RESUMEN

In a cell line, stably expressing α1A-adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced concentration-dependent increases in intracellular calcium. All of these agents increase α1A-adrenoceptor phosphorylation and internalization. Transient co-expression of these receptors with Rab proteins tagged with the enhanced Green Fluorescent Protein was employed to estimate α1A-adrenoceptor-Rab interaction using Förster Resonance Energy Transfer. Noradrenaline and methoxamine increased α1A-adrenoceptor interaction with Rab5 and Rab7 but did not modify it with Rab9. Oxymetazoline induced adrenoceptor interaction with Rab5 and Rab9 and only an insignificant increase in Rab7 signal. Phorbol myristate acetate increased α1A-adrenoceptor interaction with Rab5 and Rab9 but did not modify it with Rab7. The agonists and the active phorbol ester, all of which induce receptor phosphorylation and internalization, favor receptor interaction with Rab5, i.e., association with early endosomes. Cell stimulation with phorbol myristate acetate induced the α1A-adrenoceptors to interact with the late endosomal marker, Rab9, suggesting that the receptors are directed to slow recycling endosomes once they have transited to the Trans-Golgi network to be retrieved to the plasma membrane. The agonists noradrenaline and methoxamine likely induce a faster recycling and might direct some of the adrenoceptors toward degradation and/or very slow recycling to the plasma membrane. Oxymetazoline produced a mixed pattern of interaction with the Rab proteins. These data indicate that α1A-adrenoceptor agonists can trigger different vesicular traffic and receptor fates within the cells.

Asunto(s)

Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Ésteres del Forbol/farmacología , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Proteínas de Unión al GTP rab/efectos de los fármacos , Calcio/metabolismo , Línea Celular , Endosomas/efectos de los fármacos , Humanos , Proteínas Luminiscentes , Metoxamina/farmacología , Norepinefrina/farmacología , Oximetazolina/farmacología , Fosforilación , Acetato de Tetradecanoilforbol/farmacología , Proteínas de Unión al GTP rab5/efectos de los fármacos , Red trans-Golgi/efectos de los fármacos , Proteína Fluorescente Roja

RESUMEN

In LNCaP cells that stably express α1A-adrenergic receptors, oxymetazoline increased intracellular calcium and receptor phosphorylation, however, this agonist was a weak partial agonist, as compared to noradrenaline, for calcium signaling. Interestingly, oxymetazoline-induced receptor internalization and desensitization displayed greater effects than those induced by noradrenaline. Phorbol myristate acetate induced modest receptor internalization and minimal desensitization. α1A-Adrenergic receptor interaction with ß-arrestins (colocalization/coimmunoprecipitation) was induced by noradrenaline and oxymetazoline and, to a lesser extent, by phorbol myristate acetate. Oxymetazoline was more potent and effective than noradrenaline in inducing ERK 1/2 phosphorylation. Mass spectrometric analysis of immunopurified α1A-adrenergic receptors from cells treated with adrenergic agonists and the phorbol ester clearly showed that phosphorylated residues were present both at the third intracellular loop and at the carboxyl tail. Distinct phosphorylation patterns were observed under the different conditions. The phosphorylated residues were: a) Baseline and all treatments: T233; b) noradrenaline: S220, S227, S229, S246, S250, S389; c) oxymetazoline: S227, S246, S381, T384, S389; and d) phorbol myristate acetate: S246, S250, S258, S351, S352, S401, S402, S407, T411, S413, T451. Our novel data, describing the α1A-AR phosphorylation sites, suggest that the observed different phosphorylation patterns may participate in defining adrenoceptor localization and action, under the different conditions examined.

Asunto(s)

Señalización del Calcio/efectos de los fármacos , Proteolisis , Receptores Adrenérgicos alfa 1/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Espectrometría de Masas , Norepinefrina/farmacología , Oximetazolina/farmacología , Fosforilación/genética , Proteína Quinasa C/genética , Receptores Adrenérgicos alfa 1/metabolismo , Acetato de Tetradecanoilforbol/farmacología

RESUMEN

Loss of response on repetitive drug exposure (i.e., tachyphylaxis) is a particular problem for the vasoconstrictor effects of medications containing oxymetazoline (OXY), an α1-adrenoceptor (AR) agonist of the imidazoline class. One cause of tachyphylaxis is receptor desensitization, usually accompanied by phosphorylation and internalization. It is well established that α1A-ARs are less phosphorylated, desensitized, and internalized on exposure to the phenethylamines norepinephrine (NE), epinephrine, or phenylephrine (PE) than are the α1B and α1D subtypes. However, here we show in human embryonic kidney-293 cells that the low-efficacy agonist OXY induces G protein-coupled receptor kinase 2-dependent α1A-AR phosphorylation, followed by rapid desensitization and internalization (∼40% internalization after 5 minutes of stimulation), whereas phosphorylation of α1A-ARs exposed to NE depends to a large extent on protein kinase C activity and is not followed by desensitization, and the receptors undergo delayed internalization (∼35% after 60 minutes of stimulation). Native α1A-ARs from rat tail artery and vas deferens are also desensitized by OXY, but not by NE or PE, indicating that this property of OXY is not limited to recombinant receptors expressed in cell systems. The results of the present study are clearly indicative of agonist-directed α1A-AR regulation. OXY shows functional selectivity relative to NE and PE at α1A-ARs, leading to significant receptor desensitization and internalization, which is important in view of the therapeutic vasoconstrictor effects of this drug and the varied biologic process regulated by α1A-ARs.

Asunto(s)

Agonistas de Receptores Adrenérgicos alfa 1/metabolismo , Norepinefrina/metabolismo , Oximetazolina/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Animales , Células HEK293 , Humanos , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Norepinefrina/farmacología , Oximetazolina/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Ratas Wistar

RESUMEN

It has been recently shown that the supersensitivity of distal segments of the rat tail artery to phenylephrine after chemical sympathectomy with reserpine results from the appearance of alpha(1D)-adrenoceptors. It is known that both alpha(1A)- and alpha(1D)-adrenoceptors are involved in the contractions of proximal portions of the rat tail artery. Therefore, this study investigated whether sympathectomy with reserpine would induce supersensitivity in proximal segments of the rat tail artery, a tissue in which alpha(1D)-adrenoceptors are already functional. Proximal segments of tail arteries from reserpinised rats were three- to sixfold more sensitive to phenylephrine and methoxamine than were arteries from control rats (n = 6-2; p < 0.05). The imidazolines N-[5-(4,5-Dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide hydrobromide (A-61603) and oxymetazoline, which activate selectively alpha(1A)-adrenoceptors, were equipotent in tail arteries from control and reserpinised rats (n = 4-2; p < 0.05), whereas buspirone, which activates selectively alpha(1D)-adrenoceptor, was approximately 4-fold more potent in tail arteries from reserpinised rats (n = 4-6; p < 0.05). Prazosin (nonselective) and 5-methylurapidil (alpha(1A)-selective), were competitive antagonists of contractions induced by phenylephrine and were equipotent in tail arteries from control and reserpinised rats (n = 4-6). The selective alpha(1D)-adrenoceptor antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride (BMY-7378) presented similar complex antagonism in tail arteries from control and reserpinised rats, with Schild slopes much lower than 1.0 (p < 0.05, n = 4-6). Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) revealed that mRNA encoding alpha(1A)-and alpha(1B)-adrenoceptors are similarly distributed in tail arteries from control and reserpinised rats, whereas mRNA for alpha(1D)-adrenoceptors is twice more abundant in the tail artery from reserpinised rats. In conclusion, the supersensitivity induced by reserpine is related only to alpha(1D)-adrenoceptors, even in tissues where this receptor subtype is already present and functional. Only the use of subtype-selective alpha(1)-adrenoceptor agonists detected the increased alpha(1D)-adrenoceptor component after reserpinisation, as the antagonists behaved similarly in tail arteries from control and reserpinised rats.

Asunto(s)

Arterias/inervación , Músculo Liso Vascular/fisiología , Receptores Adrenérgicos alfa 1/biosíntesis , Cola (estructura animal)/irrigación sanguínea , Agonistas de Receptores Adrenérgicos alfa 1 , Antagonistas de Receptores Adrenérgicos alfa 1 , Animales , Arterias/efectos de los fármacos , Arterias/metabolismo , Buspirona/farmacología , Expresión Génica , Imidazoles/farmacología , Técnicas In Vitro , Masculino , Metoxamina/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Oximetazolina/farmacología , Fenilefrina/farmacología , Piperazinas/farmacología , Prazosina/farmacología , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Reserpina/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Simpatectomía , Tetrahidronaftalenos/farmacología

RESUMEN

1 Several imidazolines were examined for the antagonism of muscarinic (M3) and other receptors on the isolated ileum of guinea-pig. The effect of the muscarinic agonist, carbachol was competitively antagonized by oxymetazoline at 10(-5) m. A dissociation constant (KB) of 3.6 microm for the antagonist was calculated. At higher concentrations, 3 x 10(-5) and 10(-4) m, of the antagonist, the agonist dose-response curve was shifted to the right with a decrease in the maximum effect. Thus, a non-competitive block occurred at higher concentrations of oxymetazoline. Blockade of histamine H, and serotonin receptor-mediated responses by oxymetazoline were also of a non-competitive type. 2 Naphazoline at 10(-4) m shifted the dose-response curves of carbachol and serotonin to the right by two- and 15-fold, respectively. The maximum contraction of the agonist was not affected. Tolazoline also had a weak antihistaminic activity. At similar concentration; tetrahydrozoline clonidine and phentolamine at 10(-5) m produced two-, three- and four-fold shift of the carbachol dose-response curve without significant changes in the maxima. Neither methoxamine, p-amino-clonidine nor cimetidine blocked the responses of carbachol. 3 The isosteric nature of the alpha-adrenoceptor agonist, oxymetazoline and some imidazolines with carbachol, in part, explains its molecular competition at the muscarinic M3 receptor of the guinea-pig ileum. Surprisingly, contractile effects of carbachol (M3), histamine (H1) or serotonin (5HT3/5HT4) were not influenced by methoxamine, tetrahydrozoline, p-amino clonidine and cimetidine.

Asunto(s)

Íleon/efectos de los fármacos , Imidazolinas/farmacología , Antagonistas Muscarínicos/farmacología , Animales , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Relación Dosis-Respuesta a Droga , Cobayas , Histamina/farmacología , Íleon/metabolismo , Imidazolinas/química , Técnicas In Vitro , Masculino , Metoxamina/farmacología , Antagonistas Muscarínicos/química , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Oximetazolina/farmacología , Receptores Histamínicos H1/efectos de los fármacos , Receptores Histamínicos H1/metabolismo , Receptores de Serotonina/efectos de los fármacos , Receptores de Serotonina/metabolismo , Serotonina/farmacología

RESUMEN

We investigated whether or not surgical denervation of the rat vas deferens changes the alpha(1)-adrenoceptor subtypes involved in the contractions to noradrenaline. Denervated vas deferens was approximately 22 times more sensitive to noradrenaline (pD(2)=7.35+/-0.04) than control vas (pD(2)=6.01+/-0.03). This difference in noradrenaline potency was eliminated when cocaine (6 microM) was added to control vas (pD(2)=7.22+/-0.04). The noradrenaline-induced contractions of control and denervated vas deferens were insensitive to the alpha(1B)/alpha(1D)-adrenoceptor alkylating agent chloroethylclonidine (100 microM, 45 min). The concentration-response curves to noradrenaline in control and denervated vas deferens were competitively antagonised by prazosin (pA(2) approximately equal to 9.6), WB-4101 (pA(2) approximately equal to 9.5), 5-methyl urapidil (pA(2) approximately equal to 8.4), phentolamine (pA(2) approximately equal to 8.7), yohimbine (pA(2) approximately equal to 6.9), BMY 7378 (pA(2) approximately equal to 6.9) and indoramin (pA(2) approximately equal to 8.7). After the treatment of control and denervated vas deferens with phenoxybenzamine, the partial agonist oxymetazoline antagonised competitively the concentration-response curves to noradrenaline showing pA(2) values approximately 7.4 in both groups. We conclude that noradrenaline-induced contractions in control and denervated rat vas deferens are mediated by alpha(1A)-adrenoceptors and that surgical denervation of the rat vas deferens is not able to change the alpha(1)-adrenoceptor subtypes involved in the contractions to noradrenaline.

Asunto(s)

Antagonistas Adrenérgicos alfa/farmacología , Receptores Adrenérgicos alfa 1/efectos de los fármacos , Conducto Deferente/efectos de los fármacos , Agonistas alfa-Adrenérgicos/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/metabolismo , Masculino , Contracción Muscular/efectos de los fármacos , Desnervación Muscular , Músculo Liso/efectos de los fármacos , Norepinefrina/farmacología , Oximetazolina/farmacología , Ratas , Ratas Wistar , Receptores Adrenérgicos alfa 1/metabolismo , Conducto Deferente/inervación , Conducto Deferente/metabolismo

RESUMEN

We investigated the alpha(1)-adrenoceptor subtype(s) involved in contraction of the isolated rat mesenteric artery by the use of the agonists noradrenaline (NA), phenylephrine (PHE), oxymetazoline (OXY), and methoxamine (MET), the competitive antagonists 8-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4.5)decane-7,9-dione dihydrochloride (BMY 7378) and 5-methylurapidil, and the alkylating agent chloroethylclonidine (CEC). Agonists showed the potency order NA> or =PHE>OXY>MET; pA(2) values for 5-methylurapidil and BMY 7378 were 7.74+/-0.11 and 8.72+/-0.28, respectively, while Schild slopes were not different than unity; alpha(1)-adrenoceptor alkylation with CEC showed a drastic decrease in maximal agonists-induced contraction and a shift to the right of about 46-, 122-, 2-, and 15-fold higher than controls for NA, PHE, OXY, and MET, respectively. Data suggest that alpha(1D)-adrenoceptors predominate for contraction in mesenteric artery of the Wistar rat, with a second population of alpha(1A)-adrenoceptors responding at high agonist concentrations.

Asunto(s)

Clonidina/análogos & derivados , Arterias Mesentéricas/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Receptores Adrenérgicos alfa 1/fisiología , Agonistas alfa-Adrenérgicos/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Animales , Clonidina/farmacología , Técnicas In Vitro , Masculino , Arterias Mesentéricas/fisiología , Metoxamina/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Norepinefrina/farmacología , Oximetazolina/farmacología , Fenilefrina/farmacología , Piperazinas/farmacología , Ratas , Ratas Wistar , Receptores Adrenérgicos alfa 1/efectos de los fármacos

RESUMEN

In the present paper, the cloning and expression of the guinea pig alpha(1A)-adrenoceptor is presented. The nucleotide sequence had an open reading frame of 1401 bp that encoded a 466 amino-acid protein with an estimated molecular mass of approximately 51.5 kDa. When the clone was expressed in Cos-1 cells, specific high-affinity binding of [(3)H]prazosin and [(3)H]tamsulosin was observed. Chloroethylclonidine treatment of membranes slightly decreased the total binding with both radioligands. Binding competition experiments using [(3)H]tamsulosin showed the following potency order: (a) for agonists: oxymetazoline >>epinephrine>norepinephrine>methoxamine, and (b) for antagonists: prazosin> or 5-methyl-urapidil=benoxathian>phentolamine>>BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione). Photoaffinity labeling using [(125)I-aryl]azido-prazosin revealed a major broad band with a molecular mass between 70 and 80 kDa. The receptor was functional, as evidenced by an epinephrine-increased production of [(3)H]inositol phosphates that was blocked by prazosin.

Asunto(s)

Receptores Adrenérgicos alfa 1/genética , Agonistas Adrenérgicos/farmacología , Antagonistas Adrenérgicos/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Unión Competitiva , Células COS , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Epinefrina/farmacología , Expresión Génica , Cobayas , Metoxamina/farmacología , Datos de Secuencia Molecular , Norepinefrina/farmacología , Oxatiinas/farmacología , Oximetazolina/farmacología , Fentolamina/farmacología , Piperazinas/farmacología , Prazosina/metabolismo , Prazosina/farmacología , Receptores Adrenérgicos alfa 1/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Sulfonamidas/metabolismo , Tamsulosina , Tritio

RESUMEN

Arginine vasopressin (AVP) increases water permeability in the collecting duct of the nephron via activation of adenylyl cyclase. Alpha-2 (alpha2) agonists inhibit AVP-stimulated water permeability via binding to alpha2 adrenoceptors that have been divided into 3 subtypes- alpha2A, alpha2B, and alpha2C. Some biological effects mediated by alpha2 agonists result from nonadrenergic imidazoline receptors that exist in the rat kidney. Thus, alpha2-inhibition of AVP-stimulated water permeability in the rat collecting duct could be caused by imidazoline receptors. The purpose of this study was to test agonists and antagonists selective for alpha2 and imidazoline receptors on AVP-stimulated water permeability in the rat inner medullary collecting duct (IMCD). Some experiments were conducted where water permeability was stimulated by a nonhydrolyzable analog of adenosine 3', 5'-cyclic monophosphate (cAMP). Agonists included dexmedetomidine, clonidine, oxymetazoline, agmatine and rilmenidine. The latter two are selective imidazoline agonists. Antagonists included yohimbine, RX821002, atipamezole, prazosin, WB4101, idazoxan, and BU239. Prazosin and WB4101 demonstrate selectivity for the alpha2B and alpha2C subtypes, respectively, and oxymetazoline and RX821002 are selective for the alpha2A subtype. BU239 is selective for imidazoline receptors. Wistar rat terminal IMCDs were isolated and perfused to determine the osmotic water permeability coefficient (Pf). All agonists except agmatine inhibited AVP-stimulated Pf. Inhibition by rilmenidine indicated a different mechanism of action from other agonists. Dose-response data show dexmedetomidine to be the most potent inhibitor. Oxymetazoline and clonidine inhibited cAMP-stimulated Pf indicating that the mechanism involves postcAMP cellular events. It was reported previously that dexmedetomidine inhibits cAMP-stimulated Pf (1). All antagonists except prazosin and WB4101 reversed alpha2-inhibition of AVP-stimulated Pf. BU239 was effective at 1 microM but not at 100 nM. Results suggest that alpha2A adrenoceptors modulate water permeability in the IMCD. The involvement of imidazoline receptors is inconclusive.

Asunto(s)

Agonistas alfa-Adrenérgicos/farmacología , Túbulos Renales Colectores/metabolismo , Agua/metabolismo , Agonistas de Receptores Adrenérgicos alfa 2 , Antagonistas de Receptores Adrenérgicos alfa 2 , Animales , Arginina Vasopresina , Clonidina/farmacología , AMP Cíclico , Relación Dosis-Respuesta a Droga , Imidazoles/farmacología , Túbulos Renales Colectores/efectos de los fármacos , Medetomidina , Oxazoles/farmacología , Oximetazolina/farmacología , Permeabilidad/efectos de los fármacos , Ratas , Ratas Wistar , Rilmenidina

RESUMEN

A rinomanometria anterior ativa foi considerado o método de escolha para avaliaçäo da permeabilidade nasal em crianças e adolescentes que apresentavam obstruçäo nasal e foram submetidos a algum tipo de cirurgia para sua correçäo. Desta maneira, pode ser realizado, uma comparaçäo objetiva entre os resultados pré e pós-operatórios

Asunto(s)

Niño , Adolescente , Humanos , Masculino , Femenino , Manometría/instrumentación , Cavidad Nasal , Ventilación Pulmonar/fisiología , Obstrucción Nasal/cirugía , Oximetazolina/farmacología , Periodo Posoperatorio , Respiración/fisiología

RESUMEN

The rat vas deferens has been considered to be the tissue of choice to study alpha-adrenergic drugs. However, some of these agonists have elicited complex responses in this organ. Therefore, detailed characterization of alpha-adrenoceptor-mediated responses of the rat vas deferens was the aim of this work. Experiments were designed to study the contractile response of this tissue to phenylethanolamine (noradrenaline) and to two imidazolines (oxymetazoline and naphazoline). These responses were related to receptor occupancy and to other parameters of drug action, i.e. dissociation constants, relative efficacies, ED50 and maximal responses. A theoretical model was used to check the experimental data. There was a non-linear relationship between response and receptor occupancy with all three agonists. The dissociation constants for noradrenaline, oxymetazoline and naphazoline were 11.06, 0.15 and 0.10 microM, respectively. The rat vas deferens had 75-80% spare receptors for noradrenaline. Oxymetazoline and naphazoline were shown to be partial agonists with low relative efficacies as compared to noradrenaline (0.0063 and 0.0056 respectively). The dose-response curves generated by the model for partial agonists were similar to the curves obtained experimentally in vitro.

Asunto(s)

Receptores Adrenérgicos alfa/fisiología , Conducto Deferente/fisiología , Animales , Dibencilcloretamina/metabolismo , Dibencilcloretamina/farmacología , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Cinética , Masculino , Modelos Biológicos , Contracción Muscular/efectos de los fármacos , Nafazolina/administración & dosificación , Nafazolina/metabolismo , Nafazolina/farmacología , Norepinefrina/administración & dosificación , Norepinefrina/metabolismo , Norepinefrina/farmacología , Oximetazolina/administración & dosificación , Oximetazolina/metabolismo , Oximetazolina/farmacología , Ratas , Receptores Adrenérgicos alfa/efectos de los fármacos , Conducto Deferente/efectos de los fármacos