31P nuclear magnetic relaxation studies of phosphocreatine in intact muscle: determination of intracellular free magnesium.

Abstract

ABSTRACT31P nuclear magnetic relaxation rates for phosphocreatine in intact frog gastrocnemius were compared with those observed in model solutions at 40, a temperature at which muscle maintains its physiological state for at least 5 hr. Both nuclear Overhauser effect and spin-lattice relaxation rate(1/Tj) experiments indicate that dirle-dipole interactions form the dominant relaxation path for P in intact muscle and model solutions, independent of phosphocreatine and Mg concentrations. Spin-spin relaxation rates (1/T2) measured by modified Carr-Purcell-Melboom-Gill spin-echo experiments suggest the importance of scalar coupling modulated by chemical exchange with free Mg. From these results, we estimate the free intracellular Mg in intact muscle as 4.4 mM and demonstrate that 31p T2 experiments can be used as a tool for studying free Mg levels with minimum disturbance of the intact cell. 31P nuclear magnetic resonance (NMR) studies of intact cells to date have concentrated on high-resolution spectra of metabolites in intact muscle (1, 2), erythrocytes (3), yeast (4), and Escherichia coli and Ehrlich ascites tumor cells (5, 6). We report here on the feasibility of measuring the relaxation parameters of phosphocreatine (PCr) in intact frog muscle at 4°. The spin-lattice relaxation time (TI) and the spin-spin relaxation time (T2) of PCr in intact muscle are compared with those observed in model solutions at 4°. Both relaxation rates are valuable as dynamic probes of the internal environment in muscle because T2 can be sensitive to processes that do not affect T, at all. These relaxation studies also enable us to assign a value for the free intracellular Mg concentration in intact gastrocnemius via an intrinsically nonintrusive method.It is now clear that metal ions are of central importance in many biological reactions and knowledge of free Mg levels has particular significance. For (10). Spinning 10-mm samples were used; the field was locked either to a capillary of D20 (muscles and PCr/H20 solutions) or D20 solvent. Typical conditions included a 900 pulse of ca 20 lisec, 32-128 scans of 4000 data points each, sweep width of 1 kHz, exponential multiplication introducing 0.6-Hz line broadening, and 'H irradiation.T, measurements were made with the following pulse sequence: 900 pulse, spoil field homogeneity, wait delay time r, 90°pulse, sample free-induction decay, and then spoil field homogeneity. This sequence (11), which was repeated n times, afforded a considerable saving of time over the inversion-recovery method in the T, regimen of 31P in PCr, thus making it possible to carry out T, measurements on muscle during the interval in which there was no more than a 10% change in PCr.The Carr-Purcell-Meiboom-Gill (CPMG) (12) T2 measurements were performed on a Bruker B-KR pulsed spectrometer operating in conjunction with the same Nicolet data system and a 21.14 kG magnet used for high-resolution spectra. Two home-built modifications to the probe of this pulsed spectrometer were made so that satisfactory results ...

Intracellular free magnesium in frog skeletal muscle fibres measured with ion‐selective micro‐electrodes.

“…The results from such measurements vary considerably. Cohen & Burt (1977) Gupta et al (1984) have questioned the accuracy of such an estimate because sizeable effects of steady-state phosphoryl transfer via creatine kinase on the T2 of phosphocreatine were neglected in the study of Cohen & Burt (1977), and therefore their value of 3 mm must be considered only as an upper limit on possible values of [Mg2+]j. Moreover, their experiments were performed at 4 0C, a point which has to be borne in mind since there is evidence that low temperatures can increase…”

“…[Mg2+]i has been estimated from measurements obtained through indirect methods such as tracer flux measurements (Gilbert, 1960); nuclear magnetic resonance (Cohen & Burt, 1977;Gupta & Moore, 1980; reviewed by Gupta, Gupta & Moore, 1984) and liquid-sampling and X-ray spectroscopy (Maughan, 1983). Estimated values for [Mg2+]i using these methods range from 02 to 3 0 mm with a median of around 1P2 mM.…”

Analysis of Intact Tissue with 31P NMR

“…Tl OP l p in phosph�creatine (PCr) in frog muscle was measured by Cohen & Burt (19) at 4°C to be 4.8 sec. The NOE of PCr in muscle was determined to be 2.04, indicating that inter-and intramolecular dipole-dipole interac tions with protons provided the dominate relaxation mechanism for 31 P nuclei of P C r in muscle, as was found for model solutions (19).…”

Free magnesium in sheep, ferret and frog striated muscle at rest measured with ion‐selective micro‐electrodes

“…Several attempts have been made to estimate indirectly the level of the ionized cytoplasmic Mg2+ in contractile tissue. With the use of a variety of methods including tracer fluxes, binding of Mg2+ to phosphonucleotides or enzymes and nuclear magnetic resonance techniques, values between 0-6 and 6 mm were reported for skeletal muscle of frog (Gilbert, 1960;Cohen & Burt, 1977;Gupta & Moore, 1980), barnacle (Ashley & Ellory, 1972) and rat (Gunther, 1967). In cardiac tissue, a value of 3-6 mm was inferred from flux studies (Paradise et al 1978).…”