. 89:2545–2551.[Medline]
13. Bartelink ML, Wollersheim H, Theeuwes D, Thien T. 1990 Changes in skin blood flow during the menstrual cycle. Clin Sci (Lond). 78:527–532.[Medline]
14. Gorski J, Welshons WV, Sakai D, et al. 1986 Evolution of a model of estrogen action. Recent Prog Horm Res. 42:297–321.
15. Wehling M, Kasmayr J, Theisen K. 1991 Rapid effects of mineralocorticoids on sodium-proton exchanger: genomic or nongenomic pathway? Am J Physiol 260:E719–E726.
16. McEwen BS. 1991 nongenomic and genomic effects of steroids on neural activity. Trends Pharmacol Sci. 12:141–147.[CrossRef][Medline]
17. Mueck AO, Seeger H, Lippert TH. 1996 Calcium antagonistic effect of natural and synthetic estrogens - investigations on a nongenomic mechanism of vascular action. Int J Clin Pharmacol Ther. 34:424–426.[Medline]
18. Sudhir K, Chou TM, Mullen WL, et al. 1995 Mechanisms of estrogen-induced vasodilation: in vivo studies in canine coronary conductance and resistance arteries. J Am Coll Cardiol. 26:807–814.[Abstract]
19. Reis SE, Gloth ST, Blumenthal RS, et al. 1994 Ethinyl estradiol acutely attenuates abnormal coronary vasomotor responses to acetylcholine in postmenopausal women. Circulation. 89:52–60.[Medline]
20. Morris SJ, Shore AC. 1996 Skin blood flow responses to the iontophoresis of acetylcholine and sodium nitroprusside in man: possible mechanisms. J Physiol. 496:531–542.[Medline]
21. Carlsson I, Sollevi A, Wennmalm A. 1987 The role of myogenic relaxation, adenosine and prostaglandins in human forearm reactive hyperaemia. J Physiol. 389:147–161.[Abstract/Free Full Text]
22. Sagach VF, Tkachenko MN. 1991 On the mechanisms of the involvement of endothelium in reactive hyperemia. Experientia. 47:828–830.[CrossRef][Medline]
23. Sudhir S, Chou TM, Messina LM, et al. 1997 Endothelial dysfunction in a man with disruptive mutation in oestrogen-receptor gene. Lancet. 347:1146–1147.

This article has been cited by other articles:

				M. R. I. Williams, T. Dawood, S. Ling, A. Dai, R. Lew, K. Myles, J. W. Funder, K. Sudhir, and P. A. Komesaroff Dehydroepiandrosterone Increases Endothelial Cell Proliferation in Vitro and Improves Endothelial Function in Vivo by Mechanisms Independent of Androgen and Estrogen Receptors J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4708 - 4715. [Abstract] [Full Text] [PDF]

				G. A. Head, V. R. Obeyesekere, M. E. Jones, E. R. Simpson, and Z. S. Krozowski Aromatase-Deficient (ArKO) Mice Have Reduced Blood Pressure and Baroreflex Sensitivity Endocrinology, September 1, 2004; 145(9): 4286 - 4291. [Abstract] [Full Text] [PDF]

				L. Maffei, Y. Murata, V. Rochira, G. Tubert, C. Aranda, M. Vazquez, C. D. Clyne, S. Davis, E. R. Simpson, and C. Carani Dysmetabolic Syndrome in a Man with a Novel Mutation of the Aromatase Gene: Effects of Testosterone, Alendronate, and Estradiol Treatment J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 61 - 70. [Abstract] [Full Text] [PDF]

				A. C. Calkin, K. Sudhir, S. Honisett, M. R. I. Williams, T. Dawood, and P. A. Komesaroff Rapid Potentiation of Endothelium-Dependent Vasodilation by Estradiol in Postmenopausal Women Is Mediated via Cyclooxygenase 2 J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 5072 - 5075. [Abstract] [Full Text] [PDF]

				M. R. I. Williams, R. A. Westerman, B. A. Kingwell, J. Paige, P. A. Blombery, K. Sudhir, and P. A. Komesaroff Variations in Endothelial Function and Arterial Compliance during the Menstrual Cycle J. Clin. Endocrinol. Metab., November 1, 2001; 86(11): 5389 - 5395. [Abstract] [Full Text] [PDF]

				P. A. Komesaroff, M. Fullerton, M. D. Esler, A. Dart, G. Jennings, and K. Sudhir Low-Dose Estrogen Supplementation Improves Vascular Function in Hypogonadal Men Hypertension, November 1, 2001; 38(5): 1011 - 1016. [Abstract] [Full Text] [PDF]

				S. F. Palter, A. B. Tavares, A. Hourvitz, J. D. Veldhuis, and E. Y. Adashi Are Estrogens of Import to Primate/Human Ovarian Folliculogenesis? Endocr. Rev., June 1, 2001; 22(3): 389 - 424. [Abstract] [Full Text] [PDF]

				S. C. Manolagas and S. Kousteni Perspective: Nonreproductive Sites of Action of Reproductive Hormones Endocrinology, June 1, 2001; 142(6): 2200 - 2204. [Full Text] [PDF]

				J Dupont and D Le Roith Insulin-like growth factor 1 and oestradiol promote cell proliferation of MCF-7 breast cancer cells: new insights into their synergistic effects Mol. Pathol., June 1, 2001; 54(3): 149 - 154. [Abstract] [Full Text]

				M. T. Littleton-Kearney, D. M. Agnew, R. J. Traystman, and P. D. Hurn Effects of estrogen on cerebral blood flow and pial microvasculature in rabbits Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H1208 - H1214. [Abstract] [Full Text] [PDF]

				K. Sudhir and P. A. Komesaroff Cardiovascular Actions of Estrogens in Men J. Clin. Endocrinol. Metab., October 1, 1999; 84(10): 3411 - 3415. [Full Text] [PDF]

				A. M. McNeill, N. Kim, S. P. Duckles, D. N. Krause, and H. A. Kontos Chronic Estrogen Treatment Increases Levels of Endothelial Nitric Oxide Synthase Protein in Rat Cerebral Microvessels • Editorial Comment Stroke, October 1, 1999; 30(10): 2186 - 2190. [Abstract] [Full Text] [PDF]

				S. Ling, P. J. Little, M. R. I. Williams, A. Dai, K. Hashimura, J.-P. Liu, P. A. Komesaroff, and K. Sudhir High glucose abolishes the antiproliferative effect of 17beta -estradiol in human vascular smooth muscle cells Am J Physiol Endocrinol Metab, April 1, 2002; 282(4): E746 - E751. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Komesaroff, P. A. Articles by Westerman, R. A. Search for Related Content PubMed PubMed Citation Articles by Komesaroff, P. A. Articles by Westerman, R. A.