Distinct Alpha 1-adrenoceptor Subtypes Research Articles

Identification of α1 adrenoceptor subtypes in the rabbit prostate

1. The alpha 1-adrenoceptor subtypes of rabbit prostate were characterized in binding and functional experiments. 2. In saturation experiments, [3H]-prazosin bound to two distinct affinity sites in the rabbit prostate (pKD = 11.20 +/- 0.22 and 8.39 +/- 0.11, Bmax = 15.3 and 736 fmol mg protein-1). 3. In the displacement experiments, the binding was inhibited with shallow displacement curves by unlabelled prazosin, WB4101, and 5-methylurapidil, suggesting the presence of two distinct affinity sites for prazosin, WB4101, or 5-methylurapidil. On the other hand, HV723 displaced the [3H]-prazosin binding monophasically with a low affinity. From the results, the presence of two distinct alpha 1-adrenoceptor subtypes was suggested; presumably one is the classical alpha 1A (cloned alpha 1C) subtype with high affinity for prazosin, WB4101 and 5-methylurapidil but not for HV723 and the other corresponds to the alpha 1L subtype, which shows low affinity for the four antagonists. 4. In the functional experiments, prazosin, WB4101, HV723 and 5-methylurapidil competitively antagonized the contractile response to noradrenaline with low affinities close to those for the alpha 1L subtype determined in binding experiments. These results suggest that contractile response to noradrenaline in the rabbit prostate is predominantly mediated through the alpha 1L subtype.

Alpha-1 adrenoceptor subtypes involved in noradrenaline-induced contraction of snake ( Bothrops jararaca) isolated aorta

The effects of chloroethylclonidine (CEC) and calcium channel antagonists on noradrenaline-induced contractions of Bothrops jararaca aorta were investigated, in order to characterize the alpha-1 adrenoceptor subtypes present in this preparation. CEC (5 × 10 −5or 10 −4M) displaced to the right, in the same order of magnitude, the concentration-response curve of noradrenaline without affecting its maximum response. The residual response was blocked by prazosin (10 −8 M) but not by idazoxan (10 −5 M). Nifedipine (10 −5 M) or diltiazem (10 −5 M), in the absence of cocaine, did not have any effect on the concentration-response curve of noradrenaline; however, in the presence of cocaine, both the drugs were equipotent in displacing the curve, though diltiazem produced a non-parallel displacement. These results suggest the presence of two distinct alpha-1 adrenoceptor subtypes in Bothrops jararaca aorta: one CEC-sensitive (alpha-1B-like) and another CEC-insensitive, though the possibility of the presence of a single but different subtype cannot be excluded.

Pharmacological characterization of α1‐adrenoceptor subtypes in rat heart: a binding study

1. The alpha 1-adrenoceptor subtypes of rat heart were characterized in binding experiments performed with [3H]-prazosin as the radiolabel. The specific binding to the alpha 1-adrenoceptors was determined with 0.3 microM prazosin, because phentolamine (10 microM) was insufficient to inhibit completely the specific binding of high concentrations of [3H]-prazosin. 2. In saturation experiments, [3H]-prazosin bound to two distinct affinity sites (pKD = 10.39 and 8.19). The proportion of the low affinity sites was approximately 84% of total specific binding. Membranes pretreated with chloroethylclonidine (CEC, 10 microM) also showed two distinct affinity sites for [3H]-prazosin, although the maximum numbers of high and low affinity sites were reduced by 86 and 64%, respectively. 3. In competition experiments, [3H]-prazosin (100 pM) binding was inhibited by WB4101 (2-(2,6-dimethoxy-phenoxyethyl)aminomethyl-1,4-benzodioxane) and 5-methylurapidil. The inhibition curves displayed shallow slopes which could be subdivided into high and low affinity components (pKi = 10.43 and 8.36 for WB4101, 8.62 and 6.61 for 5-methylurapidil). However, unlabelled prazosin or HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)-ethyl)amin o) propyl)benzeneacetonitrile fumarate) competed for [3H]-prazosin binding monophasically (pKi = 10.34 and 8.28, respectively). In CEC-pretreated membranes, prazosin, WB4101, 5-methylurapidil and HV723 antagonized the [3H]-prazosin (100 pM) binding monophasically (pKi = 9.70, 9.56, 8.60 and 8.82, for each antagonist). 4. On the other hand, 1000 pM [3H]-prazosin binding was inhibited by unlabelled prazosin biphasically (pKi = 10.49 and 8.49). HV723 did not discriminate both prazosin-high and low affinity sites (pKi = 8.18). 5. These results suggest the presence of at least three distinct alpha1-adrenoceptor subtypes in rat hearts(two prazosin-high affinity sites and one prazosin-low affinity site). According to the recent alpha l-adrenoceptor subclassifications, one of the former two sites corresponds to the alpha 1B subtype with low affinities for WB4101 and 5-methylurapidil and sensitive to CEC, while another site with relatively high affinities for WB4101 and 5-methylurapidil may be classical alpha 1A, cloned alpha 1c, alpha 1D subtypes or their mixture. The prazosin-low affinity site corresponds to putative alpha 1L subtype with low affinity for HV723,which may be predominantly involved in the positive inotropic response to phenylephrine.

Pharmacological characterization of alpha 1-adrenoceptor subtypes in the human prostate: functional and binding studies.

To characterize the alpha 1-adrenoceptor subtypes of the human benignly enlarged prostate using functional and binding studies. Strips of prostatic tissue taken from nine patients with benign prostatic hypertrophy who were undergoing open prostatectomy were used in the study. The strips isolated from five prostates produced a large contraction in response to noradrenaline and phenylephrine but not to clonidine. The contractile response induced by noradrenaline was competitively antagonized by representative alpha 1-adrenoceptor antagonists (prazosin, WB4101, 5-methylurapidil and HV723), the dissociation constants (pKB) being < 8.5. Pre-treatment with chloroethylclonidine was without effect on the contractile response to noradrenaline. In saturation experiments with five prostates, [3H]-prazosin bound to the prostate membranes with two distinct affinities (pKD = 9.95 +/- 0.07 and 8.71 +/- 0.04, Bmax = 151 +/- 8 and 138 +/- 3 fmol/mg protein, respectively). Unlabelled prazosin and WB4101 biphasically displaced the binding of 200 pM [3H]-prazosin; the resulting high and low pKI values for each of the antagonists were consistent with the two pKD values obtained for [3H]-prazosin in the saturation experiments. 5-Methylurapidil and HV723 displaced the [3H]-prazosin binding monophasically with an affinity (pKI) close to 8.5. These results suggest the presence of at least two distinct alpha 1-adrenoceptor subtypes (presumably an alpha 1C subtype with a high affinity for prazosin and WB4101, and a putative alpha 1L subtype with a low affinity for the antagonists) in the human prostate, in which the latter subtype may be predominantly involved in the contractile response to noradrenaline.

Pharmacological evidence of distinct alpha 1-adrenoceptor subtypes mediating the contraction of human prostatic urethra and peripheral artery.

1. The alpha 1-adrenoceptor subtypes mediating contractions of the smooth muscle in human prostatic urethra and branches of internal iliac artery were characterized in isometric contraction experiments. 2. Phenylephrine produced concentration-dependent contractions in both the urethra and artery. These responses were competitively inhibited by prazosin, WB4101 and 5-methyl-urapidil, and the slopes of Schild plots for all these antagonists were close to unity. 3. The pA2 values for prazosin were not significantly different between the urethra (9.42 +/- 0.11; mean +/- s.d.) and artery (9.50 +/- 0.27), while the pA2 values for WB4101 and 5-methyl-urapidil in the human prostatic urethra (8.94 +/- 0.19 and 8.42 +/- 0.14, respectively) were significantly greater than in the branches of human internal iliac artery (7.94 +/- 0.21 and 7.43 +/- 0.22, respectively; P < 0.01). 4. Pretreatment with chlorethylclonidine (CEC) at concentrations ranging from 0.1 microM to 100 microM attenuated the maximum contraction to phenylephrine in a concentration-dependent manner in both the urethra and artery. However, the urethra was significantly less affected by CEC than the artery. The pD'2 values (negative logarithm of the molar concentration of antagonist which reduced the maximum contraction to one half) in the urethra and artery were 4.35 +/- 0.27 and 5.20 +/- 0.37, respectively (P < 0.01). 5. The present results indicate that there are distinct populations of alpha 1-adrenoceptor subtypes in the human prostatic urethra and branches of the internal iliac artery. The alpha 1-adrenoceptors responsible for the contraction of the human internal iliac artery branches are predominantly alpha 1 B-subtype, whereas those in the human prostatic urethra are considered to be not alpha 1 B, but alpha 1 c or possibly alpha 1 A or alpha 1A/D-subtype.

Pharmacological characterization of two distinct alpha 1-adrenoceptor subtypes in rabbit thoracic aorta.

1. alpha 1-Adrenoceptor subtypes in rabbit thoracic aorta have been examined in binding and functional experiments. 2. [3H]-prazosin bound to two distinct populations of alpha 1-adrenoceptors (pKD,high = 9.94, Rhigh = 79.2 fmol mg-1 protein; pKD,low = 8.59, Rlow = 215 fmol mg-1 protein). Pretreatment with chloroethylclonidine (CEC, 10 microM) almost inactivated the prazosin-high affinity sites and reduced the number of the low affinity sites without changing the pKD value. 3. In the displacement experiments with CEC-untreated membranes, unlabelled prazosin, WB4101 and HV723 displaced the binding of 200 pM [3H]-prazosin monophasically; the affinities for WB4101 (pK1 = 8.88) and HV723 (8.49) were about 10 times lower than that for prazosin (9.99). In the CEC-pretreated membranes also, the antagonists inhibited the binding of 1000 pM [3H]-prazosin monophasically; the pK1 values for prazosin, WB4101 and HV723 were 9.09, 8.97 and 8.17, respectively. These results suggest that the prazosin-high and low affinity sites can be independently appraised in the former and latter experimental conditions. Noradrenaline, but not methoxamine, showed slightly higher affinity for the prazosin-high affinity site than for the low affinity site. 4. In the functional experiments, noradrenaline (0.001-100 microM) and methoxamine (0.1-100 microM) produced concentration-dependent contractions. Pretreatment with CEC inhibited the contractions induced by low concentrations of noradrenaline but without effect on the responses to methoxamine. Prazosin inhibited the concentration-response curves for noradrenaline in the CEC-untreated aorta in a manner which was not consistent with competitive antagonism at a single site, and two distinct affinity constants(pKB = 9.71 and 8.74) were obtained. However, after CEC-pretreatment, Schild plots for prazosin were not significantly different from unity (pKB = 8.50). WB4101 and HV723 competitively inhibited the noradrenaline-induced contraction with low pKB values (approximately 8.30), irrespective of CEC pretreatment.Methoxamine-induced contractions were competitively inhibited by prazosin, WB4101 and HV723 with low pKB values similar to those obtained when noradrenaline was used as the agonist.These were not affected by CEC-pretreatment.5. The affinity constant for noradrenaline (pKA = 6.40) in CEC-untreated aorta was slightly greater than that obtained in CEC-pretreated aorta (5.78). On the other hand, methoxamine showed a similar affinity in CEC-untreated and pretreated aortae (pKA = approximately 4.5).6. Nifedipine (1 microM) slightly attenuated the contractile responses to noradrenaline and methoxamine in CEC-untreated and pretreated aortae, suggesting that nifedipine cannot discriminate between alpha 1-adrenoceptors involved in CEC-sensitive and -resistant contractions.7. From these results it is suggested that in the rabbit thoracic aorta there are two distinct alpha 1-adrenoceptor subtypes (presumably alpha 1B and alpha 1L subtypes according to recently proposed subclassification),both of which are involved in noradrenaline-induced contraction. The alpha 1L subtype predominantly mediates the contraction induced by methoxamine.

Identification of alpha 1-adrenoceptor subtypes in the dog prostate.

The alpha 1-adrenoceptor subtypes of dog prostate were characterized in binding and functional experiments. In saturation experiments, [3H]prazosin bound to alpha 1-adrenoceptors with high affinity. In the displacement experiments, unlabelled prazosin and WB4101 biphasically inhibited the binding of 400 pM [3H]prazosin, suggesting the presence of at least two distinct affinity sites for prazosin or WB4101. The proportion of high-affinity sites was approximately 10%. HV723 also recognized two distinct affinity sites but the proportion of high-affinity sites was approximately 20%. From these results the presence of three distinct alpha 1-adrenoceptor subtypes was suggested: presumably subtypes alpha 1A (high affinity for prazosin and WB4101), alpha 1N (high affinity for only HV723) and alpha 1L (low affinity for the three antagonists) according to the recently proposed alpha 1-adrenoceptor subclassification. The density of subtype alpha 1L was much higher than that of subtypes alpha 1A and alpha 1N subtypes. In the functional experiments, prazosin, WB4101 and HV723 competitively antagonized the contractile response to noradrenaline with low affinities close to those estimated for the alpha 1L subtypes. These results suggest that the contractile response to noradrenaline in the dog prostate is mediated predominantly through alpha 1L subtype alpha-adrenoceptors.

Drug-receptor interactions in single smooth muscle cells

Smooth muscle tissues were contracted by excitation of each muscle cell. Single cells prepared from guinea pig taenia caecum and trachea were contracted by extracellular application of acetylcholine and/or carbachol, whose concentrations were the same as those in the tissues. The concentration-response curve was shifted in a parallel fashion by competitive antagonists. The pA2-values of the antagonists were in good agreement with those estimated using the intact tissue. The apparent dissociation constants of cholinergic drugs estimated from inhibition of the specific binding of [3H]QNB (quinuclidinyl benzilate) to the single cells by the cholinergic drugs were also in agreement with the values in other membrane preparations. Similar findings were obtained in the single cells, microsomal fractions and isolated tissues from the guinea pig tracheal smooth muscles. In rabbit aortic single cells, the existence of two pharmacologically distinct alpha 1-adrenoceptor subtypes, alpha 1A and alpha 1B, in vascular smooth muscle cells was supported. Furthermore, the amount of prostaglandin F2 alpha released from guinea pig tracheal single cells was increased through activation by alpha 2-adrenoceptor agonists. The amount of prostaglandin F2 alpha released by norepinephrine decreased with age, while the total amount of alpha 2-adrenoceptors and the dissociation constants of the alpha 2-adrenergic drugs from the receptor did not change. The relaxation induced by beta-adrenoceptors did not alter with age. The total amount of beta-adrenoceptor and the dissociation constants of beta-adrenergic drugs from their receptor did not alter with age. An excellent relationship between the potency of isoprenaline and the maximum binding of [3H]dihydroalprenolol estimated in the single cells from 6- to 40-week-old guinea pigs was found, suggesting that the increase in the potency of isoprenaline is due to the increase in the maximum binding. The value in the single cells from 100-week-old guinea pigs deviated significantly from the regression line. This result suggests that the decrease in potency in the single cells from 100-week-old animals is due to a change in post beta-receptor processes in responsiveness. The smooth muscle single cells are useful for the study of drug-receptor interactions. Furthermore, post-receptor processes in responsiveness were discussed.

Activation of a single alpha-1-adrenoceptor subtype in rat aorta mobilizes intracellular and extracellular pools of calcium.

The calcium channel antagonist, nifedipine (0.1 microM), only slightly inhibited the response to the full alpha 1-adrenoceptor agonist, (-)-norepinephrine, in rat aorta, but nearly completely inhibited the response to the partial alpha 1-adrenoceptor agonist, (-)-dobutamine, indicating that (-)-norepinephrine primarily utilizes intracellular stores of calcium to produce vasoconstriction, whereas (-)-dobutamine relies primarily upon the translocation of extracellular calcium. It has been proposed that the different pools of calcium mobilized by (-)-norepinephrine and (-)-dobutamine in rat aorta result from the activation of two different alpha 1-adrenoceptor subtypes by these agonists. Because the irreversible alpha 1-adrenoceptor antagonist, phenoxybenzamine, has been proposed to inactivate an alpha 1-adrenoceptor subtype coupled specifically to the mobilization of intracellular stores of calcium, this compound should selectively inhibit the response of (-)-norepinephrine, and not the response of (-)-dobutamine, if two distinct alpha 1-adrenoceptor subtypes exist in rat aorta. In the present study, phenoxybenzamine produced concentration-dependent, noncompetitive inhibition of the responses to both agonists in rat aorta, and the pA2' values for phenoxybenzamine were 8.12 +/- 0.23 and 7.74 +/- 0.27 against (-)-dobutamine and (-)-norepinephrine, respectively, which are not significantly different from each other (p greater than 0.05). In addition, the phasic and tonic components of the response to (-)-norepinephrine, which are mediated by the mobilization of intracellular calcium and the translocation of extracellular calcium, respectively, are also inhibited to the same extent by phenoxybenzamine. The results do not support the hypothesis of two alpha 1-adrenoceptor subtypes in rat aorta, one of which being coupled to the translocation of extracellular calcium, and the other to the mobilization of intracellular calcium. Rather, the results support our previous hypothesis that a single alpha 1-adrenoceptor subtype exists in vascular smooth muscle, and this alpha 1-adrenoceptor is coupled to two distinct signal transduction processes, one of which elicits the mobilization of intracellular calcium, and the other opens membrane calcium channels to permit the influx of extracellular calcium.

Subtypes of .ALPHA.1-Adrenoceptors Involved in Noradrenaline-Induced Contractions of Rat Thoracic Aorta and Dog Carotid Artery.

We tried to determine alpha 1-adrenoceptor subtypes involved in noradrenaline-induced contractions of rat thoracic aorta and dog carotid artery. Prazosin competitively antagonized the contractions induced by noradrenaline in both the arteries with a high pKB value (approximately 9.7). WB4101, benoxathian, phentolamine, HV723 and 5-methylurapidil also competitively antagonized the responses to noradrenaline in both the arteries: however, the affinities for the antagonists were significantly higher in the rat thoracic aorta than in the dog carotid artery. The affinities for the competitive antagonists were not changed by treatment with nifedipine. In the rat thoracic aorta, chlorethylclonidine (CEC) elicited either a persistent contraction with rhythmic activities before treatment with nifedipine or partial inactivation of alpha 1-adrenoceptors in the presence of nifedipine. On the other hand, CEC produced only inactivation of alpha 1-adrenoceptors in the dog carotid artery. These results suggest that noradrenaline-induced contractions of the rat thoracic aorta and dog carotid artery are respectively mediated through distinct alpha 1-adrenoceptor subtypes. According to the alpha 1A, alpha 1B subclassification, the alpha 1-adrenoceptor of dog carotid artery is like the alpha 1B subtype, while that of rat thoracic aorta is atypical. Both subtypes are also identified as a high affinity site for prazosin (alpha 1H subtype) in the alpha 1H, alpha 1L and alpha 1N subclassification.

Different desensitization mechanisms of two alpha 1‐adrenoceptor subtypes in the contraction of rabbit aorta.

Using alpha 1-adrenoceptor selective antagonists chlorethylclonidine (CEC) and WB4101, both functional and radioligand binding studies showed that rabbit aorta contains at least two pharmacologically distinct alpha 1-adrenoceptor subtypes of approximately 10% alpha 1a and 90% alpha 1b receptors, and that each receptor subtype has a distinct role in the alpha 1-adrenoceptor-mediated vasoconstrictive response through different biochemical mechanisms for increasing intracellular Ca2+; alpha 1a receptors cause tonic response predominantly dependent on the influx of extracellular Ca2+, while alpha 1b receptors stimulate phosphoinositides (PI) hydrolysis/intracellular Ca2+ mobilization and cause phasic response mainly independent of extracellular Ca2+. Incubation of rabbit aorta with 10 microM noradrenaline (NA) for 2 h resulted in a reduction in the phasic response and PI hydrolysis to NA with no change in the tonic response. Similar to the NA incubation, the protein kinase C stimulator PMA (1 microM) selectively attenuated alpha 1b-receptor mediated PI hydrolysis and phasic contraction but had little effect on alpha 1a-receptor-mediated tonic response. The protein kinase C inhibitor H-7 (10 microM) blocked these inhibitory effects of PMA. Treatment with H-7 (10 microM) prevented the NA-induced alpha 1b receptor desensitization in inositol monophosphate (IP) formation and phasic response. The results suggest that activation of C kinase may be involved in the development of selective desensitization of alpha 1b receptors by a short-time in vitro incubation of NA.