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Transcript
600th MEETING, OXFORD 399 31Pn.m.r. analysis of the renal response to respiratory acidosis DOMINIQUE FREEMAN,* MARTIN LOWRY,? GEORGE RADDA* and BRIAN ROSS? *Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 JQU, and tlvufleld Department of Medicine, John Radclixe Hospital, Headington, Oxford OX3 9DU, U.K. The kidney is the key to the biochemical response in acidosis, since renal adaptation to excrete excess H+ ions and enhance ammoniagenesis protects other pH-sensitive tissues. The trigger to this metabolic response in acute acidosis is unknown; the favoured candidate is intracellular H + ion, but HC0,- or some other metabolite with allosteric effects cannot be excluded (Warnock & Rector, 1981). It has hitherto been impossible to measure intracellular pH directly in a functioning kidney. ,lP n.m.r. offers a possibility, by following the chemical shift of Pi, but heterogeneity and compartmentation within the kidney limit the interpretation of such measurements. The results of experiments designed to follow the intrarenal response to acute metabolic acidosis (elevated IH+l; low [HCO,-I) have been reported (Ackerman et al., 1981). No attempt was made to quantify the adenine nucleotides in that study. By combining quantitative n.m.r. with conventional enzymic or chemical assays, the present study extends the understanding of the role of cytosolic pH changes in the metabolic and physiological response to acidosis. Here we report experiments involving acute respiratory acidosis, which provides a controlled and reversible method of changing pH at constant [HCO,-]. In the isolated perfused kidney from fed rats, tubular transport function, urinary acidification, ammonia and glucose metabolism and intrarenal pH and adenine nucleotide content were measured continuously. pH of the perfusion medium was altered after a 30min control period, by substituting a gas containing 27% CO, in 0, for the O,/CO, (19: 1) used during the control period. An immediate response in perfusate pH, which reached its steadystate minimum within 2-3min, was observed. After 30min at the lower pH, normal pH was restored, and at the end of 2 h perfusion the kidney was freeze-clamped for analysis of adenine nucleotides, P, and other metabolites. Control perfusions were carried out outside the spectrometer, and gave comparable results. Pi was omitted from the normal Krebs-Henseleit albumin perfusion medium, supplemented with amino acids, to allow determination of only intrarenal pH. Function was not altered by this, and stability of the preparation was much improved by the addition of amino acids (Brosnan et al., 1981). The spectra of the kidney collected under quantitative conditions IT1 relaxation values for the isolated perfused kidney were: a-phosphate of ATP 0.98s, P-phosphate of ATP 1.0s, yphosphate of ATP 0.94s, P, 0.99s, glycerophospho choline 2.2s, unidentified peak at 11.1 p.p.m. (including AMP) 1 . 9 ~ 1 gave absolute values for adenine nucleotides and phosphate which differ significantly from those obtained by enzymic assay in the same kidney. The differences cannot be explained as artifacts from breakdown of the normal renal constituents, and reflect compartmentation of metabolites. In particular it is likely, but not yet proven, that the Pi that is observed by n.m.r. is confined to cytosol. Hence, pH measurements reported below reflect cytosolic pH and not that of the mitochondrion. Intracellular pH, initially 7.2 (perfusate pH 7.4), fell to 6.8 (perfusate 6.85) when acidosis was introduced. No changes in ATP or other phosphorylated intermediates were detectable by n.m.r. In particular, sugar phosphates did not accumulate during the marked acidosis, presumably because the bulk of the renal tissue (cortex) is not glycolytic. pH recovered to control values within 2-4min of restoring 5% CO, to the gassing mixture. Intrarenal oxoglutarate concentration fell from 3.1 to pmol/g dry wt. in normal kidney to 1.5 ,umol/g in acidotic kidneys (freeze-clamped without reversing the pH change). Ammonia production increased from 1.3 to 2.0pmol/min per g. Urine pH fell from 7.3 to 6.7. With the exception of urine pH, which in metabolic acidosis fell from 7.3 to 5.8, these results are identical with those obtained in metabolic acidosis. The difference is that intrarenal [HCO,-] calculated from the pH value obtained by ,lP n.m.r. and prevailing [CO,] in respiratory acidosis was 4 times (29.5 versus 6 . 9 m ~that ) in metabolic acidosis. These results indicate that: (1) adaptation of the kidney to ammoniagenesis in acidosis depends only on intracellular pH, rather than the intracellular concentration of HC0,-; (2) in contrast, urinary acidification is limited by the high intracellular concentration of HC0,-; (3) compartmentation of phosphate occurs in the functioning kidney, with ,IP n.m.r. likely to throw light on the properties of a cytosolic pool that is metabolically active. Ackerman, J. J. H., Lowry, M., Radda, G. K., Ross, B. D. & Wong, G. G. (1981) J. Phpiol. (London) 319.65-79 Brosnan, J. T., Green, Y. S. & Ross, B. D. (1981) Proc. Int. Congr. Nephrol. 8th p. 056 Warnock, D. G. & Rector, F. (1981) in The Kidnev (Brenner, B. & Rector, F., eds.), 2nd edn., vol. 1, pp. 4 4 0 4 9 5 , Saunders, Philadelphia. Inhibition of rat liver protein kinases by Be2+ MATTHEW R. KASER, GAIL WIGGINS, MARGERY G. O R D and LLOYD A. STOCKEN Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 JQU, U.K. BeZ+ given intravenously to rats as 1 :1 BeSO,/sulphosalicylic acid is toxic to rats: (LD,, about 40ymol/kg). It accumulates in the liver and the animals die after 3-4 days (Skilleter & Price, 1980). Soluble cytoplasmic protein kinase, histone phosphorylation (Kaser et al., 1980), enzyme induction (Witschi & Marchand, 1971; Ord & Stocken, 1981) and compensatory growth after partial hepatectomy (Witschi, 1970) are all inhibited in vivo. The kinase sensitive to Be2+ has now been identified as a cyclic AMP-independent enzyme which preVOl. 10 ferentially used casein, rather than phosvitin, protamine or histones, as substrate. The inhibition was competitive with respect to its protein substrate. Nuclear and cytoplasmic phosvitin kinase(s) and the catalytic subunit of cyclic AMPdependent kinase were insignificantly affected by 0.1 mM-Be2+. Three sources of kinase were examined: beef heart was used to prepare cyclic AMP-dependent kinase and its catalytic subunit (Chen & Walsh, 1974). These enzymes were also obtained commercially from Sigma. Rat liver cytoplasm ( IO’g supernatant) was used to prepare phosvitin kinase (Baggio et al., 1970); the material precipitating between 25-50% saturation with (NH,),SO, was also used as a source of casein kinase I (Dahmus, 1981). Rat liver nuclei, isolated in 2.2M-SUCrOSe (Chauveau et al., 1956) and subsequently extracted with