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Ikunobu Muramatsu

Publications and source records attributed to Ikunobu Muramatsu.

At least 19 recordsLinked to original sources
PubMed2007

Identification of alpha-1L and alpha-1A adrenoceptors in human prostate by tissue segment binding.

PURPOSE: Silodosin (KMD-3213 or [(-)-1-(3-hydroxypropyl)-5-[(2R)-2-({2-[2-(2,2,2trifluoroethoxy)phenoxy]ethyl}amino)propyl]-2,3-dihydro-1H-indole-7-carboxamide]) (Kissei Pharmaceutical Co., Ltd., Matsumoto, Japan) is a selective antagonist for alpha-1A and alpha-1L adrenoceptors. Using this tritiated ligand the 2 alpha-1 adrenoceptors were examined in binding studies with intact tissue segments and membrane preparations of human prostate, and compared with functionally identified alpha-1 adrenoceptor. MATERIALS AND METHODS: Binding assays with tissue segments and membrane preparations of human prostate samples were performed using [3H]-silodosin and binding affinities for various drugs were estimated. In functional experiments antagonist affinities were evaluated from the inhibitory potency against the contractile response to noradrenaline. RESULTS: [3H]-silodosin bound to intact segments and membrane preparations of human prostate with subnanomolar affinity. [3H]-silodosin binding sites in intact segments were divided into 2 distinct components with different affinities for prazosin and RS-17053 (N-[2(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha, alpha-dimethyl1H-indole-3-ethanamine hydrochloride) (Research Biochemicals International, Natick, Massachusetts), while binding in membrane preparations showed single high affinity for these drugs. [3H]-silodosin binding sites also showed high affinity for silodosin and tamsulosin but low sensitivity to BMY 7378 (8-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4.5)decane-7,9-dione) (Research Biochemicals International) in intact segments and in membrane preparations. In functional experiments silodosin and tamsulosin potently inhibited the contractile response to noradrenaline but prazosin, RS-17053 and BMY 7378 showed low antagonistic affinity. CONCLUSIONS: The current binding studies in human prostate samples clearly show that alpha-1L and alpha-1A adrenoceptors coexist as pharmacologically distinct entities in intact tissues but not in crude membrane preparations. Also, alpha-1 adrenoceptors involved in the contractile response to noradrenaline are the alpha-1L subtype.

Humans↗
PubMed2006

Beta-blockers show inverse agonism to a novel constitutively active mutant of beta1-adrenoceptor.

We obtained a new mutant of the beta(1)-adrenergic receptor (beta(1)-AR) by point mutations that can constitutively activate beta(1)-AR. Aspartate104 of the beta(1)-AR in the 2nd transmembrane was replaced with alanine. The beta(1)-AR mutant expressed in human embryonic kidney (HEK)-293 cells displayed high level of constitutive activity with respect to wild-type (P<0.05), which could be partially inhibited by some beta-blockers. The constitutive activity of the mutant was confirmed by the finding that the enhanced activity is dependent on the level of receptor expression. The results of this study might have interesting implications for future studies aiming at elucidating the activation process of the beta(1)-AR as well as the mechanism of action of beta-blockers.

Adrenergic beta-1 Receptor Agonists↗
PubMed2006

Pharmacological evaluation of plasma membrane beta-adrenoceptors in rat hearts using the tissue segment binding method.

This study evaluates beta-adrenoceptors in rat atria and ventricle using the tissue segment binding method and compares the results with those obtained using conventional homogenate binding assays. In studies with tissue segment binding, the hydrophilic radioligand [(3)H]-CGP12177 selectively bound to plasma membrane beta-adrenoceptors, and the B(max) levels were significantly higher than those obtained with homogenate binding. However, both binding approaches revealed similar proportions of beta(1)- and beta(2)-adrenoceptors. The regional distribution of plasma membrane beta(1)- and beta(2)-adrenoceptors in rat hearts were also determined using tissue segment binding. Abundance of beta-adrenoceptors and proportion of beta(1)-adrenoceptors were higher in atria than in ventricle, but there was no significant difference between right and left atria or within ventricle (right and left ventricle free walls, apex, and interventricular septum). To establish the ability of the tissue segment binding method to study beta-adrenoceptor regulation such as the internalization of receptors, the effect of prolonged exposure of rat ventricle to (-)-isoprenaline was also investigated by using tissue segments and homogenate binding. Incubation with (-)-isoprenaline for 1 h in vitro caused a concentration-dependent decrease in the density of beta-adrenoceptors, predominantly beta(2)-adrenoceptors, when assessed with tissue segment binding method. In contrast, the subtype-specific change after treatment with (-)-isoprenaline was not detected using homogenate binding. In summary, the tissue segment binding method with [(3)H]-CGP12177 enables a more precise quantitation of plasma membrane beta(1)- and beta(2)-adrenoceptors in rat hearts and is suitable for studying their regulation.

Adrenergic beta-Agonists↗
PubMed2006

The inhibitory effects of local anesthetics on primary sensory nerve and parasympathetic nerve in rabbit eye.

Primary sensory nerves transmit information to both the periphery and central nervous systems, and they mediate neurogenic inflammation by release of neurotransmitters, such as tachykinins, in the periphery. Because the effect of local anesthetics on neurogenic inflammation is a subject of controversy, we investigated the direct effect of local anesthetics on tachykininergic neurotransmission, comparing it with cholinergic neurotransmission in the rabbit iris sphincter muscle. Rabbit iris sphincter muscle is innervated by trigeminal tachykininergic and parasympathetic cholinergic nerves, and the electrical transmural stimulation produces tachykininergic and cholinergic contractions. Cocaine and lidocaine (1-300 microM) attenuated tachykininergic and cholinergic contractions induced by electrical transmural stimulation in concentration- and stimulus frequency-dependent manner. However, the sensitivity to both local anesthetics was slightly, but significantly, higher in tachykininergic than in cholinergic responses. Exogenous neurokinin A and carbachol produced contractions that were not inhibited by 100 microM of cocaine and lidocaine. These results show that local anesthetics have a direct inhibitory effect on tachykininergic neurotransmission of the trigeminal sensory nerve, and the effect on this nerve is more potent than on the parasympathetic nerve and suggests that local anesthetics may have antineurogenic inflammatory effects via the inhibitory effects on the peripheral transmission of primary sensory nerve.

Anesthetics, Local↗
PubMed2006

[Alpha1-adrenoceptor subtypes and alpha1-adrenoceptor antagonists].

Alpha(1)-adrenoceptors are widely distributed in the human body and play important physiologic roles. Three alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B) and alpha(1D)) have been cloned and show different pharmacologic profiles. In addition, a putative alpha(1)-adrenoceptor (alpha(1L) subtype) has also been proposed. Recently, three drugs (tamsulosin, naftopidil, and silodosin) have been developed in Japan for the treatment of urinary obstruction in patients with benign prostatic hyperplasia. In this review, we describe recent alpha(1)-adrenoceptor subclassifications and the pharmacologic characteristics (subtype selectivity and clinical relevance) of alpha(1)-adrenoceptor antagonists.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed2005

Characteristics of acid extrusion from Chinese hamster ovary cells expressing different prostaglandin EP receptors.

Acid extrusion responses to prostaglandin E2 were investigated in Chinese hamster ovary (CHO) cells heterologously expressing human EP1, EP2, and EP3I receptors (hEP1, hEP2 and hEP3I) by using a microphysiometer that detected small pH changes in the extracellular microenvironment. In the cells expressing hEP1, which is known to increase intracellular Ca2+, prostaglandin E2 (1 and 10 nM) slowly accelerated acid extrusion, but at higher concentrations an initial transient phase (approximately 5 times greater than the basal acidification) overlapped the slowly developing phase. In contrast, the cells expressing hEP2, which evokes cAMP production, showed dual responses to prostaglandin E2: an initial reduction followed by an acceleration of acid extrusion. In the cells expressing hEP3I, which is known to produce both a decrease in cAMP and a modest increase in intracellular Ca2+, acid extrusion was gradually accelerated by prostaglandin E2 and reached a plateau at around 2 min. Elimination of extracellular Ca2+ diminished the responses to prostaglandin E2 in hEP1 cells, but had little effect on the responses in hEP2 and hEP3I cells. Forskolin mimicked the dual effects of prostaglandin E2 observed in the hEP2 cells. Pretreatment with pertussis toxin inhibited the response to prostaglandin E2 in hEP3I cells, but the responses in hEP1 and hEP2 cells were not affected. Na+/H+ exchanger (NHE) inhibitors (EIPA and HOE642) suppressed all the responses induced by prostaglandin E2 in hEP1, hEP2, and hEP3I cells. These results suggest that EP receptor subtypes regulate acid extrusion mainly via NHE-1 through distinct signal transduction pathways in CHO cells.

Animals↗
PubMed2005

Quantifying receptor properties: the tissue segment binding method - a powerful tool for the pharmacome analysis of native receptors.

The radioligand binding assay technique is an extremely powerful tool for studying receptors. It allows an analysis of the interactions of hormones, neurotransmitters, and related drugs with their receptors. Most of the binding assays have widely been applied to crude membrane fractions prepared from many tissues, but in the conventional method, there are some limitations such as a yield loss of receptor-bearing membranes and a change in receptor environment upon homogenization and fractionation. Recently, in order to overcome these problems, a binding assay has been developed using intact tissue segments. This article presents a brief overview of the tissue segment binding assay that has been developed mainly in our department. Practical guidelines for setting up this new assay are presented, including segment preparation, choice of appropriate radioligand, optimizing assay conditions, and appropriate methods for data analysis. The unique advantages and disadvantages of the tissue segment binding method are discussed in comparison with those of conventional membrane binding methods. We suggest that the tissue segment binding method is a powerful tool for detecting the native properties of receptors occurring in tissues and cells without altering their environment.

Animals↗
PubMed2005

Coupled K+-water flux through the HERG potassium channel measured by an osmotic pulse method.

The streaming potential (V(stream)) is a signature feature of ion channels in which permeating ions and water molecules move in a single file. V(stream) provides a quantitative measure of the ion and water flux (the water-ion coupling ratio), the knowledge of which is a prerequisite for elucidating the mechanisms of ion permeation. We have developed a method to measure V(stream) with the whole-cell patch-clamp configuration. A HEK293 cell stably expressing the HERG potassium channel was voltage clamped and exposed to hyperosmotic solutions for short periods of time (<1 s) by an ultrafast solution switching system (the osmotic pulse [quick jump-and-away] method). The reversal potentials were monitored by a series of voltage ramps before, during, and after the osmotic pulse. The shifts of the reversal potentials immediately after the osmotic jump gave V(stream). In symmetrical K+ solutions (10 mM), the V(stream)s measured at different osmolalities showed a linear relationship with a slope of -0.7 mV/DeltaOsm, from which the water-ion coupling ratio (n, the ratio of the flux of water to the flux of cations; Levitt, D.G., S.R. Elias, and J.M. Hautman. 1978. Biochim. Biophys. Acta. 512:436-451) was calculated to be 1.4. In symmetrical 100 mM K+ solutions, the coupling ratio was decreased significantly (n = 0.9), indicating that the permeation process through states with increased ion occupancy became significant. We presented a diagrammatic representation linking the water-ion coupling ratio to the mode of ion permeation and suggested that the coupling ratio of one may represent the least hydrated ion flux in the single-file pore.

Cell Membrane Permeability↗
PubMed2005

Comparison of the binding affinity of some newly synthesized phenylethanolamine and phenoxypropanolamine compounds at recombinant human beta- and alpha1-adrenoceptor subtypes.

We evaluated six new compounds, SWR-0065HA ([4-[2-[3-[[(3,4-dihydro-4-oxo-[1,2,4]-triazino(4,5-a)indol)-lyl]oxy]-2-hydroxypropylamino]ethoxy]phenyl]acetic acid methyl ester hydrochloride), SWR-0098NA ((R*R*-UE)-(E)-[4-[3-[(2-phenyl-2-hydroxyethyl)amino]-1-butenyl]phenoxy]acetic acid sodium salt), SWR-0315NA ((E, Z)-[4[[1-[2-[(3-phenoxy-2-hydroxy propyl)]amino]ethyl]-1-propenyl]phenoxy]acetic acid sodium), SWR-0338SA ((E)-[4-[5-[(2-phenyl-2-hydroxyethyl)amino]-2-pentene-3-yl]phenoxy] acetic acid ethanedioic acid), SWR-0342SA ((S)-(Z)-[4-[[1-[2-[(2-hydroxy-3-phenoxypropyl)]amino] ethyl]-1-propenyl]phenoxy]acetic acid ethanedioic acid) and SWR-0345HA ((E)-2-methyl-3-[4-[2-(2-phenyl-2-hydroxyethylamino)ethoxy]phenyl]-2-propenoic acid ethyl ester hydrochloride) for their potencies as selective ligands at human beta-adrenoceptors expressed in COS-7 cells and compared the binding affinities for human alpha(1)-adrenoceptors expressed in Chinese hamster ovary (CHO) cells using a radioligand-binding assay. Phenoxypropanolamine derivatives SWR-0315NA and SWR-0342SA showed higher binding affinities for beta-adrenoceptor subtypes; SWR-0065HA, however, showed a higher affinity for only beta-adrenoceptors, accounting for 3-fold and 6-fold selectivity against beta(1)- and beta(3)-adrenoceptors. Compounds SWR-0315NA and SWR-0342SA did not show any binding selectivity for any of the subtypes. However, functionally these two compounds are selective for beta(3)-adrenoceptors. Among the phenylethanolamine derivatives, SWR-0338SA and SWR-0345HA showed 9-fold and 16-fold higher binding selectivity for beta(3)-adrenoceptors against beta(1)-adrenoceptors, respectively, whereas they both showed a 7-fold higher binding selectivity for beta(3)-adrenoceptors against beta(2)-adrenoceptors. SWR-0098NA did not show any significant binding affinity for any of the beta-adrenoceptor subtypes. These compounds, except for SWR-0098NA, were not found to possess any significant binding affinity for alpha(1)-adrenoceptor subtypes over that for beta-adrenoceptor subtypes. However, SWR-0098NA has about a 3-fold to 22-fold higher binding selectivity for alpha(1)-adrenoceptor subtypes against beta-adrenoceptor subtypes, making it difficult for use in a beta-adrenoceptor receptor study. Compounds SWR-0315NA and SWR-0342SA have similar binding potency for alpha(1)-adrenoceptors as adrenaline (epinephrine), proving the finding of this manuscript that this phenoxypropanolamine group of beta-adrenoceptor ligands could also be used as alpha(1)-adrenoceptor ligands. Functional assays have to be performed to confirm their agonistic activity.

Adrenergic beta-Agonists↗
PubMed2004

An EP4 receptor agonist prevents indomethacin-induced closure of rat ductus arteriosus in vivo.

Indomethacin exerts a strong tocolytic effect by suppressing uterine contractions mediated by prostaglandins. However, indomethacin also induces in utero closure of fetal ductus arteriosus (DA), leading to serious neonatal consequences. Using rats, we tested the effect of an agonist for a subtype of prostaglandin E2 receptor (EP4), ONO-AE1-437 and its prodrug ONO-4819, as a DA dilator during indomethacin treatment. In vitro, ONO-AE1-437 exhibited a potent dilatory effect on DA against O(2)- and indomethacin-induced contractions in a concentration-dependent manner. In vivo, rat dams were given indomethacin (10 mg/kg, p.o.) alone or with ONO-4819 (0.3 micrograms/kg/h, s.c.) on d 21 of gestation and pups were delivered 4 h later through cesarean section to evaluate the ratio of diameter of DA to that of pulmonary artery. Pups from dams with no drug had DA/PA ratio of 0.9 +/- 0.05, whereas those from dams with indomethacin alone had a decreased ratio of 0.2 +/- 0.03. When ONO-4819 was co-administered to the dams, the ratio recovered significantly to 0.7 +/- 0.06. The administration of ONO-4819 to the dams did not induce any increase in the uterine activity. These results suggest that administration of an EP4 agonist in addition to indomethacin might prevent adverse reactions of indomethacin on fetal DA without restricting its tocolytic effects.

Animals↗
PubMed2004

Alpha-1D adrenoceptors are involved in reserpine-induced supersensitivity of rat tail artery.

1. We examined reserpine-induced chemical denervation supersensitivity with special reference to alpha-1 adrenoceptor (AR) subtypes. 2. Chronic treatment with reserpine for 2 weeks depleted noradrenaline in the tail artery and spleen of rats. Noradrenaline in the thoracic aorta was negligible before and after reserpine treatment. 3. The treatment with reserpine produced supersensitivity in the contractile responses of the rat tail artery to phenylephrine, 5-HT and KCl, resulting in leftward shift of concentration-response curves (11.6-, 2.5- and 1.1-fold at EC(50) value, respectively). These results suggest a predominant sensitization of the alpha-1 AR-mediated response by reserpine treatment. 4. BMY 7378 at a concentration (30 nm) specific for blocking the alpha-1D AR subtype, but not KMD-3213 at a concentration (10 nm) selective for blocking the alpha-1A AR subtype, inhibited the supersensitivity of the phenylephrine-induced response in the reserpine-treated artery. On the other hand, the response to phenylephrine in reserpine-untreated artery was selectively inhibited by the same concentration of KMD-3213, but not by BMY 7378. Prazosin, a subtype-nonselective antagonist, blocked the responses to phenylephrine with the same potency, regardless of reserpine treatment. 5. In the thoracic aorta and spleen, no supersensitivity was produced in the responses to phenylephrine by reserpine treatment. 6. In a tissue segment-binding study using [(3)H]-prazosin, the total density and affinity of alpha-1 ARs in the rat tail artery were not changed by treatment with reserpine. However, alpha-1D AR with high affinity for BMY 7378 was significantly detected in reserpine-treated tail artery, in contrast to untreated artery. Decreases in alpha-1A AR with high affinity for KMD-3213 and alpha-1B AR with low affinities for KMD-3213 and BMY 7378 were also estimated in reserpine-treated tail artery. 7. Alpha-1D AR mRNA in rat tail artery increased to three-folds by reserpine treatment, whereas the levels of alpha-1A and 1B mRNAs were not significantly changed. 8. The present results suggest that chronic treatment with reserpine affects the expression of alpha-1 AR subtypes of rat tail artery and that the induction of alpha-1D ARs with high affinity for catecholamines is in part associated with reserpine-induced supersensitivity.

Animals↗
PubMed2004

Identification of alpha-1L adrenoceptor in rabbit ear artery.

The alpha-1L adrenoceptor (AR) was identified in rabbit ear artery by both functional and ligand binding studies. In functional studies using arterial rings, the contractile response to NS-49 [(R)-(-)-3'-(2-amino-1-hydroxyethyl)-4'-fluorometh-anesulfonanilide hydrochloride] (alpha-1A and alpha-1L AR-selective agonist) was competitively antagonized with low affinities by prazosin, RS-17053 [N-[2-(2-cyclopropylmethoxyphenoxy) ethyl]-5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethamine hydrochloride], and 5-methylurapidil but with high affinities by tamsulosin and KMD-3213 [(-)-1-(3-hydroxypropyl)-5-[(2R)-2-([2-[(2,2,2-trifluoroethoxy)phenoxy]ethyl]amino)propyl]-2,3-dihydro-1H-indole-7-carboxamide]. In contrast, the response to noradrenaline (nonselective alpha-1 AR agonist) was inhibited noncompetitively by these antagonists (except 5-methylurapidil) with Schild slopes different from unity. These results suggest that the response to NS-49 was mediated predominantly via alpha-1L ARs, whereas the response to noradrenaline was produced through two distinct alpha-1 AR subtypes (presumably alpha-1B and alpha-1L ARs). In binding studies with intact segments of rabbit ear artery, [3H]KMD-3213 bound with high affinity (pKD=9.7) to alpha-1 ARs, which were subdivided by prazosin, RS-17053, and 5-methylurapidil into two subtypes (alpha-1A and alpha-1L ARs). In contrast, [3H]prazosin binding sites in ear artery segments (pKD = 9.8) were identified as alpha-1A and alpha-1B ARs. In conventional binding studies using isolated rabbit ear artery microsomal membranes, [3H]KMD-3213 binding sites were identified as alpha-1A ARs with high affinities for prazosin, RS-17053, and 5-methylurapidil. Our study indicates that an alpha-1L AR having a unique pharmacological profile coexists with alpha-1A and alpha-1B ARs in rabbit ear artery and can be identified either functionally or by binding studies using intact tissues but not microsomal membrane preparations.

Adrenergic alpha-Antagonists↗
PubMed2004

Pharmacological characterization and cross talk of alpha1a- and alpha1b-adrenoceptors coexpressed in human embryonic kidney 293 cells.

We established three human embryonic kidney (HEK) 293 cell lines stably expressing alpha(1)-adrenoceptor (AR) subtypes, one (alpha(1A), (1B)-AR) coexpressing both receptors and the other two (alpha(1A)-AR and alpha(1B)-AR) expressing each receptor in isolation. In the alpha(1A), (1B)-AR cells, both receptors were clearly distinguished by the alpha(1A)-selective ligands (-)-1(3-hydroxypropyl)-5-((2R)-2-([2-(2,2,2-trifluoroethyl]oxy]phenyl)oxy)ethyl]amino)propyl)-2,3-dihydro-1H-indole-7-carboxamide (KMD-3213) and methoxamine, but not by the subtype-nonselective ligands prazosin and phenylephrine. In all three cell lines, phenylephrine caused a concentration-dependent increase in inositol phosphates and an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) activation. However, there was a 2-fold or greater maximal response to phenylephrine and a somewhat higher agonist potency in ERK1/2 activation in the alpha(1A,1B)-AR cells, compared with the responses of cells expressing either receptor individually (alpha(1A)-AR or alpha(1B)-AR). Furthermore, the antagonistic affinities of prazosin (pK(b) of 10.1) and KMD-3213 (9.4) for inhibiting the phenylephrine response were intermediate between the values for inhibition in alpha(1A)-AR cells (prazosin, 9.3; KMD-3213, 10.5) and alpha(1B)-AR cells (prazosin, 11.0; KMD-3213, 8.1). The inhibitor pK(b) values in alpha(1A), (1B)-AR also differed from their ligand binding affinities measured in alpha(1A)-AR and alpha(1B)-AR cells. In contrast, the alpha(1A)-selective agonist methoxamine, which did not activate alpha(1B)-AR cells, stimulated either alpha(1A,) (1B)-AR or alpha(1A)-AR cells with a comparable potency and maximum effectiveness. Our data indicate that when coexpressed in the same cell, the activation of common pathways by individual AR receptor subtypes by a nonselective agonist can exhibit enhanced responsiveness and a distinct antagonist affinity compared with the parameters for the same receptors, when expressed alone in the same cell background.

Adrenergic alpha-Agonists↗
PubMed2004

Alpha-1 adrenoceptors: evaluation of receptor subtype-binding kinetics in intact arterial tissues and comparison with membrane binding.

The binding kinetics of [3H]-prazosin were measured using intact segments of rat tail artery (RTA) and thoracic aorta (RAO), and the data were compared with those obtained using a conventional membrane ligand-binding method. In intact RTA and RAO segments, [3H]-prazosin bound reversibly in a time-dependent and receptor-specific manner at 4 degrees C to alpha-1 adrenoceptors (ARs) of the plasma membrane, with affinities (pKD): 9.5 in RTA; 9.9 in RAO) that were in agreement with values estimated by a conventional membrane ligand-binding method. However, nonspecific binding was considerably higher in RAO than RTA, failing to detect clearly the specific binding at high concentrations (>300 pm) of [3H]-prazosin in binding experiments with RAO segments and membranes. The abundance of receptor in the RTA and RAO (Bmax mg-1) of total tissue protein), estimated using the tissue segment-binding approach (527+/-14 fmol mg-1 for RTA; 138+/-4 fmol mg-1 for RAO), was about 25-fold higher than values estimated using a conventional membrane-binding method (22+/-5 fmol mg-1) for RTA; 5+/-1 fmol mg-1 for RAO). Binding competition experiments using intact tissue segments or membranes derived from RTA tissue yielded comparable data, indicating a coexistence of alpha-1A AR (high affinity for prazosin, KMD-3213 and WB4101 and low affinity for BMY 7378) and alpha-1B AR (high affinity for prazosin but low affinity for KMD-3213, WB4101 and BMY 7378). In RAO tissue, careful evaluation of the tissue segment-binding assay revealed the coexpression of alpha-1B AR (high affinity for prazosin, but low affinity for KMD-3213 and BMY 7378) and alpha-1D AR (high affinity for prazosin and BMY 7378, but low affinity for KMD-3213), whereas the membrane-binding approach failed to detect these receptor subtypes with certainty. The present study indicates that previous estimates of alpha-1 AR density and alpha-1 AR subtypes obtained by a conventional membrane-binding approach, as opposed to our improved tissue segment-binding assay, may have substantially underestimated the abundance of receptors present in arterial tissues, and may have failed to identify accurately the presence of receptor subtypes. Advantages and disadvantages of the tissue segment-binding approach are discussed.British Journal of Pharmacology (2004) 141, 468-476. doi:10.1038/sj.bjp.0705627

Adrenergic alpha-1 Receptor Antagonists↗
PubMed2003

Pharmacological knockout of endothelin ET(A) receptors.

We employed newly developed antagonists, which are specific for endothelin ET(A) receptors, to test whether this drug could mimic the phenotype of the mouse with corresponding gene knock out. Newborn rats, whose dams were given the ET(A) antagonist from day 7 of gestation, exhibited the typical ET(A)-lacking phenotypes like craniofacial abnormalities and major vessel anomalies. Interestingly, craniofacial abnormality was seen in the pups that were exposed to the drug in the mid-gestational period, while another phenotype, patent ductus arteriosus (DA), was seen in the pups that were exposed to the drug in the late gestation. We have focused on the function of the ET system in DA closure after birth because the animals with a genetic defect of ET(A) would die of suffocation shortly after birth. Rat pups were delivered by Caesarean section and were given the antagonist intraperitoneally. The antagonists caused an inhibition of DA closure in vivo at 3 h after birth when DA closure was completed in the control pups. Next, we tested the potential utilities of the ET(A) specific antagonists in tocolysis with NSAIDs which sometimes leads to a closure of fetal DA in utero. Indomethacin administration to rat dams resulted in the constriction of DA in utero which was cancelled by the co-administration of the antagonists. These results suggested that ET(A) plays a physiological role in the postnatal closure of the rat DA in vivo and that ET(A) specific antagonists may be able to leave fetal DA intact during tocolysis with NSAIDs.

Abnormalities, Drug-Induced↗
PubMed2003

Nicotine induces human neutrophils to produce IL-8 through the generation of peroxynitrite and subsequent activation of NF-kappaB.

Leukocytosis in tobacco smokers has been well recognized; however, the exact cause has not been elucidated. To test the hypothesis that tobacco nicotine stimulates neutrophils in the respiratory tract to produce IL-8, which causes neutrophilia in vivo, we examined whether nicotine induces neutrophil-IL-8 production in vitro; the causative role of NF-kappaB in its production, in association with the possible production of reactive oxygen intermediates that activate NF-kappaB; and the nicotinic acetylcholine receptors (nAChRs) involved in IL-8 production. Nicotine stimulated neutrophils to produce IL-8 in both time- and concentration-dependent manners with a 50% effective concentration of 1.89 mM. A degradation of IkappaB-alpha/beta proteins and an activity of NF-kappaB p65 and p50 were enhanced following nicotine treatment. The synthesis of superoxide and the oxidation of dihydrorhodamine 123 (DHR) were also enhanced. The NOS inhibitor, nomega-Nitro-l-arginine methyl ester, prevented nicotine-induced IL-8 production, with an entire abrogation of DHR oxidation, IkappaB degradation, and NF-kappaB activity. Neutrophils spontaneously produced NO whose production was not increased, but rather decreased by nicotine stimulation, suggesting that superoxide, produced by nicotine, generates peroxynitrite by reacting with preformed NO, which enhances the NF-kappaB activity, thereby producing IL-8. The nAChRs seemed to be involved in IL-8 production. In smokers, blood IL-8 levels were significantly higher than those in nonsmokers. In conclusion, nicotine stimulates neutrophil-IL-8 production via nAChR by generating peroxynitrite and subsequent NF-kappaB activation, and the IL-8 appears to contribute to leukocytosis in tobacco smokers.

Humans↗
PubMed2003

Pharmacological characterization of unique prazosin-binding sites in human kidney.

In human kidney, we found unique prazosin-binding sites that were insensitive to phentolamine and were thus unlikely to be alpha(1)-adrenoceptors. As the binding of [(3)H]prazosin to phentolamine-insensitive sites was prevented by 100 microM guanabenz, the insensitive sites were evaluated by subtracting [(3)H]prazosin binding in the presence of 100 microM guanabenz from that in the presence of 10 microM phentolamine. [(3)H]Prazosin bound to the phentolamine-insensitive sites monophasically with a high affinity (pK(d); 9.1+/-0.08, n=8), and the B(max) value (814+/-204 fmol mg(-1) protein, n=8) was more than ten times that of the phentolamine-sensitive alpha(1)-adrenoceptor (pK(d)=9.9+/-0.13, B(max)=66+/-23 fmol mg(-1) protein, n=7). The phentolamine-insensitive sites in human kidney were highly sensitive to other quinazoline derivatives such as terazosin and doxazosin. However, other alpha(1)-adrenoceptor antagonists (tamsulosin, WB4101 and corynanthine) did not inhibit the binding at a range of concentrations that generally exhibit alpha(1)-adrenoceptor antagonism, and noradrenaline, rauwolscine and propranolol were without effect on the [(3)H]prazosin binding. On the other hand, ligands for the renal Na(+)-transporter (amiloride and triamterene) and for imidazoline recognition sites (guanabenz, guanfacine and agmatine) displaced the binding of [(3)H]prazosin to phentolamine-insensitive sites at micromolar concentrations. Photoaffinity labeling with [(125)I]iodoarylazidoprazosin showed phentolamine-insensitive labeling at around 100 kDa, a molecular size larger than that of human alpha(1a)- and alpha(1b)-adrenoceptors expressed in 293 cells (50-60 and 70-80 kDa, respectively) on electrophoresis. In contrast, there was no detectable phentolamine-insensitive binding site but were phentolamine-sensitive alpha(1)-adrenoceptors in human liver (pK(d)=10.0+/-0.06, B(max)=44+/-6 fmol mg(-1) protein, n=3). Phentolamine-insensitive prazosin binding sites were also detected in rabbit kidney (approximately 50% of specific binding sites) but were minor in rat kidney (less than 20%). In conclusion, there are unique prazosin-binding sites in human kidney, the pharmacological profiles of which were distinct from those of known adrenoceptors.

Adrenergic alpha-Agonists↗