Cold constricts cutaneous blood vessels by increasing the reactivity of smooth muscle α2-adrenergic receptors (α2-ARs). Experiments were performed to determine the role of α2-AR subtypes (α2A-, α2B-, α2C-ARs) in this response. Stimulation of α1-ARs by phenylephrine or α2-ARs by UK-14,304 caused constriction of isolated mouse tail arteries mounted in a pressurized myograph system. Compared with proximal arteries, distal arteries were more responsive to α2-AR activation but less responsive to activation of α1-ARs. Cold augmented constriction to α2-AR activation in distal arteries but did not affect the response to α1-AR stimulation or the level of myogenic tone. Western blot analysis demonstrated expression of α2A- and α2C-ARs in tail arteries: expression of α2C-ARs decreased in distal compared with proximal arteries, whereas expression of the glycosylated form of the α2A-AR increased in distal arteries. At 37°C, α2-AR-induced vasoconstriction in distal arteries was inhibited by selective blockade of α2A-ARs (BRL-44408) but not by selective inhibition of α2B-ARs (ARC-239) or α2C-ARs (MK-912). In contrast, during cold exposure (28°C), the augmented response to UK-14,304 was inhibited by the α2C-AR antagonist MK-912, which selectively abolished cold-induced amplification of the response. These experiments indicate that cold-induced amplification of α2-ARs is mediated by α2C-ARs that are normally silent in these cutaneous arteries. Blockade of α2C-ARs may prove an effective treatment for Raynaud's Phenomenon.
- Raynaud's Phenomenon
- vascular smooth muscle
cold-induced vasoconstriction in the cutaneous circulation is a protective physiological response that acts to reduce heat loss (12, 51). Cold exposure causes vasoconstriction by a reflex increase in sympathetic output and by a local direct action to increase the activity of the adrenergic neurotransmitter norepinephrine (12,51). The direct effect of cold is mediated by a rapid augmentation of α2-adrenergic receptor (α2-AR) activity (8,9, 12, 14). This effect is selective for α2-ARs, and cold can actually inhibit constriction to other stimuli, including α1-AR activation (12, 14, 20). The unique, cold sensitivity of α2-ARs may explain their increased prominence in the cutaneous circulation and their role in the heightened cold-induced vasoconstriction that occurs in Raynaud's Phenomenon (12, 22). The mechanism underlying the cold-induced augmentation of α2-AR function has not been defined.
α2-ARs are known to comprise three subtypes: α2A, α2B, and α2C (2, 34,41). In some species, including mice, the α2A-AR is homologous to the human protein (92% identity) but displays distinct ligand-binding characteristics (2, 38). Therefore, although these sites are still designated as α2A-AR proteins, their pharmacology is defined as α2D-ARs (2, 38). The role of α2-AR subtypes in the vascular system has not yet been clearly defined. Studies of the intact cardiovascular system in mice with targeted deletion or mutation in these receptors suggests that α2A/2D- and α2B-ARs can mediate vasopressor responses to α2-AR stimulation, α2A/2D-ARs mediate the central antihypertensive response, and α2C-ARs are not involved in vascular regulation (39, 41,42). The aim of the present study was to investigate the role of α2-AR subtypes in cold-induced modulation of α2-AR vasoconstriction using a new model of the cutaneous circulation, namely the mouse tail artery.
Blood vessel chamber.
Male mice (C57BL6) were killed by CO2 asphyxiation. Proximal and/or distal segments of tail artery were then rapidly removed and placed in cold Krebs-Ringer bicarbonate solution containing (in mM) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 25.0 NaHCO3, and 11.1 glucose (control solution). The small arteries were cannulated at both ends with glass micropipettes, secured using 12-0 nylon monofilament suture, and placed in a microvascular chamber (Living Systems, Burlington, VT). The arteries were maintained at a constant transmural pressure (PTM) of 60 mmHg in the absence of flow (40). The chamber was superfused with control solution, maintained at 37°C, pH 7.4, and gassed with 16% O2-5% CO2-balance N2. The chamber was placed on the stage of an inverted microscope (×20; Nikon TMS-F) connected to a video camera (CCTV camera; Panasonic). The vessel image was projected on a video monitor, and the internal diameter was continuously determined by a video dimension analyzer (Living Systems Instrumentation; see Ref. 40
Small arteries were allowed to equilibrate for 30–40 min at a PTM of 60 mmHg before commencing experiments (40). Concentration-effect curves to the selective α1-AR agonist phenylephrine (14, 17) or the selective α2-AR agonist UK-14,304 (14, 17) were generated by increasing the concentration of the agonists in half-log increments once the constriction to the previous concentration had stabilized. After completion of the concentration-effect curve, the influence of the agonists was terminated by repeatedly exchanging the buffer solution and allowing the artery to return to its stable baseline level. In some experiments, concentration-effect curves to UK-14,304 were determined under control conditions and in the presence of the selective α2A/2D-AR antagonist BRL-44408 (100 and 1,000 nM), the selective α2B-AR antagonist ARC-239 (50 nM), or the selective α2C-AR antagonist MK-912 (0.3 nM; Table1; see Refs. 2, 48, 49). When these receptor antagonists were used, the preparations were incubated for 30 min with the drugs before, and also during, exposure of the arteries to the agonists. When analyzing the influence of cold on α-AR responsiveness, the temperature of the superfusate was decreased to 28°C for 30 min before commencing a concentration-effect curve to the constrictor agonists (14, 51). This provides sufficient time for the effect of cold on adrenergic reactivity to stabilize (14, 20).
Analysis of α2-AR expression.
COS-7 cells were transiently transfected with human α2-ARs using the DEAE-dextran method (expression constructs kindly supplied by Dr. Herve Paris, Toulouse, France). After transfection (48 h), cells were scraped in 50 mM Tris ⋅ HCl, 2 mM MgCl2, and 1 mM EDTA containing antiproteases [chymostatin, antipain, pepstatin each at 15.7 μg/ml; 57.7 μg/ml leupeptin; 250 μg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride]. Membrane fractions were then prepared as previously described (25). Membrane fractions from Sf9 cells expressing human α2-ARs were obtained from Research Biochemicals International (Natick, MA). Deglycosylation of membrane-bound proteins was obtained by incubating membrane fractions with peptide N-glycosidase F (PNGaseF, 500,000 U/ml, 30 min, 37°C) according to the manufacturer's instructions (New England BioLabs). Because of the small size of tail arteries, α2-AR expression was assessed using total lysates. Arteries were first denuded of endothelial cells by gently rubbing the lumen of the vessels with a fine wire. Samples of proximal, middle, and distal tail arteries were then homogenized (Dounce, 30 strokes, 4°C) in sample buffer containing 60 mM Tris ⋅ HCl and 2% SDS (pH = 6.8) at room temperature. The homogenates were cleared by centrifugation (14,000 g, 10 min), and the supernatant was collected. Protein concentrations were determined using the bincinchoninic acid assay (Pierce).
For Western blot analysis, 0.03 μg of protein from membrane fractions of transfected cells or 5 μg of total protein from tail arteries were separated on 8% SDS-polyacrylamide gels and then transferred to Immobilon-P membranes (Millipore). The membranes were blocked with 10% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20. The membranes were incubated with affinity-purified, rabbit polyclonal antibodies (6) directed against the α2-ARs (1:500 dilution, 60 min, room temperature). Western blots were developed using the SuperSignal detection system (Pierce) and were quantified using a Molecular Dynamics Personal Densitometer (Molecular Dynamics, Sunnyvale, CA).
ARC-239 was a gift from Boehringer Ingelheim (Ridgefield, CT), BRL-44408 was a gift from SmithKline Beecham (Harlow, UK), MK-912 was a gift from Merck (West Point, PA), phenylephrine and sodium nitroprusside were obtained from Sigma (St. Louis, MO), and UK-14,304 was from Research Biochemicals International. Stock solutions of drugs were prepared fresh each day and were stored at 4°C during the experiment. Drugs were dissolved in distilled water with the exception of 1) UK-14,304, which was dissolved in DMSO (highest chamber concentration of 0.001%), 2) BRL-44408, which was dissolved in 0.1 N HCl (highest chamber concentration of 0.04%), and 3) ARC-239, which was dissolved in methanol (highest chamber concentration of 0.004%). At these concentrations, the solvents did not alter reactivity of the blood vessels. All drug concentrations are expressed as final molar concentration (M) in the chamber superfusate.
Vasomotor responses were expressed as a percentage change in ID before administrating the agent. Because of the phasic behavior of the vasomotion in distal tail arteries (Fig.1n number of experiments, where n equals the number of animals from which blood vessels were studied. Concentration-effect curves were analyzed by determining the maximal response (or response to the highest concentration of agonist used) and the concentration of agonist evoking 15 or 20% constriction (EC15 and EC20, respectively). Antagonist dissociation constants (K D) were determined either from Arunlakshana and Schild plots or according to the formula: K D = [Ant]/(CR-1), where [Ant] is the concentration of antagonist, and CR is the ratio of agonist concentrations producing equal responses in the presence and absence of the antagonist (17, 20). In all cases, slopes of Arunlakshana and Schild plots were not significantly different from unity, consistent with competitive antagonism (17). Statistical evaluation of the data was performed by Student's t-test for either paired or unpaired observations. When more than two means were compared, ANOVA was used. If a significant F-value was found, Scheffé's test for multiple comparisons was employed to identify differences among groups. Values were considered to be statistically different when P was less than 0.05.
When PTM was increased from 10 to 60 mmHg, distal arteries immediately dilated and then gradually constricted (Fig. 1 B). The pressure-induced constriction or myogenic response comprised both tonic and phasic components. These responses are characteristic of arterioles. Administration of the vasodilator sodium nitroprusside (10−5 M), abolished both constrictor components (Fig.1, A and B). Under these conditions, an increase in PTM caused only a passive increase in arterial diameter (Fig. 1 B). In contrast to the distal arteries, proximal arteries did not constrict in response to increases in PTMand, under quiescent conditions, did not dilate to sodium nitroprusside (10−5 M), indicating the absence of myogenic tone (Fig. 1, A and B). Once the blood vessels had stabilized at 60 mmHg, the ID was 334 ± 9 μm in proximal and 158 ± 15 μm in distal arteries.
α1- And α2-AR activation.
Stimulation of α1-ARs by phenylephrine (10−9 to 3 × 10−7 M) or α2-ARs by UK-14,304 (10−9 to 3 × 10−7 M) caused concentration-dependent constriction of proximal and distal arteries (Fig. 2). Distal arteries were significantly more responsive to activation of α2-ARs but significantly less responsive to stimulation of α1-ARs compared with proximal arteries (Fig. 2). Higher concentrations of phenylephrine were required to evoke vasoconstriction in distal compared with proximal arteries (log EC15 values of −6.86 ± 0.21 and −7.86 ± 0.11, respectively, P < 0.05), and the maximal observed response to the agonist (at 3 × 10−7M) was decreased in distal compared with proximal arteries (constriction of 25 ± 7 and 69 ± 4%, respectively, P < 0.005). In contrast, lower concentrations of UK-14,304 were required to evoke vasoconstriction in distal compared with proximal arteries (log EC15 values of −8.68 ± 0.09 and −7.82 ± 0.13, respectively, P < 0.05), and the maximal response to the agonist was greater in distal compared with proximal arteries (constriction of 36 ± 2 and 25 ± 2%, respectively, P < 0.05).
Influence of cold on α-AR constriction.
The influence of cold on adrenergic constrictor responsiveness was evaluated on distal arteries. Cold did not significantly affect the baseline diameter or myogenic tone in distal arteries (IDs of 153 ± 12 and 158 ± 15 μm at 37 and 28°C, respectively). Cold dramatically and reversibly increased the vasoconstriction caused by activation of α2-ARs with UK-14,304 (Fig.3; maximal responses of 23 ± 1 and 41 ± 2% at 37 and 28°C, respectively, P < 0.001; log EC15 values of −8.11 ± 0.07 and −9.22 ± 0.22 at 37 and 28°C, respectively, P < 0.05). However, cold did not affect the constrictor response to stimulation of α1-ARs by phenylephrine (Fig.4; maximal observed responses, at 10−6 M, of 37 ± 4 and 39 ± 5% constriction at 37 and 28°C, respectively; log EC15 values of −6.76 ± 0.14 and −7.05 ± 0.21 at 37 and 28°C, respectively).
α2-AR subtype expression.
Western blot analysis, using rabbit polyclonal antibodies specific for the α2-AR subtypes, demonstrated expression of α2A/2D-ARs and α2C-ARs in the smooth muscle of mouse tail arteries (Fig. 5).
For α2A/2D-ARs, COS-7 cells expressed a high-molecular-weight glycosylated form of the receptor, whereas Sf9 cells expressed predominantly a low-molecular-weight form of the receptor (Fig. 5 A). After deglycosylation of COS-7 cell membranes with PNGaseF, only the low-molecular-weight species was observed (Fig. 5 A), indicating that it represents the native, nonglycosylated receptor. In tail arteries, expression of the glycosylated form of the receptor increased, whereas expression of the native receptor decreased in distal compared with proximal arteries (Table 2, Fig. 5 A).
For α2C-ARs, COS-7 cells expressed two distinct glycosylated forms of the receptor, whereas Sf9 cells expressed predominantly a low-molecular-weight nonglycosylated receptor (Fig.5 B). After deglycosylation of COS-7 cell membranes with PNGaseF, only this low-molecular-weight species was observed (Fig.5 B). In tail arteries, the α2C-AR was expressed predominantly as the lower-molecular-weight glycosylated form of the receptor (Fig. 5 B). Expression of the α2C-AR was decreased in distal arteries compared with proximal arteries (Table 2and Fig. 5 B).
Cold and α2-AR subtypes.
At 37°C, vasoconstriction to the α2-AR agonist UK-14,304 was inhibited by the selective α2A/2D-AR antagonist BRL-44408 (100 and 1,000 nM; Fig.6) but was not inhibited by the selective α2B-AR antagonist ARC-239 (50 nM; data not shown) or the selective α2C-AR antagonist MK-912 (0.3 nM; Fig.7). Based on the K D for ARC-239 and MK-912 (Table 1), these antagonists would be expected to cause ∼11- and ∼6-fold shifts in concentration-effects curves generated by α2B-AR and α2C-AR stimulation, respectively. The Arunlakshana and Schild plot for the inhibitory effect of BRL-44408 generated a −log K D of 7.69 ± 0.13 (K D of 20 nM), consistent with antagonism of α2A/2D-ARs (Table 1). These results suggest that, at 37°C, α2A/2D-ARs, but not α2B-AR or α2C-AR, contribute to α2-AR vasoconstriction.
During exposure to cold (28°C), the augmented vasoconstrictor response to UK-14,304 was dramatically inhibited by the α2C-AR antagonist MK-912 (3 × 10−10 M; Fig. 7). The inhibitory effect generated a −log K D value of 10.9 ± 0.17 (K D of 14 pM), consistent with inhibition of α2C-ARs (Table 1). The vasoconstrictor response was not inhibited by the α2B-AR antagonist ARC 239 (50 nM, data not shown) but was reduced by the α2A/2D antagonist BRL-44408 (100 and 1,000 nM; Fig. 6). The Arunlakshana and Schild plot for the inhibitory effect of BRL-44408 generated a −log K D of 7.54 ± 0.10 (K D value of 29 nM), which was not significantly different from that observed at 37°C. These results suggest that, at 28°C, α2C- and α2A/2D-ARs contribute to α2-AR vasoconstriction.
Inhibition of α2C-ARs by MK-912 (3 × 10−10 M) attenuated the α2-AR-induced vasoconstriction only at low temperatures and selectively abolished cold-induced amplification of the α2-AR response (Fig.8). In the presence of MK-912 (3 × 10−10 M), cold did not increase the response to low concentrations of UK-14,304 (log EC15 values of −7.85 ± 0.10 and −7.78 ± 0.12 at 37 and 28°C, respectively) but continued to increase the response to high concentrations of the agonist (maximal responses of 28 ± 1 and 37 ± 2% constriction at 37 and 28°C, respectively, P < 0.05; Figs. 3 and 8). In contrast to MK-912, blockade of α2A/2D-ARs with BRL-44408 inhibited α2-AR-induced constriction to a similar degree at warm and cold temperatures and did not reduce the cold-induced amplification of the response (Table 1 and Fig. 6).
Activation of α2-ARs located on vascular smooth muscle evokes vasoconstriction (15, 17). Pharmacological and molecular analysis of α2-ARs have identified three subtypes of the receptor: α2A/2D-, α2B-, and α2C-ARs (2). These homologous proteins (50–60% identity) are separate gene products and are uniquely sensitive to physiological regulation (34, 41). The role of these receptor subtypes in the regulation of the vascular system has not been clearly defined. Studies of the intact cardiovascular system in mice with either a point (α2A/2D-ARs) or null (α2B-ARs/α2C-ARs) mutation (“knock-out”) suggested that α2A/2D- and α2B-ARs can contribute to the vasopressor response to α2-AR stimulation, that α2A/2D-ARs mediate the central antihypertensive response, and that α2C-ARs are not involved in vascular regulation (39, 41, 42). Indeed, α2C-ARs were originally considered to be silent or vestigial receptor sites (41), although they are now thought to regulate memory processing in the central nervous system (47). A key physiological response associated with vascular α2-ARs is cold-induced vasospasm in the cutaneous circulation (8, 9, 12, 14). Direct analysis of cutaneous arteries in the present study confirms that α2C-ARs do not normally contribute to vasoconstriction. However, during cold-induced vasoconstriction, α2C-ARs are no longer silent and mediate the remarkable cold-induced augmentation of α2-AR responsiveness. Inhibition of α2C-ARs may provide an effective and selective therapy for the vasospastic episodes in Raynaud's disease.
Unlike α1-ARs, functional vascular smooth muscle α2-ARs are not widely distributed in the vascular system (12, 13, 17). α2-AR constrictor activity is not present in large arteries but is generally restricted to small arteries/arterioles and to the venous circulation (10, 13, 16, 19, 35). This pattern was also evident in the tail circulation. The constrictor activity of α2-ARs increased in distal compared with proximal arteries, whereas the opposite pattern was observed for α1-ARs. The altered functional activity of vascular smooth muscle α2-ARs was associated with a remarkable change in the posttranslational modification of α2A/2D-ARs. The expression of the glycosylated form of the α2A/2D-ARs increased in distal compared with proximal arteries. This occurred with a corresponding reduction in the native form of the receptor, suggesting that the α2A/2D-AR becomes increasingly glycosylated in more distal segments of the mouse tail artery. As with the native form of the α2A/2D-AR, the expression of the α2C-AR decreased in distal compared with proximal arteries. The role of glycosylation on α2A/2D-AR function has not been defined. However, with other G protein-coupled receptors, including β-ARs, glycosylation is associated with enhanced receptor signaling (1, 11, 44). Therefore, the increased glycosylation of the α2A/2D-ARs may contribute to the increased vasoconstrictor activity resulting from stimulation of α2-ARs in distal arteries. The regulation of this process within different regions of the vascular system has not been described previously. These results are consistent with previous reports that constrictor α2A/2D-ARs are functional in the microcirculation (35), whereas, in large arteries, the receptors are expressed but are not functional (18, 21, 43).
In the mouse tail artery, cold caused a dramatic and selective increase in the ability of α2-ARs to induce vasoconstriction. Indeed, vasoconstriction to stimulation of α1-ARs or the inherent myogenic tone of the artery was not affected by cold. Although this is the first demonstration of this phenomenon in an isolated cutaneous artery, it confirms previous observations in isolated cutaneous veins and in the intact human cutaneous circulation that these receptors function as cutaneous thermosensors (8, 12, 14, 20). At 37°C, the response to activation of α2-ARs was inhibited by the selective α2A/2D-AR antagonist BRL-44408 but was not affected by inhibition of α2B (ARC-239)- or α2C (MK-912)-ARs. Furthermore, the calculatedK D (20 nM) for BRL-44408 was consistent with its affinity for α2A/2D-ARs (Table 1). Therefore, at 37°C, α2-AR-induced vasoconstriction was mediated by the α2A/2D-AR subtype, with no or minimal contribution from α2C-ARs. However, at 28°C, the augmented vasoconstrictor response to α2-AR activation was highly sensitive to the α2C-AR antagonist MK-912, and theK D (14 pM) for MK-912 was consistent with its affinity for α2C-ARs (Table 1). This indicates that, although α2C-ARs do not appear to contribute to vasoconstriction at 37°C, they are active during cold-induced amplification of the α2-AR response. Indeed, inhibition of α2C-ARs by MK-912 selectively abolished the cold-induced augmentation of α2-AR-mediated vasoconstriction, indicating that this phenomenon is mediated by an effect of cold to amplify α2C-AR activity. Under control conditions, cold increased the response evoked by low and high concentrations of UK-14,304 (Fig. 3). However, after inhibition of α2C-ARs by MK-912, cold no longer increased responses evoked by low concentrations of UK-14,304 but continued to increase the responses evoked by high concentrations of the agonist (Fig. 8). This likely reflects the competitive nature of the inhibition produced by MK-912 and the ability of high concentrations of the agonist to overcome the inhibitory effect (e.g., Fig. 7 B).
α2-AR-dependent vasoconstriction was inhibited by the α2A/2D-AR antagonist BRL-44408 at 37 and 28°C. In each case, the K D for the antagonist indicated an interaction with α2A/2D-ARs (Table 1), suggesting that α2A/2D-ARs were not modulated by temperature and were active at 28 and 37°C. Inhibition of α2A/2D-ARs did not prevent cold-induced amplification of the α2-AR response, suggesting that cold-induced modulation of α2A/2D-ARs does not contribute to cold vasoconstriction. However, because inhibition of α2A/2D-ARs and α2C-ARs reduced vasoconstriction at 28°C, α2-adrenergic vasoconstriction at low temperatures may represent an interaction between these two receptor systems. Indeed, α2A/2D-ARs may play a permissive role for α2C-AR-induced vasoconstriction at low temperatures. This may be analogous to other nonhuman, cutaneous arteries that require permissive stimulation by another constrictor to observe α2-AR vasoconstriction (rat tail artery, porcine digital artery; see Ref. 26).
The original concept of α2C-ARs as silent receptors (41) is consistent with their inability to contribute to α2-AR-dependent vasoconstriction, except during cold exposure. The mechanism underlying the effect of warm temperatures to selectively silence or reduce α2C-AR activity in the mouse tail artery is not known. The effect of cold was too rapid to be mediated by transcriptional regulation and likely reflects improved signaling of the α2C-AR. Although the α2-AR subtypes are homologous, they are uniquely sensitive to physiological regulation (41). α2C-ARs have a prominent intracellular distribution (endoplasmic reticulum and Golgi apparatus), whereas α2A/2D-AR are localized on the cell membrane (6, 41, 45). Furthermore, these receptor subtypes are reported to have differing sensitivities to desensitization and also differ in their efficiency to couple to G proteins (5, 7, 31, 33). Therefore, the specific temperature modulation of the α2C-AR could reflect altered membrane targeting or processing of the receptor or altered activity of α2C-AR-specific signaling or amplification processes.
The α2-AR agonist used in the present study, UK-14,304, is generally used as a non-subtype-selective α2-AR agonist (3, 46). However, reports have suggested that the agonist is somewhat selective for human α2A-ARs, demonstrating increased affinity and efficacy at these sites compared with α2B-AR and α2C-ARs (29, 30, 32). If UK-14,304 selectively activated α2A/2D-ARs located on tail artery smooth muscle cells, then this could have contributed to the lack of an α2C-AR response at 37°C. However, the reported selectivity of UK-14,304 for human α2A-ARs is not evident at α2D-ARs, the mouse homologue of the human α2A-AR. UK-14,304 has similar affinity and efficacy for α2D-AR and α2C-AR (28, 30, 50). Therefore, it is unlikely that the decreased activity of α2C-AR at 37°C resulted from any pharmacological selectivity of UK-14,304.
The results obtained in the present study may explain the abnormal cutaneous vasoconstriction that occurs in Raynaud's Phenomenon. This condition results from vasospasm of the digital arteries in response to cold, causing episodic, sharp demarcated cutaneous pallor and cyanosis of the digits (52). Indeed, the mouse tail artery may represent an important new model for the human digital circulation. In the digital circulation, as occurred in the tail artery, α2-AR stimulation evoked constriction that was increased in digital arteries, compared with the more proximal palmer arch (13). Previous models of the human cutaneous arterial system either do not express functional α2-ARs (rabbit ear artery; see Ref. 27) or need permissive stimulation by another constrictor to observe α2-AR-dependent responses (rat tail artery, porcine digital artery; unpublished observations and Ref. 26). In 1929, Lewis (36) postulated that Raynaud's Phenomenon resulted from a “local fault” of the blood vessel wall. The local fault likely represents abnormal regulation of α2-ARs. Nonselective blockade of α2-ARs (α2A-, α2B-, and α2C-ARs) abolished cold-induced vasospastic attacks in patients with primary Raynaud's disease, whereas α1-AR blockade was ineffective (22). However, analysis of responses to exogenous agonists has demonstrated only small increases or no change in α2-AR-induced vasoconstriction in subjects with primary Raynaud's disease (4, 23, 24, 37). Therefore, although α2-ARs have been implicated in the disease, there is no consistent evidence of abnormal reactivity of these receptors. The results of the present study suggest that an increased activity of α2C-ARs could profoundly influence cold-induced vasospasm, without greatly affecting the α2-AR response at ambient temperatures. Therefore, the local fault in Raynaud's Phenomenon may represent increased expression or effectiveness of α2C-ARs. Because these receptors appear to be silent in the normal regulation of vascular function (39, 41), selective blockade of these receptors may provide a highly selective therapeutic intervention for this condition.
We thank the drug companies mentioned in methods for the kind gift of α2-AR antagonists.
Address for reprint requests and other correspondence: N. A. Flavahan, Heart and Lung Institute, Medical Research Facility, R524, 420 West 12th Ave., Columbus, OH 43210 (E-mail:).
This work was supported by grants from the Scleroderma Research Foundation and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-46126.
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. §1734 solely to indicate this fact.
- Copyright © 2000 the American Physiological Society