Peter G. Morris

Design of an indicator of intracellular free Na+ concentration using 19F-NMR.

The development is described of an Na+ chelator with appropriate properties for an indicator of intracellular free Na+ concentration ([Na+]i). The new indicator, FCryp-1, is a tribenzo derivative of the parent (2:2:1) cryptand structure, incorporating the same F-substituted dibenzo 19F-NMR reporter group as the free [Ca2+] indicator, 5FBAPTA (Smith, G.A., Hesketh, T.R., Metcalfe, J.C., Feeney, J. and Morris, P.G. (1983) Proc. Natl. Acad. Sci., USA 80, 7178-7182). FCryp-1 has appropriate affinity for Na+ (KNa = 10(1.3) M-1) and selectivity over other intracellular cations (KK; KCa; K Mg less than 10(-1) M(-1)) for a [Na]i indicator. There is an 19F-NMR chemical shift of 2.00 ppm between free FCryp-1 and the Na-FCryp-1 complex which provides a direct read out of free [Na+]. FCryp-1 carries four carboxylate groups to confer aqueous solubility which can be esterified with acetoxymethyl groups to render the indicator membrane permeant. Experiments on pig lymphocytes loaded with FCryp-1 gave an indicated [Na+]i of 13.8 +/- 1.8 mM (n = 4). The FCryp-1 structure can also be readily modified to provide fluorescent [Na+]i indicators.

Measurement of free intracellular calcium in the brain by 19F-nuclear magnetic resonance spectroscopy.

Abstract: We report the first measurement of the free intracellular calcium level in an actively metabolising intact cerebral tissue preparation. To this end, we applied the recently developed 19F‐nuclear magnetic resonance calcium chelator, 5,5′‐F2‐1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (5FBAPTA), in superfused cerebral cortical slices to give values for the intracellular Ca2+ concentration of 350 and 480 nM, at external calcium concentrations of 1.2 and 2.4 mM, respectively. Under both conditions, the intracellular Ca2+ concentration was increased by depolarisation using a high external K+ concentration. Interleaved 31P spectra showed that the presence of the 5FBAPTA had a deleterious effect on the metabolic state of the tissue with an external Ca2+ concentration of 1.2 mM, but normal viability was maintained using 2.4 mM.

Use of 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA) in the measurement of free intracellular calcium in the brain by 19F-nuclear magnetic resonance spectroscopy

Abstract: We have applied the 19F‐nuclear magnetic resonance (NMR) calcium indicator 1,2‐bis(2‐amino‐5‐fluorophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (5FBAPTA) to the measurement of the free intracellular calcium concentration ([Ca2+] i) in superfused brain slices. A mean ± SD control value of 380 ± 71 nM(n = 18) was obtained at 37°C using 2.4 mM extracellular Ca2+. Subcellular fractionation studies using [3 H] 5FBAPTA showed that after loading of its tetraac‐etoxymethyl ester, ∼55% was de‐esterified, with the other 45% remaining as the tetraester bound to membranes. Of the de‐esterified 5FBAPTA, >90% was in the cytosolic fractions, with >1% in the mitochondria or microsomes. The NMR‐visible de‐esterified 5FBAPTA slowly disappeared from the tissue with a t1/2 of 4 h. A time course after loading confirmed that the calculated [Ca2+], was constant over a 5‐h period, although the scatter of individual results was ±20%. The [Ca2+]; was increased by a high extracellular K+ concentration ([K+]e), by a low extracellular concentration of Na+, and by the calcium ionophore A23187. On recovery from high [K+]e. the [Ca2+]i“overshot” to values lower than the original control value. The [Ca2+]i was surprisingly resistant to changes in extracellular Ca2+ concentration.

NMR pulse symmetry

Abstract Symmetry properties of the total magnetic field experienced by a system of noninteracting spins can give rise to symmetry in the magnetization response of the system. These effects are analyzed by introducing operations which represent reflections of rotation axes across the x = 0, y = 0, and z = 0 planes. The effects of reflections in time or in any component of the applied field are determined. The symmetry rules derived fall into two principal categories: (A) comparisons between the magnetization responses of isochromats experiencing different applied fields related by symmetry, and (B) analyses of the rotation experienced by a single isochromat in a field of high symmetry. Under category (A), for example, it is shown (i) that symmetric responses require only that the B 1 field be real, not that it be symmetric, and (ii) that the flip angle response to a fixed-phase pulse applied in the presence of a time-varying gradient field is unaffected by time reflection. A simple group-theoretical formalism is developed which enables all possible symmetry rules of the types considered to be tabulated. Extensions of the method to the symmetry analysis of composite pulses and multiple selective pulses are indicated.

Effects of N-methyl-D-aspartate on [Ca2+]i and the energy state in the brain by 19F- and 31P-nuclear magnetic resonance spectroscopy.

Abstract: The effects of N‐methyl‐d‐aspartate (NMDA) on the free intracellular Ca2+ concentration ([Ca2+]i) and the energy state in superfused cerebral cortical slices have teen studied using 19F‐ and 31P‐nuclear magnetic resonance spectroscopy. [Ca2+]i was measured using the calcium indicator 1,2‐bis(2‐amino‐5‐fluorophenoxy)ethane‐N.N.N′.N′‐tetraacetic acid (5FBAPTA). NMDA (10 μM) in the absence of extracellular Mg2+ caused the expected rise in [Ca2+]i but produced an impairment of the energy state: the phosphocreatine (PCr) content was decreased by 42%, and the Pi/PCr ratio was increased by 55%. There was no detectable change in ATP or free intracellular Mg2+ concentration. Increasing the NMDA concentration in the superfusing medium to 100 or 400 μM caused no further increase in [Ca2+]i or further decrease in PCr content, but the Pi/PCr ratio continued to rise. The impairment of the energy state preceded the effect on [Ca2+]i, and these changes were irreversible on return to control conditions. Repeating the experiments in the presence of 1.2 mM extracellular Mg2+ resulted in similar changes in the energy state, with no change in [Ca2+]i. The possibilities that the effects were due to membrane depolarisation or to the presence of 5FBAPTA within the tissues were eliminated. The results suggest that low concentrations (10 μM) of NMDA produce an impaired energy state independent of the presence of extracellular Mg2+ and that the decreased energy state is not due to the changes in [Ca2+]i, which are seen only in the absence of extracellular Mg2+.

NMR Spectroscopy in Living Systems

Publisher Summary This chapter reviews that nuclear magnetic resonance (NMR) spectra has been recorded from suspensions of intact red blood cells. NMR imaging techniques were developed as the most powerful diagnostic modality. In vivo spectroscopy too developed into a full-blown clinical diagnostic technique. The main thrust of scientific endeavor in this case has been concerned with the development of a biochemical understanding of normal and pathological states. These more fundamental objectives have meant that isolated tissue and animal models have retained their importance. Interest in them is increasing rapidly, stimulated by possible future clinical applications. The chapter discusses that the development of MRI and in vivo spectroscopy have proceeded in parallel, with rather little in the way of cross-fertilization. Thus on several occasions scientists developing MRI methods have rediscovered pulse sequences well known to high-resolution spectroscopists. Equally, the value of selective excitation methods for conventional NMR is only just beginning to be appreciated with the advent of the first generation of high-resolution spectrometers equipped with this capability.