|Home | About | Journals | Submit | Contact Us | Français|
Cold-induced vasoconstriction is mediated in part by selective enhancement of local α2C–adrenoceptor (α2C-AR) activity. A common insertion-deletion variant in the α2C-AR gene (ADRA2C del322-325) results in an approximately 85% reduction of agonist-mediated function in vitro. We tested the hypothesis that individuals with the ADRA2C del322-325 variant have attenuated vasoconstriction in response to cold.
Cutaneous digital blood flow (flux) was measured by laser Doppler flowmetry in a controlled environment at room temperature and during two cycles of graduated local heat and cold exposure in 31 subjects. Temperature-response curves were analyzed to estimate the following measures: Emin (minimal flux during cooling), and ET50 and ET90 (the local temperature at which flux decreased by 50% and 90% respectively).
We found no significant genotypic differences in Emin (24.3±19.5, 30.0±20.5, and 21.5±25.9 AU for ins/ins, ins/del, and del/del genotypes, respectively; P=0.48), ET50 (25.5±6.0, 25.1±6.7, and 25.1±7.1 °C; P=0.99), or ET90 (20.5±4.7, 22.1±4.0, and 20.8±6.7 °C; P=0.77) in either the first or second heating and cooling cycle (cycle 1 values presented).
The ADRA2C del322-325 variant did not affect vascular sensitivity to local cold exposure.
Cold-induced vasoconstriction of peripheral blood vessels serves as a physiologic reflex to conserve central body heat during cold exposure. This occurs via a reflex increase in sympathetic activity coupled with local alterations resulting in increased vascular sensitivity to norepinephrine [8, 11]. Vasoconstriction in response to cold is mediated by the rapid selective enhancement of local α2–adrenergic receptor (α2-AR) activity [19, 12] and is inhibited by an α2-AR antagonist . Excessive cold-induced vasoconstriction is the hallmark of Raynaud's phenomenon, a clinical disorder that affects between 8-11% of the general population , and Raynaud's phenomenon has been linked to α2-AR dysfunction [14, 15].
There are three subtypes of α2-ARs, α2A, α2B, and α2C [4, 28]. At 37°C, α2-AR mediated vasoconstriction is conveyed primarily by the α2A subtype , and the α2C-AR has minimal activity, as it is not expressed at the cell surface, but sequestered predominantly in the Golgi apparatus . Hence, the α2C-AR was originally thought to be a “silent receptor .” However, cooling activates rho-kinase signaling  and evokes a redistribution of α2C-ARs to the cell surface, enhancing vasoconstrictor responses to norepinephrine  and mediating vasoconstriction in response to cold. Furthermore, selective inhibition of the α2C-AR attenuates the increased sensitivity to adrenergic agonists during cold exposure . Thus, the α2C-AR is a key mediator of vasoconstriction in response to cold and, therefore, has become a drug target for the development of subtype selective antagonists. Indeed, pharmacological treatment with such an investigational selective α2C–AR antagonist has been explored as a potential therapy for Raynaud's phenomenon .
A common genetic variant in the human α2C-AR gene (ADRA2C) results in the deletion of four amino acids, del322-325 . The frequency of the deletion allele is 42.5% and 6.3% in African-Americans and Caucasians, respectively , and it results in a reduction of approximately 85% of agonist-mediated function in vitro [30, 31]. In view of the α2C-AR's key role in cold-induced vasoconstriction, the decreased function of the receptor encoded by the del322-325 variant may result in attenuation of cold-induced vasoconstriction. However, to date, the effects of this variant on vascular sensitivity to cold have not been defined.
We previously developed a method to measure changes in skin blood flow in response to controlled incremental local cold exposure , and we used it in the present study to examine the hypothesis that subjects with the ADRA2C del322-325 variant have decreased vascular sensitivity to cooling. Since exposure to cold is required for α2C-ARs to translocate to the cell surface and become active, we performed a second cold exposure test by repeating the heating/cooling cycle to examine whether previous cold exposure would change subsequent skin flux responses to cold.
The study was approved by the Vanderbilt University Institutional Review Board, and all subjects provided written informed consent. To enrich the study population for the genotype of interest, we studied 31 African-American subjects, since the ADRA2C del322-325 variant is rare in Caucasians. Unrelated males and females were eligible to participate if they were African-American, between 18-40 years of age, nonsmokers, and had no clinically significant abnormality based on medical history and physical examination. No laboratory screening was performed for this study, though for 29 of the 31 subjects, red blood cell (RBC) count values were available from previous recent studies, and these are included in Table 1. Ethnicity was determined by self-report. Subjects were free of medications for at least 2 weeks. Since cutaneous blood flow varies with the menstrual cycle , all women were studied in the luteal phase of the menstrual cycle.
Subjects were admitted to the General Clinical Research Center (GCRC) of the Vanderbilt University Medical School at 8:00 am on the morning of the study day after an overnight fast. All studies were performed in a temperature-controlled room (24.0±1.0°C). After 30 minutes of acclimatization, a 20 G intravenous cannula was inserted into an antecubital arm vein for blood sampling. After an additional 30 minutes of supine comfortable rest, resting blood pressure and heart rate were obtained by a semi-automated device (Dinamap MPS; GE Medical Systems, Waukesha, Wis, USA), and a venous blood sample (12 mL) was drawn from the uncuffed arm for determination of plasma norepinephrine concentration.
The subject's contralateral hand was then inserted to a fixed location inside a thermally inert plexi-glass box. Laser probes (DP1T-V2, Moor Instruments, Devon, UK) were attached to the pulp of the distal phalanges of the third and fourth digits. Skin blood flow was assessed by laser Doppler flowmetry (DRT4, Moor Instruments, Devon, UK), and skin temperature by a thermocouple. The laser Doppler flowmeter produces an output signal based on the reflection of light off moving red blood cells (RBCs). This signal is proportional to the microvascular blood flow (flux), a product of the number of RBCs in the sampling volume and the mean velocity . Laser Doppler flux is proportional to blood flow, though the absolute value of the arbitrary units depends on many factors. There is considerable inter-individual variability in flux measurements; therefore the greatest utility of laser Doppler lies in assessing relative within-subject changes in flow, rather than absolute flux differences between subjects . Skin flux and skin temperature were recorded continuously on an attached computer. The box temperature was controlled by a modified heating and air conditioning unit (Whirlpool ACE082XR). A metal baffle placed before the airflow inlet ensured even temperature distribution within the box, as confirmed in preliminary studies .
After an acclimatization period of at least 60 minutes, baseline skin flux and temperature measurements were recorded at room temperature for 10 minutes. The temperature in the box enclosing the hand was then increased gradually to 40.5° within 5 minutes and maintained at that temperature for 20 minutes (Figure 1) . During the following 60 minutes, the temperature in the box was decreased stepwise at 2 minute intervals in approximately 2.5°C decrements to a minimum temperature of 8°C. We then performed a second cold exposure test by repeating the heating/cooling cycle to examine whether previous cold exposure would change subsequent skin flux responses to cold (Figure 1). During each cooling cycle, if skin flux did not change over three stepwise temperature decrements, this value was considered to represent maximum vasoconstriction in response to cold, and the cooling cycle was ended.
Blood samples (12 mL each) were drawn, and blood pressure was measured after 20 minutes at 40.5 °C during the 2 heating cycles, and at the minimum temperature during the 2 cooling cycles.
Blood was collected into cooled heparinized tubes, immediately placed on ice, and centrifuged at 4°C for 10 minutes at 3,000 rpm. Plasma was harvested and stored in tubes containing 40 μL of reduced glutathione (6%) at -20°C until assayed. Norepinephrine concentrations were measured by high-performance liquid chromatography using electrochemical detection with dihydroxybenzylamine as the internal standard .
We performed a series of studies to optimize the heating/cooling protocol and to assess reproducibility. Preliminary studies on within-subject reproducibility of ET50 (n=4) yielded a day-to-day difference of 1.6±2.9°C.
After amplification of DNA fragments by polymerase chain reaction (PCR), ADRA2C del322-325 genotyping was performed by DNA fragment analysis as described previously . In brief, a fluorescently labeled forward primer (5′-6-FAM-AGACGGACGAGAGCAGCGCA-3′) and a reverse primer (5′-AGGCCTCGCGGCAGATGCCGTACA-3′) were used to amplify DNA fragments by PCR. Amplicons were denatured at 95°C for 5 min, and fragment analysis was performed on an ABI 3730 Genetic Analyzer and its Genotyper V.1.0.1 software. The success rate for genotyping was 100%. We use the term “ADRA2C” when referring to the gene and “α2C-AR” when referring to the receptor encoded by the gene. Individuals homozygous for the ADRA2C insertion (wild type) allele, homozygous for the deletion allele, and heterozygous are termed ADRA2C “ins/ins,” “del/del,” or “ins/del,” respectively.
Skin temperature and skin blood flow (flux) were calculated as the average of the readings in the two fingers. Baseline skin temperature and flux were calculated as the average of values obtained over 10 minutes after subjects had been acclimated in the room for at least 60 minutes. For the heating/cooling cycles, for each subject, average flux during every 2 minute temperature step was plotted against the box temperature, and nonlinear regression analysis was used to fit a sigmoidal curve (Prism v4.0, GraphPad Software Inc., San Diego, CA) (Figure 2). The software provides estimates for the steady-state blood flow during the heating stimulation (Estim), the minimum blood flow achieved during cooling (Emin), the box temperature at which flux decreased by half the maximal effect (ET50), and the box temperature at which flux decreased by 90% (ET90) (Figure 2). We interpreted Emin to reflect maximal cold-induced vasoconstriction, and ET50 and ET90 to reflect the sensitivity to cooling. The software also calculates the R2 value as a measure of goodness of curve fit.
Skin temperature, which is determined by both ambient temperature and flux, was analyzed with the following parameters: Tstim (the maximum skin temperature during heating stimulation), Tmin (the skin temperature at the time point of ET90, at which flux had decreased to 90% of the maximum change (Estim - Emin)), and Tmid (the skin temperature at the time when box temperature measured 24.5 °C, a midpoint, approximately 71 minutes into the study). Mean arterial blood pressure was calculated as the diastolic blood pressure plus one third of the systolic minus the diastolic pressure. All values are reported as mean ± standard deviation. A Kruskal-Wallis or chi square test was used to compare values in subjects with different ADRA2C genotypes. A repeated measures analysis of variance (ANOVA) was used to compare plasma norepinephrine concentrations and mean arterial pressure readings, using ADRA2C genotype as a covariate. All tests were two-tailed, and a P-value of < 0.05 was considered significant. Analyses were performed with the statistical software SPSS (SPSS v14.0, SPSS Inc., Chicago, IL).
Demographic characteristics arranged by ADRA2C genotype are shown in Table 1. There were no differences among subjects of different genotypes in any of the variables.
The mean (± SD) goodness-of-fit for the temperature-flux response curves, expressed as R2, was 0.89 ± 0.09. There were no significant differences in baseline skin blood flow (flux), Emin, ET50, or ET90 in either the first or second heating or cooling cycle between genotypes (Table 2). Similarly, genotype was not associated with the skin temperature responses measured by Tstim, Tmin, and Tmid. The distributions of ET50 and ET90 for the first heating/cooling cycle are shown in Figure 3. The stimulated flux during the first heating cycle (Estim) was lower in the ADRA2C del/del genotype group than the ins/del and ins/ins genotype groups (P=0.01), but there was no difference in Estim during the second cycle (P=0.64). There were no differences in the flux or skin temperature parameters between cycles 1 and 2.
Among subjects with different ADRA2C genotypes, there were no significant differences in mean arterial blood pressure (MAP) at baseline (Table 1, P=0.69) or during the course of the study (all P>0.21), and MAP did not change over time (P=0.78). Similarly, plasma norepinephrine concentrations were not different at baseline (390.5±258.4, 347.9±165.3, and 327.0±146.7 pg/dL, for ins/ins, ins/del, and del/del genotypes respectively, P=0.99), and the values did not change significantly during the study (P=0.98).
This is the first study to examine the effects of a common, functionally significant deletion polymorphism in the α2C-adrenergic receptor on cold-induced vasoconstriction. Baseline skin blood flow and skin temperature were not associated with ADRA2C genotype, and neither the magnitude of vasoconstriction during cooling (Emin), nor the sensitivity of vasoconstriction to cold (ET50) differed among ADRA2C genotypes.
Cold-induced vasoconstriction is an intricate physiologic defense mechanism that serves to reduce heat loss. Cooling induces vasoconstriction of arterioles via rapid augmentation of α2-AR activity. Both in vitro and in vivo studies have shown that inhibition of α2-ARs impairs the cold-induced vasoconstriction response [8, 9]. α2C-ARs are located predominantly in small arteries, arterioles, and veins [10, 13, 24] and, although “silent” at 37°C, become the major effectors of vasoconstriction during cooling . In response to cold, the α2C-ARs translocate from the Golgi apparatus, where they are sequestered at physiologic temperature, to the cell surface, amplifying vasoconstriction in response to norepinephrine . The response is likely further magnified by increased local sympathetic activation, resulting in greater norepinephrine release from the nerve terminal . In vivo studies suggest that α2–AR upregulation is quite rapid and that α2–AR mediated vasoconstriction can be seen as early as 1-5 minutes into cooling . While the important role of α2C-ARs in cold-induced vasoconstriction is well established, other mechanisms are also involved. For example, the production of reactive oxygen species in the smooth muscle cell mitochondria appears to function as a temperature sensing mechanism initiating the cascade . Subsequently, rho-kinase acts as a mediator whereby the α2C-AR translocates to the plasma membrane [1, 35].
Recent studies have shown that both adrenergic and non-adrenergic mechanisms contribute to varying degrees, at different times, to the vascular response to cooling. The adrenergic mechanism, mediated by α2C-AR, is responsible primarily for the early response to cooling (within the first 10 minutes). After prolonged cooling (>30 minutes), non-adrenergic mechanisms become more prominent [21, 38]. Adrenergic blockade does not prevent this late phase vasoconstriction. Inhibition of nitric oxide synthase appears to play a role in this late response, though the sympathetic cotransmitter neuropeptide Y  and endothelin  may potentially be involved.
The ADRA2C del322-325 variant results from the in-frame deletion of 12 nucleotides in the coding region. The variant is located in the third intracellular loop of the α2C-AR, in close proximity to the Gi coupling domain. The deletion variant of the α2C-AR exhibited impaired agonist-receptor-G protein coupling in transfected cells, resulting in approximately 85% loss of function . Studies to elucidate the significance of this variant in man are limited. One case-control study showed that African-American subjects who were homozygous for the ADRA2C del322-325 variant had an increased risk of heart failure, compared to heterozygotes and non-carriers . Additionally, a small controlled study in healthy subjects suggested that individuals homozygous for the deletion variant had higher blood pressure and norepinephrine secretion . However, more recent studies in larger cohorts did not show an association of the ADRA2C del322-325 variant with blood pressure and heart rate [23, 25], more specific markers of resting sympathetic activity , or early markers of congestive heart failure . Despite the importance of the α2C-AR in the vascular response to cold, there have been no studies to assess the effect of ADRA2C genetic variants on cold-induced vasoconstriction.
Given the finding that ADRA2C del322-325 leads to a marked loss of function in vitro, our hypothesis was that the ADRA2C variant del322-325 would be associated with a decreased vasoconstrictor response to local cooling. Our main outcome was the cooling response, assessed by the variables Emin, ET50, and ET90, as well as Tmin and Tmid. There were no differences in these variables in either cycle among genotype groups. During the first cycle, there was a trend to a lower baseline flux and a statistically significant lower stimulated flux (Estim) in individuals with the homozygous deletion, findings that were not observed during the second cycle. These findings were of marginal significance and are likely to reflect incomplete acclimatization of skin flux responses to the experimental conditions. Moreover, there was no difference in the maximum skin temperature during cycle 1. Additionally, as multiple factors lead to inter-individual variability in flux values, laser Doppler is most informative when assessing relative within-subject changes (such as ET50) .
We expected carriers of the deletion variant to show an attenuated vasoconstrictor response to cold. The study was sufficiently powered to detect physiologically important differences; the sample size provided 80% power with α=0.05 to detect a 6±5 °C difference in ET50 between subjects homozygous for the insertion allele and subjects with at least one deletion allele. Though non-adrenergic mechanisms contribute to the late cold-induced vasoconstriction response, in our study, we believe that in using the temperature-response curves and ET50 measurements, we selected for adrenergic-dependent responses. The ET50 of our temperature-response curves corresponded to a skin temperature typically in the range of 29-31°C, measured after approximately 10-15 minutes of gradual cooling. The contribution of α2C-AR activity should have been near maximal during this time of our cooling protocol.
Possible explanations for the lack of effect of the ADRA2C del322-325 variant on cold-induced vasoconstriction could be functional redundancy in the vasoconstrictor and vasodilator systems , opposing effects of pre- and postsynaptic hypofunctional α2C-ARs on vasoconstriction, and that the α2C-AR may be sufficient, but not necessary for cold-induced vasoconstriction in vivo. Additionally, it is also possible that despite 85% reduced agonist-mediated function in vitro, residual activity of α2C-AR del/del may be sufficient to effect in vivo cooling responses.
Compensatory variation in factors such as α2A-ARs, rho-kinase [29, 36], or nitric oxide , may mask any apparent loss-of-function associated with α2C-AR variants. For example, the vasodilation in response to warming is in part related to nitric oxide release . In our study, there was a trend toward decreased Estim flux in individuals with the ADRA2C del/del genotype (Table 2). One could theorize that this lower plateau vasodilation reflects decreased nitric oxide production. If this decreased nitric oxide production carried forward into the cooling phase as well, this could mask effects of the ADRA2C deletion. In such a situation, the deletion variant α2C-AR could still be hypofunctional, trending toward decreased vasoconstrictor activity, but if coupled with an attenuated nitric oxide response, this could shift the balance and restore vasoconstriction to “normal” levels.
Our study was designed to apply local heat and cold stimuli while avoiding extreme temperatures. Cutaneous blood flow is often low at room temperature. Therefore, after obtaining baseline measures, we applied a standardized local heat exposure to achieve vasodilation, reflected by an elevation in flux to steady-state. For the heat stimulation, a box temperature of 40.5°C was selected, as this temperature has been shown to cause vasodilation, while higher temperatures may elicit nociceptive responses that increase skin blood flow via a mechanism independent of temperature itself [22, 26]. After heating, we gradually decreased the ambient temperature until flux reached a minimum. All temperatures in our testing sequence were within a range typically experienced in a temperate climate. The gradual cooling process allowed us to determine skin blood flow responses over the whole range of temperatures, such that we could accurately quantify the sensitivity of an individual's vasoconstrictor response to cold. We repeated the heating and cooling cycle to account for any residual baseline environmental effects present in the first cycle, as well as to assess whether upregulation of α2C-AR mediated signaling during cooling in the first cycle would affect responses during the second cooling cycle.
There are several limitations of our study. Our in vivo study design allowed us to examine local vascular responses and the effects of genetic variants within a physiologic system. A potential drawback to any in vivo study is that the effect of a genetic variant may be attenuated by local and systemic homeostatic and counterregulatory systems. Central sympathetic nervous system activity, partially regulated by α2C-ARs, could also affect peripheral vasoconstriction. Thus, we made an effort to avoid systemic responses triggered by heat or cold pain, and the lack of changes in mean arterial blood pressure and plasma norepinephrine concentrations during the study indicates that systemic effects, if present, were minimal.
We believe that our parameters extrapolated from the temperature-response curves reflect adrenergic, α2C-AR-mediated mechanisms, since the ET50 was measured at a temperature and time point at which cold-induced vasoconstriction has been shown to be most adrenergically mediated. However, validating this using an α2-AR antagonist could have been very informative. Additionally, pharmacologic inhibition of nitric oxide production or rho kinase could have helped elucidate whether there were compensatory changes in these pathways masking effects of the ADRA2C del/del genotype.
In conclusion, in this study, the ADRA2C del322-325 variant did not affect vascular sensitivity to cold. Future exploration should involve in vitro studies to examine the effect of the deletion variant on cold-induced receptor translocation to the cell surface and in vivo studies using specific α2C-AR antagonists. Moreover, the effect of genetic variants in associated proteins, e.g. rho-kinase, on cold-induced vasoconstriction will be of interest.
The authors would like to acknowledge Norman G. Beaty and James Morton of Whirlpool, Inc. for supplying the modified heating/cooling unit and hand box and providing valuable advice on design of the unit. This study was supported in part by US Public Health Service grants P01 HL56693, a Pharmacogenetics Research Network Grant (U01 HL65962), and a Vanderbilt CTSA grant 1 (UL1 RR024975) from the National Center for Research Resources, National Institutes of Health. Dr. Kurnik was a recipient of a Merck Sharp and Dohme International Fellowship in Clinical Pharmacology.
Disclosures: None of the authors has a conflict of interest relevant to this manuscript.