Intermediate-conductance Ca²⁺-activated Potassium And Volume-sensitive Chloride Channels In Endothelial Progenitor Cells From Rat Bone Marrow Mononuclear Cells

Coronary artery disease (CAD) continues to be the leading cause of mortality in the world. The economic cost of CAD in the world is tremendous per year. Despite great advances obtained in interventional cardiology and pharmacological treatments, the therapeutics themselves cann't stimulate the growth of new blood vessels in myocardial infarction, thus do not have the potential to reverse and cure coronary artery disease thoroughly. Cellular cardiomyoplasty developed in recent years, is a promising technology possible to fundamentally solve the problem and improve life quality in patients with CAD. Endothelial progenitor cells (EPCs) are believed to be a good cell source for regenerative medicine and cell-based cardiomyoplasty; however, their electrophysiology properties are not fully understood, some challenge still remains to be overcomed: little in number,transplantation with high mortality,unknown arrhythmia mechanism,suspicious of differentiation.In recent year ,electrophysiology property in stem cells has become a popular research .accumulating data shows that functional ion channels in stem cells was associated with proliferation and mortarity of stem cell .There are also some articles show that ion channels participate in the course of stem cells'differentiation and cell cycle. Studies in electrophysiology of endothelial progenitor cells will help to explain the real reason of arrhythmia causing by cell transplantation therapy.Objectives (1) To investigate what kinds of ion channels exist in rat endothelial progenitor cell(2)To explore electrophysiology features as well mRNA expression of ion channels in endothelial progenitor cells in order to provide a basis for further physiological functions studies.Methods (1) Separation, culture and identification of bone marrow endothelial progenitor cells. Mononuclear cells were separated using density gradient centrifugation, then cultured with endothelial progenitor cell conditioned medium. Attached cells were identified with dil-acLDL and UEA lectin. (2) Expression and characteristics of functional ion channels in rat bone marrow progenitor cells. The present study was designed to record transmembrane ion currents with the whole-cell voltage clamp technique and investigate mRNA expression of corresponding ion channel subunits in rat Bone marrow endothelial progenitor cells(BMEPCs) using RT-PCR analysis.Results (1)Separation, culture and identification of bone marrow endothelial progenitor cells Stainning of cultured cells showed acLDL uptake and FITC-ulex-lectin double positive and was supposed to be characteristics of bone marrow progenitor. (2) I_Kca and I_cl were present on the endothelial progenitor cells. We observed two types of ionic currents in undifferentiated rat BMEPCs. One was detected when 0.1μmol/L Ca²⁺ was employed in pipette solution and was inhibited by 4-AP, which was similar to I_Kca. The other was chloride channel current, when BMEPCs were subjected to no K⁺ hypotonic extracellular solution and isotonic free K⁺ pipette solution .This current could be mostly inhibited by NPPB (3) I_Kca was intermediate-conductance Ca²⁺-activated potassium. Calcium-activated potassium channels can not be inhibited by large conductance and small conductance I_Kca blockers inberiotoxin and apamin block, but it was almost inhibited by intermediate conductance blocker of I_Kca clotrimazole. RT-PCR results showed that gene responsible for this current was KCNN4, which provided further proof that the calcium-activated potassium channel seen was the conductance I_Kca. (4)I_clwas volume-sensitive chloride channel current. Chloride currents recorded in hypotonic bath solution without potassium was 4 times larger than under isotonic conditions, and gene encoding this current was Clcn3, which illustrated that chloride channels in BMEPCs were sensitive to cell volume.Conclusion Almost BMEPCs show outward current:intermediate-conductance Ca²⁺-activated potassium current and volume-sensitive chloride current, and genes encoding these two channels are KCNN4 and Clcn3 respectivly.