Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The ₂-adrenergic receptor (₂-AR) is prominently involved in blood flow regulation and is widely expressed in sympathetic afferents to the peripheral vascular system (presynaptic and postsynaptic sites) and on vascular endothelial and smooth muscle cells (Ruffolo, 1985; Kable et al., 2000). The precise function of each subtype (_2A, _2B, and _2C, rat contains _2D-AR, which is a species ortholog of human _2A-AR) in the regulation of blood flow, as well as the explanation for their distribution is not fully understood due to the lack of availability of subtype-specific agonists or antagonists. The development of drugs selectively targeting the ₂-adrenoceptor subtypes may have therapeutic potential. OPC-28326, 4-(N-methyl-2-phenylethylamino)-1-(3,5-dimethyl-4-propionylaminobenzoyl) piperidine hydrochloride monohydrate, is a newly synthesized compound, which has been shown to be a vasodilator, selective for the femoral artery. OPC-28326 has been reported to selectively increase femoral blood flow in anesthetized open-chest dogs and autoperfused canine femoral artery preparations (Orito et al., 1999). Initial ligand binding studies, together with other pharmacological analyses, have suggested that OPC-28326 mediates its effect through antagonism of the ₂-adrenoceptor. To confirm previous findings in vivo, we examined the selectivity and efficacy of OPC-28326 in the anesthetized rat. To further define the pharmacological properties of OPC-28326 on the ₂-adrenoceptor subtypes, we established Chinese hamster ovary (CHO) cell lines that stably express functional rat _2D-, _2B-, or _2C-adrenoceptor and coexpress luciferase as a reporter system. The expression of luciferase was driven by a cAMP response element in its promoter region. The level of luciferase expression therefore reflects the intracellular cAMP concentration. It is known that all three subtypes of ₂-adrenoceptors are coupled to G_i proteins (Pepperl and Regan, 1993); therefore, the activation or inhibition of these receptors is reflected in the level of luciferase expression, which was determined by luciferase activity assay. Using these cell lines, the effect and potency of OPC-28326 were evaluated by both radioligand membrane binding assay and luciferase assay, and compared with the well known ₂-adrenoceptor-specific antagonist yohimbine. In addition, in situ hybridization was performed to investigate the expression of each subtype of ₂-adrenoceptor in rat hind limb skeletal muscles.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

In Vivo Pharmacology Studies. Male Sprague-Dawley rats were anesthetized using isoflurane (IsoFlo, Abbott Diagnostics, Abbott Park, IL), delivered via a positive pressure ventilator (Hallowell AWS; Hallowell EMC, Pittsfield, MA), 3% (v/v) during surgical preparation of the animal and reduced to 2.5% thereafter. Positive pressure ventilation was initiated after placement of an endotracheal tube; and ventilation rate adjusted to give an arterial blood PO₂ of 150 to 200 mm Hg, PCO₂ of 30 to 45 mm Hg, and pH of 7.35 to 7.45. The body temperature of animal was maintained on a 37°C heating pad and a rectal thermometer-controlled infrared lamp. A midline longitudinal incision was made in the cervical area and the carotid artery freed from its connective tissue sheath by blunt dissection. A flow probe (1RB; Transonic, Ithaca, NY) was attached to the carotid artery and secured. A small piece of skin was removed in the inguinal area of the right leg and the femoral artery and vein dissected free of connective tissue. A catheter was placed in the artery to record blood pressure and heart rate. The left femoral artery was also exposed in a similar manner and a flow probe positioned, as for the carotid. The catheter was attached to two pressure transducers, which were connected to a blood pressure/heart rate monitor (Stoelting, Wood Dale, IL) and to a flowmeter (Transonic T206, Ithaca, NY). In experiments where PGE₁ was to be administered, a catheter was also placed into the right femoral vein. For administration of OPC-28326, the stomach was removed from the abdomen via a midline incision using ring-tipped forceps. A catheter was introduced into the duodenum and secured. The stomach was returned to the abdominal cavity and the incision sutured.

cDNA Cloning and Vector Construction. Total RNA was extracted from a fresh Sprague-Dawley rat brain, and 5 µg was reverse-transcribed into cDNA and used as a template for the polymerase chain reaction (PCR). Specific primers with a Kozak sequence (CCCACC) for each subtype of ₂-adrenoceptors were designed (_2D, forward primer: 5'-CCCACCATGGGCTCCCTGCAGCCG-3', reverse primer: 5'-CACACGATGCGCTTTCTGTCC-3'; _2B, forward primer: 5'-CCCACCATGTCCGGCCCCACCATG-3', reverse primer: 5'-CACCAGCCAGTCTGGGTCC-3' and _2C, forward primer: 5'-CCCACCATGGCGTCCCCAGCGCTG-3', reverse primer: 5'-CACTGCCTGAAGCCCCTTC-3'), and synthesized by Invitrogen (Carlsbad, CA). Using these primers, full coding regions were amplified by PCR and further recombined into cloning vector (pCR2.1; Invitrogen). The DNA sequences of the inserts were confirmed by automatic DNA sequencing before inserting into mammalian expression vector (pcDNA3.1+; Invitrogen). An expression vector (pCRE-Luc) containing a cAMP-response element in promoter region that drives the expression of luciferase was purchased from Stratagene (La Jolla, CA).

Establishment of Overexpression CHO Cell Lines. Coexpression of the luciferase reporting vector with the vectors containing ₂-adrenergic subtype receptors was carried out by calcium phosphate precipitation into Chinese hamster ovary cells, which do not express native - and -adrenergic receptors. CHO cells were maintained at 37°C in F12K medium plus 10% fetal calf serum, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) under humidified 5% CO₂, 95% air. Stable transfectants were selected with 1.0 mg/ml G418 (Invitrogen) for 12 days on a 96-well plate. The cell clones overexpressing functional ₂-adrenoceptor were examined by cAMP radioimmunoassay (PerkinElmer Life Science Products, Boston, MA).

Radioligand Binding Assay. The cells expressing transfected gene were grown to confluence. After decanting the culture medium and rinsing the cell surface with ice-cold PBS, the culture plate was placed on ice and the cells were gently suspended in PBS with a plastic policeman. The cells were then collected and centrifuged at 400g for 10 min at 4°C. The cell pellet was frozen at 70°C for later processing. For the cell membrane preparation, frozen cells were thawed in a 37°C water bath, washed once with PBS buffer, and centrifuged at 800g for 5 min. The cells were then homogenized with a Polytron (20 s at setting 6) on ice with NaPO₄ buffer (25 mM, pH 7.4) in a volume 20 times the cell pellet. The homogenate was centrifuged at 16,000g for 30 min at 4°C. After rinsing one more time, the cell membrane pellet was resuspended in the buffer for radioligand binding studies with [³H]RX821002 (67.0 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ) (Deupree et al., 1996). A small aliquot was used for protein measurement. First, the K_d values of [³H]RX821002 binding to the membranes and receptor densities of rat _2D-, _2B-, or _2C-adrenoceptor in the transfected cells were determined with saturation binding studies (up to 12.5 nM [³H]RX821002) in the absence or presence of 10 µM yohimbine. Nonspecific binding in the presence of 10 µM yohimbine is less than 10% in all three membrane preparations. The K_d and B_max values calculated with GraphPad Prism 3.0 (GraphPad Software, San Diego, CA) were 0.76 nM and 1.24 pmol/mg of protein, 5.81 nM and 4.68 pmol/mg of protein, and 0.68 nM and 0.71 pmol/mg of protein for _2D-, _2B-, and _2C-adrenoceptor, respectively (average of two separate experiments). The K_d values of [³H]RX821002 from these cell lines are 7 to 11 times higher than previously reported on all three receptors (0.07, 0.89, and 0.10 nM for _2D-, _2B-, and _2C-adrenoceptors, respectively) (Deupree et al., 1996). We do not have a clear answer for the difference, although interlaboratory variance is a possible reason, because saturation experiments were performed with different concentration ranges. Also, nonspecific binding was defined using different nonradioactive substrates (yohimbine versus norepinephrine), and membrane homogenates were prepared from different cell lines. Nevertheless, the order of [³H]RX821002 affinity to each subtype is similar. Scatchard analysis of the data showed single binding site in each of the membrane preparations. Competition binding studies were performed with serial dilution of test compounds (OPC-28326 and yohimbine) in triplicate in the presence of 1.25 nM [³H]RX821002 and 70 µg of membrane protein in a total volume of 250 µl. After 45-min incubation at room temperature, the binding mixture was filtered rapidly through a GF/B glass fiber filter paper (Whatman, Clifton, NJ), using a Brandel harvester (Biomedical Research & Development Laboratories, Gaithersburg, MD). After washing twice with ice-cold NaPO₄ buffer, the radioactivity retained on the filter was counted by a liquid scintillation spectroscope (1209 Rackbeta; LKB, Turku, Finland). K_i values were calculated as K_i = IC₅₀/(1 + [³H]RX821002 concentration/K_d).

Luciferase Assay. To test the effect of OPC-28326, the cells were subcultured into a white 96-well plate with clear bottom (Corning Costar, Cambridge, MA) at near confluence. The next day, the cells were washed once with F12K medium plus 0.5% fetal calf serum. The cells were then incubated with the washing medium only (basal), or with forskolin (1 µM) or plus epinephrine (100 nM, control for receptor activation) in the presence of a serial dilution of OPC-28326 or yohimbine, for 4 h at 37°C. Forskolin, yohimbine, epinephrine, brimonidine, and p-aminoclonidine were purchased from Sigma Chemical (St. Louis, MO). After washing the cells twice with PBS, the cells were lysed in 20 µl of lysis buffer for 30 min at room temperature with shaking. One hundred microliters of substrate (Luciferase detection kit; Stratagene) was injected into each well for luciferase activity measurement, using a Mediators PhL luminescence plate reader (ImmTech, New Windsor, MD). The value of luminescence (arbitrary units) detected during the first second after mixing cell lysis with substrate was taken as luciferase activity. Forskolin (1 µM) increased luciferase activity of these cell lines (4-h incubation) by 1 to 2 orders of magnitude above basal levels, which were usually very low.

In Situ Hybridization. Coding regions of rat _2D (783-1352), _2B (989-1258), and _2C (1-339) cDNA were subcloned into pGEM3z (Promega, Madison, WI). cRNA probes in the sense and antisense orientation were synthesized using ³⁵S-labeled UTP and T7 and SP6 polymerase, respectively. The specificity of the probes was tested on tissue sections from rat kidney and brain, and no cross-hybridization was observed.

Data Analysis. Because of variability between animals, statistical comparisons were made using normalized values rather than absolute values. Normalization was performed by expressing the variable as a percentage of change from baseline value before drug administration. Statistical analysis of the effect of OPC-28326 or PGE₁ on percentage of change in blood flow in each artery, blood pressure, and heart rate was performed compared with the corresponding values in the control group, using Student's t test. Differences with a P value less than 0.05 were considered to be statistically significant. All data are presented as mean ± S.D. of the mean.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

In previous experiments, the effect OPC-28326 was studied in rat under ketamine/xylazine anesthesia. It was demonstrated that OPC-28326 caused a dose-dependent increase in femoral blood flow, whereas it had no effect on carotid flow at 1 and 3 mg/kg. At 10 mg/kg, the increase in femoral blood flow is similar to 1 mg/kg; however, no effect on carotid blood flow was observed (data not shown). Due to the possibility that OPC-28326 exerts its action through antagonism of the peripheral ₂-adrenoceptors, as we found out later, and that xylazine is also a nonspecific ₂-adrenoceptor agonist (Virtanen and MacDonald, 1985), we decided to use isoflurane anesthesia, which has been previously used to study the hemodynamic effect of ₂-adrenoceptor agonist dexmedetomidine (Bloor et al., 1992). Based on previous data, we selected one dose (3 mg/kg), which gave the maximal elevation of femoral blood flow and examined its effects. It was found that the effect of 3 mg/kg under the two anesthetic regimes gave quantitatively similar results. At 30 min after administration, the increase in femoral blood flow from baseline was 33.0 and 42.2% under ketamine/xylazine and isoflurane, respectively; the change in carotid blood flow at the same time point was 0 and 1.1%.

As shown in Fig. 1, A and B, OPC-28326 caused a statistically significant increase in femoral blood flow at all time points from 10 min after drug administration (maximum increase 44.7%, from 3.22 ± 0.68 ml/min at t = 0 min to 4.63 ± 1.31 ml/min at t = 45 min), compared with control, whereas there was no significant change in carotid flow (maximum increase of 3.6% at t = 35 min). PGE₁, which is a vasodilator known to increase intracellular cAMP in vascular smooth muscle (Dembinska-Kiec et al., 1980), caused a dose-dependent increase in both femoral and carotid blood flow. The higher doses of PGE₁ tested (1.25 and 2.5 µg/kg/min) caused an increase in femoral flow that exceeded that caused by OPC-28326 (68.3 and 70.5%, respectively, corresponding to an increase from 2.5 ± 1.4 ml/min basal line to 4.0 ± 1.9 ml/min at t = 45 min and 2.6 ± 1.0 ml/min basal line to 4.4 ± 1.6 ml/min at t = 45 min, respectively). The lowest dose (0.63 µg/kg/min) caused an increase of 35.3% (from 2.7 ± 1.0 ml/min basal line to 3.8 ± 1.7 ml/min at t = 45 min), which was slightly less than that caused by OPC-28326 (44.7%). The increases in flow at all three doses were statistically significant at all time points beyond 15 min, compared with control. In contrast to OPC-28326, PGE₁ also increased carotid blood flow dose dependently. Although PGE₁-induced (0.63 µg/kg/min) increases in carotid flow did not reach statistically significance, the maximum percentage of increase was much higher (14.7% at t = 40 min) compared with OPC-28326 (3.6% at t = 35 min). PGE₁ at 1.25 and 2.5 µg/kg/min caused significant increase in carotid blood flow from control 20 min after administration (21.8 and 38.2%, respectively, corresponding to 4.8 ± 1.2 ml/min basal line to 5.8 ± 1.6 ml/min at t = 45 min and 4.6 ± 1.2 ml/min basal line to 6.4 ± 1.9 ml/min at t = 45 min).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Comparison of OPC-28326 (3 mg/kg intraduodenally) and PGE1 (0.63, 1.25, and 2.5 µg/kg/min i.v.) on femoral (A) and carotid (B) artery blood flow, mean blood pressure (C), and heart rate (D) in anesthetized rats (n = 6, mean ± S.D.). The same amount of saline was used in control group. , control; , OPC-28326; , PGE1 0.63 µg/kg/min; , PGE1 1.25 µg/kg/min; , PGE1 2.5 µg/kg/min.

Blood pressure was significantly decreased from 5 to 45 min after administration of OPC-28326 (maximum decrease 18.0%, from 74 ± 9 mm Hg basal line to 61 ± 8 mm Hg at t = 15 min; Fig. 1C). Longer recording in some animals showed that blood pressure returned to 2.6% of control at 1 h. Heart rate was significantly less than control only at 10 min (by 6.3%, from 323 ± 35 to 302 ± 31 min¹; Fig. 1D). During PGE₁ infusion, blood pressure exhibited a transient, dose-dependent decrease, which recovered toward baseline. The effect of PGE₁ on blood pressure was significantly different from control for a portion of the duration of infusion (at 0.625 µg/kg/min from 5 to 15 min, maximum decease by 7.8%, from 74 ± 9-68 ± 10 mm Hg at 5 min; at 1.25 µg/kg/min from 5-30 min, maximum decrease by 17.6%, from 82 ± 15-67 ± 13 mm Hg at 5 min; and at 2.5 µg/kg/min at 5 min only, decreased by 23.7%, from 74 ± 7-56 ± 4 mm Hg). The effect of PGE₁ on heart rate was minimum, significant change (decrease by 3.1%, P < 0.05) observed only at 2.5 µg/kg/min at 5 min after administration.

OPC-28326 was tested by luciferase assay on the ₂ adrenoceptor-expressing cells to see whether it has any agonistic effects. This was done by adding OPC-28326 (final concentration from 10 nM to 10 µM) to the cells expressing each subtype of ₂-adrenoceptors in the absence or presence of 1 µM forskolin. No change of luciferase activity was detected for OPC-28326 at the basal level or under forskolin stimulation on any ₂-subtypes (data not shown). Therefore, these results ruled out any agonistic effect of OPC-28326 on ₂-adrenoceptors.

The antagonistic effect of OPC-28326 on ₂-adrenoceptors was tested by both radioligand membrane binding with [³H]RX821002 and luciferase assay in the presence of epinephrine (100 nM). Using the membranes prepared from CHO cells expressing the subtypes of rat ₂-adrenoceptors, OPC-28326 and yohimbine (as control drug) displaced the binding of [³H]RX821002 in a specific and concentration-dependent manner. On all three ₂-adrenoceptor subtypes, the displacement with OPC-28326 was complete with one binding site. As shown in Fig. 2, the affinity of OPC-28326 at _2C-adrenoceptors was similar to yohimbine, with a calculated K_i of 13.7 ± 1.9 nM (n = 3; Table 1). The affinity of OPC-28326 at _2D-adrenoceptors and _2B-adrenoceptors was much lower (3840 ± 887 and 633 ± 46 nM, respectively; n = 3). In the same experiments, the affinities of yohimbine on the three ₂-adrenoceptor subtypes were found to be within 1 order of magnitude in difference (Table 1). When OPC-28326 was used to inhibit ₂-adrenoceptor activation by epinephrine (100 nM) in luciferase assay, similar levels of affinity and order to the three ₂-adrenoceptor (i.e., _2C > _2B > _2D) were found (n = 5) (Fig. 3). Epinephrine (100 nM) inhibited forskolin-induced (1 µM) luciferase increase by 51, 48, and 16% on average on the _2D-, _2B-, and _2C-AR-expressing cells, respectively. Calculated K_i values for OPC-28326 and yohimbine are summarized in Table 1. Similar results were obtained when the cells were activated with p-aminoclonidine or brimonidine, a specific ₂-adrenoceptor agonist (data not shown). The data clearly indicate that OPC-28326 is different from yohimbine, with a high affinity for _2C- followed by _2B-, and by _2D-adrenoceptor.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Competition radioligand binding with OPC-28326 and yohimbine on 2D-adrenoceptors (A), 2B-adrenoceptors (B), and 2C-adrenoceptors in the presence of 1.25 nM [3H]RX821002. Membrane protein (70 µg) prepared from the CHO cells overexpressing rat 2D-adrenoceptors, 2B-adrenoceptors, or 2C-adrenoceptors was incubated with the drugs at room temperature for 40 min. Data are the mean ± S.D. of triplicate measurements of three experiments. , OPC-28326; , yohimbine.

View this table: [in this window] [in a new window]   TABLE 1 Ki values of OPC-28326 and yohimbine determined by ligand binding assay with [3H]RX821002 (n = 3) and by luciferase assay with epinephrine (n = 5, mean ± S.D.), using the CHO cells overexpressing rat 2D-adrenoceptors, 2B-adrenoceptors, or 2C-adrenoceptors

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Luciferase assay of OPC-28326 and yohimbine on the concentration-dependent inhibition of epinephrine (100 nM) effect on 2D-adrenoceptors (A), 2B-adrenoceptors (B), and 2C-adrenoceptors in the presence of forskolin (1 µM). Basal luciferase activity from control (without forskolin) was subtracted. Data are the mean ± S.D. of triplicate measurements of five experiments and expressed as the percentage of inhibition of epinephrine (100 nM). , OPC-28326; , yohimbine.

The expression of all three ₂-adrenoceptor subtypes in rat skeletal muscle from hind limb can be detected by reverse transcription and PCR, using total RNA extracted from the tissues and specific primers (data not shown). To further locate the expression site, in situ hybridization on tissue section of m. gastrocnemius was performed using subtype-specific probes. A strong signal for _2C-adrenoceptor was detected in areas corresponding to vascular tissue (both of arteries and veins; medium and small vessels), together with moderate expression of _2D-adrenoceptor and _2B-adrenoceptor over the entire tissue section (Fig. 4). In the rat brain sections examined, no labeled blood vessels with any of the probes could be seen, although the expression of _2A-adrenoceptor was detected in cerebral cortex and hypothalamus and _2C-adrenoceptor messenger was detected in hippocampus region as reported previously (Nicholas et al., 1996).

View larger version (60K): [in this window] [in a new window]   Fig. 4.   In situ hybridization of tissue sections from rat m. gastrocnemius, with 2-adrenoceptor subtype-specific antisense cRNA probes. The cRNA probes with sense orientation were used as negative controls (only 2C is presented). One tissue section was stained with neutral red for histological examination. Scale bar, 1 mm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

OPC-28326 (0.3 and 1.0 µg/kg i.v.) has been reported to selectively increase femoral artery blood flow in anesthetized open-chest dogs, with only minimal action on systemic blood pressure, heart, and coronary, carotid, vertebral, renal, and mesenteric blood flows (Orito et al., 1999). The mechanism of action of OPC-28326 has been suggested to be due to blockade of ₂-adrenoceptors, because OPC-28326 inhibited the decrease in perfusion flow of rat hind limb preparations induced by brimonidine, a selective ₂-adrenoceptor agonist. In canine autoperfused femoral artery preparations, the potency of OPC-28326 was similar to that of yohimbine in increasing femoral blood flow (clear effect was observed at 1 nM), but 14 times higher than that of prazosin (10 nM) (Orito et al., 1999). In addition, screening with radioligand binding studies showed competitive displacement of OPC-28326 on ₂-adrenoceptor (Orito et al., 1999). In the present study, the selectivity of OPC-28326 in increasing femoral over carotid blood flow was confirmed in vivo in another species (rat) under anesthesia and compared with the effects of PGE₁. In previous experiments, under ketamine/xylazine anesthesia, we initially used yohimbine (0.1 and 0.5 mg/kg i.v., n = 4 for each group) as control. Although much less effective in increasing femoral blood flow (9.7 and 23.0%, respectively, versus 42.2% for OPC-28326 at 30 min after administration), we reasoned that yohimbine may not serve as the most appropriate in vivo control for OPC-28326, due to profound hemodynamic effects (blood pressure decreased by 32.0% compared with 18.0% for OPC-28326 and heart rate increased by 16.2%). These effects of yohimbine may be mediated either by direct central nervous system or peripheral nonselective blockade of ₂-adrenoceptors coupled with secondary sympathetic regulation (Goldberg et al., 1983), whereas OPC-28326 does not cross the blood-brain barrier (T. Imaizumi, K. Orito, and T. Mori, unpublished observation). After a literature search, we chose PGE₁ as an alternative control. As the results indicate, PGE₁ dose dependently and nonselectively increased blood flow in both femoral and carotid arteries, whereas OPC-28326 only increased femoral blood flow.

To understand the mechanism of selectivity of OPC-28326 on femoral vasculature, we performed membrane ligand binding studies and functional luciferase assays on overexpressed rat ₂-adrenoceptor subtypes and the distribution of ₂-adrenoceptor in skeletal muscle. Compared with yohimbine, OPC-28326 was shown to be a selective antagonist for _2C-adrenoceptors. OPC-28326, with an affinity at the _2C-adrenoceptor at least 30-fold higher than the other two ₂-adrenoceptor subtypes, is probably the most _2C-adrenoceptor-selective antagonist reported so far, compared with ARC239, MK912, or rauwolscine (Bylund et al., 1988; Uhlen et al., 1998). The similar affinity of yohimbine and OPC-28326 at _2C-adrenoceptors is in agreement with the previous observation that both OPC-28326 and yohimbine, at the same concentration, induced about the same level of increase in femoral blood flow in a canine autoperfused model (Orito et al., 1999). The selectivity of OPC-28326 in increasing femoral blood flow is further supported by the abundance of _2C-adrenoceptors expressed in the hind limb skeletal muscle vasculature, as demonstrated by in situ hybridization. This is the first report of _2C-adrenoceptor expression in skeletal muscle, although the expression of only _2A-adrenoceptor has been detected previously by Northern blot analysis (Lorenz et al., 1990).

Multiple -adrenoceptors subtypes (both ₁ and ₂) are expressed in vascular smooth muscle and are involved in various aspects of blood vessel function, including contraction. ₂-Adrenoceptors have been reported to play a major role in mediating vasoconstriction in certain type of vessels or tissues. Faber (1988) demonstrated in the rat skeletal muscle that both ₁- and ₂-adrenoceptors regulate agonist-induced constriction in large arterioles and venules, but that the small precapillary arterioles are primarily under the control of ₂-adrenoceptors. The selective ₂-adrenergic agonist B-HT 920 was shown to cause greater contractile responses in human distal digital arteries compared with proximal arteries (Flavahan et al., 1987). Small contractile responses to B-HT 920 were also seen in rat aorta and femoral artery preparations (Dyke and Widdop, 1987). It has been shown that the ₂-adrenoceptor is the sole adrenergic receptor subtype involved in mediating the vasoconstrictive response to neurally released norepinephrine in rat saphenous vein (Cheung, 1985), tail artery (Medgett, 1985) and small terminal arterioles of the rat cremaster skeletal microcirculation (Ohyanagi et al., 1991). Postjunctional ₂-adrenoceptor-mediated vasoconstriction has also been demonstrated in canine and human saphenous vein by using SK&F 104856 (Hieble et al., 1991). However, no subtype of ₂-adrenoceptors in these tissues was identified due to lack of any subtype-selective agonist or antagonist. There are also difficulties in using the classical radioligand binding approach, or immunohistochemical staining to study the distribution of ₂-adrenoceptors in peripheral cardiovascular system, due to the lack of selective ligands, or antibodies and low expression levels for each subtype. Little is known about the expression and function of _2C-adrenoceptor in the vascular tissues. Recently, a few reports demonstrated the specific role of _2C-adrenoceptors by in vitro pharmacological studies. The _2C-adrenoceptor was shown to mediate contractile responses to noradrenaline in the human saphenous vein (Gavin et al., 1997) and porcine nasal mucosa (Rizzo et al., 2001). Chotani et al. (2000) demonstrated that _2C-adrenoceptor-mediated constriction of mouse tail arteries was augmented during cold exposure. Recent studies with -adrenoceptor subtype knockout mice suggested that both the _2A- and _2B-adrenoceptor may play a role in vasoconstriction. The immediate vasoconstrictive effect of ₂-agonists was absent in both _2A-adrenoceptor knockout mice (Altman et al., 1999) or the mice with a mutant _2A-adrenoceptor (MacMillan et al., 1996), and _2B-adrenoceptor knockout mice (Link et al., 1996). The response to ₂-agonist was not altered in the _2C-adrenoceptor-deficient mice (Link et al., 1996). Although the exact subtype of ₂ adrenoceptors expressed in the femoral vascular bed is not known, yohimbine has been shown to inhibit pressor responses to noradrenaline (Polonia et al., 1986). The present studies not only demonstrate the selectivity of OPC-28326 at the _2C-adrenoceptor and the presence of _2C in rat hind limb skeletal muscles but also suggest a possible functional role for the _2C-adrenoceptor in the regulation of femoral vascular tone.

OPC-28326, which selectively increases blood flow in the femoral artery, may be a good candidate as therapeutic agent to treat peripheral arterial occlusive disease or other vasospasm disorders, such as intermittent claudication and Raynaud's syndrome. Because no hemodynamic effect was produced by disruption of the _2C-subtype (Link et al., 1996) and a minimum effect of OPC-28326 on blood pressure and heart rate was seen in the present and previous studies, selective _2C-adrenoceptor antagonists, such as OPC-28326, may offer beneficial clinical use with few side effects.