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REGULATION OF ION TRANSPORT AND INTRACELLULAR pH 
Sergio Grinstein, Ph.D., International Research Scholar 
Coordination of the activity of the myriad cellular 
enzymes requires accurate and continuous regula- 
tion of the intracellular pH. The cytosolic compart- 
ment tends to become acidic as a result of metabolic 
generation of protons (H^). In addition, the trans- 
membrane potential drives the passive electropho- 
retic accumulation of equivalents inside the cell. 
These processes must be effectively counteracted in 
order to maintain pH homeostasis. Dr. Grinstein's 
laboratory is studying the mechanisms responsible 
for the regulation of intracellular pH. 
Na+/H+ Antiports 
A family of electroneutral exchangers utilize the 
inward Na^ gradient to drive the extrusion of H"^ 
across the plasma membrane. These so-called Na^/ 
H"^ exchangers, or antiports, play a central role in 
the maintenance of intracellular pH, but have also 
been implicated in the control of cellular volume 
and in the stimulatory effects of some hormones and 
growth factors. 
When activated by cell shrinkage or growth pro- 
moters, the internal pH dependence of the ex- 
change reaction undergoes an alkaline shift. The mo- 
lecular basis of this alteration in kinetic behavior is 
poorly understood. To assess the role of protein 
phosphorylation, lymphoid cells were exposed to 
okadaic acid, a selective inhibitor of phosphatases 1 
and 2A. In otherwise untreated cells, okadaic acid 
promoted a rapid activation of the antiport, suggest- 
ing the presence of constitutively active kinases. In- 
deed, a sizable increase in protein phosphorylation 
was detectable under comparable conditions. 
The enhancement of antiport activity by okadaic 
acid was not additive with the osmotically induced 
stimulation, suggesting common pathways. How- 
ever, the two effects were differentially affected by 
protein kinase A, consistent with separate, yet con- 
vergent, signaling pathways. Immunoprecipitation 
experiments revealed that treatment with okadaic 
acid increased the phosphorylation of the antiport, 
and the use of inhibitors indicated that this effect 
was not due to protein kinases A, C, or CaM. Al- 
though phosphorylation of ancillary proteins may 
also be involved, direct phosphorylation of the anti- 
port seems a likely mechanism to account for the 
alkaline shift in its pH dependence. 
Channels 
Passive conductive mechanisms for or its 
equivalents (OH", HCO^") have not been consid- 
ered important regulators of intracellular pH, be- 
cause the electrochemical gradient would normally 
drive acid equivalents into the cells. Phagocytes are 
unique, however, in that activation by invading mi- 
croorganisms not only tends to lower cytosolic pH, 
but also depolarizes the plasma membrane, thus re- 
versing the proton motive force. Experiments were 
therefore undertaken to determine whether an H^ 
conductance is activated in neutrophils when ex- 
posed to agents that mimic microbial infection. Us- 
ing fluorimetric determinations of intracellular pH, 
Dr. Grinstein and his colleagues found the conduc- 
tance in unstimulated cells to be low and unaffected 
by moderate changes in membrane potential. In 
contrast, a sizable H^ conductance was unmasked 
when the cells were stimulated with protein kinase 
C agonists or with chemoattractant peptides. 
The existence of an H"'"-selective conductance was 
verified electrophysiologically, using the whole- 
cell configuration of the patch clamp. In human 
neutrophils and in HL-60 cells differentiated along 
the granulocytic lineage, an outward H"*" current was 
detected. This current was voltage-gated and was 
exquisitely sensitive to the intracellular pH. Prelimi- 
nary results indicate that the permeability pathway 
mediating the H^ current is extremely selective and 
displays a sharply rectifying behavior, allowing the 
passage of acid equivalents out of, but not into, the 
cells. The equivalence of the fluxes measured elec- 
510 
