Protein carboxyl methylation controls intracellular pH in human platelets.
OBJECTIVE
Carboxyl methylation is a reversible post-translational event which regulates the function of several cellular proteins. Because the human Na+-H+ antiporter (NHE-1) possesses a C-terminal consensus sequence for carboxyl methylation, we examined the role of protein carboxyl methylation in the regulation of intracellular pH homeostasis.


METHODS
Experiments were conducted using human platelets and N-acetyl-S-trans,trans-farnesyl-L cysteine (AFC), a specific prenylcysteine methyltransferase inhibitor. The effect of AFC on both basal intracellular pH (pHi) and on the kinetic properties of the Na+-H+ antiporter was characterized.


METHODS
pHi was determined in cell suspensions using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein tetraacetoxymethyl ester, a fluorescent pH indicator. The kinetics properties of the Na+-H+ antiporter activity were determined using platelets acidified with nigericin and challenged with varying extracellular concentrations of Na+.


RESULTS
AFC (20 micromol/l) decreased basal pHi significantly (7.047 +/- 0.011 versus 7.133 +/- 0.012 for control, P< 0.001). The acidification was dose-dependent and reached steady state 3 min after AFC addition. In the absence of extracellular Na+, the platelets were acidified to the same extent with AFC or with ethanol (control): 6.530 +/- 0.031 versus 6.532 +/- 0.031 (P= 0.97). However, upon addition of Na+, the platelets treated with AFC showed a significant decrease in the maximal value for initial pHi recovery compared with controls: 0.788 +/- 0.041 versus 0.983 +/- 0.047 pH/min (P< 0.02). AFC also increased the Hill coefficient (2.89 +/- 0.22 versus 2.14 +/- 0.16, P < 0.03), and tended to decrease K0.5, the [Na+] corresponding to half-maximal activation (51.3 +/- 1.8 versus 60.5 +/- 3.9 mmol/l, P = 0.06) of the antiporter.


CONCLUSIONS
Our data indicate that inhibition of carboxyl methylation reduces basal pHi and alters the kinetic properties of the Na+-H+ antiporter in human platelets, suggesting that carboxyl methylation is implicated in the regulation of intracellular pH homeostasis.
Otsuka
K
K
Division of Nephrology, Hypertension and Clinical Pharmacology, Oregon Health Sciences University, Portland 97201-3098, USA.
Roullet
C M
CM
McDougal
P
P
McCarron
D A
DA
Roullet
J B
JB
D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D008745:Methylation
D004791:Enzyme Inhibitors; D006859:Hydrogen; C068268:N-acetyl-S-farnesylcysteine; D000111:Acetylcysteine