Heterogeneity of neurogenic responses in intra- and extrameningeal arteries of dogs.

1. Neurogenic responses to transmural electrical stimulation were examined in endothelium-denuded extrameningeal (vertebral and carotid) and intrameningeal (spinal, basilar and middle cerebral) arteries isolated from dogs. 2. In the extrameningeal arteries, transmural electrical stimulation produced a phasic contraction. This contraction was abolished by tetrodotoxin, prazosin and guanethidine. However, alpha,beta-methylene ATP and NG-nitro-L-arginine (L-NOARG) had no significant effect on the contractile responses. 3. In the intrameningeal arteries, the neurogenic responses to electrical stimulation were composed of a transient contraction and relaxation. The transient contraction was selectively inhibited by guanethidine L-NOARG abolished the relaxation but not the contraction induced by electrical stimulation. Prazosin had no effect on either neurogenic response. 4. Noradrenaline produced a large contraction in the extrameningeal arteries which was selectively inhibited by prazosin. alpha,beta-Methylene ATP produced neither contraction nor inhibition of the response to noradrenaline in the extrameningeal arteries. 5. In the intrameningeal arteries, alpha,beta-methylene ATP produced a greater contraction than noradrenaline. The response to alpha,beta-methylene ATP was selectively abolished by desensitization of P2x-purinoceptors with alpha,beta-methylene ATP itself. The contractile response to noradrenaline was inhibited by rauwolscine but not by prazosin. 6. ATP produced endothelium-dependent relaxations in the extrameningeal and intrameningeal arteries, which were attenuated by endothelium removal. 7. NADPH diaphorase-positive fibres were dense in the middle cerebral and basilar arteries but rare or absent in the spinal artery. In the extrameningeal arteries diaphorase-positive traces were observed in the vasa vasorum. 8. The present findings indicate that the neurogenic responses of intrameningeal arteries of dogs are composed of NO-ergic and sympathetic purinergic components, while the extrameningeal arteries tested produced only sympathetic adrenergic responses, suggesting that regional heterogeneity may be associated with a sudden transition in innervation and receptor expression at the meninx.

[The effects of treatment with lipopolysaccharide on responsiveness of rat blood vessels].

Effects of treatment with lipopolysaccharide (LPS) both in vivo (intraperitoneal administration of 20 mg.kg-1 LPS for 6 hours) and in vitro (incubation with 1 microgram.kg-1 LPS for 6 hours) on the responsiveness of the rat thoracic aorta, carotid, renal, femoral, mesenteric, pulmonary arteries, and the femoral and mesenteric veins were examined. Intraperitoneal administration of LPS did not change the blood pressure of rats, but increased the heart rate significantly. The same amplitude of relaxation was produced by L-arginine in the aortic strips treated by LPS in vivo and in vitro, and the responses were inhibited by NG-nitro-L-arginine (L-NOARG). The contractile responses to phenylephrine in the aortic strips were reduced by LPS-treatment in vivo or in vitro, but the extent of inhibition was greater in vivo than in vitro. Further, the attenuation of contractile responses to phenylephrine was completely reversed by L-NOARG in the strips treated with LPS in vitro, whereas the reversal by L-NOARG was incomplete in the strips treated with LPS in vivo. Different amplitudes of relaxations were also produced by L-arginine or SNP in the blood vessels treated by LPS in vivo or in vitro. However, the tail artery treated with LPS in vivo or in vitro did not relax in response to L-arginine but did produce a relaxation to SNP. These results suggest that the hyporesponsiveness of rat blood vessels after treatment with LPS in vivo or in vitro may be related to an enhanced NO production in the smooth muscle cells and that there is a possible heterogeneity of regional induction or activation of L-arginine/NO pathway by LPS in rat blood vessels.

Pharmacological characterization of alpha 1-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.

Effects of propranolol and atenolol on immobilization stress-induced hypertension and down-regulation of central beta-adrenoceptors in rats.

Effects of chronic treatment with propranolol or atenolol on stress-induced changes in blood pressure, body weight, and cerebral beta-adrenoceptors in rats were examined and compared with the effects of chronic treatment with prazosin. Immobilization stress (2 h daily for 2 weeks) induced a moderate elevation of blood pressure, loss of body weight gain, and downregulation of cerebral beta-adrenoceptors, but produced no changes in the cerebral alpha 1-adrenoceptors. Chronic administration of propranolol (5 or 50 mg.kg-1), atenolol (5 or 50 mg.kg-1) or prazosin (2 or 20 mg.kg-1) inhibited stress-induced hypertension but did not affect loss of body weight gain. Propranolol increased the density of cerebral beta-adrenoceptors by 77% and reduced the downregulation induced by stress. Atenolol also increased the density of cerebral beta-adrenoceptors by 34% and abolished the stress-induced downregulation in cerebral beta-adrenoceptor density. In contrast, prazosin had no effect on the cerebral beta-adrenoceptors in nonstressed or stressed rats. These results suggest that the antihypertensive action of propranolol and atenolol may be partly associated with the inhibition of stress-activated central beta-adrenoceptor transmission.

Pharmacological profiles of a novel alpha 1-adrenoceptor agonist, PNO-49B, at alpha 1-adrenoceptor subtypes.

The effects of a newly synthesized compound, PNO-49B, (R)-(-)-3'-(2-amino-1-hydroxyethyl)-4'-fluoromethanesulfonanilide hydrochloride, on alpha 1-adrenoceptor subtypes were examined in various tissues in which the following distribution of alpha 1-adrenoceptor subtypes has been suggested: dog carotid artery (alpha 1B), dog mesenteric artery (alpha 1N), rabbit thoracic aorta (alpha 1B + alpha 1L), rat liver (alpha 1B), rat vas deferens (alpha 1A + alpha 1L), rat cerebral cortex (alpha 1A + alpha 1B) and rat thoracic aorta (controversial subtype). PNO-49B (0.1-100 microM) produced concentration-dependent contractions in dog mesenteric artery, rabbit thoracic aorta, rat thoracic aorta and rat vas deferens; and the maximal amplitudes of contraction were almost the same as or slightly less than those of noradrenaline. By contrast, the maximal response to PNO-49B in dog carotid artery was markedly smaller than the response to noradrenaline. In rabbit thoracic aorta, the contractile response to PNO-49B was not affected by inactivation of the alpha 1B subtype with chloroethylclonidine (CEC), although the response to noradrenaline was attenuated by that treatment. The dissociation constants (KA) of PNO-49B were not different among the rat thoracic aorta, dog carotid and mesenteric arteries and rabbit thoracic aorta (CEC-pretreated). The contractile responses to PNO-49B were inhibited competitively by prazosin, HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)-ethyl)- amino(propyl)benzeneacetonitrile fumarate) and by WB4101 (2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4- benzodioxane).(ABSTRACT TRUNCATED AT 250 WORDS)

Selective inhibition of sympathetic nerve-mediated contraction by L-arginine in lipopolysaccharide-treated tail artery of rats.

The effects of L-arginine on the adrenergic responses to either electrical transmural stimulation or phenylephrine were studied in isolated endothelium-denuded strips of rat tail arteries treated with lipopolysaccharide for 6 h in vitro. L-arginine did not relax the strips precontracted by phenylephrine. However, the adrenergic contractions induced by electrical transmural stimulation were significantly inhibited by the addition of L-arginine. This inhibitory effect was reversed by NG-nitro-L-arginine (a nitric oxide synthase inhibitor) or methylene blue (a soluble guanylate cyclase inhibitor) but was not affected by hemoglobin (a scavenger of nitric oxide). These results indicate that the adrenergic neurogenic contractions may be directly modulated by nitric oxide derived from the sympathetic nerves and/or neighboring cells in the lipopolysaccharide-treated rat tail arteries, and the nitric oxide production may be associated with the reduction of sympathetic tone in sepsis.

Attenuation of sodium nitroprusside responses after prolonged incubation of rat aorta with endotoxin.

We investigated the effects of prolonged treatment with Escherichia coli lipopolysaccharide (LPS) on the responses to sodium nitroprusside (SNP) in endothelium-denuded rat aortic strips. Incubation of the aortic strips with LPS for 24 h dramatically attenuated relaxation and guanosine 3',5'-cyclic monophosphate (cGMP) formation by SNP, which were significantly restored by the inhibition of nitric oxide (NO) production with N omega-nitro-L-arginine. In the aorta coincubated with LPS and protein synthesis inhibitor (dexamethasone or cycloheximide, which prevents induction of endotoxin-inducible NO synthase), no attenuation of the relaxation was observed and the cGMP formation was significantly restored. Relaxation response to 8-bromo-cGMP or papaverine was not attenuated, even after 24 h of incubation. These results suggest that the attenuation of SNP responses is mainly associated with a decrease in the activation of guanylate cyclase (GC) as a consequence of the prolonged exposure to muscle-derived NO. Moreover SNP in the presence of methylene blue evoked a small but apparent relaxation of 24-h-incubated aorta without significant elevation of cGMP, suggesting the involvement of cGMP-independent pathways in the remaining relaxation produced by SNP.

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

OBJECTIVE: To characterize the alpha 1-adrenoceptor subtypes of the human benignly enlarged prostate using functional and binding studies. MATERIALS AND METHODS: Strips of prostatic tissue taken from nine patients with benign prostatic hypertrophy who were undergoing open prostatectomy were used in the study. RESULTS: 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. CONCLUSIONS: 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.

Effects of chronic diabetes on the responsiveness to endothelin-1 and other agents of rat atria and thoracic aorta.

1. Plasma endothelin-1 level as well as plasma lipid and glucose levels was markedly elevated in the rats treated with streptozotocin 8 weeks earlier. 2. Positive inotropic response, which was significantly greater than that in the age-matched control, was produced by endothelin-1 in the left atria isolated from diabetic rats. On the other hand, the contractile response of thoracic aorta to endothelin-1 was conversely attenuated in the diabetic rats. Such contrasting effects of diabetes between the atrial and aortic muscles were observed in the responsiveness to other contractile drugs. Endothelium-dependent relaxation in the thoracic aorta was also significantly attenuated in diabetic rats. However, the basal twitch contraction of left atria, the chronotropic responses to endothelin-1, isoproterenol and carbachol and the relaxation of the aorta by papaverine were not affected by diabetes. 3. These results suggest that the contractile responsiveness of atrial and aortic muscles and the endothelial functions significantly alter during diabetes for 8 weeks.

Evidence for sympathetic, purinergic transmission in the iris dilator muscle of the rabbit.

Electrical transmural stimulation of isolated iris dilator muscle of the rabbit produced a transient contraction that consisted of adrenergic and nonadrenergic components. In contrast to the adrenergic component, the nonadrenergic component was resistant to prazosin and other adrenoceptor antagonists. However, both components were completely blocked by guanethidine or tetrodotoxin. Among some tested compounds including neuropeptide Y, both ATP and 2-methylthio ATP produced a transient contraction in the dilator muscle and the sustained treatment with each markedly attenuated the nonadrenergic responses to electrical stimulation and to ATP. Suramin had no effect on and alpha,beta-methylene ATP potentiated the responses to electrical stimulation and to ATP. These results suggest that the nonadrenergic contraction induced by electrical transmural stimulation is a sympathetic purinergic response that may be mediated through unique purinoceptors.

Heterogeneity of alpha 1-adrenoceptor subtypes involved in adrenergic contractions of dog blood vessels.

1. We determined the alpha 1-adrenoceptor subtypes involved in adrenergic contractions of eight different blood vessels isolated from the dog. 2. Noradrenaline produced concentration-dependent contractions in all the blood vessels tested, which were competitively inhibited by prazosin, WB4101, HV723 and 5-methylurapidil. However, there was considerable difference between the vessels with regard to the pKB values for all the antagonists. The alpha 1-adrenoceptors of dog vertebral and carotid arteries had high affinity for prazosin (pKB > 9.0) but low affinity for WB4101 (< 8.5), 5-methylurapidil (< 7.5) and HV723 (< or = 8.5). By contrast, HV723 had higher affinity (> 9.0) than prazosin (< 8.3), WB4101 (< 8.7) and 5-methylurapidil (< 8.2) in the portal vein, mesenteric artery and vein, and renal artery. In the femoral artery and vein, however, the four antagonists showed pKB values in the range 8.0-8.7. 3. Chloroethylclonidine (10 microM) produced a remarkable reduction of the contractile responses to noradrenaline in the vertebral and carotid arteries as compared with those in the other vessels. Nifedipine inhibited the responses to noradrenaline in all the tissues tested, and had marked effects in the portal vein. 4. Sympathetic adrenergic contractions induced by transmural electrical stimulation were also inhibited by prazosin and HV723 at different potencies among tissues. The relative potencies of both the antagonists paralleled the relationship in inhibiting the responses to exogenous noradrenaline in each vessel. 5. According to recent alpha l-adrenoceptor subclassification, the present results suggest that the contractions of blood vessels induced by endogenous and exogenous noradrenaline are mediated through different alpha l-adrenoceptor subtypes heterogeneously distributed in each vessel; presumably, the alpha 1 B subtype in the carotid and vertebral arteries, the alpha IN subtype in the visceral region and the alpha IL subtype in the femoral region. Regionally different expression of alpha 1-adrenoceptor subtypes may be in part associated with the regional heterogeneity of sympathetic responses in the blood vessels.

Change in the contractility of rat atrial muscle by cold preservation.

Cold preservation of isolated mammalian hearts attenuates cardiac contractility. We examined the effect of intracellular and extracellular Ca ion on the contractility of cold-preserved atria of rat. Rat hearts were isolated and stored in Collins' solution at 4 degrees C for either 0 or 12 h. Mechanical responses were then examined in electrically driven left and beating right atrial muscle preparations. When stimulus frequencies were increased stepwise from 0.1 to 3 Hz, the twitch contractions initially decreased (Woodworth phenomenon: WP) and then increased (Bowditch phenomenon: BP) in 0 hour-preserved (fresh) left atrial preparations. However, BP, but not WP, was abolished in 12 hour-preserved preparations. The absence of BP was also observed when fresh preparations were exposed to a low-Ca medium (0.9 mM). Ryanodine (3 x 10(-8) M) abolished the twitch contractions at low stimulus frequencies, but did not inhibit BP in fresh preparations. In 12 hour-preserved preparations, both ryanodine and low-Ca medium completely inhibited the twitch contraction. The positive inotropic effects of isoproterenol in right and left atria were markedly attenuated after 12-hour cold preservation. However, the rate of spontaneous beating and the positive chronotropic effect of isoproterenol in right atria were not changed in the cold-preserved preparations or in fresh preparations that had been treated with ryanodine or exposed to a low-Ca medium. These results clearly show that cold-preserved atrial muscle is more susceptible to changes in intracellular and extracellular Ca ion and suggest that the attenuation of cardiac contractility by cold preservation may be due to the impairment of Ca2+ influx which may result from membrane deterioration.

Comparison between the effects of treatment in vitro and in vivo with lipopolysaccharide on responsiveness of rat thoracic aorta.

Effects of treatment in vivo and in vitro with lipopolysaccharide (LPS) on the responsiveness of rat thoracic aorta were examined. The endothelium-denuded aortic strips isolated 6 hr after intraperitoneal (i.p.) administration of LPS (20 mg/kg), which could produce hemodynamic changes, relaxed in response to L-arginine. The same amplitude of relaxation was produced by L-arginine in the aortic strips incubated with a lower dose of LPS (1 microgram/ml) in vitro for 6 hr. Both relaxing responses were inhibited by NG-nitro-L-arginine (L-NOARG). The contractile responses to phenylephrine and KCl were reduced by LPS treatment in vivo or in vitro, but the extent of inhibition was greater in vivo than in vitro. Further, the attenuation of contractile responses was completely reversed by L-NOARG in the strips treated in vitro, whereas the reversal by L-NOARG was incomplete in the strips treated with LPS in vivo. Endothelium-dependent relaxation induced by acetylcholine was attenuated by LPS in vivo but not in vitro. These results suggest that the hyporesponsiveness of rat thoracic aorta after treatment in vitro with LPS, which can produce hemodynamic changes, may be related to an enhanced NO production in the smooth muscle cells, while not only the NO pathway but also additional factors may be involved in the vascular hyporesponsiveness of the sepsis rats.

Identification of alpha 1-adrenoceptor subtypes in rat lung by binding of [3H]-prazosin and [3H]-WB4101.

The alpha 1-adrenoceptor subtypes in rat lung were characterized according to their binding of [3H]-prazosin or [3H]-WB4101 and were compared with that in rat liver. [3H]-prazosin bound with high affinity to an apparently homogeneous population of sites in rat lung. The binding of [3H]-prazosin was inhibited by WB4101, benoxathian and 5-methylurapidil biphasically but the proportions differed between WB4101 or benoxathian and 5-methylurapidil. In the lung membranes pretreated with chloroethylclonidine a single population with high affinity for WB4101 and benoxathian was detected while 5-methylurapidil still discriminated two sites of distinctly different affinities. These results suggest that the WB4101-high affinity sites of rat lung were subdivided further into two subclasses according to 5-methylurapidil binding affinity. In fact, [3H]-WB4101 bound to lung membranes with two different affinities and the high affinity binding sites were subdivided by 5-methylurapidil into two classes. By contrast, [3H]-prazosin or [3H]-WB4101 binding sites of liver membranes were detected as a single population with high affinity for prazosin but with low affinity for WB4101, benoxathian and 5-methylurapidil. These results suggest that the alpha 1-adrenoceptors of rat lung are composed of three distinct subtypes (alpha 1A, alpha 1B and unknown subtypes) while that of liver is of alpha 1B subtype. Two radioligands with different affinities may be used as powerful probes to identify receptor subclasses.

Ca(2+)- and Na(+)-dependent depolarization induced by maitotoxin in the crayfish giant axon.

1. Maitotoxin (MTX) depolarized the membrane of the crayfish giant axon and decreased the amplitude and maximum rate of rise of action potentials in an irreversible manner. 2. The depolarizing action of MTX was attenuated in low Ca (1 mM; 1/10 of normal concentration) solution and was also inhibited by 2 mM Co2+, 300 microM Ni/+, 1 microM nifedipine, 10 microM verapamil or 1 microM tetrodotoxin. 3. These results suggest that the depolarization by MTX in the crayfish giant axon may be related to changes not only in Ca permeability but also in Na permeability, possibly through the modification of existing Na or Ca channels and/or a new type of channel or pore induced by MTX.

Alpha 1-adrenoceptor subtype involved in the positive inotropic response to phenylephrine in rat atria.

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.

Effects of bevantolol HCl on immobilization stress-induced hypertension and central beta-adrenoceptors in rats.

Effects of chronic treatment with bevantolol, a beta-adrenoceptor blocker, and of repeated immobilization stress on blood pressure, body weight, and [3H]dihydroalprenolol ([3H]DHA) binding to the cerebral cortex were examined in rats. Systolic blood pressure increased to approximately 150 mmHg when stress was applied for 14 days (2 h day-1). This increase was inhibited by chronic treatment with bevantolol (250 mg kg-1 daily). However, bevantolol did not suppress the inhibition of body weight gain by stress. The maximum number of [3H]DHA binding sites (Bmax) in the cerebral cortex was decreased by stress without changing the affinity, and the decrease in Bmax mainly reflected the reduction of beta 1-adrenoceptors. Bevantolol treatment (250 mg kg-1) increased the Bmax to 137% and completely inhibited the downregulation of beta-adrenoceptors by stress. These results show that bevantolol can inhibit both the hypertension and downregulation of the central beta 1-adrenoceptors induced by stress.

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.

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.

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.