RESUMEN
1. The goal of the present study was to investigate differences in calcium movements between normal and Duchenne muscular dystrophy (DMD) human contracting myotubes co-cultured with explants of rat spinal cord with attached dorsal root ganglia. Membrane potential, variations of intracellular calcium concentration and T- and L-type calcium currents were recorded. Further, a descriptive and quantitative study by electron microscopy of the ultrastructure of the co-cultures was carried out. 2. The resting membrane potential was slightly less negative in DMD (-61.4 +/- 1.1 mV) than in normal myotubes (-65.5 +/- 0.9 mV). Both types of myotube displayed spontaneous action potentials (mean firing frequency, 0.42 and 0.16 Hz, respectively), which triggered spontaneous calcium transients measured with Indo-1. 3. The time integral under the spontaneous Ca(2+) transients was significantly greater in DMD myotubes (97 +/- 8 nM s) than in normal myotubes (67 +/- 13 nM s). 4. The L- and T-type current densities estimated from patch-clamp recordings were smaller in DMD cells (2.0 +/- 0.5 and 0.90 +/- 0.19 pA pF(-1), respectively) than in normal cells (3.9 +/- 0.7 and 1.39 +/- 0.30 pA pF(-1), respectively). 5. The voltage-dependent inactivation relationships revealed a shift in the conditioning potential at which inactivation is half-maximal (V(h,0.5)) of the T- and L-type currents towards less negative potentials, from -72.1 +/- 0.7 and -53.7 +/- 1.5 mV in normal cells to -61.9 +/- 1.4 and -29.2 +/- 1.4 mV in DMD cells, respectively. 6. Both descriptive and quantitative studies by electron microscopy suggested a more advanced development of DMD myotubes as compared to normal ones. This conclusion was supported by the significantly larger capacitance of the DMD myotubes (408 +/- 45 pF) than of the normal myotubes (299 +/- 34 pF) of the same apparent size. 7. Taken together, these results show that differences in T- and L-type calcium currents between normal and DMD myotubes cannot simply explain all observed alterations in calcium homeostasis in DMD myotubes, thus suggesting that other transmembrane calcium transport mechanisms must also be altered in DMD myotubes compared with normal myotubes.
Asunto(s)
Calcio/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citología , Distrofia Muscular de Duchenne/metabolismo , Animales , Canales de Calcio/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Ganglios Espinales/citología , Homeostasis/fisiología , Humanos , Potenciales de la Membrana/fisiología , Microscopía Electrónica , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/ultraestructura , Distrofia Muscular de Duchenne/patología , Técnicas de Placa-Clamp , Ratas , Médula Espinal/citologíaRESUMEN
The K(+) channel currents are important modulators of smooth muscle membrane potential and excitability. We assessed whether voltage-gated K(+) currents from human myometrium are regulated by placental steroid hormones during pregnancy and labor. Pregnant human myometrial cells were isolated from samples obtained at cesarean section. Primary cultured cells were treated with 100 nM 17beta-estradiol, 1 microM progesterone, or both hormones in combination for 24 h. Acute effects of the two hormones were also determined. The K(+) currents were recorded using the standard whole-cell, patch-clamp technique. Primary cultures possessed both delayed rectifier (I(KV)) and A-like (I(KA)) voltage-gated K(+) currents. The 24-h 17beta-estradiol treatment caused a hyperpolarizing shift in the steady-state inactivation of both I(KV) and I(KA). Progesterone treatment also shifted the inactivation of I(KA) and increased I(KV) amplitude by 60%-110%. Conversely, the combined treatment had no effect on these currents. Neither 17beta-estradiol (0.1-1 microM) nor progesterone (1-5 microM) had any effect on the K(+) current when applied acutely. These results show that 17beta-estradiol should inhibit myometrial K(+) channel activity, whereas progesterone is likely to have the opposite effect. These results are consistent with the respective procontractile and proquiescence roles for 17beta-estradiol and progesterone in human uterus during pregnancy.
Asunto(s)
Estradiol/farmacología , Miometrio/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Progesterona/farmacología , Células Cultivadas , Cesárea , Conductividad Eléctrica , Femenino , Humanos , Trabajo de Parto/fisiología , Miometrio/fisiología , Canales de Potasio/fisiología , EmbarazoRESUMEN
There is increasing evidence that gamma-sarcoglycan is absent and other sarcoglycans are reduced in patients with the limb-girdle muscular dystrophy type 2C (LGMD2C) form of severe childhood autosomal recessive muscular dystrophy. In the present investigation, we combined microspectrofluorimetry and electron microscopy techniques to investigate the physiological function and the ultrastructure of control and LGMD2C myotubes. Results obtained from Ca2+ measurements showed that the resting level of the cytosolic free calcium ([Ca2+ ]i ) in control myotubes was 73+/-3.4 nmol/l (mean+/-se, n=35) and in LGMD2C myotubes was 69+/-4 nmol/l (n=44). Carbachol (CCh, 10 micromol/l ) induced a 335+/-10 nmol/l (n=8) rise in [Ca2+ ]i in control myotubes and 531.9+/-32 nmol/l (n=23) in LGMD2C myotubes. Similarly, elevations of [Ca2+ ]i by 35 mmol/l K+ were 324+/-32 nmol/l (n=8) in control myotubes and 442.8+/-24 nmol/l (n=22) in LGMD2C myotubes. Caffeine (10 mmol/l) activated similar [Ca2+]i peaks in control and LGMD2C myotubes but induced a biphasic response in LGMD2C in four out of 12 myotubes and only a monophasic response in control myotubes. The ultrastructural results showed that the plasma membrane was abnormally indented and convoluted in both the LGMD2C biopsy and the LGMD2C cultured myotubes. It is suggested that the reduction in components of the dystrophin-glycoprotein complex results in the instability and an increase in the surface area of the plasma membrane, which may result in a higher population of Ca2+ channels in the LGMD2C myotubes.
Asunto(s)
Calcio/metabolismo , Homeostasis/fisiología , Distrofias Musculares/metabolismo , Cafeína/farmacología , Carbacol/farmacología , Niño , Técnicas de Cultivo , Colorantes Fluorescentes , Fura-2 , Humanos , Inmunohistoquímica , Masculino , Microscopía Electrónica , Agonistas Muscarínicos/farmacología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/ultraestructura , Distrofias Musculares/patología , Inhibidores de Fosfodiesterasa/farmacología , Potasio/sangreRESUMEN
1. Cis-9,10-octadecenoamide (cOA) accumulates in the CSF of sleep-deprived cats and may represent a novel signalling molecule. Synthetic cOA has been shown to induce physiological sleep when injected into laboratory rats. Here we assess the cellular mode of action of cOA in vitro. 2. In all rat cultured cortical neurones (pyramidal cells) examined, the synthetic brain lipid (3.2-64 microM) enhanced the responses to subsaturating GABA concentrations (up to circa 2x) in a concentration-dependent manner (EC50, circa 15 microM). 3. (20 microM) cOA significantly enhanced the affinity of exogenous GABA for its receptor without changing the Hill slope or the maximal response. These effects were not voltage-dependent or secondary to shifts in E(Cl). 4. In the absence of GABA, cOA directly evoked small inhibitory currents in a subpopulation (<7%) of sensitive cells. 5. 20 microM cOA reversibly enhanced the duration of spontaneous inhibitory post synaptic currents (circa 2 fold) without significantly altering their amplitude. 6. At 32-64 microM, cOA reversibly reduced the incidence and amplitude of both inhibitory post synaptic currents (i.p.s.cs) and excitatory post synaptic currents (e.p.s.cs) in the cultured neuronal circuits in common with other depressant drugs acting at the GABA(A) receptor. 7. 32 microM Oleic acid did not modulate exogenous GABA currents or synaptic activity suggesting that cOAs actions are mediated through a specific receptor. 8. A specific, protein-dependent interaction with GABA(A) receptors was confirmed in Xenopus oocytes. Recombinant human receptors were modulated by 10 microM cOA (and diazepam) only when a gamma2 subunit was co-expressed with alpha1beta2: the cOA response was not sensitive to the specific benzodiazepine antagonist flumazenil (1 microM). 9. cOA may represent an endogenous ligand for allosteric modulatory sites on isoforms of GABA(A) receptors which are crucial for the regulation of arousal and have recently been implicated in the circadian control of physiological sleep.
Asunto(s)
Cerebrósidos/fisiología , Canales de Cloruro/fisiología , Ácidos Oléicos/fisiología , Receptores de GABA-A/fisiología , Sueño/fisiología , Sinapsis/fisiología , Animales , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/fisiología , Cerebrósidos/farmacología , Canales de Cloruro/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Femenino , Flumazenil/farmacología , Moduladores del GABA/farmacología , Antagonistas de Receptores de GABA-A , Humanos , Ácidos Oléicos/farmacología , Oocitos , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Células Piramidales/fisiología , Ratas , Receptores de GABA-A/biosíntesis , Receptores de GABA-A/efectos de los fármacos , Proteínas Recombinantes/biosíntesis , Sinapsis/efectos de los fármacos , Xenopus laevisRESUMEN
The control of Cl- conductance in rat parotid isolated acinar cells was studied by combined use of whole-cell recording and flash photolysis techniques. Cells were voltage-clamped either at a membrane potential of -40 mV or stepped between -85 mV and 0 mV. Bath-applied carbachol and noradrenaline evoked Cl- current at -85 mV and K+ current at 0 mV. Similar current activations resulted from the photolytic release of either inositol trisphosphate (InsP3) or Ca2+ by a brief near-UV flash. The peak amplitudes of the Cl- conductance (at -85 mV), measured relative to the K+ conductance (at 0 mV), evoked by application of carbachol, noradrenaline or direct manipulation of cytosolic free calcium ([Ca2+]i), were very similar, being 0.56 +/- 0.09 (mean +/- SEM, n = 9), 0.52 +/- 0.01 (n = 7) and 0.46 +/- 0.06 (n = 7). In contrast, the relative amplitude of the Cl- conductance evoked by InsP3 was much larger: 1.49 +/- 0.24 (n = 9). Neither bath application of isoprenaline nor photolysis of "caged" cAMP induced any detectable membrane current. The most probable interpretation of these results is that the observed activation of Cl- conductance by agonists can be explained by the elevation of [Ca2+]i alone. In addition, the present results provide further support for the previously reported suggestion that the Cl- channels and the Ca(2+)-release sites are co-localised [10].
Asunto(s)
Cloruros/fisiología , Glándula Parótida/fisiología , Agonistas Adrenérgicos/farmacología , Animales , Separación Celular , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Conductividad Eléctrica , Inositol 1,4,5-Trifosfato/análogos & derivados , Inositol 1,4,5-Trifosfato/farmacología , Luz , Masculino , Glándula Parótida/citología , Glándula Parótida/efectos de los fármacos , Ratas , Ratas Wistar , Receptores Muscarínicos/fisiologíaRESUMEN
1. The temporal relationship between cytosolic free Ca2+ concentration ([Ca2+]i) and activation of membrane current responses in single rat parotid acinar cells has been examined. Activation of muscarinic receptors by carbachol (CCh) at -40 mV (midway between EK and ECl under our experimental conditions) frequently evoked biphasic current responses, application of 2 microM CCh leading to rapid activation of an inward current followed by a slower outward current. 2. Photochemical release of inositol 1,4,5-trisphosphate (InsP3), from 'caged' InsP3, by a brief near-UV flash, evoked similar biphasic current responses at -40 mV. In contrast, elevation of [Ca2+]i by photolysis of the caged calcium compound nitr-5 at -40 mV activated only monophasic current responses. 3. These results can be explained by a model in which the InsP3-sensitive Ca2+ release sites are localized at the luminal pole of the cell, combined with a relative preponderance of Ca(2+)-activated Cl- channels at that pole, and a relative preponderance of Ca(2+)-activated K+ channels at the basal end.
Asunto(s)
Calcio/metabolismo , Glándula Parótida/metabolismo , Fotólisis , Animales , Carbacol/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/farmacología , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Agonistas Muscarínicos/farmacología , Glándula Parótida/citología , Glándula Parótida/efectos de la radiación , Técnicas de Placa-Clamp , Ratas , Receptores Muscarínicos/efectos de los fármacos , Rayos UltravioletaRESUMEN
1. Intracellular recordings were made from identified Helix central neurones, sensitive to L-glutamate. 2. Out of a range of substituted glutamate analogues, only the L- and D-isomers of thio-glutamate possessed clear glutamate-like activity. 3. On neurones excited by L-glutamate, the EC50 values for L-glutamate, gamma-thio-L-glutamate and gamma-thio-D-glutamate were 30 microM, 20 microM and greater than 1 mM, respectively. 4. On neurones inhibited by L-glutamate, the EC50 values for L-glutamate, gamma-thio-L-glutamate and gamma-thio-D-glutamate were 6.0 microM, 0.7 microM and greater than 200 microM, respectively. 5. It is concluded that, unlike the situation with thio derivatives of GABA, thio derivatives of glutamate possess potent glutamate-like activity.
Asunto(s)
Glutamatos/farmacología , Caracoles Helix/efectos de los fármacos , Neuronas/efectos de los fármacos , Receptores de Neurotransmisores/efectos de los fármacos , Animales , Electrofisiología , Glutamatos/metabolismo , Neuronas/fisiología , Receptores de Glutamato , Receptores de Neurotransmisores/metabolismo , Relación Estructura-ActividadRESUMEN
1. Intracellular recordings were made from identified neurones in the suboesophageal ganglionic mass of the snail, Helix aspersa. Neurones were classified as either "H" cells, inhibited by acetylcholine or "D" cells, excited by acetylcholine. 2. The actions of levamisole, morantel, pyrantel, amidantel, deacylated amidantel and hycanthone were investigated on these neurones and compared to that of acetylcholine. 3. Levamisole was 10.85 +/- 0.56 times less active than acetylcholine on "H" cells but more than 100 times less active on "D" cells. On "H" cells levamisole had a secondary gradual depolarizing effect which was irreversible and resulted in the loss of cell activity. 4. Morantel and pyrantel were 1.12 +/- 0.13 and 2.56 +/- 0.26 times respectively less active than acetylcholine on "D" cells and 5.16 +/- 0.6 and 3.53 +/- 0.63 times respectively less active than acetylcholine on "H" cells. 5. Amidantel was more than 100 times less active than acetylcholine on both "D" and "H" cells while its deacylated derivative was 26.0 +/- 1.0 and 76.0 +/- 3.25 times respectively less active than acetylcholine on "D" and "H" cells. 6. Hycanthone possessed weak inhibitory effects on "H" cells but also appeared to reduce the duration of acetylcholine inhibitory responses when applied immediately after the acetylcholine response had reached its maximum.
Asunto(s)
Antihelmínticos/farmacología , Caracoles Helix , Neuronas/efectos de los fármacos , Receptores Colinérgicos/efectos de los fármacos , Animales , Hicantona/farmacología , Levamisol/farmacología , Morantel/farmacología , Fenilendiaminas/farmacología , Pirantel/farmacologíaRESUMEN
Strips of muscle, approximately 12 segments in length, were prepared from the body wall of the earthworm, Lumbricus terrestris, from which the nerve cord and viscera had been removed. Contractions to electrical stimulation and acetylcholine agonists were recorded using an isometric transducer. A range of nicotinic and muscarinic agonists and antagonists were tested on this preparation and the results indicate that the acetylcholine receptor on this muscle cannot be classified as either nicotinic or muscarinic. Hemicholinium-3 abolished electrically induced muscle twitches at concentrations which had no effect on the acetylcholine response. Alpha-Bungarotoxin blocked the responses to both electrical stimulation and acetylcholine while beta-bungarotoxin blocked the contractions induced by electrical stimulation but potentiated the acetylcholine contraction.