Influence of Neurones and Contractile Activity on Acetylcholinesterase and Acetylcholine Receptors in Muscle Cell Cultures

plexus is regulated via presynaptic inhibitory muscarinic receptors. Heart. lsolated guinea-pig hearts were perfused with Tyrode solution in the presence of eserine. The ACh overflow during a 2-min perfusion with 108 mM K t was not changed by atropine. Likewise, atropine did not modify the ACh output caused by 2-min perfusions of isolated chicken hearts with high K’ (45 or 108 mM) or with DMPP ( 3 0 0 p M ) either in the absence or presence of eserine. Urinary bladder. Strips of the guinea-pig urinary bladder were incubated in eserinecontaining Tyrode solution. The release of ACh evoked by a 10-min incubation of the strips with 108 mM K* was not changed by atropine. It is concluded that only the ACh release from the guinea-pig myenteric plexus can be modulated via presynaptic muscarinic-receptors. Apparently, the terminals of the postganglionic parasympathetic nervesof guinea-pig and chicken hearts and of guinea-pig urinary bladder are not equipped with inhibitory muscarinic-receptors. -

Influence of Neurones and Contractile Activity on Acetylcholinesterase and Acetylcholine Receptors in Muscle Cell Cultures J . KOENIG

and M. VIGNY

Unite‘Biologie e t Pathologie neuromusculaires (U 163), 75005 Paris and Laboratoire de Neurocytologie, Universite P. et M. Curie, 75005 Paris and * Laboratoire de Neurobiologie, Ecole Normale Supe‘rieure, 75230 Paris (France) The influence of neurones and of muscle contractility o n t h e synthesis of the 16 S (synaptic) (Hall, Z., J . Neurobiol., 4, 343, 1973; Vigny, M., Koenig, J . and Rieger, F., J. Neurochem., 27, 1347, 1976) molecular form of AChE, the focalisation of this enzyme and of ACh receptors was investigated in cell cultures of muscle, mixed o r n o t with spinal cord neurones. Tetrodoxin was used t o block muscle contractile activity. We find: (1) Muscle cells devoid of 16 S activity before the plating (13-14 day-old embryos) are unable t o synthesize this form of AChE if neurones are not added t o the cultures. With muscles already contacted by axons in vivo, the synthesis of 16 S AChE continues in vitro in the absence of neurones. Thus, the synthesis of this peculiar, “synaptic” form of AChE is induced under neural influence (Koenig, J. and Vigny, M., Nature, (Lond.), 2 7 1 , 7 5 , 1978). (2) Focalisation of AChE does not occur in mixed cultures in the presence of tetrodoxin or in myotubes cultivated without neurones, even if they contract spontaneously. It is concluded that focalisation of AChE i s dependent both on neurones and o n muscle contractile activity, which is also suggested by experiments in vivo (Ldmo, T. and Slater, M., in Synaptogenesis, Gif Lectures in Neurobiology, 1976, p. 930). ( 3 ) Focalisation of ACh receptors is only neurone-dependent; inhibition of the myotube contractions does not prevent it. In conclusion, in our conditions of culture, muscle contractile activity exerts different effects o n the 3 synaptic markers studied.

Miniature End-Plate Potentials are Composed of Subunits M.E. KRIEBEL, F. LLADOS and D.R. MATTESON

Upstate Medical Center, Syracuse, N Y 13210 (U.S.A.) Miniature end-plate potential (MEPP) amplitude histograms from frog and mouse muscle cells show a number of peaks that are integral multiples of the smallest peak (SMEPP). Kriebel, M.E. and Gross, C.E. (J. Gen. Physiol., 64,85-103, 1974) found that in unstressed frog cells, SMEPP composed only 2-5% of the total but formed a distinct peak 1/7 that of the major peak. They also showed that repetitive nerve stimulation, heat and/or calcium challenges and nerve degeneration increased the percentage of SMEPPs and decreased the