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Am J Physiol Heart Circ Physiol 292: H1700-H1705, 2007. First published December 15, 2006; doi:10.1152/ajpheart.01078.2006
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Cold-induced cutaneous vasoconstriction is mediated by Rho kinase in vivo in human skin

Caitlin S. Thompson-Torgerson,1 Lacy A. Holowatz,1 Nicholas A. Flavahan,3 and W. Larry Kenney1,2

1Noll Laboratory, Department of Kinesiology and 2Graduate Program in Physiology, The Pennsylvania State University, University Park, Pennsylvania; and 3Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland

Submitted 2 October 2006 ; accepted in final form 7 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cutaneous vasoconstriction (VC) is the initial thermoregulatory response to cold exposure and can be elicited through either whole body or localized skin cooling. However, the mechanisms governing local cold-induced VC are not well understood. We tested the hypothesis that Rho kinase participates in local cold-induced cutaneous VC. In seven men and women (20–27 yr of age), up to four ventral forearm skin sites were instrumented with intradermal microdialysis fibers for localized drug delivery during cooling. Skin blood flow was monitored at each site with laser-Doppler flowmetry while local skin temperature was decreased and maintained at 24°C for 40 min. Cutaneous vascular conductance (CVC; laser-Doppler flowmetry/mean arterial pressure) was expressed as percent change from 34°C baseline. During the first 5 min of cooling, CVC decreased at control sites (lactated Ringer solution) to –45 ± 6% (P < 0.001), increased at adrenoceptor-antagonized sites (yohimbine + propranolol) to 15 ± 14% (P = 0.002), and remained unchanged at both Rho kinase-inhibited (fasudil) and adrenoceptor-antagonized + Rho kinase-inhibited sites (yohimbine + propranolol + fasudil) (–9 ± 1%, P = 0.4 and –6 ± 2%, P = 0.4, respectively). During the last 5 min of cooling, CVC further decreased at all sites when compared with baseline values (control, –77 ± 4%, P < 0.001; adrenoceptor antagonized, –61 ± 3%, P < 0.001; Rho kinase inhibited, –34 ± 7%, P < 0.001; and adrenoceptor antagonized + Rho kinase inhibited sites, –35 ± 3%, P < 0.001). Rho kinase-inhibited and combined treatment sites were significantly attenuated when compared with both adrenoceptor-antagonized (P < 0.01) and control sites (P < 0.0001). Rho kinase mediates both early- and late-phase cold-induced VC, supporting in vitro findings and providing a putative mechanism through which both adrenergic and nonadrenergic cold-induced VC occurs in an in vivo human thermoregulatory model.

fasudil; local cooling; vascular function; adrenergic; norepinephrine

CUTANEOUS VASOCONSTRICTION (VC) is the initial thermoregulatory response to defend against cold exposure, effectively minimizing heat loss to the environment. Whole body skin cooling evokes reflex VC, which involves the release of norepinephrine and sympathetic cotransmitters from sympathetic adrenergic axon terminals (38–40, 42), whereas localized cooling of the cutaneous blood vessels and surrounding tissue engages local (i.e., nonreflex) VC mechanisms that are mediated, in part, by {alpha}2-adrenoceptors (12, 14, 27, 34). Reflex and local VC are not mutually exclusive responses and often operate in concert during cold exposure to maximize VC (1). However, whereas reflex VC mechanisms are relatively well understood, the mechanisms that govern local cold-induced VC have not been fully elucidated.

Localized cooling of the skin results in immediate VC that is sustained as long as the cold stimulus persists. During the early phase of localized cooling (0–10 min), cold-induced VC is mediated by norepinephrine released from sympathetic axon terminals in the absence of a reflex stimulus. However, during prolonged localized cooling (>10 min), cold-induced VC is driven primarily by nonsympathetic mechanisms (24, 34) that include a putative downregulation of the nitric oxide synthase (NOS) pathway (19, 44) but that still remain largely unclear.

Rho kinase has been implicated as a key intracellular mechanism contributing to cold-induced VC. In vitro, cutaneous vessel cooling leads to increased mitochondrial production of reactive oxygen species, which stimulate Rho kinase activity (2, 3). Cold-induced Rho kinase activity in cutaneous vascular smooth muscle cells can drive VC via two distinct pathways: 1) stimulation of {alpha}2C-adrenoceptor translocation from intracellular storage in the Golgi to the vascular smooth muscle cell surface (2, 3, 10, 22) and 2) enhanced intracellular Ca2+ sensitization (2, 4, 15, 17, 36, 37). These findings suggest that Rho kinase may participate in early and/or later phases of cutaneous cold-induced VC in vivo. Accordingly, the purpose of the present study was to test the hypothesis that Rho kinase participates in cold-induced VC in human skin in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subjects. Seven subjects (20–27 yr of age; 4 men and 3 women) participated in the present study. All women were tested during days 1–7 of the menstrual cycle, and none were taking oral contraceptives. All subjects underwent a standardized medical screening and were healthy, normotensive, nonobese nonsmokers. No subjects were taking any medications that might alter cardiovascular responses to cooling. They abstained from alcohol and caffeine for 12 h before coming to the laboratory but were permitted to eat on the morning of the experiment. Approval was obtained from the Institutional Review Board at The Pennsylvania State University. Each subject gave verbal and written informed consent before participation in the study, and all procedures conformed to the standards of the Declaration of Helsinki.

Instrumentation. Subjects arrived at the laboratory at 0800. In two subjects, three microdialysis fibers (model MD-2000, Bioanalytical Systems, West Lafayette, IN) were placed into the ventral surface of the right forearm by using sterile technique, whereas four fibers were placed into the forearm dermis of the remaining five subjects. For each fiber, a 25-gauge needle was inserted into unanesthetized skin and guided horizontally through the skin such that entry and exit points were ~2 cm apart. The fiber, consisting of a cylindrical 10-mm membrane (320 µm OD, 20-kDa molecular mass cutoff) and connective tubing attached to either end of the membrane, was threaded through the needle. The needle was then withdrawn, leaving the membrane in the skin. After fiber insertion, subjects rested quietly for ~90 min to allow local hyperemia due to insertion trauma to subside, during which time lactated Ringer solution was perfused through all fibers at a rate of 2.0 µl/min (Bee Hive controller and Baby Bee microinfusion pumps, Bioanalytical Systems).

Skin blood flow was measured by using laser-Doppler flowmetry (MoorLAB, Moor Instruments) to provide an index of cutaneous red blood cell flux. Laser-Doppler probes were placed on the skin directly over each microdialysis site, and red blood cell flux data were collected continuously throughout the experiment. Arterial blood pressure was monitored periodically via brachial auscultation approximately every 20 min and during experimental interventions. Mean arterial pressure was calculated as [(1/3 systolic blood pressure) + (2/3 diastolic blood pressure)]. Skin blood flow was converted to cutaneous vascular conductance (CVC; red blood cell flux/mean arterial pressure) and expressed as percent change from baseline CVC values (%{Delta}CVCbase). Local skin temperature (Tloc) at each site was controlled within ±0.02°C by using Peltier elements (TecThermo Temperature Controller 1575, Menlo Park, CA) with a small aperture in the center to accommodate the laser-Doppler probe.

Protocol. Following the resolution of needle insertion trauma, microdialysis sites were randomly assigned to continuously receive combinations of yohimbine (competitively antagonizes {alpha}-adrenoceptors), propranolol (competitively antagonizes beta-adrenoceptors), and fasudil (competitively binds to ATP-binding domain to inhibit Rho kinase) as follows: site 1, lactated Ringer (control); site 2, 5 mM yohimbine + 1 mM propranolol (Y + P); and site 3, Y + P + 3 mM fasudil (Y + P + fasudil). Although yohimbine is typically used as an {alpha}2-adrenoceptor-specific antagonist, it is effective as a nonspecific {alpha}-adrenoceptor antagonist in higher concentrations, including those used in this study (18, 38–40, 42). In a subset of subjects (n = 5), data were also collected at a fourth site treated only with 3 mM fasudil. In in vivo human pilot studies, varying concentrations of fasudil (0.1–5 mM) were delivered at 2 µl/min for at least 1 h before and then throughout a localized cooling protocol. Although all doses of fasudil inhibited cold-induced VC to varying degrees, inhibition reached a plateau at doses 3 mM and higher. Therefore, 3 mM fasudil was perfused in the present study as the minimum effective inhibitory dose during localized cooling.

After solutions perfused the sites for ~60 min, Tloc was clamped at 34°C for a 15-min baseline period. Following baseline, 1 µM norepinephrine + 1 mg/ml L-ascorbate (preservative) (20) were added to the perfusate through all fibers for 4 min to test vascular responsiveness and the integrity of Y + P adrenoceptor antagonism. After a washout period (~30 min) to allow CVC to recover to pre-norepinephrine baseline values, all sites were cooled at a rate of 3°/min, and Tloc was clamped at 24°C for 40 min, after which sites were rewarmed back to 34°C.

All drugs were obtained from Sigma-Aldrich (St. Louis, MO), except fasudil, which was obtained from Tocris Bioscience (Ellisville, MO). All drug solutions were mixed just before usage, dissolved in lactated Ringer solution, and sterilized by using syringe microfilters (Acrodisc; Pall, Ann Arbor, MI).

Data collection and analysis. Data were recorded and stored as 1-min averages by using computer software (LabView) and a data acquisition system (National Instruments, Austin, TX). Baseline values for normalization were determined by averaging the last 5 min of baseline data. Representative time course data are expressed as 5-min averages throughout the entire 40-min cooling protocol. Mean summary cooling data are expressed as averages of the first (early phase) or last (late phase) 5 min of cooling. These time points were chosen to clearly illustrate discrete early- and late-phase mechanisms of VC, unobscured by transition-phase mechanisms (~10–25 min). Data were analyzed by using repeated-measures, two-way analysis of variance, with planned comparison post hoc tests when significant differences were detected. Statistical significance was set at {alpha} = 0.05. Values are expressed at means ± SE, unless otherwise noted.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The seven subjects who participated in the study were young, healthy, normotensive, and nonobese (Table 1). No subjects were excluded due to a failure of Y + P to fully antagonize adrenoceptors. No sex differences in CVC responses were detected, so data from men and women were pooled for analysis.


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  		Table 1. Subject characteristics

 
In a representative time course tracing of cold-induced VC from a single subject (Fig. 1), all pharmacological treatments blocked at least a portion of cold-induced VC. Adrenergic antagonism alone (Y + P) mildly attenuated VC compared with control throughout the cooling protocol, whereas both Rho kinase inhibition (fasudil) and combined adrenoceptor antagonism and Rho kinase inhibition (Y + P + fasudil) blocked VC to a greater degree throughout the entire cooling protocol.


Figure 1
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  		Fig. 1. Representative tracing of cutaneous vascular effects of 40-min localized cooling to 24°C at sites treated with lactated Ringer solution (control), adrenoceptor antagonists yohimbine and propranolol (Y + P), a Rho kinase inhibitor (fasudil), and combined adrenoceptor antagonists + Rho kinase inhibitor (Y + P + fasudil). All time points are 5-min averages. {Delta}CVC, change in cutaneous vascular conductance; Tloc, local skin temperature.

 
During the first 5 min of skin cooling, mean early-phase VC in all subjects (n = 7; fasudil, n = 5) was abolished by adrenoceptor antagonism and Rho kinase inhibition, administered both alone and in combination (Fig. 2A). CVC at control sites decreased to –45 ± 6% {Delta}CVCbase (P < 0.0001 vs. 34°C baseline). CVC at Y + P sites increased to 15 ± 14% {Delta}CVCbase (P = 0.002 vs. 34°C baseline), whereas CVC at fasudil and Y + P + fasudil sites remained unchanged (fasudil, –9 ± 1% {Delta}CVCbase, P = 0.4 vs. 34°C baseline; Y + P + fasudil, –6 ± 3% {Delta}CVCbase, P = 0.4 vs. 34°C baseline).


Figure 2
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  		Fig. 2. Average cutaneous vasoconstriction after 5 min (A) and 40 min (B) of localized skin cooling to 24°C at control, adrenoceptor-antagonized (Y + P), Rho kinase-inhibited (fasudil) and combined adrenoceptor-antagonized + Rho kinase-inhibited (Y + P + fasudil) sites. Control, Y + P, and Y + P + fasudil, n = 7; fasudil, n = 5. *P < 0.05 vs. baseline; {dagger}P < 0.05 vs. control site.

 
During the last 5 min of cooling, all sites exhibited greater VC when compared with the early phase of cooling (Fig. 2B). CVC at control sites further decreased to –77 ± 4% {Delta}CVCbase (P < 0.0001 vs. 34°C baseline). CVC at Y + P sites decreased to –61 ± 3% {Delta}CVCbase (P < 0.0001 vs. 34°C baseline), but the late-phase VC response at this site was significantly attenuated when compared with control (P = 0.03). CVC at fasudil and Y + P + fasudil sites decreased to –34 ± 7% {Delta}CVCbase and –35 ± 3% {Delta}CVCbase (both P < 0.0001 vs. 34°C baseline) and exhibited significantly less VC compared with the Y + P and control sites (P < 0.001).

In response to 4 min of 1 µM norepinephrine infusion under noncooled conditions (Tloc = 34°C), control and Rho kinase-inhibited sites exhibited significant VC, whereas CVC at adrenoceptor-antagonized sites remained unchanged (Fig. 3). CVC at control and fasudil sites significantly decreased from baseline (control, –33 ± 6% {Delta}CVCbase, P < 0.0001 vs. 34°C baseline; fasudil, –22 ± 8% {Delta}CVCbase, P = 0.001 vs. 34°C baseline); there was no statistical difference between the sites (P = 0.09). CVC at Y + P and Y + P + fasudil sites did not significantly change from preinfusion baseline (Y + P, 7 ± 6% {Delta}CVCbase, P = 0.2; Y + P + fasudil, –6 ± 2% {Delta}CVCbase, P = 0.2).


Figure 3
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  		Fig. 3. Average maximal cutaneous vasoconstriction in response to 34°C 1 µM norepinephrine infusion at control, adrenoceptor-antagonized (Y + P), Rho kinase-inhibited (fasudil), and combined adrenoceptor-antagonized + Rho kinase-inhibited (Y + P + fasudil) sites. Control, Y + P, and Y + P + fasudil, n = 7; fasudil, n = 5. *P < 0.05 vs. baseline; {dagger}P < 0.05 vs. control site.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The primary findings of the present study were that 1) early-phase cold-induced VC is inhibited by yohimbine + propranolol (adrenoceptor antagonism) or fasudil (inhibition of Rho kinase), 2) late-phase cold-induced VC is minimally attenuated by adrenergic antagonists and is significantly attenuated by a Rho kinase inhibitor, and 3) Rho kinase inhibition more effectively attenuates VC at cold, compared with thermoneutral, local skin temperatures.

Rho kinase is a key intracellular mediator directly involved in VC, operating through inhibition of myosin light chain phosphatase, ultimately resulting in an increase in vascular smooth muscle sensitivity to extant intracellular Ca2+ (2, 4, 15, 17, 36, 37). In healthy resting vessels, tonically active RhoA/Rho kinase downregulates the endothelial NOS (eNOS) pathway (13, 29, 30, 41), whereas the nitric oxide pathway conversely downregulates RhoA/Rho kinase function (6, 7, 35). This mutual antagonism effectively maintains a healthy and necessary balance between dilator and constrictor influences in the vasculature. However, Rho kinase activity is disproportionately increased when vessels are exposed to either a particular physiological stress, such as stretch or a change in redox status (3, 11), or a specific disease state, such as hypertension, hypercholesterolemia, or diabetes (6, 16, 21, 23, 28, 31, 43). Recently, Bailey and colleagues (2, 3) established that RhoA/Rho kinase is also activated by direct cooling of cutaneous arteriolar smooth muscle cells and mediates cold-induced VC in vitro. Specifically, cold-induced Rho kinase activation mediates VC via two distinct pathways: Ca2+ sensitization and the characteristic {alpha}2C-adrenoceptor translocation observed with the onset of cutaneous tissue cooling (significantly augmenting the adrenoceptor population on vascular smooth muscle cells), with the latter effect predominating. Thus Rho kinase activation in the vasculature seems to be predominantly associated with either physiological (including cooling) or pathophysiological stressors; it is likely that superimposing these two types of stressors would elicit even greater Rho kinase activation, although this speculation requires further validation. Conversely, Rho kinase participation in vascular regulation may be less pronounced in healthy, resting states (25, 28, 32, 33).

This study tested the hypothesis that Rho kinase also contributes to cold-induced VC in vivo during localized skin cooling. During the first 10 min of local cooling, an immediate and pronounced VC occurs that can be inhibited by either presynaptic sympatholytics or adrenoceptor antagonists (specifically {alpha}2) but not with proximal nerve blockade, suggesting a localized (i.e., nonreflex) mechanism dependent on norepinephrine of sympathetic origin binding to {alpha}2-adrenoceptors (9, 12, 14, 19, 24, 27, 34). The results from the present study help to further clarify the mechanisms underlying this early-phase response to cooling. Adrenergic antagonism abolished VC, whereas both Rho kinase inhibition and simultaneous adrenergic antagonism + Rho kinase inhibition blocked 85–90% of the VC response. These results indicate that VC during the first few minutes of cooling is very sensitive to both adrenergic blockade and Rho kinase inhibition, suggesting that both pathways are integrally involved in early-phase cold-induced VC and most likely interact, as has been shown in vitro (2, 3).

As localized skin cooling progresses past 10–15 min, CVC continues to decrease, albeit at a slower rate than during the first 10 min. During this prolonged phase of VC, pre- and postsynaptic sympathetic inhibitors only block ~20% of the VC response, suggesting that nonadrenergic mechanisms helps mediate late-phase cold-induced VC (19, 24, 34, 44). The results from the present study confirm that prolonged cooling is predominantly mediated by nonadrenergic mechanisms and provide direct evidence identifying cellular mechanisms centrally involved in this response. Whereas adrenergic antagonism only blocked 20% of late-phase VC in this study, Rho kinase inhibition or concurrent administration of both adrenoceptor antagonists and a Rho kinase inhibitor blocked ~60% of late-phase VC. Attenuation with Rho kinase inhibition indicates that Rho kinase participates in late-phase cold-induced VC, and similar attenuation with combined adrenoceptor antagonism + Rho kinase inhibition suggests that, although Rho kinase and norepinephrine are both involved in late-phase VC, their effects are not additive. Rather, these data cumulatively indicate that a smaller portion of late-phase VC is mediated by an interaction between adrenergic and Rho kinase pathways, whereas the majority of the response is mediated directly by Rho kinase, possibly through Ca2+ sensitization.

To establish whether the Rho kinase contribution to cold-induced VC was specifically elicited by cooling per se, norepinephrine was infused at all treatment sites when Tloc = 34°C (thermoneutral). CVC at adrenoceptor-antagonized sites did not significantly change from preinfusion baseline, verifying the integrity of the adrenoceptor antagonism at those sites. However, control and Rho kinase-inhibited sites both exhibited significant VC in response to thermoneutral norepinephrine administration. Specifically, Rho kinase inhibition only attenuated norepinephrine-mediated VC by ~33% (indicating only modest Rho kinase activity at rest), compared with 85–90% reduction of cold-induced VC. These data preclude the argument that fasudil nonspecifically suppresses VC by indicating that Rho kinase inhibition much more effectively blocked VC during cooling, suggesting that Rho kinase is significantly more involved in mediating VC under cold (compared with thermoneutral) conditions. This conclusion is consistent with the cold-induced increase in Rho kinase activity that has been observed in vitro (2, 3). This conclusion also supports data in the literature indicating that vessels that have been exposed to a specific physiological and/or pathophysiological stressor tend to exhibit greater Rho kinase-mediated VC when compared with healthy, resting vessels in vivo (25, 28, 32, 33).

Cumulatively, the data from this study indicate that the interaction of norepinephrine and Rho kinase plays an integral role in both early- and late-phase cold-induced VC. During early-phase cooling, inhibition of either the adrenergic or the Rho kinase pathway fully abolished cold-induced VC, suggesting that these two pathways interactively mediated early-phase VC. During late-phase cooling, this interaction accounted for a smaller, but still significant, portion of the VC response, ~20%. Although we cannot conclusively articulate the point(s) of interaction between the two pathways based on the current data, there at least two possible explanations. It is feasible that norepinephrine-mediated VC is relatively Rho kinase independent until the onset of tissue cooling, whereupon the cold-induced increase in Rho kinase activity provides an additional intracellular mechanism through which norepinephrine can elicit VC. Alternatively, it is possible that cold-induced Rho kinase activity augments norepinephrine-mediated VC through its actions on {alpha}2C-adrenoceptors. Taking into consideration 1) the results of the present study suggesting a cold-specific Ca2+-dependent element in Rho kinase-mediated VC, 2) the established role of Rho kinase in mediating cold-induced {alpha}2C-adrenoceptor translocation in vitro (2, 3, 10, 22), and 3) the preponderance of in vivo human literature suggesting that early-phase cold-induced VC is mediated almost exclusively by {alpha}2-, but not {alpha}1-, adrenoceptors (despite the presence of functional {alpha}1-adrenoceptors in skin), it is plausible that Rho kinase may mediate VC, at least in part, through stimulation of {alpha}2C-adrenoceptor translocation. However, this speculation requires further testing to verify its validity in an in vivo model.

In addition to its reliance on Rho kinase putatively working through/interacting with adrenergic mechanisms, late-phase VC also comprises at least two other mechanisms. A Rho kinase-dependent mechanism that was independent of adrenoceptors (i.e., a direct VC effect of Rho kinase) accounted for an additional ~40% of the overall VC response. In view of the long-established role of Rho kinase in Ca2+-independent VC via inhibition of myosin light chain phosphatase (15, 36, 37), it is likely that this portion of late-phase VC was mediated by Ca2+ sensitization. This conclusion is further supported by the observation that Rho kinase-mediated Ca2+ sensitization can contribute to the maintenance of prolonged VC (36, 37). These results also suggest that there is at least one additional mechanism contributing to late-phase cold-induced VC that is not sensitive to either adrenoceptor antagonism or Rho kinase inhibition.

Rho kinase activation during localized skin cooling also supports a recently articulated role of the eNOS system in cold-induced VC, suggesting a putative third mechanism through which Rho kinase may mediate cold-induced VC. Hodges and colleagues (19) found that full expression of cold-induced VC involves a decrease in eNOS activity and/or nitric oxide production but did not speculate as to the mechanism(s) through which localized skin cooling might regulate the eNOS pathway. Just as nitric oxide (through cyclic GMP-dependent protein kinase) counters Rho kinase-mediated VC by activating myosin light chain phosphatase (7) and inhibiting activation of upstream RhoA (35), RhoA and Rho kinase similarly downregulates several steps in the eNOS pathway: decreased eNOS gene expression (30), decreased eNOS mRNA expression (13), eNOS mRNA destabilization (26, 41), decreased eNOS protein expression (6, 13, 41), decreased activation of eNOS (6, 8, 30), and increased arginase activity, which decreases nitric oxide bioavailability (5, 29). With a consideration for the interaction of these two systems under other conditions and in other vascular beds, it is plausible that they may interact in the cutaneous circulation during cooling, as well. Thus the combined findings of the present study and Hodges et al. (19) may illustrate one of two possible regulatory scenarios during cooling: 1) cold-induced reduction in eNOS activity through an unidentified mechanism leads to a reciprocal increase in Rho kinase activity and subsequent Rho kinase-mediated VC or 2) cold-induced activation of Rho kinase downregulates the eNOS pathway, leading to both active and passive VC (via withdrawal of basal dilatory vascular effects). However, further testing with simultaneous manipulation of eNOS and Rho kinase pathways is required to determine whether decreased eNOS activity or increased Rho kinase activity might be the initial step in this cycle of increasingly proconstrictor activity, as well as whether this mutual antagonism between dilator and constrictor systems functionally contributes to cutaneous vascular regulation in vivo.

Limitations. In the present study, fasudil, which was administered to determine the role of Rho kinase in cold-induced VC, presented two potential sources of uncertainty. Although fasudil is a selective inhibitor of Rho kinase, when administered in high concentrations it can also affect the activity of other protein kinases that may affect cold-induced VC. Therefore, it is possible that 3 mM fasudil may have affected other protein kinases in the cutaneous vasculature, and Rho kinase may have participated in cold-induced VC to a lesser extent than indicated by the data. However, more selective inhibitors (e.g., Y-27632) could not be substituted in this study, because fasudil is currently the only Rho kinase inhibitor available for in vivo administration in humans. Conversely, it was not possible to unequivocally determine whether the dose of fasudil administered in the study fully inhibited Rho kinase. Therefore, it is also possible that the contribution of Rho kinase to cold-induced VC was underestimated due to incomplete enzyme inhibition.

It is also possible that the ascorbate used in the present study as a norepinephrine preservative could have attenuated Rho kinase-mediated VC. Reactive oxygen species activity increases in response to cold and subsequently activates the RhoA/Rho kinase pathway (3); therefore, the antioxidant properties of ascorbate could have limited Rho kinase activity in the present study. However, given that the dose and duration of ascorbate delivery in the present study are significantly less than what is required for adequate antioxidant administration in the vasculature, it is unlikely that sufficient ascorbate accumulated before washout to exert significant antioxidant effects on the cutaneous arterioles. Furthermore, if the minute dose of ascorbate did lead to any longer-term reduction in reactive oxygen species activity, its effects would be accounted for at the control site and would not confound the interpretation of data relative to control or other treated sites.

In summary, this study presents direct evidence that Rho kinase mediates both early and late phases of cold-induced cutaneous VC. During early-phase skin cooling, both adrenergic antagonism and Rho kinase inhibition abolished adrenergic VC, suggesting the involvement of both pathways in this early response. During late-phase skin cooling, adrenergic antagonism attenuated VC by ~20%, whereas Rho kinase inhibition and combined adrenoceptor antagonism + Rho kinase inhibition both attenuated VC by ~60%, suggesting that Rho kinase mediates both adrenergic and nonadrenergic portions of late-phase cold-induced VC. Additionally, although Rho kinase can be activated by a variety of physiological and pathophysiological stimuli, Rho kinase contributed significantly more to VC after the onset of skin cooling in this study, lending support for its putative role in human thermoregulation. The results of this study confirm the extensive in vitro work investigating Rho kinase and cold-induced VC and extend those findings to an in vivo human thermoregulatory model.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health Grants R01-AG-07004-15 (to W. L. Kenney) and M01-RR-10732 (General Clinical Research Center).


   ACKNOWLEDGMENTS
 
The authors thank Jane Pierzga for research assistance, the General Clinical Research Center for providing medical consultation and screenings, and the research subjects for participation.

Present address for C. S. Thompson-Torgerson: Department of Anesthesiology and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21287.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. S. Thompson-Torgerson, 370 Ross Research Bldg., 720 Rutland Ave., The Johns Hopkins Univ. School of Medicine, Baltimore, MD 21205 (e-mail: cthomp44{at}jhmi.edu)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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