Amino acids of the human alpha1d-adrenergic receptor involved in antagonist binding

Computer simulations of the human alpha(1d)-adrenergic receptor (alpha(1d)-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. Our results indicate that the amino acids Asp176 in the third transmembrane domain (TMD), Glu237 in TMD IV, and Ser258 in TMD V of alpha(1d)-AR were directly involved in prazosin and tamsulosin binding. The Asp176Ala mutant did not exhibit any affinity for [(3)H]prazosin and neither did it show agonist-stimulated inositol phosphates (IP) formation. On the other hand, the Glu237Ala and Ser258Ala mutant alpha(1d)-AR showed increased binding affinity for [(3)H]prazosin. Competition binding experiments showed that prazosin affinity had increased to 5-fold and 3-fold in the Glu237Ala and Ser258Ala mutants, respectively, versus wild-type; and tamsulosin affinity only increased in the Ser258Ala mutant (2-fold vs wild-type). It seems that these two residues constrain the receptor by interaction with other residues and this disruption of the interaction increased the receptor's binding affinity towards antagonists. However, the Glu237Ala and Ser258Ala mutant receptors retained the ability to stimulate the formation of myo-[(3)H]inositol but had activities lower than that of the wild-type receptor. The present results provide direct evidence that these amino acid residues are responsible for the interactions between alpha(1d)-AR and the radioligand [(3)H]prazosin as well as tamsulosin.     

Asp125 and Thr130 in transmembrane domain 3 are major sites of alpha1b-adrenergic receptor antagonist binding

Site-directed mutagenesis was used to investigate the molecular interactions involved in prazosin binding to the human alpha(1b)-adrenergic receptor (alpha(1b)-AR) receptor. Based on molecular modeling studies, Thr130 and Asp125 in transmembrane region III of the alpha(1b)-AR receptor were found to interact with prazosin. Thr130 and Asp125 were mutated to alanine (Ala) and expressed in HEK293 cells. The radioligand [(3)H]prazosin did not show any binding to Asp125Ala mutant of alpha(1b)-AR. Therefore, it was not possible to find any prazosin affinity to the mutant using the radioligand [(3)H]prazosin. The mutation also abolished phenylephrine-stimulated inositol phosphate (IP) formation of [(3)H]myo-inositol. On the other hand, the Thr130Ala mutant showed reduced binding affinity for [(3)H]prazosin (dissociation constant, K(d) 674.27 pM versus 90.27 pM for the wild-type receptor) and had reduced affinity for both tamsulosin and prazosin (11-fold and 9-fold, respectively). However, the Thr130Ala mutant receptor retained the ability to stimulate the formation of [(3)H]myo-inositol. The results provide direct evidence that Asp125 and Thr130 are responsible for the interactions between alpha(1b)-AR receptor and radioligand [(3)H]prazosin as well as tamsulosin.     

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

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

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.     

Selective and sustained occupancy of prostatic alpha1-adrenoceptors by oral administration of KMD-3213 and its plasma concentration in rats

This study examined the ex-vivo occupancy by KMD-3213 of alpha1-adrenoceptors in the prostate and other tissues of rats in terms of tissue selectivity and duration of occupancy in relation to plasma concentration. Oral administration of KMD-3213 (0.2-20.2 micromol kg(-1), 0.5 h) dose-dependently decreased [3H]prazosin binding sites (Bmax) in the prostate (42-74%) and submaxillary gland (54-88%) compared with the control value. In contrast, there was only a slight change in the Bmax values in the spleen and cerebral cortex of KMD-3213-treated rats. The alpha1-adrenoceptor occupancy in the prostate and submaxillary gland was increased, with plasma free concentration of KMD-3213 at 0.5 h after oral administration of KMD-3213 (0.6-20.2 micromol kg(-1)). The receptor occupancy in these tissues was much greater than that in the spleen, heart or cerebral cortex. After oral administration of KMD-3213 (6.1 micromol kg(-1)), the alpha1-adrenoceptor occupancy in the prostate and submaxillary gland occurred rapidly, in parallel with the rise in the plasma concentration of the drug, and it lasted for at least 24 h, despite a remarkable decrease in the plasma concentration. It is concluded that KMD-3213 may produce fairly selective and sustained occupancy of alpha1-adrenoceptors in the prostate, a target organ for treatment of bladder outlet obstruction in patients with benign prostatic hyperplasia.     

Effects of KMD-3213, a uroselective alpha 1A-adrenoceptor antagonist,on the tilt-induced blood pressure response in normotensive rats

KMD-3213 ((-)-1-(3-hydroxypropyl)-5-((2R)-2-[[2-([2-[(2,2,2-trifluoroethyl)oxy]phenyl]oxy)ethyl]amino]propyl)-2,3-dihydro-1H-indole-7-carboxamide), an alpha(1A)-adrenoceptor antagonist with potency similar to that of tamsulosin, is under development for the treatment of bladder outlet obstruction in patients with benign prostatic hypertrophy. In the present study, we investigated the effects of KMD-3213 on the tilt-induced blood pressure response in anesthetized normotensive rats. Male normotensive Sprague-Dawley rats were placed in the supine position on a board under cocktail anesthetization (alpha-chloralose, urethane and sodium pentobarbital). The arterial blood pressure was measured from the carotid artery. The animals were given consistent 45 degrees head-up tilt from the horizontal position, following the transient decrease in the blood pressure, and then recovery of the blood pressure to the normal level. Significant orthostatic hypotension was seen with intravenous administration of both prazosin and tamsulosin at doses over 3 micro g/kg, and these drugs completely blocked the tilt-induced blood pressure responses at 30 micro g/kg. On the other hand, these responses were still retained when KMD-3213 was administered intravenously at a dose up to 75 micro g/kg of KMD-3213. Moreover, KMD-3213 showed the highest uroselectivity of the test drugs. These results indicate that KMD-3213 is not likely to induce orthostatic hypotension and would be a useful compound for the treatment of urinary outlet obstruction in patients with benign prostatic hyperplasia.     

Relationship between prostatic alpha(1)-adrenoceptor binding and reduction in intraurethral pressure following continuous infusion of KMD-3213 in rats

The relationship between alpha(1)-adrenoceptor binding in rat tissues and pharmacodynamic effects of continuous infusion of KMD-3213 was examined. In vivo specific binding of [(3)H]KMD-3213 after continuous intravenous infusion of the ligand (100 pmol/kg/min for 10 min, followed by 30 pmol/kg/min for 60 or 90 min) differed largely among the tissues examined. Specific binding of [(3)H]KMD-3213 in aorta, heart, lung, and kidney was not different in terms of infusion time in the case of continuous infusion for 10, 70 and 100 min, whereas the binding in prostate, vas deferens, and submaxillary gland by 70- and/or 100-min infusion was significantly greater than that by the 10-min infusion. A similar extent of specific binding in the prostate was observed by the infusion (100 min) of a three-fold higher dose of [(3)H]KMD- 3213. Continuous intravenous infusion of KMD-3213 (100 pmol/kg/min for 10 min, followed by 30 pmol/kg/min) for 70 or 100 min significantly reduced the phenylephrine-induced increase in the mean blood pressure and that in the intraurethral pressure of anesthetized rats. Extent and time course of the KMD-3213 effect reduction in the phenylephrine-induced increase in intraurethral pressure were closely associated with those in prostatic [(3)H]KMD-3213 binding after continuous infusion of the corresponding dosage of the radioligand. The reduction in the phenylephrine-induced increase by the infusion of a three-fold higher dose of KMD-3213 was significantly greater in the case of the intraurethral pressure than in that of the mean blood pressure, thereby suggesting a greater selectivity for the alpha(1)-adrenoceptor in the lower urinary tract than for that in the vascular tissue. In conclusion, the present study has shown that specific binding of [(3)H]KMD-3213 in the rat prostate after the continuous intravenous infusion of the radioligand may be closely associated with the pharmacological effect of this drug on the lower urinary tract.     

Comparative study on pharmacokinetics and in vivo alpha1-adrenoceptor binding of [3H]tamsulosin and [3H]prazosin in rats.

The plasma concentration, total radioactivity and in vivo alpha1-adrenoceptor binding in rat tissues after intravenous (i.v.) injection of [3H]tamsulosin were measured and they were compared with those obtained after the injection of [3H]prazosin. The plasma concentration of [3H]tamsulosin was consistently higher than that of [3H]prazosin, with 1.4 times greater areas under the curve (AUC(0-infinity)) of plasma concentration. As there was a significantly lower value of apparent volume of central compartment (Vd(c)) and distribution volume at steady state (Vd(ss)) for [3H]tamsulosin than [3H]prazosin with little difference in elimination rate constant (beta), the higher concentration of [3H]tamsulosin in plasma might be associated mainly with the smaller volume of distribution. The ratio of total radioactivity in tissues to the plasma unbound concentration of [3H]tamsulosin after i.v. injection of the ligand was consistently lower than that of [3H]prazosin. These observations suggest that [3H]tamsulosin is distributed in rat tissues in a more limited manner than [3H]prazosin. A significantly lower level of in vivo specific binding of [3H]tamsulosin than [3H]prazosin was observed in the spleen, heart and liver. Further, the apparent dissociation constant (Kd) and maximal number of binding sites (Bmax) for in vivo specific [3H]tamsulosin binding were considerably lower than those for [3H]prazosin binding. Therefore, these findings suggest that [3H]tamsulosin labels preferentially a subpopulation of the alpha1-adrenoceptor sites in rat tissues labeled by [3H]prazosin. In conclusion, the present study has shown that there is a significant difference in the pharmacokinetics and in vivo alpha1-adrenoceptor binding characteristics between tamsulosin and prazosin.     

Alpha-1 adrenoceptor up-regulation induced by prazosin but not KMD-3213 or reserpine in rats

1. We have investigated the effects of chronic administration of prazosin (a subtype-nonspecific alpha-1 AR antagonist), KMD-3213 (an alpha-1A AR subtype-specific antagonist) and reserpine (a catecholamine depletor) on the density of alpha-1 AR subtypes in various rat tissues (liver, kidney, submaxillary gland, heart and spleen). 2. Administration of prazosin (2 mg kg(-1) day(-1), i.p.) for 2 weeks did not affect K(D) values for [(3)H]-prazosin or [(3)H]-KMD-3213 of alpha-1 ARs in five rat tissues tested. However, it caused 52% up-regulation of alpha-1B AR in the spleen, and 84% and 107% up-regulation of alpha-1A- and alpha-1B ARs, respectively, in the heart. Although major subtypes of alpha-1 AR are alpha-1A AR in the submaxillary gland, alpha-1B AR in the liver, and alpha-1A and alpha-1B ARs in the kidney, these tissues showed no up-regulation. The mRNA levels of alpha-1 AR subtypes were not affected by prazosin administration in any tissue tested. 3. Neither administration of KMD-3213 (2 mg kg(-1) day(-1), i.p.) nor reserpine (0.5 - 1 mg kg(-1) day(-1), i.p.) for 2 weeks caused any change in either the binding affinity for [(3)H]-prazosin or [(3)H]-KMD-3213 or the density of the alpha-1 AR subtypes in the five rat tissues. 4. Neither prazosin nor KMD-3213 treatment reduced the noradrenaline content in the five rat tissues, in contrast to reserpine treatment, which markedly reduced it. 5. The findings of the present study demonstrated that up-regulation of alpha-1 AR is selectively caused by prazosin treatment in some tissues but neither by KMD-3213 treatment nor by chemical denervation with reserpine. These results suggest that up-regulation of alpha-1 ARs is not caused by a simple blockade of sympathetic tone.     

Site-directed mutagenesis of the serotonin 5-Hydroxytryptamine2c receptor: identification of amino acids responsible for sarpogrelate binding

Site-directed mutagenesis was used to investigate the molecular interactions involved in sarpogrelate binding to the human 5-Hydroxytryptamine(5-HT)2C receptor. Based on molecular modeling studies, Aspartic acid (Asp)155[3.32] in transmembrane region III and Serine(Ser)361[7.46] in transmembrane region VII of the 5-HT2C receptor were found to interact with sarpogrelate. Asp3.32 and Ser7.46 were mutated to alanine (Ala) and expressed in COS-7 cells. The radioligand [3H]mesulergine did not show any binding to Asp3.32Ala mutant of 5-HT2C receptor. Therefore, it was not possible to find any sarpogrelate affinity to the mutant using [3H]mesulergine. The mutation also abolished agonist-stimulated IP formation of [3H]myo-inositol. Introduction of dual mutation at position Ser7.46 (Asp3.32Ala-Ser7.46Ala) could not restore the function disrupted by the first mutation (Asp3.32Ala). On the other hand, the Ser7.46Ala mutant showed reduced binding affinity for [3H]mesulergine (Kd 3557 pM versus 573 pM for the wild-type receptor) and had reduced affinity for sarpogrelate. Moreover, the Ser7.46Ala mutant receptor also showed a great loss of potency for sarpogrelate in inhibiting 5-HT-stimulated IP formation of [3H]myo-inositol. The results provide direct evidence that Asp3.32 and less importantly, Ser7.46 are responsible for the interaction between 5-HT2C receptor and [3H]mesulergine as well as sarpogrelate. More interestingly, Ser7.46Ala increases the receptor expression (20-fold vs. wild-type) of the mutant receptors and basal [3H]myo-inositol formation (2.5-fold vs. wild-type), which indicates that the 5-HT2C receptor could be rendered constitutively active by mutating the amino acid serine at position 7.46 to alanine.     

Binding and functional characterization of alpha1-adrenoceptor subtypes in the rat prostate

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.     

Competitive interaction of amiloride and verapamil with alpha 1-adrenoceptors in vascular smooth muscle

We have characterized [3H]prazosin binding to purified plasma membranes isolated from bovine carotid arteries and studied the effects of Na+ and Ca2+ channel blockers on [3H]prazosin binding to alpha 1-adrenoceptors. Amiloride and verapamil competitively inhibited the specific binding of [3H]prazosin to purified plasma membranes isolated from bovine carotid artery in a dose-dependent manner. The Ki values of verapamil and amiloride for alpha 1-adrenergic receptor were 1.04 +/- 0.037 microM and 32.6 +/- 0.59 microM, respectively. Verapamil (10 microM) and amiloride (100 microM) caused a 6-fold and 2.7-fold decrease in affinity of [3H]prazosin binding, respectively, with no change in the number of binding sites. The inhibition of [3H]prazosin binding by amiloride and verapamil could be reversed after the membranes were washed. Another Ca2+-channel blocker, nifedipine, and a Na+-channel blocker, furosemide, did not significantly inhibit [3H]prazosin binding up to 0.1 mM concentrations. Our results suggest that amiloride and verapamil may produce vascular smooth muscle relaxation by modulating alpha 1-adrenoceptor affinity in addition to blocking Na+ and Ca2+ channels, respectively.     

Pharmacological properties of naftopidil, a drug for treatment of the bladder outlet obstruction for patients with benign prostatic hyperplasia

Naftopidil, a phenylpiperazine derivative, is a novel alpha 1-adrenoceptor antagonist and is new drug for the bladder outlet obstruction in patients with benign prostatic hyperplasia (BPH). Naftopidil competitively inhibited specific [3H]prazosin binding in prostatic membranes of humans, and its Ki value was 11.6 nM. Using cloned human alpha 1-adrenoceptor subtypes (alpha 1a, alpha 1b and alpha 1d), naftopidil was selective for the alpha 1d-adrenoceptor with approximately 3- and 17-fold higher affinity than for the alpha 1a- and alpha 1b-adrenoceptor subtypes, respectively. In anesthetized dogs, naftopidil selectively inhibited the phenylephrine-induced increase in prostatic pressure compared with mean blood pressure. The selectivity of naftopidil for prostatic pressure was more potent than those of tamsulosin and prazosin. In conscious rabbits, the effect of naftopidil on the blood pressure reactions following the tilting was less potent than those of tamsulosin and prazosin. In clinical studies, naftopidil has been demonstrated to be effective in the treatment of bladder outlet obstruction in patients with BPH. In Japan, naftopidil has been already approved for clinical use as a drug for BPH.     

Characterization of alpha 1-adrenergic receptor subtypes in rat brain: a reevaluation of [3H]WB4104 and [3H]prazosin binding

[3H]Prazosin and [3H]WB4101 [2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4 benzodioxane] have both been proposed to label alpha 1-adrenergic receptors in the rat central nervous system. As many discrepancies between the binding of these two ligands have arisen, we conducted these studies in order to reevaluate their binding characteristics and resolve the similarities and differences in the pharmacological characteristics of their respective binding sites. [3H]Prazosin binding is characterized by a monophasic saturation isotherm. Prazosin, indoramine, and dihydroergocryptine competitions with [3H]prazosin are steep and monophasic, and model best to a single binding site. In contrast, phentolamine and WB4101 competition curves are shallow in rat cortex, exhibiting Hill coefficients significantly less than 1.0, and model to two binding sites of approximately equal proportions. The higher and lower affinity components are defined as alpha 1A and alpha 1B, respectively. [3H]WB4101 also labels two binding sites in rat cortex and hippocampus with picomolar and nanomolar affinity, respectively. However, the nanomolar binding site is serotonergic and not adrenergic. The picomolar site (KD = 150 pm) has characteristics of an alpha 1-receptor binding site: prazosin, WB4101, and phentolamine affinities for this [3H]WB4101 binding site correlate with their affinities for the highest affinity component (alpha 1A) of [3H]prazosin binding. In addition, the Bmax of this [3H] WB4101-labeled site is equal to one-half of the total [3H]prazosin Bmax. Agonist competitions with [3H]prazosin binding are multiphasic with pseudo-Hill slopes less than 1.0 and with a rank order of affinity of epinephrine greater than norepinephrine greater than phenylephrine. When binding to the alpha 1A component is blocked by a 30 nM phentolamine mask, the same rank order of agonist affinities is preserved. Although the affinities of epinephrine and norepinephrine at the two subtypes are identical, phenylephrine is weaker at the alpha 1B site. The ratio of the potency of phentolamine versus prazosin is about 4 at the alpha 1A component but about 80 at the alpha 1B binding site. We discuss these data in relation to the reported potencies of these antagonists in blocking alpha 1-receptor-mediated responses which may correlate with our designation of alpha 1A or alpha 1B binding sites.     

Identification of amino acid residues important for sarpogrelate binding to the human 5-hydroxytryptamine2A serotonin receptor

The purpose of the present study was to examine 5-hydroxytryptamine (5-HT)(2A)-receptor sarpogrelate interactions by site-directed mutagenesis. Based on molecular modeling studies, aspartic acid (Asp)155[3.32] and tryptophan (Trp)151[3.28] in transmembrane helix (TMH) III and Trp336[6.48] in TMH VI of the 5-HT(2A) receptor were found to interact with sarpogrelate. All of these residues were mutated to alanine (Ala). The Asp3.32Ala mutant did not exhibit any affinity for [(3)H]ketanserin, whereas the Trp3.28Ala mutant showed a markedly decrease in the binding affinity for [(3)H]ketanserin (K(d) >10,000 nM). Therefore, it was not possible to find any sarpogrelate affinity to the mutants using [(3)H]ketanserin. The mutation also abolished agonist-stimulated formation of [(3)H]inositol phosphates (IP) in both cases. On the other hand, the Trp6.48Ala mutant showed reduced binding affinity for [(3)H]ketanserin (K(d) 2.0 nM vs 0.8 nM for the wild-type receptor) and had reduced affinity for sarpogrelate (pK(i) value of 5.71 vs 9.06 for the wild-type receptor). The Trp6.48Ala mutant also showed the greatest decrease in sensitivity to sarpogrelate (pK(b) value 8.81 for wild-type and 5.11 for mutant) in inhibiting agonist-stimulated IP formation. These results provide direct evidence that Asp3.32, Trp3.28, and less importantly, Trp6.48 are responsible for the interaction between the 5-HT(2A) receptor and sarpogrelate. In addition, these results support the findings obtained from molecular modeling studies.     

Age-related change of the role of alpha1L-adrenoceptor in canine urethral smooth muscle.

To examine age-related alteration of the role of alpha1L-adrenoceptor in the urethra, young non-parous and aged parous female dogs were used. In a functional study, we evaluated phenylephrine-induced contraction and antagonistic effects of JTH-601, a newly synthesized alpha1-adrenoceptor antagonist, and prazosin; in a localization survey using autoradiographic technique, we investigated specific [3H]JTH-601 and [3H]tamsulosin binding. Concentration-response curves were obtained for phenylephrine (pD2 = 5.0-5.3). JTH-601 and prazosin antagonized this contraction with pA2 values of 8.2-8.3 and 8.0-8.1, respectively. Specific binding of both [3H]JTH-601 and [3H]tamsulosin were observed in the bladder neck and proximal section of urethra. There were no significant differences of the pD2, pA2, and radio ligand binding between young non-parous and aged parous dogs.     

Alpha 2-adrenoceptor subtypes and imidazoline-like binding sites in the rat brain.

1. The binding of [3H]-yohimbine and [3H]-idazoxan to rat cortex and hippocampus is rapid, reversible and of high affinity. Saturation data indicate that a single population of binding sites exist for [3H]-yohimbine in the cortex (Bmax 121 +/- 10 fmol mg-1, protein; Kd 5.2 +/- 0.9 nM) and hippocampus (Bmax 72 +/- 6 fmol mg-1 protein; Kd 5.8 +/- 0.7 nM). [3H]-idazoxan labels one site in the cortex (Bmax 87 +/- 8 fmol mg-1 protein; Kd 4.1 +/- 0.9 nM) and hippocampus (Bmax 30 +/- 6 fmol mg-1 protein; Kd 3.5 +/- 0.5 nM), when 3 microM phentolamine is used to define non-specific binding. A second distinct [3H]-idazoxan binding site (Bmax 110 +/- 21 fmol mg-1 protein; Kd 3.6 +/- 0.07 nM) is identified in rat cortex if 0.3 microM cirazoline is used to define non-specific binding and 3 microM yohimbine is included to prevent binding to alpha 2-adrenoceptors. 2. Displacement studies indicate that the alpha 1-adrenoceptor antagonist prazosin and the 5-HT1 ligands 8-OH-DPAT, RU 24969 and methysergide differentiate [3H]-yohimbine binding into two components; a high and low affinity site. In contrast the displacement of [3H]-idazoxan by each ligand was monophasic. 3. The affinities of 8-OH-DPAT, RU 24969 and methysergide determined against [3H]-idazoxan binding to the cortex and hippocampus correlate significantly with the binding site displaying low affinity for prazosin and previously designated alpha 2A. In contrast, a poor correlation exists for the high affinity site for prazosin designated alpha 2B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased affinity and preference of halogenated derivatives of BE 2254 for alpha 1-adrenoceptors demonstrated by functional and binding experiments

The effects of congeners of BE 2254 (2-[beta-(4'-hydroxyphenyl)-ethyl-aminomethyl]-tetralone), an alpha 1-adrenoceptor antagonist, were compared in functional and binding experiments. In detail, we studied the effects of the compounds on the electrically evoked 3H overflow (alpha 2-adrenoceptor-mediated) and on the evoked contractions (alpha 1-adrenoceptor-mediated) of strips from rabbit pulmonary artery preincubated with [3H]noradrenaline as well as their effects on [3H]yohimbine binding to human platelet membranes (alpha 2-adrenoceptors) and [3H]prazosin binding to rat liver plasma membranes (alpha 1-adrenoceptors). The potencies of the drugs at the presynaptic alpha 2-adrenoceptors of the rabbit pulmonary artery and their affinities for [3H]yohimbine binding sites of human platelets were closely correlated. The same held true for their potencies at the postsynaptic alpha 1-adrenoceptors of the rabbit pulmonary artery and their affinities for [3H]prazosin binding sites of rat hepatic membranes. Compared with the parent compound, the monohalogenated derivatives of BE 2254 (i.e., the 3'-I, 3'-Br, and 3'-Cl analogues) exhibited an even higher affinity (equal to that of prazosin) and a higher selectivity for alpha 1-adrenoceptors (inferior to that of prazosin, but similar to that of corynanthine).     

Site-directed mutagenesis studies of human A(2A) adenosine receptors: involvement of glu(13) and his(278) in ligand binding and sodium modulation

To provide insights into interactions between ligands and A(2A) adenosine receptors, site-directed mutagenesis was used to test the roles of a glutamic acid residue in the first transmembrane domain (Glu13) and a histidine residue in the seventh transmembrane domain (His278). The two residues, which have been suggested to be closely linked in molecular modeling studies, were mutated to glutamine (E13Q) and tyrosine (H278Y), respectively. Saturation experiments revealed that [(3)H]ZM241385 (4-2-[7-amino-2-(2-furyl)-1,2, 4-triazolo[1,5-a][1,3,5]triazin-5-yl-amino]ethylphenol) bound wild-type and mutant receptors in membranes from COS-7 cells expressing human A(2A) adenosine receptors with high affinity and low non-specific binding. It was found from the competition experiments that the affinity of the A(2A) adenosine receptor agonists for the mutant receptors was 3- to 200-fold lower than for the wild-type receptor. Among antagonist competitors of binding at E13Q and H278Y mutant receptors, there was variation in the affinity depending on their different structures, although changes were relatively minor (<3-fold) except in the case of theophylline, whose affinity was decreased approximately 20 times on the H278Y mutant. The possible involvement of the two residues in sodium ion regulation was also tested. The agonist competition curves for [(3)H]ZM241385 were shifted to the right in both wild-type and mutant receptors in the presence of 1 M sodium ions, but the extent of shift (2- to 27-fold) in wild-type receptor was generally larger than for the mutant receptors. Sodium ions also decreased [(3)H]ZM241385 dissociation from both wild-type and mutant receptors, being more influential on the former than the latter. The results suggest that the two closely linked residues Glu13 and His278 in A(2A) adenosine receptor are most important for agonist recognition and are partly responsible for the allosteric regulation by sodium ions.