Glucose metabolism in cat outer retina. Effects of light and hyperoxia.

Glucose Metabolism in Cat Outer RetinaEffects of Light and Hyperoxia

Lin Wang,* Mitsuhiro Kondo,-\ and Anders Bill*

Purpose. To determine the roles of oxidation and glycolysis with aerobic and anaerobic lactateformation in the glucose metabolism of the cat outer retina in light and darkness.

Methods. Blood was collected from a choroidal vein and from an artery, and veno-arterialdifferences in lactate concentration (Lacv<1) were determined at increasing light intensities.Blood also was sampled under conditions of darkness, light, and hyperoxia and were analyzedfor oxygen, glucose, and lactate concentrations with or without blood flow determinations.

Results. When the dark-adapted eye was subjected to increasing light intensities, there was areduction in the Lacv.a, indicating reduced glycolysis in the outer part of the retina as therods saturated. In darkness, the mean lactate formation per retina was 0.409 /Ltmol/minute,oxygen consumption was 0.198 //mol/minute, and glucose consumption 0.236 //mol/minute.In light, the corresponding figures were 0.253, 0.166, and 0.123 yumol/minute. Hyperoxiareduced lactate formation and increased oxygen consumption in light and in darkness.

Conclusions. Approximately 80% of the glucose consumed by the outer retina is used primarilyin aerobic lactate formation. Because it is more efficient, oxidation of glucose still accountsfor most of the energy production in light and in darkness. Light reduces oxidation as wellas aerobic and anaerobic lactate formation. Invest Ophthalmol Vis Sci. 1997;38:48-55.

An most tissues, anaerobic glycolysis is an importantsource of energy under conditions of hypoxia. How-ever, some cells produce lactate even under normalconditions, suggesting that glycolysis and oxidativephosphorylation are weakly coupled or that the physi-ological oxygen tension is close to zero. Early in vitrostudies by Warburg et al1 and Craig and Beecher2 indi-cated that the retina has a high rate of oxygen con-sumption but, surprisingly, considerable lactate for-mation as well. Later in vivo studies in rabbits3'4 andpigs5 indicated the same phenomenon.

In some animals, such as rabbits, most of the ret-ina is supplied by diffusion from the choroid becausethere are no true retinal vessels. In such animals, theoxygen tension can be expected to be the lowest inthe inner parts of the retina, with the prospect of

From the * Department of Physiology and Medical Biophysics, University of Uppsala,Uppsala, Sweden, and the f Department of Ophthalmology, Aichi MedicalUniversity, Aichi-Ken, Japan.Supported by National Institutes of Health grant EY00475 and Swedish MedicalResearch Council grant 00147.Received for publication September 25, 1995; revised July 1, 1996; accepted August14, 1996.Proprietary interest category: N.Reprint requests: Anders Bill, Department of Physiology and Medical Biophysics,Uppsala Universitet, BMC, Box 572, S-751 23 Uppsala, Sweden.

anaerobic glycolysis in this region. In others, such ascats, pigs, and primates, which have a system of retinalvessels, the lowest values can be expected in the avas-cular outer part of the retina supplied mainly by diffu-sion from the choroid. Studies using oxygen-sensitivemicroelectrodes in cats have demonstrated that theoxygen tension in the nuclear region of the photore-ceptors is approximately 20 mm Hg in light and ap-proaches zero in darkness.6'7 It was assumed that thiseffect of darkness resulted from the extra energy pro-duction required to maintain the dark current of thephotoreceptors.

Thus, in darkness, some anaerobic glycolysis maytake place because of hypoxia, which would result inacidification of the tissue. Light then should causea rise in pH, which, in fact, has been observed inamphibious animals8'9 and cats10 in vitro or in vivo.Similarly, hyperoxia in darkness is expected to inhibitacidification of retina, as has been observed." How-ever, recent studies in vivo4 and in vitro12 in rabbitsfailed to demonstrate clearly enhanced lactic acid for-mation in darkness, suggesting that the rabbit is a poormodel for glucose metabolism in higher mammals.Therefore, the current in vivo study has been con-

48Investigative Ophthalmology & Visual Science, January 1997, Vol. 38, No. 1Copyright © Association for Research in Vision and Ophthalmology

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Retinal Glucose Metabolism in Cat 49

ducted in cats. Blood drained from the choroid of catscan be collected by cannulation of choroidal veinsthrough a large intrascleral venous plexus.13 We havesampled blood in this way under different conditionsand determined the choroidal blood flow.

MATERIALS AND METHODS

Twenty-nine adult domestic cats of either sex, eachweighing 2.1 to 4.5 kg, were used. Anesthesia was in-duced with an intramuscular injection of a mixtureof xylazin, 8 mg (Rompun; Bayer AG, Leverkusen,Germany) and ketamine, 20 mg (Ketalar; Parke-Davis, Barcelona, Spain). Anesthesia was maintainedwith alpha-chloralose (Merck, Darmstadt, West Ger-many); approximately 50 mg/kg body weight was ad-ministered intravenously at the beginning, and 25mg/kg body weight was administered during the ex-periments. All animals were tracheotomized and arti-ficially ventilated with normal air initially. In someexperiments they were later ventilated with 5% CO2 inO2 so we could study the effects of hyperoxia withoutretinal vasoconstriction. Body temperatures weremaintained at 37°C to 38°C. Two femoral arteries andtwo femoral veins were cannulated with polyethylenetubes. One artery was used for the collection of arterialblood, the other for continuous recording of the arte-rial blood pressure; veins were used for the injectionof drugs. For blood flow determination, performed insome experiments, the left heart ventricle was cannu-lated through the right brachial artery for the injec-tion of microspheres. During the experiments, acid-base balance and blood gas were determined intermit-tently by analyzing arterial blood samples in an acid-base analyzer (ABL300; Radiometer, Copenhagen,Denmark). During normoxia, disturbances of theacid-base balance were corrected by the administra-tion of sodium bicarbonate, changing the ventilationvolume, or both. During hyperoxia, no correctionswere made. All experimental procedures compliedwith the National Institutes of Health Guidelines forthe Care and Use of Laboratory Animals and theARVO Statement for the Use of Animals in Ophthal-mic and Vision Research.

Choroidal Vein Cannulation

The upper eyelid was lifted either by a retractor orby sutures. If necessaiy, an external canthotomy wasperformed. A parallel incision in the upper bulbarconjunctiva 5 to 6 mm behind the corneoscleral lim-bus was made, and the subconjunctival tissue was sepa-rated until the intrascleral venous plexus was identi-fied. Usually, a few minutes after exposing the area tothe air, the plexus became clearly visible. A branchoriginating in the choroid was identified, and, withthe help of a stereoscopic dissecting microscope, a

small opening was made into the vein. A tube with atapered tip was introduced approximately 3 to 4 mminto the vein in the upstream direction. Heparin (1mg/kg) was administered intravenously to prevent co-agulation, and indomethacin (5 mg/kg) body weightwas administered intravenously to prevent clotting atthe tip of the tube. The free end of the tube was placedbelow the level of the cat, which resulted in a freeflow of approximately 100 to 150 fj,\ per minute.

Determination of Arteriovenous ConcentrationDifferences

Oxygen and glucose arteriovenous concentration dif-ferences (Oxya.v and Glua.v, respectively) and Lacv.:,were determined by paired arterial and venous bloodsamples collected concomitantly from a femoral arteryand the choroidal vein through small glass capillaries.Average values under each experimental conditionwere calculated from at least three pairs of samples.Samples were analyzed immediately in the acid-baseanalyzer to determine Pco2, Po2, pH, and hemoglo-bin concentration. Oxygen concentration was mea-sured in the analyzer calibrated with an oxygen satura-tion meter (OSM3; Radiometer, Denmark). Hemato-crit levels were calculated from hemoglobinconcentrations using data from measurements of bothin another group of cats. Samples were centrifugedand stored in a cold room (4C°) for later determina-tion of plasma glucose and lactate concentration witha GOD-PAP method kit (14365; Merckotest, Darm-stadt, Germany) and a lactate diagnostic kit (No. 735;Sigma, St. Louis, MO), respectively. In some of theexperiments, lactate and glucose concentrations weredetermined using a dual analyzer (model 2700; YSI,Yellow Springs, OH). Comparison between blood andplasma lactate concentrations indicated that there wasa rapid equilibration of lactate across the red cellmembrane and that the blood concentration, Cb,could be calculated from plasma concentration by theequation: Cb = Cp-(1 - Ht) + Cp-Hf0.7l. Here,Cp was plasma concentration, and Ht was hematocrit.Separate experiments indicated that when Glu:i.v wascalculated, the intracellular glucose could be disre-garded because of a slow glucose transport across themembrane in this species.14

Dark and Light Adaptation

Dark adaptation was induced by covering the head ofthe animal with a box and a black cloth for at least60 minutes. Light adaptation was effected by placinga white screen 15 cm in front of the eyes for 5 to 10minutes with a light intensity, close to the cat's cornea,of approximately 180 lux. Ordinary fluorescent lightwas used as the light source. The horizontal pupildiameter was 6 to 8 mm. Different light intensitiesused to stimulate the eye in some experiments were


50 Investigative Ophthalmology & Visual Science, January 1997, Vol. 38, No. 1

monitored at the position described above, and atro-pine (0.04 mg/kg body weight, injected subcutane-ously) was used in these experiments to preventchanges in pupil size.

Determination of Blood FlowRegional blood flow was determined in some experi-ments by the microsphere method described by Aimand Bill.15 Briefly, 2 X 106 microspheres 15 /zm indiameter labeled with either 141Ce, 113Sn, or 10SRu(New England Nuclear, Boston, MA) were used foreach left ventricle injection. This enabled three bloodflow determinations in each animal. The injectionswere made during a span of 10 to 15 seconds. A refer-ence blood sample from the femoral artery was takenduring a 1-minute period, starting at the beginningof the microsphere injection. In some of the experi-ments, additional lactate measurements were per-formed before and after the flow determination toobserve the possible metabolic effect of the micro-spheres impacted in the tissue. The animals werekilled with an overdose of anesthetic and approxi-mately 10 ml of saturated potassium chloride solutionadministered intravenously, and the eyes were dis-sected. All blood and tissue samples were weighed,and the radioactivities were analyzed in a three-chan-nel gamma-spectrometer (Nuclear-Chicago, Chi-cago, IL). Oxygen consumption and lactate formationwere calculated on the basis of blood flow in millilitersper minute, and Oxya.v and Lacv.a were calculated asmentioned. Glucose consumption was calculated fromdata for plasma flow and the plasma arteriovenousdifference.

Experiments

Three groups of experiments were performed (A, B,and C).

A. The time course of the Lacv.a was observed un-der the following conditions:1. (n = 11) Stepwise increased light intensity

after dark adaptation. In the first four ani-mals, we determined the lowest light inten-sities giving a clear effect and observed thiseffect for 30 minutes. We then increasedthe intensity to 110 lux for 20 minutes.Blood samples were taken every 3 to 5 min-utes. In the other seven experiments, sev-eral light intensity increments were used.Each step lasting for approximately 5 min-utes, and blood samples were taken duringthe last 2 minutes of each period.

2. (n = 5) Light adaptation for 20 minutesfrom an initially dark-adapted conditionand then 1 hour of dark adaptation.

B. (n = 8) To calculate lactate production and

1.6-

1.4-

1.2-

1.0-

0.8-

0.6-

0.4-

0.2-

0.0-

HHit \U

10

Light intensity

100 (lux)

FIGURE l. Lacv.:> (mean ± SE; n = 7) at increasing light inten-sities (O) after 1 hour of dark adaptation (•). Each lightintensity lasted for approximately 5 minutes, and measure-ments were made during the last 2 minutes. Mean arterialpressure during the experiment was 84 ± 2 mm Hg at thebeginning and 74 ± 4 mm Hg at the end. *P < 0.05 com-pared with that in darkness.

oxygen and glucose consumption, we mea-sured Lacv.a, Oxya.v, Glua.v, and blood flow inthe intraocular tissues. The measurementswere carried out under dark adaptation, thenlight adaptation, and then a combination oflight adaptation and hyperoxia.

C. (n = 5) To compare the response to darknessand light under normoxia and hyperoxia, wemeasured Lacv.a, Oxya.v, and Glua.v during darkadaptation, dark adaptation under hyperoxia,light adaptation under hyperoxia, and lightunder normoxia.

In experiments B and C, the blood samples werecollected at stable arterial Po2 after dark adaptationfor 1 hour or more or after light adaptation for atleast 10 minutes.

StatisticsResults are presented as the mean ± SE; n representsthe number of animals used in a given experiment.Paired data were analyzed with the two-tailed Stu-dent's /-test. Analysis of variance using the 7 t̂est andcomparisons of all means to one control were per-formed for repeated measurements. P < 0.05 was con-sidered significant.

RESULTS

Effect of light Intensity on the Lacv.aFrom experiment Al, it was ascertained that the lowestlight intensity causing a detectable change in theLacv.a ranged from 0.3 to 1 lux. The effect was sus-tained for more than 30 minutes until the eye was


Retinal Glucose Metabolism in Cat

E,(0

1

1.4-

1.2-

i .o-

0.8-

0.6-

0.4-

0.2"

n .U

n

I-O-I

0

\

10

i

*

I :i

20 30

*

1

40

**I 1i

50 60 (min)

FIGURE 2. Lacv.;i in light (O) followed by 1 hour of darkadaptation (•) (mean ± SE, n = 5). *P < 0.05 comparedwith that in light.

exposed to a higher light intensity (110 lux). Thechange induced by dim light was approximately onethird that induced by stronger light. The experimentusing stepwise increments of light intensity was de-signed from these observations. In these experiments,there was a graded response at low intensities and afull effect at 5 to 10 lux (Fig. 1). The Lacv.a changedsignificantly (P < 0.05) when the light intensity was>2 lux.

The decrease in Lacv.a caused by light was detectedwithin a few minutes with little further change. Goingfrom light to darkness, the increase in Lacv.a startedvery soon but continued for approximately 30 minutesuntil a steady value was reached (Fig. 2).

Metabolism of the Outer Retina in Darknessand Light, Effect of Hyperoxia

Blood flow values in different intraocular tissues deter-mined in experiment B are shown in Table 1. Therewas no significant difference in blood flow betweencannulated and intact eyes. When ventilation was ad-justed from air to 5% CO2 in O2, there were no clear

51

changes in blood flow in most of the observed tissues.However, in the iris, the blood flow increased by 60%.Choroidal blood flow tended to decline, probably asa result of falling blood pressure; 79 ± 3 mm Hg indarkness, 73 ± 3 mm Hg in light, and 70 ± 4 mm Hgin light under hyperoxia.

Average arterial oxygen, glucose, and lactate con-centrations at the beginning of the experiments areshown in Table 2. Oxya.v and Glua.v were very small,which made methods errors a greater problem thanin most other studies of this kind.

Based on the measurements of choroidal bloodflow and Lacv.a, Oxya.v, and Glua.v, lactate productionand oxygen and glucose consumption were calculated(Table 2). The ratio of lactate formation in hyperoxiato that in normoxia was 0.6; for glucose consumptionand oxygen consumption, the corresponding valueswere 0.8 and 1.4, respectively. Blood samples collectedbefore and after the injection of microspheres did notindicate that impaction of spheres caused a significantchange in lactate formation.

Within the glucose range of these experiments(10 to 25 raM), there was no indication of a Crab treeeffect—decreasing oxygen consumption with increas-ing glucose concentration. There was no correlationbetween lactate production and arterial lactate con-centration in the 0.6 to 2.6 mM range.

Effects of Darkness and Light and Hyperoxiaon Oxya.v, Glua.v, and Lacv.a

In experiment C, the transition from darkness to lightwas under hyperoxia, and no blood flow was deter-mined. Oxy^,, Glua_v, and Lacv.a showed changes simi-lar to those in experiment B (Fig. 3). The transitionfrom darkness to light in hyperoxia caused a statisti-cally significant reduction in Lacv.a; the average reduc-tion was 57% ± 6% (P < 0.01). The reduction ofOxya.vwas 19% ± 2.2% (P < 0.01).

Changing from normoxia to hyperoxia in darknesscaused effects similar to those in light in experimentB. The hyperoxia-normoxia ratios for the Lacwl,

TABLE l. Blood Flow* in Different Intraocular Tissues Determined by the MicrosphereMethod in Darkness, Light, and Light With Hyperoxia

Darkness

Experiment^ Control^ Experiment

Light

Control

Light +

Experiment

Hyperoxia

Control

RetinaChoroidIris

0.018 ± 0.0020.540 ± 0.0540.030 ± 0.006

0.016 ± 0.0040.678 ± 0.1230.033 ± 0.012

0.023 ± 0.0030.481 ± 0.0410.031 ± 0.006

0.018 ± 0.0040.539 ± 0.07850.036 ± 0.010

Ciliary body 0.190 ± 0.015 0.187 ± 0.015 0.200 ± 0.017 0.208 ± 0.017

0.019 ± 0.0030.413 ± 0.043§0.049 ± 0.010§0.219 ± 0.015

0.016 ± 0.0030.435 ± 0.0750.058 ± 0.01450.233 ± 0.015

* ml/minute. Values are mean ± SE; n = 8.f Experiment = choroidal vein cannulated.X Control = untouched eyes.§ Significant difference compared with the preceding measurement.



TABLE 2. Lactate Production and Oxygen and Glucose Consumption (/^mol/minute) andthe Arteriovenous pH Difference (pHa.v) in Darkness (D), Light (L), and Light UnderHyperoxia (LH) in Experiment B*

Lactate productionOxygen consumptionGlucose consumptionpH,,. (U)pCO2 (kPa)Lactate concentrationO2 (mM)Glucose concentration

(mM)

(mM)

D

0.409 ± 0.0490.198 ± 0.0370.236 ± 0.0290.055 ± 0.0104.94 ± 0.140.99 ± 0.07

16.05 ± 1.3019.59 ± 0.86

L

0.253 ± 0.0230.166 ± 0.0180.123 ± 0.0240.032 ± 0.0054.94 ±0.121.04 + 0.07

16.40 ± 1.5719.15 ± 0.84

(0.156(0.032(0.113(0.023

(D-L)

± 0.035)1± 0.022)± 0.023)t± 0.006)t————

LH

0.164 ± 0.0200.236 ± 0.0160.102 ± 0.0210.031 ± 0.0036.97 ± 0.130.83 ± 0.06

18.24 ± 1.9119.18 ± 0.88

(L-LH)

( 0.089 ±(-0.070 ±( 0.021 ±(-0.002 ±

————

0.018)f0.018)t0.010)0.002)

* The corresponding changes after transition from darkness to light, and from normoxia to hyperoxia in light arc shown in parentheses.The arterial PCO2 and the concentrations of lactate, oxygen, and glucose also are presented. \ P < 0.01.

Glua.v, and Oxy.,.v were 0.7, 0.6, and 1.3, respectively.Similar changes, but in the opposite direction, wereobserved when changing from hyperoxia to normoxiain light.

Energy Production

Adenosine triphosphate (ATP) production in nor-moxia was calculated from the data for lactate forma-tion and oxygen consumption. Table 3 shows that thetotal ATP production was reduced by 22% from dark-ness to light in normoxia and that glycolysis wirii lac-tate formation accounted for 20% to 26% of the en-ergy production in both light and darkness.

mM1.2-

i.o-

0.8-

0.6-

0.4-

o.o-i

T

X

. X .v^

El dark

H dark+hyperoxia

• llght+hyperoxia

• light

T

I

-

>

Ii•

>

t

tT

Lacv GlUa-v Oxya-v

FIGURE 3. Blood LacWi and Oxyu.v and plasma Glua.v underfour different experimental conditions in experiment C(mean ± SE; n = 5). In light, the intensity was 180 lux infront of the experimental eye. Oxygen tension in choroidalvenous blood during hyperoxia in light was 36.3 ± 1.63kPa, and oxygen saturation was 99.6%. Mean arterial bloodpressure during the different experimental conditions were88 ± 5 mm Hg, 83 ± 4 mm Hg, 79 ± 3 mm Hg, and 76± 4 mm Hg (in chronological order). Marks indicate thatdifferences between the indicated and preceding values arestatistically significant (paired Student's Hest). *P < 0.05;T/>< 0.01; %P< 0.001.

DISCUSSION

Effects of Light on Lactate Formation in theOuter Retina

Previous experiments in monkeys16 have shown thatconstant light tends to reduce glucose consumptionin the outer retina but does not affect choroidal bloodflow. Table 1 and 2 indicate that the situation is similarin cats. It is clear then that stepwise reductions inlactate production, rather than increasing choroidalblood flow, were the reasons for die results presentedin Figure 1. Most of the reduction took place fromdarkness to approximately 5 lux. According to Linsen-meier (personal communication, 1994), this is therange within which there is saturation of the rods, asunder our study conditions, and, most interestingly,it is also the range within which light causes alkaliniza-tion of the extracellular space in the outer retina.10

There can be little doubt that much of the alkaliniza-tion is caused by the inhibition of glycolysis, as sug-gested by Yamamoto et al.10 In their experiments, ittook approximately 3 minutes for the light-evoked al-kalinization to reach a plateau and 8 to 12.5 minutesfor the pH to return to the dark-adapted level, when

TABLE 3. Adenosine TriphosphateProduction in the Outer Retina inDarkness and Light*

ATP (byoxidation)

ATP (byglycolysis) Total

Glycolysis

Dark 1.19 ± 0.233 0.409 ± 0.049 1.599 26Light 0.995 ± 0.109 0.253 ± 0.023 1.248 20

* Calculated using the data for oxygen and glucose consumptionand lactate formation in Table 2. It was assumed thai theoxidation of glucose results in 36 mol ATP/mol glucose, thatglucose used in glycolysis with lactate formation results in 2 molATP/mol glucose, and that all the oxygen consumed was usedfor energy production. The ATP production (//mol/min) byoxidation and glycolysis in normoxia and the percentagesresulting from glycolysis were calculated.



the light was switched off. Similar time courses wereobserved in the current experiments, lending furthersupport to the hypothesis that, in cats, light inhibitsglycolysis in the outer retina.

Aerobic and Anaerobic Glycolysis

In experiments by Linsenmeier and Yancey,17 it wasfound with oxygen-sensitive microelectrodes that hy-peroxia resulted in very high values of oxygen tensionin all layers of the retina in light and in darkness. Inour experiments, lactate formation was reduced butnot abolished by hyperoxia, indicating that under nor-moxia some lactate formation was in fact caused byhypoxia in light and in darkness. Assuming that allthe lactate formation remaining in hyperoxia was aer-obic and that the difference between normoxic andhyperoxic lactate formation was caused by anaerobiclactate formation, the results presented in Table 2 andFigure 3 can be used to estimate the relative impor-tance of aerobic and anaerobic lactate formation inlight and in darkness. Table 2 shows that in the experi-ments in which blood flow was determined, total lacticacid formation in light was 0.253 /umol/minute. Aero-bic lactate formation (under hyperoxia) was 0.164//mol/minute, and the difference between normoxiaand hyperoxia, 0.089 ^/mol/minute, represents anaer-obic lactic acid formation. Figure 3 shows that in dark-ness the Lacv.a under normoxia was 1.080 //mol/mland that under hyperoxia it was 0.748 /^mol/ml. Themean difference, caused by anaerobic lactate forma-tion, was 0.332 //mol/ml. Assuming constant bloodflow, anaerobic lactate formation in darkness was ap-proximately one third the total (0.089/0.253), whichis similar to the conditions in light (0.332/1.08). Evi-dence for insufficient oxygen supply to the outer ret-ina in normoxia has been observed in pigs18; in cats,oxygen consumption in the photoreceptors in hyper-oxia has been reported to be similar to that in nor-moxia.19

Using the data presented in the Table 2, we calcu-lated the amount of oxygen required to produce thesame amount of ATP by oxidation as that producedby anaerobic glycolysis, and we found that, in light,oxygen consumption during hyperoxia seemed to in-crease five times more than would be required. Thissuggests that in hyperoxia, some oxygen was diffusedfrom the choroid and retina into the adjacent tissues.

Site of Light-Sensitive Lactic Acid Production

In the experiment by Yamamoto et al,10 the lowest pHvalues in darkness and the greatest change in lightwere observed in the outer nuclear layer, indicatingthat acid formation in this region is influenced by thedark current. Studies with oxygen-sensitive microelec-trodes7 indicated high oxygen consumption in the in-ner segments, which were rich in mitochondria, and

no oxygen consumption in the outer nuclear layer.There can be little doubt that there is light-dependentaerobic glycolysis in the nuclear layer of the photore-ceptors. However, this is not the only possible sourceof lactate. Under aerobic in vitro conditions, there islactate formation in the Miiller cells and metabolismof lactate to fuel oxidative metabolism in the attachedphotoreceptors.20 Lactate is produced also in theouter segment21'22 and in the pigment epithelium.2'"'1

In the current study, the protons produced in theretina seemed to be eliminated by the pigment epithe-lium. Measurements of arteriovenous pH differencesin the current study indicated less acidification ofblood passing through the choriocapillaris duringlight exposure than in darkness. Lactate and protontransport mechanisms, which may be involved in suchtransport, have been demonstrated in frog and humanfetal pigment epithelium.2a~27

Oxygen Consumption in Darkness and Light

In this study, oxygen consumption in the choroid andouter retina in darkness, measured from the choroidalside, was 0.198 ± 0.037 //mol/minute, correspondingto 4.4 ± 0.8 /il/minute. The figure in light was 3.7 ±0.4 //1/minute, which is a more realistic value than aprevious estimate of 5.6 /il/minute, based on bloodflow determinations in one set of experiments15 andOxya_v estimated in another.28 Oxygen consumption inthe choroid is much lower than that in the retina,probably approximately 0.1 //1/minute.5 Oxygen pro-files in the retina indicate that in the dark, 90% ofthe oxygen consumed by the photoreceptors comesfrom the choroid, and the rest comes from the ret-ina.29 It becomes possible to give an estimate of theoxygen consumption of the photoreceptors. In ourexperiments, the wet weight of the retina was 0.151 ±0.046 g (n — 11). On the assumption that the outeravascular retina represents 50% of the total weight,and that 90% of its oxygen consumption is suppliedby the choroid, the oxygen consumption of the photo-receptors in the dark can be calculated to be approxi-mately 6.3 ml/100 g per minute. This is between thevalues based on oxygen profiles in normoxia, whichrange from 3.7 to 7 ml/100 g per minute,6'719'30'31 andit is higher than in vitro observations in dark-adaptedrabbit retina (lacking pigment epithelium) of approxi-mately 4.4 ml/100 g per minute12 and in dark-adaptedphotoreceptors from rats of approximately 4.2 ml/100 g per minute.32 However, if the differences inmethods, assumptions, and structures are taken intoaccount, the agreement is reasonable.

In our study, the amount of oxygen delivered bythe choroid was reduced by 16% from darkness tolight under normoxia. Under hyperoxia, there was a19% reduction in the Oxya.v, indicating a similarchange in oxygen delivery. Only the change under



hyperoxia was statistically significant. The reason forthe greater variability under normoxia may be agreater methodological error: The error in the deter-mination of oxygen saturation played a greater roleunder normoxia than under hyperoxia, when the ef-fect was calculated primarily from changes in physi-cally dissolved oxygen.

It is interesting to compare our figures for oxygensupply from the choroid in light and darkness withfigures for oxygen consumption in the photoreceptorsunder these conditions. The percentage reduction inoxygen consumption by the photoreceptors in light,calculated from oxygen profiles, ranged from 40%7 to50%29 and, most recently, 64%.l9 These values clearlyare higher than our values for the reduction in oxygensupply from the choroid. This might be the effect ofdifferences in anesthetics and other drugs used in theexperiments. However, in three control experimentsthat used the same anesthetic and muscle relaxant asin the studies with oxygen electrodes619 and withoutindomethacin, light caused reductions in oxygen con-sumption of 19%, 31%, and 42%. It seems clear thenthat light reduces the oxygen consumption of the pho-toreceptors a good deal more than it reduces the oxy-gen supply from the choroid. Part of this differencemay be explained by the observation that light causesan increase in the region supplied by the choroid.29

It is interesting, too, to consider some recent ob-servations in other species. Observations in rats dem-onstrated that in the pharmacologically isolated outerretina, light caused a 37% reduction in oxygen con-sumption.32 Experiments by Winkler22 indicated onlya small reduction in oxidation for the whole retina.In monkeys, a 16% to 36% reduction was observedwith the lower value in the fovea.33 In rabbits, Stefans-son34 found an 8% reduction in retinal oxygen con-sumption in light, and experiments with determina-tions of Oxya.v and blood flow4 failed to demonstrateany clear effect. Ames et al,12 in isolated retina, founda reduction by 28%; this seemed to be the net effectof inhibitory and stimulatory effects.

Adenosine Triphosphate Production andGlucose Consumption

The methods used in this study to evaluate ATP pro-duction and the contributions by glycolysis were notidentical to those used in some previous studies. Nev-ertheless, as shown in Table 3, the total ATP produc-tion generated by oxidation and glycolysis in light was22% lower than that in darkness, similar to that dem-onstrated in rabbits12 and rats.22 In the current study,the percentage of ATP production generated by gly-colysis in cats was close to that seen in rabbits,12 andfrogs35 but lower than that reported in rats.22

It was calculated from the data of the currentstudy that under conditions of normoxia and dark-

ness, 78% of the glucose was used for glycolysis withlactate formation, 14% was oxidized, and 8% was leftunaccounted for. The amounts of glucose unac-counted for may have been used in the synthesis ofamino acids.20'36 In light, 25% more glucose was calcu-lated to be used for oxidation and glycolysis than wasobserved experimentally. This result is a reminder thatthe assumptions underlying the calculations are provi-sional. As mentioned, part of the carbohydrates con-sumed by the outer retina may derive from sourcesother than the choroid, including the Muller cells,20

by a gluconeogenic pathway,37 and oxygen may beused for purposes other than oxidation of glucose;Cohen and Noell38 estimated that, at most, 30% of theoxygen uptake of the rabbit retina in vitro was usedin oxidation of noncarbohydrate material. In pigs,5

26% of the glucose consumed in the outer retina inlight was oxidized, whereas in rabbits,39 the corre-sponding figure for the nonvascularized part in dark-ness was 10%, with 39% glycolyzed and 51% unac-counted for. In rats, aerobic glycolysis accounts forapproximately 90% of the glucose consumption.22

It is clear from this discussion that there are con-siderable differences between species in their use ofglucose in the outer retina but that lactate formationin the retina is a feature in common. The effects oflight on oxidation and glycolysis seem to vary withmore marked net effects in cats than in rats and rab-bits. However, in all the species discussed, most ofthe energy release from glucose in the outer retina iscaused by oxidation.

Key Words

glucose, glycolysis, lactate, oxygen, retinal metabolism

References

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Glucose metabolism in cat outer retina. Effects of light and hyperoxia.

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