Download 31P n.m.r. analysis of the renal response to respiratory acidosis

600th MEETING, OXFORD
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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