Alteration of sarcoplasmic reticulum Ca2+ ATPase expression in lower limb ischemia caused by atherosclerosis obliterans.

Atherosclerosis is a disease caused by a build-up of fatty plaques and cholesterol in the arteries. The lumen of the vessels is obliterated resulting in restricted blood supply to tissues. In ischemic conditions, the cytosolic Ca2+ level of skeletal muscle may increase, indicating the alteration of Ca2+ removal mechanisms. Ca2+ is transported from cytosol into the sarcoplasmic reticulum by Ca2+ ATPase (SERCA), with its 1a isoform expressed in adult, while its 1b isoform in neonatal and regenerating fast-twitch skeletal muscle. To investigate the role of these isoforms in ischemic skeletal muscle, biopsies from musculus biceps femoris of patients who underwent amputation due to atherosclerosis were examined. Samples were removed from the visibly healthy and hypoxia-affected tissue. Significantly increased SERCA1a expression was detected under the ischemic conditions (246 ± 69%; p < 0.05) compared with the healthy tissue. Furthermore, the ratio of SERCA1a-positive fibers was slightly increased (46 ± 4% in healthy tissue and 60 ± 5% in ischemic tissue; p > 0.05), whereas SERCA2a did not change. In addition, in primary cultures derived from hypoxia-affected tissue, the diameter and fusion index of myotubes were significantly increased (30 ± 1.6 µm vs. 41 ± 2.4 µm and 31 ± 4% vs. 45 ± 3%; p < 0.05). We propose that the increased SERCA1a expression indicates the existence and location of compensating mechanisms in ischemic muscle.

Effects of fluvastatin and coenzyme Q10 on skeletal muscle in normo- and hypercholesterolaemic rats.

Myalgia and muscle weakness may appreciably contribute to the poor adherence to statin therapy. Although the pathomechanism of statin-induced myopathy is not completely understood, changes in calcium homeostasis and reduced coenzyme Q10 levels are hypothesized to play important roles. In our experiments, fluvastatin and/or coenzyme Q10 was administered chronically to normocholesterolaemic or hypercholaestherolaemic rats, and the modifications of the calcium homeostasis and the strength of their muscles were investigated. While hypercholesterolaemia did not change the frequency of sparks, fluvastatin increased it on muscles both from normocholesterolaemic and from hypercholesterolaemic rats. This effect, however, was not mediated by a chronic modification of the ryanodine receptor as shown by the unchanged ryanodine binding in the latter group. While coenzyme Q10 supplementation significantly reduced the frequency of the spontaneous calcium release events, it did not affect their amplitude and spatial spread in muscles from fluvastatin-treated rats. This indicates that coenzyme Q10 supplementation prevented the spark frequency increasing effect of fluvastatin without having a major effect on the amount of calcium released during individual sparks. In conclusion, we have found that fluvastatin, independently of the cholesterol level in the blood, consistently and specifically increased the frequency of calcium sparks in skeletal muscle cells, an effect which could be prevented by the addition of coenzyme Q10 to the diet. These results support theories favouring the role of calcium handling in the pathophysiology of statin-induced myopathy and provide a possible pathway for the protective effect of coenzyme Q10 in statin treated patients symptomatic of this condition.

The role of the different neoglottis forms in the development of esophageal voice.

UNLABELLED: Evaluation of voice quality parameters of esophageal speech in different neoglottis forms after total laryngectomy. METHODS: Presentation of voice analysis of 20 patients, who underwent total laryngectomy. The success of acquiring this technique was estimated by means of a voice analyzing program (pitch, sound-holding, loudness, spectrogram),and by the intelligibility via the telephone. Shape of the different types of neoglottis that developed and its functioning during vocalization and continuous speech were observed by nasal endoscopy. Data obtained from the voice analysis were compared among the observed three different neoglottis forms. RESULTS: The average dysphonia index of the 20 patients was 1.67 ± 0.38 (mean ± SD). Nasal fiberoscopic examination revealed three different types of neoglottis forms ­ a small mucosal button, two mucosal battens, and a mucosal lip. Voice quality of the esophageal speech of the patients with the mucosal button was found to be the closest to normal by subjective and objective acoustical evaluation. CONCLUSIONS: These findings emphasize the importance of the proper wound closure technique which can facilitate the development of a special button shape neoglottis form and help to acquire esophageal speech with the best quality parameters shortly after total laryngectomy.

Melanoma cells exhibit strong intracellular TASK-3-specific immunopositivity in both tissue sections and cell culture.

Amplification of the kcnk9 gene and overexpression of the encoded channel protein (TASK-3) seems to be involved in carcinogenesis. In the present work, TASK-3 expression of melanoma cells has been studied. For the investigation of TASK-3-specific immunolabelling, a monoclonal antibody has been developed and applied along with two, commercially available polyclonal antibodies targeting different epitopes of the channel protein. Both primary and metastatic melanoma cells proved to be TASK-3 positive, showing prominent intracellular TASK-3-specific labelling; mostly concentrating around or in the proximity of the nuclei. The immunoreaction was associated with the nuclear envelope, and with the processes of the cells and it was also present in the cell surface membrane. Specificity of the immunolabelling was confirmed by Western blot and transfection experiments. As TASK-3 immunopositivity of benign melanocytes could also be demonstrated, the presence or absence of TASK-3 channels cannot differentiate between malignant and non-malignant melanocytic tumours.

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.

Differences in electrophysiological and contractile properties of mammalian cardiac tissues bathed in bicarbonate - and HEPES-buffered solutions.

AIM: The aim of this study was to compare the action potential configuration, contractility, intracellular Ca2+ and H+ concentrations in mammalian cardiac tissues bathed with Krebs and Tyrode solutions at 37 degrees C. RESULTS: In Langendorff-perfused guinea-pig hearts, loaded with the fluorescent Ca2+-indicator Fura-2, or H+-sensitive dye carboxy-SNARF, shifts from Krebs to Tyrode solution caused intra-cellular acidification, increased diastolic pressure and [Ca2+]i, decreased systolic pressure and [Ca2+]i, leading to a reduction in the amplitude of [Ca2+]i transients and pulse pressure. Contractility was also depressed in canine ventricular trabeculae when transferred from Krebs to Tyrode solution. Shifts from Krebs to Tyrode solution increased the duration of action potentials in multicellular cardiac preparations excised from canine and rabbit hearts but not in isolated cardiomyocytes. All these changes in action potential morphology, contractility, [Ca2+]i and [H+]i were readily reversible by addition of 26 mmol L(-1) bicarbonate to Tyrode solution. Effects of dofetilide and CsCl, both blockers of the delayed rectifier K current, on action potential duration were compared in Krebs and Tyrode solutions. Dofetilide lengthened rabbit ventricular action potentials in a significantly greater extent in Tyrode than in Krebs solution. Exposure of canine Purkinje fibres to CsCl evoked early after depolarizations within 40 min in all preparations incubated with Tyrode solution, but not in those bathed with Krebs solution. CONCLUSION: It is concluded that the marked differences in action potential morphology, [Ca2+]i, [H+]i and contractility observed between preparations bathed with Krebs and Tyrode solutions are more likely attributable to differences in the intracellular buffering capacities of the two media.

Intramembrane charge movement and L-type calcium current in skeletal muscle fibers isolated from control and mdx mice.

Dystrophin-deficient muscle fibers from mdx mice are believed to suffer from increased calcium entry and elevated submembranous calcium level, the actual source and functional consequences of which remain obscure. Here we compare the properties of the dihydropyridine receptor as voltage sensor and calcium channel in control and mdx muscle fibers, using the silicone-voltage clamp technique. In control fibers charge movement followed a two-state Boltzmann distribution with values for maximal charge, midpoint voltage, and steepness of 23 +/- 2 nC/ micro F, -37 +/- 3 mV, and 13 +/- 1 mV (n = 7). Essentially identical values were obtained in mdx fibers and the time course of charge recovery from inactivation was also similar in the two populations (tau approximately 6 s). In control fibers the voltage dependence of the slow calcium current elicited by 100-ms-long pulses gave values for maximal conductance, apparent reversal potential, half-activation potential, and steepness factor of 156 +/- 15 S/F, 65.5 +/- 2.9 mV, -0.76 +/- 1.2 mV, and 6.2 +/- 0.5 mV (n = 17). In mdx fibers, the half-activation potential of the calcium current was slightly more negative (-6.2 +/- 1.2 mV, n = 16). Also, when using longer pulses, the time constant of calcium current decay was found to be significantly larger (by a factor of 1.5-2) in mdx than in control fibers. These changes in calcium current properties are unlikely to be primarily responsible for a dramatic alteration of intracellular calcium homeostasis. They may be speculated to result, at least in part, from remodeling of the submembranous cytoskeleton network due to the absence of dystrophin.

Effects of dantrolene on steps of excitation-contraction coupling in mammalian skeletal muscle fibers.

The effects of the muscle relaxant dantrolene on steps of excitation-contraction coupling were studied on fast twitch muscles of rodents. To identify the site of action of the drug, single fibers for voltage-clamp measurements, heavy SR vesicles for calcium efflux studies and solubilized SR calcium release channels/RYRs for lipid bilayer studies were isolated. Using the double Vaseline-gap or the silicone-clamp technique, dantrolene was found to suppress the depolarization-induced elevation in intracellular calcium concentration ([Ca2+]i) by inhibiting the release of calcium from the SR. The suppression of [Ca2+]i was dose-dependent, with no effect at or below 1 microM and a 53 +/- 8% (mean +/- SEM, n = 9, cut fibers) attenuation at 0 mV with 25 microM of extracellularly applied dantrolene. The drug was not found to be more effective if injected than if applied extracellularly. Calculating the SR calcium release revealed an equal suppression of the steady (53 +/- 8%) and of the early peak component (46 +/- 6%). The drug did not interfere with the activation of the voltage sensor in as much as the voltage dependence of both intramembrane charge movements and the L-type calcium currents (I(Ca)) were left, essentially, unaltered. However, the inactivation of I(Ca) was slowed fourfold, and the conductance was reduced from 200 +/- 16 to 143 +/- 8 SF(-1) (n = 10). Dantrolene was found to inhibit thymol-stimulated calcium efflux from heavy SR vesicles by 44 +/- 10% (n = 3) at 12 microM. On the other hand, dantrolene failed to affect the isolated RYR incorporated into lipid bilayers. The channel displayed a constant open probability for as long as 30-50 min after the application of the drug. These data locate the binding site for dantrolene to be on the SR membrane, but be distinct from the purified RYR itself.

Altered inhibition of the rat skeletal ryanodine receptor/calcium release channel by magnesium in the presence of ATP.

Magnesium-induced inhibition of the skeletal ryanodine receptor/calcium-release channel (RyR) was studied in the presence and absence of ATP under isolated conditions and in situ, by examining the RyR incorporated into a planar lipid bilayer and the calcium release flux (Rrel) in isolated single fibres mounted in the double Vaseline gap system. When the incorporated RyR had been activated by calcium (50 microM) in the absence of ATP, the magnesium-induced inhibition showed co-operativity with a Hill coefficient (N) of 1.83 and a half-inhibitory concentration (IC50) of 635 microM. When the open probability was measured in the presence of 5 mM ATP and at a low calcium concentration, the magnesium-induced inhibition was non-cooperative (N=1.1, IC50= 860 microM). In isolated muscle fibres, in the presence of ATP, lowering the intracellular magnesium concentration ([Mg2+]i) increased the maximal Rrel and shifted its voltage dependence to more negative membrane potentials. Increasing [Mg2+]i had the opposite effect. The concentration dependence was described with an IC50 of 174 microM, N=1, under depolarized conditions and showed a tenfold increase in affinity in polarized fibres. At the concentration required for the measurements from isolated fibres, ATP had a full activatory effect on the isolated channel. At a low calcium concentration, the RyR had two ATP-binding sites with half-activatory concentrations of 19 and 350 microM, respectively.

Deterministic inactivation of calcium release channels in mammalian skeletal muscle.

Enzymatically dissociated fibres from the extensor digitorum communis muscle of rats were mounted into a double Vaseline gap chamber. The rate of calcium release (R(rel)) from the sarcoplasmic reticulum (SR) and changes in SR permeability to Ca2+ (PSR) were calculated from measured changes in intracellular calcium concentration. Calcium release during a prepulse attenuated the inactivating component of PSR of the subsequent test pulse. The suppression was graded, larger release causing greater suppression, as expected from a calcium-dependent inactivation process. However, if the dissociation constant of the putative inhibitory calcium binding site (Kd) was estimated using different test pulses different affinities were obtained: a smaller test pulse yielded a smaller Kd. Comparing the suppression of the inactivatable component of PSR during the test pulse (suppression) with the inactivatable component during the prepulse (pre-inactivation) revealed a linear relationship with a regression coefficient close to unity. Lowering intracellular magnesium by decreasing its concentration to 25 microM in the internal solution altered the time course of PSR. The maximal peak-to-steady-level ratio was increased to 6.3 +/- 0.4 (n = 10, mean +/- s.e.m.) from a control value of 3.0 +/- 0.2 (n = 19). Despite the apparent change in steady-state inactivation, suppression remained equal to that pre-inactivation. Our results support the view that a depolarizing pulse always recruits the same set of calcium release channels and a portion of these channels undergoes a deterministic inactivation process.

Regulation of the rat sarcoplasmic reticulum calcium release channel by calcium.

The regulation by calcium of the ryanodine receptor/SR calcium release channel (RyR) from rat skeletal muscle was studied under isolated conditions and in situ. RyRs were either solubilized and incorporated into lipid bilayers or single fibres were mounted into a Vaseline gap voltage clamp. Single channel data were compared to parameters determined from the calculated calcium release flux. With K+ (250 mM) being the charge carrier the single channel conductance was 529 pS at 50 microM Ca2+ cis and trans, and decreased with increasing cis [Ca2+]. Open probability showed a bell shaped calcium dependence revealing an activatory and an inhibitory Ca2+ binding site (Hill coefficients of 1.18 and 1.28, respectively) with half activatory and inhibitory concentrations of 9.4 and 298 microM. The parameters of the inhibitory site agreed with the calcium dependence of channel inactivation deduced from the decline in SR calcium release in isolated fibres. Mean open time showed slight [Ca2+] dependence following a single exponential at every Ca2+ concentration tested. Closed time histograms, at high [Ca2+], were fitted with three exponentials, from which the longest was calcium independent, and resembled the recovery time constant of SR inactivation (115+/-15 ms) obtained in isolated fibres. The data are in agreement with a model where calcium binding to the inhibitory site on RyR would be responsible for the calcium dependent inactivation in situ.

Interacting effects of capsaicin and anandamide on intracellular calcium in sensory neurones.

Capsaicin (100 nM to 1 microM) and anandamide (200 nM to 10 microM) caused a transient increase in fluorescence of fura-2 loaded cultured small trigeminal neurones of rats measured with a ratiometric technique. The percentage of cells responding to capsaicin at 100 nM, 330 nM and 1 microM was 47.4%, 45.3%, and 70.4%, respectively. Averaged peak value of fluorescense ratio (R) at 340 and 380 nm excitation was slightly dose dependent. Peaks of anandamide-induced transients were R = 0.2 at 200 nM and 0.16 at 10 microM. Near 40% of capsaicin-sensitive cells responded also to anandamide. Anandamide (200 nM) inhibited the capsaicin-induced calcium influx. The results suggest that anandamide increases intracellular calcium and inhibits capsaicin-evoked calcium transients.

Calcium release activated calcium entry in a human skin derived cell line (HaCaT).

Using isolated cells from a spontaneously immortalized human keratinocyte cell line (HaCaT) loaded with Fura-2 the regulation of intracellular Ca2+ concentration ([Ca2+]i) was studied. The continuous presence of ATP induced a biphasic response in high external Ca2+. The first component reflected the release of calcium from intracellular stores since it was present after the removal of external calcium with ethylene-glycol-bis-N,N,N',N'-tetraacetic acid (EGTA). The second phase of [Ca2+]i increase was not detectable in the absence of external calcium and raising the extracellular [Ca2+] increased the rate of rise in [Ca2+]i suggesting that it was influenced by the external environment. Furthermore, after adenosine triphosphate (ATP) had emptied the intracellular store in a calcium-free milieu, the elevation of external Ca2+ induced a secondary increase in [Ca2+]i that did not require the presence of ATP. Depleting the intracellular calcium store with a Ca-ATP-ase inhibitor (cyclopiasonic acid, 10 microM) also induced calcium entry. The depletion induced calcium entry was inhibited by econazole (100 microM). These findings indicate the presence of a calcium release activated calcium influx pathway in HaCaT keratinocytes.

Biphasic effect of bimoclomol on calcium handling in mammalian ventricular myocardium.

1. Concentration-dependent effects of bimoclomol, the novel heat shock protein coinducer, on intracellular calcium transients and contractility were studied in Langendorff-perfused guinea-pig hearts loaded with the fluorescent calcium indicator dye Fura-2. Bimoclomol had a biphasic effect on contractility: both peak left ventricular pressure and the rate of force development significantly increased at a concentration of 10 nM or higher. The maximal effect was observed between 0.1 and 1 microM, and the positive inotropic action disappeared by further increasing the concentration of bimoclomol. The drug increased systolic calcium concentration with a similar concentration-dependence. In contrast, diastolic calcium concentration increased monotonically in the presence of bimoclomol. Thus low concentrations of the drug (10 - 100 nM) increased, whereas high concentrations (10 microM) decreased the amplitude of intracellular calcium transients. 2. Effects of bimoclomol on action potential configuration was studied in isolated canine ventricular myocytes. Action potential duration was increased at low (10 nM), unaffected at intermediate (0.1 - 1 microM) and decreased at high (10 - 100 microM) concentrations of the drug. 3. In single canine sarcoplasmic calcium release channels (ryanodine receptor), incorporated into artificial lipid bilayer, bimoclomol significantly increased the open probability of the channel in the concentration range of 1 - 10 microM. The increased open probability was associated with increased mean open time. The effect of bimoclomol was again biphasic: the open probability decreased below the control level in the presence of 1 mM bimoclomol. 4. Bimoclomol (10 microM - 1 mM) had no significant effect on the rate of calcium uptake into sarcoplasmic reticulum vesicles of the dog, indicating that in vivo calcium reuptake might not substantially be affected by the drug. 5. In conclusion, the positive inotropic action of bimoclomol is likely due to the activation of the sarcoplasmic reticulum calcium release channel in mammalian ventricular myocardium.

Frog skeletal muscle fibers recovering from fatigue have reduced charge movement.

Following prolonged exercise, muscle force production is often impaired. One possible cause of this force deficit is impaired intracellular activation. We have used single skeletal muscle fibers from the lumbrical muscle of Xenopus laevis to study the effects of fatigue on excitation-contraction coupling. Fatigue was induced in 13 intact fibers. Five fibers recovered in normal Ringer only (fatigued-only fibers). The remaining eight fibers were subjected to a brief hypotonic treatment (F-H fibers) that is known to prolong the effects of fatigue. Intramembrane charge movement, changes in intracellular calcium concentration ([Ca2+]i) and force transients were measured in a single Vaseline gap chamber under voltage clamp. In F-H fibers, membrane capacitance was reduced. Confocal microscopy showed that this was not due to closure of the transverse tubules. The amount of normalized intramembrane charge was reduced from 21.0 +/- 2.8 nC/microF (n = 10) in rested fibers to 12.2 +/- 1.1 nC/microF in F-H fibers. However, the voltage dependence of intramembrane charge movement was unchanged. In F-H fibers, force production was virtually abolished. This was the consequence of the greatly reduced [Ca2+]i accompanying a depolarizing pulse. In recovering fatigued-only fibers, while the maximal available charge was not significantly smaller (18.3 +/- 1.1 nC/ microF), both calcium and force were reduced, albeit to a lesser extent than in F-H fibers. The data are consistent with a model where fatigue reduces the number of voltage sensors in the t-tubules and, in addition, alters the coupling between the remaining functional voltage sensors and the calcium channels of the sarcoplasmic reticulum.

Differential effects of caffeine and perchlorate on excitation-contraction coupling in mammalian skeletal muscle.

1. Enzymatically dissociated single muscle fibres of the rat were studied under voltage clamp conditions in a double Vaseline gap experimental chamber. Intramembrane charge movement and changes in intracellular calcium concentration ([Ca2+]i) were measured and the rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) was calculated. This enabled the determination of SR permeability and thus the estimation of the transfer function between intramembrane charge movement and SR permeability. 2. Perchlorate (3 mM) shifted the membrane potential dependence of intramembrane charge movement to more negative voltages without any effect on the steepness or on the maximal available charge. The drug increased SR permeability at every membrane potential but did not alter the peak-to-steady level ratio. It also increased the slope of the transfer function, indicating a more efficient coupling between the voltage sensors and the ryanodine receptors. 3. Caffeine (1 mM), on the other hand, increased SR permeability without altering the voltage dependence of intramembrane charge movement. It neither prolonged the depolarization-induced increase in [Ca2+]i at short pulse durations nor altered the time to peak of Rrel. The augmentation of SR permeability by the drug was more pronounced during the peak caffeine response than during its steady level. This was manifested in a leftward shift of the transfer function rather than an increase in its slope. 4. These observations indicate that perchlorate and caffeine alter the coupling between the voltage sensors and SR calcium release channels in mammalian skeletal muscle. They do not, however, share a common mechanism for enhancing the depolarization-induced release of calcium from the SR.

Effects of tetracaine on sarcoplasmic calcium release in mammalian skeletal muscle fibres.

1. Single muscle fibres were dissociated enzymatically from the extensor digitorum communis muscle of rats. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation-contraction coupling were studied under voltage clamp conditions in the presence and absence of the local anaesthetic tetracaine. 2. Changes in intracellular calcium concentration ([Ca2+]i) were monitored using the calcium sensitive dyes antipyrylazo III and fura-2 and the rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) was calculated. Tetracaine decreased the maximal attained [Ca2+]i and suppressed, in a dose-dependent manner, both the early peak and the steady level of Rrel in the voltage range examined. 3. The concentration dependence of the effects on the two kinetic components of Rrel were almost identical with a half-effective concentration (K50) of 70 and 71 microM and a Hill coefficient (nH) of 2.7 and 2.3 for the peak and the steady level, respectively. Furthermore, the drug did not alter the peak to steady level ratio up to a concentration (50 microM) that caused a 35 +/- 5 % reduction in calcium release. Higher concentrations did suppress the ratio but the degree of suppression was voltage independent. 4. Tetracaine (50 microM) neither influenced the total available intramembrane charge nor altered its membrane potential dependence. It shifted the transfer function, the normalized SR permeability versus normalized charge to the right, indicating that similar charge transfer caused a smaller increase in SR permeability. 5. To explore the site of action of tetracaine further the ryanodine receptor (RyR) calcium release channel of the SR was purified and reconstituted into planar lipid bilayers. The reconstituted channel had a conductance of 511 +/- 14 pS (n = 8) in symmetric 250 mM KCl that was not affected by tetracaine. Tetracaine decreased the open probability of the channel in a concentration-dependent manner with K50 = 68 microM and nH = 1.5. 6. These experiments show that tetracaine suppresses SR calcium release in enzymatic isolated mammalian skeletal muscle fibres. This effect is due, presumably, to the decreased open probability of the RyR in the presence of the drug. Since both the inactivating peak and the steady level of Rrel were equally affected by tetracaine, our observations suggest that there is a tight coupling between these kinetic components of SR calcium release in mammalian skeletal muscle.

Regulation of the ryanodine receptor calcium release channel of the sarcoplasmic reticulum in skeletal muscle.

In striated muscle contraction is under the tight control of myoplasmic calcium concentration ([Ca2+]i): the elevation in [Ca2+]i and the consequent binding of calcium to troponin C enables, while the decrease in [Ca2+]i prevents the actin-myosin interaction. Calcium ions at rest are stored in the sarcoplasmic reticulum (SR) from which they are rapidly released upon the depolarisation of the sarcolemmal and transverse (T-) tubular membranes of the muscle cell. The protein responsible for this controlled and fast release of calcium is the calcium release channel found in the membrane of the terminal cisternae of the SR. This review focuses on the physiological and pharmacological modulators of the calcium release channel and tries to draw an up-to-date picture of the events that occur between T-tubular depolarisation and the release of calcium from the SR.

Distinct subpopulations in HaCaT cells as revealed by the characteristics of intracellular calcium release induced by phosphoinositide-coupled agonists.

Intracellular calcium release induced by transient applications of phosphoinositide agonists was measured using adherent single HaCaT keratinocytes loaded with the acetoxymethyl derivative of fura-2. Application of ATP, bradykinin and formyl-Met-Leu-Phe (fMLP) resulted in a transient increase in intracellular calcium concentration ([Ca2+]i) with an average half-width of 40+/-21 s and a decay time constant of 15+/-10 s (mean +/- SD, n = 108), irrespective of the agonist applied. The cells could be classified into two groups: in 53% of the cells repeated stimulation brought about a progressively smaller change in [Ca2+]i (type 1 cells), whereas in the remaining cells the amplitude of the calcium transients was essentially unchanged (type 2 cells). Furthermore, calcium transients in type 1 cells had broader half-widths and slower decays. No difference was found between the agonists in respect of the characteristics of the evoked calcium transient within each subpopulation. However, bradykinin and fMLP desensitized some cells. These results indicate that the activation of the inositol trisphospate transduction pathway by different agonists induces a characteristic elevation of [Ca2+]i within a given cell. Our results demonstrate that cultured HaCaT keratinocytes are heterogeneous in respect of the calcium transients evoked by the activators of this second messenger system.

Measurements of intracellular Mg2+ concentration in mouse skeletal muscle fibers with the fluorescent indicator mag-indo-1.

Measurements of intracellular free magnesium concentration ([Mg2+]i) were performed on enzymatically isolated skeletal muscle fibers from mice, using the fluorescent ratiometric indicator mag-indo-1. An original procedure was developed to calibrate the dye response within the fibers: fibers were first permeabilized with saponin in the presence of a given extracellular magnesium concentration and were then embedded in silicone grease. The dye was then pressure microinjected into the saponin-permeabilized silicone-embedded fibers, and fluorescence was measured. The results show that for all tested [Mg2+], the value of the measured fluorescence ratio was higher than that found in aqueous solutions. Furthermore, the apparent binding curve that could be fit to the in vivo ratio data was shifted toward higher [Mg2+] by a factor of approximately 2. Using the in vivo calibration parameters, the mean resting [Mg2+]i was found to be 1.53 +/- 0.16 mM (n = 7). In an attempt to gain insight into the myoplasmic magnesium buffering capacity, we measured, together with mag-indo-1 fluorescence, the current elicited by the application of carbamylcholine (CCh) to the endplate of isolated fibers, in the presence of a high extracellular magnesium concentration. The results show that, under these conditions, a change in [Mg2+]i displaying a time course and amplitude qualitatively consistent with the CCh-induced inward current can be measured.