Is chloride passively distributed in skeletal muscle in vivo

Boyle and Conway (1941) and Hodgkin and Horowicz (1959) first provided evidence that chloride, CI, is passively distributed in frog skeletal muscle in vitro. This conclusion has been supported by chemical measurements of intracellular C1 concentration, [Cl]i , (Adrian, 1961) and direct measurements of intracellular C1 i (Bolton and Vaughan-Jones, 1977; activity, aCl Baumgarten and Fozzard, 1978). On the other hand some chemical measurements and electron microprobe analysis of [Cl]i (Harris, 1965; Somlyo et i al., 1977), as well as some measurements of aCl (Kernan et al., 1974; Armstrong et al., 1977) suggest that muscle cells contain more C1 than expected for a passive distribution, and i~ly that C1 is actively accunulated by the cells. To date the issue remains unresolved for muscles studied in vitro. Recently, using improved methods for determining C1 content, ~lacchia et al. (1978) have shown that [Cl]i of both frog and toad semitendinosus muscles studied in vivo is much lower than reported for these preparations in vitro. [Cl]i was as expected from a passive distribution assuming that in vivo membrane potential (F m) is similar to that found in vitro and tha t intraand extracellular activity coefficients are equal. Here we report the in vivo determination of Em' ac li , and [Cl]i in toad semitendinosus muscle. These measurements provide further evidence for the passive distribution of Cl. Toads, Bufo marinus, were pithed, the semitendinosus muscle partially exposed by blunt dissection, and a small 'Nell" created around the exposed portion. The exposed muscles were superfused at 10 ml/min with Ringer ' s so lu t ion (temperature 21-23 ~ which was cont inuously bubbled with 100% 02 . The composition of the Ringer's solution was (in n~4): 70 NaCI; 3.I KCI; 2.0 CaCI 2 ; 0.5 MgCI 2 ; 45 Na methane sulfonate; 5.0 HEPES (N-2-hydroxyethylpiperazine-N' -2ethanesulfonic acid); and 5.0 glucose. The pH was adjusted to 7.5 with approximately 2 r~l NaOH. The muscle preparation was thus superfused by an isotonic solution whose Na, K, and C1 concentrations and pH were matched to the average composition of toad plasma (Danielson, 1964; Macchia et 8/., 1978; Macchia and Page, unpublished observations) and at the same time was perfused through the vascular system of the toad. 1 determined with C1 ion-selective was microelectrodes (Walker, 1971) made using Coming 477515 liquid ion-exchange resin (Coming Glass, Medfield, MA) and dimethyldichlorosilane to render the glass hydrophobic. The ion-selective microelectrodes were calibrated both before and after the experiment in pure solutions of KCI (i-300 m~0 and Na methanesulfonate (5-300 raM). each preparation, _ @I was calculated accordFor ing to the equations of Walker (1971) from the average potentials of the ion-selective and conventional electrodes. After the electrophysiological measurements, both semitendinosus muscles were excised. The [Cl]i of each muscle was calculated from the wet and dry weights of the muscle, water and C1 content of the plasma, the muscle C1 content, and the extracellular volume, ECV ~4acchia, et al. , 1978). The ECV of each specimen was estimated from