title: Functional comparison of the reverse mode of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells.
abstract: OBJECTIVE
  To investigate the reverse mode function of Na(+)/Ca(2+) exchangers NCX1.1 and NCX1.5 expressed in CHO cells as well as their modulations by PKC and PKA.
  
  
  METHODS
  CHO-K1 cells were transfected with pcDNA3.1 (+) plasmid carrying cDNA of rat cardiac NCX1.1 and brain NCX1.5. The expression of NCX1.1 and NCX1.5 was examined using Western blot analysis. The intracellular Ca(2+) level ([Ca(2+)]i) was measured using Ca(2+) imaging. Whole-cell NCX currents were recorded using patch-clamp technique. Reverse mode NCX activity was elicited by perfusion with Na(+)-free medium. Ca(2+) paradox was induced by Ca(2+)-free EBSS medium, followed by Ca(2+)-containing solution (1.8 or 3.8 mmol/L CaCl2).
  
  
  RESULTS
  The protein levels of NCX1.1 and NCX1.5 expressed in CHO cells had no significant difference. The reverse modes of NCX1.1 and NCX1.5 in CHO cells exhibited a transient increase of [Ca(2+)]i, which was followed by a Ca(2+) level plateau at higher external Ca(2+) concentrations. In contrast, the wild type CHO cells showed a steady increase of [Ca(2+)]i at higher external Ca(2+) concentrations. The PKC activator PMA (0.3-10 μmol/L) and PKA activator 8-Br-cAMP (10-100 μmol/L) significantly enhanced the reverse mode activity of NCX1.1 and NCX1.5 in CHO cells. NCX1.1 was 2.4-fold more sensitive to PKC activation than NCX1.5, whereas the sensitivity of the two NCX isoforms to PKA activation had no difference. Both PKC- and PKA-enhanced NCX reverse mode activities in CHO cells were suppressed by NCX inhibitor KB-R7943 (30 μmol/L).
  
  
  CONCLUSIONS
  Both NCX1.1 and NCX1.5 are functional in regulating and maintaining stable [Ca(2+)]i in CHO cells and differentially regulated by PKA and PKC. The two NCX isoforms might be useful drug targets for heart and brain protection.
authors.affiliation: State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Long
authors.forename: Wei-ping
authors.initials: WP
authors.lastname: Wang
authors.forename: Hui
authors.initials: H
authors.lastname: Yuan
authors.forename: Shi-ping
authors.initials: SP
authors.lastname: Ma
authors.forename: Nan
authors.initials: N
authors.lastname: Feng
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.forename: Xiao-liang
authors.initials: XL
authors.lastname: Wang
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015870:Gene Expression; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D019831:Sodium-Calcium Exchanger / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D015124:8-Bromo Cyclic Adenosine Monophosphate; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D002118:Calcium
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