Regulation of the Na+/Ca2+ exchanger (NCX) in the murine embryonic heart.
OBJECTIVE
The Na+/Ca2+ exchanger (NCX) is involved in embryonic heart development and function demonstrated by the abnormal myofibrillar organization, defects in heartbeat, and early embryonic death of NCX-null embryos. It was therefore the aim of our study to identify key functional regulators of the embryonic NCX.


METHODS
NCX current (I(NCX)) density was measured as the Ni2+ (5 mM)-sensitive current applying the whole-cell patch-clamp technique in early (EDS, E10.5V) and late developmental stage (LDS, E16.5V) mouse ventricular cardiomyocytes.


RESULTS
Compared to LDS, cardiomyocytes derived from EDS showed a significantly higher basal I(NCX) density for the I(NCX) forward (-120 mV: -4.1+/-1 pA/pF, n=15 versus -1.7+/-0.4, n=11, p<0.05) and reverse modes (+60 mV: 4.0+/-0.9 pA/pF, n=15 versus 1.8+/-0.4, n=11, p<0.05). There was 2-3-fold elevation of forward and reverse current in LDS on application of ATP-gamma-S (2 mM) together with forskolin (1 microM) as well as intracellular application of the catalytic subunit of cAMP-dependent protein kinase (cPKA, 200 U/mL), cAMP (200 microM), phorbol 12-myristate-13-acetate (PMA), a direct activator of protein kinase C (PKC), and 8-Br-cGMP, a membrane permeable analog of cGMP. The specific PKC inhibitor Ro 31-8220 significantly reduced I(NCX) by 70%. Co-application of 20 microM PKA inhibitor Fragment 14-22 (PKI), a specific inhibitor of PKA, and cAMP significantly reduced the exchanger activity by approx 60%. Despite these obvious effects in LDS we could not detect a significant impact of these compounds on I(NCX) in EDS-derived cardiomyocytes. Application of the alkaline phosphatase to test for constitutive phosphorylation of NCX did not affect I(NCX) density in LDS but led to an approx 80% reduction of I(NCX) in EDS.


CONCLUSIONS
In EDS cardiomyocytes I(NCX) density is upregulated, at least in part by the high phosphorylation of the exchanger protein. At LDS, embryonic cardiomyocytes showed a strong increase of I(NCX) density upon stimulation by PKC- and PKA-dependent signalling pathways.
Reppel
Michael
M
Institute of Neurophysiology, University of Cologne, Cologne, Germany. akp72@uni-koeln.de
Fleischmann
Bernd K
BK
Reuter
Hannes
H
Sasse
Philipp
P
Schunkert
Heribert
H
Hescheler
Jürgen
J
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000494:pharmacology; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D047109:Fetal Development / Q000502:physiology*; D005865:Gestational Age; D006321:Heart / Q000196:embryology*; D006352:Heart Ventricles; D007211:Indoles / Q000494:pharmacology; D051379:Mice; D032383:Myocytes, Cardiac / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011758:Pyrroles / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002227:Carbazoles; D007211:Indoles; D011758:Pyrroles; D019831:Sodium-Calcium Exchanger; D005576:Colforsin; C016276:8-bromocyclic GMP; C057416:KT 5720; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000469:Alkaline Phosphatase; D006152:Cyclic GMP; D013755:Tetradecanoylphorbol Acetate; C064758:Ro 31-8220