RESUMEN
Hospitalized patients often receive oxygen supplementation, which can lead to a supraphysiological oxygen tension (hyperoxia). Hyperoxia can have hemodynamic effects, including an increase in systemic vascular resistance. This increase suggests hyperoxia-induced vasoconstriction, yet reported direct effects of hyperoxia on vessel tone have been inconsistent. Furthermore, hyperoxia-induced changes in vessel diameter have not been studied in mice, currently the most used mammal model of disease. In this study we set out to develop a pressure-myograph model using isolated vessels from mice for investigation of pathways involved in hyperoxic vasoconstriction. Isolated conduit and resistance arteries (femoral artery and gracilis arteriole, respectively) from C57BL/6 mice were exposed to normoxia (PO2 of 80 mmHg) and three levels of hyperoxia (PO2 of 215, 375 and 665 mmHg) in a no-flow pressure myograph setup. Under the different PO2 levels, dose-response agonist induced endothelium-dependent vasodilation (acetylcholine, arachidonic acid), endothelium-independent vasodilation (s-nitroprusside), as well as vasoconstriction (norepinephrine, prostaglandin F2α) were examined. The investigated arteries did not respond to oxygen by a change in vascular tone. In the dose-response studies, maximal responses and EC50 values to any of the aforementioned agonists were not affected by hyperoxia either. We conclude that arteries and arterioles from healthy mice are not intrinsically sensitive to hyperoxic conditions. The present ex-vivo model is therefore not suitable for further research into mechanisms of hyperoxic vasoconstriction.
Asunto(s)
Arteria Femoral/fisiopatología , Hiperoxia/fisiopatología , Acetilcolina/farmacología , Animales , Ácido Araquidónico/farmacología , Evaluación Preclínica de Medicamentos , Arteria Femoral/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiopatología , Nitroprusiato/farmacología , Norepinefrina/farmacología , Oxígeno/farmacología , Vasoconstricción , Vasoconstrictores/farmacología , Vasodilatación , Vasodilatadores/farmacocinéticaRESUMEN
Maurotoxin is a 34-residue toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus palmatus and contains four disulfide bridges that are normally found in long-chain toxins of 60-70 amino acid residues, which affect voltage-gated sodium channels. However, despite the unconventional disulfide-bridge pattern of maurotoxin, the conformation of this toxin remains similar to that of other toxins acting on potassium channels. Here, we analyzed the effects of synthetic maurotoxin on voltage-gated Shaker potassium channels (ShB) expressed in Xenopus oocytes. Maurotoxin produces a strong, but reversible, inhibition of the ShB K+ current with an IC50 of 2 nM. Increasing concentrations of the toxin induce a progressively higher block at saturating concentrations. At nonsaturating concentrations of the toxin (5-20 nM), the channel block appears slightly more pronounced at threshold potentials suggesting that the toxin may have a higher affinity for the closed state of the channel. At the single channel level, the toxin does not modify the unitary current amplitude, but decreases ensemble currents by increasing the number of depolarizing epochs that failed to elicit any opening. A point mutation of Lys23 to alanine in maurotoxin produces a 1000-fold reduction in the IC50 of block by the toxin suggesting the importance of this charged residue for the interaction with the channel. Maurotoxin does not affect K+ currents carried by Kir2.3 channels in oocytes or Na+ currents carried by the alphaIIa channel expressed in CHO cells.
Asunto(s)
Bloqueadores de los Canales de Potasio , Canales de Potasio/metabolismo , Venenos de Escorpión/farmacología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Disulfuros , Relación Dosis-Respuesta a Droga , Expresión Génica , Microinyecciones , Datos de Secuencia Molecular , Oocitos , Técnicas de Placa-Clamp , Conformación Proteica , ARN Complementario/metabolismo , Venenos de Escorpión/química , Venenos de Escorpión/metabolismo , Canales de Potasio de la Superfamilia Shaker , Canales de Sodio/metabolismo , Xenopus laevisRESUMEN
SPC3 is a multibranched peptide containing eight identical GPGRAF motifs which are derived from the human immunodeficiency virus (HIV)-1 gp120 V3 loop consensus sequence. This molecule was reported to prevent the infection of CD4+ cells by various HIV-1 and HIV-2 strains. However, the molecular mode of action of SPC3 remains unclear. Here, we investigated the possibility that SPC3 could interact with alpha/beta-chemokine receptors following observations that, first, the V3 loop is likely to be involved in alpha/beta-chemokine receptor-dependent HIV entry and, second, natural ligands of these receptors are potent inhibitors of cell infection. To address this point, we examined the effects of SPC3 on Xenopus oocytes either uninjected or expressing exogenous human CXCR4 alpha-chemokine receptors. Extracellular applications of micromolar concentrations of SPC3 onto Xenopus oocytes trigger potent inward chloride currents which can be inhibited by increasing extracellular Ca2+ concentration. This effect can be blocked by chloride channel antagonists and is highly specific to SPC3 as it is not triggered by structural analogs of SPC3. The SPC3-induced chloride conductance in oocytes is alpha/beta-chemokine receptor dependent because: (i) SPC3 alters the sensitivity of this channel to external applications of human recombinant MIP-1alpha, a natural ligand of human CCR5 receptor, and (ii) the amplitude of the inward current could be increased by the expression of exogenous human CXCR4 chemokine receptor. The effect of SPC3 appears to rely on the activation of a phospholipase A2 signaling pathway, but is not affected by changes in cytosolic Ca2+ concentration, or by alterations in Gi/Go protein, adenylate cyclase, phospholipase C or protein kinase C activity. Altogether, the data indicate that SPC3 is capable of activating a surface alpha/beta-chemokine-like receptor-mediated signaling pathway in competent cells, thereby triggering, either directly or indirectly, a Ca2+-inactivated chloride conductance.
Asunto(s)
Quimiocinas/química , Proteínas Fúngicas/química , Proteína gp120 de Envoltorio del VIH/química , Canales Iónicos/química , Iones/química , Péptidos/química , Proteínas de Saccharomyces cerevisiae , Animales , Calcio/química , Calcio/farmacología , Quelantes/farmacología , Relación Dosis-Respuesta a Droga , Electrofisiología , Inhibidores Enzimáticos/farmacología , Humanos , Oocitos/química , Biosíntesis de Péptidos , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Potasio/química , Potasio/farmacología , ARN Complementario/química , Receptores CXCR4/química , Transducción de Señal , Factores de Tiempo , XenopusRESUMEN
The voltage-gated calcium channel beta subunit is a cytoplasmic protein that stimulates activity of the channel-forming subunit, alpha 1, in several ways. Complementary binding sites on alpha 1 and beta have been identified that are highly conserved among isoforms of the two subunits, but this interaction alone does not account for all of the functional effects of the beta subunit. We describe here the characterization in vitro of a second interaction, involving the carboxyl-terminal cytoplasmic domain of alpha 1A and showing specificity for the beta 4 (and to a lesser extent beta 2a) isoform. A deletion and chimera approach showed that the carboxyl-terminal region of beta 4, poorly conserved between beta isoforms, contains the interaction site and plays a role in the regulation of channel inactivation kinetics. This is the first demonstration of a molecular basis for the specificity of functional effects seen for different combinations of these two channel components.
Asunto(s)
Canales de Calcio/metabolismo , Animales , Sitios de Unión , Canales de Calcio/genética , Citoplasma/metabolismo , Mapeo Peptídico , Unión Proteica , Conejos , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
1. Voltage-dependent Ca2+ channels are multi-protein complexes composed of at least three subunits: alpha 1, alpha 2 delta and beta. Ba2+ currents were recorded in Xenopus oocytes expressing the neuronal alpha 1A Ca2+ channel, using the two-electrode voltage-clamp technique. Various subunit combinations were studied: alpha 1A, alpha 1A alpha 2 delta b, alpha 1A beta or alpha 1A alpha 2 delta b beta. 2. The alpha 1A subunit alone directs the expression of functional Ca2+ channels. It carries all the properties of the channel: gating, permeability, voltage dependence of activation and inactivation, and pharmacology. The alpha 1A channel is activated by low voltages when physiological concentrations of the permeant cation are used. Both ancillary subunits alpha 2 delta and beta induced considerable changes in the biophysical properties of the alpha 1A current. The subunit specificity of the changes in current properties was analysed for all four beta gene products by coexpressing beta 1b, beta 2a, beta 3 and beta 4. 3. All beta subunits induce a stimulation in the current amplitude, a change in inactivation kinetics, and two hyperpolarizing shifts--one in the voltage dependence of activation and a second in the voltage dependence of steady-state inactivation. The most significant difference in regulation among beta subunits is the induction of variable rate constants of current inactivation. Rates of inactivation were induced in the following order (fastest to slowest): beta 3 > beta 1b = beta 4 > beta 2a. 4. The alpha 2 delta b subunit does not modify the properties of alpha 1A Ca2+ channels in the absence of beta subunits. However, this subunit increases the beta-induced stimulation in current amplitude and also regulates the beta-induced change in inactivation kinetics. 5. Of all the subunit combinations tested, Ca2+ channels that included a beta subunit were the most prone to decrease in activity. It is concluded that beta subunits are the primary target for the inhibitory mechanisms involved in Ca2+ channel run-down. 6. Both alpha 2 delta b and beta 1 b subunits slightly modified the sensitivity of the alpha 1A subunit to the snail peptide omega-conotoxin MVIIC. 7. The subunit-induced changes in properties of the alpha 1A channel are surprisingly similar to changes reported for other alpha 1 subunits. These modifications in channel activity should therefore represent important functional landmarks in the on-going characterization of subunit-subunit interactions.
Asunto(s)
Canales de Calcio/metabolismo , Oocitos/metabolismo , Animales , Bario/metabolismo , Canales de Calcio/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Femenino , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , ARN Complementario/farmacología , Xenopus laevisRESUMEN
Postmitotic cerebellar granule cells, maintained for 5 - 6 days in Dulbecco's modified essential medium supplemented with 25 mM KCl, have been studied in whole-cell recording conditions to characterize calcium currents. With 10 mM Ba2+ as the divalent charge carrier, and using a pipette solution highly buffered for Ca2+ (30 mM EGTA, 100 mM HEPES - Tris, pH 7.2), only a high-threshold voltage-activated barium current was recorded from a holding potential of -90 mV. The addition of 1 mM ATP to the pipette medium allowed stable recording for an average duration of 10 min, compatible with pharmacological studies of the barium current. Ninety-six per cent of the current was half-inactivated at low negative holding potential (-76 mV). A total block of current was obtained with 1 microM Cd2+. Sixty-three per cent of the mean current was abolished by 3 microM omega-conotoxin (omega-CgTx; Ki=10 nM for a 15 min application), but individual cells showed either full sensitivity to this toxin or incomplete sensitivity. Seventy-eight per cent of the mean current was also abolished by 10 microM nicardipine but with a higher Ki of 0.5 microM. After exposure to omega-CgTx, BAY K 8644 had no effect on the remaining current, though it was suppressed by nicardipine. No sensitivity to diltiazem, desmethoxyverapamil or flunarizine could be detected. Our major conclusion is that at least half of the channels have a mixed pharmacology, showing sensitivity to both omega-CgTx and dihydropyridine antagonists.