Relaxation evoked by extracellular Ca(2+) in rat aorta is nerve-independent and involves sarcoplasmic reticulum and L-type Ca(2+) channell

The perivascular nerve network expresses a Ca(2+) receptor that is activated by high extracellular Ca(2+) concentrations and causes vasorelaxation in resistance arteries. We have verified the influence of perivascular nerve fibers on the Ca(2+)-induced relaxation in aortic rings. To test our hypothesis, either pre-contracted aortas isolated from rats after sensory denervation with capsaicin or aortic rings acutely denervated with phenol were stimulated to relax with increasing extracellular Ca(2+) concentration. We also studied the role of the endothelium on the Ca(2+)-induced relaxation, and we verified the participation of endothelial/nonendothelial nitric oxide and cyclooxygenise-arachidonic acid metabolites. Additionally, the role of the sarcoplasmic reticulum, K(+) channels and L-type Ca(2+) channels on the Ca(2+)-induced relaxation were evaluated. We have observed that the Ca(2+)-induced relaxation is completely nerve independent, and it is potentiated by endothelial nitric oxide (NO). In endothelium-denuded aortic rings, indomethacin and AH6809 (PGF(2 alpha) receptor antagonist) enhance the relaxing response to Ca(2+). This relaxation is inhibited by thapsigargin and verapamil, while was not altered by tetraethylammonium. In conclusion, we have shown that perivascular nervous fibers do not participate in the Ca(2+)-induced relaxation, which is potentiated by endothelial NO. In endothelium-denuded preparations, indomethacin and AH6809 enhance the relaxation induced by Ca(2+). The relaxing response to Call was impaired by verapamil and thapsigargin, revealing the importance of L-type Ca(2+) channels and sarcoplasmic reticulum in this response. (c) 2008 Elsevier Inc. All rights reserved.

Effects of beta-escin and saponin on the transverse-tubular system and sarcoplasmic reticulum membranes of rat and toad skeletal muscle

Mechanically skinned skeletal muscle fibres from rat and toad were exposed to the permeabilizing agents beta-escin and saponin. The effects of these agents on the sealed transverse tubular system (t-system) and sarcoplasmic reticulum (SR) were examined by looking at changes in the magnitude of the force responses to t-system depolarization, the time course of the fluorescence of fura-2 trapped in the sealed t-system, and changes in the magnitude of caffeine-induced contractures following SR loading with Ca2+ under defined conditions. In the presence of 2 mu g ml(-1) beta-escin and saponin, the response to t-system depolarization was not completely abolished, decreasing to a plateau, and a large proportion of fura-2 remained in the sealed t-system. At 10 mu g ml(-1), both agents abolished the ability of both rat and toad preparations to respond to t-system depolarization after 3 min of exposure, but a significant amount of fura-2 remained in sealed t-tubules even after exposure to 100 mu g ml(-1) beta-escin and saponin for 10 min. beta-Escin took longer than saponin to reduce the t-system depolarizations and fura-2 content of the sealed t-system to a similar level. The ability of the SR to load Ca2+ was reduced to a lower level after treatment with beta-escin than saponin. This direct effect on the SR occurred at much lower concentrations for rat (2 mu g ml(-1) beta-escin and 10 mu g ml(-1) saponin) than toad (10 mu g ml(-1) beta-escin and 150 mu g ml(-1) saponin). The reverse order in sensitivities to beta-escin and saponin of t-system and SR membranes indicates that the mechanisms of action of beta-escin and saponin are different in the two types of membrane. In conclusion, this study shows that: (1) beta-escin has a milder action on the surface membrane than saponin; (2) beta-escin is a more potent modifier of SR function; (3) simple permeabilization of membranes is not sufficient to explain the effects of beta-escin and saponin on muscle membranes; and (4) the t-system network within muscle fibres is not a homogeneous compartment.

Effects of Mg2+ on Ca2+ release from sarcoplasmic reticulum of skeletal muscle fibres from yabby (crustacean) and rat

1. The role of myoplasmic [Mg2+] on Ca2+ release from the sarcoplasmic reticulum (SR) was examined in the two major types of crustacean muscle fibres, the tonic, long sarcomere fibres and the phasic, short sarcomere fibres of the fresh mater decapod crustacean Cherax: destructor (yabby) and in the fast-twitch rat muscle fibres using the mechanically skinned muscle fibre preparation. 2. A robust Ca2+-induced Ca2+-release (CICR) mechanism was present in both long and short sarcomere fibres and 1 mM Mg2+ exerted a strong inhibitory action on the XR Ca2+ release in both fibre types. 3. The XR displayed different properties with respect to Ca2+ loading in the long and the short sarcomere fibres and marked functional differences were identified with respect to Mg2+ inhibition between the two crustacean fibre types. Thus, in long sarcomere fibres, the submaximally loaded XR was able to release Ca2+ when [Mg2+] was lowered from 1 to 0.01 mw in the presence of 8 mM ATP(total) and in the virtual absence of Ca2+ (< 5 nM) even when the CICR was suppressed. In contrast, negligible Ca2+ was released from the submaximally loaded SR of short sarcomere yabby fibres when [Mg2+] was lowered from 1. to 0.01 mM under the same conditions as for the long sarcomere fibres. Nevertheless, the rate of XR Ca2+ release in short sarcomere fibres increased markedly when [Mg2+] was lowered in the presence of [Ca2+] approaching the normal resting levels (50-100 nM). 4. Rat fibres were able to release SR Ca2+ at a faster rate than the long sarcomere yabby fibres when [Mg2+] was lowered from 1 to 0.01 mM in the virtual absence of Ca2+ but, unlike with yabby fibres, the net rate of Ca2+ release was actually increased for conditions that were considerably less favourable to CICR. 5. In summary it is concluded that crustacean skeletal muscles have more that one functional type of Ca2+-release channels, that these channels display properties that are intermediate between those of mammalian skeletal and cardiac isoforms, that the inhibition exerted by Mg2+ at rest on the crustacean SR Ca2+-release channels must be removed during excitation-contraction coupling and that, unlike in crustacean fibres, CICR cannot play the major role in the activation of XR Ca2+-release channels in the rat skeletal muscle.

Elevated plasma membrane and sarcoplasmic reticulum Ca2+ pump mRNA levels in cultured aortic smooth muscle cells from spontaneously hypertensive rats

We have observed previously that Ca2+ pump-mediated Ca2+ efflux is elevated in cultured aortic smooth muscle cells from spontaneously hypertensive rats compared to those from Wistar-Kyoto rat controls. The objective of this work was to determine if these strains differ in mRNA levels for the PMCA1 isoform of the plasma membrane Ca2+-ATPase and the SERCA2 isoform of the sarcoplasmic reticulum Ca2+-ATPase. mRNA levels were compared in cultured aortic smooth muscle cells from 10-week-old male rats. PMCA1 and SERCA2 mRNA levels were elevated in SHR compared to WKY. Angiotensin II increased the level of PMCA1 and SERCA2 mRNA in both strains. These studies provide further evidence for alterered Ca2+ homeostasis in hypertension at the level of Ca2+ transporting ATPases in the spontaneously hypertensive rat model. These data are also consistent with the hypothesis that the expression of these two Ca2+ pumps may be linked. (C) 1997 Academic Press

Effect of saponin treatment on the sarcoplasmic reticulum of rat, cane toad and crustacean (yabby) skeletal muscle

1. Mechanically skinned fibres from skeletal muscles of the rat, toad and yabby were used to investigate the effect of saponin treatment on sarcoplasmic reticulum (SR) Ca2+ loading properties. The SR was loaded submaximally under control conditions before and after treatment with saponin and SR Ca2+ was released with caffeine. 2. Treatment with 10 mu g ml(-1) saponin greatly reduced the SR Ca2+ loading ability of skinned fibres from the extensor digitorum longus muscle of the rat with a rate constant of 0.24 min(-1). Saponin concentrations up to 150 mu g ml(-1) and increased exposure time up to 30 min did not further reduce the SR Ca2+ loading ability of the SR, which indicates that the inhibitory action of 10-150 mu g ml(-1) saponin is not dose dependent. The effect of saponin was also not dependent on the state of polarization of the transverse-tubular system. 3. Treatment with saponin at concentrations up to 100 mu g ml(-1) for 30 min did not affect the Ca2+ loading ability of SR in skinned skeletal muscle fibres from the twitch portion of the toad iliofibularis muscle but SR Ca2+ loading ability decreased markedly with a time constant of 0.22 min(-1) in the presence of 150 mu g ml(-1) saponin. 4. The saponin dependent increase in permeability could be reversed in both rat and toad fibres by short treatment with 6 mu M Ruthenium Red, a potent SR Ca2+ channel blocker, suggesting that saponin does affect the SR Ca2+ channel properties in mammalian and anuran skeletal muscle. 5. Treatment of skinned fibres of long sarcomere length (> 6 mu m) from the claw muscle of the yabby (a freshwater decapod crustacean) with 10 mu g ml(-1) saponin for 30 min abolished the ability of the SR to load Ca2+, indicating that saponin affects differently the SR from skeletal muscles of mammals, anurans and crustaceans. 6. is concluded that at relatively low concentrations, saponin causes inhibition of the skeletal SR Ca2+ loading ability in a species dependent manner, probably by increasing the Ca2+ loss through SR Ca2+ release channels.

Sarcoplasmic reticulum calcium ATPase is inhibited by organic vanadium coordination compounds: pyridine-2,6-dicarboxylatodioxovanadium(V), BMOV, and an amavadine analogue

Inorg Chem. 2008 Jul 7;47(13):5677-84. doi: 10.1021/ic702405d

Inhibition of the sarcoplasmic reticulum Ca2+ pump with thapsigargin to estimate the contribution of Na+-Ca2+ exchange to ventricular myocyte relaxation

Relaxation in the mammalian ventricle is initiated by Ca2+ removal from the cytosol, which is performed by three main transport systems: sarcoplasmic reticulum Ca2+-ATPase (SR-A), Na+-Ca2+ exchanger (NCX) and the so-called slow mechanisms (sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uptake). To estimate the relative contribution of each system to twitch relaxation, SR Ca2+ accumulation must be selectively inhibited, usually by the application of high caffeine concentrations. However, caffeine has been reported to often cause changes in membrane potential due to NCX-generated inward current, which compromises the reliability of its use. In the present study, we estimated integrated Ca2+ fluxes carried by SR-A, NCX and slow mechanisms during twitch relaxation, and compared the results when using caffeine application (Cf-NT) and an electrically evoked twitch after inhibition of SR-A with thapsigargin (TG-TW). Ca2+ transients were measured in 20 isolated adult rat ventricular myocytes with indo-1. For transients in which one or more transporters were inhibited, Ca2+ fluxes were estimated from the measured free Ca2+ concentration and myocardial Ca2+ buffering characteristics. NCX-mediated integrated Ca2+ flux was significantly higher with TG-TW than with Cf-NT (12 vs 7 µM), whereas SR-dependent flux was lower with TG-TW (77 vs 81 µM). The relative participations of NCX (12.5 vs 8% with TG-TW and Cf-NT, respectively) and SR-A (85 vs 89.5% with TG-TW and Cf-NT, respectively) in total relaxation-associated Ca2+ flux were also significantly different. We thus propose TG-TW as a reliable alternative to estimate NCX contribution to twitch relaxation in this kind of analysis.

Probing the SERCA1a sarcoplasmic reticulum Ca2+-ATPase phosphorylation-site mutant D351E with inorganic phosphate

The expression of sarcoplasmic reticulum SERCA1a Ca2+-ATPase wild-type and D351E mutants was optimized in yeast under the control of a galactose promoter. Fully active wild-type enzyme was recovered in yeast microsomal membrane fractions in sufficient amounts to permit a rapid and practical assay of ATP hydrolysis and phosphoenzyme formation from ATP or Pi. Mutant and wild-type Ca2+-ATPase were assayed for phosphorylation by Pi under conditions that are known to facilitate this reaction in the wild-type enzyme, including pH 6.0 or 7.0 at 25ºC in the presence of dimethylsulfoxide. Although glutamyl (E) and aspartyl (D) residue side chains differ by only one methylene group, no phosphoenzyme could be detected in the D351E mutant, even upon the addition of 40% dimethylsulfoxide and 1 mM 32Pi in the presence of 10 mM EGTA and 5 mM MgCl2. These results show that in the D351E mutant, increasing hydrophobicity of the site with inorganic solvent was not a sufficient factor for the required abstraction of water in the reaction of E351 with Pi to form a glutamylphosphate (P-E351) phosphoenzyme moiety. Mutation D351E may disrupt the proposed alignment of the reactive water molecule with the aspartylphosphate (P-D351) moiety in the phosphorylation site, which may be an essential alignment both in the forward reaction (hydrolysis of aspartylphosphate) and in the reverse reaction (abstraction of water upon formation of an aspartylphosphate intermediate).

Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban (PLB), and ryanodine channel (RYR2) mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats) or 50% diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50% food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 ± 0.48 vs food-restricted group = 4.84 ± 0.33, P < 0.01). The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 ± 0.44 vs food-restricted group = 7.96 ± 0.45, and control = 1.52 ± 0.06 vs food-restricted group = 1.53 ± 0.10, respectively). Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

Down-regulation of the cardiac sarcoplasmic reticulum ryanodine channel in severely food-restricted rats

We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban (PLB), and ryanodine channel (RYR2) mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats) or 50% diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50% food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 +/- 0.48 vs food-restricted group = 4.84 +/- 0.33, P < 0.01). The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 +/- 0.44 vs food-restricted group = 7.96 +/- 0.45, and control = 1.52 +/- 0.06 vs food-restricted group = 1.53 +/- 0.10, respectively). Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.

Ca(2+) release from the sarcoplasmic reticulum activated by the low affinity Ca(2+) chelator TPEN in ventricular myocytes

The Ca(2+) content of the sarcoplasmic reticulum (SR) of cardiac myocytes is thought to play a role in the regulation and termination of SR Ca(2+) release through the ryanodine receptors (RyRs). Experimentally altering the amount of Ca(2+) within the SR with the membrane-permeant low affinity Ca(2+) chelator TPEN could improve our understanding of the mechanism(s) by which SR Ca(2+) content and SR Ca(2+) depletion can influence Ca(2+) release sensitivity and termination. We applied laser-scanning confocal microscopy to examine SR Ca(2+) release in freshly isolated ventricular myocytes loaded with fluo-3, while simultaneously recording membrane currents using the whole-cell patch-clamp technique. Following application of TPEN, local spontaneous Ca(2+) releases increased in frequency and developed into cell-wide Ca(2+) waves. SR Ca(2+) load after TPEN application was found to be reduced to about 60% of control. Isolated cardiac RyRs reconstituted into lipid bilayers exhibited a two-fold increase of their open probability. At the low concentration used (20-40muM), TPEN did not significantly inhibit the SR-Ca(2+)-ATPase in SR vesicles. These results indicate that TPEN, traditionally used as a low affinity Ca(2+) chelator in intracellular Ca(2+) stores, may also act directly on the RyRs inducing an increase in their open probability. This in turn results in an increased Ca(2+) leak from the SR leading to its Ca(2+) depletion. Lowering of SR Ca(2+) content may be a mechanism underlying the recently reported cardioprotective and antiarrhythmic features of TPEN.

Abnormal junctions between surface membrane and sarcoplasmic reticulum in skeletal muscle with a mutation targeted to the ryanodine receptor.

Junctions that mediate excitation-contraction (e-c) coupling are formed between the sarcoplasmic reticulum (SR) and either the surface membrane or the transverse (T) tubules in normal skeletal muscle. Two structural components of the junctions, the feet of the SR and the tetrads of T tubules, have been identified respectively as ryanodine receptors (RyRs, or SR calcium-release channels), and as groups of four dihydropyridine receptors (DHPRs, or voltage sensors of e-c coupling). A targeted mutation (skrrm1) of the gene for skeletal muscle RyRs in mice results in the absence of e-c coupling in homozygous offspring of transgenic parents. The mutant gene is expected to produce no functional RyRs, and we have named the mutant mice "dyspedic" because they lack feet--the cytoplasmic domain of RyRs anchored in the SR membrane. We have examined the development of junctions in skeletal muscle fibers from normal and dyspedic embryos. Surprisingly, despite the absence of RyRs, junctions are formed in dyspedic myotubes, but the junctional gap between the SR and T tubule is narrow, presumably because the feet are missing. Tetrads are also absent from these junctions. The results confirm the identity of RyRs and feet and a major role for RyRs and tetrads in e-c coupling. Since junctions form in the absence of feet and tetrads, coupling of SR to surface membrane and T tubules appears to be mediated by additional proteins, distinct from either RyRs or DHPRs.