Sex- and limb-specific differences in the nitric oxide-dependent cutaneous vasodilation in response to local heating

Abstract

Local heating of the skin is commonly used to assess cutaneous microvasculature function. Controversy exists as to whether there are limb or sex differences in the nitric oxide (NO)-dependent contribution to this vasodilation, as well as the NO synthase (NOS) isoform mediating the responses. We tested the hypotheses that 1) NO-dependent vasodilation would be greater in the calf compared with the forearm; 2) total NO-dependent dilation would not be different between sexes within limb; and 3) women would exhibit greater neuronal NOS (nNOS)-dependent vasodilation in the calf. Two microdialysis fibers were placed in the skin of the ventral forearm and the calf of 19 (10 male and 9 female) young (23 ± 1 yr) adults for the local delivery of Ringer solution (control) or 5 mM N^ω-propyl-l-arginine (NPLA; nNOS inhibition). Vasodilation was induced by local heating (42°C) at each site, after which 20 mM N^G-nitro-l-arginine methyl ester (l-NAME) was perfused for within-site assessment of NO-dependent vasodilation. Cutaneous vascular conductance (CVC) was calculated as laser-Doppler flux/mean arterial pressure and normalized to maximum (28 mM sodium nitroprusside, 43°C). Total NO-dependent vasodilation in the calf was lower compared with the forearm in both sexes (Ringer: 42 ± 5 vs. 62 ± 4%; P < 0.05; NPLA: 37 ± 3 vs. 59 ± 5%; P < 0.05) and total NO-dependent vasodilation was lower in the forearm for women (Ringer: 52 ± 6 vs. 71 ± 4%; P < 0.05; NPLA: 47 ± 6 vs. 68 ± 5%; P < 0.05). NPLA did not affect total or NO-dependent vasodilation across limbs in either sex (P > 0.05). These data suggest that the NO-dependent component of local heating-induced cutaneous vasodilation is lower in the calf compared with the forearm. Contrary to our original hypothesis, there was no contribution of nNOS to NO-dependent vasodilation in either limb during local heating.

the cutaneous microvasculature is an easily accessible vascular bed for minimally invasive investigation of vascular control mechanisms in health and disease (15, 21). While several methods have been developed to induce vasodilation in the cutaneous vessels, direct local heating of the skin to 42°C has been increasingly used to examine microvascular dysfunction in clinical populations (8, 9, 14, 24) due to its highly reproducible vasodilatory response and large nitric oxide (NO)-dependent contribution. The skin blood flow response to local heating is biphasic, consisting of an initial rapid sensory nerve-mediated rise in skin blood flow, followed by a slower secondary rise to a plateau that is mediated primarily by NO-dependent mechanisms (16, 18, 22).

Despite the wealth of data detailing the involvement of NO during local thermal hyperemia, it remains unclear whether there are regional differences in the NO-dependent contribution to this response or in the nitric oxide synthase (NOS) isoform responsible for NO production. The majority of the local heating data has been collected from the cutaneous vasculature of the ventral forearm, and these data suggest that in healthy, young (<30 yr) subjects 1) ∼70% of the local heating plateau is mediated by NO-dependent mechanisms (18) and 2) that endothelial NOS (eNOS) is the primary NOS isoform responsible for NO synthesis (1, 19). However, Stewart et al. (25) have routinely examined the cutaneous vasculature of the calf, and their data suggest that in young, healthy women, the local heating response is ∼90% mediated by NO-dependent mechanisms, with neuronal NOS (nNOS) serving as the isoform responsible for NO synthesis (25). The methodologies employed and subject populations examined in these studies varied considerably, and it is unclear whether the disparity in the findings reflects 1) regional differences in microvascular control mechanisms, 2) posture-induced differences in the cutaneous vessels, or 3) sex-specific differences in NOS isoforms and NO-dependent vasodilation.

There is also a third NOS isoform, inducible NOS (iNOS), which has been implicated in aberrant NO production in vascular pathologies, such as hypertension (24). Previously, our laboratory group has found that iNOS does not contribute to local thermal hyperemia in the forearm cutaneous vasculature of healthy young and middle-aged adults (2). As such, we have not examined it in the present study. Rather, we have used a selective nNOS inhibitor (N^ω-propyl-l-arginine, NPLA) to eliminate nNOS production of NO. This isoform-specific inhibition, followed by nonspecific NOS inhibition with N^G-nitro-l-arginine methyl ester (l-NAME), allows us to methodologically partition out the nNOS-specific contribution to the NO-dependent cutaneous vasodilation response to local heating in vivo.

Recently, Del Pozzi et al. (7) reported that the contribution of NO to local thermal hyperemia is consistent between the forearm and the calf. Using nonspecific NOS inhibition in a group of healthy young subjects, the authors concluded that the contribution of NO to local thermal hyperemia was ∼23% and ∼24% in the arm and leg, respectively. Further, because most studies are not appropriately powered to detect small differences in the NO contribution to the local heating-induced plateau, questions remain as to possible sex differences. Most studies conducted on women, utilizing varying methodologies, have concluded that estrogen and progesterone have minimal influence on the magnitude of the plateau observed with local heating (3, 4). However, to date, few studies have specifically examined NO-dependent cutaneous vasodilation in healthy women, and whether there is a sex-specific difference in NO-dependent dilation in response to local heating is unclear.

Given the widespread use of local thermal hyperemia for the examination of vascular dysfunction in clinical populations, a more complete understanding of the mechanisms mediating this response is needed. Specifically, as this methodology is employed to examine the cutaneous vasculature of various anatomical regions and to explore the efficacy of novel therapeutic targets and intervention strategies, it is pertinent to fully elucidate the mechanisms mediating the NO-dependent vasodilatory response to local heating in a healthy, young cohort of subjects. Previously, sex and limb comparisons of the mechanisms mediating the rise in cutaneous vascular conductance observed with local heating of the skin have been performed indirectly, drawing from different studies using widely varying methodologies and subject populations. To this end, we aimed to provide a direct comparison of the mechanisms mediating the forearm and leg cutaneous vascular responses to local heating in men and women using a standardized local heating protocol. We examined regional and sex differences in NO-mediated vasodilation in a group of healthy, young subjects. We further examined the contribution of eNOS and nNOS isoforms to NO production in the local thermal hyperemia response of the forearm and the calf. We hypothesized that NO-dependent vasodilation would be greater in the calf compared with the forearm. We further hypothesized that the magnitude of the regional NO-dependent dilation would not be different between sexes; however, on the basis of the findings of Stewart et al. (25), women would exhibit greater nNOS-dependent dilation in the cutaneous vasculature of the calf.

METHODS

Subjects.

Experimental protocols were approved by the institutional review board of The Pennsylvania State University. Written and verbal consent were obtained voluntarily from all subjects prior to participation, according to the Declaration of Helsinki. Prior to recruitment, we determined by power analysis (power = 0.8, α = 0.05) that, on the basis of previously reported values in the literature, nine subjects in each group (men vs. women) would be sufficient to detect sex and limb differences in the cutaneous vascular response to local heating. Studies were performed on 19 healthy subjects (23 ± 1 yr, 10 men and 9 women). Subjects were screened for neurological, cardiovascular, and dermatological diseases and underwent a complete medical screening, including resting ECG, physical examination, lipid profile, and blood chemistry (Quest Diagnostics, Pittsburgh, PA). All subjects were normally active, nonhypertensive, nondiabetic, healthy nonsmokers, who were not taking over-the-counter or prescription medications with primary or secondary vascular effects (e.g., statins, antihypertensives, anticoagulants, antidepressants). None of the women who participated in this study were using hormonal contraceptives. All women were normally menstruating and were tested during the early follicular phase (days 1–7) of their menstrual cycle.

Instrumentation.

All protocols were performed in a thermoneutral laboratory with the subjects in a supine position and the experimental arm and leg supported at heart level. Two intradermal microdialysis fibers (10 mm, 20-kDa cutoff membrane, MD 2000; Bioanalytical Systems, West Lafayette, IN) were placed into the dermal layer of the ventral forearm and the calf for the local delivery of pharmacological agents (1). Microdialysis sites were randomly assigned to receive 1) 5 mM NPLA (Tocris, Ellisville, MO) for selective inhibition of nNOS (1, 24) or 2) lactated Ringer solution to serve as a control. Pharmacological agents were mixed just before use, dissolved in lactated Ringer solution, sterilized using syringe microfilters (Acrodisc; Pall, Ann Arbor, MI), and wrapped in foil to prevent degradation due to light exposure. Site-specific pharmacological solutions were perfused through the microdialysis fibers at a rate of 2 μl/min (Bee Hive controller and Baby Bee microinfusion pumps; Bioanalytical Systems). We and others have previously demonstrated the efficacy of NPLA for selectively inhibiting nNOS. This concentration of NPLA selectively inhibits nNOS with a K_i of 57 nM and inhibits the NO component to the reflex vasodilatory response in vivo (1, 5, 29).

Local heating.

Sixty to ninety minutes were allowed for hyperemia to cease before a standard local heating protocol to induce local vasodilation (1, 22). Following ∼20 min of baseline measurements, local heater temperature was increased from the baseline-clamped temperature of 33°C to 42°C at a rate of 0.1°C every second and then clamped at 42°C for the remainder of the heating protocol (1, 10, 22). After ∼30–40 min, when skin blood flow reached an established plateau, 20 mM l-NAME was perfused at a rate of 4 μl/min to nonselectively inhibit NOS and quantify NO-dependent vasodilation at all sites. After infusion of l-NAME and subsequent stabilization of a post-l-NAME plateau in skin blood flow, 28 mM sodium nitroprusside (SNP; USP, Rockville, MD) was perfused, and local temperature was increased to 43°C to elicit maximal dilation (CVC_max) (17).

Brachial arterial blood pressure was measured every 5 min throughout the protocol with an automated blood pressure cuff placed on the left arm (Cardiocap 5; GE Healthcare, New York, NY). Cutaneous red blood cell flux was continually measured directly over each microdialysis site with an integrated laser-Doppler flowmetry probe placed in a local heating unit (Moor Instruments SHO2). Cutaneous vascular conductance (CVC) was calculated as red blood cell flux divided by mean arterial pressure (MAP) and expressed as a percentage of site-specific maximal vasodilation (%CVC_max).

A two-way repeated-measures ANOVA was used to detect sex and local treatment differences in baseline, local heating plateau, NO-dependent vasodilation, and maximal CVC (version 9.1.3; SAS, Cary, NC). Post hoc comparisons with Bonferroni corrections were performed when appropriate. The level of significance was set at α = 0.05 for main effects. Values are presented as means ± SE.

RESULTS

Subject characteristics are presented in Table 1. There were no significant differences between sexes for body mass index, resting MAP, total cholesterol, or fasting blood glucose. Mean arterial blood pressure did not change throughout the protocol. Table 2 presents baseline and maximal absolute CVC values in the forearm and the calf in Ringer and NPLA-treated microdialysis sites in both men and women. There was no effect of microdialysis treatment, region, or sex on baseline or maximal absolute CVC (all P > 0.05).

Table 1.

Subject characteristics

	Men	Women
Age, yr	23 ± 1	23 ± 1
BMI, kg/m2	24 ± 1	23 ± 1
Resting MAP, mmHg	90 ± 2	82 ± 3
Total cholesterol, mg/dl	165 ± 12	187 ± 10
Fasting glucose, mg/dl	88 ± 2	85 ± 2

Values are expressed as means ± SE.

BMI, body mass index; MAP, mean arterial pressure.

Table 2.

Baseline and maximal CVC values in the forearm and the calf in Ringer's and NPLA-treated microdialysis sites in both men and women

	Men		Women
	Forearm	Calf	Forearm	Calf
Baseline CVC
Ringer's	0.19 ± 0.02	0.15 ± 0.02	0.23 ± 0.04	0.16 ± 0.02
NPLA	0.19 ± 0.02	0.21 ± 0.03	0.16 ± 0.01	0.33 ± 0.09
Maximal CVC
Ringer's	1.51 ± 0.13	1.39 ± 0.17	1.67 ± 0.13	1.59 ± 0.24
NPLA	1.50 ± 0.13	1.34 ± 0.19	1.71 ± 0.16	1.34 ± 0.19

Values are expressed as means ± SE.

CVC, cutaneous vascular conductance; NPLA, N^ω-propyl-l-arginine.

Figure 1 shows a representative tracing of the local heating response in the control site in the forearm of one subject.

Fig. 1.

A representative tracing of the local heating response in the control site in the forearm of one subject. The arrow denotes the decrease in skin blood flow with NOS-inhibition. l-NAME, N^G-nitro-l-arginine methyl ester.

Regional differences.

Figure 2 depicts mean skin blood flow at the local heating plateau (Fig. 2A) and within-site NO-dependent vasodilation (Fig. 2B) in Ringer solution and NPLA-treated microdialysis sites in the forearm and calf of all subjects. The local heating plateau was lower in the Ringer solution's site in the calf compared with the forearm (calf: 90 ± 2 vs. forearm: 97 ± 1% CVC_max, P < 0.05; Fig. 2A). NO-dependent vasodilation was lower in the calf compared with the forearm (Ringer solution: 42 ± 5 vs. 62 ± 4%; P < 0.05 and NPLA: 37 ± 3 vs. 59 ± 5%; P < 0.05; Fig. 2B).

Fig. 2.

Values are expressed as means ± SE vasodilation response (percent maximum cutaneous vascular conductance, %CVC_max) at the local heating plateau (A) and within site NO-dependent vasodilation (B) in Ringer's and NPLA-treated microdialysis sites in the forearm and calf of all subjects (n = 19). *P < 0.05% CVC_max different from Ringer's site in the forearm. †P < 0.05 NO-dependent dilation different from forearm within site.

Sex differences.

Figure 3 shows mean %CVC_max at the local heating plateau (Fig. 3, A and B) and within-site NO-dependent vasodilation (Fig. 3, C and D) for men and women in Ringer solution- and NPLA-treated microdialysis sites in the forearm and calf. The local heating plateau was lower in the Ringer solution site in the calf compared with the forearm (calf: 89 ± 4 vs. forearm: 97 ± 1% CVC_max; P < 0.05; Fig. 3A) of men. There was no difference between NPLA- and Ringer solution-treated sites in the calf of men. There were no sex differences in the magnitude of the local heating plateau in either the forearm or the calf (Fig. 3, A and B). However, NO-dependent vasodilation was lower in the forearm for women compared with men (Ringer solution: 52 ± 6 vs. 71 ± 4%, P < 0.05; NPLA: 47 ± 6 vs. 68 ± 5%, P < 0.05; Fig. 3, C and D). In addition, total NO-dependent vasodilation was lower in the calf compared with the forearm in both men (Ringer solution: 49 ± 7 vs. 71 ± 4%; P < 0.05; NPLA: 43 ± 4 vs. 68 ± 5%; P < 0.05; Fig. 3C) and women (Ringer solution: 35 ± 6 vs. 52 ± 6%; P = 0.07; NPLA: 30 ± 4 vs. 47 ± 6%, P < 0.05; Fig. 3D).

Fig. 3.

Values are expressed as means ± SE vasodilation response (%CVC_max) at the local heating plateau (A and B) and within site NO-dependent vasodilation (C and D) separated by sex in Ringer's and NPLA-treated microdialysis sites in the forearm and calf. *P < 0.05 %CVC_max different from control site in the forearm. ‡P < 0.05 NO-dependent dilation different from men within site. †P < 0.05 NO-dependent dilation different from forearm within sex. ††P = 0.07 NO-dependent dilation different from forearm within sex.

eNOS and nNOS isoforms.

nNOS-inhibition (NPLA-treated microdialysis sites) had no effect on either the local heating plateau or NO-dependent vasodilation across regions (Fig. 2) or between sexes (Fig. 3) (all P > 0.05).

DISCUSSION

The primary findings of this study were that, while there are no regional or sex differences in the local heating-induced vasodilatory plateau, 1) the NO-dependent portion of this plateau is reduced in the calf compared with the forearm skin in both men and women, 2) the total NO-dependent vasodilation was lower in the forearm of women compared with the forearm of men, and 3) there was no significant contribution of nNOS to the local thermal hyperemia response in the forearm or the calf of young, healthy adults. Although we observed a statistically significant difference in the local heating plateau of the Ringer solution site of the calf compared with the forearm when the groups were collapsed (Fig. 2A) and in the men when the groups were divided by sex (Fig. 3A), this difference (∼91% CVC_max vs. ∼96% CVC_max) is likely not physiologically relevant in the context of our young, healthy subject population.

In the cutaneous vasculature of young, healthy subjects, there is controversy regarding regional differences in the NO-dependent contribution to the local heating plateau and the NOS isoform primarily responsible for NO production (2, 19, 25). The use of varying methodologies, subject populations, and pharmacological inhibitors may contribute to these inconsistent findings. To date, only one study has examined regional differences in local thermal hyperemia using the same methodologies in the forearms and the calves of young, healthy subjects, and while this study provided important data regarding variability in NO-dependent dilation across regions, it did not explore potential variation due to sex differences or the NOS isoforms responsible for NO production (7). Our study provides novel data directly comparing the mechanisms mediating the forearm and leg cutaneous vascular responses to local heating in men and women using a standardized local heating protocol, which allows for within-site assessment of NO-dependent vasodilation.

Regional differences in NO-mediated vasodilation.

Our results show that although there is no difference in the absolute magnitude of the local heating plateau, there is a region-specific difference in the relative contribution of NOS-derived NO during local thermal hyperemia. Contrary to our original hypothesis, we found that the contribution of NO was lower in the calf compared with the forearm (Fig. 2B). Most local heating data have been collected in the cutaneous vasculature of the ventral forearm. These studies have consistently found that ∼70% of the secondary rise to a plateau in vasodilation is mediated by NO-dependent mechanisms (1, 18). Stewart et al. (26, 27) examined the cutaneous vasculature of the calves in women and concluded that ∼90% of the local thermal hyperemia plateau was NO-dependent. Recently, Del Pozzi et. al. (7) explored the relative contributions of NO to local thermal hyperemia in the forearm and calf cutaneous circulation with and without NOS inhibition in nine (five men, four women) healthy subjects. They concluded that the relative contribution of NO to the vasodilator response was consistent between limbs. Although that study provided data in both the forearm and calf of the same subjects, the study design did not allow for a direct within-site assessment of NO-dependent vasodilation. Furthermore, the percent contribution reported in that study—26 ± 3% and 27 ± 2% in the forearm and calf, respectively—was substantially lower than values previously reported in either regional circulation (2, 13, 18, 19, 25).

Because we did not observe differences in the local heating plateau, our data suggest that secondary NO-independent mechanisms play a larger role in the overall response of the cutaneous vasculature of the calf. We speculate that this difference may reflect, in part, differences in chronic hydrostatic pressure and possibly shear stress exposure between the cutaneous vessels of the forearm and the calf. Studies examining the conduit vessels of the arm and the leg have found structural and functional differences and suggest that there is no relation between brachial artery and superficial femoral or popliteal artery flow-mediated dilation (11, 28). Moreover, these studies postulate that between-limb differences in physiological stimuli (e.g., shear stress, blood pressure, physical activity) contribute to these differences. However, whether structural differences in the cutaneous vessels of the forearm and calf exist is currently unknown. Utilizing spectral analysis, Hodges and Del Pozzi (12) recently reported differences in myogenic, sympathetic, and endothelial vasomotion between the forearm and the calf during local skin heating, providing evidence for regional variation in cutaneous vascular control. Our data similarly suggest that regional differences exist in the mechanisms mediating cutaneous vasodilation in response to local heating of the skin. However, the structural and/or molecular origins of those differences remain to be explored.

Sex differences in NO-mediated vasodilation.

The current study was specifically designed and sufficiently powered to examine potential sex differences and regional differences in microvascular function between men and women. Our results demonstrate that women may have a decreased reliance on NO-dependent mechanisms to increase blood flow in the forearm skin during local heating. We did not detect a difference between men and women in the absolute magnitude of the local heating plateau, suggesting that our female subjects may have a decreased reliance on NO-dependent mechanisms in the cutaneous vasculature of the forearm. Interestingly, these sex-specific differences were absent in the cutaneous vasculature of the calf. However, in the context of our finding that the vasculature of the calf relies more heavily on NO-independent mechanisms in both sexes, the absence of sex-specific differences in the calf may be explained by regional variation in the control of vasodilation in response to local skin heating.

It is important to note that all of the women who participated in this study were not using hormonal contraceptives and were tested during the early follicular phase of their menstrual cycle, when female reproductive hormones are at their lowest circulating concentrations. Although there is little role of female hormone status on the local heating plateau (3, 4), given the important role of estrogen for endothelial function and eNOS-dependent production of NO (20), our results may reflect variation in NO-dependent vasodilation explained by cyclic changes in circulating endogenous hormones.

Endothelial vs. neuronal NOS-mediated cutaneous vasodilation.

Consistent with our previous study (2), the results from the current study suggest that eNOS is the primary NOS isoform responsible for NO production in the cutaneous vasculature of both the forearm and the calf in men and women. Previously, Bruning et al. (2) and Kellogg et al. (19) showed that eNOS is the primary isoform mediating NO production in response to local heating of the forearm skin (2, 19). In contrast, Stewart et al. (25) concluded that nNOS is the primary isoform responsible for NO production in the cutaneous vasculature of the calves of women. Previously, our laboratory group has found that iNOS does not contribute to local thermal hyperemia in the forearm cutaneous vasculature of healthy young and middle-aged adults (2). Because iNOS has not been shown to contribute to NO production in response to local heating in the healthy cutaneous vasculature, we have not examined it in the present study. In the current study, we found that inhibition of nNOS did not affect the local heating plateau or the within-site measure of NO-dependent vasodilation, in the forearm or the calf (Fig. 2). Furthermore, there was no effect of nNOS inhibition in the forearm or the calf in either men or women (Fig. 3). These findings suggest that there is little to no contribution of nNOS-derived NO to local thermal hyperemia across regions or between sexes.

Perspectives and Significance

In summary, our results suggest that there are regional differences in the relative contribution of NO to the cutaneous vasodilation response to local heating. There was no difference in the absolute magnitude of the local heating plateau between regions or across sexes, but the forearm had a greater NO-dependent contribution compared with the calf. Furthermore, women had a smaller reliance on NO-dependent mechanisms to increase skin blood flow in the forearm; however, this sex-specific difference was not apparent in the calf. Finally, nNOS does not contribute to NO production in either the forearm or the calf of men or women, suggesting that eNOS is the primary NOS isoform responsible for NO production in local thermal hyperemia.

Given the growing use of the cutaneous vasculature and the local heating response as a means to investigate endothelial dysfunction and intervention efficacy in an expansive range of clinical populations (6, 8–10, 14, 23–25), it is important to characterize regional and sex-specific differences in the control of local thermal hyperemia. Specifically, given the high reliance on NO for cutaneous vasodilation in response to local heating and the importance of NO as a vasoprotective molecule and molecular target for intervention strategies, this technique is frequently employed to investigate NO-dependent vasodilation. Our results suggest that though the NOS isoform mediating NO production is similar, the contribution of NO may differ, making comparison across regions and sexes difficult. Further, possible effects of hormone status may confound NO-dependent vasodilation data collected in the forearm of women, suggesting that particular attention should be paid to clinical populations in which this may be a factor. Collectively, our findings provide new insights into the direct comparison of sex and limb differences in the cutaneous vascular response to local heating, contribute to the body of work characterizing the role of NO in the cutaneous vasodilatory response, and provide new insights into the relative contribution and source of NO in the cutaneous vasculature of the forearm and the calf of young, healthy men and women.

AKNOWLEDGMENTS

The authors are grateful for the assistance of Susan Slimak and Jane Pierzga.

GRANTS

This research was supported by The National Institutes of Health Grants HL-120471-01 (to J. L. Greaney), AG-007004-23 (to W. L. Kenney), and HL-093238-04 (to L. M. Alexander).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: A.E.S. and J.L.G. conception and design of research; A.E.S. and J.L.G. performed experiments; A.E.S. and J.L.G. analyzed data; A.E.S., J.L.G., W.L.K., and L.M.A. interpreted results of experiments; A.E.S. and J.L.G. prepared figures; A.E.S. and J.L.G. drafted manuscript; A.E.S., J.L.G., W.L.K., and L.M.A. edited and revised manuscript; A.E.S., J.L.G., W.L.K., and L.M.A. approved final version of manuscript.