Electrophysiology Of VSOR Cl-channel Induced By STS And Its Relationship With PI3K/Akt

Cloride ion (Cl-) is the most important and abundant anion in the organism, and the volume-sensitive outwardly rectifying (VSOR) Cl- channels which principally mediate the anion current take part not only in a variety of physiological and pathological process including cell volume regulation, cell proliferation and differentiation, cell apoptosis, and providing action potential, but also play a substantial role in the process of multi-cardiovascular diseases such as heart failure, myocardial infarction, arrhythmias.Apoptosis is also named programmed cell death (PCD). The early cell shrinkage before apoptosis namely apoptotic volume decrease (AVD) is an important morphological characteristic. For the past few years, AVD and the activated Cl- channels were demonstrated by generous evidence playing important roles in cell apoptosis as a perquisite of cell apoptosis. Under apoptosis stimuli including mitochondrion-mediated intrinsic and death- mediated extrinsic apoptosis ways, cell apoptosis can be inhibited by suppressing activated VSOR Cl- channel and the cell shrinkage in a variety of cell types. In the same time volume-sensitive Cl- currents (ICl,Vol) or swelling- activated Cl- current (ICl,Swell) activated accompanied with the apoptotic process, was also proved by patch-clamp tests, furthermore, apoptotic cell death can be rescued by blocking ICl,Vol. However, the underlying mechanism remains unclear. Staurosporine (STS) was a kind of inhibitor of protein kinase C (PKC). Our previous molecular biological studies had proved that VSOR Cl- channel inhibitors inhibited cardiac myocytes apoptosis induced by STS and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signal molecule participated in the ptrotection of cardiac myocytes abscent of direct electrophysiological evidences. This is just the objective of this study.Aims:1. To observe whether ICl,Vol can be induced by STS on cardiomyocytes using whole-cell patch clamp techniques and the electrophysiological characteristics of ICl,Vol.2. To rescord the effects of Cl- channel inhibitor DIDS and the specific VSOR Cl- channel inhibitor DCPIB on ICl,Vol induce by STS.3. To observe whether PI3K/Akt signal molecule participate in the modulation of ICl,Vol induce by STS by applying with the specific inhibitor and excitor of PI3K/Akt LY294002 and Insulin, respectively.Methods:1. Primary cultured SD rat neonatal cardiacmyocytes, set up negative control group (isotonic group), normal control group (hypotonic group), positive control group (STS group) and experimental groups (hypotonic+DIDS/ DCPIB group, STS+DIDS/DCPIB group, STS+LY294002/Insulin group). There are at least 5 cells subjecting to the same treatment in each group.2. Recording the production and electrophysiological properties of ICl,Vol and the inhibition effects of two kinds of Cl- channel blockers DIDS and DCPIB on cardiacmyocytes perfusing with hypotonic extrinsic fluid, using whole-cell patch clamp techniques.3. Recording the production and electrophysiological properties of Cl- currents on cardiacmyocytes perfusing with isotonic extrinsic fluid contained STS, and the inhibition effects of two kinds of Cl- channel blockers DIDS and DCPIB.4. Pretreatment with the specific inhibitor and excitor of PI3K/Akt LY294002 and Insulin for 30min, using the pach- clamp techniques as above, to observe the effects of LY294002 and Insulin on Cl- currents.Results:1. ICl,Vol was produced by perfusing 220 Osmol/kgH2O hypotonic extrinsic fluid. It showed us time-dependent inactivation when the depolarization potential reached to +80mV. The desitity of Cl- currents at +100mV is 19.45±1.76 pA/pF (n=5) and it could be voltage-dependently blocked by DIDS and DCPIB at +40mV, +60mV, +80mV, +100mV, and I/V curves indicated outwardly rectification of the Cl- current.2. We firstly recorded Cl- currents induced by STS (4μmol/L) in primary cultured cardiomyocytes showing the electrophysiological characteristics resembling ICl,Vol : outward rectification, time-dependentive inactivation at high positive potentials, and the sensitivity to Cl- channel blockers corresponding with the electrophysiological characteristics of ICl,Vol. The desitity of Cl- currents at +100mV is 18.73±2.32 pA/pF (n=5, P>0.05, STS vs. hypotonic). Also, we investigated DIDS (500μmol/L) and DCPIB (10μmol/L) both inhibited the current remarkably by 92.6%±7.3%( n=5, P<0.01, STS+DIDS vs. STS) and 78.4%±5.5% (n=5, P<0.01, STS+ DCPIB vs. STS) at +100mV, respectively, with significant difference (n=5, P<0.01, STS+DIDS vs. STS+DCPIB). Thus, the results indicated that the Cl- currents induced by STS were mainly ICl,Vol.3. The specific inhibitor of PI3K/Akt LY294002 (20μmol/L) facilitated STS-induced Cl- currents and the desitity of Cl- currents at +100mV was 19.54±2.70 pA/pF (n=5, P>0.05, STS+LY294002 vs. STS), while the time of the current′s presentation was shorter than STS group (n=5,P<0.01, STS +LY294002 vs. STS); Insulin as the excitor of PI3K/Akt (100nmol/L) inhibited STS-induced Cl- currents and the desitity of Cl- currents at +100mV was 15.40±2.13 pA/pF(n=6, P<0.01, STS+Insulin vs. STS and STS+LY294002), which indicated that PI3K/Akt signal molecule take part in the modulation of VSOR Cl- channel.Conclusions:1. ICl,Vol was induced by hypotonic conditions with outward rectification, time-dependentive inactivation at high positive potentials, and can be blocked by Cl- channel blockers DIDS and DCPIB.2. Analogical ICl,Vol was induced by STS according with the electrophysilogi- cal characterizations of ICl,Vol.3. ICl,Vol can be faciliated and decreased by LY294002 and Insulin respectively,these demonstrated that PI3K/Akt sigal molecule take part in the regulation of ICl,Vol.