Regional quantification of muscarinic acetylcholine receptors and ß-adrenoceptors in human airways.

BACKGROUND AND PURPOSE Muscarinic acetylcholine receptors (mAChRs) and ß-adrenoceptors in the airways and lungs are clinically important in chronic obstructive pulmonary disease (COPD) and asthma. However, the quantitative and qualitative estimation of these receptors by radioligand binding approaches in human airways has not yet been reported because of tissue limitations. EXPERIMENTAL APPROACH The regional distribution and relative proportion of mAChR and ß-adrenoceptor subtypes were evaluated in human bronchus and lung parenchyma by a tissue segment binding method with [(3)H]-N-methylscopolamine ([(3)H]-NMS) for mAChRs and [(3)H]-CGP-12,177 for ß-adrenoceptors. Functional responses to carbachol and isoprenaline were also analysed in the bronchus. KEY RESULTS The M(3) subtype predominantly occurred in the bronchus, but the density decreased from the segmental to subsegmental bronchus, and was absent in lung parenchyma. On the other hand, the M(1) subtype occurred in the lung only, and the M(2) subtype was distributed ubiquitously in the bronchus and lungs. ß(2)-adrenoceptors were increased along the airways, and their densities in the subsegmental bronchus and lung parenchyma were approximately twofold higher than those of mAChRs in the same region. ß(1)-adrenoceptors were also detected in lung parenchyma but not in the bronchus. The muscarinic contractions and adrenoceptor relaxations in both bronchial regions were mediated through M(3)-mAChRs and ß(2)-adrenoceptors, respectively. CONCLUSIONS AND IMPLICATIONS From the present radioligand binding approach with intact tissue segments, we constructed a distribution map of mAChRs and ß-adrenoceptors in human bronchus and lung parenchyma for the first time, providing important evidence for future pharmacotherapy and new drug development for respiratory disorders.

Phenotype pharmacology of lower urinary tract α(1)-adrenoceptors.

α(1)-Adrenoceptors are involved in numerous physiological functions, including micturition. However, the pharmacological profile of the α(1)-adrenoceptor subtypes remains controversial. Here, we review the literature regarding α(1)-adrenoceptors in the lower urinary tract from the standpoint of α(1L) phenotype pharmacology. Among three α(1)-adrenoceptor subtypes (α(1A), α(1B) and α(1D)), α(1a)-adrenoceptor mRNA is the most abundantly transcribed in the prostate, urethra and bladder neck of many species, including humans. In prostate homogenates or membrane preparations, α(1A)-adrenoceptors with high affinity for prazosin have been detected as radioligand binding sites. Functional α(1)-adrenoceptors in the prostate, urethra and bladder neck have low affinity for prazosin, suggesting the presence of an atypical α(1)-adrenoceptor phenotype (designated as α(1L)). The α(1L)-adrenoceptor occurs as a distinct binding entity from the α(1A)-adrenoceptor in intact segments of variety of tissues including prostate. Both the α(1L)- and α(1A)-adrenoceptors are specifically absent from Adra1A (α(1a)) gene-knockout mice. Transfection of α(1a)-adrenoceptor cDNA predominantly expresses α(1A)-phenotype in several cultured cell lines. However, in CHO cells, such transfection expresses α(1L)- and α(1A)-phenotypes. Under intact cell conditions, the α(1L)-phenotype is predominant when co-expressed with the receptor interacting protein, CRELD1α. In summary, recent pharmacological studies reveal that two distinct α(1)-adrenoceptor phenotypes (α(1A) and α(1L)) originate from a single Adra1A (α(1a)-adrenoceptor) gene, but adrenergic contractions in the lower urinary tract are predominantly mediated via the α(1L)-adrenoceptor. From the standpoint of phenotype pharmacology, it is likely that phenotype-based subtypes such as the α(1L)-adrenoceptor will become new targets for drug development and pharmacotherapy.

Expression of distinct alpha 1-adrenoceptor phenotypes in the iris of pigmented and albino rabbits.

BACKGROUND AND PURPOSE: The expression of multiple pharmacological phenotypes including alpha(1L)-adrenoceptor has recently been reported for alpha(1)-adrenoceptors. The purpose of the present study was to identify alpha(1)-adrenoceptor phenotypes in the irises of pigmented and albino rabbits. EXPERIMENTAL APPROACH: Radioligand binding and functional bioassay experiments were performed in segments or strips of iris of pigmented and albino rabbits, and their pharmacological profiles were compared. KEY RESULTS: [(3)H]-silodosin at subnanomolar concentrations bound to intact segments of iris of pigmented and albino rabbits at similar densities (approximately 240 fmol x mg(-1) protein). The binding sites in the iris of a pigmented rabbit were composed of a single component showing extremely low affinities for prazosin, hydrochloride [N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethamine hydrochloride (RS-17053)] and 5-methylurapidil, while two components with high and low affinities for prazosin, RS-17053 and 5-methylurapidil were identified in irises from albino rabbits. In contrast, specific binding sites for [(3)H]-prazosin were not clearly detected because a high proportion of non-specific binding and/or low affinity for prazosin occurred. Contractile responses of iris dilator muscle to noradrenaline were antagonized by the above ligands, and their antagonist affinities were consistent with the binding estimates at low-affinity sites identified in both strains of rabbits. CONCLUSIONS AND IMPLICATIONS: A typical alpha(1L) phenotype with extremely low affinity for prazosin is exclusively expressed in the iris of pigmented rabbits, while two distinct phenotypes (alpha(1A) and alpha(1L)) with high and moderate affinities for prazosin are co-expressed in the iris of albino rabbits. This suggests that a significant difference in the expression of phenotypes of the alpha(1)-adrenoceptor occurs in the irises between the two strains of rabbits.

Identification of alpha 1L-adrenoceptor in mice and its abolition by alpha 1A-adrenoceptor gene knockout.

BACKGROUND AND PURPOSE: The alpha(1L)-adrenoceptor has pharmacological properties that distinguish it from three classical alpha(1)-adrenoceptors (alpha(1A), alpha(1B) and alpha(1D)). The purpose of this was to identify alpha(1L)-adrenoceptors in mice and to examine their relationship to classical alpha(1)-adrenoceptors. EXPERIMENTAL APPROACH: Radioligand binding and functional bioassay experiments were performed on the cerebral cortex, vas deferens and prostate of wild-type (WT) and alpha(1A)-, alpha(1B)- and alpha(1D)-adrenoceptor gene knockout (AKO, BKO and DKO) mice. KEY RESULTS: The radioligand [(3)H]-silodosin bound to intact segments of the cerebral cortex, vas deferens and prostate of WT, BKO and DKO but not of AKO mice. The binding sites were composed of two components with high and low affinities for prazosin or RS-17053, indicating the pharmacological profiles of alpha(1A)-adrenoceptors and alpha(1L)-adrenoceptors. In membrane preparations of WT mouse cortex, however, [(3)H]-silodosin bound to a single population of prazosin high-affinity sites, suggesting the presence of alpha(1A)-adrenoceptors alone. In contrast, [(3)H]-prazosin bound to two components having alpha(1A)-adrenoceptor and alpha(1B)-adrenoceptor profiles in intact segments of WT and DKO mouse cortices, but AKO mice lacked alpha(1A)-adrenoceptor profiles and BKO mice lacked alpha(1B)-adrenoceptor profiles. Noradrenaline produced contractions through alpha(1L)-adrenoceptors with low affinity for prazosin in the vas deferens and prostate of WT, BKO and DKO mice. However, the contractions were abolished or markedly attenuated in AKO mice. CONCLUSIONS AND IMPLICATIONS: alpha(1L)-Adrenoceptors were identified as binding and functional entities in WT, BKO and DKO mice but not in AKO mice, suggesting that the alpha(1L)-adrenoceptor is one phenotype derived from the alpha(1A)-adrenoceptor gene.

Native profiles of alpha(1A)-adrenoceptor phenotypes in rabbit prostate.

BACKGROUND AND PURPOSE: alpha(1)-Adrenoceptors in the rabbit prostate have been studied because of their controversial pharmacological profiles in functional and radioligand binding studies. The purpose of the present study is to determine the native profiles of alpha(1)-adrenoceptor phenotypes and to clarify their relationship. EXPERIMENTAL APPROACH: Binding experiments with [3H]-silodosin and [3H]-prazosin were performed using intact tissue segments and crude membrane preparations of rabbit prostate and the results were compared with alpha(1)-adrenoceptor-mediated prostate contraction. KEY RESULTS: [3H]-Silodosin at subnanomolar concentrations bound specifically to intact tissue segments of rabbit prostate. However, [3H]-prazosin at the same range of concentrations failed to bind to alpha(1)-adrenoceptors of intact segments. Binding sites of [3H]-silodosin in intact segments were composed of alpha(1L) phenotype with low affinities for prazosin (pKi=7.1), 5-methyurapidil and N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethamine hydrochloride (RS-17053), and alpha(1A)-like phenotype with moderate affinity for prazosin (pKi=8.8) but high affinity for 5-methyurapidil and RS-17053. In contrast, both radioligands bound to a single population of alpha(1)-adrenoceptors in the membrane preparations at the same density with a subnanomolar affinity, showing a typical profile of 'classical' alpha(1A)-adrenoceptors (pKi for prazosin=9.8). The pharmacological profile of alpha(1)-adrenoceptor-mediated prostate contraction was in accord with the alpha(1L) phenotype observed by intact segment binding approach. CONCLUSIONS AND IMPLICATIONS: Three distinct phenotypes (alpha(1L) and alpha(1A)-like phenotypes in the intact segments and a classical alpha(1A) phenotype in the membranes) with different affinities for prazosin were detected in rabbit prostate. It appears that the three phenotypes are phenotypic subtypes of alpha(1A)-adrenoceptors, but are not genetically different subtypes.

Identification of the alpha1L-adrenoceptor in rat cerebral cortex and possible relationship between alpha1L- and alpha1A-adrenoceptors.

BACKGROUND AND PURPOSE: In addition to alpha1A, alpha1B and alpha1D-adrenoceptors (ARs), putative alpha1L-ARs with a low affinity for prazosin have been proposed. The purpose of the present study was to identify the alpha1A-AR and clarify its pharmacological profile using a radioligand binding assay. EXPERIMENTAL APPROACH: Binding experiments with [3H]-silodosin and [3H]-prazosin were performed in intact tissue segments and crude membrane preparations of rat cerebral cortex. Intact tissue binding assays were also conducted in rat tail artery. KEY RESULTS: [3H]-silodosin at subnanomolar concentrations specifically bound to intact tissue segments and membrane preparations of rat cerebral cortex at the same density (approximately 150 fmol mg(-1) total tissue protein). The binding sites in intact segments consisted of alpha1A and alpha1L-ARs that had different affinities for prazosin, while the binding sites in membranes showed an alpha1A-AR-like profile having single high affinity for prazosin. [3H]-prazosin also bound at subnanomolar concentrations to alpha1A and alpha1B-ARs but not alpha1L-ARs in cerebral cortex; the binding densities being approximately 200 and 290 fmol mg(-1) protein in the segments and the membranes, respectively. In the segments of tail artery, [3H]-silodosin only recognized alpha1A-ARs, whereas [3H]-prazosin bound to alpha1A and alpha1B-ARs. CONCLUSIONS AND IMPLICATIONS: The present study clearly reveals the presence of alpha1L-ARs as a pharmacologically distinct entity from alpha1A and alpha1B-ARs in intact tissue segments of rat cerebral cortex but not tail artery. However, the alpha1L-ARs disappeared after tissue homogenization, suggesting their decomposition and/or their pharmacological profile changes to that of alpha1A-ARs.

Endothelin-1-endothelin receptor type A mediates closure of rat ductus arteriosus at birth.

1. The ductus arteriosus (DA) undergoes rapid closure after birth as pulmonary circulation is established. The involvement of endothelin-1 (ET1) in this closure mechanism is controversial. 2. The effect of ATZ1993 (ATZ), a non-peptide antagonist for the ET(A) and ET(B) receptors, on postnatal closure and O2-induced contraction of the rat DA was investigated both in vivo and in vitro. Rat pups were delivered by Caesarean section and were given ATZ intraperitoneally. The minimum external DA diameter and the extent of DA constriction in vivo were evaluated at 2.5 h after birth. ATZ caused a dose-dependent inhibition of DA closure in vivo. When rat pups were given ATZ at 2.5 h after birth, re-opening of the DA was observed. 3. In vitro, ATZ also caused a marked inhibition of O2-induced and ET1-induced DA contractions as did BQ123, an ET(A)-specific antagonist. In contrast, sarafotoxin S6c, an ET(B)-specific agonist, did not cause DA contraction and BQ788, an ET(B)-specific antagonist, did not affect O2-induced DA contraction. 4. In conclusion, ET1 and its cognate receptor ET(A) may play a physiological role in the postnatal closure of the rat DA in vivo.

Rapid acid extrusion response triggered by alpha(1) adrenoceptor in CHO cells.

1. Using a microphysiometer with synchronized valve switching, we investigated real-time acid extrusion from Chinese hamster ovary (CHO) cells in which human alpha(1) adrenoceptor (AR) is stably expressed, in response to noradrenaline (NA). 2. In the cells expressing alpha(1a) AR, the time course of extracellular acidification after stimulation had two phases; in the first phase it transiently reached a rate several times greater than the base rate with a peak at around 10 s, and in the second it increased to 2 times the base rate and reached a plateau in 2 min. Both phases showed a concentration-dependent increase of acidification rate in response to NA, but had distinct pEC(50) values; 5.6 for the transient phase and 7.2 for the steady phase. 3. In the cells expressing alpha(1b) AR, the transient phase was not detected but the steady phase was observed. The pEC(50) value was 7.1, although the magnitude of the response was much smaller than that with alpha(1a) AR. 4. Both 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and HOE642 inhibited the acid extrusion response by either AR in a concentration-dependent manner. EIPA and HOE642 had high pIC(50) values (7.4 and 7.3, respectively) for inhibition of the transient phase response via alpha(1a) AR. In the inhibition of the steady phase response via alpha(1a) AR, both drugs revealed the presence of two components in the response; one had high pIC(50) values (8.1 and 8.2 for EIPA and HOE642, respectively) and the other had low pIC(50) values (5.6 and 6.0, respectively). In contrast, the steady phase response via alpha(1b) AR was inhibited by EIPA and HOE642 with low pIC(50) values (5.3 and 5.9, respectively). 5. As Ca2+ was depleted, the alpha(1a) AR-induced transient phase disappeared, while the steady phase was not affected. 6. These results suggest that alpha(1a) AR drives two acid extrusion systems in CHO cells upon stimulation; one elicits the transient response, which is largely mediated by an EIPA/HOE642-sensitive and Ca(2+)-dependent Na(+)-H(+) exchanger (NHE), presumably NHE1, and the other induces the steady acid extrusion that is mediated by NHE1 and another NHE which has low sensitivity to both EIPA and HOE642. alpha(1b) AR drives only the steady phase acid extrusion response, which is mainly mediated by NHEs other than NHE1.

Anterior interosseous nerve palsy after cardiopulmonary resuscitation in a resuscitator with undiagnosed muscle anomaly.

IMPLICATIONS: We present a case of nerve palsy after cardiopulmonary resuscitation in a resuscitator with undiagnosed muscle anomaly. Effort-related nerve palsy may occur after prolonged performance of CPR.

Affinity of serotonin receptor antagonists and agonists to recombinant and native alpha1-adrenoceptor subtypes.

Binding affinities of serotonin (5-HT)-receptor antagonists and agonists at human recombinant alpha1-adrenoceptor subtypes (alpha1a-, alpha1b- and alpha1d-subtypes) were examined and compared with the functional affinities obtained in rat caudal artery (alpha1A-subtype), dog carotid artery (alpha1B-subtype) and rat thoracic aorta (alpha1D-subtype). Most of the 5-HT-receptor antagonists and agonists tested showed relatively high affinity to three alpha1-adrenoceptor subtypes. The highest affinity close to 1 nM was seen for NAN-190 (5-HT1A antagonist) in binding and functional studies. 5-Methylurapidil (5-HT1A agonist) and BMY7378 (5-HT1A agonist) showed, respectively, alpha1a(alpha1A)- or alpha1d(alpha1D)-subtype selectivity in both binding and functional affinities, but spiperone (5-HT2A antagonist) showed alpha1b-selectivity only in binding affinity. Functional affinity of ritanserin (5-HT2A antagonist) to the alpha1B-subtype was approximately 500-fold lower than that of affinity to the alpha1b-subtype. The present results show that many 5-HT-receptor antagonists and agonists have high affinity to alpha1-adrenoceptors, but suggest that there is deviation between their functional affinities and binding affinities for some drugs.

No evidence for AT2R gene derangement in human urinary tract anomalies.

BACKGROUND: It has been recently found that mice, especially males, with a disrupted angiotensin type 2 receptor (AT2R) gene, which is located on the X-chromosome, often have a range of congenital anomalies of the kidney and urinary tract (CAKUT), including renal hypoplasia, and that Caucasian male patients with ureteropelvic junction stenosis (UPJ) and multicystic dysplastic kidneys frequently have A-G transition in intron 1 of the AT2R gene. We have previously found that renal hypoplasia is remarkably predominant in Japanese boys. METHODS: We investigated sex ratios for the frequency of each CAKUT. The frequency of the A-G transition between the controls and 66 Japanese boys with CAKUT were compared. There was renal hypoplasia in 16, UPJ in 17, vesicoureteral in 20, and other anomalies in 13. We also investigated whether any mutations in AT2R genes were detectable in patients with renal hypoplasia. RESULTS: In contrast to mice with a disruption of the AT2R gene, the male-to-female ratios in human patients proved to be considerably variable: 16 for renal hypoplasia, 2.1 for UPJ, 0.8 for vesicoureteral, and 1.2 for others. The frequency of the A-G transition was not different between the control population and the patients with CAKUT [31 of 102 (30%) vs. 23 of 66 (35%), respectively]. A sequencing study disclosed no mutations in nine boys with renal hypoplasia. CONCLUSIONS: These findings indicate that the AT2R gene may not play a major role in the development of renal hypoplasia and other CAKUT in humans, at least in the Japanese population.

Pharmacological characterization of [3H]-JTH-601, a novel alpha1-adrenoceptor antagonist: binding to recombinant human alpha1-adrenoceptors and human prostates.

Several alpha1-adrenoceptor (AR) selective antagonists are now widely used to improve lower urinary tract symptoms in benign prostatic hyperplasia patients. However, these drugs often result in orthostatic hypotension, because of their poor uroselectivity; the blockade of alpha1-AR not only in prostate but also in vasculature. Here we have investigated uroselectivity of JTH-601, a newly developed antagonist, in radioligand binding experiment using recombinant human alpha1-AR subtypes and human prostate. In saturation experiments, [3H]-JTH-601 showed subtype selectivity: high affinity to alpha1a-AR (pKd; 9.88+/-0.09), lower affinity to alpha1b-AR (pKd; 8.96+/-0.17) and no specific binding at concentrations up to 3000 pM to alpha1d-AR. In competition experiments, JTH-601 and its metabolic compound (JTH-601-G1) also showed alpha1a-AR selectivity, exhibiting approximately 5 times higher affinity for alpha1a-AR than for alpha1b-AR, 10 to 20 times higher affinity than for alpha1d-AR, respectively. [3H]-JTH-601 also bound to human prostate membranes in monophasic manner with high affinity constant (pKd; 9.89+/-0.12, Bmax=123.6+/-16 fmol/mg protein). JTH-601 is a unique alpha1-AR antagonist that shows high affinity and selectivity for human recombinant alpha1a- and human prostate. This new compound is useful for understanding alpha1-AR pharmacology and may have a therapeutic value.

Characterization of the endothelin receptor subtypes in human prostate.

Endothelin (ET) receptor subtypes in human prostate with benign prostatic hyperplasia were investigated by binding and functional studies. In the displacement experiment, LU224332 [endothelin-A/-B (ET(A)/ET(B)) nonselective antagonist] competed for [125I]ET-1 binding with a monophasic curve. On the other hand, LU135252 (ET(A)-selective antagonist) and sarafotoxin S6c (S6c, ET(B)-selective agonist) competed for [125I]ET-1 binding with shallow and biphasic curves. The analysis of the displacement curves for LU135252 and S6c showed that both ET(A) and ET(B) subtypes coexist but that ET(A) is the dominantly expressed receptor. In human prostate strips, 10 microM of both LU135252 and LU224332 strongly inhibited the contractile response to ET-1 with equal potency. However, 10 microM of BQ788 (ET(B)-selective antagonist) did not show a clear inhibition. S6c also produced a contractile response, which was potently inhibited by LU224332 or BQ788, and slightly suppressed by LU135252. These results suggest that in human prostate both ET(A) and ET(B) subtypes are functional receptors mediating contraction, but that ET-1-mediated contractions are predominantly mediated by activation of dominant receptor subtype, ET(A).

Homeobox gene Hex is essential for onset of mouse embryonic liver development and differentiation of the monocyte lineage.

Disruption of the mouse Hex gene resulted in embryonic lethality around embryonic age (E) 10.5, due to no substantial liver formation. Expression of albumin was detectable in heterozygous (Hex(+/-)) but not in homozygous (Hex(-/-)) [corrected] embryos at E8.5. Instead of liver bud formation at E9.5, a liver-like capsule structure was observed in Hex(-/-) [corrected] embryos. In Hex(-/-) [corrected] mutant liver, we found no hepatocytes but no signs of apoptotic cell death in the area. Expression of transcription factors involved in hepatocyte differentiation, hepatocyte nuclear factor (Hnf)3beta, Hnf6, Hnf4alpha and Hnf1alpha, were restricted to the capsule and internal matrix-like structure in the mutant liver and expression of a subset of these factors were reduced. Hematopoiesis of monocytes was impaired in mutant embryos while erythroid lineage was unaffected. These results indicate that Hex plays an essential role in progenitor cells which commit to the hepatic endoderm and in the hematopoietic differentiation of the monocyte lineage.

Effects of perinatal nicotine exposure on development of [3H]hemicholinium-3 binding sites in rat neonate brain.

In this study, [3H]hemicholinium-3 ([3H]HC-3) binding, which labels the presynaptic high affinity-choline transport sites, was examined in two brain regions, cerebral cortex and midbrain, of nicotine-treated and -untreated rat neonates. In nicotine-untreated neonates, [3H]HC-3 binding sites of cerebral cortex increased from 64 fmol/mg protein at postnatal day 7 to 142 fmol/mg protein at postnatal day 35. In nicotine-treated neonates, the development of [3H]HC-3 binding sites in cerebral cortex was significantly retarded, compared with control neonates on the 7th, 14th and 21st postnatal days. In parallel with this, the development of muscarinic receptor in cerebral cortex, which was detected by [3H]quinuclidinyl benzylate ([3H]QNB) binding, was also retarded by nicotine treatment. However, in midbrain, neither [3H]HC-3 nor [3H]QNB binding sites at postnatal day 14 was affected by nicotine treatment. These results strongly suggest that perinatal treatment with nicotine inhibits presynaptic and postsynaptic development of the cholinergic pathway in cerebral cortex but not in midbrain of rat neonate.

Inverse agonism and neutral antagonism at a constitutively active alpha-1a adrenoceptor.

We have studied the antagonist action of prazosin and KMD-3213 in a constitutively active mutant of the human alpha-1a adrenoceptor in which Ala(271) was substituted to Thr and was expressed in CHO cells. Inverse agonism was characterized by up-regulation of receptor density, a decrease in basal GTPgammaS binding, and a reduction in basal inositol-1,4,5-trisphosphate (IP(3)) level. According to the above criteria, prazosin acted as an inverse agonist, whilst KMD-3213 behaved as a neutral antagonist. Compared with the wild-type receptor, mutant receptor exhibited single affinity sites for [(3)H]-prazosin, [(3)H]-KMD and the non-radioactive ligands tested, and displayed significantly higher affinities for several agonists but not for the two antagonists. Administration of KMD-3213 to prazosin-treated CHO cells expressing the mutant receptor reversed the inverse agonism of prazosin resulting in rapid increases in cellular IP(3), in intracellular [Ca(2+)] and in the rate of extracellular acidification. These results indicated that a neutral antagonist can reverse the action of an inverse agonist at the receptor site. The distinct properties of inverse agonist and neutral antagonist in affecting receptor function may be important for the clinical use of such antagonists.

Tissue selectivity of KMD-3213, an alpha(1)-adrenoreceptor antagonist, in human prostate and vasculature.

PURPOSE: We evaluated the binding and functional affinity of KMD-3213 and other alpha 1-adrenoceptor (AR) antagonists such as prazosin or tamsulosin, to compare the tissue selectivity of these antagonists between human prostate and vasculature. MATERIALS AND METHODS: In the binding experiments, saturation experiments using [3H]-KMD and [3H]-prazosin (PZ) were performed, and competition of [3H]-PZ binding by antagonists was also examined in human prostatic and aortic membranes. In the functional study, contractile responses to noradrenaline were evaluated in human prostate and mesenteric artery. RESULTS: [3H]-PZ bound to human prostatic and aortic membranes with subnanomolar affinity. [3H]-KMD also bound to human prostate, with higher affinity than [3H]-PZ; whereas it did not bind sufficiently to human aorta. Competition of [3H]-PZ binding revealed that KMD-3213 had more than 200-fold higher affinity for human prostate than for aorta. Binding profiles of antagonists revealed that human prostate predominantly expressed alpha 1A-AR, whereas human aorta expressed alpha 1B-AR mainly. In functional experiments, KMD-3213 potently inhibited the noradrenaline-induced contraction in human prostate as potently as tamsulosin, although prazosin showed relatively low affinity. Comparing these functional affinities with those in the mesenteric artery, only KMD-3213 exhibited substantial tissue selectivity, showing more than 100-fold higher affinity for human prostate than for mesenteric artery. Functional affinity of each antagonist suggested that noradrenaline-induced contractions were mainly mediated by alpha 1L-AR in the human prostate and by alpha 1B-AR in the mesenteric artery. CONCLUSION: These results suggest that KMD-3213 is a substantially prostate-selective alpha 1-AR antagonist in human tissues compared with other alpha 1-AR antagonists.

Pharmacological analysis of neurogenic, sympathetic responses mediated through alpha-1-, alpha-2-adrenergic and purinergic receptors in the dog saphenous vein.

Electrical transmural stimulation evoked a sympathetic contraction in the isolated dog saphenous vein. This contraction consisted of three components (alpha(1)-adrenergic, alpha(2)-adrenergic and purinergic), which were separately observed under combined treatments either with yohimbine (blockade of alpha(2)-adrenoceptor) and alpha,beta-methylene ATP (desensitization of P(2X)-purinoceptors), with prazosin (blockade of alpha(1)-adrenoceptor) and alpha,beta-methylene ATP, or with prazosin and yohimbine, respectively. The alpha(1)-adrenergic and purinergic contractions immediately developed after the start of stimulation and reached a peak rapidly. In contrast, the alpha(2)-adrenergic contraction developed slowly, thus the time to peak contraction was longer than the other two components. The relationship between the peak amplitudes of contraction and stimulus frequencies were similar between alpha(1)- and alpha(2)-adrenergic components, but the purinergic contraction was smaller than the other components at all frequencies (0.1-30 Hz). Cocaine, a neuronal uptake inhibitor of noradrenaline, significantly potentiated alpha(1)- and alpha(2)-adrenergic components and prolonged their duration with a relatively greater effect on the alpha(2)-adrenergic component. In contrast, the purinergic component was not affected by cocaine. Exogenous noradrenaline produced concentration-dependent contraction, which was inhibited more effectively after combined treatment with combination of prazosin and yohimbine than either blocker given alone. Cocaine potentiated the attenuated contractile response to noradrenaline in the presence of prazosin, resulting in the recovery of response to the control level. Exogenous ATP produced a transient contraction, which was abolished under conditions where postjunctional P(2X)-purinoceptors were desensitized with alpha,beta-methylene ATP. Cocaine did not affect the ATP-induced contraction. These results suggest that sympathetic contraction of the dog saphenous vein is caused through three distinct routes (alpha(1)- and alpha(2)-adrenoceptors and P(2X)-purinoceptors).

Adsorption of lidocaine into a plastic infusion balloon.

An infusion balloon is a well established device used to continuously supply drugs for pain management. In this study, we determined the concentration of lidocaine that flowed out of a balloon because the balloon is made from plastics that adsorb local anesthetics.

Cloning of rabbit alpha(1b)-adrenoceptor and pharmacological comparison of alpha(1a)-, alpha(1b)- and alpha(1d)-adrenoceptors in rabbit.

We have isolated a cDNA clone of the rabbit alpha(1b)-adrenoceptor which has an open reading frame of 1557 nucleotides encoding a protein of 518 amino acids. The sequence shows higher identity to those of hamster, human, and rat alpha(1b)-adrenoceptors than to those of rabbit alpha(1a)- and alpha(1d)-adrenoceptors. The pharmacological binding properties of this clone expressed in Cos-7 cells showed a characteristic profile as alpha(1b)-adrenoceptor; high affinity for prazosin (pK(i)=10.3), relatively high affinity for tamsulosin (9.5) and low affinity for (-)-(R)-1-(3-hydroxypropyl)-5-[2-[[2-[2-(2,2, 2-trifluoroethoxy)phenoxy]ethyl]amino]propyl]indoline-7-carboxamid e (KMD3213) (8.5), 2-(2,6-dimethoxy-phenoxyethyl)-aminomethyl-1, 4-benzodioxane hydrochloride (WB4101) (8.7), and 8-[2-[4-(2-methoxy-phenyl)-L-piperazinyl]-8-azaspiro[4,5]decane-7, 9-dione dihydrochloride (BMY7378) (7.3). We have compared the levels of mRNA expression of three alpha(1)-adrenoceptor subtypes in rabbit tissues using the competitive reverse transcription/polymerase chain reaction (RT/PCR) assay. In most rabbit tissues except heart, alpha(1a)-adrenoceptor mRNA was expressed 10 folds more than the other two subtypes. However, binding experiments with [3H]prazosin and [3H]KMD3213 in rabbit tissues revealed a poor relationship between binding density and mRNA level. Especially, alpha(1b) binding sites were exclusively predominant in spleen, whereas the alpha(1b) subtype was minor at the mRNA level. These results indicate a high identity of structural and pharmacological profiles of three distinct alpha(1)-adrenoceptor subtypes between rabbit and other species, but there are species differences in their distribution.