We Chemical analyses of single-fiber segments dissected from resting and stimulated rat plantaris muscle and SOL (21) showed correlations among fiber types with metabolite content at rest as well as with the extent of PCr and ATP splitting during stimulation. The mechanistic basis for the differences in metabolite concentrations was attributed to the greater average rate of neural activation of type 1 fibers in the animal compared with type 2 fibers. Thus the reduced content ofPCr and ATP was explained by increased muscle activity, not by a phenotypic characteristic. An alternative possibilitynamely, that there are characteristic differences among fiber types in their content of PCr, ATP, and other metabolites at rest-has not been fully considered. We therefore designed experiments to test the hypothesis that there are significant differences in bioenergetically important metabolites in the major classes of fiber types. METHODSMuscle Preparation. We studied the extensor digitorum longus muscle (EDL), the SOL, the tensor fascia lata muscle (TFL), and the diaphragm muscle (DPH) dissected from 150-to 250-g Sprague-Dawley rats anesthetized with pentobarbital (60 mg/kg i.p.). We also studied EDL and SOL from 25-to 40-g Swiss-Webster mice. The component fiber types in the adult muscles that we chose for study were identified and classified into types 2b, 2a, 2x, or 1; the volume fraction of each muscle studied is listed in Table 1. Because the wet weight of some of the rat muscles exceeded the range of 30 mg, which can be kept in good physiological condition by superfusion with oxygenated physiological salt solution (PSS), and because their shape was inappropriate for our NMR methods, the rat DPH and TFL were split by blunt dissection along the axis of the fibers. All preparations had a maximal thickness of < 1 mm. Only the middle portions ofthe muscle were laid in the sensitive volume of the coil such that attachment regions did not contribute to the spectral signal. All muscles were kept in PSS equilibrated with 100% 02 and contained 116 mM NaCl, 4.6 mM KCl, 26.2 mM Mops (titrated to pH 7.4 with NaOH), 2.5 mM CaCl2, 1.2 mM MgSO4, and gentamycin (10 mg/l) at pH 7.4.Muscles were frozen between brass blocks cooled to -1960C after the completion of spectral acquisition. When the muscles were put into the NMR probe, muscles from the Abbreviations: DPH, diaphragm muscle; EDL, extensor digitorum longus muscle; PCr, phosphocreatine; TCr, total creatine (the sum of creatine plus phosphocreatine); SOL, soleus muscle; TFL, tensor fascia lata muscle. tTo whom reprint requests should be addressed at: Department of Radiology, SB-O5,

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We investigated whether the creatine kinase-catalyzed phosphate exchange between PCr and gamma ATP in vivo equilibrated with cellular substrates and products as predicted by in vitro kinetic properties of the enzyme, or was a function of ATPase activity as predicted by obligatory "creatine phosphate shuttle" concepts. A transient NMR spin-transfer method was developed, tested, and applied to resting and stimulated ex vivo muscle, the soleus, which is a cellularly homogeneous slow-twitch mammalian muscle, to measure creatine kinase kinetics. The forward and reverse unidirectional CK fluxes were equal, being 1.6 mM.s-1 in unstimulated muscle at 22 degrees C, and 2.7 mM.s-1 at 30 degrees C. The CK fluxes did not differ during steady-state stimulation conditions giving a 10-fold range of ATPase rates in which the ATP/PCr ratio increased from approximately 0.3 to 1.6. The observed kinetic behavior of CK activity in the muscle was that expected from the enzyme in vitro in a homogeneous solution only if account was taken of inhibition by an anion-stabilized quaternary dead-end enzyme complex: E.Cr.MgADP.anion. The CK fluxes in soleus were not a function of ATPase activity as predicted by obligatory phosphocreatine shuttle models for cellular energetics.

Measurements of the intracellular diffusion coefficients (Di) of ATP and creatine phosphate (PCr) in stable, isolated preparations of skeletal muscle were made by means of pulsed field gradient (PFG) 31P NMR. Experiments used a PFG NMR probe specifically designed for small, superfused biological samples. This provided a magnetic field gradient in the z axis of up to 195 G/cm with minimal eddy currents. DiATP and DiPCr in white (fast, glycolytic) skeletal muscle from goldfish (Carassius auratus) were determined to be 2.48 +/- 0.33 and 3.49 +/- 0.33 x 10(-6) cm2/s, respectively, at 25 degrees C and a diffusion time of approximately 19 ms. For comparison with Di values, diffusion coefficients of ATP and PCr also were measured in solutions of ionic composition similar to that of fish muscle cytosol. The in vitro diffusion coefficients of ATP and PCr were 3.54 +/- 0.11 and 5.28 +/- 0.08 x 10(-6) cm2/s, respectively, at 25 degrees C.

The time-and orientational-dependence of phosphocreatine (PCr) diffusion was measured using pulsed-field gradient nuclear magnetic resonance (PFG-NMR) as a means of non-invasively probing the intracellular diffusive barriers of skeletal muscle. Red and white skeletal muscle from fish was used because fish muscle cells are very large, which facilitates the examination of diffusional barriers in the intracellular environment, and because they have regions of very homogeneous fiber type. Fish were cold-acclimated (5°C) to amplify the contrast between red and white fibers. Apparent diffusion coefficients, D, were measured axially, D k , and radially, D c , in small muscle strips over a time course ranging from 12 to 700 ms. Radial diffusion was strongly time dependent in both fiber types, and D decreased with time until a steady-state value was reached at a diffusion time 9100 ms. Diffusion was also highly anisotropic, with D k being higher than D c for all time points. The time scale over which changes in D c occurred indicated that the observed anisotropy was not a result of interactions with the thick and thin filament lattice of actin and myosin or restriction within the cylindrical sarcolemma, as has been previously suggested. Rather, the sarcoplasmic reticulum (SR) and mitochondria appear to be the principal intracellular structures that inhibit mobility in an orientation-dependent manner. This work is the first example of diffusional anisotropy induced by readily identifiable intracellular structures.