Postsynaptic production of nitric oxide implicated in long-term depression at the mature amphibian (Bufo marinus) neuromuscular junction.
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We report here evidence for endogenous NO signalling in long-term (>1 h) synaptic depression at the neuromuscular junction induced by 20 min of 1 Hz nerve stimulation. Synaptic depression was characterized by a 46% reduction in the end-plate potential (EPP) amplitude and a 21% decrease in miniature EPP (MEPP) frequency, but no change to MEPP amplitude, indicating a reduction in evoked quantal release. Both the membrane-impermeant NO scavenger cPTIO and the NOS inhibitor L-NAME blocked depression, suggesting that it is induced by NO originating from a source outside the terminal. The depression was dependent on activation of muscle-type, but not neuronal-type, nAChRs and was still observed when Ca2+ release from the sarcoplasmic reticulum and muscle contraction were blocked with dantrolene. These data suggest that the depression depends on transmission, but not muscle contraction. The calcineurin inhibitors cyclosporin A and FK506, as well as ODQ, an inhibitor of NO-sensitive soluble guanylyl cyclase, Rp-8-pCPT-cGMPS, an inhibitor of cGMP-dependent protein kinase, and the calmodulin antagonist phenoxybenzamine also blocked depression. We propose that low frequency synaptic transmission leads to production of NO at the synapse and depression of transmitter release via a cGMP-dependent mechanism. The NO could be generated either directly from the muscle, or possibly from the Schwann cell in response to an unidentified muscle-derived messenger. We showed that the long-lasting depression of transmitter release was due to sustained activity of the NO signalling pathway, and suggest dephosphorylation of NOS by calcineurin as the basis for continued NO production. (+info)
Ca(2+) spark sites in smooth muscle cells are numerous and differ in number of ryanodine receptors, large-conductance K(+) channels, and coupling ratio between them.
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Ca(2+) sparks are highly localized Ca(2+) transients caused by Ca(2+) release from sarcoplasmic reticulum through ryanodine receptors (RyR). In smooth muscle, Ca(2+) sparks activate nearby large-conductance, Ca(2+)-sensitive K(+) (BK) channels to generate spontaneous transient outward currents (STOC). The properties of individual sites that give rise to Ca(2+) sparks have not been examined systematically. We have characterized individual sites in amphibian gastric smooth muscle cells with simultaneous high-speed imaging of Ca(2+) sparks using wide-field digital microscopy and patch-clamp recording of STOC in whole cell mode. We used a signal mass approach to measure the total Ca(2+) released at a site and to estimate the Ca(2+) current flowing through RyR [I(Ca(spark))]. The variance between spark sites was significantly greater than the intrasite variance for the following parameters: Ca(2+) signal mass, I(Ca(spark)), STOC amplitude, and 5-ms isochronic STOC amplitude. Sites that failed to generate STOC did so consistently, while those at the remaining sites generated STOC without failure, allowing the sites to be divided into STOC-generating and STOC-less sites. We also determined the average number of spark sites, which was 42/cell at a minimum and more likely on the order of at least 400/cell. We conclude that 1) spark sites differ in the number of RyR, BK channels, and coupling ratio of RyR-BK channels, and 2) there are numerous Ca(2+) spark-generating sites in smooth muscle cells. The implications of these findings for the organization of the spark microdomain are explored. (+info)
Using total fluorescence increase (signal mass) to determine the Ca2+ current underlying localized Ca2+ events.
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The feasibility of determining localized Ca(2+) influx using only wide-field fluorescence images was explored by imaging (using fluo-3) single channel Ca(2+) fluorescence transients (SCCaFTs), due to Ca(2+) entry through single openings of Ca(2+)-permeable ion channels, while recording unitary channel currents. Since the image obtained with wide-field optics is an integration of both in-focus and out-of-focus light, the total fluorescence increase (DeltaF(total) or "signal mass") associated with a SCCaFT can be measured directly from the image by adding together the fluorescence increase due to Ca(2+) influx in all of the pixels. The assumptions necessary for obtaining the signal mass from confocal linescan images are not required. Two- and three-dimensional imaging was used to show that DeltaF(total) is essentially independent of the position of the channel with respect to the focal plane of the microscope. The relationship between Ca(2+) influx and DeltaF(total) was obtained using SCCaFTs from plasma membrane caffeine-activated cation channels when Ca(2+) was the only charge carrier of the inward current. This relationship was found to be linear, with the value of the slope (or converting factor) affected by the particular imaging system set-up, the experimental conditions, and the properties of the fluorescent indicator, including its binding capacity with respect to other cellular buffers. The converting factor was used to estimate the Ca(2+) current passing through caffeine-activated channels in near physiological saline and to estimate the endogenous buffer binding capacity. In addition, it allowed a more accurate estimate of the Ca(2+) current underlying Ca(2+) sparks resulting from Ca(2+) release from intracellular stores via ryanodine receptors in the same preparation. (+info)
Disruption of excitation-contraction coupling and titin by endogenous Ca2+-activated proteases in toad muscle fibres.
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This study investigated the effects of elevated, physiological levels of intracellular free [Ca(2+)] on depolarization-induced force responses, and on passive and active force production by the contractile apparatus in mechanically skinned fibres of toad iliofibularis muscle. Excitation-contraction (EC) coupling was retained after skinning and force responses could be elicited by depolarization of the transverse-tubular (T-) system. Raising the cytoplasmic [Ca(2+)] to approximately 1 microm or above for 3 min caused an irreversible reduction in the depolarization-induced force response by interrupting the coupling between the voltage sensors in the T-system and the Ca(2+) release channels in the sarcoplasmic reticulum. This uncoupling showed a steep [Ca(2+)] dependency, with 50% uncoupling at approximately 1.9 microm Ca(2+). The uncoupling occurring with 2 microm Ca(2+) was largely prevented by the calpain inhibitor leupeptin (1 mm). Raising the cytoplasmic [Ca(2+)] above 1 microm also caused an irreversible decline in passive force production in stretched skinned fibres in a manner graded by [Ca(2+)], though at a much slower relative rate than loss of coupling. The progressive loss of passive force could be rapidly stopped by lowering [Ca(2+)] to 10 nm, and was almost completely inhibited by 1 mm leupeptin but not by 10 microm calpastatin. Muscle homogenates preactivated by Ca(2+) exposure also evidently contained a diffusible factor that caused damage to passive force production in a Ca(2+)-dependent manner. Western blotting showed that: (a) calpain-3 was present in the skinned fibres and was activated by the Ca(2+)exposure, and (b) the Ca(2+) exposure in stretched skinned fibres resulted in proteolysis of titin. We conclude that the disruption of EC coupling occurring at elevated levels of [Ca(2+)] is likely to be caused at least in part by Ca(2+)-activated proteases, most likely by calpain-3, though a role of calpain-1 is not excluded. (+info)
The role of the third extracellular loop of the Na+,K+-ATPase alpha subunit in a luminal gating mechanism.
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Na+,K+-ATPase is responsible for maintaining the cross-membrane Na+ and K+ gradients of animal cells. This P-type ATPase works via a complex transport cycle, during which it binds and occludes three intracellular Na+ ions and then two extracellular K+ ions, which it then releases on the other side of the membrane. The cation pathway through the protein, and the structures responsible for occluding cations inside the protein, have not yet been definitely identified. We used cysteine mutagenesis to explore the accessibility and the role of five conserved residues in the short third extracellular loop, between the fifth and the sixth transmembrane helices. The P801C and L802C mutants were not affected by extracellular sulfhydryl reagents. The presence of cysteine residues at three positions (G803C, T804C and V805C) conferred sensitivity to omeprazole, a known inhibitor of the gastric proton pump, and to [2-(trimethylammonium)-ethyl]methanethiosulphonate bromide (MTSET). The effects of omeprazole and MTSET were modulated by the presence of extracellular K+, indicating that the accessibility of these positions depended on the conformational state of the protein. MTSET binding to cysteine at position 803 partially inhibited the Na+,K+-pump function by decreasing its affinity towards extracellular K+, suggesting a restriction of the access of extracellular K+ ions to their binding sites. In contrast, MTSET binding to cysteine at position 805 partially inhibited the Na+,K+-pump function by reducing its maximum turnover rate, probably by slowing a rate-limiting conformational change. These residues occupy positions that are critical for either the cation pathway or the conformational modifications. (+info)
Fully tuneable stochastic resonance in cutaneous receptors.
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Stochastic resonance describes a phenomenon whereby the addition of "noise" to the input of a nonlinear system can improve sensitivity. "Fully tuneable stochastic resonance" is a particular form of the phenomenon that requires the matching of two time scales: one being that of the subthreshold periodic stimulus of the system and the other being the noise-induced response of the system. First proposed in 1981, stochastic resonance has been reported in a wide range of biological systems; however, conclusive experimental evidence for fully tuneable stochastic resonance in biological systems is limited. Evidence of fully tuneable stochastic resonance in the response of slowly adapting type I mechanoreceptors in the toad is presented. The results are extended to include the first evidence supporting fully tuneable stochastic resonance in psychophysical experiments, namely tactile detection thresholds, indicating that the human CNS is capable of accessing the improved information available via fully tuneable stochastic resonance. (+info)
Inverted photocurrent responses from amphibian rod photoreceptors: role of membrane voltage in response recovery.
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We recorded photocurrent responses of retinal rods isolated from cane toads Bufo marinus and clawed frogs Xenopus laevis. With the outer segment drawn part way into the suction pipette, presentation of flashes to the base of the outer segment (outside the pipette) elicited a slow inverted response. Stimulation of the same region, with the outer segment drawn fully in, gave a response of conventional polarity. For moderate to bright flashes a fast transient preceded the slow inverted response. Upon bleaching the tip of the outer segment, the slow inverted response was abolished but the fast initial transient remained, and we attribute this fast component to a capacitive current. Experiments employing simultaneous whole-cell patch-clamp and suction pipette recording revealed that both the fast and slow components of the inverted responses were absent in voltage-clamped cells. In current-clamped cells the slow inverted current response was delayed substantially with respect to the voltage response. We present a computational model for the slow component, in which hyperpolarization leads to increased activity of the Na+ -Ca2+, K+ exchanger, hence lowering the cytoplasmic Ca2+ concentration, activating guanylyl cyclase, raising cyclic GMP concentration, opening cyclic nucleotide-gated channels, and increasing circulating current in the unstimulated region. For the measured voltage response to stimulation of the base, we solve these equations to predict the photocurrent in the tip, and obtain an adequate explanation of the inverted responses. Our work suggests a novel role for membrane voltage in accelerating the inactivation phase of the response to light. (+info)
Facilitation of transmitter secretion from toad motor nerve terminals during brief trains of action potentials.
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1. End-plate potentials produced by brief trains of action potentials (5-7 at 50-100 Hz) were recorded at toad sciatic-sartorius neuromuscular junctions. When transmitter secretion was depressed in solutions containing magnesium, the increase in amplitude (growth pattern) of successive end-plate-potentials was greater than could be accounted for by arithmetic summation of facilitation (arithmetic model) as proposed by Mallart & Martin (1967). 3. With e.p.p.s of normal quantral content or in solutions in which the calcium concentration was lowered, growth patterns were occasionally reasonably close to those predicted by the arithmetic model but there was always some degree of disparity. 4. A simple, two-step, kinetic model is described which is more consistent with the varied growth patterns of end-plate potentials that have been recorded. The model can predict growth patterns of e.p.p.s with high or low quantal content. (+info)