Transmembrane helices 1 and 6 of the human breast cancer resistance protein (BCRP/ABCG2): identification of polar residues important for drug transport.

The human breast cancer resistance protein (BCRP/ABCG2) mediates efflux of drugs and xenobiotics. In this study, we investigated the role of polar residues within or near the predicted transmembrane α-helices 1 and 6 of BCRP in drug transport. We substituted Asn(387), Gln(398), Asn(629), and Thr(642) with Ala, Thr(402) with Ala and Arg, and Tyr(645) with Phe, and the mutants were stably expressed in human embryonic kidney-293 or Flp-In-293 cells. Immunoblotting and confocal microscopy analysis revealed that all of the mutants were well expressed and predominantly targeted to the plasma membrane. While T402A and T402R showed a significant global reduction in the efflux of mitoxantrone, Hoechst 33342, and BODIPY-prazosin, N629A exhibited significantly increased efflux activities for all of the substrates. N387A and Q398A displayed significantly impaired efflux for mitoxantrone and Hoechst 33342, but not for BODIPY-prazosin. In contrast, T642A and Y645F showed a moderate reduction in Hoechst 33342 efflux only. Drug resistance profiles of human embryonic kidney-293 cells expressing the mutants generally correlated with the efflux data. Furthermore, N629A was associated with a marked increase, and N387A and T402A with a significant reduction, in BCRP ATPase activity. Mutations of some of the polar residues may cause conformational changes, as manifested by the altered binding of the 5D3 antibody to BCRP in the presence of prazosin. The inward-facing homology model of BCRP indicated that Thr(402) within transmembrane 1 may be important for helical interactions, and Asn(629) may be involved in BCRP-substrate interaction. In conclusion, we have demonstrated the functional importance of some of these polar residues in BCRP activity.

Doxazosin-related alpha1-adrenoceptor antagonists with prostate antitumor activity.

Doxazosin analogues 1-3 and 1a were synthesized and investigated at alpha1-adrenoceptors and PC-3, DU-145, and LNCaP human prostate cancer cells. Compound 1 (cyclodoxazosin) was a potent alpha(1B)-adrenoceptor antagonist displaying antiproliferative activity higher than that of doxazosin in cancer cells in vitro and in vivo, respectively. Because of its antitumor efficacy at low concentrations, lower apoptotic activity in NHDF vs tumor cells, and antiangiogenetic effect, 1 showed a better therapeutic profile relative to doxazosin.

The involvement of urothelial alpha1A adrenergic receptor in controlling the micturition reflex.

The current study was undertaken in an attempt to characterize the functional properties of urothelial alpha1A adrenergic receptors, especially in modulating the micturition reflex. The expression of alpha1A receptors in rat bladder was analyzed by immunohistochemistry and Western blotting. As a functional study, we obtained continuous infusion cystometrograms in conscious rats using noradrenaline (NA) and subtype selective alpha1 adrenergic receptor antagonists, tamsulosin (alpha1A/alpha1D selective) and silodosin (alpha1A superselective). Alpha1A receptors were immunohistochemically detected in rat urothelium. Intravesical infusion of NA (60 microM) significantly shortened the intercontraction interval (ICI). Pretreatment with tamsulosin at a dose of 0.4 microg/kg i.v. abolished intravesical NA infusioninduced reduction of ICI. Neither intravesical infusion of tamsulosin (20 microM) nor that of silodosin (0.2 microM) significantly altered ICI. After intravesical infusion of silodosin, intravesical NA infusion did not affect ICI. Urothelial alpha1A receptors might modulate bladder afferent activity under pathophysiological conditions with augmented concentrations of NA in blood or urine.

Alpha-1 adrenergic receptor transactivates signal transducer and activator of transcription-3 (Stat3) through activation of Src and epidermal growth factor receptor (EGFR) in hepatocytes.

Hepatocytes express adrenergic receptors (ARs) that modulate several functions, including liver regeneration, hepatocyte proliferation, glycogenolysis, gluconeogenesis, synthesis of urea and fatty acid metabolism. Adrenergic hepatic function in adults is mainly under the control of alpha(1)-ARs; however, the mechanism through which they influence diverse processes remains incompletely understood. This study describes a novel alpha(1)-AR-mediated transactivation of signal transducer and activator of transcription-3 (Stat3) in primary and transformed hepatocytes. Treatment of primary rat hepatocytes with the alpha(1)-AR agonist, phenylephrine (PE), induced a rapid phosphorylation of Stat3. PE also increased Stat3 phosphorylation, DNA binding and transcription activity in transformed human hepatocellular carcinoma cells (Hep3B). The PE-induced Stat3 phosphorylation, DNA binding and reporter activity were completely blocked by the selective alpha(1)-AR antagonist, prazosin. In addition, transfection of Hep3B cells with human alpha(1B)-AR expression vector also enhanced Stat3 phosphorylation and reporter activity. Moreover, overexpression of RGS2, a protein inhibitor of G(q/11) signaling, blocked PE-induced Stat3 phosphorylation and reporter activity. The observations that PE induced the formation of c-Src-Stat3 binding complex and phosphorylation of epidermal growth factor receptor (EGFR) and that inhibiting Src and EGFR prevented PE-induced Stat3 activation indicate the involvement of Src and EGFR. Taken together, these observations demonstrate a novel alpha(1)-AR-mediated Stat3 activation that involves G(q/11), Src, and EGFR in hepatic cells.

Metabolism of prazosin in rat, dog, and human liver microsomes and cryopreserved rat and human hepatocytes and characterization of metabolites by liquid chromatography/tandem mass spectrometry.

Prazosin (2-[4-(2-furanoyl)-piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline) is an antihypertensive agent that was introduced to the market in 1976. It has since established an excellent safety record. However, in vitro metabolism of prazosin has not been investigated. This study describes the in vitro biotransformation of prazosin in liver microsomes from rats, dogs, and humans, as well as rat and human cryopreserved hepatocytes and characterization of metabolites using liquid chromatography/tandem mass spectrometry. The major in vivo biotransformation pathways reported previously in rats and dogs include demethylation, amide hydrolysis, and O-glucuronidation. These metabolic pathways were also confirmed in our study. In addition, several new metabolites were characterized, including a stable carbinolamine, an iminium species, and an enamine-all formed via oxidation of the piperazine ring. Two ring-opened metabolites generated following oxidative cleavage of the furan ring were also identified. Using semicarbazide hydrochloride as a trapping agent, an intermediate arising from opening of the furan ring was captured as a pyridazine product. In the presence of glutathione, three glutathione conjugates were detected in microsomal incubations, although they were not detected in cryopreserved hepatocytes. These data support ring opening of the furan via a reactive gamma-keto-alpha,beta-unsaturated aldehyde intermediate. In the presence of UDP-glucuronic acid, prazosin underwent conjugation to form an N-glucuronide not reported previously. Our in vitro investigations have revealed additional metabolic transformations of prazosin and have shown the potential of prazosin to undergo bioactivation through metabolism of the furan ring to a reactive intermediate.

Bloqueo beta y alfa adrenérgico con carvedilol en el tratamiento de hipertensos esenciales no complicados: estudio multicéntrico abierto no comparativo / Beta and alfa adrenergic blockade with carvedilol in the treatment of essential hypertensive patients: open, multicenter, non comparative study

Antecedentes: Los beneficios de algunos antihipertensivos están limitados por sus efectos adversos y baja adhesividad a terapia. El Carvedilol es una beta y alfa-bloqueador, sin efectos metabólicos adversos y además, utilizable como monoterapia y muchas veces en monodosis, mejorando adherencia al tratamiento. Objetivos: Evaluar los efectos del Carvedilol, en hipertensos esenciales no complicados sobre presión arterial (PA), glicemia, lípidos, su tolerabilidad, posibilidad de ser usado en monodosis y adherencia a la terapia. Método: Estudio multicéntrico prospectivo, abierto, de 12 semanas. Los resultados fueron sometidos a análisis estadísticos. Se contemplaron normas éticas de investigación clínica. Resultados: 285 enfermos en 79 consultorios, 66 por ciento mujeres, 53,6 ± 12 años de edad. La PA basal mostró que el 28,8 por ciento tenían HT etapa 1; 49,8 por ciento etapa 2 y el 21,4 por ciento etapa 3, clasificación JNC VI, siendo el 90,6 por ciento de los pacientes HT sisto-diastólicos. En promedio la PAS bajó de 159,9 ± 14,8 a 131,3 ± 13.5 mmHg y la PAD de 98,5 ± 8,1 a 80,9 ± 8,6 mmHg. El 4,2 por ciento alcanzó normotensión. El colesterol total bajó de 219,2 a 202,4 mg/100ml (p<0,001). La glicemia no se modificó. El 90,2 por ciento de los pacientes usó 25 mg diarios en dosis única. Los efectos adversos fueron escasos, los más comunes mareos, cefalea, rubor facial, edema e hipotensión. La adhesividad al tratamiento fue de 85,5 por ciento. Como hallazgos secundarios, el 77 por ciento de los hipertensos tenían IMC > 25 kg/m². Conclusiones: Carvedilol mostró buena eficacia antihipertensiva, sin efectos metabólicos adversos y buena tolerabilidad.

Multidrug transporter activity in lymphocytes.

Multidrug transporters play a dual role in haematopoietic cells, mediating the efflux of xenobiotics and regulating cell migration. For several reasons including the lack of specific antibodies, reports of multidrug transporter distribution on lymphocytes conflict. Murine B cells have been reported to completely lack transporter activity. Through analysis of parental and 'knockout' mice we show that, contrary to previous studies, murine B and T lymphocytes possess at least three active multidrug transporters and also a hitherto unrecognised drug-specific import activity. Surprisingly, the drug specificity of P-glycoprotein appears cell type dependent. The data indicate that a range of developmentally regulated, multidrug transporters can impose a barrier to treatment of immune disorders.

Effects of topically instilled bunazosin, an alpha1-adrenoceptor antagonist, on constrictions induced by phenylephrine and ET-1 in rabbit retinal arteries.

PURPOSE: To examine the inhibitory effects of topically instilled bunazosin hydrochloride (bunazosin), a selective alpha1-adrenoceptor antagonist, on the retinal artery constrictions induced by intravitreous phenylephrine hydrochloride (phenylephrine) and endothelin (ET)-1 in rabbits. METHODS: Phenylephrine or ET-1 (20 microL) was injected into the central part of the vitreous in both eyes in pigmented rabbits. Color fundus photographs were taken at 5 minutes before and 60 minutes after the injection. The average diameter of the major retinal arteries at the rim of the optic nerve head (ONH) was normalized with respect to ONH diameter. Bunazosin was instilled into one eye (chosen randomly) and vehicle into the fellow eye at 60 minutes before the intravitreous injection. To examine any interaction between the alpha1-adrenoceptor and ET receptor, phenylephrine and ET-1 were co-injected at individually ineffective doses. In addition, ET-1-induced vasoconstriction was examined after unilateral superior cervical ganglionectomy. The binding affinities of bunazosin for ETA and ETB receptors were also evaluated. The series of experiments was performed as masked tests. RESULTS: Retinal arteries were dose-dependently constricted by both intravitreous phenylephrine and intravitreous ET-1. Topically instilled bunazosin at 0.01% partly inhibited both of these vasoconstrictions on the ipsilateral side, but not on the contralateral side. Bunazosin did not bind to ET receptors. Co-injection of phenylephrine and ET-1 at individually ineffective doses constricted retinal arteries significantly. An adrenergic supersensitivity in retinal arteries was observed after superior cervical ganglionectomy only on the ganglionectomized eye. The ET-1-induced vasoconstriction was significantly weaker in cervical ganglionectomized eyes than in sham-surgery eyes. CONCLUSIONS: The present findings suggest that topically instilled bunazosin reaches the posterior retina by local penetration at concentrations sufficient to attenuate the phenylephrine- or ET-1-induced constriction of retinal arteries in normal rabbit eyes, and that the inhibitory effect of bunazosin on the ET-1-induced vasoconstriction in this tissue may be partly attributable to an interaction between the alpha1-adrenoceptor and ET receptor.

Estrogen controls lipolysis by up-regulating alpha2A-adrenergic receptors directly in human adipose tissue through the estrogen receptor alpha. Implications for the female fat distribution.

Estrogen seems to promote and maintain the typical female type of fat distribution that is characterized by accumulation of adipose tissue, especially in the sc fat depot, with only modest accumulation of adipose tissue intraabdominally. However, it is completely unknown how estrogen controls the fat accumulation. We studied the effects of estradiol in vivo and in vitro on human adipose tissue metabolism and found that estradiol directly increases the number of antilipolytic alpha2A-adrenergic receptors in sc adipocytes. The increased number of alpha2A-adrenergic receptors caused an attenuated lipolytic response of epinephrine in sc adipocytes; in contrast, no effect of estrogen on alpha2A-adrenergic receptor mRNA expression was observed in adipocytes from the intraabdominal fat depot. These findings show that estrogen lowers the lipolytic response in sc fat depot by increasing the number of antilipolytic alpha2A-adrenergic receptors, whereas estrogen seems not to affect lipolysis in adipocytes from the intraabdominal fat depot. Using estrogen receptor subtype-specific ligands, we found that this effect of estrogen was caused through the estrogen receptor subtype alpha. These findings demonstrate that estrogen attenuates the lipolytic response through up-regulation of the number of antilipolytic alpha2A-adrenergic receptors only in sc and not in visceral fat depots. Thus, our findings offer an explanation how estrogen maintains the typical female sc fat distribution because estrogen seems to inhibit lipolysis only in sc depots and thereby shifts the assimilation of fat from intraabdominal depots to sc depots.

Spinal gabapentin and antinociception: mechanisms of action.

Spinal gabapentin has been known to show the antinociceptive effect. Although several assumptions have been suggested, mechanisms of action of gabapentin have not been clearly established. The present study was undertaken to examine the action mechanisms of gabapentin at the spinal level. Male SD rats were prepared for intrathecal catheterization. The effect of gabapentin was assessed in the formalin test. After pretreatment with many classes of drugs, changes of effect of gabapentin were examined. General behaviors were also observed. Intrathecal gabapentin produced a suppression of the phase 2 flinching, but not phase 1 in the formalin test. The antinociceptive action of intrathecal gabapentin was reversed by intrathecal NMDA, AMPA, D-serine, CGS 15943, atropine, and naloxone. No antagonism was seen following administration of bicuculline, saclofen, prazosin, yohimbine, mecamylamine, L-leucine, dihydroergocristine, or thapsigargin. Taken together, intrathecal gabapentin attenuated only the facilitated state. At the spinal level, NMDA receptor, AMPA receptor, nonstrychnine site of NMDA receptor, adenosine receptor, muscarinic receptor, and opioid receptor may be involved in the antinociception of gabapentin, but GABA receptor, L-amino acid transporter, adrenergic receptor, nicotinic receptor, serotonin receptor, or calcium may not be involved.

Hormonal influence on expression and functionality of alpha1-adrenoceptor in rat submandibular gland.

In this work, we characterized alpha(1)-adrenoceptor expression and functionality in rat submandibular gland. Cumulative dose-response curve of methoxamine was constructed to determine the peroxidase secretion by glands from proestrous, estrous, metestrous and diestrous rats. They were compared to those from animals untreated or treated with sex hormones, estradiol and progesterone. The sensitivity of glands to an alpha(1)-adrenoceptor agonist varied depending on hormonal state, i.e. glands from proestrous and estrous were more sensitive to the stimulatory action of methoxamine than those from metestrous, diestrous and ovariectomised animals. The efficacy of the alpha(1) agonist was enhanced in glands from ovariectomised estrogen-treated rats but it was ineffective in glands from ovariectomised progesterone-treated rats. The functional studies correlated with 3H-prazosin binding assays in which estrogen increased alpha(1)-adrenoceptor density while progesterone decreased it. The results demonstrated that alpha(1)-adrenoceptor expression and functionality in rat submandibular glands are apparently under hormonal control and probably represent other examples of bidirectional interactions between neuronal and exocrine systems.

Different sensitivities to alpha1 adrenoceptor blockade on periarterial sympathetic nerve-induced constriction by low and high frequencies in canine isolated splenic arteries.

The periarterial nerve electrical stimulation at 4 and 10 Hz induced a monophasic vasoconstriction of the canine splenic artery in a pulse number-related manner (1-30 pulses). The responses at 4 Hz were not significantly affected by 0.1 microM prazosin, but abolished by 1 microM alpha, beta-methylene ATP. Prazosin (0.1 microM) partially but significantly inhibited responses at 10 Hz, and the remaining responses were blocked by 1 microM alpha, beta-methylene ATP. It indicates that the monophasic vasoconstrictor response to short pulses of stimulation at a low frequency is mediated by P2X-receptors, whereas the response at a high frequency may be due to activation of not only P2X-receptors but also alpha1 adrenoceptors.

High level of alpha2-adrenoceptor in rat foetal liver and placenta is due to alpha2B-subtype expression in haematopoietic cells of the erythrocyte lineage.

1. Rat foetal liver contains large amounts of alpha2-adrenoceptors. The present work aimed to identify the receptor subtype and the cell type accounting for high expression and to clarify the mechanisms responsible for the sharp decrease in hepatic receptivity occurring during the late stage of foetal development. 2. Binding experiments indicated that the density of alpha2-adrenoceptors in the foetal liver (embryonic day 18; 615+/-155 fmol mg(-1) of protein) is 18 fold higher than in newborn or adult (35.2+/-4.3 fmol mg(-1)). A high amount of receptor is also found in the placenta (443+/-53 fmol mg(-1)). In both tissues, the rank order of antagonists to inhibit radioligand binding matched the pharmacological profile of the alpha2B-adrenoceptor and exclusively RNG transcripts were detected by RNase protection assays. 3. Isolation of cell fractions from foetal liver showed that alpha2B-adrenoceptor is primarily expressed by haematopoietic cells. Consistent with this view, the receptor is found to be abundant in foetal blood, carried by reticulocytes. The expression in blood gradually declines to zero at 3 weeks of age and it is not recovered following induction of reticulocytosis in adults. 4. In foetal reticulocytes, a low proportion of the receptor population is coupled to G-protein. The alpha2-agonist UK14304 has a marginal effect on cyclic AMP level but significantly increases arachidonic acid release. The function of the receptor remains to be elucidated. However, together with observations on alpha2B-knockout mice, the current finding strongly suggests a role for alpha2B-adrenoceptor during foetal haematopoiesis in rodents.

Receptor phosphorylation mediates estradiol reduction of alpha2-adrenoceptor coupling to G protein in the hypothalamus of female rats.

Estrogen increases evoked norepinephrine release in the hypothalamus of female rodents, in part by reducing the ability of alpha2-adrenoceptors to act as negative feed-back inhibitors of norepinephrine release. Estrogen enhancement of norepinephrine release in the hypothalamus correlates with decreased coupling of the alpha2-adrenoceptor to G protein. To determine the mechanism by which estrogen uncouples alpha2-adrenoceptors from G protein, we tested the hypothesis that estrogen increases alpha2-adrenoceptor phosphorylation. Short-term activation of endogenous serine/threonine phosphatases with protamine or treatment with exogenous phosphatase restored alpha2-adrenoceptor coupling to G protein to control levels in hypothalami from estrogen-exposed female rats. Additional experiments examined whether estrogen alters G protein-coupled receptor kinase expression or activity or serine/threonine phosphatase activity. These proteins are involved in G protein-coupled receptor phosphorylation, internalization, and recycling. Estrogen exposure reduced G protein-coupled receptor kinase mRNA, protein, and activity in the hypothalamus. Furthermore, estrogen treatment reduced serine/threonine phosphatase activity in the hypothalamus. Analysis of ligand binding in subcellular fractions demonstrated that estrogen decreases the fraction of internalized alpha2-adrenoceptors in the hypothalamus.Therefore, estrogen promotes norepinephrine release in the hypothalamus by stabilizing alpha2-adrenoceptor phosphorylation, uncoupling the receptor from G protein. Estrogen may stabilize alpha2-adrenoceptor phosphorylation by inhibiting receptor internalization and dephosphorylation.

[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.

Phentolamine prevents the adverse effects of adenosine on glycolysis and mechanical function in isolated working rat hearts subjected to antecedent ischemia.

Adenosine inhibits glycolysis from exogenous glucose, reduces proton production and enhances post-ischemic left ventricular minute work (LV work) following ischemia in isolated working rat hearts perfused with glucose and fatty acids. In hearts partially depleted of glycogen by antecedent ischemic stress (AIS)--two cycles of ischemia (10 min) and reperfusion (5 min)--adenosine stimulates rather than inhibits glycolysis, increases proton production and worsens recovery of post-ischemic LV work. We determined if the switch in adenosine effect on glycolysis and recovery of LV work following ischemia in hearts subject to AIS was due to the reduction in glycogen content per se or because of alpha-adrenoceptor stimulation. One series of hearts underwent a 35-min period of substrate-free Langendorff perfusion (substrate-free glycogen depletion; SFGD) and a second series of hearts was subjected to AIS. Both series of hearts had a similar glycogen content (approximately 70 micromol/g dry wt) prior to drug treatment. In SFGD hearts perfused aerobically, adenosine (500 microM) inhibited glycolysis from exogenous glucose and reduced proton production. In SFGD hearts reperfused after prolonged ischemia, adenosine exerted similar effects on glucose metabolism and enhanced recovery of post-ischemic LV work (87.2 +/- 2.2% of preischemic values) relative to untreated hearts (25.9 +/- 13.3% of preischemic values). In AIS hearts perfused aerobically or subject to ischemia and reperfusion, phentolamine (1 microM) given in combination with adenosine, prevented adenosine-induced stimulation of glycolysis from exogenous glucose and reduced calculated proton production from glucose. Recoveries of post-ischemic LV work in AIS hearts for untreated, adenosine, phentolamine and adenosine/phentolamine groups were 34.4 +/- 11.4%, 8.6 +/- 3.9%, 16.3 +/- 13.5% and 73.2 +/- 13.1% respectively, of preischemic values. Glycogen depletion in the absence of ischemia does not switch the effect of adenosine from inhibition to stimulation of glycolysis or alter the cardioprotective properties of adenosine in hearts subject to ischemia and reperfusion. The detrimental switch in the metabolic and cardioprotective effects of adenosine, in hearts subject to AIS, can be prevented by phentolamine, an alpha-adrenoceptor antagonist. These data support the concept that modulation of glucose metabolism is an important factor in the mechanical functional recovery of the post-ischemic heart.

Structure-activity studies for a novel series of tricyclic substituted hexahydrobenz[e]isoindole alpha(1A) adrenoceptor antagonists as potential agents for the symptomatic treatment of benign prostatic hyperplasia (BPH).

In search of a uroselective agent that exhibits a high level of selectivity for the alpha(1A) receptor, a novel series of tricyclic hexahydrobenz[e]isoindoles was synthesized. A generic pharmacophoric model was developed requiring the presence of a basic amine core and a fused heterocyclic side chain separated by an alkyl chain. It was shown that the 6-OMe substitution with R, R stereochemistry of the ring junction of the benz[e]isoindole and a two-carbon spacer chain were optimal. In contrast to the highly specific requirements for the benz[e]isoindole portion and linker chain, a wide variety of tricyclic fused heterocyclic attachments were tolerated with retention of potency and selectivity. In vitro functional assays for the alpha(1) adrenoceptor subtypes were used to further characterize these compounds, and in vivo models of vascular vs prostatic tone were used to assess uroselectivity.

Characterisation of alpha1B-adrenoceptors linked to inositol phosphate formation and calcium mobilisation in human astrocytoma U373 MG cells.

The human U373 MG astrocytoma cell line has been widely used as a model system for the investigation of astrocyte function. The aim of this study was to establish which alpha1-adrenoceptors are present on these cells. The specific binding of [3H]prazosin to membranes of U373 MG cells (Bmax 32+/-3 fmol mg(-1) protein, Kd 0.27+/-0.03 nM) was inhibited in a monophasic manner by alpha1-antagonists that have different affinities for alpha1A-, alpha1B- and alpha1D-adrenoceptors. Estimates for pKi values were: prazosin 9.69+/-0.06, 5-methylurapidil 7.10+/-0.21; (+)-niguldipine 7.06+/-0.26; WB 4101 8.26+/-0.16; and BMY 7378 6.60+/-0.21. The specific binding of [3H]prazosin was reduced to low levels by pretreatment of cells with 10 microM chloroethylclonidine for 15 min. In the presence of 30 mM LiCl, 100 microM noradrenaline stimulated [3H]inositol phosphate accumulation by 2.1+/-0.1-fold of basal after 30-min incubation. The EC50 for the accumulation of [3H]IP1, the major product detected (85+/-2% of total [3H]IP1 + [3H]IP2 + [3H]IP3), was 0.38+/-0.05 microM. Noradrenaline-induced [3H]IP1 accumulation was also inhibited by alpha1-antagonists. Estimates for pKi values were: 5-methylurapidil 6.95+/-0.01; WB 4101 8.31+/-0.07; and BMY 7378 6.71+/-0.28. The accumulation of [3H]IP1 in response to 100 microM noradrenaline was not significantly affected by raising the extracellular Ca2+ concentration from 1.3 to 4 mM. Noradrenaline (100 microM) also produced an increase in intracellular Ca2+ (mean peak 86+/-5 nM above basal). Pretreatment with chloroethylclonidine (10 microM, 15 min) abolished noradrenaline-induced [3H]IP1 accumulation and Ca2+ mobilisation. Activation of the alpha1B-adrenoceptors by 10 microM phenylephrine increased [3H]thymidine uptake to 140+/-5% of control uptake. Taken together, these results indicate that U373 MG cells express a single class of alpha1-adrenoceptors, the alpha1B-subtype, which are coupled to phosphoinositide hydrolysis and calcium mobilisation, and which mediate a mitogenic response to alpha1-agonists.

WB 4101-related compounds. 2. Role of the ethylene chain separating amine and phenoxy units on the affinity for alpha(1)-adrenoreceptor subtypes and 5-HT(1A) receptors.

WB 4101 (1)-related benzodioxanes were synthesized by replacing the ethylene chain separating the amine and the phenoxy units of 1 with a cyclopentanol moiety, a feature of 6, 7-dihydro-5-[[(cis-2-hydroxy-trans-3-phenoxycyclopentyl)amino]meth yl] -2-methylbenzo[b]thiophen-4(5H)-one that was reported to display an intriguing selectivity profile at alpha(1)-adrenoreceptors. This synthesis strategy led to 4 out of 16 possible stereoisomers, which were isolated in the case of (-)-3, (+)-3, (-)-4, and (+)-4 and whose absolute configuration was assigned using a chiral building block for the synthesis of (-)-3 starting from (+)-(2R)-2, 3-dihydro-1,4-benzodioxine-2-carboxylic acid ((+)-9) and (1S,2S, 5S)-2-amino-5-phenoxycyclopentan-1-ol ((+)-10). The aim of this project was to further investigate whether it is possible to differentiate between these compounds with respect to their affinity for alpha(1)-adrenoreceptor subtypes and the affinity for 5-HT(1A) receptors, as 1 binds with high affinity at both receptor systems. The biological profiles of reported compounds at alpha(1)-adrenoreceptor subtypes were assessed by functional experiments in isolated rat vas deferens (alpha(1A)), spleen (alpha(1B)), and aorta (alpha(1D)) and by binding assays in CHO and HeLa cells membranes expressing the human cloned alpha(1)-adrenoreceptor subtypes and 5-HT(1A) receptors, respectively. Furthermore, the functional activity of (-)-3, (+)-3, (-)-4, and (+)-4 toward 5-HT(1A) receptors was evaluated by determining the induced stimulation of [(35)S]GTPgammaS binding in cell membranes from HeLa cells transfected with human cloned 5-HT(1A) receptors. The configuration of the cyclopentane unit determined the affinity profile: a 1R configuration, as in (+)-3 and (-)-4, conferred higher affinity at alpha(1)-adrenoreceptors, whereas a 1S configuration, as in (-)-3 and (+)-4, produced higher affinity for 5-HT(1A) receptors. For the enantiomers (+)-4 and (-)-4 also a remarkable selectivity was achieved. Functionally, the stereoisomers displayed a similar alpha(1)-selectivity profile, that is alpha(1D) > alpha(1B) > alpha(1A), which is different from that exhibited by the reference compound 1. The epimers (-)-3 and (+)-4 proved to be agonists at the 5-HT(1A) receptors, with a potency comparable to that of 5-hydroxytryptamine.

Patterns of adrenoceptor change during liver regeneration of the Wistar Kyoto rat: a binding study.

BACKGROUND: Adrenoceptors have been involved in the regulation of hepatocyte proliferation after partial hepatectomy, as well as in primary culture. This report characterizes alpha 1- and beta-adrenoceptor change during the time-course of liver regeneration in adult Wistar Kyoto rats. METHODS: Saturation binding assays with [3H]prazosin or [3H]dihydroalprenolol (for alpha 1- and beta-adrenoceptors, respectively) were done in liver plasma membranes from 6-month-old rats subjected to 70% hepatectomy followed by hepatic regeneration. RESULTS: [3H]Prazosin and [3H]dihydroalprenolol binding gave control Bmax values of 101 +/- 10 and 12 +/- 1 fmol/mg protein and Kd of 0.50 +/- 0.10 and 4.1 +/- 0.4 nM for alpha 1- and beta-adrenoceptors, respectively. alpha 1-Adrenoceptor number and Kd increased at 24 and 48 h and returned to control values at 72 and 96 h after surgery, whereas beta-adrenoceptors augmented at 48 and 72 h, with a Kd change at 24 and 48 h posthepatectomy. CONCLUSIONS: These results suggest that dual control of alpha 1- and beta-adrenoceptor membrane expression could be involved in different steps during hepatocyte proliferation, and that Wistar Kyoto rats have a different adrenoceptor pattern expression from other rat strains.