Oscillations in NF-kappaB signaling control the dynamics of gene expression.

Signaling by the transcription factor nuclear factor kappa B (NF-kappaB) involves its release from inhibitor kappa B (IkappaB) in the cytosol, followed by translocation into the nucleus. NF-kappaB regulation of IkappaBalpha transcription represents a delayed negative feedback loop that drives oscillations in NF-kappaB translocation. Single-cell time-lapse imaging and computational modeling of NF-kappaB (RelA) localization showed asynchronous oscillations following cell stimulation that decreased in frequency with increased IkappaBalpha transcription. Transcription of target genes depended on oscillation persistence, involving cycles of RelA phosphorylation and dephosphorylation. The functional consequences of NF-kappaB signaling may thus depend on number, period, and amplitude of oscillations.

Novel cobalt complex inhibitors of mitochondrial calcium uptake.

Reperfusion of the ischaemic myocardium leads to intracellular calcium overload followed by mitochondrial dysfunction, resulting in insufficient energy supply and ultimately myocardial necrosis. Ruthenium red (RR), a potent mitochondrial calcium uptake inhibitor, prevents this disruption to mitochondrial metabolism and improves post reperfusion recovery. This therefore suggested that mitochondrial calcium influx is an attractive target for the treatment of reperfusion injury. However, RR is unsuitable for therapeutic use, so we undertook a search for novel compounds which inhibit mitochondrial calcium uptake. The most potent compounds discovered were simple tris(ethylenediamine) transition metal complexes and dinuclear Co complexes. The structure-activity relationship (SAR) of these small molecules has helped to define the structural requirements for inhibition of calcium transport by outlining the size and charge dependency of the interactive site on the mitochondrial calcium uniporter.

Intracellular [Ca2+] staircase in the isovolumic pressure--frequency relationship of Langendorff-perfused rat heart.

Fluorescence and 31P magnetic resonance spectroscopy have been used to monitor simultaneously, the [Ca2+]i staircase and high energy phosphate metabolism in isolated Langendorff-perfused rat heart paced at 2, 4 and 6 Hz. In order to investigate further the relationship between high energy phosphate metabolism and the calcium staircase we perturbed the intracellular phosphocreatine (PCr)/creatine concentration with dietary beta-guanidinopropionic acid (beta-GPA). We have observed that: (a) At 2 Hz stimulation, the ventricular -Ca2+-i-dependent fluorescence decay is biexponential and continues to decay throughout the interstimulus interval; (b) at 4 Hz and 6 Hz, the [Ca2+]i decay is monoexponential; (c) end-diastolic [Ca2+]i is elevated at higher stimulation frequencies; (d) net [Ca2+]i flux per cycle is reduced at higher stimulation frequencies and is therefore correlated inversely with stimulation frequency and end-diastolic [Ca2+]i; (e) "heart rate * [Ca2+]i flux product" which is a measure of the work done in cycling calcium, is directly proportional to stimulation frequency; (f) the hysteresis between peak ventricular isovolumic pressure and peak fluorescence is decreased at higher stimulation frequencies; (g) no correlation was detected between the PCr/ATP ratio and stimulation frequency; (h) despite a 60% decrease in the myocardial PCr/ATP ratio after beta-GPA feeding, rat heart is able to maintain the end-diastolic [Ca2+]i-dependent fluorescence, and therefore the [Ca2+]i staircase relationship, similar to that of normal rat heart. In conclusion, using a physiological stimulation range and substrate supply we have observed a negative staircase of both [Ca2+]i and isovolumic pressure in whole heart which is not hypoxic. We propose that the inability of the sarcoplasmic reticulum to sequester sufficient cytosolic calcium at high stimulation frequencies leads to an elevation in end-diastolic [Ca2+]i, decreased net calcium flux per cycle resulting in a negative [Ca2+]i staircase and thus a negative isovolumic pressure-frequency relationship. We did not detect any correlation between steady-state high energy phosphate metabolism and stimulation frequency.

The acute effects of the creatine analogue, beta-guanidinopropionic acid, on cardiac energy metabolism and function.

1. Perfusion of isolated rat hearts with 150 mM beta GPA led to the linear accumulation of intracellular P beta GPA (approx. 150 nmol/min per g (dry wt.)) after an initial lag period of 20 min. 2. This accumulation of intracellular P beta GPA was accompanied by a decrease in PCr (30%) and an increase in total phosphagen content (20%). These results show that PCr was not equally replaced by P beta GPA, but was degraded at the expense of beta GPA phosphorylation to produce a net increase in cardiac phosphagen content. Correspondingly, total phosphate (the sum of PCr, P beta GPA, Pi and ATP) was increased, indicating that there was no cellular necrosis and that the sarcolemma remained intact throughout the perfusion. 3. An increase in Pi and decrease in ATP also occurred concomitantly with P beta GPA accumulation, indicating that ATP synthesis was not keeping up with demand. This may be due to the gradual replacement of PCr by the less efficient phosphagen, P beta GPA, resulting in inadequate transduction of energy and hence an imbalance between energy demand and supply. However, the increased hyperosmolarity of the perfusate may be partly responsible for these effects on cardiac energy metabolism, as perfusion with 150 mM mannitol produced a similar decrease in ATP, but a smaller rise in Pi. 4. Perfusion with either 150 mM beta GPA or mannitol led to a significant intracellular alkalosis (max. pHi 7.3), which was reversed on returning to normal perfusate. In addition, both hyperosmolar perfusions led to a significant reduction in cardiac frequency (40 and 15%, respectively). However, only beta GPA caused significant negative inotropism. The time-courses for the changes in cardiac frequency and pHi did parallel the increase in P beta GPA. This suggests that both hyperosmolarity and the production of P beta GPA during beta GPA perfusions determine the degree of negative chronotropism, but that hyperosmolarity alone causes alkalosis and beta GPA phosphorylation, a decrease in developed tension. 5. When hearts, acutely loaded with P beta GPA were perfused with control medium, the levels of ATP, PCr and P beta GPA stabilised to produce a new steady state. There was no decrease in P beta GPA concentration during this procedure, implying that beta GPA efflux was negligible.

Determination of free creatine and phosphocreatine concentrations in the isolated perfused rat heart by 1H- and 31P-NMR.

To measure free creatine in the isolated perfused rat heart, the concentration of phosphocreatine, and phosphocreatine plus creatine (sigma Cr) were measured by 31P- and 1H-NMR, respectively. Quantification was performed in the presence and absence of an intraventricular balloon filled with a known amount of PCr, which acted as an external standard. Total (free plus bound) phosphocreatine and creatine were measured by HPLC analysis of extracts from the same hearts, freeze-clamped at the end of the perfusions. A greater concentration of creatine (mumol/g dry wt.) in the perfused rat heart was measured by HPLC analysis (40.3 +/- 2.38 (11)) as compared to NMR (34.6 +/- 1.95 (11)), whilst no significant difference was observed in the measurement of phosphocreatine between the two assay methods. Consequently, a greater sigma Cr was measured by HPLC. This work suggests that the majority of Cr in the heart is NMR visible and unbound, so available to interact with creatine kinase. The lower free ADP concentration calculated from NMR measurements (53.3 +/- 3.80 microM (9)) was not significantly different from that determined by HPLC analysis (56.9 +/- 5.90 microM (9)). This suggests that the concentration of free ADP in the heart is higher than values where it can regulate oxidative phosphorylation most effectively.

Direct evidence for a role of intramitochondrial Ca2+ in the regulation of oxidative phosphorylation in the stimulated rat heart. Studies using 31P n.m.r. and ruthenium red.

1. The concentrations of free ATP, phosphocreatine (PCr), Pi, H+ and ADP (calculated) were monitored in perfused rat hearts by 31P n.m.r. before and during positive inotropic stimulation. Data were accumulated in 20 s blocks. 2. Administration of 0.1 microM-(-)-isoprenaline resulted in no significant changes in ATP, transient decreases in PCr, and transient increases in ADP and Pi. However, the concentrations of all of these metabolites returned to pre-stimulated values within 1 min, whereas cardiac work and O2 uptake remained elevated. 3. In contrast, in hearts perfused continuously with Ruthenium Red (2.5 micrograms/ml), a potent inhibitor of mitochondrial Ca2+ uptake, administration of isoprenaline caused significant decreases in ATP, and also much larger and more prolonged changes in the concentrations of ADP, PCr and Pi. In this instance values did not fully return to pre-stimulated concentrations. Administration of Ruthenium Red alone to unstimulated hearts had minor effects. 4. It is proposed that, in the absence of Ruthenium Red, the transmission of changes in cytoplasmic Ca2+ across the mitochondrial inner membrane is able to maintain the phosphorylation potential of the heart during positive inotropic stimulation, through activation of the Ca2+-sensitive intramitochondrial dehydrogenases (pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases) leading to enhanced NADH production. 5. This mechanism is unavailable in the presence of Ruthenium Red, and oxidative phosphorylation must be stimulated primarily by a fall in phosphorylation potential, in accordance with the classical concept of respiratory control. However, the full oxidative response of the heart to stimulation may not be achievable under such circumstances.