A"? 0 7 933} I / APaWsT‘flfhf ABSTRACT AEROBIC AND ANAEROBIC GLYCOLYSIS IN THE BETINA OF A TELEOST (Salmo gairdneri) by Dennis A. Baeyens Carbohydrate metabolism was investigated in the rainbow trout retina by measuring glucose utilization and lactic acid production under aerobic and anaerobic condi- tions and varying glucose concentrations. The retinas were incubated in various culture media for varying time periods at 130. The teleost retina was found to be capable of pro- ducing lactic acid in the presence of high oxygen tensions, thereby confirming the occurrence of aerobic glycolysis. More glucose was utilized (hl.5$) and more lactic acid was produced (33%) under an anaerobic environment than an aero- bic environment. The inhibition of glycolysis by oxygen (Pasteur effect) was thus substantiated in the teleost retina. The inhibition of the tricarboxylic acid (TCA) cycle by high levels of glucose (Crabtree effect) was verified in the teleost retina by varying the glucose con- centration of the incubation media. Under physiological levels of glucose in the media all carbohydrate metabolism could be-accounted for by glycolysis. At increasing levels of glucose in the media, up to 100 mg%. the TCA cycle be- came predominant in carbohydrate metabolism. At levels in excess of 150 mg% glucose an inhibition of TCA cycle acti- vity was demonstrated. Glycolysis in the teleost retina was affected by the type of incubation media employed. In modified Medium 199 (pH 7.0) there was more glucose utilized and lactic acid produced than in modified Mammalian Krebs Saline Medium (pH 7.6). It was further demonstrated that glycolysis is dependent upon the integrity of the retinal cells. being almost completely abolished by cellular disruption. Finally, it.was shown that an exogenous source of lactic acid could not be utilized by the teleost retina as-a substrate for the TCA cycle. The Crabtree effect is only evident under glucose concentrations in excess of those normally encountered in ‘zizgpand probably does not have a great deal of physiologi- cal significance in the teleost retina. The inability of the retina to utilize lactic acid as a substrate coupled with the aerobic.glycolysis occurring in the retina would result in the accumulation of acid metabolites. The acid metabolites would shift the oxygen dissociation curve to the left causing the release of oxygen from hemoglobin in.the choroid region. This release of 02 might explain in part the high P02 encountered in the vicinity cf the retina. The Pasteur effect may function as a possible component of a negative feedback control loop functioning to control the set point for the oxygen concentrations generated in the teleost eye. AEROBIC AND ANAEROBIC GLYCOLYSIS IN THE RETINA OF A TELEOST (Salmo gairdneri) By :) .e-J‘ Dennis A? Baeyens A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1970 Dedicated to my parents. Hector and Marion Baeyens. whose moral and financial support made this study possible. 11 ACKNOWLEDGEMENTS The author would like to extend his sincere grati- tude to Dr. J. B. Hoffert for his helpful suggestions which contributed greatly to this work. The author would further like to express his appre- ciatiOn to Beverly Baeyens, Esther Brenke, Kay Johnson and Glenn.Knight for their assistance in the typing of this 6.1 8 8 ertati on“. The author is also indebted to the National Insti-- tutes of Health for the support of this work through grant No. EY 00009 from the National Advisory Eye Council. 111 TABLE OF CONTENTS LIST OF TABLES. e o . e . . . o o o o e o e . . LIST OF FIGURES e INTBOIDCTIONoecoococcocoococoa. LITERATUREREVIEVOOoececcocoocco Anatomy of the Retina and Related Structures Chemical Environment of the Retina . . . . General Aspects of Retinal Metabolism . The Contribution of the HMP Pathway to Retinal ”Subclism e e o o o o e o 0 Factors Effecting Retinal Metabolism Pasteur Effect . . . . . . . . . . o Crabtree Effect m . . . . . . . . . . umms AND nmons . . Experimental Rationale Experimental Animals . Preparation of Tissues Gassing Procedure . . . Analysis of the Media . Incubation Procedure . Procedure for Examining the Effect of Varying Gas Concentrations . . . . Procedure for Studying the Effect of Sonification Procedure for Examining the Effect of Various Glucose Concentrations - . . . . . . . . . Procedure to Study the Effect of Iodoacetate Statistical Analysis 0 v - 0 . - . . . . . . BESULTSeocoeceooeoooooeoooo Effect of varying 02 Concentrations on Retinal HetBbOIIBMooooocccocooecooo One-Half Hour Incubations . . . . . . . One Hour Incubations. o o o o e e o e 0 Three Hour Incubations. . . . . . . . . Effect of Aerobic and Anaerobic Treatment. 0n Cardiac and Kidney Tissue Metabolism 0 Effect of Sonification on Retinal Metabolism The Effect of Varying Glucose Concentrations on Retinal Metabolism e c o c o o o o o 0 Effect of Iodoacetate on Retinal Metabolism. ..... iv 3 ....... Page vi vii 33 35 37 37 37 38 1&1 Mt “5 ”DISCUSSION 0 0 SUMMARY AND CONCLUSIONS 0 o . . . LITERATURE CITED . . . O . O O O 0 APPENDIX I APPENDIX II Incubation Solutions Regression Analyses Page 56 7O 72 76 79 Tabl 5. 7. Table l. 8. 9. LIST OF TABLES Glucose utilization and lactic acid pro- duction by retinal tissue_utilizing modified Medium 199 for i hour at 13c . . . . . . . . Glucose utilization and lactic acid pro-. duction by retinal tissue utilizing modified Medium 199 for 1 hour at.l3C . . . . . . . . Glucose utilization and lactic acid pro- duction by retinal tissue utilizing modified Medium 199 for 3 hours at 130 . . . . . . . Metabolism of cardiac tissue under anaerobic and aerobic conditions in modified Medium 199 for 1 hour at 130 e o o o o o o o o o c Metabolism of kidney tissue under anaerobic and aerobic conditions in modified Medium 199 for 1 hour at 130 . . . . . . . . . . . Effect of sonification on anaerobic retinal metabolism in PBS with 100 mg% glucose for 5 hours at 13C 0 e c o c c o c o e e e c o 0 Effect of varying glucose concentration on retinal metabolism in modified Mammalian Krebs Saline Medium with bicarbonate buffer for 1 hour at 130 o c o e o o e e e e c c 0 Summary of the effects of aerobic and anaerobic conditions and differing glucose concentrations on retinal metabolism at 130 Chan es in lactic acid concentration in modi ied Mammalian Krebs Saline Medium forti- fied with lactic acid in the presence of iodoacetate at 130 for 1 hour . . . . . . . Page 39 #0 #2 “3 an 46 50 53 55 Fig; l. 9. 10 ll, Figure l. 2. 3. 4. LIST OF FIGURES Scheme demonstrating the three metabolic pathways occurring in the teleost retina. . Tissue incubation apparatus . . . . . . . . Gassing apparatus . . . . . . . . . . . . . Regression line showing glucose utilization in a modified Mammalian Kreb Saline Medium ranging in concentration from 50-200 mg% glucose for 1 hour at 130 . . . . . . . . . Regression line showing lactic acid pro- duction in a modified Mammalian.Krebs Saline Medium ranging in concentration from 50-200 ng for 1 hour at 130 , , . . Regression line showing glucose/lactate ratios in a modified Mammalian Krebs Saline Medium ranging in concentration from 50-100 glucose for 1 hour at 130 . . . . . . . Regression line showing glucose/lactate ratios in a modified Mammalian Krebs Saline Medium ranging in concentration from 100-200 glucose for 1 hour at 130 . . . . . . . Graphs in Appendix 2 8. 9. 10. 11. Aerobic glucose utilization(95$ 02 - 55 002) by retinal tissue at 130 o e c o o o o e o Aerobic lactate production (95$ 02 - 5x 002) by retinal tissue at 13C c e c c o o c o e Anaerobic glucose utilization(95% N2 - 5% 002) by retinal tissue at 13C 0 c o c e o o c o Page 12 27 30 48 49 51 52 80 81 82 Anaerobic lactate production (95% N2 - 51 002) 8 3 by retinal tissue at 13C 0 e o m e e e c 0 vii INTRODUCTION Harburg (1927) showed that the mammalian retina had the highest rate of respiration (Q02) and anaerobic glycolys- is, as measured by lactate production, of any tissue he studied. It is now known that no other tissue in the body, with the possible exception of the brain and the gonads, has a higher rate of anaerobic glycolysis than the retina. Since the discovery of the high rate of glycolysis by the retina, considerable interest has been attached to this as a possible source of energy for the photochemical visual processes. Moreover the retina has a unique capacity for aerobic glycolysis with the production of lactic acid in the presence of oxygen that is only equaled by neoplastic tissue. It is well known that the retina, which is primarily nervous tissue, utilizes glucose as the predominant sub- strate for its energy metabolism. Glucose is catabolized in the retina by two major pathways. The predominant metabolic pathway employed is the Embden-Meyerhof-Parnas (EMP) scheme. Under anaerobic conditions the glycolytic pathway (EMP) results in the breakdown of glucose to lactate as the end product. Under aerobic conditions, those.nor- mally occurring in zizg, glucose can be completely oxidized by the second major pathway, the tricarboxylic acid cycle (TCA), with the formation of carbon dioxide and-water and the liberation of substantially more energy than via the EMP pathway. A third minor metabolic scheme employed by the retina to break down glucose is the hexose monophosphate shunt (RMP). The reactions of the HMP shunt result in the formation of 002, reduced pyridine nucleotides (NADPE), various intermediates of the EMP pathway, and pentose sugars. Oxygen tension (Pasteur effect) and glucose avail- ability (Crabtree effect) in-the immediate environment of a tissue are-known to determine its predominant carbohydrate metabolic pathways. Within the past fifteen years consider- able interest has been generated in the study of these two factors. Due to its high metabolic rate-the avascular teleost retina-is-an ideal tissue for the in;zitgg study of nervous tissue metabolism. Even at the reduced temperatures.nor- mally encountered by this poikilothermic organism the high metabolic activity of the retina allows one to easily quantitate glucose metabolism. It can be readily removed from-the eye-with minimal injury to the cells and is thin enough to allow for adequate diffusion of metabolites and therefore negates the necessity of slicing and injury re- sulting therefrom. It is known that the mammalian retina is capable_of aerobic glycolysis, but few attempts have been made to demon- strate aerobic glycolysis in the retina of poikilotherms. Oxygen tensions have been measured and were found to be high in the choroid region of the teleost eye (Fairbanks, Hoffert, and Fromm, 1969). It was hypothesized that these high oxy- gen tensions were due to a shift of the oxygen dissociation curve to the left which caused a release of oxygen from hemoglobin coupled with a counter current diffusion multi- plying system. A shift in the oxygen dissociation curve to the left could be accounted for by the formation of acid metabolites. These workers further speculated that lactic acid derived from glycolysis and 002 from the TCA cycle were the acid metabolites causing this shift. To help explain these phenomena a high rate of aerobic glycolysis which would provide the needed lactic acid was assumed to be present in the teleost retina. In only one study, in the abundance of work on re- tinal metabolism, has the measurement of aerobic glycolys- is in the teleost retina been attempted. In this study de Vincentiis (1951) measured lactic_acid production in a marine teleost and found that aerobic glycolysis was lhfi less than anaerobic glycolysis. Due to the paucity.of data presented in this study it is impossible to draw any definite conclusions as to the statistical significance of the magnitude of either aerobic or anaerobic glycolysis. In an erroneous interpretation of de Vincentiis' data Davson (1962) concluded that there was no aerobic glycolysis occurr: study I retina the £02 113m 0: occurring in the teleost retina. The above facts justify a study of both aerobic and anaerobic glycolysis in the trout retina c The objectives of this study were to investigate the following questions concerning the carbohydrate metabo- lism of the teleost retina: l. 2. 3. what effects do gas mixtures of different.oxygen concentrations have on.glucose utilization and lactic acid production by the teleost retina? Does the teleost retina demonstrate a.Pasteur effect? what is the rate of glucose utilization and latic acid production by the teleost retina as affected by duration of incubation and tissue disruption? Does the teleost retina demonstrate.a Crabtree effect? Is the teleost retina in the absence of glucose capable of utilizing lactate? LITERATURE REVIEW Anatomy of the Retina and Related Structures In no other vertebrate class does the retina vary more in thickness than in the teleost (Walls, l9h2). The nervous elements of the retina including cell bodies, dend- rites, and axons which are usually mixed together throughout the CNS, have been sorted into discrete layers. Much of the variation in thickness of the teleost retina is caused by variation in the number of conductive elements per number of visual cells. The horizontal cells of the teleost retina have small bodies and are slender. Where cones predominate, as in the case in trout, conductive and integrative elements pile up resulting in a thick inner nuclear layer and a com- pact ganglion cell layer. In almost all teleosts there are three types of visual cells. rods, single cones, and twin cones. The twin cones are peculiar to the teleosts and appear to function on exposure to bright light. The teleost retina in contrast to the mammalian reti- na is poorly vascularized. However, due to the high metabolic rate of the retina, it seems obvious that this nervous tissue would be sensitive to any interference with its supplies of raw metabolic materials and oxygen. These come in the most part from the direction of the choroid which is devoted to the nutrition of the visual and neural cells. The choroid, in addition to its pigmented vascular layers, characteris- tically contains the choroid gland. The choroid gland is horseshoe shaped and surrounds the optic nerve. It only exists when the remnant of the first gill arch, the pseudo- branch, is present. Blood flows into the pseudobranch, is directed into an efferent artery (opthalmic artery) which passes through the sclera, along with the optic nerve, and breaks up into a set of capillaries (rete mirabile) in the choroid gland (Barnett, 1951). From these capillaries the blood enters the chorio-capillaris circulation, returning to the rate mirabile and leaving the eye by way of the opthalmic vein. The falciform process is a ridge, formed of pigmented and vascular choroidal tissue, which projects upward.into the vitreous cavity from the floor of the eyeball. The arterial blood supply of the falciform process comes from the lentiform body. The blood supply to these structures is derived from the retinal artery and does not pass through the pseudobranch (Barnett, 1951). A possible function of the falciform process is to provide the inner layer of the retina with nutrients. Glucose passes out of the falciform process, diffuses in all directions through the vitreous, to be absorbed by the retina. Chemical Environment of the Retina lngizg P02 measurements were made in the eye of rainbow trout (Fairbanks, Hoffert and Fromm, 1969jdand oxy- gen tensions behind the retina were found that were 201 higher than arterial blood and 3.5x those of the environ- mental water. This ability to concentrate oxygen in the eye is associated with the development of the choroidal_ rete mirabile (Wittenburg and Wittenburg, 1962). Eairbanks et a1. (1969) suggest the participation of the choroid gland, erythrocyte and retinal carbonic anhydrase as the major components in the oxygen concentrating mechanism of the teleost eye. Adler (1931) found that the sugar content of the normal vitreous was always considerably lower than that of the aqueous or blood serum in cats. All but the anterior portion of the vitreous is bounded by the retina.. The fact that the retina has such high glycolytic activity explained the low concentration of sugar found in the vitreous. The major sources of glucose to the mammalian retina.are derived from the choroidal and retinal circulation, whereas in the teleost the falciform process functions in place of the re- tinal circulation. Adler in the same study demonstrated that degenerative changes took place in the retina, and the aqueous.and vitreous glucose concentrations were higher than normal after the optic nerve had been severed. The glycolytic activity of the atrophied retinas was considerably less than that of the normal. These facts support the hypotheses previously made that the low concentration of glucose in the vitreous is due to the high rate of glycolysis of the normal retina. Futterman and.Kinoshita (1959) found that whole cattle retinas could use lactic acid in the presence of iodoacetate for cellular respiration in zitrg. There are two exogenous sources of lactic acid which the teleost retina may use in zizg. One source is from the blood. Blood levels of lactic acid have been measured in unexercised two year old sockeye salmon and were found to average 19.5 mg% (Black, 1957). The value goes as high as 200 mgfl in exercised fish. Adler (1959) found that changes in the chemical environment of the blood are reflected, after a time lag, in the vitreous body. Therefore the retina has a supply of lactic acid from the blood. A second source of lactic acid for the retina comes from the aqueous humor. Kronfeld and Bothman (1928) observed that anaerobic gly- colysis occurs in the mammalian lens. Anaerobic glycolysis was also found to occur in the trout lens (Hoffert and Fromm, 1970). It can therefore be assumed that lactic acid is present in the aqueous humor of the trout. Adler (1959) cites data which indicates that substances which are manu- factured by the tissues of the eye freely pass into and out of t any {or lact 1301 vitl int: am] {01” rat. of the anterior and posterior chambers. This indicates that any lactic acid in the aqueous may pass into the vitreous for utilization by the retina. The two exogenous sources of lactic acid would only be available if the concentration of lactic acid in the retina is lower than that found in the vitreous or plasma, thus setting up a diffusion gradient into the retina. Glycogen was first detected in the retina of the frog by Ehrlich in 1833, and has now been found in the re- tinas of all vertebrate phyla. A comprehensive attempt was made to compare the content and distribution of glycogen in the retinas of a variety of species by Kuwabara and Cogan in 1961. They found that the variations in glycogen con- tent in different species depended on the blood supply, and the amount of glial tissue in the retina. Highest glycogen contents were found in those retinas which had a.high tissue to capillary ratio. These retinas accumulated glycogen dur- ing periods of low glucose utilization to be used subsequently when they were unable to meet their substrate requirements by diffusion of glucose from the blood stream. In the same study Kuwabara and Cogan, arranging animals in increasing retinal tissue to capillary ratios, found abundant glycogen in the guinea pig and rabbit; mode- rate glycogen in the human, cat, and fish (unknown.species); little glycogen in the rat: and none in the mouse. Glycogen 10 was more abundant in the peripheral than central portions of the vascular retinas. This result is attributed to the poorer vascular supply in the periphery of the retina. It was found that the glycogen in the fish retina was associated with Muller's cells and fibers, and was also present in the horizontal cells. It was further shown that fish retinas were capable of synthesizing glycogen when incubated in‘zitrg in a media high in glucose and potassium. The synthesis of glycdgen occurred predominantly in Muller's cells and fibers and in the horizontal cells. The problem still remains to discover if glycogen can be used as an endogenous substrate for glucose formation in the teleost retina. General Aspects of Retinal Metabolism Many measurements have been made of the RQ of the retina, and the values found closely approximate 1. An RQ of 1 implies the oxidation of carbohydrates. The major scheme for glucose degradation in the retina, both aerobic- ally and anaerobically, is glycolysis. Under anaerobic conditions glucose is converted to lactic acid via the EMP pathway with little production of energy. .Aerobically glucose may be oxidized to carbon dioxide and water via the TCA cycle with a much higher energy output. A third pathway for glucose catabolism, which is probably of minor.signifi- canoe in the retina, is the HMP shunt. The HMP shunt diverges 11 from glycolysis at the level of glucose-6-phosphate (Fig. l). A great deal of work has gone into the determination of the contribution of the visual cells to the overall metab- olism of the retina. Noell (1952) used retinas in.which the visual cells had been inhibited by iodoacetate and found that half of the in vitro 002 and half the lactic acid production of the adult rabbit's retina was contributed by the visual cells, whereas anaerobically about two thirds of the lactic acid comes from the inner layers. Noell used these obser- vations to explain why the inner layers are relatively high in glycolytic enzymes and relatively low in enzymes of the citric acid cycle. Fonner, Hoffert and Fromm (1969) used histochemical evidence to show that the EMP and TCA pathways are concentrated in the photoreceptor ellipsoids of the rainbow trout. They suggest that the HMP shunt is in the inner layers of the retina and not the ellipsoids. The Contribution of the HMP Pathway to Retinal Metabolism There has been some controversy concerning the im- portance of the HMP pathway in retinal metabolism. .A few workers have attributed as much as 25$ of the metabolism of the retina to the HMP shunt, while others have found it operating to a negligible extent. The method used to test the occurrence of this metabolic pathway consists of com- paring the rate of appearance of label in the respired 002 12 Glucose “W e——c-c-c-LL:-c-P——+ ’m‘m HMP SHUNT ) TPNE 4///// ‘A_ 3-phgppho- I'D-glycorate A ‘ cs -co-coo- [was I * an... TCA CICLB .” .—-’ FIGURE 1.--Scheme demonstrating the three metabolic pathways occurring in the teleost retina. 13 derived from l-luC glucose with labeled 002 produced from 'other positions. The molecules of 002 produced from the' HMP shunt pathway all have their origin in 0-1 of glucOSe. The rate of glucose 6-140 oxidation is used as a standard for comparison with glucose 1_lhc because the l-C and 6-0 carbons, when only the TCA cycle is operational, appear in 002 at equal rates. Any deviation of the ratio l-C/6-C above unity indicates some participation of the non-glycoly- ltic pathway (HMP) (Hoar and Randall, 1969). ' To determine the significance of the HMP pathway in .the aerobic metabolism of glucose by the retina, intact cattle retinas were incubated with glucose-l-lnc cr.glucose- 6-1uCC.- No evidence of any significant preferential oxida- 'tion of the carbon 1 atom of glucose was observed by Futterman and.Kinoshita (1959). These results suggest that- the HMP pathway does not play a significant role in.the ' utilization cf glucose by the retina. In‘a study by Kerley and Rahman (1961) it is shown that the ratio of luCCZ- I 1-1“0 to formed during incubation with glucose labeled at that formedwith.6-luc was near unity in ox retinas. How- ever, these workers found that the difference in the isotope content of the lactate produced from the two glucose samples .was greater than that found by Putterman and.Kinoshita (1959). ‘This, together with low valuestund for the specific.aotivity of 002, has led Kerley and Rahman to conclude that the HMP 14 shunt may be of more importance for glucose metabolism in the retina than has been previously reported. Although the HMP shunt does not hold the importance it has in corneal 1 epithelium or in the lens, some glucose is metabolised by this route, and under aerobic conditions it is not suppressed completely as has been suggested by Futterman and Kinoshita (1959). Kerley and Rahman conclude that one glucOsecmole- cule in four is metabolized by the HMP shunt pathway in the ox retina. 1 A The capacity for‘retinal oxidation of glucose by the HMP shunt was also investigated by Cohen and Noell (1960). . The HMP shunt path operated in both adult and young rabbit retinas under in.zitgg conditions but was severly restricted 'by the availability of electron acceptors. The conclusion . was drawn that at both ages the capacity for glucose oxida-_ tion.by the HMP shunt pathway was small. Von Holt et a1. '(1959) used bovine retinas to see if there was a difference in the oxidation time for 1-1uC glucose and 6-1uC glucose. It was found that the retina shows only a small preponderance of C-1 oxidation which again indicates that the HMP shunt_ pathway holds little significance in retinal-metabolism. -Hoffert and Fromm (l970),using both diseased and ' normal retinas, studied retinal metabolism in rainbow and lake trout. Their results indicate that slightly more glucose I_was metabolizedby the EMP pathway than the TCA cycle. In 15 both the diseased and normal trout retinas the HMP shunt accounted for less than 5.5 i of the total glucose metabo- lizede Factors Affecting Retinal Metabolism Kornblueth, Yardini-Yaron and Wertheimer (1953), using the retinas of white rats found that glucose.utiliza- tion is dependent on the integrity of the cells. It was discovered that after the retina was cut into.20npieces or more, the utilization of glucose was not appreciably dif- ferent from that found in intact retina. When.the tissue was homogenized, however, the glucose utilization fell to zero. In the study of retinal metabolism, when one re- moves the retina by severing the optic nerve, the ganglion cell bodies themselves are not directly injured because only the axone is cut. This injury to the retinal ganglion cell axone occurs at some distance from the cell body, and does not cause severe trauma to the neural elements of the retina. Retinal metabolism is known to vary 12.21322 with the type of incubation media used. As early as.l936, Laser used rat retinas and found that respiration is about twice as high in bicarbonate buffered Ringer as it is in phosphate buffered Ringer. In a later study, Craig and Beecher.(1943) 16 used the retinas of mongrel white rats to study lactic acid production in a medium containing phosphate.. They found that retinal 002 in a phosphate medium were sensitive to P02. Craig and Beecher in the same study experimented.with retinal metabolism in a bicarbonate medium subjected to varying oxygen tensions and found no significantlchange in respiration but glycolysis increased to nearly the anaerobic level when the oxygen tension was lowered from 95 $.to 5 fl. Kornblueth et al. (1953) found that serum and.Krebs-Ringer bicarbonate were the best media for glucose utilization by the retina of white rats. They surmised that bicarbonate was necessary for the synthesis of the Krebs cycle inter- mediates. Differing gaseous environments may affect.retinal metabolism. In 1936, Laser measured the respiration of rat retinas under varying gas concentrations. In 95 % nitrogen with 5 5 oxygen, he found the respiration.to be normal. It was also demonstrated that 95 % carbon menoxide with 5 Z oxygen leads to no inhibition of retinal respira- tion. In a later study Craig and Beecher (19h3) discovered‘ oxygen uptake of the nearly intact rat retina ithhe presence of glucose was doubled by raising the carbon dioxide tension- from 1 f to 5 x. This result led the investigators to speculate that carbon dioxide may be important attan.inter- mediate stage in metabolism for the synthesis of dicarboxylic acids. 17 The effect of light on retinal metabolism.was studied by Lindeman in 19h0. The Warburg manometric method was used to measure oxygen consumption of frog retinas. The results indicated that there was no consistent difference in the rate of oxygen consumption in the dark as compared with the light when the retinas were observed under alternat- ing periods of darkness and light. Iodoacetate is a metabolic inhibitor which is known to block the EMP pathway. The site of action is.the in- hibition of glyceraldehyde-P-dehydrogenase (Mahler.and.Cordes, 1966). Lenti (l9#0) used minced calf retina suspended in Ringer solution to study the effects of iodoacetate on retinal metabolism. A 0.02M solution of iodoacetate was found to completely inhibit glycolysis. The fact that iodoacetate inhibits glycolysis has proved to be an im- portant analyticaltool in the study of retinal metabolism. Noell (1952) used iodoacetate in an attempt to demonstrate that aerobic glycolysis predominates over the TCA.cycle in yielding the energy for certain functions-of the retina. His findings suggest that in the higher vertebrates (rabbit and cat) glycolysis does in fact provide the predominate support of retinal functions. In the lower vertebrates (frog), however, aerobic glycolysis is of minor importance compared with the TCA cycle in maintaining retinal functions. To demonstrate experimentally that glucose or glycogen was 18 not the principle endogenous substrate of bovine retinas, a study of the effect of iodoacetate was undertaken (Futterman ,and.Kinoshita, 1959). These workers discovered that during the first hour of incubation 002 could be supported by en- dogenous substrate even in the absence of an-active glycolys- is. These experiments suggested that lactic acid was a major endogenous substrate contributing to respiration in the presence of iodoacetate. various chemical substances have pronounced effects on retinal metabolism. Using the Summerson manometric technique, Bobbie and Leinfelder (1998) did simultaneous determinations-of Q02 and glycolysis in white rat retinas. They attempted to simulate an anaerobic environment through the action of cyanide which is an inhibitor of the cytochrome oxidase system. They found that when a concentration of k 10'3n cyanide was added to the media there was an inhibition of the Pasteur effect-and an increased rate of anaerobic glycolysis. When a high concentration of cyanide was added (lo-2n) there was a decrease in anaerobic glycolysis as measured indirectly by C02 production. Lenti (1940) found that phlorizin (0.1 S) did not affect glycolysis in calf retinas ig‘zitgg. In the same study it was found that although glyceraldehyde was con- verted to lactic acid it had an overall inhibitory effect on glycolysis when added to the medium. Von Holt et a1. 19 (1959) experimented with the effect of insulin on bovine re- tinal metabolism and found that insulin caused a significant increase in glucose oxidation. Pasteur Effect In all cells that are capable of degrading glucose both in the presence and absence of oxygen, the sugar will disappear, and the lactic acid will be formed more rapidly under anaerobic than under aerobic conditions. This in- hibition of glycolysis by oxygen was first recognized by Pasteur and later confirmed by Heyerhof and Warburg, and is known as the Pasteur-effect. Aerobiosis will remove inorganic orthophosphate (P1) and ADP, a great deal more effectively than will anaerobiosis. A decrease in the availability of P1 and ADP leads to a diminution in the rate of glycolysis and an increase in the rate of gluconeo- genisis (Mahler and Cordes, 1966). The presence of a Pasteur effect has.been sub- stantiated in the mammalian retina, but there is.much controversy concerning its existence in the retina of poikilotherms. Craig and Beecher (1943) subjected the retinas of mongrel white rats to varying oxygen tensions. In a bicarbonate medium, when the oxygen tension was lowered from 95 S to 5 Z there was no significant change in the rate of glucose oxidation by the retinal TCA cycle, but glycolysis 20 was increased to nearly the anaerobic level, suggesting a Pasteur effect. They concluded that the rate of glycolysis is controlled by oxygen tension rather than by the rate of glucose oxidation via the TCA cycle. Using the.Barker and Summerson method for lactic acid determination, de Vincentiis (1951) looked at the effect of high oxygen tension on gly- colysis in the fish retina in zitgg. Two varieties of marine fish were used (gghzlliorhinus and‘ggggpgggg scrofa) and it was found that less lactic acid was produced under aerobic conditions in the retinas of these fish than under anaerobic conditions. This result led de Vincentiis to con- clude that oxygen inhibits glycolysis in the fish retina. thereby confirming the existence of the Pasteur effect. Crabtree Effect The inhibition of the TCA cycle by glucose is re- ferred to as the Crabtree effect. The only nonsnaoplastic tissues for which the Crabtree effect has bean.shown are the . retina and leukocytes (Cohen and Noell, 1959). . Hu and Backer (1957) proposed a possible mechanism for the action of the Crabtree effect. These workers determined the intracellular concentrations of adenine nucleotides, hexose phosphates and inorganic P1 in ascites tumor cells. The addition of glucose to washed cells resulted in a marked fall in intracellular P1 and ADP. Cohen (1957) postulated that the drop in 21 inorganic P1 and ADP observed in the Crabtree effect was caused by a stimulation of glycolysis. A reconstructed system consisting of the glycolytic enzymes and actively respiring liver mitochondria was studied by Gatt. Krimsky and Backer (1956). The addition of glucose to this system resulted in a pronounced inhibition of mito- chondrial respiration. These findings suggested.that the limiting amounts of ADP are shared by the intrae and extra- mitochondrial systems,-and shuttle back and forth between Anthem. An active glycolytic system may deprive mitochondrial respiration of essential ADP. The Crabtree effect is absent in the adult rabbit's retina despite the high mitochondrial respiration (Cohen, 1957). Furthermore, it was observed that during postnatal development of the retina, prior to the formation of the visual cells, glucose inhibited mitochondrial respiration as much as #0 S. The data indicated that the adult retina contains a highly active component of mitochondrial res~ piration which does not share its phosphoralative coefactors with a system of glycolysis of high capacity. MATERIALS AND METHODS Experimental Rationale The experiments were designed to study the carbo- hydrate metabolism of the teleost retina under different conditions. The contribution of glycolysis to retinal meta- bolism was determined by measuring lactic acid production. Total glucose utilization was measured to quantitate the contribution of the TCA cycle to retinal metabolism with the assumption that 1 mole of glucose produces 2 moles of lactic acid in the glycolytic scheme. The total amount of glucose utilized subtracted from the amount that could be attributed to glycolysis would give a quantitative estimate of TCA activity. In all determinations a sample-containing media without tissue was employed to determine the initial level of glucose and lactic acid in the culture media. The values obtained from the media blank were compared with values obtained from the media in which the tissues were incubated to determine the amount of glucose utilized.and lactic acid produced. Glycolysis was measured at varying time periods under both anaerobic and aerobic conditions. The Pasteur effect was studied by subjecting the retinas to aerobic conditions and measuring the amount of glucose utilized and lactic acid produced and then comparing these results with 22 23 glucose utilization and lactic acid production of retinas subjected to anaerobic conditions. The Crabtree effect, in- hibition of the TCA cycle by glucose, was studied by measur- ing glucose utilization and lactic acid production by the retinas during incubation in media of different.g1ucose concentrations. The contribution of lactic acid as-a,snb- strate for retinal metabolism was studied. This determination was made by measuring the disappearance of an exogenous source of lactic acid from the culture media in the presence of iodoacetate which blocks the utilization of both exogenous and endogenous glucose. Experimental Animals Rainbow trout (gglmg‘gairdneri) used in this study were obtained from the Michigan Department of Natural Re- sources at Grayling, Michigan. The 2-2; year old trout. selected were between 9 and 11 inches in length and weighed from 380-h80 grams. The fish were kept in fiberglass lined plywood tanks in a constant temperature room at 13 #_1 C. Dechlorinated tap water constantly flowed into the tanks and was aerated with compressed air filtered through activated charcoal. The photo-period consisted of 15 hours of light and 9 hours of darkness. 24 Preparation of Tissues The trout were killed by cervical dislocation and selected tissues removed. The eye was extracted from its orbit by severing the ocular muscles and optic nerve with scissorsa The eye was then immediately immersed in a petri dish filled with sterile Ringer solution (Appendix 1). While holding the eye with rat tooth forceps. a small incision was made into the periphery of the cornea with the blade of an iris scissors, the incision being continued along the cir- cumference of the cornea. After removing the cornea, the lens was lifted out of the aqueous humor with small curved forcepsi Next, the optic nerve was firmly grasped with iris forceps while a small circular incision was made into the posterior surface of the sclera to one side of the optic nerve with iris scissors. Eye dressing forceps were care- fully inserted into the incision and the retina and choroid were worked free from the sclera by gentle scraping. In some instances cardiac and kidney tissue were re- moved from the trout. To remove the heart an incision was made from the base of the operculum along the mid-ventral line. After the aorta and vena cavae were cut, the heart was lifted from the pericardial cavity by means of forceps and.placed in sterile Ringer solution. Using iris_scissors, four thin slices, less than 1 mm in thickness, were cut from the posterior surface of the ventricle. Slicing of the large 25 tissue mass was necessary to insure the adequate diffusion of the nutrients. The dorsally located opisthonephros kidney was found by continuing the incision posteriorally to the cloacas Scissors were used to cut through the swim bladder and a piece of the middle portion of the kidney,.approximately 10 mm in length and 5 mm in width, was removed and placed in sterile Ringer solution. The loose arrangement of the con- nective tissue-elements and the general flatness of the tissue made further sectioning unnecessary. All surgical ,.instruments which came in contact with the tissues studied were sterilized under 15 psi at 121 C. for 15 minutes in a N04 777 Speed Clave (Uilmot Castle Co.. Rochester, N.Y.). After removal.from the Ringer solution the tissues were blotted dry on sterile No. l Whatman filter paper. During the blotting process as much of the vitreous body was removed from the retinas as was possible without damaging the retinal cells. All tissues were then weighed on a Roller- Smith (0-500 mg) precision balance (Roller-Smith Co., Newark, U.Je) to the nearest 0.1 mg, and placed in 0.5 ml of the .appropriate media. The retinal weights ranged between 290 u.and.450 mg with never more than 50 mg variation between re- tinas from the individual fish. The cardiac tissue slices ‘weighed between 70 and 100 mg while kidney tissue weighed between 90 and lho.mg. The media was contained.in a steri- lized.center well (Fig. 2) constructed of Pyrex glass and 26 FIGURE 2.--Tissue incubation apparatus. 1. 2. Graduated milk dilution bottle (flask) (165 ml). Center well for tissue and media (200 m1)e Black rubber stopper (No. 2). Syringe needle (18 guage, 2.5“ long). 3-way plastic stopcock. FIGURE 3 28 having a volume of approximately 2.0 ml. The center well was attached to a No. 2 black rubber stopper which was fitted into a 165 ml capacity graduated milk dilution bottle (No. 1370.' Corning Glassware Co., Corning, N.Y.). An 18 guage needle fitted with a 3 way plastic stopcock (No. k-75 Pharmaseal Laboratories. Glendale,,California) was permanently inserted _into the.stoppar (Fig. 2). The needle provided the means for gas to enter the incubation flask. The flasks were secured in a wire test tube basket. Gassing Procedure Gas mixtures were made by means of.a water dis- placement spirometer having.a capacity of 1.9 liters (Fig. 3). The spirometer was attached to a mercury manometer which allowed an accurate determination of the pressure within the incubation flasks. The threeeway.plastic-stopcock,was utilized to attach the incubation flasks to a multigoutlet exhaust .manifolde The entrance.of gas from the spirometer.into the partially evacuated incubation flasks was controlled by means of a.three-way glass stopcock (Fig. 3). The vacuum.was created in the flasks by means.of a Cenco-nagavac Vacuum Pump (Central Scientific-Co.,.Boston-Chicago-Toronto). Approxi- mately 95 1 of the.air was evacuated from.the incubation flasks before gas was.readmitted. After the flasks were evacuated and the vacuum stabilized at approximately 720 mm 29 ,FIGURE 3.--Gassing apparatus. 1. Gas inlet. 2. 3-way glass stopcock. 3. Water displacement spirometer (1.9 liters). h. Pressure bottle. 5. Pressure bulb. 6. Cenco-hagavac vacuum pump. 7. Acidified water 8. Multi-outlet exhaust manifold. 9. Mercury vacuum gauge. 10. Gas outlet. 30 nullnlllllllllldllLl 1 L)\\‘ K‘ \3.\\ J1 .415 ere/f x “\\\\\\\\ \ \ \ x 1 [IIAAIILIIIIIJIII \_ :L 1 FIGURE 3 31 Hg below atmospheric pressure. gas was admitted from the spirometer.» As the gas mixture entered the incubation flasks the vacuum was reduced to zero so atmospheric pressure existed in the flasks. The gassing procedure as outlined above was ._repeated three times- when the pressure in the bottles reached atmospheric pressure the plastic.stopcocks were closed and the tissues were ready for incubation. Analysis of the Media The determination.of glucose was by the Glucostat _enzymatic microemethod (Worthington Biological Corp..Ereehold, N.J.). During each glucose assay, a glucose blank.containing, 0 mgf’ glucose and three standards.consisting of 16.6,.25.0 and 33.3 mg} glucose were used for comparison with the un- .knowns.‘ The color development was carried outwat.37_c for ”exactly 10 minutes. The method.ofHBarker andwfiwnmereon(l9hl) _ was employed for the determinationwof.lactic acid. 1A #0 mg‘ ,.1 lactic acid standard solution (Sigma.Chemica1 Co.. St. Louis,.uissouri) was utilized.to.make.up.three.standards having concentrations.-.of.,3.33,...6.66 and 13.33..mg$ lactate. .A'blank containing no lactic acid was also.prepared. The .lactic acid and glucose standard and blank tubes were combined to facilitate the assays. 32 Incubation Procedure Immediately after the samples were gassed_they were incubated.for variable time periods. All incubations were carried out in a constant temperature room at l3.C. .Shaking was accomplishedsbyrplacing the test tube basket.on a.slid- ing platform attached to a steel rod which in turn was attached to,a 1/18.H.P. electric speed reducer motori(Bodine Electric Cos. Chicago, 111.). The speed at which the plat- form.was driven wasmcontrolled by a.Powerstat (Superior Electric 00.. Bristol,aConn.) attached to the.motor. The speed was adjustedito provide maximum agitation (0.7.cm stroke length at 1.5.cps) of the media without splashing it from the center wells. At the completion of the incubation period, 0.3 m1 samples of the media were removed from each center well by means of disposable prothrombin pipettes (Scientific Products,mEranston.-Ill.).and placed in 15 ml Pyrex.centrifuge tubes. The samples were then treated with 2 m1 of ZnSOu and 2 m1 of.Ba(OR)2 to precipitate any protein present. After the addition of 3.8 ml of distilled 320, a 26 fold dilution was produced, bringing the unknowns and standards on to the linear portion of the standard curve. The resulting pH was 7.0. ' 33 Procedurenforgggaminigg the Effect of Varying Gas Concentrations Both retinas.were removed from the trout.and weighed. Each.retina was separately placed in 0.5 ml of Modified Medium 199 (Grand Island Biological.Co., Grand Island, N.Y.). Modified Medium 199 consists of an Earles base which contains 1.25 gm/1.Naflcoa, 0-5qgm/l glucose and no phenol red. .The center wells were then tightly.stoppered into the milk dilu- tionbottlesxmfiextf the samples were.gassed with a gas mix- ture having a compositionaof 95 z ,Nz-s X cog or 95% 02-5 at 002. The volume of gas used during gassing wasuapproxi- matelyil355 ml.. In the first set of experimentsaanrincuba- tion time of & hour.was used. After this, 1 hour and finally 3 hour incubation periods were tested. Samples of cardiac and kidney tissue were weighed and placed in 0-5 m1 Of modified Medium 199 in separate-center wells.A Cardiac and kidney tissue-were gassed under the same conditions-as the-retinas. The tiesues~were~ehensineutated for 1 hour. ,Procedure foy Studying the Effect of Sonification The two retinas were removed from the trout, blotted dry and weighed. One intact retina.was transferred to a center well containing 0.5 ml of a Phosphate Buffered Saline 3“ (PBS) (Grand Island Biological Co., Grand.Island,.N.I.), solution with a 100 mg! glucose.concentration. The second.retina was.placed in arthickewalled Pyrex test tube containing 1.0 ml ofPBSJIith a 100 mg‘ concentration of glucose. This retina.was then homogenized by means of.a No. U185C Sonifier Cell.Disrupter (Heat Systems Co., Melville, L.I., N.Y.). During the sonification.process the test tube was cooled by immersion in ice water. After cellup lar disruption was completed, 0.5 ml of the solution was transferred to a center well. The center wells were placed in milk dilution bottles and.gassed with a.mixture of 95 % N2-5 fl C02. After gassing, the samples were incubated for a period of 5 hours. Procedure.for Examiniggithe Effect of Various Glucose Concentrations In this experiment the retinas were.incubated in a modified.Mammalian.Krebs Saline Medium.(Appsndix l). The medium.was made up to have.an osmolarity of 289 mflsm/kg. Enough NaH603 was added to the medium.to.give a pH of 7.6 after the solution was saturated with.99.5 S 02-0.5 % C02 at 13 C. The mediumnwas than divided.into five.100 m1 portions. To eachiportion, exceptwthe-first,.rarying- quantities of anhydrous glucose were.added, resulting in five concentrations: 0, 50, 100. 150, and 200 mg% glucose. 35 The retinas were weighed and placed in. 0.5 ml of media of known glucose concentration. After gassing thetissues with 99.5 f 02-0.5 S 002, they were incubated for aperiod of 1 hour . Procedure to Study the Effect of Iodoacetate A solution of 0. 5 ml of modified Mammalian Krebs Saline Medium. containing 10 mg % lactic acid and90.001 moles, of iodoaceticacidtsodium-.-sa1t (Mathesonmcmu Cincinnati, Ohio) .was placed in three. separate center wells. The amountiof iodoacetate employed inthis experi- ment ranged from 0.001ito 0.0.1 moles. The retinas were removed, wet weight. determined, and placed in.-100 ml of Ringer solution withlo mg. 1 .lactic.acid..and..-0.001 moles of, iodoacetate. Afterwarpreincubation period of one—half hour the retinas were removedfrom theRinger solution. and placed in two of the center wells. Thesamples were then exposed to a 99.5 % 02-0.5 % C02 gas- mixture. Afters. one hour incubation, 0.3 m1 of the media was. removed from each flaskand analyzed for glucose and lactic acid. Statistical. Analysis ' ’ Glucose utilizationandllactic acidproduction of the tissues studied were expressed in yg/hr or yg/hr/gm. 36 During the weighing procedure it was foundithat notall of the vitreous body. could be removed from the retina without destroying the integrity of the cells. Thus, fora more complete interpretationof thadata, yg/hr as wall as )18/ hr/gm wet weightswere -determined. Statisticallanalysis to determine - the. significant. difference between . the. name was carriedoutbyremploying the student "'t'.'..test. -Values considered significantin. this study havens, calculated a( value of 0.0.5 or. .less.. All regression lines were «plotted by the method of least squares. L RESULTS The project of elucidating the metabolism.of the retina was carried outeby measuring the glucoselutiliza- tion and lactic acid production.of this tissueeunder differing experimental conditions. The measurements of glucose utilizationsgaremawquantitativerestimatewof.carbo- hydrate metabolismap Thewmeasurementwof.lacticlacidwpro— duction determined the degree to which the EMPupathway contributed to metabolism. ' Effect of Varyigg 02 Concentrations on Retinal Metaboligm, Oneyfialf Hour Incubations In the first series of experiments,-utilizing an incubation period of k hour, retinas from the trout were exposed to a gaseous environment of 95 S N2-5 S 002 or 95 S 0245 z 002. There was no apparent change in the physical appearance of the retinasafteruincubationf but in some cases the modified Medium 199.was grayish in color after 5 hour, probably due towa disruption of thespigmented epithelium.of.the.retina. .Under anaerobic conditions (95 S N2-5 S C02) glucose utilization was higher.andithere was a greater.production of lactic acidtthan-under aerobic conditions (95 S 02-5 S C02). The results of the anaerobic 37 38 and aerobic experiments were statistically different at the p40..001 (Table. 1). One ..Hour Incubations Utilizing a- lhour incubation period, there -was more glucose utilized and more. lactiaacid produced under both anaerobicancLaerobic. conditions . than. when .an. incuba- tion period of.ahourmwas used. The results. alsoindicate that. when glucose utilization and, lactic.-acid.-production are expressed.-in_pg/hr/gm there is less glucosautilization and lactic. acichroduction under incubationperiods of 1 hour. than undersidentical..treatments of} houri...indioating a slowing of the-rata..of..g1ycolysis.as the incubation time increases (Table 2). Using a l hourincubation timetherewas more glu- cose utilized and more lactic-.acid,produced-under anaerobic than under aerobic conditions. .The results of glucose . utilization under anaerobic- and-aerobic . conditions . were statistically di.fferent..at_the 1140-001 level- Lactic. acid. production showed a significant difference of p( 0.001 between anaerobic and aerobic levels. Three Hour. Incubations Retinal metabolismunder differentgaseous environ- ments was tested utilizing an incubation period of 3 hours. 39 «00.0 v a as successes» canoaom one oHooaomom monsoon oomoaohuad unmadhdowfim a .m.m 4 use: Assadpo>aomoo ho nonessv z a.mn « a.aam m.ma « e.aa~ m.mn a n.ees o.e~ 4 e.mem Noo an . No who on oacoso< .m.mm 4 s.nee~ er.o~ a e.sme .a.mm 4 s.eHmH .a.ea « m.emm Noo an u «a mom on eaconoos< Aaw\as\me0 Aac\waa Aaa\naxw:0 Asr\w4a coosuoaa couscous dosdadus consadps mOauwausoosoo 2 possesses suspend corpora emoosao omoosao moo one as use: e.aoc mas sauce: assesses mmauaadns comma» Hmoapoa an scaposuoaa odes capowa use ooauwuaadus omoosaoni.d mqm canons “no.0 ”van peooaunnwau one ocean» uncommon accrues oeoooeeuudn «encunm>ueeno eons». .m.m « one: L’! No.0 « Hm.“ no.0 4 n:.« :o.o a no.m No.0 4 00.0 sw\an\m,2‘ E euoosao m.mm w H.m:ma «.mm « m.~oo« w.o« 4 o.mhw come 4 0.0mm amino... deosdonn ouduoqq m.m~ i o.m~m« o.en « a.meaa 5.0m a m.nmn« H.na 4 w.oem EM\Hn\w4< Goudadps omOOSHU emoosao “macaw omoosao swoon” emoosao «wooed enoosac swoon and am too: a poo acumen osmoonawodn and: season condom moons goddesses uosodooa ea luaaonmpes Hosanna no meanwhpeoosoo omoosaw wedhamp mo pooummau.m mqm c: 200‘ 81 y: -22.4092 (x) + 966.160 Time (minutes) FIGURE 9.--Aerobic lactate production (95% 02-51 002) by retinal tissue at 13C. E" 1600«~ 1400;- 1200 1000. 800. 3°... 9 .s \ o C o 3400. r1 u) u) A. 200+ 82 l I 1 1 I I 30 60 90.. 120 150 180 Time (minutes) FIGURE 10.--Anaerobic glucose utilization (95$ Nz-Sfi 002) by retinal tissue at 130 e 83 1600 - ' y=-40.7878 (x) 4 1523.549 1400 1200 1000 800 600 400 pg lactate/hr/gm 200 l l I 0 50‘ 86‘ 90 120 150 180 Time (minutes) FIGURE ll.--Anaerobic lactate production by retinal tissue at 130.