Characteristics of Ca2+ oscillations in ileal longitudinal muscle cells of guinea pig.

We studied the mechanisms and characteristics of the spontaneously evoked intracellular Ca2+ changes (Ca2+ oscillations) in ileal longitudinal smooth muscle from guinea pig. Two-dimensional images of Ca2+ oscillations were obtained at 33-ms intervals with a Ca2+-sensitive fluorescence probe, fluo-3 using the intensified CCD camera. Nicardipine (10-7 M) significantly decreased the maximum level of fluorescence intensity of the Ca2+ oscillations, inhibited the frequency of the oscillations and tended to decrease the basal level of fluorescence intensity. However, tetrodotoxin (3 x 10-7 M) did not affect these oscillations. Phorbol 12,13-dibutyrate (10-7 M) significantly increased the maximum level of fluorescence intensity and the frequency of Ca2+ oscillations, and it changed them to steady and chronometric Ca2+ oscillations. Cyclopiazonic acid (3 x 10-5 M) also significantly increased the frequency of Ca2+ oscillations. Acetylcholine (10-8 M) increased the basal and maximum level of fluorescence intensity and the frequency of Ca2+ oscillations, and accelerated their onset. The increase of basal level of fluorescence intensity was then decreased by cyclopiazonic acid treatment. These results suggest that the augmentation of Ca2+ oscillations is mainly due to the activation of L-type Ca2+ channels, which is modulated by protein kinase C, and that the emptying of intracellular Ca2+ stores may activate the Ca2+ oscillations mediated through the increase of Ca2+ influx in ileal smooth muscle of guinea pig.

Analysis of alpha1-adrenoceptor subtypes in rabbit aorta and arteries: regional difference and co-existence.

This study was done to determine the alpha1-adrenoceptor subtypes and to characterize the functional role of alpha1D-adrenoceptors in the following rabbit arteries: thoracic and abdominal aorta, mesenteric, renal and iliac arteries. In all arteries, selective alpha1D-adrenoceptor antagonist BMY 7378 (8-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspirol(4,5) decane-7,9-dione dihydrochloride) dose dependently shifted the concentration-response curves for norepinephrine to the right. Schild plots of the results obtained from the inhibition by BMY 7378 for norepinephrine yielded a straight line with a slope of unity in thoracic (pA2 6.54+/-0.02) and abdominal (pA2 6.73+/-0.03) aorta. Slopes of Schild plots obtained from the inhibition by BMY 7378 for norepinephrine were significantly different from unity in mesenteric, renal and iliac arteries. Slopes of Schild plots for BMY 7378 were not different from unity in chloroethylclonidine-treated thoracic (pA2 6.49+/-0.14) and abdominal (pA2 6.61+/-0.11) aorta. Slopes of Schild plots for BMY 7378 were significantly different from unity in chloroethylclonidine-treated mesenteric, renal and iliac arteries. On the other hand, in Ca2+-free physiological saline solution (Ca2+-free PSS) slopes obtained from Schild plots for BMY 7378 were not different from unity in thoracic (pA2 6.41+/-0.09) and abdominal (pA2 6.28+/-0.07) aorta and mesenteric (pA2 6.55+/-0.06), renal (pA2 6.24+/-0.10) and iliac (pA2 6.64+/-0.13) arteries. BMY 7378 inhibited [3H]prazosin binding to thoracic (pKi 6.44+/-0.08) and abdominal (pKi 6.59+/-0.02) aorta with low potency, and mesenteric (pKi High 8.66+/-0.28, pKi Low 6.34+/-0.14), renal (pKi High 8.71+/-0.33, pKi Low 6.45+/-0.03) and iliac artery (pKi High 8.60+/-0.24, pKi Low 6.56+/-0.13). These results suggest that alpha1D-adrenoceptors play a significant role for contractile responses in renal and iliac artery, but play virtually no role in thoracic and abdominal aorta and that an alpha1-adrenoceptor subtype, which is pharmacologically distinguishable from the alpha1A-, alpha1B- and alpha1D-adrenoceptor subtype, may co-exist in mesenteric artery.

Dimensions of schizophrenic positive symptoms: an exploratory factor analysis investigation.

Current psychopathology classifies schizophrenic positive symptoms into four groups: delusions, hallucinations, formal thought disorder, and catatonic symptoms. The present study explores the factor structure of different positive symptoms to refine this classification. The 35 positive symptoms of 429 psychiatric patients, consecutively admitted to any of 95 mental hospitals, with diagnosis of the ICD-10 F20 schizophrenia, were studied. After excluding those items with a base rate of 10% or less, factor analysis yielded six factors. The first factor was loaded by most of Schneider's first-rank symptoms and two specific auditory hallucinations; the second by all the catatonic symptoms and incoherence; the third by bodily delusions/hallucinations; the fourth by delusions of persecution and reference; the fifth by grandiose and religious delusions; and the sixth by visual and miscellaneous hallucinations. The finding that schizophrenic positive symptoms may have more than four dimensions suggests the need for reclassification of schizophrenic symptoms and for reconsideration of evidence-based diagnostic criteria for the disorder.

Beta-funaltrexamine discriminates between two subtypes of mu2-opioid receptors in electrically stimulated longitudinal muscle of guinea pig ileum.

1. Morphine inhibited twitch responses of the longitudinal muscle of the guinea pig to electrical stimulation. A concentration-response curve of morphine was partly inhibited by a 60-min pretreatment of ileal strip with 10(-10) M of beta-funaltrexamine (beta-FNA). However, the 60-min pretreatment with a higher concentration (10(-9) M) of beta-FNA had no further significant inhibitory effect on the curve of morphine, suggesting that beta-FNA discriminated between two subtypes of mu2 receptors, beta-FNA-sensitive and -resistant receptors. 2. The mu2 receptors in synaptosomal fraction were selectively labeled by [3H]naloxone, and the labeled receptors were competitively inhibited by morphine. The competitive-inhibition curve of morphine showed the presence of high- and low-affinity sites. Beta-FNA eliminated the high-affinity site only when endogenous GTP or GTPgamma-S was present. 3. Beta-FNA discriminated between two subtypes of mu2 receptors.

The characteristics of endothelin receptor subtypes on muscle contraction and neuro-transmission in rat vas deferens.

We observed endothelin (ET)-induced contractile responses on prostatic and epididymal segments, as well as the facilitation of an electrically stimulated tone on prostatic segments of isolated rat vas deferens. In both segments, the selective ET(B)-receptor agonists, IRL 1620 and sarafotoxin S6c, produced only a small contraction or no contraction at a concentration of 1 microM. The rank order of contraction potencies (pD2 value) was ET-1 = ET-2 > ET-3 >> sarafotoxin S6c = IRL 1620. The maximum responses of ET-induced contractions in the prostatic segments were larger than those in the epididymal segments. The contractile response to ET-3 was antagonized by pretreatment for 30 min with BQ-123 (10 nM), a selective ET(A) receptor antagonist, and BQ-788 (1 microM), a selective ET(B) receptor antagonist. The contractile responses to ET-1 were antagonized by pretreatment with BQ-123 (10 microM), but not with BQ-788 (1 microM). The ET-3-induced facilitation on the twitch response to electrical stimulation in the prostatic segment of the vas deferens was antagonized by BQ-123 (0.1 microM) and BQ-788 (1 microM). The ET-1-induced facilitation was antagonized by pretreatment with BQ-123 (3 microM), but not with BQ-788 (10 microM). These results suggest that in rat vas deferens the ET(A) receptors are divided into BQ-123-sensitive ET(A1) and BQ-123-insensitive ET(A2) subtypes, and the production of a contractile response of smooth muscle as well as the facilitation of neurotransmission are accomplished through mediation by ET(A1)- and ET(A2)-subtypes.

Regional differences in alpha1-adrenoceptor subtypes and mechanisms in rabbit arteries.

Contractility mediated through alpha1-adrenoceptor subtypes and the maximum binding site (Bmax value) and the dissociation constant (Kd value) for [125I]HEAT ([125I]iodo-2-(beta-(4-hydroxyphenyl)ethylaminomethyl)tetralone) were determined in the following rabbit arteries: thoracic and abdominal aorta, mesenteric, renal and iliac arteries, and the alpha1-adrenoceptor subtypes mediating contractile mechanisms in vascular smooth muscle were studied. The pD2 values for norepinephrine differed considerably among the arteries in the presence of nicardipine (10(-5) M), while the pA2 values for 5-methylurapidil against norepinephrine were identical at low affinity in all the arteries used. In Ca2+-free physiological saline solution (Ca2+-free PSS), the pA2 values for 5-methylurapidil were also similar except for the renal artery, in which there were no stable contractions. In normal PSS, the concentration-response curves for norepinephrine with chloroethylclonidine-pretreatment were shifted to the right (pD2 values of 5.58, 5.70, 5.74, 5.98 and 6.38 for thoracic and abdominal aorta, mesenteric, renal and iliac arteries, respectively). In the [125I]HEAT binding study using membrane preparations obtained from chloroethylclonidine-treated strips, the Bmax values (33.2-105.2 fmol/mg protein) for [125I]HEAT varied considerably among arteries, while the Kd values (0.20-0.26 nM) were identical. The logarithm of Bmax values is proportional to the pD2 values for norepinephrine (slope=0.69, r=0.961). These observations suggest that the regional differences in potency (pD2 value) of the alpha1-adrenoceptor agonist, norepinephrine, are a result of the differences in population and density of alpha1-adrenoceptor subtypes in rabbit arteries.

Beta3-adrenoceptor mechanisms in guinea-pig taenia caecum.

Beta-adrenoceptor-mediated relaxation of guinea-pig taenia caecum was investigated by studying the effects of BRL37344, CGP12177 and norepinephrine. These drugs caused graded relaxation of the guinea-pig taenia caecum. The concentration-response curves for these drugs were unaffected by propranolol, atenolol, butoxamine, prazosin, yohimbine and phentolamine. Bupranolol produced shifts of the concentration-response curves for these drugs. Schild regression analyses carried out for bupranolol against BRL37344, CGP12177 and norepinephrine gave pA2 values of 5.79, 5.61 and 5.53, respectively. CGP12177 and norepinephrine significantly increased cyclic AMP levels in this preparation. Bupranolol significantly decreased cyclic AMP levels elicited by CGP12177 and norepinephrine, whereas propranolol produced no effect. These results suggest that the relaxant responses to BRL37344, CGP12177 and norepinephrine in the guinea-pig taenia caecum are mediated by beta3-adrenoceptors.

Protein kinase C mediates increase of Ca2+ sensitivity for contraction by cholinoceptor partial agonist in ileal longitudinal muscle of guinea pig.

1. Experiments were designed to study the roles of protein kinase C in carbachol- and pilocarpine-induced contraction and the increase in cytosolic Ca2+ concentration ([Ca2+]i) in guinea pig ileal longitudinal muscle. 2. The protein kinase C inhibitors, GF 109203X (10 microM), calphostin C (10 microM) and H-7 (10 microM), reduced the maximum of the concentration response curve produced by pilocarpine more effectively than that produced by carbachol. 3. The slopes of the regression lines between [Ca2+]i and tension development for pilocarpine and carbachol in tissues treated with GF 109203X were significantly gentler than those for untreated tissues. 4. The protein kinase C alpha- and beta 1 selective inhibitor Goe 6976 (1 microM) decreased both [Ca2+]i and contraction, but did not affect the slopes of the regression lines for pilocarpine and carbachol. 5. These results suggest that protein kinase C (both n- and/or a-type) plays an important role in the increase of Ca2+ sensitivity of the contractile element, and that pilocarpine mainly activates the protein kinase C-dependent pathways for contractile mechanisms in guinea pig ileal longitudinal muscle.

Differences of antagonism for a selective alpha1D-adrenoceptor antagonist BMY 7378 in the rabbit thoracic aorta and iliac artery.

Based on the affinity of alpha1D adrenoceptor subtype for a selective antagonist BMY 7378, we studied its functional role in rabbit thoracic aorta and iliac artery, and evaluated the subtypes of the alpha1-adrenoceptors that are activated by phenylephrine (a full agonist) and tizanidine (a partial agonist). In thoracic aorta, the concentration response curves of phenylephrine and tizanidine were antagonized by BMY 7378 with low potency (pA2 values 6.68+/-0.06 and 6.67+/-0.06, slopes of Schild plot 1.06+/-0.04 and 1.01+/-0.04, respectively). On the other hand, in iliac artery concentration response curves for phenylephrine were potently antagonized by a low concentration of BMY 7378, and the slope (0.75+/-0.02) of the Schild plot was significantly different from unity. In iliac artery, a concentration response curve of tizanidine was antagonized by BMY 7378 with low potency (pA2 value 6.64+/-0.08, slope of Schild plot 1.01+/-0.05). These results suggest that an alpha1D-adrenoceptor subtype contributes to alpha1-adrenoceptor mediating muscle contraction in iliac artery, but not in thoracic aorta of rabbit, and that it is activated by a full agonist phenylephrine but not by a partial agonist tizanidine.

Beta3-adrenoceptors mediate relaxation of guinea pig taenia caecum by BRL37344A and BRL35135A.

Beta-adrenoceptor-mediated relaxation of guinea pig taenia caecum was investigated by studying the effects of the beta3-adrenoceptor agonists, BRL37344A [(R*,R*)-(+/-)-4-[2'-[2-hydroxy-2-(3-chlorophenyl) ethylamino] propyl] phenoxyacetic acid sodium salt sesquihydrate] and BRL35135A [(R*,R*)-(+/-)-methyl-4-[2-[2-hydroxy-2-(3-chlorophenyl) ethylamine] propyl] phenoxyacetate hydrobromide]. BRL37344A and BRL35135A caused dose-dependent relaxation of the guinea pig taenia caecum. The concentration-response curves for BRL37344A and BRL35135A were unaffected by propranolol, ICI118551 [erythro-1-(7-methylindan-4-yloxy)-3-(isopropylamine)-but an-2-ol], atenolol, butoxamine, prazosin, yohimbine and phentolamine. Bupranolol produced shifts of the concentration-response curves for BRL37344A and BRL35135A. Schild regression analyses carried out for bupranolol against BRL37344A and BRL35135A gave pA2 values of 5.79 and 5.84, respectively. These results suggest that the relaxant response to BRL37344A and BRL35135A of the guinea pig taenia caecum is mediated by beta3-adrenoceptors.

Inhibitory effect of a fullerene derivative, monomalonic acid C60, on nitric oxide-dependent relaxation of aortic smooth muscle.

1. Effects of a fullerene C60 derivative, monomalonic acid C60 (MMA C60), on endothelium-containing or denuded aorta of rabbit, trachea and ileum of guinea pig, and stomach (fundus), vas deferens and uterus of rat were studied pharmacologically. 2. MMA C60 (10(-5) M) significantly reduced the maximum response of the relaxation induced by acetylcholine in endothelium-containing thoracic aorta of rabbit, and the acetylcholine-induced relaxation was recovered in the presence of superoxide dismutase (SOD, 250 units/ml). 3. Nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, caused the relaxation of aorta without endothelium in a concentration-dependent manner, and the concentration-response curve was shifted to the right in the presence of MMA C60. This inhibitory effect of the derivative was also masked in the presence of superoxide dismutase (SOD). 4. Sodium nitroprusside-induced relaxation was not affected by either MMA C60 or SOD. In the other tissues, this C60 derivative had no effect on the responses induced by any agonist. 5. These observations indicate that MMA C60 inhibits the endothelium-dependent relaxation induced by acetylcholine but does not affect the agonist-induced contractile response of smooth muscle.

The beta2- and beta3-adrenoceptor-mediated relaxation induced by isoprenaline and salbutamol in guinea pig taenia caecum.

To understand the receptor subtypes responsible for beta-adrenoceptor-mediated relaxation of guinea pig taenia caecum, we investigated the effects of isoprenaline and salbutamol. Isoprenaline and salbutamol caused dose-dependent relaxation of the guinea pig taenia caecum. Propranolol, bupranolol and butoxamine produced shifts of the concentration response curves for isoprenaline and salbutamol. Schild regression analyses carried out for propranolol against isoprenaline and salbutamol gave pA2 values of 8.43 and 8.88, respectively. Schild regression analyses carried out for butoxamine against isoprenaline and salbutamol gave pA2 values of 6.46 and 6.68, respectively. Schild regression analyses carried out for bupranolol against isoprenaline and salbutamol gave pA2 values of 8.60 and 8.69, respectively. However, in the presence of 3 x 10(-4) M atenolol, 10(-4) M butoxamine and 10(-6) M phentolamine to block the beta1-, beta2- and alpha-adrenoceptor effects, respectively, Schild regression analyses carried out for bupranolol against isoprenaline and salbutamol gave pA2 values of 5.77 and 5.97, respectively. These results suggest that the relaxant responses to isoprenaline and salbutamol in the guinea pig taenia caecum are mediated by both the beta2- and the beta3-adrenoceptors.

Inhibitory effects of a fullerene derivative, dimalonic acid C60, on nitric oxide-induced relaxation of rabbit aorta.

Dimalonic acid C60 (10(-5) M), a new fullerene derivative, produced an augmentation of phenylephrine-induced tone and reduced both the acetylcholine-induced maximum relaxation and the amplitude of substance P (10(-8) M)-induced relaxation in endothelium-containing thoracic aorta of rabbit; the acetylcholine- and substance P-induced relaxation was restored in the presence of superoxide dismutase (250 U/ml). Dimalonic acid C60 (10(-5) M) did not influence the phenylephrine-induced contractile response in the absence of endothelium, but the acetylcholine-induced relaxation was eliminated by removal of the endothelium. Superoxide anion generation, using hypoxanthine (1 mM)/xanthine oxidase (16 mU/ml), reduced the acetylcholine-induced relaxation and produced an augmentation of phenylephrine-induced tone in endothelium-containing strips; these effects were negated by the addition of superoxide dismutase (250 U/ml). A nitric oxide-generating agent, S-nitroso-N-acetylpenicillamine, caused relaxation of aorta without endothelium in a concentration-dependent manner, and the concentration-response curve was shifted to the right in the presence of dimalonic acid C60. This inhibitory effect of dimalonic acid C60 was also masked in the presence of superoxide dismutase. Sodium nitroprusside-induced relaxation was not affected by either dimalonic acid C60 or superoxide dismutase. These observations suggest that dimalonic acid C60 inhibits endothelium (nitric oxide)-dependent agonist-induced relaxation through the production of superoxide.

The significant role of endothelin-3 in potentiating electrically stimulated contractions in the rat iris sphincter.

1. We determined the endothelin (ET) receptor subtype involved in the facilitation of electrical field stimulation-(EFS) induced contraction using the ETB receptor agonist sarafotoxin S6c (STX S6c) and the ET receptor antagonists TTA-386 and bosentan. 2. ET-3-and ETB-receptor-selective agonist STX S6c enhanced EFS-induced contractions. The increasing effect of ET-3 was partially reduced by the desensitization to STX S6c or the ETA receptor antagonist TTA-386. After simultaneous treatment with TTA and desensitization, ET-3-induced potentiation was completely abolished. The combined ETA/B receptor blocker bosentan (Ro 47-0203) eliminated the ET-3-evoked neuronal effect. Both ETB and ETA receptors are involved in the facilitating effects of ETs on EFS contraction. 3. ET-3 immunoreactivity occurs densely in this tissue and was released from neuronal sites by electrical stimulation. We suggest that endogenous ET-3 has a more predominant role than ET-1.

Characteristics of [3H]CGP12177 binding sites at beta 2- and beta 3-adrenoceptors in the guinea pig taenia caecum.

1. Experiments were carried out to characterize the [3H]CGP12177 binding sites in the microsomal fraction from the guinea pig taenia caecum. 2. The Scatchard plot in the absence of butoxamine (10(-4) M) or bupranolol (10(-4) M) was concave, suggesting two affinity sites: high and low affinity binding sites. 3. The Scatchard plot in the presence of butoxamine gave a straight line, indicating only the low affinity site. 4. The Scatchard plot in the presence of bupranolol was undetectable. 5. These results suggest that the high affinity binding sites for [3H]CGP12177 correspond to beta 2-adrenoceptors in the microsomal fraction from the guinea pig taenia caecum, whereas the low affinity binding sites represent beta 3-adrenoceptors.

Drug receptor mechanisms in smooth muscle: beta-chloroethylamine-sensitive and -resistant receptor mechanisms.

Both alpha1-adrenoceptors and M3-cholinoceptors can be divided into two subtypes discriminated by the beta-chloroethylamines, chloroethylclonidine and propylbenzilylcholine mustard (PrBCM), only in the presence of GTP. The full agonists interact with both subtypes to induce responses. The partial agonists activate one of them to induce responses but behave as competitive antagonists when they interact with the other. The responses mediated through the receptors that are activated by the partial agonists are resistant to myosin light chain kinase inhibitors, while the response through the activation of the other receptors are suppressed by the inhibitors. The receptor stimulations through alpha1A-adrenoceptor and PrBCM-sensitive M3-cholinoceptor subtypes mainly activate the myosin light chain-phosphorylation-independent pathway mediated through protein kinase C and low molecular weight GTP-binding protein, whereas the stimulations through alpha1B-adrenoceptors and the PrBCM-phosphorylation-dependent pathway are directly related to Ca2+/calmodulin.

Phenoxybenzamine discriminates between two distinct populations of histamine H1-receptors in longitudinal muscle of guinea-pig ileum.

The longitudinal muscle of guinea-pig ileum was used as a test tissue. Progressive (up to 70 min) treatment with phenoxybenzamine (10(-6) M) inhibited progressively the response to histamine and progressively decreased the density of histamine H1-receptors. However, the 90 min treatment has no further significant inhibitory effect on the contractile response and did not decrease the density of the receptors. These results suggest that phenoxybenzamine discriminates between two distinct populations of H1-receptors. Furthermore, it was found that the presence of GTP gamma-S made some populations of H1-receptors resistant to phenoxybenzamine and facilitated an interaction of mepyramine with H1-receptors.

Relaxant responses by optical isomers of ephedrine and methylephedrine in guinea pig tracheal smooth muscle.

The effects of optical isomers of ephedrine and methylephedrine on the guinea pig tracheal smooth muscle were studied. l-Ephedrine markedly caused a graded relaxation of the guinea pig trachea where the tone had been raised spontaneously. A rightward shift of the l-ephedrine concentration-response curve was observed for propranolol and butoxamine, and the pA2 values for propranolol and butoxamine were 8.55 and 6.38, respectively. d-Methylephedrine markedly caused a graded relaxation of the guinea pig trachea contracted with histamine. Propranolol and bupranolol did not affect the relaxant response to d-methylephedrine. d-Methylephedrine competitively antagonized the contractile responses to histamine, and the pA2 value for d-methylephedrine was 5.12. These results suggest that l-ephedrine-induced relaxation of the guinea pig trachea is mediated through beta 2-adrenoceptors, whereas d-methylephedrine relaxes the guinea pig trachea by blocking histamine receptors.

Pharmacological characteristics of indoline derivatives in muscarinic receptor subtypes.

The present study was designed to investigate which muscarinic receptors the indoline derivatives interact with and also their pharmacological properties. Compounds I and II contracted guinea-pig ileum in a concentration-dependent manner, whereas compounds III-IX behaved as antagonists and Schild plots gave straight lines. 4-DAMP antagonized the contractile responses to compounds I and II, and the pA2 values for 4-DAMP were 8.96 +/- 0.23 and 9.09 +/- 0.06, respectively. In guinea-pig left atrium, compound I partly inhibited twitch responses, whereas compound II did not have any effect. In rabbit vas deferens, compounds I and II produced inhibitory effects on twitch responses evoked by field stimulation. Pirenzepine antagonized the inhibitory responses of compounds I and II, and the pA2 values for pirenzepine were 7.90 +/- 0.13 and 8.12 +/- 0.06, respectively. Compound I has about 150-fold higher affinity to M1 receptors than to M3 receptors, while compound II has about 360-fold higher affinity to M1 receptors. Our results indicate that compounds I and II show 7- and 16-fold higher M1 receptor selectivity than McN-A-343, respectively. Therefore, compound II is selective for M1 receptors over M2 or M3 receptors.

Differentiation of binding sites of CGP12177, a beta 3-adrenoceptor partial agonist, and carteolol, a beta 1/beta 2-adrenoceptor partial agonist, to the beta-adrenoceptors in guinea-pig taenia caecum.

Differentiation of binding sites of CGP12177 and carteolol to the beta-adrenoceptors in the guinea-pig taenia caecum was investigated. Carteolol and CGP12177 competitively antagonized the relaxation responses to isoprenaline, and the pA2 values were 9.87 and 9.33, respectively. Butoxamine, a beta 2-selective antagonist, caused competitive antagonism of the relaxant responses to carteolol, and the pA2 value for butoxamine was 6.22. However, butoxamine (10(-4) M) did not significantly affect the relaxant responses to CGP12177. CGP12177 caused competitive antagonism of the relaxant responses to carteolol, and the pA2 value for CGP12177 was 9.32. However, carteolol (10(-6) M) did not significantly affect the relaxant responses to CGP12177. The competitive inhibition curve for specific binding of 50 nM [3H]befunolol by carteolol showed a biphasic shape, although the curve by CGP12177 was monophasic. Moreover, the competitive inhibition curve for specific binding of 100 nM [3H]CGP12177 by CGP12177 showed a biphasic shape, although the curve by carteolol indicated partial inhibition. These results suggest that the low affinity site of beta-adrenoceptor and beta 3-adrenoceptors are different from each other.