Introduction

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There have been numerous reports on the presence of an unusual class of alpha adrenoceptors in the dog saphenous vein (DSV) Constantine et al., 1982; Flavahan and Vanhoutte, 1986; Guimaraes et al., 1987; Hicks et al., 1991). Rauwolscine, a selective alpha-2 adrenoceptor antagonist, competitively antagonized contractions induced by phenylephrine (PE), an alpha-1 agonist, in the DSV with a pA₂ of 8.5 (Daniel et al., 1996) but not in the dog mesenteric vein (DMV). However, antagonism by rauwolscine of PE responses in DSV did not show classic competition (i.e., the slope of Schild plot was <1.00). DeMey and Vanhoutte (1981) had also previously reported a slope of <1 and a pA₂ of 7.6 using yohimbine against norepinephrine.

Radioligand-binding studies in the DSV showed that prazosin competed with [³H]rauwolscine for higher (K_iH = 1.5 µM) and lower (K_iL = 95 µM) affinity-binding sites (Daniel et al., 1996), but neither value suggested that these agents interacted at their sites of competition with PE. Pretreatment with chloroethylclonidine (CEC), an alkylating agent, thought to selectively inactivate alpha-1B and alpha-1D adrenoceptor sites, abolished prazosin-binding sites from DSV and from DMV, which appears to have alpha-1D adrenoceptors (Daniel et al., 1997). CEC also reduced the very high density of rauwolscine binding (B_max or K_D) by 55% in DSV but did not affect rauwolscine binding sites (B_max) in DMV. In the DSV, CEC pretreatment reduced the potency of PE in competition with rauwolscine for binding. Based on these observations and on studies with additional selective antagonists (Daniel et al., 1996), we suggested that the unusual adrenoceptor subtype in the DSV may be related to the subtype of alpha-1D adrenoceptor that has high affinity for WB 4101 but not for 5 methyl-urapidil. In this blood vessel, alpha-2 antagonists such as yohimbine or rauwolscine may also bind to it and inhibit responses to PE.

To further understand the unusual nature of the alpha adrenoceptor subtype in the DSV, we examined the interaction of the alkylating agent, phenoxybenzamine (PBZ), with sites of action of PE and selective alpha-2 adrenoceptor agonists using the receptor protection protocol of Furchgott (1972). This technique measured the potency of PBZ to inactivate each response and the ability of prazosin and rauwolscine to protect these receptors from PBZ inactivation. We also examined whether there was any alteration in sensitivity of the residual receptors responding to alpha-1 and alpha-2 selective agonists and antagonists after partial receptor inactivation by PBZ to determine whether there was an overlap between them.

	Materials and Methods

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Muscle Bath Procedures. Mongrel dogs (10-30 kg) were sacrificed with an overdose of sodium pentobarbital (100 mg/kg i.v.). The procedure was approved by our University Animal Care Committee in keeping with the guidelines of the Canadian Council of Animal Care. The lateral branch of the DSV on both legs was removed and placed in Krebs' solution at pH 7.4 containing 119 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 2 mM MgCl₂, 25 mM NaHCO₃, 1 mM NaH₂PO₄, and 11 mM glucose. After fat and connective tissue were removed under a dissecting microscope, 5- to 6-mm rings of DSV were prepared. The endothelium was removed with the teeth of a pair of forceps, and the rings were mounted in a 15-ml organ bath connected to a force transducer (Grass FT03C; Grass Instruments Co., Quincy, MA) and a chart recorder (R611; Beckman, Mississauga, Ontario, Canada). The absence of endothelial cells was confirmed by testing for lack of relaxation by 1 µM carbachol in rings precontracted with 60 mM K⁺.

PBZ Treatment. PBZ was dissolved in 100% EtOH to make a stock appropriate for use with 10 µl in 10 ml of Krebs' in the muscle bath. Preliminary studies have shown that responses to this final concentration (0.1%) of EtOH vehicle alone were no different from those in untreated controls. After a 20-min incubation, a time shown in pilot studies to be sufficient for optimal PBZ inactivation, unreacted or unbound PBZ was removed with three 10-min washes before constructing the next series of concentration-response curves.

Receptor Protection Protocol. The DSV rings were incubated with prazosin or rauwolscine for 15 min before the addition of PBZ. Preliminary studies showed that the complete washout time for 10 and 30 nM prazosin was 120 min. The length of time taken to wash out 1 µM rauwolscine also was 120 min. These washout times were applied before construction of the second concentration-response curve.

Data Handling for Contractions. All tension measurements were expressed as a percentage of the response to 100 mM K⁺. The effective concentrations that produced 50% of maximum response (EC₅₀) were estimated by sigmoidal curve fitting of each concentration-response curve (Origin v4.1; MicroCal Software, Northampton, MA). The fraction of residual receptors not inactivated by PBZ (q) was calculated using Furchgott's method (1972).

Chemicals. PE, PBZ, and prazosin were obtained from Sigma Chemical (St. Louis, MO). UK-14,304 and B-HT 920 were purchased from Research Biochemicals (Natick, MA). Rauwolscine was obtained from Carl Roth KG (Karlsruhe, Germany). Prazosin and UK-14,304 were dissolved in dimethyl sulfoxide and shielded from light. PE, B-HT 920, and rauwolscine were dissolved in double-distilled deionized water.

Statistics. Data are expressed as mean ± S.E.M. Mean values were compared using Student's t test, the Mann-Whitney U test (two-tailed), and one-way analysis of variance where appropriate. Statistical significance was accepted at p < .05. In analysis of variance, significant differences were interpreted using post hoc t testing of differing pairs with the Bonferroni correction.

	Results

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Effects of Varying PBZ Concentrations on PE Concentration-Response Curves. Increasing concentrations of PE concentration-dependently initiated contractile responses. PBZ pretreatment caused concentration-dependent inhibition of PE E_max (p < .05) and a rightward shift in the EC₅₀ value (p < .05), which was statistically significant for PBZ concentrations of 30 nM (Fig. 1). At 100 and 300 nM, PBZ caused 40% to 50% inhibition of E_max, whereas 1000 nM PBZ completely abolished responses, precluding determination of EC₅₀ values. Estimates of EC₅₀ values, E_max calculations, K_A for PE, and residual receptors (q) are shown in Table 1 for all concentrations of PBZ. When PBZ concentration changed from 10 to 300 nM, there was a 16-fold reduction in the fraction of receptors available for interaction with PE.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Effects of PBZ (10-1000 nM) on concentration responses to PE in the DSV expressed as a percentage of 100 mM K+ contraction. Concentration-dependent inhibition of Emax of PE was observed with increasing concentrations of PBZ. Control responses (, n = 7), vehicle (0.1% EtOH)/time controls (, n = 10), PBZ 10 nM (, n = 8), PBZ 30 nM (, n = 7), PBZ 100 nM (, n = 12), PBZ 300 nM (, n = 10), and PBZ 1000 nM (, n = 6). Values are mean ± S.E.M., and n is the number of tissues.

Antagonism of PE Responses by Rauwolscine (50 nM) after PBZ Treatment. To test whether the adrenoceptors that recognize rauwolscine and PE were selectively inactivated or preserved among those inactivated by PBZ, we constructed a series of PE concentration-response curves after PBZ treatment in the presence of rauwolscine (50 nM). Results are summarized in the two columns on the right of Table 1. Rauwolscine shifted the EC₅₀ value to the right by at least 1 log unit in tissues pretreated with PBZ (p < .05). The K_B value of rauwolscine did not vary significantly, ranging only from 3.6 to 7 nM for tissues treated with all concentrations of PBZ (10-300 nM). These results suggest that PBZ did not selectively inactivate or preserve receptors at which rauwolscine antagonized PE, unlike CEC. Pretreatment with this agent (Daniel et al., 1996) reduced B_max for rauwolscine binding, increased the IC₅₀ value of PE displacement (8-94 µM) of rauwolscine binding, and shifted the EC₅₀ value for PE-induced contraction slightly but significantly from 1.4 to 3.5 µM. These earlier results suggested that some of the alpha-1 adrenoceptors recognizing both PE and rauwolscine with higher affinity for PE were selectively inactivated by CEC. The present results show that PBZ had no such selective inactivating effect.

Antagonism of PE Responses by Prazosin (30 and 100 nM) after PBZ Treatment. PBZ may also inactivate selectively adrenoceptors at which prazosin acts with high affinity. We examined the effect of prazosin (30 and 100 nM) on PE concentration-response curves in PBZ (30 and 100 nM)-treated tissues. In vessels used as vehicle controls, prazosin (30 nM) caused 1 log unit rightward shift in the EC₅₀ values (Table 2). PBZ (30 and 100 nM) caused a significant reduction in the E_max values and a rightward shift in EC₅₀. In tissues that had been preincubated with PBZ, the EC₅₀ values for PE and the K_B values for 30 nM prazosin did not differ significantly between the groups treated with 30 or 100 nM PBZ. However, treatment with 100 nM prazosin produced a change in the EC₅₀ values in those tissues pretreated with 30 nM PBZ (Table 2) but not in those pretreated with 100 nM PBZ. K_B values for prazosin increased significantly only in the former case.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effects of prazosin and rauwolscine on residual receptors not inactivated by PBZ are shown. Prazosin (100 nM, , n = 6) did not have an additional effect on the residual receptors after PBZ inactivation, whereas rauwolscine (100 nM, , n = 6) shifted the concentration-response curve 1 log unit to the right. Untreated controls (, n = 6), PBZ 300 nM (, n = 17), and PBZ vehicle/time control (, n = 17).

Are the Unusual Adrenoceptors Protected from PBZ Inactivation by Rauwolscine? K_A values for PE were unaltered by the proportion of alpha-1 adrenoceptors that were inactivated by PBZ. Furthermore, rauwolscine shifted the concentration-response curves of PE farther to the right after PBZ treatment, yielding unchanged K_B values (Table 1; Fig. 2). These findings suggest that the PBZ-inactivated and residual responses used the same populations of receptors on the basis of PE and rauwolscine sensitivity. To test this further, we attempted to protect the receptors with 30, 100, and 1000 nM rauwolscine 15 min before PBZ (300 nM) inactivation. If rauwolscine and PBZ interact at the same site on a receptor recognizing PE, then the protection afforded by rauwolscine against PBZ inactivation should occur in a concentration-dependent manner. If they bind at different sites, there should be either no protection or noncompetitive interaction. A summary of the results from these experiments is shown in Fig. 3, and data from them are summarized in Table 3. After complete washout (2 h) of the "protective" rauwolscine, an unchanged rightward shift of EC₅₀ values for PE by PBZ was observed. The fractions of receptors that survived inactivation by PBZ (q), like the B_max responses, also were not changed by the presence of rauwolscine with PBZ. Therefore, rauwolscine in DSV did not protect the alpha adrenoceptors responding to PE against inactivation by PBZ, even though it antagonized contractile responses to PE (Table 1) (Daniel et al., 1996). This suggests that PBZ and rauwolscine do not interact at the same site on these receptors.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Concentration-response curves to PE were constructed and expressed as a percentage of 100 mM K+ contraction. To determine whether rauwolscine protected against inactivation by PBZ (300 nM), varying concentrations of rauwolscine were added to the baths 15 min before the addition of PBZ. If rauwolscine bound to receptors to which PBZ inactivate, then these receptors could be protected from inactivation by PBZ. Curves generated with rauwolscine protection protocol were not different from the curve without rauwolscine protection (PBZ alone). Little protection against inactivation by 300 nM PBZ was observed for all three concentrations of rauwolscine used. Control responses (, n = 24), vehicle (0.1% EtOH)/time controls (, n = 3), PBZ (300 nM) alone (, n = 3), 300 nM rauwolscine given before 300 nM pBZ (, n = 5), 100 nM given before 300 nM PBZ (, n = 5), and 1000 nM rauwolscine given before 300 nM PBZ (, n = 5). Values are mean ± S.E.M., and n is the number of tissues.

Are the Unusual Adrenoceptors Protected from PBZ Inactivation by Prazosin? Preincubation of PBZ with prazosin at different concentrations was used to evaluate protection of adrenoceptors from PBZ inactivation. Concentration-response curves for controls and vehicle/time controls were very similar (Fig. 4), but E_max was reduced by 60% after receptor inactivation by 300 nM PBZ.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Concentration-response curves to PE were constructed and expressed as a percentage of 100 mM K+ contraction. To determine whether prazosin pretreatment protected against inactivation by PBZ (300 nM), receptors were allowed to be in contact with 10 nM and 30 nM prazosin before 300 nM PBZ was added. Controls (, n = 10) and vehicle (0.1%EtOH)/time controls (, n = 10) were not different from each other. PE responses were significantly improved with the presence of 10 () or 30 nM () prazosin compared with responses in the presence of PBZ 300 nM alone (, n = 5), suggesting partial protection against PBZ inactivation by prazosin. Values are mean ± S.E.M., and n is the number of tissues.

Characterization of Alpha-2 Adrenoceptors Using PBZ against B-HT 920 and UK-14,304. The effects of PBZ on concentration-response curves to B-HT 920 and UK-14,304 were examined. Although as noted earlier, 100 nM PBZ reduced the E_max of PE concentration-response curves by ~50%, it had little or no effect on responses to B-HT 920 and UK-14,304 (compare Figs. 1 and 5). E_max values of B-HT 920 and UK-14,304 were reduced by 50% with 300 nM PBZ concentration (Fig. 5). At 1 µM PBZ, E_max of B-HT 920 was inhibited with a residual response of 5%. E_max of UK-14,304 was reduced to 25% after receptor inactivation by 1 µM PBZ. Lower concentrations of PBZ than 100 nM did not have any significant effect.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Concentration-responses to B-HT 920 (A) and UK-14,304 (B) and the effects of PBZ (3-1000 nM). Pooled controls (, n = 24-28), vehicle/time controls for PBZ (, n = 7 or 8), 3 nM PBZ (, n = 3), 30 nM PBZ (, n = 3), 100 nM PBZ (, n = 3), 300 nM PBZ (, n = 3), and 1000 nM (, n = 3). Emax values for B-HT 920 and UK-14,304 were significantly inhibited at 300 and 1000 nM PBZ. Values are mean ± S.E.M., and n is the number of experiments.

Competitive Inhibition by Rauwolscine. Concentration-response curves to UK-14,304 and B-HT 920 were constructed in the presence of 0, 30, 100, and 300 nM rauwolscine in three independent experiments. Time-parallel control PE concentration-response curves were similar to control curves. However, in the presence of rauwolscine, the curves were shifted to the right in a classic competitive manner. Schild plots estimated slopes not significantly different from unity and yielded a pA₂ of 9.12 and slope of 0.89 for rauwolscine against UK-14,304 (r = .79608, p = .00113) and 8.91 and 0.97, respectively, for rauwolscine against B-HT 920 (r = .75956, p = .00416). These values were comparable with those previously reported: pA₂ of 8.9 for rauwolscine against B-HT 920 (Fowler et al., 1984), 8.1 for yohimbine against B-HT 920 (Eskinder et al., 1988), and 8.6 for rauwolscine against UK-14,304 (Alabaster et al., 1985). Thus, rauwolscine apparently competitively inhibited UK-14,304 and B-HT 920 responses at typical alpha-2 adrenoceptors.

Effects of Rauwolscine Protection against PBZ Alkylation on B-HT 920 and UK-14,304 Responses. The effectiveness of rauwolscine (100 and 300 nM) in protecting B-HT 920 and UK-14,304 responses against PBZ inactivation (1 µM) was examined (Fig. 6). Rauwolscine (100 and 300 nM) offered complete protection of B-HT responses against PBZ inactivation. UK-14,304 responses were protected partially, by ~50% by 100 nM rauwolscine and ~30% by 300 nM rauwolscine. These findings were also consistent with the suggestion that PBZ at higher concentrations than for alpha-1 adrenoceptors inactivated typical alpha-2 adrenoceptors in DSV, activated by B-HT 920 and UK-14,304, and protected by rauwolscine.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Concentration-response curves to UK 14,304 (A) and B-HT 920 (B) were constructed and expressed as a percentage of 100 mM K+ contraction. Receptor protection by rauwolscine from receptor inactivation by PBZ (1 µM) was examined. Vehicle/time controls (, n = 4), 1 µM PBZ alone (, n = 4), 100 nM rauwolscine introduced before 1 µM PBZ (, n = 4), and 300 nM rauwolscine introduced before 1 µM PBZ (, n = 4). Protection of UK-14,304 and B-HT 920 responses from inactivation were observed with both concentrations of rauwolscine used. Values are mean ± S.E.M., and n is the number of experiments.

	Discussion

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The pharmacology of alpha adrenoceptors in the DSV is an interesting but a confusing topic. These receptors do not easily fall into traditional classification schemes (Daniel et al., 1996; see Hicks et al., 1991, for review and comparative data) because alpha-2 as well as alpha-1 antagonists competitively inhibit responses to alpha-1 agonists. Also, CEC inactivates half of [³H]rauwolscine-binding sites under conditions that inactivate nearly all of [³H]prazosin-binding sites. In this study, we observed that PBZ caused concentration-dependent inhibition of PE responses. However, inactivation of increasing fractions of receptors mediating contractile responses to PE by increasing concentrations of PBZ did not unmask any receptors with altered affinity for rauwolscine (i.e., the residual responses showed unchanged K_B values). Also, prazosin, but not rauwolscine, partially protected the PE-sensitive sites from PBZ inactivation. Higher concentrations of PBZ were required for inactivation of B-HT 920 and UK-14,304 responses than for inactivation of PE responses. Rauwolscine, however, protected UK-14,304 and B-HT 920 responses from PBZ inactivation.

There have been previous reports that PBZ can selectively inactivate alpha-1 more potently than alpha-2 adrenoceptors in DSV (Constantine et al., 1982; Flavahan et al., 1986; Ruffolo and Zeid, 1985; Hicks et al., 1991), as confirmed here. CEC inactivated 55% of rauwolscine binding sites and markedly decreased the affinity of PE to interact with rauwolscine binding sites and significantly increased the EC₅₀ for PE-induced contractions (Daniel et al., 1996). Thus, CEC selectively inactivated sites of PE and rauwolscine-binding interactions and, to a lesser degree, reduced the potency of PE to induce contractions, in contrast to PBZ, which did not selectively inactivate receptors at which rauwolscine competed with PE.

Competitive antagonism of B-HT 920, UK-14,304, and PE responses showed pA₂ for rauwolscine typical of the alpha-2 adrenoceptor subtype. The sites activated by PE and functionally antagonized by rauwolscine among the unusual alpha adrenoceptor subtype had PBZ sensitivity similar to typical alpha-1 sites and were more sensitive to PBZ than the typical alpha-2 subtype sites (alpha-2A according to Hicks et al., 1991) responding to B-HT 920 or UK-14,304.

Because PBZ did not selectively inactivate rauwolscine-sensitive PE sites of action and rauwolscine did not protect PE responses from PBZ inactivation, rauwolscine cannot interact at the same site as PBZ alkylation on any alpha-1 adrenoceptor. On the other hand, prazosin did protect partially PE responses against PBZ alkylation. By inference, then, rauwolscine did not compete at the same interaction site for PE as prazosin and PBZ. This is consistent with our observations (Daniel et al., 1996; unpublished observations) that prazosin and rauwolscine competed for each others' binding sites with very low (micromolar) affinity.

Our original study did not exclude the possibility that the sites of rauwolscine interaction with PE were also sites at which alpha-2 adrenoceptor agonists acted. This study shows that the DSV had some typical alpha-2 adrenoceptors that were sensitive to alpha-2 agonists such as B-HT 920 and UK-14,304 and could be competitively antagonized by rauwolscine (pA₂ values of 8.9 and 9.1, respectively; Schild slopes near 1.0). In contrast to its inability to protect PE sites of action from PBZ inactivation, rauwolscine at 100 and 300 nM afforded close to full protection of UK-14,304 and B-HT 920 sites of action against PBZ (1 µM) alkylation. These typical alpha-2 sites are thus likely to be different from the unusual rauwolscine-sensitive PE interaction sites that have a Schild plot slope of 0.52 and an apparent pA₂ of 8.5 (Daniel et al., 1996). These findings show that typical alpha-2 adrenoceptors are present in DSV but that the PE does not use them to produce rauwolscine-sensitive responses.

PBZ (100 nM) inactivated 89% to 98% of PE sites of action (Tables 1 and 2) and markedly reduced the prazosin-sensitive sites of PE antagonism (Table 2) but did not significantly affect B-HT 920 and UK-14,304 concentration-effect curves. The B-HT 920- and UK-14,304-sensitive sites were protected by rauwolscine from inactivation by high concentrations of PBZ. Rauwolscine could also competitively antagonize the effects of these agonists. Such behavior is typical of alpha-2 adrenoceptors and implies not only that alpha-2 adrenoceptors were independent from the rauwolscine-sensitive sites at which PE acted but also that inactivation of PE sites by 100 nM PBZ eliminated most of the unusual alpha-1 adrenoceptors but not alpha-2 adrenoceptors.

These data suggest that the unusual alpha-1 adrenoceptors at which PE or methoxamine produced rauwolscine-sensitive contractions are the same receptors at which prazosin and other alpha-1 adrenoceptor antagonists such as WB 4101 also acted (Daniel et al., 1996). In these earlier experiments, CEC abolished prazosin binding and reduced rauwolscine binding by 55%, caused a persistent, rauwolscine-sensitive contraction (Low et al., 1994), shifted the EC₅₀ for responses to PE and methoxamine, and reduced the affinity of PE competition for [³H]rauwolscine binding (Daniel et al., 1996). We propose that the sites at which CEC activated persistent contraction and partially inactivated rauwolscine binding are the atypical alpha-1 adrenoceptors. The residual rauwolscine-binding sites would then be identified as typical alpha-2 adrenoceptors and sites of rauwolscine antagonism. The reduction in B_max for [³H]rauwolscine binding after CEC was approximately equal to the B_max of [³H]prazosin binding, allowing the possibility that there is one rauwolscine binding site on each receptor. The atypical rauwolscine binding sites apparently have no affinity for B-HT 920 or UK-14,304. The atypical alpha-1 adrenoceptors have PE and PBZ interactions that are competitive with prazosin and noncompetitive (at the same site) with rauwolscine. Rauwolscine, unlike prazosin, fails to protect the unusual sites against PBZ inactivation, and its functional antagonism of PE or methoxamine contractions leads to Schild plot slopes of <1 (Daniel et al., 1996).

Thus, it is likely that there are receptors containing sites of rauwolscine binding that are not alpha-2 adrenoceptors, at which CEC interacts to inhibit rauwolscine binding, and at which PE initiates contractions. They are likely to be the same receptors at which CEC also binds to initiate contraction as outlined above, and they may be antagonized by 30 nM PBZ. Observations (Nunes and Guimaraes, 1993; present study) that 30 nM PBZ did not block UK-14,304 responses, as well other studies showing the lack of efficacy of PBZ to affect responses to other alpha-2 adrenoceptor agonists (Ruffolo and Zeid, 1985), provide additional support that the sites at which CEC binds to initiate contraction and prevent rauwolscine binding are not the typical alpha-2 adrenoceptors. Also, the contraction to CEC was much more sensitive to blockade by rauwolscine or yohimbine compared with prazosin (Nunes and Guimaraes, 1993; Low et al., 1994).

The phenomenon of receptor promiscuity has been shown to occur in cell lines when high numbers of alpha-2 adrenoceptors were expressed and shown to couple functionally to both G_s and G_i proteins (Eason et al., 1992; Zhu et al., 1994; Kenakin, 1995). Receptor promiscuity can be considered in DSV because of its extremely high density of [³H]rauwolscine binding sites (exceeding 800 fmol/mg of microsomal protein), where it would be the first such example in naturally occurring blood vessels. Because the K_B for rauwolscine did not change as receptor number decreased in tissues pretreated with increasing concentrations of PBZ (Table 1), we concluded that receptor promiscuity can be excluded as an explanation for the unusual actions of rauwolscine in this blood vessel.

Our studies agreed with the earlier study of Ruffolo and Zeid (1985), which showed that PBZ was less potent to inactivate alpha-1 compared with alpha-2 adrenoceptors. In the earlier study, the authors used cirazoline as a selective alpha-1 adrenoceptor agonist and B-HT 933 as a selective alpha-2 adrenoceptor agonist. They, like ourselves (Shi et al., 1989; Daniel et al.,1996) and others (e.g., Hicks et al., 1991), found that E_max was always less for full alpha-2 compared with alpha-1 adrenoceptor agonists in DSV. However, they failed to find any antagonism of cirazoline contractions by 10 nM rauwolscine. Possibly, cirazoline is more selective at alpha-1 adrenoceptor sites than PE and interacts only with receptors that are typical in not being antagonized by rauwolscine or sensitive to CEC. This remains to be tested, and there are few studies of the relative selectivity of agonists in which binding and/or functional selectivities have been compared. Ruffolo and Zeid (1985) also found evidence that spare receptors existed for the alpha-2 agonist, B-HT 933, as for cirazoline. Our goals were not focused on spare receptors, but we also found evidence of spare receptors for PE. Our data did not exclude a similar conclusion for B-HT 920 and UK-14,304 because mean K_A values were usually larger than mean EC₅₀ values, but the variation in EC₅₀ and K_A values was too great to confirm the presence of spare receptors at alpha-2 adrenoceptors.

Knowledge of the subtypes of alpha adrenoceptors in DSV is important because it shows close similarities to human saphenous vein (Beckeringh et al., 1987; Eskinder et al., 1988), which acts as a conduit in human coronary artery bypass graft surgery. This study shows that PE acts on receptors represented by those prazosin binding sites previously shown to have some characteristics of alpha-1D adrenoceptors (Daniel et al., 1996) but are atypical in their interactions with rauwolscine and CEC. In addition, the present study confirms that typical alpha-2 adrenoceptors are present. The unusual alpha-1 adrenoceptor subtype, although it binds rauwolscine like the typical alpha-2 adrenoceptor also present, can be clearly distinguished from the latter by its greater sensitivity to inactivation by PBZ and the inability of rauwolscine to protect it from inactivation in contrast to the protection of alpha-2 adrenoceptors by this agent. The structural changes in these receptors or changes in their membrane environment making them available to rauwolscine binding and antagonism remain to be determined.