Loss of Acetylcholine Receptor Clusters Induced by Treatment of Cultured Rat Myotubes with Carbachol
Total Page：16
File Type：pdf, Size：1020Kb
The Journal of Neuroscience March 1966, 6(3): 691-700 Loss of Acetylcholine Receptor Clusters Induced by Treatment of Cultured Rat Myotubes with Carbachol Robert J. Bloch Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201 Prolonged exposure to carbachol disrupts the acetylcholine re- of carbachol; (2) the cation flux into myotubes following the ceptor (AChR) clusters of cultured rat myotubes without causing conformational change; (3) the depolarization following cation myotube loss. The effect is reversible, and is dependent on tem- flux. I show that the second possibility, cation flux through the perature. Half-maximal cluster loss is achieved at 3 PM car- receptor channel, accounts for the destabilizing effect of car- bachol. Cluster loss is also caused by other agonists of the AChR bachol on AChR clusters. These results are consistent with the and is blocked by receptor antagonists. QX314 (a lidocaine de- idea that the stability of the AChR clusters of cultured rat myo- rivative), meproadifen, and fluphenazine also completely block tubes is dependent on the intracellular ionic milieu. cluster loss caused by carbachol. These results are consistent with the idea that cluster loss caused by carbachol and other Materials and Methods receptor agonists results from their interaction with the AChR, Methods for preparing myotube cultures, for staining them with tetra- and the consequent influx of cations into the myotubes. Several methylrhodamine-cu-bungarotoxin (R-BT; Ravdin and Axelrod, 1977), experiments suggest that extracellular Na+ and Ca*+ are re- and for analyzing AChR clusters have been presented in detail elsewhere quired, and that at least Na+ must permeate the AChR ion chan- (Bloch, 1979, 1983). In brief, rat myotube cultures, grown on glass nel for full cluster loss to occur in the presence of carbachol. coverslips, are washed with Dulbecco-Vogt modified Eagle’s medium (DMEM) buffered with HEPES, then covered with DME&I containing Depolarization alone is not sufficient to cause cluster loss, how- 5% fetal calf (Gibco. Grand Island. NY) or cadet calf serum (Biocell ever. Ca*+-activated proteases do not play a significant role in Laboratories, barson, CA), and incibateh with or without the addition carbachol-induced cluster loss. of drugs for 6 hr at 37°C in an atmosphere of 94% air, 6% CO,. Cultures are washed extensivelv with HEPES-buffered DMEM. stained with R-BT. The clustering of acetylcholine receptors (AChR) at sites of nerve- fixed, and observed-under fluorescence optics to hetermine the per: muscle contact is one of the earliest events in the differentiation centage of the total myotubes displaying large, flat AChR clusters on of the postsynaptic region of the neuromuscular junction (Fam- their ventral surfaces. brough, 1979). To understand the synaptogenic interactions be- A cluster is defined as a discrete region of the myotube surface, -40 tween nerve and muscle, 1 have been studying AChR clustering, pm2 in area, that stains brightly with R-BT, is neither linear nor an in vivo and in vitro, in aneural cultures of rat myotubes. The artifact of the overlapping of two or more cells, and is not located on a cellular process isolated from the muscle cell or detached from the organization and properties of the AChR clusters in newly in- tissue culture substrate (Bloch. 1979). Usuallv 50 mvotubes ner culture nervated muscle and in cultured myotubes seem to be similar were analyzed, and eacd expehmental value Is the average ok duplicate in the rat (Steinbach and Bloch, in press). One similarity is that or triplicate determinations. Values generally vary by f 10% myotubes AChR clusters in cultured rat myotubes and at the embryonic with clusters (e.g., Tables 3 and 5, below). postsynaptic membrane are susceptible to disruption by pro- Influx experiments with 22Na+ were performed as described by Bloch longed treatment with carbachol, an AChR agonist (Bloch, 1979; (1979) and Stallcup and Cohn (1976), except that the temperature was Bloch and Steinbach, 198 1). To learn more about this phenom- 35-37“C, and incubations were performed for 15 min. Longer exposure enon, I have studied the factors involved in carbachol-induced to carbachol before assay also resulted in increased 22Na+ influx, but AChR cluster loss in cultured cells. was not as quantitatively reproducible and so was not pursued further. The sources of most materials have been reported earlier (Bloch, In the experiments reported here, I show, first, that carbachol 1979). Nicotinic and muscarinic cholinergic drugs and other neuro- does not exert its effect by causing loss or irreversible damage transmitter receptor ligands were purchased from Sigma Chemical Com- to myotubes, and, second, that it destabilizes AChR clusters by pany (St. Louis, MO) or were gifts from Dr. J. Patrick (Neurobiology interacting with the nicotinic receptor. I then consider three Laboratory, The Salk Institute, La Jolla, CA). Leupeptin, aprotinin, actions of carbachol that could destabilize AChR clusters: (1) pepstatin, and L-seryl-L-leucine were purchased from Sigma. The pro- the conformational change induced in the receptor by binding tease inhibitor, Ep475, was kindly provided by Dr. L. Waxman (De- partment of Physiology, Harvard Medical School). Fluphenazine and meproadifen were the gifts of Drs. B. Krueger and E. Albuquerque Received Feb. 27, 1985; revised May 20, 1985; accepted June 11, 1985. (Departments of Physiology, and Pharmacology, respectively, Univer- I thank Dr. Stephen Heinemann, in whose laboratory at the Salk Institute (La sity of Maryland School of Medicine). Jolla, CA) much of this work was performed, for his encouragement and financial support. I also thank Dr. P. Brehm for his measurements of membrane potential, and Drs. J. Patrick, J. H. Steinbach, and R. Gruener for their useful suggestions. Results Drs. D. Burt. L. Waxman. B. Krueaer, and E. X. Albuquerque generously offered their reagents and advice, and Ms. w. tiesneck assisted ih some ofthe experiments. Cluster losscaused by carbachol I began this work as a Fellow of the San Diego Chapter of the American Heart Association, with support from the Samuel Roberts Noble Foundation of Ard- In cultures of rat myotubes, 70-90% of the myotubes normally more, OK, and from grants to Dr. Heinemann from the National Institutes of display large clusters of AChR, which can be visualized after Health (NS 11549) aid the Muscular Dystrophy Association. More recently, I staining with R-BT. Upon exposure to 10m4M carbachol, the have been supported by grants from the National Institutes of Health (NS 17282) and the Muscular Dystrophy Association, as well as by a M&night Scholar’s fraction of myotubes displaying clusters decreasesto ~30% Award and a Research Career Development Award (NS 00679). (Bloch, 1979). This loss of clusters occurs as a linear function Copyright 0 1986 Society for Neuroscience 0270-6474/86/030691-10$02.00/O of time up to 6 hr (Fig. 1). As the effects of carbachol on AChR 691 692 Bloch Vol. 6, No. 3, Mar. 1986 Table 1. Effect of carbachol on AChR clusters and number of myotubes Carbachol No carbachol Experiment Experiment 1 2 Total O/o 1 2 Total % Myotubes t=O 37 52 88 100 56 53 106 100 t=6hr 32 41 73 83 43 50 93 88 Myotubeswith clusters 2 4 6 t=6hr 30 3 4 35 38 73 70 Time (hr) Myotube cultures on glass coverslips were mounted into a special incubation chamber (Anderson et al., 1977). Selected fields were photographed at low Figure 1. The time course of AChR clusterloss in the presenceof magnification, and their grid positions were noted. These photographs subsequently carbachol.Myotube cultureswere incubated in medium(open circles) gave the number of myotubes present in the selected fields at the beginning of the or in mediumcontaining either 10e4M carbachol(crosses) or 10m4 M experiment (t = 0). The cultures were then placed into a solution consisting of d-tubocurarine(triangles). At the times indicated,the cultureswere DMEM plus 5% cadet calf serum. Half of the chambers also contained 10m4 M removedfrom the incubator,washed extensively with bufferedmedium, carbachol. Cultures were incubated for 6 hr at 37”C, washed free of the incubation medium and stained with R-BT. After staining, the cultures were fixed in 2% stainedwith R-BT, and observedunder fluorescence.Myotubes with paraformaldehyde. The same grid positions were relocated and photographed. AChR clusterswere quantitated as described in Methods.The results These photographs subsequently gave the number of myotubes present in the showthat clusterloss is inducedby carbachol,but not by d-tubocurarine, same fields at the termination of the experiment (t = 6 hr). The myotubes in these and that it proceedsas a linearfunction of time of treatment. selected fields were then observed with fluorescence optics under high magnification to determine whether AChR clusters were present, as outlined in Methods. These data are contained in the row labeled “Myotubeswith clusters,” t = 6 hr. clustersare apparent at 6 hr, this time was routinely usedin the experiments presented here. Data in Figure 1 further suggest that the failure to detect AChR clusters was not due to incom- bated for an additional 18 hr. Upon staining with R-BT, most plete removal of carbachol before staining with R-BT. For ex- of the myotubes present at the end of the experiment contained ample, similar amounts of carbachol should remain after wash- AChR clusters(see Table 2). Similar resultswere obtained when ing cells treated for 2 and 6 hr, but the percentageof myotubes myotube cultures were stainedwith R-BT after the 6 hr exposure with clustersat 6 hr is one-third that seenafter 2 hr oftreatment. to carbachol but before the 18 hr recovery period (seeTable 2). Also, d-tubocurarine at 1O-4 M causesno lossof clusters,despite Thus, all the clusters seenafter the 18 hr recovery contained the fact that it has a higher affinity for AChR than does car- AChRs that were present before recovery began.