Differences between the alpha 2-adrenoceptor in rat submaxillary gland and the alpha 2A-and alpha 2B-adrenoceptor subtypes.

1. The alpha 2-adrenoceptors of rat submaxillary gland, labelled with [3H]-rauwolscine, were characterized by use of a range of subtype selective ligands and were compared to rabbit spleen alpha 2A-adrenoceptors and rat kidney alpha 2B-adrenoceptors. 2. In rat submaxillary gland, [3H]-rauwolscine labelled an apparently homogeneous population of binding sites with relatively low affinity (Kd = 11.65 nM) compared to the affinity in rat kidney (Kd = 2.18 nM) and rabbit spleen (Kd = 4.64 nM). 3. In competition studies using 16 ligands the alpha 2-adrenoceptors in rat submaxillary gland appeared to differ from both the alpha 2A-adrenoceptor of rabbit spleen (r = 0.62) and also the alpha 2B-adrenoceptor of rat kidney (r = 0.28). 4. The affinity data obtained with benoxathian, imiloxan and WB 4101 indicated the presence of an alpha 2B-adrenoceptor in rat submaxillary gland. However, data for chlorpromaxine, oxymetazoline, spiroxatrine and xylometazoline indicated that submaxillary gland alpha 2-adrenoceptors were of the alpha 2A subtype. The affinity estimate for prazosin in rat submaxillary gland was intermediate between its affinity at the alpha 2A- and alpha 2B-adrenoceptors while affinity estimates for idazoxan and phentolamine in rat submaxillary gland were greater than those obtained at either the alpha 2A- or alpha 2B-adrenoceptor. 5. These data indicate that rat submaxillary gland alpha 2-adrenoceptors differ from the alpha 2A- and alpha 2B-adrenoceptors found in rabbit spleen and rat kidney, respectively.     

Assessment of imiloxan as a selective alpha 2B-adrenoceptor antagonist

1. The alpha 2-adrenoceptor binding sites of rabbit spleen and rat kidney, labelled with [3H]-rauwolscine, were characterized using a range of subtype selective ligands. 2. In rabbit spleen, the alpha-2-adrenoceptor binding sites displayed high affinity for oxymetazoline and WB 4101 and low affinity for prazosin and chlorpromazine suggesting the presence of an alpha 2A subtype. 3. There was evidence for heterogeneity of the alpha 2-adrenoceptor binding sites present in rabbit spleen. The results obtained with oxymetazoline and WB 4101 indicated that at least 75% of the [3H]-rauwolscine binding sites in this preparation displayed a pharmacology consistent with the presence of an alpha 2A subtype. 4. In rat kidney, the alpha 2-adrenoceptor binding sites displayed high affinity for prazosin and chlorpromazine and low affinity for oxymetazoline and WB 4101 suggesting the presence of an alpha 2B subtype. 5. The inclusion of guanylylimidodiphosphate (Gpp(NH)p, 0.1 mM) did not modify the pharmacology of the alpha 2-adrenoceptor binding sites present in the two preparations. Furthermore, when the two membrane preparations were combined, the resultant pharmacology was still consistent with the presence of two receptors that retained the characteristics of the alpha 2A and alpha 2B subtypes. 6. Imiloxan was identified as a selective alpha 2B ligand while benoxathian displayed a high degree of selectivity for the alpha 2A-adrenoceptor binding site. The selectivity of imiloxan for the alpha 2B-adrenoceptor binding site, coupled with its specificity for alpha 2-adrenoceptors, should make it a valuable tool in the classification of alpha 2-adrenoceptor subtypes.     

Determination of alpha 1-adrenoceptor subtype selectivity by [3H]-prazosin displacement studies in guinea-pig cerebral cortex and rat spleen membranes.

1. [3H]-prazosin homogeneously labels alpha 1-adrenoceptors in guinea-pig cerebral cortex and rat spleen membranes with dissociation constants of 1.28 and 1.49 x 10(-10) M respectively. 2. Phentolamine and WB 4101 displacement studies show that guinea-pig cerebral cortex contains 30% alpha 1A- and 70% alpha 1B-adrenoceptor subtypes, whereas rat spleen contains a virtually homogeneous alpha 1B-adrenoceptor subtype population. The alpha 1-adrenoceptor population of rat thoracic aorta is predominantly of the alpha 1A-adrenoceptor subtype, and in guinea-pig thoracic aorta it is mainly of the alpha 1B-adrenoceptor subtype. 3. Half of the compounds displacing [3H]-prazosin bound to guinea-pig cerebral cortex membranes display alpha 1A-adrenoceptor selectivity. Among these compounds, WB 4101 and methoxamine are most selective, displaying selectivity ratios of approximately 38 and approximately 26 respectively. 4. The affinity constants of the non-selective compounds for the alpha 1-adrenoceptor in guinea-pig cerebral cortex membranes correlate well with the affinity constants obtained for alpha 1B-adrenoceptors in rat spleen membranes. The affinities of selective compounds for the alpha 1B-adrenoceptor subtype in guinea-pig cerebral cortex correlate very well with their affinity for alpha 1B-adrenoceptor in the rat spleen homogenate. Both regression lines coincide with the line of identity. The affinity constants of selective compounds for the alpha 1A-adrenoceptors in guinea-pig cerebral cortex only apparently correlate with the affinity for either the alpha 1B-adrenoceptors in guinea-pig cerebral cortex or in the rat spleen.(ABSTRACT TRUNCATED AT 250 WORDS)     

A possible structural determinant of selectivity of boldine and derivatives for the alpha 1A-adrenoceptor subtype.

1. The selectivity of action of boldine and the related aporphine alkaloids, predicentrine (9-O-methylboldine) and glaucine (2,9-O-dimethylboldine) and alpha 1-adrenoceptor subtypes was studied by examining [3H]-prazosin competition binding in rat cerebral cortex. WB 4101 and benoxathian were used as selective alpha 1A-adrenoceptor antagonists. 2. In the competition experiments [3H]-prazosin (0.2 nM) binding was inhibited by WB 4101 and benoxathian. The inhibition curves displayed shallow slopes which could be subdivided into high and low affinity components (pKi = 9.92 and 8.29 for WB 4101, 9.35 and 7.94 for benoxathian). The two antagonists recognized approximately 37% of the sites with high affinity from among the total [3H]-prazosin specific binding sites. 3. Boldine, predicentrine and glaucine also competed for [3H]-prazosin (0.2 nM) binding with shallow and biphasic curves recognizing 30-40% of the sites with high affinity. Drug affinities (pKi) at the high and low affinity sites were, 8.31 and 6.50, respectively, for boldine, 8.13 and 6.39 for predicentrine, and 7.12 and 5.92 for glaucine. The relative order of selectivity for alpha 1A-adrenoceptors was boldine (70 fold alpha 1A-selective) = predicentrine (60 fold, alpha 1A-selective) > glaucine (15 fold, alpha 1A-selective). 4. Pretreatment of rat cerebral cortex membranes with chloroethylclonidine (CEC, 10 microM) for 30 min at 37 degrees C followed by thorough washing out reduced specific [3H]-prazosin binding by approximately 70%. The CEC-insensitive [3H]-prazosin binding was inhibited by boldine monophasically (Hill slope = 0.93) with a single pKi value (7.76). 5. These results suggest that whereas the aporphine structure shared by these alkaloids is responsible for their selectively of action for the alpha 1A-adrenoceptor subtype in rat cerebral cortex, defined functional groups, namely the 2-hydroxy function, induces a significant increase in alpha 1A-subtype selectivity and affinity.     

Rat pineal alpha 1-adrenoceptor subtypes: studies using radioligand binding and reverse transcription-polymerase chain reaction analysis.

1. The pharmacological characteristics of alpha 1-adrenoceptor binding sites in rat pineal gland membranes, detected by use of a selective alpha 1-adrenoceptor antagonist ([125I]-iodo-2-[beta-(4-hydroxyphenyl) ethylaminomethyl]tetralone, [125I]-HEAT), were investigated with the alkylating agent, chloroethylclonidine (CEC), and in competition experiments with a number of adrenoceptor agonists and antagonists. 2. Chloroethylclonidine (CEC) treatment (10 microM, 10 min) of rat pineal membranes inactivated approximately 70% of specific [125I]-HEAT binding sites. Higher concentrations of CEC (up to 100 microM) or longer treatment periods (up to 40 min) were no more effective. 3. Adrenoceptor agonists and antagonists competitively inhibited [125I]-HEAT binding with Hill coefficients close to unity indicating a single alpha 1-adrenoceptor subtype is present. The affinity (Ki) of subtype selective agonists (oxymetazoline, SDZ NVI-085) and antagonists (5-methylurapidil, WB4101, benoxathian, phentolamine) was consistent with binding to an alpha 1B-adrenoceptor subtype. 4. The (-)- and (+)-enantiomers of niguldipine had an equal and low affinity for alpha 1-adrenoceptor binding sites both in untreated (log Ki-6.66 and -6.90 respectively) and CEC-treated membranes in which approximately 70% of sites had been inactivated (log Ki-6.41 and -6.86 respectively). This indicates that the small proportion of alpha 1-adrenoceptors insensitive to CEC are not alpha 1A-adrenoceptors. 5. mRNA was isolated from rat pinealocytes, cDNA was synthesized and then amplified by the polymerase chain reaction with alpha 1-adrenoceptor subtype specific primers. These experiments identified both alpha 1A- and alpha 1B-adrenoceptor mRNA, but not alpha 1D-mRNA in rat pinealocytes, although all three adrenoceptor subtypes were readily identified in rat brain cortex. 6. These data indicate that although both alpha 1A- and alpha 1B-adrenoceptor mRNAs are present in the pineal the major subtype of alpha 1-adrenoceptor expressed is the alpha 1B.     

5-Methylurapidil may discriminate between alpha 1-adrenoceptors with a high affinity for WB4101 in rat lung.

The alpha 1-adrenoceptors of rat lung with a high affinity for [3H]-prazosin were subdivided into two populations (high and low affinity sites) by WB4101 and 5-methylurapidil but the proportions were different between both drugs. After pretreatment with chlorethylclonidine, WB4101 recognized only high affinity sites, while 5-methylurapidil still detected high and low affinity sites. These results indicate that alpha 1-adrenoceptors with a high affinity for WB4101 are not homogeneous in the rat lung, suggesting the possible existence of a new alpha 1-adrenoceptor subtype in addition to alpha 1A and alpha 1B subtypes.     

Identification and structural characterization of alpha 1-adrenergic receptor subtypes

Rat liver and brain membrane alpha 1-adrenergic receptors were purified greater than 500-fold by successive chromatographic steps using heparin-agarose, an affinity matrix constructed by coupling a novel derivative of the alpha 1-selective antagonist prazosin to Affigel-102 and wheat germ agglutinin-agarose. Several lines of evidence were obtained for the existence in brain of an alpha 1-adrenergic receptor subtype that is structurally distinct from that previously characterized in liver and other tissues using photoaffinity labeling, protein purification, and DNA cloning techniques. The alpha 1-selective ligand chlorethylclonidine (CEC) (an alkylating agent) irreversibly inactivates 100% of [3H]prazosin binding sites in partially purified preparations of rat liver. Under identical conditions, only 50% of brain receptors are irreversibly inactivated. Computer modeling of data obtained from the competition by the alpha antagonists WB4101 and phentolamine for [3H]prazosin binding to partially purified preparations of rat liver is best fit by assuming a single class of low affinity sites for both ligands. However, analysis of partially purified brain preparations indicates the presence of two binding sites with different affinities for these antagonists. Additionally, prior alkylation of brain receptors with CEC results in the loss of low affinity phentolamine and WB4101 binding sites. The CEC-insensitive site in brain, which displays high affinity for phentolamine and WB4101, is resistant to photoaffinity labeling by [125I]azidoprazosin. This is not due to a markedly lower affinity of the CEC-insensitive sites for the photoaffinity label, because competition studies with [127I]azidoprazosin revealed a single class of high affinity sites in partially purified brain samples. Photoaffinity labeling of partially purified liver and brain samples not treated with CEC results in the specific labeling of a single protein of Mr 80,000. No specifically labeled protein is observed for partially purified brain samples that had previously been incubated with CEC. Treatment of photoaffinity-labeled liver and brain receptors with N-glycanase to cleave N-linked oligosaccharides results in a single Mr 55,000 protein. Taken together, these data provide evidence for the existence of a single receptor subtype (alpha 1b) in rat liver and for two subtypes (alpha 1a and alpha 1b) in rat brain. Furthermore, the insensitivity of the alpha 1a subtype to CEC and the resistance of the alpha 1a subtype to covalent labeling by an alpha 1b-selective photoaffinity probe suggest that the primary structures of the two receptor subtypes differ, such that an amino acid(s) in the alpha 1b subtype that incorporates CEC and the photoaffinity label is lacking in the alpha 1a subtype.     

Mechanisms underlying the differential sensitivity to alpha 1-adrenoceptor activation in the bisected rat vas deferens.

1. The factors underlying the different responsiveness of the prostatic and epididymal portions of rat vas deferens to alpha 1-adrenoceptor stimulation were investigated. 2. The alpha 1-adrenoceptors in membranes of both halves of rat vas deferens were labelled with [3H]-prazosin and the affinities of agonists and antagonists for these receptors were determined. In saturation studies, the Bmax and KD values for [3H]-prazosin in membranes of both portions were the same. 3. In competition studies, the inhibition curves for phentolamine were biphasic and consistent with the presence of both alpha 1a- and alpha 1b-adrenoceptor subtypes. The proportions of binding sites with high and low affinity for phentolamine in both halves of rat vas deferens were similar and in good agreement with the percentages of binding sites for WB-4101 and phentolamine previously reported in the whole rat vas deferens. 4. The phenylethylamines displaced [3H]-prazosin with a shallow inhibition curve. The data are compatible with the assumption of two affinity states for the binding sites. For the imidazoline compounds no such distinct affinity states could be demonstrated. 5. The affinity for, and the relative intrinsic efficacy on postsynaptic alpha 1-adrenoceptors of both portions of rat vas deferens were studied for noradrenaline, phenylephrine and methoxamine by irreversible inactivation of the alpha 1-adrenoceptors by phenoxybenzamine. The parameters for partial agonists were determined by comparing the responses to the partial agonist to those of a full agonist in the same tissue. Homogeneous estimates of the equilibrium dissociation constants (Ka) were obtained, indicating that these agonists bind to the receptors of both tissues in an identical manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of guinea-pig,bovine and rat alpha 1-adrenoceptor subtypes.

1. To elucidate a possible role of species differences in the classification of alpha 1-adrenoceptor subtypes, we have characterized the alpha 1-adrenoceptors in guinea-pig spleen, kidney and cerebral cortex and in bovine cerebral cortex using concentration-dependent alkylation by chloroethylclonidine and competitive binding with 5-methlurapidil, methoxamine, (+)-niguldipine, noradrenaline, oxymetazoline, phentolamine, SDZ NVI-085, tamsulosin and (+)-tamsulosin. Rat liver alpha 1B-adrenoceptors were studied for comparison. Chloroethylclonidine-sensitivity and (+)-niguldipine affinity were also compared at cloned rat and bovine alpha 1a-adrenoceptors. 2. Chloroethylclonidine concentration-dependently inactivated alpha 1-adrenoceptors in all five tissues. While chloroethylclonidine inactivated almost all alpha 1-adrenoceptors in rat liver and guinea-pig kidney and brain, 20-30% of alpha 1-adrenoceptors in guinea-pig spleen and bovine brain were resistant to alkylation by 10 microM chloroethylclonidine. With regard to concentration-dependency guinea-pig kidney and brain were approximately 10 fold less sensitive than guinea-pig spleen or rat liver. 3. In rat liver, all drugs tested competed for [3H]-prazosin binding with steep and monophasic curves. Drug affinities were relatively low and resembled most closely those of cloned rat alpha 1b-adrenoceptors. 4. In guinea-pig spleen, all drugs tested competed for [3H]-prazosin binding with steep and monophasic curves. Drug affinities were relatively low and resembled most closely those of cloned rat alpha 1b-adrenoceptors. 5. In guinea-pig kidney most drugs tested competed for [3H]-prazosin binding with steep and monophasic curves and had relatively low drug affinities close to those of cloned rat alpha 1b- and alpha 1d-adrenoceptors. However, noradrenaline and tamsulosin had consistently biphasic competition curves recognizing 36-39% high and 61-64% low affinity sites. 6. In guinea-pig cerebral cortex, all drugs tested competed for [3H]-prazosin binding with shallow and biphasic curves. While most drugs recognized approximately 25% high affinity sites, tamsulosin and noradrenaline recognized approximately 50% high affinity sites. Drug affinities at the high and low affinity sites except those for tamsulosin and noradrenaline resembled those at cloned alpha 1a- and alpha 1b-adrenoceptors, respectively. 7. In bovine cerebral cortex all drugs tested except for noradrenaline competed for [3H]-prazosin binding with shallow and biphasic curves. All drugs recognized approximately 70% high affinity sites. Drug affinities at the high and low affinity sites resembled those at cloned alpha 1a- and alpha 1b-adrenoceptors, respectively. Noradrenaline competition curves in bovine cerebral cortex were steep and monophasic. 8. When cloned rat and bovine alpha 1a-adrenoceptors transiently expressed in COS cells were studied in a direct side-by-side comparison, both species homologues had similar chloroethylclonidine-sensitivity and (+)-niguldipine affinity. 9. We conclude that properties of bovine alpha 1A- and alpha 1B-adrenoceptors are very similar to those of other species such as rat. alpha 1-Adrenoceptor subtypes in guinea-pigs resemble alpha 1A- and alpha 1B-adrenoceptors in other species but chloroethylclonidine sensitivity and competition binding profiles of noradrenaline and tamsulosin are not compatible with previously established alpha 1-adrenoceptor subtype classification.     

Pharmacological characterization of alpha 1-adrenoceptor subtypes in rat heart: a binding study.

1. The alpha 1-adrenoceptor subtypes of rat heart were characterized in binding experiments performed with [3H]-prazosin as the radiolabel. The specific binding to the alpha 1-adrenoceptors was determined with 0.3 microM prazosin, because phentolamine (10 microM) was insufficient to inhibit completely the specific binding of high concentrations of [3H]-prazosin. 2. In saturation experiments, [3H]-prazosin bound to two distinct affinity sites (pKD = 10.39 and 8.19). The proportion of the low affinity sites was approximately 84% of total specific binding. Membranes pretreated with chloroethylclonidine (CEC, 10 microM) also showed two distinct affinity sites for [3H]-prazosin, although the maximum numbers of high and low affinity sites were reduced by 86 and 64%, respectively. 3. In competition experiments, [3H]-prazosin (100 pM) binding was inhibited by WB4101 (2-(2,6-dimethoxy-phenoxyethyl)aminomethyl-1,4-benzodioxane) and 5-methylurapidil. The inhibition curves displayed shallow slopes which could be subdivided into high and low affinity components (pKi = 10.43 and 8.36 for WB4101, 8.62 and 6.61 for 5-methylurapidil). However, unlabelled prazosin or HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)-ethyl)amin o) propyl)benzeneacetonitrile fumarate) competed for [3H]-prazosin binding monophasically (pKi = 10.34 and 8.28, respectively). In CEC-pretreated membranes, prazosin, WB4101, 5-methylurapidil and HV723 antagonized the [3H]-prazosin (100 pM) binding monophasically (pKi = 9.70, 9.56, 8.60 and 8.82, for each antagonist). 4. On the other hand, 1000 pM [3H]-prazosin binding was inhibited by unlabelled prazosin biphasically (pKi = 10.49 and 8.49). HV723 did not discriminate both prazosin-high and low affinity sites (pKi = 8.18).(ABSTRACT TRUNCATED AT 250 WORDS)     

Subtypes of alpha 1-adrenoceptors in hippocampus of pigs, guinea-pigs, calves and humans: regional differences

Radioligand binding studies were performed with membranes of guinea-pig, pig, calf and human hippocampus using [125I]BE 2254 (also known as [125I]HEAT) as the radioligand. [125I]BE 2254 bound with similar high affinity to saturable populations of recognition sites in all four membrane preparations. Competition curves obtained with a variety of ligands (e.g., WB 4101, benoxathian, 5-methyl-urapidil) were biphasic and the profiles of the high- and low-affinity components of [125I]BE 2254 binding were similar in all four membrane preparations. The data suggest that [125I]BE 2254 labels two subtypes of alpha 1-adrenoceptors in the hippocampus of these species. [3H]WB 4101 was used to label alpha 1A recognition sites in pig hippocampus membranes. [3H]WB 4101 recognized with high affinity an apparently homogeneous class of sites, as suggested by monophasic saturation and competition experiments. The rank order of affinity of the compounds for the high-affinity component of [125I]BE 2254 binding was similar to the rank order of affinity of these drugs for [3H]WB 4101 sites. The pharmacological profile of the low-affinity component of [125I]BE 2254 binding was similar to that described recently for the alpha 1B-adrenoceptor cloned from DDT1 cells. In autoradiographic studies with human hippocampal slices, CEC (chloroethylclonidine), an alkylating agent described to show selectivity for alpha 1B-adrenoceptors, displaced preferentially [125I]BE 2254 binding from the molecular layer of the dentate gyrus. In contrast, WB 4101 an alpha 1A-adrenoceptor-selective ligand, displaced preferentially [125I]BE 2254 binding in the hilus and the CA3 region. The data show that 2 subtypes of alpha 1-adrenergic recognition sites can be identified in the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of alpha 1-adrenoceptor subtypes in the rat vas deferens: binding and functional studies.

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).(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological properties of the cloned alpha 1A/D-adrenoceptor subtype are consistent with the alpha 1A-adrenoceptor characterized in rat cerebral cortex and vas deferens.

1. The pharmacological characteristics of cloned mammalian alpha 1A/D-, alpha 1B- and alpha 1C-adrenoceptor subtypes expressed in rat 1 fibroblasts were determined in comparison to the binding and functional properties of these subtypes in rat tissues. 2. Analysis of [3H]-prazosin binding to membrane homogenates from rat 1 fibroblast cells expressing each of the alpha 1-subtypes indicated high affinity binding to a single population of binding sites. Binding affinities were similar for alpha 1A/D-, alpha 1B- and alpha 1C-subtypes (Kds: 0.13, 0.10 and 0.15 nM respectively) although a higher density of alpha 1B- and alpha 1C-receptors (Bmax: 4068 and 10,323 fmol mg-1 protein respectively) were expressed in comparison to alpha 1A/D (838 fmol mg-1). 3. Displacement of [3H]-prazosin from membranes expressing cloned alpha 1-adrenoceptor subtypes revealed that 5-methyl-urapidil, WB 4101, benoxathian and phentolamine displayed high affinity and selectivity for alpha 1A/D- over alpha 1B-subtypes. These compounds also had high affinity and selectivity for alpha 1C- over alpha 1B-subtypes. 5-Methyl-urapidil showed selectivity for alpha 1C (Ki 0.60 +/- 0.16 nM) over both alpha 1A/D (Ki, 9.8 +/- 2.8 nM) and alpha 1B (Ki 57.2 +/- 12 nM) subtypes. Prazosin and doxazosin were not subtype selective. 4. In comparison to [3H]-prazosin a similar pharmacological profile was obtained with [125I]-HEAT using cloned alpha 1A/D-, alpha 1B- and alpha 1C-adrenoceptors expressed in rat 1 fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of an alpha 1D-adrenoceptor mediating the contractile response of rat aorta to noradrenaline.

1. The affinities of a number of alpha 1-adrenoceptor antagonists were determined by displacement of [3H]-prazosin binding from cloned human alpha 1A-adrenoceptors (previously designated cloned alpha 1c subtype), alpha 1B alpha 1D and rat alpha 1D-adrenoceptors, stably expressed in rat-1 fibroblasts. Functional affinity estimates for these compounds were also determined from noradrenaline-mediated contractions of rat aorta. 2. BMY 7378 displayed high affinity for cloned human alpha 1D-adrenoceptors (pKi = 8.2 +/- 0.10) and was selective over alpha 1A (pKi = 6.2 +/- 0.10) and alpha 1B subtypes (6.7 +/- 0.11). WB 4101, benoxathian and phentolamine displayed high affinity for alpha 1A and alpha 1D adrenoceptors compared to the alpha 1B subtype. Spiperone displayed high affinity and selectivity for alpha 1B adrenoceptors (pKi 8.8 +/- 0.16). 5-Methyl-urapidil was selective for cloned alpha 1A adrenoceptors. 3. Comparative binding affinities (pKi) for compounds at cloned human and rat1D adrenoceptors were almost identical (r = 0.99, slope = 1.08). 4. Prazosin, doxazosin and 5-methyl-urapidil were potent, competitive antagonists of noradrenaline-mediated contractions of rat aorta (pA2 values of 9.8, 8.8 and 7.8 respectively). The selective alpha 1D antagonist BMY 7378 was also a potent antagonist on rat aorta (pKB = 8.3 +/- 0.1) but the interaction of this compound was not consistent with competitive antagonism at a single population of receptors. 5. Functional affinities for compounds determined against noradrenaline-mediated contractions of rat aorta correlated well with binding affinities at cloned alpha 1D-adrenoceptors (r = 0.96), but not with alpha 1A (r = 0.61) or alpha 1B (r = 0.46) subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subclassification of alpha 1-adrenoceptor recognition sites by urapidil derivatives and other selective antagonists

1. The affinities of urapidil derivatives and other antagonists for alpha 1-adrenoceptors labelled by [3H]-prazosin were determined on membranes of six different rat tissues. 2. Urapidil and its 5-acetyl-, 5-formyl- and 5-methyl-derivative displaced [3H]-prazosin from alpha 1-adrenoceptor binding sites in a concentration-dependent manner which varied with tissue. IC50 values were lower in vas deferens, hippocampus and cerebral cortex than in heart, liver and spleen. For 5-methyl-urapidil, binding to two distinct sites could be demonstrated with mean K1 values of about 0.6 and 45 nM. Saturation binding studies with [3H]-prazosin in the presence of 5-methyl-urapidil indicated a competitive type of interaction between 5-methyl-urapidil and [3H]-prazosin. 3. The proportion of [3H]-prazosin binding sites with high affinity for 5-methyl-urapidil was 58% in vas deferens, 69% in hippocampus, 41% in cerebral cortex and 23% in myocardium. In liver and spleen virtually no high affinity sites were found. These values were in good agreement with the percentages of binding sites with high affinities for WB-4101 and phentolamine, indicating that all these antagonists bind to the same subtype of alpha 1-recognition sites, whereas other alpha-antagonists like BE 2254, yohimbine and unlabelled prazosin did not discriminate between two binding sites. 4. Preincubating membranes of the cerebral cortex with chloroethylclonidine preferentially inactivated [3H]-prazosin binding sites with low affinity for 5-methyl-urapidil. 5. The antagonist potencies of 5-methyl-urapidil and WB-4101 against alpha 1- adrenoceptor-mediated contractile responses were higher in vas deferens than in myocardium. The alpha 1-mediated effects in vas deferens but not in the heart were highly susceptible to nitrendipine. 6. Using 5-methyl-urapidil, the existence of two distinct alpha 1-adrenoceptor recognition sites could be demonstrated which correspond to the proposed alpha 1A- and alpha 1B-subtypes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Subtypes of alpha 1-adrenoceptors involved in noradrenaline-induced contractions of rat thoracic aorta and dog carotid artery.

We tried to determine alpha 1-adrenoceptor subtypes involved in noradrenaline-induced contractions of rat thoracic aorta and dog carotid artery. Prazosin competitively antagonized the contractions induced by noradrenaline in both the arteries with a high pKB value (approximately 9.7). WB4101, benoxathian, phentolamine, HV723 and 5-methylurapidil also competitively antagonized the responses to noradrenaline in both the arteries: however, the affinities for the antagonists were significantly higher in the rat thoracic aorta than in the dog carotid artery. The affinities for the competitive antagonists were not changed by treatment with nifedipine. In the rat thoracic aorta, chlorethylclonidine (CEC) elicited either a persistent contraction with rhythmic activities before treatment with nifedipine or partial inactivation of alpha 1-adrenoceptors in the presence of nifedipine. On the other hand, CEC produced only inactivation of alpha 1-adrenoceptors in the dog carotid artery. These results suggest that noradrenaline-induced contractions of the rat thoracic aorta and dog carotid artery are respectively mediated through distinct alpha 1-adrenoceptor subtypes. According to the alpha 1A, alpha 1B subclassification, the alpha 1-adrenoceptor of dog carotid artery is like the alpha 1B subtype, while that of rat thoracic aorta is atypical. Both subtypes are also identified as a high affinity site for prazosin (alpha 1H subtype) in the alpha 1H, alpha 1L and alpha 1N subclassification.     

Pharmacological subclassification of alpha 1-adrenoceptors in vascular smooth muscle

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).(ABSTRACT TRUNCATED AT 250 WORDS)     

alpha 1-adrenoceptors can mediate chronotropic responses in the rat heart.

In the pithed rat, amidephrine a "selective" alpha 1-adrenoceptor agonist, evokes a positive chronotropic response. This response can be antagonized by prazosin but not by propranolol or by rauwolscine. Similarly, part of the chronotropic response to cardioaccelerator nerve stimulation is resistant to blockade by propranolol but is sensitive to prazosin or WB4101. We conclude that alpha 1-adrenoceptors can mediate a chronotropic response in the rat heart either to exogenous agonists or to the endogenous neurotransmitter noradrenaline.     

Pharmacological characterization of two distinct alpha 1-adrenoceptor subtypes in rabbit thoracic aorta.

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.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selectivity of benzodioxane alpha-adrenoceptor antagonists for alpha 1- and alpha 2-adrenoceptors determined by binding affinity

A series of benzodioxane derivatives, structurally related to WB 4101 and piperoxan as well as prazosin and its two analogues UK-18,596 and UK-33,274, was studied with respect to their affinity for alpha 1-and alpha 2-adrenoceptors identified by 3H-prazosin (specific activity 33 Ci/mmol) and 3H-clonidine (specific activity 26.7 Ci/mmol), respectively, in isolated rat brain membranes. The structural variations made in these molecules gave rise to pronounced differences in affinity for alpha 1-adrenoceptors, whereas their binding affinity for alpha 2-adrenoceptors only slightly varied. Apart from piperoxan and its analogues, which showed some preference for alpha 2-adrenergic binding sites, all benzodioxane-like structures displayed a general selectivity for the alpha 1-adrenoreceptor sites labeled with 3H-prazosin. The drugs were 5-50 times more potent in inhibiting 3H-prazosin than 3H-clonidine from their specific binding sites in rat brain membranes. The highest alpha 1 selectivity was found for prazosin and UK-33,274. Within the present series of WB 4101-related benzodioxane compounds, the affinity for alpha 1-adrenoceptors is greatly reduced by alkyl substitution at the secondary amino nitrogen in the side chain. Ortho substitution of the phenyl moiety with methoxy increased affinity as did hydroxy at the para position. The side chain oxygen atom can be deleted or substituted by methylene without great loss in 3H-prazosin displacing effectiveness. The affinity for alpha 1-adrenoceptors was profoundly influenced by the configuration of the molecule. Upon introducing a second chiral center through a methyl group, the two resulting racemates differ 10-fold in activity and selectivity towards alpha 1-adrenoceptors. One of these racemates was even slightly more selective than WB 4101 itself. The selectivity of the drugs to bind to alpha 1-and alpha 2-adrenoceptors corresponded well with their in vivo selectivity to antagonize alpha 1- and alpha 2-adrenoceptor-mediated vasoconstriction in pithed normotensive rats. It is suggested that a systematic study of the structure-affinity relationship in benzodioxane antagonists may provide potent and selective blocking drugs of alpha 1-adrenoceptors.