Search

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

Nicotinic acetylcholine receptors (nAChRs) of the cerebral cortex and cerebellum of rats were evaluated by a radioligand binding assay, employing tissue segments, or homogenates as materials. [(3)H]-epibatidine specifically bound to nAChRs in rat cortex or cerebellum, but the dissociation constants for [(3)H]-epibatidine differed between segments and homogenates (187 pM for segments and 42 pM for homogenates in the cortex and 160 pM for segments and 84 pM for homogenates in the cerebellum). The abundance of total nAChRs was approximately 310 fmol/mg protein in the segments of cortex and 170 fmol/mg protein in the segments of cerebellum, which were significantly higher than those estimated in the homogenates (115 fmol/mg protein in the homogenates of the cortex and 76 fmol/mg protein in the homogenates of the cerebellum). Most of the [(3)H]-epibatidine binding sites in the cortex segments (approximately 70% of the population) showed high affinity for nicotine (pK(i) = 7.9), dihydro-β-erythroidine, and cytisine, but the binding sites in the cerebellum segments had slightly lower affinity for nicotine (pK(i) = 7.1). An upregulation of nAChRs by chronic administration of nicotine was observed in the cortex segments but not in the cerebellum segments with [(3)H]-epibatidine as a ligand. The upregulation in the cortex was caused by a specific increase in the high-affinity sites for nicotine (probably α4β2). The present study shows that the native environment of nAChRs is important for a precise quantitative as well as qualitative estimation of nAChRs in rat brain.

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

1. alpha 1-Adrenoceptor subtypes in rabbit thoracic aorta have been examined in binding and functional experiments. 2. [3H]-prazosin bound to two distinct populations of alpha 1-adrenoceptors (pKD,high = 9.94, Rhigh = 79.2 fmol mg-1 protein; pKD,low = 8.59, Rlow = 215 fmol mg-1 protein). Pretreatment with chloroethylclonidine (CEC, 10 microM) almost inactivated the prazosin-high affinity sites and reduced the number of the low affinity sites without changing the pKD value. 3. In the displacement experiments with CEC-untreated membranes, unlabelled prazosin, WB4101 and HV723 displaced the binding of 200 pM [3H]-prazosin monophasically; the affinities for WB4101 (pK1 = 8.88) and HV723 (8.49) were about 10 times lower than that for prazosin (9.99). In the CEC-pretreated membranes also, the antagonists inhibited the binding of 1000 pM [3H]-prazosin monophasically; the pK1 values for prazosin, WB4101 and HV723 were 9.09, 8.97 and 8.17, respectively. These results suggest that the prazosin-high and low affinity sites can be independently appraised in the former and latter experimental conditions. Noradrenaline, but not methoxamine, showed slightly higher affinity for the prazosin-high affinity site than for the low affinity site. 4. In the functional experiments, noradrenaline (0.001-100 microM) and methoxamine (0.1-100 microM) produced concentration-dependent contractions. Pretreatment with CEC inhibited the contractions induced by low concentrations of noradrenaline but without effect on the responses to methoxamine. Prazosin inhibited the concentration-response curves for noradrenaline in the CEC-untreated aorta in a manner which was not consistent with competitive antagonism at a single site, and two distinct affinity constants(pKB = 9.71 and 8.74) were obtained. However, after CEC-pretreatment, Schild plots for prazosin were not significantly different from unity (pKB = 8.50). WB4101 and HV723 competitively inhibited the noradrenaline-induced contraction with low pKB values (approximately 8.30), irrespective of CEC pretreatment.Methoxamine-induced contractions were competitively inhibited by prazosin, WB4101 and HV723 with low pKB values similar to those obtained when noradrenaline was used as the agonist.These were not affected by CEC-pretreatment.5. The affinity constant for noradrenaline (pKA = 6.40) in CEC-untreated aorta was slightly greater than that obtained in CEC-pretreated aorta (5.78). On the other hand, methoxamine showed a similar affinity in CEC-untreated and pretreated aortae (pKA = approximately 4.5).6. Nifedipine (1 microM) slightly attenuated the contractile responses to noradrenaline and methoxamine in CEC-untreated and pretreated aortae, suggesting that nifedipine cannot discriminate between alpha 1-adrenoceptors involved in CEC-sensitive and -resistant contractions.7. From these results it is suggested that in the rabbit thoracic aorta there are two distinct alpha 1-adrenoceptor subtypes (presumably alpha 1B and alpha 1L subtypes according to recently proposed subclassification),both of which are involved in noradrenaline-induced contraction. The alpha 1L subtype predominantly mediates the contraction induced by methoxamine.

In the presence of adrenergic and cholinergic blocking agents, transmural electrical stimulation evoked a relaxation in isolated dog urethra precontracted with histamine. The response was abolished by tetrodotoxin, indicating its neurogenic origin. The non-adrenergic and non-cholinergic relaxation developed rapidly and was transient at low stimulation frequencies (⪯ 1 Hz). However, at higher frequencies (⪰ 5 Hz ) the recovery phase of the relaxation became slow and often showed a notch, suggesting the presence of transient and slow components. NG-Monomethyl-L-arginine, a nitric oxide synthase inhibitor, inhibited the transient relaxation but did not affect the relaxation evoked at high stimulation frequencies. NG-Nitro-L-arginine, a more potent nitric oxide synthase inhibitor, abolished the transient relaxation produced at low stimulation frequencies and markedly attenuated the transient component at high frequencies. However, NG-nitro-L-arginine did not affect the slow component. The inhibition by NG-monomethyl-L-arginine and NG-nitro-L-arginine was reversed by the addition of L- but not D-arginine. Exogenously applied vasoactive intestinal polypeptide (VIP) produced a slowly developing relaxation. The slow relaxation induced by transmural electrical stimulation and VIP was not affected by [4-Cl-D-Phe6, Leu17]VIP, a reportedly competitive VIP antagonist. NG-Nitro-L-arginine did not effect the relaxation induced by VIP and sodium nitroprusside. These results suggest that the non-adrenergic and non-cholinergic relaxation induced by transmural electrical stimulation is composed of nitric oxide-dependent and -independent components in the isolated dog urethra.

The alpha 1-adrenoceptor subtypes of dog prostate were characterized in binding and functional experiments. In saturation experiments, [3H]prazosin bound to alpha 1-adrenoceptors with high affinity. In the displacement experiments, unlabelled prazosin and WB4101 biphasically inhibited the binding of 400 pM [3H]prazosin, suggesting the presence of at least two distinct affinity sites for prazosin or WB4101. The proportion of high-affinity sites was approximately 10%. HV723 also recognized two distinct affinity sites but the proportion of high-affinity sites was approximately 20%. From these results the presence of three distinct alpha 1-adrenoceptor subtypes was suggested: presumably subtypes alpha 1A (high affinity for prazosin and WB4101), alpha 1N (high affinity for only HV723) and alpha 1L (low affinity for the three antagonists) according to the recently proposed alpha 1-adrenoceptor subclassification. The density of subtype alpha 1L was much higher than that of subtypes alpha 1A and alpha 1N subtypes. In the functional experiments, prazosin, WB4101 and HV723 competitively antagonized the contractile response to noradrenaline with low affinities close to those estimated for the alpha 1L subtypes. These results suggest that the contractile response to noradrenaline in the dog prostate is mediated predominantly through alpha 1L subtype alpha-adrenoceptors.

Two distinct alpha(1)-adrenoceptor phenotypes (alpha(1A)- and alpha(1L)-ARs) are known to originate from a single ADRA1A(alpha(1a)) gene by an as-yet-unknown mechanism. We hypothesized that an alpha(1a)-AR-interacting protein could generate the alpha(1L)-AR phenotype and we sought to identify such a protein and to examine its effects on the expression of alpha(1A) and alpha(1L) phenotypes. Cysteine-rich epidermal growth factor-like domain 1alpha (CRELD1alpha) was first identified using a yeast two-hybrid approach as an alpha(1a)-AR-interacting protein. Transfection of alpha(1a)-AR cDNA alone yielded Chinese hamster ovary (CHO) cells expressing alpha(1A)-ARs having a predominant high affinity site for prazosin, with a low proportion (

Muscarinic acetylcholine receptors (mAChRs) of rat cerebral cortex were evaluated using a tissue segment radioligand binding assay. [3H]-Quinuclidinyl benzilate (QNB, a hydrophobic ligand) specifically bound to mAChRs in the cortex segments. The total mAChRs level was approximately 2,000 fmol/mg protein, which was estimated after incubation for 120 min at 37°C or for 8 h at 4°C. These mAChRs were a mixture of high- and low-affinity sites for N-methylscopolamine (NMS) in a 70:30 ratio. In contrast, only a single high-affinity site for NMS was detected following incubation for 30 min at 37°C, whose abundance was about 70% of that of the total mAChRs. Atropine showed a single affinity for mAChRs under all conditions. These indicate that mAChRs are constitutively expressed not only on plasma membrane sites but also at intracellular sites in rat cerebral cortex and that the receptors at both sites have different affinities for NMS. Acetylcholine completely inhibited [3H]-QNB binding to both mAChRs without any change in the subcellular distribution, suggesting the possibility that acetylcholine can access, and bind to, both mAChRs in intact tissue. Two different affinity states for acetylcholine were detected only in plasma membrane mAChRs at 37°C. The present study demonstrates a unique subcellular distribution, and distinct pharmacological profiles, of mAChRs in rat cerebral cortex. Keywords:: muscarinic acetylcholine receptor, intracellular receptor, quinuclidinyl benzilate, rat cerebral cortex, tissue segment binding assay

1. The alpha 1-adrenoceptor subtypes of the prostatic and epididymal portion of rat vas deferens were characterized in binding and functional experiments. 2. In saturation experiments, [3H]-prazosin bound to two distinct affinity sites in the epididymal portion of rat vas deferens (pKD = 10.1 +/- 0.13 and 9.01 +/- 0.15, Bmax = 507 and 1231 fmol mg-1 protein, respectively). In the prostatic portion [3H]-prazosin bound to a single affinity site (pKD = 9.82 +/- 0.04, Bmax = 924 fmol mg-1 protein). 3. In the displacement experiments, unlabelled prazosin displaced biphasically the binding of 200 pM [3H]-prazosin to the epididymal portion; the resulting two pKI values were consistent with the affinity constants obtained in the saturation experiments. WB4101 (2-(2,6-dimethoxy-phenoxyethyl)-amino-methyl-1,4-benzodioxane) and benoxathian also discriminated the two affinity sites in the epididymal portion and the population of low affinity sites for the three antagonists was approximately 40%. On the other hand, the prostatic portion predominantly showed a single affinity site for prazosin, WB4101 and benoxathian, although the presence of a small proportion (less than 10%) of the low affinity site could be detected. HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-a min o)- propyl) benzeneacetonitrile fumarate) displaced the [3H]-prazosin binding monophasically with a low affinity in both halves. 4. Pretreatment with chlorethylclonidine (CEC) at concentrations higher than 1 microM inhibited 700 pM [3H]-prazosin binding to the prostatic portion by approximately 50%. However, the inhibition in the epididymal portion was much less (approximately 21% at 50 microM CEC).5. In the functional study, the contractile response to noradrenaline was competitively inhibited by prazosin, WB4101, benoxathian and HV723 with similar and low affinities (pKB value ranging from 8.0to 9.0) in the epididymal portion of rat vas deferens. In the prostatic portion of rat vas deferens,noradrenaline also produced a contraction, but the maximal amplitude of contraction developed was approximately one-fourth of that in the epididymal portion. Prazosin and WB4101 also inhibited the contractile response of the prostatic portion with the pKB values similar to those obtained in the epididymal portion. The contractions to noradrenaline in both portions were potently attenuated by 1 LM nifedipine but were not affected by pretreatment with 1O LM CEC.6. Under conditions where P2x-purinoceptors and prejunctional M2-adrenoceptors were blocked, electrical transmural stimulation produced a rapidly developing phasic contraction and a subsequent tonic contraction in the epididymal portion of rat vas deferens. The phasic and tonic contractions were inhibited in a concentration-dependent manner by prazosin (ICs = 25.7 and 25.9 nm, respectively),WB4101 (ICo= 7.27 and 7.58 nM), benoxathian (ICs = 10.9 and 8.66 nM) and HV723 (ICs = 15.9 and 14.9 nM). Nifedipine selectively attenuated the tonic contraction induced by electrical stimulation, and the residual phasic response was inhibited by the antagonists mentioned above with similar affinities to those in the absence of nifedipine. CEC (10 gM) had little effect on the adrenergic neurogenic contractions.7. The present results indicate the presence of two distinct alpha&-adrenoceptor subtypes in the rat vas deferens, which show respectively high and low affinities for each of prazosin, WB4101 and benoxathian,and presumably correspond to putative MIA and alL subtypes according to the recent am-adrenoceptorsubclassifications. The contractions induced by exogenous and endogenous noradrenaline seem to be predominantly mediated through the alL subtype. The heterogeneous distribution of the low affinity sites(alL subtype) may well explain differences in functional responsiveness between the two portions of rat vas deferens.

We have examined the action of the thrombin receptor-derived polypeptide, S42FLLRNPNDKYEPF55 (TRP42–55), in rat and guinea pig aortic rings and helical arterial strips, and we have compared the actions of the peptide with those of thrombin. In rat preparations, both TRP 42–55 and thrombin caused a concentration-dependent endothelium-dependent relaxation that was blocked by Nω-nitro-L-arginine methyl ester; the relaxation response of the intact rat aortic strip preparation to concentrations of the peptide in the range 30–60 μg/mL (17–34 μM) was equivalent to the response to 0.03–0.1 U/mL of thrombin (about 0.3–0.9 nM), yielding a potency ratio (TRP 42–55:thrombin) of about 38 000: 1. In contrast with the complete desensitization of thrombin-treated rat aortic preparations to a second administration of the enzyme, the rat aortic tissue was not desensitized by repeated exposures to TRP 42–55 and remained responsive to the peptide even after treatment of the tissue by thrombin. In contrast with the rat aortic tissue, in either intact or endothelium-free guinea pig aortic preparations both TRP 42–55 and thrombin caused a concentration-dependent endothelium-independent contraction. The contractile action of 60 μg/mL of receptor peptide (34 μM) in guinea pig aortic strip preparations was equivalent to the contractile action of 0.1–0.3 U/mL thrombin (0.9–3 nM), yielding a potency ratio of about 17 000: 1. In guinea pig aortic preparations with an intact endothelium that were precontracted with noradrenaline, neither thrombin nor TRP 42–55 caused relaxation, whereas substance P did so. As with the rat aortic preparation, thrombin treatment of the guinea pig aorta rendered the tissue refractory to a second exposure to the enzyme, whereas treatment with the receptor peptide did not desensitize the tissue to a subsequent exposure to thrombin. Nonetheless, as opposed to the rat aortic preparation, wherein repeated exposure to the peptide yielded a constant response, repeat doses of TRP 42–55 caused a modest desensitization of its contractile action in the guinea pig aortic strip preparation. None of the effects of TRP 42–55 were affected by the thrombin inhibitor hirudin, which blocked the action of thrombin in both the rat and guinea pig aortic preparations. We conclude that the distinct effects of TRP 42–55 in both rat and guinea pig aortic tissue, which mimic the actions of thrombin in both preparations, are due to the presence of a thrombin receptor in these tissues, akin to the one previously characterized in platelet tissue. Further, the data indicating different TRP 42–55:thrombin potency ratios and different desensitizing properties in the rat and guinea pig aortic tissue support the notion that there may be distinct thrombin receptor signal transduction systems that regulate vascular contractility either directly by acting on smooth muscle elements or indirectly via an action on endothelial cells.Key words: thrombin, thrombin receptor, vasoconstriction, vasorelaxation, aorta, endothelium.

Alpha(1)-adrenoceptors are involved in physiological functions such as urinary excretion and ejaculation in the lower urinary tract (LUT). Several alpha(1) antagonists are clinically used for the treatment of urinary obstruction in patients with benign prostatic hyperplasia. At present, three classical alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) have been identified, among which the alpha(1A) and alpha(1D)-adrenoceptor subtypes have been regarded as the main targets of alpha(1) antagonist therapy for LUT symptoms. Prazosin has been used as a prototypic, classical antagonist, to characterize alpha(1)-adrenoceptors pharmacologically, (i.e. all classical alpha(1)-adrenoceptor subtypes show high-affinity for the drug). However, we found that alpha(1)-adrenoceptors in the LUT show atypical low-affinity for prazosin. Therefore, the concept alpha(1L)-receptor, which indicates alpha(1)-adrenoceptor(s) showing low-affinity for prazosin has been introduced. A recent study demonstrated that the alpha(1L)-adrenoceptor is a specific phenotype present in the many intact tissues including human LUT, and that it originates from the ADRA1A gene. Therefore, the alpha(1L)-adrenoceptor in the LUT is now re-defined as alpha(1A(L))-adrenoceptor. The physiological and pharmacological difference between classical alpha(1A(H),) and alpha(1A(L)) which is the native receptor expressed in the LUT is of special interest as it provides fundamental bases for urological alpha(1A)-adrenoceptor blocking pharmacotherapy. Here, we briefly review the alpha(1)-adrenoceptors in the LUT with special reference to phenotype-based (pharmacome) analysis.

[3H]-CGP12177 biphasically bound to β-adrenoceptors with high and low affinities in the segments and crude membranes of rabbit left ventricle. The low-affinity sites for [3H]-CGP12177 in the segments was double in density, compared to the density of high-affinity sites. Total abundance of the β-adrenoceptors decreased to approximately 10% upon tissue homogenization, and the proportion of low-affinity sites was the same as that of the high-affinity sites in the membranes. The majority of the high-affinity binding sites of [3H]-CGP12177 in the segments and the membranes were β1H-adrenoceptor, being highly sensitive to propranolol and β1-antagonists (atenolol and ICI-89,406), whereas the low-affinity binding sites showed a β1L-profile (less sensitive to propranolol and β1-, β2-, and β3-antagonists). Furthermore, a part of the β1L-adrenoceptors was insensitive to atenolol, ICI-89,406, and/or isoproterenol. The present binding study clearly shows that β1L-adrenoceptors occur as a distinct phenotype from β1H-adrenoceptors in rabbit ventricle. However, quantitative imbalance between β1H- and β1L-adrenoceptors and divergent ligand–β1L-adrenoceptor interactions suggest a possibility that the β1L-adrenoceptor may not reflect a simple conformational change or allosteric state in the β1-adrenoceptor molecule. Keywords:: β1L-adrenoceptor, β1H-adrenoceptor, CGP12177, rabbit ventricle, tissue segment binding assay

Although alpha(1L)-adrenoceptor is recognized as a target of alpha(1) antagonist therapy for benign prostatic hyperplasia, the most common techniques, such as immunohistochemistry and in situ hybridization, are not applicable to examine alpha(1L)-AR vs alpha(1A)-AR tissue distribution because alpha(1L)-AR is now considered another phenotype sharing the alpha(1A)-AR gene and protein molecule. We labeled the alpha(1A) and alpha(1L)-adrenoceptor selective antagonist silodosin (Kissei Pharmaceutical, Matsumoto, Japan) with the fluorophore Alexa Fluor(R) 488 (Alexa-488-silodosin) to visualize alpha(1L)-AR expression.Radioligand binding and functional bioassay experiments were done to assess alpha(1)-AR expression in Chinese hamster ovary cells and human prostate tissues. Confocal imaging was subsequently performed.Although Alexa-488-silodosin had about 10 times lower affinity for all alpha(1)-AR subtypes than silodosin in binding and functional studies, it had high selectivity to alpha(1A) and alpha(1L)-ARs. Confocal imaging revealed clear localization of fluorescence on the membrane of Chinese hamster ovary cells expressing alpha(1A)-AR but not alpha(1B)-and alpha(1D)-ARs, and in the muscle layer of the human prostate. The fluorescent signal in Chinese hamster ovary cells disappeared in the presence of 3 nM prazosin but fluorescence was observed in the human prostate even in the presence of 100 nM prazosin.Alexa-488-silodosin is a powerful fluorescent probe with high selectivity to alpha(1A) and alpha(1L)-ARs. Thus, Alexa-488-silodosin successfully visualizes the site of alpha(1L)-ARs in the muscle layer of the human prostate without losing its distinct pharmacological profile.

The susceptibilities of atria and ventricles to cold preservation were studied in rat hearts. Rat hearts were stored in Collins' solution at 4 degrees C for 0, 12, or 24 hours, and the atrial and ventricular function was measured in a working heart model and in isolated atrial and papillary muscle preparations. In working heart preparations, cardiac output decreased to 0 ml/min and other parameters of ventricular function (systolic and end-diastolic pressure and dP/dt of the left ventricle) markedly deteriorated after 12 hours of preservation. After 24 hours, no contraction of the left ventricle was observed despite the presence of atrial contraction. In isolated atrial muscle preparations, the rate of spontaneous beating of the right atrium was not affected by cold preservation. The twitch contractions of right and left atria were attenuated but elicited after 24 hours. In electrically driven papillary muscles, twitch contractions were also markedly attenuated by 12 hours of preservation and were abolished after 24 hours. The positive inotropic effect of isoproterenol was also markedly attenuated in the atrial preparations by cold preservation. However, the positive chronotropic response to isoproterenol and the negative chronotropic and inotropic responses to carbachol were little affected by cold preservation. Intramural cholinergic and adrenergic nerve stimulation produced first negative and then positive effects on the beating rate and twitch contraction in the isolated right atria. Cold preservation selectively attenuated and finally abolished the adrenergic responses. In the ventricles, the adenosine triphosphate and creatine phosphate content significantly decreased and the lactate content increased with an increase in the preservation period. On the other hand, changes of such metabolites in the atria were either not observed or were much smaller. These results suggest that atrial function is maintained better than ventricular function in the cold-preserved heart.

This study examines the functional role of Na+/H+ exchanger (NHE) in Ca2+ influx mediated by human endothelin type A receptor (ETAR) expressed in Chinese hamster ovary (CHO) cells. Endothelin-1 (ET-1) increased extracellular acidification rate (ECAR), which was abolished by 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an NHE inhibitor. EIPA and KB-R7943, a Na+/Ca2+ exchanger (NCX) inhibitor, inhibited ET-1-induced sustained increases in intracellular Ca2+ concentration ([Ca2+]i), and EIPA had no effect on [Ca2+]i after KB-R7943 treatment. ET-1-elicited sustained [Ca2+]i increase was suppressed by reducing extracellular Na+ concentration. These results suggest that possible coupling of NHE with NCX via Na+ transport is involved in ETAR-mediated sustained [Ca2+]i increase. Keywords:: endothelin type A receptor, Na+/H+ exchanger, intracellular free Ca2+ concentration

Computer simulations of the human alpha 1a-adrenergic receptor (alpha 1a-AR) based on the crystal structure of rhodopsin have been combined with experimental site-directed mutagenesis to investigate the role of residues in the transmembrane domains in antagonist binding. Previous molecular dynamics studies from our laboratory indicated that the amino acids Asp106 in the third transmembrane domain (TMD), Gln167 in TMD IV of alpha 1a-AR were directly involved in prazosin, tamsulosin and KMD-3213 binding. The Asp106Ala mutant did not exhibit any affinity for [3H]prazosin. On the other hand, the Gln167Phe mutant alpha 1a-AR showed reduced binding affinity for [3H]prazosin. In competition binding experiment the binding affinities of prazosin and tamsulosin were increased 11-fold and 33-fold respectively to Gln167Phe mutant in comparison with wild type receptor. It seems that mutation of this residue by phenylalanine has offered more interaction for the ligands with its aromatic ring. The results provide direct evidence that these amino acid residues are responsible for the interactions between alpha 1a-AR and radioligand [3H]prazosin as well as tamsulosin and KMD-3213.

Muscarinic receptors in the human and rat lower urinary tract (urinary bladder detrusor muscle and mucosa, and prostate) were identified by intact tissue segment binding assays with two radioligands, and the effects of prolonged receptor activation in vitro on muscarinic receptors were examined. Hydrophilic [3H]-NMS and hydrophobic [3H]-QNB bound to the detrusor muscle segments with the same density, suggesting that the muscarinic receptors were localized at the plasma membrane. While the density of muscarinic receptor was higher in detrusor muscle than in the bladder mucosa and prostate, there was no species-specific difference either in density or in subtype distribution (M1, M2, and M3subtypes in detrusor; M2and M3subtypes in bladder mucosa; and M1and M2subtypes in prostate). Incubation of detrusor strips with carbachol decreased [3H]-NMS binding sites within 20 min, followed by a reduction of [3H]-QNB binding sites after a 60-min lag phase. The loss of the binding sites over 3 h after carbachol treatment was the same (approximately 40%) for both radioligands. The present intact tissue segment binding assay reveals tissue-specific and plasma membrane distribution of distinct muscarinic receptor subtypes and their dynamic changes (internalization and down-regulation) in lower urinary tract of humans and rats. Keywords:: urinary bladder (detrusor, mucosa), prostate, muscarinic receptor, tissue segment binding assay, down-regulation

Anthopleurin-A produced two distinct responses in crayfish giant axons: depolarization and prolongation of action potentials. The depolarization was prominent at low concentrations of anthopleurin-A, while a remarkable prolongation of the action potential was produced by higher concentrations of anthopleurin-A. Both effects of anthopleurin-A were inhibited by pre- or posttreatment with tetrodotoxin or lidocaine at concentrations which had not effect on the normal sodium channel. The prolongation of the action potential was more sensitive to tetrodotoxin and lidocaine than the depolarization was. These results suggest that sodium channels of the crayfish giant axon are modified by anthopleurin-A in at least two ways, thereby resulting in sensitization to tetrodotoxin and lidocaine.

1. The effects of TYB-3823 (B-GYKI 38233), a new antiarrhythmic drug, were electrophysiologically examined in the guinea-pig ventricular muscles. 2. TYB-3823 at concentrations of 1-3 x 10(-7) M significantly prolonged the action potential duration (APD) of the papillary muscle. However, the resting potential, action potential amplitude and maximum rate of depolarization (Vmax) were unaffected by the drug at such concentrations. At a higher concentration (1 x 10(-4) M) TYB-3823 reduced Vmax. 3. Voltage clamp experiments with single ventricular cells revealed that TYB-3823 at concentrations higher than 1 x 10(-7) M reduced the outward potassium currents, especially the time-dependent outward current, but that TYB-3823 failed to affect the calcium current. 4. These results suggested that TYB-3823 at low concentrations reduces the outward potassium current to give rise to a prolongation of APD and that at higher concentrations it additionally inhibits the sodium channels; both the effects may be related to the antiarrhythmic action of this drug.

1. We examined whether alpha 1-adrenoceptors in various blood vessels can be divided into subtypes by antagonist affinity or by susceptibility to chloroethylclonidine or nifedipine. 2. Noradrenaline or phenylephrine produced concentration-dependent contractions in all the tissues tested, which were competitively inhibited by phentolamine, yohimbine, prazosin, WB4101 and HV723. However, there were large differences between the tissues in the pA2 values for all the antagonists except phentolamine. 3. The blood vessels could be classified into three groups (I, II and III) on the basis of their affinity variation. In group I (dog mesenteric artery and vein, saphenous vein), the pA2 values for HV723 were greater than 9, and those for HV723 and WB4101 were approximately 1 log unit higher than for prazosin. This rank order of affinity reversed in group II (dog carotid artery and rat thoracic aorta), where prazosin was more potent (pA2 values greater than 9.5) than HV723 or WB4101. In group III (rabbit mesenteric artery, thoracic aorta and carotid artery and guinea-pig thoracic aorta), on the other hand, prazosin, HV723 and WB4101 inhibited the noradrenaline response with a similar affinity (pA2 values ranging from 8 to 9). 4. Yohimbine inhibited the responses to noradrenaline and phenylephrine with a lower affinity than prazosin, HV723 or WB4101. The pA2 values for yohimbine were similar in groups I and II (the values greater than 6.5), which were greater than those in group III (values less than 6.4). 5. The alpha l-adrenoceptors in group II were selectively affected by chlorethylclonidine, resulting in an irreversible attenuation of noradrenaline responses in the dog carotid artery and a persistent contraction in the rat thoracic aorta. 6. Nifedipine either produced no effect or a slight inhibition of alpha l-adrenoceptor-mediated contractions in all the blood vessels; these effects were not correlated to the above groups. 7. These results suggest that alpha,-adrenoceptors of blood vessels can be divided into three subtypes (designated as alpha 1H, alpha4L and alpha 1N) by antagonist affinity and their susceptibility to chloroethylclonidine but not to nifedipine: the characteristics of each subtype are summarized in Table 3. Subtypes alpha lH, alpha 1L and alpha lN may be predominantly involved in the contractile responses to noradrenaline or phenylephrine of the blood vessels in groups II, III and I, respectively.

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.

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.

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.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.[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.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.

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.

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.

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.

Supersensitivity is one of classical and pharmacological phenomena, and may be caused by a variety of procedures, including surgical and chemical denervation and chronic treatment with antagonists. We examined reserpine-induced chemical denervation supersensitivity with special reference to arterial alpha 1-adrenoceptor (alpha 1-AR) subtypes. Chronic treatment with reserpine for 2 weeks produced supersensitivity in the contractile response to phenylephrine of isolated rat tail artery, resulting a leftward shift of concentration-response curve (10 fold shift at EC50 value). This supersensitivity in reserpine-treated artery was selectively inhibited by BMY7378 (alpha 1D-AR selective antagonist) but not by KMD-3213 (alpha 1A-AR selective antagonist). On the other hand, the response to phenylephrine in reserpine-untreated artery was inhibited by KMD-3213 but not by BMY7378. Tissue segment binding study with 3H-prazosin revealed that high affinity binding sites for BMY7378 were detected with a proportion of 32% in reserpine-treated tail artery but absent in untreated artery, although total density of alpha 1-ARs was not changed by reserpine-treatment. The present results strongly suggest that reserpine-induced supersensitivity of rat tail artery is caused by a selective induction of alpha 1D-AR subtype.

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

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-κB in its production, in association with the possible production of reactive oxygen intermediates that activate NF-κB; 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 IκB-α/β proteins and an activity of NF-κB 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, nω-Nitro-l-arginine methyl ester, prevented nicotine-induced IL-8 production, with an entire abrogation of DHR oxidation, IκB degradation, and NF-κB 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-κB 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-κB activation, and the IL-8 appears to contribute to leukocytosis in tobacco smokers.

The pharmacological profile of N-(6-(2-(5-bromopyrimidine-2-yloxy)ethoxy)-5-(4-methylphenyl)pyrimidin-4-yl)-4-(2-hydroxy-1,1-dimethylethyl) benzensulfonamide sodium salt sesquihydrate (TA-0201), a new antagonist of endothelin receptors, was examined, using human recombinant and prostate endothelin receptors. In binding experiments with [125I]endothelin-1, TA-0201 showed extremely high affinity for recombinant endothelin ET(A) receptors (pK(i)=10.7), as compared with that for recombinant endothelin ET(B) receptors (pK(i)=7.8). Endothelin ET(A) and ET(B) receptors coexisted in human prostate with different proportions (endothelin ET(A) receptor: approximately 70%), which were distinguished by TA-0201 in the same manner as with recombinant receptors. Human prostate strips contracted in response to endothelin-1 and sorafotoxin S6c, but the maximum contraction induced by endothelin-1 was approximately three times greater than that induced by sarafotoxin S6c. The response to endothelin-1, but not to sarafotoxin S6c, was inhibited by TA-0201 and cyclo(D-Asp-Pro-D-Val-Leu-D-Trp) (BQ123) (endothelin ET(A) receptor antagonist) but not by BQ788 (endothelin ET(B) receptor antagonist). These results suggest that TA-0201 is a highly selective endothelin ET(A) receptor antagonist and will be useful for understanding the physiological and pathological roles of the endothelin ET(A) receptor in human prostate and other tissues.

The functional characteristics of purinoceptors in Chinese hamster ovary (CHO) cells were investigated using a microphysiometer which detects small metabolic changes to living cells in real-time as variations of pH in the extracellular microenvironment. Uridine 5'-triphosphate (UTP) increased the extracellular acidification rate biphasically, namely a transient and a steady response were observed. The transient phase reached a peak (four- to fivefold the basal extracellular acidification rate in amplitude) within 20 s and was followed by the steady phase which was sustained for more than 1 min at an amplitude less than twofold the basal extracellular acidification rate. Both phases showed a concentration-dependent increase in response to UTP. However, there was a significant difference in the pEC(50) value for UTP between the transient (4.8) and steady phases (6.1). Like UTP, ATP increased the extracellular acidification rate, but alpha,beta-methyleneATP (alpha,beta-MeATP), 2-methylthioATP (2-MeSATP), ADP, UDP and adenosine did not. This result suggests that the acid is extruded through a P2Y(2) or P2Y(2)-like purinoceptor. 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and 4-isopropyl-3-methylsulphonylbenzoyl-guanidine methanesulphonate (HOE642) suppressed both phases of the UTP-stimulated extracellular acidification rate response with high affinity (pIC(50): approximately 7.0). This result suggests that the Na(+)/H(+) exchanger 1 (NHE-1) predominantly mediates the UTP-induced acid extrusion response in CHO cells. Elimination of extracellular Ca(2+) or treatment with thapsigargin diminished both phases of the UTP-stimulated extracellular acidification rate. In addition, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride (W-7) also abrogated the two phases. These results are consistent with the involvement of NHE-1 which is activated via Ca(2+)/calmodulin. Persistent exposure to UTP reduced both extracellular acidification rate phases, causing desensitization of the P2Y purinoceptor. This desensitization did not affect the acid extrusion response mediated by the alpha(1)-adrenoceptor.

The pharmacological profile of (+/-)-domesticine, a novel alpha(1)-adrenoceptor antagonist, was examined in animal tissues and Chinese hamster ovary (CHO) cells expressing cloned human alpha(1)-adrenoceptor subtypes and compared with the properties of BMY-7378 ([8-(2-[4-(2-methoxy-phenyl)-1-piperazinyl]ethyl)-8-azaspirol [4.5]decane-7,9-dione dihydrochloride], the prototypical alpha(1D)-adrenoceptor antagonist. Both (+/-)-domesticine and BMY-7378 were more potent in inhibiting the phenylephrine-induced contraction in rat thoracic aorta than tail artery or spleen. The selectivity of (+/-)-domesticine to inhibit phenylephrine-induced contraction in rat thoracic aorta was 32- and 17-fold higher than that in tail artery and spleen, respectively, while that of BMY-7378 it was 125- and 11-fold, respectively. The functional affinity profiles of these compounds for the alpha(1)-adrenoceptor subtypes in animal tissues were consistent with the respective binding affinity profiles in cloned human alpha(1)-adrenoceptor subtypes. (+/-)-Domesticine displayed a 34- and 9-fold higher selectivity for alpha(1d)-adrenoceptor than for alpha(1a)- and alpha(1b)-adrenoceptor, respectively, while BMY-7378 showed a selectivity for alpha(1d)-adrenoceptor of 102-fold higher than that of alpha(1a)-adrenoceptor and 21-fold higher than that of alpha(1b)-adrenoceptor. Interestingly, in [3H]8-OH-DPAT (8-hidroxy-2-(di-n-propyl-amino)tetraline hidrobromide) binding to 5-HT(1A) receptors of rat cerebral cortex, (+/-)-domesticine showed a 183-fold higher selectivity for alpha(1D)-adrenoceptor relative to 5-HT(1A) receptor, whereas BMY-7378 displayed a similar affinity at this receptor with respect to the alpha(1D)-adrenoceptor (0.89-fold). Both compounds, however, showed a weak affinity for 5-HT(2A)/5-HT(2C) receptors in rat frontal cortex. These results suggest that (+/-)-domesticine is more potent for alpha(1D)-adrenoceptor than for alpha(1A)- or alpha(1B)-adrenoceptor subtypes and it is highly selective compared to 5-HT(1A) and other receptors.

The ductus arteriosus (DA) is a shunt vessel that, in the fetal period of some animals including mammals, connects the pulmonary artery (PA) and systemic circulation, bypassing the unexpanded lungs. After birth, the DA is exposed to an increase in O2 concentration in the blood and undergoes rapid closure as the pulmonary circulation is established. Two major factors are thought to be involved in the closure: an attenuation of dilator activity and an increase of endogenous constrictor activity (Smith, 1998). Endothelin-1 (ET1) has been suggested as a likely candidate to mediate O2-induced DA constriction (Coceani et al. 1992; Smith, 1998). However, the role of ET1 is still controversial. One group has provided several lines of evidence supporting a role for ET1 in DA closure (Coceani et al. 1989; Coceani & Kelsey, 1991). However, other work, employing ET receptor blockade, did not support a role for ET1 in the closure of the DA after birth (Fineman et al. 1998). Recently, Coceani et al. (1999) have reported that O2-induced contraction was absent in the DA isolated from ET receptor (ETA)-null mice, but that closure of the DA after birth was not affected in the ETA-null mice. The role of ET1 and/or the extent of ET1 involvement in physiological closure of the DA after birth therefore remains an open question. To further address this question, we used ATZ1993 (ATZ), an orally active non-peptide antagonist for the ETA and ETB receptors (Azuma et al. 1999), to investigate the contribution of the ET system to the postnatal closure and O2-induced contraction of the rat DA in vivo and in vitro.

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.

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).

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.

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.

Using microphysiometer, we have investigated acid expulsion from CHO cells expressing human alpha-1a adrenoceptor. Time course of extracellular acidification rate after noradrenaline stimulation had two phases; one with a peak within 10 s reached several folds of base rate, and another increased gradually to two folds of base rate and reached plateau around two min. Both phases showed concentration-dependent increase of acidification rates in response to noradrenaline but had distinct pEC50 values; 6.0 for rapid phase and 6.6 for late phase. Amiloride and its analogs inhibited both phases entirely, suggesting that Na/H exchanger mainly mediated these acid expulsion responses. Elimination of Ca by BAPTA/EGTA treatment resulted in extensive reductions of the rapid phase response but small decrease of the late phase response. Several Ca channel blockers, Ni and LOE908 also suppressed the rapid phase while nifedipine, verapamil, SKF96365 and omega-conotoxin GIVA did not. Repeated stimulation with noradrenaline enhanced inhibitory effect of blockers. These results indicate that Ca is one of the elements in the rapid phase but not in the late phase of acid expulsion from CHO cells in response to alpha-1 adrenoceptor stimulation and suggest that Ca from both intracellular storage and some type of Ca channel dominantly participates in the rapid phase.

The alpha1-adrenoceptor subtypes of rat prostate were characterized in binding and functional experiments. In binding experiments, [3H]tamsulosin bound to a single class of binding sites with an affinity (pKD) of 10.79+/-0.04 and Bmax of 87+/-2 fmol mg(-1) protein. This binding was inhibited by prazosin, 2-(2,6-dimethoxy-phenoxyethyl)-aminomethyl-1,4-benzodioxane hydrochloride (WB4101), 5-methylurapidil, alpha-ethyl-3,4,5,-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amin o)-propyl)benzeneacetonitrile fumarate (HV723) and oxymetazoline with high efficacy, resulting in a good correlation with the binding characteristics of cloned alpha1a but not alpha1b and alpha1d-adrenoceptor subtypes. In functional studies, noradrenaline and oxymetazoline produced concentration-dependent contractions. These contractions were antagonized by tamsulosin, prazosin, WB4101 and 5-methylurapidil with an efficacy lower than that exhibited by these agents for inhibition of [3H]tamsulosin binding. The relationship between receptor occupancy and contractile amplitude revealed the presence of receptor reserve for noradrenaline, but the contraction induced by oxymetazoline was not in parallel with receptor occupation and developed after predicted receptor saturation. From these results, it is suggested that alpha1A-adrenoceptors are the dominant subtype in the rat prostate which can be detected with [3H]tamsulosin, but that the functional subtype mediating adrenergic contractions has the characteristics of the alpha1L-adrenoceptor subtype, having a lower affinity for prazosin and some other drugs than the alpha1A-adrenoceptor subtype.

In the presence of prazosin and propranolol, electrical transmural stimulation of isolated dog mesenteric artery produced a sympathetic purinergic contraction, which was followed by a relaxation in PGF2 alpha-contracted arteries. Such purinergic responses were mimicked by brief exposure to alpha, beta-methylene ATP (alpha, beta-Me ATP) and were completely inhibited after desensitization of P2x-purinergic receptors. However, exogenous ATP predominantly evoked a relaxation in PGF2 alpha-contracted artery. These results suggest that the sympathetic purinergic response may be caused by a P2x-purinergic receptor-selective mechanism or substance, rather than ATP.

Selectivity of tamsulosin and terazosin to functional alpha1-adrenoceptors was examined. Both drugs competitively inhibited the contractile responses to noradrenaline in different tissues where the responses were mediated through the alpha1D-, alpha1B- or alpha1L-subtype. Together with the affinities obtained in the binding study with cloned (alpha1a, alpha1b, alpha1d) and native (alpha1A and alpha1B) subtypes, the selectivity of tamsulosin was alpha1Aalpha1L, alpha1Dalpha1B. Terazosin had lower affinity at various subtypes than tamsulosin, but showed relatively high selectivity to the alpha1D-subtype. In the human prostate, tamsulosin was more than 30-fold higher in affinity than terazosin in functional and binding studies.

Anthracycline derivatives including 4'-epidoxorubicin are known to cause cardiovascular side effects. In this study we examined the effects of 4'-epidoxorubicin on sympathetic nerves of rat tail artery in vitro. Treatment with 4'-epidoxorubicin at concentrations higher than 10 microM gradually increased the resting tension of the arterial strips, an effect which was greatly enhanced by subsequent addition of 10 microM cocaine. This increase of the resting tension by 4'-epidoxorubicin was prevented by prazosin, suppressed in the arterial strips of reserpine-pretreated rats, and reduced by superoxide dismutase. However, tetrodotoxin and histamine receptor antagonists (diphenhydramine and cimetidine) failed to influence it. The contractile response to electrical sympathetic stimulation was slightly attenuated by 30 microM 4'-epidoxorubicin. 4'-epidoxorubicin did not shift the concentration-response curve for noradrenaline. In the superfusion experiments, the basal release of noradrenaline was increased approximately five-fold by 30 microM 4'-epidoxorubicin, and this increase was not inhibited by 0.1 microM prazosin, 0.5 microM tetrodotoxin, 10 microM cocaine or Ca2+-free medium. Noradrenaline release evoked by electrical stimulation was gradually suppressed by 30 microM 4'-epidoxorubicin treatment. These results suggest that 4'-epidoxorubicin directly acts on the sympathetic nerve to cause persistent release of noradrenaline in rat tail artery.

The subtypes of alpha1-adrenoceptor are coexpressed in many tissues. We examined the relationship between coexpressed alpha1-adrenoceptor subtypes and their functions in blood vessels. Rat and rabbit aortas coexpressed three subtypes (alpha1A, alpha1B, alpha1D) and four subtypes (alpha1A, alpha1B, alpha1D, alpha1L), respectively. In rat aorta however, noradrenaline-induced contraction was mediated predominantly through the alpha1D subtype, and oxymetazoline produced alpha1B-mediated contraction. In rabbit aorta, concentration-response curves for noradrenaline were composed of two components (alpha1B and alpha1L-mediated), while oxymetazoline produced alpha1L-mediated contraction. Therefore, the inhibitory actions of some antagonists varied markedly among tissues and agonists. These results demonstrate diversity of the two receptor systems and suggest that the heterogeneity of physiological responses reflects the differences in functional subtypes among tissues and in their sensitivities to agonists and antagonists.

A highly cytotoxic extract from marine sponge, polytheonamide B, is a linear 48-residue peptide. Alternative D- and L-forms of unusual amino acids suggest formation of beta-helix that is stable in membrane and serves for ion conducting pore. The NMR study indicated that polytheonamide B forms beta-helix in methanol/chloroform solution. Channel activity of polytheonamide B was examined using planar lipid bilayers. Ionic current appeared from pM concentration. Measurements of the reversal potentials revealed that the channel showed cation selectivity. Single channel current was recorded in symmetrical 1 M solutions. The selectivity sequence was: H+ > Cs+ > Rb+ > K+ > Na+. Single-channel I-V curve exhibited slight inward rectification. Voltage-dependent transitions between brief openings and long closures were observed. Orientation of the peptide in the membrane was fixed when the peptide was added to one side of the chamber. The asymmetric behaviors, such as single channel rectification, voltage-dependent gating and oriented incorporation into the membrane, must be correlated to the molecular structure of polytheonamide B.