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Am J Physiol Heart Circ Physiol 278: H1075-H1083, 2000;
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Vol. 278, Issue 4, H1075-H1083, April 2000

Silent alpha 2C-adrenergic receptors enable cold-induced vasoconstriction in cutaneous arteries

Maqsood A. Chotani1, Sheila Flavahan1, Srabani Mitra1, David Daunt2, and Nicholas A. Flavahan1

1 Heart and Lung Institute, Ohio State University, Columbus, Ohio 43210; and 2 Department of Comparative Medicine, Stanford University, Stanford, California 94305


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cold constricts cutaneous blood vessels by increasing the reactivity of smooth muscle alpha 2-adrenergic receptors (alpha 2-ARs). Experiments were performed to determine the role of alpha 2-AR subtypes (alpha 2A-, alpha 2B-, alpha 2C-ARs) in this response. Stimulation of alpha 1-ARs by phenylephrine or alpha 2-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 alpha 2-AR activation but less responsive to activation of alpha 1-ARs. Cold augmented constriction to alpha 2-AR activation in distal arteries but did not affect the response to alpha 1-AR stimulation or the level of myogenic tone. Western blot analysis demonstrated expression of alpha 2A- and alpha 2C-ARs in tail arteries: expression of alpha 2C-ARs decreased in distal compared with proximal arteries, whereas expression of the glycosylated form of the alpha 2A-AR increased in distal arteries. At 37°C, alpha 2-AR-induced vasoconstriction in distal arteries was inhibited by selective blockade of alpha 2A-ARs (BRL-44408) but not by selective inhibition of alpha 2B-ARs (ARC-239) or alpha 2C-ARs (MK-912). In contrast, during cold exposure (28°C), the augmented response to UK-14,304 was inhibited by the alpha 2C-AR antagonist MK-912, which selectively abolished cold-induced amplification of the response. These experiments indicate that cold-induced amplification of alpha 2-ARs is mediated by alpha 2C-ARs that are normally silent in these cutaneous arteries. Blockade of alpha 2C-ARs may prove an effective treatment for Raynaud's Phenomenon.

Raynaud's Phenomenon; scleroderma; microcirculation; thermoregulation; vascular smooth muscle


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha 2-adrenergic receptor (alpha 2-AR) activity (8, 9, 12, 14). This effect is selective for alpha 2-ARs, and cold can actually inhibit constriction to other stimuli, including alpha 1-AR activation (12, 14, 20). The unique, cold sensitivity of alpha 2-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 alpha 2-AR function has not been defined.

alpha 2-ARs are known to comprise three subtypes: alpha 2A, alpha 2B, and alpha 2C (2, 34, 41). In some species, including mice, the alpha 2A-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 alpha 2A-AR proteins, their pharmacology is defined as alpha 2D-ARs (2, 38). The role of alpha 2-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 alpha 2A/2D- and alpha 2B-ARs can mediate vasopressor responses to alpha 2-AR stimulation, alpha 2A/2D-ARs mediate the central antihypertensive response, and alpha 2C-ARs are not involved in vascular regulation (39, 41, 42). The aim of the present study was to investigate the role of alpha 2-AR subtypes in cold-induced modulation of alpha 2-AR vasoconstriction using a new model of the cutaneous circulation, namely the mouse tail artery.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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) and was monitored using a BIOPAC (Santa Barbara, CA) data acquisition system (Gateway Dimensions Pentium Computer).

Experimental protocol. 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 alpha 1-AR agonist phenylephrine (14, 17) or the selective alpha 2-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 alpha 2A/2D-AR antagonist BRL-44408 (100 and 1,000 nM), the selective alpha 2B-AR antagonist ARC-239 (50 nM), or the selective alpha 2C-AR antagonist MK-912 (0.3 nM; Table 1; 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 alpha -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).

                              
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  		Table 1.   KD values for alpha 2-AR antagonists

Analysis of alpha 2-AR expression. COS-7 cells were transiently transfected with human alpha 2-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 alpha 2-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, alpha 2-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 alpha 2-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).

Drugs. 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.

Data analysis. 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. 1), the signal was electronically averaged (BIOPAC software, smoothing factor of 2,000) to obtain diameter measurements. Functional data are expressed as means ± SE for n 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 (KD) were determined either from Arunlakshana and Schild plots or according to the formula: KD = [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. 


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  		Fig. 1.   Representative tracing demonstrating myogenic activity of mouse tail arteries. A: when administered under quiescent conditions, the vasodilator sodium nitroprusside (SNP, 10-5 M) abolished phasic contractile activity and increased the mean diameter of distal tail arteries without affecting diameter of proximal arteries. B: an abrupt increase in transmural pressure (from 10 to 60 mmHg) evoked a contractile response in distal but not proximal arteries. The pressure-induced contraction was inhibited by SNP (10-5 M). C, control. In A and B, vertical bars represent 20-µm (distal artery) or 40-µm (proximal artery) changes in diameter, whereas the horizontal bar represents 1 min.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Baseline characteristics. When PTM was increased from 10 to 60 mmHg, distal arteries immediately dilated and then gradually constricted (Fig. 1B). 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. 1B). In contrast to the distal arteries, proximal arteries did not constrict in response to increases in PTM and, 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.

alpha 1- And alpha 2-AR activation. Stimulation of alpha 1-ARs by phenylephrine (10-9 to 3 × 10-7 M) or alpha 2-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 alpha 2-ARs but significantly less responsive to stimulation of alpha 1-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 × 10-7 M) 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).


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  		Fig. 2.   Vasoconstrictor effects of the selective alpha 1-adrenergic receptor (AR) agonist phenylephrine (10-9 to 3 × 10-7 M; A) or the selective alpha 2-AR agonist UK-14,304 (10-9 to 3 × 10-7 M; B) in proximal and distal tail arteries of the mouse. Vasoconstriction was expressed as a percentage of the stable baseline diameter and is presented as means ± SE [n = 4 (UK-14,304) or 3 (phenylephrine) experiments].

Influence of cold on alpha -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 alpha 2-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 alpha 1-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).


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  		Fig. 3.   Effect of cold (from 37 to 28°C) on the vasoconstrictor response to the selective alpha 2-AR agonist UK-14,304 (3 × 10-10 to 3 × 10-7 M) in distal tail arteries of the mouse. Vasoconstriction was expressed as a percentage of the stable baseline diameter and is presented as means ± SE (n = 4).



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  		Fig. 4.   Effect of cold (from 37 to 28°C) on the vasoconstrictor response to the selective alpha 1-AR agonist phenylephrine (10-9 to 10-6 M) in distal tail arteries of the mouse. Vasoconstriction was expressed as a percentage of the stable baseline diameter and is presented as means ± SE (n = 4).

alpha 2-AR subtype expression. Western blot analysis, using rabbit polyclonal antibodies specific for the alpha 2-AR subtypes, demonstrated expression of alpha 2A/2D-ARs and alpha 2C-ARs in the smooth muscle of mouse tail arteries (Fig. 5).


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  		Fig. 5.   Western blot analysis of alpha 2-AR expression in mouse tail arteries. Blots are representative of at least 4 experiments. A: analysis of alpha 2A-AR expression. Lane 1, COS-7 membranes expressing human alpha 2A-ARs; lane 2, COS-7 membranes expressing human alpha 2C-ARs; lane 3, COS-7 membranes expressing alpha 2A-ARs after deglycosylation with peptide N-glycosidase F (PNGaseF); lane 4, Sf9 membranes expressing human alpha 2A-ARs; lanes 5, 6, and 7, lysates from distal, middle, and proximal tail arteries, respectively. B: analysis of alpha 2C-AR expression. Lane 1, COS-7 membranes expressing human alpha 2A-ARs; lane 2, COS-7 membranes expressing human alpha 2C-ARs; lane 3, COS-7 membranes expressing alpha 2C-ARs after deglycosylation with PNGaseF; lane 4, Sf9 membranes expressing human alpha 2C-ARs; lanes 5, 6, and 7, lysates from distal, middle, and proximal tail arteries, respectively.

For alpha 2A/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. 5A). After deglycosylation of COS-7 cell membranes with PNGaseF, only the low-molecular-weight species was observed (Fig. 5A), 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. 5A).

                              
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  		Table 2.   Expression of alpha 2A-ARs and alpha 2C-ARs in mouse tail artery

For alpha 2C-ARs, COS-7 cells expressed two distinct glycosylated forms of the receptor, whereas Sf9 cells expressed predominantly a low-molecular-weight nonglycosylated receptor (Fig. 5B). After deglycosylation of COS-7 cell membranes with PNGaseF, only this low-molecular-weight species was observed (Fig. 5B). In tail arteries, the alpha 2C-AR was expressed predominantly as the lower-molecular-weight glycosylated form of the receptor (Fig. 5B). Expression of the alpha 2C-AR was decreased in distal arteries compared with proximal arteries (Table 2 and Fig. 5B).

Cold and alpha 2-AR subtypes. At 37°C, vasoconstriction to the alpha 2-AR agonist UK-14,304 was inhibited by the selective alpha 2A/2D-AR antagonist BRL-44408 (100 and 1,000 nM; Fig. 6) but was not inhibited by the selective alpha 2B-AR antagonist ARC-239 (50 nM; data not shown) or the selective alpha 2C-AR antagonist MK-912 (0.3 nM; Fig. 7). Based on the KD 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 alpha 2B-AR and alpha 2C-AR stimulation, respectively. The Arunlakshana and Schild plot for the inhibitory effect of BRL-44408 generated a -log KD of 7.69 ± 0.13 (KD of 20 nM), consistent with antagonism of alpha 2A/2D-ARs (Table 1). These results suggest that, at 37°C, alpha 2A/2D-ARs, but not alpha 2B-AR or alpha 2C-AR, contribute to alpha 2-AR vasoconstriction.


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  		Fig. 6.   Effect of the alpha 2A/2D-AR antagonist BRL-44408 (100 and 1,000 nM) on the vasoconstrictor response to the alpha 2-AR agonist UK-14,304 (10-10 to 3 × 10-6 M) in distal tail arteries of the mouse. Inhibitory effect of BRL-44408 was assessed at 37°C (A) and at 28°C (B). Vasoconstriction was expressed as a percentage of the stable baseline diameter and is presented as means ± SE (n = 4). Absence of error bar indicates the SE was less than the size of the symbol.



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  		Fig. 7.   Effect of the alpha 2C-AR antagonist MK-912 (0.3 nM) on the vasoconstrictor response to the alpha 2-AR agonist UK-14,304 (10-10 to 3 × 10-7 M) in distal tail arteries of the mouse. Inhibitory effect of MK-912 was assessed at 37°C (A) and at 28°C (B). Vasoconstriction was expressed as a percentage of the stable baseline diameter and is presented as means ± SE (n = 4). Absence of error bar indicates the SE was less than the size of the symbol.

During exposure to cold (28°C), the augmented vasoconstrictor response to UK-14,304 was dramatically inhibited by the alpha 2C-AR antagonist MK-912 (3 × 10-10 M; Fig. 7). The inhibitory effect generated a -log KD value of 10.9 ± 0.17 (KD of 14 pM), consistent with inhibition of alpha 2C-ARs (Table 1). The vasoconstrictor response was not inhibited by the alpha 2B-AR antagonist ARC 239 (50 nM, data not shown) but was reduced by the alpha 2A/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 KD of 7.54 ± 0.10 (KD value of 29 nM), which was not significantly different from that observed at 37°C. These results suggest that, at 28°C, alpha 2C- and alpha 2A/2D-ARs contribute to alpha 2-AR vasoconstriction.

Inhibition of alpha 2C-ARs by MK-912 (3 × 10-10 M) attenuated the alpha 2-AR-induced vasoconstriction only at low temperatures and selectively abolished cold-induced amplification of the alpha 2-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 alpha 2A/2D-ARs with BRL-44408 inhibited alpha 2-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).


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  		Fig. 8.   Effect of the alpha 2C-AR antagonist MK-912 (0.3 nM) on cold-induced augmentation of alpha 2-AR vasoconstriction in distal tail arteries of the mouse. Vasoconstrictor responses to the alpha 2-AR agonist UK-14,304 (3 × 10-9 to 3 × 10-7 M) were assessed as described in Fig. 3. However, in contrast to Fig. 3, alpha 2C-ARs were blocked by treating the arteries with MK-912 (0.3 nM) before and during each of the concentration-effect curves. Vasoconstriction was expressed as a percentage of the stable baseline diameter and is presented as means ± SE (n = 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of alpha 2-ARs located on vascular smooth muscle evokes vasoconstriction (15, 17). Pharmacological and molecular analysis of alpha 2-ARs have identified three subtypes of the receptor: alpha 2A/2D-, alpha 2B-, and alpha 2C-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 (alpha 2A/2D-ARs) or null (alpha 2B-ARs/alpha 2C-ARs) mutation ("knock-out") suggested that alpha 2A/2D- and alpha 2B-ARs can contribute to the vasopressor response to alpha 2-AR stimulation, that alpha 2A/2D-ARs mediate the central antihypertensive response, and that alpha 2C-ARs are not involved in vascular regulation (39, 41, 42). Indeed, alpha 2C-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 alpha 2-ARs is cold-induced vasospasm in the cutaneous circulation (8, 9, 12, 14). Direct analysis of cutaneous arteries in the present study confirms that alpha 2C-ARs do not normally contribute to vasoconstriction. However, during cold-induced vasoconstriction, alpha 2C-ARs are no longer silent and mediate the remarkable cold-induced augmentation of alpha 2-AR responsiveness. Inhibition of alpha 2C-ARs may provide an effective and selective therapy for the vasospastic episodes in Raynaud's disease.

Unlike alpha 1-ARs, functional vascular smooth muscle alpha 2-ARs are not widely distributed in the vascular system (12, 13, 17). alpha 2-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 alpha 2-ARs increased in distal compared with proximal arteries, whereas the opposite pattern was observed for alpha 1-ARs. The altered functional activity of vascular smooth muscle alpha 2-ARs was associated with a remarkable change in the posttranslational modification of alpha 2A/2D-ARs. The expression of the glycosylated form of the alpha 2A/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 alpha 2A/2D-AR becomes increasingly glycosylated in more distal segments of the mouse tail artery. As with the native form of the alpha 2A/2D-AR, the expression of the alpha 2C-AR decreased in distal compared with proximal arteries. The role of glycosylation on alpha 2A/2D-AR function has not been defined. However, with other G protein-coupled receptors, including beta -ARs, glycosylation is associated with enhanced receptor signaling (1, 11, 44). Therefore, the increased glycosylation of the alpha 2A/2D-ARs may contribute to the increased vasoconstrictor activity resulting from stimulation of alpha 2-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 alpha 2A/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 alpha 2-ARs to induce vasoconstriction. Indeed, vasoconstriction to stimulation of alpha 1-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 alpha 2-ARs was inhibited by the selective alpha 2A/2D-AR antagonist BRL-44408 but was not affected by inhibition of alpha 2B (ARC-239)- or alpha 2C (MK-912)-ARs. Furthermore, the calculated KD (20 nM) for BRL-44408 was consistent with its affinity for alpha 2A/2D-ARs (Table 1). Therefore, at 37°C, alpha 2-AR-induced vasoconstriction was mediated by the alpha 2A/2D-AR subtype, with no or minimal contribution from alpha 2C-ARs. However, at 28°C, the augmented vasoconstrictor response to alpha 2-AR activation was highly sensitive to the alpha 2C-AR antagonist MK-912, and the KD (14 pM) for MK-912 was consistent with its affinity for alpha 2C-ARs (Table 1). This indicates that, although alpha 2C-ARs do not appear to contribute to vasoconstriction at 37°C, they are active during cold-induced amplification of the alpha 2-AR response. Indeed, inhibition of alpha 2C-ARs by MK-912 selectively abolished the cold-induced augmentation of alpha 2-AR-mediated vasoconstriction, indicating that this phenomenon is mediated by an effect of cold to amplify alpha 2C-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 alpha 2C-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. 7B).

alpha 2-AR-dependent vasoconstriction was inhibited by the alpha 2A/2D-AR antagonist BRL-44408 at 37 and 28°C. In each case, the KD for the antagonist indicated an interaction with alpha 2A/2D-ARs (Table 1), suggesting that alpha 2A/2D-ARs were not modulated by temperature and were active at 28 and 37°C. Inhibition of alpha 2A/2D-ARs did not prevent cold-induced amplification of the alpha 2-AR response, suggesting that cold-induced modulation of alpha 2A/2D-ARs does not contribute to cold vasoconstriction. However, because inhibition of alpha 2A/2D-ARs and alpha 2C-ARs reduced vasoconstriction at 28°C, alpha 2-adrenergic vasoconstriction at low temperatures may represent an interaction between these two receptor systems. Indeed, alpha 2A/2D-ARs may play a permissive role for alpha 2C-AR-induced vasoconstriction at low temperatures. This may be analogous to other nonhuman, cutaneous arteries that require permissive stimulation by another constrictor to observe alpha 2-AR vasoconstriction (rat tail artery, porcine digital artery; see Ref. 26).

The original concept of alpha 2C-ARs as silent receptors (41) is consistent with their inability to contribute to alpha 2-AR-dependent vasoconstriction, except during cold exposure. The mechanism underlying the effect of warm temperatures to selectively silence or reduce alpha 2C-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 alpha 2C-AR. Although the alpha 2-AR subtypes are homologous, they are uniquely sensitive to physiological regulation (41). alpha 2C-ARs have a prominent intracellular distribution (endoplasmic reticulum and Golgi apparatus), whereas alpha 2A/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 alpha 2C-AR could reflect altered membrane targeting or processing of the receptor or altered activity of alpha 2C-AR-specific signaling or amplification processes.

The alpha 2-AR agonist used in the present study, UK-14,304, is generally used as a non-subtype-selective alpha 2-AR agonist (3, 46). However, reports have suggested that the agonist is somewhat selective for human alpha 2A-ARs, demonstrating increased affinity and efficacy at these sites compared with alpha 2B-AR and alpha 2C-ARs (29, 30, 32). If UK-14,304 selectively activated alpha 2A/2D-ARs located on tail artery smooth muscle cells, then this could have contributed to the lack of an alpha 2C-AR response at 37°C. However, the reported selectivity of UK-14,304 for human alpha 2A-ARs is not evident at alpha 2D-ARs, the mouse homologue of the human alpha 2A-AR. UK-14,304 has similar affinity and efficacy for alpha 2D-AR and alpha 2C-AR (28, 30, 50). Therefore, it is unlikely that the decreased activity of alpha 2C-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, alpha 2-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 alpha 2-ARs (rabbit ear artery; see Ref. 27) or need permissive stimulation by another constrictor to observe alpha 2-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 alpha 2-ARs. Nonselective blockade of alpha 2-ARs (alpha 2A-, alpha 2B-, and alpha 2C-ARs) abolished cold-induced vasospastic attacks in patients with primary Raynaud's disease, whereas alpha 1-AR blockade was ineffective (22). However, analysis of responses to exogenous agonists has demonstrated only small increases or no change in alpha 2-AR-induced vasoconstriction in subjects with primary Raynaud's disease (4, 23, 24, 37). Therefore, although alpha 2-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 alpha 2C-ARs could profoundly influence cold-induced vasospasm, without greatly affecting the alpha 2-AR response at ambient temperatures. Therefore, the local fault in Raynaud's Phenomenon may represent increased expression or effectiveness of alpha 2C-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.


    ACKNOWLEDGEMENTS

We thank the drug companies mentioned in METHODS for the kind gift of alpha 2-AR antagonists.


    FOOTNOTES

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.

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: flavahan.1{at}osu.edu).

Received 9 July 1999; accepted in final form 19 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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