Exercise-induced decreases in the 1 H transverse relaxation rate (R 2 ) of muscle have been well documented, but the mechanism remains unclear. In this study, the hypothesis was tested that R 2 decreases could be explained by pH decreases and apparent intracellular volume (V i ) increases. 31 P and 1 H spectroscopy, biexponential R 2 analysis, and imaging were performed prior to and following fatiguing exercise in iodoacetate-treated (IAA, to inhibit glycolysis), NaCN-treated (to inhibit oxidative phosphorylation), and untreated frog gastrocnemii. In all exercised muscles, the apparent intracellular R 2 (R 2i ) and pH decreased, while intracellular osmolytes and V i increased. These effects were larger in NaCN-treated and untreated muscles than in IAAtreated muscles. Multiple regression analysis showed that pH and V i changes explain 70% of the R 2i variance. Separate experiments in unexercised muscles demonstrated causal relationships between pH and R 2i and between V i and R 2i . These data indicate that the R 2 change of exercise is primarily an intracellular phenomenon caused by the accumulation of the end-products of anaerobic metabolism. In the NaCN-treated and untreated muscles, the R 2i change increased as field strength increased, suggesting a role for pH-modulated chemical exchange. Key words: skeletal muscle; exercise; hydrogen-ion concentration; osmolarity; nuclear magnetic resonance Exercise-induced increases in the apparent 1 H transverse relaxation time (T 2 ) of muscle water have been well documented. The amount of T 2 change increases as exercise intensity increases; T 2 plateaus 3-4 min after the onset of exercise (1). Following isometric leg extension to fatigue, T 2 recovery follows an approximately exponential time course and is complete after ϳ35 min. (2). The increase in signal intensity from active muscles in T 2 -weighted images allows muscle activity to be detected reliably and noninvasively, which may aid in the placement of regions of interest or surface coils in spectroscopic studies (2,3). With a full understanding of the mechanism(s) of the T 2 change, this phenomenon may also be useful in functional studies of muscle activation during exercise (e.g., Refs. 4 -6), noninvasive studies of pathologic conditions in which the T 2 response to exercise is altered (e.g., Refs. 7 and 8), and as a means of studying noninvasively those physiological changes that increase T 2 .The most universally offered and best-studied explanation for the T 2 increase during exercise is the concomitant increase in muscle volume. However, the mechanism of the T 2 increase is more complex than total water accumulation. For example, recovery of the muscle's anatomical cross-sectional area is faster than T 2 recovery (2), and enhancement of the extracellular fluid volume increase during exercise is not proportional to the T 2 increase (9). A further complication is that both ex vivo amphibian (10) and in vivo mammalian (11) studies suggest that exchange between the intra-and extracellular spaces in muscle is sl...

The appearance of new peaks in the 7.7-8.6 and 6.8 -7.4 ppm regions of the postexercise 1 H spectrum of frog muscle is reported. These new peaks result from the splitting of single pre-exercise carnosine C-2 and C-4 peaks into two peaks, representing the intracellular pH (pH I ) of oxidative and glycolytic fibers. The following data support this conclusion: 1) comparison of means and regression analysis indicates equivalence of the pH I measurements by 1 H and 31 P NMR; 2) the pre-and poststimulation concentrations of carnosine are equal; 3) in ischemic rat hindlimb muscles, the presence of a single, more acidic peak in the plantaris; a single, less acidic peak in the soleus; and two peaks (more and less acidic) in the gastrocnemius correspond to published values for the fiber-type composition of these muscles; and 4) in muscles treated with iodoacetate prior to and during stimulation, a second peak never appears. These data indicate that it is feasible to measure separately the pH I of oxidative and glycolytic fibers using 1 H NMR spectroscopy. Measuring intracellular pH (pH I ) is important in studies of many cellular processes. In studies of muscle metabolism and exercise, it is desirable to measure separately the pH I of oxidative and glycolytic fibers. pH I measurements are commonly made using 31 P NMR spectroscopy, using the inorganic phosphate (P i ) peak's chemical shift. Following intense muscle exercise, the single pre-exercise P i peak frequently splits into two or more poststimulation P i peaks (P i,A and P i,B ), which are believed to correspond to P i in glycolytic and oxidative fibers, respectively (1-7). However, because of the low NMR sensitivity of the phosphorus nucleus, it is sometimes not possible to distinguish a P i peak from noise when the concentration of P i is low. Extreme broadening of the P i peak during exercise has also been observed (5), interfering with the ability to resolve individual peaks.The C-2 and C-4 protons on the imidazole ring of carnosine (␤-alanyl histidine) have pK A 's of approximately 7.0 and chemical shifts that are sensitive to pH in the physiological range, providing an alternative means of measuring pH noninvasively (8). Several authors have taken advantage of this pH sensitivity to measure the pH I of muscle in vivo and ex vivo (8 -13), and of muscle extracts (8,14). Specific applications have been made to muscle in resting (8,10,11,(13)(14)(15)(16), diseased (16), caffeinetreated (14), and exercised (9,11,12) states. Pan et al. (11) and Hitzig et al. (13) found high correlations between pH I measurements by the C-2 and P i methods.While studying exercising ex vivo frog gastrocnemii, we also observed the C-2 peak of carnosine at ϳ8.0 ppm prior to stimulation. However, we noted two peaks (one at ϳ8.53 and one at ϳ8.37 ppm) following exercise. In this study, we tested the hypothesis that they both originate from C-2 protons of carnosine. We also tested the hypothesis that the downfield peak (which we call carnosine C-2 A ) originates from a more acidic compartm...