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Department of Neurobiology, Civitan International Research Center, University of Alabama School of Medicine, Birmingham, Alabama 35294–0021
Submitted 9 September 2003; accepted in final form 5 February 2004
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ABSTRACT |
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INTRODUCTION |
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; Krebs et al. 1975). As a result, homeostatic mechanisms of pH control must deal with the constant production of H+ by living cells and with changes in acid production due to alterations in cellular activity. Many metabolic reactions generate H+, and many others consume it (see Mellergard and Siesjo 1998). However, taking into consideration only the initial substrates and final products, the production of protons during energy metabolism exceeds their consumption; cells import pH-neutral substances, such as glucose and O2, but release acidic substances, such as lactic acid and CO2.
It has been suggested that the steady production of H+ during energy metabolism can account for the difference in pH between the superfusate and living tissue, such as has been observed in various in vitro brain preparations (Balestrino and Somjen 1988; Brockhaus et al. 1993; Chen and Chesler 1992; Walz 1989). A similar pH gradient in which the tissue has a lower pH than the superfusate has also been recorded when pH-selective microelectrodes were used to measure pH in isolated vertebrate retinas (Dmitriev and Mangel 2000, 2001; Oakley and Wen 1989). In vivo measurements also show that intact retina is more acidic than the choroidal blood supply (Padnick-Silver and Linsenmeier 2002; Yamamoto et al. 1992). In fact, a consistent extrusion of acid can be detected even in the case of individual isolated retinal cells (Malchow et al. 1998). However, although the steady production of H+ during energy metabolism can account for the lower extracellular pH of living tissue compared with that of the surrounding milieu, such a relationship has never been directly demonstrated.
Even if the steady difference in pH between superfusate and tissue is primarily due to the sustained production of H+ during energy metabolism, the relation between the production of energy and the associated production of acid may not be proportional. This is because the two major metabolic pathways, glycolysis and oxidative phosphorylation, are different in their energetic and acidic outputs. Glycolysis is a much more acidic way to produce energy than oxidative phosphorylation, and it is widely accepted that glycolysis is the main, if not the only, source of metabolically generated acid. Nevertheless, the contribution of oxidative phosphorylation to acid production may be significant, as has been demonstrated in in vitro brain preparations by direct measurements with CO2-selective microelectrodes (Voipio and Bellanyi 1997; Voipio and Kaila 1993).
The largest change in pH that has been reported to occur under normal physiological conditions is that due to a circadian clock in the vertebrate retina (Dmitriev and Mangel 2000, 2001). A circadian clock is a type of biological oscillator with a period of 24 h (Pittendrigh 1981). In both fish and rabbit retinas, the steady difference in pH between the tissue and superfusate increases in the night and decreases during the day (Dmitriev and Mangel 2000, 2001). It has been suggested that the clock regulates pH by modulating the rate of energy metabolism. In fact, studies in which 2-deoxyglucose has been used to measure metabolic activity have indicated that a circadian clock regulates metabolic activity in the suprachiasmatic nucleus (Schwartz and Gainer 1977). However, because glycolysis is a much more acidic way to produce energy than oxidative phosphorylation, it is possible that the circadian rhythm in retinal pH reflects a change in the proportion between glycolysis and oxidative phosphorylation rather then a change in metabolic rate.
In this study, we examined whether and to what extent the suppression of different components of energy metabolism altered the steady pH gradient between living in vitro retina and the superfusion medium. We also compared the results of suppressing different components of energy metabolism during the subjective day and subjective night to investigate whether changes in the relative contributions of glycolysis and oxidative phosphorylation are responsible for circadian-induced changes in retinal pH. Our results show that 1) both glycolysis and oxidative phosphorylation contribute to acid production, and 2) the circadian clock does not affect the proportion of glycolysis and oxidative phosphorylation. Thus we conclude that circadian clock regulation of retinal pH reflects circadian regulation of the rate of energy metabolism in the retina.
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METHODS |
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2 mM) of test drugs (iodoacetate, cyanide, and ouabain) were added to the Ringer. All chemicals and test drugs were purchased from Sigma (St. Louis, MO). The superfusion solution was constantly bubbled by a gas mixture of CO2 and O2. In one series of experiments, O2 was replaced with N2. In most experiments, the proportion of the gases was adjusted to 2.6% CO2-97.4% O2 (e.g., the partial pressure of CO2 was 2.5 kPa) so that the solution in the experimental chamber had a pH = 7.55. In separate series of experiments, the partial pressure of CO2 was increased to 4.5 kPa or decreased to 2.0 kPa with the same amount of NaHCO3 in the solution so that the Ringer pH was 7.30 or 7.65, respectively. The pH was measured with a conventional glass pH-selective electrode in the source jar and with a pH-selective microelectrode in the experimental chamber. All experiments were performed at room temperature (21–23° C).
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). The diameter of the microelectrode tip was 
5 µm. The resistance of the pH-selective barrel was 5–10 GOhm, and the reference barrel had a resistance of 20–50 MOhm. Microelectrodes were calibrated in the experimental chamber before and after measurements on tissue. The calibration solutions contained the same concentrations of the principal cations (Na+, K+, Ca2+, Mg2+, all Cl– salts) of the Ringer solution plus 5 mM Tris. Different amounts of HCl were added to the calibration solution so that its pH was equal to 7.0 or 8.0. All microelectrodes used in the experiments had slopes of 54–56 mV/pH unit before and after retinal pH measurements. The superfusion solution, which flowed over the photoreceptor side of the isolated retina, was grounded by an Ag-AgCl macroelectrode. Another Ag-AgCl macroelectrode was in contact with the vitreal surface of the retina. This pair of macroelectrodes allowed us to record the electroretinogram (ERG). The pH-selective microelectrodes were always positioned in the photoreceptor layer, just distal to the outer limiting membrane. Voltage signals were digitized and sent to computer memory via a DigiData 1200 Interface under the control of AxoScope 1.1 software (Axon Instruments).
The isolated retinal tissue was always maintained in darkness for 1 h after surgery. The physiological condition of the tissue was assessed by recording the ERG to brief (10 s) monochromatic (500 nm) light flashes of scotopic intensity that were delivered once every 2 min. Specifically, the b-wave of the ERG, which is primarily generated by bipolar cells (Stockton and Slaughter 1989), and sPIII, which is generated by Muller glial cells in response to light-induced, photoreceptor-mediated changes in the extracellular K+ concentration (Hanitzsch 1973; Witkovsky et al. 1975), were used to determine the extent of physiological viability.
Most data were obtained during the subjective night [Zeitgeber Time (ZT) 14–18, where ZT = 0 is dawn], but two series of experiments were performed during the subjective day (ZT 7–10) for comparison (see Figs. 8 and 9). Under conditions of constant darkness, subjective day refers to the time of the circadian cycle during which illumination was previously present; subjective night refers to the time of the circadian cycle during which illumination was not previously present.
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RESULTS |
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When H+-sensitive microelectrodes were moved through the superfusate to the retina, a decrease of pH in the vicinity of the tissue was always recorded (Dmitriev and Mangel 2000, 2001). Retinal extracellular pH also changed from layer to layer, but this intraretinal pH difference was an order of magnitude less than the difference between the tissue and the superfusion solution. The minimum value of pH was usually at the level of the inner segments of the photoreceptors, and the pH in this layer is defined here as the retinal pH. All of our pH data were obtained with the pH-selective microelectrodes positioned in this layer.
The pH of the superfusion solution greatly affected retinal pH, but not the pH difference between the superfusate and the retina. When the pH of the superfusion solution was 7.65, retinal pH was 7.36 ± 0.007 (SE, n = 34), i.e., 0.29 pH units less than in the Ringer. When the pH of the superfusion solution was decreased to 7.30, i.e., lower than the pH in the retina in the preceding experiments, retinal pH was 7.16 ± 0.005 (n = 10), i.e., 0.14 pH units less than in the Ringer. Interestingly, when the H+ concentrations were recalculated in nanomolar (instead of pH units), the difference between the retina and the Ringer was about the same regardless of the pH of the superfusate (
[H+]o = 21.3 ± 1.2 nM in Ringer with pH = 7.65 and [H+]o = 19.2 ± 1.2 nM in Ringer with pH = 7.3).
If the source of the H+ production is the energy metabolism of retinal cells, suppression of energy metabolism should decrease the difference in pH between the retina and Ringer, resulting in an increase of retinal pH. To test this idea, we suppressed energy metabolism by different methods while simultaneously monitoring retinal pH and the ERG.
Figure 1 shows the effects of suppressing energy metabolism by removing glucose from the superfusion solution. For the experiments described here and for all below, the pH of the Ringer was 7.55. The results show that the in vitro fish retina is not very dependent on an external glucose supply over the course of almost 1 h. After 50 min in glucose-free Ringer, light stimulation still evoked an ERG with a b-wave that was 82 ± 8% (n = 5) of control in amplitude and a sPIII component that was 68 ± 13% (n = 5) of control in amplitude. At the same time, the difference in pH between the retina and superfusion solution was 84 ± 6% (n = 5) of control. It is thus likely that the fish retina has enough internal energy resources, probably from glycogen stores, to support its function at a satisfactory level for a prolonged time without an external glucose supply.
In contrast to the relatively small effects of a glucose-free Ringer on the fish retina, application of iodoacetate (IAA, 2 mM), which blocks glycolysis, quickly and dramatically suppressed both light-induced retinal activity and acid production (Fig. 2). Following IAA application, the b-wave disappeared in 2–3 min, the sPIII was reduced to one-half of its control value after 7 min, and the ERG was completely eliminated within 20–25 min. IAA also quickly and dramatically reduced the difference in pH between the retina and superfusion solution. The effects of IAA application were the same with or without glucose added to the superfusate (data not shown).
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). In fact, the addition of pyruvate (20 mM) markedly diminished the effects of IAA, and the ERG was practically unaffected after 5–7 min of perfusion with IAA plus pyruvate. However, following a longer time in such solution, the b-wave of the ERG disappeared, and the sPIII dramatically decreased. Finally, after 25–30 min, light stimulation evoked only some very weak responses with unclear components. All of these effects were practically the same when we used lactate instead of pyruvate as a substrate for oxidative metabolism (data not shown). The experiments described so far and summarized in Fig. 4 show that pharmacological suppression of energy metabolism produces a decrease in light-induced activity and a reduction of acid production in the retina and therefore a decrease in the difference in pH between the retina and superfusion solution. The strongest suppression of energy metabolism using IAA alone produced the strongest decrease of both sPIII (1.4 ± 1.2% of control after 25 min, n = 4) and the pH gradient (31 ± 4.2% of control after 25 min, n = 4). Mild suppression of energy metabolism using a glucose-free solution had little effect on sPIII (79 ± 6.6% of control after 25 min, n = 5) and the pH gradient (95 ± 3.6% of control after 25 min, n = 5). Moderate suppression of energy metabolism using IAA and pyruvate produced an intermediate effect on sPIII (36 ± 3.4% of control after 25 min, n = 6) and the pH gradient (56 ± 4.2% of control after 25 min, n = 7). Therefore the greater the suppression of energy metabolism, the greater the decrease of light-induced retinal activity, and the greater the reduction in acid production in the retina.
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In other experiments, a decrease in energy production was induced by reducing energy consumption. In the retina, almost one-half of all the energy that is produced is used to maintain transmembrane ionic gradients (Ames et al. 1992). If this is so, inhibition of the Na+/K+-ATPase using ouabain (Winkler 1983) should significantly reduce the amount of ATP required, and accordingly, the amount of ATP produced. Typical results of the effects of ouabain (0.1 mM) on retinal electrical activity and production of acid are presented in Fig. 5. Ouabain usually had a marked effect within a minute, and after 3 min, the sPIII component of the ERG was completely suppressed. The b-wave was a little less sensitive to ouabain, but was also completely eliminated after 10 min of superfusion with ouabain. During these initial 10 min, the difference in pH between the retina and superfusion solution decreased to 67 ± 1.5% of control (n = 4). Following this initial 10-min period, acid production in the retina did not decrease significantly, and after 25 min of drug application, the difference in pH was 62 ± 4.7% of control. Application of ouabain at higher concentrations (e.g., 0.5 mM) did not have stronger or faster effects.
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In all of the above experiments, the suppression of retinal energy metabolism was always accompanied by a reduction in acid production. However, in contrast to the suppression of glycolysis, the suppression of oxidative metabolism commonly results in acidification of living tissue, a phenomenon known as the Pasteur effect. The reason for this increase in acid production is the compensatory activation of glycolysis under conditions in which the tissue cannot use oxidative phosphorylation. Because glycolysis produces more H+ per ATP generated compared with oxidative phosphorylation, living tissue becomes more acidic when oxidative metabolism is blocked, even if the total production of ATP decreases (see DISCUSSION). We observed the Pasteur effect in our experiments, that is, application of sodium cyanide (NaCN 1 mM), which disrupts oxidative phosphorylation (Ackrell et al. 1978; Ohnishi and King 1978), increased acid production (Fig. 6). Similar results were obtained when oxidative metabolism was eliminated by replacement of O2 with N2 in the gas mixture, which was constantly bubbled through the superfusion solution (data not shown). Both the b-wave and sPIII of the ERG were quickly decreased in the presence of NaCN or N2, and after 15–20 min, were completely eliminated (Fig. 6). Acidification of the retina to the extent shown here (for 0.15–0.20 pH units) by decreasing the pH of the superfusate reduced the amplitude of the ERG, but did not eliminate it (data not shown). Therefore the elimination of the ERG by NaCN or N2 apparently results from energy deficiency.
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, 2001) could result either from an increase in the rate of energy metabolism or from the selective stimulation of glycolysis. To determine the means by which the circadian clock regulates retinal pH, we compared the effects of the selective suppression of oxidative phosphorylation (Fig. 7) and glycolysis (Fig. 8) in the subjective day and night. If the increase in acid production during the subjective night results from an increase in the metabolic rate without a change in the proportion between glycolysis and oxidative phosphorylation, selective suppression of either metabolic pathway should have the same proportional effect in the subjective day and night. In contrast, if the circadian clock affects the proportion between glycolysis and oxidative phosphorylation by stimulating glycolysis at night, selective suppression of glycolysis should have a stronger proportional effect during the subjective night, while selective suppression of oxidative phosphorylation should have a stronger proportional effect during the subjective day.
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The effects of selectively suppressing glycolysis on the pH difference between the retina and superfusion solution were also the same when measured at different circadian times (Fig. 8). Here again, we have normalized the results with 1.0 representing the predrug value. In the test solution, which "shunted" glycolysis by suppressing it with IAA and supported oxidative metabolism with pyruvate, the pH difference decreased to the same value in the subjective day (55 ± 4.9%, n = 5) and subjective night (56 ± 4.2%, n = 7; both after 25 min).
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DISCUSSION |
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In all measurements reported here, as well as in our previous work (isolated retina of fish: Dmitriev and Mangel 2000; eyecup of rabbit: Dmitriev and Mangel 2001; "retina-pigment epithelium-choroid" preparation of chick: A. V. Dmitriev, unpublished observations), the extracellular pH in every retinal layer was always lower than the pH of the superfusion solution regardless of the species, the type of preparation, or the type of experimental chamber. In addition, a gradient of H+ from the retina to the superfusion solution was recorded in this work with different superfusate pH (7.65–7.30). A similar pH gradient, in which the retina is more acidic than the superfusate, has routinely been reported in the literature, beginning with the very first attempts to characterize the retinal pH (Oakley and Wen 1989; Tsacopoulos and Levy 1976). In vivo measurements on intact cat (Padnick-Silver and Linsenmeier 2002; Yamamoto et al. 1992) have also shown that the pH in the retina is lower than in the choroidal blood supply. The vertebrate retina is not unique in this respect: the brain slice preparation is also more acidic than the superfusate (Balestrino and Somjen 1988; Brockhaus et al. 1993; Chen and Chesler 1992; Walz 1989).
To maintain the retinal pH at a lower level than the pH of the surrounding fluid, all H+ that the tissue loses by diffusion has to be replaced by an equivalent amount of new acid. The only known steady source of acid in living tissue is energy metabolism (Alberti and Cuthbert 1982; Krebs et al. 1975). Our findings are consistent with the idea that energy metabolism is responsible for the pH difference between the retina and superfusate. Suppression of energy metabolism by removing glucose from the Ringer (Fig. 1) or by adding IAA (Fig. 2) leads to a decrease in acid production in the retina and to an appropriate reduction in the difference in pH between the retina and the superfusion solution. In addition, energy production was also decreased indirectly by reducing ATP utilization via inhibiting the Na+/K+-ATPase with ouabain. As shown in Fig. 5, decreasing energy metabolism in this fashion also reduced acid production, and consequently, the pH difference between the retina and the superfusate.
It should be emphasized that the pH gradient between the retina and superfusate described in this paper is a steady standing gradient. Experimental disturbances of energy metabolism will also lead to the redistribution of H+ between the internal compartments of cells and between cells and the extracellular space. Although these H+ movements could have some effect on retinal pH, the effect will be transient since diffusion would equilibrate such changes in H+ concentration with a time constant of tens of seconds (Govardovskii et al. 1994). The slow changes in the steady pH gradients described here took tens of minutes to occur and resulted from changes in the steady source of H+. Taking into consideration all of the above, we can conclude that acid production that determines the tissue pH is linked to energy metabolism.
The H+ concentration is significantly affected by the presence of substances that contribute to pH buffering (for reviews see Chesler 1998; Voipio 1998). Because of buffering, the measurable pH changes represent just a fraction of the changes in acid concentration that produce them. Thus buffering diminishes temporal changes of pH. However, buffering by itself does not influence the spatial distribution of H+, since the concentrations of H+ and acid-base pairs involved in buffering are constant in time at all locations, if the pH gradient is steady. In fact, the pH gradient between living tissue and the surrounding solution results from the unequal distribution of substances that form buffers. The main buffer in the brain and retina is the 
system (Chesler 1998; Voipio 1998), and CO2 is constantly produced during aerobic metabolism. Lactic acid, which also participates in pH buffering (Voipio 1998), is constantly produced during glycolysis. As a result, H+ buffering is a complex system that includes CO2, 
, lactate, and H+, which are produced, diffuse, and interact with each other. The quantitative analysis of the system of H+ buffering is beyond the scope of this paper. In any event, the final result of all of these complex interactions is the pH gradient, and the magnitude of the pH gradient between the retina and superfusate is proportional to the intensity of acid production in the retina.
Contributions of glycolytic and oxidative metabolism to acid production
Glycolysis is the "classic" acid generator. During glycolysis, a molecule of glucose is reduced to two molecules of pyruvic acid, and two ATP molecules are formed from two ADP and two phosphates. A certain portion of produced pyruvic acid is used in the subsequent phase of energy metabolism— oxidative phosphorylation. The remaining portion of pyruvic acid is quickly converted to lactic acid. At physiological pH, lactic acid almost completely dissociates to lactate anion and H+. Thus considering glycolysis not as a preliminary phase for oxidative phosphorylation, but as a complete process (anaerobic glycolysis), one observes that to synthesize two ATPs, one molecule of pH-neutral glucose is consumed and two molecules of completely dissociated lactic acid are released. It has been shown that under normal conditions mammalian retina produces lactate at a rate of 1.2–1.3 mol/mg dry weight per hour (Winkler 1981). Two H+ have to be added to two lactate anions to equilibrate them with one glucose molecule.
Glycolysis is not the only producer of metabolic acid. Another phase of energy metabolism, oxidative phosphorylation, can also contribute to H+ generation. During oxidative phosphorylation, one molecule of pyruvate converts into three CO2 (and 3 H2O) molecules. Some CO2 simply diffuses out of cells, and other CO2 turns into and H+. The spontaneous hydration of CO2 is a relatively slow process, but it becomes very fast in the presence of carbonic anhydrase (Ridderstrale and Wistrand 1998). In fact, an effective 
buffer is not possible without carbonic anhydrase (Chesler 1998; Voipio 1998). Both intra- and extracellular forms of this enzyme have been found in the retina, and it has been shown by direct measurements with H+-selective electrodes that suppression of carbonic anhydrase markedly affected retinal pH both in vitro (Oakley and Wen 1989; Wolfensberger et al. 1999) and in vivo (Yamamoto and Steinberg 1992). However, to contribute to the H+ gradient between the tissue and surrounding solution, the production of CO2 must be strong enough to generate a CO2 tissue-solution gradient despite the rapid diffusion of this gas. Such a CO2 gradient has been recorded when the CO2 concentration was directly measured with specially constructed microelectrodes (Voipio and Ballanyi 1997; Voipio and Kaila 1993).
Our results (Figs. 2, 3, 4) suggest that oxidative phosphorylation contributes to the generation of protons in the retina. Although measurements of CO2 with CO2-selective microelectrodes have not been performed on the vertebrate retina, it has been known for years that the vertebrate retina utilizes O2 at a very high rate, especially in the photoreceptor layer (Linsenmeier 1986). Each molecule of O2 consumed during oxidative phosphorylation is converted into CO2, and in the presence of carbonic anhydrase, potentially most of the CO2 is converted into and H+. Our experiments have demonstrated that oxidative phosphorylation contributes to H+ generation. Suppression of glycolysis with IAA (Fig. 2) prevents generation of pyruvate and consequently suppresses oxidative phosphorylation too. However, oxidative phosphorylation can be maintained by the addition of pyruvate to the superfusion solution, which already has IAA, producing "a shunt" of glycolysis. Fish retina, as well as mammalian retina (Winkler 1981), requires both glycolysis and oxidative phosphorylation to produce enough energy for normal function, and when IAA is applied, the tissue dies with or without pyruvate. However, the addition of pyruvate apparently helps during the first 10–12 min, when only small changes in the ERG are observed (Fig. 3). At the same time, retinal production of acid was also significantly higher, than occurred in a solution with IAA only (Fig. 4 for comparison). All of the differences between the effects of IAA plus pyruvate and the effects of IAA alone were due to the activity of oxidative phosphorylation. Thus the 20–22% of extra acid production that was recorded with the addition of pyruvate resulted from the hydration of CO2 that was generated during oxidative phosphorylation.
As we can see, both major energy pathways— glycolysis and oxidative phosphorylation— contribute to acid production in the in vitro fish retina. However, these two processes are different with respect to acid production and energy effectiveness. When glucose is used anaerobically, each glucose molecule is converted to two lactates and two H+. In aerobic metabolism, one molecule of glucose degrades to six CO2 and six H2O. Part of this CO2 diffuses out of the tissue, but another portion is hydrated, producing and H+. The presence of carbonic anhydrase significantly accelerates the hydration of CO2 (Oakley and Wen 1989; Wolfensberger et al. 1999; Yamamoto and Steinberg 1992; for reviews, Chesler 1998; Ridderstrale and Wistrand 1998; Voipio 1998). Thus the acidic output of aerobic metabolism can potentially be close to six H+ per one glucose molecule.
However, if we take into consideration the ratio of H+ generated per ATP synthesized, it is clear that anaerobic glycolysis is a far more acidic way to produce energy than oxidative metabolism, as revealed by the Pasteur effect (see Fig. 6). In glycolysis, one molecule of glucose is used to generate two ATP, and the process is accompanied by releasing two H+, i.e., the ratio is one H+ per one ATP. When one molecule of glucose is completely utilized in oxidative metabolism, 38 ATP are generated and <6 H+ are released, i.e., the ratio is less then 0.16 of H+ per 1 ATP. Thus aerobic metabolism is not only a more efficient means of energy production, but it also produces at least six times less acidic waste than glycolysis. Nevertheless, the retina intensively exploits glycolysis. Oxidative metabolism and glycolysis contribute equally to energy production in the rabbit retina (Ames et al. 1992). This means that only 1 of 20 molecules of glucose is used in oxidative phosphorylation, and the remainder is utilized in glycolysis. Thus under conditions in which oxidative phosphorylation and glycolysis generate equal amounts of ATP, 19 glucose molecules will produce 38 H+ during glycolysis and 1 glucose molecule will produce <6 H+ during oxidative phosphorylation. As a result, more than 86% of all "metabolic" H+ comes from glycolysis (38 "glycolytic" H+ of 44 total H+), and <14% from the aerobic phase of energy metabolism (maximum 6 "aerobic" H+ of 44).
Circadian clock, cellular work, and H+ generation
As has been shown here, steady H+ production during energy metabolism determines the magnitude of the pH gradient from the retina to the superfusion solution. This means that with a constant superfusate pH, energy metabolism determines the value of retinal pH. Consequently, changes in retinal pH can be used as an indicator of changes in energy metabolism, but only if the proportion of glycolysis and oxidative phosphorylation is kept constant. Our measurements demonstrated that this proportion was not affected during circadian-induced changes in H+ generation in the fish retina (Figs. 7 and 8). Therefore we can conclude that the larger pH gradient during the subjective night compared with the subjective day is a consequence of the higher rate of metabolic activity of the retina during the night and not due to the selective activation of glycolysis by the clock.
The intensification of energy metabolism indicates that a greater amount of work (e.g., ionic transport, biosynthesis, etc.), which requires more energy, is performed by retinal cells during the subjective night. Energy metabolism generates ATP to support the work of cells and also releases H+ as an inevitable byproduct. The amount of ATP synthesized in energy metabolism is equal to the amount of ATP used by retinal cells, and therefore the ATP concentration stays constant. The stability of the ATP concentration is achieved by negative feedback from ATP to the very first biochemical reactions in energy metabolism at the level of glucose phosphorylation. This relationship places cellular work in control of its own energy supply. When the amount of energy utilized for cellular work increases, the concentration of ATP starts to decrease. This in turn lessens the negative feedback to energy metabolism, and the production of ATP rapidly increases to satisfy the new higher energy demand and to prevent depletion of the ATP pool. When the amount of energy utilized for cellular work diminishes, the concentration of ATP starts to increase. That in turn strengthens the negative feedback to energy metabolism, and the production of ATP decreases. This simple mechanism adjusts energy production with energy consumption, and makes H+ production proportional not only to the rate of energy metabolism, but also to the total amount of cellular work performed. Because of all of these interactions, the circadian clock, which regulates the activity of retinal cells, influences H+ production and retinal pH.
In summary, our results show that the suppression of energy metabolism results in a reduction of the pH difference between the retina and superfusate and that the magnitude of this reduction in the pH gradient is correlated with the extent of energy metabolism suppression. These results support the idea that the pH gradient between the tissue and the surrounding solution is due to the steady production of acid that accompanies energy metabolism, and consequently, that the rate of energy metabolism determines tissue pH. Our data also show that the circadian clock does not affect the relative proportion of glycolysis and oxidative phosphorylation. Thus the circadian-induced increase in acid production during the subjective night results from an increase in the rate of energy metabolism, and not from the selective activation of glycolysis, compared with oxidative phosphorylation. These findings suggest that a circadian clock regulates retinal energy metabolism affecting cellular functions, such as the maintenance of transmembrane ionic currents and the biosynthesis of macromolecules, in the retina.
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ACKNOWLEDGMENTS |
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This investigation was supported in part by National Institutes of Health Grants EY-005102 and EY-014235 to S. C. Mangel, National Science Foundation Grant IBN-9819981 to S. C. Mangel, Grant HD-38985, and Core Grant EY-03039 to the University of Alabama at Birmingham.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. V. Dmitriev, Dept. of Neurobiology, Univ. of Alabama School of Medicine, 1719 6th Ave. S., CIRC-425, Birmingham, AL 35294–0021 (E-mail: anvadmi{at}nrc.uab.edu).
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, glucose-free solution; 
, solution with IAA; 
, solution with IAA and Pyr. Amplitude of sPIII responses from the different experiments were normalized by defining the average amplitude of 3 responses before application of the test solution as 1.0. To normalize the pH difference between the retina and superfusate, the pH gradient before application of the test solution was defined as 1.0. Each data point indicates the mean ± SE.
