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We have applied the transition state theory of Eyring et al. (The Theory of Rate Processes, McGraw-Hill, 1941) to water transport across cell membranes. We have then evaluated free energy (Delta F(not equal)), enthalpy (Delta H(not equal)) and entropy (Delta S(not equal)) of activation for water permeation across membranes, such as Arbacia eggs, Xenopus oocytes with or without aquaporin water channels, mammalian erythrocytes, aquaporin proteoliposomes, liposomes and collodion membrane. Delta H(not equal) was found to be correlated with Delta S(not equal). This is so-called Delta H(not equal) and Delta S(not equal) compensation over the ranges of Delta H(not equal) and Delta S(not equal) from 2 to 22 kcal/mol and from -26 to 45 e.u., respectively, indicating that low Delta H(not equal) values correspond to negative Delta S(not equal). Large positive Delta S(not equal) and high Delta H(not equal) values might be accompanied by reversible breakage of secondary bonds in the membrane, presumably in membrane lipid bilayer. Largely negative Delta S(not equal) and low Delta H(not equal) values for aquaporin water channels, aquaporin proteoliposomes and porous collodion membrane could be explained by the immobilization of permeating water molecules in the membrane, i.e., the partial loss of rotational and/or translational freedoms of water molecules in water channels.

The physical state of water in mouse lenses (2-, 4- or 8-wk-old) and soft contact lenses (SCLs, water content from 18.4 to 79.2%) were studied by measuring spin-lattice relaxation times (T1) and apparent intermolecular cross-relaxation times (TIS) from irradiated protein or polymer protons to water protons, using 360 MHz 1H-NMR spectrometer at 25 degrees C. (1) 1/T1 values of SCLs increased gradually with increasing dry weight (W(%)). 1/TIS values of SCLs were approximately zero at W of 20.8 and 26.8%, increased gradually from 26.8% and then steeply above approximately 50%. (2) A plot of 1/T1 vs. W(%) of mouse lenses was almost equal to that of SCLs. However, a plot of 1/TIS vs. W(%) was an approximately straight line with the intercept at W of 23% and with the slope which is almost equal to that of SCLs above W of approximately 50%. The plot of 1/TIS vs. W(%) of mouse lenses might indicate the significant change in the physical state of water and/or protein-water interactions above W of 23%.

23Na in the prefused rat mandibular salivary gland was measured by spin-echo double quantum filter 23Na-NMR spectroscopy at 8.45 T. Resonances due to the intracellular 23Na and the interstitial 23Na were observed in the perfused gland at 25 degrees C. The resonance due to intracellular 23Na consisted of two Lorentzian signals stemming from the [1/2 mean value of -1/2[ coherence (sharp resonance) and the [-1/2 mean value of -3/2[ and [3/2 mean value of 1/2[ coherences (broad resonance). The transverse relaxation rate constant corresponding to the [1/2 mean value of -1/2[ coherence was 95 +/- 4 s-1 and that corresponding to the [-1/2 mean value of -3/2[ and [3/2 mean value of 1/2[ coherences was 1360 +/- 75 s-1 (mean +/- S.E., n = 5). The resonance due to the interstitial 32Na had longer relaxation rate constants, and disappeared upon administration of dysprosium triethylenetetramine-N,N',N",N",N"'-hexaacetic acid.

To evaluate the energy-shuttle hypothesis of the phosphocreatine/creatine kinase system, diffusion rates for ATP, phosphocreatine and flux through the creatine kinase reaction were determined by 31P-NMR in resting bullfrog biceps muscle. The diffusion coefficient of phosphocreatine measured by 31P-pulsed gradient NMR was 1.4-times larger than ATP in the muscle, indicating the advantage of phosphocreatine molecules for the intracellular energy transport. The flux of the creatine kinase reaction measured by 31P-saturation transfer NMR was 3.6 mmol/kg wet wt. per s in the resting muscle. The flux is equal to the turnover rate of ATP, ADP, phosphocreatine and creatine molecules, therefore, the life-times of these substrates and the average distance traversed after the life-times by the diffusing molecules were calculated using the diffusion coefficients obtained by 31P-NMR. The mean square length of one-dimensional diffusion was 22 microns in ATP molecules and the minimum diffusion length was 1.8 microns in ADP molecules. The latter was calculated using free ADP concentration, 30 mumol/kg wet wt., obtained from the equilibrium constant of the creatine kinase reaction and the diffusion coefficient assumed to be the same of ATP in muscle. Similar diffusion lengths of ADP were calculated using the reported values for the flux of the creatine kinase reaction in heart and smooth-muscle. The diffusion lengths of all substrates involved in the creatine kinase reaction were larger than the radii of myofibrils. Therefore, in the muscles with an alternating arrangement of mitochondria and myofibrils, such as heart and certain skeletal muscles, ATP and ADP molecules can move freely between myofibrils and mitochondria without the aid of the creatine kinase reaction; thus, we conclude that the energy-shuttle hypothesis is not obligatory for energy transport between the mitochondria and the myofibrils.

Phosphorus nuclear magnetic resonance (31P-NMR) was used for the sequential measurement of phosphorus energy metabolites in perfused canine submandibular gland. Under resting conditions, ATP and creatine phosphate levels were 0.42 +/- 0.11 mM and 0.62 +/- 0.16 mM (mean +/- S.D., in nine glands). When perfusion of the gland was stopped, the tissue contents of ATP and creatine phosphate decreased, that of ADP increased and tissue pH decreased. Restarting perfusion led to recoveries of the tissue content of the phosphorus compounds and tissue pH to normal. Acetylcholine administration induced secretion of saliva, decreased the level of ATP, creatine phosphate and tissue pH, and increased the ADP level.