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1Department of Physiology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas; 2Department of Clinical Pathophysiology, School of Health Sciences, University of Occupational and Environmental Health, Kitakyushu; and 3Department of Exercise and Health Science, Faculty of Education, University of Yamaguchi, Yamaguchi, Japan
Submitted 2 February 2005 ; accepted in final form 15 September 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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skin blood flow; skin temperature; bretylium; laser-Doppler flowmetry; microdialysis
One local factor important to the cutaneous vasoconstriction is the temperature of the blood vessels and surrounding tissues themselves. The mechanisms for the vascular effects of local temperature (Tloc) include both adrenergic and nonadrenergic elements. With respect to adrenergic participation, it has been reported that an adrenergically dependent vasoconstriction can be locally initiated through an axon reflex during local cooling (21, 32, 33). This is the major mechanism responsible for the immediate vasoconstrictor response to local cooling (21, 33). It has also been shown that the affinity of postsynaptic 
2-adrenergic receptors for norepinephrine is enhanced, whereas norepinephrine synthesis and vascular contractility are reduced by local cooling (1, 7, 11, 13, 14, 41, 42).
The cutaneous vasomotor effects of local cooling can be explained in part as being due to nonadrenergic mechanisms. Those mechanisms are, however, not known. On the basis of responses after inhibition of sympathetic vasoconstrictor function, an initial nonadrenergic vasodilator and a later vasoconstrictor response to local cooling have been identified (21, 33), but their mechanisms have not. There is reason to hypothesize an involvement of nitric oxide (NO) in one or both of these responses to direct cooling of the tissue. The NO synthase (NOS) enzymes, especially neuronal NOS and inducible NOS, have been shown to be temperature sensitive such that enzyme activity is reduced by mild cooling (43). Thermal effects may influence blood viscosity and shear stress, which, coupled with changes in blood flow, can moderate NO production (2, 9, 26), although there is some question about the importance of shear stress in NO production in human skin (44). Also, NO does play an important role in the cutaneous vasodilator response to local heating (24, 28) as well as several other cutaneous vasodilator responses (22, 24, 25, 28, 36). Thus it is possible that NO production could be enhanced by local cooling, contributing to the early nonadrenergic vasodilation, and/or reduced, serving as a mechanism for the later nonadrenergic vasoconstrictor phase. Following from these considerations, NOS inhibition should reduce the above initial vasodilation if it is important there. Also, NOS inhibition should reduce blood flow, dependent on the tonic level of enzyme activity and, by doing so, limit the vasoconstriction with subsequent local cooling inasmuch as the enzyme activity was already reduced by the inhibitor.
To optimize our exploration of the role of NO in these responses to local cooling, we first aimed to optimize the occurrence of the initial vasodilator response. We know from earlier work from this laboratory that more aggressive local cooling made that vasodilator response more evident, but the distinction between the rate of cooling and the degree of cooling was not clear from that work (30).
The present studies were designed to better understand the cutaneous vascular responses to decreases in Tloc and to elucidate the role of NO in those responses. We first examined the rate dependence of the responses to local cooling (protocol 1), hypothesizing that the early nonadrenergic vasodilator phase would be dependent on the rate of local cooling. Next, we examined the effect of NOS inhibition on the initial and longer term responses to local cooling (protocols 2 and 3). We hypothesized that NOS inhibition would reduce the initial nonadrenergic vasodilator and the sustained vasoconstrictor responses during local skin cooling.
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METHODS |
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The protocol for this study was approved by the Institutional Review Board of the University of Texas Health Science Center at San Antonio. Seven female and five male subjects were recruited for this study. Mean age, height, and weight were 36 ± 2 yr, 171 ± 2 cm, and 72 ± 4 (SE) kg. All subjects were healthy nonsmokers and did not consume caffeine within 12 h of any experiment. The menstrual cycle was not considered in the experiments in female subjects because a previous report showed that the vasoconstrictor response to local cooling was unaffected by reproductive hormone status (6). Furthermore, differences in response between the male and female subjects were not seen in the present studies, and their data were combined for analysis. The informed consent of each subject was obtained before his/her participation in this study.
Measurements
The following methods were used in the three protocols of the study. Whole body skin temperature was recorded from six thermocouples placed on the body surface and controlled by means of a water-perfused suit (40). The suit covered the entire body with the exception of the head, feet, and the arm from which measurements were taken. Cold stress (3-min duration, aggressive cooling) was induced by perfusing the suit with cold water to lower whole body skin temperature quickly from 34 to 30–32°C to test for adequate blockade of the vasoconstrictor nerves (Ref. 23; see Protocols 1 and 3). Otherwise, whole body skin temperature remote from the area of blood flow measurement was maintained at 34°C (thermoneutral) in all experiments. Skin blood flow was measured by the laser-Doppler method (LaserFlo, Vasamedics, St. Paul, MN) and expressed as laser-Doppler flow (LDF) (18, 31). LDF measurements are specific to the skin and are not influenced by blood flow to underlying skeletal muscle (34). Tloc of the 6.3-cm2 area surrounding the site of LDF measurement was controlled by a metal sleeve for the flow probe that has both a heating element and a Peltier cooling element. A thermocouple between the skin surface and the sleeve served for measurement and feedback control. Tloc can be easily maintained within 0.1°C and can be rapidly changed with this controller. Arterial blood pressure was recorded noninvasively and continuously from the left middle finger (Finapres blood pressure monitor, Ohmeda, Madison, WI). Mean arterial pressure was obtained from the electrical integration of the continuous blood pressure signal. Cutaneous vascular conductance (CVC) was calculated as the ratio of LDF to mean arterial pressure (in arbitrary units). All studies were performed with the subject at supine rest.
Experiments were carried out according to the following protocols, conducted on separate days.
Protocols
Protocol 1: rate dependency in the skin vascular responses to local cooling. This protocol was performed to test whether the nonadrenergic skin vascular response to local cooling is dependent on the rate of reduction of Tloc. LDF was monitored at two control untreated sites and at two bretylium tosylate (BT)-treated sites, all on the ventral surface of the left forearm. BT (Schweizerhall, South Plainfield, NJ), which blocks the release of transmitter from adrenergic terminals (15), was applied iontophoretically to a 0.64-cm2 area of skin at least 1.5 h before the study (23). This method produces a selective local blockade of the cutaneous adrenergic vasoconstrictor system (21, 23, 33).
Six subjects were studied in this protocol. Approximately 90 min after the application of BT, data collection began. To control for effects of previously applied cooling, two different procedures of local cooling were performed. One untreated site and one BT-treated site were simultaneously cooled to 24°C at the rate of –0.33°C/min (slow cooling), maintained at 24°C for 5 min, and then rewarmed to 34°C (recovery period). During the slow cooling procedure, Tloc at the other two sites was maintained at 34°C. After a recovery period of 25 min, Tloc at all LDF measurement sites was decreased to 24°C at the rate of –4.0°C/min (fast cooling), maintained at 24°C for 32.5 min, and then returned to 34°C. After a recovery period of 20 min, whole body cold stress was performed to verify the persistence of vasoconstrictor nerve blockade at the BT-treated sites.
Protocol 2: role of NO in the vascular responses in intact skin during local cooling. This protocol was performed to examine the effect of NOS inhibition on the vascular response to local cooling in the skin with intact sympathetic function. Seven subjects were studied in this protocol. We chose intradermal microdialysis for delivery of an inhibitor of NOS activity (NG-nitro-L-arginine methyl ester; L-NAME, Sigma Chemical, St. Louis, MO), because it allows local and continuous delivery of L-NAME to the cutaneous interstitium at high concentrations without confounding systemic effects (22, 24). Each microdialysis probe consisted of a microdialysis membrane with an 18-kDa cutoff (Spectrum, Laguna Hills, CA), 1 cm in length and 200 µm in diameter, connected to polyimide tubing with a 0.0049-in. internal diameter. The probe was reinforced with 0.0015-in.-diameter coated stainless steel wire (3, 24).
Subjects had three intradermal microdialysis probes placed 
2.5 h before the start of data collection. Before insertion of each microdialysis probe, a cold pack was applied to the ventral surface of the left forearm as a temporary anesthetic. A needle (25 gauge) was inserted intradermally into the arm for 2.5 cm. The probe was then fed through the lumen of the needle. Probes were aligned such that the microdialysis membranes were centered within the dermis. The needle was then removed, leaving the probe in place.
Sterile saline was perfused (4 µl/min) at all three sites for a baseline period of 15–20 min at a Tloc of 34°C (Fig. 1B). LDF was monitored using LDF probes placed directly over the center of microdialysis probes. Subsequently, two sites received 20 mM L-NAME (L-NAME sites) or saline (control site). This concentration of L-NAME was based on earlier studies in which lower concentrations, similarly administered, were sufficient to inhibit markedly the NO-dependent phase of the vasodilator response to local warming (24, 28). It was also based on preliminary studies in which the effects of 50 mM L-NAME were not seen to differ from those of 20 mM L-NAME. This phase of the experiment lasted 40–50 min, until a steady-state blood flow was observed. The results from protocol 1 showed that the early nonadrenergic vasodilator response to local cooling to be more obvious with moderately rapid changes in Tloc. Therefore, Tloc at all LDF measurement sites was cooled to 24°C at the rate of –4.0°C/min, maintained at 24°C for 32.5–42.5 min, returned to 34°C, and followed by a recovery period of 20 min.
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Data Processing and Statistical Analysis
All measurements were recorded once per second (Labview, National Instruments) and averaged into 20-s periods by a laboratory computer. Data for CVC from BT-treated and untreated sites were further compiled into 1-min averages. Data averaged over the 5 min just before the beginning of local cooling were used for precooling baseline values. The changes in CVC were expressed as percent changes from the precooling baseline. In protocols 2 and 3, changes in CVC were also expressed as percent changes from the baseline values before the infusion of L-NAME. To characterize the CVC responses to local cooling in protocols 2 and 3, we separately analyzed the responses from the early and late phases of cooling. Peak reduction (vasoconstriction) or peak increase (vasodilation) in CVC during the first 15 min of cooling was defined as the CVC response in the early phase. In the late phase, CVC responses were averaged over last 3 min of cooling. Values for CVC from pairs of identically treated sites were averaged for each subject. To analyze changes in CVC from the precooling values and to test for differences in response in CVC between BT-treated and untreated sites in protocol 1, data were analyzed by two-way analysis of variance with repeated measures and a Bonferroni post hoc test when a significant difference was identified. Changes in CVC from baseline before infusion of L-NAME and from the precooling values during L-NAME infusion were each compared by paired t-test in protocols 2 and 3. Also, a paired t-test was used to test for differences in response in CVC between L-NAME treated and untreated sites. The level of significance was set at P < 0.05. All data are expressed as means ± SE.
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RESULTS |
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Protocol 1
Figure 2 shows changes in CVC in response to different rates of cooling in protocol 1. When Tloc was maintained at 34°C, CVC at BT-treated and untreated sites remained unchanged. Subsequent fast cooling increased (P = 0.001) CVC at BT-treated sites in the early phase (Fig. 2A). CVC at untreated sites showed a triphasic change in the early phase of fast cooling. That is, CVC decreased (P < 0.01) rapidly, recovered to precooling levels, and again decreased (P < 0.01) slowly until the end of cooling. Slow cooling did not significantly increase CVC at BT-treated sites in the early phase (P = 0.55; Fig. 2B). The reduction in CVC at BT-treated sites was more slowly initiated than that at untreated sites during the slow cooling protocol (Fig. 2B). That is, a significant decrease of CVC from the baseline was obtained at 15 min (Tloc = 29.0°C) of cooling at untreated sites, but this was not observed until 20 min (Tloc = 27.3°C) of cooling at BT-treated sites. When fast cooling was subsequently applied to the same skin areas previously subjected to the slow cooling trial (Fig. 2C), CVC at untreated sites showed the triphasic pattern in the early phase of fast cooling as described above (Fig. 2A), whereas the tendency for CVC to increase at BT-treated sites did not reach statistical significance in the early phase of fast cooling. However, CVC at BT-treated sites was higher than that at control sites for minutes 3–11 of local cooling (P < 0.05). In the later phase of cooling, CVC at BT-treated and untreated sites was decreased (P < 0.01) by cooling. The reductions did not differ significantly (P > 0.05) between the slow and fast cooling trials or between BT-treated and untreated sites.
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Changes in CVC from a representative subject in this protocol are shown in Fig. 3. Application of L-NAME by microdialysis decreased (P < 0.05) CVC by 28.3 ± 3.8% from the preinfusion baseline value (P < 0.05); infusion of saline did not change CVC, suggesting a role for NOS in blood flow in normothermic skin. Local cooling rapidly decreased CVC at both saline and L-NAME sites in the early phase. In some cases, the CVC reduction in the early phase was followed by a transient increase (see saline site in Fig. 3) and then by a gradual reduction for the remainder of the cooling period. Changes in CVC in this protocol are summarized in Fig. 4. When CVC is expressed as a percentage of the baseline value before infusion of L-NAME, CVC decreased to similar levels at saline (39.2 ± 3.2%) and L-NAME (42.7 ± 3.7%) sites in the late phase of cooling (Fig. 4A), with a smaller reduction at the L-NAME-treated sites (P < 0.05). When the changes in CVC are expressed relative to the precooling baseline values, CVC in the late phase of cooling decreased less (P < 0.05) at L-NAME-treated sites (to 60.7 ± 6.2% of baseline) than at saline-treated sites (to 40.8 ± 4.1% of baseline; Fig. 4B). The smaller reduction in CVC with L-NAME during local cooling, regardless of which baseline was chosen, suggests that NOS inhibition plays a role in the long-term reduction in CVC with local cooling.
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This protocol was the same as protocol 2 with the exception that all sites were pretreated with BT to block sympathetic function. Responses in CVC from one representative subject in this protocol are shown in Fig. 5. Infusion of L-NAME decreased (P < 0.05) CVC by 46.9 ± 6.3% from the baseline value before the infusion (P < 0.05); infusion of saline did not significantly change CVC. Local cooling transiently increased (P < 0.05) CVC at both BT + saline and BT + L-NAME sites in the early phase (Figs. 5 and 6). When CVC is expressed as a percent change from the baseline value before infusion of L-NAME, the cooling-induced vasodilation was smaller (P < 0.05) at BT + L-NAME sites (an increase of 65 ± 15%) than at BT sites (an increase of 107 ± 29%) (Fig. 6A). The difference in the responses between sites was also observed when the changes of CVC were expressed as absolute values. However, when CVC is expressed as a percent change from the precooling baseline value, the vasodilator responses in this phase of cooling did not differ between that at BT + L-NAME sites (an increase of 129 ± 29%) and that at BT + saline sites (an increase of 129 ± 46%) (Fig. 6B). After the vasodilator responses reached a peak at 10 min of cooling, CVC decreased gradually until the end of cooling. In the late phase, local cooling decreased (P < 0.05) CVC at BT + saline sites relative to the earlier peak and to the precooling values, but it did not reduce CVC below precooling values at BT + L-NAME sites (P = 0.29; Fig. 6). The smaller changes in CVC, regardless of the normalization scheme, suggest a role for NO in the longer term vasoconstrictor response to local cooling. The comparison between the results of protocols 2 and 3, specifically that CVC fell below precooling levels only in protocol 2, suggests a role for adrenergic nerve function in prolonged cooling.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Rate Dependency in the Nonadrenergic Cutaneous Vascular Response to Local Cooling
Blockade of norepinephrine release from adrenergic terminals delayed the initiation of the cutaneous vasoconstrictor response to local cooling regardless of the rate of cooling. This finding agrees with earlier reports (21, 33), which showed that locally evoked norepinephrine release is the major mechanism responsible for the immediate vasoconstrictor response to direct local cooling. In those reports, the initial vasoconstrictor response to local cooling was reversed to a vasodilation by blockade of transmitter release from vasoconstrictor nerves, adrenergic receptor antagonism, or sensory nerve block (21, 33). In the present study, the initial vasodilation at BT-treated skin sites observed during fast local cooling was absent during slow local cooling, showing that the nonadrenergic vasodilation in nonglabrous skin is dependent on the rate of local cooling. The delay in the vasoconstrictor response (Fig. 2B) to slow local cooling at BT-treated sites suggests some element of the vasodilator mechanism to be involved at the slower cooling rate but that the effect is not the net increase in CVC seen with more rapid cooling. The mechanisms for nonadrenergic vasodilation also appear to be evoked in the skin vasculature with functionally intact adrenergic nerve terminals because, as noted earlier by Pérgola et al. (33), rapid cooling transiently increased CVC at untreated control sites after the initial reduction (Fig. 2, A and C). Thus it appears that nonadrenergic vasodilator mechanisms compete with adrenergic vasoconstrictor mechanisms in this phase of rapid local cooling. When both mechanisms are present, the latter appears to dominate. An advantage of this protocol is that it provided a comparison between slow (Fig. 2B) and rapid cooling (Fig. 2A) for the first cooling exposure at each of those sites. Rapid cooling after slow cooling (Fig. 2C) shows an attenuated vasodilator response.
What physical and/or physiological mediators are affected by the different rates of cooling? Transmitters such as calcitonin gene-related peptide released from sensory nerve terminals via an axon reflex (17, 35) are unlikely candidates for the mediators of nonadrenergic vasodilation because sensory nerve blockade with topical anesthesia at the cooling site with adrenergic function intact also revealed the initial vasodilator response to direct local cooling (21). Thus intact sensory function is not required for the nonadrenergic vasodilation. One possible mechanism for the nonadrenergic vasodilation is the direct effect of cooling on myogenic tone, as postulated from studies of pulmonary arterial and aortic smooth muscle preparations (29). Indeed, it may represent a general reduction in vascular smooth muscle contractility associated with direct cooling (13, 41, 42). However, there are regional differences in the myogenic responses of smooth muscle to direct cooling. For example, the ear artery in rabbit (16) and gastrointestinal tract in rat (30) show a constriction rather than a dilatation to cooling in in vitro studies. In addition, for this mechanism to explain the vasodilation in the initial phase of cooling, the direct effect of cooling on vascular smooth muscle in the skin has to be rate-sensitive. The rate-sensitive mechanisms for vasodilation are still unclear. The smaller response with repeated rapid cooling (Fig. 2C) and the rate sensitivity suggest receptor or enzyme mediation, but biophysical effects of cooling cannot be dismissed. It is unclear whether the mechanism(s) causing the initial vasodilation persist throughout the duration of local cooling. If they do persist, then vasoconstrictor mechanisms override that tonic vasodilator mechanisms and act to decrease the vasodilation during prolonged local cooling.
CVC fell with decreasing Tloc during slow local cooling. Moreover, the decreases of CVC in the latter phase of cooling did not differ between the slow- and fast-cooling trials. These findings show that the final vasoconstrictor responses to prolonged local cooling depend on the level but not the previous rate of cooling.
Role of NO in the Skin Vascular Responses to Local Cooling
Protocol 1 provided a pattern of local cooling that consistently yielded the initial vasodilation in sympathetically inhibited skin and a longer term vasoconstriction. In protocols 2 and 3, we tested for a role for NO in the nonadrenergic mechanisms for skin vasomotor control at thermoneutral and sites locally cooled according to the rapid cooling portion of protocol 1. At neutral skin temperature (34°C), inhibition of NOS activity decreased basal CVC by 28% at untreated sites (protocol 2) and by 47% at BT-treated sites (protocol 3). These findings imply an important role for NOS activity in basal vascular tone in the skin. The finding from protocol 2 that infusion of L-NAME significantly decreased CVC in thermoneutral conditions is consistent with previous reports (24, 25), although some (22, 28, 44) did not describe an L-NAME-induced reduction of CVC. This inconsistency may be due to differences in experimental conditions (e.g., differences in Tloc, concentration or infusion rate of L-NAME, timing of baseline measurement during the infusion). In the present study, NOS activity was inhibited by a higher concentration (20 mM) and a higher infusion rate (4 µl/min) of L-NAME than most previous studies, and the data were analyzed after 40–50 min of infusion. The ability to measure a vasoconstrictor response to NOS inhibition requires both an effective level of the NOS inhibitor as well as tonic NOS activity. Kellogg et al. (25) recently reported that the interstitial concentration of NO measured by an amperometric electrode technique showed a measurable level in normothermia that was increased during hyperthermia. Thus it appears that functional NOS plays a role for control of basal vascular tone in intact skin in a temperature-dependent manner.
The reduction in baseline CVC with NOS inhibition causes some uncertainty in the quantitative evaluation of a role for NOS in the subsequent vasodilator response to local cooling at BT-treated sites. When CVC at BT-treated sites was expressed relative to the baseline value before the infusion of L-NAME (protocol 3), NOS inhibition decreased the subsequent vasodilator response by 40% (Fig. 6A). On the other hand, when CVC was expressed relative to the precooling value, L-NAME did not significantly alter the nonadrenergic vasodilator response in the early phase of cooling (Fig. 6B). It is not clear which is the best expression of CVC changes to determine quantitatively the effect of L-NAME on the response to local cooling in this portion of study. In either case, however, these findings suggest that NOS does not play a primary role in the nonadrenergic vasodilation. It is most likely that other substances, such as prostaglandins released from the vascular endothelium (5, 12), participate independently or synergistically with NO in this vasodilator mechanism.
Prolonged local cooling of sites with intact sympathetic function decreased CVC to similar levels (40% of the initial baseline) at saline- and L-NAME-treated sites (protocol 2, Fig. 4A). In both cases, the final level of CVC was significantly less than the precooling baseline. This vasoconstriction in the presence of significant or complete NOS inhibition indicates NOS independent mechanisms to be important in this response to prolonged local cooling. The evidence from this study indicates a role for adrenergic participation in this mechanism because the addition of adrenergic nerve blockade with bretylium to NOS inhibition (protocol 3) prevented CVC from being reduced below the precooling baseline, whereas, in protocol 2, with intact vasoconstrictor nerve function, cooling caused CVC to fall to 60% of the precooling levels in the presence of NOS inhibition. A role for NOS in the vasoconstrictor response to longer term local cooling is also implicated by our findings. In both protocol 2 and protocol 3, NOS inhibition greatly reduced the reduction in CVC with subsequent local cooling. In both cases, CVC was reduced by NOS inhibition. With local cooling, the level of CVC at L-NAME-treated sites and untreated sites fell to levels not statistically distinguishable from one another (Figs. 4A and 6A). This is consistent with achieving part of the vasoconstriction through inhibition of NOS by the lower temperature. L-NAME accomplished that inhibition ahead of time, thereby reducing the influence of cooling. In this model, some of the influence of local cooling would be through the inhibition of NOS and part of the remainder being due to adrenergic function. This interpretation is in keeping with earlier observations of adrenergic involvement in the initial vasoconstrictor response to local cooling (21, 32, 33), the reduction in the effect of NOS blockade by exposure to a cold environment (8), and the documented role for increased NOS in the cutaneous vasodilator response to Tloc increases (24, 28).
While we favor the above model for the cutaneous vascular response to local cooling, there are several caveats that must be acknowledged. First, the recent observation that NO can inhibit adrenergically mediated vasoconstriction (10) raises the possibility that, in the present study, inhibition of NOS with L-NAME might increase the apparent effect of released norepinephrine induced by local cooling beyond the effect in unblocked skin. If this contributes to the present study, it might imply a somewhat smaller role for adrenergic mechanisms than implied by the data. However, if NOS activity is reduced by local cooling, that effect should be small. Second, we have described the vasodilation at the beginning of local cooling in adrenergically blocked skin as transient, but it is conceivable that the vasodilator mechanism is sustained and is slowly overridden by a competing vasoconstrictor mechanism. If that were the case, the vasoconstriction during prolonged local cooling would include the rather marked decrease from the early peak level of CVC. In that scenario, neither adrenergic activity nor NOS inhibition would appear to be importantly involved in that phase of the reduction in CVC. In that case, one might expect a greater ultimate vasoconstriction at control sites than at sites pretreated with BT, but that was not the case here or earlier (21). Nevertheless, that scenario would modify the quantitative estimates of the roles of NOS and adrenergic nerves in the vasoconstriction with local cooling, but it would not negate them. Third, could it be that the limited vasoconstrictor response to prolonged local cooling in the presence of NOS inhibition is because the vasculature is at or near maximal vasoconstriction? Indeed, the 47% reduction in CVC with L-NAME treatment at BT-treated sites is impressive. However, this consideration should include the fact that BT treatment or local cutaneous nerve block, by removing the effects of tonic adrenergic tone, generally raises CVC to about twice the previous level (e.g., Refs. 20, 32, 33). Hence, the 47% reduction in CVC with L-NAME treatment at those sites should restore CVC to near normal resting levels (i.e., levels without BT or L-NAME treatment), clearly outside of such a "basement" limitation. Related to this potential baseline dependence of the response, past studies noted that the reduction in CVC (or its equivalent) with a vasoconstrictor stimulus was greater from higher initial baseline levels (see Ref. 19). In those cases, however, the final values of CVC remained very different, unlike the results after NOS inhibition and local cooling with the present data. Therefore, although a role for initial values may exist here, it clearly cannot explain our observation that local cooling alone achieved the same final value as local cooling in the presence of L-NAME. Fourth, vasoconstriction originating from other than adrenergic or NOS-dependent mechanisms probably also participates in the response to local cooling. Sympathetic cotransmitters such as neuropeptide Y and ATP may contribute, although neuropeptide Y Y1-receptor antagonism had no effect on the cutaneous vasoconstrictor response to local cooling (21). Similarly, endothelin produced by vascular endothelial cells is known to have increased plasma levels accompanying severe cooling of the hand (27, 45), but whether milder cooling of a more limited area of forearm stimulates release of effective amounts of endothelin is unknown.
We postulate that the mechanism through which local cooling involves the NOS system is through inhibition by cold of NOS enzyme(s). This is known to occur, especially, for neuronal and inducible NOS (43). Whether endothelial NOS has more cold sensitivity in vivo than what might be expected for in vitro studies, or if one of the other NOS enzymes participates in this response, is not currently known. The mechanisms of the influence of local cooling on adrenergic function appears to involve enhanced affinity of 2-adrenergic receptors (1, 7, 11, 13, 14), which may include transfer of 2C-adrenergic receptors to the plasma membrane through a Rho kinase system stimulated by the local cooling (1, 7).
Limitations of This Study
We used a higher concentration (20 mM) and a higher microdialysis flow rate for delivery of L-NAME to inhibit NOS activity compared with those in most other studies of the role of NO in the skin vascular response to local warming (24, 28), to ensure a significant inhibition of NOS activity. Nevertheless, it is difficult to confirm whether complete blockade of NOS activity was accomplished. This is a limitation for interpreting the results in this study. In pilot studies, we found the initial vasodilator and later vasoconstrictor responses to local cooling during infusion of 50 mM L-NAME not to differ from those during infusion of 20 mM L-NAME. We take this to indicate that 20 mM L-NAME is appropriate to inhibit adequately NOS in the skin under the conditions of the present study. Nevertheless, this potential problem with the use of NOS inhibitors is still unresolved.
In summary, local cooling involves at least two nonadrenergic mechanisms. A nonadrenergic vasodilator mechanism that dominates the initial phase is rate sensitive. Functional NOS plays at best a minor role in this early-phase vasodilation during local cooling. A nonadrenergic vasoconstrictor mechanism can dominate the latter phase of local cooling whether or not adrenergic function is blocked. This mechanism appears to require functional NOS for its full expression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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. The role of myogenic relaxation, adenosine and prostaglandins in human forearm reactive hyperaemia. J Physiol 389: 147–161, 1987.2C-adrenergic receptors enable cold-induced vasoconstriction in cutaneous arteries. Am J Physiol Heart Circ Physiol 278: H1075–H1083, 2000.-Adrenoceptors and cold-induced vasoconstriction in human finger skin. Am J Physiol Heart Circ Physiol 255: H1000–H1003, 1988.1- and 2-adrenergic responses in cutaneous veins: role of receptor reserve. Am J Physiol Heart Circ Physiol 249: H950–H955, 1985.This article has been cited by other articles:
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  G. J. Hodges, D. N. Jackson, L. Mattar, J. M. Johnson, and J. K. Shoemaker Neuropeptide Y and neurovascular control in skeletal muscle and skin Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2009; 297(3): R546 - R555. [Abstract] [Full Text] [PDF]
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 G. J. Hodges, C. Chiu, W. A. Kosiba, K. Zhao, and J. M. Johnson The effect of microdialysis needle trauma on cutaneous vascular responses in humans J Appl Physiol, April 1, 2009; 106(4): 1112 - 1118. [Abstract] [Full Text] [PDF]
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 G. J. Hodges, W. A. Kosiba, K. Zhao, and J. M. Johnson The involvement of heating rate and vasoconstrictor nerves in the cutaneous vasodilator response to skin warming Am J Physiol Heart Circ Physiol, January 1, 2009; 296(1): H51 - H56. [Abstract] [Full Text] [PDF]
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G. J. Hodges, W. A. Kosiba, K. Zhao, and J. M. Johnson The involvement of norepinephrine, neuropeptide Y, and nitric oxide in the cutaneous vasodilator response to local heating in humans J Appl Physiol, July 1, 2008; 105(1): 233 - 240. [Abstract] [Full Text] [PDF]
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F. Yamazaki, K. Takahara, R. Sone, and J. M. Johnson Influence of hyperoxia on skin vasomotor control in normothermic and heat-stressed humans J Appl Physiol, December 1, 2007; 103(6): 2026 - 2033. [Abstract] [Full Text] [PDF]
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G. J. Hodges, W. A. Kosiba, K. Zhao, G. E. Alvarez, and J. M. Johnson The role of baseline in the cutaneous vasoconstrictor responses during combined local and whole body cooling in humans Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H3187 - H3192. [Abstract] [Full Text] [PDF]
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G. J. Hodges, J. A. Traeger III, T. Tang, W. A. Kosiba, K. Zhao, and J. M. Johnson Role of sensory nerves in the cutaneous vasoconstrictor response to local cooling in humans Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H784 - H789. [Abstract] [Full Text] [PDF]
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R. J. Widmer, R. H. Stewart, M. F. Young, J. E. Laurinec, G. A. Laine, and C. M. Quick Application of local heat induces capillary recruitment in the Pallid bat wing Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2007; 292(6): R2312 - R2317. [Abstract] [Full Text] [PDF]
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C. S. Thompson-Torgerson, L. A. Holowatz, N. A. Flavahan, and W. L. Kenney Cold-induced cutaneous vasoconstriction is mediated by Rho kinase in vivo in human skin Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1700 - H1705. [Abstract] [Full Text] [PDF]
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J. M. Johnson Mechanisms of vasoconstriction with direct skin cooling in humans Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1690 - H1691. [Full Text] [PDF]
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 G. J. Hodges, K. Zhao, W. A. Kosiba, and J. M. Johnson The involvement of nitric oxide in the cutaneous vasoconstrictor response to local cooling in humans J. Physiol., August 1, 2006; 574(3): 849 - 857. [Abstract] [Full Text] [PDF]
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G. E. Alvarez, K. Zhao, W. A. Kosiba, and J. M. Johnson Relative roles of local and reflex components in cutaneous vasoconstriction during skin cooling in humans J Appl Physiol, June 1, 2006; 100(6): 2083 - 2088. [Abstract] [Full Text] [PDF]
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P < 0.05 vs. untreated site.
P < 0.05 vs. precooling baseline. 
