Postjunctional α-adrenoceptors located on vascular smooth muscle belong to two major subtypes: α1- and α2-adrenoceptors (1-4). Based on analysis of antagonist potency, vascular α1-adrenoceptors were subclassified as α1H for receptors with high affinity for prazosin (−log KB ≥9.5) and α1L for receptors with low affinity (−log KB < 9.0) for the antagonist (5,6). α1-Adrenoceptors with high affinity for prazosin have subsequently been subdivided by pharmacologic, biochemical, and molecular approaches into α1A, α1B, and α1D-adrenoceptors (7-9). Although α1L-adrenoceptors have been identified by pharmacologic (5,10-12) and biochemical techniques (6,11,13,14), they have not yet been characterized by molecular approaches (e.g., 8).
There is little information regarding the distribution, regulation, and physiological function of α1L-adrenoceptors. Previous studies suggested that they mediate contraction of rabbit and guinea-pig aorta and of human prostate (5,10-12,15). The aim of this study was to determine whether α1L-adrenoceptors were present on smooth muscle of the canine pulmonary artery and if these receptors could mediate contractile responses to the physiologic agonist, norepinephrine.
Blood vessel preparation
Intralobar pulmonary arteries were obtained from lower lung lobes of mongrel dogs of either sex (15-30 kg) that had been anesthetized with sodium pentobarbital (30 mg/kg intravenously) and exsanguinated. The vessels were placed in modified Krebs-Ringer bicarbonate solution (control solution, in mM: NaCl, 118.3; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2; NaHCO3, 25.0; calcium disodium edetate, 0.016; glucose, 11.1), cleaned of connective tissue, and cut into rings 4-5 mm long. The endothelium was mechanically removed from all rings by inserting forceps into the vessel and rolling the vessel over damp filter paper. Endothelial denudation was later confirmed by the absence of relaxation to acetylcholine (3 × 10−7M) during a submaximal contraction to norepinephrine. The vessels were suspended between two stainless-steel wire clips for isometric tension recording (Grass FTO3 transducers, Grass Instrument Co., Quincy, MA, U.S.A.) in organ chambers filled with 25 ml of control solution (37°C, 95% O2/5% CO2). The rings were stretched gradually to their optimal basal tension (16,17), determined in preliminary experiments to be 5 g. They were then allowed to equilibrate for 30 min. The rings were then contracted with norepinephrine (10−4M) to determine their maximal contractile ability, and subsequent responses were expressed as a percentage of this maximal contraction (e.g., 18). After washout of norepinephrine and the return of tension to basal levels, the preparations were allowed to equilibrate for a further 30 min.
Concentration-response curves (CRCs) to adrenergic agonists were obtained by increasing the agonist concentration by half-log increments in a noncumulative manner. Concentrations were increased only when the contractile response to the preceding stimulus had stabilized. Before exposing the tissues to adrenergic agonists, the rings were treated with cocaine (5 × 10−6M), hydrocortisone (3 × 10−5M), and propranolol (5 × 10−6M) to inhibit neuronal uptake, extraneuronal uptake, and β-adrenoceptors, respectively (18). In addition, rauwolscine (10−7M) was present to inhibit α2-adrenergic stimulation by the agonists (18). CRCs to the agonists were determined under control conditions and in the presence of either prazosin (3 × 10−10-3 × 10−8M), yohimbine (10−6-3 × 10−5M), BMY7378 (3 × 10−8-3 × 10−6M), or risperidone (3 × 10−9-3 × 10−7M). When these receptor antagonists or inhibitors of disposition mechanisms were used, the preparations were incubated for 45 min with the drugs before and also during exposure of the tissues to the agonists. Only one CRC was obtained on each ring, and up to eight rings of the same pulmonary artery were studied in parallel to obtain data under control conditions and in the presence of antagonist(s) (e.g., 18-20). Preliminary studies demonstrated that contractile responses to α-adrenergic agonists did not vary along the length of pulmonary artery used in this study. This protocol is different from the more common approach using two CRCs (i.e., before and after antagonist). Both approaches are based on the same assumption that a control arterial ring, studied in parallel with the preparations exposed to antagonists, is representative of the other rings. The control ring either provides a measure of the control activity (in the single-curve protocol, this study) or of the time/exposure-dependent change in reactivity that is used to estimate the theoretic change in reactivity of the antagonist-treated rings (sequential protocol, this study).
In some experiments, the influence of the irreversible adrenergic antagonists, chloroethylclonidine (10−5M) or SZL49 (10−8 and 3 × 10−8M) were determined. Arterial rings were incubated with chloroethylclonidine or SZL49 for 30 min and then removed during a 45-min washout period. The rings were then incubated with inhibitors of disposition pathways/competitive antagonists (45 min) and a concentration-effect curve to adrenergic agonists performed as described.
The following drugs were used: BMY7378 (Research Biochemicals International, RBI, Natick, MA, U.S.A.), chloroethylclonidine (RBI), cocaine HCl (Yar Lang, La Crosse, WI, U.S.A.), hydrocortisone 21-hemisuccinate (sodium salt; Sigma Chemical Co., St. Louis, MO, U.S.A.), methoxamine HCl (Burroughs Wellcome, Research Triangle Park, NC, U.S.A.), L-norepinephrine bitartrate (Sigma), L-phenylephrine HCl (Sigma), prazosin HCl (Sigma), dl-propranolol (Sigma), rauwolscine HCl (Carl Roth, Karlsruhe, Federal Republic of Germany), risperidone (gift from D.A. Schwinn), SZL49 (RBI), and yohimbine HCl (Sigma). Except for prazosin, drugs were prepared and diluted by using distilled water. Stock solutions of prazosin were prepared by using dimethylsulfoxide (highest bath concentration, 0.0002% vol/vol%) with further dilution in distilled water. Concentrations of drugs are expressed as final molar concentrations in the organ chamber.
Data are expressed as mean ± SEM; n refers to the number of dogs from which blood vessels were taken. Contractions are expressed as a percentage of the maximal response to norepinephrine obtained at the beginning of the experiment, before treatment of the rings with adrenergic antagonists. To calculate EDx (expressed as −log EDx) for the agonists, in the presence and absence of the antagonists, the concentration of the agonist producing a contraction of X% of the maximal response to norepinephrine was interpolated from concentration-effect curve. pA2 values were calculated from Arunlakshana and Schild plots (21) by linear-regression analysis. Arunlakshana and Schild plots were generated for each experiment, and the means ± SEM for the slopes and pA2 values are reported in the text. A graph of the Arunlakshana and Schild plot was generated by determining the mean of the individual values of log (DR-1), where DR is the dose ratio. Statistical analysis was performed by using Student's t test for paired or unpaired observations. When more than two means were compared, analysis of variance was used. If a significant F value was found, Scheffé's test for multiple comparisons was used to identify differences among groups. Differences between means were considered to be statistically significant when p was <0.05.
Pharmacologic demonstration of α1H- and α1L-adrenoceptors in canine pulmonary artery
Phenylephrine activates α1H-adrenoceptors. Prazosin (3 × 10−10-3 × 10−8 M) caused concentration-dependent, parallel, and rightward shifts in the CRC to phenylephrine (Fig. 1). The slope of the Arunlakshana and Schild plot did not differ significantly from unity (0.86 ± 0.08; n = 5), and the pA2 value for prazosin was 9.68 ± 0.14 (n = 5).
SZL49, 10−8 and 3 × 10−8 M, caused rightward shifts in the CRC to phenylephrine of 4.7-fold (log shift, 0.67 ± 0.05; n = 5) and 13.6-fold (log shift, 1.13 ± 0.09; n = 5), respectively (Fig. 2). SZL49 did not inhibit the maximal response of arterial rings to phenylephrine (Fig. 2).
Chloroethylclonidine (10−5 M) caused a rightward shift in the CRC to phenylephrine (log shift of 1.70 ± 0.27; 50-fold; n = 4) and reduced the maximal response to the agonist (by 50.8 ± 8.2%; n = 4; Fig. 3).
Yohimbine (10−6-3 × 10−5 M) produced concentration-dependent, parallel, and rightward shifts in the CRC to phenylephrine (data not shown). The slope of the Arunlakshana and Schild plot did not differ significantly from unity (0.83 ± 0.06; n = 5), and the pA2 value for yohimbine was 6.77 ± 0.08 (n = 5).
Methoxamine activates α1L-adrenoceptors. In contrast to phenylephrine, low concentrations of prazosin (3 × 10−10 M and 10−9 M) did not significantly affect responses evoked by methoxamine. Higher concentrations of prazosin (3 × 10−9-3 × 10−8 M) caused concentration-dependent parallel and rightward shifts in the CRC to the agonist (Fig. 4). The slope of the Arunlakshana and Schild plot for the inhibitory effect of prazosin did not differ significantly from unity (0.94 ± 0.06; n = 5), and the pA2 value was 8.37 ± 0.11 (n = 5). This was significantly lower than the value obtained against phenylephrine.
SZL49 (10−8 and 3 × 10−8 M) did not significantly affect the CRC to methoxamine (Fig. 5).
Chloroethylclonidine (10−5 M) caused a rightward shift in the CRC to methoxamine (log shift of 0.96 ± 0.06; ninefold; n = 4; significantly less than against phenylephrine, p < 0.05) and reduced the maximal response to the agonist (by 41.5 ± 4.0%; n = 4; Fig. 6).
Yohimbine (10−6-3 × 10−5 M) caused concentration-dependent parallel and rightward shifts in the CRC to methoxamine (data not shown). The slope of the Arunlakshana and Schild plot did not differ significantly from unity (1.03 ± 0.11; n = 6), and the pA2 value for yohimbine was 6.26 ± 0.10 (n = 6). This was significantly lower than the value obtained against phenylephrine.
Norepinephrine activates α1H- and αIL-adrenoceptors. Low concentrations of prazosin (3 × 10−10 and 10−9 M) caused concentration-dependent shifts in the CRC to norepinephrine (Fig. 7). A higher concentration of the antagonist (3 × 10−9 M) produced no further shift in the curve. However, increasing the concentration of prazosin (to 10−8 and 3 × 10−8 M) caused further concentration-dependent shifts in the CRC to norepinephrine (Fig. 7). Despite causing parallel shifts in the CRC to methoxamine and phenylephrine, prazosin produced nonparallel shifts in the CRC to norepinephrine, causing greater antagonism at low compared with high levels of tension. For example, prazosin (3 × 10−8 M) was more potent at the ED25 compared with the ED75 level of tension [log shifts of 1.72 ± 0.12 (52-fold) and 0.97 ± 0.20 (ninefold), respectively; n = 5; p < 0.05; Fig. 7). Arunlakshana and Schild plots for the inhibitory effects of prazosin at the ED25, ED50, and ED75 levels of the response produced a family of biphasic curves, with inflections occurring at 3 × 10−9 M prazosin (Fig. 8). The component of the Arunlakshana and Schild plots occurring at low concentrations of the antagonist (3 × 10−10-10−9 M) was similar to the plot obtained when phenylephrine was the agonist (Fig. 8). Indeed, if analyzed separately, this component was associated with slopes that were not significantly different from unity and pA2 values that were similar to those attained against phenylephrine (e.g., at ED25 level of response, slope of 1.16 ± 0.19 and a pA2 value of 9.77 ± 0.10; n = 5). The inflection in the Arunlakshana and Schild plot became more severe at the ED75 level of the response compared with the ED25 or ED50. Indeed, at the ED75 level of the response, the component of the curve occurring at high concentrations of the antagonist (10−8-3 × 10−8 M) was similar to the plot obtained when methoxamine was the agonist (Fig. 8). If analyzed separately, this component was associated with a slope that was not significantly different from unity and a pA2 value that was similar to that obtained against methoxamine (slope of 0.96 ± 0.03 and pA2 value of 8.45 ± 0.21; n = 5).
SZL49 (10−8 and 3 × 10−8 M) caused rightward shifts in the CRC to norepinephrine without affecting the maximal response to the agonist (Fig. 9). As with prazosin, SZL49 caused nonparallel shifts in the curve and was most effective at the ED25 compared with the ED75 level of the response [e.g., at 10−8 M, SZL49 caused a log shift of 0.46 ± 0.05 (2.9-fold) at ED25 but no significant change at ED75; n = 5; Fig. 9). After treatment of the arterial rings with SZL49 (3 × 10−8 M), prazosin (3 × 10−9-3 × 10−8 M) caused parallel, rightward shifts in the CRC to norepinephrine (Fig. 10). After SZL49, Arunlakshana and Schild plots for the inhibitory effect of prazosin (3 × 10−9-3 × 10−8 M) at different levels of tension (ED25, ED50, and ED75) were similar to each other and to the plot generated when methoxamine was the agonist (Fig. 11). For example, at the ED50 level of tension, the plot, which did not differ significantly from unity (0.91 ± 0.14; n = 5) generated a pA2 value of 8.34 ± 0.26 (n = 5; Fig. 11), similar to that obtained with methoxamine.
Yohimbine (10−6-3 × 10−5 M) caused concentration-dependent shifts in the concentration-effect curve to norepinephrine (data not shown). The slope of the Arunlakshana and Schild plot did not differ significantly from unity (0.87 ± 0.05; n = 6), and the pA2 value for yohimbine was 6.38 ± 0.10 (n = 6). This was not significantly different from the value obtained against methoxamine but was different from that obtained with phenylephrine.
Pharmacologic characterization of α1H-adrenoceptors activated by phenylephrine in canine pulmonary artery
As described, phenylephrine caused contraction that was highly sensitive to inhibition by chloroethylclonidine (10−5M;Fig. 4), which at this concentration is selective for α1B- and α1D-adrenoceptors (22,23). Low concentrations of BMY7378 (3 × 10−8 and 3 × 10−7M), a selective α1D-adrenoceptor antagonist, did not affect the concentration-effect curve to phenylephrine, whereas a higher concentration of the antagonist (3 × 10−6M) caused a significant rightward shift in the curve (data not shown). The −log KB value for this interaction was 6.07 ± 0.12 (n = 4), which is consistent with interaction of the antagonist with α1B- (but not α1D-) adrenoceptors (e.g., 24,25). The α1B-adrenoceptor antagonist, risperidone (3 × 10−9-3 × 10−7M) caused concentration-dependent, parallel shifts in the CRC to phenylephrine (data not shown). Arunlakshana and Schild (1949) analyses generated a plot with a slope that was not significantly different from unity (0.84 ± 0.12; n = 4) and a pA2 value of 8.51 ± 0.25 (n = 4), consistent with interaction of the antagonist with α1B-adrenoceptors (26,27).
Vascular α1-adrenoceptors have been subclassified as α1H for receptors with high affinity for prazosin (−log KB ≥ 9.5) and α1L for receptors with low affinity (−log KB < 9.0) for the antagonist (5,6,10-12). Although α1-adrenoceptors with high affinity for prazosin have subsequently been subdivided by pharmacologic, biochemical, and molecular approaches into α1A-, α1B-, and α1D-adrenoceptors (7-9), α1L-adrenoceptors have been identified solely by pharmacologic (5,10-12) and biochemical techniques (6,8,11,13,14). In our study, prazosin and yohimbine inhibited contractions to phenylephrine and methoxamine in canine pulmonary artery in a manner that suggested competitive interaction at α1-adrenoceptors. However, the pA2 values for the inhibitory influence of these antagonists were higher for phenylephrine than for methoxamine. The results suggests that two distinct α1-adrenoceptors are located on the vascular smooth muscle of this blood vessel: one with high affinity (i.e., α1H) for prazosin (pA2 of ∼9.8) and yohimbine (pA2 of ∼6.8) that was stimulated by phenylephrine, and a secondary site with lower affinity (i.e., αIL) for these two α-adrenergic antagonists (pA2 values of ∼8.4 and 6.3, respectively) that was stimulated by methoxamine. Prazosin had a greater degree of selectivity for the high-affinity site with a selectivity ratio (KB low-affinity site/KB high-affinity site) of ∼20 compared with yohimbine with a ratio of ∼3. These values are consistent with previous analyses of α1H- and αIL-adrenoceptors in other blood vessels (5,10-12).
Muramatsu et al. (11) proposed that the irreversible antagonist, chloroethylclonidine, caused selective inactivation of α1H-adrenoceptors with little influence on α1L-adrenoceptors. In our study, although chloroethylclonidine was more potent against responses to phenylephrine, it also inhibited responses to methoxamine, suggesting that the antagonist inhibits both α1H- and α1L-adrenoceptors. Indeed, at the concentration used in this study, chloroethylclonidine inhibited the binding of [3H]prazosin to α1L-adrenoceptors (12,13). Therefore chloroethylclonidine can inactivate α1L- and α1H-adrenoceptors. The selectivity of this antagonist will be determined by the magnitude of the receptor-reserve (e.g., 19,28), as well as the identity of the α1H-adrenoceptors (α1A, α1B, or α1D; 7-9,22).
Because α1H- and α1L-adrenoceptors have differing sensitivities to prazosin, experiments were performed to determine whether the irreversible prazosin analog, SZL49 (29), could discriminate between responses mediated by these adrenoceptors. SZL49 in concentrations up to 3 × 10−8M inhibited contractile responses to phenylephrine but did not significantly affect those to methoxamine, suggesting that SZL49 may be a selective, irreversible antagonist of α1H compared with α1L-adrenoceptors. Previous studies demonstrated that SZL49 was ∼10-fold less potent at antagonizing α1-adrenergic contractions in rabbit (29) compared with rat aorta (30). α1L-Adrenoceptors contribute to contractile responses of rabbit but not rat aorta and could be responsible for this difference in inhibitory potency (5,6,11,12,31). However, the α1H-adrenoceptors mediating contraction of these blood vessels are also different: α1D-adrenoceptors predominate in rat aorta and α1B-adrenoceptors in rabbit aorta (25,32,33). Previous reports suggested that SZL49 might distinguish between α1A-, α1B-, and α1D-adrenoceptors (34-36). However, analysis of native and cloned α1-adrenoceptors indicates that SZL49 can inactivate all α1-adrenoceptors with high affinity for prazosin but appears to have decreased activity at α1B/b- compared with α1A/a- or α1D/d-adrenoceptors (23,37,38).
Norepinephrine caused contraction of the pulmonary artery by stimulating α1H- and α1L-adrenoceptors. At concentrations of prazosin that would be selective for α1H-adrenoceptors, the antagonist at first inhibited (3 × 10−10 and 10−9M) and then failed to inhibit (3 × 10−9M) the CRC to norepinephrine. Further inhibition was achieved only when the concentration of prazosin was sufficient to antagonize α1L-adrenoceptors (10−8 and 3 × 10−8M). Arunlakshana and Schild plots for this interaction were biphasic and had overall slopes that were less than unity. Biphasic plots can result from a number of causes including the presence of a saturable disposition mechanism (e.g., 39). However, in our study, the biphasic nature of the plot resulted from activation of two α1-adrenoceptor subtypes by norepinephrine. When considered separately, the two components of the Arunlakshana and Schild plot were linear (i.e., slopes of unity) and generated pA2 values consistent with the interaction of prazosin with α1H- and α1L-adrenoceptors. Furthermore, the irreversible antagonist, SZL49, which appears to be relatively selective for α1H-adrenoceptors, abolished the α1H-like component of the Arunlakshana and Schild plot. After SZL49, the interaction between prazosin and norepinephrine generated a family of linear Arunlakshana and Schild plots with pA2 values expected of α1L-adrenoceptors (8.33 to 8.47). These results indicate that at low concentrations, norepinephrine contracts the pulmonary artery by activating α1H-adrenoceptors, whereas at higher concentrations, it activates α1H- and α1L-adrenoceptors. In contrast to the effects of prazosin, the Arunlakshana and Schild plot for the inhibitory effect of yohimbine against norepinephrine was not biphasic and did not differ significantly from unity. The inability of yohimbine to distinguish between the two components uncovered by prazosin most likely reflects the decreased selectivity expressed by yohimbine for these receptor subtypes.
Although prazosin caused parallel shifts in the CRC to phenylephrine and methoxamine, this was not the case with norepinephrine, and the antagonist was more potent at the ED25 compared with ED75 level of tension. Similar results were obtained with SZL49. The Arunlakshana and Schild plot for the inhibitory effect of prazosin against norepinephrine (in rings not treated with SZL49) was also dependent on the level of tension at which the dose ratios (DRs) were calculated. The component of the Arunlakshana and Schild plot resulting from activation of α1L-adrenoceptors by norepinephrine increased markedly at the ED75 compared with ED25 level of tension, suggesting an increased role of the α1L-adrenoceptor (or decreased role of the α1H-adrenoceptor) at high levels of tension. A decreased influence of α1H-adrenoceptors at the ED75 level of tension could occur if the α1H-adrenergic response was a low-maximal effect, as is often observed with α2-adrenoceptors (18-20,28). However, the maximal response to phenylephrine did not differ significantly from that produced by norepinephrine, suggesting this is not the case. The most likely explanation is that the concentration-effect curve for the α1H-component of norepinephrine-induced contraction is characterized by a reduced slope when compared with the α1L-component.
Although our results might suggest that phenylephrine and methoxamine are selective agonists at α1H- and α1L-adrenoceptors, respectively, whereas norepinephrine is relatively nonselective, this need not be the case. Because of the selectivity ratio of prazosin for α1H/α1L-adrenoceptors, it may not detect an α1L-adrenergic component in the response to an agonist if there is a >20-fold difference between the α1H- and α1L-components of the response. The concentration of prazosin needed to shift the α1H-adrenergic component would then be large enough to also shift the αIL-adrenergic response. Estimation of the selectivity of an agonist that is more effective at α1L-adrenoceptors is even more problematic. Because prazosin is relatively selective at inhibiting α1H-adrenergic responses, concentrations of the antagonist required to inhibit α1L-adrenergic responses will have produced an ∼20-fold shift in the concentration-effect curve to an α1H-adrenergic response. Therefore even if methoxamine activated α1H-adrenoceptors at concentrations immediately higher than those required to activate α1L-adrenoceptors, this may not be detected by using this antagonist. Indeed, in blood vessels that appear to lack functional α1L-adrenoceptors, methoxamine is still an effective agonist (5).
In a previous study, Docherty and Ruffolo (40) obtained two different pA2 values for the inhibitory effect of prazosin against methoxamine in canine pulmonary artery: a value of 9.35 by using a double CRC protocol (comparing CRCs before and after antagonist administration) and a value of 8.50 by using a variation of the single-CRC protocol (comparing only the second group of CRCs). Because these values were similar to those expected of α1H- and α1L-adrenoceptors, Docherty and Ruffolo (40) proposed that α1L-adrenoceptors were an erroneous result of using the single-CRC protocol. In their study, Docherty and Ruffolo (40) performed only partial CRCs to methoxamine after prazosin and did not allow the agonist to generate a full maximal response. Because Docherty and Ruffolo (40) assumed that the single-CRC protocol provided no information on maximal responses when the authors assessed that protocol, they merely assumed that a partial CRC to methoxamine represented the full CRC. Therefore a response to methoxamine in the presence of prazosin that had attained only 50% of the maximal response was now assumed to be equal to 100% (their Fig. 3). This caused inappropriate leftward shifts in the CRC for the antagonist-treated tissues, resulting in a downward shift in the Arunlakshana and Schild plot and the low pA2 value for prazosin. According to the data obtained by Docherty and Ruffolo (40), if the maximal response was accurately determined in each protocol (as was done in our study), then both the single- and double-CRC protocols generated similar KB values. Therefore α1L-adrenoceptors are not an experimental artifact of the single-CRC protocol.
The pharmacologic characterization of the α1H-adrenoceptors mediating contraction of the pulmonary artery suggests that these receptors belong to the α1B-adrenoceptor subtype. This conclusion is prompted by the findings that contractions to phenylephrine were highly sensitive to inhibition by chloroethylclonidine, suggesting α1B- or α1D-involvement, and that the KB values obtained for the inhibitory effects of risperidone (8.5) and BMY737 8 (6.1) are consistent with their activity at α1B-rather than α1A- or α1D-adrenoceptors (e.g., 24-27).
In conclusion, the results of our study suggest that α1-adrenoceptors located on vascular smooth muscle of the canine pulmonary artery, and that mediate contraction, belong to two subtypes: α1L- and α1B-adrenoceptors. The physiologic agonist, norepinephrine, appears to be a relatively nonselective activator of both receptors. The irreversible antagonist, SZL49, which selectively inhibited α1-adrenoceptors with high affinity for prazosin, may be a useful tool to analyze the role of these receptors in the physiologic and pathophysiologic control of this vascular bed.
Acknowledgment: We thank the drug companies listed under Materials and Methods for their kind contributions of drugs.
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