RESUMEN
Hypoxic inhibition of K(+) channels in type I cells is believed to be of central importance in carotid body chemotransduction. We have recently suggested that hypoxic channel inhibition is mediated by AMP-activated protein kinase (AMPK). Here, we have further explored the modulation by AMPK of recombinant K(+) channels (expressed in HEK293 cells) whose native counterparts are considered O(2)-sensitive in the rat carotid body. Inhibition of maxiK channels by AMPK activation with AICAR was found to be independent of [Ca(2+)](i) and occurred regardless of whether the alpha subunit was co-expressed with an auxiliary beta subunit. All effects of AICAR were fully reversed by the AMPK inhibitor compound C. MaxiK channels were also inhibited by the novel AMPK activator A-769662 and by intracellular dialysis with the constitutively active, truncated AMPK mutant, T172D. The molecular identity of the O(2)-sensitive leak K(+) conductance in rat type I cells remains unclear, but shares similarities with TASK-1 and TASK-3. Recombinant TASK-1 was insensitive to AICAR. However, TASK-3 was inhibited by either AICAR or A-769662 in a manner which was reversed by compound C. These data highlight a role for AMPK in the modulation of two proposed O(2) sensitive K(+) channels found in the carotid body.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Oxígeno/metabolismo , Canales de Potasio/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Línea Celular , Diálisis , Conductividad Eléctrica , Activación Enzimática , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Ribonucleótidos/farmacologíaAsunto(s)
Cuerpo Carotídeo/metabolismo , Hipoxia de la Célula/fisiología , Complejos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Animales Recién Nacidos , Señalización del Calcio/efectos de los fármacos , Cuerpo Carotídeo/citología , Cuerpo Carotídeo/efectos de los fármacos , Células Quimiorreceptoras/metabolismo , Electrofisiología , Inmunohistoquímica , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Modelos Neurológicos , Fosforilación Oxidativa/efectos de los fármacos , Ratas , Ribonucleótidos/farmacologíaRESUMEN
Inhibitors of mitochondrial energy metabolism have long been known to be potent stimulants of the carotid body, yet their mechanism of action remains obscure. We have therefore investigated the effects of rotenone, myxothiazol, antimycin A, cyanide (CN(-)) and oligomycin on isolated carotid body type I cells. All five compounds caused a rapid rise in intracellular Ca(2+), which was inhibited on removal of extracellular Ca(2+). Under current clamp conditions rotenone and CN(-) caused a rapid membrane depolarization and elevation of [Ca(2+)](i). Voltage clamping cells to -70 mV substantially attenuated this rise in [Ca(2+)](i). Rotenone, cyanide, myxothiazol and oligomycin significantly inhibited resting background K(+) currents. Thus rotenone, myxothiazol, cyanide and oligomycin mimic the effects of hypoxia in that they all inhibit background K(+) current leading to membrane depolarization and voltage-gated calcium entry. Hypoxia, however, failed to have any additional effect upon membrane currents in the presence of CN(-) or rotenone or the mitochondrial uncoupler p-trifluoromethoxyphenyl hydrazone (FCCP). Thus not only do mitochondrial inhibitors mimic the effects of hypoxia, but they also abolish oxygen sensitivity. These observations suggest that there is a close link between oxygen sensing and mitochondrial function in type I cells. Mechanisms that could account for this link and the actions of mitochondrial inhibitors are discussed.
Asunto(s)
Cuerpo Carotídeo/fisiología , Mitocondrias/fisiología , Neuronas/fisiología , Animales , Calcio/metabolismo , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Cuerpo Carotídeo/citología , Cuerpo Carotídeo/efectos de los fármacos , Transporte de Electrón/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Hipoxia/fisiopatología , Membranas Intracelulares/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Metacrilatos , Mitocondrias/efectos de los fármacos , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Neuronas/efectos de los fármacos , Oligomicinas/farmacología , Concentración Osmolar , Ratas , Ratas Sprague-Dawley , Rotenona/farmacología , Cianuro de Sodio/farmacología , Tiazoles/farmacología , Desacopladores/farmacologíaRESUMEN
1. The effect has been examined of the accessory alpha2-delta and beta subunits on the properties of alpha1G currents expressed in monkey COS-7 cells and Xenopus oocytes. 2. In immunocytochemical experiments, the co-expression of alpha2-delta increased plasma membrane localization of expressed alpha1G and conversely, the heterologous expression of alpha1G increased immunostaining for endogenous alpha2-delta, suggesting an interaction between the two subunits. 3. Heterologous expression of alpha2-delta together with alpha1G in COS-7 cells increased the amplitude of expressed alpha1G currents by about 2-fold. This finding was confirmed in the Xenopus oocyte expression system. The truncated delta construct did not increase alpha1G current amplitude, or increase its plasma membrane expression. This indicates that it is the exofacial alpha2 domain that is involved in the enhancement by alpha2-delta. 4. Beta1b also produced an increase of functional expression of alpha1G, either in the absence or the presence of heterologously expressed alpha2-delta, whereas the other beta subunits had much smaller effects. 5. None of the accessory subunits had any marked influence on the voltage dependence or kinetics of the expressed alpha1G currents. These results therefore suggest that alpha2-delta and beta1b interact with alpha1G to increase trafficking of, or stabilize, functional alpha1G channels expressed at the plasma membrane.
Asunto(s)
Canales de Calcio/genética , Canales de Calcio/fisiología , Animales , Células COS , Canales de Calcio/química , Canales de Calcio Tipo T , Chlorocebus aethiops , Femenino , Sustancias Macromoleculares , Potenciales de la Membrana/fisiología , Oocitos/fisiología , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfección , Xenopus laevisRESUMEN
1. High voltage activated (HVA) Ca2+ channels are composed of a pore-forming alpha 1 subunit and the accessory beta and alpha2-delta subunits. However, the subunit composition of low voltage activated (LVA), or T-type, Ca2+ channels has yet to be elucidated. We have examined whether native calcium channels in NG108-15 mouse neuroblastoma x rat glioma hybrid cells, which express predominantly LVA currents when undifferentiated, are modulated by overexpression of accessory calcium channel subunits. 2. Endogenous alpha 1A, B, C, C, and E, and low levels of beta and alpha 2-delta subunit protein were demonstrated in undifferentiated NG108-15 cells. 3. The alpha 2-delta, beta 2a or beta 1b accessory subunits were overexpressed by transfection of the cDNAs into these cells, and the effect examined on the endogenous Ca2+ channel currents. Heterologous expression, particularly of alpha 2-delta but also of beta 2a subunits clearly affected the profile of these currents. Both subunits induced a sustained component in the currents evoked by depolarizing voltages above -30 mV, and alpha 2-delta additionally caused a depolarization in the voltage dependence of current activation, suggesting that it also affected the native T-type currents. In contrast, beta 1b overexpression had no effect on the endogenous Ca2+ currents, despite immunocytochemical evidence for its expression in the transfected cells. 4 These results suggest that in NG108-15 cells, overexpression of the Ca2+ channel accessory subunits alpha 2-delta and beta 2a induce a sustained component of HVA current, and alpha 2-delta also influences the voltage dependence of activation of the LVA current. It is possible that native T-type alpha 1 subunits are not associated with beta subunits.
Asunto(s)
Canales de Calcio/biosíntesis , Canales de Calcio/metabolismo , Animales , Neoplasias Encefálicas/metabolismo , Canales de Calcio/genética , Estimulación Eléctrica , Electrofisiología , Glioma/metabolismo , Células Híbridas , Inmunohistoquímica , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Ratones , Neuroblastoma/metabolismo , Técnicas de Placa-Clamp , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
1. The presence of calcium channel alpha 1D subunit mRNA in cultured rat dorsal root ganglion (DRG) neurones and guinea-pig cardiac myocytes was demonstrated using the reverse transcriptase-polymerase chain reaction. 2. An antipeptide antibody targeted at a region of the voltage-dependent calcium channel alpha 1D subunit C-terminal to the pore-forming SS1-SS2 loop in domain IV (amino acids 1417-1434) only bound to this exofacial epitope if the DRG neurones and cardiac myocytes were depolarized with 30 mM K+. 3. Incubation of cells under depolarizing conditions for 2-4 h with the antibody resulted in a maximal inhibition of inward current density of 49% (P < 0.005) for DRGs and 30% (P < 0.05) for cardiac myocytes when compared with controls. 4. S-(-)-Bay K 8644 (1 microM) enhanced calcium channel currents in DRGs by 75 +/- 19% (n = 5) in neurones incubated under depolarizing conditions with antibody that had been preabsorbed with its immunizing peptide (100 micrograms ml-1). This was significantly (P < 0.05) larger than the enhancement by S-(-)-Bay K 8644 that was seen with cells incubated under identical conditions but with antibody alone, which was 15 +/- 4% (n = 5). 5. These results demonstrate the presence of calcium channel alpha 1D subunits in rat DRG neurones and guinea-pig cardiac myocytes. They also show that amino acids 1417-1434 of the alpha 1D subunit are only exposed to the extracellular face of the membrane following depolarization and that the binding of an antibody to these amino acids attenuates calcium channel current and reduces the ability of S-(-)-Bay K 8644 to enhance this current, indicating that it is an L-type current that is attenuated.
Asunto(s)
Canales de Calcio/fisiología , Ganglios Espinales/fisiología , Corazón/fisiología , Neuronas/fisiología , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Secuencia de Bases , Sitios de Unión de Anticuerpos , Canales de Calcio/biosíntesis , Canales de Calcio/química , Células Cultivadas , Cartilla de ADN , Electrofisiología , Epítopos , Cobayas , Ventrículos Cardíacos , Inmunohistoquímica , Masculino , Potenciales de la Membrana , Microscopía Confocal , Datos de Secuencia Molecular , Miocardio/citología , Neuronas/citología , Reacción en Cadena de la Polimerasa/métodos , Ratas , Ratas Sprague-DawleyRESUMEN
Whole-cell patch-clamp recordings were used to study voltage-gated Ca2+ channel currents in type I carotid body cells of young rats born and reared in normoxia or in a chronically hypoxic (CH) environment (10% O2). Currents activated at potentials of -40 mV and more positive, and typically peaked at 0 mV in both groups of cells. Steady-state inactivation curves were similar in the two populations. Ca2+ currents were significantly larger in CH type I cells, but this was accounted for by the increased size of CH cells: current density was similar in both cell types. Nifedipine (5 microM) always partially inhibited currents and Bay K 8644 (2-5 microM) always enhanced currents, indicating the presence of L-type channels. In a small number of cells from each group, the N-type channel blocker omega-conotoxin GVIA caused partial, irreversible inhibition, but in most cells was without discernible effect. These results indicate that type I cells possess L-type Ca2+ channels, that N-type are expressed in some cells and that non-L, non-N-type channels are also present. Furthermore, chronic hypoxia does not appear to cause specific adaptive changes in the properties of Ca2+ channels in type I cells.
Asunto(s)
Agonistas de los Canales de Calcio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Cuerpo Carotídeo/fisiología , Hipoxia Encefálica/fisiopatología , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Animales , Animales Recién Nacidos , Canales de Calcio/efectos de los fármacos , Cuerpo Carotídeo/citología , Cuerpo Carotídeo/efectos de los fármacos , Enfermedad Crónica , Hipoxia Encefálica/patología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Nifedipino/farmacología , Técnicas de Placa-Clamp , Péptidos/farmacología , Ratas , Ratas Wistar , Valores de Referencia , omega-Conotoxina GVIAAsunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Cuerpo Carotídeo/metabolismo , Hipoxia/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/clasificación , Canales de Calcio/efectos de los fármacos , Canales de Calcio Tipo L , Cuerpo Carotídeo/efectos de los fármacos , Cuerpo Carotídeo/patología , Hipoxia de la Célula , Células Cultivadas , Enfermedad Crónica , Hipoxia/congénito , Transporte Iónico/efectos de los fármacos , Proteínas del Tejido Nervioso/efectos de los fármacos , Nifedipino/farmacología , Péptidos/farmacología , Ratas , omega-Conotoxina GVIARESUMEN
1. Ca(2+)-activated K+ (K+Ca) channels in neonatal rat type I carotid body cells were studied using single channel patch clamp techniques. In outside-out patches, using symmetrical 120 mM [K+] solutions, channels were observed with a slope conductance of 190 pS and a reversal potential of 0 mV. Reducing [K+]o to 5 mM shifted the reversal potential as expected for a K(+)-selective channel. 2. With 100 nM Ca2+ bathing the cytosolic aspect of patches, channel activity (number of active channels in a patch x open probability, NPo) increased with depolarization. NPo also increased with increasing 'cytosolic' [Ca2+] at a fixed membrane potential (0 mV). Using outside-out patches, bath application of 20 or 100 nM charybdotoxin reduced NPo by > 85%. These data indicate the presence of K+Ca channels in type I cells. 3. At 0 mV, using solutions of identical composition (1 microM Ca2+ bathing the cytosolic aspect of the channels), NPo was higher in outside-out patches than in inside-out patches. NPo was greatest in recordings using the perforated-vesicle technique. 4. Hypoxia and anoxia were without effect on K+Ca channels in outside-out patches, but caused significant, reversible reductions of NPo in channels recorded in perforated vesicles. 5. The whole-cell perforated-patch technique was used to record membrane potential at 35-37 degrees C. Hypoxia, anoxia and charybdotoxin all depolarized type I cells. 6. Our results suggest an important role for K+Ca channels in type I carotid body cells, and their activity in relation to a model for chemotransduction is discussed.
Asunto(s)
Calcio/farmacología , Cuerpo Carotídeo/metabolismo , Canales de Potasio/efectos de los fármacos , Animales , Animales Recién Nacidos , Cuerpo Carotídeo/citología , Cuerpo Carotídeo/fisiología , Separación Celular , Caribdotoxina , Hipoxia/fisiopatología , Potenciales de la Membrana/efectos de los fármacos , Oxígeno/metabolismo , Presión Parcial , Ratas , Venenos de Escorpión/farmacologíaRESUMEN
Carotid body-mediated ventilatory increases in response to acute hypoxia are attenuated in animals reared in an hypoxic environment. Normally, O2-sensitive K+ channels in neurosecretory type I carotid body cells are intimately involved in excitation of the intact organ by hypoxia. We have therefore studied K+ channels and their sensitivity to acute hypoxia (PO2 12-20 mmHg) in type I cells isolated from neonatal rats born and reared in normoxic and hypoxic environments. When compared with cells from normoxic rats, K+ current density in cells from hypoxic rats was significantly reduced, whereas Ca2+ current density was unaffected. Charybdotoxin (20 nM) inhibited K+ currents in cells from normoxic rats by approximately 25% but was without significant effect in cells from hypoxic rats. However, hypoxia caused similar, reversible inhibitions of K+ currents in cells from the two groups. Resting membrane potentials (measured at 37 degrees C using the perforated-patch technique) were similar in normoxic and hypoxic rats. However, although acute hypoxia depolarized type I cells of normoxic rats, it was without effect on membrane potential in type I cells from hypoxic animals. Charybdotoxin (20 nM) also depolarized cells from normoxic rats. Our results suggest that type I cells from chronically hypoxic rats, like normoxic rats, possess O2-sensing mechanisms. However, they lack charybdotoxin-sensitive K+ channels that contribute to resting membrane potential in normoxically reared rats, and this appears to prevent them from depolarizing (and hence triggering Ca2+ influx and neurosecretion) during acute hypoxia.
Asunto(s)
Cuerpo Carotídeo/fisiología , Células Quimiorreceptoras/fisiología , Hipoxia/fisiopatología , Neuronas/fisiología , Oxígeno/farmacología , Canales de Potasio/fisiología , Transducción de Señal , Animales , Animales Recién Nacidos , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Cuerpo Carotídeo/fisiopatología , Células Cultivadas , Caribdotoxina , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Ratas , Ratas Wistar , Valores de Referencia , Venenos de Escorpión/farmacologíaRESUMEN
Diphenyleneiodonium (DPI) blocks hypoxic vasoconstriction in the pulmonary vasculature. Because one of the actions of DPI is the inhibition of NADPH oxidase, this has led to the suggestion that NADPH oxidase acts as an oxygen tension sensor in pulmonary smooth muscle cells. We investigated the effects of DPI on potassium and calcium currents in freshly isolated pulmonary artery smooth muscle cells by using whole cell patch-clamp recordings, since these ionic currents are known to be involved in hypoxic pulmonary vasoconstriction. DPI (3 and 10 microM) reversibly inhibited potassium currents, and in its presence, residual currents appeared markedly more transient than under control conditions. The actions of DPI could not be reversed by 4.4 mM hydrogen peroxide, the product of NADPH oxidase. Calcium channel currents were also reversibly inhibited by 3 microM DPI. Thus DPI is a nonselective blocker of ionic channels in pulmonary smooth muscle cells, and its mechanism of action does not appear to involve inhibition of hydrogen peroxide formation. The ability of DPI to block calcium currents can explain its inhibition of hypoxic pulmonary vasoconstriction.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Músculo Liso Vascular/metabolismo , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , Compuestos Onio/farmacología , Canales de Potasio/efectos de los fármacos , Animales , Femenino , Peróxido de Hidrógeno/farmacología , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/enzimología , NADH NADPH Oxidorreductasas/metabolismo , Arteria Pulmonar/citología , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Ratas , Ratas Wistar , Vasoconstricción/fisiologíaRESUMEN
Diphenylene iodonium (DPI), an inhibitor of NAD(P)H oxidase, blocks hypoxic excitation of the carotid body. We used the whole-cell patch-clamp technique to investigate the actions of DPI on ionic currents in isolated type I carotid body cells. DPI (10 microM) caused reversible blockade of K+ and Ca2+ currents in these cells, indicating that DPI is a non-selective ion channel blocker. Since hypoxic excitation of the carotid body is dependent on Ca2+ influx into type I cells, our observation that DPI blocks Ca2+ currents in type I cells can account for the ability of this compound to inhibit hypoxic excitation of the intact organ.
Asunto(s)
Animales Recién Nacidos/metabolismo , Compuestos de Bifenilo/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Cuerpo Carotídeo/metabolismo , Neuronas/metabolismo , Compuestos Onio/farmacología , Canales de Potasio/efectos de los fármacos , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Cuerpo Carotídeo/citología , Cuerpo Carotídeo/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , RatasAsunto(s)
Calcio/farmacología , Cuerpo Carotídeo/fisiología , Canales de Potasio/fisiología , Animales , Animales Recién Nacidos , Cuerpo Carotídeo/citología , Caribdotoxina , Doxapram/farmacología , Epitelio/fisiología , Técnicas In Vitro , Cinética , Potenciales de la Membrana/efectos de los fármacos , Bloqueadores de los Canales de Potasio , Ratas , Venenos de Escorpión/farmacologíaAsunto(s)
Calcio/metabolismo , Cuerpo Carotídeo/fisiología , Yoduro de Dimetilfenilpiperazina/farmacología , Potenciales Evocados/efectos de los fármacos , Nicotina/farmacología , Agonistas Nicotínicos , Animales , Animales Recién Nacidos , Cuerpo Carotídeo/citología , Epitelio/efectos de los fármacos , Epitelio/fisiología , Técnicas In Vitro , Técnicas de Placa-Clamp , RatasRESUMEN
The actions of two structurally related tricyclic antidepressants on neuronal nicotinic acetylcholine receptors were investigated in human neuroblastoma (SY-SY5Y) cells, using whole-cell patch-clamp recordings. Both desipramine and imipramine reversibly inhibited inward currents evoked by application of the nicotinic receptor agonist dimethylphenylpiperazinium iodide (30-300 microM) with IC50 values of 0.17 microM and 1.0 microM respectively (holding potential -70 mV). The degree of current inhibition caused by either tricyclic compound was unaffected by agonist concentration (30-300 microM). The effects of desipramine were voltage-independent over the range -40 mV to -100 mV, and inhibition caused by imipramine only increased very slightly with membrane hyperpolarization over the same range. These results indicate that tricyclic antidepressants can inhibit neuronal nicotinic acetylcholine receptors by mechanisms which are distinct from their actions at non-neuronal nicotinic acetylcholine receptors.
Asunto(s)
Desipramina/farmacología , Imipramina/farmacología , Neuronas/metabolismo , Antagonistas Nicotínicos , Yoduro de Dimetilfenilpiperazina/farmacología , Electrofisiología , Humanos , Canales Iónicos/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Neoplasias del Sistema Nervioso/metabolismo , Neuroblastoma/metabolismo , Neuronas/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Electrophysiological responses of enzymatically isolated type I cells from the neonatal rat carotid body to cholinergic agonists were examined using the whole-cell patch-clamp technique. Inward currents were evoked in cells clamped at -70 mV in response to bath-applied carbachol and two selective nicotinic agonists, nicotine and dimethylphenylpiperazinium. Muscarine failed to produce any change in membrane current. Responses to nicotine were concentration-dependent and also voltage-dependent, showing strong rectification positive to -40 mV. Currents evoked by nicotine were reduced or abolished in the presence of mecamylamine and also by high concentrations of atropine (10 or 100 microM). Under "current-clamp", nicotine was shown to depolarize type I cells, an effect which was only slowly reversible, but which could be rapidly attenuated by introduction of mecamylamine to the perfusate. In voltage-clamped cells, nicotine could evoke inward currents when extracellular Na+ was replaced by Ca2+. Our results demonstrate the presence of functional nicotinic acetylcholine receptors on type I cells of the neonatal rat carotid body. Activation of these receptors could lead to excitation of the intact carotid body by either of two possible mechanisms: depolarization of type I cells sufficient to open voltage-gated Ca2+ channels, or Ca2+ influx through the receptor pore itself. Either (or both) mechanisms could trigger catecholamine release from type I cells, which is a fundamental step in chemotransmission.
Asunto(s)
Cuerpo Carotídeo/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Animales Recién Nacidos , Calcio/metabolismo , Cuerpo Carotídeo/citología , Cuerpo Carotídeo/fisiología , Separación Celular , Electrofisiología , Nicotina/farmacología , Concentración Osmolar , Parasimpaticomiméticos/farmacología , Ratas , Ratas Sprague-Dawley , Ratas WistarRESUMEN
The sulphydryl reagent, p-chloromercuribenzenesulfonic acid (PCMBS), irreversibly excites the carotid body in vivo. We tested the effects of PCMBS on ionic currents in isolated type I cells, using the whole-cell patch-clamp technique. PCMBS selectively and irreversibly inhibited the Ca(2+)-activated K+ current (IKCa) in a dose-dependent manner (0.01-1 mM). The same concentrations of PCMBS did not affect the Ca(2+)-independent K+ current (IKv), but caused a transient enhancement of the Ca2+ current (ICa). The inhibition of IKCa by PCMBS is similar to the previously reported effects of hypoxia, and suggests a central role for the channels underlying this current in chemotransduction.