The effects of Mg2+ and adenine nucleotides on the sensitivity of the heart mitochondrial Na+-Ca2+ carrier to extramitochondrial Ca2+. A study using arsenazo III-loaded mitochondria

Abstract

The technique of reversible Ca2+-induced permeabilization [Al Nasser & Crompton (1986) Biochem. J. 239, 19-29, 31-40] has been applied to the preparation of heart mitochondria loaded with the Ca2+ indicator arsenazo III (2 nmol of arsenazo III/mg of mitochondrial protein). The loaded mitochondria ('mitosomes') were used to study the control of the Na+-Ca2+ carrier by extramitochondrial Ca2+ mediated by putative regulatory sites. The Vmax. of the Na+-Ca2+ carrier and the degree of regulatory-site-mediated inhibition were similar to normal heart mitochondria. Ca2+ occupation of the sites in mitosomes yields partial inhibition, which is half-maximal with 0.8 microM external free Ca2+. The inhibition consists of a small decrease in Vmax. and a relatively large increase in apparent Km for internal Ca2+. Mg2+ also appears to interact with the sites, but this is largely abolished by ATP and ADP (but not AMP) under conditions in which the free [Mg2+] is maintained constant. The results indicate that the regulatory sites are effective in controlling the Na+-Ca2+ carrier at physiological concentrations of adenine nucleotides, Mg2+, intra- and extra-mitochondrial free Ca2+.

Dependence of cardiac mitochondrial pyruvate dehydrogenase activity on intramitochondrial free Ca2+ concentration

“…matrix adenine nucleotide pool, but also allowed the presence of free Mg2" to modulate the activity of the Ca2+-transport cycle. It is known that Mg2" ions serve profoundly to inhibit the electrophoretic Ca21 uniport of heart mitochondria (Sordahl, 1974;Crompton et al, 1976a); Mg2+ also inhibits the Na+/Ca2+ antiport (Hansford & Castro, 1981;Crompton, 1985;Rizzuto et al, 1987), although this inhibition may be relieved by ATP at constant free Mg2+ concentration (Hayat & Crompton, 1987 (Chance & Williams, 1957). Although the PDHA content is substantially higher at any given value of [Ca2+]m in the presence of ADP (Fig.…”

“…Previous experiments have documented the progressive appearance of Ca2+ in the suspending medium under these conditions (Crompton et al, 1976b), the obverse of the present finding. By contrast, the null-point technique (Coll et al, 1982;Hansford & Castro, 1982) Hayat & Crompton (1987) involving the loading of resealed mitochondria with Arsenazo III does have that capability. It is noted that Na+ was present at 10 mm in this experiment ( Fig.…”

“…Each of these dehydrogenase systems is present within the permeability barrier of the mitochondrial inner membrane, leading (Sheu & Fozzard, 1982;McCormack & England, 1983), would elicit enhanced dehydrogenase activity, and enhanced energy transduction by oxidative phosphorylation. Evidence in favour of this proposal has been recently reviewed (Denton & McCormack, 1985;Hansford, 1985).Although there is good agreement on the relationship between the fraction of pyruvate dehydrogenase existing in the active dephosphorylated form (PDHA; see reviews by Reed, 1981;Wieland, 1983) in isolated cardiac mitochondria and the concentration of free ionized Ca2" ([Ca2+]O) in the suspending medium (Hansford & Cohen, 1978;Hansford, 1981) al., 1982;Hansford & Castro, 1982), and the insertion of the metallochromic indicator Arsenazo III into the mitochondria by a permeabilization/resealing protocol (Hayat & Crompton, 1987). The precision of the 'nullpoint' technique is limited by the small absorbance changes seen with the extramitochondrial metallochromic indicator when mitochondrial Ca2" loads low enough to be of relevance to dehydrogenase regulation are investigated, whereas the permeability/resealing protocol may, theoretically at least, perturb the matrix composition, especially with respect to metal ions and low-molecular-mass cofactors.…”

Mitochondrial Transport of Cations: Channels, Exchangers, and Permeability Transition

“…This pathway, the "Na ϩ -dependent pathway for Ca 2ϩ efflux" (NCE), has likewise been the subject of a large number of studies (e.g., Refs. 8,9,22,74,75,93,135,136,[159][160][161]164,182,283,296,303,308,318,363) and of a recent debate about the Na ϩ -Ca 2ϩ stoichiometry (23,180,361). The two pathways are distinct, since their kinetics are completely different, in that NICE is second order whereas NCE is first order in [Ca 2ϩ ] (360, 361).…”

Evidence for the presence of a reversible Ca2+-dependent pore activated by oxidative stress in heart mitochondria

“…Recent studies with liver mitochondria in this laboratory indicate that the lesion may reflect the presence of a non selective Ca2"-activated pore, which opens when matrix Ca2" exceeds normal limits and closes on Ca2" removal (Al Nasser & Crompton, 1986a). Although its natural role is as yet obscure, pore reversibility has proved useful practically in allowing the entrapment of Ca2" indicators and Ca2" buffers in the matrix space (Al Nasser & Crompton, 1986b; Hayat & Crompton, 1987).…”