Masafumi Oshita

Pharmacological subclassification of α1‐adrenoceptors in vascular smooth muscle

1 We examined whether α1‐adrenoceptors in various blood vessels can be divided into subtypes by antagonist affinity or by susceptibility to chloroethylclonidine or nifedipine. 2 Noradrenaline or phenylephrine produced concentration‐dependent contractions in all the tissues tested, which were competitively inhibited by phentolamine, yohimbine, prazosin, WB4101 and HV723. However, there were large differences between the tissues in the pA2 values for all the antagonists except phentolamine. 3 The blood vessels could be classified into three groups (I, II and III) on the basis of their affinity variation. In group I (dog mesenteric artery and vein, saphenous vein), the pA2 values for HV723 were greater than 9, and those for HV723 and WB4101 were approximately 1 log unit higher than for prazosin. This rank order of affinity reversed in group II (dog carotid artery and rat thoracic aorta), where prazosin was more potent (pA2 values greater than 9.5) than HV723 or WB4101. In group III (rabbit mesenteric artery, thoracic aorta and carotid artery and guinea‐pig thoracic aorta), on the other hand, prazosin, HV723 and WB4101 inhibited the noradrenaline response with a similar affinity (pA2 values ranging from 8 to 9). 4 Yohimbine inhibited the responses to noradrenaline and phenylephrine with a lower affinity than prazosin, HV723 or WB4101. The pA2 values for yohimbine were similar in groups I and II (the values greater than 6.5), which were greater than those in group III (values less than 6.4). 5 The α1‐adrenoceptors in group II were selectively affected by chlorethylclonidine, resulting in an irreversible attenuation of noradrenaline responses in the dog carotid artery and a persistent contraction in the rat thoracic aorta. 6 Nifedipine either produced no effect or a slight inhibition of α1‐adrenoceptor‐mediated contractions in all the blood vessels; these effects were not correlated to the above groups. 7 These results suggest that α1 ‐adrenoceptors of blood vessels can be divided into three subtypes (designated as α1H, α1L and α1N) by antagonist affinity and their susceptibility to chloroethylclonidine but not to nifedipine: the characteristics of each subtype are summarized in Table 3. Subtypes α1H, α1L and α1N may be predominantly involved in the contractile responses to noradrenaline or phenylephrine of the blood vessels in groups II, III and I, respectively.

Identification of α1‐adrenoceptor subtypes in the rat vas deferens: binding and functional studies

1 The α1‐adrenoceptor subtypes of the prostatic and epididymal portion of rat vas deferens were characterized in binding and functional experiments. 2 In saturation experiments, [3H]‐prazosin bound to two distinct affinity sites in the epididymal portion of rat vas deferens (pKD = 10.1 ± 0.13 and 9.01 ± 0.15, Bmax = 507 and 1231 fmol mg−1 protein, respectively). In the prostatic portion [3H]‐prazosin bound to a single affinity site (pKD = 9.82 ± 0.04, Bmax = 924 fmol mg−1 protein). 3 In the displacement experiments, unlabelled prazosin displaced biphasically the binding of 200 pm [3H]‐prazosin to the epididymal portion; the resulting two pKI values were consistent with the affinity constants obtained in the saturation experiments. WB4101 (2‐(2,6‐dimethoxy‐phenoxyethyl)‐aminomethyl‐1,4‐benzodioxane) and benoxathian also discriminated the two affinity sites in the epididymal portion and the population of low affinity sites for the three antagonists was approximately 40%. On the other hand, the prostatic portion predominantly showed a single affinity site for prazosin, WB4101 and benoxathian, although the presence of a small proportion (less than 10%) of the low affinity site could be detected. HV723 (α‐ethyl‐3,4,5‐trimethoxy‐α‐(3‐((2‐(2‐methoxyphenoxy)ethyl)‐amino)‐propyl) benzeneacetonitrile fumarate) displaced the [3H]‐prazosin binding monophasically with a low affinity in both halves. 4 Pretreatment with chlorethylclonidine (CEC) at concentrations higher than 1 μm inhibited 700 pm [3H]‐prazosin binding to the prostatic portion by approximately 50%. However, the inhibition in the epididymal portion was much less (approximately 21% at 50 μm CEC). 5 In the functional study, the contractile response to noradrenaline was competitively inhibited by prazosin, WB4101, benoxathian and HV723 with similar and low affinities (pKB value ranging from 8.0 to 9.0) in the epididymal portion of rat vas deferens. In the prostatic portion of rat vas deferens, noradrenaline also produced a contraction, but the maximal amplitude of contraction developed was approximately one‐fourth of that in the epididymal portion. Prazosin and WB4101 also inhibited the contractile response of the prostatic portion with the pKB values similar to those obtained in the epididymal portion. The contractions to noradrenaline in both portions were potently attenuated by 1 μm nifedipine but were not affected by pretreatment with 10 μm CEC. 6 Under conditions where P2x‐purinoceptors and prejunctional α2‐adrenoceptors were blocked, electrical transmural stimulation produced a rapidly developing phasic contraction and a subsequent tonic contraction in the epididymal portion of rat vas deferens. The phasic and tonic contractions were inhibited in a concentration‐dependent manner by prazosin (IC50 = 25.7 and 25.9 nm, respectively), WB4101 (IC50 = 7.27 and 7.58 nm), benoxathian (IC50 = 10.9 and 8.66 nm) and HV723 (IC50 = 15.9 and 14.9 nm). Nifedipine selectively attenuated the tonic contraction induced by electrical stimulation, and the residual phasic response was inhibited by the antagonists mentioned above with similar affinities to those in the absence of nifedipine. CEC (10 μm) had little effect on the adrenergic neurogenic contractions. 7 The present results indicate the presence of two distinct α1‐adrenoceptor subtypes in the rat vas deferens, which show respectively high and low affinities for each of prazosin, WB4101 and benoxathian, and presumably correspond to putative α1A and α1L subtypes according to the recent α1‐adrenoceptor subclassifications. The contractions induced by exogenous and endogenous noradrenaline seem to be predominantly mediated through the α1L subtype. The heterogeneous distribution of the low affinity sites (α1L subtype) may well explain differences in functional responsiveness between the two portions of rat vas deferens.

Pharmacological characterization of two distinct α1‐adrenoceptor subtypes in rabbit thoracic aorta

1 α1‐Adrenoceptor subtypes in rabbit thoracic aorta have been examined in binding and functional experiments. 2 [3H]‐prazosin bound to two distinct populations of α1‐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 μm) 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 μm) and methoxamine (0.1–100 μm) 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 μm) slightly attenuated the contractile responses to noradrenaline and methoxamine in CEC‐untreated and pretreated aortae, suggesting that nifedipine cannot discriminate between α1‐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 α1‐adrenoceptor subtypes (presumably α1B and α1L subtypes according to recently proposed subclassification), both of which are involved in noradrenaline‐induced contraction. The α1L subtype predominantly mediates the contraction induced by methoxamine.

Three distinct binding sites for [3H]-prazosin in the rat cerebral cortex

1 The putative α1‐adrenoceptor subtypes of rat cerebral cortex membranes were characterized in binding experiments with [3H]‐prazosin. 2 Specific binding of [3H]‐prazosin was saturable between 20–5000 pm. Scatchard plots of the binding data were non‐linear, indicating the presence of two distinct affinity sites for prazosin (pKD, high = 10.18, Rhigh = 308 fmol mg−1 protein; pKD, low = 8.96, Rlow = 221 fmol mg−1 protein). 3 In the membranes pretreated with chlorethylclonidine (CEC) two affinity sites for prazosin were also observed: the affinities were similar to those without CEC pretreatment, but the maximum numbers of binding sites were reduced by CEC pretreatment to 23 and 62% for prazosin‐high (Rhigh) and low affinity sites (Rlow), respectively. 4 The prazosin‐high affinity sites were further subdivided into two subclasses by WB4101(2‐(2,6‐dimethoxyphenoxyethyl)aminomethyl‐1,4‐benzodioxane) and phentolamine; the low affinity sites for WB4101 and phentolamine were more potently inactivated by CEC as compared with the high affinity sites. On the other hand, prazosin, HV723 (α‐ethyl‐3,4,5‐trimethoxy‐α‐(3‐((2‐(2‐methoxyphenoxy)ethyl)‐amino)‐propyl)benzeneacetonitrile fumarate) and yohimbine inhibited [3H]‐prazosin binding to prazosin‐high affinity sites monophasically. 5 In addition to the high affinity sites, the prazosin‐low affinity sites were labelled at high concentrations of [3H]‐prazosin. Thus, prazosin and WB4101 showed shallow displacement curves. On the other hand, HV723 and yohimbine did not discriminate between prazosin‐high and low affinity sites. 6 Two distinct α1‐adrenoceptor subclassifications have been recently proposed (α1A, α1B subtypes and α1H, α1L, α1N subtypes). According to the criteria defined with competitive antagonists in both the subclassifications, the present results indicate that the α1‐adrenoceptors of rat cerebral cortex consist of three different subtypes, presumably α1A, α1B and α1L, and suggest that the α1A and α1B subtypes are identified as a single affinity site for prazosin (α1H). The results also indicate that care must be taken in the use of CEC for α1‐adrenoceptor subclassification because of its low selectivity.

Two distinct α1‐adrenoceptor subtypes involved in noradrenaline contraction of the rabbit thoracic aorta

1 Recently, α1‐adrenoceptors in blood vessels have been classified into three subtypes (α1H, α1L and α1N). We examined which subtype (or subtypes) is involved in the noradrenaline‐induced contraction of rabbit thoracic aorta. 2 Noradrenaline produced a concentration‐dependent contraction in the rabbit isolated thoracic aorta. Prazosin antagonized the contractions to noradrenaline, resulting in a rightward displacement of the concentration‐response curve. However, the shift was not proportional to the concentration of prazosin; Schild plots showed that the inhibition by prazosin was biphasic, implying that noradrenaline acted through two receptor populations. Two affinity constants (pKB values of 10.02 and 8.83) were determined for prazosin at these sites. 3 However, under continuous treatment with 1 nm prazosin, or in strips pretreated with chlorethylclonidine (CEC; an α1H inactivating agent) to remove the contribution of one receptor population, prazosin showed a single pKB or pA2 value of approximately 8.3. 4 Yohimbine also produced biphasic antagonism of noradrenaline‐induced contractions, resulting in two affinity constants (pKB = 6.52 and 6.17). However, a monophasic Schild plot was obtained for yohimbine either in the presence of 1 nm prazosin (pA2 = 6.08) or in strips pretreated with CEC (pA2 = 6.03). 5 The Schild plot for HV723 (a selective α1N‐antagonist) yielded a monophasic slope (pKB = 8.47) and the inhibition was not affected by 1 nm prazosin or CEC‐pretreatment. 6 [3H]‐prazosin bound to α1‐adrenoceptors of the aortic membrane preparations with two different affinities (pKD = 9.94 and 8.37). The high but not the low affinity site was completely masked by 1 nm prazosin and inactivated by pretreatment with CEC. 7 These results strongly suggest that noradrenaline‐induced contraction of the rabbit thoracic aorta is mediated through two distinct α1‐adrenoceptor subtypes, designated α1H and α1L.

α1-Adrenoceptor subtypes and two receptor systems in vascular tissues

The subtypes of alpha1-adrenoceptor are coexpressed in many tissues. We examined the relationship between coexpressed alpha1-adrenoceptor subtypes and their functions in blood vessels. Rat and rabbit aortas coexpressed three subtypes (alpha1A, alpha1B, alpha1D) and four subtypes (alpha1A, alpha1B, alpha1D, alpha1L), respectively. In rat aorta however, noradrenaline-induced contraction was mediated predominantly through the alpha1D subtype, and oxymetazoline produced alpha1B-mediated contraction. In rabbit aorta, concentration-response curves for noradrenaline were composed of two components (alpha1B and alpha1L-mediated), while oxymetazoline produced alpha1L-mediated contraction. Therefore, the inhibitory actions of some antagonists varied markedly among tissues and agonists. These results demonstrate diversity of the two receptor systems and suggest that the heterogeneity of physiological responses reflects the differences in functional subtypes among tissues and in their sensitivities to agonists and antagonists.

Binding and functional characterization of α1-adrenoceptor subtypes in the rat prostate

The alpha1-adrenoceptor subtypes of rat prostate were characterized in binding and functional experiments. In binding experiments, [3H]tamsulosin bound to a single class of binding sites with an affinity (pKD) of 10.79+/-0.04 and Bmax of 87+/-2 fmol mg(-1) protein. This binding was inhibited by prazosin, 2-(2,6-dimethoxy-phenoxyethyl)-aminomethyl-1,4-benzodioxane hydrochloride (WB4101), 5-methylurapidil, alpha-ethyl-3,4,5,-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amin o)-propyl)benzeneacetonitrile fumarate (HV723) and oxymetazoline with high efficacy, resulting in a good correlation with the binding characteristics of cloned alpha1a but not alpha1b and alpha1d-adrenoceptor subtypes. In functional studies, noradrenaline and oxymetazoline produced concentration-dependent contractions. These contractions were antagonized by tamsulosin, prazosin, WB4101 and 5-methylurapidil with an efficacy lower than that exhibited by these agents for inhibition of [3H]tamsulosin binding. The relationship between receptor occupancy and contractile amplitude revealed the presence of receptor reserve for noradrenaline, but the contraction induced by oxymetazoline was not in parallel with receptor occupation and developed after predicted receptor saturation. From these results, it is suggested that alpha1A-adrenoceptors are the dominant subtype in the rat prostate which can be detected with [3H]tamsulosin, but that the functional subtype mediating adrenergic contractions has the characteristics of the alpha1L-adrenoceptor subtype, having a lower affinity for prazosin and some other drugs than the alpha1A-adrenoceptor subtype.

Characterization of β‐adrenoceptors in urinary bladder: comparison between rat and rabbit

1 β‐Adrenoceptor subtypes in rat and rabbit urinary bladder were investigated in functional experiments by use of several agonists and antagonists. 2 All agonists tested produced concentration‐dependent relaxation, but the relative potencies varied between both species: BRL  37344 (pD2:8.0)>isoprenaline (7.3)>adrenaline (6.7)=noradrenaline (6.6) in rat bladder, and isoprenaline (8.7)=adrenaline (8.5)>noradrenaline (7.7)=BRL  37344 (7.4) in rabbit bladder. 3 The relaxation response to isoprenaline in rat bladder was relatively resistant to propranolol and ICI  118551, and the slopes of Schild plot for both antagonists were different from unity. The apparent pKB values estimated by single concentrations of propranolol (1, 10 μm) and ICI  118551(10 μm) were 6.6 and 5.4, respectively. 4 On the other hand, the relaxation response to isoprenaline in rabbit bladder was antagonized by lower concentrations (1 nm–100 nm) of propranolol and ICI  118551 in a competitive manner, resulting in pA2 values of 8.7 and 8.6, respectively. 5 These results suggest species‐heterogeneity of β‐adrenoceptors in urinary bladder; β3 and β2 subtypes in rat and β2 subtype in rabbit.

Cloning, functional expression and tissue distribution of rabbit alpha 1d-adrenoceptor.

We have cloned a cDNA encoding rabbit alpha 1d-adrenoceptor from the rabbit liver cDNA library. The deduced amino-acid sequence of this clone encodes a protein of 576 amino acids that shows strong sequence homology to previously cloned human, rat and mouse alpha 1d-adrenoceptors. The pharmacological radioligand binding properties of this clone expressed in COS-7 cells were similar to those of rat alpha 1d-adrenoceptors. Competitive RT/PCR assays revealed wide tissue distribution of the alpha 1d-adrenoceptor mRNA in rabbit, especially abundant in vas deferens, aorta, prostate and cerebral cortex.

Alteration of alpha and muscarinic receptors in rat brain and heart following chronic nicotine treatment

Adrenergic and muscarinic binding sites in 4 brain regions (cerebral cortex, corpus striatum, hypothalamus/thalamus and brainstem) and in heart ventricles were measured in rats chronically treated with nicotine added to the drinking water in doses ranging from 6 to 8 mg/kg/day, for 4 weeks. Control rats received only tap water. The nicotine treatment led to increases in the specific binding of both [3H]prazosin and [3H]clonidine in the cerebral cortex. An increase in [3H]prazosin binding was also observed in the hypothalamus/thalamus of nicotine-treated rats. These changes were all due to an increase of about 23% in Bmax. In the brainstem and heart left ventricle, respectively, an increase and a decrease in the affinity of [3H]quinuclidinyl benzilate binding were observed. There were no changes of the binding parameters for the 3 radioligands in other regions tested, and no alteration of [3H]dihydroalprenolol binding was detected in any region examined. These results indicate that chronic administration of nicotine causes an increase in the density of alpha 1- and alpha 2-binding sites in some brain regions and reciprocal changes of the affinity of muscarinic binding sites in the brain and in the heart.