Evidence for rapid consumption of millimolar concentrations of cytoplasmic ATP during rigor-contracture of metabolically compromised single cardiomyocytes

Abstract

Cytoplasmic ATP can be measured continuously in single cardiac myocytes by monitoring the luminescence from microinjected firefly luciferase. We show here that the signals are markedly influenced by changes in cytoplasmic pH, and the calibration of the signals as ATP concentration is markedly affected by cytoplasmic protein. Measurements with a pH-sensitive fluorescent dye show that intracellular pH (pHi) can be clamped at pH 7.08 by perfusing cells with a modified bicarbonate-buffered Krebs saline containing 92 mM NaHCO3 and equilibrated with 20% CO2. Calibration of the firefly luciferase signal in vitro in the presence of high concentrations of BSA (180 mg/ml), to simulate the cytoplasmic protein concentration, revealed a shift in Km (ATP) to 2 mM, from approx. 400 microM in the absence of albumin in an identical ionic milieu. Luciferase measurements in pH-clamped cells show that metabolically poisoned isolated rat ventricle cardiomyocytes enter rigor at a cytoplasmic ATP concentration of between 1 and 2 mM. As the cells shorten in rigor, a process that is complete in 30-40 s, the cytoplasmic ATP concentration falls simultaneously to a level of typically 20 microM. When cyanide is removed 10 min later, to simulate reoxygenation, the signal recovers over a period of 2-3 min to a level approx. 70% of the original in the healthy cell. These studies indicate that rigor-mediated depletion of cytoplasmic ATP in metabolically poisoned cardiomyocytes is considerably more extreme than hitherto indicated.

Effects of bisindolylmaleimide PKC inhibitors on p90RSK activity in vitro and in adult ventricular myocytes

“…The present data demonstrate that GF109203X exhibits approximately 30-fold selectivity for PKCa and PKCe when compared to RSK2, with IC 50 values for PKC isoform inhibition (8-12 nM) close to that described in the original study describing this inhibitor (14 nM) (Toullec et al, 1991). The selectivity of GF109203X for PKC isoforms was retained at an elevated ATP concentration of 5 mM, which is akin to that estimated in ventricular myocytes (Allue et al, 1996). Ro31-8220 exhibited considerably lower (approximately six-fold) selectivity for PKC isoforms over RSK2.…”

“…We also determined the in vitro potency of GF109203X and Ro31-8220 as inhibitors of PKCa, PKCe and RSK2 (the predominant p90 RSK isoform expressed in myocardium (Wagner, 2004)) at an ATP concentration (5 mM) that is akin to the estimated intracellular ATP concentration in ARVM (Allue et al, 1996). At this ATP concentration, the inhibitory potencies of both bisindolylmaleimides against all three Figure 1 Time-dependent phosphorylation of (a) GST-MARCKS by the PKC isoforms PKCa and PKCe and (b) GST-NHE1 by the p90 RSK isoforms RSK1, RSK2 and RSK3.…”

Sperm-triggered [Ca2+] oscillations and Ca2+homeostasis in the mouse egg have an absolute requirement for mitochondrial ATP production

“…In order to assess the use of ATP at fertilisation, we measured the variations of bulk cytosolic [ATP] by measuring the luminescent signal emitted by injected firefly luciferase (Allue et al, 1996) in fertilised eggs. (Fig.…”

“…The luminescent ATP indicator firefly luciferase (Calbiochem) (Allue et al, 1996;Jouaville et al, 1999) was injected into unfertilised mouse eggs as described above. The concentration of recombinant luciferase protein in the injection pipette was 2 mg ml -1 .…”

Mitochondrial Calcium Homeostasis: Mechanisms and Molecules

“…Remarkably, these predictions could be veri ed experimentally in living cells. Indeed, mobilization of cytosolic Ca 2C is paralleled by an increase in NAD(P)H levels (7,8,35,36), by transient depolarisation [because of Ca 2C uptake (26)] followed by hyperpolarisation of the inner mitochondrial membrane (32), and by an increase in ATP levels (29,37,38 ] i enhances the synthesis of aldosterone, partly by activating the transport of its precursor, cholesterol, into the mitochondria. On the other hand, also in these cells, a Ca 2C signal is transferred to the mitochondrial matrix, and enhances the reduction of NAD C and NADP C (40).…”