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. 1988 Sep 1;107(3):1147–1156. doi: 10.1083/jcb.107.3.1147

Metabolic stability and antigenic modulation of nicotinic acetylcholine receptors on bovine adrenal chromaffin cells

PMCID: PMC2115298  PMID: 3417779

Abstract

Bovine adrenal chromaffin cells have nicotinic acetylcholine receptors (AChRs) that are activated by the splanchnic nerve, resulting in release of catecholamines from the cells. Examination of the AChRs can provide information about the regulation and turnover of synaptic components on neurons and endocrine cells. Previous studies have shown that mAb 35 recognizes the AChR on the cells. Here we show that mAb 35 can remove AChRs from the surface of the cells by antigenic modulation, and that the modulation can be used together with other methods to examine the stability and turnover of the receptors in the plasma membrane. Unexpectedly, the results indicate a disparity between the rate at which AChRs reappear on the cells and the rate at which the ACh response recovers after preexisting AChRs have been removed. Exposure of bovine adrenal chromaffin cultures to mAb 35 results in a parallel decrease in the magnitude of the nicotinic response and the number of AChRs on the cells. The decrease depends on the concentration and divalence of mAb 35, and on the time and temperature of the incubation. The antibody induces receptor aggregation in the plasma membrane under conditions where receptor loss subsequently occurs. After binding to receptor, mAb 35 appears to be internalized, degraded, and released from the cells through a temperature sensitive pathway that requires lysosomal function. These features are characteristic of antigenic modulation. Appearance of new AChRs on the cells either after antigenic modulation or after blockade of existing AChRs with monovalent antibody fragments occurs at a rate equivalent to 3% of the receptors present on control cells per hour. The rate of receptor loss from the cells was measured in the presence of either tunicamycin or puromycin to block appearance of new receptors. Both conditions indicated a receptor half- life of approximately 24 h and a rate of loss of approximately 3%/h. The finding that the rate of receptor loss equaled the rate of receptor appearance was consistent with the observation that the total number of AChRs on untreated cells did not increase with time. In the presence of tunicamycin, loss of receptor-mediated response to nicotine also occurred with a half-time of 24 h. Paradoxically, the rate of recovery of the nicotinic response, determined using two procedures, was more than twice as great as the rate at which new AChRs appeared on the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

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Selected References

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  1. Albuquerque E. X., Rash J. E., Mayer R. F., Satterfield J. R. An electrophysiological and morphological study of the neuromuscular junction in patients with myasthenia gravis. Exp Neurol. 1976 Jun;51(3):536–563. doi: 10.1016/0014-4886(76)90179-5. [DOI] [PubMed] [Google Scholar]
  2. Baccaglini P. I., Cooper E. Influences on the expression of acetylcholine receptors on rat nodose neurones in cell culture. J Physiol. 1982 Mar;324:441–451. doi: 10.1113/jphysiol.1982.sp014123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boulter J., Connolly J., Deneris E., Goldman D., Heinemann S., Patrick J. Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7763–7767. doi: 10.1073/pnas.84.21.7763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boulter J., Evans K., Goldman D., Martin G., Treco D., Heinemann S., Patrick J. Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor alpha-subunit. 1986 Jan 30-Feb 5Nature. 319(6052):368–374. doi: 10.1038/319368a0. [DOI] [PubMed] [Google Scholar]
  5. Bourguignon L. Y., Singer S. J. Transmembrane interactions and the mechanism of capping of surface receptors by their specific ligands. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5031–5035. doi: 10.1073/pnas.74.11.5031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brenner H. R., Martin A. R. Reduction in acetylcholine sensitivity of axotomized ciliary ganglion cells. J Physiol. 1976 Aug;260(1):159–175. doi: 10.1113/jphysiol.1976.sp011509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Devreotes P. N., Fambrough D. M. Acetylcholine receptor turnover in membranes of developing muscle fibers. J Cell Biol. 1975 May;65(2):335–358. doi: 10.1083/jcb.65.2.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Devreotes P. N., Gardner J. M., Fambrough D. M. Kinetics of biosynthesis of acetylcholine receptor and subsequent incorporation into plasma membrane of cultured chick skeletal muscle. Cell. 1977 Mar;10(3):365–373. doi: 10.1016/0092-8674(77)90023-x. [DOI] [PubMed] [Google Scholar]
  9. Drachman D. B., Angus C. W., Adams R. N., Kao I. Effect of myasthenic patients' immunoglobulin on acetylcholine receptor turnover: selectivity of degradation process. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3422–3426. doi: 10.1073/pnas.75.7.3422. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dunn P. M., Marshall L. M. Lack of nicotinic supersensitivity in frog sympathetic neurones following denervation. J Physiol. 1985 Jun;363:211–225. doi: 10.1113/jphysiol.1985.sp015705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fenwick E. M., Marty A., Neher E. Sodium and calcium channels in bovine chromaffin cells. J Physiol. 1982 Oct;331:599–635. doi: 10.1113/jphysiol.1982.sp014394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Halvorsen S. W., Berg D. K. Affinity labeling of neuronal acetylcholine receptor subunits with an alpha-neurotoxin that blocks receptor function. J Neurosci. 1987 Aug;7(8):2547–2555. [PMC free article] [PubMed] [Google Scholar]
  13. Heinemann S., Bevan S., Kullberg R., Lindstrom J., Rice J. Modulation of acetylcholine receptor by antibody against the receptor. Proc Natl Acad Sci U S A. 1977 Jul;74(7):3090–3094. doi: 10.1073/pnas.74.7.3090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Higgins L. S., Berg D. K. A desensitized form of neuronal acetylcholine receptor detected by 3H-nicotine binding on bovine adrenal chromaffin cells. J Neurosci. 1988 Apr;8(4):1436–1446. doi: 10.1523/JNEUROSCI.08-04-01436.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Higgins L. S., Berg D. K. Cyclic AMP-dependent mechanism regulates acetylcholine receptor function on bovine adrenal chromaffin cells and discriminates between new and old receptors. J Cell Biol. 1988 Sep;107(3):1157–1165. doi: 10.1083/jcb.107.3.1157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Higgins L. S., Berg D. K. Immunological identification of a nicotinic acetylcholine receptor on bovine chromaffin cells. J Neurosci. 1987 Jun;7(6):1792–1798. doi: 10.1523/JNEUROSCI.07-06-01792.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jacob M. H., Berg D. K. Effects of preganglionic denervation and postganglionic axotomy on acetylcholine receptors in the chick ciliary ganglion. J Cell Biol. 1987 Oct;105(4):1847–1854. doi: 10.1083/jcb.105.4.1847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kilpatrick D. L., Slepetis R., Kirshner N. Ion channels and membrane potential in stimulus-secretion coupling in adrenal medulla cells. J Neurochem. 1981 Mar;36(3):1245–1255. doi: 10.1111/j.1471-4159.1981.tb01724.x. [DOI] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Leprince P. Chemical modification of the nicotinic cholinergic receptor of PC-12 nerve cell. Biochemistry. 1983 Nov 22;22(24):5551–5556. doi: 10.1021/bi00293a015. [DOI] [PubMed] [Google Scholar]
  21. Lindstrom J., Einarson B. Antigenic modulation and receptor loss in experimental autoimmune myasthenia gravis. Muscle Nerve. 1979 May-Jun;2(3):173–179. doi: 10.1002/mus.880020304. [DOI] [PubMed] [Google Scholar]
  22. Livett B. G. Adrenal medullary chromaffin cells in vitro. Physiol Rev. 1984 Oct;64(4):1103–1161. doi: 10.1152/physrev.1984.64.4.1103. [DOI] [PubMed] [Google Scholar]
  23. Loring R. H., Zigmond R. E. Characterization of neuronal nicotinic receptors by snake venom neurotoxins. Trends Neurosci. 1988 Feb;11(2):73–78. doi: 10.1016/0166-2236(88)90168-3. [DOI] [PubMed] [Google Scholar]
  24. Margiotta J. F., Berg D. K., Dionne V. E. Cyclic AMP regulates the proportion of functional acetylcholine receptors on chicken ciliary ganglion neurons. Proc Natl Acad Sci U S A. 1987 Nov;84(22):8155–8159. doi: 10.1073/pnas.84.22.8155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Margiotta J. F., Berg D. K., Dionne V. E. The properties and regulation of functional acetylcholine receptors on chick ciliary ganglion neurons. J Neurosci. 1987 Nov;7(11):3612–3622. doi: 10.1523/JNEUROSCI.07-11-03612.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Merlie J. P., Heinemann S., Einarson B., Lindstrom J. M. Degradation of acetylcholine receptor in diaphragms of rats with experimental autoimmune myasthenia gravis. J Biol Chem. 1979 Jul 25;254(14):6328–6332. [PubMed] [Google Scholar]
  27. Merlie J. P., Heinemann S., Lindstrom J. M. Acetylcholine receptor degradation in adult rat diaphragms in organ culture and the effect of anti-acetylcholine receptor antibodies. J Biol Chem. 1979 Jul 25;254(14):6320–6327. [PubMed] [Google Scholar]
  28. Mizobe F., Kozousek V., Dean D. M., Livett B. G. Pharmacological characterization of adrenal paraneurons: substance P and somatostatin as inhibitory modulators of the nicotinic response. Brain Res. 1979 Dec 14;178(2-3):555–566. doi: 10.1016/0006-8993(79)90714-5. [DOI] [PubMed] [Google Scholar]
  29. Olson E. N., Glaser L., Merlie J. P., Sebanne R., Lindstrom J. Regulation of surface expression of acetylcholine receptors in response to serum and cell growth in the BC3H1 muscle cell line. J Biol Chem. 1983 Nov 25;258(22):13946–13953. [PubMed] [Google Scholar]
  30. Pestronk A. Intracellular acetylcholine receptors in skeletal muscles of the adult rat. J Neurosci. 1985 May;5(5):1111–1117. doi: 10.1523/JNEUROSCI.05-05-01111.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ravdin P. M., Berg D. K. Inhibition of neuronal acetylcholine sensitivity by alpha-toxins from Bungarus multicinctus venom. Proc Natl Acad Sci U S A. 1979 Apr;76(4):2072–2076. doi: 10.1073/pnas.76.4.2072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ravdin P. M., Nitkin R. M., Berg D. K. Internalization of alpha-bungarotoxin on neurons induced by a neurotoxin that blocks neuronal acetylcholine sensitivity. J Neurosci. 1981 Aug;1(8):849–861. doi: 10.1523/JNEUROSCI.01-08-00849.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Role L. W., Perlman R. L. Both nicotinic and muscarinic receptors mediate catecholamine secretion by isolated guinea-pig chromaffin cells. Neuroscience. 1983 Nov;10(3):979–985. doi: 10.1016/0306-4522(83)90236-1. [DOI] [PubMed] [Google Scholar]
  34. Schmidt J., Rossie S., Catterall W. A. A large intracellular pool of inactive Na channel alpha subunits in developing rat brain. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4847–4851. doi: 10.1073/pnas.82.14.4847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Smith M. A., Stollberg J., Lindstrom J. M., Berg D. K. Characterization of a component in chick ciliary ganglia that cross-reacts with monoclonal antibodies to muscle and electric organ acetylcholine receptor. J Neurosci. 1985 Oct;5(10):2726–2731. doi: 10.1523/JNEUROSCI.05-10-02726.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stall A. M., Knopf P. M. The effect of inhibitors of protein synthesis on the reexpression of surface immunoglobulin following antigenic modulation. Cell. 1978 May;14(1):33–42. doi: 10.1016/0092-8674(78)90298-2. [DOI] [PubMed] [Google Scholar]
  37. Stollberg J., Berg D. K. Neuronal acetylcholine receptors: fate of surface and internal pools in cell culture. J Neurosci. 1987 Jun;7(6):1809–1815. doi: 10.1523/JNEUROSCI.07-06-01809.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Trifaró J. M., Bourne G. W. Differential effects of concanavalin A on acetylcholine and potassium-evoked release of catecholamines from cultured chromaffin cells. Neuroscience. 1981;6(9):1823–1833. doi: 10.1016/0306-4522(81)90216-5. [DOI] [PubMed] [Google Scholar]
  39. Trifaró J. M., Lee R. W. Morphological characteristics and stimulus-secretion coupling in bovine adcrenal chromaffin cell cultures. Neuroscience. 1980;5(9):1533–1546. doi: 10.1016/0306-4522(80)90018-4. [DOI] [PubMed] [Google Scholar]
  40. Tuttle J. B. Interaction with membrane remnants of target myotubes maintains transmitter sensitivity of cultured neurons. Science. 1983 May 27;220(4600):977–979. doi: 10.1126/science.6133352. [DOI] [PubMed] [Google Scholar]
  41. Tzartos S. J., Rand D. E., Einarson B. L., Lindstrom J. M. Mapping of surface structures of electrophorus acetylcholine receptor using monoclonal antibodies. J Biol Chem. 1981 Aug 25;256(16):8635–8645. [PubMed] [Google Scholar]
  42. Tzartos S. J., Seybold M. E., Lindstrom J. M. Specificities of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies. Proc Natl Acad Sci U S A. 1982 Jan;79(1):188–192. doi: 10.1073/pnas.79.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wan K. K., Lindstrom J. M. Effects of monoclonal antibodies on the function of acetylcholine receptors purified from Torpedo californica and reconstituted into vesicles. Biochemistry. 1985 Feb 26;24(5):1212–1221. doi: 10.1021/bi00326a024. [DOI] [PubMed] [Google Scholar]
  44. Whiting P. J., Lindstrom J. M. Purification and characterization of a nicotinic acetylcholine receptor from chick brain. Biochemistry. 1986 Apr 22;25(8):2082–2093. doi: 10.1021/bi00356a037. [DOI] [PubMed] [Google Scholar]
  45. Whiting P., Lindstrom J. Purification and characterization of a nicotinic acetylcholine receptor from rat brain. Proc Natl Acad Sci U S A. 1987 Jan;84(2):595–599. doi: 10.1073/pnas.84.2.595. [DOI] [PMC free article] [PubMed] [Google Scholar]

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