Rates of both 14CO2 fixation and 14C-glutamate appearance are a function of pyruvate levels in the retina
In preliminary experiments, 14CO2 fixation, levels of ATP and creatine phosphate were examined as a function of time (10, 20, 40 and 60 min) in retinas incubated with 0.2 mm pyruvate. Creatine phosphate and ATP levels were 16 ± 1.1 nmol/mg protein and 20 ± 0.2 nmol/mg protein, respectively, and remained constant for 60 min. These results indicate that oxygenation and pH control in the retinal preparation are adequate for incubation periods of 1 h. Total CO2 fixation (5.50 ± 0.14 nmol/mg protein) peaked between 20 and 40 min but was already 76% of maximal (4.2 ± 0.1 nmol/mg protein) at 10 min. Because total CO2 fixation did not change or decreased slightly (10–15%) after 20 min, the kinetic pattern indicates that the rate of CO2 fixation was constant, but that by 20 min continuous breakdown of non-volatile 14C products to 14CO2 + H2O produced a near steady-state level of glutamate and glutamine specific radioactivity. Thus, the total amount of 14C-glutamate and glutamine achieved at steady state is influenced not only by the rate of synthesis of 14C-glutamate but also by its rate of breakdown.
To assess the control of pyruvate carboxylation over glutamate synthesis, retinas were incubated for 20 min with either 0.2 or 5 mm added pyruvate and intracellular and extracellular metabolites were analyzed. The results are summarized in Table 1 and Fig. 3. Raising the pyruvate concentration resulted in an 80% increase in the total 14CO2 fixed from 5.27 ± 0.45 to 9.50 ± 0.74 nmol H14CO3− per mg protein (p < 0.002). Almost all of the 14C-glutamate was intracellular, whereas almost all of the 14C-lactate was in the extracellular compartment. At least 90% of the 14C-aspartate and all 14C-labeled TCA cycle intermediates were intracellular. Figure 3 shows the sum of intracellular plus extracellular 14C-glutamate plus 14C-glutamine (Fig. 3a) and 14C-lactate (14C-lactate + 14C-pyruvate, see Materials and methods) (Fig. 3b). Increasing unlabeled pyruvate increased the sum of the intracellular plus extracellular 14C-glutamate plus 14C-glutamine by 70% (Fig. 3a), whereas H14CO3− incorporation into pyruvate plus lactate increased by 107% with citric acid cycle intermediates increasing by 126%. The 14C cycled into pyruvate and lactate represents glial recycling through the pyruvate/malate cycle enzymes (pyruvate carboxylase, malic enzyme and phosphoenolpyruvate carboxykinase). We conclude that the incorporation of 14CO2 into pyruvate (pyruvate cycling) takes place in the glia because decarboxylation of malate or oxaloacetate in the neurons could not have produced 14C-lactate or 14C pyruvate if the source of the 14C was CO2. As shown in Fig. 2, the neuronal glutamate is labeled by H14CO3 only in the carboxyl group adjacent to the carbonyl (1-C). When the glutamate is transaminated to α-ketoglutarate and subsequently oxidized, the labeled carbon is converted to 14CO2 and all subsequent citric acid cycle intermediates: succinate, fumarate, malate, and oxaloacetate are unlabeled and therefore could not produce 14C-lactate.
Table 1. The influence of medium pyruvate concentration on incorporation of H14CO3− into retinal metabolites
|Pyruvate concentration||Compartment||Gln*||Glu*||Asp*||Lact*||TCA cycle*||Total|
|0.2 mm||Intracellular||0.59 ± 0.05||1.47 ± 0.29||0.85 ± 0.06||0.16 ± 0.06||1.02 ± 0.26||4.08 ± 0.41|
|Extracellular||0.61 ± 0.08||0.04 ± 0.01||0.08 ± 0.01||0.55 ± 0.03||0||1.19 ± 0.13|
|Sum||1.20 ± 0.09||1.51 ± 0.29||0.93 ± 0.08||0.71 ± 0.06||1.02 ± 0.26||5.27 ± 0.45|
|5.0 mm||Intracellular||0.91 ± 0.13||2.67 ± 0.29a||1.08 ± 0.12||0.29 ± 0.04||2.31 ± 0.13b||7.26 ± 0.61b|
|Extracellular||0.96 ± 0.14||0.06 ± 0.01||0.08 ± 0.01||1.18 ± 0.11b||0||2.24 ± 0.21b|
|Sum||1.87 ± 0.23a||2.73 ± 0.28a||1.16 ± 0.12||1.47 ± 0.13b||2.31 ± 0.13||9.50 ± 0.74b|
Figure 3. Effect of pyruvate concentration on incorporation of H14CO3− into (a) glutamate plus glutamine and (b) pyruvate plus lactate by ex vivo rat retinas. Hemi-retinas were incubated at 37°C with 25 mm H14CO3− for 20 min as described in Materials and methods. Values shown are mean ± SEM (n = 4, *p < 0.02 and **p < 0.001).
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Figure 2. Diagram of the labeling of metabolites by the pyruvate carboxylase (PC) reaction in the de novo synthesis of glutamate. The position of the 14C-label from H14CO3− is shown by the solid circle. Additional abbreviations: Ala, alanine; fum, fumarate; Gln, glutamine; Glu, glutamate; KG, α-ketoglutarate; mal, malate; OAA, oxaloacetate; pyr, pyruvate; PDH, pyruvate dehydrogenase; suc, succinate.
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These results demonstrate for the first time that the citric acid intermediates malate and oxaloacetate are decarboxylated by glial enzymes in intact neural tissue. Active pyruvate cycling has been shown previously in isolated cultured astrocytes by many workers (Yu et al. 1983; Sonnewald et al. 1993, 1996; Hutson et al. 1998). The results of Fig. 3 also show that the increase in flux through pyruvate carboxylase induces a larger change in pyruvate recycling than in de novo synthesis of retinal glutamate, suggesting that incorporation of 14CO2 into glutamate can be slower than into citric acid cycle intermediates, and into pyruvate and lactate.
A large fraction of total glutamine synthesis is derived from pyruvate carboxylation
The importance of pyruvate carboxylation as a source for replenishing the glutamate released from retinal nerve endings during neuronal transmission was assessed by determining the specific radioactivity of glutamine in retinal tissue incubated with medium containing H14CO3−. A fixed proportion of glutamine synthesis in the retina derives from unlabeled glutamate released by neurons and taken up by Müller glia. The remainder comes from glial α-ketoglutarate synthesized anaplerotically from pyruvate and 14CO2. Therefore, the fraction of total glutamine derived from H14CO3− provides an estimate of the proportion of glutamine from pyruvate carboxylation, i.e. de novo synthesis.
As shown in Fig. 2, the product of pyruvate carboxylase in the glia is [1,4-14C]-oxaloacetate. Both the 1 and 4 positions of oxaloacetate are labeled because the 14C initially incorporated into the 4-C position of oxaloacetate is randomized between the 1 and 4 positions by equilibration of oxalocetate with symmetrical fumarate (Sterniczuk et al. 1991; Hassel and Brathe, 2000a,b). The labeled oxaloacetate condenses with pyruvate to form citrate, which is then isomerized to isocitrate. Next [1,4-14C]isocitrate is oxidized and decarboxylated to [1-14C]α-ketoglutarate. Therefore the α-ketoglutarate synthesized from 14CO2 and pyruvate has approximately half of the specific radioactivity of the original substrate H14CO3−, depending on the degree of randomization of the label in oxaloacetate. This is also true of the glutamate and glutamine synthesized from the [1-14C]α-ketoglutarate.
The specific radioactivity of glutamine was measured in the retinal tissue and medium extracts incubated with 0.2 mm pyruvate and H14CO3− for 10, 20, 40 and 60 min. Although approximately half of the 14C-glutamine is found in the medium, a much larger fraction of the total glutamine mass was found in the medium. We found that much of the glutamine present in the in vivo retina leaks out of the retina during the 3-min incubation prior to addition of H14CO3− (data not shown). This conclusion is also supported by the recent study by Winkler et al. (1999). Specific radioactivity of the intraretinal pool of glutamine was constant between 10 and 60 min. After 20 min of incubation the mass amount of glutamine in the tissue was 4.50 ± 0.44 nmol/mg protein (n = 4), and its specific radioactivity was 1970 ± 115 d.p.m./nmol or 15.8 ± 0. 9% of the bicarbonate specific radioactivity. By taking into account the randomization of the label in the 1-C and 4-C positions of oxaloacetate, which results in a loss of approximately 50% of the 14C-label in the TCA cycle, we calculated that about 32% of the glutamine was synthesized from H14CO3− via pyruvate carboxylase.
The specific radioactivity of retinal glutamate (269 ± 8 d.p.m./nmol) was lower than that of glutamine (1970 d.p.m./nmol), but the mass amount of glutamate was higher (66.0 ± 1.0 nmol/mg protein) and constant as a function of time. Thus, glutamate specific radioactivity was only 14% of the specific radioactivity of glutamine. This implies that formation of 14C-glutamate from 14C-labeled glutamine may be about 14% of the rate of formation of unlabeled glutamate from the neuronal citric acid cycle.
To calculate absolute fluxes rather than percentages, the rate of turnover of the neuronal glutamate is needed. The H14CO3− labels only the 1-C of glutamate and glutamine (see Fig. 2). Therefore, when glutamate equilibrates with α-ketoglutarate and is oxidized in the citric acid cycle, the 14C in the 1-C position is irreversibly lost in the first turn of the cycle. Therefore, a knowledge of the first-order rate constant of glutamate turnover (kA) and the steady-state fraction of glutamine derived from pyruvate carboxylation allows one to calculate flux through the glutamine cycle. The details of the calculation are as follows:
First-order rate constant of glutamate turnover (per min) = Ka
Measured mass of glutamate (nmol per mg protein) = A
Rate of turnover of the neuronal glutamate pool (nmol per min) = Ka (A)
First order rate constant of glutaminase (per min) = kB
Measured mass of glutamine (nmol per mg protein) = B
nmol of glutamate derived from H14CO3− (nmol H14CO3−in glutamate/mg protein)=A*
nmol of glutamine derived from H14CO3− (nmol H14CO3 −in glutamine/mg protein) = B*
The fraction of glutamine derived from CO2 fixation = B*/B. [See above and Fig. 1 for why glutamate and glutamine d.p.m./mg protein is multiplied by 2 and divided by specific radioactivity of H14CO3− to obtain A* and B*.]
kA(A) = nmol of glutamate (mass) exiting the glutamate pool per min
kA(A*) = nmol of glutamate derived from H14CO3− exitingglutamate pool per min
kB(B*) = rate of entry of H14CO3− into glutamate pool (via14C − glutamine and neuronal glutaminase)
At isotopic steady state:
kB(B) = kB(B*) · B/B* = the rate of the glutamate/ glutamine cycle.
Subsequent experiments were carried out to estimate the first-order rate constant for turnover of the retinal neuronal glutamate pool (kA). The pool was labeled for 20 min by incubating retinas with H14CO3− and 0.2 mm pyruvate, then retinas were transferred to fresh buffer containing unlabeled HCO3−. The decline in retina glutamate specific radioactivity versus time shown in Figs 4(a) and (b) shows the first-order decline of 14C-label in the retinal glutamate pool. The mass amount of glutamate was constant at 66 ± 3 nmol/mg protein. The first-order rate constant of glutamate turnover (kA) calculated from the data shown in Fig. 4 was 0.068 per min. Multiplying 2 × 14C-labeled glutamate (A*) at t = 0 (1.8 × 2 nmol/mg protein), the rate of loss of 14C-glutamate from the glutamate pool was 0.24 nmol/min. Because at t = 0 the radioactivity of glutamate and glutamine were at steady state, the rate of 14C-glutamine conversion to glutamate must also be 0.24 nmol/min. B* was 0.81 nmol/mg protein at t = 0. Because kA (A*) = kB (B*) the value of kB was 0.15. Since 32% of the glutamine is derived from H14CO3− while 68% enters the glutamine pool from unlabeled neuronal glutamate, the total rate of conversion of intraretinal glutamine to glutamate kB(B*)(B/B*) is 0.68 nmol/min/mg protein. Therefore, at steady state this is the approximate rate of the glutamate/glutamine cycle. Compared with total glutamate turnover (0.068 × 66 nmol/mg protein = 4.5 nmol/min/mg protein), the glutamate/glutamine cycle accounts for 15% of the glutamate turnover. The results are summarized in Table 2.
Figure 4. Determination of the first-order rate constant for glutamate turnover in the ex vivo retina. Half-retinas were incubated for 20 min with 25 mm H14CO3− as described in Fig. 3. Then the retinas were transferred to buffer containing unlabeled HCO3−. Incubations were stopped at the times shown and the H14CO3− incorporated into retinal glutamate (Glu) determined as described under Materials and methods. (a) nmol of H14CO3− in glutamate plotted as a function of time; (b) natural log (ln) of the nmol of H14CO3− in glutamate plotted as function of time.
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Table 2. Calculated metabolic fluxes for glutamate metabolism in brain and retina
| ||Equation||Brain nmol/min/ mg protein||Retina nmol/min/ mg protein|
|Turnover neuronal glutamate pool||kA(A)||10.5||4.5|
|Synthesis of neuronal glutamate from H14CO3−||kA(A)*||1.42||0.24|
|Rate of entry of H14CO3− into glutamate pool (via 14C glutamine)||kB (B*)||1.42||0.24|
|Glutamate/glutamine cycle flux||(kBB*)/(B/B*)||6.17||0.68|
|Fraction of total glutamine derived from H14CO3−||B*/B||0.23||0.32|
Synthesis of retinal glutamate and glutamine is partially dependent on BCAA transamination
Our recent studies of glutamate synthesis in cultured astrocytes suggested that the conversion of α-ketoglutarate to glutamate could act as a rate-controlling step in anaplerotic synthesis of glutamate (Gamberino et al. 1997). Several groups (Yudkoff 1997; Kanamori et al. 1998) including our own (Gamberino et al. 1997) have indicated that the α-amino group of glutamate cannot be supplied as ammonia and must therefore be supplied by transamination of α-ketoglutarate with another amino acid. BCAA are likely nitrogen contributors because they can cross the blood–brain and blood–retinal barriers efficiently. Hutson et al. (1998) have proposed that a nitrogen shuttle between neurons and glia that involves BCAA and two BCAT isoenzymes, one in neuronal cytosol (BCATc) and one in glial mitochondria (BCATm), can provide the needed nitrogen. According to this hypothetical shuttle, which is a modification of the BCAA shuttle proposed by Yudkoff and coworkers (Yudkoff et al. 1996; Yudkoff 1997) and Bixel et al. (1997), neuronal BCATc facilitates glial BCATm in conversion of α-ketoglutarate to glutamate by regenerating BCAA in the neurons (Fig. 1).
As a first step towards investigating the role of the BCAT isoenzymes in de novo glutamate synthesis in the retina, immunoblotting was used to determine whether the BCAT isoenzymes are expressed in rat retina. As shown in Fig. 5, both BCATc and BCATm are expressed in the retina at levels that are comparable to those found in whole brain homogenates. Therefore, freshly dissected rat retina is an appropriate intact neural system in which to test whether or not BCAA and BCAT are involved in nitrogen shuttling between neurons and glia.
Figure 5. Expression of branched-chain aminotransferase (BCAT) isoenzymes in rat retina. Tissue extraction, SDS-PAGE and immunoblotting were performed as described in Materials and methods. Immunoaffinity purified rabbit rat cytosolic branched-chain aminotransferase (BCATc) peptide antibody (1 : 1000) and rabbit immunoaffinity purified human mitochondrial branched-chain aminotransferase (BCATm) antibody (1 : 1000) were used.
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The leucine analog gabapentin is a neuroactive drug that inhibits BCATc but not BCATm (Hutson et al. 1998). We confirmed that gabapentin inhibits the transamination of leucine in freshly dissected rat retinas (data not shown). We measured the effect of gabapentin and BCAA on retina synthesis of 14C-glutamate plus 14C-glutamine from H14CO3−. As shown in Fig. 6, addition of all three BCAA (200 µm each) alone had no effect on 14C-glutamate plus 14C-retina glutamine synthesis. However, addition of gabapentin (1.0 mm) reduced the formation of 14C-glutamate plus 14C-glutamine to 71% of control values (2.26 ± 0.05 versus 3.18 ± 0.12 nmol/mg protein, p < 0.001). The addition of the three BCAA to retinas incubated with gabapentin (BCAA/gabapentin ratio of 0.6) clearly antagonized the inhibitory effect of gabapentin on 14C-glutamate plus 14C-glutamine synthesis (89% of control without gabapentin). There was no effect of gabapentin on either ATP or creatine phosphate concentrations, indicating that gabapentin did not impair retinal bioenergetic processes.
Figure 6. The effect of gabapentin (GP) and branched-chain amino acids (BCAA) on incorporation of H14CO3− into glutamate plus glutamine (14C-Glu + 14C-Gln) by ex vivo rat retinas. Half-retinas were incubated at 37°C under standard conditions with 25 mm H14CO3− and 0.2 mm pyruvate. When added, GP was 1 mm and BCAA – leucine, isoleucine and valine – were 0.2 mm each. Values shown are means ± SEM (n = 3–8, *p < 0.001).
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Further experiments were carried out to investigate dose–response properties of gabapentin. As shown in Fig. 7, the inhibition of de novo glutamate synthesis by gabapentin was nearly maximal at 0.2 mm drug, concentrations that are reached in vivo in the rat brain (Welty et al. 1995). The tendency of gabapentin to increase citric acid cycle intermediates and lactate is also consistent with the hypothesis. Gabapentin slows the conversion of α-ketoglutarate to glutamate, probably by decreasing the supply of leucine to the Müller cells (see Fig. 1).
Figure 7. Influence of gabapentin concentration on incorporation of H14CO3− into glutamate and glutamine (Glu + C-Gln) the sum of lactate (Lac) and citric acid cycle metabolites (TCA) in ex vivo rat retinas. Conditions were the same as in Fig. 6 except for the concentration of gabapentin. Values shown are means ± SEM (n = 6–7, *p < 0.001).
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De novo synthesis of glutamate and glutamine in whole brain is similar to that observed in the retina
In order to estimate the likelihood that studies of retinal glutamate metabolism might be relevant to glutamate metabolism in the whole brain, incorporation of H14CO3− into metabolites of the brains of intact, awake rats infused for 1 h with H14CO3− was measured. Specific radioactivities of H14CO3− in the blood were measured at 15-min intervals, and the non-volatile 14C remaining in the plasma were determined. The non-volatile radiolabel increased linearly in serum so that it accounted for approximately 19% of the total counts at the end of 1 h. Ninety-five per cent of the non-volatile counts were in glucose, and only negligible amounts in glutamine and lactate. Their specific activities were so low they would not have influenced the results. Hepatic gluconeogenesis accounts for the conversion of H14CO3− to glucose in the blood. The serum glucose specific radioactivity per three carbon unit remained below 20% of the H14CO3− specific activity. Analysis of brain metabolites indicated that the specific radioactivities of brain and serum glucose were the same. The average specific radioactivity of the brain lactate/pyruvate was also the same per three carbon unit and was 18% of the mean serum H14CO3− specific radioactivity (162.6 ± 8.0 d.p.m./nmol, n = 5 rats). Mitochondrial pyruvate specific radioactivity might have been slightly higher than the average because of pyruvate recycling, but we assumed an average value in our calculations
Because the (1-14C)-labeled lactate and the H14CO3− contribute 14C equally to the anaplerotic synthesis of oxalacetate from 14CO2, we used the sum of the constant specific radioactivity of H14CO3− plus the average lactate specific activity to calculate the nmol of pyruvate carboxylase product in each metabolite. To determine the fraction of each metabolite derived from 14CO2 fixation (pyruvate carboxylase reaction), the total nmol H14CO3− in each metabolite fraction was divided by the mass amount of each metabolite (see Table 3).
Table 3. Incorporation of the products of pyruvate carboxylase into brain metabolites
| ||14C-labeled metabolite|
|Product||H14CO3− incorporated(14C-labeled metabolite) (nmol/mg protein)||Metabolite mass (nmol/mg protein)||Metabolite mass|
|Glutamate||7.91 ± 0.22||116.7 ± 1.1||0.068 ± 0.0014|
|Glutamine||7.53 ± 0.23||66.32 ± 3.8||0.114 ± 0.0054|
|Aspartate||2.57 ± 0.14||34.74 ± 1.1||0.074 ± 0.002|
|α-ketoglutarate||0.26 ± 0.10||–||–|
|Citrate||1.21 ± 0.12||–||–|
|Unknown neutral metabolite||5.51 ± 0.85||–||–|
|Alanine||n.s.||3.52 ± 0.13|| |
Assuming steady state and using a published value for neuronal glutamate turnover in whole brain of 0.09 nmol/mg protein per min (Sibson et al. 1998), fluxes for the glutamate/glutamine cycle and entry of the 14C-labeled products of pyruvate carboxylase into brain glutamate were calculated. The results for retina and whole brain are compared in Table 2. As in the retina, in brain, anaplerotic synthesis of glutamate from H14CO3− constitutes a significant fraction of the input into the glutamate/glutamine cycle. Also, glutamate/glutamine cycle flux is faster relative to total glutamate turnover in the intact in vivo brain than in the excised retina (Table 2).