Tunicamycin inhibits the expression of surface Na+ channels in cultured muscle cells

✍ Scribed by Dafna Bar-Sagi; Joav Prives

• Publisher: John Wiley and Sons
• Year: 1983
• Tongue: English
• Weight: 552 KB
• Volume: 114
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.1041140113

✦ Synopsis

Cell culture

Primary cultures of skeletal muscle cells were prepared from breast of 12 day old chick embryos as described (O'Neill and Stockdale, 1972;Paterson and Prives, 1973). Cells were plated on collagen-coated culture dishes at an initial density of 1.8 x lo6 cells per 60-mm culture dish. The cultures were grown in Dulbecco's modified Eagle's medium (DME), supplemented with 25 mM Hepes (pH 7.4), 10% horse serum, and 2% embryo extract, at 37°C in an atmosphere of 92% air/8% COz. At 2 days after plating, cultures were fed with growth medium containing 10 pM cytosine arabinoside for a 48-hour period, to minimize fibroblast proliferation (Fischbach and Cohen, 1973).

Measurement of 22Na+ uptake

Rate of 22Na+ uptake was measured essentially as described by Catterall (1977Catterall ( , 1980a)). Cultures were preincubated in 1 ml of Na+-free medium consisting of 135.4 mM KC1,50 mM Hepes (adjusted to pH 7.4 with Tris base), 5.5 mM glucose, 0.8 mM MgS04, 0.1 mM ouabain, 1 pM BTX, and 1 mglml bovine serum albumin (BSA) for 30 min at 37°C. At the end of this period, medium was removed and cells were rinsed twice within 10 sec with 2.5 ml of medium consisting of 5.4 mM KCl, 130 mM choline chloride, 50 mM Hepes (pH 7.4), 5.5 mM glucose, 0.8 mM