Introduction

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Shi et al. (1989a,b, 1990) showed that receptors of dog mesenteric and saphenous veins (DMVs and DSVs, respectively) had similar K_D (dissociation constant in saturation ligand binding studies) values for and densities of [³H]prazosin (PR) binding sites. However, in dog mesenteric arteries (DMAs), the K_D value for prazosin binding was lower at a similar receptor density. Only DMVs and DSVs, which had higher densities of [³H]rauwolscine binding sites than DMAs, responded by contraction to ₂-adrenoceptor agonists. All three vessels responded to phenylephrine (PE) with similar pD₂ values and similar efficacies. Responses to norepinephrine were effected only through ₁-adrenoceptors in DMAs, but ₂-adrenoceptor activation also contributed to the norepinephrine-induced contraction of DMVs and DSVs. Later, Shimamoto et al. (1992) showed that responses of DMAs to UK 14304, an ₂-adrenoceptor-selective agonist, were promoted by agents that produced threshold contractile stimulation by enhanced Ca²⁺ entry, but ₁-adrenoceptors as well as ₂-adrenoceptors mediated these responses.

However, the question of which of the various subtypes of ₁-adrenoceptors mediate contraction in DMAs remains unanswered. After the subclassification of ₁-adrenoceptors into 1A and 1B subtypes based on greater sensitivity of the latter to inactivation by chloroethylclonidine (CEC; Han et al., 1987a,b), molecular biological studies have defined three subtypes, now known as 1A, 1B, and 1D, all with high affinity for prazosin (Lomasney et al., 1991a,b). The _1B-adrenoceptor defined pharmacologically proved to be similar to the cloned _1b-adrenoreceptor, with a lower affinity for 2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane (WB 4101) and 5-methylurapidil (5-MU) than the _1A-adrenoreceptor, which had high affinity for these antagonists, as reviewed by Ford et al. (1994).

The _1D-adrenoceptor is now recognized to be a distinct subtype (Schwinn and Lomasney, 1992; Perez et al., 1994; see reviews in Ford et al., 1994; Hieble et al., 1995a), distinguished from the _1A-adrenoceptor by a low affinity for 5-MU and a high affinity for recently described agents such as BMY 7378, 8-[2-(1,4-benzodioxan-2-ylmethylamino)ethyl]8-azaspirol[4,5]decane-7,9-dione HCl (MDL 73005EF; Saussy et al., 1994; Goetz et al., 1995), and 7-chloro-2-bromo-3,4,5,6-tetrahydro-4-methylfurol[4,3,2-ef]3-benzapine (SK&F 105854; Hieble et al., 1995b) in cloned and expressed rat and human receptors.

All these subclasses of receptors have high binding affinity (pK_i > 9) for prazosin when expressed in cell lines. Naturally occurring receptors have been found to have lower affinities (pK_B or pK_i < 9) in some tissues (Muramatsu et al., 1990; Ohmura et al., 1992) and have been classified as _1L-adrenoceptors in contrast to the high-affinity types, _1H-adrenoceptors. Receptors with low affinity for prazosin have not been cloned, but some antagonists [e.g., SB216469; 8-3-[4-(2-methoxyphenyl)-1-piperazinyl]propylcarbamoyl)-3-methyl-4-oxo-22-phenyl-4H-1-benzopyran 2HCl (Rec 15/2739)] with high affinity for _1A-adrenoceptors have been reported to distinguish between them and other ₁- (perhaps _1L-) adrenoceptor subtypes (Testa et al., 1996, 1997; Leonardi et al., 1997). Recently, Ford et al. (1997) showed that the _1A-adrenoceptor expressed in CHO-K1 cells demonstrated binding properties [high affinity to prazosin, 5-MU, N-[2-(2-cyclopropyl methoxy phenoxy)ethyl]5-chloro-,-dimethyl-1H-indole-3-ethanamine HCl (RS-17053), Rec 15/2739, WB 4101, and (+)-niguldipine] expected of _1A-adrenoceptors. However, when a functional property, inhibition of production of inositol phosphates, was evaluated, many antagonists gave lower affinity interactions, as expected for _1L-adrenoceptors. Rec 15/2739 showed the same functional as binding affinity for the _1A-adrenoceptor. The authors suggested that the _1L-adrenoceptor was a pleiotropic expression of this receptor.

The goal of this study was to characterize the ₁-adrenoceptor subtypes of DMAs by using functional interactions as well as immunostaining studies. The results can be compared with those from other canine blood vessels that have different -adrenoceptors (Daniel et al., 1996, 1997; Low et al., 1998).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animal and Tissue Preparation. Mongrel dogs of either sex, weighing 10 to 25 kg, were kept under standard conditions in our animal quarters, fasted 24 h before use, and euthanized with an i.v. overdose (100 mg/kg) of pentobarbital. These procedures were approved by the University Animal Care Committee following the guidelines of the Canadian Council on Animal Care. Segments to be used for functional studies were placed in Krebs-Ringer solution (see below for composition).

Functional Studies. Rings of DMAs from the first or, occasionally, the second branch (3 mm wide) were mounted individually in 10-ml organ baths filled with modified Krebs' solution composed of 115.5 mM NaC1, 4.6 mM KC1, 1.16 mM MgSO₄, 1.16 mM NaH₂PO₄, 2.5 mM CaC1₂, 21.9 mM NaHCO₃, and 11.1 mM glucose, pH 7.4; gassed with a 95% O₂/5% CO₂ gas mixture; and kept at 37°C. The endothelium was removed by rubbing with forceps, confirmed by showing that carbachol could not relax a contraction induced by PE (3 µM) or 60 mM KCl. The tissues were subjected to a 3g preload tension, which gave the maximum contractile response, and allowed to equilibrate for 2 h. Rings were subjected to repeated exposures to 100 mM KCl, followed by washing, until contractions were regular. Cumulative dose-response curves were constructed before and 30 min after incubation with increasing concentrations of antagonists (except prazosin incubated for 45 min) were added. The concentration of agonist in the bath was increased approximately 3-fold at each step after the response to the previous dose had plateaued. Data were discarded if a 2-fold shift or more in the EC₅₀ value for PE occurred in concomitant time controls.

Data Analysis for Functional Studies. Data were expressed in terms of the initial responses to 100 mM KCl as 100%. In all experiments, time controls were run, and corrections were made to C-E curves if needed. EC₅₀ values were estimated by fitting each concentration-response curve (logistic function) using MicroCal Origin Software (Northampton, MA). Changes in dose ratios from EC₅₀ values with antagonist concentration were evaluated using ANOVA. In some experiments after CEC pretreatment, subsequent exposure to other antagonists resulted in C-E curves that at very high PE concentrations began to show increased responses after a flex point at the expected plateau level. We used the response level that corresponded to the EC₅₀ response before the antagonist exposure to determine the new EC₅₀ value. K_B (calculated antagonist dissociation constant in functional studies) values (expressed and analyzed as pK_B) were calculated for antagonist effects at each antagonist concentration (Furchgott, 1972). When K_B values increase (pK_B values decrease) significantly with antagonist concentration, Schild plots have slopes of less than 1. We used pK_B values to emphasize the occurrence or nonoccurrence of decreases with antagonist concentration and presented mean values of dose ratios, the dependent variable determining K_B values. In these studies, each n value refers to the mean value in a study of two or more arterial rings from a single animal. For data presentation in the tables, pK_B values were expressed as mean ± S.E.

CEC Pretreatment. CEC pretreatment of de-endothelized arterial rings involved exposure for 30 min to several concentrations of CEC (0.3-100 µM) at 37°C, followed by washing for several exchanges of bath fluid. Then, C-E curves to PE were constructed. Finally, the shifts on C-E curves of endothelium-free arterial rings to BMY 7378 or to RS-17053 before and, in other rings, after exposure to 100 µM CEC for 30 min were determined.

Immunocytochemical Studies. Four healthy dogs of either sex were euthanized, and blood vessels were collected from aorta and mesenteric arteries as described above. Blood vessels were opened, rinsed free of blood, pinned out on Sylgard silicon rubber-coated dishes, and fixed with 4% paraformaldehyde with 0.1 M phosphate buffer, pH 7.4. The tissues to be used for cryostat sectioning were cut into small pieces and then stored in 15% sucrose containing PBS for cryoprotection at 4°C for 24 h and sectioned into 15-µM-thick slices in a cryostat (Leitz 1720 digital, Wetzlar, Germany). The sections were collected on the slides coated with gelatin. Cryostat sections were incubated overnight at 4°C in 1:300 dilutions of rabbit anti-sera raised against residues 506 to 515 at the carboxyl terminus of the hamster _1B-adrenoceptor, which had been coupled to keyhole limpet hemocyanin (Fonseca et al., 1995). The antibody was visualized with CY3-labeled goat anti-rabbit goat anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). Specificity of staining was determined using preimmune serum and by saturation of the antibody with the peptide epitope during exposure of cryostat sections against which it was raised at 5 µg/ml. After washing with PBS, the sections were mounted in 80% glycerol in PBS (pH 10) and viewed on a Leitz microscope equipped with fluorescence epiluminator and I₂ filter. Kodak T-MAX 400 film was used for black-and-white photography.

Drugs and Chemicals. BMY 7378 was purchased from Research Biochemicals Inc. (Natick, MA). RS-17053, MDL 73005EF, and 8-[4-(1,4-benzodioxan-2-ylmethylamino)butyl]8-azaspirol[4,5]decane-7,9-dione HCl (MDL 72832) were purchased from Tocris Cookson Chemicals (Bristol, UK). Prazosin was a gift from Pfizer Canada Inc. (Kirkland, Quebec, Canada). SK&F 105854 was a gift from Dr. J. P. Hieble (SmithKline Beecham, King of Prussia, PA). Rec 15/2739 was a gift from Dr. A. Leonardi (Recordati, S.p.a., Milan, Italy). Other chemicals, all of analytical grade, were purchased from Sigma Chemical Co. (St. Louis, MO) or Research Biochemicals Inc. except for Tris, which was purchased from Boehringer Mannheim Co. (Indianapolis, IN), and dimethyl sulfoxide (DMSO; BDH Inc., Toronto, Canada). Drug solutions were prepared in deionized water or DMSO (for prazosin, RS-17053, Rec 15/2739, and 5-MU). In all cases, the final DMSO concentration was no more than 0.1%, and time controls received the diluent solutions.

Statistical Analysis. Unpaired or paired (as appropriate) Student's t tests were used, and significance of difference was accepted at P < .05. Changes in antagonist pK_B values with concentration were calculated and subjected to ANOVA with Bonferroni's correction (Version 5; GraphPAD, San Diego, CA).

	Results

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Functional Studies

Effects of Selective ₁-Adrenoceptor Antagonists: Nonselective or _1D-Selective. PE was used as a ₁-adrenoceptor agonist; it is poorly selective among subtypes of this receptor and is not a substrate for reuptake up by sympathetic nerves. The EC₅₀ values varied only slightly in different experimental series [e.g., 2.6 ± 0.6 × 10⁶ M (n = 7) versus 1.2 ± 0.2 × 10⁶ (n = 7)].

View this table: [in this window] [in a new window]   TABLE 1 pKB values for non- or 1D-selective adrenoceptor antagonists against PE-induced contractions of DMA

View this table: [in this window] [in a new window]   TABLE 2 pKB values for 1A or 1L antagonists against PE-induced contractions of DMA

7

7

6

5

7

7

6

6

_1A-Adrenoceptor Selective Antagonists. 5-MU was a potent antagonist (Table 2), with a pK_B value of 9.12 at 3 nM. Values of pK_B decreased slightly but significantly to 8.64 at both the concentrations of 30 and 300 nM. The dose ratios increased significantly from 5.23 ± 0.81 at 3 nM to 14.84 ± 2.41 and 176.48 ± 81.31 at 30 and 300 nM, respectively. All of these pK_B values were within the range expected for _1A-adrenoceptors (Hieble et al., 1995). Because we found no precedent in the published literature for changes in pK_B values with increasing 5-MU concentrations, we repeated these studies 3 years later and obtained similar results (Table 2 and Fig. 1). The dose ratios were also similar (no significant differences from the previous study), increasing from 4.57 ± 0.60 at 3 nM to 13.82 ± 2.41 and 74.67 ± 26.22 at 30 and 300 nM, respectively.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Effects of increasing 5-MU concentrations on responses to PR from second studies (see Table 2). After control responses to cumulatively added concentrations of PE and subsequent washout, 5-MU was added in different concentrations (3, 30, or 300 nM) in different strips 30 min before a repetition of the C-E curves. Time controls were carried out simultaneously in each experiment. The n value was 4 (four dogs with results from two strips at each concentration of 5-MU averaged).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Effects of increasing RS-17053 (RS) concentrations on responses to PE. After control responses to cumulatively added concentrations of PE and subsequent washout, RS-17053 was added in different concentrations (0.3, 3, 30, or 300 nM) in different strips 30 min before a repetition of the C-E curves. Time controls were carried out simultaneously in each experiment. See Table 2.

_1L/A-Adrenoceptor Selective Antagonist. Rec 15/2739, a compound highly selective for _1A-adrenoceptors and for the putative _1L-adrenoceptors (Testa et al., 1996, 1997; Leonardi et al., 1997), had pK_B values of 9.63, 9.63, and 9.66 at 3, 10, and 30 nM (Fig. 3). Thus, over a 10-fold antagonist concentration range and a concentration ratio of ~200, this antagonist recognized a homogeneous group of receptors (i.e., most receptors present, including _1A-adrenoceptors, had the same functional affinity for this compound).

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Effects of increasing Rec 15/2739 concentrations on responses to PE. After control responses to cumulatively added concentrations of PE and subsequent washout, Rec 15/2739 was added in different concentrations (3, 10, or 30 nM) in different strips 30 min before a repetition of the C-E curves. Time controls were carried out simultaneously in each experiment. See Table 2.

Effects of CEC. The effects of increasing concentrations of CEC on responses of DMAs to PE are summarized in Fig. 4. Note that the effects of CEC are small until 10 µM was applied and that even after 100 µM, there were large residual responses to PE. The vessels were pretreated with 100 µM CEC to reduce or eliminate any contribution from _1D- or _1B-adrenoceptors, and the antagonism at residual receptors by RS-17053 (1A selective) or BMY 7378 (_1D selective) was reexamined. If the CEC-sensitive receptors were _1D-adrenoceptors, the pK_B values for BMY 7378 should be reduced to those expected from its interaction with _1A-adrenoceptors, whereas those for RS-17053 should be enhanced to those expected for _1A-adrenoceptors.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effects of increasing concentrations of CEC on responses to PE. After 30-min exposure to increasing concentrations of CEC (0.3, 1, 3, 10, and 100 µM), concentration-response curves to PE were obtained and compared with control values. Only concentrations of CEC greater than 3 µM significantly affected responses. After 10 µM, the concentration-response curve was shifted rightward, and the maximum response was decreased. After exposure to 100 µM CEC, the concentration-response curve was also shifted rightward. The maximum response was not determined because at 1 mM PE, the responses had not plateaued. Responses are normalized in relation to the stable responses to 100 mM KCl. The n values (determined as in Fig. 1) were 38 for controls, 5 for 0.3 µM, 7 for 1 µM, 6 for 3 µM, 6 for 10 µM, and 6 for 100 µM.

Effects of PBZ Inactivation of ₁-Adrenoceptors on Responses to Selective Antagonists

The effects of various concentrations of PBZ on responses to PE are shown in Fig. 5. Note that 6 nM PBZ for 30 min reduced E_max (maximum response to PE) values by more than 60% and shifted the response curve rightward by ~2 log units. -Adrenoceptors on DMAs were much more sensitive to PBZ inactivation than were those of DSV (Low et al., 1998b) or dog aorta. In DSVs, 100 nM PBZ was required to comparably shift the PE concentration-response curve and reduce E_max. In five experiments in dog aorta, 100, 1000, and 10,000 nM PBZ reduced the E_max value to 78.9 ± 9.8, 49.9 ± 8.9, and 20.5 ± 7.1%, respectively, of control E_max and shifted EC₅₀ values from 4.1 ± 0.5 to 10.1 ± 2.6, 13.7 ± 4.1, and 69.2 ± 15.3 µM, respectively. Lower concentrations had no significant effects.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Effects of increasing concentrations of PBZ on concentration-response curves to PE. As little as 1 nM PBZ shifted the C-E to PE rightward and reduced maximal responses. At 10 nM, PBZ reduced the maximum response by more than 50%, and 100 nM PBZ abolished responses. From these findings, we chose 6 nM PBZ for studies of receptor protection (see Fig. 6) to achieve ~50% inactivation of maximum responses.

As shown in Fig. 6, BMY 7378 (300 nM), and Rec 15/2739 (10 nM) were tested as to the effects of each alone and with PBZ (receptor protection). BMY 7378 had no significant antagonistic effect by itself after washout and failed to affect the location of the concentration-response curve after PBZ. It did restore the E_max level from ~40 to ~60% of control. In contrast, Rec 15/2739 alone after washout shifted the concentration-response curve ~6-fold (apparent pK_B = 8.7). Moreover, it restored the concentration-response curve after PBZ to the value with Rec 15/2739 alone. In control experiments, we found that Rec 15/2739 alone did not wash out over the experimental time period. We interpret these experiments to suggest that BMY 7378 protects only a small subset of ₁-adrenoceptors, responding to high concentrations of PE, from PBZ inactivation, whereas Rec 15/2739 protects nearly all receptors.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Ability of Rec 15/2739 but not RS-17053 to protect receptors against inactivation by 6 nM PBZ. Top, Rec 15/2739 (REC; 10 nM) even after washout shifted the control C-E curve () for to PE rightward ~6-fold (). PBZ alone shifted the curve rightward and decreased the maximum responses to ~40% of control (). When PBZ and Rec 15/2739 were present together, the final C-E curve () resembled that for Rec 15/2739 alone. Bottom, BMY 7378 (BMY; 300 nM) alone had no effect after washout (, control; , after BMY 7378) but when present with PBZ () increased the maximum response to PE from 40 (with PBZ alone, ) to ~60 to 65% of control.

Immunochemistry for _1B-Adrenoceptors in DMAs and Aorta

Figure 7 shows the staining of aorta as a positive control for recognition of _1B-adrenoceptors. Canine aorta appears to contain predominantly _1B-adrenoceptors based on functional and ligand binding studies (Hoo et al., 1994; Leonardi et al., 1997; Low et al., 1998). Aortic cells were stained in particulate fashion (Fig. 6A), and the preimmune serum failed to stain these cells (Fig. 6B). Saturation of the antibody with the peptide epitope also abolished staining (Fig. 6C). In contrast, DMAs did not stain with the antibody to _1B-adrenoceptors (Fig. 6D).

View larger version (145K): [in this window] [in a new window]   Fig. 7.   Immunoreactivities of canine aorta and mesenteric artery to antibody against 1B-adrenoceptors. A, cryostat section of canine aorta exposed to antibody against 1B-adrenoceptors. Note particulate nature of staining where cells cut tangentially. B, similar section exposed to preimmune serum form rabbit used to make antibody in A. C, similar section as a except that antibody was preabsorbed with 5 µg/ml concentration of the peptide epitope used to raise the antibody. D, cryostat section of mesenteric artery exposed to antibody under same conditions as in a. Length bars, 12.5 µM in A to C and 25 µM in D.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we used several approaches to identify the ₁-adrenoceptors that mediate PE-induced contractions of DMAs in vitro. We quantified antagonisms by various selective and nonselective antagonists: prazosin was expected to have high and nonselective affinity at all subtypes of cloned receptors, WB 4101 had a higher affinity for _1A- and _1D-adrenoceptors; 5-MU, MDL 72832, RS-17053, and Rec 15/2739 had a higher affinity for _1A-adrenoceptors, but Rec 15/2739 possibly had a special high affinity for _1L-adrenoceptors, presently defined only by their low affinity for prazosin (Hieble et al., 1995a; Testa et al., 1996, 1997; Leonardi et al., 1997). BMY 7378, MDL 73005EF, and SK&F 105854 have higher affinity for _1D-adrenoceptors (Hieble et al., 1995a; Saussy et al., 1996).

We used CEC, expecting it to inactivate _1B-adrenoceptors nearly completely and _1D-adrenoceptors mostly, whereas sparing most _1A-adrenoceptors. Thus, we expected pretreatment with CEC to eliminate or reduce any effects of _1B- or _1D-adrenoceptors. However, it might inhibit agonist action at _1A-adrenoceptors without receptor inactivation (Michel et al., 1993). In the human prostate, which may contain _1L- or _1A-adrenoceptors, 50 µM CEC for 30 min had little effect compared with vehicle control (Testa et al., 1996).

We also used receptor protection by selective antagonists of PBZ inactivation of _1A-adrenoceptors because we have found that both _1B- and _1D-adrenoceptors are relatively resistant to this agent (Low et al., 1998, 1999; current study).

Our initial main findings were that at low agonist concentrations, all antagonists behaved as if they interacted with _1A-adrenoceptors, but some of them also appeared to interact at higher antagonist concentrations with another receptor, likely unidentifiable because of interference from the most sensitive pathway.

This last receptor subtype was identified by Muramatsu et al. (1990) after finding that ₁-adrenoceptors included those with a varied affinities for prazosin (1H, 1L, and 1N, in which 1H has a pK_D value of 9 and 1L and 1N have a pK_D value of 8 but 9). Muramatsu et al. (1990, 1992) and Ohmura et al. (1992) reported that WB 4101 and 5-MU did not distinguish _1N- and _1L-adrenoceptors and that the _1A-, _1B-, and _1D-adrenoceptors were all members of the _1H-adrenoceptor class. Bevan et al. (1989) reported that functional ₁-adrenoceptors have varied affinities for prazosin in different blood vessels. In our study, receptors with both high and low affinity for prazosin seem to function in DMAs. However, high-affinity receptors are not _1B-adrenoceptors.

Evidence Excluding Participation of _1B-Adrenoceptors in Contraction of DMAs. Piascik et al. (1997) reported that the _1B-adrenoceptor and its mRNA were present and that the receptors contributed to the contractile responses of rat mesenteric arteries. Zhu et al. (1999) reported that contractile responses of rat mesenteric arteries were mediated primarily by _1A-adrenoceptors, suggesting that the situation is similar to that in DMAs. Multiple evidence rules out _1B-adrenoceptors playing a major functional role in canine DMAs. First, the high potency of WB 4101 to inhibit PE responses with subnanomolar K_B values over the concentration range of 3 to 300 nM is inconsistent with interactions with _1B-adrenoceptors (Schwinn and Lomasney, 1992). Second, immunocytochemical studies with an antibody to _1B-adrenoceptors stained cells of canine aorta but not DMA. Aorta appeared to contain predominantly _1B-adrenoceptors but not _1A-adrenoceptors (Hoo et al., 1994; Leonardi et al., 1997; Low et al., 1998) and immunostained strongly with the antibody to that receptor, whereas the DMA did not contain antigens that recognized this antibody. Third, in DMAs, CEC was less potent than canine aorta, in which 10 µM CEC reduced the maximum response to PE to 11% of the control value and 100 µM abolished all responses (Low et al., 1998). At 10 µM, CEC reduced the maximum response of DMA rings to 73% of control, and in 100 µM, the maximal response to PE was 53% of control and had not reached a plateau. Fourth, PBZ was much more potent to inactivate receptors in DMAs compared with canine aorta. We conclude that DMA has few or no functional _1B-adrenoceptors and that the persistent responses after CEC likely reflect effects on _1A-adrenoceptors (see below). In the absence of a tool to selectively and irreversibly eliminate _1A-adrenoceptors, we could not characterize residual adrenoceptors after CEC in DMAs.

Identification of ₁-Adrenoceptor Subtypes in DMAs. Most of the prazosin interaction sites in DMAs had pK_D or pK_B values for prazosin and other antagonists near the values expected from binding studies of cloned human and rat _1A-adrenoceptors (Hieble et al., 1995). DMA had a K_D value of 0.8 nM for prazosin receptors in earlier studies (Shi et al., 1989a). To evaluate functional receptors, we discounted K_B values derived from concentration ratios of less than 2-fold more than for time controls as subject to large errors (e.g., values for 3 and 30 nM MDL 73005EF and 0.3 or 3 nM RS-17053). The remaining values for functional receptors also corresponded to _1A-adrenoceptors in their high-affinity interactions with prazosin, WB 4101, MDL 72832, RS-17053, Rec 15/2739, and 5-MU and their resistance to inactivation by CEC. However, at higher agonist concentrations, lower-affinity interactions occurred with prazosin and possibly with 5-MU but not with WB 4101 or Rec 15/2739.

Possible Contributions of _1D- or _1L-Adrenoceptors to DMA Contractions. If _1D-adrenoceptors are present in DMAs along with _1A-adrenoceptors, removing or minimizing their contribution of non_1A-adrenoceptor with CEC should leave classic _1A-adrenoceptors. As shown in Table 3, elimination of CEC-sensitive receptors did not reduce the pK_B value of BMY 7378 to levels expected for _1A-adrenoceptors, nor was there a major effect to increase the pK_B of higher concentrations of RS-17053. Thus, the CEC-resistant and CEC-sensitive receptors behaved similarly to antagonists that were selective for both the 1D and 1A subtypes. The effects of CEC to shift C-E curves to PE for DMAs might result from noncompetitive antagonism of the _1A-adrenoceptors rather than inactivation of ₁-adrenoceptors. These results are inconsistent with a model in which there are two functional receptors: the _1A-adrenoceptor and the _1D-adrenoceptor. A model in which all receptors recognize the same receptor population before and after CEC was suggested. Thus, either CEC has no selectivity for _1D-adrenoceptors over _1A-adrenoceptors in DMA, or the receptors are all the same and the differences in pK_B values at higher concentrations of some antagonists result in nonclassic, possibly pleiotropic behavior of the receptor. However, the presence of another receptor, the _1L-adrenoceptor subtype, has not been excluded by these data.

Both _1A- and _1L-Adrenoceptors in DMAs? The occurrence of blood vessels with relatively low affinity for prazosin at high concentrations of agonist but many pharmacological characteristics of the 1A subtype is not confined to canine blood vessels. Recently, van der Graaf et al. (1996) reported a similar result for rat mesenteric arteries. They suggested that the pharmacologically defined 1L subtype operated in that resistance vessel. Testa et al. (1996) suggested that human mesenteric artery contained either exclusively _1A- or _1L-adrenoceptors. Lachnit et al. (1997) suggested that rat caudal artery contained at least two subtypes of ₁-adrenoceptors: mostly _1A-adrenoceptors and another of lower affinity to RS-17053. If the findings of Ford et al. (1997) with recombinant _1A-adrenoceptors apply to resistance blood vessels, it is possible that those in DMA have _1A-adrenoceptors in an environment in which their functional behavior at high agonist levels corresponds to the _1L-adrenoceptors.

Conclusions and Future Perspectives. The results of this study suggest that the main functional adrenoceptor subtype in DMA are _1A-adrenoceptors and that DMA lacks functional _1B- or _1D-adrenoceptors. Low-affinity interactions of these receptors may reflect the functional behavior of the _1A-adrenoceptor at high agonist concentrations.