RESUMEN
In order to gain insight into the regulation of vascular tone by mitochondria, the effects of mitochondrial complex III inhibitors on contractile responses in porcine isolated coronary arteries were investigated. Segments of porcine coronary arteries were set up for isometric tension recording and concentration response curves to contractile agents were carried out in the absence or presence of the complex III inhibitors antimycin A or myxothiazol. Activity of AMP kinase was determined by measuring changes in phosphorylation of AMP kinase at Thr172. Pre-incubation with 10 µM antimycin A (Qi site inhibitor), or myxothiazol (Qo site inhibitor) led to inhibition of the contraction to the thromboxane receptor agonist U46619. Similar effects were seen on contractile responses to extracellular calcium, and the L-type calcium channel opener BAY K 8644, suggesting that both antimycin A and myxothiazol inhibit calcium-dependent contractions. The inhibitory effect of antimycin A was still seen in the absence of extracellular calcium, indicating an additional effect on a calcium independent pathway. The AMP kinase inhibitor dorsomorphin (10 µM) prevented the inhibitory of antimycin A but not myxothiazol. Furthermore, antimycin A increased the phosphorylation of AMP kinase, indicating an increase in activity, suggesting that antimycin A also acts through this pathway. These data indicate that inhibition of complex III attenuates contractile responses through inhibition of calcium influx. However, inhibition of the Qi site can also inhibit the contractile response through activation of AMP kinase.
Asunto(s)
Antimicina A/farmacología , Vasos Coronarios/efectos de los fármacos , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Mitocondrias/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Señalización del Calcio , Vasos Coronarios/enzimología , Complejo III de Transporte de Electrones/metabolismo , Femenino , Técnicas In Vitro , Masculino , Metacrilatos/farmacología , Mitocondrias/enzimología , Fosforilación , Sus scrofa , Tiazoles/farmacologíaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Asphodelus tenuifolius Cav. (Asphodelaceae), a wild, terrestrial, annual stemless herb, is widely used in traditional medicine for the treatment of hypertension, diabetes, atherosclerosis and circulatory problems. A previous research study from our laboratory revealed that A. tenuifolius has beneficial effects in reducing blood pressure and improves aortic endothelial dysfunction in chronically glucose fed rats. Despite the fact that A. tenuifolius reduces blood pressure and improves endothelial function in vivo, there are no detailed studies about its possible mechanism of action. AIM OF THE STUDY: This study was designed to provide pharmacological basis and mechanism of action for the traditional use of A. tenuifolius in hypertension and circulatory problems. We explored the vasorelaxant effect of A. tenuifolius and its underlying vasorelaxation mechanism in porcine coronary artery rings. MATERIALS AND METHODS: Aqueous methanolic crude extract of A. tenuifolius was prepared by maceration process and then activity guided fractionation was carried out by using different polarity based solvents. Phytochemical studies were carried out using LC-DAD-MS. Segments of porcine distal coronary artery were set up in a wire myograph for isometric force measurements. Extract/fractions of A. tenuifolius seeds were tested for vasodilator activity by measurement of changes in tone after pre-contraction with the thromboxane mimetic U46619 in the presence or absence of inhibitors of intracellular signaling cascades. RESULTS: Crude extract/fractions of A. tenuifolius produced dose dependent endothelium independent vasorelaxant response in coronary rings, whereas, the butanol fraction of A. tenuifolius (BS-AT) produced the largest relaxation response with 100% relaxation at 1 mg/ml, therefore the mechanism of relaxation of this fraction was determined. The relaxation to BS-AT was unaffected by removal of the endothelium, pre-contraction with KCl, or the presence of the non-selective potassium channel blocker tetraethylammonium, indicating that the relaxation was endothelium-independent, and does not involve activation of potassium channels. BS-AT (1 mg/ml) inhibited the contractile response to calcium,the L-type calcium channel activator BAY K8664,and ionomycin, indicating that it inhibits calcium-induced contractions. The relaxation response to BS-AT was attenuated in the absence of extracellular calcium. However, relaxations to BS-AT were also reduced after deletion of calcium from intracellular stores with cyclopiazonic acid. Incubation with 1 mg/ml BS-AT also inhibited phosphorylation of myosin light chains in homogenates of coronary artery. CONCLUSION: The butanol extract of Asphodelus tenuifolius produces a large endothelium-independent relaxation of the porcine coronary artery through inhibition of calcium-induced contractions. The effect appears to be downstream of calcium influx, possibly through inhibition of myosin light chain kinase. This study supports previous studies demonstrating that A. tenuifolius reduces blood pressure. Future studies will aim to determine the active compounds underlying this response.
Asunto(s)
Asphodelaceae , Vasos Coronarios/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Extractos Vegetales/farmacología , Vasodilatadores/farmacología , Animales , Vasos Coronarios/enzimología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/enzimología , Inhibidores Enzimáticos/aislamiento & purificación , Inhibidores Enzimáticos/farmacología , Técnicas de Cultivo de Órganos , Extractos Vegetales/aislamiento & purificación , Porcinos , Vasodilatadores/aislamiento & purificaciónRESUMEN
KEY POINTS: The fat surrounding blood vessels (perivascular adipose tissue or PVAT) releases vasoactive compounds that regulate vascular smooth muscle tone. There are sex differences in the regulation of vascular tone, but, to date, no study has investigated whether there are sex differences in the regulation of blood vessel tone by PVAT. This study has identified that the cyclooxygenase products thromboxane and PGF2α are released from coronary artery PVAT from pigs. Thromboxane appears to mediate the PVAT-induced contraction in arteries from females, whereas PGF2α appears to mediate the contraction in arteries from males. These sex differences in the role of these prostanoids in the PVAT-induced contraction can be explained by a greater release of thromboxane from PVAT from female animals and greater sensitivity to PGF2α in the porcine coronary artery from males. ABSTRACT: Previous studies have demonstrated that perivascular adipose tissue (PVAT) causes vasoconstriction. In this present study, we determined the role of cyclooxygenase-derived prostanoids in this contractile response and determined whether there were any sex differences in the regulation of vascular tone by PVAT. Contractions in isolated segments of coronary arteries were determined using isolated tissue baths and isometric tension recording. Segments were initially cleaned of PVAT, which was then re-added to the tissue bath and changes in tone measured over 1 h. Levels of PGF2α and thromboxane B2 (TXB2 ) were quantified by ELISA, and PGF2α (FP) and thromboxane A2 (TP) receptor expression determined by Western blotting. In arteries from both male and female pigs, re-addition of PVAT caused a contraction, which was partially inhibited by the cyclooxygenase inhibitors indomethacin and flurbiprofen. The FP receptor antagonist AL8810 attenuated the PVAT-induced contraction in arteries from males, whereas the TP receptor antagonist GR32191B inhibited the PVAT-induced contraction in arteries from females. Although there was no difference in PGF2α levels in PVAT between females and males, PGF2α produced a larger contraction in arteries from males, correlating with a higher FP receptor expression. In contrast, release of TXB2 from PVAT from females was greater than from males, but there was no difference in the contraction by the TXA2 agonist U46619, or TP receptor expression in arteries from different sexes. These findings demonstrate clear sex differences in PVAT function in which PGF2α and TXA2 antagonists can inhibit the PVAT-induced vasoconstriction in male and female PCAs, respectively.
Asunto(s)
Tejido Adiposo/metabolismo , Ácido Araquidónico/metabolismo , Vasos Coronarios/fisiología , Fosfolipasas A2/metabolismo , Vasoconstricción , Tejido Adiposo/fisiología , Animales , Dinoprost/metabolismo , Femenino , Masculino , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo , Receptores de Tromboxano A2 y Prostaglandina H2/agonistas , Receptores de Tromboxano A2 y Prostaglandina H2/genética , Receptores de Tromboxano A2 y Prostaglandina H2/metabolismo , Factores Sexuales , Porcinos , Tromboxano B2/metabolismoRESUMEN
BACKGROUND AND PURPOSE: As there is sexual dimorphism in the regulation of vascular tone, the aim of this present study was to determine whether there are sex differences in perivascular adipose tissue (PVAT)-mediated regulation of the porcine coronary artery (PCA) tone. EXPERIMENTAL APPROACH: Isometric tension recording system was used to record changes in tone in PCAs. Western blot analysis was performed to examine the expression of adiponectin in PVAT and adiponectin receptors and adiponectin binding protein (APPL1) in PCA. The level of adiponectin released from PVAT was measured using elisa. KEY RESULTS: In the presence of adherent PVAT, contractions to the thromboxane mimetic U46619 and endothelin-1 were significantly reduced in PCAs from females, but not males. In PCAs pre-contracted with U46619, re-addition of PVAT caused relaxation in PCAs from females, but not males. This relaxant response in females was attenuated by combined inhibition of NOS (with L-NAME) and COX (with indomethacin). Pre-incubation with an anti-adiponectin antibody abolished the relaxant effects of PVAT. The adiponectin receptor agonist (adipoRon) produced a greater relaxation in PCAs from females compared with males. However, there was no difference in either the expression or release of adiponectin from PVAT between sexes. Similarly, there was no difference in the expression of adiponectin receptors or the adiponectin receptor adaptor protein APPL1 in PCAs. CONCLUSION AND IMPLICATIONS: These findings demonstrate a clear sex difference in the regulation of coronary arterial tone in response to adiponectin receptor stimulation, which may underlie the anticontractile effects of PVAT in females.
Asunto(s)
Adiponectina/fisiología , Tejido Adiposo/fisiología , Vasos Coronarios/fisiología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Vasos Coronarios/efectos de los fármacos , Endotelina-1/farmacología , Femenino , Técnicas In Vitro , Masculino , Caracteres Sexuales , Porcinos , Vasoconstricción , Vasoconstrictores/farmacologíaRESUMEN
Despite pharmacological treatment, bronchial hyperresponsiveness continues to deteriorate as airway remodelling persists in airway inflammation. Previous studies have demonstrated that the phytocannabinoid Δ9-tetrahydrocannabinol (THC) reverses bronchoconstriction with an anti-inflammatory action. The aim of this study was to investigate the effects of THC on bronchial epithelial cell permeability after exposure to the pro-inflammatory cytokine, TNFα. Calu-3 bronchial epithelial cells were cultured at air-liquid interface. Changes in epithelial permeability were measured using Transepithelial Electrical Resistance (TEER), then confirmed with a paracellular permeability assay and expression of tight junction proteins by Western blotting. Treatment with THC prevented the TNFα-induced decrease in TEER and increase in paracellular permeability. Cannabinoid CB1 and CB2 receptor-like immunoreactivity was found in Calu-3 cells. Subsequent experiments revealed that pharmacological blockade of CB2, but not CB1 receptor inhibited the THC effect. Selective stimulation of CB2 receptors displayed a similar effect to that of THC. TNFα decreased expression of the tight junction proteins occludin and ZO-1, which was prevented by pre-incubation with THC. These data indicate that THC prevents cytokine-induced increase in airway epithelial permeability through CB2 receptor activation. This highlights that THC, or other cannabinoid receptor ligands, could be beneficial in the prevention of inflammation-induced changes in airway epithelial cell permeability, an important feature of airways diseases.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Bronquios/efectos de los fármacos , Agonistas de Receptores de Cannabinoides/farmacología , Dronabinol/farmacología , Alucinógenos/farmacología , Receptor Cannabinoide CB2/agonistas , Mucosa Respiratoria/efectos de los fármacos , Algoritmos , Antiinflamatorios no Esteroideos/metabolismo , Bronquios/inmunología , Bronquios/metabolismo , Agonistas de Receptores de Cannabinoides/metabolismo , Antagonistas de Receptores de Cannabinoides/farmacología , Línea Celular Tumoral , Permeabilidad de la Membrana Celular/efectos de los fármacos , Dronabinol/metabolismo , Impedancia Eléctrica , Alucinógenos/metabolismo , Humanos , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/metabolismo , Cinética , Ligandos , Ocludina/agonistas , Ocludina/antagonistas & inhibidores , Ocludina/metabolismo , Permeabilidad/efectos de los fármacos , Receptor Cannabinoide CB2/antagonistas & inhibidores , Receptor Cannabinoide CB2/metabolismo , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismo , Proteínas de Uniones Estrechas/agonistas , Proteínas de Uniones Estrechas/antagonistas & inhibidores , Proteínas de Uniones Estrechas/metabolismo , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Proteína de la Zonula Occludens-1/agonistas , Proteína de la Zonula Occludens-1/antagonistas & inhibidores , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
In recent years, it has become apparent that the gaseous pollutant, hydrogen sulphide (H2S) can be synthesised in the body and has a multitude of biological actions. This review summarizes some of the actions of this 'gasotransmitter' in influencing the smooth muscle that is responsible for controlling muscular activity of hollow organs. In the vasculature, while H2S can cause vasoconstriction by complex interactions with other biologically important gases, such as nitric oxide, the prevailing response is vasorelaxation. While most vasorelaxation responses occur by a direct action of H2S on smooth muscle cells, it has recently been proposed to be an endothelium-derived hyperpolarizing factor. H2S also promotes relaxation in other smooth muscle preparations including bronchioles, the bladder, gastrointestinal tract and myometrium, opening up the opportunity of exploiting the pharmacology of H2S in the treatment of conditions where smooth muscle tone is excessive. The original concept, that H2S caused smooth muscle relaxation by activating ATP-sensitive K(+) channels, has been supplemented with observations that H2S can also modify the activity of other potassium channels, intracellular pH, phosphodiesterase activity and transient receptor potential channels on sensory nerves. While the enzymes responsible for generating endogenous H2S are widely expressed in smooth muscle preparations, it is much less clear what the physiological role of H2S is in determining smooth muscle contractility. Clarification of this requires the development of potent and selective inhibitors of H2S-generating enzymes.
Asunto(s)
Sulfuro de Hidrógeno/farmacología , Músculo Liso/efectos de los fármacos , Animales , Humanos , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
The present study examined whether vascular function, expression and activity of NADPH oxidases differ between sexes in porcine isolated coronary arteries (PCAs) using selective Nox inhibitors, ML-171 and VAS2870. Vascular responses of distal PCAs were examined under myographic conditions in the presence of a range of inhibitors. Nox activity in PCA homogenates was assessed using lucigenin-enhanced chemiluminescence. Protein expression of Nox1, Nox2 and Nox4 was compared using Western immunoblotting. The presence of ML-171 or DPI had no effect on the bradykinin-induced vasorelaxation in PCAs from females. In males, DPI shifted the EC50 2.8-fold to the right. In the presence of L-NAME and indomethacin, DPI and ML-171 had no effect in females, but enhanced the bradykinin-induced vasorelaxation in males. ML-171 had no effect on the forskolin-induced vasorelaxation but decreased the potency of U46619-induced tone in both sexes in the absence or presence of endothelium. VAS2870 had no effect on the bradykinin-induced vasorelaxation in both sexes but reduces the EDH-type response in males only. Nox activity was reduced by DPI and ML-171, but not VAS2870 in PCAs from both sexes. Protein expression of Nox1 and Nox2 in PCAs was higher in males compared to females whereas Nox4 was higher in females. Inhibition of Nox with ML-171 enhances while VAS2870 reduces the EDH-type response in PCAs from males but not females. This indicates that Nox-generated ROS play a role in the EDH-type response in males with differences attributed to the differential expression of Nox isoforms. This may underlie the greater oxidative stress observed in males.
Asunto(s)
Vasos Coronarios/metabolismo , Endotelio Vascular/metabolismo , NADPH Oxidasas/metabolismo , Vasodilatación/fisiología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Animales , Benzoxazoles/farmacología , Bradiquinina/farmacología , Vasos Coronarios/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Femenino , Indometacina/farmacología , Masculino , NG-Nitroarginina Metil Éster/farmacología , Oxidación-Reducción , Caracteres Sexuales , Porcinos , Triazoles/farmacología , Vasodilatación/efectos de los fármacosRESUMEN
Endothelial and smooth muscle Transient Receptor Potential (TRP) channels contribute to regulation of vascular tone. We have previously reported sex differences in the endothelial function in porcine isolated coronary arteries (PCAs). The present study examined the role of TRP channels in endothelium-dependent and H2O2-induced vasorelaxations in male and female PCAs. Distal PCAs were mounted in a wire myograph and precontracted with U46619. Concentration-response curves to bradykinin, H2O2 and A23187 were constructed in the presence of TRP channel antagonists with or without L-NAME and indomethacin to inhibit NO synthase and cyclooxygenase respectively. 2-APB (TRPC & TRPM antagonist) inhibited the maximum relaxation (Rmax) of the bradykinin-induced vasorelaxation and abolished the EDH-type response in PCAs from both sexes. SKF96365 (TRPC antagonist) inhibited the Rmax of bradykinin-induced vasorelaxation in males, and inhibited Rmax of the EDH-type response in both sexes. Pyr3 (TRPC3 antagonist) inhibited both the NO and EDH components of the bradykinin-induced vasorelaxation in males, but not females. RN1734 (TRPV4 antagonist) reduced the potency of the NO component of the bradykinin-induced vasorelaxation in females only, but inhibited the Rmax of the EDH-type component in both sexes. 2-APB, SKF96365 and RN1734 all reduced the H2O2-induced vasorelaxation, whereas Pyr3 had no effect. No differences in expression level of TRPC3 and TRPV4 between sexes were detected using Western blot. Present study demonstrated a clear sex differences in the role TRP channels where TRPC3 play a role in the NO- and EDH-type response in males and TRPV4 play a role in the NO-mediated response in females.
Asunto(s)
Vasos Coronarios/fisiología , Endotelio Vascular/metabolismo , Caracteres Sexuales , Porcinos , Canales de Potencial de Receptor Transitorio/metabolismo , Vasodilatación , Animales , Compuestos de Boro/farmacología , Bradiquinina/farmacología , Calcimicina/farmacología , Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/metabolismo , Endotelio Vascular/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Imidazoles/farmacología , Indometacina/farmacología , Masculino , NG-Nitroarginina Metil Éster/farmacología , Pirazoles/farmacología , Sulfonamidas/farmacología , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Vasodilatación/efectos de los fármacosRESUMEN
Oxygenation with 95%O2 is routinely used in organ bath studies. However, hyperoxia may affect tissue responses, particularly in studies which involve reactive oxygen species (ROS). Here, the effects of the antioxidant, Tiron, were investigated under different gassing conditions in the porcine isolated coronary artery (PCA). Distal PCAs from male and female pigs were mounted in a wire myograph gassed with either 95%O2/5%CO2 or 95% air/5%CO2 and pre-contracted with U46619. Concentration-response curves to bradykinin were constructed in the presence of Tiron (1mM), a cell permeable superoxide scavenger and catalase (1000Uml(-1)) to breakdown H2O2. The H2O2 level in Krebs'-Henseleit solution was detected using Amplex Red. Bradykinin produced concentration-dependent vasorelaxations in male and female PCAs when gassed with either 95%O2 or air, with no differences in the Rmax or EC50. Tiron increased the potency of bradykinin only when gassed with 95%O2 in PCAs from both sexes. At 95%O2, catalase prevented the leftward shift caused by Tiron in both sexes indicating that catalase prevented the formation of H2O2 by Tiron. In female PCAs, addition of catalase to Tiron significantly reduced the Rmax. In the EDH-type response (using L-NAME and indomethacin), Tiron enhanced the potency of the bradykinin-induced vasorelaxation when gassed with 95%O2 in PCAs from both sexes. Biochemical analysis using Amplex Red demonstrated that H2O2 was generated in Krebs'-Henseleit solution when gassed with 95%O2, but not with air. Therefore, hyperoxic gassing conditions could alter the environment generating superoxide within the Krebs'-Henseleit buffer, which may, in turn, influence the in vitro pharmacological responses.
Asunto(s)
Sal Disódica del Ácido 1,2-Dihidroxibenceno-3,5-Disulfónico/farmacología , Vasos Coronarios/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Hiperoxia , Vasodilatación/efectos de los fármacos , Animales , Bradiquinina , Vasos Coronarios/fisiología , Endotelio Vascular/fisiología , Femenino , Peróxido de Hidrógeno/metabolismo , Hiperoxia/metabolismo , Hiperoxia/fisiopatología , Técnicas In Vitro , Masculino , Superóxidos/metabolismo , PorcinosRESUMEN
HMG-CoA reductase inhibitors, or statins, are widely used as cholesterol-lowering agents in the treatment of dyslipidemias. Statins have also been reported to have pleiotropic effects, independent of their effects on cholesterol synthesis, possibly through inhibition of the monomeric G proteins Ras and Rho, which are able to signal through ERK and Rho kinase activities, respectively. We have previously demonstrated that inhibition of ERK activation enhances ß-adrenoceptor-mediated vasodilatation in the porcine isolated coronary artery. As statins can also inhibit ERK activation, the initial aim of this study was to determine whether statins have a similar influence on ß-adrenoceptor-evoked vasodilatation. Segments of porcine distal coronary artery were mounted in a Mulvany wire myograph and bathed in Krebs-Henseleit buffer gassed with 95% O(2)/5% CO(2) and maintained at 37 °C. Tissues were pre-contracted with the thromboxane mimetic U46619 prior to cumulative concentration-response curves to the ß-adrenoceptor agonist salbutamol in the absence or presence of simvastatin (1, 5 or 10 µM), pravastatin (10 µM), or lovastatin (10 µM). Simvastatin inhibited the salbutamol-induced relaxation of the coronary artery. Similar effects were seen with lovastatin, but not pravastatin or the sodium salt of simvastatin. Simvastatin, but not pravastatin also inhibited the relaxations to the Ca(2+)-activated K(+) channel opener NS1619 and the K(ATP) channel opener pinacidil. Unexpectedly, these data indicate that, rather than enhancing ß-adrenoceptor-mediated vasodilatation, lipophilic statins impair these responses. This is likely to be due to effects on K(+) channels.
Asunto(s)
Vasos Coronarios/efectos de los fármacos , Vasos Coronarios/fisiología , Receptores Adrenérgicos beta/metabolismo , Simvastatina/farmacología , Vasodilatación/efectos de los fármacos , Animales , Colforsina/farmacología , Vasos Coronarios/metabolismo , Femenino , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Masculino , Canales de Potasio Calcio-Activados/metabolismo , PorcinosRESUMEN
Hypertension is a risk factor for myocardial infarction, stroke, renal failure, heart failure, and peripheral vascular disease. One feature of hypertension is a hyperresponsiveness to contractile agents, and inhibition of vasoconstriction forms the basis of some of the treatments for hypertension. Hypertension is also associated with an increase in the growth and proliferation of vascular smooth muscle cells, which can lead to a thickening of the smooth muscle layer of the blood vessels and a reduction in lumen diameter. Targeting both the enhanced contractile responses, and the increased vascular smooth muscle cell growth could potentially be important pharmacological treatment of hypertension. Extracellular signal-regulated kinase (ERK) is a member of the mitogen-activated protein kinase family that is involved in both vasoconstriction and vascular smooth muscle cell growth and this, therefore, makes it attractive therapeutic target for treatment of hypertension. ERK activity is raised in vascular smooth muscle cells from animal models of hypertension, and inhibition of ERK activation reduces both vascular smooth muscle cell growth and vasoconstriction. This review discusses the potential for targeting ERK activity in the treatment of hypertension.
RESUMEN
Extracellular signal regulated kinase (ERK) is known to regulate vascular smooth muscle contraction. However, a role for ERK in airway smooth muscle contraction has yet to be demonstrated conclusively, although contractile agents increase ERK activity in airway smooth muscle. Rather than initiating contraction, ERK could regulate airway tone by interfering with relaxation. Therefore, the aim of this study was to determine whether ERK regulates contraction or relaxation of airway smooth muscle. Segments of porcine peripheral bronchioles were mounted in an isolated tissue bath in Krebs-Henseleit buffer and maintained at 37°C. Cumulative concentration-response curves to histamine, endothelin-1, or the muscarinic agonist carbachol were then carried out in the absence or presence of the MEK inhibitor PD98059. In separate experiments, cumulative concentration response curves to the ß-adrenoceptor agonist isoprenaline or the adenylyl cyclase activator forskolin were carried out in the absence or presence of the MEK inhibitors PD98059 or U0126. ERK activity was measured by Western blotting. All three contractile agents increased ERK activity, but the contractile responses were unaffected by PD98059. On the other hand, both PD98059 and U0126 enhanced the relaxations to isoprenaline but not relaxations to the adenylyl cyclase activator forskolin. The enhancement of isoprenaline-induced relaxations with PD98059 was prevented by the K(+) channel blocker tetraethylammonium. These data suggest that ERK regulates airway smooth muscle tone by inhibiting ß-adrenoceptor-mediated relaxations, rather than an initiation of contraction. The effect on ß-adrenoceptor-mediated responses appears to be through a cAMP-independent mechanism, possibly through an interaction with K(+) channels.
Asunto(s)
Bronquiolos/efectos de los fármacos , Bronquiolos/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Animales , Bronquiolos/metabolismo , Carbacol/farmacología , Colforsina/farmacología , Inhibidores de la Ciclooxigenasa/farmacología , Endotelina-1/farmacología , Histamina/farmacología , Técnicas In Vitro , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Músculo Liso/fisiología , Canales de Potasio/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Receptores Adrenérgicos beta/metabolismo , PorcinosRESUMEN
Alpha2-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon activation of the extracellular signal-regulated kinase-mitogen-activated protein (ERK-MAP) kinase signal transduction pathway. Recent studies have shown that alpha2-adrenoceptor-mediated vasoconstriction in the rat aorta is also dependent upon activation of Rho kinase. The aim of this study was to determine whether Rho kinase and ERK-MAP kinase are part of the same signaling pathway. The Rho kinase inhibitor Y27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride) (10 microM) almost completely inhibited the contractile response to the alpha2-adrenoceptor agonist UK14304 (5-bromo-6-[2-imidazolin-2-ylamine]-quinoxaline bitartrate) in segments of porcine palmar lateral vein [maximum response 2.9 +/- 2.3% of 60 mM KCl response (mean +/- S.E.M.) in the presence of Y27632, compared with 64.9 +/- 7.1% in control tissues, n = 4]. However, Y27632 had no effect on alpha2-adrenoceptor-mediated ERK activation, as measured by Western blotting. Alpha2-adrenoceptor-mediated vasoconstriction was associated with an increase in phosphorylation of the myosin phosphatase-targeting subunit (MYPT) at Thr696 (the Rho kinase phosphorylation site). This phosphorylation was inhibited by 10 microM Y27632. In contrast, inhibition of ERK activation with the MAP kinase kinase inhibitor PD98059 (2-amino-3-methoxyflavone) (50 microM) had no effect on MYPT phosphorylation. Both Y27632 and PD98059 inhibited myosin light chain phosphorylation. These data indicate that alpha2-adrenoceptor-mediated vasoconstriction in the porcine palmar lateral vein is dependent upon both Rho kinase and ERK activation, although these are separate pathways. Rho kinase causes vasoconstriction through inhibition of myosin phosphatase and an increase in myosin light chain phosphorylation, whereas ERK causes vasoconstriction through a myosin phosphatase-independent pathway.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Adrenérgicos alfa 2/fisiología , Vasoconstricción/fisiología , Amidas/farmacología , Animales , Tartrato de Brimonidina , Interacciones Farmacológicas , Flavonoides/farmacología , Péptidos y Proteínas de Señalización Intracelular , Cadenas Ligeras de Miosina/metabolismo , Fosforilación/efectos de los fármacos , Proteína Quinasa C/metabolismo , Piridinas/farmacología , Quinoxalinas/farmacología , Porcinos , Vasoconstricción/efectos de los fármacos , Venas/efectos de los fármacos , Venas/fisiología , Quinasas Asociadas a rhoRESUMEN
Continuous infusion of lipopolysaccharide (LPS) into conscious rats elicits regionally selective cardiovascular disturbances. The aim of the present study was to assess contractile function in different vascular preparations (renal, mesenteric, and thoracic aorta) taken from rats infused with LPS for 2 or 24 h. Sustained responses to continuous infusion of methoxamine but not to KCl were reduced in the aorta (at 2 and 24 h LPS) and mesentery (at 24 h LPS) but not in the renal vascular bed. In contrast, transient responses to bolus doses of methoxamine were unchanged in the mesentery. In Ca2+-imaging experiments with fura-2, challenge with a single concentration of methoxamine (10 microM, which showed an impaired contractile response at 24 h LPS) induced a rise in intracellular Ca2+ in the mesenteric artery that was not different from the control. Furthermore, in the aorta, the contractile response to caffeine was attenuated only in the 2 h LPS group. These results show that there is regional heterogeneity in in vitro vascular responsiveness in preparations taken from LPS-infused rats. Thus, in mesenteric beds and aortae, but not renal beds, there is hypocontractility to methoxamine that is not due to a generalized inability of the smooth muscle to contract, which is evident with sustained but not transient application of agonist (mesentery) and which, in late endotoxemia (24 h LPS), does not appear to involve abnormalities in Ca2+ mobilization or entry.