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Mothers and Babies Research Centre (R.C., V.L.C., R.S., A.T.B., M.A.R., I.M.R.W.), Hunter Medical Research Institute; Discipline of Reproductive Medicine (A.T.B.), University of Newcastle; and Division of Obstetrics and Gynaecology (M.A.R.) and Neonatal Intensive Care Unit (I.M.R.W.), John Hunter Hospital, Newcastle NSW 2310, Australia
Address all correspondence and requests for reprints to: Dr. Ian M.R. Wright, Neonatal Intensive Care Unit, John Hunter Hospital, Locked Bag #1, Hunter Region Mail Centre, Newcastle, NSW 2310, Australia. E-mail: iwright{at}mail.newcastle.edu.au.
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Abstract |
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CRH-induced vasodilation in human skin was examined using laser Doppler flowmetry and iontophoresis in adult females. CRH (1 nM) was administered iontophoretically to the forearm, and blood flow was measured simultaneously in the same area by laser Doppler. CRH-induced dilation of the skin microvasculature was significantly reduced in the presence of the mast cell degranulation inhibitor, sodium cromoglycate, the histamine H1-antagonist, promethazine, or the H2-antagonist, ranitidine. CRH-induced dilation was also significantly reduced in the presence of the nitric oxide synthase inhibitor, N
-nitro-L-arginine methyl ester, or the cyclooxygenase inhibitor, piroxicam.
These findings provide novel evidence that CRH-induced vasodilation in human skin occurs via mast cell degranulation and is principally mediated by histamine and, to a lesser extent, by prostacyclin and nitric oxide.
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Introduction |
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The effects of CRH are mediated through two major classes of receptors, designated CRH-R1 and CRH-R2 (15). These two receptors are products of separate genes, but both comprise seven putative transmembrane domains characteristic of G protein-coupled receptors, and they typically are positively coupled to adenylate cyclase (15). The hemodynamic effects of CRH are complex. The hypotensive effects caused by parenteral CRH administration are believed to be largely mediated through a direct action on peripheral CRH-R2 receptors in vascular endothelium and smooth muscle (16, 17, 18, 19, 20, 21, 22). CRH-induced effects on skin have been most extensively studied in the rat, in which it has been demonstrated that CRH increases vascular permeability through the degranulation of mast cells, an action mediated via CRH-R1 receptors (3).
Histamine is a major factor released during mast cell degranulation. Histamine is a vasodilator and has the capacity to contribute locally to sc blood flow control under normal and pathologic conditions (23). Therefore, histamine is likely to be an important but indirect mediator of the vasodilator action of CRH in the skin microcirculation. CRH and related peptides do not appear to act exclusively by cAMP-dependent mechanisms. They can also act directly on blood vessels to cause vasodilation mediated by CRH-R2 receptors via a nitric oxide (NO)-cyclic GMP-dependent pathway (9, 12, 24). Therefore, it is possible that CRH causes dilation in human skin via a combination of direct and indirect mechanisms, mediated by a combination of CRH-R1 and CRH-R2 receptor subtypes and second messenger pathways.
The aim of the present study is to examine potential mechanisms by which CRH causes dilation of the human skin microvasculature with particular reference to mast cell/histamine-dependent events. We have shown that CRH causes mast cell degranulation in human skin, as has been previously observed in rats (13, 25). Furthermore, mast cell-derived histamine appears to be the principal mediator of the vasodilatory effects of CRH in human skin. Our results are consistent with CRH acting predominantly via a CRH-R1 receptor subtype. However, the relative contribution of CRH-R1 and CRH-R2 receptor-mediated pathways to the overall vasoactive effect of CRH in human skin remains to be determined.
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Subjects and Methods |
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Nonsmoking, premenopausal females (n = 54), who were not using an oral contraceptive, were recruited under a protocol approved by the Hunter Area Health Human Ethics Committee. Subjects with dermatitis or essential hypertension were excluded from the study. All subjects were tested at the middle of the menstrual cycle between d 11 and 16 in an effort to minimize effects due to changes in sex hormone profiles. Subject weight, height, age, medications, and day of menstrual cycle were recorded. The participants refrained from coffee and food for at least 1 h before the investigations.
Laser Doppler and iontophoresis
Microvascular laser Doppler assesses the function of blood vessels of the peripheral microvasculature and skin tissues (26). Low-intensity laser light is reflected from moving blood cells in the skin circulation, and a measurement of blood flow is obtained. We used the Periflux 5001 Laser Doppler (Perimed AB, Järfälla, Sweden) with a single temperature-regulated iontophoresis probe and a single temperature-regulated control probe sited on the volar aspect of the same forearm, approximately 10 cm apart. The PeriIont micropharmacology system was used (Perimed). This system is described elsewhere (14). Briefly, a transdermal current is applied to cause movement of drugs into the skin from a disposable electrode surrounding the temperature-controlled laser Doppler head. 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 identical doses of CRH (1 nM) or control solutions 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. Due to its chemical charge, sodium nitroprusside (1 nM) was administered in six doses at a current of 0.06 mA for 30 sec/dose with a negative polarity. The repeated administration of iontophoretic current causes an increased concentration of the drug in the skin and the local circulation. Blood flow is recorded by laser Doppler after each medication dose.
Standard provocations were performed to allow for comparison between different studies and subjects, as previously described (27). After the final period of iontophoretic and when skin microvascular blood flow had returned to a stable level, the forearm blood flow was occluded using a standard sphygmomanometer cuff 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 (27). Re-perfusion after the cuff was released was studied at the control probe. Flow was allowed to stabilize before a standard thermal provocation was then used. A small heater around the head of the control probe increased the temperature setting from 40–44 C in 1-degree increments at 60-sec intervals. The reactive hyperemia after the heat provocation was monitored by the laser Doppler.
To elucidate the mechanisms involved in producing the vasoactive effects of CRH in the microcirculation, potential pathways by which CRH could cause vasodilation were examined using antagonists of a number of pathways involved in vascular function. A pad containing each of the potential antagonists was applied to the skin surface, on separate occasions, of each midcycle female subject for 1 hr before the iontophoresis of CRH using the experimental protocol described previously.
To investigate whether the vasodilatory effects of CRH in human skin were mediated via mast cell degranulation, a mast cell inhibitor (1% sodium cromoglycate solution, n = 6) was applied to the skin. To determine whether histamine plays a role in mediating the CRH-induced dilation, a histamine-H1 antagonist (2% promethazine hydrochloride, n = 6) or H2 antagonist (2.5% ranitidine hydrochloride, n = 6) was also applied to the skin on separate occasions.
CRH-induced dilation has been shown to be mediated via NO in other tissues (6), and therefore, we examined this pathway in the skin by applying a NO synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME) (100 nM, n = 6), or the control, which is the inactive stereoisomer, N-nitro-D-arginine methyl ester (D-NAME) (100 nM, n = 4). A pretreatment of the skin with L-NAME was also followed by the iontophoresis of six doses of sodium nitroprusside (1 nM, n = 4), an endothelium independent dilator, as a control to determine for any nonspecific effects of L-NAME.
To test whether CRH-induced dilation may be mediated via prostacyclin (PGI2), a cyclooxygenase inhibitor, piroxicam gel (n = 8), was used as a 1-h pretreatment on the skin before CRH administration. As a control for these pretreatments, distilled water was added to a dressing pad.
Histamine-induced dilation was assessed to examine whether gender differences in CRH-induced dilation were mediated via an enhanced dilator response to histamine. Midcycle females (n = 6) were compared with age-matched males (n = 5), and histamine (100 mM in distilled water) was administered by iontophoresis at a current of 0.06 mA for 30 sec/dose with a positive polarity until maximal dilation was reached.
To determine whether microvascular blood flow was functional after administration of the inhibitors, each subject was exposed to the inhibitors or vehicle for 1 h, and endothelial-independent dilation was examined using sodium nitroprusside (1 nM), a NO donor that acts directly on the vascular smooth muscle.
Drugs
Human CRH was obtained from Auspep (Melbourne, Australia); L-NAME and D-NAME were obtained from Sigma Chemical Co. (St. Louis, MO); 0.5% wt/wt piroxicam gel was obtained from Pfizer (Sydney, Australia); 2% wt/wt promethazine hydrochloride cream was obtained from Novartis (Dorval, Quebec, Canada); 2.5% wt/wt ranitidine hydrochloride solution was obtained from Glaxo-Wellcome (Melbourne, Australia); and 1% wt/vol sodium cromoglycate inhalation solution was obtained from Pharmacia & Upjohn (Sydney, Australia). Histamine (10 mg/ml wt/vol) was obtained from Stallergenes (Antony, France), and sodium nitroprusside (50 mg, crystalline) was obtained from Faulding (Melbourne, Australia).
Data analysis
Dose-response curves were compared using generalized estimating equations (GEE) using the STATA statistical software (Version 7, 2001; STATA Press, College Station, TX). Box and longitudinal plots of the individual vascular responses were generated using the same software to determine the variability of the response to CRH and the other treatments used in the study (data not shown). The Student’s t test was used for comparison of height and weight between test and control groups as appropriate. All values were expressed as means ± SEM except for subject age, which was expressed as a mean ± SD. P 
0.05 was considered significant unless stated otherwise.
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Results |
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Human CRH (1 nM) caused a dose-dependent vasodilation in the female skin circulation during midstage of the menstrual cycle. CRH-induced dilation was significantly reduced in the presence of the mast cell inhibitor, sodium cromoglycate (n = 6; GEE, P 0.001; Fig. 1
). It was also significantly reduced by the histamine-H1 receptor antagonist, promethazine hydrochloride (n = 6; GEE, P 0.001; Fig. 2). In the presence of the H2 receptor antagonist, ranitidine (n = 6), there was also a significant inhibition of CRH-induced dilation (GEE, P 0.001; Fig. 2). The NO synthase inhibitor, L-NAME, significantly inhibited CRH-induced dilation (n = 6; GEE, P < 0.001; Fig. 3); however, in the presence of D-NAME (n = 4), CRH-induced dilation was unchanged (Fig. 3). CRH-induced dilation was decreased by the cyclooxygenase inhibitor, piroxicam (n = 8; GEE, P 0.001; Fig. 4). Histamine-induced dilation was not significantly different between midcycle females (n = 6) and age-matched males (n = 5; GEE, P > 0.05, data not shown).
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Discussion |
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Human skin is a known target for CRH and pro-opiomelanocortin (POMC) peptides. Immunohistochemical and in situ hybridization studies have demonstrated that CRH and POMC proteins and genes are expressed in the basal layer of epidermis and pilosebaceous cells (28). Similarly, expression of the urocortin gene and its corresponding peptide, a member of the CRH peptide family, have been shown in human skin and in cultures of normal and malignant keratinocytes and melanoma cells (29, 30, 31). Urocortin has been localized to the epidermal and follicular keratinocytes, sweat glands, nevocytes, malignant melanocytes, blood vessel walls, dermal smooth muscle, mononuclear inflammatory cells, and dermal spindle cells (31). This indicates that human skin cells locally produce CRH, urocortin, and POMC peptides and supports the proposal by Slominski and Wortsman (29) that a stress response system similar to the hypothalamic-pituitary-adrenal axis may exist in the skin.
CRH-R1 has been identified in follicular and epidermal keratinocytes, mast cells, melanocytes, peripheral lymphocytes, and dermal blood vessel endothelial cells in human skin (32). The CRH-R2 receptor is also expressed in human skin (31). Because CRH-induced dilation in human skin was completely blocked in the presence of the mast cell inhibitor, sodium cromoglycate, in our study, it is likely that CRH acts predominantly via CRH-R1 receptors on the mast cell to cause dilation. Nevertheless, CRH-mediated effects on the endothelium, smooth muscle, and other secretory cells, such as eosinophils, also need to be considered.
Mast cells are found in large numbers in the skin (7,000–20,000 mast cells/mm2) (22) and are located in the subpapillary region, around blood vessels, lymphatic structures, epithelial appendages, and nerves (33, 34, 35). This suggests that skin mast cells could have multiple roles in the skin including the control of skin blood flow. Previous studies indicate that mast cells are responsive to neuropeptides such as substance P, vasoactive intestinal polypeptide, somatostatin (36, 37), and CRH (3).
Mast cells have a number of roles in the regulation of vascular function. In the canine liver, electron microscopy techniques clearly depict mast cells attached to both endothelium and smooth muscle of the venous circulation, (38) suggesting functional association with vascular tissue. Theoharides et al. (13) and Singh et al. (3) have previously reported that both CRH and urocortin increase vascular permeability in rat skin via mast cell degranulation. Mast cells may also be important in the pathogenesis of atherosclerotic disease by releasing a number of pro-inflammatory cytokines, which aid in the recruitment of other inflammatory cells, such as monocytes and lymphocytes, into vascular tissue. These events lead to vascular inflammation, endothelial dysfunction, and macrophage foam-cell formation, subsequently contributing to the development of atherosclerosis (39). In contrast to these deleterious effects, mast cells express proteases such as mast cell tryptase (40), which has been suggested to provide a general anticoagulant function and thus may slow thrombus formation at the sites of plaque rupture. Mast cells may also have a role in angiogenesis, through the production of growth factors (40). Collectively these studies indicate that mast cells have multifactorial roles in the regulation of vascular function, especially in the microvasculature in both the physiological and pathophysiological environment.
Mast cell degranulation involves the release and stimulation of numerous vasoactive molecules including histamine and NO (41). CRH-induced vasodilation in human skin appears to be mediated, at least in part, by mast cell-derived histamine because we demonstrated that promethazine hydrochloride, a H1 receptor antagonist, and ranitidine, a H2 antagonist, significantly reduced CRH-mediated effects. Previous studies have shown that histamine is a vasodilator in the skin, and this response is mediated via H1 and H2 receptors (23, 42). However, neither H1 nor H2 receptor antagonists in our study completely inhibited CRH-induced dilation, suggesting that other vasodilators may be involved. These findings are consistent with a study by Theoharides et al. (13), whereby partial inhibition of the CRH-induced vascular permeability in rat skin was observed in the presence of the H1 receptor antagonist, diphenhydramine.
A number of studies, although not in the microvasculature, have indicated that CRH-induced dilation is mediated via the NO pathway. Clifton et al. (9) reported that CRH was a potent dilator in the human fetal-placental circulation and that its effects were mediated via the NO-cyclic GMP-dependent pathway. Futhermore, Jain et al. (5) demonstrated that CRH caused dilation in rat mesenteric aorta via NO.
In the present study, inhibition of NO synthase using L-NAME blocked CRH-induced vasodilation in the human skin circulation. In contrast, Theoharides et al. (13) demonstrated that inhibition of NO synthesis potentiated CRH-induced vascular permeability in rat skin. Histamine-induced vasodilation in skin microvasculature is at least partially NO dependent (43), which is consistent with the endothelium playing a role in CRH-induced vasodilation. It is possible that CRH-induced dilation in human skin could be mediated via histamine and NO derived from mast cells and/or by histamine-induced NO released from the vascular endothelium.
We have previously reported gender differences in the dilator response to CRH where females have a more enhanced vasodilation when compared with males (14). Our present data indicate that histamine may be an important mediator of CRH-induced vasodilation after mast cell degranulation, and therefore, we questioned whether this CRH-related gender difference was due to alterations in the vascular response to histamine. Histamine-induced vasodilation was not significantly different between males and females, suggesting that the gender difference in CRH-induced dilation may occur at the level of the mast cell rather than at the endothelium or vascular smooth muscle.
Prostaglandins are potent vasoactive mediators and are produced by mast cells in skin and elsewhere, such as human lung, intestine, and liver (44, 45, 46). Cyclooxygenase, which exists in two isoforms, COX-1 and COX-2, is the rate-limiting enzyme in the biosynthesis of prostaglandins. Kawata et al. (47) has localized cyclooxygenase in mouse mast cells, and Roberts et al. (48) demonstrated that PGI2, a known vasodilator, is produced in rat mast cells in vitro. Vascular endothelium is also a major source of PGI2. In our study, the inhibition of cyclooxygenase activity partially decreased CRH-induced vasodilation in the human skin circulation. These findings suggest that CRH may act via PGI2 to cause dilation. However, because CRH has the potential to variously stimulate PGI2 production, including via a mast cell CRH-R1 receptor and/or an endothelium-dependent CRH-R2 receptor-mediated pathway, further studies will be required to determine the relative importance or the separate mechanisms involved.
In summary, this study demonstrates that the mechanisms by which CRH causes vasodilation in human skin are primarily via a mast cell-dependent pathway and are principally mediated by histamine. PGI2 and NO, with likely contributions from both the mast cell and vascular endothelium, also contribute to the microvascular effects of CRH. Mast cells have been reported to play a role in both the physiological and pathophysiological function of vascular tissue. Regulation of mast cell activity or inhibition of the effects of mast cell-derived mediators could therefore provide novel therapeutic agents for the prevention of cardiovascular disease in humans. This present study contributes to our understanding of vasoregulatory mechanisms operating in the peripheral circulation and how CRH may contribute to these events.
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Acknowledgments |
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Footnotes |
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Abbreviations: GEE, Generalized estimating equation; D-NAME, N-nitro-D-arginine methyl ester; L-NAME, N-nitro-L-arginine methyl ester; NO, nitric oxide; PGI2, prostacyclin; POMC, pro-opiomelanocortin; PU, perfusion unit.
Received March 4, 2003.
Accepted July 30, 2003.
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). Microvascular skin blood flow was measured in PU, and CRH was administered by six 30-sec pulses of a positive electrical current at 0.06 mA after a 1-h pretreatment of skin with sodium cromoglycate. All values are expressed as mean ± SEM.
). CRH-induced dilation (1 nM, n = 8) (•) was also significantly reduced in the presence of ranitidine hydrochloride (2.5% wt/wt, n = 6) (GEE, P 
