AJP - Renal Physiology, Vol 267, Issue 1 153-F159, Copyright © 1994 by American Physiological Society
Stimulatory effect of calcium on metabolism and its sensitivity to pH in kidney mitochondria
K. Drewnowska and A. C. Schoolwerth
Department of Internal Medicine, Medical College of Virginia, Richmond 23298.
The relationship between mitochondrial matrix free Ca2+ concentration
([Ca2+]m) and pH was evaluated by incubating isolated rat kidney
mitochondria with different extramitochondrial Ca2+ concentrations
([Ca2+]e) at medium pH (pHe) 7.0 and 7.4. [Ca2+]m was monitored using the
fluorescent signal from mitochondria loaded with the Ca2+ indicator fura 2.
The changes in [Ca2+]m were compared with alpha-ketoglutarate dehydrogenase
(alpha-KGDH) flux, measured as O2 consumption (nmol.min-1.mg protein-1)
from 185 microM alpha-ketoglutarate (alpha-KG). The apparent dissociation
constant of the matrix fluorescent probe for Ca2+ was determined in each
experiment and was 323 +/- 45 nM (n = 14). When mitochondria were exposed
to [Ca2+]e below 160 nM, [Ca2+]m was greater at pHe 7.0 than at pHe 7.4.
However, above 160 nM [Ca2+]e, [Ca2+]m plateaued at pHe 7.0 but rose
progressively at pHe 7.4. Increasing [Ca2+]m by consecutive additions of
Ca2+ to the medium had a significantly more pronounced acceleratory effect
on alpha-KG oxidation at pHe 7.0 than at pHe 7.4. Kinetic analysis of
alpha-KGDH revealed a 45% decrease in the Michaelis constant (Km) for
alpha-KG at pHe 7.0, but the Km was unchanged at pHe 7.4 with elevation of
[Ca2+]m from 32 to 751 nM. Maximal velocity (Vmax) increased significantly
at both pHe values. Half-maximal alpha-KG oxidation occurred at [Ca2+]m of
76 +/- 11 nM and 105 +/- 31 nM at pHe 7.0 and 7.4, respectively. These
studies demonstrate a direct, pH-sensitive correlation between [Ca2+]e and
[Ca2+]m; [Ca2+]m changed over a range that may regulate alpha-KGDH flux in
intact kidney mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)
