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. 1987 Jun;84(12):4313–4317. doi: 10.1073/pnas.84.12.4313

Activation of a muscarinic receptor selectively inhibits a rapidly inactivated Ca2+ current in rat sympathetic neurons.

E Wanke, A Ferroni, A Malgaroli, A Ambrosini, T Pozzan, J Meldolesi
PMCID: PMC305075  PMID: 2438697

Abstract

Sympathetic neurons dissociated from the superior cervical ganglion of 2-day-old rats were studied by whole-cell patch clamp and by fura-2 measurements of the cytosolic free Ca2+ concentration, [Ca2+]i. Step depolarizations in the presence of tetrodotoxin and hexamethonium triggered two Ca2+ currents that differed in the voltage dependence of activation and kinetics of inactivation. These currents resemble the L and N currents previously described in chicken sensory neurons [Nowycky, M. C., Fox, A. P. & Tsien, R. W. (1985) Nature (London) 316, 440-442]. Treatment with acetylcholine resulted in the rapid (within seconds), selective, and reversible inhibition of the rapidly inactivated, N-type current, whereas the long-lasting L-type current remained unaffected. The high sensitivity to blocker drugs (atropine, pirenzepine) indicated that this effect of acetylcholine was due to a muscarinic M1 receptor. Intracellular perfusion with nonhydrolyzable guanine nucleotide analogs or pretreatment of the neurons with pertussis toxin had profound effects on the Ca2+ current modulation. Guanosine 5'-[gamma-thio]triphosphate caused the disappearance of the N-type current (an effect akin to that of acetylcholine, but irreversible), whereas guanosine 5'-[beta-thio]diphosphate and pertussis toxin pretreatment prevented the acetylcholine-induced inhibition. In contrast, cAMP, applied intracellularly together with 3-isobutyl-1-methylxanthine, as well as activators and inhibitors of protein kinase C, were without effect. Acetylcholine caused shortening of action potentials in neurons treated with tetraethylammonium to partially block K+ channels. Moreover, when applied to neurons loaded with the fluorescent indicator fura-2, acetylcholine failed to appreciably modify [Ca2+]i at rest but caused a partial blunting of the initial [Ca2+]i peak induced by depolarization with high K+. This effect was blocked by muscarinic antagonists and pertussis toxin and was unaffected by protein kinase activators. Thus, muscarinic modulation of the N-type Ca2+ channels appears to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein and independent of both cAMP-dependent protein kinase and protein kinase C.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Belluzzi O., Sacchi O., Wanke E. Identification of delayed potassium and calcium currents in the rat sympathetic neurone under voltage clamp. J Physiol. 1985 Jan;358:109–129. doi: 10.1113/jphysiol.1985.sp015543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carbone E., Lux H. D. A low voltage-activated, fully inactivating Ca channel in vertebrate sensory neurones. Nature. 1984 Aug 9;310(5977):501–502. doi: 10.1038/310501a0. [DOI] [PubMed] [Google Scholar]
  3. Cheng Y., Prusoff W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973 Dec 1;22(23):3099–3108. doi: 10.1016/0006-2952(73)90196-2. [DOI] [PubMed] [Google Scholar]
  4. DeRiemer S. A., Strong J. A., Albert K. A., Greengard P., Kaczmarek L. K. Enhancement of calcium current in Aplysia neurones by phorbol ester and protein kinase C. Nature. 1985 Jan 24;313(6000):313–316. doi: 10.1038/313313a0. [DOI] [PubMed] [Google Scholar]
  5. Di Virgilio F., Pozzan T., Wollheim C. B., Vicentini L. M., Meldolesi J. Tumor promoter phorbol myristate acetate inhibits Ca2+ influx through voltage-gated Ca2+ channels in two secretory cell lines, PC12 and RINm5F. J Biol Chem. 1986 Jan 5;261(1):32–35. [PubMed] [Google Scholar]
  6. Dunlap K., Fischbach G. D. Neurotransmitters decrease the calcium conductance activated by depolarization of embryonic chick sensory neurones. J Physiol. 1981 Aug;317:519–535. doi: 10.1113/jphysiol.1981.sp013841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Forscher P., Oxford G. S. Modulation of calcium channels by norepinephrine in internally dialyzed avian sensory neurons. J Gen Physiol. 1985 May;85(5):743–763. doi: 10.1085/jgp.85.5.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Galvan M., Adams P. R. Control of calcium current in rat sympathetic neurons by norepinephrine. Brain Res. 1982 Jul 22;244(1):135–144. doi: 10.1016/0006-8993(82)90911-8. [DOI] [PubMed] [Google Scholar]
  9. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  10. Hamill O. P., Marty A., Neher E., Sakmann B., Sigworth F. J. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981 Aug;391(2):85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  11. Hartzell H. C., Fischmeister R. Opposite effects of cyclic GMP and cyclic AMP on Ca2+ current in single heart cells. Nature. 1986 Sep 18;323(6085):273–275. doi: 10.1038/323273a0. [DOI] [PubMed] [Google Scholar]
  12. Hidaka H., Inagaki M., Kawamoto S., Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry. 1984 Oct 9;23(21):5036–5041. doi: 10.1021/bi00316a032. [DOI] [PubMed] [Google Scholar]
  13. Holz G. G., 4th, Rane S. G., Dunlap K. GTP-binding proteins mediate transmitter inhibition of voltage-dependent calcium channels. Nature. 1986 Feb 20;319(6055):670–672. doi: 10.1038/319670a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Horn J. P., McAfee D. A. Alpha-drenergic inhibition of calcium-dependent potentials in rat sympathetic neurones. J Physiol. 1980 Apr;301:191–204. doi: 10.1113/jphysiol.1980.sp013198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kao L. S., Schneider A. S. Muscarinic receptors on bovine chromaffin cells mediate a rise in cytosolic calcium that is independent of extracellular calcium. J Biol Chem. 1985 Feb 25;260(4):2019–2022. [PubMed] [Google Scholar]
  16. Katada T., Bokoch G. M., Northup J. K., Ui M., Gilman A. G. The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Properties and function of the purified protein. J Biol Chem. 1984 Mar 25;259(6):3568–3577. [PubMed] [Google Scholar]
  17. MacDonald R. L., Skerritt J. H., Werz M. A. Adenosine agonists reduce voltage-dependent calcium conductance of mouse sensory neurones in cell culture. J Physiol. 1986 Jan;370:75–90. doi: 10.1113/jphysiol.1986.sp015923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Marchetti C., Carbone E., Lux H. D. Effects of dopamine and noradrenaline on Ca channels of cultured sensory and sympathetic neurons of chick. Pflugers Arch. 1986 Feb;406(2):104–111. doi: 10.1007/BF00586670. [DOI] [PubMed] [Google Scholar]
  19. Milligan G., Gierschik P., Spiegel A. M., Klee W. A. The GTP-binding regulatory proteins of neuroblastoma x glioma, NG108-15, and glioma, C6, cells. Immunochemical evidence of a pertussis toxin substrate that is neither Ni nor No. FEBS Lett. 1986 Jan 20;195(1-2):225–230. doi: 10.1016/0014-5793(86)80165-x. [DOI] [PubMed] [Google Scholar]
  20. Misgeld U., Calabresi P., Dodt H. U. Muscarinic modulation of calcium dependent plateau potentials in rat neostriatal neurons. Pflugers Arch. 1986 Nov;407(5):482–487. doi: 10.1007/BF00657504. [DOI] [PubMed] [Google Scholar]
  21. Morita K., North R. A., Tokimasa T. Muscarinic presynaptic inhibition of synaptic transmission in myenteric plexus of guinea-pig ileum. J Physiol. 1982 Dec;333:141–149. doi: 10.1113/jphysiol.1982.sp014444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nowycky M. C., Fox A. P., Tsien R. W. Three types of neuronal calcium channel with different calcium agonist sensitivity. Nature. 1985 Aug 1;316(6027):440–443. doi: 10.1038/316440a0. [DOI] [PubMed] [Google Scholar]
  23. Paupardin-Tritsch D., Hammond C., Gerschenfeld H. M., Nairn A. C., Greengard P. cGMP-dependent protein kinase enhances Ca2+ current and potentiates the serotonin-induced Ca2+ current increase in snail neurones. 1986 Oct 30-Nov 5Nature. 323(6091):812–814. doi: 10.1038/323812a0. [DOI] [PubMed] [Google Scholar]
  24. Pozzan T., Di Virgilio F., Vicentini L. M., Meldolesi J. Activation of muscarinic receptors in PC12 cells. Stimulation of Ca2+ influx and redistribution. Biochem J. 1986 Mar 15;234(3):547–553. doi: 10.1042/bj2340547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rane S. G., Dunlap K. Kinase C activator 1,2-oleoylacetylglycerol attenuates voltage-dependent calcium current in sensory neurons. Proc Natl Acad Sci U S A. 1986 Jan;83(1):184–188. doi: 10.1073/pnas.83.1.184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Reuter H. Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature. 1983 Feb 17;301(5901):569–574. doi: 10.1038/301569a0. [DOI] [PubMed] [Google Scholar]
  27. Scott R. H., Dolphin A. C. Regulation of calcium currents by a GTP analogue: potentiation of (-)-baclofen-mediated inhibition. Neurosci Lett. 1986 Aug 15;69(1):59–64. doi: 10.1016/0304-3940(86)90414-3. [DOI] [PubMed] [Google Scholar]
  28. Stevens C. F. Modifying channel function. Nature. 1986 Feb 20;319(6055):622–622. doi: 10.1038/319622a0. [DOI] [PubMed] [Google Scholar]
  29. Tsien R. Y., Rink T. J., Poenie M. Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths. Cell Calcium. 1985 Apr;6(1-2):145–157. doi: 10.1016/0143-4160(85)90041-7. [DOI] [PubMed] [Google Scholar]
  30. Vicentini L. M., Ambrosini A., Di Virgilio F., Meldolesi J., Pozzan T. Activation of muscarinic receptors in PC12 cells. Correlation between cytosolic Ca2+ rise and phosphoinositide hydrolysis. Biochem J. 1986 Mar 15;234(3):555–562. doi: 10.1042/bj2340555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Vicentini L. M., Ambrosini A., Di Virgilio F., Pozzan T., Meldolesi J. Muscarinic receptor-induced phosphoinositide hydrolysis at resting cytosolic Ca2+ concentration in PC12 cells. J Cell Biol. 1985 Apr;100(4):1330–1333. doi: 10.1083/jcb.100.4.1330. [DOI] [PMC free article] [PubMed] [Google Scholar]

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