The Search Of Regulation Of The Na～+/Ca～(2+) Exchanger By Metabolic Inhibition Pretreatment In Rat Ventricular Myocytes

Cytosolic Ca²⁺ plays a key role in intracellular signaling in virtually all types of animal cells. In myocytes, neurons, and nephron cells, The Na⁺/Ca²⁺ exchanger (NCX) is thought to play an important role in the regulation of intracellular Ca²⁺ concentration. NCX is an ion transporter that exchanges Na⁺ and Ca²⁺ in either Ca²⁺ efflux or Ca²⁺ influx mode, depending on membrane potential and transmembrane ion gradients. Under physiological conditions, the primary function of NCX is thought to be to pump Ca²⁺ to the outside of the cell using the Na⁺ concentration gradient across the cell membrane. Under pathological conditions, the exchanger is thought to cause Ca²⁺ overload, which causes cardiac ischemia/reperfusion injury (I/R). Protection against ischemia by IP is seen in many tissues and organs. The phenomenon of ischemic preconditioning (IP) is a period of sublethal ischemia that can profoundly protect the cell from infarction during a subsequent ischemic insult, which is a very powerful protective mechanism against repeat lethal ischemia. Early studies revealed that IP causes two windows of cardioprotection: a classical form lasting ² h after the preconditioning ischemia followed a day later by a second window of protection lasting ³ days, and subsequent efforts to detect cardioprotective factors have identified various triggers, mediators, and potent effectors of IP. Today we know that IP is an immensely complex process involving multiple signalling systems that are still not fully understood. Pathophysiological conditions that affect myocardial metabolism contained hypoxia and ischemia.In hypoxia and ischemia, where the rate of anaerobic glycolysis is increased. This progress leads to an accumulation of lactate, and to an intracellular acidosis. The purpose of this study is: 1)whether the IonOptix Photometry System can record the forward mode of the Na⁺/Ca²⁺ exchange or the reverse mode of the Na⁺/Ca²⁺ exchange, 2) to study the activity of reverse-mode Na⁺/Ca²⁺ exchanger (NCX) during metabolic inhibition pretreatment (MIP) of rat ventricular myocytes through lactate mimic IP and to determine whether the cardioprotection of metabolicinhibition preconditioning can be prevented by reverse-mode NCX inhibitor, so that supply experimental evidence that the relationship between NCX and IP.Single ventricular myocytes were enzymatically isolated and loaded with Fura-2/AM for 30min in dark at room temperature, and the change of intracellular fluorescence intensity assessed by the IonOptix Photometry System indirectly indicated the forward mode of the Na+/Ca2+ exchange and the reverse mode of the Na /Ca exchange. The activity of reverse-mode NCX was assessed by withdrawal extracellular Na+ and the changes of [Ca2+]i were measured. The cell viability is used as indices of cell injury and death.The fist part of experiments results showed, using the IonOptix Photometry System can real-time beat-to-beat observe and record: 1) [Ca2+]i was increased by the increasing of [Ca2+]0 after ventricular myocytes exposed to Na+-free solution (NMDG solution), and returned to baseline following washout. Nifedipine (lumol/L), the selective inhibitor of L-type calcium channel, did not affect NMDG solution-induced increases of [Ca2+];, while both Ni2+ (lmmol/L), the non-selective inhibitor of NCX and KB-R7943(15umol/L),the selective inhibitor of NCX, ahnost completely blocked the effect, an indication of the reverse mode of the Na+/Ca2+ exchange. 2) The decline of caffeine-induced [Ca2+]i transient, an indication of the forward mode of the Na+/Ca2+ exchange.The second part of experiments results showed, 1) In myocyte subjected to 20 mmol/L lactate and 10 mmol/L Deoxy-glucose treatment(LD treatment) for 5, 10 and 30 min respectively, and followed by 10 min normal Tyrode solution perfusion, NMDG solution-induced increases of [Ca2+]j was time-dependently inhibited. 2) when 20 mmol/L lactate and 10 mmol/L Deoxy-glucose treatment subjected for 30min, and followed by 10 min normal Tyrode solution perfusion, as metabolic inhibition pretreatment(MIP), NMDG solution-induced increases of [Ca2+]i was inhibited completely, and the cell viability is increased after I/R. Administration of Ni2+ and KB-R7943 before 5min and during MIP, which can cause increases of [Ca2+]i intensively, and decreases of the cell viability. 3) In myocyte, only subjected to 20 mmol/L lactate and 10 mmol/L Deoxy-glucose treatment for 30min(LD treatment), not subjected to following 10 min normal Tyrode solution perfusion, NMDG solution-induced increases of [Ca2+]i was further increased, and the cell viability was decreased acutely. Administration of Ni2+ and KB-R7943 before 5min and during LDtreatment, the increment of [Ca2+]i the cell viability were both as same as the control condition.The conclusion: 1) Indirect measurement Na+/Ca2+ exchange using the IonOptix Photometry System is a new technique that could provide much more information on the effect of Na+/Ca2+ exchange in Ischemia/Reperfusion injury.2) Metabolic inhibition pretreatment (MIP) could mimic ischemic preconditioning (IP) to protect myocytes from ischemic/reperfusion injury (I/R).However, only subjected to LD treatment did not finish cardioprotection. MIP can regulate the function of the reverse-mode of NCX, for during the course of LD treatment, the reverse-mode NCX was more acted. LD treatment was one of important indispensable parts of MIP. NCX acted as a trigger to promote MIP, which plays an important role in cardioprotection.