Using lipid bilayer reconstitution technique, we investigated the oxidation effect of t-butyl hydrogen peroxide (tBHP) on the single channel activity of the sarcoplasmic reticulum (SR) calcium release channels isolated from canine latissimus dorsi muscles. When 0.7% tBHP was added in the cytosolic side, the channel activity became suppressed (n = 7), and it was recovered by changing the solution to the control solution. The suppression was due to the change in the gating mode of the channel: before tBHP the channel opened to four sub-conductance levels, but it opened to only one level after tBHP. These effects by tBHP were different from the previous finding using hydrogen peroxide (H2O2), which may be explained by different oxidation patterns between the two oxidants.
Animal ; Calcium Channels/drug effects* ; Dogs ; Hydrogen Peroxide/pharmacology ; Peroxides/pharmacology* ; Sarcoplasmic Reticulum/metabolism ; Sarcoplasmic Reticulum/drug effects* ; tert-Butylhydroperoxide

OBJECTIVETo investigate the effects of curcumin on sarcoplasmic reticulum Ca2+-ATPase in heart failure rabbits.

METHODSRabbit heart failure model was made with aortic regurgitation and abdominal aorta constriction and 40 rabbits were randomly divided into 4 groups including: (1) heart failure treated with curcumin; (2) heart failure treated with placebo; (3) healthy control treated with curcumin and (4) healthy control treated with placebo. All rabbits were administrated with curcumin capsules or placebo capsules 100 mg x kg(-1) x d(-1), respectively. All groups were sacrificed after eight weeks. Myocardial ultrastructural organization was detected by transmission electron microscope. RT-PCR and Western blot were used to measure the expression of sarcoplasmic reticulum Ca2+-ATPase in mRNA and protein levels, respectively. Malachite green colorimetric assay was used to evaluate the activity of sarcoplasmic reticulum Ca2+-ATPase.

RESULTSAll detected parameters were similar between control curcumin group and control placebo group. Compared with the control groups (Groups 3 and 4), the heart/body weight ratio was significantly increased in the heart failure-curcumin group (Group 1) and the heart failure-placebo group (Group 2, all P < 0.05), but the ratio was significantly lower in heart failure-curcumin group than in heart failure-placebo group (P < 0.05). The degree of heart failure was decreased by curcumin. Activity and mRNA and protein expression for sarcoplasmic reticulum Ca2+-ATPase were significantly reduced in the heart failure-placebo group and which could be significantly attenuated by curcumin (all P < 0.05).

CONCLUSIONCurcumin could improve cardiac function via upregulating the expression of sarcoplasmic reticulum Ca2+-ATPase in this model.

Animals ; Calcium ; metabolism ; Curcumin ; pharmacology ; Heart Failure ; metabolism ; RNA, Messenger ; genetics ; Rabbits ; Sarcoplasmic Reticulum ; drug effects ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism

OBJECTIVEThe explore the mechanism responsible for diaphragmatic contractile and relaxation dysfunction in a rat model of sepsis.

METHODSThirty-six adult male Sprague-Dawley rats were randomized equally into a sham-operated group and two model groups of sepsis induced by cecal ligation and puncture (CLP) for examination at 6 and 12 h following CLP (CLP-6 h and CLP-12 h groups). The parameters of diaphragm contractile and relaxation were measured, and the calcium uptake and release rates of the diaphragmatic sarcoendoplasmic reticulum (SR) and the protein expressions of SERCA1, SERCA2 and RyR in the diaphragmatic muscles were determined.

RESULTSThe half-relaxation time of the diaphragm was extended in both the CLP-6 h and CLP-12 h groups with significantly reduced maximum tension declinerate and the peek uptake rate of SERCA (P<0.01). Diaphragmatic maximum twitch force development rate, the maximal twitch, tetanus tensions and the peek release rate of SR decreased only at 12h after CLP (P<0.01). The expression levels of SERCA1 protein decreased significantly in the diaphragmatic muscles at 12h following CLP (P<0.01) while SERCA2 expression level and SERCA activity showed no significant changes.

CONCLUSIONIn the acute stage of sepsis, both the contractile and relaxation functions of the diaphragm are impaired. Diaphragmatic relaxation dysfunction may result from reduced calcium uptake in the SR and a decreased level of SERCA1 in the diaphragmatic muscles.

Animals ; Calcium ; metabolism ; Cecum ; Diaphragm ; drug effects ; metabolism ; Endoplasmic Reticulum ; metabolism ; Ligation ; Male ; Muscle Contraction ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism ; Sepsis

OBJECTIVETo observe the influences of quercetin (Que) on the contraction of small intestine smooth muscle of rabbits in vitro and explore the mechanism.

METHODSWith the isothermal perfusion of small intestine in vitro. The influences of quercetin on the spontaneous contraction of small intestine and contraction induced by Ach, histamine and Bacl2 were observed and the mechanism of quercetin was studied.

RESULTSQuercetin reduced the tension of contraction of small intestine smooth muscle in rabbits in a dose-depended manner. Quercetin could completely block the contraction of Bay K8644. Heparin could also block the inhibition of quercetin on small intestine smooth muscle but ruthenium red (RR) had no effect on the relaxation of quercetin. Nitro-L-arginine methylester(L-NAME) inhibited the relaxation of quercetin.

CONCLUSIONQuercetin inhibits the contraction of small intestine smooth muscle of rabbits in vitro. The mechanism may be related to increase NO concentration in small intestine smooth muscle so that it inhibits extracellular Ca2+ inflowing via cell membrane. And quercetin has effect on intracellular Ca2+ releasing via IP3 of sarcoplasmic reticulum.

Animals ; Calcium ; metabolism ; In Vitro Techniques ; Intestine, Small ; drug effects ; Muscle, Smooth ; drug effects ; physiology ; Quercetin ; pharmacology ; Rabbits ; Sarcoplasmic Reticulum ; drug effects ; metabolism

Resveratrol (trans-3, 4', 5-trihydroxy stilbene), a phytoalexin found in grape skins and red wine, has been reported to have a wide range of biological and pharmacological properties. It has been speculated that resveratrol may have cardioprotective activity. The objective of our study was to investigate the effects of resveratrol on intracellular calcium concentration ([Ca(2+)](i)) in rat ventricular myocytes. [Ca(2+)](i) was detected by laser scanning confocal microscopy. The results showed that resveratrol (15~60 mumol/L) reduced [Ca(2+)](i) in normal and Ca(2+)-free Tyrode's solution in a concentration-dependent manner. The effects of resveratrol on [Ca(2+)](i) in normal Tyrode's solution was partially inhibited by pretreatment with sodium orthovanadate (Na3VO4, 1.0 mmol/L, P<0.01), an inhibitor of protein tyrosine phosphatase, or L-type Ca(2+) channel agonist Bay K8644 (10 mumol/L, P<0.05), but could not be antagonized by NO synthase inhibitor L-NAME (1.0 mmol/L). Resveratrol also markedly inhibited the ryanodine-induced [Ca(2+)](i) increase in Ca(2+)-free Tyrode's solution (P<0.01). When Ca(2+) waves were produced by increasing extracellular Ca(2+) concentration from 1 to 10 mmol/L, resveratrol (60 mumol/L) could reduce the velocity and duration of propagating waves, and block the propagating waves of elevated [Ca(2+)](i). These results suggest that resveratrol may reduce the [Ca(2+)](i) in isolated rat ventricular myocytes. The inhibition of voltage-dependent Ca(2+) channel and tyrosine kinase, and alleviation of Ca(2+) release from sarcoplasmic reticulum (SR) are possibly involved in the effects of resveratrol on rat ventricular myocytes. These findings could help explain the protective activity of resveratrol against cardiovascular disease.
Animals ; Calcium ; metabolism ; Calcium Channels ; drug effects ; Heart Ventricles ; cytology ; metabolism ; Intracellular Fluid ; drug effects ; metabolism ; Male ; Myocytes, Cardiac ; drug effects ; metabolism ; Protein-Tyrosine Kinases ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum ; drug effects ; metabolism ; Stilbenes ; pharmacology

Receptor proteins in both eukaryotic and prokaryotic cells often form regular lattice or array in the membrane. Recent theoretical analyses indicate that such arrays may provide a novel mechanism for receptor signaling regulation in cells. The functional coupling between neighboring receptors could improve the signaling performance. The ryanodine receptors (RyR)/calcium release channels usually form 2-D regular lattice in the endoplasmic/sarcoplasmic reticulum membranes. Thus, RyR is a potentially good model to study the function of receptor 2-D array. In this article, we briefly review recent progresses in this research field, including RyR-RyR interaction, RyR array's function and working mechanisms. The investigations performed by new methods in our laboratory are summarized. We demonstrate that the RyR-RyR interaction is modulated by the functional states of RyRs. Accordingly, the mechanism of "dynamic coupling" of RyR array is proposed. Its possible role in RyR-mediated Ca(2+) release is discussed.
Animals ; Calcium ; metabolism ; Cations ; Humans ; Muscle, Skeletal ; drug effects ; metabolism ; Receptor Cross-Talk ; physiology ; Ryanodine Receptor Calcium Release Channel ; physiology ; Sarcoplasmic Reticulum ; metabolism

The present study is aimed to study the effect of sarcoplasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) gene transfer on the contractile function of isolated cardiomyocytes of canines. The cardiomyocytes were isolated with collagenases. The isolated cardiac cells were divided into untransfected group, empty vector group and SERCA2a-transfected group. Recombinant adenovirus vector carrying enhanced green fluorescent protein gene was used for SERCA2a gene delivery. The expression of SERCA2a protein in cardiomyocytes was determined by Western blot. Contractile function of cardiomyocytes was measured with motion edge-detection system of single cell at 48 h after transfection. The results showed, compared with untransfected group, SERCA2a protein level, percentage of peak contraction amplitude under normal condition, percentages of peak contraction amplitude under Ca(2+) or isoproterenol stimulation, time-to-peak contraction (TTP) and time-to-50% relaxation (R50) in SERCA2a-transfected group all increased significantly. While all the above indices in empty vector group did not show any differences with those in untransfected group. These results suggest that the overexpression of SERCA2a by gene transfer may enhance the contraction function of canine myocardial cells.
Adenoviridae ; genetics ; metabolism ; Animals ; Dogs ; Male ; Myocardial Contraction ; drug effects ; physiology ; Myocytes, Cardiac ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics ; metabolism ; Transfection

The study was designed to explore the alteration of intracellular calcium concentration ([Ca²⁺]i), induced by transient receptor potential melastatin 8 (TRPM8) channel-specific agonist menthol, in pulmonary arterial smooth muscle cells (PASMCs) between control and pulmonary hypertensive (PH) rats. PH rat models were established by means of chronic hypoxia (CH) and monocrotaline (MCT) injection, respectively. PASMCs from control and PH rats were cultured. The change of [Ca²⁺]i in PASMCs induced by menthol, and the effect of TRPM8 channel-specific antagonist BCTC on the change of [Ca²⁺]i, were observed. Cellular localization of TRPM8 was examined by using immunohistochemistry. Results showed that menthol increased [Ca²⁺]i in the control PASMCs both in Ca²⁺ -normal and Ca²⁺ - free Tyrode's solutions, and at the same time BCTC could inhibit these two kinds of elevations. Compared with the control group, elevations of [Ca²⁺]i were decreased notably in CH- and MCT-pretreated PASMCs superfused with 2 mmol/L Ca²⁺ - or 0 Ca²⁺ -Tyrode's solutions. Immunohistochemical localization experiments showed that the whole PASMCs were dyed brown except for the nucleus. This study verified that TRPM8 exists both in membrane and sarcoplasmic reticulum of PASMCs. In addition, CH- and MCT-pretreatment could independently down-regulate the Ca²⁺ influx and Ca²⁺ release mediated by TRPM8 channel.
Animals ; Calcium ; metabolism ; Cells, Cultured ; Menthol ; pharmacology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Pulmonary Artery ; cytology ; Rats ; Sarcoplasmic Reticulum ; metabolism ; TRPM Cation Channels ; metabolism

OBJECTIVETo investigate the effects of losartan on mRNA expression of myocardial sarcoplasmic reticulum calcium handling proteins (SERCA2, RyR2 and PLB) and the role of which in prevention of chronic heart failure in rabbit.

METHODSAfter chronic heart failure was induced by ligation of the left anterior descending artery in rabbits, the animals were treated with losartan. At 8 weeks after ligation, left ventricular function, hemodynamic parameters, and SERCA2, RyR2, PLB mRNA expressions were observed.

RESULTSCompared with the control group (group C), LVEDP in the infarcted group (group I) increased (P < 0.01), while +dp/dt(max) and -dp/dt(max) decreased significantly (P < 0.01). LVEDP was lower but +dp/dt(max) and -dp/dt(max) significantly higher in the losartan treated group (group L) than those in group I (P < 0.05). SERCA2, RyR2, and PLB mRNA expressions in group I were remarkably lower than those in group L (P < 0.01) and group C (P < 0.01), respectively.

CONCLUSIONLosartan can improve cardiac function, probably owing to its upregulating mRNA expressions of myocardial sarcoplasmic reticulum Ca(2+) handling proteins (RyR2, SERCA2 and PLB) in the prevention of heart failure.

Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Animals ; Calcium ; metabolism ; Calcium Channels ; drug effects ; Calmodulin ; biosynthesis ; genetics ; Female ; Heart Failure ; drug therapy ; metabolism ; Losartan ; pharmacology ; Male ; Rabbits ; Ryanodine Receptor Calcium Release Channel ; biosynthesis ; Sarcoplasmic Reticulum ; drug effects ; metabolism

BACKGROUNDStudies have shown that β-blockers can improve cardiac performance in heart failure (HF) by reversing protein kinase A (PKA)-mediated sarcoplasmic reticulum (SR) Ca2+ leak. However, it is being strongly questioned as to whether the PKA-mediated ryanodine receptor (RyR2) hyper-phosphorylation is a critical regulator of SR Ca2+ leak. In this study, we used a rabbit HF model to investigate whether β-blockers affect SR Ca2+ leak by other potential mechanisms.

METHODSNew Zealand white rabbits were randomly divided in three groups (n=7 in each group): normal group, metoprolol-untreated group and metoprolol-treated group. Cardiac function was determined by echocardiography and hemodynamic assays. The SR Ca2+ leak was measured by a calcium-imaging device, and the expression and activities of related proteins were evaluated by Western blotting and auto-phosphorylation.

RESULTSIn the metoprolol-untreated group, there was significantly increased ventricular cavity size, reduced systolic function, increased SR Ca2+ leak, reduced associated amount of FK506 binding protein 12.6 (FKBP12.6), increased expression and activity of PKA and Ca2+/calmodulin-dependent protein kinase II (CaMKII), and increased phosphorylated RyR2 phosphorylation sites (with unchanged RyR2-P2030). In the treated group, there was partly increased ventricular cavity size with preserved systolic function, but no prominent Ca2+ leak, with unchanged expression and activity of PKA, CaMKII and their RyR2 phosphorylation sites.

CONCLUSIONChronic administration of metoprolol prevented the SR Ca2+ leak by restoring not only PKA-dependent but also CaMKII-dependent hyper-phosphorylation of RyR2, which may be one of the potential mechanisms by which β-blockers improve cardiac function and reduce the incidence of fatal arrhythmia in HF.

Animals ; Calcium ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Cyclic AMP-Dependent Protein Kinases ; metabolism ; Echocardiography ; Heart Failure ; drug therapy ; metabolism ; Hemodynamics ; drug effects ; Metoprolol ; therapeutic use ; Rats ; Sarcoplasmic Reticulum ; drug effects ; metabolism