Current Eye Research
Volume 9 nunibcr 7 1990
Lactate-proton cotransport in rabbit corneal epithelium
Joseph A.Bonanno
Morton D.Sarver Center for Cornea and Contact Lens Research, University of California, School o f Optometry, Berkeley, C A 94720, USA
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ABSTRACT The presence of the membrane transport mechanism, lactateH + cotransport, was tested in explants of rabbit corneal epithelium. Basal corneal epithelial cells were loaded with the pH sensitive fluorescent dye BCECF. lntracelluiar pH (pHi) was measured by ratioing the fluorescence emission output following excitation at 490 and 440 nm. Perfusion of explants in lactatecontaining Ringer’s, pH 7.40, produced a reversible decrease in pHi. The lactate induced proton influx (mM/min) followed saturating kinetlcs, Km = 10.7 mM lactate, Vmax = 10.2 mM/min. Proton influx following addition of 10 mM lactate was inhibited 36, 60 and 47% by pre-perfusion in 1 mM CHC (cyanohydroxycinammic acid), 500 pM H2DIDS (4,4’-diisothiocyanato -
be facilitated by a membrane carrier mechanism involving the lactate- anion (5,6). A lactate-H + cotransport mechanism has been described in erythrocytes (5),Ehrlich ascites tumor cells (6) and skeletal muscle (7) all of which are highly glycolytic. In addition, the cotransporter has been descrlbed for the serosal side of the kidney proximal tubule (8)and for hepatocytes (9) as a mechanism to help reabsorb lactate into the blood and into the liver for gluconeogenesis, respectively. This study examines the rabbit corneal epithelium for the presence of the lactate-H + cotransporter by measuring: (1)
dihydrostllbene- 2,2’-disulfonic acid) and 1 mM LAlE (lactic acid
changes in intracellular pH due to lactate addition, (2) lnhlbition of
isobutylester), respectively. These inhibitors of lactate-H + cotransport were reversible. Mersalyl acid (500 pM) inhibited proton flux from 10 mM lactate addition by nearly 1OO%, but was Irreversible. Stimulation of lactate production by perfusion in Np
lactate-Induced changes in pHi by known lactate-H + cotransporter inhibitors and (3) pHi changes due to stimulation of glycolysis (i.e. lactate production) by hypoxia and the effects of cotransporter
equilibrated Ringer’s (hypoxia) or the addition of 1 rnM NaCN led to a slow alkalinization (0.1 pH unit in 10 min). Pre-perfusionwith the reversible inhibitors slowed the hypoxic alkalinization by
Inhibitors on hypoxia-induced pHi changes.
approximately 40%. It is concluded that lactate-H + cotransport is present in the corneal epithelium and that it contributes to pHi regulatlon during hypoxia.
Chemicals and solutions
INTRODUCTION
BCECF-AM [2’,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxy-methylester] and H2-DIDS [ 4,4’-dlisothiocyanato-
MATERIALS AND METHODS
dihydrostilbene-2,2’-disulfonicacid] were obtained from Molecular
In the presence of air the corneal epithelium metabolizes approximately 65 - 85% of the glucose it consumes to lactate (1 -3).
Probes (Eugene, OR). All other chemicals were obtalned from
The reason for the high glycolytic activity is not known, however
Sigma (St. Louis, MO). Stock solutions of BCECF-AM(10 mM In
the relative paucity of mitochondria in the corneal epithelium may
DMSO) and nigericln (1 0 mM in absolute ethanol) were stored at
be a strategy to keep light scattering to a minimum. When the
-2O’C. The basic NaCl Ringer’s contained in mM: 100 NaCI, 10
tissue is made hypoxic, as happens during contact lens wear, lactate production can double (2, 3) and an equivalent amount of
HEPES (N-2-hydroxyethyl-piperazlne-N’-2-ethanesulfonlc acid), 2.4 K2HP04, 1.2 Ca-gluconate, 0.6 MgC12,26 glucose, 40.5Na-
protons are generated causing stromai acidification (4). Efficient
gluconate and 5 NaOH to adjust pH to 7.40, osmolarlty
removal of lactate and protons from the epithelial cells is imperative
mOSM/Kg. Lactate Ringer’s (pH 7.40) was prepared by
If glycolysis is not to be inhibited in either the normoxic or hypoxic
substituting some of the Na-gluconate with an equlmolar amount of
condition.
300 k 5
Na-lactate (equimolar L-( +)-Lactic acid and NaOH). Lactate
At physiologic pH. the lactate-/lactic acid ratio Is greater than 1OOO:l (pK lactic acid
=
=
solutions were made fresh on the day of the experiment. Sodiumfree Ringer’s (pH 7.40) was prepared by omitting Na-gluconate,
3.86).Whereas lactic acid is uncharged
and permeable to the plasma membrane, total lactate transport can ~
~
substitution of NaCl with NMDG-CI (N-methyl-D-glucamine base
~~
Received on May 10. 1990; accepted on June 28, 1990 ~-
@ Oxford University Press
707
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Current Eye Research dissolved in equlmolar HCL), pH adjusted with NMDG base and
motorized filter shuttle, data sampling and storage are controlled
adjusting osmoiarity to 300 with sucrose. Calibration Ringer's was
by UMANS software (C. Regan, Urbana, IL).
prepared by omlttlng NaCi and Na-giuconate,adding 105 KCI and adjusting osmoiarity to 300 with sucrose. All drugs, H2DIDS, CHC
Calibration
(cyano-4-hydroxyclnnamic acid), LAiE (lactlc acid isobutylester)
intraceiiuiar pH (pHi) Is set equal to solution pH (pHo) by perfusion in Callbration-Ringer's ([KO]approximately equal to [Ki])
and Mersalyi acid ([3-hydroxymercuri 2'-methoxypropyl)
contalnlng 50 pM nigericin (K+/H+ ionophore) (10). Solution pH
carbamoyi] phenoxyacetic acid, were dissolved directly in Rlnger's
Is then varied by adding small amounts of HCI or NMDG base, and
on the day of the experiment. Bicarbonate-freeRinger's was used
the steady-state fluorescence ratio is recorded. Three calibratlon
throughout to minimize lntraceliuiar buffering In order to produce
polnts are obtained for each preparation, and a linear regression is
greater and faster pHi changes when small amounts of lactate were
performed to obtain a caiibratlon curve for the experimental data.
introduced.
Background fluorescence at 490 and 440 was measured at the
Corneal eDitheiiai P
r
beginning of an experimental day using tissue prepared as above
m
Preparationof the epithellal expiant has been previously descrlbed in detail (10). Animals were treated in accordance with
however without dye loading. These background values were subtracted from the raw data.
the ARVO resolution on the use of animals in research. Briefly, eyes are enucleated from New Zealand albino rabbits immediately foilowing death by overdose with pentobarbltai. Corneas are
RESULTS Figure 1 shows that additlons of lactate produce rapid
extracted, and the superficial and wlng cells are removed by gentle
reversible cellular acldiflcatlon. The initial rate of acidification
abrasion with a cotton appilcator. The epithelial cells are then
Increases with Increasing [lactate]. The proton flux (mM/min)
loaded with BCECF by incubation in NaCl Ringer's contalnlng 10
across the plasma membrane can be estimated from the initial
pM BCECF-AM, 45 min., RT. One by four mm sllces are then cut
velocity of pH change (dpH/min) and the buffering capacity of the
with a razor blade; the posterior stroma
+ endothellurn Is dissected
cytosoi, 0 (mM/pH), i.e. Proton Flux = (dpH/min) X (0). Buffering
away using jeweler's forceps and discarded. The exposed stromai
capacity has been previously shown (10) to be negatlvely
surface of the remaining epitheilai piece is affixed to a round
correlated to PHI, 0 = 218 - 25.5 (pHi). Proton flux was calculated
coversllp with cyanoacrylate glue and placed in a mlcroscope-
for each [lactate], taking into account the starting pHi. Figure 2
stage perfusion chamber, 35'C. Perfusionsolutions were placed in
shows that proton flux is a simple saturating functlon of [lactate];
hanging syringes enclosed in a warming box (37'C) and bubbled
Km = 10.7 mM, Vmax = 10.2 mM/min, determined byfltting the
with room air. Solutionswere easily changed by means of an eight-
data to the Mlchaelis-Mentenequation, non-hear regression, r2 =
way valve (Hamilton, Reno, NV) and perfused via standard PE
,963. These data show saturable lactate flux, characteristic of a
tublng. Gas impermeable Saran tubing was used throughout when solutlons were bubbled wlth 100% N2 gas.
"carrier" mediated process.
MicroscoDe fluorimeter
coupled to Na, a series of experiments was performed in Na-free
The microscope fluorimeter has been previously described in
Since lactate Is known to move across some membranes solutions. Expiants were perfused In Na-free Ringer's, pHo 7.40.
detail (10). Briefly, the pHi is determined by the ratlo of
By itself thls maneuver acldlfies cells (pH1 < 6.7) due to reversal of
fluorescence emission (520-560 nm) due to excitation of BCECF at
the Na/H exchanger (10). However, pHi could then be raised by
490 and 440 nm. Excitation Is provided by a Xenon arc
substltutlng various amounts of NMDG with K + (mechanism
epifiuorescenceattachment on a Nikon Diaphot inverted
unknown, possibly due to K +/H+ exchange). The pHi was raised
microscope. Interference fliters (490 and 440) are alternately
uslng this maneuver to 6.9 - 7.1, 7.0 - 7.2, 7.1 - 7.3, and 7.3 - 7.6 by
placed in the light path by a motorized filter shuttle, 2 changes/sec
changlng to 20, 40, 70 or 105 mM KCI Ringer's. respectively (data
or 1 ratio/sec. The excitation llght is directed to the mlcroscope
not shown). When pHi reached a new steady-state, the Ringer's
objective O OX, water immersion) by a fluorescein dichroic mirror.
was changed to the same K-containing solution with the addition of
The fluorescent light from the epithelial cells Is collected by the
10 mM NMDG-lactate(all at pHo 7.40). Figure 3 shows that the
objective, filtered (520-560 nm barrier filter) and directed to a
initial velocity of acidiflcatlon was similar to that seen in Na-
photon-counting photomultiplier tube. Photomuitlpller digital
containing Rlnger's at normal resting pHi (7.2 - 7.6). At lower pH1
output is inputed to an IBM-AT compatible computer. The
(6.9-7.2) however, proton influx was reduced by as much as two-
708
Current Eye Research
5 MIN
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Figure 1: Effects of various [lactate] on pHi in continually perfused basal corneal epithelial cells. The numbers above the trace indicate the [lactate] in millimolar.
thirds. These results show that lactate transport is Na independent
10
but is inversely related to [H ]i,which most likely indicates the
T
presence of a membrane lactate-H cotransport mechanism. n
A number of agents have been identified as inhibitors of
.-It
lactate-H cotransport In various systems. The effectivity of three as of these, CHC (5.8, 9),H2DIDS (5, 8), and Mersalyl acid (6, 1 l),
\
well as LAlE were tested in corneal epithelium. Figure 4a shows a
W
control response to 10 mM lactate and a subsequent response to
3 LL
10 mM lactate following perfusion with 1 mM CHC. CHC slowed the initial acidification velocity by 36 ? 15% (+ SE, p < .05, paired ttest, n = 5). Figure 4b shows that .5mM H2DIDS produced 60
+
25% inhibition (+ SE.p < .05,n = 5) only after a minimum of 10 min.
I
E
8
6
X
4
C
0
Y
e
a
2
exposure. Figure 4c shows that 1 mM LAlE inhibited proton flux by 47 f 20 % (+ SE, p < ,051, n =5). All of these inhibitors were
H
0
reversible. By contrast, 0.5 mM Mersalyl acid produced nearly
1
1
10
20
100%inhibition after 5 min. exposure (figure 5), butwas apparently
IACFLUX.SER I
30
I
40
[Lactate)
irreversible. Initial exposure to Mersalyl or LAlE led to a small drop in pHi of about .05 units, which is consistent with their action on lactate-H efflux. CHC produced small drops or essentially no change in pHi, while initial exposure to H2DIDS gave inconsistent
Figure 2: Lactate induced proton flux as a function of added [lactate] in millimolar. Proton flux was determined from the initial rate of acidification and the intrinsic buffering capacity. See text for details.
results presumably due to other non-specific transport inhibition. Corneal epithelial hypoxia has been shown to double lactate
cell-generated H equivalents may at first be expected to decrease
Figure 7 shows that the three reversible cotransport inhibitors partially inhibit the hypoxic alkalinization. CHC, H2DIDS and LAlE
pHi, however this may be blunted by the presence of an efficient
each slowed the rate of alkalinization by 40 f 20% (k SE, p < .05,
lactate-H cotransporter and the Na/H exchanger. Figure 6 shows that perfusion of explants with either Np gas equilibrated
paired t-test, n
Ringer's (hypoxia) or air-equilibrated Ringer's containing 1 mM
with hypoxia since the agent caused pHi to drift up to 7.4 after
NaCN leads to a slow alkalinization. The average pHi increase from
approximately 10 mln exposure. This may be related to its
hypoxiaafter lOminwas0.095f .01 pHunits(+SE, n=31);the
inhibition of glycolysis (1 1) and consequent loss of ATP
production (2,3) and acidfy the stromal space (4). The increase in
range was 0 to
t 0.20.
Cyanide produced a similar change, 0.093
(+
=
8), 49
+ 14% (k SE, p < .05,n = 4) and 42 f 14%
SE,p < .05.n = 4), respectively. Mersalyl could not be tested
productlon, due to the high intracellular [lactate]. The loss of ATP
f .04 pH units, (+ SE, n =3). These surprising results will be
will inhibit the Na/K pump and collapse all Ion gradlents so pH/ wlll
discussed below.
tend to go to pHo. This is precisely what happens when corneal
709
Current Eye Research
T
7.20A
\I
B
w
El
.5 mM dlhvdro-DIDS
w
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pHi Figure 3: Lactate Induced proton flux as a function of PHI. Cells were perfused In a Na-free Rlnger’s and steady-state pH1was varied by addition of [K]. Proton flux was determinedfrom the Initialrate of acidlflcatlon due to addltlon of 10 mM lactate. Error bars lndlcate f SE; n = 3 for each pH1 Interval.
epithelial cells are perfused wlth ouabaln for 10-15 mln (data not shown).
DISCUSSION
7.20J
1 mM LAlE
5 MIN
Flgure 4: Inhibition of lactate inducedacidification by CHC, DlDS and LAIE. a: Control response to 10 mM lactate followed by perfuslonIn 1 mM CHC and subsequent addition of lactate In the presence of CHC. b: .5 mM HzDIDS had no effect shortly after exposure; break in trace lndlcates passage of 10 mln then lnhlbltory effect of H2DIDS. c: Effect of 1 mM LAlE on pHi, lnhlbltlon of lactate acldlfication,also showing reverslblllty.
Lactate coupled proton transport In highly glycolytlc cells facliltates removal of these metabolic waste products so that further production of ATP Is not Inhibitedby high cellular [lactate] (mass action) or acidlc pH (pH sensitlvlty of giycolytlc enzymes).
coupled transport (8). Both Na coupled and H coupled lactate
Thus It Is not surprlslng that the corneal epithelium, which Is
transport could exist In the corneal eplthellurn. The lactate Induced
predominantly giycoiytlc (1), possesses lactate-H cotransport.
acldlficationwould be blunted If lactate simultaneously
The evidence provided In thls study for the membrane
+
accumulated by a Na coupled mechanism. Removal of Na from the
cotransporter In corneal eplthellum Is fourfold: (1) rapid
Rlnger’s would eiimlnate Na-lactate uptake and enhance iactate-
acldlflcatlon upon lactate addltlon, (2) saturatlon of proton Influx at
H + uptake. in the corneal epithelium however, lactate Induced
hlgh [lactate], (3) slowlng or ellmlnation of acldlflcatlon by
acldlflcatlon In Na free Rlnger’s was the same as in Na containing
Inhibitors of lactate-H cotransport and (4) sensitlvlty of lactate
Ringer’s. argulng agalnst the presence of a Na coupled lactate
induced proton influx to the lnltlal membrane [ H
1 gradlent. A
recent study examlning lactate efflux from whole rabbit corneas
transporter.
also showed a sensltlvlty to mersalyl and to proton gradients (11).
The incomplete lnhlbltion of lactate Inducedacldlfication by CHC or H2DiDS at the concentratlonsused Is consistent wlth other
Simple dlffusion of lactic acid, although only about 2 pM at pH 7.40
studles (5, 8,9). CHC is a competltlve Inhlbltor, and higher
(10 mM lactate), could also account for points (1) and (4) above,
concentratlons(5 mM) are more effective (9). Unfortunately CHC
however it Is the saturation klnetlcs and lnhibltlon speclflclty whlch
soiutlons are yellow at pH 7.4. which producesan artifactual
lndlcates a carrler mechanism (5,8). The Km for lactate in corneal
fluorescence ratlo change due to selectlve absorbtlon of the 440
eplthellum was 10.7 mM. Thls Is higher than that found for
nm excltatlon light (note small upward blip when 1 mM CHC was
hepatocytes (3 mM) (9). but It Is slmllar to that seen In kidney
added In flg. 4a and 7). Attempts to conslstently compensate for
proxlmal tubule cells (10 mM) (8) and RBC’s (13.4 mM) under
thls fllter effect at higher [CHC) were unsuccessful and may be due
comparable condltions (5).
to non-speclflcblndlng of the compound to the explant. Thls Is the
Lactate uptake has also been described as occurring by Na
710
flrst report of LAlE as a lactate-H+ cotransport blocker.
Current Eye Research
.I
7.4-
Q
7.3-
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5 Min
Figure 5: inhibitory effect of mersalyl. L, indicates time perfused in 10 mM lactate Ringer’s.
Presumably it can bind to the lactate transport slte, but is not
7.501
transportable. By contrast, mersalyl (a thiol group reagent) was
Air b
apparently 100%effective in inhibiting transport in corneal epithelium (1 1), as has also been shown for Erlich ascites tumor cells (6).
7.20-1
Hypoxia stimulates lactate and proton production by respiring cells . Analysis of the stoichiometry has shown that one proton is generated for each lactate generated (12). Given the presence of a
-
lactate-H cotransporter one would predict that pHi may acidify or not change at all during hypoxia depending on the speed and
5 MIN
capacity of the cotransporter. Thus it was surprising that corneal epithelium alkalinized when made hypoxic. Cyanide perfused squid axons (13) and N2 perfused corneal endothellum (Bonanno, unpublished observations), both highly oxidative cell types, cause
Figure 6: Upper trace indicates effect of perfusion In N2 equilibrated Ringer’s. Lower trace shows the effect of 1mM NaCN. All Ringer’s solutions were at pH 7.40.
pHi to drop. These cells may have inadequate lactate-H+ cotransport capacity since they normally would be producing The pH1 change elicited by hypoxia in wiwo may be quite
relatlvely little lactate. Slowing of the hypoxia Induced alkalinization in corneal epithelium by lactate-H+ cotransport
different than that observed here. In the in witro experiments
inhibitors points out that part of the response is contributed by the
protons and lactate are rapidly washed away, however in viwo
cotransporter. If this were the only transport phenomenon
extracellular pH decreases (4) due to slower diffusion of H+ from
responsible for the hypoxia- induced pHi changes, then pH1 should
the epithelium to the strorna and then to the aqueous humor. Thus
not have increased and inhibition of cotransport should lead to a
the drop In extracellular pH in wiwo will slow both lactate-H +
net acidification. Na/H exchange, the only other presently
cotransport and Na/H exchange, which could lead to a net drop in
identified acid extruding mechanism in corneal epithelium (lo), Is
PHI.
not affected over the course of these experiments. This was tested
Previous reports have Indicated that the stoichiometry of the
by comparing recovery rates from acid loads under control and
lactate-H + cotransporter Is 1:1, i.e. electroneutral(5,E). In the
hypoxic conditions. Since ATP levels are declining under hypoxla,
corneal epithelium, lactate Induced acidification in the presence of
Na/K pump activity is depressed, ion gradients, e. g. K + , and
high K + (where it is expected that the membrane potential will be
membrane potential will be perturbed, which may lead to a slowing
depoiarized) yields the same proton influx as in normal Rlnger’s. In
of H influx via H + channels or K/H exchange.
addltion, the stromal acld load induced by hypoxia In vlvo
71 1
Current Eye Research Air
2mM CHC
CHC
+ Nitm
CORRESPONDINGAUTHOR Joseph A. Bonanno, School of Optometry, Universlty of California, Berkeley, CA 94720
5 MIN
+ Nitro
7.50,
- Nitro
DlDS
REFERENCES 1. Riley, M.V. (1969) Glucose and oxygen utillzatlon by the rabbit cornea. Exp. Eye. Res. 8, 193-200. 2. Klyce, S.D. (1981) Stromal lactate accumulatlon can account for corneal oedema osmotically following 49-64. eplthellal hypoxla In the rabblt. J. Physlol. 3. Smelser, G. and Chen, D. (1955) Physiological changes In cornea induced by contact lenses. Arch. Ophthalmol. 52,676-679. 4. Bonanno, J. A. and Poise. K.A. (1987) Corneal acldosls durlng contact lens wear: Effects of hypoxla and COP.
w,
I-
7.d
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7.35
.7.15
5 UIN 7
Figure 7: Effects of 2 mM CHC. .5 mM H2DIDS, and 1 mM LAlE on Np Induced alkailnlzatlon.
(approx. 10 mM) (4) Is very similar to the change In stromal [lactate] found in virro (2). These results are consistent wlth an electroneutral, 1:1 cotransporter In the corneal epithelium. These results have a number of Interestinglmpllcatlons. The lactate-H + cotransporter allows for either efflclent removal or uptake of lactate and H +, depending on the trans-membrane [lactate] and [ H + ] gradients. The lactate accumulation between epithelial cells that occurs durlng hypoxla could lead to local fluid accumulatlon and an Increase In light scatter, whlch Is observed during epithelial hypoxia (14). Lactate diffusion Into the stroma then leads to osmotically induced stromai swelling (2), and the pH of the extracelluiar space and stroma drops (4). Therefore It would
be expected that the stromal lactate and pH changes would cause a drop In pHI of the neighborlng corneal endothellum. Thls may then actlvate Na/H exchange In the endotheliumwhlch could ellcit a cell volume change (15) providlng a possible explanationfor the appearance of "blebs" in the acidlfied In vlvo endotheiial mosaic (16).
ACKNOWLEDGEMENTS The author gratefully acknowledges the support of Terry E. Machen In whose laboratory much of this work was performed. Thls work was also supported in part by NIH grant EY 06037 and EY 08347.
71 2
Invest. Ophthalmol. Vis. Scl. 2& 1514-1520. 5. Deutlcke, B. (1982) Monocarboxylatetransport In erythrocytes. J. Mem. Blol. ZQ,89-103. 6. Spencer, T. L. and Lehnlnger, A. L. (1976) L-Lactate transport In Erllch ascites-tumorcells. Blochem. J. 405-414. 7. Flshbeln. W. N. (1986) Lactate transporter defect: A new disease of muscle. Science, 23.4,1254-1256. 8. Slebens, A. W. and Boron W. F. (1987) Effect of electroneutral lumlnal and basolateral lactate transport on intraceliular pH In salamander proximal tubules. J. Gen. PhySlOl. 9p,799-831. 9. Edlund, C. and Halestrap, A. (1988) The kinetics of transport of lactate and pyruvate Into rat hepatocytes. Blochem. J. 249, 117-126. 10. Bonanno, J. A. and Machen, T. E. (1989) lntraceilular pH regulation In basal corneal epithelial cells measured in corneal explants: Characterlzatlonof Na/H exchange. Exp. 129-142. Eye Res. B9 11. Chen, C. H. and Chen, S. C. (1990) Lactate transport and glycolytlc actlvlty in the freshly isolated rabblt cornea. Arch. Blochem. Blophys. 226.70-76. 12. Hochachka, P. and Mommsen, T. (1983) Protons and anaeroblosls. Science, U ,1391-1397. 13. Boron, W. F. and De Weer, P. (1976) intracellular pH transients In squid giant axons caused by Cop, NH3 and metabollc Inhlbitors. J. Gen. Physlol. u,91-112. 14. Lambert, S. and Kiyce, S. D. (1981) Theorlglns of Sattler's veil. Am. J. Ophthalmol. 91,51-55. 15. Larsen, M. and Sprlng, K. R. (1987) Volume reguiatlon In epithelia. Cur.Top. Mem. Transport 105-123. 16. Holden, B., Williams, L. and Zantos, S. (1985) Etiology of transient endothellai changes in the human cornea. Invest. Ophthalmol.Vis. Sci. 23, 522-527.
a,
Volume 9 nunibcr 7 1990
Lactate-proton cotransport in rabbit corneal epithelium
Joseph A.Bonanno
Morton D.Sarver Center for Cornea and Contact Lens Research, University of California, School o f Optometry, Berkeley, C A 94720, USA
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ABSTRACT The presence of the membrane transport mechanism, lactateH + cotransport, was tested in explants of rabbit corneal epithelium. Basal corneal epithelial cells were loaded with the pH sensitive fluorescent dye BCECF. lntracelluiar pH (pHi) was measured by ratioing the fluorescence emission output following excitation at 490 and 440 nm. Perfusion of explants in lactatecontaining Ringer’s, pH 7.40, produced a reversible decrease in pHi. The lactate induced proton influx (mM/min) followed saturating kinetlcs, Km = 10.7 mM lactate, Vmax = 10.2 mM/min. Proton influx following addition of 10 mM lactate was inhibited 36, 60 and 47% by pre-perfusion in 1 mM CHC (cyanohydroxycinammic acid), 500 pM H2DIDS (4,4’-diisothiocyanato -
be facilitated by a membrane carrier mechanism involving the lactate- anion (5,6). A lactate-H + cotransport mechanism has been described in erythrocytes (5),Ehrlich ascites tumor cells (6) and skeletal muscle (7) all of which are highly glycolytic. In addition, the cotransporter has been descrlbed for the serosal side of the kidney proximal tubule (8)and for hepatocytes (9) as a mechanism to help reabsorb lactate into the blood and into the liver for gluconeogenesis, respectively. This study examines the rabbit corneal epithelium for the presence of the lactate-H + cotransporter by measuring: (1)
dihydrostllbene- 2,2’-disulfonic acid) and 1 mM LAlE (lactic acid
changes in intracellular pH due to lactate addition, (2) lnhlbition of
isobutylester), respectively. These inhibitors of lactate-H + cotransport were reversible. Mersalyl acid (500 pM) inhibited proton flux from 10 mM lactate addition by nearly 1OO%, but was Irreversible. Stimulation of lactate production by perfusion in Np
lactate-Induced changes in pHi by known lactate-H + cotransporter inhibitors and (3) pHi changes due to stimulation of glycolysis (i.e. lactate production) by hypoxia and the effects of cotransporter
equilibrated Ringer’s (hypoxia) or the addition of 1 rnM NaCN led to a slow alkalinization (0.1 pH unit in 10 min). Pre-perfusionwith the reversible inhibitors slowed the hypoxic alkalinization by
Inhibitors on hypoxia-induced pHi changes.
approximately 40%. It is concluded that lactate-H + cotransport is present in the corneal epithelium and that it contributes to pHi regulatlon during hypoxia.
Chemicals and solutions
INTRODUCTION
BCECF-AM [2’,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxy-methylester] and H2-DIDS [ 4,4’-dlisothiocyanato-
MATERIALS AND METHODS
dihydrostilbene-2,2’-disulfonicacid] were obtained from Molecular
In the presence of air the corneal epithelium metabolizes approximately 65 - 85% of the glucose it consumes to lactate (1 -3).
Probes (Eugene, OR). All other chemicals were obtalned from
The reason for the high glycolytic activity is not known, however
Sigma (St. Louis, MO). Stock solutions of BCECF-AM(10 mM In
the relative paucity of mitochondria in the corneal epithelium may
DMSO) and nigericln (1 0 mM in absolute ethanol) were stored at
be a strategy to keep light scattering to a minimum. When the
-2O’C. The basic NaCl Ringer’s contained in mM: 100 NaCI, 10
tissue is made hypoxic, as happens during contact lens wear, lactate production can double (2, 3) and an equivalent amount of
HEPES (N-2-hydroxyethyl-piperazlne-N’-2-ethanesulfonlc acid), 2.4 K2HP04, 1.2 Ca-gluconate, 0.6 MgC12,26 glucose, 40.5Na-
protons are generated causing stromai acidification (4). Efficient
gluconate and 5 NaOH to adjust pH to 7.40, osmolarlty
removal of lactate and protons from the epithelial cells is imperative
mOSM/Kg. Lactate Ringer’s (pH 7.40) was prepared by
If glycolysis is not to be inhibited in either the normoxic or hypoxic
substituting some of the Na-gluconate with an equlmolar amount of
condition.
300 k 5
Na-lactate (equimolar L-( +)-Lactic acid and NaOH). Lactate
At physiologic pH. the lactate-/lactic acid ratio Is greater than 1OOO:l (pK lactic acid
=
=
solutions were made fresh on the day of the experiment. Sodiumfree Ringer’s (pH 7.40) was prepared by omitting Na-gluconate,
3.86).Whereas lactic acid is uncharged
and permeable to the plasma membrane, total lactate transport can ~
~
substitution of NaCl with NMDG-CI (N-methyl-D-glucamine base
~~
Received on May 10. 1990; accepted on June 28, 1990 ~-
@ Oxford University Press
707
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Current Eye Research dissolved in equlmolar HCL), pH adjusted with NMDG base and
motorized filter shuttle, data sampling and storage are controlled
adjusting osmoiarity to 300 with sucrose. Calibration Ringer's was
by UMANS software (C. Regan, Urbana, IL).
prepared by omlttlng NaCi and Na-giuconate,adding 105 KCI and adjusting osmoiarity to 300 with sucrose. All drugs, H2DIDS, CHC
Calibration
(cyano-4-hydroxyclnnamic acid), LAiE (lactlc acid isobutylester)
intraceiiuiar pH (pHi) Is set equal to solution pH (pHo) by perfusion in Callbration-Ringer's ([KO]approximately equal to [Ki])
and Mersalyi acid ([3-hydroxymercuri 2'-methoxypropyl)
contalnlng 50 pM nigericin (K+/H+ ionophore) (10). Solution pH
carbamoyi] phenoxyacetic acid, were dissolved directly in Rlnger's
Is then varied by adding small amounts of HCI or NMDG base, and
on the day of the experiment. Bicarbonate-freeRinger's was used
the steady-state fluorescence ratio is recorded. Three calibratlon
throughout to minimize lntraceliuiar buffering In order to produce
polnts are obtained for each preparation, and a linear regression is
greater and faster pHi changes when small amounts of lactate were
performed to obtain a caiibratlon curve for the experimental data.
introduced.
Background fluorescence at 490 and 440 was measured at the
Corneal eDitheiiai P
r
beginning of an experimental day using tissue prepared as above
m
Preparationof the epithellal expiant has been previously descrlbed in detail (10). Animals were treated in accordance with
however without dye loading. These background values were subtracted from the raw data.
the ARVO resolution on the use of animals in research. Briefly, eyes are enucleated from New Zealand albino rabbits immediately foilowing death by overdose with pentobarbltai. Corneas are
RESULTS Figure 1 shows that additlons of lactate produce rapid
extracted, and the superficial and wlng cells are removed by gentle
reversible cellular acldiflcatlon. The initial rate of acidification
abrasion with a cotton appilcator. The epithelial cells are then
Increases with Increasing [lactate]. The proton flux (mM/min)
loaded with BCECF by incubation in NaCl Ringer's contalnlng 10
across the plasma membrane can be estimated from the initial
pM BCECF-AM, 45 min., RT. One by four mm sllces are then cut
velocity of pH change (dpH/min) and the buffering capacity of the
with a razor blade; the posterior stroma
+ endothellurn Is dissected
cytosoi, 0 (mM/pH), i.e. Proton Flux = (dpH/min) X (0). Buffering
away using jeweler's forceps and discarded. The exposed stromai
capacity has been previously shown (10) to be negatlvely
surface of the remaining epitheilai piece is affixed to a round
correlated to PHI, 0 = 218 - 25.5 (pHi). Proton flux was calculated
coversllp with cyanoacrylate glue and placed in a mlcroscope-
for each [lactate], taking into account the starting pHi. Figure 2
stage perfusion chamber, 35'C. Perfusionsolutions were placed in
shows that proton flux is a simple saturating functlon of [lactate];
hanging syringes enclosed in a warming box (37'C) and bubbled
Km = 10.7 mM, Vmax = 10.2 mM/min, determined byfltting the
with room air. Solutionswere easily changed by means of an eight-
data to the Mlchaelis-Mentenequation, non-hear regression, r2 =
way valve (Hamilton, Reno, NV) and perfused via standard PE
,963. These data show saturable lactate flux, characteristic of a
tublng. Gas impermeable Saran tubing was used throughout when solutlons were bubbled wlth 100% N2 gas.
"carrier" mediated process.
MicroscoDe fluorimeter
coupled to Na, a series of experiments was performed in Na-free
The microscope fluorimeter has been previously described in
Since lactate Is known to move across some membranes solutions. Expiants were perfused In Na-free Ringer's, pHo 7.40.
detail (10). Briefly, the pHi is determined by the ratlo of
By itself thls maneuver acldlfies cells (pH1 < 6.7) due to reversal of
fluorescence emission (520-560 nm) due to excitation of BCECF at
the Na/H exchanger (10). However, pHi could then be raised by
490 and 440 nm. Excitation Is provided by a Xenon arc
substltutlng various amounts of NMDG with K + (mechanism
epifiuorescenceattachment on a Nikon Diaphot inverted
unknown, possibly due to K +/H+ exchange). The pHi was raised
microscope. Interference fliters (490 and 440) are alternately
uslng this maneuver to 6.9 - 7.1, 7.0 - 7.2, 7.1 - 7.3, and 7.3 - 7.6 by
placed in the light path by a motorized filter shuttle, 2 changes/sec
changlng to 20, 40, 70 or 105 mM KCI Ringer's. respectively (data
or 1 ratio/sec. The excitation llght is directed to the mlcroscope
not shown). When pHi reached a new steady-state, the Ringer's
objective O OX, water immersion) by a fluorescein dichroic mirror.
was changed to the same K-containing solution with the addition of
The fluorescent light from the epithelial cells Is collected by the
10 mM NMDG-lactate(all at pHo 7.40). Figure 3 shows that the
objective, filtered (520-560 nm barrier filter) and directed to a
initial velocity of acidiflcatlon was similar to that seen in Na-
photon-counting photomultiplier tube. Photomuitlpller digital
containing Rlnger's at normal resting pHi (7.2 - 7.6). At lower pH1
output is inputed to an IBM-AT compatible computer. The
(6.9-7.2) however, proton influx was reduced by as much as two-
708
Current Eye Research
5 MIN
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Figure 1: Effects of various [lactate] on pHi in continually perfused basal corneal epithelial cells. The numbers above the trace indicate the [lactate] in millimolar.
thirds. These results show that lactate transport is Na independent
10
but is inversely related to [H ]i,which most likely indicates the
T
presence of a membrane lactate-H cotransport mechanism. n
A number of agents have been identified as inhibitors of
.-It
lactate-H cotransport In various systems. The effectivity of three as of these, CHC (5.8, 9),H2DIDS (5, 8), and Mersalyl acid (6, 1 l),
\
well as LAlE were tested in corneal epithelium. Figure 4a shows a
W
control response to 10 mM lactate and a subsequent response to
3 LL
10 mM lactate following perfusion with 1 mM CHC. CHC slowed the initial acidification velocity by 36 ? 15% (+ SE, p < .05, paired ttest, n = 5). Figure 4b shows that .5mM H2DIDS produced 60
+
25% inhibition (+ SE.p < .05,n = 5) only after a minimum of 10 min.
I
E
8
6
X
4
C
0
Y
e
a
2
exposure. Figure 4c shows that 1 mM LAlE inhibited proton flux by 47 f 20 % (+ SE, p < ,051, n =5). All of these inhibitors were
H
0
reversible. By contrast, 0.5 mM Mersalyl acid produced nearly
1
1
10
20
100%inhibition after 5 min. exposure (figure 5), butwas apparently
IACFLUX.SER I
30
I
40
[Lactate)
irreversible. Initial exposure to Mersalyl or LAlE led to a small drop in pHi of about .05 units, which is consistent with their action on lactate-H efflux. CHC produced small drops or essentially no change in pHi, while initial exposure to H2DIDS gave inconsistent
Figure 2: Lactate induced proton flux as a function of added [lactate] in millimolar. Proton flux was determined from the initial rate of acidification and the intrinsic buffering capacity. See text for details.
results presumably due to other non-specific transport inhibition. Corneal epithelial hypoxia has been shown to double lactate
cell-generated H equivalents may at first be expected to decrease
Figure 7 shows that the three reversible cotransport inhibitors partially inhibit the hypoxic alkalinization. CHC, H2DIDS and LAlE
pHi, however this may be blunted by the presence of an efficient
each slowed the rate of alkalinization by 40 f 20% (k SE, p < .05,
lactate-H cotransporter and the Na/H exchanger. Figure 6 shows that perfusion of explants with either Np gas equilibrated
paired t-test, n
Ringer's (hypoxia) or air-equilibrated Ringer's containing 1 mM
with hypoxia since the agent caused pHi to drift up to 7.4 after
NaCN leads to a slow alkalinization. The average pHi increase from
approximately 10 mln exposure. This may be related to its
hypoxiaafter lOminwas0.095f .01 pHunits(+SE, n=31);the
inhibition of glycolysis (1 1) and consequent loss of ATP
production (2,3) and acidfy the stromal space (4). The increase in
range was 0 to
t 0.20.
Cyanide produced a similar change, 0.093
(+
=
8), 49
+ 14% (k SE, p < .05,n = 4) and 42 f 14%
SE,p < .05.n = 4), respectively. Mersalyl could not be tested
productlon, due to the high intracellular [lactate]. The loss of ATP
f .04 pH units, (+ SE, n =3). These surprising results will be
will inhibit the Na/K pump and collapse all Ion gradlents so pH/ wlll
discussed below.
tend to go to pHo. This is precisely what happens when corneal
709
Current Eye Research
T
7.20A
\I
B
w
El
.5 mM dlhvdro-DIDS
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pHi Figure 3: Lactate Induced proton flux as a function of PHI. Cells were perfused In a Na-free Rlnger’s and steady-state pH1was varied by addition of [K]. Proton flux was determinedfrom the Initialrate of acidlflcatlon due to addltlon of 10 mM lactate. Error bars lndlcate f SE; n = 3 for each pH1 Interval.
epithelial cells are perfused wlth ouabaln for 10-15 mln (data not shown).
DISCUSSION
7.20J
1 mM LAlE
5 MIN
Flgure 4: Inhibition of lactate inducedacidification by CHC, DlDS and LAIE. a: Control response to 10 mM lactate followed by perfuslonIn 1 mM CHC and subsequent addition of lactate In the presence of CHC. b: .5 mM HzDIDS had no effect shortly after exposure; break in trace lndlcates passage of 10 mln then lnhlbltory effect of H2DIDS. c: Effect of 1 mM LAlE on pHi, lnhlbltlon of lactate acldlfication,also showing reverslblllty.
Lactate coupled proton transport In highly glycolytlc cells facliltates removal of these metabolic waste products so that further production of ATP Is not Inhibitedby high cellular [lactate] (mass action) or acidlc pH (pH sensitlvlty of giycolytlc enzymes).
coupled transport (8). Both Na coupled and H coupled lactate
Thus It Is not surprlslng that the corneal epithelium, which Is
transport could exist In the corneal eplthellurn. The lactate Induced
predominantly giycoiytlc (1), possesses lactate-H cotransport.
acldlficationwould be blunted If lactate simultaneously
The evidence provided In thls study for the membrane
+
accumulated by a Na coupled mechanism. Removal of Na from the
cotransporter In corneal eplthellum Is fourfold: (1) rapid
Rlnger’s would eiimlnate Na-lactate uptake and enhance iactate-
acldlflcatlon upon lactate addltlon, (2) saturatlon of proton Influx at
H + uptake. in the corneal epithelium however, lactate Induced
hlgh [lactate], (3) slowlng or ellmlnation of acldlflcatlon by
acldlflcatlon In Na free Rlnger’s was the same as in Na containing
Inhibitors of lactate-H cotransport and (4) sensitlvlty of lactate
Ringer’s. argulng agalnst the presence of a Na coupled lactate
induced proton influx to the lnltlal membrane [ H
1 gradlent. A
recent study examlning lactate efflux from whole rabbit corneas
transporter.
also showed a sensltlvlty to mersalyl and to proton gradients (11).
The incomplete lnhlbltion of lactate Inducedacldlfication by CHC or H2DiDS at the concentratlonsused Is consistent wlth other
Simple dlffusion of lactic acid, although only about 2 pM at pH 7.40
studles (5, 8,9). CHC is a competltlve Inhlbltor, and higher
(10 mM lactate), could also account for points (1) and (4) above,
concentratlons(5 mM) are more effective (9). Unfortunately CHC
however it Is the saturation klnetlcs and lnhibltlon speclflclty whlch
soiutlons are yellow at pH 7.4. which producesan artifactual
lndlcates a carrler mechanism (5,8). The Km for lactate in corneal
fluorescence ratlo change due to selectlve absorbtlon of the 440
eplthellum was 10.7 mM. Thls Is higher than that found for
nm excltatlon light (note small upward blip when 1 mM CHC was
hepatocytes (3 mM) (9). but It Is slmllar to that seen In kidney
added In flg. 4a and 7). Attempts to conslstently compensate for
proxlmal tubule cells (10 mM) (8) and RBC’s (13.4 mM) under
thls fllter effect at higher [CHC) were unsuccessful and may be due
comparable condltions (5).
to non-speclflcblndlng of the compound to the explant. Thls Is the
Lactate uptake has also been described as occurring by Na
710
flrst report of LAlE as a lactate-H+ cotransport blocker.
Current Eye Research
.I
7.4-
Q
7.3-
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5 Min
Figure 5: inhibitory effect of mersalyl. L, indicates time perfused in 10 mM lactate Ringer’s.
Presumably it can bind to the lactate transport slte, but is not
7.501
transportable. By contrast, mersalyl (a thiol group reagent) was
Air b
apparently 100%effective in inhibiting transport in corneal epithelium (1 1), as has also been shown for Erlich ascites tumor cells (6).
7.20-1
Hypoxia stimulates lactate and proton production by respiring cells . Analysis of the stoichiometry has shown that one proton is generated for each lactate generated (12). Given the presence of a
-
lactate-H cotransporter one would predict that pHi may acidify or not change at all during hypoxia depending on the speed and
5 MIN
capacity of the cotransporter. Thus it was surprising that corneal epithelium alkalinized when made hypoxic. Cyanide perfused squid axons (13) and N2 perfused corneal endothellum (Bonanno, unpublished observations), both highly oxidative cell types, cause
Figure 6: Upper trace indicates effect of perfusion In N2 equilibrated Ringer’s. Lower trace shows the effect of 1mM NaCN. All Ringer’s solutions were at pH 7.40.
pHi to drop. These cells may have inadequate lactate-H+ cotransport capacity since they normally would be producing The pH1 change elicited by hypoxia in wiwo may be quite
relatlvely little lactate. Slowing of the hypoxia Induced alkalinization in corneal epithelium by lactate-H+ cotransport
different than that observed here. In the in witro experiments
inhibitors points out that part of the response is contributed by the
protons and lactate are rapidly washed away, however in viwo
cotransporter. If this were the only transport phenomenon
extracellular pH decreases (4) due to slower diffusion of H+ from
responsible for the hypoxia- induced pHi changes, then pH1 should
the epithelium to the strorna and then to the aqueous humor. Thus
not have increased and inhibition of cotransport should lead to a
the drop In extracellular pH in wiwo will slow both lactate-H +
net acidification. Na/H exchange, the only other presently
cotransport and Na/H exchange, which could lead to a net drop in
identified acid extruding mechanism in corneal epithelium (lo), Is
PHI.
not affected over the course of these experiments. This was tested
Previous reports have Indicated that the stoichiometry of the
by comparing recovery rates from acid loads under control and
lactate-H + cotransporter Is 1:1, i.e. electroneutral(5,E). In the
hypoxic conditions. Since ATP levels are declining under hypoxla,
corneal epithelium, lactate Induced acidification in the presence of
Na/K pump activity is depressed, ion gradients, e. g. K + , and
high K + (where it is expected that the membrane potential will be
membrane potential will be perturbed, which may lead to a slowing
depoiarized) yields the same proton influx as in normal Rlnger’s. In
of H influx via H + channels or K/H exchange.
addltion, the stromal acld load induced by hypoxia In vlvo
71 1
Current Eye Research Air
2mM CHC
CHC
+ Nitm
CORRESPONDINGAUTHOR Joseph A. Bonanno, School of Optometry, Universlty of California, Berkeley, CA 94720
5 MIN
+ Nitro
7.50,
- Nitro
DlDS
REFERENCES 1. Riley, M.V. (1969) Glucose and oxygen utillzatlon by the rabbit cornea. Exp. Eye. Res. 8, 193-200. 2. Klyce, S.D. (1981) Stromal lactate accumulatlon can account for corneal oedema osmotically following 49-64. eplthellal hypoxla In the rabblt. J. Physlol. 3. Smelser, G. and Chen, D. (1955) Physiological changes In cornea induced by contact lenses. Arch. Ophthalmol. 52,676-679. 4. Bonanno, J. A. and Poise. K.A. (1987) Corneal acldosls durlng contact lens wear: Effects of hypoxla and COP.
w,
I-
7.d
Curr Eye Res Downloaded from informahealthcare.com by Nyu Medical Center on 12/13/14 For personal use only.
7.35
.7.15
5 UIN 7
Figure 7: Effects of 2 mM CHC. .5 mM H2DIDS, and 1 mM LAlE on Np Induced alkailnlzatlon.
(approx. 10 mM) (4) Is very similar to the change In stromal [lactate] found in virro (2). These results are consistent wlth an electroneutral, 1:1 cotransporter In the corneal epithelium. These results have a number of Interestinglmpllcatlons. The lactate-H + cotransporter allows for either efflclent removal or uptake of lactate and H +, depending on the trans-membrane [lactate] and [ H + ] gradients. The lactate accumulation between epithelial cells that occurs durlng hypoxla could lead to local fluid accumulatlon and an Increase In light scatter, whlch Is observed during epithelial hypoxia (14). Lactate diffusion Into the stroma then leads to osmotically induced stromai swelling (2), and the pH of the extracelluiar space and stroma drops (4). Therefore It would
be expected that the stromal lactate and pH changes would cause a drop In pHI of the neighborlng corneal endothellum. Thls may then actlvate Na/H exchange In the endotheliumwhlch could ellcit a cell volume change (15) providlng a possible explanationfor the appearance of "blebs" in the acidlfied In vlvo endotheiial mosaic (16).
ACKNOWLEDGEMENTS The author gratefully acknowledges the support of Terry E. Machen In whose laboratory much of this work was performed. Thls work was also supported in part by NIH grant EY 06037 and EY 08347.
71 2
Invest. Ophthalmol. Vis. Scl. 2& 1514-1520. 5. Deutlcke, B. (1982) Monocarboxylatetransport In erythrocytes. J. Mem. Blol. ZQ,89-103. 6. Spencer, T. L. and Lehnlnger, A. L. (1976) L-Lactate transport In Erllch ascites-tumorcells. Blochem. J. 405-414. 7. Flshbeln. W. N. (1986) Lactate transporter defect: A new disease of muscle. Science, 23.4,1254-1256. 8. Slebens, A. W. and Boron W. F. (1987) Effect of electroneutral lumlnal and basolateral lactate transport on intraceliular pH In salamander proximal tubules. J. Gen. PhySlOl. 9p,799-831. 9. Edlund, C. and Halestrap, A. (1988) The kinetics of transport of lactate and pyruvate Into rat hepatocytes. Blochem. J. 249, 117-126. 10. Bonanno, J. A. and Machen, T. E. (1989) lntraceilular pH regulation In basal corneal epithelial cells measured in corneal explants: Characterlzatlonof Na/H exchange. Exp. 129-142. Eye Res. B9 11. Chen, C. H. and Chen, S. C. (1990) Lactate transport and glycolytlc actlvlty in the freshly isolated rabblt cornea. Arch. Blochem. Blophys. 226.70-76. 12. Hochachka, P. and Mommsen, T. (1983) Protons and anaeroblosls. Science, U ,1391-1397. 13. Boron, W. F. and De Weer, P. (1976) intracellular pH transients In squid giant axons caused by Cop, NH3 and metabollc Inhlbitors. J. Gen. Physlol. u,91-112. 14. Lambert, S. and Kiyce, S. D. (1981) Theorlglns of Sattler's veil. Am. J. Ophthalmol. 91,51-55. 15. Larsen, M. and Sprlng, K. R. (1987) Volume reguiatlon In epithelia. Cur.Top. Mem. Transport 105-123. 16. Holden, B., Williams, L. and Zantos, S. (1985) Etiology of transient endothellai changes in the human cornea. Invest. Ophthalmol.Vis. Sci. 23, 522-527.
a,
