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Microvascular Effects of CRH in Human Skin Vary in Relation to Gender

Mothers and Babies Research Centre, John Hunter Hospital, University of Newcastle, Newcastle, New South Wales, Australia 2310

Address all correspondence and requests for reprints to: Dr. Ian Wright, Neonatal Intensive Care Unit, John Hunter Hospital, Locked Bag #1, Hunter Region Mail Center, Newcastle, New South Wales 2310, Australia. E-mail: iwright{at}mail.newcastle.edu.au

	Abstract

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Males and females have a significantly different life expectancy because of the cardioprotective effects of estrogen. The mechanisms that result in this difference between the sexes are not fully understood. However, stress is a contributing factor to the development of cardiovascular disease, and stress-related factors derived from central or peripheral sources may have differential effects in the modulation of cardiovascular function in males and females. CRH is a central modulator of the stress response and is known to have vasodilator effects in a number of vascular beds. We have examined whether CRH has vasodilator effects in human skin and whether this effect is different between males and females using laser Doppler and iontophoresis. CRH (1 nM) had vasodilatory effects in the skin circulation of both premenopausal females (n = 6) and age-matched males (n = 5), but CRH-induced dilation was significantly more potent in females than males. Acetylcholine-(1 nM) and sodium nitroprusside- (0.74 nM) induced dilation was not significantly different between males (n = 6) and females (n = 6). This is the first study to demonstrate that CRH acts locally as a vasodilator in human skin circulation and that this response is augmented in premenopausal females. The mechanism by which CRH causes dilation in human skin is presently unknown. However, these data suggest that CRH-induced dilation may be one mechanism by which cardiovascular risk is reduced in premenopausal women.

	Introduction

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MALES AND FEMALES have a significantly different life expectancy because of the cardioprotective effects of the female sex hormone, estrogen (1). The mechanisms that result in this difference between the sexes are not fully understood. However, stress is a contributing factor for the development of hypertension, a known risk factor of cardiovascular disease (2). Because males are more at risk of developing cardiovascular disease at a younger age, it is possible that factors involved in the stress response may have differential effects on cardiovascular function in males and females at a central level and at peripheral sites in which stress factors are produced locally.

CRH, first identified in ovine hypothalami, is a 41 amino acid peptide, produced predominantly in the central nervous system and plays a coordinating role in the modulation of the pituitary-adrenal axis (3) and its response to stress. CRH is also produced at a number of peripheral sites including the human skin (4) and is known to have vascular effects at both central and peripheral sites (5, 6, 7, 8, 9, 10), including skin in the rat (11). Because CRH is present in human skin and is known to act as a vasodilator in rat skin, we have hypothesized that CRH has vasodilatory effects in human skin.

	Experimental Subjects

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Nonsmoking, premenopausal females (n = 21, 21–35 yr) who were not using an oral contraceptive and age-matched nonsmoking males (n = 7, 21–40 yr) were recruited to the study using a protocol approved by the Hunter Area Health Human Ethics Committee. Tests were performed on the volar aspect of the forearm. Subjects with any generalized dermatitis or essential hypertension were excluded. All female subjects were tested at the middle of the menstrual cycle between d 11 and 15. Subject weight, height, age, and day of menstrual cycle were recorded. Subjects refrained from coffee and food for at least an hour before the investigation.

	Materials and Methods

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Drugs

Human CRH was obtained from Auspep (Melbourne, Australia), -helical CRH 9–41 was obtained from Peninsula Laboratories, Inc. (San Carlos, CA). Acetylcholine chloride and sodium nitroprusside were obtained from Sigma (St. Louis, MO).

Laser Doppler and iontophoresis

Microvascular laser Doppler is an established method of assessing the function of blood vessels of the peripheral microvasculature and skin tissues (12). Low-intensity laser light is reflected from moving blood cells in the skin circulation, and a measurement of blood flow is thus obtained. We used the Periflux 5001 laser Doppler (Perimed AB, Järfälla, Sweden) with one temperature-regulated iontophoresis probe and one temperature-regulated control probe sited on the same aspect of the forearm. The PeriIont micropharmacology system was used (Perimed AB). This system is described elsewhere (13), but in brief a transdermal current is applied to cause movement of drugs from a disposable electrode, surrounding the temperature-controlled laser Doppler head, into the skin. Blood flow readings are expressed as arbitrary perfusion units (PU).

Experimental protocol

Subjects were placed in a semisupine position and skin basal blood flow was recorded for 5 min, after which six doses of CRH, CRH antagonist, acetylcholine, or controls were administered to the skin circulation, on separate occasions, by iontophoresis at a current of 0.06 mA for 30 sec/dose with a positive polarity. Because of its chemical charge, sodium nitroprusside was administered in six doses at a current of 0.06 mA for 30 sec/dose with a negative polarity. It has been previously shown that a negative electrical charge causes nonspecific dilation in the skin circulation via the activation of the afferent nociceptive C fibers (14). As a control in the nitroprusside experiments, nonspecific dilation was induced with saline administered by six doses of a negative electrical charge at 0.06 mA for 30 sec/dose. At the final analysis, nonspecific dilation values for each individual were subtracted from the corresponding dose of sodium nitroprusside-induced dilation. The repeated administration of iontophoretic current caused a cumulative increase of the drug in the skin and its circulation. Blood flow was recorded by laser Doppler after each medication dose.

As described previously (13), certain standard provocations were performed to allow for comparison among different studies and subjects. After the last iontophoretic stimulation and when skin perfusion returned to a stable level, a blood pressure cuff was used to test the response to a short period of absent blood flow. This allowed for a biological zero to be obtained in each experiment. This zero was subtracted from the blood flow values obtained in each experiment (13). Reperfusion commenced after the cuff was released and was studied at the control probe. Flow was allowed to stabilize before a standard thermal provocation was used. A small heater around the head of the control probe increased the temperature setting from 40–44 C in one-degree increments at 60-sec intervals. The reactive hyperemia following the heat provocation was monitored by the laser Doppler.

Data analysis

Microvascular blood flow data were analyzed using a custom-designed macroanalysis program on an Excel spreadsheet (Microsoft Corp., Redmond, WA). Differences in the linear portions of the curves were compared by linear regression analysis and compared and tested for significant displacement and deviation from parallelism as described by Bowman and Rand (15). Differences between the response curves were calculated by determining the degree of displacement between parallel concentrations in the curve where appropriate. Nonparallel curves and multiple comparisons of means were tested with one-way ANOVA and Tukey-Kramer for postanalysis tests using InStat software version 2.04a (GraphPad Software, Inc., San Diego, CA). For comparison of male and female parameters such as height and weight, t tests were used. All values are expressed as means ± SEM (SEM) unless otherwise stated. P less than 0.05 was considered significant.

	Results

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The average age of women in this study was 25.9 plus or minus 1.3 yr (n = 21) and men was 24.4 plus or minus 4.9 yr (n = 7) (t test P > 0.05). The male subjects were significantly heavier (75.7 ± 5.8 vs. 61.6 ± 1.6 kg) and taller (1.80 ± 1.7 vs. 1.64 ± 1.4 m) than the female subjects (t test P < 0.05).

Biological zero was not significantly different between males and females (t test P > 0.05). Basal skin microvascular flow was significantly higher in men (n = 7, 6.01 ± 1.02 PU) than women (n = 21, 4.15 ± 0.43 PU, t test P < 0.05). Postocclusive reperfusion was not significantly different between the sexes (t test P > 0.05). The response to heat-induced hyperemia was not significantly different between age-matched males and females (ANOVA, regression analysis, P > 0.05).

Human CRH (1 nM) caused a dose-dependent vasodilation in human skin circulation (Fig. 1). The degree of dilation was significantly greater in female subjects (27.0 ± 2.7 yr, n = 6) when compared with male subjects (23.6 ± 1.7 yr, n = 5) (ANOVA, regression analysis, P < 0.05, Fig. 1). CRH-induced dilation was inhibited when the CRH antagonist, -helical-CRH_(9–41) (10 nM) was coadministered with CRH (1 nM, n = 6) (Fig. 2, ANOVA, regression analysis, P < 0.05). -Helical-CRH_(9–41) (10 nM) when administered alone had weak vasodilatory effects (n = 4, Fig. 2).

View larger version (18K): [in this window] [in a new window]   Figure 1. CRH-induced vasodilation in human skin circulation. CRH-induced dilation (1 nM) was greater in premenopausal females () (n = 6) than age-matched males () (n = 5). Microvascular skin blood flow was measured in perfusion units, and CRH was administered by six 30-sec pulses of a positive electrical current at 0.06 mA. All values are expressed as mean ± SEM.

View larger version (16K): [in this window] [in a new window]   Figure 2. Comparison of CRH-induced vasodilation in female human skin circulation in the presence and absence of the CRH antagonist, -helical CRH(9–41). CRH-induced dilation (1 nM, n = 6) () was significantly reduced in the presence of -helical CRH(9–41) (10 nM, n = 6) (). -Helical CRH(9–41) (10 nM, n = 4) when administered alone () has some vasodilatory effects. Microvascular skin blood flow was measured in perfusion units, and each drug was administered by six 30-sec pulses of a positive electrical current at 0.06 mA. All values are expressed as mean ± SEM.

View larger version (18K): [in this window] [in a new window]   Figure 3. The vasodilator effects of acetylcholine in human skin circulation. Acetylcholine-induced dilation (1 nM) was the same in premenopausal females () (n = 6) and age-matched males () (n = 6). Microvascular skin blood flow was measured in perfusion units and acetylcholine was administered by six 30-sec pulses of a positive electrical current at 0.06 mA. All values are expressed as mean ± SEM.

View larger version (18K): [in this window] [in a new window]   Figure 4. The vasodilator effects of sodium nitroprusside (SNP) in human skin circulation. SNP-induced dilation (0.74 nM) was the same in pre-menopausal females () (n = 6) and age-matched males () (n = 6). Microvascular skin blood flow was measured in perfusion units, and SNP was administered by six 30-sec pulses of a negative electrical current at 0.06 mA. All values are expressed as mean ± SEM.

	Discussion

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Our data suggest that the response to CRH is mediated via a CRH receptor because we were able to suppress CRH-induced dilation by the administration of the CRH antagonist, -helical CRH_(9–41). Both CRH and the CRH receptor subtype 1 have been identified in human skin in follicular keratinocytes, epidermal cells, mast cells, and melanocytes (4). It is possible that the type 2 CRH receptor is also expressed in human skin; however, this receptor has been identified only in rodent skin (16). Previous studies in the human placental circulation suggest that CRH-induced dilation is mediated by a type 2 CRH receptor (17) and the NO pathway ( 18). In rat skin, Singh et al. (11) reports that both CRH and urocortin increase vascular permeability through the degranulation of mast cells via a type 1 CRH receptor. The mechanism by which CRH causes dilation in human skin may be through a direct effect on vascular pathways or indirectly through a mast cell pathway to cause dilation. However, this mechanism requires further investigation.

We examined whether there were differences in endothelial cell function between the sexes using acetylcholine, which is known to act via an endothelial-dependent pathway (19, 20). Acetylcholine-induced dilation was not significantly different in its vasoactive effects in age-matched males and females in our study. A previous iontophoretic study (21) confirms that acetylcholine vascular responses do not vary significantly with age or gender. Acetylcholine-induced dilation in the skin circulation is via an endothelium-mediated pathway involving multiple factors including NO, prostacyclin, and hyperpolarizing factor (19, 20). Our data suggests there may be no gender-related differences in skin dilator pathways activated by endothelium-derived substances such as hyperpolarizing factor, NO, or prostacyclin in young adult subjects.

Conversely, there is a large amount of evidence that indicates that there are gender-related differences in vascular responses that are dependent on the NO pathway alone. For example, Algotsson et al. (21) suggest that estrogenic alterations of vascular function may be through changes in the smooth muscle NO pathway. Darkow et al. (22) report that estrogen-relaxed porcine coronary artery smooth muscle by the up-regulation of the NO-cGMP pathway. It has been reported that skin vascular dilation to heat-induced hyperemia is mediated by NO (23) and is enhanced in females with high circulating concentrations of estrogen (24). Our study demonstrates that the heat-induced hyperemic response in females was not significantly different from age-matched males. Furthermore, there were no significant differences in the response to NO donor, sodium nitroprusside, in our study group. It is likely that the young age of the participants may have influenced the results we have observed in this study because Algotsson et al. (21) report there are no differences in the vascular response to sodium nitroprusside between young men and women. It can be concluded from these findings that there may be no differences in NO-induced dilation in this particular age group.

In summary, there were no differences in vascular function between young adult males and females when we examined dilator pathways that were dependent on either the endothelium or the vascular smooth muscle. However, we observed that CRH-induced dilation was more potent in the female circulation, suggesting there may be some involvement of the sex hormone estrogen and that the effect of estrogen on the CRH pathway may occur upstream from the vascular tissue. This is the first study to demonstrate that CRH acts as a vasodilator in human microvascular circulation and that this response is augmented in premenopausal females. The mechanism by which CRH causes dilation in human skin is presently unknown. However, these data suggest that CRH-induced dilation may be one mechanism by which cardiovascular risk is reduced in premenopausal women by reducing peripheral resistance.

	Acknowledgments

 

	Footnotes

 
This work was supported by grants from the John Hunter Hospital Charitable Foundation.

Abbreviations: NO, Nitric oxide; PU, perfusion unit.

Received August 3, 2001.

Accepted October 4, 2001.

	References

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1. Maxwell SR 1998 Women and heart disease. Basic Res Cardiol 93(Suppl 2): 79–84
2. Everson SA, Lynch JW, Kaplan GA, Lakka TA, Sivenius J, Salonen JT 2001 Stress-induced blood pressure reactivity and incident stroke in middle-aged men. Stroke 32:1263–1270[Abstract/Free Full Text]
3. Vale W, Spiess J 1981 Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin or beta-endorphin. Science 213:1394–1397[Free Full Text]
4. Slominski A, Wortsman J 2000 Neuroendocrinology of the skin. [Review] [361 refs]. Endocr Rev 21:457–487[Abstract/Free Full Text]
5. Fisher LA 1993 Central actions of corticotropin-releasing factor on autonomic nervous activity and cardiovascular function. Ciba Found Symp 172:243–253, discussion 253–257[Medline]
6. MacCannell KL, Hamilton PL, Lederis K, Newton CA, Rivier J 1984 Corticotropin releasing factor-like peptides produce selective dilatation of the dog mesenteric circulation. Gastroenterology 87:94–102[Medline]
7. Jain V, Vedernikov YP, Saade GR, Chwalisz K, Garfield RE 1997 The relaxation responses to corticotropin-releasing factor in rat aorta are endothelium dependent and gestationally regulated. Am J Obstet Gynecol 176:234–240[CrossRef][Medline]
8. Udelsman R, Gallucci WT, Bacher J, Loriaux DL 1986 Hemodynamic effects of corticotropin releasing hormone in the anesthetized cynomologus monkey. Peptides 7:465–471[CrossRef][Medline]
9. Clifton VL, Read MA, Leitch IM, Boura AL, Robinson PJ, Smith R 1994 Corticotropin-releasing hormone-induced vasodilatation in the human fetal placental circulation. J Clin Endocrinol Metab 79:666–669[Abstract]
10. Briscoe RJ, Cabreera CL, Baird TJ, Rice KC, Woods JH 2000 Antalarmin blockade of corticotropin-releasing hormone-induced hypertension in rats. Brain Res 881:204–207[CrossRef][Medline]
11. Singh LK, Boucher W, Pang X, Letourneau R, Seretakis D, Green M, Theoharides TC 1999 Potent mast cell degranulation and vascular permeability triggered by urocortin through activation of corticotropin-releasing hormone receptors. J Pharmacol Exp Ther 288:1349–1356[Abstract/Free Full Text]
12. Kubli S, Waeber B, Dalle-Ave A, Feihl F 2000 Reproducibility of laser Doppler imaging of skin blood flow as a tool to assess endothelial function. J Cardiovasc Pharmacol 36:640–648[CrossRef][Medline]
13. Hu J, Norman M, Wallensteen M, Gennser G 1998 Increased large arterial stiffness and impaired acetylcholine induced skin vasodilatation in women with previous gestational diabetes mellitus. Br J Obstet Gynaecol 105:1279–1287[Medline]
14. Wardell K, Naver HK, Nilsson GE, Wallin BG 1993 The cutaneous vascular axon reflex in humans characterized by laser Doppler perfusion imaging. J Physiol 460:185–190[Abstract/Free Full Text]
15. Bowman WC, Rand MJ 1980 Textbook of pharmacology, ed 2. Carlton, Victoria, Australia: Blackwell Scientific Publication
16. Slominski A, Wortsman J, Pisarchik A, Zbytek B, Linton EA, Mazurkiewicz JE, Wei ET 2001 Cutaneous expression of corticotropin-releasing hormone, urocortin and CRH receptors. FASEB J 15:1678–1693[Abstract/Free Full Text]
17. Leitch IM, Boura AL, Botti C, Read MA, Walters WA, Smith R 1998 Vasodilator actions of urocortin and related peptides in the human perfused placenta in vitro. J Clin Endocrinol Metab 83:4510–4513[Abstract/Free Full Text]
18. Clifton VL, Read MA, Leitch IM, Giles WB, Boura AL, Robinson PJ, Smith R 1995 Corticotropin-releasing hormone-induced vasodilatation in the human fetal-placental circulation: involvement of the nitric oxide-cyclic guanosine 3',5'-monophosphate-mediated pathway. J Clin Endocrinol Metab 80:2888–2893[Abstract/Free Full Text]
19. Buus NH, Simonsen U, Pilegaard HK, Mulvany MJ 2000 Nitric oxide, prostanoid and non-NO, non-prostanoid involvement in acetylcholine relaxation of isolated human small arteries. Br J Pharmacol 129:184–192[CrossRef][Medline]
20. Coats P, Johnston F, MacDonald J, McMurray J, Hillier C 2001 Endothelium-derived hyperpolarizing factor: identification and mechanisms of action in human subcutaneous resistance arteries. Circulation 103:1702–1708[Abstract/Free Full Text]
21. Algotsson A, Nordberg A, Winblad B 1995 Influence of age and gender on skin vessel reactivity to endothelium-dependent and endothelium-independent vasodilators tested with iontophoresis and a laser Doppler perfusion imager. J Gerontol A Biol Sci Med Sci 50A:M121–M127
22. Darkow DJ, Lu L, White RE 1997 Estrogen relaxation of coronary artery smooth muscle is mediated by nitric oxide and cGMP. Am J Physiol 272:H2765–H2773
23. Kellogg Jr DL, Liu Y, Kosiba IF, O’Donnell D 1999 Role of nitric oxide in the vascular effects of local warming of the skin in humans. J Appl Physiol 86:1185–1190[Abstract/Free Full Text]
24. Charkoudian N, Stephens DP, Pirkle KC, Kosiba WA, Johnson JM 1999 Influence of female reproductive hormones on local thermal control of skin blood flow. J Appl Physiol 87:1719–1723[Abstract/Free Full Text]

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