Effect of Carbonic Anhydrase Inhibition and Acetoacetate on Anaplerotic Pyruvate Carboxylase Activity in Cultured Rat Astrocytes

Abstract

In peripheral tissues, carbonic anhydrase (CA) inhibition secondarily decreases the anaplerotic activity of pyruvate carboxylase activity leading to a decline in citric acid cycle intermediates and glutamate. In view of the important role of pyruvate carboxylase in the brain, we examined the effects of CA inhibition on pyruvate-carboxylase-mediated [¹⁴C]CO₂ fixation in cultured astrocytes from postnatal rat brains. Incubation with H[¹⁴C]O3̄ led to radiolabeling of metabolites found both in the cells and in the medium. These were separated by ion exchange chromatography for identification. Ethoxyzolamide (ETZ), a sulfonamide CA inhibitor (SCAT) with a heterocyclic side group, caused a 43–73% decrease in cell lysate [α-ketoglutarate] and ¹⁴C incorporation into major products of pyruvate carboxylation in the cell lysates and cell medium (i.e., released products). Half-maximal inhibition of [¹⁴C]CO₂ fixation was observed between 1 and 3 x l0^–7M. This is similar to the IC₅₀ value for ETZ inhibition of events in other cells that are thought to be mediated by CA. Inhibition was also observed with trifluor-methanesulfonamide, an aliphatic SCAI, providing further evidence that this effect is mediated by CA. Western blot analysis using isozyme-specific anti-sera indicated that astrocytes contain CA II, a cytosolic isozyme, but CA in, CA IV and CA V could not be detected. This finding is unusual since the effects of SCAIs on pyruvate carboxylation in other tissues have been attributed to inhibition of the intramitochondrial isozyme, CA V. [¹⁴C]CO₂ fixation was also decreased by lowering media [pyruvate] or by addition of 5 mM acetoacetate. It is hypothesized that SCAIs may inhibit pyruvate carboxylation in astrocytes by limiting the supply of bicarbonate to this enzyme while ketone bodies, by inhibiting glucose oxidation, may limit the supply of pyruvate. Interestingly, both SCAIs and ketogenic diets are used to treat adolescent forms of epilepsy. The possibility that these treatments might ultimately work by affecting anaplerotic pyruvate carboxylase activity in the brain is discussed.

Role of Pyruvate Carboxylase in Facilitation of Synthesis of Glutamate and Glutamine in Cultured Astrocytes

“…Citric acid was also exported in large amounts. This has been noted previously by us (Hazen et al, 1997) and by others Hasse! and Sonnewald.…”

“…The results ofunknowns are compared with a standard curve. This luminometric procedure for measuring a-ketog!utarate has been reported by us previously (Hazen et al, 1997). However, the PCA interference was not noted in the previous report.…”

“…In a previous study from this laboratory, "'CO2 fixation was measured, the products were separated and identified on Dowex chloride columns, and the effect of inhibiting pyruvate carboxylase via carbonic anhydrase inhibitors was assessed (Hazen et a!., 1997).…”

“…2 (Hazen et a!., 1997). The purpose of the present study was to investigate the possi- Cultured astrocytes (0.3-0.5 mg) were incubated in 2 ml of medium A containing 2 mM glucose, 25 mM HCO3 and either 0.1 (A) or 0.5 mM (B) pyruvate in the presence of varied levels of added NH3 as shown in different data columns.…”

Targeted mutagenesis of mitochondrial carbonic anhydrases VA and VB implicates both enzymes in ammonia detoxification and glucose metabolism

“…The most limited by CA V deficiency appears to be carbamoyl phosphate synthase 1, which catalyzes the synthesis of carbamoyl phosphate from NH 3 and HCO 3 − , the first committed step in ureagenesis. The second enzyme is pyruvate carboxylase, which generates oxaloacetate from pyruvate and HCO 3 − for gluconeogenesis, oxidative metabolism of glucose (30), and anapleurosis of the Kreb's cycle (31). The third enzyme is propiony CoA carboxylase, which catalyzes an important step in the breakdown of branched chain amino acids and provides succinate for the TCA cycle.…”