Biochemical SocietyTransactions (1 992) 20
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Activation by anserine and inhibition by carnosine of Caz+-uptake by mammalian mitochondria RACHAEL L. DANIEL, NICOLA J. OSBALDESTON and JAMES G. McCORMACK' Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K. ('present address, Department of Pharmacology, Syntex Research Centre, Heriot-Watt University Research Park, Riccarton, Edinburgh EH14 4AP, U.K.) The Ca2+-transport system in the inner membrane of mammalian mitochondria consists of an electrophoretic CaZ+-uniporterfor uptake and an electroneutral ZNa+/CaZ+exchanger which is the major egress mechanism, though there may also be Na+-independent egress pathway(s) which are of lesser activity and remain poorly characterised (see [1,2] for reviews). Both aspects are driven by the respiratory protonmotive gradient, however under normal cellular conditions the cost of cycling of Caz+ across the inner membrane is likely to be 4 % of the mitochondrial respiratory capacity [3]. The primary function of the mitochondrial Ca2+-transport system under normal physiological conditions is to relay changes in cytosolic CaZ+ brought about by hormones and other agents into the mamx where it also has an important second-messenger role [1,4]. This involves its activation of.severa1 key enzymes in oxidative metabolism (see [ 1.4]), and hence ATP production can be stimulated to match the increases in ATP demand which are brought about by the increases in CP+in the cytosol by its stimulation of processes such as contraction, secretion etc. The mitochondrial Ca2+-transport system has, from necessity, largely been studied at the level of isolated mitochondria, however several potential physiological regulators have been identified. These include extramitochondrial Mg2+ which inhibits both uptake and egress, spermine which activates uptake, and extramitochondrial Ca2+ which inhibits the NdCa egress mechanism; artificial regulators include ruthenium red, an inhibitor of uptake, and agents such as diltiazem and similar compounds which, rather weakly, inhibit the N d C a pathway (see [ 1-31]. Clearly the presence or absence of such agents will influence the distribution of Caz+ across the inner membrane, and hence could alter the relationship between energy supply and demand noted above. The imidazole dipeptides anserine and camosine and their acetylated derivatives have been found to be present at relatively high concentrations (up to several mM) in various mammalian tissues, and have also been found to affect the Caz+-sensitivity, and hence energy requirements, of the contractile apparatus in several skinned muscle preparations (see [5,6]).These and other imidazoles are also known to affect CaZ+-handling by other cellular systems such as the sarcoplasmic reticulum (see 171). This prompted us to see whether either of these compounds affected any aspects of the mitochondrial CaZ+-transport system. Mitochondria were prepared from rat heart and loaded with the fluorescent CP+-indicator fura-2, or prepared from rat liver or kidney and set up to assay for the activity of the matrix Ca2+sensitive 2-oxoglutarate dehydrogenase, in both cases monitoring changes in matrix [Caz+] to study CaZ+-transport across their inner membranes, as has been fully described previously [8-lo]. Lcamosine and L-anserine (Sigma) were added as neutralised salts and were checked to have no effects on either fura-2 or extracted 2oxoglutarate dehydrogenase, or on intact mitochondrial parameters measured in the absence of added Ca2+ (i.e. with EGTA alone, see Fig. 1) ; appropriate control salt additions were always performed. Fig. 1 shows the effects of both anserine and camosine added at 5mM to isolated rat heart mitochondria loaded with fura-2 to monitor changes in matrix [Caz+]. It can be clearly seen that the presence of anserine increases the concentration of matrix Caz+ under these conditions, whereas conversely camosine causes a decrease. Further studies (not shown) established that these effects were both on the Caz+-uptake pathway and that neither agent affected Caz+-egress in either the absence or presence of Na+ (at I-lOmM). No effects were found with uncoupled mitochondria. It was also found (not shown) that the effects of these two agents appeared to be independent of, and hence additive to, the effects of the other physiological effectors of mitochondrial CaZ+-transpon mentioned above. Again within the
Fig. 1 Effects of anserine and carnosine on the increases in matrix Ca2+ of isolatedfura-2-loaded rat heart mitochondria exposed to an increase in extramitochondria1Ca2+. Mitochondria were initially incubated in a fluorirneter with excitation at 340nm and emission at 500nm and at 300C and pH 7.3 in buffered KC1-based media containing phosphate, respiratory substrates, 1mM EGTA (see 181) and either (a) no further additions (control), (b) 5mM anserine or (c) 5mM camosine, and then 1mM CaCIzplus IrnM EGTA (as a buffer solution to then give about 50nM free Ca2+) was added at the arrow i as shown. The upward deflection of the trace is indicative of increases i n matrix [Caz+].
concentration ranges of these two compounds which have been measured in different tissues, substantial effects on Caz+-uptake were also found with isolated rat liver and kidney mitochondna. The physiological relevance of the present findings has yet to be established, however such a statement could largely be made about all likely physiological regulators of this key transport process as the environment of mitochondria within the cell can only be guessed at. What is clear, however, is that both anserine and camosine exhibit substantial and, perhaps surprisingly i n terms of their similar structures, opposing effects on the Ca*+-uptakepathway of isolated mammalian mitochondna, and at concentrations at which they have been found to be present in mammalian tissues. Another possibility is that they are interacting at a site on the transporter for which there are other, perhaps more potent ligands which are the real physiological effectors, which have yet to be identified and which could have important regulatory function through effects at this site. These studies were supported by a SERC studentship (RLD) and by a project grant from the MRC. 1. McCormack, J.G., Halestrap, A.P. & Denton, R.M. (1990) Physiol. Rev. 70, 391-42 2. Gunter, T.E. & Pfeiffer, D.R. (1990) Am. J. Physiol. 258, C755-C786 3. Crompton, M. (1985) Curr. Top. Membr. Transp. 25, 231-276 4. McCormack, J.G. & Denton, R.M.(1986) Trends Biochem. Sci. 11, 258-262 5. O'Dowd, J.J., Robins, D.J. & Miller, D.J. (1988) Biochim. Biophys. Acta 967, 241-249 6. Harrison, S.M., Lamont, C. & Miller, D.J. (1985) J. Physiol. 371, 197P 7. Lopina, O.D. & Boldyrev, A.A. (1975) Dokl. Akad. Nauk SSSR 220, 1218-1221 8. McCormack, J.G., Browne, H.M. & Dawes, N.J. (1989) Biochim. Biophys. Acta 973, 420-427 9. McCormack. J.G. (1985) Biochem. J . 231, 581-595 10. McCormack, J.G., Bromidge, E.S. & Dawes, N.J. (1988) Biochim. Biophys. Acta 934, 282-292
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Activation by anserine and inhibition by carnosine of Caz+-uptake by mammalian mitochondria RACHAEL L. DANIEL, NICOLA J. OSBALDESTON and JAMES G. McCORMACK' Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K. ('present address, Department of Pharmacology, Syntex Research Centre, Heriot-Watt University Research Park, Riccarton, Edinburgh EH14 4AP, U.K.) The Ca2+-transport system in the inner membrane of mammalian mitochondria consists of an electrophoretic CaZ+-uniporterfor uptake and an electroneutral ZNa+/CaZ+exchanger which is the major egress mechanism, though there may also be Na+-independent egress pathway(s) which are of lesser activity and remain poorly characterised (see [1,2] for reviews). Both aspects are driven by the respiratory protonmotive gradient, however under normal cellular conditions the cost of cycling of Caz+ across the inner membrane is likely to be 4 % of the mitochondrial respiratory capacity [3]. The primary function of the mitochondrial Ca2+-transport system under normal physiological conditions is to relay changes in cytosolic CaZ+ brought about by hormones and other agents into the mamx where it also has an important second-messenger role [1,4]. This involves its activation of.severa1 key enzymes in oxidative metabolism (see [ 1.4]), and hence ATP production can be stimulated to match the increases in ATP demand which are brought about by the increases in CP+in the cytosol by its stimulation of processes such as contraction, secretion etc. The mitochondrial Ca2+-transport system has, from necessity, largely been studied at the level of isolated mitochondria, however several potential physiological regulators have been identified. These include extramitochondrial Mg2+ which inhibits both uptake and egress, spermine which activates uptake, and extramitochondrial Ca2+ which inhibits the NdCa egress mechanism; artificial regulators include ruthenium red, an inhibitor of uptake, and agents such as diltiazem and similar compounds which, rather weakly, inhibit the N d C a pathway (see [ 1-31]. Clearly the presence or absence of such agents will influence the distribution of Caz+ across the inner membrane, and hence could alter the relationship between energy supply and demand noted above. The imidazole dipeptides anserine and camosine and their acetylated derivatives have been found to be present at relatively high concentrations (up to several mM) in various mammalian tissues, and have also been found to affect the Caz+-sensitivity, and hence energy requirements, of the contractile apparatus in several skinned muscle preparations (see [5,6]).These and other imidazoles are also known to affect CaZ+-handling by other cellular systems such as the sarcoplasmic reticulum (see 171). This prompted us to see whether either of these compounds affected any aspects of the mitochondrial CaZ+-transport system. Mitochondria were prepared from rat heart and loaded with the fluorescent CP+-indicator fura-2, or prepared from rat liver or kidney and set up to assay for the activity of the matrix Ca2+sensitive 2-oxoglutarate dehydrogenase, in both cases monitoring changes in matrix [Caz+] to study CaZ+-transport across their inner membranes, as has been fully described previously [8-lo]. Lcamosine and L-anserine (Sigma) were added as neutralised salts and were checked to have no effects on either fura-2 or extracted 2oxoglutarate dehydrogenase, or on intact mitochondrial parameters measured in the absence of added Ca2+ (i.e. with EGTA alone, see Fig. 1) ; appropriate control salt additions were always performed. Fig. 1 shows the effects of both anserine and camosine added at 5mM to isolated rat heart mitochondria loaded with fura-2 to monitor changes in matrix [Caz+]. It can be clearly seen that the presence of anserine increases the concentration of matrix Caz+ under these conditions, whereas conversely camosine causes a decrease. Further studies (not shown) established that these effects were both on the Caz+-uptake pathway and that neither agent affected Caz+-egress in either the absence or presence of Na+ (at I-lOmM). No effects were found with uncoupled mitochondria. It was also found (not shown) that the effects of these two agents appeared to be independent of, and hence additive to, the effects of the other physiological effectors of mitochondrial CaZ+-transpon mentioned above. Again within the
Fig. 1 Effects of anserine and carnosine on the increases in matrix Ca2+ of isolatedfura-2-loaded rat heart mitochondria exposed to an increase in extramitochondria1Ca2+. Mitochondria were initially incubated in a fluorirneter with excitation at 340nm and emission at 500nm and at 300C and pH 7.3 in buffered KC1-based media containing phosphate, respiratory substrates, 1mM EGTA (see 181) and either (a) no further additions (control), (b) 5mM anserine or (c) 5mM camosine, and then 1mM CaCIzplus IrnM EGTA (as a buffer solution to then give about 50nM free Ca2+) was added at the arrow i as shown. The upward deflection of the trace is indicative of increases i n matrix [Caz+].
concentration ranges of these two compounds which have been measured in different tissues, substantial effects on Caz+-uptake were also found with isolated rat liver and kidney mitochondna. The physiological relevance of the present findings has yet to be established, however such a statement could largely be made about all likely physiological regulators of this key transport process as the environment of mitochondria within the cell can only be guessed at. What is clear, however, is that both anserine and camosine exhibit substantial and, perhaps surprisingly i n terms of their similar structures, opposing effects on the Ca*+-uptakepathway of isolated mammalian mitochondna, and at concentrations at which they have been found to be present in mammalian tissues. Another possibility is that they are interacting at a site on the transporter for which there are other, perhaps more potent ligands which are the real physiological effectors, which have yet to be identified and which could have important regulatory function through effects at this site. These studies were supported by a SERC studentship (RLD) and by a project grant from the MRC. 1. McCormack, J.G., Halestrap, A.P. & Denton, R.M. (1990) Physiol. Rev. 70, 391-42 2. Gunter, T.E. & Pfeiffer, D.R. (1990) Am. J. Physiol. 258, C755-C786 3. Crompton, M. (1985) Curr. Top. Membr. Transp. 25, 231-276 4. McCormack, J.G. & Denton, R.M.(1986) Trends Biochem. Sci. 11, 258-262 5. O'Dowd, J.J., Robins, D.J. & Miller, D.J. (1988) Biochim. Biophys. Acta 967, 241-249 6. Harrison, S.M., Lamont, C. & Miller, D.J. (1985) J. Physiol. 371, 197P 7. Lopina, O.D. & Boldyrev, A.A. (1975) Dokl. Akad. Nauk SSSR 220, 1218-1221 8. McCormack, J.G., Browne, H.M. & Dawes, N.J. (1989) Biochim. Biophys. Acta 973, 420-427 9. McCormack. J.G. (1985) Biochem. J . 231, 581-595 10. McCormack, J.G., Bromidge, E.S. & Dawes, N.J. (1988) Biochim. Biophys. Acta 934, 282-292
