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Vol. 295, Issue 2, 627-633, November 2000


An alpha 1A/alpha 1L-Adrenoceptor Mediates Contraction of Canine Subcutaneous Resistance Arteries1

Sally Anne Argyle and John Christie McGrath

Autonomic Physiology Unit, Division of Neuroscience and Biomedical Systems, Institute of Biomedical & Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom

    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

To determine the characteristics of the alpha 1-adrenoceptor subtypes involved in adrenergic regulation of peripheral vascular resistance, contraction of canine subcutaneous resistance arteries was studied using wire myographs. The potencies of agonists and antagonists, chosen for their ability to discriminate between alpha 1-adrenoceptor subtypes, were assessed in the presence of cocaine (3 µM), corticosterone (30 µM), and propranolol (1 µM). The rank order of agonist potency (pEC50 ± S.E.) was (R)-A-61603 (7.88 ± 0.1) > norepinephrine (6.41 ± 0.1) > phenylephrine (5.83 ± 0.1). The high sensitivity to (R)-A-61603 relative to phenylephrine is inconsistent with the presence of the alpha 1D-adrenoceptor and most consistent with an alpha 1A-adrenoceptor response. This is supported by the low affinity for the alpha 1D-selective antagonist BMY 7378 (pKB 6.51 ± 0.47). The low pA2 values for prazosin (8.36) and HV723 (8.81), by definition, indicate the involvement of the putative alpha 1L-adrenoceptor, a hypothesis supported by the pA2 values for WB4101 (8.42) and 5-methyl-urapidil (8.08). Pre-exposure to 1 µM CEC had little effect, whereas 100 µM CEC reduced the maximum contraction but not the sensitivity to norepinephrine. This low sensitivity to CEC argues against the presence of the alpha 1B-adrenoceptor. We conclude that, by current definitions, an alpha 1A-/alpha 1L-adrenoceptor causes contraction of these vessels. This does not support the concept that selectivity for the alpha 1A-adrenoceptor is the basis for the effectiveness of some alpha -blockers in some tissues, such as prostate, but not in other tissues such as blood vessels. Rather, the generally low potency of alpha -blockers in some tissues may be due to a tissue-specific property of the receptors.

    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The objective of this study was to determine the alpha 1-adrenoceptor subtypes involved in adrenergic regulation of peripheral vascular resistance in the dog. This may provide an explanation for the ability of some alpha -blockers to be efficacious in some organs while preserving other functions such as blood pressure. It is now known that there are three alpha 1-adrenoceptor phenotypes, alpha 1A, alpha 1B, and alpha 1D, which correspond to and are encoded by the three cloned alpha 1-adrenoceptors denoted by lowercase letters, alpha 1a, alpha 1b, and alpha 1d, in accordance with the adrenoceptor nomenclature committee of the International Union of Pharmacology (Hieble et al., 1995).

The pattern of functional expression of these subtypes in the vascular system is unresolved. It has been argued that the role of alpha 1A-adrenoceptors may be greater in resistance than in conduit arteries (Kong et al., 1994). Early work, published before the alpha 1-adrenoceptor nomenclature was standardized, frequently refers to alpha 1b- or alpha 1B-adrenoceptors in blood vessels, particularly aorta of several species, based on susceptibility of these tissues to chloroethylclonidine. However, using the standardized nomenclature, these examples would be redesignated as alpha 1D-adrenoceptors (Kenny et al., 1995). Thus it is possible to argue that most resistance arteries utilize alpha 1A-adrenoceptors, that some conduit arteries utilize alpha 1D-adrenoceptors, and that there are no noncontroversial examples of alpha 1B-adrenoceptors mediating arterial contraction. However, some relatively selective alpha 1A-adrenoceptor blockers, are used to treat heart failure and benign prostatic hyperplasia, partly on the basis that they do not block adrenergic control of resistance vessels and thus do not affect blood pressure (McGrath et al., 1996). This implies that alpha 1A-adrenoceptors are not critical to vascular control.

The three cloned alpha 1-adrenoceptor subtypes are characterized by a high affinity for prazosin in both functional and radioligand binding experiments, whereas, in functional experiments, alpha 1-adrenoceptors with a relatively low affinity for prazosin (pA2 < 9) have been found. These have been termed alpha 1L-adrenoceptors, as opposed to alpha 1H-adrenoceptors for those with a high affinity for prazosin (Holck et al., 1983; Drew, 1985; Flavahan and Vanhoutte, 1986). This alpha 1L-adrenoceptor phenotype has been further classified as either an alpha 1L-adrenoceptor or alpha 1N-adrenoceptor, based on either a low or a high affinity for HV723, respectively (Flavahan and Vanhoutte, 1986; Muramatsu et al., 1990). A separate gene encoding for this low-affinity receptor has not been identified, but there is now evidence to support the idea that the alpha 1L-adrenoceptor may be a phenotype of the cloned alpha 1a-adrenoceptor. When all four isoforms of the human alpha 1a-adrenoceptor are expressed in cell lines, radioligand binding studies have revealed a profile typical of the alpha 1A-adrenoceptor. However, functional experiments, measuring inositol phosphate accumulation, have shown that the affinities of these receptor isoforms are all lower for prazosin, RS 17053, WB4101 , and 5-methyl-urapidil, giving a profile more typical of the alpha 1L-adrenoceptor (Ford et al., 1997; Chang et al., 1998).

The few published functional studies of alpha 1-adrenoceptors in resistance arteries have failed to demonstrate that a particular alpha 1-adrenoceptor subtype is of primary importance in the adrenergic control of these vessels. Smith and McGrath (1996) concluded that the alpha 1-adrenoceptors in the rat mesenteric resistance artery were consistent with the presence of the alpha 1A-/alpha 1L-adrenoceptor or alpha 1B-adrenoceptor subtypes or a mixture of these, whereas Van der Graaf et al. (1996) concluded that the alpha 1L-adrenoceptor subtype was present in this tissue.

In rabbit cutaneous resistance arteries, Smith et al. (1997) proposed that both the alpha 1L-adrenoceptor and the alpha 1B-adrenoceptor subtypes were present on the basis of antagonist potency, although agonists were consistent with the alpha 1A-adrenoceptor.

The dog has provided some of the lowest pA2 values for prazosin, providing compelling arguments for the existence of the alpha 1L-adrenoceptor phenotype (Muramatsu et al., 1995; Flavahan et al., 1998). However, these studies focused on larger vessels, with no information currently available for resistance vessels, which are arguably the most important for adrenergic hemodynamic regulation. In addition, the dog has been a prominent model for the testing of drugs used in the cardiovascular system and urinary tract, highlighting the need for information concerning control of the peripheral vasculature in this species.

We characterized the alpha 1-adrenoceptor causing contraction of canine subcutaneous resistance arteries. Sensitivities to the agonists norepinephrine, phenylephrine, UK14304, and (R)-A-61603 were studied. In addition, the potencies of five reversible antagonists and the irreversible antagonist CEC were examined. The competitive antagonists used were the nonsubtype-selective antagonist prazosin, the alpha 1A-selective ligand 5-methyl-urapidil (Hanft and Gross, 1989), the alpha 1D-selective ligand BMY 7378 (Goetz et al., 1995), the alpha 1A/D-selective ligand WB4101 (Morrow and Creese, 1986), and HV723, which has a higher affinity for the alpha 1N-adrenoceptor subtype compared with prazosin, as opposed to the alpha 1L-adrenoceptor subtype where both antagonists have a low affinity (Muramatsu, 1991).

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Vessel Collection and Preparation. Dogs were euthanized at the local dog and cat shelter using pentobarbitone sodium (Euthatal, 200 mg/ml; Merial, Essex, UK) at a dose of 150 mg kg-1 of body weight, administered by intravenous injection. Dogs of any breeds, ages, sexes, and weights were used. Immediately after euthanasia, a patch of skin overlying the gluteal musculature was dissected free and placed in ice-cold Krebs' solution of the following composition: 112 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 2 mM CaCl2, 25 mM NaHCO3, 1.2 mM NaHPO4, and 11.5 mM glucose. Na2EDTA (0.023 mM) was also included in the Krebs' solution at all times to prevent degradative oxidation of NE.

Resistance-sized arteries were removed with the aid of a dissecting microscope and were used within 24 h of removal. At all times, vessels were stored at 4°C in Krebs' solution. To standardize protocols, all agonist studies were performed on vessels collected on the same day, and all antagonist studies were performed on vessels stored overnight (they would have been harvested, cleaned, and dissected 18 h previously).

Mounting. Experiments were carried out using a four-chamber Mulvany-Halpern wire myograph (Danish Myo Technology, Aarhus, Denmark) (Mulvany and Halpern, 1976).

Resistance arteries [255 ± 7 µm in diameter (n = 89)] were cut into approximately 2-mm lengths and mounted between two 40-µm wires. One wire was attached to a fixed head while the other was attached to a moveable head, connected to a force transducer. The force transducer was connected to a Linseis (TYP 2066; Linseis, Selb, Germany) pen recorder to allow recordings of isometric force. Tissues were maintained at 37°C and continually gassed with 95%O2, 5%CO2 in Krebs' solution. Cocaine (3 µM) (Aboud et al., 1993), corticosterone (30 µM) (Blue et al., 1995), and propranolol (1 µM) (Forster, 1996) were added to block neuronal uptake, non-neuronal uptake, and beta -adrenoceptors, respectively. These drugs were present in the Krebs' solution during all experiments.

Normalization Procedure. After a 30-min rest period, the resistance arteries were stretched at 1-min intervals to determine the exponential passive wall tension/internal circumference (L) relationship, as previously described (Mulvany and Halpern, 1977; Smith et al., 1997). This allowed the calculation of the circumference at 0.9× L100, where L100 is the circumference of the relaxed vessel if it were exposed to a transmural pressure of 100 mm Hg. Normalized vessel internal diameter was then set at 0.9× L100 for the remainder of the experiment. From the known length-tension relationship, it was then possible to calculate the wall tension and the active effective pressure produced by the vessel throughout the course of the experiment.

Thirty minutes after normalization, arteries were exposed to 10 µM NE until maximal contraction was reached. Vessels were then washed until baseline was achieved. Twenty minutes later, vessels were exposed to 125 mM potassium chloride solution (composition: 117.9 mM KCl, 1.2 mM MgCl2, 2 mM CaCl2, 25 mM NaHCO3, 1.2 mM NaHPO4, and 11.5 mM glucose) until a plateau contraction was achieved and again washed until they returned to baseline. Exposure to the 125 mM potassium chloride solution was repeated 10 min later and, following washing, vessels were allowed a 30-min period of recovery before commencement of the following protocols. This starting procedure was found to reduce the variability in subsequent responses (data not shown).

Agonist Study. Three consecutive CCRCs were performed to the following agonists using half-log increments: NE (1 nM-1 mM) followed by PE (1 nM-1 mM) and then followed by (R)-A-61603 (3 nM-30 µM) or UK14304 (1 nM-0.1 µM). This agonist sequence was kept constant for all experiments. Vessels were washed back to baseline, and a 40-min rest period was allowed between curves.

Antagonist Study. Four vessels were set up in parallel. One was assigned as a time control, and each other ring was assigned one of the five competitive antagonists chosen to distinguish between alpha 1-adrenoceptor subtypes.

In the agonist study, although there were small and inconsistent responses to UK14304, there was still the possibility that NE might be exerting part of its action via an alpha 2-adrenoceptor. To eliminate this possibility, when antagonists were tested versus NE, the alpha 2-adrenoceptor antagonist RS-15385-198 (0.1 µM) (Brown et al., 1993) was present in the Krebs' solution.

An initial CCRC to NE was performed in all rings. Subsequent to this, a concentration of antagonist was added to each ring with the exception of the time control. After a 40-min incubation period, a second CCRC to NE was performed. This procedure was repeated with increasing concentrations of the designated antagonist (three concentrations always starting with the lowest antagonist concentration and ending with the highest antagonist concentration). A maximum of four consecutive CCRCs were performed. Experiments were excluded from the study if there was any significant alteration in the maximum or pEC50 values for the time control vessel. In this series of experiments using these criteria, no vessels were excluded. The alpha 1-adrenoceptor antagonists used were prazosin, 5-methyl-urapidil, WB4101, HV723, and BMY 7378.

Chloroethylclonidine. After an initial CCRC to NE, 1 or 100 µM CEC was added to the bath and allowed to incubate for 60 min. This was followed by 40 min of washing with Krebs' solution (10 washes) and a second CCRC to NE (O'Rourke et al., 1995).

Analysis of Data. For the agonist studies, CCRC data were expressed as a percentage of the maximal contraction to the 10 µM concentration of NE. For the antagonist studies, CCRC data were expressed as a percentage of the control curve maximum. Data were analyzed on Microsoft Excel spreadsheets, and pEC50 values were derived from interpolation. The pEC50 was defined as the negative log of the concentration of agonist required to achieve 50% of the maximal response. Data for each concentration of agonist were averaged, and results were plotted as mean ± S.E. for graphical representation of CCRCs.

Schild analysis was performed by plotting log (DR - 1) values for individual vessels against the antagonist concentration (log [B]), where DR is defined as the ratio of the EC50 values in the presence and absence of the antagonist (Arunlakshana and Schild, 1959). Analysis was performed using GraphPad Prism 2.01 (GraphPad Software, Inc., San Diego, CA). Antagonist pA2 values were taken to be the x-intercept of the Schild slope. In addition, pKB values were derived from individual antagonist concentrations using the equation, log (DR - 1) = log [B] + log KB (Arunlakshana and Schild, 1959; Kenakin, 1982; Jenkinson, 1991). This assumes a slope of unity.

Comparisons were made using one-way ANOVA. In all cases a P < .05 was taken as indicating a significant difference. A Bonferroni post test allowing multiple comparisons was used to determine the origin of any significant differences.

In all cases, n = the number of experiments. For each set of experiments, each artery segment used was taken from a different animal unless stated otherwise.

Drugs. The following drugs were used: (R)-A-61603 [N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]-methanesulfonamide hydrobromide; a gift from Dr. A. Hancock, Abbott Laboratories, Chicago, IL]; (-)-noradrenaline bitartrate (Sigma, Dorset, UK); RS-15385-198 [Roche Bioscience (formerly Syntex), Palo Alto, CA; a gift from Dr. R. Whiting]; WB4101 [2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride; Research Biochemicals International, Natick, MA]; HV723 [alpha -ethyl-3,4,5-trimethoxy-alpha -(3-((2-(2-methoxyphenoxy)ethyl)-amino)propyl)benzeneacetonitrile fumarate; a gift from I. Muramatsu, Fukui Medical School, Japan]; 5-methyl-urapidil (Research Biochemicals International); chloroethylclonidine (Research Biochemicals International); prazosin HCl (Pfizer, Sandwich, UK); cocaine HCl (MacCarthy's, Glasgow, Scotland); propranolol HCl (Sigma); corticosterone 2-acetate (Sigma); BMY 7378 [dihydrochloride 8-[2-[4-(2-methoxyphenyl)-1-piperozynl]ethyl]-8-azaspiro[4.5]decone-7,9-dione; Research Biochemicals International]; (-)-phenylephrine HCl (Sigma); UK14304 (Research Biochemicals International).

All drugs were prepared daily from salts and dissolved in deionized water, with the exception of NE, which was dissolved in 0.023 mM Na2EDTA. The concentrations given are the final concentration of the drug in the bath.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Agonists. NE produced concentration-dependent contractions of the subcutaneous resistance artery with a pEC50 of 6.41 ± 0.1 (n = 8). PE and (R)-A-61603 also produced concentration-dependent increases in tension. The pEC50 and maximum values are summarized in Table 1, and the mean CCRC data are illustrated in Fig. 1. The rank order of agonist potency was (R)-A-61603 > NE > PE, with (R)-A-61603 being 27 times more potent than NE and 112 times more potent than PE. Maximal values for the agonists were significantly different (P = .04), and a Bonferroni post test showed that this resulted from a significantly greater maximum response to NE (114.9 ± 2.48%, n = 8) compared with PE (99.82 ± 11.3%, n = 10). The maximal response to (R)-A-61603 was intermediate (106.4 ± 4.52%, n = 6). Thus, PE and (R)-A-61603 are almost full agonists relative to NE.


                              
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  		TABLE 1
Agonist potencies and maximum responses in canine subcutaneous resistance arteries

Potencies and maximal responses for NE, (R)-A-61603, PE, and UK14304 in canine subcutaneous resistance artery. Maximum responses are expressed as a percentage of an initial contraction to 10 µM NE. n represents the number of experiments. The weak and inconsistent response to UK14304 cannot entirely exclude alpha 2-adrenoceptor involvement.


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  		Fig. 1.   CCRC data for NE, PE, (R)-A-61603, and UK14304 in canine subcutaneous resistance arteries. Points on the graph represent mean values, and vertical bars represent S.E. It should be noted that the weak response to UK14304 cannot entirely exclude alpha 2-adrenoceptor involvement.

UK14304 was used only in four animals. In three of these animals, UK14304 caused concentration-dependent contractions with a mean pEC50 value of 7.29 ± 0.22 (n = 3) and a mean maximal contraction of 42.33 ± 11.3% (n = 3). In the fourth animal, UK14304 failed to contract the vessels within the range of agonist concentrations used.

Antagonists. Graphs illustrating the mean CCRC data for the five reversible antagonists are illustrated in Fig. 2 (a-e). Schild regressions are illustrated in Fig. 3 (a-d).


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  		Fig. 2.   Canine subcutaneous resistance artery CCRC to NE, in the presence and absence of the following antagonists: prazosin (a); BMY 7378 (b); HV723 (c); WB4101 (d); 5-methyl-urapidil (5 MeU) (e). Values represent mean values, and error bars represent S.E.


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  		Fig. 3.   Schild plots for the antagonists prazosin (a), HV723 (b), WB4101 (c), and 5-methyl-urapidil (d). Points represent values from individual experiments.

With the exception of BMY 7378, all the competitive antagonists caused concentration-dependent rightward shifts in the NE CCRCs. Maximal values, in the presence of the highest concentrations of antagonists used, were significantly reduced for all antagonists, with the exception of BMY 7378. In time controls, neither maximum nor pEC50 values were significantly altered. The reduced maximal values were most marked for 5-methyl-urapidil (P < .0001) and HV723 (P = .0051). In the case of 5-methyl-urapidil, the response reached a plateau in the presence of 1 µM of the antagonist, whereas in the presence of 0.1 µM HV723, a maximum response was not achieved within the concentration range of NE used. Nevertheless, Schild analysis was performed. Slopes for WB4101 and HV723 were not significantly different from negative unity, whereas those for prazosin and 5-methyl-urapidil were. Schild slopes and pA2 values are summarized in Table 2. When pKB values were calculated from individual antagonist concentrations, values were not significantly different over the range of antagonists used when compared using ANOVA. The pKB values are also summarized in Table 2.

                              
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  		TABLE 2
Summary of pA2, slope, and pKB values for the competitive antagonists in dog subcutaneous resistance arteries

In the case of the alpha 1D-selective antagonist BMY 7378, maximum contractions to NE were not significantly different in the presence of the antagonist. However, even the highest concentration of antagonist used (0.1 µM) produced only a small rightward displacement of the CCRC. A pKB value of 6.51 ± 0.47 (n = 4) was calculated for 0.1 µM BMY 7378. The lack of effect of the two lower concentrations of antagonist (1 nM and 10 nM) meant that Schild analysis using data from these concentrations was not appropriate.

The irreversible antagonist CEC, at a concentration of 1 µM, had no significant effect on the CRC to NE. However, 100 µM CEC caused a significant decrease in the maximum NE contraction to 50.8 ± 8% (n = 5) of control. In addition, this concentration of CEC caused a baseline contraction of 4 and 50% of control maximum in vessels from two of the five animals tested. CCRC data for NE in the presence of CEC are illustrated in Fig. 4.


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  		Fig. 4.   CCRCs to NE before and after pre-exposure to 1 and 100 µM CEC. Points represent mean values, and error bars represent S.E.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Agonists

The rank order of agonist potency was (R)-A-61603 > NE > PE. The logic of studying the agonists PE and A61603 is that their potency ratio has been shown to distinguish the alpha 1A- and alpha 1B-adrenoceptor subtypes from the alpha 1D-adrenoceptor. In canine prostate strips (considered to possess the alpha 1A-adrenoceptor), A-61603 was 165-fold more potent than PE and 128-fold more potent than NE. At the alpha 1B-adrenoceptor in rat spleen, A-61603 was only 40-fold more potent than PE. In contrast, at alpha 1D-adrenoceptor sites in rat aorta, A-61603 was less potent than PE (Knepper et al., 1995). In the present study and studies by Smith et al. (1997) and Pediani et al. (1998), the more potent and selective R-enantiomer of A-61603 rather than the racemic mixture was used. Knepper et al. (1995) report a 590-fold greater potency versus PE when using the R-enantiomer of A-61603 in canine prostatic strips. Radioligand binding studies in our laboratory, with the same samples of the compound used in the present study, demonstrate a 141-fold ratio of affinity for the recombinant human alpha 1a-adrenoceptor for (R)-A-61603 over PE (Pediani et al., 1998). On this basis, the high potency of (R)-A-61603 relative to PE in this present study (112 times) indicates an alpha 1a/A-adrenoceptor phenotype.

The weak and inconsistent response to UK14304, although not entirely excluding the involvement of alpha 2-adrenoceptors, suggests that there is not a substantial alpha 2-adrenoceptor contribution in these vessels and is consistent with UK14304's known partial agonism at alpha 1-adrenoceptors (Nagadeh et al., 1994; McGrath et al., 1999).

Antagonists

Contractions to NE were inhibited by prazosin, WB4101, HV723, and 5-methyl-urapidil. All tended to reduce the maximum, although this effect was most marked for HV723 and 5-methyl-urapidil. For prazosin and 5-methyl-urapidil, there was evidence of a noncompetitive interaction evidenced not only by the reduction in maximum but also by Schild slopes that did not encompass negative unity. This could indicate the involvement of more than one receptor subtype. In an alternative analysis, the pKB values calculated from individual antagonist concentrations were not significantly different over the range of antagonist concentrations used, although there was a trend toward a decrease in pKB value with the highest antagonist concentrations tested. This may suggest a heterogeneous receptor population with one receptor subtype demonstrating a higher affinity for the two antagonists, identified by the pKB value derived from the lowest concentration used, and a lower affinity site identified by the higher antagonist concentrations. With this proviso, the relative antagonist potencies indicate the presence of the alpha 1A-adrenoceptor. Absolute values for antagonist affinities were low (see discussion of alpha 1L-adrenoceptors below).

Since BMY 7378 has a high affinity for alpha 1D-adrenoceptors, we used low concentrations of this antagonist to detect the alpha 1D-adrenoceptor subtype (Goetz et al., 1995). The failure to detect antagonism at any but the highest concentration of BMY 7378 (pKB of 6.51) reflects the low affinity of this antagonist for the alpha 1-adrenoceptors in this blood vessel and suggests that the alpha 1D-adrenoceptor subtype is not present. The pKB value of BMY 7378 in the present study is similar to the pA2 value of 6.3 obtained in rat tail artery (Lachnit et al., 1997), which is considered to possess the alpha 1A-adrenoceptor.

The usefulness of CEC to identify alpha 1-adrenoceptor subtypes is controversial (Zhong and Minneman, 1999), although it is frequently used in alpha -adrenoceptor classification. It is considered to bind to all alpha 1-adrenoceptors and to alkylate and subsequently inactivate these receptors in a subtype-selective manner. CEC produces alpha 1- and alpha 2-adrenoceptor agonism in the dog saphenous vein and rat aorta (Nunes and Guimaraes, 1993). The baseline contraction to CEC in this study is likely to be due to alpha 1-adrenoceptor activation since an alpha 2-adrenoceptor antagonist was present. CEC has the greatest effect at the alpha 1B-adrenoceptor subtype, least effect at the alpha 1A-adrenoceptor subtype, and an intermediate effect at the alpha 1D-adrenoceptor subtype (Michel et al., 1993). Rat splenic strips are the only noncontroversial example of smooth muscle contraction mediated by alpha 1B-adrenoceptors. Here CEC causes shifts in sensitivity to alpha 1-adrenoceptor agonists of 2 to 3 orders of magnitude (Burt et al., 1995). Similar sensitivity shifts occur in rat aorta (Kong et al., 1994), which contains alpha 1D-adrenoreceptors (Kenny et al., 1995). In contrast, at vascular alpha 1A-adrenoreceptors, CEC commonly reduces maximum contraction with little or no sensitivity shifts (Fagura et al., 1997). This latter finding corresponds to the present study, suggesting the presence of alpha 1A-adrenoceptors and adding to the evidence that alpha 1A-adrenoceptors rather than alpha 1B-adrenoceptors are the major mediators of contraction of these resistance arteries.

Based on our findings with BMY 7378 and CEC, we believe that if there is more than one receptor subtype mediating contraction in this blood vessel, the second is unlikely to be either the alpha 1D-adrenoceptor or the alpha 1B-adrenoceptor.

Relationship to the alpha 1L-Adrenoceptor Hypothesis

Agonists. The putative alpha 1L-adrenoceptor is also sensitive to A-61603. Smith et al. (1997) observed a very high sensitivity of A-61603 (944-fold over PE) in rabbit cutaneous resistance arteries, which had a pKB for 10 µM prazosin of 8.6 (defining an alpha 1L-adrenoceptor). The canine prostate, a definitive example of an alpha 1A-adrenoceptor according to Knepper et al. (1995) (A-61603 165-fold over PE) has also been described as possessing alpha 1L-adrenoceptors (Muramatsu et al., 1995). Thus, the high potency of (R)-A-61603 relative to PE in this present study (112 times) is consistent with an alpha 1L-adrenoceptor as well as an alpha 1a/A-adrenoceptor.

Antagonists. Based on the criteria used to define the alpha 1L adrenoceptor of low affinity for prazosin and equivalent affinity of prazosin and HV723 (Muramatsu et al., 1990), the individual pKB values for prazosin in dog cutaneous arteries suggest that the alpha 1L-adrenoceptor subtype is present.

The low affinities of WB4101 and HV723 (pA2 of 8.42 and 8.81, respectively) would also support the presence of the alpha 1L-adrenoceptor and are consistent with the potency of these antagonists at other tissues considered to possess the alpha 1L-adrenoceptor (Table 3).

                              
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  		TABLE 3
Comparison of pA2/pKB values in dog subcutaneous resistance arteries to previously published data

The data described above suggest that the NE-induced contraction of the dog cutaneous resistance arteries is mediated by alpha 1A- or alpha 1L-adrenoceptors. In the present study, as in others, the receptor with low affinity for antagonists (alpha 1L-adrenoceptor) possesses general pharmacological characteristics of the alpha 1A-adrenoceptor with little further difference apart from relatively low antagonist affinity. This is consistent with the hypothesis of Ford et al. (1997) that alpha 1L-adrenoceptor pharmacology may be a phenotype of the alpha 1a-adrenoceptor genotype. We believe that this is the explanation for the present results and for other examples of resistance blood vessels where low-affinity subtypes have been found (Smith and McGrath, 1996; Van der Graaf et al., 1996; Smith et al., 1997).

The affinities of antagonists in dog cutaneous resistance arteries were similar to those of cloned human alpha 1a-adrenoceptors and to examples of alpha 1L-adrenoceptors in tissues from human lower urinary tract in that sensitivity to prazosin was similar to that of WB4101 and pA2 values for these antagonists were between 8 and 9. This contrasts with tissues that contain alpha 1D-adrenoceptors and alpha 1B-adrenoceptors where the pA2 values for prazosin are greater than 9 and the values for prazosin are substantially greater than for WB4101. This puts this dog blood vessel clearly into the alpha 1a-/A-adrenoceptor or alpha 1L-adrenoceptor category. The receptors in these dog blood vessels are, therefore, not substantially different from human comparators. The pA2 value for prazosin of 8.4 is low but still clearly higher than the low-affinity values for prazosin found in other dog blood vessels, for example, 7.9 in dog saphenous vein (Muramatsu et al., 1990). The simplest conclusion is that the dog resistance arteries in this study, like other dog blood vessels, have a low affinity for prazosin, but this is less extreme than in larger canine vessels and is not radically different from human alpha 1A/lL-adrenoceptors.

Tissue-Specificity of alpha -Blockers

Our conclusion that an alpha 1A-/alpha 1L-adrenoceptor causes contraction of dog resistance vessels does not support the concept that selectivity for a particular alpha 1-adrenoceptor subtype is the basis for the effectiveness of alpha -blockers in some tissues, particularly prostate, while preserving blood pressure control, since it is these same receptor subtypes that have been proposed as mediating adrenergic responses in prostate (McGrath et al., 1996). Rather, the variable potency of antagonists may be due to a tissue-specific property of the receptors. Ford et al. (1997) showed that human cloned alpha 1a-adrenoceptor isoforms re-expressed in cell cultures can display alpha 1L-adrenoceptor properties in functional studies and that their affinity measured by radioligand binding was lower when measured in whole cells than in membrane preparations. Mackenzie et al. (2000) have now shown in single human prostate cells, using fluorescent ligand binding, that the affinity of a prazosin analog for native human alpha 1A-adrenoceptors is higher than for human cloned alpha 1a-adrenoceptors expressed in cell cultures. This suggests that tissue-specific affinity states of the same receptor genotype exist and that this, rather than different subtypes, could be the basis for differences in antagonist effectiveness in different tissues.

To our knowledge the sequence for the dog alpha 1a-adrenoceptor has not been published nor have recombinant receptors been studied, so we cannot tell whether there is a structural reason for the generally low affinity for some ligands in this species. At present, we have determined a partial sequence for the canine alpha 1a-adrenoceptor (submitted to the GenBank with accession no. AF068283) and are currently in the process of determining the full sequence of this receptor together with the sequence of the other dog alpha 1-adrenoceptor subtypes. This will enable us to use isolated dog alpha 1-adrenoceptors subtypes to determine whether the dog alpha 1A/L-adrenoceptor is a true receptor subtype, a species homolog, or a tissue-specific affinity state of the alpha 1A-adrenoceptor.

    Footnotes

Accepted for publication June 21, 2000.

Received for publication December 7, 1999.

1 This work was funded by a Glasgow University Scholarship and the Clinical Research Initiative in heart failure. The work was presented in part at the IUPHAR Congress 1998 (Argyle et al., 1998).

Send reprint requests to: Sally Anne Argyle, Division of Veterinary Pharmacology, Glasgow University Veterinary School, Bearsden, Glasgow, G61 1QH, Scotland, UK. E-mail: saa6k{at}udcf.gla.ac.uk

    Abbreviations

CEC, chloroethylclonidine; CCRC, cumulative concentration-response curve; NE, norepinephrine; PE, phenylephrine.

    References
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Abstract
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Materials and Methods
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