Determination of creatine kinase kinetic parameters in rat brain by NMR magnetization transfer. Correlation with brain function

A Sauter,M Rudin

doi:10.1016/s0021-9258(19)38633-8

A Sauter, M Rudin

Open Access

https://doi.org/10.1016/s0021-9258(19)38633-8

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Abstract

The pseudo first-order rate constant kf of the creatine kinase (CK) forward reaction as well as the CK forward flux FCK,f have been shown to correlate better with cardiac performance than the steady-state levels of ATP and PCr (Bittl, J. A., and Ingwall, J. S. (1985) J. Biol. Chem. 260, 3512-3517). In order to elucidate the relationship between the CK kinetic parameters and brain activity, we have determined, using the non-invasive NMR technique of magnetization transfer, kf and FCK,f in rats, in which brain activity was experimentally varied by administration of either thiopental sodium or bicuculline to decrease or increase electro-encephalogram (EEG) intensity, respectively. The steady-state levels of ATP and PCr, as well as the accumulation of deoxyglucose 6-phosphate (DG-6P) in brain following intraperitoneal administration of deoxyglucose, were determined simultaneously by the NMR technique, whereas the cortical EEG was recorded in a separate experiment. The EEG intensity (range, 1-20 Hz), taken as a measure for brain performance, as well as the amount of DG-6P formed in brain, reflecting the synthesis rate of high energy phosphates (ATP and PCr), linearly correlated with kf. Despite large changes in both EEG intensity (50-250%) and kf (0.12-0.69 s-1) between thiopental sodium- and bicuculline-treated rats, the ATP levels remained constant, whereas the PCr levels decreased with high EEG activity. In contrast to the expectation based on model calculations of CK kinetics, the PCr levels did not increase above control values at reduced EEG intensity (50% of controls). At EEG intensities exceeding control values (bicuculline-treated rats) FCK,f increased as predicted by CK equilibrium. In conclusion, we have shown that in the rat brain, like in the heart, the CK forward rate constant kf, in contrast to ATP and PCr levels, is a sensitive reliable indicator of both increased and reduced function.

Highlights

NMR technique,whereasthecortical EEG wasre- stratesotherthan glucose maybe used
Based on model calculations of CK kinetics, the PCr turnover is the mitochondrial FIFO-ATP synthase (ADPPi levels did not increase above control valuaetsreduced HATP)
In tiontransfertechniquesis difficult inbrain.Inaddition, conclusion, we have shown that in the rat brain, like magnetization transfer flux studies yielded somewhat controin the heart, theCK forward rate constantkf,in con- versial results, depending on the tissue studied

Summary

MATERIALS ANDMETHODS

N M R Spectros~opy-~~sPpectra were recorded on a Biospec 47/15 spectrometer (Bruker, Karlsruhe, Germany), equipped with a 4.7T/ 15-cm horizontal bore magnet. Comparison with intensity values derived from data processing using Lorentzian deconvolution yielded identical results as thepeak height quantification within error limits. This illustrates that (a)the line width of control and saturation transfer spectra are identical, and ( b ) quantification errors due to overlapping lines did not constitute aproblem. Knowing T1,pcrfrom a different set of saturation transfer experiments with varying transfer time (see “Results”), the rate constant can be derived from relative PCr signal PCr(m)/PCr(O) undercon&tions of steady state saturation transfer according to the following equation. EEG signals were recorded in blocks of 8 8,stored to a disk, and immediately Fourier-transformed yielding the power spectrum pi(u,t), u being the frequency and i indicatingthe channel ( i = 1 or 2). The animals received the following treatments:thiopental sodium (Abott, Zug, Switzerland), injected intraperitoneally (20 and 40 mg/kg) immediately after ST1; or bicuculline, infused intravenously (0.4,0.8, and 1.2 mg/kgduring the first min, 1/10 of the dose over the 14 rnin), starting with ST2; or the corresponding solvent (=control, Ctl). 2-Deoxyglucose (DG) was injected intraperitoneally (0.5 g/kg) 1min before ST2

RESULTS

Correlation of Creatine Kinase Rate with Brain Function

DISCUSSION

EEG Intensity

Treatment n