Katsuo Koike

α1-Adrenoceptors are required for normal male sexual function

α1‐Adrenoceptor antagonists are extensively used in the treatment of hypertension and lower urinary tract symptoms associated with benign prostatic hyperplasia. Among the side effects, ejaculatory dysfunction occurs more frequently with drugs that are relatively selective for α1A‐adrenoceptors compared with other drugs of this class. This suggests that α1A‐adrenoceptors may contribute to ejaculation. However, this has not been studied at the molecular level.

NEW INSIGHTS INTO β-ADRENOCEPTORS IN SMOOTH MUSCLE: DISTRIBUTION OF RECEPTOR SUBTYPES and MOLECULAR MECHANISMS TRIGGERING MUSCLE RELAXATION

1. The β‐adrenoceptor is currently classified into β1, β2 and β3 subtypes and all three subtypes are expressed in smooth muscle. Each β‐adrenoceptor subtype exhibits tissue‐specific distribution patterns, which may be a determinant controlling the mechanical functions of corresponding smooth muscle. Airway and uterine smooth muscles abundantly express the β2‐adrenoceptor, the physiological significance of which is established as a fundamental regulator of the mechanical activities of these muscles. Recent pharmacomechanical and molecular approaches have revealed roles for the β3‐adrenoceptor in the gastrointestinal tract and urinary bladder smooth muscle.

Characterization of α1-adrenoceptor-mediated contraction in the mouse thoracic aorta

In the mouse thoracic aorta, noradrenaline, adrenaline, phenylephrine and methoxamine behaved as full agonists. The pA(2) values for 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione dihydrochloride (BMY 7378) against each agonist were in good agreement with the generally accepted affinity value of alpha(1D)-adrenoceptors. 5-Methylurapidil, 2-[2,6-dimethoxyphenoxyethyl]aminomethyl-1,4-benzodioxane hydrochloride (WB 4101) and prazosin inhibited the contraction in response to noradrenaline. A significant correlation was obtained between the antagonist affinities in mouse thoracic aorta and those of native alpha(1D)-adrenoceptors in rat thoracic aorta or with those of cloned alpha(1d)-adrenoceptors, but not with those for either alpha(1a)- or alpha(1b)-adrenoceptors. Buspirone behaved as a partial agonist in mouse thoracic aorta, the contraction of which was antagonized by BMY 7378 with a pA(2) value (8.49) consistent with that found against noradrenaline (8.43). Clonidine acted as a partial agonist (pD(2)=5.94). The pK(p) value for clonidine against noradrenaline was similar to the pD(2) value for clonidine. The apparent pK(B) value for BMY 7378 against clonidine was similar to the pA(2) value against other full agonists used in the present study. These results suggest that the alpha(1D)-adrenoceptor subtype exists, and that the full agonists and the partial agonists evoke the contraction mediated through the alpha(1D)-adrenoceptor in mouse thoracic aorta.

Possible mechanisms of β-adrenoceptor-mediated relaxation induced by noradrenaline in guinea pig taenia caecum

The mechanisms of the beta-adrenoceptor-mediated relaxation induced by noradrenaline in guinea pig taenia caecum were investigated. Noradrenaline caused graded relaxation of this preparation. However, the concentration-response curves for noradrenaline were unaffected by propranolol (approximately 10(-5) M) or phentolamine (approximately 10(-5) M). The responses to noradrenaline were antagonized in a concentration-dependent manner by bupranolol, and Schild plots of the data revealed a pA2 value of 5.53. Also, bupranolol antagonized responses to isoprenaline, and Schild plots of the data revealed the pA2 value to be 8.53. Noradrenaline significantly increased the cyclic AMP level in this preparation. Bupranolol (10(-4) M) significantly decreased the cyclic AMP response elicited by noradrenaline, whereas propranolol (10(-5) M) produced no effect. These results suggest that the relaxant response to noradrenaline in guinea pig taenia caecum is mainly mediated by beta 3-adrenoceptors (or atypical beta-adrenoceptors) and that in guinea pig taenia caecum noradrenaline behaves as a beta 3-selective adrenoceptor agonist.

α1-Adrenoceptor subtypes in the mouse mesenteric artery and abdominal aorta

Subtypes of α1‐adrenoceptor‐mediated contractions to noradrenaline in mouse mesenteric artery and abdominal aorta were examined. In mesenteric artery, BMY7378, 5‐methylurapidil, WB4101 and prazosin were inhibited contraction to noradrenaline The good correlation for pA2 values of antagonists in native α1D‐ (rat thoracic aorta) adrenoceptor subtype and pKi values in rat cloned α1d‐adrenoceptor with the pA2 values estimated in the mouse mesenteric artery was obtained. However, the pA2 value for BMY7378 is significantly lower than the accepted value against the α1D‐adrenoceptor subtype. In the abdominal aorta, it was obtained the regional difference for the sensitivity for noradrenaline. In the upper abdominal aorta, the good correlation for the pA2 values of the antagonists in the native α1D‐adrenoceptor subtype and pKi values in the cloned α1d‐adrenoceptor with the pA2 values estimated in the upper abdominal aorta was obtained, and regression line was close to the line of identity. In the lower abdominal aorta, the good correlation for the reported pKi values in the cloned α1a‐adrenoceptor subtype with the pA2 values estimated in the mouse lower abdominal aorta was obtained, and regression line was close to the line of identity. In conclusion, the present functional data in the mouse suggest that (1) α1D‐like adrenoceptors are present in the mesenteric artery, (2) there is the regional difference for the sensitivity for noradrenaline in the abdominal aorta and (3) noradrenaline evokes the contraction mediated through α1D‐adrenoceptor in the upper abdominal aorta, whereas there is α1A‐adrenoceptor‐mediated contraction in the lower abdominal aorta.

Correlation between vasoconstrictor roles and mRNA expression of α1‐adrenoceptor subtypes in blood vessels of genetically engineered mice

We examined the contribution of each α1‐adrenoceptor (AR) subtype in noradrenaline (NAd)‐evoked contraction in the thoracic aortas and mesenteric arteries of mice. Compared with the concentration–response curves (CRCs) for NAd in the thoracic aortas of wild‐type (WT) mice, the CRCs of mutant mice showed a significantly lower sensitivity. The pD2 value in rank order is as follows: WT mice (8.21)>α1B‐adrenoceptor knockout (α1B‐KO) (7.77)>α1D‐AR knockout (α1D‐KO) (6.44)>α1B‐ and α1D‐AR double knockout (α1BD‐KO) (5.15). In the mesenteric artery, CRCs for NAd did not differ significantly between either WT (6.52) and α1B‐KO mice (7.12) or α1D‐KO (6.19) and α1BD‐KO (6.29) mice. However, the CRC maximum responses to NAd in α1D‐ and α1BD‐KO mice were significantly lower than those in WT and α1B‐KO mice. Except in the thoracic aortas of α1BD‐KO mice, the competitive antagonist prazosin inhibited the contraction response to NAd with high affinity. However, prazosin produced shallow Schild slopes in the vessels of mice lacking the α1D‐AR gene. In the thoracic aorta, pA2 values in WT mice for KMD‐3213 and BMY7378 were 8.25 and 8.46, respectively, and in α1B‐KO mice they were 8.49 and 9.13, respectively. In the mesenteric artery, pA2 values in WT mice for KMD‐3213 and BMY7378 were 8.34 and 7.47, respectively, and in α1B‐KO mice they were 8.11 and 7.82, respectively. These pharmacological findings were in fairly good agreement with findings from comparison of CRCs, with the exception of the mesenteric arteries of WT and α1B‐KO mice, which showed low affinities to BMY7378. We performed a quantitative analysis of the mRNA expression of each α1‐AR subtype in these vessels in order to examine the correlation between mRNA expression level and the predominance of each α1‐AR subtype in mediating vascular contraction. The rank order of each α1‐AR subtype in terms of its vasoconstrictor role was in fairly good agreement with the level of expression of mRNA of each subtype, that is, α1D‐AR>α1B‐AR>α1A‐AR in the thoracic aorta and α1D‐AR>α1A‐AR>α1B‐AR in the mesenteric artery. No dramatic compensatory change of α1‐AR subtype in mutant mice was observed in pharmacological or quantitative mRNA expression analysis.

Proliferative effects of angiotensin II and endothelin-1 on guinea pig gingival fibroblast cells in culture.

We investigated whether phenytoin (PHT) and nifedipine (NIF) induce angiotensin II (Ang II) and endothelin-1 (ET-1) generation by cultured gingival fibroblasts derived from guinea pigs and whether Ang II and ET-1 induce proliferation of these cells. Immunohistochemical experiments showed that PHT (250 nM) and NIF (250 nM) increased the immunostaining intensities of immunoreactive Ang II and ET-1 (IRET-1) in these cells. Captopril (3 microM), an angiotensin-converting enzyme inhibitor, reduced these enhanced intensities to control levels. Ang II (100 nM) enhanced the immunostaining intensity of IRET-1. PHT (250 nM) and NIF (250 nM)-induced cell proliferation. Both PHT- and NIF-induced proliferation was inhibited by captopril (3 microM). Ang II (100 nM) and ET-1 (100 nM) also induced cell proliferation. Ang II-induced proliferation was inhibited by CV11974 (1 microM), an AT(1) receptor antagonist and saralasin (1 microM), an AT(1)/AT(2) receptor antagonist, but not by PD123,319 (1 microM), an AT(2) receptor antagonist. ET-1-induced proliferation was inhibited by BQ123 (10 microM), an ET(A) receptor antagonist, but not by BQ788 (1 microM), an ET(B) receptor antagonist. These findings suggest that PHT- and NIF-induced gingival fibroblast proliferation is mediated indirectly through the induction of Ang II and ET-1 and probably mediated through AT(1) and ET(A) receptors present in or on gingival fibroblasts.

Piperazine analog of vesamicol: In vitro and in vivo characterization for vesicular acetylcholine transporter

The probes to detect vesicular acetylcholine transporter (VAChT) in vivo are important to evaluate the mapping and function in cholinergic system. To develop high‐specific and high‐affinity radiotracer for single photon emission computed tomography, we investigated piperazine analogs which replaced the piperidine ring of (‐)‐vesamicol with a piperazine ring. We found that the piperazine analog of iodobenzovesamicol, trans‐5‐iodo‐2‐hydroxy‐3‐[4‐phenylpiperazinyl] tetralin (DRC140), had high affinity for VAChT in rat brain. We carried out binding assay in subcellular fraction of the rat brain. The highest Bmax for [125I]‐DRC140 binding was observed in the synaptic vesicle fraction (1,751 fmol/mg protein), followed by the crude vesicle (821 fmol/mg protein) and the P2 fraction (187 fmol/mg protein). These Kd values were similar to the affinity of highly purified synaptic vesicular fraction (Kd = 0.3 nM) with a one‐site model. The possibility that [125I]‐DRC140 recognizes sigma receptor was excluded by our finding large inhibition constants (Ki = 849 nM for haloperidol, Ki = 3,052 nM for 1,3‐di(2‐tolyl)guanidine). In vivo distribution studies with the [123I]‐DRC140 in rats showed a rapid brain uptake. The highest brain area was in striatum, followed by frontal cortex, occipital cortex, and hippocampus. The lowest brain area was cerebellum. The radioactivity of high‐accumulated areas in ex vivo autoradiography was reduced by a preinjection of (‐)‐vesamicol and these levels were reduced to the radioactivity in cerebellum. These results show that [125I]‐DRC140 can provide extremely high specific tracer with excellent brain permeability as a ligand for single photon emission computed tomography. Synapse 38:27–37, 2000.

Cyclic AMP-independent relaxation mediated by β3-adrenoceptors on guinea pig gastrointestine

In this study, we investigated the signal transduction pathway involved in beta(3)-adrenoceptor-mediated relaxations of guinea pig gastric fundus and duodenum. In the presence of beta1- and beta2-adrenoceptor blockade, the potency (pD2 value) of catecholamines ((-)-isoprenaline, (-)-noradrenaline and (-)-adrenaline) and beta(3)-adrenoceptor agonists ((R*, R*)-(+/-)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]phenoxyacetic acid sodium (BRL37344) and (+/-)-[4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one] hydrochloride ((+/-)-CGP12177A)) to induce relaxation was not affected by the adenylate cyclase inhibitor, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ-22,536, 100 microM). Catecholamines induced an elevation of cyclic AMP and SQ-22,536 significantly abolished the responses of gastric fundus. However, cyclic AMP levels were unaltered by the beta3-adrenoceptor agonists in gastric fundus and by the five agonists in duodenum. Furthermore, the relaxant responses to catecholamines and to beta3-adrenoceptor agonists were unaffected by the cyclic AMP-dependent protein kinase inhibitor, N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89, 10 microM) in gastric fundus. These results suggest that beta3-adrenoceptor-induced relaxation is mediated through both cyclic AMP-dependent and cyclic AMP-independent pathways in gastric fundus and through a cyclic AMP-independent pathway in duodenum.

Agonistic activity of SR59230A at atypical β-adrenoceptors in guinea pig gastric fundus and duodenum

We have recently suggested that atypical beta-adrenoceptors are present in guinea pig gastric fundus and duodenum. In the present study, we have shown that SR59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate), a selective beta(3)-adrenoceptor antagonist, possesses agonistic activities at atypical beta-adrenoceptors in these tissues. SR59230A caused concentration-dependent relaxations. However, (+/-)-propranolol (1 microM) did not affect SR59230A-induced relaxations. Pretreatment of with a combination of (+/-)-propranolol (1 microM) and the non-selective beta(1)-, beta(2)-, beta(3)- and beta(4)-adrenoceptor antagonist, (+/-)-bupranolol (30 microM), significantly antagonized the relaxant effects induced by SR59230A. The results clearly indicate that SR59230A acts as an atypical beta-adrenoceptor agonist on guinea pig gastric fundus and duodenum.