Ionic Homeostasis in White Blood Cells 
Sergio Grinstein, Ph.D. — International Research Scholar 
Dr. Grinstein is Head of the Division of Cell Biology at the Research Institute of the Hospital for Sick 
Children, Toronto, and Professor of Biochemistry at the University of Toronto. He received his Ph.D. degree 
at the National Polytechnic Institute, Mexico City, where he studied with David Erlij. His postdoctoral 
training was in two stages: initially at the Hospital for Sick Children under the supervision of Aser 
Rothstein, and later at the Federal Institute of Technology in Zurich with Giorgio Semenza. He has received 
the Ayerst Award of the Canadian Biochemical Society. 
LEUKOCYTES constitute the body's first line of 
defense against invading microorganisms. 
These white blood cells first detect and engulf 
bacteria and other microbes, then secrete lytic 
enzymes and synthesize reduced oxygen metabo- 
lites to kill them. 
These microbicidal processes call for pro- 
nounced changes in the generation and intracel- 
lular distribution of acid equivalents. First, the 
phagocytic vacuole wherein the microorganisms 
are trapped becomes markedly acidic. This evi- 
dently promotes the activity of the lytic enzymes 
released into the phagosome and may also facili- 
tate its fusion with the vesicles containing bacteri- 
cidal agents. Then too, the leukocytes' rate of 
metabolic acid production increases greatly dur- 
ing infection, threatening the stability of the cyto- 
solic compartment, which must remain slightly 
alkaline to preserve optimal cell function. 
The purpose of our research is to understand 
the mechanisms underlying phagosomal acidifi- 
cation, the pathways responsible for excess meta- 
bolic acid during leukocyte activation, and partic- 
ularly the processes involved in the maintenance 
of the cytoplasmic pH under both resting and ac- 
tivated conditions. 
Antiports and Channels in the Regulation 
of Cytosolic pH 
The pronounced metabolic burst that leuko- 
cytes undergo when confronted by microorgan- 
isms or their products can be largely attributed to 
activation of an otherwise quiescent enzyme, the 
NADPH oxidase. The one-electron reduction of 
oxygen catalyzed by this enzyme is accompanied 
by oxidation of NADPH to NADP^ and release of 
protons. Regeneration of NADPH through the 
hexose monophosphate shunt is a source of fur- 
ther proton production. If uncompensated, the 
proton production by these pathways would pro- 
duce a massive intracellular acidification, incom- 
patible with normal leukocyte function and possi- 
bly even their viability. 
Three primary pathways appear to be involved 
in proton (equivalent) extrusion in activated leu- 
kocytes. The first and perhaps most important is 
an electroneutral exchanger (antiport) that trans- 
ports protons out of the cells in exchange for ex- 
tracellular sodium. A major isoform of this anti- 
port has been identified in fibroblasts as a 
1 10-kDa membrane glycoprotein. We have found 
that the antiport is active not only after stimula- 
tion but also in resting cells. Its activity, however, 
is greatly enhanced following the addition of bac- 
terial chemoattractants or of molecules that 
mimic events in the intracellular signaling cas- 
cade triggered by microorganisms. 
Our current and future research eff'orts in this 
area deal with the molecular characterization of 
the antiport in leukocytes and of the mechanisms 
underlying its activation during infection and in- 
flammation. We are particularly interested in the 
subcellular localization of the antiports before 
and after stimulation, in the mechanisms 
whereby chemoattractants signal activation, and 
in the segregation and/or inactivation of anti- 
ports in compartments where sodium/proton ex- 
change activity is counterindicated (such as the 
phagosome) . 
We have recently detected a second pathway 
that appears to be important in the extrusion of 
protons from activated leukocytes, namely a pro- 
ton conductance, possibly a channel. This con- 
ductive path is essentially undetectable in quies- 
cent cells but becomes clearly apparent when the 
cells are stimulated. Preliminary data indicate 
that this putative channel is present in neutro- 
phils, macrophages, and the human leukemic 
cell line HL60. The conductive proton pathway 
could serve two important functions in the stimu- 
lated leukocyte: it could contribute to the extru- 
sion of net acid equivalents from the cell, and it 
could also serve as a source for counterions to 
neutralize the voltage generated by the NADPH 
oxidase, proposed to be electrogenic. 
Little is known at present about the conductive 
pathway. We are interested in defining its molec- 
ular identity, physiological significance, intra- 
cellular distribution, developmental pattern, and 
the molecular basis of its activation. In this re- 
gard, it is noteworthy that activation of the con- 
ductance closely mirrors the behavior of the 
497 
