hv-723 and chlorethylclonidine

This study was designed to further discriminate alpha1-adrenoceptor subtypes in rat aorta and prostate using functional experiments. Responses induced by phenylephrine were equilibrated in both tissues. The pA2 values and slope factors of several alpha1-antagonists were assessed using concentration-response curves. The antagonists used were prazosin, WB-4101, 5-methylurapidil (5-MU), HV-723, and tamsulosin. In addition, the effects of chloroethylclonidine (CEC) and nifedipine on phenylephrine-induced contractions were investigated. A high pA2 value for prazosin was observed in both tissues (aorta 9.84, prostate 9.19) and the ranking of each drug's pA2 value is as follows: tamsulosin > prazosin > WB-4101 > HV-723 > 5-MU in the aorta, and tamsulosin > prazosin > 5-MU > WB-4101 = HV-723 in the prostate. A significant difference between the pA2 value of each drug except for tamsulosin in the aorta and in prostate was observed (p < 0.01). Inhibition of contraction by pretreatment with CEC was 83.9 +/- 2.42% in the aorta, and 6.17 +/- 0.94% in the prostate. On the other hand, inhibition of maximal response by pretreatment with nifedipine (1 micromol/l) was 35.1 +/- 2.2% in the aorta and 24.5 +/- 3.1% in the prostate. A good correlation between these pA2 values and pKi values for recombinant human alpha1b-adrenoceptor expressed in CHO cells (aorta) and alpha1a-subtypes of CEC pretreated rat hippocampus (prostate) were observed. In conclusion, these results suggest that: (1) the contraction of these two tissues is mediated by alpha1H-adrenoceptor with a high affinity for prazosin; (2) alpha1H-adrenoceptors correspond to alpha1b-(aorta) and alpha1a-subtypes (prostate), and (3) each alpha1-adrenoceptor subtype in the aorta and prostate may be alpha1b-(aorta) and alpha1a-subtypes (prostate), respectively.

Topics: Acetonitriles; Adrenergic alpha-1 Receptor Antagonists; Adrenergic alpha-Antagonists; Animals; Aorta; Clonidine; Dioxanes; Dose-Response Relationship, Drug; Male; Muscle Contraction; Muscle, Smooth; Nifedipine; Piperazines; Prazosin; Prostate; Rats; Rats, Wistar; Receptors, Adrenergic, alpha-1; Sulfonamides; Tamsulosin; Vasoconstriction; Vasodilator Agents

1. Cutaneous resistance arteries (c.r.a.) (internal diameter=240.94+/-5.42 microm, n=67/25 (number arteries/number animals)) from New Zealand white rabbits were mounted in wire myographs and a normalization procedure followed. 2. Cumulative concentration-response curves (CCRCs) were constructed for the alpha-adrenoceptor agonists noradrenaline (NA), (R)A61603 and phenylephrine (PE) in the presence of cocaine (3 microM), propranolol (1 microM) and corticosterone (10 microM). The effects of competitive alpha1-adrenoceptor antagonists, prazosin, WB4101, 5-methyl-urapidil, HV723, BMY7378 and the irreversible alpha1B selective compound chloroethylclonidine (CEC) were examined versus the potency and maximum response of the c.r.a.s to noradrenaline. 3. The high potency of A-61603 relative to PE has been shown to differentiate both functional and binding site alpha1A- or alpha1B-adrenoceptors from alpha1D-adrenoceptors: A-61603 was 944 times more potent than phenylephrine (at EC50) suggesting the presence of a functional alpha1A or alpha1B as opposed to an alpha1D-subtype. 4. Exposure to chloroethylclonidine (CEC; 100 microM) decreased the maximum response to noradrenaline but did not significantly change noradrenaline sensitivity indicating that a substantial part of noradrenaline-induced vasoconstriction in rabbit cutaneous arteries is CEC-insensitive. 5. The potencies of prazosin (pA2=9.14) and WB4101 (pA2=9.30) indicate the involvement of prazosin-sensitive functional alpha1-adrenoceptors. The slopes of corresponding Schild plots for prazosin and WB4101 did not include negative unity which implies the possible involvement of more than one functional alpha1-adrenoceptor subtype in noradrenaline-induced vasoconstriction in rabbit cutaneous resistance arteries. In contrast to this, in the case of 5-methyl-urapidil and HV723, the Schild plot slope parameters were not significantly different from negative unity over the range of concentrations used; the low pA2 value for 5-methylurapidil (7.27) suggests the non-involvement of an alpha1A- or an alpha1D-adrenoceptor; the low pA2 value for HV723 (8.47) was similar to that against responses postulated as alpha1L. 6. We conclude that rabbit cutaneous resistance arteries express a prazosin-sensitive functional alpha1-adrenoceptor resembling the alpha1B and another low affinity site for prazosin which on the basis of the functional antagonism produced by HV723 most closely resembles the alpha1L-adrenoceptor; the low pA2

Topics: Acetonitriles; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Arteries; Clonidine; Dioxanes; Dose-Response Relationship, Drug; Imidazoles; In Vitro Techniques; Male; Norepinephrine; Phenylephrine; Piperazines; Prazosin; Rabbits; Receptors, Adrenergic, alpha-1; Skin; Tetrahydronaphthalenes; Vascular Resistance; Vasoconstriction

Phenylephrine produced positive inotropic responses in isolated rat right and left atria. The responses were competitively inhibited by alpha 1-adrenoceptor antagonists (prazosin, WB4101 and HV723) with relatively low affinities (pA2 values close to 8.0). Chloroethylclonidine had no significant effect on the responses to phenylephrine. These results suggest that the positive inotropic response to phenylephrine in rat atria is mediated through alpha 1-adrenoceptors which cannot be defined by the alpha 1A, alpha 1B subclassification.

Topics: Acetonitriles; Adrenergic alpha-Antagonists; Animals; Clonidine; Dioxanes; Heart Atria; In Vitro Techniques; Male; Myocardial Contraction; Phenylephrine; Prazosin; Rats; Rats, Wistar; Receptors, Adrenergic, alpha

1. In vascular smooth muscle, alpha 1-adrenoceptors have been classified recently into two or three subtypes. We examined which alpha 1-adrenoceptor subtypes are involved in the noradrenaline-induced contraction of rat portal vein smooth muscle. 2. Binding studies with [3H]-prazosin in membranes from equine portal vein smooth muscle revealed the presence of two distinct affinity binding sites. The high-affinity site for [3H]-prazosin was also identified in intact strips of rat portal vein. Prazosin, HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amin o)- propyl) benzene-acetonitrile fumarate), WB4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane), 5-methylurapidil, phentolamine and yohimbine antagonized [3H]-prazosin binding at both types of sites. Pretreatment with 50 microM chloroethylclonidine (CEC) eliminated the high-affinity sites for prazosin but had no effect on the low-affinity sites. 3. Noradrenaline produced a concentration-dependent contraction in the rat portal vein. Pretreatment with 50 microM CEC induced a slight rightward displacement of the concentration-response curve but the maximal contraction was not significantly affected suggesting that the CEC-sensitive alpha 1-adrenoceptors played a minor role in the noradrenaline-induced contraction. Prazosin, WB4101 and HV723 produced a concentration-dependent inhibition of noradrenaline-induced contractions. The inhibition curves were little affected by CEC-pretreatment and yielded a relative order of potency of WB4101 > prazosin > HV723. 4. In the presence of 0.1 microM isradipine to block voltage-dependent Ca2+ channels, the noradrenaline-induced contraction is due to release of Ca2+ ions from agonist-sensitive intracellular Ca2+ stores. Under these conditions, the noradrenaline-induced contraction was not significantly affected by pretreatment with 50 microM CEC but was inhibited by the antagonists mentioned above with affinities different from those in the absence of isradipine. The rank order of potency became HV723 > WB4101 > prazosin.5. The present results indicate the existence of two distinct o1-adrenoceptor subtypes in rat portal vein smooth muscle, which show high- and low-affinities respectively for each of prazosin, WB4101 andHV723 and correspond to alphalH- and alphalL-adrenoceptor subtypes. According to recent alpha1-adrenoceptor subclassifications, the alpha l H-adrenoceptor subtype which is sensitive to inactivation by CEC may correspond

Topics: Acetonitriles; Adrenergic alpha-Antagonists; Alkylating Agents; Animals; Clonidine; Dioxanes; Horses; In Vitro Techniques; Isometric Contraction; Kinetics; Microsomes; Muscle Contraction; Muscle, Smooth, Vascular; Norepinephrine; Portal Vein; Prazosin; Rats; Receptors, Adrenergic, alpha-1

1. The alpha 1-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)-amino-methyl-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 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-a min o)- 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 microM 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 microM 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 8.0to 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 LM nifedipine but were not affected by pretreatment with 1O LM CEC.6. Under conditions where P

Topics: Acetonitriles; Adrenergic alpha-Antagonists; Animals; Clonidine; Dioxanes; Electric Stimulation; Epididymis; In Vitro Techniques; Kinetics; Male; Muscle, Smooth; Nifedipine; Norepinephrine; Oxathiins; Prazosin; Prostate; Rats; Rats, Wistar; Receptors, Adrenergic, alpha; Vas Deferens

1. We examined the distribution of alpha 1-adrenoceptor subtypes and their relation to adrenergic neurogenic contraction induced by electrical transmural stimulation in the dog mesenteric and carotid arteries and the rabbit carotid artery. 2. In the dog mesenteric artery, contraction to noradrenaline was competitively inhibited by HV723 (pKB = 9.37) and prazosin (pKB = 8.40). Pretreatment with chlorethylclonidine (CEC) slightly attenuated only the contractions induced by low concentrations of noradrenaline. Contraction induced by electrical transmural stimulation was inhibited at lower concentrations of HV723 than of prazosin. 3. In the dog carotid artery, contraction to noradrenaline was inhibited with higher affinity by prazosin (pKB = 9.82) than by HV723 (pKB = 8.47). Prazosin was also more potent than HV723 in inhibiting the contraction to electrical stimulation. Pretreatment with CEC markedly attenuated or abolished contraction to noradrenaline and electrical stimulation. 4. In the rabbit carotid artery, prazosin inhibited noradrenaline-induced contraction biphasically (pKB = 9.91 and 8.60). After CEC pretreatment, contraction to noradrenaline was attenuated moderately and the high affinity site for prazosin was abolished. HV723 competitively inhibited the noradrenaline response with a similar pKB value (approximately 8.5) regardless of CEC treatment. Contraction to electrical stimulation was inhibited by prazosin more effectively than by HV723 in preparations not treated with CEC, while it was equipotently inhibited by both antagonists in CEC-treated preparations. 5. These results suggest that the contractions induced by endogenous and exogenous noradrenaline are mediated through the same subtypes of alpha,-adrenoceptor distributed in each artery; according to our recent subclassification: alpha 1N subtype in the dog mesenteric artery, alpha 1H subtype in the dog carotid artery and alpha lH and alpha 1L subtypes in the rabbit carotid artery. Different susceptibility to alpha l-adrenoceptor antagonists of sympathetic adrenergic responses in various blood vessels may be related to heterogeneous involvement of distinct alpha,-adrenoceptor subtypes in the sympathetic response.

Topics: Acetonitriles; Adrenergic alpha-Antagonists; Animals; Carotid Arteries; Clonidine; Dogs; Electric Stimulation; Female; In Vitro Techniques; Male; Mesenteric Arteries; Muscle Contraction; Muscle, Smooth, Vascular; Norepinephrine; Prazosin; Rabbits; Receptors, Adrenergic, alpha; Sympathetic Nervous System

1. We examined whether alpha 1-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 alpha l-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 alpha l-adrenoceptor-mediated contractions in all the blood vessels; these effects were not correlated to the above groups. 7. These results suggest that alpha,-adrenoceptors of blood vessels can be divided into three subtypes (designated as alpha 1H, alpha4L and alpha 1N) by antagonist affinity and their susceptibility to chloroethylclonidine but not to nifedipine: the characteristics of each subtype are summarized in Table 3. Subtypes alpha lH, alpha 1L and alpha lN may be predominantly involved in the contractile responses to noradrenaline or phenylephrine of the blood vessels in groups II, III and I, respectively.

Topics: Acetonitriles; Adrenergic alpha-Antagonists; Animals; Blood Vessels; Clonidine; Dioxanes; Dogs; Female; Guinea Pigs; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Nifedipine; Norepinephrine; Phentolamine; Prazosin; Rabbits; Rats; Receptors, Adrenergic, alpha; Species Specificity; Yohimbine

1. Recently, alpha 1-adrenoceptors in blood vessels have been classified into three subtypes (alpha 1H, alpha 1L and alpha 1N). 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 alpha 1H 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 alpha 1N-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 alpha 1-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 alpha l-adrenoceptor subtypes, designated alpha 1H and (alpha lL*

Topics: Acetonitriles; Adrenergic alpha-Antagonists; Animals; Aorta, Thoracic; Clonidine; Female; In Vitro Techniques; Male; Norepinephrine; Prazosin; Rabbits; Receptors, Adrenergic, alpha; Receptors, Adrenergic, alpha-1; Vasoconstriction; Yohimbine