The modulatory effects of heat shock protein 70 on membrane calcium homeostasis of PC12 cells induced by ischemic/hypoxia / 中华危重病急救医学

Objective To investigate the effects of lentivirus-mediated heat shock protein 70 (HSP70) gene on voltage-gated calcium channels on the cell membrane of pheochromocytoma cell 12 (PC 12 cells) induced by ischemic/hypoxia and its mechanisms.Methods PC12 cells at logarithmic phase were collected,which were challenged with hypoxia for 6 hours and reoxygenation for 12 hours to stimulate the nerve cells suffered ischemia/reperfusion pathological process in vitro.PC12 cells were divided into non-infection group,infected by lentivirus containing green fluorescent protein (GFP) without HSP70 gene lentivirus control group (GFP lentivirus control group),and infected by lentivirus containing HSP70 and GFP gene recombined lentiviral infection group (HSP70 recombined lentiviral infection group).Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot were used to determine mRNA and protein expressions of L-type voltage-gated Ca2+ channel subunits cav1.2 and cav1.3,receptor gated channel subunits NR1 and NR2,and Na+/Ca2+ exchange (NCX) in PC12 cells.Results After being challenged with hypoxia/reoxygenation,the mRNA and protein expressions of cav1.2,NR1 and NR2 in the PC12 cells were significantly lower in HSP70 recombined lentiviral infection group than those of GFP lentivirus control group and non-infection group [cav1.2 mRNA (2-△△Ct):3.13 ± 0.46 vs.5.12 ± 0.52,5.13 ± 0.66;NR1 mRNA (2-△△△Ct):1.61 ± 0.44 vs.3.23 ±0.82,3.31 ±0.78;NR2 mRNA (2-△△Ct):2.09±0.41 vs.3.91 ±0.64,3.88±0.62;cav1.2 protein (gray value):2.82±0.39 vs.3.98±0.23,3.96±0.24;NR1 protein (gray value):1.84±0.35 vs.2.79±0.21,2.86±0.23;NR2 protein (gray value):0.87±0.24 vs.1.57±0.31,1.33±0.44;all P ＜ 0.01].But there were no statistical differences in the mRNA and protein expressions of cav1.2,NR1 and NR2 between GFP lentivirus control group and non-infection group (all P ＞ 0.01).There were no statistical differences in the mRNA and protein expressions of cav1.3 and NCX among non-infection group,GFP lentivirus control group and HSP70 recombined lentiviral infection group [cav1.3 mRNA (2-△△Ct):4.82 ± 0.32,4.72 ± 0.36,4.82 ± 0.29;NCX mRNA (2-△△Ct):3.49 ± 0.78,3.47 ± 0.71,3.56 ± 0.65;cav 1.3 protein (gray value):2.63±0.40,2.64±0.39,2.68±0.39;NCX protein (gray value):3.27±0.48,3.34±0.48,3.31 ±0.42;all P ＞ 0.01].Conclusion Exogenous HSP70 affects the expression of L-type voltage-gated Ca2+ channel subunit cav1.2 and receptor gated channel subunits NR1 and NR2,which may protect PC12 cells from the injury caused by ischemic/hypoxia.

PKC-Independent Stimulation of Cardiac Na+/Ca2+ Exchanger by Staurosporine

[Ca2+]i transients by reverse mode of cardiac Na+/Ca2+ exchanger (NCX1) were recorded in fura-2 loaded BHK cells with stable expression of NCX1. Repeated stimulation of reverse NCX1 produced a long-lasting decrease of Ca2+ transients ('rundown'). Rundown of NCX1 was independent of membrane PIP2 depletion. Although the activation of protein kinase C (PKC) was observed during the Ca2+ transients, neither a selective PKC inhibitor (calphostin C) nor a PKC activator (PMA) changed the degrees of rundown. By comparison, a non-specific PKC inhibitor, staurosporine (STS), reversed rundown in a dose-dependent and reversible manner. The action of STS was unaffected by pretreatment of the cells with calphostin C, PMA, or forskolin. Taken together, the results suggest that the stimulation of reverse NCX1 by STS is independent of PKC and/or PKA inhibition.

Ca2+-dependent Long-term Inactivation of Cardiac Na+/Ca2+ Exchanger

Using BHK cells with stable expression of cardiac Na+/Ca2+ exchanger (BHK-NCX1), reverse mode (i.e. Ca2+ influx mode) of NCX1 current was recorded by whole-cell patch clamp. Repeated stimulation of reverse NCX1 produced a cytosolic Ca2+-dependent long-term inactivation of the exchanger activity. The degrees of inactivation correlated with NCX1 densities of the cells and were attenuated by reduced Ca2+ influx via the reverse exchanger. The inactivation of NCX1 was attenuated by (i) inhibition of Ca2+ influx with reduced extracellular Ca2+, (ii) treatment with NCX1 blocker (Ni2+), and (iii) increase of cytoplasmic Ca2+ buffer (EGTA). In BHK-NCX1 cells transiently expressing TRPV1 channels, Ca2+ influx elicited by capsaicin produced a marked inactivation of NCX1. We suggest that cytoplasmic Ca2+ has a dual effect on NCX1 activities, and that allosteric Ca2+ activation of NCX1 can be opposed by the Ca2+-dependent long-term inactivation in intact cells.

Na+-Ca2+ Exchange Curtails Ca2+ before Its Diffusion to Global Ca2+i in the Rat Ventricular Myocyte

In the heart, Na+-Ca2+ exchange (NCX) is the major Ca2+ extrusion mechanism. NCX has been considered as a relaxation mechanism, as it reduces global [Ca2+]i raised during activation. However, if NCX locates in the close proximity to the ryanodine receptor, then NCX would curtail Ca2+ before its diffusion to global Ca2+i. This will result in a global [Ca2+]i decrease especially during its ascending phase rather than descending phase. Therefore, NCX would decrease the myocardial contractility rather than inducing relaxation in the heart. This possibility was examined in this study by comparing NCX-induced extrusion of Ca2+ after its release from SR in the presence and absence of global Ca2+i transient in the isolated single rat ventricular myocytes by using patch-clamp technique in a whole-cell configuration. Global Ca2+i transient was controlled by an internal dialysis with different concentrations of BAPTA added in the pipette. During stimulation with a ramp pulse from +100 mV to -100 mV for 200 ms, global Ca2+i transient was suppressed only mildly, and completely at 1 mmol/L, and 10 mmol/L BAPTA, respectively. In these situations, ryanodine-sensitive inward NCX current was compared using 100micromol/L ryanodine, Na+ depletion, 5 mmol/L NiCl2 and 1micromol/L nifedipine. Surprisingly, the result showed that the ryanodine-sensitive inward NCX current was well preserved after 10 mmol/L BAPTA to 91 % of that obtained after 1 mmol/L BAPTA. From this result, it is concluded that most of the NCX-induced Ca2+ extrusion occurs before the Ca2+ diffuses to global Ca2+i in the rat ventricular myocyte.

Heterogeneity of the SR-dependent Inward Na+-Ca2+ Exchange Current in the Heavily Ca2+-buffered Rat Ventricular Myocytes

Voltage-sensitive release mechanism was pharmacologically dissected from the Ca2+-induced Ca2+ release in the SR Ca2+ release in the rat ventricular myocytes patch-clamped in a whole-cell mode. SR Ca2+ release process was monitored by using forward-mode Na+-Ca2+ exchange after restriction of the interactions between Ca2+ from SR and Na+-Ca2+ exchange within micro-domains with heavy cytosolic Ca2+ buffering with 10 mM BAPTA. During stimulation every 10 s with a pulse roughly mimicking action potential, the initial outward current gradually turned into a huge inward current of -12.9+/-0.5 pA/pF. From the inward current, two different inward INCXs were identified. One was 10 muM ryanodine-sensitive, constituting 14.2+/-2.3%. It was completely blocked by CdCl2 (0.1 mM and 0.5 mM) and by Na+-depletion. The other was identified by 5 mM NiCl2 after suppression of ICaL and ryanodine receptor, constituting 14.8+/-1.6%. This latter was blocked by either 10 mM caffeine-induced SR Ca2+-depletion or 1 mM tetracaine. IV-relationships illustrated that the latter was activated until the peak in 30~35 mV lower voltages than the former. Overall, it was concluded that the SR Ca2+ release process in the rat ventricular myocytes is mediated by the voltage-sensitive release mechanism in addition to the Ca2+-induced-Ca2+ release.

Effect of dofetilide on Na~+/Ca~(2+) exchange in ventricular myocytes of adult guinea pigs / 中国药理学通报

Aim This paper aims at investigating the effect of dofetilide on Na+/Ca 2+exchange in ventricular myocytes of adult guinea pigs. Methods Rapid enzymatic isolation procedure was performed to get single ventricular myocytes from adult guinea pigs. After the whole cell configuration was formed, a ramp voltage-clamp pulse depolarized from a holding potential of -40 mV to +60 mV, then hyperpolarized to -120 mV at a rate of 90 mV/s to record the Na+/Ca 2+ exchange current (I Na/Ca) and the influence of dofetilide from 0.03～1.0 ?mol?L -1. Result Dofetilide from 0.03～1.0 ?mol?L -1 concentration-dependently increased the I Na/Ca in both outward and inward mode. The EC 50 of dofetilide on I Na/Ca in outward and inward mode of NCE was 0.178 ?mol?L -1 (95% confidence interval was 0.040～0.787 ?mol?L -1) and 0.178 ?mol?L -1 (95% confidence interval was 0.038～0.842 ?mol?L -1), respectively. Conclusion Dofetilide concentration-dependently increased the outward and inward Na+/Ca 2+ exchange current in ventricular myocytes of adult guinea pigs.

Roles of Na+-Ca2+ exchange in the negative force-frequency relationship

Frequency-force relationships (FFR) were studied in electrically field stimulated rat left atria (LA) by reducing the stimulation frequency from resting 3 Hz to test frequencies (0.1-1 Hz) for 5 minutes. The twitch amplitudes of LA elicited the typical negative staircases with 3-phased changes: the initial rapid increase, the second decrease and the following plateau at test frequencies. Verapamil (3 X 10-5 M) pretreatment elicited frequency-dependent suppression of the twitch amplitudes, exaggerating the negative staircase. Monensin pretreatment enhanced not the peak but the plateau amplitudes in a concentration-dependent manner. When the Na+-Ca2+ exchange was blocked by Na+ and Ca2+ depletion in the Krebs Hensleit buffer (0 Na+-0 Ca2+ KHB), the twitch amplitudes increased in a frequency-dependent manner, changing the negtive staircase into the positve one. Meanwhile, the 0 Na+-0 Ca2+ KHB applicationinduced enhancement was strongly suppressed by caffeine (5 mM) pretreatment. Only dibucaine among the local anesthetics increased the basal tone during frequency reduciton. There were no differences in 45Ca uptakes between 0.3 Hz and 3 Hz stimulation except at 1 min when it was significantly low at 0.3 Hz than 3 Hz, illustrating net Ca2+ losses. Monensin pretreatment enhanced the rate of this Ca2+ loss. Taken together, it is concluded that Na+-Ca2+ exchange extrudes more SR released Ca2+ out of the cell in proportion to the frequency, resulting in the negative rate staircase in the rat LA.

Intracellular acidosis decreases the outward Na(+)-Ca2+ exchange current in guinea pig ventricular myocytes

The Na(+)-Ca2+ exchange transport operating in outward mode has been suggested to cause Ca2+ entry during reperfusion or reoxygenation, exchanging extracellular Ca2+ for intracellular Na+ that has accumulated during ischemia or cardioplegia. During cardioplegia, however, an increase in Ca2+ entry via this mechanism can be decreased due to increased intracellular H+ activity and a decrease in cellular ATP content. In this study giant excised cardiac sarcolemmal membrane patch clamp technique was employed to investigate the effect of cytosolic pH change on the Na(+)-Ca2+ exchanger, excluding the effect of ATP, in guinea pig cardiac myocytes. The outward Na(+)-dependent current, which has a characteristics of Hill equation, was decreased as pH was decreased in the range of 7.5-6.5. The current density generated by the Na(+)-Ca2+ exchange transport was 56.6 +/- 4.4 pA/pF (Mean +/- S.E.M.) at pH 7.2 and decreased to 42.9 +/- 3.0 pA/pF at pH 6.9. These results imply that Na(+)-Ca2+ exchange transport, operating in a reverse mode during cardioplegia, decreases due to increased intracellular H+, and further suggest that consequent intracellular Na+ accumulation is one of aggravating factors for Ca2+ influx during reoxygenation or reperfusion.

Effects of Hyperosmolar Solution on the Twitch Force, Membrane Potential, and Intracellular Sodium Activity in Purkinje Fibers and Ventricular Muscles

BACKGROUND: Hypertonic solutions are using in emergency patients including refractory shock. The effects of the hyperosmotic solutions for the cardiac contractile effect has remained unclear. To study the mechanism of increase in twitch force by hypertonic solution, memberane potential, intracellular sodium activities(aNia), and twitch force were measured simultaneously in 1 Hz-driven canine Purkinje fibers and guinea pig papillary muscles. METHODS: To increase osmolarity, 20, 40, and 80 mOsm glucose, NaCl or mannitol was added to normal Tyrode solution. We used the conventional and Na(+)-selective microelectrodes, to study the membrane potential and intracellular sodium activity. Changes in twitch force were evaluated also by tension tranducer. RESULTS: 1) Hyperosmolar glucose or NaCl added to normal Tyrode solution produced membrane pontential hyperpolarization, increase in aNia, and increase in twitch force in dog Purkinje fibers. Increase in twitch force was related to decrease in the ratio of aNia to extracellular sodium activity(aNoa). NaCl-inducedd aNia increase was not blocked by 10(-5)M tetrodotoxin, a fast sodium channel blocker. 2) Hyperosmolar glucose or mannitol added to normal Tyrode solution produced membrane potential hyperpolarization, increase in aNia, and increase in twitch force in guinea pig papillary muscles. However, the addition of hyperosmolar NaCl did not affect on membrane potential, but produced increase in aNia, and decrease in twitch force. 3) Prolonging effect of hyperosmolar glucose on duration of action potential was smaller than that of NaCl or mannitol in Purkinje fibers and papillary muscles. 4) Increase in twich force produced by ECF Na+reduction or by hyperosmotic solution was reated to decrase in the aNia ratio. 5) Relationship curve between increase in twitch force and aNoa/aNia ratio in hyperosmolr solution was less steeper than that in ECF Na(+)-reduced solution. CONCLUSION: The above results suggested that hyperosmolar solution-induced twitch force change was related to aNoa/aNia ratio change which influenced intracellular calcium activity via Na(+)-Ca(2+)exchange.

Effect of hypothermic cardioplegia on cardiac protection--I. Effect of hypothermic cardioplegia on the cytosolic Ca2+ concentration in rat ventricular myocytes

Cytosolic Ca2+ concentration of rat ventricular cells was measured under varying experimental conditions by using a fluorescent Ca2+ indicator, Fura-2. Resting [Ca2+]i of rat myocyte was 150 +/- 30 nM (n = 39), and this value was compatible with others. The Perfusion of cardioplegic solution significantly increased [Ca2+]i, and this effect was further augmented by hypothermia (p<0.05). Application of nifedipine (5 x 10(-7) M) to the perfusate or pretreatment of caffeine (10 mM) had no apparent effect on this cardioplegia-induced [Ca2+]i change. But Ni2+ (5 mM), an antagonist of Na+/Ca2+ exchange mechanism, prevented the [Ca2+]i change during cardioplegia (p<0.05). Magnitude of cardioplegia-induced [Ca2+]i increase was also dependent on the Ca2+ concentration of cardioplegic solution. These results suggest that Na+/Ca2+ exchange may play an important role in cardioplegia-induced [Ca2+]i change. To rule out the possibility whether the protective effect of hypothermic cardioplegia is due to the preservation of high-energy phosphate store or decreasing the transmembrane ionic fluxes by phase transition, we exhausted a energy store of cardiac cell by application of 2,4 dinitrophenol to the bath and measured its effect on [Ca2+]i change during cardioplegia. Hypothermic cardioplegia delayed the onset of [Ca2+]i increase and decreased its amplitude compared to those of normothermic cardioplegia. From the above results, hypothermic cardioplegia may protect the cardiac cells from ischemic insult by preserving a high-energy phosphate store. Application of Ni2+ to the cardioplegic solution or reduction of external Ca2+ concentration also had some protective effect, since it prevented [Ca2+]i increase during cardioplegia.

Effects of Na+ and Ca2+ concentration in cardioplegic and reperfusion solutions on the intracellular Ca2+ of cardiac muscle cells

The removal of Ca2+ from the cardioplegic solutions could cause the danger of inducing a "calcium paradox" during reperfusion. Since intracellular Ca2+ activities are coupled to Na+ activities via Na(+)-Ca2+ exchange, an increase in intracellular Na+ activities during the cardioplegia could cause an abrupt Ca2+ influx when reperfused. To study the effects of Na+ and Ca2+ concentrations in cardioplegic solutions on intracellular Ca2+ activities during the cardioplegia and subsequent recovery period, the membrane potential and intracellular Na+ and Ca2+ activities of guinea pig ventricular papillary were measured. 1) A cardioplegia with low Ca2+ cardioplegic solution significantly decreased the overshoot and duration of the first action potential after cardioplegia, but the changes in action potential configuration were minimized after a cardioplegia with Ca2+ concentration adjusted according to the Na(+)-Ca2+ exchange mechanism. 2) Intracellular Na+ activity was continuously decreased during the cardioplegia, and the intracellular Na+ activity 20 minutes after cardioplegia was the highest with low Ca2+ cardioplegic solution. 3) Intracellular Na+ and Ca2+ activities were continuously decreased during the cardioplegia with Ca2+ concentration adjusted according to the Na(+)-Ca2+ exchange mechanism. 4) During a reperfusion of Tyrode solution after cardioplegia intracellular Na+ and Ca2+ activities were increased. Intracellular Ca2+ activity was increased more rapidly than intracellular Na+ activity. 5) The rate of increase in intracellular Ca2+ activity with reperfusion of Tyrode solution was dependent upon intracellular Na+ activity during cardioplegia, in such a way that the higher the intracellular Na+ activity was, the faster the intracellular Ca2+ activity increased. These data suggest that Na(+)-Ca2+ exchange mechanism may play an important role in the regulation of intracellular Ca2+ activity during recovery after cardioplegia.

Na+-Ca2+ exchange transport and pacemaker activity of the rabbit SA node

Recent electrophysiological data have provided the evidences that background currents such as Na(+)-Ca2+ exchange can significantly modulate cardiac pacemaker activity. In this study, the effects of extracellular Na+ and Ca2+ concentrations on the pacemaker activity were investigated by measuring the intracellular Na+ activity (aiNa) with Na(+)-selective microelectrodes and the results are summarized as follows. 1) In the rabbit SA node, aiNa was 3.2 +/- 0.3 mM and mean MDP (maximal diastolic potential) was -63.3 +/- 1.4 mV. 2) Graded decreases of external Na+ concentration resulted in the loss of spontaneous beating, hyperpolarization and the decrease of aiNa. 3) An increase in extracellular Ca2+ concentration in low Na+ solution augmented the transient decrease of aiNa, about 3 minutes in low Na+ solution, until aiNa started to increase. 4) In low Na+ solution, which had extracellular Ca2+ concentration according to the calculation based on the equilibrium state of Na+-Ca2+ exchange, aiNa was continuously decreased. It was concluded that intracellular Na+ activity modulated by Na+-Ca2+ exchange could play an important role in the initiation of pacemaker potential.

Effect of Caffeine on Calcium Flux across the Sarcolemma of Guinea Pig Atrial Trabeculae during Contracture

The changes in extracellular calcium activities during contractures of guinea pig atrial trabecular muscles were measured with Ca2+-selective electrodes. When the tissue was superfused with Na+-free Tyrode solution extracellular Ca2+ activities were decreased and contractures were induced with some delay. When the contracture was relaxed with Na+-containing Tyrode solution, extracellular Ca2+ activities were increased transiently and recovered in a Na+-dependent manner. The magnitude of extracellular Ca2+ activity decreased was proportional to the maximum magnitude of contracture induced by Na+-free solution. Addition of caffeine (10 mM) to Na+-free solution induced transient contracture following slow development of contracture and an increase in extracellular Ca2+ activity. Removal of caffeine from Na+-free solution caused a slow relaxation of contracture and a decrease in extracellular Ca2+ activity. These results confirm that caffeine blocks Ca2+ uptake by the sarcoplasmic reticulum (SR) resulting in an increase in sarcoplasmic Ca2+ activity. Ca2+ activity in the extracellular space, the amount of Ca2+ transported into the cell(Ca2+ depletion in the extracellular space), and the magnitude of contracture are well correlated. Present experiments suggest that extracellular use of Ca2+-selective electrodes provides continuous and quantitative monitoring of Na+-dependent Ca2+ flux across the cardiac cell membrane.

Effects of Vanadate on Cellular Ca2+ Movements in Guinea Pig Papillary Muscles

The effects of vanadate on cellular Ca2+ movements across the sarcolemma of cardiac muscle cells were investigated by measuring the intracellular and extracellular Ca2+ activities of guinea pig papillary muscle with Ca2+-selective electrodes. During the rest period following a steady-state of 2 contractions per second the extracellular Ca2+ concentration was increased over the basal level within a minute. During the rest period Ca2+ was transported across the sarcolemma into the extracellular space. Vanadate decreased the change in extracellular Ca2+ concentration during the rest period implying that the Ca2+ efflux across the sarcolemma was decreased by vanadate. Vanadate increased intracellular Ca2+ activities significantly (from 1.9 X 10(-7) M to 10(-6)M) resulting in an increase in resting tension. These results suggest that vanadate decreases Ca2+ efflux from the cells into the extracellular space by blocking Ca2+ transport across the sarcolemma, possibly blocking the Na+-Ca2+ exchange transport.

Postconditioning protective effect of KB-R7943 on cardiac ischemia/reperfusion injury in isolated rat heart / 中国药理学通报

Aim This study aimed at investigating the postconditioning protective effect of KB-R7943 and the proper administration time on cardiac ischemia-reperfusion injury in isolated rat heart. Methods Rat heart was isolated quickly when the animal was anesthetized. With Langendorff perfusion, the left anterior descending coronary artery was ligated for 30 min, then released for 120 min in isolated rat heart. Left ventricular function (LVF) and the infarct size of left ventricle were determined. Result Compared with control, KB-R7943 at 1 ?mol?L-1 perfused during the early period of the reperfusion prevented the post-ischemic depression of LVF, decreased the infarct size by 77 %(P