Shigeru Kigoshi

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.

Nitric oxide-dependent and -independent neurogenic relaxation of isolated dog urethra

In the presence of adrenergic and cholinergic blocking agents, transmural electrical stimulation evoked a relaxation in isolated dog urethra precontracted with histamine. The response was abolished by tetrodotoxin, indicating its neurogenic origin. The non-adrenergic and non-cholinergic relaxation developed rapidly and was transient at low stimulation frequencies (< or = 1 Hz). However, at higher frequencies (> or = 5 Hz) the recovery phase of the relaxation became slow and often showed a notch, suggesting the presence of transient and slow components. NG-Monomethyl-L-arginine, a nitric oxide synthase inhibitor, inhibited the transient relaxation but did not affect the relaxation evoked at high stimulation frequencies. NG-Nitro-L-arginine, a more potent nitric oxide synthase inhibitor, abolished the transient relaxation produced at low stimulation frequencies and markedly attenuated the transient component at high frequencies. However, NG-nitro-L-arginine did not affect the slow component. The inhibition by NG-monomethyl-L-arginine and NG-nitro-L-arginine was reversed by the addition of L- but not D-arginine. Exogenously applied vasoactive intestinal polypeptide (VIP) produced a slowly developing relaxation. The slow relaxation induced by transmural electrical stimulation and VIP was not affected by [4-Cl-D-Phe6,Leu17]VIP, a reportedly competitive VIP antagonist. NG-Nitro-L-arginine did not affect the relaxation induced by VIP and sodium nitroprusside. These results suggest that the non-adrenergic and non-cholinergic relaxation induced by transmural electrical stimulation is composed of nitric oxide-dependent and -independent components in the isolated dog urethra.

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.

Single ionic channels induced by palytoxin in guinea-pig ventricular myocytes.

1 Mechanisms of palytoxin‐induced ion permeability were examined in isolated single ventricular cells of guinea‐pig under whole‐cell‐attached patch clamp conditions. 2 Palytoxin (1–2 × 10−11 m, dissolved in Tyrode solution and put in the patch electrode) induced an elementary current flowing through single channels. Direction of the current was inward and the amplitude was 0.65 ± 0.03 pA (mean ± s.e. mean) at the resting membrane potential. The amplitude increased linearly with membrane hyperpolarization and decreased with depolarization; the single channel conductance was 9.5 ± 0.5 pS. 3 Palytoxin‐induced single channel current was resistant to tetrodotoxin (5 × 10−5 m) or cobalt ions (2 × 10−3 m) and was observed under Ca‐free conditions. However, no channel current was induced by palytoxin (10−11‐10−9 m) dissolved in Na+‐free, choline‐Tyrode solution. 4 Palytoxin also induced single channel currents in Na+‐free, NH+4‐, Li+‐ or Cs+‐Tyrode solution, and the slope conductances were 16.5 ± 1.6 pS, 9.2 ± 0.7 pS and 11.0 ± 0.7 pS, respectively. 5 These results indicate that palytoxin forms a new type of ionic channel with unique ion selectivity and gating behaviour.

Neurogenic responses of urethra isolated from the dog

Electrical transmural stimulation evoked contraction and relaxation in isolated urethral circular muscle of the dog. The responses were abolished by tetrodotoxin, indicating their neurogenic origin. The contractile force in the middle urethra was greater than that in the proximal and distal urethra. The contractions were not affected by atropine and propranolol, but were completely inhibited by phenoxybenzamine, prazosin and guanethidine. In preparations contracted with prostaglandin F2 alpha, electrical stimulation induced frequency-dependent relaxation in all urethral portions. Atropine, phenoxybenzamine, prazosin and guanethidine had no effect on the relaxation, while propranolol slightly attenuated the relaxation induced at the highest frequency used (5 Hz). The non-adrenergic, non-cholinergic relaxation was also not affected by ketanserin, methysergide, diphenhydramine, alpha,beta-methylene ATP or capsaicin. Exogenously applied phenylephrine and clonidine both produced contractions but the maximal response to clonidine was much smaller than that to phenylephrine. Acetylcholine produced no or feeble contractions. In the preparations contracted with prostaglandin F2 alpha, isoproterenol and vasoactive intestinal polypeptide (VIP) produced relaxation. These results suggest that the circular muscle of dog urethra is reciprocally innervated by sympathetic adrenergic and non-adrenergic, non-cholinergic nerves, and that the neurogenic responses are markedly affected by muscle tension and the portion of the urethra examined.

Purinergic and non‐purinergic innervation in the cerebral arteries of the dog

1 Possible involvement of sympathetic purinergic transmission in the neurogenic response of dog cerebral and basilar arteries was examined with the use of α,β‐methylene ATP and adrenoceptor, cholinoceptor blocking agents. 2 In the isolated basilar arteries, electrical transmural stimulation produced a transient contraction which was frequently followed by a relaxation. This transient contraction was abolished after desensitization of P2‐purinoceptors with α,β‐methylene ATP or by treatment with guanethidine. The relaxant response induced by electrical stimulation was also attenuated but was not abolished by such treatments. Prazosin, propranolol and atropine had no significant effect on the responses to electrical stimulation. Yohimbine augmented both the contractile and relaxant responses. 3 In most preparations of the dog middle cerebral arteries, electrical transmural stimulation produced only a relaxation. This relaxation was little affected after treatment with α,β‐methylene ATP or guanethidine, and was not inhibited by the other adrenoceptor and cholinoceptor blocking agents. 4 Tetrodotoxin abolished the responses induced by electrical transmural stimulation in both the basilar and middle cerebral arteries. 5 Exogenous ATP (10−6 and 10−5 m) produced a transient contraction followed by a relaxation of the basilar arteries and a relaxation of the middle cerebral arteries. Desensitization of P2‐purinoceptors abolished the contractile response to ATP without affecting the amplitude of relaxation. 6 In the basilar and middle cerebral arteries preincubated with [3H]‐noradrenaline, electrical transmural stimulation evoked an increase in 3H‐efflux and this response was markedly inhibited by guanethidine or tetrodotoxin but was not affected by α,β‐methylene ATP. Yohimbine increased the evoked 3H‐efflux. 7 These findings indicate that cerebral arteries of the dog are innervated by sympathetic purinergic nerves and non‐sympathetic nerves which liberate unknown vasodilator substance(s), and that the former nerves are more dominant in the neurogenic response to electrical stimulation of the dog basilar artery than in the middle cerebral artery.

Electrophysiological and mechanical effects of calcitonin gene‐related peptide on guinea‐pig atria

1 The effects of calcitonin gene‐related peptide (CGRP) on mechanical and electrophysiological responses were studied in the guinea‐pig atrial muscle preparations and in single cells. 2 CGRP (>10−9m) enhanced the twitch contraction in a concentration‐dependent manner in electrically driven left atria and increased heart rate in spontaneously beating right atria. The positive inotropic and chronotropic effects of CGRP were not inhibited by propranolol but were attenuated by reduction of the calcium concentration in the bathing medium. 3 In single left atrial cells, CGRP slightly hyperpolarized the resting potential but did not affect the other action potential parameters significantly. 4 Under whole‐cell voltage‐clamp conditions, CGRP increased the calcium inward current. The peptide also increased the steady inward current elicited by hyperpolarization and the late outward current by depolarization. 5 These results suggest that CGRP may produce the positive inotropic and presumably chronotropic effects by increasing calcium inward current. CGRP also increases the potassium permeability. Such effects on ionic currents may not produce any apparent change in the action potential conformation, due to their opposite directional actions and relatively weak potencies.