Differential influence of electrical blocking agents on embryonic acetylcholine receptor mRNA levels in long‐term cultures of aneural mammalian myotubes

Abstract

The influence of spontaneous muscle activity on acetylcholine receptor (AChR) expression was examined by exposing long-term cultures of mammalian myotubes to two pharmacological agents that have similar effects on the rate of spontaneous contractile activity but pharmacologically distinct actions on voltage gated Na+ channels. Previous studies by other investigators have shown that tetrodotoxin upregulates and that veratridine downregulates surface AChR expression in short-term mammalian muscle cultures. In order to determine whether these drugs have disparate actions on AChR mRNA levels, myotubes were exposed to either tetrodotoxin or veratridine for a period of 10 days, and measurements of the relative levels of embryonic AChR subunit mRNAs (alpha, beta, gamma, delta) were obtained during and following the period of drug exposure. Veratridine produced a substantial decrease (between 33% and 50% reduction), while tetrodotoxin produced a relatively small increase (between 17% and 23%), in each of the AChR subunit mRNAs after 6 days of drug exposure. At 23 days in culture, spontaneously active myotubes exhibited a decrease in the relative levels of each of the AChR subunit mRNAs. Myotubes previously exposed to either veratridine or tetrodotoxin exhibited elevated levels of beta, gamma, and delta AChR subunit mRNAs 6 days after cessation of drug treatment, thus suggesting that a period of muscle inactivity can induce sustained influences on some AChR mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous changes in acetylcholine receptor and calcium leakage activity in cell-attached patches from cultured dystrophic myotubes

“…Primary cultures of nondystrophic (C57B16J and C57Bl10/SnJ) and mdx (C57Bl10-mdx; Jackson Laboratories) myotubes were prepared from the fore-and hindlimb muscles of embryonic (embryonic days [16][17][18][19] and newborn (postnatal days 0-3) mice and maintained on a laminin substrate as described previously [7,9,10]. The cultures were treated with veratridine for a period of about 10 days in order to reduce spontaneous contractile activity, and were maintained for up to 34 days in 50:50 Dulbecco's Modified Eagles Medium:Hams Nutrient Mixture F12 (plus 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin; [7]) in 5% CO 2 at temperatures between 35 and 37°C.…”

Agents Which Inhibit NF‐κB Signaling Block Spontaneous Contractile Activity and Negatively Influence Survival of Developing Myotubes

“…Primary cultures of both nondystrophic and mdx myotubes undergo identical periods of spontaneous contractile activity between CD 7 and CD 20 (Carlson and Feng, ; Carlson et al, ). Since the nondystrophic and mdx myotubes also exhibited no differences in p65 expression, the effects of NF‐κB inhibitors on spontaneous contractile activity were first examined only on primary cultures of nondystrophic myotubes.…”

“…The day of plating is defined as culture day (CD) 1. By allowing fibroblast proliferation in association with the proliferation of myoblasts, the myotubes eventually develop within an extracellular matrix and therefore can spontaneously contract without detaching from the substrate (Rubin, ; Carlson et al, ). In this culture system, myoblasts reach confluence by CD 5 and undergo fusion largely between CD 5 and 7.…”

“…In this culture system, myoblasts reach confluence by CD 5 and undergo fusion largely between CD 5 and 7. Spontaneous contractile activity of individual myotubes is observed between CD 7 and 20 (Carlson and Feng, ; Carlson et al, ).…”

“…The present study was therefore designed to first assess whether primary cultures of spontaneously contracting nondystrophic and mdx myotubes (Carlson and Feng, ; Carlson et al, ) exhibit differences in NF‐κB signaling. The results indicated that nondystrophic and mdx myotubes exhibited no differences in p65 distribution or expression, and responded with equivalent increases in the nuclear expression of p65 following exposure to the cytokine TNF‐α.…”