Solubilization of γ-aminobutyric acid receptor from rat brain

Gamma-aminobutyric acid (GABA) binding sites were solubilized from rat brain synaptosomal fractions by extraction with a combination of sodium deoxycholate and potassium chloride. Specific ³H-GABA binding to the solubilized fraction was saturable with the apparent dissociation constant, Kd = 23.4 ± 0.2 nM. GABA agonists and an antagonist inhibited the binding. The relative potencies of these drugs in competing for ³H-GABA binding to the solubilized fraction are in good agreement with findings with the membrane fraction, suggesting that the binding sites in the solubilized fraction retain the characteristics of membrane-bound GABA receptor. The sedimentation coefficient value of ³H-GABA binding site was estimated to be 11.3S by sucrose density gradient centrifugation, and this value was identical with that of ³H-flunitrazepam binding site in the same solubilized fraction.     

Properties of γ-aminobutyric acid synthesis by rat renal cortex

Substantial synthesis of γ-aminobutyric acid occurs in rat renal cortex. Renal glutamate decarboxylase activity (24.3±2.9 (S.E.) nmols/mg protein per h) is 15% of that in brain; renal γ-aminobutyric acid content (39.5±5.3 (S.E.) nmols/g wet wt.) is 5% of the whole brain concentration. Properties of glutamate decarboxylase were studied in homogenates of rat renal cortex and rat brain under conditions for which γ-aminobutyric acid formation from [2,3-³H]glutamate and CO₂ release from [1-¹⁴C]glutamate were equal. Several properties of renal glutamate decarboxylase distinguish it from the corresponding brain enzyme: (1) renal glutamate decarboxylase is selectively inhibited by cysteine sulfinic acid (K_i = 5·10^?5 M) ; (20 renal glutamate decarboxylase is less sensitive (K_i = 3–5·10^?5 M)_to inhibition by aminooxyacetic acid than is the brain enzyme (K_i = 1·10^?6 M); (3) brain but not renal glutamate decarboxylase activity can be substantially stimulated in vitro by the addition of exogenous pyridoxal 5′-phosphate; (4) renal glutamate decarboxylase is significantly decreased in renal cortex from rats on a low-salt diet. Proximal tubules are enriched in glutamate decarboxylase compared to the activity in whole renal cortex or glomeruli (42, 22 and 14 nmols/mg protein per h, respectively). We speculate that renal γ-aminobutyric acid synthesis does not reflect the presence of GABAergic renal nerves, but may serve a function in proximal tubular cells.     

Prolyl 3-hydroxylase and 4-hydroxylase activities in certain rat and chick-embryo tissues and age-related changes in their activities in the rat.

Prolyl 3-hydroxylase activity, expressed per unit of extract protein, was much higher in rat kidney cortex than in the lung, liver or skin. A marked decrease in activity was found in the kidney cortex, liver and skin beyond 10 days of age. The ratio of prolyl 3-hydroxylase to 4-hydroxylase activity in the kidney cortex was 13--17 times that in the skin, that in the liver 6--8 times, and that in the lung about twice the value for the skin, there being no changes in this ratio with age. In 16-day chick embryos the highest ratios of prolyl 3-hydroxylase to 4-hydroxylase activity were found in the liver, heart, lens, aorta and kidney, and the lowest ratios in tendon, cartilage, cartilaginous and membranous bone and skin. The results suggest that the differences in the extent of prolyl 3-hydroxylation between various collagens can in part be explained by differences in the amount of prolyl e-hydroxylase activity among different cells.     

The relationship of 4-aminobutyric acid metabolism to ammoniagenesis in renal cortex

Mitochondrial 4-aminobutyrate aminotransferase in rat kidney can utilize pyruvate as the acceptor for the amino group of 4-aminobutyrate. Renal 4-aminobutyrate aminotransferase activity at saturating equimolar concentration of 4-aminobutyrate and 5 mM pyruvate is 42.8 ± 2.5 μmol/g protein per h (mean ± S.E.M.) or 70% of 4-aminobutyrate aminotransferase activity with equimolar α-ketoglutarate. 4-Aminobutyrate aminotransferase in brain does not transaminate with pyruvate. Since pyruvate is an important mitochondrial metabolite in kidney, net disposal of glutamate via the 4-aminobutyrate pathway is possible. The renal 4-aminobutyrate pathway in the rat has other distinctive features when compared with the pathway in rat brain. Most inhibitors of rat neuronal glutamate decarboxylase were ineffective against the renal form of the enzyme, but 20 mM semicarbazide inhibited the latter form by 80% (P < 0.001) in vitro and reduced renal 4-aminobutyrate content by 75% (P < 0.001) in vivo. In the presence of 20 mM semicarbazide, ammoniagenesis by rat renal cortex slices incubated in 1 mM glutamine was inhibited 26% (P < 0.01). Semicarbazide was proportionately less effective (15% inhibition) when ammoniagenesis was stimulated (+243%) in slices prepared from chronically acidotic animals, and was no deterrant to ammoniagenesis when non-acidotic slices were incubated in supraphysiologic concentrations of 10 mM glutamine. We conclude that whereas integrity of the renal 4-aminobutyrate pathway may contribute to glutamate disposal and thus ammoniagenesis under physiologic conditions, the pathway is a passive participant in the overall process of ammoniagenesis.