APPS November 2002 Meeting Abstract 1358


Giuseppe S. Posterino, Graham. D. Lamb, Department of Zoology, Faculty of Science & Technology, La Trobe University, Melbourne Victoria 3086, Australia.

It is not clear if increasing the sarcoplasmic reticulum (SR) Ca2+ content will increase, decrease or have no effect on the skeletal muscle ryanodine receptor Ca2+ efflux rates1, 2. We examined this using single mechanically-skinned fast-twitch muscle fibres from the rat that retain functional E-C coupling. Skinned fibres were mounted on a force transducer and then transferred to a bath containing a solution that mimicked the normal intracellular milieu (ie. (mM): K+, 125; Na+, 37; hexamethylene-diamine-tetraacetate (HDTA2-), 50; total ATP, 8: total Mg2+, 8.6; free Mg2+, 1; creatine phosphate, 10; total EGTA, 0.05; Hepes, 90; NaN3, 1; pH 7.10 ▒ 0.01 and free [Ca2+], 0.0001). Twitch responses were elicited by electric field stimulation as previously described3.

To quantify the amount of Ca2+ released with each twitch, Ca2+ reaccumulation by the SR was prevented by inhibiting the SR Ca2+-ATPases with 50 ÁM TBQ and Ca2+ release was rapidly buffered with a known quantity of the fast Ca2+ buffer, BAPTA (250 ÁM), such that the twitch size was submaximal and a sensitive indicator of Ca2+ release. In fibres that initially contained the endogenous level of SR Ca2+, the first twitch elicited in TBQ/BAPTA was ~40% of the maximum Ca2+-activated force (and was indicative of ~300 ÁM Ca2+ released). Thereafter, 2-3 more twitches were elicited (each progressively smaller) before the SR was almost fully depleted. When the SR of fibres was then near-maximally loaded with Ca2+ (~3-4 times endogenous level), the first and as many as 12 subsequent twitches (total of 15) were virtually unchanged (~40% of maximum force) with the last 3-4 twitches declining as the SR became depleted as above. This data shows that raising the SR Ca2+ content does not affect the amount of Ca2+ released with each twitch.

(1) Kurebayashi N, Ogawa Y. Biophysical Journal. 1998;74:1795-1807.

(2) Donoso P, Prieto H, Hidalgo, C. Biophysical Journal. 1995;68:507-515.

(3) Posterino GS, Lamb GD, Stephenson DG. Journal of Physiology. 2000;527.1:131-137.

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