trically and by fluorescence, the molecular details 
of their activation, and their role in the regulation of 
internal pH are being actively investigated. 
Vacuolar-type Pumps 
Studies in Dr. Grinstein's laboratory also ad- 
dressed the role of pumps in the regulation of the 
pH of the cytosol and other intracellular organelles. 
Like antiports and channels, vacuolar pumps 
were found to be active in cytosolic pH regulation 
in phagocytes. Recovery from an acid load was 
found to be accompanied by extrusion of acid from 
the cells, consistent with a plasmalemmal location 
of the pumps. In neutrophils, pumping was only 
evident following stimulation with phorbol esters 
or chemoattractants, suggesting migration of intra- 
cellular pumps to the membrane or functional un- 
masking of resident plasmalemmal pumps. 
Vacuolar-type H"^ pumps were also found to be 
central to the generation and maintenance of an 
acidic interior in phagosomes. A low phagosomal 
pH is essential for effective microbial killing. The 
determinants of the intraphagosomal pH were stud- 
ied in situ, recording the emission of covalently 
fluoresceinated bacteria ingested by macrophages. 
The phagosomal membrane was found to be compar- 
atively tight, although not entirely impermeant to 
H^. A counterion conductance, which supports the 
rheogenic movement of via leaks or pumps, was 
also detected. Both anions and cations were found to 
contribute to the counterion pathway. The magni- 
tude of the counterion flux suffices and, in fact, 
greatly exceeds the rate of pumping in the steady 
state. 
These findings imply that the phagosomal mem- 
brane potential is negligible and therefore not a ma- 
jor contributor to the establishment of intraphago- 
somal pH. The rate of active pumping was found 
to decrease steeply as the phagosomal lumen be- 
came acidified. Therefore it appears that the intrin- 
sic pH sensitivity of the vacuolar pumps, which 
likely reflects a kinetic or allosteric effect, is the 
primary determinant of the intraphagosomal pH. It 
is thus conceivable that the differential pH of dis- 
tinct endomembrane compartments is dictated by 
the pumps themselves, as a result of the exis- 
tence of varying pump isoforms or the presence of 
allosteric inhibitors or activators. Experiments are 
in progress to resolve these alternatives. 
Dr. Grinstein is Professor of Biochemistry at the 
University of Toronto and Head of the Division of 
Cell Biology at the Hospital for Sick Children, 
Toronto. 
Articles 
Bianchini, L., Woodside, M., Sardet, C, Pouyssegur, 
J., Takai, A., and Grinstein, S. 1991. Okadaic 
acid, a phosphatase inhibitor, induces activation 
and phosphorylation of the NaVH"^ antiport. / 
Biol Chem 266:15406-15413. 
Bourgoin, S., and Grinstein, S. 1992. Peroxides of 
vanadate induce activation of phospholipase D in 
HL-60 cells. /fi/o/ Chem 267:11908-11916. 
Downey, G.P., Chan, C.K., Lea, P., Takai, A., and 
Grinstein, S. 1992. Phorbol ester-induced actin 
assembly in neutrophils: role of protein kinase C. 
/ Cell Biol 1 16:695-706. 
Lu, D.J., Takai, A., Leto, T.L., and Grinstein, S. 
1992. Modulation of neutrophil activation by 
okadaic acid, a protein phosphatase inhibitor. Am 
f Physiol 262:C39-C49. 
Lukacs, G.L., Rotstein, O.D., and Grinstein, S. 
1991. Determinants of the phagosomal pH in mac- 
rophages. In situ assessment of vacuolar H*^^^- 
ATPase activity, counterion conductance, and 
"leak.'' f Biol Chem 266:24540-24548. 
Nanda, A. , and Grinstein, S. 1991. Protein kinase C 
activates an (equivalent) conductance in the 
plasma membrane of human neutrophils. Proc 
Natl Acad Sci USA 88:10816-10820. 
INTERNATIONAL RESEARCH SCHOLARS 
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