Metabolic requirements for anaerobic active Cl and Na transport in the bullfrog cornea PETER S. REINACH, HOWARD F. SCHOEN, AND OSCAR A. CANDIA Departments of Ophthalmology and Physiology, l&u& Sinai School of Medicine, City Uniuersity of New York, New York, New York 10029

REINACH, PETER S., HOWARD F. SCHOEN, AND OSCAR A. CANDIA. Metabolic requirements for anaerobic active CL and Nu transport in the bullfrog cornea. Am. J. Physiol. 236(5): C268-C276, 1979 or Am. J. Physiol.: Cell Physiol. 5(3): C268C276, 1979.-In the bullfrog cornea, the relationships between the rates of aerobic and anaerobic glycolysis and active Cl and Na transport were studied. In NaCl Ringer (glucose-free), the short-circuit current (SCC) declined much more slowly under aerobic than under anaerobic conditions. The aerobic lactate effluxes in glucose-free and glucose-rich NaCl Ringer were 0.08 and 0.23 pmol/h cm’, respectively. The transition to anoxia caused these values to increase significantly and was accompanied by depletion of endogenous glycogen in glucose-free Ringer. In Na2S04 Ringer, amphotericin B (lo-” M) stimulation of the aerobic SCC was not dependent on the presence of glucose but under anoxia, SCC stimulation required glucose. In Na&04 (glucose-rich) Ringer, amphotericin B stimulated the aerobic lactate efflux from 0.26 to 0.36 pmol/h*cm2 and anoxia increased it to 0.55 pmol/h* cm2. In NaCl Ringer, the addition of either 0.5 mM adenosine or 1 mM ATP with 26 mM glucose restored the anaerobic-inhibited SCC and lactate efflux of glucose-depleted corneas. The results show that the reactions of glycolysis are a sufficient energy source for supporting active Na and Cl transport. l

Rana catesbeiana; energetics

adenosine;

adenosine

triphosphate;

glucose;

thatinthebullfrogcornea there is a coupling between respiration and active transepithelial transport of sodium and chloride: active Cl transport inhibitors and ouabain reduced oxygen consumption (15). Although respiration is reduced by active ion transport inhibitors, it has been shown in the bullfrog cornea that anoxia results in only partial inhibition of the rates of active transepithelial transport of sodium and chloride (22). The fact that active Na and Cl transport persists under anoxia suggests that there is a significant amount of glycolytic activity in the bullfrog cornea. In the rabbit cornea, a high rate of glycolysis appears to exist since 84% of the glucose metabolized is converted to lactate (16). Therefore, it is of interest to quantitate glycolysis and its coupling with active transepithelial Cl and Na transport in the bullfrog cornea. We report here on the results of experiments in the bullfrog cornea wherein the relationship between aerobic and anaerobic glycolysis and active transepithelial Cl and Na transport was studied. The rates of glycolysis and active Cl and Na transport were determined by simultaWEHAVEPREVIOUSLYSHOWN

C268

neously measuring the lactate efflux and short-circuit current, Some of the requirements for the support of glycolysis and active Cl and Na transport were studied. This was done by measuring the effects of the addition of substrates to substrate-free bathing solutions. An estimation of the contributory roles of glycolysis and respiration for the support of active Cl and Na transport was attempted with the use of various metabolic inhibitors, The endogenous glucose reserves of aerobic and anaerobic corneas were also measured. MATERIALS

AND METHODS

Corneas were dissected from bullfrogs, Rana catesbeiana, and mounted as a membrane between two halves of a Ussing-Zerahn type chamber fabricated from Lucite following a procedure previously described (4). Each side of the chamber was filled with 5 ml of a Ringer solution containing (in mM): Na+, 104; K’, 2.5; Ca”, 1.0; Mg2+, 1.2; Cl, 74.5; HCOa-, 25; Sod’-, 1.8; HP0d2-, 2.9; gluconate, 2.0; and glucose, 26. The total osmolarity was 241 mM. The solutions were bubbled either with air or nitrogen and the pH, measured with a glass electrode, was 8.6. The Ringer either contained glucose as a substrate or was substrate-free and the difference in osmdlarity was compensated for with sucrose. In experiments where the bathing solution contained 20 mM pyruvate, sucrose was omitted from the Ringer solution. In the experiments where a Cl-free solution was used, each mole of Cl was replaced by 0.5 mol of sulfate and the difference in osmolarity was compensated for with sucrose, Anaerobiosis was provided by equilibrating the Ringer solution with nitrogen (Union Carbide, New York) and with the addition of neutralized sodium cyanide at a final concentration of 2 mM. The following substrates and drugs were used: glucose (Fisher Scientific, New York); adenosine, ATP, pyruvate, iodoacetate, and iodoacetamide (Sigma Chemical, St, Louis, MO); and amphotericin B (Squibb, New York). Substrate additions were made simultaneously to both bathing solutions to avoid any osmotic effects. Transcorneal potential difference (PD) was monitored and current was sent across the cornea by means of agarRinger-filled polyethylene tubing. PD bridges were kept close to the cornea1 surfaces so that the solution resistance was negligible. The PD bridges were connected to the measuring and recording equipment through calomel cells (Keitheley ZOOBmillivoltmeters and Heath EU 20B

0363-6143/79/OOOO-OOOO$U1.25

Copyright

0 1979 the American

Physiological

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Society

GLYCOLYSIS

AND IONIC

TRANSPORT

IN

C269

CORNEA

recorders). Short-circuit current (KC) was obtained and measured with an automatic voltage clamp apparatus. Resistance was determined by periodically measuring the additional current necessary to further depolarize by 22.5 or 9.25 mV the PD from the short-circuited condition. Once the SCC and PD had stabilized lactate production by the cornea was measured by removing 3-ml aliquots from the bathing solutions and then replacing with an equal volume every 30 min. In preliminary experiments, it was determined that no measurable amounts of lactate appeared in the epithelial bathing solution. Therefore, in d subsequent experiments only the lactate efflux into the endothelial bathing solution was measured. Lactate was determined fluorometrically with beef heart lactate dehydrogenase (Sigma Chemical) (2). Either O.l- or 0.2-ml aliquots were used for the assay. To determine the lactate content of the cornea, the tissue was removed from the chamber and extracted for 12 h in 3.7 ml of HC104 (final concentration 8%) and neutralized to pH 7 with 1.3 ml of a 2.5 M K&OS. The solution was centrifuged at 1,000 g for 15 min and the resulting supernatant was lyophilized. The lyophilized fraction was dissolved in 1 ml of distilled water and 0.1 ml was taken for the lactate assay. Known amounts of lactate were added to the tissue extract and their recovery was measured. The lactate recovery was 95%, indicating that the extraction was complete enough to detect approximately 95% of the corneal lactate. Samples for obtaining the lactate standard curve were treated similarly in the absence of tissue. The amount of lactate remaining in the cornea after it had been incubated for 2 h in NaCl Ringer solution containing 26 mM glucose was measured to determine whether any substantial amounts of lactate were retained by the cornea. The extracts of 10 corneas contained 41.08 t 12.38 nmol; this amount was about l,OOO-fold less than the amount of lactate appearing in the bathing solution and indicates that there is hardly any accumulation of lactate prior to its appearance in the bathing solution. Oxygen consumption was measured with Yellow Springs polarographic electrodes (model YSI 53) in a stirrer bath assembly and the measuring vessels contained 5 ml of a NaCl Ringer solution (Tris buffer, pH 8.6). The compositioti and osmolarity of the NaCl Ringer has been previously described (7). The decline in oxygen tension at 25°C was recorded in the range of 9O-100% saturation and the values are expressed in microliters of oxygen per gram dry weight per minute at standard temperature and pressure. Cl fluxes were measured according to a previously described method (7). In those experiments where the epithelial-to-endothelial side Cl fluxes were measured, the sample volume was 3.2 ml instead of 3 ml. Three milliliters were used to measure 36C1activity appearing in the unlabeled side of the Ussing chamber and 0.2 ml was used in the lactate assay. The glycogen content of the cornea was measured using a method described by Riley (16). The corneas were dissected and either immediately placed in cold 0.6 N perchloric acid or incubated in glucose-free NaCl Ringer for 120 min and every 30 min aliquots of the bathing solution were assayed for lactate. After extrac-

tion in 0.6 N perchloric acid at 4°C for 16 h, the cornea was hydrolyzed in 1 N HCl for 2 h at 100°C. After neutralization of the two extracts and treatment of the perchlorate extract with amyloglucosidase, the glucose contents of the extracts were estimated by the hexokinase method. RESULTS

Effects of Anaerobiosis and Glucose on Active Cl Transport In NaCl Ringer solution 95% of the SCC is due to active transepithelial Cl transport (21). Corneas incubated in aerated NaCl Ringer solution maintain their active Cl transport for extended periods of time. Table 1A shows that after 6 h the SCC, PD, and lactate efflux changed gradually. In contrast, the switch to anaerobic conditions had striking inhibitory effects on the electrical parameters and an acceleratory effect on lactate efflux (Table 1B). Regardless of whether anoxia was provided by Nz or by 2 mM NaCN + Nz, the effects on the electrical parameters and lactate efflux were equivalent. Thus, 90 min after switching from air to nitrogen the SCC and PD declined by 82% and 7996,respectively. The lactate efflux during the initial 30-min period after the transition to nitrogen nearly tripled and was almost twice as large as the aerobic value 90 min after the switch to anoxia. The fact that the electrical parameters were maintained under aerobic conditions suggests that there -is an endogenous reserve of substrate. Accordingly, the glucose contents of three different sets of eight corneas were measured under three different conditions: 1) immediately after dissection from the animal; 2) after incubation in the glucose-free NaCl Ringer solution for 2 h; and 3) 2 h after incubation in Nx. The results are summarized in Table 2 and indicate that there was no difference between conditions 1 and 2 but in the anoxic corneas the glucose content was appreciably smaller than in the aerobic corneas. The lactate efflux from the latter group of corneas was measured as well. The total lactate efflux for 2 h in the anaerobic corneas was 0.68 pmol and 1 mol of glucose is metabolized to 2 mol of lactate. If no other substrate besides glucose is metabolized to lactate, TABLE 1. Effects of absence of exogenous glucose on aerobic and anaerobic bullfrog corneas bathed in NuCl Ringer A: Air* 30 min in air

360 min in air

11.78 t 1.61 21.5 AI 4.3

SSC, PA/cm2 PD, mV R, Mb cm2. LE, pmol/h cm2

7.53 * 14.0 k 2.21. t 0.06 k

1.79 t 0.24

0.10 t 0.02

l

0.94 2,8 0.35 0.01

B: Air + Nzt 30 min in air

SCC,

PA/cm2 PD, mV R, k%cm2 LE, pmol/h . cm2 SCC, short-circuit LE, lactate efflux. t Values are means

12.34 -+ 0.94

18,5 k 1.8 1.65 rk 0.09 0.07

* 0.01

30 min in Nt

7.80 6.8 1.74 0.20

current; PD, potential * Values are means -+ SE of 14 experiments.

t, 0,65 * 1.1

* 0.17 AI 0.02

90 min in N2

2.16 3.9 1.87 0.13

AI 0.36 t 1.2 t 0.30 t, 0.02

difference; R, resistance; t SE of 8 experiments.

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c270

REINACH,

-I

60

90

120

150

180 Minutes

210

240

270

300

FIG. 1. Effects of anoxia and 2 mM iodoacetamide on Cl-originated SCC and lactate efflux of isolated bullfrog cornea in glucose-rich Ringer.

TABLE

aerobic

2. Endugenous

glucose reserues of bullfrog curneas . -

and anaerobic

Air

Glucosyl units, pmol/cm2 Values

are means

2 h incubation

2 h incubation

0.38

0.40

0.16

0.06

CANDIA

and Glucose

The coupling between glycolysis and active transepithelial Na transport was studied in corneas bathed in NazSOd (Cl-free) Ringer solution containing 26 mM glucose. Inasmuch as the SCC of these corneas is less than 1 pA/cm2, their SCC was stimulated with amphotericin B at a final concentration of 10m5 M in the tear-side bathing solution (6). Two different experimental procedures were used to delineate the individual effects of amphotericin B stimulation and anoxia on the Na-originated SCC and the lactate efflux. With the first procedure, the corneas were anoxic upon the addition of amphotericin B; with the second procedure, the stimulation of the SCC occurred before switching from aerobic to anaerobic conditions. The results of these procedures on the electrical parameters and lactate efflux are shown in Tables 4 and 5. When amphotericin B was added to epithelial bathing solution of anaerobic corneas, it stimulated the SCC and PD to values similar to those observed under aerobic conditions and increased the lactate 3. Effects of anaerobiosis on bullfrog cwneas in NaCl Ringer containing 26 mMglucose - _-.-n Air -N’L, 80 min

TABLE

--

No incubation

t

AND

ence of glucose in the bathing solution was more than twice as large as the lactate efflux in the absence of glucose. In the presence of glucose, the lactate efflux increased by 71% upon the transition to anoxia. Furthermore, the aerobic and anaerobic lactate efflux in the presence of glucose was stable for at least 120 min. In six other corneas, we found that 1 mM glucose was sufficient to maintain active Cl transport under anoxia since the declines in the SCC and PD were 14 * 4% and 8 t 3%, respectively, values similar to those found when the NaCl Ringer solution contained 26 mM glucose. Effects of Anaerobiosis on Active Na Transport

0

SCHOEN,

k 0.05

t 0.01

& SE of 8 corneas.

0.34 pmol of glucose should be metabolized to 0.68 pmol of lactate and 0.06 pmol of glucose should remain after 2 h of anaerobiosis. However, 0.16 pmol were found. This inconsistency may be partially due to tissue variability of glucose content. The presence of glucose in NaCl Ringer solution prevented the striking decline in SCC and PD caused by anaerobiosis. In Table 3 are summarized the effects of changing from aerobic to anaerobic conditions on the electrical parameters, chloride fluxes, and lactate efflux of corneas bathed in glucose-rich NaCl Ringer solution. Figure I illustrates the time course of the changes in the SCC (solid line) and lactate efflux. The results show that after 80 min under anaerobiosis the XC and PD decreased by 17% and 996, respectively; the electrical resistance increased by 21%. In air and under anoxia the net Cl flux of the two groups of corneas (calculated from the backward and forward Cl fluxes) was within 10% of the SCC. Therefore, under anoxia the near identity between the SCC and the net active transepithelial Cl transport was maintained. The aerobic lactate efflux in the pres-

SCC, peq/hocm2 PD, mV R, k%cm2 FF, peq/h* cm2 BF, peq/h+cm2 LE, pmol/h cm2

11

0.65 21.2 1.35 0.96 0.24 0.23

11 11 6 5 5

l

t * k t t, k

0.05 1.9 0.08 0.04 0.02 0.02

0.54 19.3 1.63 0.82 0.23 0.42

* 0.04

k 1.6 t -tt t

0.10 0.02 0,02 0.04

Values are means & SE; n, number of experiments. FF, forward BF, backward flux; see Table I footnote for other abbreviations.

4. Effects of IF5 M amphotericin on anaerobic corneas bathed in Na804 Ringer containing 26 mM glucose -- -.----

B

TABLE

+ Amphotericin

SCC, PA/cm2 PD, mV R, k%cm2 LE, pmol/h

Wcm2

1

2

0.5 kO.4

0.4 to.4

1.1

0.7

kO*4 6.08 to.95 0.39 kO.03

kO.4 5.66 t-O.86 0.47 ~~0.04

3

0.4 kO.4 0.5 kO.31 5.5 k0.85 0,45 &0,02

4

9.9 H.28 30 &2.56 3,02 to.53 0.45 kO.03

flux;

5

10.24 kl.0 30 t2.15 3.63 k0.37 0.54 kO.008

Values are means t SE of 5 experiments. Periods 1-7 30-min intervals. See Table 1. footnote for abbreviations,

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B 6

9.68 -+0.90 29 k2.09 3.85 k0.46 0.50 to.03

7

9.24 , k0.89 29 k2.20 j 3.74 k0.51 0.56 to.02

are successive

GLYCOLYSIS

AND

IONIC

TRANSPORT

IN

CORNEA

5. Effects of anoxia on bullfrog corneas bathed in Na2S04 Ringer (26 mM glucose) and stimulated with 1W5 M amphotericin B T + Amphotericin B, Air t + Amphotericin Period

TABLE

SCC, PA/cm’ PU, mV R, kSt*cm” LE, pmol/h

acm”

Control

1

0.6 kO.4 1.8 Ito. 5.6 *1.0 0.26 kO.03

11.04 Ztl.1 29.7 f4,9 2.61 *0.33 0.27 zto.02

11.16 kO.94 28 ~~4.6 2.42 k0.27 0.29 LO.06

Values are means f SE of 5 experimenti. Table 1 footnote for abbreviations.

I 27 14.5 2.81 kO.4 0.36 *0.05

Periods

25 k5.3 2.67 ~0.32 0.34 20.05 1-8

5

6

7

8

9.96 kl.15 24 k4.4 2.96 k0.42 0.36 kO.04

9.52 kO.60 22 k4.2 2.75 kO.29 0.51 kO.03

9.48 kO.60 21 k2.1 2.76 kO.26 0.50 kO.04

9.48 kO.60 19.75 k4.7 2.84 k0.28 0.46 kO.03

are succesive

6. Effects of IF5 M amphotericin in glucose-free Na2SO4 Ringer on toad corneas under anaerobiosis Amphotericin Peak

SCC, PA/cm2 PD, mV R, k%cm2 LE, pmol/h+ cm2 Values are means abbreviations.

0.4 t 1* 3,4 t 0.16 k

OJ 0.2 0.08 0.02

11.3 * 32 4 2.5 k 0.22 *

t SE of 5 experiments.

30-min

intervals.

See

B

TABLE

Initial

B, Nz

B

0.4 0.8 0.1 0.07 See Table

Amphotericin 2 h later

B

1.6 -t- 0.1 2 t OJ 3.1 t 0.1 0.06 t 0.03 f footnote

for

efflux by 21%. The net active transepithelial Na transport increased by 0.35 peq/h*cm2 and the lactate efflux by 0.11 pmol/h cm2. The results of the second experimental procedure are summarized in Table 5. The results of the second experimental procedure show that anoxia produced a small decrease of the amphotericin R-stimulated SCC and increased the lactate efflux by 43%. The electrical resistance did not change appreciably. The results of this procedure show that during anaerobiosis amphotericin B further stimulated lactate efflux. Under aerobic conditions, the amphotericin B stimulated SCC of eight corneas only declined by 10% after 2 h. This rate of decline was independent of the presence of glucose. In contrast, when anaerobic conditions were induced in the absence of glucose there was a marked inhibition of the SCC after 2 h (see Table 6). Both under control conditions and during the stimulation by amphotericin B the lactate efflux in glucose-free NazS04 Ringer solution was lower than in glucose-rich Na2S04 Ringer solution (compare Tables 5 and 6). Under glucose-free conditions amphotericin B addition had only a transient stimulatory effect on the electrical parameters and lactate efflux. The large decreases of the SCC and PD and the parallel decrease of the lactate efflux suggest that in the absence of exogenous glucose the energetic requirements of active Na transport could not be supported by a decreased rate of glycolysis. l

bition of glycolysis was attempted with iodoacetamide and iodoacetate as it is known that they inhibit the activity of 3-phosphoglyceraldehyde dehydrogenase (1). If these inhibitors are selective for glycolysis, then they should not block transport in corneas incubated under aerobic conditions provided enough substrates are available (see Tables 7 and 8). However, it was found that iodoacetamide produced quick and complete inhibition of active Cl transport regardless of whether the corneas were aerobic or anaerobic. In Fig. I is shown the time course of the inhibitory effects of 2 mM iodoacetamide on the SCC and lactate efflux of anoxic corneas bathed in glucose-rich NaCl Ringer solution. In Table 7 are shown the effects of iodoacetamide on the electrical parameters and the forward and backward Cl fluxes and the lactate efflux. The inhibitory effect of iodoacetamide on the SCC was accounted for by the decline in the forward Cl flux since the backward Cl flux did not change. The electrical resistance did not change during this time period, indicating that the integrity of the tissue was maintained. In another set of eight anoxic corneas, the inhibitory effects of 2 mM iodoacetate were similar to those of iodoacetamide. Under aerobic conditions the inhibitory effect of iodoacetate was more gradual because only after 210 min were the electrical parameters completely inhibited. In Na2SOd Ringer solution, after 45 min 0.2 mM iodoacetamide nearly completely inhibited the electrical parameters of aerobic corneas in which SCC had been stimulated by amphotericin B (Table 8). It is interesting to note that iodoacetamide was an effective inhibitor of the electrical parameters at l/lo its effective concentration in NaCl Ringer solution. The effects of 0.2 mM iodoacetamide were identical to those of 0.2 mM iodoacetate. As shown in Table 8, iodoacetamide had similar inhibitory effects on the electrical parameters under aerobic and anaerobic conditions. Corneas were preincubated with 20 mM pyruvate so

7. Effects uf 2 mil4 iodoacetamide on anoxic bullfrog curneas bathed in NaCl glucose-rich Ringer

TABLE

n

SCC, peq/h cm2 PD, mV R, k!&cm’ FF, peq/hocm2 BF, pq/h* cm2 LE, pmol/h . cm2 l

Control

0.54 t 0.04 19.3 k 1.6 1.63 t 0.1 0.82 k 0.02 0.23 t 0.02 0.42 -t- 0.02

11 11

11 6 5 5

We were interested in separating the roles of glycolysis and respiration as energy sources for maintaining active transepithelial Cl and Na transport. The selective inhi-

0.02 & 0.01 0*7 t 0.2 1.66 t 0.3

0.23 t 0.03 0.24 t 0.03 0.08 k 0.02

Values are means & SE; n, number of experiments. Values iodoacetamide were obtained 1 h after drug addition. See footnotes Tables 1 and 3 for abbreviations.

for of

8. Effects of 0.2 mM iodoacetamide on curneas in Na2SQ4 Ringer under aerobic and anaero bit conditions --_--,“-.. -_.- ---.-~_-. ---- A -- --~-_ -~---I.._-. _I_TABLE

Air (n = 6)

Effects of lMetaboLic Inhibitors on Cl and Na Transport and Lactate Efflux

Iodoacetamide

SCC, PA/cm2 PD, mV R, k%cm2 t, min Values See Table

are percent 1 footnote

-97%

-97% -70% 45 decline from control; n, number for abbreviations; t, time.

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NZ (n = 6)

-76% -100%

-77% 45 of experiments.

C272

REINACH,

as to determine whether the inhibition of glycolysis by iodoacetamide could be bypassed with pyruvate as a substrate for respiration. In spite of the presence of pyruvate, iodoacetamide had the same me of inhibitory effects on the electrical parameters with the same time course as observed under aerobic and anaerobic conditions in the absence of pyruvate. The effect of 2 mM iodoacetamide on respiration was studied since iodoacetamide’s inhibitory effects did not appear to be restricted to glycolysis. The oxygen consumption of six corneas bathed in NaCl Ringer solution (pyruvate rich) was measured before and after incubation with 2 mM iodoacetamide. After 20 min of incubation, the oxygen consumption rate declined from 6.43 t 0.54 to 2.07 t 0.09 ~1 Ox/g dry wt per min. These experiments show that iodoacetate and iodoacetamide are not sufficiently specific to selectively inhibit glycolysis. Dinitrophenol, an uncoupler of electron transport, was used to eliminate the energetic contribution of respiration in corneas incubated in glucose-rich NaCl Ringer solution (8, 19). This agent produced an inhibition of the SCC that was concentration dependent; in four corneas the amount of inhibition increased from 21% to 60% when the dinitrophenol concentration was increased from 10B4 to lo-” M. The inhibitory effect of 10v2 M dinitrophenol on the SCC may be due to effects other than the uncoupling of electron transport. Langham and Taylor (14) showed in the rabbit cornea that 10B4M dinitrophenol clearly accelerated respiration whereas 10m2M dinitrophenol was markedly inhibitory. The poor inhibitory effects of 10W4M dinitrophenol on the Cl-originated SCC resemble the effects of anoxia and support the suggestion that glycolysis is capable of maintaining active Cl transport at a rate close to the aerobic level. It was of interest to determine whether fluorocitrate had any inhibitory effect on active Cl transport inasmuch as fluorocitrate is an effective inhibitor of cis-aconitase and respiration. The effects of this metabolic inhibitor were studied on four corneas bathed in glucose-rich NaCl Ringer solution. Successive doses of 2.5 and 25 mM fluorocitrate had no inhibitory effects on the SCC after I h, which again supports the fact that glycolysis can support active Cl transport. Restoration of Active Cl and Na Transport in Glucose- Depleted Corneas Glucose addition, Under aerobic conditions in glucosefree NaCl Ringer solution, the SCC and PD gradually declined (cf. Table 1). The possibility of reversing these declines by the addition of glucose to glucose-free NaCl Ringer solution was studied and the results are shown in Table 9. The values of the electrical parameters and the lactate efflux of eight corneas after in cubation in glucosefree Ringer solution are compared with the effect of incubating with glucose for 3b min. Even though the lactate efflux increased more than threefold to a value similar to glucose-rich NaCl Ringer solution under aerobic conditions, both the SCC and PD continued to decline gradually. Under anaerobic conditions in glucose-free NaCl Ringer solution, the SCC and PD declined rapidly (cf.

SCHOEN,

AND

CANDIA

9. Effects of glucose addition on bullfrog corneas in glucose-free NaCl Ringer under aero bit and anaero bit conditions TABLE

Initial

SCC, PA/cm’ PD, mV R, k&m2 lactate efflux, pmol/h cm2

7.53 14.0 2.21 0.058

t -t* *

+ Glucose

0.94 2.76 0.35 0.01

5.78 11.55 2.07 0.20

t & k 2

0.73 2.86 0.48 0.02

l

B:

Nz, CNT

Depleted

SCC, PA/cm’ PD, mV R, k&m2 lactate efflux, pmol/h* cm2

1.6 t 0.6 3.8 k 1.6 1.9 * 0.4 0.13 * 0.03

+ Glucose

2.81 5.1 2.01 0.37

k * * t

0.8 1.5 0.3 0.05

+ Glucose, values obtained 30 min after glucose addition. See Table I footnote for abbreviations. * Values are means t SEof8 experimen ts. t Values are means t SE of 14 experiments.

Table 1). In 14 other corneas, anoxia caused the SCC and PD to decline by 87% after 90 min to the values shown in Table 9B under the heading depleted. The subsequent addition of glucose slightly stimulated the SCC and PD after 30 min (seecolumn headed glucose). The restorative effect was small since after 30 min the SCC increased to a value that was only 22% of the value measured in air. After longer periods, no further appreciable restoration was seen because at I and 1.5 h the restoration had only increased to 25%. Glucose + ATP. The effect of adding 1 mM ATP in combination with 26 mM glucose to glucose-free NaCl Ringer solution on the electrical parameters and lactate efflux was studied. The results are shown in Fig. 2. Initially the corneas were aerobic, and once the SCC stabilized, they were made anoxic. Anaerobiosis resulted, after 90 min, in a 61% decrease of the SCC and a nearly threefold increase of the lactate efflux. Glucose addition to the bathing solution did not significantly change the SCC after 30 min, even though the lactate efflux increased to a value larger than in glucose-rich NaCl Ringer solution under anaerobiosis (see also Table 3). ATP addition, however, immediately stimulated the SCC after 60 min to values larger than the aerobic value. The lactate efflux also increased after ATP addition and this increase was somewhat larger than that observed in corneas where bathing solutions were only supplemented with glucose. In four corneas, the lactate efflux increased only slightly in the second 30-min period after glucose addition to the bathing solution. In Fig. 3 are shown the consecutive effects of the addition of ATP (1 mM) and glucose (26 mM) on the SCC and lactate efflux. In glucose-free NaCl Ringer solution, anoxia caused a progressive decline of the SCC and lactate efflux where the pattern of decline was practically unaltered by the addition of ATP. After 3 h of anoxia, the addition of glucose had profound and immediate stimulatory effects on the SCC and lactate efflux. Sixty minutes after the addition of glucose the lactate efflux increased to a value more than twice as large as the value at 30 min and the SCC was nearly fivefold

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GLYCOLYSIS

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‘z 0

12

-

- 0.6

I E N

‘, ., ,. ., ,.., . ;. .‘.‘..:~.‘.:‘.‘.:‘.

- 0.5

10 -

F - 0.4 (YE

-8

Y 5

E l3 26

2

0

60

90

120

150

100 Minutes

210

FIG. 2. Effects of consecutive addition ATP in restoring inhibited Cl-originated isolated bullfrog cornea.

240

270

300

0

of 26 mM glucose and 1 mM SCC and lactate efflux of

- 0.6

12

10

4

2

0

FIG.

glucose bullfrog

30

60

90

12Q

3. Effects of consecutive in restoring Cl-originated cornea.

150 Minutes

180

210

240

addition of 1 mM SCC and lactate

270

4 0.1

300

ATP and 26 mM efflux of isolated

30

60

90

120

FIG. 4. Effects of consecutive mM glucose in restoring inhibited of isolated bullfrog cornea.

150 Minutes

180

210

240

270

300

addition of 0.5 mM adenosine and 26 Cl-originated SCC and lactate efflux

greater than the value at the time glucose was added to the bathing solution. Therefore the order of addition of these substances appeared not to be important inasmuch as the restoration of the SCC was observed regardless of their sequence of addition. It was of interest to study whether the restoration of the SCC was specific for glucose and ATP addition. Therefore, another adenine derivative, adenosine, was used in place of ATP. The results are shown in Fig. 4 and indicate that 5 x low4 M adenosine like ATP had no stimulatory effect on the lactate efflux and only a transient stimulatory effect on the SCC. Adenosine had the same effects as ATP in the presence of glucose in restoring the SCC and lactate efflux. Because the restorative effect was not ATP specific, the possibility of restoring the SCC with adenine, ribose, and glucose was studied. Five corneas were mounted aerobically in glucose-free NaCl Ringer solution and then were glucose depleted anaerobically. After 90, 120, and 150 min, 5 x low4 M adenine, 5 x 10e4 M ribose, and glucose were added consecutively to the bathing solution, and as when glucose was added alone the percent of restoration was 26%. The results of these experiments indicate that full restoration of the SCC and lactate efflux is dependent on the addition of either adenosine or ATP in combination with glucose. The restorative effects of glucose and adenosine additions were studied in anoxic glucose-depleted corneas that were stimulated with amphotericin B and bathed in glucose-free NazS04 Ringer solution. In contrast with the

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effects in NaCl Ringer solution, After 90 min their SCC was not restored by the addition of glucose and adenosine and the electrical resistance continued to decline. DISCUSSION

Our results show that in the bullfrog cornea, under anaerobiosis, glucose can support for long periods active transepithelial Cl and Na transport. With glucose present, the transition from aerobic to anaerobic conditions resulted in substantial increases of the lactate efflux and only slight decreases of the Na and Cl originated SCC. However, in the absence of exogenous glucose the SCC did not stabilize but decreased to small values after 2 h. Similarly, the lactate efflux did not increase but rather declined in some instances to values that were unmeasurable after 2 h. All these results suggest that there is a positive correlation between anaerobic glycolytic rate and active transepithelial Cl and Na transport. It is of interest to attempt to estimate whether the rates of the energy-conserving reactions of glycolysis are rapid enough to support anaerobic active Cl and Na transport. If it is assumed that these energy-conserving reactions involve the synthesis of ATP, then the energetic requirement for active Cl transport can be estimated from the results of earlier studies where the oxygen consumption of isolated corneas was measured (15). From the results of this study, it appeared that active Cl transport consumes about 0.19 pmol/h cm2 of ATP. Under anaerobiosis in the presence of glucose the lactate efflux was about 0.4 pmol/h cm2 in NaCl Ringer solution (cf. Table 3) and this efflux corresponds to an ATP yield of 0.4 pmol/h cm2. Therefore, since the energetic requirement of active Cl transport is less than the ATP yield from anaerobic glycolysis this metabolic pathway seemed to be sufficiently accelerated by anoxia to maintain active Cl transport. It has been reported that the ox and rabbit corneas contain endogenous reserves of glucose (16). We measured these reserves in the bullfrog cornea and found that there was a significant decline of these reserves after 2 h of incubation under anoxia in the absence of exogenous glucose. The glucose content of these corneas declined from 0.4 to 0.16 pmol/cm2, whereas under aerobic conditions there was no significant decline after 2 h. As we estimated that a lactate efflux of about 0.4 pmol/ha cm2 was sufficient to maintain active anaerobic Cl transport, a glucose reserve of 0.4 pmol/cm’ would appear to be sufficient to maintain the Cl-originated SCC for up to 4 h. However, it was found that the Cl-originated SCC declined immediately under anoxia in the absence of exogenous glucose and after 90 min it decreased by 82% (cf. Table 1). A possible explanation for this decline, in spite of what appeared to be sufficient glucose reserves, is that other processes are competing with active Cl transport for energy-rich substrates. One of these other energy consuming processes may be active Na transport. We earlier estimated on the basis of the effects of ouabain on the oxygen consumption of isolated corneas bathed in NaCl Ringer solution or Na2S04 Ringer solution that the energetic requirements for active Na and Cl transport are similar (15). Therefore the combined enl

l

l

SCHOEN,

AND

CANDIA

ergetic requirements of these two active processes may be about 0.4 pmol ATP/h. cm2 and because the endogenous glucose reserve was 0.4 pmol/dm2, this reserve would appear to be only sufficient for maintaining anaerobic active Na and Cl transport for up to 2 instead of 4 h. In any case, the rapid decline of the SCC cannot be explained on the basis of inadequate glucose reserves for maintaining the active transport of Na and Cl. The marked stimulation of the lactate efflux and the SCC by adenosine or ATP in the presence of glucose is an interesting result and suggests that adenosine or its metabolites may have a regulatory role in active Cl transport. This regulatory role seems to be related to glycolysis since sustained stimulation of active Cl transport only occurred in the presence of glucose when glycolysis was accelerated. Once glycolysis was accelerated by the addition of glucose to glucose-free NaCl Ringer solution, the addition of adenosine or ATP further accelerated glycolysis at the same time that active Cl transport was restored. A cooperative interaction between adenosine or ATP and glucose i.n accelerating glycolysis is further indicated by the fact that neither of these adenine derivatives could serve as substrates for glycolysis. This interaction was specific in the sensethat neither adenine nor ribose could substitute for adenosine or ATP in the presence of glucose in stimulating active Cl transport. Our results suggest that the restoration and maintenance of active Cl transport requires that the cellular ATP pool size be above a minimum level and this level is attainable by sufficiently accelerating glycolysis. Generally it is thought that phosphorylated intermediates (ATP) do not penetrate the cell membrane readily. The fact that the effects of ATP and adenosine were equivalent in the presence of glucose ma.y suggest that ATP could permeate the cell membrane of the cornea. In human red blood cells, it has been shown that adenosine readily penetrates the cell mem brane and is phosphorylated to ATP by acting as a phosphate acceptor in glycolysis (20) . An alternative explanation for* the effects of adenosine and ATP in stimulating the Cl-originated SCC is that their effect may result from membrane interactions, even if adenosine penetrates the cells. In other systems, adenosine and ATP appear to have membrane effects. In guinea Pig ventricular preparations, adenosine stimulated CAMP accumulation presumably due to an activation of membrane bound adenylate cyclase (9). Cyclic AMP and adenosine are potent stimulatdrs of active Cl transport in the bullfrog cornea (3,23). A membrane interaction for ATP has been shown in canine red blood cells where it increased Na influx by either chelation of membrane charges or by a direct interaction with membrane proteins (18). Under aerpbic conditions, in the absence of exogenous glucose neither amphotericin B-stimulated active Na nor active Cl transport were immediately inhibited. The SCC of aerobic corneas were comparable regardless of whether glucose was present in the bathing solution. However, after longer periods of incubation (6-18 h) the SCC usually declined more rapidly in corneas bathed in glucose-free NaCl Ringer solution. The decreased lactate efflux in a glucose-free Ringer solution may not only stem from a decreased rate of glucose utilization but also from

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GLYCOLYSIS

AND

IONIC

TRANSPORT

IN

c275

CORNEA

differences in rates of lactate oxidation depending on whether glucose was present in the bathing solution. Kuhlman and Resnick (13) showed by means of variously labeled [14C]lactate that this compound was readily utilized in the citric acid cycle. A surprising result was that the addition of glucose to the bathing solution of aerobic corneas had no stimultatory effect on the SCC. The lactate efflux, however, increased to a level similar to that observed in glucose-rich solution. This result is different from that observed in toad bladders where active transepithelial Na transport appears to be coupled to respiration. Maffly and Edelman (10) showed that the addition of glucose to the bathing solution of isolated short-circuited toad bladders immediately stimulated the SCC to levels observed in the control preparation. The cellular ATP pool under aerobic conditions in the absence of exogenous glucose may not be much less than the value in the presence of glucose. From the lactate effluxes under these two conditions, and the fact that respiration is not affected by the presence or absence of glucose, the calculated ATP yield in the presence of glucose was about 1.4 pmol/h* cm2 and decreased to about 1.25 pmol/h cm2 in the absence of glucose (1 7) Therefore a decrease of about 10% in the ATP yield in glucose-free solutioti does not appear to have any large inhibitory effects on the short-term SCC. An interesting result of this study has been that, regardless of the presence of Cl in the bathing solution, the lactate effluxes under aerobic and anaerobic conditions were very similar. Because anaerobic active Cl transport accounts for about 95% of the SCC in NaCl Ringer solution, and appears to be positively correlated with the rate of glycolysis, one might have predicted a smaller lactate efflux in corneas only actively transporting Na. On the contrary, in different sets of corneas the lactate efflux under aerobic conditions in NaCl and Na2S04 Ringer solution (prior to stimulation of the SCC with amphotericin B) was 0.23 and 0.26 pmol/h*cm”, respectively (cf. Tables 3 and 5). Similarly under anaerobic conditions the correspondence in NaCl and Na2S04 Ringer solution was 0.42 vs. 0.44 pmol/h* cm2, respecl

l

tively. Under both conditions the correspondence between the lactate efflux in NaCl and NazS04 Ringer solution disappeared once the small Na-originated SCC in NazS04 Ringer solution was stimulated by amphotericin B. From the stimulatory effects of amphotericin B under anaerobiosis on the SCC and lactate efflux, have calculated that the energetic requirement for active transepithelial transport-of 3.18 mol of Na requires the hydrolysis of I mol of ATP. The fact that glycolytic flux in NazS04 Ringer solution was stimulated by amphotericin B as well as by anoxia suggests that glycolytic flux may be modulated by intracellular Na levels that presumably change upon stimulating active Na transport with amphotericin B. The reason for the correspondence between the lactate effluxes in Na2S04 Ringer solution, in the absence of amphotericin B, and in NaCl Ringer solution cannot be presently explained. The use of metabolic inhibitors, iodoacetamide and iodoacetate, did not clearly separate the roles of aerobic and anaerobic metabolism in the support of active transepithelial Na and Cl transport. Under aerobic and anaerobic conditions the effects of iodoacetamide were particularly confusing as this inhibitor was equally effective in abolishing active Na and Cl transport regardless of any attempt to bypass the inhibitory effects of this compound on glycolysis by preincubation with 20 mM pyruvate. Similar observations have been made in the lens and kidney slices as to the lack of specificity of these inhibitors. Kinoshita et al. (12) observed that 3 x 10m5 M iodoacetate inhibited Na extrusion and K reaccumulation in the lens prior to any inhibitory effect on anaerobic glycolysis. Mudge (11) obtained a 50% inhibition of respiration as well as the abolishment of Na:K transport in kidney slices with 0.33 mM iodoacetate. We thank Lawrence Alvarez for his most excellent technical assistante. Dr. David Erlij’s careful reading of this manuscript is highly appreciated . L -This work was supported by National Eye Institute Research Grants EY-01867, EY-01976, and EY-00160. H. Schoen was supported by Research Training Grant EY-07014. Received

24 July

1978; accepted

in final

form

15 December

1978.

REFERENCES 1. ADLER, E., H. VON EULER, AND G. GUNTHER, Dehydrasen und Jodessigsaure, Skand. Arch. Physiol. 80: 1-18, 1938. 2, BERGMEYER, H. (Editor). Methods of Enzymatic AnaZysis, New York: Academic, 1974, p. 1468-1472. 3. BEITCH, B., I. BEITCH, AND J. ZADUNAISKY. The stimulation of chloride transport by prostaglandins and their interaction with epinephrine, theophylline and cyclic AMP in the corneal epithelium. J. Membr. Biol. 19: 381-396, 1974, 4. CANDIA, 0. A, Ouabain and sodium effects on chloride fluxes across the isolated bullfrog cornea. Am, J. PhysioZ, 223: 1053-1057, 1972, 5. CANDIA, 0. A., AND W. A. ASKEW. Active sodium transport in the isolated bullfrog cornea. Biochim. Biophys. Acta 163: 262-265,1968. 6, CANDIA, 0. A., P. J. BENTLEY, AND P. I. COOK. Stimulation by amphotericin B of active Na transport across amphibian cornea. Am. J. PhysioZ. 226: 1438-1444, 1974. 7. CANDIA, 0. A., AND H. F. SCHOEN, Selective effects of bumetanide on chloride transport in bullfrog cornea. Am. J. Physiol. 234: F297F30i, 1978 or Am. J. PhysioZ.: RenaZ Fluid EZectroZyte Physiol. 3: F297-F301, 1978. 8. CHANCE, B., G. R. WILLIAMS, AND G. HOLLUNGER, Inhibition of electron and energy transfer in mitochondria. J. BioZ. Chem. 238: 439-446,1963.

9. HUANG, M., AND G. DRUMMOND. Interaction between adenosine and catecholamines on cyclic AMP accumulation in guinea pig ventricular myocardium. Biochem. PharmacoZ. 27: 187-191, 1978. 10. MAFFLY, R., AND I. S, EDELMAN. The coupling of the short circuit current to metabolism in the urinary bladder of the toad. J. Gen. PhysioZ. 46: 733-754, 1963. II. MUDGE, G. H. Electrolyte and water metabolism of rabbit kidney slices: effect of metabolic inhibitors, Am. J. Physiol. 167: 206-233, 1951, 12. KINOSHITA, J. H., H. KERN, AND 0. MEROLAFactors affecting the cation transport of calf lens, Biochim. Biophys. Acta 47: 458-466, 1961, 13. KUHLMAN, R. E., AND R. A. RESNIK. The oxidation of C14-labeled glucose and lactate by the rabbit cornea. Arch. Biochem, Biophys. 85: 29-36, 1959. 14. LANGHAM, M. E., AND I. S. TAYLOR. Factors affecting the hydration of the cornea in the excised eye and the living animal. Br. J. OphthaZmoZ.40: 321-340, 1956. 15, REINACH, P. S., H. SCHOEN, AND 0. A. CANDXA. Effect of Na and Cl transport inhibitors on oxygen consumption in the bullfrog cornea. Exp. Eye Res. 24: 493-500, 1977. 16. RILEY, M. V. Glucose and oxygen utilization by the rabbit cornea.

Downloaded from www.physiology.org/journal/ajpcell at Tulane University (129.081.226.078) on February 14, 2019.

C276 17.

REINACH,

Exp. Eye Res. 8: 193-200, 1969. ROETTH, A. DE. Respiration of the cornea.

623,

Arch.

OphthaZmoZ.

44:

666-676,195O.

ROMUALDEZ, A,, M. VOLPI, AND R.I. SHAAFI. Effect of exogenous ATP on sodium in mammalian red cells. J. CeZZ PhysioZ. 87: 297306, 1976. 19. SLATER, E. C. In: Metabolic Inhibitors, edited by R. M. Hochster and J, H. Quastel. New York: Academic, 1963, vol, 2, p. 503-524. 20. WHITTAM, R. The high permeability of human red cells to adenine and hypoxanthine and their ribosides. J. PhysioZ. London 154: 61418.

SCHOEN,

AND

CANDIA

1960.

ZADUNAISKY, J. A. Active transport of chloride in frog cornea. Am. J. PhysioZ. 211: 506-512, 1966. 22. ZADUNAISKY, J. A.,M. A. LANDE,AND J, HAFNER. Further studies on chloride transport in the frog cornea. Am. J. Physiol. 221: 1832-

21.

1836, 23,

1971.

ZADUNAISKY, J. A., AND B. SPINOWITZ. Drugs affecting the transport and permeability of the cornea1 epithelium. In: Drugs and Ocular Tissues, edited by S. Dikstein. New York: Karger, 1977, p. 57-72.

Downloaded from www.physiology.org/journal/ajpcell at Tulane University (129.081.226.078) on February 14, 2019.