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

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

It was previously found that the efficacy of synaptic transmission between retinal cone systems and luminosity-type horizontal cells (LHCs) was activity-dependent. Repetitive activation of red-cone pathway increased the LHCos hyperpolarizing response to red light, and the response enhancement was reversible. In this study, intracellular recording and pharmacological method were applied to investigate the mechanism(s) underlying red-flickering-induced response enhancement. Lowering intracellular Ca(2+) in the LHC by intracellular injection of Ca(2+) chelator EGTA prevented the development of red-flickering-induced response enhancement, which implicates the importance of postsynaptic calcium signal. The response enhancement could also be eliminated by a potent antagonist of Ca(2+)-permeable AMPA receptor (CP-AMPAR), which suggests the possibility that Ca(2+) influx via glutamate-gated calcium channels is related to the changes of [Ca(2+)](i). Furthermore, the administration of ryanodine or caffeine also attenuated the phenomenon, which gives evidence that the local calcium signal caused by intracellular calcium-induced calcium release (CICR) may be involved. Taken together, our data implicate that postsynaptic CICR and CP-AMPAR are related to the activity-dependent response enhancement.
Animals ; Caffeine ; pharmacology ; Calcium ; metabolism ; Carps ; Neuronal Plasticity ; physiology ; Receptors, AMPA ; physiology ; Retina ; cytology ; Retinal Cone Photoreceptor Cells ; physiology ; Ryanodine ; pharmacology ; Ryanodine Receptor Calcium Release Channel ; physiology ; Signal Transduction ; physiology ; Synapses ; physiology

OBJECTIVETo investigate the underlying mechanism for the selective modulation of the permeability of blood-tumor barrier (BTB) by small dose of bradykinin (BK).

METHODSC6 glioma cells were treated with BK, and changes of intracellular nitric oxide (NO) and intracellular calcium level were measured with fluorescent spectrophotometer.

RESULTSThe initial application of BK easily triggered extracellular calcium influx, which resulted in intracellular calcium store release in C6 glioma cells. The above mechanism was also named ryanodine mediated calcium induced calcium release (CICR). We also detected a long-lasting intracellular NO elevation in C6 glioma cells upon BK treatment. Further study showed that ryanodine mediated CICR contributed greatly to the secondary NO elevation induced by BK treatment.

CONCLUSIONThese results suggested that BK triggered CICR in C6 glioma cells and the associated NO generation might be the underlying mechanism for the selective modulation of BTB permeability by BK.

Animals ; Bradykinin ; pharmacology ; Calcium ; metabolism ; Cell Line, Tumor ; Glioma ; pathology ; Intracellular Fluid ; drug effects ; Nitric Oxide ; metabolism ; Rats ; Ryanodine ; pharmacology ; Spectrometry, Fluorescence ; methods ; Time Factors

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 explore change of ryanodine receptor (RyR) in junior mouse with heart failure (HF) and the effect of β-adrenoreceptor blocker and Radix astragali on RyR in HF in this experiment.

METHODThe animal model of congestive heart failure was established by coarctation of abdominal aorta. Five weeks old mice were randomly divided into 4 groups: (1) HF group without treatment (n = 30); (2) HF group treated with carvedilol (n = 30); (3) HF group treated with carvedilol and Radix astragali(n = 30); (4) Sham-operated group (n = 30). Carvedilol and Radix astragali were administered through direct gastric gavage. After 4 weeks of treatment the high frequency ultrasound was performed. Myocardial sarcoplasmic reticulum (SR) was fractionated with ultra centrifugation. The time courses of Ca(2+) uptake and leak were determined by fluorescent spectrophotometry. The levels of expression of RyR2 in the 4 groups were detected by semi-quantitative reverse transcription-polymerase chain reaction.

RESULTCompared with the sham-operated group, left ventricular diastolic dimension (LVEDD) (P < 0.05), left ventricular systolic dimension (LVESD), interventricular septal thickness at end-diastole (IVSTd), interventricular septal thickness at end-systole (IVSTs), left ventricular posterior wall thickness at end-diastole (LVPWTd), and left ventricular posterior wall thickness at endsystole (LVPWTs) were all significantly increased (P < 0.01), ejection fraction (EF)(%) (HF group without treatment 51.60 ± 1.15, HF treated with carvedilol 72.06 ± 1.39, HF treated with carvedilol and Radix astragali 79.06 ± 1.09, sham-operated group 85.86 ± 1.45) and fractional shortening (FS) (HF group without treatment 44.55 ± 1.20, HF treated with carvedilol 44.55 ± 1.20, HF treated with carvedilol and Radix astragali 53.58 ± 1.30, sham-operated group 59.03 ± 1.67) were decreased (P < 0.01) in HF group without treatment. LVEDD (P < 0.05), LVESD, IVSTd, IVSTs, LVPWTd and LVPWTs were all significantly decreased (P < 0.01), EF and FS were increased (P < 0.01) in the cases with HF treated with carvedilol and carvedilol and Radix astragali when compared with HF group without treatment. EF and FS were much more increased in the group treated with carvedilol and Radix astragali than in those treated with carvedilol (P < 0.05). After adding thapsigargin to the buffer including SR of the four groups, there were fewer Ca(2+) leak (%) in sham-operated group (11.5 ± 4.3), HF group treated with carvedilol (15.6 ± 5.8) and treated with carvedilol and Radix astragali (13.6 ± 4.8) than that of HF group without treatment (65.6 ± 6.2) (P < 0.01), while after adding FK506 and thapsigargin together to the buffer including SR of four groups, there were marked Ca(2+) leak in sham-operated group (60.6 ± 7.8), HF group treated with carvedilol (66.2 ± 4.5)and those treated with carvedilol and Radix astragali (70.2 ± 5.5, P < 0.01). However, there was no additional increase in Ca(2+) leak in HF group (67.3 ± 7.5) compared with that of the group where only thapsigargin was added (P > 0.05). The levels of expression of RyR2 were significantly decreased in HF group and increased in the group treated with carvedilol and the group treated with carvedilol and Radix astragali.

CONCLUSIONThere was more cardiac Ca(2+) leak and the expression of RyR2 mRNA decreased in HF. Carvedilol and Radix astragali can increase expression of RyR2 mRNA and inhibit Ca(2+) leak by restoring the binding of FKBP12.6 back to RyR in HF to improve cardiac function and prevent left ventricle from remodeling.

Adrenergic beta-Antagonists ; pharmacology ; Animals ; Astragalus Plant ; Carbazoles ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Heart Failure ; metabolism ; Male ; Propanolamines ; pharmacology ; Rats ; Rats, Wistar ; Ryanodine Receptor Calcium Release Channel ; drug effects ; metabolism

OBJECTIVE: Effects of ATP and acetylcholine (ACh) on intracellular Ca2+ concentrations ([Ca2+]i) and possible mechanism of Ca2+-induced Ca2+ release (CICR) of the isolated outer hair cells (OHCs) in the guinea pig cochlea were studied with confocal microscopy. METHOD: OHCs were isolated from guinea pig cochlea by enzymatic and mechanical methods. The effects of ATP, ACh, Ryanodine + ATP (or ACh) and Thapsigargin + ATP (or ACh) in the presence or absence of extracellular Ca2+ on [Ca2+]i in OHCs were examined by confocal microscopy. RESULT: In the presence of ATP, Ryanodine + ATP, Thapsigargin + ATP, ACh, Ryanodine + ACh and Thapsigargin + ACh increased [Ca2+]i and evoked an evident wave, respectively, the relative magnitude of fluorescence were 1.60 +/- 0.01(ATP), 1.644 +/- 0.005 (Ryanodine + ATP), 1.491 +/- 0.005 (Thapsigargin + ATP), 1.43 +/- 0.01 (ACh), 1.58 +/- 0.02 (Ryanodine + ACh), 1.398 +/- 0.003 (Thapsigargin + ACh) in OHCs in the presence of extracellular Ca2+ respectively. In the absence of extracellular Ca2+, ATP and Ryanodine + ATP induced a gradual and small [Ca2+]i wave, the relative magnitude of fluorescence were 1.341 +/- 0.006 and 1.386 +/- 0.008, however, ACh, Ryanodine + ACh, Thapsigargin + ACh and Thapsigargin + ATP can not induce wave but a gradual [Ca2+]i elevation. ACh can not increase [Ca2+]i. CONCLUSION In the presence of extracellular Ca2+, ATP and ACh increased [Ca2+]i in OHCs not only by Ca2+ influx through ion channel on cell membrane but also a release of Ca2+ from IP3-sensitive calcium reservoir and CICR. In the absence of extracellular Ca2+, ATP activated IP3 sensitive calcium reservoir and Ca2+ release through IP3 sensitive calcium reservoir, in turn CICR was induced. ACh can not activate IP3 sensitive calcium reservoir and CICR in the absence of extracellular Ca2+, therefore, the effect of ACh was dependent of extracellular Ca2+.
Acetylcholine ; pharmacology ; Adenosine Triphosphate ; pharmacology ; Animals ; Calcium ; metabolism ; Calcium Channels ; drug effects ; metabolism ; Cells, Cultured ; Cochlea ; cytology ; metabolism ; Guinea Pigs ; Hair Cells, Auditory, Outer ; metabolism ; Microscopy, Confocal ; Ryanodine ; pharmacology ; Thapsigargin ; pharmacology

Effect of strophanthidin (Str) on intracellular calcium concentration ([Ca2+]i) was investigated on isolated ventricular myocytes of guinea pig. Single ventricular myocytes were obtained by enzymatic dissociation technique. Fluorescent signal of [Ca2+]i was detected with confocal microscopy after incubation of cardiomycytes in Tyrode' s solution with Fluo3-AM. The result showed that Str increased [Ca2+]i in a concentration-dependent manner. The ventricular myocytes began to round-up into a contracture state once the peak level of [Ca2+]i was achieved in the presence of Str (10 micromol L(- 1)), but remained no change in the presence of Str (1 and 100 nmol L(-1)). Tetrodotoxin (TTX), nisodipine, and high concentration of extracellular Ca2+ changed the response of cardiomycytes to Str (1 and 100 nmol L(-1)) , but had no obvious effects on the action of Str (10 micromol L(-1)). The elevation of [Ca2+]i caused by Str at all of the detected concentrations was partially antagonized by rynodine (10 micromol L(-1)) or the removal of Ca2+ from Tyrode's solution. In Na+, K+ -free Tyrode' s solution, the response of cardiomycytes in [Ca2+]i elevation to Str (10 micromol L(-1)) was attenuated, while remained no change to Str (1 and 100 nmol L(-1)). TTX, nisodipine, and high concentration of extracellular Ca2+ changed the response of cardiomycytes to Str at all of the detected concentrations in Na+, K+ -free Tyrode's solution. The study suggests that the elevation of [Ca2+]i by Str at the low (nomomolar) concentrations is partially mediated by the extracellular calcium influx through Ca2+ channel or a "slip mode conductance" of TTX sensitive Na+ channel. While the effect of Str at high (micromolar) concentrations was mainly due to the inhibition of Na+, K+ -ATPase. Directly triggering the release of intracellular Ca2+ from sarcoplasmic reticulum (SR) by Str may be also involved in the mechanism of [Ca2+]i elevation.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ; pharmacology ; Aequorin ; pharmacology ; Animals ; Calcium ; metabolism ; Calcium Channel Blockers ; pharmacology ; Calcium Channels ; metabolism ; Fura-2 ; pharmacology ; supply & distribution ; Guinea Pigs ; Myocardium ; pathology ; Nifedipine ; pharmacology ; Ryanodine ; pharmacology ; Sarcolemma ; metabolism ; pathology ; Sarcoplasmic Reticulum ; drug effects ; metabolism ; Sodium-Calcium Exchanger ; Sodium-Potassium-Exchanging ATPase ; antagonists & inhibitors ; Strophanthidin ; pharmacology ; Tetrodotoxin ; pharmacology ; Thapsigargin ; pharmacology

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

The aim of the present study was to investigate the effects of inositol 1,4,5-trisphosphate (IP(3))-generating agonist UTP on spontaneous transient outward currents (STOCs), and explore the role of intracellular Ca(2+) release in the current response mediated by IP(3) in porcine coronary artery smooth muscle cells (CASMCs). The coronary artery was excised from the fresh porcine heart and cut into small segments (2 mm × 5 mm) and then transferred to enzymatic dissociation solution for incubation. Single CASMCs were obtained by two-step enzyme digestion at 37 °C. STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration in freshly isolated porcine CASMCs. The currents were amplified and filtered by patch-clamp amplifier (Axopatch 200B), and then the digitized data were recorded by pClamp 9.0 software and further analyzed by MiniAnalysis 6.0 program. The results were as follows: (1) UTP led to conspicuous increases in STOC amplitude by (57.54±5.34)% and in frequency by (77.46±8.42)% (P<0.01, n=38). (2) The specific blocker of phospholipase C (PLC) - U73122 (5 μmol/L) remarkably reduced STOC amplitude by (31.04±7.46)% and frequency by (41.65±16.59)%, respectively (P<0.05, n=10). In the presence of U73122, UTP failed to reactivate STOCs (n=7). (3) Verapamil (20 μmol/L) and CdCl2 (200 μmol/L), two blockers of L-type voltage-dependent Ca(2+) channels, had little effects on STOCs initiated by UTP (n=8). (4) 1 μmol/L bisindolylmaleimide I (BisI), a potent blocker of protein kinase C (PKC), significantly increased STOC amplitude by (65.44±24.66)% and frequency by (61.35±21.47)% (P<0.01, n=12); UTP (40 μmol/L), applied in the presence of 1 μmol/L BisI, could further increase STOC activity (P<0.05, P<0.01, n=12). Subsequent application of ryanodine (50 μmol/L) abolished STOC activity. (5) In the presence of UTP (40 μmol/L), inhibition of IP(3) receptors (IP(3)Rs) by 2-aminoethoxydiphenyl borate (2-APB, 40 μmol/L) reduced STOC amplitude by (24.08±3.97)% (P<0.05, n=8), but had little effect on STOC frequency (n=8). While application of 2-APB (80 μmol/L) significantly reduced STOC amplitude by (31.43±6.34)% and frequency by (40.59±19.01)%, respectively (P<0.05, P<0.01, n=6). Subsequent application of ryanodine (50 μmol/L) completely blocked STOC activity. Pretreatment of cells with 2-APB (40 μmol/L) or ryanodine (50 μmol/L), UTP (40 μmol/L) failed to reactivate STOCs. The results suggest that UTP activates STOCs mainly via PLC and IP(3)-dependent mechanisms. Complex Ca(2+)-mobilization pathways are involved in UTP-mediated STOC activation in porcine CASMCs.
Animals ; Boron Compounds ; pharmacology ; Calcium ; metabolism ; Coronary Vessels ; cytology ; Inositol 1,4,5-Trisphosphate ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; Protein Kinase C ; metabolism ; Ryanodine ; pharmacology ; Signal Transduction ; Swine ; Type C Phospholipases ; metabolism ; Uridine Triphosphate ; metabolism