RESUMEN
BACKGROUND AND PURPOSE: The aim of the study was to determine whether KCNQ channels are functionally expressed in bladder smooth muscle cells (SMC) and to investigate their physiological significance in bladder contractility. EXPERIMENTAL APPROACH: KCNQ channels were examined at the genetic, protein, cellular and tissue level in guinea pig bladder smooth muscle using RT-PCR, immunofluorescence, patch-clamp electrophysiology, calcium imaging, detrusor strip myography, and a panel of KCNQ activators and inhibitors. KEY RESULTS: KCNQ subtypes 1-5 are expressed in bladder detrusor smooth muscle. Detrusor strips typically displayed TTX-insensitive myogenic spontaneous contractions that were increased in amplitude by the KCNQ channel inhibitors XE991, linopirdine or chromanol 293B. Contractility was inhibited by the KCNQ channel activators flupirtine or meclofenamic acid (MFA). The frequency of Ca²âº-oscillations in SMC contained within bladder tissue sheets was increased by XE991. Outward currents in dispersed bladder SMC, recorded under conditions where BK and KATP currents were minimal, were significantly reduced by XE991, linopirdine, or chromanol, and enhanced by flupirtine or MFA. XE991 depolarized the cell membrane and could evoke transient depolarizations in quiescent cells. Flupirtine (20 µM) hyperpolarized the cell membrane with a simultaneous cessation of any spontaneous electrical activity. CONCLUSIONS AND IMPLICATIONS: These novel findings reveal the role of KCNQ currents in the regulation of the resting membrane potential of detrusor SMC and their important physiological function in the control of spontaneous contractility in the guinea pig bladder.
Asunto(s)
Expresión Génica , Canales de Potasio KCNQ/metabolismo , Contracción Muscular , Músculo Liso/metabolismo , Vejiga Urinaria/metabolismo , Animales , Animales Endogámicos , Señalización del Calcio , Células Cultivadas , Fenómenos Electrofisiológicos/efectos de los fármacos , Cobayas , Inmunohistoquímica , Técnicas In Vitro , Canales de Potasio KCNQ/agonistas , Canales de Potasio KCNQ/antagonistas & inhibidores , Canales de Potasio KCNQ/genética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Moduladores del Transporte de Membrana/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Miografía , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análisis de la Célula Individual , Vejiga Urinaria/citología , Vejiga Urinaria/efectos de los fármacosRESUMEN
BACKGROUND AND PURPOSE: Voltage-gated potassium (K(v)) channels contribute to resting membrane potential in pulmonary artery smooth muscle cells and are down regulated in patients with pulmonary arterial hypertension (PAH) and a contribution from K(v)7 channels has been recently proposed. We investigated the effect of the K(v)7 channel activator, flupirtine, on PAH in two independent mouse models: PAH induced by hypoxia and spontaneous PAH in mice over-expressing the 5-HT transporter (SERT(+) mice). EXPERIMENTAL APPROACH: Right ventricular pressure was assessed in vivo in mice chronically treated with flupirtine (30 mg.kg(-1).day(-1)). In separate in vitro experiments, pulmonary arteries from untreated mice were mounted in a wire myograph. Relaxations to acute administration of flupirtine and contractions to K(v) channel blocking drugs, including the K(v)7 channel blocker linopirdine, were measured. KEY RESULTS: In wild-type (WT) mice, hypoxia increased right ventricular pressure, pulmonary vascular remodelling and right ventricular hypertrophy. These effects were attenuated by flupirtine, which also attenuated these indices of PAH in SERT(+) mice. In the in vitro experiments, flupirtine induced a potent relaxant response in arteries from untreated WT and SERT(+) mice. The relaxation was fully reversed by linopirdine, which potently contracted mouse pulmonary arteries while other K(v) channel blockers did not. CONCLUSIONS AND IMPLICATIONS: Flupirtine significantly attenuated development of chronic hypoxia-induced PAH in mice and reversed established PAH in SERT(+) mice, apparently via K(v)7 channel activation. These results provide the first direct evidence that drugs activating K(v)7 channels may be of benefit in the treatment of PAH with different aetiologies.
Asunto(s)
Aminopiridinas/uso terapéutico , Hipertensión Pulmonar/tratamiento farmacológico , Canales de Potasio con Entrada de Voltaje/agonistas , Animales , Modelos Animales de Enfermedad , Femenino , Hipertensión Pulmonar/etiología , Hipertensión Pulmonar/fisiopatología , Hipoxia/complicaciones , Técnicas In Vitro , Ratones , Contracción Muscular/efectos de los fármacos , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiopatología , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/fisiopatología , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genéticaRESUMEN
Pulmonary vascular tone is strongly influenced by the resting membrane potential of smooth muscle cells, depolarization promoting Ca2+ influx, and contraction. The resting potential is determined largely by the activity of K+-selective ion channels, the molecular nature of which has been debated for some time. In this study, we provide strong evidence that the two-pore domain K+ channel, TASK-1, mediates a noninactivating, background K+ current (IKN), which sets the resting membrane potential in rabbit pulmonary artery smooth muscle cells (PASMCs). TASK-1 mRNA was found to be present in PASMCs, and the membranes of PASMCs contained TASK-1 protein. Both IKN and the resting potential were found to be exquisitely sensitive to extracellular pH, acidosis inhibiting the current and causing depolarization. Moreover, IKN and the resting potential were enhanced by halothane (1 mmol/L), inhibited by Zn2+ (100 to 200 micromol/L) and anandamide (10 micromol/L), but insensitive to cytoplasmic Ca2+. These properties are all diagnostic of TASK-1 channels and add to previously identified features of IKN that are shared with TASK-1, such as inhibition by hypoxia, low sensitivity to 4-aminopyridine and quinine and insensitivity to tetraethylammonium ions. It is therefore concluded that TASK-1 channels are major contributors to the resting potential in pulmonary artery smooth muscle. They are likely to play an important role in mediating pulmonary vascular responses to changes in extracellular pH, and they could be responsible for the modulatory effects of pH on hypoxic pulmonary vasoconstriction.
Asunto(s)
Músculo Liso Vascular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio/metabolismo , Arteria Pulmonar , Animales , Ácidos Araquidónicos/farmacología , Calcio/metabolismo , Calcio/farmacología , Moduladores de Receptores de Cannabinoides/farmacología , Separación Celular , Citoplasma/metabolismo , Endocannabinoides , Halotano/farmacología , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Alcamidas Poliinsaturadas , Potasio/metabolismo , Canales de Potasio/efectos de los fármacos , Canales de Potasio/genética , Arteria Pulmonar/citología , ARN Mensajero/metabolismo , Conejos , Zinc/farmacologíaRESUMEN
We report a robust and reliable platform source for visible-wavelength multiphoton microscopy that is based on nonlinear optical methods. We demonstrate a synchronously pumped, singly resonant optical parametric oscillator with simultaneous intracavity third-order quasi-phase matching in a single crystal that generates continuously tunable, visible, and femtosecond-pulsed radiation. The application of the system is demonstrated by two-photon laser-scanning fluorescence microscopy of rabbit cardiac myocytes loaded with the fluorescent Ca2+ indicator fura-2.
Asunto(s)
Rayos Láser , Microscopía , Miocitos Cardíacos/ultraestructura , Fotones , Animales , Colorantes Fluorescentes , Fura-2 , Microscopía Fluorescente , Conejos , Factores de TiempoRESUMEN
The P2 receptors that mediate contraction of the rat isolated small (SPA, 200-500 micro m i.d.) and large (LPA, 1-1.5 mM i.d.) intrapulmonary arteries were characterized. 2 In endothelium-denuded vessels the contractile order of potency was alpha,beta-methyleneATP (alpha,beta-meATP)>>UDP=UTP=ATP=2-methylthioATP>ADP in the SPA and alpha,beta-meATP=UTP>or=UDP>2-methylthioATP, ATP>>ADP in the LPA. alpha,beta-meATP, 2-methylthioATP and ATP had significantly greater effects in the SPA than the LPA (P<0.001), but there was no difference in the potency of UTP or UDP between the vessels. 3 In the SPA, P2X1 receptor desensitisation by alpha,beta-meATP (100 microM) inhibited contractions to alpha,beta-meATP (10 nM-300 microM), but not those to UTP or UDP (100 nM-300 microM). In the LPA, prolonged exposure to alpha,beta-meATP (100 microM) did not desensitize P2X receptors. 4 Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), suramin and reactive blue 2 (RB2) (30-300 microM) inhibited contractions evoked by alpha,beta-meATP. UTP and UDP were potentiated by PPADS, unaffected by RB2 and inhibited, but not abolished by suramin. 1 and 3 mM suramin produced no further inhibition, indicating suramin-resistant components in the responses to UTP and UDP. 5 Thus, both P2X and P2Y receptors mediate contraction of rat large and small intrapulmonary arteries. P2Y agonist potency and sensitivity to antagonists were similar in small and large vessels, but P2X agonists were more potent in small arteries. This indicates differential expression of P2X, but not P2Y receptors along the pulmonary arterial tree.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Arteria Pulmonar/metabolismo , Circulación Pulmonar/fisiología , Receptores Purinérgicos P2/biosíntesis , Animales , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas In Vitro , Masculino , Arteria Pulmonar/efectos de los fármacos , Circulación Pulmonar/efectos de los fármacos , Agonistas del Receptor Purinérgico P2 , Antagonistas del Receptor Purinérgico P2 , Ratas , Ratas Sprague-DawleyRESUMEN
The protective action of passive saline filled ("empty") phosphatidylcholine liposomes (PCL) on endothelial function was examined in thoracic aortas obtained from gamma irradiated (6 Gy) Chinchilla rabbits, and then verified in experiments on non-anesthetized and anesthetized rats. Acetylcholine (ACh)-induced vascular relaxant responses in isolated vascular tissues rats were used as the test of endothelial integrity and its functional ability. It was shown that when added to the bath solution (100 microg/ml), PCL effectively restored endothelium-dependent ACh relaxations of isolated vascular rings damaged resulting from gamma-irradiation but had no effect on endothelium-independent vascular responses to therapeutic nitric oxide (NO) donors. The liposomes were also without protective effect when injected to the rabbits intraperitoneally (30 mg/kg) 1 hour before irradiation. In contrast, PCL, being injected at the same dose 1 hour after radiation impact, promote normalization of both endothelium-dependent vascular responses to ACh and nitric oxide (NO) donors. PCL restored also the sensitivity of vascular tissues to authentic NO (aqueous NO solution) that was surprisingly increased after irradiation, and normalized relationship between ACh-stimulated NO release and relaxant response amplitudes in irradiated aortas. Experiments on non-anesthetized and anesthetized rats demonstrated that irradiation led to significant elevation in the level of arterial blood pressure without any changes in cardiac contractility. PCL administration (25 mg/kg, i.v.) effectively normalized an increased arterial blood pressure in irradiated animals. In conclusion, it appears that PCL due to its ability to normalize NO-dependent vascular tone control mechanisms might be worthwhile therapeutic approach in case of ionizing irradiation accident. These result support the concept that the depression of endothelium-dependent vascular responses after irradiation may be result of decreased NO bioavailability due to its conversion to less potent vasodilators during irradiation-induced oxidative attack.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/efectos de la radiación , Rayos gamma , Fosfatidilcolinas/administración & dosificación , Cloruro de Sodio/farmacología , Acetilcolina/farmacología , Adulto , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiología , Aorta Torácica/efectos de la radiación , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/efectos de la radiación , Endotelio Vascular/fisiología , Humanos , Técnicas In Vitro , Liposomas , Persona de Mediana Edad , Donantes de Óxido Nítrico/farmacología , Conejos , Ratas , Ratas Endogámicas WKY , Recuperación de la Función , Cloruro de Sodio/química , Vasoconstricción , Vasodilatadores/farmacologíaRESUMEN
Cation channels activated by Ca(2+) store depletion have been proposed to mediate Ca(2+) influx in vascular smooth muscle cells. The aim of this study was to determine if store-operated channels have a functional role in pulmonary artery smooth muscle cells (PASMCs). In intact rat pulmonary artery rings, cyclopiazonic acid (CPA) produced a sustained contraction that was resistant to inhibition by nifedipine, but abolished in Ca(2+)-free solution and 50% blocked in the presence of 6 micromol/L Cd(2+), 10 micromol/L Ni(2+), 600 micromol/L La(3+), and 7 micromol/L SKF96365. In freshly isolated PASMCs loaded with fura-2, CPA increased the intracellular Ca(2+) concentration by stimulating dihydropyridine-resistant Ca(2+) influx, which was approximately 50% blocked by 10 micromol/L Ni(2+) and 7 micromol/L SKF96365. In perforated-patch recordings, CPA activated a sustained inward current at negative membrane potentials, which persisted in cells dialyzed with BAPTA, showed a near linear dependence on membrane potential when Cs(+) was the main intracellular cation, and was blocked by Ni(2+), Cd(2+), and SKF96365 at concentrations preventing contraction. The current showed a bimodal dependence on extracellular Ca(2+), being enhanced 2-fold in the absence of Ca(2+) and around 10-fold on reducing Ca from 1.8 to 0.2 mmol/L. RT-PCR revealed the expression of Trp1, Trp3, Trp4, Trp5, and Trp6 mRNA, whereas immunostaining identified Trp1, Trp3, Trp4, and Trp6 channel proteins in isolated PASMCs. At least one of these subunits may contribute to cation channels in PASMCs, which are activated by store depletion to bring about Ca(2+) influx and contraction.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Arteria Pulmonar/metabolismo , Retículo Sarcoplasmático/metabolismo , Vasoconstricción/fisiología , Animales , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Separación Celular , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Indoles/farmacología , Transporte Iónico/efectos de los fármacos , Masculino , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Técnicas de Placa-Clamp , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Rianodina/farmacología , Retículo Sarcoplasmático/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico , Tapsigargina/farmacología , Vasoconstricción/efectos de los fármacosRESUMEN
1. The directly acting vasodilator hydralazine has been proposed to act at an intracellular site in vascular smooth muscle to inhibit Ca(2+) release. 2. This study investigated the mechanism of action of hydralazine on rabbit aorta and pulmonary artery by comparing its effects on the tension generated by intact and beta-escin permeabilized vessels and on the cytoplasmic Ca(2+) concentration, membrane potential and K(+) currents of isolated vascular smooth muscle cells. 3. Hydralazine relaxed pulmonary artery and aorta with similar potency. It was equally effective at inhibiting phasic and tonic contractions evoked by phenylephrine in intact vessels and contractions evoked by inositol 1,4,5 trisphosphate (IP(3)) in permeabilized vessels. 4. Hydralazine inhibited the contraction of permeabilized vessels and the increase in smooth muscle cell Ca(2+) concentration evoked by caffeine with similar concentration dependence, but with lower potency than its effect on IP(3) contractions. 5. Hydralazine had no effect on the relationship between Ca(2+) concentration and force generation in permeabilized vessels, but it slowed the rate at which maximal force was developed before, but not after, destroying sarcoplasmic reticulum function with the calcium ionophore, ionomycin. 6. Hydralazine had no effect on membrane potential or the amplitudes of K(+) currents recorded from isolated smooth muscle cells over the concentration range causing relaxation of intact vessels. 7. The results suggest that the main action of hydralazine is to inhibit the IP(3)-induced release of Ca(2+) from the sarcoplasmic reticulum in vascular smooth muscle cells.
Asunto(s)
Aorta Torácica/efectos de los fármacos , Hidralazina/farmacología , Arteria Pulmonar/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Animales , Aorta Torácica/fisiología , Calcio/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inositol 1,4,5-Trifosfato/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Arteria Pulmonar/fisiología , Conejos , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Vasodilatación/fisiologíaRESUMEN
The response of pulmonary arteries to hypoxia varies as a function of vessel diameter. Small intrapulmonary resistance arteries are thought to be the main site of hypoxic pulmonary vasoconstriction (HPV), with hypoxia causing minimal contraction or even dilatation in large, conduit vessels. This has been proposed to reflect a differential distribution of morphologically and electrophysiologically distinct pulmonary artery smooth muscle (PASM) cells. We investigated longitudinal heterogeneity in smooth muscle cells isolated from five regions of the rabbit pulmonary vasculature and could find no evidence of morphological heterogeneity at the level of the light microscope. PASM cells from main (8 mm outer diameter) and branch (5 mm) arteries and large ( 400 m) intrapulmonary arteries (IPA) were similar in shape and size, as indicated by cell capacitance (25 pF). PASM cells from medium (200-400 m) and small ( 200 m) IPA were significantly smaller (15 pF), but had the same classical spindle shape. Cells from all five regions also had similar resting membrane potentials and displayed voltage-activated K+ currents of similar amplitude when recorded in standard physiological solution. Longitudinal heterogeneity in K+ current became apparent when tetraethylammonium ions (TEA; 10 mM) and glibenclamide (10 M) were added. The remaining delayed rectifier current (IK(V)) doubled in amplitude upon moving down the pulmonary arterial tree from the main artery (9 pA pF-1 at 40 mV) to the large IPA (17 pA pF-1), but remained constant throughout the intrapulmonary vasculature. The O2-sensitive, non-inactivating K+ current (IK(N)) showed a similar trend, but was significantly reduced in the smallest IPA, where its amplitude was comparable with the main artery. Thus the IK(N)/IK(V) ratio was relatively constant, at around 0.14, from the main pulmonary artery to medium IPA, but fell by 50% in the smallest vessels. The amplitude of the TEA-sensitive K+ current was similar (16 pA pF-1 at 40 mV) at all levels of the pulmonary arterial tree, except in the medium sized vessels where it was 50% smaller. These variations in K+ current expression correlate with reported variations in sensitivity to hypoxia and may contribute to the regional heterogeneity of HPV in the rabbit lung.
Asunto(s)
Músculo Liso Vascular/metabolismo , Canales de Potasio/metabolismo , Potasio/metabolismo , Arteria Pulmonar/metabolismo , Anaerobiosis , Animales , Tamaño de la Célula , Células Cultivadas , Conductividad Eléctrica , Impedancia Eléctrica , Gliburida/farmacología , Potenciales de la Membrana/efectos de los fármacos , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/fisiología , Conejos , Tetraetilamonio/farmacología , VasoconstricciónRESUMEN
This study investigated the relationship between cytoplasmic, mitochondrial, and sarcoplasmic reticulum (SR) [Ca(2+)] in rabbit aorta smooth muscle cells, following cell activation. Smooth muscle cells were loaded with the Ca(2+)-sensitive fluorescent indicator Mag-Fura-2-AM, and then either permeabilized by exposure to saponin, or dialyzed with a patch pipette in the whole-cell configuration to remove cytoplasmic indicator. When the intracellular solution contained millimolar EGTA or BAPTA, activation of SR Ca(2+)release through IP(3)or ryanodine receptors induced a decrease in the [Ca(2+)] reported by Mag-Fura-2. However, when EGTA was present at < or =100 microM, the same stimuli caused an increase in the [Ca(2+)] reported by Mag-Fura-2. The increase in [Ca(2+)] caused by phenylephrine or caffeine was delayed, and prolonged, with respect to the cytoplasmic Ca(2+)transient. Evidence is presented that this Mag-Fura-2 signal reflected a rise in mitochondrial [Ca(2+)]. Agents that inhibit mitochondrial function, such as FCCP or cyanide in combination with oligomycin B, converted the increase in organelle Mag-Fura-2 fluorescence to a decrease, while also prolonging the cytoplasmic Ca(2+)transient. There was considerable similarity between the localization of Mag-Fura-2 fluorescence and the mitochondria-selective indicator tetramethylrhodamine ethyl ester. Thus, we propose that there is close functional integration between the SR and mitochondria in aorta smooth muscle cells, with mitochondria taking up Ca(2+)from the cytoplasm following cell activation.
Asunto(s)
Aorta Torácica/metabolismo , Señalización del Calcio , Calcio/metabolismo , Citoplasma/metabolismo , Fura-2/análogos & derivados , Mitocondrias Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Aorta Torácica/efectos de los fármacos , Cafeína/farmacología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Citoplasma/efectos de los fármacos , Diálisis , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Fluorescencia , Colorantes Fluorescentes/metabolismo , Fura-2/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Inositol 1,4,5-Trifosfato/farmacología , Membranas Intracelulares/metabolismo , Masculino , Mitocondrias Musculares/efectos de los fármacos , Músculo Liso Vascular/citología , Fenilefrina/farmacología , Conejos , Saponinas/metabolismo , Retículo Sarcoplasmático/efectos de los fármacosRESUMEN
Pituitary adenylyl cyclase-activating peptide (PACAP) stimulates calcium transients and catecholamine secretion in adrenal chromaffin and PC12 cells. The PACAP type 1 receptor in these cells couples to both adenylyl cyclase and phospolipase C pathways, but although phospolipase C has been implicated in the response to PACAP, the role of adenylyl cyclase is unclear. In this study, we show that PACAP38 stimulates Ca(2+) influx in PC12 cells by activating a cation current that depends upon the dual activation of both the PLC and adenylyl cyclase signaling pathways but does not involve protein kinase C. In activating the current, PACAP38 has to overcome an inhibitory effect of Ras. Thus, in cells expressing a dominant negative form of Ras (PC12asn17-W7), PACAP38 induced larger, more rapidly activating currents. This effect of Ras could be overidden by intracellular guanosine-5'-O-3-(thio)triphosphate (GTPgammaS), suggesting that it was mediated by inhibition of downstream G proteins. Ras may also inhibit the current through a G protein-independent mechanism, because cAMP analogues activated the current in PC12asn17-W7 cells, provided GTPgammaS was present, but not in PC12 cells expressing wild type Ras. We conclude that coupling of PACAP to both adenylyl cyclase and phospholipase C is required to activate Ca(2+) influx in PC12 cells and that tonic inhibition by Ras delays and limits the response.
Asunto(s)
Canales de Calcio/fisiología , Neuropéptidos/fisiología , Transducción de Señal/fisiología , Adenilil Ciclasas/metabolismo , Animales , Canales de Calcio/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/fisiología , Potenciales de la Membrana/fisiología , Neuropéptidos/metabolismo , Proteína Oncogénica p21(ras)/fisiología , Células PC12 , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Ratas , Fosfolipasas de Tipo C/metabolismoRESUMEN
Hypoxia inhibits voltage-gated K channels in pulmonary artery smooth muscle (PASM). This is thought to contribute to hypoxic pulmonary vasoconstriction by promoting membrane depolarization, Ca(2+) influx, and contraction. Several of the K-channel subtypes identified in pulmonary artery have been implicated in the response to hypoxia, but contradictory evidence clouds the identity of the oxygen-sensing channels. Using patch-clamp techniques, this study investigated the effect of hypoxia on recombinant Kv1 channels previously identified in pulmonary artery (Kv1.1, Kv1.2, and Kv1.5) and Kv3.1b, which has similar kinetic and pharmacological properties to native oxygen-sensitive currents. Hypoxia failed to inhibit any Kv1 channel, but it inhibited Kv3.1b channels expressed in L929 cells, as shown by a reduction of whole-cell current and single-channel activity, without affecting unitary conductance. Inhibition was retained in excised membrane patches, suggesting a membrane-delimited mechanism. Using reverse transcription-polymerase chain reaction and immunocytochemistry, Kv3.1b expression was demonstrated in PASM cells. Moreover, hypoxia inhibited a K(+) current in rabbit PASM cells in the presence of charybdotoxin and capsaicin, which preserve Kv3.1b while blocking most other Kv channels, but not in the presence of millimolar tetraethylammonium ions, which abolish Kv3.1b current. Kv3.1b channels may therefore contribute to oxygen sensing in pulmonary artery.
Asunto(s)
Células Quimiorreceptoras/fisiología , Neuropéptidos/metabolismo , Oxígeno/metabolismo , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Animales , Células COS , Hipoxia de la Célula/fisiología , Canales de Potasio de Tipo Rectificador Tardío , Expresión Génica/fisiología , Activación del Canal Iónico/fisiología , Canal de Potasio Kv.1.1 , Canal de Potasio Kv.1.2 , Canal de Potasio Kv1.5 , Masculino , Potenciales de la Membrana/fisiología , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/fisiología , Músculo Liso Vascular/química , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Neuropéptidos/genética , Células PC12 , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio , Canales de Potasio/genética , Canales de Potasio/fisiología , Arteria Pulmonar/química , Arteria Pulmonar/citología , Arteria Pulmonar/fisiología , Ratas , Canales de Potasio Shaw , TransfecciónRESUMEN
Vascular tone can be regulated by drugs that alter the activities of membrane ionic channels located in endothelial or smooth muscle cells in the vascular wall. This review examines the methods that are available to investigate the activities and pharmacological modulation of ion channels in vascular cells. They range from classical sucrose-gap and sharp-microelectrode techniques for studies of intact vessels, to the now widely used patch-clamp techniques for voltage-clamp recording of single-channel and macroscopic currents in isolated cells. Each method is described, along with examples of applications and discussion of potential problems and limitations.
Asunto(s)
Vasos Sanguíneos/fisiología , Animales , Calcio/metabolismo , Endotelio Vascular/citología , Uniones Comunicantes/fisiología , Humanos , Canales Iónicos/fisiología , Microelectrodos , Músculo Liso Vascular/citología , Técnicas de Placa-ClampRESUMEN
The pulmonary vasculature is sensitive to the relative components of the respiratory gases and will vasoconstrict in response to decreased oxygen (O2) levels. This hypoxic pulmonary vasoconstriction (HPV) controls pulmonary blood flow in the fetus and serves to maximize ventilation perfusion matching in the adult lung. The exact mechanism of HPV is not fully understood but it appears to involve direct effects on both the endothelium and smooth muscle cells within the vessel wall. There is growing evidence to suggest that hypoxia mediates vasoconstriction, at least in part through the inhibition of outward potassium (K+) current in smooth muscle. A number of K+ currents present in the pulmonary vasculature have been shown to be sensitive to O2, with hypoxia acting to inhibit these currents in the majority of cases. Differences in the expression of these O2-sensitive K+ channels may explain regional and generic variations observed in the HPV response. The mechanism by which these K+ channels sense changes in O2 levels may involve changes in the cellular redox state, oxidative phosphorylation or a direct effect on the channel protein itself.
Asunto(s)
Hipoxia/fisiopatología , Oxígeno/fisiología , Canales de Potasio/fisiología , Arteria Pulmonar/fisiología , Animales , Bovinos , Cricetinae , Perros , Endotelio Vascular/fisiología , Hipertensión Pulmonar/complicaciones , Hipertensión Pulmonar/fisiopatología , Enfermedades Pulmonares Obstructivas/complicaciones , Enfermedades Pulmonares Obstructivas/fisiopatología , Músculo Liso Vascular/fisiología , Arteria Pulmonar/química , Conejos , Ratas , Especificidad de la Especie , Vasoconstricción/fisiologíaRESUMEN
OBJECTIVE: An increase in stimulation frequency can facilitate or depress cardiac Ca2+ current (ICa). The aim was to examine the Ca2+ dependence of these effects, to determine if facilitation is sustained, and to elucidate the mechanism by which isoprenaline modulates facilitation. METHODS: We examined the effects of increasing the stimulation frequency for 1 min, from 0.05 to 1 Hz, on ICa recorded from guinea-pig ventricular myocytes, using the whole-cell, voltage-clamp technique. RESULTS: 1 Hz stimulation caused a facilitation of ICa that peaked in 5 s and was followed by depression towards the basal level. Metabolic inhibitors or replacement of extracellular Ca2+ with Ba2+ abolished facilitation without affecting depression, implying that they are independent processes and that facilitation required ATP and Ca2+. Subtraction of the depression observed in either condition, from the response to 1 Hz stimulation recorded under control conditions, revealed that ICa facilitation was well maintained during 1 Hz stimulation. Increased intracellular Ca2+ buffering reduced both phases of the response. Furthermore, varying the extracellular Ca2+ concentration ([Ca2+]o) revealed a Ca(2+)-dependent enhancement of depression and a bell-shaped dependence of facilitation on [Ca2+]o. Facilitation increased with [Ca2+]o up to 1 mM, then declined at higher concentrations due to partial masking by the overlaping depression. Isoprenaline produced concentration-dependent inhibition of facilitation and enhancement of depression when pipettes contained 2 mM EGTA, but not BAPTA. For an equivalent increase in ICa amplitude, the effects of isoprenaline and elevated [Ca2+]o on the response to 1 Hz stimulation were quantitatively the same. CONCLUSIONS: Facilitation is sustained during increased activity, but appears transient due to overlapping depression. Both responses are promoted by increased submembrane [Ca2+]. Isoprenaline appears to modulate facilitation and depression as a consequence of increased Ca2+ influx, rather than cAMP-dependent phosphorylation. The apparent block of facilitation by isoprenaline may result from masking by the enhanced depression.
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Agonistas de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Calcio/farmacología , Isoproterenol/farmacología , Miocardio/metabolismo , Análisis de Varianza , Animales , Bario/farmacología , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Espacio Extracelular/metabolismo , Cobayas , Masculino , Técnicas de Placa-Clamp , Retículo Sarcoplasmático/metabolismo , Estadísticas no ParamétricasRESUMEN
Exposing rats to chronic hypoxia increased the 4-aminopyridine (4-AP) sensitivity of pulmonary arteries. 1 mM 4-AP caused smooth muscle cell depolarization and contraction in arteries from hypoxic rats, but had little effect in age-matched controls. Chronic hypoxia downregulated delayed rectifier K+ current (IK(V)), which was nearly 50% blocked by 1 mM 4-AP, and non-inactivating K+ current (IK(N)), which was little affected by 1 mM 4-AP. The results suggest that IK(N) determines resting potential in control rats and that its downregulation following hypoxia leads to depolarization, which activates IK(V) and increases its contribution to resting potential. The hypoxia-induced increase in 4-AP sensitivity thus reflects a switch in the major K+ current determining resting potential, from IK(N) to IK(V). This has important implications for the actions and specificity of pulmonary vasodilator drugs.
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Hipoxia/fisiopatología , Potenciales de la Membrana/fisiología , Tono Muscular/fisiología , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Arteria Pulmonar/fisiopatología , 4-Aminopiridina/farmacología , Animales , Canales de Potasio de Tipo Rectificador Tardío , Técnicas In Vitro , Activación del Canal Iónico , Masculino , Potenciales de la Membrana/efectos de los fármacos , Tono Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , RatasRESUMEN
The pulmonary circulation changes rapidly at birth to adapt to extrauterine life. The neonate is at high risk of developing pulmonary hypertension, a common cause being perinatal hypoxia. Smooth muscle K+ channels have been implicated in hypoxic pulmonary vasoconstriction in adults and O2-induced vasodilation in the fetus, channel inhibition being thought to promote Ca2+ influx and contraction. We investigated the K+ currents and membrane potentials of pulmonary artery myocytes during development, in normal pigs and pigs exposed for 3 days to hypoxia, either from birth or from 3 days after birth. The main finding is that cells were depolarized at birth and hyperpolarized to the adult level of -40 mV within 3 days. Hypoxia prevented the hyperpolarization when present from birth and reversed it when present from the third postnatal day. The mechanism of hyperpolarization is unclear but may involve a noninactivating, voltage-gated K+ channel. It is not caused by increased Ca2+-activated or delayed rectifier current. These currents were small at birth compared with adults, declined further over the next 2 wk, and were suppressed by exposure to hypoxia from birth. Hyperpolarization could contribute to the fall in pulmonary vascular resistance at birth, whereas the low K+-current density, by enhancing membrane excitability, would contribute to the hyperreactivity of neonatal vessels. Hypoxia may hinder pulmonary artery adaptation by preventing hyperpolarization and suppressing K+ current.
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Animales Recién Nacidos/crecimiento & desarrollo , Hipoxia/fisiopatología , Potenciales de la Membrana , Desarrollo de Músculos , Músculo Liso Vascular/crecimiento & desarrollo , Canales de Potasio/fisiología , Arteria Pulmonar/crecimiento & desarrollo , Envejecimiento , Animales , Calcio/farmacología , Conductividad Eléctrica , Gliburida/farmacología , Canales de Potasio/efectos de los fármacos , PorcinosRESUMEN
1. The contributions of specific K+ currents to the resting membrane potential of rabbit isolated, pulmonary artery myocytes, and their modulation by hypoxia, were investigated by use of the whole-cell, patch-clamp technique. 2. In the presence of 10 microM glibenclamide the resting potential (-50 +/- 4 mV, n = 18) was unaffected by 10 microM tetraethylammonium ions, 200 nM charybdotoxin, 200 nM iberiotoxin, 100 microM ouabain or 100 microM digitoxin. The negative potential was therefore maintained without ATP-sensitive (KATP) or large conductance Ca(2+)-sensitive (BKCa) K channels, and without the Na(+)-K+ ATPase. 3. The resting potential, the delayed rectifier current (IK(V)) and the A-like K+ current (IK(A)) were all reduced in a concentration-dependent manner by 4-aminopyridine (4-AP) and by quinine. 4. 4-AP was equally potent at reducing the resting potential and IK(V), 10 mM causing depolarization from -44 mV to -22 mV with accompanying inhibition of IK(V) by 56% and IK(A) by 79%. In marked contrast, the effects of quinine on resting potential were poorly correlated with its effects on both IK(A) and IK(V). At 10 mM, quinine reduced IK(V) and IK(A) by 47% and 38%, respectively, with no change in the resting potential. At 100 microM, both currents were almost abolished while the resting potential was reduced < 50%. Raising the concentration to 1 mM had little further effect on IK(A) or IK(V), but essentially abolished the resting potential. 5. Reduction of the resting potential by quinine was correlated with inhibition of a voltage-gated, low threshold, non-inactivating K+ current, IK(N). Thus, 100 microM quinine reduced both IK(N) and the resting potential by around 50%. 6. The resting membrane potential was the same whether measured after clamping the cell at -80 mV, or immediately after a prolonged period of depolarization at 0 mV, which inactivated IK(A) and IK(V), but not IK(N). 7. When exposed to a hypoxic solution, the O2 tension near the cell fell from 125 +/- 6 to 14 +/- 2 mmHg (n = 20), resulting in a slow depolarization of the myocyte membrane to -35 +/- 3 mV (n = 16). The depolarization occurred without a change in the amplitude of IK(V) or IK(A), but it was accompanied by 60% inhibition of IK(N) at 0 mV. 8. Our findings suggest that the resting potential of rabbit pulmonary artery myocytes depends on IK(N), and that inhibition of IK(N) may mediate the depolarization induced by hypoxia.
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Potenciales de la Membrana/fisiología , Oxígeno/metabolismo , Canales de Potasio/fisiología , Arteria Pulmonar/fisiología , Animales , Hipoxia de la Célula , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Arteria Pulmonar/citología , ConejosAsunto(s)
Compuestos de Bis-Trimetilamonio/historia , Ganglios Parasimpáticos/efectos de los fármacos , Bloqueadores Ganglionares/historia , Canales Iónicos/historia , Fármacos Neuromusculares Despolarizantes/historia , Animales , Compuestos de Bis-Trimetilamonio/farmacología , Femenino , Ganglios Parasimpáticos/citología , Ganglios Parasimpáticos/metabolismo , Bloqueadores Ganglionares/farmacología , Historia del Siglo XX , Canales Iónicos/antagonistas & inhibidores , Masculino , Fármacos Neuromusculares Despolarizantes/farmacología , RatasRESUMEN
1. An outward current (IK(N)) was identified in rabbit pulmonary artery myocytes, which persisted after Ca(2+)-activated and ATP-sensitive K+ currents were blocked by TEA (10 mM) and glibenclamide (10 microM), respectively, and after A-like (IK(A)) and delayed rectifer (IK(V)) K+ currents were inactivated by clamping the cell at 0 mV for 10 min. It was found in smooth muscle cells at all levels of the pulmonary arterial tree. 2. The relationship between the reversal potential of IK(N) and the extracellular K+ concentration ([K+]o) was close to that expected for a K(+)-selective channel. Deviation from Nernstian behaviour at low [K+)o could be accounted for by the presence of an accompanying leakage current. 3. IK(N) is voltage gated. It has a low threshold for activation, between -80 and -65 mV, and activates slowly without delay. Activation follows an exponential time course with a time constant of 1.6 s at -60 mV. Deactivation is an order of magnitude faster than activation, with a time constant of 107 ms at -60 mV. 4. IK(N) showed a similar sensitivity to 4-aminopyridine as IK(A) and IK(V), with 49% inhibition at 10 mM. The current was not blocked by microM quinine, which did inhibit IK(A) and IK(V), by 51 and 47%, respectively. 5. Activation of IK(N) was detected at potentials close to the resting membrane potential of pulmonary artery smooth muscle cells, under physiological conditions. Thus it is likely to contribute to the resting membrane potential of these cells.