Expression, functional characterization and tissue distribution of a Na+/H+ exchanger cloned from Xenopus laevis oocytes (XL-NHE).
We examined the functional properties of a Na+/H+ exchanger cloned from Xenopus laevis oocytes (XL-NHE) upon stable transfection into PS120 fibroblasts which lack endogenous Na+/H+ exchange. In contrast to untransfected cells, XL-NHE-transfected cells displayed Na+-dependent alkalinization upon acidification with nigericin. XL-NHE activity was inhibited by amiloride, ethylisopropylamiloride, HOE694 [(3-methylsulphonyl-4-piperidinobenzoyl)-guanidine methanesulphonate] and HOE642 [4-isopropyl-3-methylsulphonylbenzoyl)-guanidine methanesulphonate], Ki values being calculated at 5 micromol/l, 25 nmol/l, 300 nmol/l and 180 nmol/l, respectively. The Na+ dependence of pHi recovery was compatible with simple Michaelis-Menten kinetics, the Km for Na+ being 22.0+/-3.2 mmol/l and the Hill coefficient for Na+ being approximately 1. XL-NHE was activated by phorbol ester, whereas forskolin exerted no effect, suggesting the involvement of phospholipase C/protein kinase C signalling pathways rather than protein kinase A signalling pathways in XL-NHE stimulation. Using reverse transcription polymerase chain reaction, XL-NHE message could be detected in various Xenopus tissues including heart, brain, skeletal muscle, reticulocytes, A6-kidney cells and oocytes.
Busch
S
S
Institut für Pharmakologie, Universitätsklinikum Essen, Hufelandstr. 55, D-45122 Essen, Germany. stefan.busch@uni-essen.de
Rosskopf
D
D
Lang
H J
HJ
Weichert
A
A
Siffert
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W
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008168:Lung; D009865:Oocytes / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014982:Xenopus laevis
D012333:RNA, Messenger; D017923:Sodium-Hydrogen Exchangers; D005576:Colforsin; D000584:Amiloride; D012964:Sodium; D013755:Tetradecanoylphorbol Acetate