AIMThe characteristics of ryanodine receptor in rat cardiac sarcoplasmic reticulum (SR) and nuclear envelope (NE) were studied.

METHODSVelocity and isopyknic gradient centrifugation was employed to fractionate rat SR and NE. Ryanodine receptor was assayed with [3H] ryanodine saturate binding to the preparations.

RESULTSThe maximal binding (Bmax) and dissociating constant (Kd) of ryanodine receptor in rat cardiac NE were, 1.7% and 60% of those in SR respectively. Phosphorylation in vitro by PKA and PKC increased Bmax of the receptors in SR by 372% and 121%, and augmented those in NE by 221% and 306%, without any effects on Kd.

CONCLUSIONRyanodine receptors were present in rat myocardial NE, with lower density and higher affinity than those located in SR, which can be activated by PKA and PKC.

Animals ; Calcium ; metabolism ; Kinetics ; Myocardium ; metabolism ; Nuclear Envelope ; metabolism ; physiology ; Phosphorylation ; Rats ; Rats, Sprague-Dawley ; Ryanodine ; metabolism ; Ryanodine Receptor Calcium Release Channel ; metabolism ; Sarcoplasmic Reticulum ; metabolism ; physiology

OBJECTIVETo examine the effects of procaine and lidocaine on intracellular Ca(2+) release from sarcoplasmic reticulum ryanodine-sensitive Ca(2+) stores.

METHODSThe experiment was performed on hippocampal slices from 60-80 g male Mongolian gerbils. Levels of intracellular Ca(2+) concentration in the slices were measured by microfluorometry. The slices were perfused with 50 mmol/L KCl containing medium for 30 seconds. Then, the medium was switched to physiological medium. After 5 min of incubation, the slice was perfused with 20 mmol/L caffeine containing physiology medium for 2 min. Following incubation, the slice was superfused with physiological medium until the end of the experiment. The effects of procaine and lidocanin (100 micro mol/L) on caffeine-evoked Ca(2+) release were evaluated by adding them to the medium after high K(+) medium perfusion.

RESULTSCaffeine induced a marked increase in intracellular Ca(2+) concentration which was then decreased 12% upon the addition of procaine (P < 0.05); however, lidocaine, did not induce a similar inhibitory reaction.

CONCLUSIONProcaine inhibits ryanodine-receptor mediated Ca(2+) release from intracellular Ca(2+) stores, while lidocaine may inhibit Ca(2+) release through other mechanisms.

Anesthetics, Local ; pharmacology ; Animals ; Calcium ; metabolism ; Gerbillinae ; Hippocampus ; drug effects ; metabolism ; Lidocaine ; pharmacology ; Male ; Procaine ; pharmacology ; Ryanodine ; pharmacology ; Ryanodine Receptor Calcium Release Channel ; physiology

Chronic Disease ; Heart Failure ; metabolism ; Humans ; Phosphorylation ; Ryanodine Receptor Calcium Release Channel ; metabolism

Glycerophosphrylocholine (GPC) is a renal medullary compatible organic osmolyte that is derived from choline via phosphatidylcholine, which is catalyzed in part by phospholipase A2 (PLA2) and its degradation by GPC: choline phosphodiesterase (GPC: choline PDE). We found that caffeine elevated intracellular free calcium ([Ca2+]i) and GPC level in cultured MDCK cells, canine kidney epithelial cells, and propose a possible biochemical mechanism. When MDCK cells were incubated for 3 h with 1 to 10 mM caffeine, cellular GPC was elevated in a dose-dependent manner, and this occurred independently of the extracellular osmolality. Caffeine stimulated the rate of [14C]choline incorporation into [14C]GPC and PLA2 activity. Whereas, GPC: choline PDE activity was accompanied by less of increase. These enzyme changes demonstrate the increased net synthesis of MDCK GPC. In order to identify what triggers the PLA2 activation, [Ca2+]i was measured by using a fluorescence dye, Fura-2. Caffeine (10 mM) resulted in a typical transient increase in MDCK [Ca2+]i concentration, and this increase was greatly inhibited by pretreatment of MDCK cells with 10 mM ryanodine for 5 min. Ryanodine (10 mM) also inhibited the caffeine-induced stimulation of PLA2 activity. These findings provide the first evidence that caffeine in MDCK cells causes a ryanodine-inhibitable increase of [Ca2+]i and PLA2 activity, resulting in cellular GPC accumulation.
Animal ; Caffeine/pharmacology* ; Calcium/metabolism* ; Carbon Radioisotopes ; Cell Line ; Choline/metabolism ; Dogs ; Glycerylphosphorylcholine/metabolism* ; Kidney/cytology ; Phospholipases A/metabolism* ; Phospholipases A/drug effects ; Phospholipases A/antagonists & inhibitors ; Phosphoric Diester Hydrolases/metabolism ; Phosphoric Diester Hydrolases/drug effects ; Ryanodine/pharmacology* ; Ryanodine/metabolism

OBJECTIVETo investigate the effects of sinusoidal magnetic field on isolated sarcoplasmic reticulum (SR) calcium release channel (RyR1) function.

METHODSWith the Ca2+ dynamic spectrum and isotope labeled methods, the Ca2+ release and [(3)H]-Ryanodine binding, the initial rates of NADH oxidation and the production of superoxide of SR exposed to 50 Hz sinusoidal magnetic field (MF) were investigated respectively.

RESULTS0.4 mT, 50 Hz sinusoidal MF exposure for 30 min increased SR Ca2+ release initial rate about 35% from (10.82 +/- 0.89) pmol.mg(-1) pro.s(-1) to (14.69 +/- 1.21) pmol.mg(-1) pro.s(-1); and the [(3)H]-Ryanodine binding by about 15% from (2.13 +/- 0.05) pmol/mg pro to (2.45 +/- 0.07) pmol/mg pro, which regulated by 1 mmol/L NADH with 1 mmol/L NAD+. Meanwhile MF upregulated the rate of NADH oxidation by about 22% from (0.88 +/- 0.11) x 10(-4) FI/s to (1.07 +/- 0.13) x 10(-4) FI/s and upregulated the production of superoxide by about 32% from (0.99 +/- 0.09) x 10(-5) FI/s to (1.31 +/- 0.06) x 10(-5) FI/s.

CONCLUSION0.4 mT sinusoidal MF increases the activity of RyR1 within the low redox potential environment, and promotes NADH oxidase activity and superoxide production.

Animals ; Calcium ; metabolism ; Magnetic Fields ; adverse effects ; Rabbits ; Ryanodine Receptor Calcium Release Channel ; metabolism ; Sarcoplasmic Reticulum ; metabolism ; radiation effects

OBJECTIVETo study the expression of the L-type calcium channel (Cav1.3) and its receptor Ryrs1 in the corpus cavernosum of the penis in aged rats, and to explore the mechanism of age-related erectile dysfunction (ED).

METHODSWe included 10 two month-old male SD rats (Group A) and another ten 18-month-old ones (Group B) in this study, measured their serum testosterone levels and analyzed the expressions of Cav1.3 and RyR1 in the corpus cavernosum of the penis by RT-PCR and immunohistochemistry.

RESULTSThe level of serum testosterone was significantly lower in Group B than in A ([1 356 +/- 424] ng/L vs [2 744 +/- 964] ng/L, P < 0.05). Compared with the young rats, the aged ones showed significant decreases in the expressions of Cav1.3 (IA = 18.65 +/- 8.47 vs 75.48 +/- 14.28, P < 0.05), RyR1 (IA = 21.37 +/- 9.64 vs 78.23 +/- 13.57, P < 0.05), Cav1.3 mRNA (mean gray value = 0.382 +/- 0.046 vs 1.137 +/- 0.415, P < 0.05), and RyR1 mRNA (mean gray value = 0.146 +/- 0.053 vs 1.215 +/- 0.261, P < 0.05).

CONCLUSIONReduced expressions of Cav1.3 and RyR1 in the corpus cavernosum of the penis may be one of the mechanisms underlying age-related ED in aged rats.

Aging ; Animals ; Calcium Channels, L-Type ; metabolism ; Erectile Dysfunction ; metabolism ; Male ; Penis ; metabolism ; Rats ; Rats, Sprague-Dawley ; Ryanodine Receptor Calcium Release Channel ; metabolism

OBJECTIVETo investigate the abnormal abundances of calcium regulatory proteins in rabbit myocytes with failing hearts.

METHODSSixteen rabbits were divided into two groups: 8 rabbits with heart failure induced by volume plus pressure overload and 8 sham-operated animals. The hemodynamic parameters and cardiac structure and function were detected via catheterization and echocardiography respectively. L-type calcium channel (LTCC), Ryanodine receptor 2 (RyR2), Sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a), and Na(+)-Ca(2+) exchanger (NCX) protein abundances were determined by Western blot analysis.

RESULTSThe ratio of left ventricular mass to body weight, heart rate and left ventricular end diastolic pressure in heart failure rabbits were significantly increased compared with sham-operated rabbits (P < 0.01), but their left ventricular shorten fraction [(21.3 +/- 4.00)% vs. (36.5 +/- 1.36)%] and ejection fraction (0.45 +/- 0.07 vs. 0.70 +/- 0.02) were decreased (P < 0.01). In heart failure rabbits, the abundances of LTCC and RyR2 were significantly decreased (R(LTCC/actin): 0.287 +/- 0.029 vs. 0.624 +/- 0.009; R(RyR2/actin): 0.106 +/- 0.001 vs. 0.203 +/- 0.011; P < 0.01), whereas the expressions of SERCA2a and NCX were markedly increased (R(NCX/actin): 0.497 +/- 0.015 vs. 0.221 +/- 0.014; R(SERCA2a/actin): 0.611 +/- 0.036 vs. 0.433 +/- 0.008; P < 0.01).

CONCLUSIONSReductions of LTCC and RyR2 might contribute to risk factors of systolic dysfunction in failing hearts. In early stage of heart failure, upregulated SERCA2a and NCX protein levels may be helpful for maintaining cardiac performance.

Animals ; Calcium ; metabolism ; Calcium-Binding Proteins ; biosynthesis ; Female ; Heart Failure ; metabolism ; Male ; Rabbits ; Ryanodine Receptor Calcium Release Channel ; metabolism ; Sarcoplasmic Reticulum ; chemistry ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism

OBJECTIVETo study the expressions of ryanodine receptor 1 (RyR1) and voltage-gated calcium channel 1.3 (CaV1.3) in the corpus cavernosum smooth muscle of castrated rats and to investigate their role in androgen deficiency-related erectile dysfunction.

METHODSForty 8-week-old SD rats were equally randomized into Groups A (2-week sham-operation), B (4-week sham-operation), C (2-week castration), and D (4-week castration). After surgery, the levels of serum testosterone in different groups of rats were determined, and the expressions of RyR1 and CaV1.3 in the corpus cavernosum were detected by immunohistochemical staining and RT-PCR.

RESULTSThe levels of serum testosterone were significantly decreased in Groups C ([15.97 +/- 5.67] nmol/L) and D ([2.03 +/- 1.57] nmol/L) as compared with A ([90.54 +/- 20.13] nmol/L) and B ([120.35 +/- 30.32] nmol/L) (P < 0.05). RyR1 and CaV1.3 expressed in all the groups. RyR1 mRNA, CaV1.3 mRNA and their proteins were remarkably reduced in Groups C (0.51 +/- 0.24, 0.50 +/- 0.12, 120.36 +/- 25.78, 103.37 +/- 39.52, respectively) and D (0.33 +/- 0.15, 0.32 +/- 0.07, 67.39 +/- 30.54, 67.56 +/- 20.12, respectively) in comparison with A (1.53 +/- 0.25, 1.33 +/- 0.05, 300.96 +/- 135.12, 298.68 +/- 126.35, respectively) and B (1.37 +/- 0.23, 1.25 +/- 0.03, 330.38 +/- 128.59, 327.35 +/- 117.37, respectively) (P < 0.05). The androgen level was positively correlated with the expressions of RyR1 and CaV1.3.

CONCLUSIONAndrogen can regulate erectile function via RyR1 and CaV1.3.

Androgens ; pharmacology ; Animals ; Calcium Channels ; metabolism ; Male ; Muscle, Smooth ; drug effects ; metabolism ; Penis ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Ryanodine Receptor Calcium Release Channel ; metabolism

OBJECTIVETo investigate changes in adenosine triphosphatase (ATPase) activity and expression of ryanodine receptor 1 (RyR1) mRNA in formation of pressure ulcer at early stage, and to analyze its mechanism.

METHODSThirty-six male Sprague-Dawley rats were divided into three groups according to the random number table as follows, with 12 rats in each group. (1) Ischemia-reperfusion (IR) for 3 times (3IR) group: unilateral gracilis of rats were loaded with 22.47 kPa pressure with a special pressure apparatus for 2.0 h to simulate ischemia, and unloaded for 0.5 h to simulate reperfusion. All rats were treated with above-mentioned manoeuvre for 3 times. (2) IR for 5 times (5IR) group: rats were treated with the same manoeuvre as that in 3IR group except for IR for 5 times. (3) CONTROL GROUP: gracilis of rats were subjected to a load of 0 kPa pressure. Rats in 3IR, 5IR groups were sacrificed, and then central part of pressured tissue was harvested for detection of activity of total ATPase, Ca(2+)-Mg(2+)-ATPase, and Na(+)-K(+)-ATPase with spectrophotometer colorimetry, the level of malondialdehyde (MDA) with enzyme linked immunosorbent assay (ELISA), and the level of RyR1 mRNA with real-time fluorescence quantitative RT-PCR. The same part of gracilis muscle of rats in control group was harvested for determination of indexes as above. Data were processed with one-way analysis of variance. Pearson correlation analysis was respectively performed between total ATPase activity and MDA level, total ATPase activity and RyR1 mRNA expression level, and RyR1 mRNA expression level and MDA level.

RESULTSActivity of total ATPase, Ca(2+)-Mg(2+)-ATPase, Na(+)-K(+)-ATPase in control group was respectively (1.629 ± 0.004), (0.907 ± 0.061), (0.697 ± 0.083) U/mg, all significantly higher than those in 3IR group [(1.365 ± 0.004), (0.784 ± 0.020), (0.581 ± 0.017) U/mg, with F value respectively 1707.0, 29.8, 15.2, P < 0.05 or P < 0.01] and 5IR group [(1.055 ± 0.049), (0.619 ± 0.016), (0.436 ± 0.039) U/mg, with F value respectively 1107.0, 169.9, 65.7, P values all below 0.01], and the values of 3 indexes in 5IR group were obviously lower than those in 3IR group (with F value respectively 322.8, 341.7, 94.0, P values all below 0.01). The level of MDA in control group [(7.5 ± 0.6) nmol/L] was lower than that in 3IR group [(9.9 ± 0.6) nmol/L, F = 53.2, P < 0.01] and 5IR group [(13.7 ± 1.3) nmol/L, F = 76.9, P < 0.01]. There was also statistical difference in MDA level between 3IR group and 5IR group (F = 82.9, P < 0.01). Expression level of RyR1 mRNA in control group (8.5 ± 4.2), which was similar to that in 3IR group (3.3 ± 2.1, F = 0.9, P > 0.05), was significantly higher than that in 5IR group (0.6 ± 0.5, F = 23.6, P < 0.05); while the RyR1 mRNA expression level was lower in 5IR group than in 3IR group (F = 39.3,P < 0.05). Activity of total ATPase was negatively correlated with MDA level (r = -0.918, P < 0.01). Activity of total ATPase was positively correlated with RyR1 mRNA expression level (r = 0.713, P < 0.01). RyR1 mRNA expression level was negatively correlated with MDA level (r = -0.702, P < 0.01).

CONCLUSIONSEnergy dysbolism may be an initial factor in the development of pressure ulcer at early stage. Calcium overload injury in pressure tissue can be identified by determination of RyR1 mRNA expression.

Adenosine Triphosphatases ; metabolism ; Animals ; Male ; Muscle, Skeletal ; metabolism ; Pressure Ulcer ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Ryanodine Receptor Calcium Release Channel ; genetics ; metabolism

AIMTo detect the expression of ryanodine receptor (RyR) subtypes in normol rat airway smooth muscle cells(ASMCs) and changes during chronic asthma formation.

METHODSASMCs were cultured with collagen enzyme digestion method. The expression of subtypes of RyR were detected by RT-PCR. Purified PCR product linked with pGEM-T vector to make DNA sequence assay. Chronic asthma model was made with OVA, the changes of RyRs detected by RT-PCR.

RESULTSAll subtypes of RyR were expressed in airway smooth muscle cells of normol rat. The expression of RyR1 increased obviously compared with control group (P < 0.05) on chronic asthma.

CONCLUSIONCo-expression of three subtypes of RyR in ASMCs of normal rat, indicate that there are complicated intercellular Ca2+ regulation mechanism in ASM, moreover RyR1 might play a role during asthma development.

Animals ; Asthma ; metabolism ; Bronchi ; Male ; Myocytes, Smooth Muscle ; metabolism ; Protein Isoforms ; genetics ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar ; Ryanodine Receptor Calcium Release Channel ; genetics ; metabolism