RESUMEN
The R-enantiomer of isovaline, an analgesic amino acid, has a chemical structure similar to glycine and GABA. Although its actions on thalamic neurons are strychnine-resistant and independent of the Cl(-) gradient, R-isovaline increases membrane conductance for K(+). The purpose of this study was to determine if R-isovaline activated metabotropic GABA(B) receptors. We used whole-cell voltage-clamp recordings to characterize the effects of R-isovaline applied by bath perfusion and local ejection from a micropipette to thalamic neurons in 250 µm thick slices of rat brain. The immunocytochemical methods that we employed to visualize GABA(B1) and GABA(B2) receptor subunits showed extensive staining for both subunits in ventrobasal nuclei, which were the recording sites. Bath or local application of R-isovaline caused a slowly developing increase in conductance and outward rectification in 70% (54/77) of neurons, both effects reversing near the K(+) Nernst potential. As with the GABA(B) agonist baclofen, G proteins likely mediated the R-isovaline effects because they were susceptible to blockade by non-hydrolyzable substrates of guanosine triphosphate. The GABA(B) antagonists CGP35348 and CGP52432 prevented the conductance increase induced by R-isovaline, applied by bath or local ejection. The GABA(B) allosteric modulator CGP7930 enhanced the R-isovaline induced increase in conductance. At high doses, antagonists of GABA(A), GABA(C), glycine(A), µ-opioid, and nicotinic receptors did not block R-isovaline responses. The observations establish that R-isovaline increases the conductance of K(+) channels coupled to metabotropic GABA(B) receptors. Remarkably, not all neurons that were responsive to baclofen responded to R-isovaline. The R-isovaline-induced currents outlasted the fast baclofen responses and persisted for a 1-2-h period. Despite some similar actions, R-isovaline and baclofen do not act at identical GABA(B) receptor sites. The binding of R-isovaline and baclofen to the GABA(B) receptor may not induce the same conformational changes in receptor proteins or components of the intracellular signaling pathways.
Asunto(s)
Agonistas de Receptores GABA-B/farmacología , Neuronas/efectos de los fármacos , Valina/farmacología , Animales , Animales Recién Nacidos , Relación Dosis-Respuesta a Droga , GABAérgicos/farmacología , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Técnicas In Vitro , Isomerismo , Potenciales de la Membrana/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Receptores de GABA-B/metabolismo , Tálamo/citología , Tionucleótidos/farmacología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The rare amino acid isovaline has analgesic properties in pain models and is a structural analogue of the inhibitory neurotransmitter glycine. Glycinergic inhibition is prevalent in pain pathways. In this paper, we examined the possibility that isovaline inhibits neurons by activating strychnine (Str)-sensitive glycine(A) receptors in ventrobasal thalamus. Sagittal brain sections containing ventrobasal nuclei were prepared from P10-P15 rats. Whole-cell recordings were made in current-clamp and voltage-clamp modes. R-isovaline (R-Iva) increased input conductance and hyperpolarized the membrane. The conductance increase shunted action potentials and low-threshold Ca(2+) spikes evoked by current pulse injection. Unlike the Cl(-)-mediated responses to glycine, isovaline responses were insensitive to Str antagonism and usually not reversible. The concentration-response curve was non-sigmoidal, rising to a maximum at approximately 100 microM, and thereafter declining in amplitude. Current-voltage relationships showed that isovaline increased inward and outward rectification. The isovaline current reversed polarity close to the K(+) equilibrium potential. The relationships were negligibly affected by tetrodotoxin (TTX), chelation of intracellular Ca(2+) or blockade of the hyperpolarization-activated current, I(h). Internal Cs(+) and external Ba(2+) or Cs(+) prevented isovaline responses. In conclusion, isovaline inhibited firing mainly by activating rectifying and possibly leak K(+) currents. Isovaline-induced changes shunted action potentials and suppressed rebound excitation in ventrobasal neurons, as expected for analgesic actions.
Asunto(s)
Analgésicos/farmacología , Neuronas/efectos de los fármacos , Dolor/tratamiento farmacológico , Canales de Potasio/efectos de los fármacos , Valina/farmacología , Núcleos Talámicos Ventrales/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Analgésicos/uso terapéutico , Animales , Animales Recién Nacidos , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Relación Dosis-Respuesta a Droga , Glicina/agonistas , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Neuronas/metabolismo , Técnicas de Cultivo de Órganos , Dolor/metabolismo , Dolor/fisiopatología , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Glicina/efectos de los fármacos , Receptores de Glicina/metabolismo , Valina/uso terapéutico , Núcleos Talámicos Ventrales/metabolismoRESUMEN
We studied the effects of pentobarbital and antagonists of glutamate, gamma-aminobutyrate (GABA), and glycine receptors on extracellular activity in ventrobasal thalamic slices. Pentobarbital at sedative-hypnotic concentration (20 microM) reversibly induced 1-15 Hz oscillations. Sustained oscillations required electrical stimulation of internal capsule, but not elevated temperature or low [Mg2+]. Anesthetic concentration (200 microM) of pentobarbital evoked only transient oscillations. Kynurenate-sensitive glutamate receptors were essential for oscillations. GABA(A) antagonism (bicuculline, 50 microM or gabazine, 20 microM) suppressed oscillations at 5-15 Hz. GABA(B) antagonism (CGP 35348, 100 nM), or antagonism of glycine receptors (strychnine, 1 microM) suppressed oscillations at 1-4 and 11-15 Hz. GABA and glycine receptors modulated oscillation frequency. For elimination, oscillations required GABA antagonists and strychnine. Receptors for glutamate and glycine mediated oscillations during GABA receptor blockade in ventrobasal nuclei, or on disconnection from nRT. Glycine receptors were critical for oscillations in dorsal thalamic network, divested of GABAergic inhibition. Glutamate and GABA receptors mediated pentobarbital-induced oscillations in nRT, disconnected from ventrobasal nuclei. Hence, pentobarbital oscillogenesis occurred in isolated networks of the ventrobasal and reticularis nuclei mediated by glutamate receptors, with frequency modulation by GABA(A), GABA(B), and glycine receptors. These stationary oscillations represent a model of sedation-hypnosis, amenable to pharmacological analysis.
Asunto(s)
Hipnóticos y Sedantes/farmacología , Pentobarbital/farmacología , Receptores de GABA/efectos de los fármacos , Receptores de Glicina/efectos de los fármacos , Tálamo/efectos de los fármacos , Animales , Ganglios Basales/efectos de los fármacos , Ganglios Basales/fisiología , Estimulación Eléctrica , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/fisiología , Análisis de Fourier , Antagonistas del GABA/farmacología , Glicinérgicos/farmacología , Técnicas In Vitro , Red Nerviosa/efectos de los fármacos , Compuestos Organofosforados/farmacología , Piridazinas/farmacología , Ratas , Receptores de Glicina/antagonistas & inhibidores , Estricnina/farmacologíaRESUMEN
1. We studied amobarbital's effects on membrane properties and currents, and electrically evoked inhibitory postsynaptic currents (IPSCs) mediated by gamma-aminobutyric acid (GABA) in rat thalamic slices. Using concentration-response relationships, we compared amobarbital's effects in nociceptive nuclei and non-nociceptive nucleus reticularis thalami (nRT). 2. Amobarbital decreased input resistance by activating GABA(A) receptors. Amobarbital produced a larger decrease in ventrobasal than nRT neurons. 3. Amobarbital depressed burst and tonic firing. Depression of burst firing was more effective, particularly in ventrobasal and intralaminar neurons. Depression was reversed by GABA(A) antagonists, and surmountable by increasing current injection, implicating a receptor-mediated shunt mechanism. 4. Amobarbital did not affect the tetrodotoxin-isolated low threshold Ca(2+) spike during GABA(A) blockade. Amobarbital reduced excitability without altering outward leak, or hyperpolarisation-activated inward currents. 5. Amobarbital increased mean conductance and burst duration of single GABA(A) channels. Consistent with this, amobarbital increased amplitude and decay time of IPSCs with distinct EC(50)s, implicating actions at two GABA(A) receptor sites. 6. Activation of GABA(A) receptors by low concentrations, fast IPSC amplitude modulation, and failure to affect intrinsic currents distinguished amobarbital's mechanism of action from previously characterised barbiturates. The selective actions of amobarbital on GABA(A) receptor may have relevance in explaining anaesthetic and analgesic uses.
Asunto(s)
Amobarbital/farmacología , Neuronas/efectos de los fármacos , Receptores de GABA-A/fisiología , Tálamo/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Neuronas/fisiología , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Tálamo/fisiologíaRESUMEN
We investigated interactions of an anesthetic barbiturate, pentobarbital, with non-ligand gated channels and identified inhibitory synaptic transmission in thalamic neurons. Using whole cell voltage-clamp, current-clamp and single channel recording techniques in rat ventrobasal neurons of slices and dispersed preparations, we determined the mechanisms of pentobarbital actions on ionic currents and inhibitory postsynaptic currents (IPSCs), mediated by aminobutyric acid (GABA). We investigated pentobarbital effects on intrinsic currents using hyperpolarizing voltage commands from rest and tetrodotoxin blockade of action potentials. At concentrations near 8 microM, pentobarbital increased input conductance and induced net outward current, I(PB), at potentials near action potential threshold. The reversal potential of I(PB) was -75 mV, implicating K+ and other ions. Cs+ (3 mM) which inhibits both K+ currents and inward rectifier (Ih), completely blocked IPB, whereas the selective Ih blocker, ZD-7288 (25 microM), or Ba2+ (2 mM) which suppresses only K+ currents, reduced IPB. Pentobarbital inhibited the Ih, consistent with a ZD-7288-induced shift in reversal potential for IPB toward K+ equilibrium potential. Pentobarbital increased the inward K+ rectifier, IKir, and leak current, Ileak. We determined the susceptibility of IPSCs, evoked by reticular stimulation, to antagonism by bicuculline, picrotoxinin and 2-hydroxysaclofen and identified their receptor subclass components. At EC50 = 53 microM, pentobarbital increased the duration of IPSCs. The prolonged IPSC duration during pentobarbital was attributable to enhanced open probability of GABAA channels, because combined with GABA, pentobarbital application increased mean channel open time without affecting channel conductance. At concentrations up to 100 microM, pentobarbital did not directly activate GABAA receptors. The concentration-response relationships for pentobarbital effects on the intrinsic currents and IPSCs overlapped, implying multiple sites of action and possible redundancy in anesthetic mechanisms. This is the first study to show that an i.v. anesthetic modulates the intrinsic currents, Ih, IKir, and Ileak, as well as IPSC time course in the same neurons. These effects likely underlie inhibition in thalamocortical neurons during pentobarbital anesthesia.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Inhibición Neural/efectos de los fármacos , Vías Nerviosas/efectos de los fármacos , Pentobarbital/farmacología , Receptores de GABA/efectos de los fármacos , Tálamo/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Relación Dosis-Respuesta a Droga , Antagonistas del GABA/farmacología , Técnicas In Vitro , Canales Iónicos/efectos de los fármacos , Canales Iónicos/fisiología , Inhibición Neural/fisiología , Vías Nerviosas/citología , Vías Nerviosas/metabolismo , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Tiempo de Reacción/fisiología , Receptores de GABA/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Tetrodotoxina/farmacología , Tálamo/citología , Tálamo/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
The role of inducible nitric oxide synthase (iNOS) in the acute activation of large-conductance, Ca(2+)-dependent K(+) channels (BK channels) by Escherichia coli endotoxin (lipopolysaccharide, LPS) was studied in murine vascular smooth muscle cells. Confocal laser scanning microscopy and patch clamp recordings were utilised. Within 2 h of donor rat sacrifice, iNOS-like immunoreactivity could be detected in cerebrovascular smooth muscle cells (CVSMCs) enzymatically dispersed from rat cerebral arteries. This staining was absent in cells fixed immediately after donor rat sacrifice. LPS was then applied to the cytoplasmic face of inside-out membrane patches excised from rat CVSMCs within 2-4 h of donor rat sacrifice. It was found that LPS (10-100 microg/ml) rapidly and reversibly increased the open probability of BK channels in these patches. This LPS response was not altered in the presence of the non-isoform specific NOS inhibitor N(omega)-nitro-L-arginine. LPS responses were then compared in aortic smooth muscle (ASMC) BK channels derived from wild-type and iNOS-knockout (iNOS-KO) mice. LPS activated BK channels in inside-out patches of ASMC membrane derived from both wild-type and iNOS-knockout mice. These studies establish that LPS can activate BK channels by a mechanism quite independent of the well-established pathway mediated by iNOS in vascular smooth muscle cells.
Asunto(s)
Lipopolisacáridos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Animales , Calcio/farmacología , Células Cultivadas , Arterias Cerebrales/efectos de los fármacos , Inmunohistoquímica , Cinética , Potenciales de la Membrana , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa/análisis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
The effects of p-nitroblue tetrazolium (NBT) on large conductance, calcium-activated potassium channels (BK channels) in enzymatically dispersed rat cerebrovascular smooth muscle cells (CVSMCs) were examined. Patch clamp methods were employed to record single BK channel currents from inside-out patches of CVMC membrane maintained at 21 - 23 degrees C. When applied to the cytoplasmic face of inside-out membrane patches (internally applied NBT), micromolar concentrations of NBT reversible reduced the mean open time of BK channels, without changing channel conductance. NBT altered the frequency distribution of BK channel open times from a two exponential to a single exponential form. In the absence of NBT, mean channel open time increased on membrane depolarization. In the presence of internally applied NBT, mean channel open became essentially independent of membrane potential. Internally applied NBT also reduced the mean closed time of BK channels when measured at membrane potentials in the range -80 mV to +20 mV. The combined effects of internal NBT on mean open and closed times resulted in the suppression of BK channel open probability when measured at positive membrane potentials. When applied to the external membrane face, micromolar concentrations of NBT reduced mean channel open time progressively as the membrane was hyperpolarized, and also reduced open probability at negative membrane potentials. A model is proposed in which NBT alters channel gating by binding to a site at or near to the cytoplasmic membrane face. Externally applied NBT suppressed BK channel open probability at concentrations which also inhibit nitric oxide synthase (NOS). Therefore, the potential role of potassium channel block in NBT actions previously attributed to NOS inhibition is discussed.
Asunto(s)
Músculo Liso Vascular/metabolismo , Nitroazul de Tetrazolio/farmacología , Bloqueadores de los Canales de Potasio , Canales de Potasio Calcio-Activados , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Arterias Cerebrales/citología , Arterias Cerebrales/efectos de los fármacos , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Cinética , Canales de Potasio de Gran Conductancia Activados por el Calcio , Potenciales de la Membrana/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
We have investigated the ability of LIPOFECTAMINE, a polycationic lipid reagent used in DNA transfection, to translocate E. coli lipopolysaccharide (LPS) into HeLa cells. Although HeLa cells did not spontaneously take up fluorescein isothiocyanate-labelled LPS (FITC-LPS) from the culture medium, the cells that were co-incubated with greater than 1 g/mL FITC-LPS and LIPOFECTAMINE showed punctate fluorescence. Virtually all cells were loaded on incubation with 100 micrograms/mL FITC-LPS. Confocal scanning laser microscopy showed extensive FITC-LPS loading in the cytoplasm of HeLa cells, but no label was evident in the nuclear regions of these cells. Loading with LPS for up to six hours had no effect on the viability of HeLa cells, beyond the 30% reduction in live cells that is attributable to the toxic effect of LIPOFECTAMINE itself. In contrast to cells treated with etoposide for six hours, LPS-loaded cells did not display apoptotic bodies. Exposure of cells to 4 beta-phorbol 12-myristate 13-acetate led to the induction of the immediate early gene c-fos and resulted in an enhanced c-Fos signal, detected by Western blot analysis. In contrast, LPS loading did not alter the c-fos expression in HeLa cells. The loading of LPS into HeLa cells by means of polycationic lipids results in relatively low acute toxicity, as judged from cell viability, morphology and c-fos expression. Therefore, our method appears well suited to the study of acute actions of LPS in the intracellular compartment of mammalian cells.
Asunto(s)
Resinas de Intercambio de Catión/farmacología , Lípidos/farmacología , Lipopolisacáridos/farmacocinética , Transporte Biológico , Genes fos , Células HeLa , HumanosRESUMEN
Patch-clamp recordings were used to study the effects of Escherichia coli bacterial endotoxin (lipopolysaccharide, LPS) on the properties of large-conductance, Ca2+-dependent K+ channels (BK channels) in the membrane of enzymatically dispersed rat cerebrovascular smooth muscle cells (CVSMCs). LPS had negligible effects on the kinetic and conductance properties of BK channels when applied to the extracellular domain of these channels. However, acute application of LPS (10-100 microg/ml) to inside-out patches of CVSMC membrane isolated in a cell-free environment rapidly and reversibly increased the open probability of BK channels, leaving the conductance of these channels unaltered. The magnitude of this effect decreased as the concentration of free Ca2+ at the cytoplasmic membrane face was lowered, but was little affected by changes in membrane potential. Kinetic analysis showed that LPS accelerated reopening of BK channels while having little effect on mean channel open time. Detoxified E. coli LPS, from which the fatty acid chains of Lipid A were partially removed, showed slightly reduced activity when compared to the parent endotoxin molecule. A purified E. coli Lipid A had negligible effects on BK channel function. These results indicate that LPS activates BK channels in cerebrovascular smooth muscle cells when present at the cytoplasmic membrane face. This novel mechanism may provide insights into the regulation of BK channels by intracellular, membrane-associated elements.
Asunto(s)
Encéfalo/irrigación sanguínea , Lipopolisacáridos/farmacología , Músculo Liso Vascular/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Escherichia coli/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio/metabolismo , RatasRESUMEN
1. In this study the role of nitric oxide synthase (NOS) in the acute activation of large conductance, Ca2+-activated K+ channels (BK channels) by internally applied E. coli lipopolysaccharide (LPS, endotoxin) was examined in vascular smooth muscle cells. 2. Cerebrovascular smooth muscle cells (CVSMCs) were enzymatically dispersed from the middle, posterior communicating and posterior cerebral arteries of adult Wistar rats and maintained at 4 degrees C for 2-4 days before recording with standard patch-clamp techniques. 3. Acute application of LPS (100 microg ml(-1)) to inside-out patches of CVSMC membrane isolated in a cell-free environment rapidly and reversibly increased the open probability, Po of BK channels in these patches by 3.3+/-0.30 fold. 4. Acute application of the nitric oxide (NO) donor sodium nitroprusside (SNP, 100 microM) to inside-out patches of CVSMC membrane, studied in the presence of intact cells, also reversibly increased Po, by some 1.8+/-0.2 fold over control. 5. Kinetic analysis showed that both LPS and SNP increased Po by accelerating the rate of BK channel reopening, rather than by retarding the closure of open channels. 6. Neither LPS nor SNP altered the reversal potential or conductance of BK channels. 7. The NOS substrate L-arginine (1 microM) potentiated the acute activation of BK channels by LPS, while the synthetic enantiomer D-arginine (1 microM) inhibited the action of LPS on BK channels. 8. The acute activation of BK channels by LPS was suppressed by pre-incubation of cells with N(omega)-nitro-L-arginine (50 microM) or N(omega)-nitro-L-arginine methyl ester (1 mM), two competitive antagonists of nitric oxide synthases. N(omega)-nitro-D-arginine (50 microM), a poor inhibitor of NOS in in vitro assays, had no effect on BK channel activation by LPS. 9. These results indicate that excised, inside-out patches of CVSMC membrane exhibit a NOS-like activity which is acutely activated when LPS is present at the cytoplasmic membrane surface. Possible relationships between this novel mechanism and the properties of known isoforms of nitric oxide synthase are discussed.
Asunto(s)
Bradiquinina/metabolismo , Arterias Cerebrales/metabolismo , Endotoxinas/farmacología , Canales Iónicos/metabolismo , Lipopolisacáridos/farmacología , Músculo Liso Vascular/metabolismo , Óxido Nítrico/fisiología , Animales , Separación Celular , Arterias Cerebrales/citología , Arterias Cerebrales/efectos de los fármacos , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Cinética , Potenciales de la Membrana/fisiología , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitroprusiato/farmacología , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
Lipopolysaccharide (LPS) may accumulate inside mammalian cells through endocytotic uptake or during the replication in invasive bacterial strains. However, the effects of intracellular LPS on cell function remain unknown. This study therefore examined the action of intracellularly applied E. coli LPS on large-conductance, Ca2+-dependent K+ channels (BK channels) in the membrane of rat cerebrovascular smooth muscle cells (CVSMCs). LPS (10-100 microg/ml) rapidly increased the open probability of BK channels when applied to the cytoplasmic face of CVSMC membrane patches. This response was reversible, dose-dependent and reflected an enhanced rate of channel opening in the presence of LPS. These results show for the first time that LPS can alter the gating behavior of ionic channels when applied to the cytoplasmic face of a eukaryotic cell membrane.
Asunto(s)
Arterias Cerebrales/química , Activación del Canal Iónico/efectos de los fármacos , Lipopolisacáridos/farmacología , Músculo Liso Vascular/química , Canales de Potasio/metabolismo , Animales , Calcio/farmacología , Arterias Cerebrales/metabolismo , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
It is presumed, but not proven, that enteropathogenic Escherichia coli (EPEC) causes secretory diarrhea by altering ion transport in enterocytes. In this study we used the whole-cell, current clamp variant of the patch clamp technique to demonstrate that EPEC infection of HeLa and Caco-2 human epithelial cells reduces cell resting membrane potential. The observed reduction of resting membrane potential in HeLa cells results from EPEC-mediated signal transduction to the host cell but is not dependent upon EPEC-mediated elevation of levels of intracellular free calcium. These findings indicate that EPEC can directly alter the relative distribution of ions across epithelial host cell membranes. This may be relevant to the etiology of diarrhea caused by EPEC infection.
Asunto(s)
Membrana Celular/fisiología , Epitelio/microbiología , Escherichia coli/fisiología , Células CACO-2 , Calcio/metabolismo , Epitelio/fisiología , Escherichia coli/patogenicidad , Células HeLa , Humanos , Potenciales de la Membrana , Técnicas de Placa-Clamp , Transducción de SeñalRESUMEN
The oral spirochete Treponema denticola is closely associated with periodontal diseases in humans. The 53-kDa major surface protein (Msp) located in the outer membrane of T. denticola serovar a (ATCC 35405) has both pore-forming activity and adhesin activity. We have used standard patch clamp recording methods to study the effects of a partially purified outer membrane complex containing Msp on HeLa cells. The Msp complex was free of the chymotrypsin-like proteinase also found in the outer membrane of T. denticola. Msp bound to several HeLa cell proteins, including a 65-kDa surface protein and a 96-kDa cytoplasmic protein. The Msp complex depolarized and increased the conductance of the HeLa cell membrane in a manner which was not strongly selective for Na+, K+, Ca2+, and Cl- ions. Cell-attached patches of HeLa cell membrane exposed to Msp complex exhibited short-lived channels with a slope conductance of 0.4 nS in physiologically normal saline. These studies show that Msp binds both a putative epithelial cell surface receptor and cytoplasmic proteins and that the Msp complex can form large conductance ion channels in the cytoplasmic membrane of epithelial cells. These properties may contribute to the cytopathic effects of T. denticola on host epithelial cells.
Asunto(s)
Proteínas Bacterianas , Membrana Celular/metabolismo , Porinas/metabolismo , Aniones/metabolismo , Cationes/metabolismo , Conductividad Eléctrica , Células HeLa , Humanos , Técnicas de Placa-Clamp , Porinas/química , Unión Proteica , Treponema , Infecciones por Treponema/etiologíaRESUMEN
The effects of intracellular magnesium ions, Mg2+i on large-conductance, Ca(2+)-dependent K+ channels (BK channels) of adult rat cerebrovascular smooth muscle cells (CVSMCs) were studied using patch clamp techniques and cells enzymatically dispersed from basilar, middle, and posterior cerebral arteries. Recordings used inside-out membrane patches and took place at 20-24 degrees C. One millimeter [Mg2+]i produced a fast block of BK channel currents, as well described by the Woodhull model of channel occlusion by a charged species. However, the affinity and voltage-sensitivity of Mg2+i block were dependent on the concentration of free intracellular calcium ions, [Ca2+]i. Calcium ions may stabilize a channel conformation in which Mg2+i binding sites are relocated closer to the inner membrane surface. In the presence of 1 microM [Ca2+]i, 0.5 mM [Mg2+]i shifted the Boltzmann curve relating BK channel open probability, Po, to membrane voltage leftward on the voltage axis, without any change in its slope. The enhancing effect of Mg2+i on Po was, therefore, insensitive to membrane potential. Quantitative considerations suggest that physiological levels of Mg2+i tonically facilitate BK channel activation. Alterations of [Mg2+]i during hyper- or hypomagnesemia may contribute to the dilation or contraction of cerebral vessels seen under these two conditions.
Asunto(s)
Calcio/fisiología , Circulación Cerebrovascular , Magnesio/metabolismo , Músculo Liso Vascular/metabolismo , Canales de Potasio/fisiología , Animales , Células Cultivadas , Conductividad Eléctrica , Membranas Intracelulares/metabolismo , Modelos Cardiovasculares , Músculo Liso Vascular/citología , Concentración Osmolar , Ratas , Ratas WistarRESUMEN
Inside-out patch clamp recordings have revealed the presence of a novel, large conductance channel of the non-selective, cation permeable type in smooth muscle cells dispersed from the cerebral arteries of adult rats. In physiologically appropriate ionic gradients, current flow in these channels reversed polarity at a membrane potential of about -42 mV. Single channel conductance in symmetrical 140 mM K+ salines was 211 pS. The channel was permeable to both K+ and Na+, with a ratio PNa/PK = 0.15, while Cl- was effectively impermeant. Calcium ions were weakly permeant (PCa/PK = 0.03, PCa/PNa = 0.20). Channel open probability increased with membrane depolarization and was weakly dependent on the concentration of free intracellular Ca2+. This channel would contribute outward membrane current at potentials more positive than about -42 mV. In concert with outward potassium currents, it may serve to limit membrane depolarization during action potential activity in cerebrovascular smooth muscle cells.
Asunto(s)
Arterias Cerebrales/metabolismo , Canales Iónicos/análisis , Músculo Liso Vascular/metabolismo , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Potenciales de la Membrana , Técnicas de Placa-Clamp , Potasio/metabolismo , Ratas , Ratas Wistar , Sodio/metabolismoRESUMEN
BACKGROUND: Patch-clamp methods were used to examine the effects of the volatile general anesthetic halothane on large-conductance calcium-dependent potassium channels (BK channels) in dispersed cerebrovascular smooth muscle cells of adult rats. METHODS: Inside-out membrane patches were used for recordings at 21-23 degrees C. Halothane was administered at aqueous concentrations of 0.5-2.8 mM in conjunction with free cytoplasmic calcium concentrations of 1 or 100 microM and at a membrane potential of -60 mV or +60 mV. RESULTS: At a free cytoplasmic calcium concentration of 1 microM, the clinically relevant dose of 0.5 mM (2 MAC) halothane reduced the open probability of large-conductance calcium-dependent potassium channels without altering the single-channel conductance. This effect was blocked by increasing the concentration of cytoplasmic free calcium to 100 microM, but was not intrinsically voltage dependent. CONCLUSION: The marked dilation of cerebral vessels seen during surgical anesthesia with halothane cannot be attributed to direct effects of the drug on large-conductance calcium-dependent potassium channels. The protective effect of calcium suggests that halothane exerts its effects at channel sites located within the cell membrane.
Asunto(s)
Calcio/fisiología , Arterias Cerebrales/efectos de los fármacos , Halotano/farmacología , Músculo Liso Vascular/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Animales , Calcio/farmacología , Arterias Cerebrales/citología , Arterias Cerebrales/fisiología , Electrofisiología , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Conducción Nerviosa/efectos de los fármacos , Conducción Nerviosa/fisiología , Ratas , Ratas Wistar , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
1. Cerebrovascular smooth muscle cells (CVSMCs) were dispersed from cerebral arteries of adult rats using collagenase and trypsin. The extracellular patch clamp technique was used to study single calcium-activated potassium channels, KCa+ channels, in these cells at 21-23 degrees C. 2. Whole-cell, current clamp recordings showed that isolated CVSMCs possessed a mean resting potential of -41 +/- 7.4 mV (n = 69), an input resistance of 3.2 +/- 0.49 G omega (n = 20) and a capacitance of 24 +/- 2.3 pF (n = 7). 3. Inside-out patches displayed a calcium-dependent potassium channel, KCa+ channel, of mean conductance 207 +/- 10 pS (n = 16) and potassium permeability 3.9 x 10(-13) cm s-1 (n = 16) in symmetrical 140 mM K+ solutions. No substate conductance level was evident. 4. This channel was highly selective for K+ over Na+ or Cs+ (permeability ratio PNa/PK < 0.05; PCs/PK < 0.05, n = 5 patches in each case). Cs+ caused a voltage-dependent block of the open channel. 5. Channel openings were detected at a threshold level of free internal calcium, [Ca2+]i = 10(-8) M, and channels were open half of the time at [Ca2+]i = 2.3 x 10(-5) M (membrane potential, Vm = +40 mV, n = 5). Over the probable physiological range of [Ca2+]i, the open probability of the KCa+ channel increased with the second power of calcium concentration. 6. Open time distributions were well fitted by the sum of two exponential terms, showing the occurrence of at least two kinetically distinguishable open states. Raising [Ca2+]i increased the time constant of the slow exponential component, but had no effect on that of the fast component. 7. At [Ca2+]i < 5 x 10(-5) M, a 14 mV depolarization in membrane potential resulted in an e-fold increase in the probability of KCa+ channels adopting an open state (n = 5). The slow time constant of the open time distributions also increased on membrane depolarization. 8. Tetraethylammonium ions applied to the cytoplasmic membrane face caused a reversible, dose-dependent reduction in current flow through the KCa+ channel. This block was characterized by a dissociation constant of 0.83 +/- 0.09 mM at Vm = +40 mV and [Ca2+]i = 10(-4) M (n = 5). 9. The lower calcium sensitivity and higher sensitivity to tetraethylammonium block distinguish this from other large conductance KCa+ channels in smooth muscle cells.
Asunto(s)
Calcio/metabolismo , Arterias Cerebrales/fisiología , Activación del Canal Iónico/fisiología , Músculo Liso Vascular/metabolismo , Canales de Potasio/fisiología , Animales , Células Cultivadas , Cesio/metabolismo , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica/fisiología , Impedancia Eléctrica , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/fisiología , Canales de Potasio/efectos de los fármacos , Ratas , Ratas Wistar , Compuestos de Tetraetilamonio/farmacologíaRESUMEN
The effects of the benzothiadiazine TAG on membrane channels activated by taurine and glycine were investigated in dissociated spinal cord neurons from embryonic mice. TAG (100-200 microM) dose-dependently and reversibly antagonized the ability of glycine and taurine to activate chloride permeable ionic channels in these patches. This effect of TAG was due to shortening of the mean open time of the glycine and taurine activated channels. In addition, TAG significantly increased the mean shut time of taurine activated channels. The mean amplitude single channel currents activated by either agonist was unaltered by TAG. Single channel currents activated by taurine were significantly more sensitive to the blocking action of TAG than currents activated by glycine.
Asunto(s)
Benzotiadiazinas/farmacología , Proteínas de la Membrana/efectos de los fármacos , Receptores de Neurotransmisores/metabolismo , Animales , Canales de Cloruro , Electrofisiología , Glicina/farmacología , Proteínas de la Membrana/fisiología , Ratones , Ratones Endogámicos , Neuronas/metabolismo , Receptores de Glicina , Médula Espinal/citología , Médula Espinal/metabolismo , Taurina/farmacologíaRESUMEN
Patch clamp methods were used to study the properties of calcium-dependent K(Ca) channels in enzyme dissociated smooth muscle cells from the cerebral arteries of adult rats. Dissociated muscle cells were maintained at 4 degrees C for up to 48 h prior to use. Inside-out membrane patches excised from these cells contained a K(Ca) channel with a conductance of 92 +/- 2.6 pS in symmetrical 140 mM potassium solutions. This channel was activated by membrane depolarization and by cytoplasmic calcium, and showed negligible permeability to sodium or cesium ions. Single channel currents were reduced by internal application of tetraethylammonium ions, with a Kd = 0.31 mM.
Asunto(s)
Calcio/fisiología , Arterias Cerebrales/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Compuestos de Tetraetilamonio/farmacología , Animales , Arterias Cerebrales/citología , Conductividad Eléctrica , Electrofisiología , Músculo Liso Vascular/citología , Canales de Potasio/fisiología , Ratas , TetraetilamonioRESUMEN
gamma-Aminobutyric acid (GABA) acts as an inhibitory transmitter in the vertebrate central nervous system, often by interacting with the GABAA type of receptor. Molecular cloning techniques have shown that GABAA receptors are assembled from at least five types of subunit, some of which are present in multiple forms. In this review, the functional properties of native GABAA receptors are compared with those of recombinant receptors, created by expression of appropriate cDNAs in frog oocytes or transfected mammalian cells. Native receptors typically display multiple conductance levels in the open state, complex kinetics, a Hill slope greater than unity, voltage dependence, and desensitization at high agonist doses. All of these features can also be exhibited by recombinant receptors, but marked qualitative and quantitative distinctions exist between receptors containing different combinations of subunits. It is argued that these functional differences are likely to be exploited in vivo by the expression of multiple, physiologically distinct GABAA receptors, distributed in an adaptive fashion throughout the nervous system.