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1 Zoology, La Trobe University, Melbourne, Victoria, Australia
* To whom correspondence should be addressed. E-mail: t.dutka{at}latrobe.edu.au.
During vigorous exercise, inorganic phosphate (Pi) levels within the cytoplasm of fast-twitch muscle fibers may reach 
30 mM. Cytoplasmic Pi may enter the sarcoplasmic reticulum (SR) and bind to Ca2+ to form a precipitate (Ca-P), thus reducing the amount of releasable Ca2+. Using mechanically-skinned fast-twitch rat muscle fibers, which retain the normal action potential (AP)-mediated Ca2+ release mechanism, we investigated what consequences Pi exposure has on normal excitation-contraction (E-C) coupling. The total amount of Ca2+ released from the SR by a caffeine-low [Mg2+] stimulus was reduced by ~20%, and the initial rate of force development slowed, following 2 min exposure to 30 mM Pi (with or without creatine phosphate, CrP present). Peak tetanic (50 Hz) force was also reduced (by ~25% and ~45% after 10 and 30 mM Pi exposures respectively). Tetanic force responses produced after 30 mM Pi exposure were nearly identical to those seen in the same fiber following depletion of total SR Ca2+ by ~35%. Ca2+ content assays revealed that the total amount of Ca2+ in the SR was not detectably changed by exposure to 30 mM Pi, indicating that Ca2+ had not leaked out of the SR but instead formed a precipitate with the Pi reducing the amount of available Ca2+ for rapid release. These results suggest that Ca-P precipitation occurring within the SR could contribute to the failure of Ca2+ release observed in the later stages of metabolic muscle fatigue. They also demonstrate that the total amount of Ca2+ stored in the SR cannot drop substantially below the normal endogenous level without reducing tetanic force responses.
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