Involvement of the endogenous opioid system in the beneficial influence of physical exercise on amphetamine-induced addiction parameters.

Psychostimulant drugs addiction is a chronic public health problem and individuals remain susceptible to relapses increasing public expenses even after withdrawal and treatment. Our research group has focused on finding new therapies to be employed in drug addiction treatment, suggesting the physical exercise as a promising tool. This way, it is necessary to know the mechanisms involved in the beneficial influences of physical exercise observing the pathway that could be explored in drug addiction treatment. Male Wistar rats were conditioned with amphetamine (AMPH) following the conditioned place preference (CPP) protocol and subsequently submitted to swimming for 5 weeks (1 h per day, 5 days per week). Half of the animals were injected with Naloxone (0.3 mg/mL/kg body weight, i.p.) 5 min prior each physical exercise day. After AMPH-CPP re-exposure, our outcomes showed that physical exercise, in addition to minimizing the relapse behavior in the CPP, it increased D1R, D2R and DAT in the Ventral Tegmental Area (VTA), but not in the Nucleus accumbens (NAc). Interestingly, while naloxone inhibited the partial beneficial influence of the exercise on drug-relapse behavior, exercise-induced changes in the dopaminergic system were not observed in the group administered with naloxone as well. Based on these evidences, besides reinforcing the beneficial influence of the physical exercise on AMPH-induced drug addiction, we propose the involvement of endogenous opioid system activation, not as a single one, but as a possible mechanism of action resulting from the physical activity practice, thus characterizing an important therapeutic approach, which may contribute to drug withdrawal consequently preventing relapse.

Binge and Subchronic Exposure to Ketamine Promote Memory Impairments and Damages in the Hippocampus and Peripheral Tissues in Rats: Gallic Acid Protective Effects.

Ketamine (KET) is a dissociative anesthetic for restrict medical use with high potential for abuse and neurotoxicity which does not prevent its recreational use. Gallic acid (GA) is a natural free radical "scavenger." We evaluated the GA protective role regarding binge or subchronic (SbChro) KET-induced toxicity in adolescent rats. In the binge protocol, animals were treated with GA (one dose of 13.5 mg/kg, p.o. every 2 h, totaling 3 doses) 12 h after KET exposure (one dose of 10 mg/kg, i.p., every 3 h, totaling 5 doses). In the SbChro, animals were treated with GA (one dose of 13.5 mg/kg/day, p.o., for 3 days) 48 h following KET exposure (one dose of 10 mg/kg/day, i.p) for 10 days. Our findings show that binge-KET impaired memory, increased pro-BDNF and TrkB levels in the hippocampus, and increased lipid peroxidation (LP) in the kidney and hippocampus, while SbChro-KET impaired memory, increased pro-BDNF, and decreased both BDNF and TrkB levels in the hippocampus, and increased LP in the kidney, liver, and hippocampus. GA treatment reversed the subchronically KET-induced harmful influences better. Interestingly, only memory impairment observed in the SbChro-KET protocol was reversed by GA. Memory impairments showed a positive correlation with hippocampal BDNF levels and negative with LP levels in the same brain area. This last hippocampal damage (LP) showed a negative correlation with BDNF levels in the hippocampus, indicating an interesting and close causal connection. Our outcomes show that the deleterious effects of SbChro-KET exposure can be attenuated or abolished with GA administration, a natural antioxidant that could be considered in KET abuse treatment.

The elusive role of store depletion in the control of intracellular calcium release.

The contractile cycle of striated muscles, skeletal and cardiac, is controlled by a cytosolic [Ca2+] transient that requires rapid movements of the ion through channels in the sarcoplasmic reticulum (SR). A functional signature of these channels is their closure after a stereotyped time lapse of Ca2+ release. In cardiac muscle there is abundant evidence that termination of release is mediated by depletion of the Ca2+ store, even if the linkage mechanism remains unknown. By contrast, in skeletal muscle the mechanisms of release termination are not understood. This article reviews measurements of store depletion, the experimental evidence for dependence of Ca2+ release on the [Ca2+] level inside the SR, as well as tests of the molecular nature of putative intra-store Ca2+ sensors. Because Ca2+ sparks exhibit the basic release termination mechanism, much attention is dedicated to the studies of store depletion caused by sparks and its relationship with termination of sparks. The review notes the striking differences in volume, content and buffering power of the stores in cardiac vs. skeletal muscle, differences that explain why functional depletion is much greater for cardiac than skeletal muscle stores. Because in skeletal muscle store depletion is minimal and reduction in store [Ca2+] does not appear to greatly inhibit Ca2+ release, it is concluded that decrease in free SR [Ca2+] does not mediate physiological termination of Ca2+ release in this type of muscle. In spite of the apparent absence of store depletion and its putative channel closing effect, termination of Ca2+ sparks is faster and more robust in skeletal than cardiac muscle. A gating role of a hypothetical "proximate store" constituted by polymers of calsequestrin and associated proteins is invoked in an attempt to preserve a role for store depletion and unify mechanisms in both types of striated muscle.

The elementary events of Ca2+ release elicited by membrane depolarization in mammalian muscle.

Cytosolic [Ca(2+)] transients elicited by voltage clamp depolarization were examined by confocal line scanning of rat skeletal muscle fibres. Ca(2+) sparks were observed in the fibres' membrane-permeabilized ends, but not in responses to voltage in the membrane-intact area. Elementary events of the depolarization-evoked response could be separated either at low voltages (near -50 mV) or at -20 mV in partially inactivated cells. These were of lower amplitude, narrower and of much longer duration than sparks, similar to 'lone embers' observed in the permeabilized segments. Their average amplitude was 0.19 and spatial half-width 1.3 microm. Other parameters depended on voltage. At -50 mV average duration was 111 ms and latency 185 ms. At -20 mV duration was 203 ms and latency 24 ms. Ca(2+) release current, calculated on an average of events, was nearly steady at 0.5-0.6 pA. Accordingly, simulations of the fluorescence event elicited by a subresolution source of 0.5 pA open for 100 ms had morphology similar to the experimental average. Because 0.5 pA is approximately the current measured for single RyR channels in physiological conditions, the elementary fluorescence events in rat muscle probably reflect opening of a single RyR channel. A reconstruction of cell-averaged release flux at -20 mV based on the observed distribution of latencies and calculated elementary release had qualitatively correct but slower kinetics than the release flux in prior whole-cell measurements. The qualitative agreement indicates that global Ca(2+) release flux results from summation of these discrete events. The quantitative discrepancies suggest that the partial inactivation strategy may lead to events of greater duration than those occurring physiologically in fully polarized cells.

Effects of the enantiomers of BayK 8644 on the charge movement of L-type Ca channels in guinea-pig ventricular myocytes.

The effects of the agonist enantiomer S(-)Bay K 8644 on gating charge of L-type Ca channels were studied in single ventricular myocytes. From a holding potential (Vh) of -40 mV, saturating (250 nm) S(-)Bay K shifted the half-distribution voltage of the activation charge (Q1) vs. V curve -7.5 +/- 0.8 mV, almost identical to the shift produced in the Ba conductance vs. V curve (-7.7 +/- 2 mV). The maximum Q1 was reduced by 1.7 +/- 0.2 nC/microF, whereas Q2 (charge moved in inactivated channels) was increased in a similar amount (1.4 +/- 0.4 nC/microF). The steady-state availability curves for Q1, Q2, and Ba current showed almost identical negative shifts of -14.8 +/- 1.7 mV, -18.6 +/- 5.8 mV, and -15.2 +/- 2.7 mV, respectively. The effects of the antagonist enantiomer R(+)BayK 8644 were also studied, the Q1 vs. V curve was not significantly shifted, but Q1max (Vh = -40 mV) was reduced and the Q1 availability curve shifted by -24.6 +/- 1.2 mV. We concluded that: a) the left shift in the Q1 vs. V activation curve produced by S(-)BayK is a purely agonistic effect; b) S(-)BayK induced a significantly larger negative shift in the availability curve than in the Q1 vs. V relation, consistent with a direct promotion of inactivation; c) as expected for a more potent antagonist, R(+)Bay K induced a significantly larger negative shift in the availability curve than did S(-)Bay K.

Ca2+ sparks and embers of mammalian muscle. Properties of the sources.

Ca2+ sparks of membrane-permeabilized rat muscle cells were analyzed to derive properties of their sources. Most events identified in longitudinal confocal line scans looked like sparks, but 23% (1,000 out of 4,300) were followed by long-lasting embers. Some were preceded by embers, and 48 were "lone embers." Average spatial width was approximately 2 microm in the rat and 1.5 microm in frog events in analogous solutions. Amplitudes were 33% smaller and rise times 50% greater in the rat. Differences were highly significant. The greater spatial width was not a consequence of greater open time of the rat source, and was greatest at the shortest rise times, suggesting a wider Ca2+ source. In the rat, but not the frog, spark width was greater in scans transversal to the fiber axis. These features suggested that rat spark sources were elongated transversally. Ca2+ release was calculated in averages of sparks with long embers. Release current during the averaged ember started at 3 or 7 pA (depending on assumptions), whereas in lone embers it was 0.7 or 1.3 pA, which suggests that embers that trail sparks start with five open channels. Analysis of a spark with leading ember yielded a current ratio ranging from 37 to 160 in spark and ember, as if 37-160 channels opened in the spark. In simulations, 25-60 pA of Ca2+ current exiting a point source was required to reproduce frog sparks. 130 pA, exiting a cylindric source of 3 microm, qualitatively reproduced rat sparks. In conclusion, sparks of rat muscle require a greater current than frog sparks, exiting a source elongated transversally to the fiber axis, constituted by 35-260 channels. Not infrequently, a few of those remain open and produce the trailing ember.

A critical approach to nutritional assessment in critically ill patients.

BACKGROUND AND AIMS: Nutritional assessment enhances quality of nutritional care, however, its practice bemuses professionals. This prospective study aimed to identify a feasible/informative nutritional parameter in intensive care. METHODS: 44 patients (APACHE II: 23.8+/-10.1), age 58.4+/-18.6 years, were evaluated at admission: clinical data, height, weight, body mass index (BMI), tricep skinfold thickness, mid-arm circumference (MAC), mid-arm muscle circumference (MAMC), albumin, total protein and lymphocyte count. Anthropometric parameters' performance was evaluated isolated or assembled according to Blackburn and McWhirter criteria. RESULTS: Oedema increased %IW and BMI (P<0.01); muscle depletion was frequent and agreed with MAC or MAMC ranked by both criteria, P=0.02. %IW and BMI overestimated well-nourished/overweight patients, whilst arm anthropometry, mostly MAC/MAMC, shifted towards +/-50% malnutrition. Patients were not equally ranked by both criteria; McWhirter's by using percentiles clarified the distribution and showed agreement between MAC and MAMC, P=0.007, unlike Blackburn's. Mortality was higher in patients with MAC<5th percentile, P=0.003; MAC;<15th percentile was able to predict mortality and major complications. In invasive ventilated patients, severe muscle depletion was associated with mortality, P=0.05. CONCLUSION: In intensive care most nutritional assessment methods are useless; MAC is simple, feasible and if classified by percentiles may prove functional with prognostic value.

Fast imaging in two dimensions resolves extensive sources of Ca2+ sparks in frog skeletal muscle.

Ca2+ sparks were monitored by confocal laser-scanning microscopy of fluo-3 at video rates, in fast twitch muscle fibres, stimulated by exposure to caffeine and/or low [Mg2+]. Scanning was in two spatial dimensions ('2D') or 2D plus time, at 4 ms per image frame. Sparks were identified in 2D images of normalized fluorescence by an automatic procedure, which also evaluated the event's location and morphometric parameters. Most sparks were circular, but some were elongated, especially in caffeine. Separation of the spark from circular symmetry was quantified by its eccentricity (length/width - 1). In an internal solution with 0.4 mM [Mg2+], sparks (989 events in 4 cells) had amplitude 0.73, width 1.94 microm, length 2.12 microm and eccentricity not significantly different from 0. Upon application of 1 mM caffeine, length (of 2578 events in the same cells) increased significantly (by 0.41 microm, or 19 %), width increased by 0.18 microm (9 %) and amplitude decreased slightly. The eccentricity became significantly different from 0, and the sparks' long axis predominantly oriented parallel to the plane of the Z disks. More than 10 % of the events in caffeine had length greater than 4 microm, a relatively flat top, and a sharp termination at both ends of the major axis. Additionally, there was only a weak correlation between eccentricity and amplitude. These properties suggest that the elongated events are produced by simultaneous opening of multiple channels within a junction, rather than anisotropic diffusion of Ca2+ or random overlap of round sparks. Elongated events often increased in eccentricity early in their evolution. Then, most remained elongated during their rise and decay, while others spread spatially in the plane of the Z disks. In 1-2 % of the events, the centre of mass migrated in space, over time, at approximately 0.1 microm x ms(-1). These spatio-temporal features require the involvement of multiple release channels, at spatially resolvable locations. Because sources often spread over distances greater than 1 or 2 microm, and arrays of junctional elements (couplons) are at most 1.2 microm long, it must be possible for activation of release to propagate between neighbouring couplons, especially under the influence of caffeine and/or low [Mg2+].

Involvement of multiple intracellular release channels in calcium sparks of skeletal muscle.

In many types of muscle, intracellular Ca(2+) release for contraction consists of brief Ca(2+) sparks. Whether these result from the opening of one or many channels in the sarcoplasmic reticulum is not known. Examining massive numbers of sparks from frog skeletal muscle and evaluating their Ca(2+) release current, we provide evidence that they are generated by multiple channels. A mode is demonstrated in the distribution of spark rise times in the presence of the channel activator caffeine. This finding contradicts expectations for single channels evolving reversibly, but not for channels in a group, which collectively could give rise to a stereotyped spark. The release channel agonists imperatoxin A, ryanodine, and bastadin 10 elicit fluorescence events that start with a spark, then decay to steady levels roughly proportional to the unitary conductances of 35%, 50%, and 100% that the agonists, respectively, promote in bilayer experiments. This correspondence indicates that the steady phase is produced by one open channel. Calculated Ca(2+) release current decays 10- to 20-fold from spark to steady phase, which requires that six or more channels be open during the spark.

Effects of 2,3-butanedione monoxime on excitation-contraction coupling in frog twitch fibres.

10 and 30 mM 2,3-butanedione monoxime (BDM) applied extracellularly to voltage-clamped frog skeletal muscle twitch fibres suppressed both Ca2+ release flux and intramembranous charge movement. Both effects could be clearly separated. The early peak of the Ca2+ release flux was suppressed at every test voltage. The steady level attained at the end of a 100 ms clamp depolarization was relatively spared for lower depolarizing pulses, but was as suppressed as the peak at voltages above -20 mV. The intramembranous charge movement was affected mainly in the I gamma component. The drug had a distinct effect on the kinetics of the intramembranous charge movement current around the threshold for Ca2+ release. The three kinetic components of I gamma were simultaneously affected. For more positive depolarizations where the kinetic effect was not evident, the oxime had no significant effect on the charge moved. Under conditions in which I gamma was absent (i.e. stretched fibres, intracellular solutions containing 6 to 10 mM BAPTA), treatment with 10 mM BDM had a small, not significant suppressive effect on the maximum charge moved (Qmax), while it affected Ca2+ release significantly. When 10 mM BDM was applied in the presence of 0.2 mM tetracaine, the local anaesthetic-resistant Ca2+ release flux was not further suppressed by the oxime.

Butanedione monoxime promotes voltage-dependent inactivation of L-type calcium channels in heart. Effects on gating currents.

The effect of 20 mM extracellularly applied 2,3-Butanedione monoxime (BDM) on L-type Ca2+ channel charge movement current was studied in whole-cell voltage-clamped guinea-pig ventricular myocytes. Intramembraneous charge movement in response to depolarizing pulses (charge 1), was reduced after the application of BDM. The effect was more pronounced at the OFF of the charge transient (41%) than at the ON (7%). The steady-state availability curve of charge 1 was shifted to the left; the magnitude of the voltage shift was similar to the shift in Ca2+ current availability. Charge movement recorded in the negative voltage range (charge 2) after conditioning depolarizing pulses of different duration, was increased by BDM. For a 300-ms conditioning pulse charge 2 measured during a negative test pulse increased 40% (in Ba2+ external solution) or 35% (in Ca2+ external solution). These results show that BDM promotes voltage-dependent inactivation of L-type Ca2+ channels in parallel with charge interconversion between intramembranous charges 1 and 2. Mechanistically they are consistent either with dephosphorylation or a dihydropyridine-like action, but argue against open channel block as the mechanism of the effect of the drug.

[The microbiological profile of nosocomial infection in a respiratory intensive care unit]. / Perfil microbiológico da infecção nosocomial numa unidade de cuidados intensivos respiratórios.

In 1987, in the Respiratory Intensive Care Unit of Santa Maria Hospital we developed a nosocomial surveillance program with specially created software that provides knowledge of NI in the ICU at any moment. The information gathered along the time is particularly useful in the correlation of the risk factors, the most frequent microorganisms and in the institution of empiric antibiotic therapy. Out of 2528 patients admitted in our ICU for the last 6 years (87/92), 10% had bacteriologically identified NI. Almost (93%) of them had occurred in mechanically ventilated patients, which corresponds to 20.7% NI in those patients. The gram-negative appeared in 63.37%, with the predominance of Enterobacteriaceae. From the gram-positives the most frequent were Staphylococcus spp. Their meticiline resistance was worrisome. NI patients had significantly higher severity and therapeutic intervention scores. Mechanical ventilation period was 4 fold higher and mortality 2.5 times higher in the patients with this complication.

Aeromonas hydrophila fulminant pneumonia in a fit young man.

A previously healthy 24 year old athletic man became ill suddenly with pneumonia the day after swimming in the sea. Despite intensive support measures in the intensive care unit he died three hours after admission and 21 hours after his first symptom. Necropsy showed bilateral haemorrhagic necrotising pneumonia. Aeromonas hydrophila was isolated from a blood culture taken at admission and from the lungs at necropsy. The infection may have come from contaminated sea water.

[Severity evaluation of ventilated patients at a respiratory intensive care unit with the APACHE II system]. / Avaliação da gravidade de doentes ventilados numa unidade de cuidados intensivos respiratórios pelo sistema APACHE II.

APACHE II system, is a simple and inexpensive method to evaluate severity of Intensive Care Patients. In a 2 years period (between 1988 and 1990), grading severity using APACHE II system, was performed on 498 consecutive mechanical Ventilated Patients in a Respiratory Intensive Care Unit. APACHE II was higher in COPD patients, but patients with Pneumonia and Organophosphate Poisoning had higher mortality. Correlating the different components of APACHE II with the results, we verified that Prognosis was not influenced by the Previous Health Status. Mortality was higher with increasing age, in patients with COPD and Organophosphate Poisoning. APS was the most important index for prognosis. Patients with Pneumonia and Organophosphate Poisoning had the highest APS. The Authors conclude that APACHE II is an objective and not time consuming method to evaluate severity in ICU Patients. However indexes measured on the first 24 hours of ICU staying are a result of severity of illness, treatment performed and time elapsed before ICU admission, and, this may be a possible source of bias when comparing different Unit results.

Voltage sensors of the frog skeletal muscle membrane require calcium to function in excitation-contraction coupling.

1. Intramembrane charge movements and changes in intracellular Ca2+ concentration (Ca2+ transients) elicited by pulse depolarization were measured in frog fast twitch cut muscle fibres under voltage clamp. 2. Extracellular solutions with very low [Ca2+] and 2 mM-Mg2+ , shown in the previous paper to reduce Ca2+ release from the sarcoplasmic reticulum (SR), were found to cause two changes in charge movement: (a) a decrease (-12 nC/microF) in the charge that moves during depolarizing pulses from -90 to 0 mV, termed here 'charge 1'; (b) an increase (+7 nC/microF) in the charge moved by hyperpolarizing pulses from -90 to -180 mV, termed 'charge 2'. 3. The increase in charge moved by hyperpolarizing pulses was correlated (r = 0.64) with the decrease in charge moved by depolarizing pulses and both were correlated with the inhibition of Ca2+ release recorded in the same fibres. 4. The low Ca2+ solutions caused a shift to more negative voltages of the dependence relating charge movement and holding potential (VH). This shift is of similar magnitude (about 22 mV) and direction as the shift in the curve relating Ca2+ release flux to VH (previous paper). 5. In solutions with normal [Ca2+] a conditioning depolarization to 0 mV, of 2 s duration, placed 100 ms before a test pulse from -70 to 0 mV, reduced by 30% the amount of charge displaced by the test pulse. Conditioning pulses of 1 s or less caused potentiation of charge movement by up to 30%. 6. In low Ca2+ solutions, reduction of charge was observed at all durations of the conditioning pulse. The duration for half-inhibition was near 200 ms. 7. An extracellular solution with no metal cations caused a more radical inhibition than the low Ca2+ solutions that contained Mg2+. The inhibition of Ca2+ release was essentially complete (90-100%). The charge moved by a pulse to 0 mV was reduced by 20 nC/microF and the charge moved by a pulse to -170 mV increased 8 nC/microF. This shows that Mg2+ supports excitation-contraction (E-C) coupling to some extent. 8. A state model of the voltage sensor of E-C coupling explains qualitatively the observations in both papers.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of extracellular calcium on calcium movements of excitation-contraction coupling in frog skeletal muscle fibres.

1. The effect of low extracellular free calcium ion concentration ([Ca2+]o) on the transient changes in cytoplasmic [Ca2+] associated with membrane depolarization (Ca2+ transients) was studied on single cut skeletal muscle fibres of the frog, voltage clamped in a double-Vaseline-gap chamber. The Ca2+ transients were monitored with the dye Antipyrylazo III diffused intracellularly. 2. The Ca2+ transients were substantially reduced in external salines with low [Ca2+] (10(-5) M or less and Mg2+ substituted for Ca2+). This decrease was more noticeable at late times during 100 ms or longer depolarizing pulses. 3. The rates of the processes that remove Ca2+ from the myoplasmic solution were not altered by the low [Ca2+]o. This implies that the input flux of Ca2+ into the myoplasm was reduced. 4. The Ca2+ input flux, equal to release flux from the sarcoplasmic reticulum (SR) plus Ca2+ influx via the T-tubule membrane Ca2+ channel, was derived from the Ca2+ transient. In low [Ca2+]o the peak input flux was reduced by 45% (n = 16 fibres) and decayed more rapidly during a depolarizing pulse. 5. The reduction in Ca2+ influx via the T-tubule membrane Ca2+ channel due to the reduced [Ca2+]o could not account for more than 5% of the reduction in Ca2- input flux, which was thus interpreted as an actual reduction of release from the SR. 6. The inward (T-tubular) Ca2+ current was not associated with this effect of extracellular Ca2+ as the effect was voltage independent at high intracellular voltages at which the Ca2+ inward current was strongly voltage dependent. 7. Low [Ca2+]o made Ca2+ release more readily inactivatable; the effect of low [Ca2-]o is best described as a left shift by 29 mV of the 'inactivation curve' of Ca2+ release, relating peak release flux to membrane holding potential. 8. The reduction of Ca2+ release by low [Ca2+]o was not accompanied by changes in the voltage dependence of Ca2+ release or in the threshold voltage for just-detectable release. 9. The results are consistent with a primary effect of Ca2+ on the T-tubular-membrane voltage sensor of excitation-contraction coupling.

Intramembrane charge movement in frog skeletal muscle fibres. Properties of charge 2.

1. Membrane currents were measured in cut skeletal muscle fibres voltage-clamped in a double Vaseline gap in solutions that had impermeant ions substituted for Na+, K+ and Cl-. The fibres were maintained at a holding potential of 0 mV. Pulses to positive voltages elicited outward currents that were proportional to voltage at all times; these were used to estimate linear capacitive currents, which in turn were used in the construction of non-linear current transients. 2. Large negative-going pulses elicited proportionally larger inward currents that decayed during the pulse with voltage-dependent kinetics. A portion of the non-linear current could be eliminated by solutions containing EGTA, as well as by large negative conditioning pulses of 200 ms or more. This portion was probably an inward Ca2+ current. 3. The non-linear current remaining in EGTA-containing solutions had characteristics of intramembrane charge movement ('charge 2'). This charge depended on voltage according to a two-state Boltzmann function of average parameters Qmax = 47.7 nC/microF, V = -115 mV, K = 21.5 mV (seven fibres). 4. The charge movement current transients were single-exponential decays (after a short rising phase) with time constants (tau) that depended on voltage (V). A single-barrier Eyring rate model described well the dependence of time constant on voltage. This fit permitted an independent estimate of a transition voltage, V, and a slope parameter K related to apparent valence of the mobile particle. The values of V and K that best fitted the kinetic data were close to the corresponding values estimated from the charge vs. voltage distribution. 5. Effective capacitance was measured by the transfer of capacitive charge by a small pulse superimposed on a variable pre-pulse. The capacitance was found to depend on pre-pulse voltage. The voltage dependence of the capacitance was as expected from the properties of charge 2 measured independently in the same fibres. 6. The presence of charge 2, defined as charge that moves in a very negative voltage range, was compared on the same fibres in a depolarized and a normally polarized (holding potential = -100 mV) situation. All fibres had less charge 2 at a holding potential of -100 mV (14 nC/microF average reduction). In these fibres charge 1, explored with pulses from -70 mV to 0 mV, was greater at a holding potential of -100 mV (18 nC/microF average increase).(ABSTRACT TRUNCATED AT 400 WORDS)

Simultaneous measurements of Ca2+ currents and intracellular Ca2+ concentrations in single skeletal muscle fibers of the frog.

The relationship between Ca2+ current amplitudes and myoplasmic Ca2+ transients was studied in single muscle fibers. Segments of muscle fibers were voltage-clamped in a double Vaseline gap chamber. Ca2+ transients were measured as an optical signal derived from the interaction between Ca2+ and the dye antipyrylazo III. The cells were maintained at -90 mV. Ca2+ currents were detected at pulse potentials to -50 mV, reached a maximum value at 0 mV, were reduced in size for larger depolarizations, and reversed at about 40 mV. Ca2+ transients were also detected at -50 Mv and progressively increased in size with larger pulse potentials up to 10 mV. Depolarizations to voltages greater than 10 mV did not further increase the size of the transient. The magnitude and time course of transients from 10 to 70 mV were almost identical Ca2+ fluxes into the myoplasm (Ca2+ input fluxes) were calculated from the Ca2+ transients applying a removal model. The size of the input fluxes increased with depolarization up to 0 mV. Between 0 and 70 mV the peak input flux slightly increased, while the flux measured at 200 ms remained unchanged. In conclusion, Ca2+ transients and input fluxes were not reduced during pulses to large positive potentials, even though a drastic reduction of Ca2+ current occurred at these potentials. These observations make it very unlikely that a voltage-dependent Ca2+ entry is the triggering signal for contraction.

Involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle.

The transduction of action potential to muscle contraction (E-C coupling) is an example of fast communication between plasma membrane events and the release of calcium from an internal store, which in muscle is the sarcoplasmic reticulum (SR). One theory is that the release channels of the SR are controlled by voltage-sensing molecules or complexes, located in the transverse tubular (T)-membrane, which produce, as membrane voltage varies, 'intramembrane charge movements', but nothing is known about the structure of such sensors. Receptors of the Ca-channel-blocking dihydropyridines present in many tissues, are most abundant in T-tubular muscle fractions from which they can be isolated as proteins. Fewer than 5% of muscle dihydropyridines are functional Ca channels; there is no known role for the remainder in skeletal muscle physiology. We report here that low concentrations of a dihydropyridine inhibit charge movements and SR calcium release in parallel. The effect has a dependence on membrane voltage analogous to that of specific binding of dihydropyridines. We propose specifically that the molecule that generates charge movement is the dihydropyridine receptor.