Thrombin induces a calcium transient that mediates an activation of the Na+/H+ exchanger in human fibroblasts.
The calcium dependence of growth factor-induced cytoplasmic alkalinization was determined in serum-deprived human fibroblasts (WS-1 cells). Intracellular pH (pHi) and intracellular calcium (Ca2+i) were measured using the fluorescent dyes 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein and fura2, respectively. Thrombin (10 nM) induced an alkalinization (0.18 +/- 0.01 pH units, n = 23) that was Na+-dependent and amiloride-sensitive, suggesting that the alkalinization was mediated by the Na+/H+ exchanger. Thrombin treatment caused a transient increase in Ca2+i (325 +/- 39 nM, n = 12) that preceded the observed increase in pHi. The increases in Ca2+i and pHi were dependent on the concentration of thrombin. The thrombin-induced increase in Ca2+i occurred in the absence of external calcium indicating that thrombin released calcium from internal stores. Inhibition of the thrombin-induced increase in Ca2+i with 8-diethylaminooctyl 3,4,5-trimethoxybenzoate hydrochloride or bis-(o-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid also inhibited the thrombin-stimulated increase in pHi. The calcium ionophore ionomycin was used to increase Ca2+i independent of growth factor stimulation. When Ca2+i was elevated with ionomycin, a concomitant increase in pHi was observed. The increase in pHi due to ionomycin was dependent on Na+ and sensitive to amiloride. The removal of external Ca2+i inhibited the ionomycin-induced elevation of both Ca2+i and pHi. The ionomycin-induced increases in Ca2+i and pHi were not inhibited by 8-diethylaminooctyl 3,4,5-trimethoxy-benzoate hydrochloride. The results suggest that thrombin treatment can activate the Na+/H+ exchanger, and this activation is mediated by an increase in Ca2+i.
Hendey
B
B
Biomedical Sciences Program, Wright State University, Dayton, Ohio 45435.
Mamrack
M D
MD
Putnam
R W
RW
D000584:Amiloride / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D001839:Bombesin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D004533:Egtazic Acid / Q000494:pharmacology / Q000031:analogs & derivatives; D005707:Gallic Acid / Q000494:pharmacology / Q000031:analogs & derivatives; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D017923:Sodium-Hydrogen Exchangers; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000502:physiology*
D002121:Calcium Channel Blockers; D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D000643:Ammonium Chloride; D004533:Egtazic Acid; D015759:Ionomycin; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D000584:Amiloride; D013917:Thrombin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D001839:Bombesin; D002118:Calcium