Prazosin Binding Sites Research Articles

Effects of age on alpha 1-adrenoceptor subtypes in the heart ventricular muscle of the rat.

The effects of ageing on alpha 1-adrenoceptor subtypes have been examined in heart ventricular muscle of young (2-3 months) and middle-aged (18 months) Sprague-Dawley rats. Radioligand binding studies with [3H]prazosin revealed an age-related loss of binding sites (Bmax 56.7 +/- 1.93 fmol (mg protein)-1 age 2 months vs 31.7 +/- 2.45 fmol (mg protein)-1 age 18 months) not followed by changes in the dissociation constant value (Kd 0.16 +/- 0.03 nM age 2 months and 0.10 +/- 0.03 nM age 18 months). Competition curves with WB 4101 showed two distinct sites with different affinities, the proportion of sites with high affinity being similar for both age groups (22.2 +/- 1.89% vs 17.8 +/- 1.96% for animals aged 2 and 18 months, respectively). Agonist displacement curves of [3H]prazosin indicate the existence of two different affinity sites for the agonist, that are maintained regardless of the ageing process (R(high) = 16.2 +/- 1.54% and R(low) = 83.8 +/- 1.89% in rats aged 2 months and R(high) = 16.3 +/- 3.23% and R(low) = 83.7 +/- 3.95% in rats aged 18 months). The fractional inactivation of alpha 1-adrenoceptors by chloroethylclonidine resulted in a loss of [3H]prazosin specific binding, and a percentage of 22.5 +/- 0.95 and 22.6 +/- 4.2 of remaining binding sites for the groups of 2 and 18 months of age, respectively. The percentage of chloroethylclonidine-insensitive [3H]prazosin binding sites was similar to those with high affinity for WB4101. The present study confirms a decline of alpha 1-adrenoceptors with increasing age and reveals that the equilibrium of the expression of the two existing subpopulations of the receptor is maintained during ageing.

Alpha 1-adrenoceptors: the ability of various agonists and antagonists to discriminate between two distinct [3H]prazosin binding sites.

Recently, it has been demonstrated that two distinct alpha 1-adrenoceptor binding sites showing high and low affinity for WB-4101 (2-(2,6-dimethoxyphenoxy)ethyl-aminomethyl-1,4-benzodioxane) and 5-methyl-urapidil can be distinguished. In the present study we examined the ability of several agonists and antagonists to discriminate between these alpha 1-adrenoceptor binding sites. [3H]Prazosin binding to membranes of rat liver, heart, cerebral cortex and hippocampus was inhibited monophasically by butanserine, I-BE 2254 (2-(3-(4-hydroxy-3-iodophenyl)ethylaminomethyl)tetralone-hydrochloride), prazosin, rauwolscine and verapamil. In contrast, competition curves of adrenaline, oxymetazoline, amidephrine and YM-12617 (5-[2-[[2-(o-ethoxy-phenoxy)ethyl]-amino]propyl]-2- methoxybenzenesulfonamide HCl) were best described by a model of two binding sites. Chloroethylclonidine (CEC), a compound shown to irreversibly eliminate binding sites with low affinity for WB-4101, increased the proportion of high affinity binding sites for oxymetazoline and amidephrine, whereas the binding data for prazosin and adrenaline remained unchanged. These results indicate that amidephrine, oxymetazoline and YM-12617, but not the other drugs tested discriminate between different alpha 1-adrenoceptor recognition sites labelled by [3H]prazosin.

Alpha-adrenoceptor interaction of tetrandrine and isotetrandrine in the rat: functional and binding assays.

The action of 1S,1'S-tetrandrine, a bisbenzyltetrahydroisoquinoline alkaloid, on alpha1-adrenoceptors has been compared with that of its isomer 1R,1'S-isotetrandrine. The work includes binding assays to analyse the affinity of these products for the [3H]prazosin binding site of rat cerebral cortical membranes and functional studies on rat isolated aorta to examine the effects of both alkaloids on intracellular calcium processes related or not to alpha-adrenoceptor activation. A radioligand receptor-binding study showed that both compounds interacted with the alpha1-adrenoceptors displacing [3H]prazosin from the specific binding site. The Ki values (inhibition constants) were 0.69+/-0.12 and 1.6+/-0.4 microM for tetrandrine and isotetrandrine, respectively. The functional studies showed that both alkaloids concentration-dependently inhibited noradrenaline-induced contraction in Ca2+-free solution (IC50 values, i.e. the concentrations needed to induce 50% inhibition, were 252.8 and 174.9 microM for tetrandrine and isotetrandrine, respectively), the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (increase in resting tone; IC50 values 11.6 and 19.6 microM for tetrandrine and isotetrandrine, respectively) and the refilling of intracellular Ca2+ stores sensitive to noradrenaline (IC50 values 7.4 and 14.9 microM for tetrandrine and isotetrandrine, respectively). The results show that tetrandrine and isotetrandrine interact with alpha1-adrenoceptors by displacing the [3H]prazosin binding site and that both compounds inhibit mainly the Ca2+-dependent process and have less action on alpha1-adrenoceptors. Tetrandrine is more potent than isotetrandrine.

α 1-Adrenoceptors in the rat cerebral cortex: New insights into the characterization of α 1L- and α 1D-adrenoceptors

Among the three α 1-adrenoceptor subtypes (α 1A, α 1B and α 1D) a peculiar intracellular localization and poor coupling to membrane signals of cloned α 1D-adrenoceptor have been reported. In addition, the α 1L-adrenoceptor (low affinity for prazosin), a functional phenotype of α 1A, has been described. The purpose of this work was to analyze the expression, cellular localization and coupling to membrane signalling (inositol phosphate accumulation) of α 1-adrenoceptor subtypes in a native tissue, the rat cerebral cortex. mRNA for the three subtypes was quantified by real-time RT-PCR (α 1D> α 1B ≫ α 1A). α 1-Adrenoceptors were also detected by immunoblotting, revealing α 1A- and α 1B-adrenoceptors to be predominantly expressed in the membrane fraction and the α 1D-adrenoceptor to be localized in the cytosolic fraction. Competitive radioligand binding studies revealed the presence of α 1D-adrenoceptor in tissue homogenates, whereas only α 1A- and α 1B-subtypes were detected in membranes. The proportion of α 1A-adrenoceptor increased after treatment with noradrenaline, suggesting differences in agonist-mediated trafficking. Saturation experiments detected high- and low (α 1A/L)-prazosin binding sites, the latter of which disappeared on incubation with GppNHp. The α 1A/L-adrenoceptor was heavily implicated in the inositol phosphate response, while the α 1D-subtype did not play a relevant role. These results suggest that the predominant cytosolic localization of α 1D-adrenoceptor lies behind its poor coupling to membrane signalling such as inositol phosphate pathway. The fact that the α 1L-adrenoceptor detected in radioligand binding studies disappeared in the presence of GppNHp implies that it represents a conformational state of the α 1A-adrenoceptor coupled to G-protein.

Comparison of ATP-Binding Cassette Transporter Interactions with the Tyrosine Kinase Inhibitors Imatinib, Nilotinib, and Dasatinib

Although the development of tyrosine kinase inhibitors (TKIs) to control the unregulated activity of BCR-ABL revolutionized the therapy of chronic myeloid leukemia, resistance to TKIs is a clinical reality. Among the postulated mechanisms of resistance is the overexpression of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2), which mediate reduced intracellular drug accumulation. We compared the interactions of the TKIs imatinib, nilotinib, and dasatinib with ABCB1 and ABCG2 in ex vivo and in vitro systems. The TKIs inhibited rhodamine 123 and Hoechst 33342 efflux mediated by endogenous expression of the transporters in murine and human hematopoietic stem cells with potency order nilotinib >> imatinib >> dasatinib. Studies with ABCB1-, ABCG2-, and ABCC1-transfected human embryonic kidney 293 cells verified that nilotinib was the most potent inhibitor of ABCB1 and ABCG2. Cytotoxicity assays in stably transduced K562-ABCG2 and K562-ABCB1 cells confirmed that the TKIs were also substrates for the two transporters. Like imatinib, both nilotinib and dasatinib decreased ABCG2 surface expression in K562-ABCG2 cells. Finally, we found that all TKIs were able to compete labeling of ABCB1 and ABCG2 by the photo-cross-linkable prazosin analog [(125)I]iodoarylazidoprazosin, suggesting interaction at the prazosin-binding site of both proteins. Our experiments support the hypothesis that all three TKIs are substrates of ABC transporters and that, at higher concentrations, TKIs overcome transporter function. Taken together, the results suggest that therapeutic doses of imatinib and nilotinib may diminish the potential of ABCB1 and ABCG2 to limit oral absorption or confer resistance. Clinical data are required to definitively answer the latter question.

Selective and sustained occupancy of prostatic α1-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.

Substrate Overlap between Mrp4 and Abcg2/Bcrp Affects Purine Analogue Drug Cytotoxicity and Tissue Distribution

The use of probe substrates and combinations of ATP-binding cassette (ABC) transporter knockout (KO) animals may facilitate the identification of common substrates between apparently unrelated ABC transporters. An unexpectedly low concentration of the purine nucleotide analogue, 9-(2-(phosphonomethoxy)ethyl)-adenine (PMEA), and up-regulation of Abcg2 in some tissues of the Mrp4 KO mouse prompted us to evaluate the possibility that Abcg2 might transport purine-derived drugs. Abcg2 transported and conferred resistance to PMEA. Moreover, a specific Abcg2 inhibitor, fumitremorgin C, both increased PMEA accumulation and reversed Abcg2-mediated PMEA resistance. We developed Mrp4 and Abcg2 double KO mice and used both single KOs of Abcg2 and Mrp4 mice to assess the role of these transporters in vivo. Abcg2 contributed to PMEA accumulation in a variety of tissues, but in some tissues, this contribution was only revealed by the concurrent absence of Mrp4. Abcg2 also transported and conferred resistance to additional purine analogues, such as the antineoplastic, 2-chloro-2'-deoxyadenosine (cladribine) and puromycin, a protein synthesis inhibitor that is often used as a dominant selectable marker. Purine analogues interact with ABCG2 by a site distinct from the prazosin binding site as shown by their inability to displace the substrate analogue and photoaffinity tag [(125)I]iodoarylazidoprazosin. These studies show that Abcg2, like Mrp4, transports and confers resistance to purine nucleoside analogues and suggest that these two transporters work in parallel to affect drug cytotoxicity and tissue distribution. This new knowledge will facilitate an understanding of how Abcg2 and Mrp4, separately and in combination, protect against purine analogue host toxicity as well as resistance to chemotherapy.

Addition of a signal peptide sequence to the α1D‐adrenoceptor gene increases the density of receptors, as determined by [3H]‐prazosin binding in the membranes

1. Both in mammalian tissues and in transfected cells, only low levels of alpha1D-adrenoceptors are detected in radioligand binding studies. It has been implicated that the comparatively long N-terminal tail of the alpha1D-adrenoceptor is responsible for the inefficient surface expression of the receptor. 2. In the present study, we created gene constructs for six N-terminally truncated variants of the human alpha1D-adrenoceptor. These constructs were used to transfect Neuro2A cells. We show that the density of alpha1D-adrenoceptors, observed by [3H]-prazosin binding, gradually increased with longer truncations of the N-terminus. This seems to indicate that the long N-terminal tail nonspecifically interferes with receptor translocation to the plasma membrane. 3. The addition of a 16 amino acids long signal peptide to the N-terminus of the wild-type alpha1D-adrenoceptor increased the density of receptor binding sites 10-fold in Neuro2A and COS-7 cells. This indicates that, after the addition of a signal peptide, the long N-terminal tail of the alpha1D-adrenoceptor does not interfere with proper translocation of the receptor to the plasma membrane. This, in turn, indicates that the N-terminal tail of the wild-type alpha1D-adrenoceptor, merely by its long length, hinders the first transmembrane helix of the receptor from being a signal anchor. 4. Neither the wild-type alpha1D-adrenoceptor (for which the expression level of [3H]-prazosin binding sites is low) nor the truncated alpha1D-adrenoceptor variant (for which the expression level of [3H]-prazosin binding sites is high) showed any constitutive activity in stimulating inositol phosphate accumulation. This indicates that the low expression level of [3H]-prazosin binding sites, after transfection with the wild-type alpha1D-adrenoceptor, is not caused by constitutive activity of the receptor and subsequent receptor downregulation.

Submandibular gland acinar cells express multiple alpha1-adrenoceptor subtypes.

We evaluated an acinar cell line (SMG-C10) cloned from rat submandibular glands as a possible model for alpha(1)-adrenoceptor regulation of submandibular function. alpha(1)-Adrenoceptors are subdivided into three subtypes called alpha(1A), alpha(1B), and alpha(1D), which can be distinguished from one another by their differential affinity values for subtype-selective alpha(1)-adrenoceptor antagonists. Thus, alpha(1)-adrenoceptor subtypes in SMG-C10 cells were characterized with reverse transcription-polymerase chain reaction (RT-PCR) and [(3)H]prazosin binding in side-by-side experiments with native submandibular glands. RT-PCR identified mRNAs for alpha(1A)-, alpha(1B)-, and alpha(1D)-adrenoceptors in SMG-C10 cells and submandibular glands. The inhibition of [(3)H]prazosin binding by 5-methylurapidil (alpha(1A)-selective) was biphasic and fit best to a two-site binding model with 40 +/- 8% high (K(iH))- and 60 +/- 10% low (K(iL))-affinity binding sites in SMG-C10 cells, and 76% high- and 24% low-affinity binding sites in submandibular glands. Respective K(iH) and K(iL) values for 5-methylurapidil were 1.9 +/- 0.4 and 100 +/- 30 nM in SMG-C10 cells and 3.2 +/- 0.8 and 170 +/- 20 nM in submandibular glands. BMY-7378 [8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (alpha(1D)-selective)] bound with low affinity in SMG-C10 cells and submandibular glands with K(i) values of 81 +/- 20 and 110 +/- 20 nM, respectively. Chloroethylclondine, an irreversible alkylating agent selective for alpha(1B) adrenoceptors, reduced the density of [(3)H]prazosin binding sites by 42 and 26% in SMG-C10 and submandibular membranes, respectively. Thus, SMG-C10 cells and submandibular glands are similar in expressing receptor protein for alpha(1A)- and alpha(1B)-adrenoceptor subtypes, establishing SMG-C10 cells as a potential model for alpha(1)-adrenoceptor-mediated secretion.

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

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

Prenatal stress changes rat arterial adrenergic reactivity in a regionally selective manner

A suboptimal fetal environment has been linked to increased risk of cardiovascular disease in adulthood. We investigated whether intrauterine stress (IUS) alters the development of adrenergic reactivity in different types of rat arteries. Intrauterine stress was induced by ligation of the uterine arteries at day 13 of pregnancy in Wistar rats. First-order mesenteric, renal, femoral and saphenous arteries of the 21-day-old male offspring were studied in a myograph. IUS in the rat changes arterial adrenergic reactivity in a regionally selective manner. Adrenoceptor-mediated responses are altered in the renal artery. Maximal contractile responses to phenylephrine were increased, while sensitivity to the α 1-adrenoceptor agonist was decreased. Intrauterine stress significantly reduced contractile responses to norepinephrine and enhanced relaxing responses to isoproterenol in the renal artery. Adrenergic responses were not modified in mesenteric, femoral and saphenous arteries. In the kidneys the densities of [ 3H]prazosin binding sites, periarterial adrenergic nerves and of the glomeruli were not altered after intrauterine stress at day 13 of gestation. The observed regionally selective alterations in arterial reactivity might link a suboptimal fetal environment to the development of cardiovascular disease in the adult.

Role of alpha-adrenergic receptors in the effect of the beta-adrenergic receptor ligands, CGP 12177, bupranolol, and SR 59230A, on the contraction of rat intrapulmonary artery.

This study investigates the effect of the aryloxypropanolamines 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one (CGP 12177), bupranolol, and 3-(2-ethylphenoxy)-1[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate (SR 59230A) [commonly used as beta(3)- and/or atypical beta-adrenergic receptors (beta-AR) ligands] on the contractile function of rat intralobar pulmonary artery. Affinities of beta-AR ligands for alpha(1)-adrenergic receptors (alpha(1)-AR) were also evaluated using [(3)H]prazosin binding competition experiments performed in rat cortical membranes. In intralobar pulmonary artery, CGP 12177 did not modify the basal tone, but antagonized the contraction induced by the alpha(1)-AR agonist phenylephrine (PHE). In arteries precontracted with PHE, CGP 12177 elicited relaxation, whereas in those precontracted with prostaglandin F(2alpha) (PGF(2alpha)), it further enhanced contraction. CGP 12177 induced an increase in intracellular calcium concentration in pressurized arteries loaded with Fura PE-3 and precontracted with PGF(2alpha). In PGF(2alpha) precontracted arteries, phentolamine (an alpha-AR antagonist) and phenoxybenzamine (an irreversible alpha-AR antagonist) antagonized the contractile responses to PHE and CGP 12177. Both responses were also decreased by bupranolol and SR 59230A. Specific [(3)H]prazosin binding was displaced by CGP 12177, bupranolol, and SR 59230A with pK(i) values of 5.2, 5.7, and 6.6, respectively. In contrast, (+/-)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium (BRL 37344) and disodium 5-[(2R)-2-([(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243) (nonaryloxypropanolamines beta(3)-AR agonists) displayed very low affinity for [(3)H]prazosin binding sites (pK(i) values below 4). These data suggest that CGP 12177 exhibits partial agonist properties for alpha(1)-AR in rat pulmonary artery. They also show that bupranolol and SR 59230A exert an alpha(1)-AR antagonist effect. As a consequence, these aryloxypropanolamine compounds should be used with caution when investigating the role of beta(3)- and atypical beta-AR in the regulation of vascular tone.

Mechanism of vascular relaxation by thaligrisine: Functional and binding assays

In the present study we examine the mechanism by which thaligrisine, a bisbenzyltetrahydroisoquinoline alkaloid, inhibits the contractile response of vascular smooth muscle. The work includes functional studies on rat isolated aorta and tail artery precontracted with noradrenaline or KCl. In other experiments rat aorta was precontracted by caffeine in the presence or absence of extracellular Ca 2+. In order to assess whether thaligrisine interacts directly with calcium channel binding sites or with α-adrenoceptors we examined the effect of the alkaloid on [ 3H]-(+)- cis diltiazem, [ 3H]-nitrendipine and [ 3H]-prazosin binding to cerebral cortical membranes. The functional studies showed that the alkaloid inhibited in a concentration-dependent manner the contractile response induced by depolarization in rat aorta (IC 50=8.9±2.9 μM, n=5) and in tail artery (IC 50=3.04±0.3 μM, n=6) or noradrenaline induced contraction in rat aorta (IC 50=23.0±0.39 μM, n=9) and in tail artery (IC 50=3.8±0.9 μM, n=7). In rat aorta, thaligrisine concentration-dependently inhibited noradrenaline-induced contraction in Ca 2+-free solution (IC 50 = 13.3 μM, n=18). The alkaloid also relaxed the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (IC 50 = 7.7 μM, n=4). The radioligand receptor-binding study showed that thaligrisine has higher affinity for [ 3H]-prazosin than for [ 3H]-(+)- cis-diltiazem binding sites, with Ki values of 0.048±0.007 μM and 1.5±1.1 μM respectively. [ 3H]-nitrendipine binding was not affected by thaligrisine. The present work provides evidence that thaligrisine shows higher affinity for [ 3H]-prazosin binding site than [ 3H]-(+)- cis-diltiazem binding sites, in contrast with tetrandrine and isotetrandrine that present similar affinity for both receptors. In functional studies thaligrisine, acted as an α 1-adrenoceptor antagonist and as a Ca 2+ channel blocker, relaxing noradrenaline or KCl-induced contractions in vascular smooth muscle. This compound specifically inhibits the refilling of internal Ca 2+-stores sensitive to noradrenaline, by blocking Ca 2+-entry through voltage-dependent Ca 2+-channels.

Alpha1-adrenoceptors involvement in painful diabetic neuropathy: a role in allodynia.

To investigate whether alpha1-adrenoceptors are involved in pain behaviors in streptozotocin (STZ)-induced diabetic rats, we measured the effects of phenylephrine or prazosin on allodynia in the diabetic rats. Phenylephrine aggravated allodynia, while prazosin alleviated allodynia in the diabetic rats. We also measured alpha1-adrenoceptors gene expression or density of [3H]-prazosin binding sites in the dorsal root ganglia (DRG) and spinal cord in painful diabetic rats. Alpha1-adrenoceptors mRNA and density of [3H]prazosin binding sites were increased in the DRG of the diabetic rats, however there were no significant differences in alpha1-adrenoceptors expression in the spinal cord between the control and diabetic rats. These results suggest increased alpha1-adrenoceptors in the DRG may play a role in the pathogenesis of painful diabetic neuropathy.

A novel prazosin binding site other than α1-adrenoceptor in human kidney

A novel prazosin binding site other than α1-adrenoceptor in human kidney

Ex vivo occupancy by tamsulosin of α1-adrenoceptors in rat tissues in relation to the plasma concentration

At 0.5–12 h after oral administration of tamsulosin (2.3 μmol/kg) in rats, there was a significant decrease in specific [ 3H]prazosin binding in the prostate as compared to the control value. The greater decrease occurred in the submaxillary gland. The effect of tamsulosin was mainly due to a marked reduction of [ 3H]prazosin binding sites (Bmax) rather than to an increase in the dissociation constant (Kd). In contrast, there was only a slight decrease or no change in the [ 3H]prazosin binding in the spleen, heart, and cerebral cortex of tamsulosin-administered rats at 0.5–12 h. Oral administration of terazosin (21.7 μmol/kg) significantly increased Kd values for [ 3H]prazosin binding with little effect on Bmax values in the rat prostate at 3 and 6 h. The greater increases in Kd values were observed in the submaxillary gland, spleen and heart at 0.5–12 h. Terazosin had a slight effect on Kd values for the cerebral cortical [ 3H]prazosin binding. Tamsulosin was absorbed rapidly after oral administration at a dose of 2.3 μmol/kg in rats, and at 6 h, plasma concentration decreased markedly to approximately one-twentieth of the 0.5 h peak level. α 1-Adrenoceptor occupancy was estimated as a percentage of decrease in Bmax values for [ 3H]prazosin binding in tissues of tamsulosin-treated rats compared with control rats. The α 1-adrenpceptor occupancy by tamsulosin in the prostate and submaxillary gland occurred rapidly in parallel with the rise in plasma concentration of tamsulosin, and lasted for over 12 h despite the marked decrease in plasma concentration. Consequently, it is suggested that tamsulosin produces more selective and sustained occupancy in vivo of α 1-adrenoceptors in the submaxillary gland and prostate of rats than in other tissues.

The alpha1B-adrenergic receptor subtype activates the phospholipase C signaling pathway in rat myometrium at parturition.

This study demonstrates that alpha1-adrenergic receptors previously identified in the pregnant rat myometrium are heterogeneous. They can be subtyped alpha1A- and alpha1B-adrenergic receptors on the basis of their affinity for the antagonists WB4101 (alpha1A > alpha1B) and chloroethylclonidine (alpha1B selective). Between Day 21 of pregnancy and term, the proportion of [3H]prazosin binding sites with low affinity for WB4101 and sensitive to inactivation by 10(-5) M chloroethylclonidine under hypotonic conditions (alpha1B subtype) remained constant. In contrast, the number of [3H]prazosin binding sites with a high affinity for WB4101 and insensitive to chloroethylclonidine (alpha1A subtype) increased by 88% at term. The effect of 5'-guanylylimidodiphosphate (Gpp[NH]p) on competition of the agonist phenylephrine for [3H]prazosin binding in the presence of WB4101 or after chloroethylclonidine pretreatment indicates that the alpha1A-adrenergic receptor underwent uncoupling whereas the alpha1B-adrenergic receptor-G protein coupled state was increased (+ 63%). Phenylephrine consistently stimulated phospholipase C activity on membrane fractions prepared from term myometria. This stimulation was completely inhibited after 10(-5) M chloroethylclonidine but was not consistently decreased with 5-methylurapidyl, a selective alpha1A-antagonist. Furthermore QL antibody (anti-G alpha(q)/G alpha11) also specifically blocked the phenylephrine-stimulated phospholipase C activity. Altogether these results strongly suggest that activation of the alpha1B-adrenergic receptor subtype in the pregnant myometrium at term may contribute to the stimulation of the G alpha(q)/G alpha11/phospholipase C signaling pathway.

Reduced responsiveness of rat mesenteric resistance artery smooth muscle to phenylephrine and calcium following myocardial infarction.

1. We evaluated responses of peripheral resistance arterial smooth muscle to alpha 1-adrenoceptor stimulation in a rat model of heart failure in relation to neurohumoral changes, wall structure, receptor density and cellular calcium handling. 2. Plasma samples and third order mesenteric artery side-branches were obtained from Wistar rats after induction of left ventricular infarction (M1) or sham surgery. Vessels were denuded of endothelium, sympathectomized, depleted of neuropeptides, and mounted in a myograph for recording of isometric force development in response to calcium, agonist and high potassium. Also, the morphology of these preparations was determined. Separate vessel segments were used in radioligand binding assays with [1H]-prazosin. 3. At 1 week after MI, circulating plasma levels of adrenaline, angiotensin II, atrial natriuretic factor (ANF) and vasopressin were significantly elevated. At 5 weeks only a significant elevation of ANF persisted. 4. At 5 weeks after MI, the structure of the vessels and responsiveness to high potassium or Bay K 8466 (10(6) mol l-1) were not modified. Yet, at this stage, sensitivity to phenylephrine was increased (pD2: 6.24 +/- 0.04 vs 5.98 +/- 0.04 for controls) while maximal contractile responses to phenylephrine in the presence of 2.5 mmol l-1 calcium (2.26 +/- 0.28 vs 3.53 +/- 0.34 N m-1) and the sensitivity to calcium in the presence of phenylephrine (pD2: 2.81 +/- 0.22 vs 3.74 +/- 0.16) were reduced. Responses to the agonist in calcium-free solution and the calcium sensitivity in the presence of 125 mmol l-1 potassium or of phorbol myristate acetate (PMA, 10(-6) mol l-1) were not altered. 5. At 5 weeks after MI, the density of prazosin binding sites was not reduced (4.04 +/- 1.40 vs 2.29 +/- 0.21 fmol microgram-1 DNA in controls). 6. In conclusion, myocardial infarction leads in the rat to a reduction of contractile responses of mesenteric resistance arterial smooth muscle to alpha 1-adrenoceptor stimulation. This seems to involve impaired agonist-stimulated calcium influx.

Sustained increase in rat myocardial alpha 1A-adrenoceptors induced by 6-hydroxydopamine treatment involves a decelerated receptor turnover.

The biochemical mechanisms involved in the alpha 1-adrenoceptor up-regulation and possible changes in subtypes of adrenoceptors in the rat heart after chemical denervation were investigated. The effects of acute 6-hydroxydopamine treatment (two increasing doses 24 h apart) on the pseudo-steady state densities and turnover rates of alpha 1-adrenoceptors were studied in ventricular myocardium of the rat. We have assessed the repopulation kinetics of [3H]prazosin binding sites after irreversible inactivation of alpha 1-adrenoceptors induced by a single dose of phenoxybenzamine (1 mg/kg i.p.) in rats acutely treated either with 6-hydroxy-dopamine or with vehicle (control animals). Seven days after the last administration of 6-hydroxydopamine an enhanced density of [3H]prazosin binding sites (Bmax 58.7 +/- 3.6 fmol/mg protein vehicle-treated rats versus 82.6 +/- 5.3 fmol/mg protein 6-hydroxydopamine-treated rats) was observed. This was not accompanied by changes in the dissociation constant value. Furthermore, the proportion of high affinity sites for WB-4101 was altered (21 +/- 2% versus 72 +/- 3% for animals treated with vehicle and 6-hydroxydopamine, respectively). In rat myocardium, alpha 1-adrenoceptor turnover, evaluated during the 6-hydroxydopamine-induced up-regulation (7-19 days after the completion of treatment with 6-hydroxydopamine) revealed an increase in the half-life of the alpha 1-adrenoceptor (t1/2 of 67.2 h versus 38.7 h in control animals). The present study confirms an increase in alpha 1-adrenoceptors in rat myocardium after chemical denervation and reveals that the effect is almost completely confined to the alpha 1A-adrenoceptor subtype. Furthermore, the up-regulation of alpha 1A-adrenoceptors is the result of a decrease in the cellular processes that control the rate of receptor degradation.

Further evidence that the classical α1A- and cloned α1c-adrenoceptors are the same subtype

We compared the inactivation of [ 3H]prazosin binding sites in membrane preparations of cell-lines expressing the cloned α 1b, α 1c and α 1d-adrenoceptors after pretreatment with the alkylating agents, 10 μM chlorethylclonidine or 10 nM SZL-49 (1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-bicyclo[2.2.2]octa-2,5-diene-Z-carbonyl)-piperazine). The cloned α 1b-adrenoceptor exhibited a similar inactivation profile to that of the classical α 1B-adrenoceptor of rat liver in that chlorethylclonidine in contrast to SZL-49 produced a marked degree of inactivation. A similarity between the cloned α 1c-adrenoceptor and the classical α 1A-adrenoceptor of rat submaxillary gland was also noted in that both subtypes were highly sensitive to SZL-49 but relatively insensitive to chlorethylclonidine. The cloned α 1d-adrenoceptor displayed a unique profile in that both chlorethylclonidine and SZL-49 produced a marked inactivation of this subtype. The similarity of the alkylation-inactivation profiles between the cloned α 1c and classical α 1A-adrenoceptors support the recent proposal that these two α 1-adrenoceptors in fact correspond to the same subtype.