Advertisement

Pflügers Archiv

, Volume 416, Issue 1–2, pp 7–16 | Cite as

Short-and long-term desensitization of serotonergic response in Xenopus oocytes injected with brain RNA: roles for inositol 1,4,5-trisphosphate and protein kinase C

  • Dafna Singer
  • Rony Boton
  • Ofira Moran
  • Nathan Dascal
Excitable Tissues and Central Nervous Physiology

Abstract

In Xenopus oocytes injected with rat brain RNA, serotonin (5HT) and acetylcholine (ACh) evoke membrane responses through a common biochemical cascade that includes activation of phospholipase C, production of inositol 1,4,5-trisphosphate (Ins1,4,5-P3), release of Ca2+ from intracellular stores, and opening of Ca-dependent Cl channels. The response is a Cl current composed of a transient component (5HT1 or ACh1) and a slow, long-lasting component (5HT2 or ACh2). Here we show that only the fast, but not the slow, component of the response is subject to desensitization that follows a previous application of the transmitter. The recovery of 5HT1 from desensitization is biphasic, suggesting the existence of two types of desensitization: short-term desensitization (STD), which lasts for less than 0.5 h; and long-term desensitization (LTD) lasting for up to 4 h. The desensitization between 5HT and ACh is heterologous and long-lasting. We searched for (a) the molecular target and (b) the cause of desensitization.(a) Pre-exposure to 5HT does not reduce the response evoked by intracellular injection of Ca2+ and by Ca2+ influx. Cl current evoked by intracellular injection of Ins1,4,5-P3 was reduced shortly after application of 5HT, but fully recovered 30 min later. Thus, the Cl channel is not a target for desensitization. Neither Ins1,4,5-P3 receptor nor the Ca2+ store is a target of LTD but they may be the targets of STD. (b) Ca2+ injection did not inhibit the 5HT response, suggesting that Ca2+ is not a sole cause of STD or LTD. An activator of protein kinase C, β-phorbol 12,13-dibutyrate (PhoOBt2), is known to inhibit the 5HT response, but this inhibition had completely subsided 30 min after washout of PhoOBt2. A protein kinase inhibitor H-7 did not prevent LTD. Thus, protein kinase C does not appear to be the cause of LTD, but its role in STD cannot be ruled out at present. Injection of Ins1,4,5-P3 caused a dose-dependent, long-lasting inhibition of subsequent Ins1,4,5-P3 and 5HT responses. Desensitization induced by Ins1,4,5-P3 affected both 5HT1 and 5HT2. Thus, Ins1,4,5-P3 is a possible cause of STD and LTD, but non-specific effects cannot be ruled out at present. The self-desensitization of Ins1,4,5-P3 response was reversed by PhoOBt2 suggesting a role for protein kinase C in recovery from desensitization.

Key words

Xenopus oocyte Desensitization Protein kinase C Serotonin Acetylcholine Ca2+ -dependent Cl channel Inositol 1,4,5-trisphosphate 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Alkon DL, Rasmussen H (1988) A spatial-temporal model of cell activation. Science 239: 998–1005Google Scholar
  2. Bader CR, Bertrand D, Schlichter R (1987) Calcium-activated chloride current in cultured sensory and parasympathetic quail neurons. J Physiol (Lond) 394: 125–148Google Scholar
  3. Bazzi MD, Nelsestuen GL (1988) Properties of membrane-inserted protein kinase C. Biochemistry 27: 7589–7593Google Scholar
  4. Berridge MJ (1987) Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem 56: 159–193Google Scholar
  5. Berridge MJ (1988) Inositol trisphosphate-induced membrane potential oscillations in Xenopus oocytes. J Physiol (Lond) 403: 589–599Google Scholar
  6. Berridge MJ, Irvine RF (1984) A novel second messenger in cellular signal transduction. Nature 312: 315–321Google Scholar
  7. Berridge MJ, Irvine RF (1989) Inositol phosphates and cell signaling. Nature 341: 197–205Google Scholar
  8. Boton R, Dascal N, Gillo B, Lass Y (1989) Two calcium-activated chloride conductances in Xenopus laevis oocytes permeabilized with the ionophore A23187. J Physiol (Lond) 408: 511–534Google Scholar
  9. Boton R, Singer D, Dascal N (1990) Inactivation of calcium-activated chloride channel in Xenopus oocytes: roles of calcium and protein kinase C. Pflügers Arch, 416: 1–6Google Scholar
  10. Burgoyne RD (1989) A role for membrane-inserted protein kinase C in cellular memory? Trends Biochem Sci 14: 87–88Google Scholar
  11. Busa WB, Ferguson JE, Joseph SK, Williamson JR, Nuccitelly R (1985) Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. 1. Characterization of Ca2+ release from intracellular stores. J Cell Biol 101: 677–6826Google Scholar
  12. Dascal N (1987) The use of Xenopus oocytes for the study of ion channels. CRC Crit Rev Biochem 22: 317–387Google Scholar
  13. Dascal N, Landau EM, Lass Y (1984) Xenopus oocytes resting potential, muscarinic responses, and the role of calcium and cyclic GMP. J Physiol (Lond) 352: 552–574Google Scholar
  14. Dascal N, Gillo B, Lass Y (1985) Role of calcium mobilization in mediation of acetylcholine-evoked chloride currents in Xenopus laevis oocytes. J Physiol (Lond) 366: 299–313Google Scholar
  15. Dascal N, Ifune C, Hopkins R, Snutch TP, Lubbert H, Davidson N, Simon M, Lester HA (1986) Involvement of a GTP-binding protein in mediation of serotonin and acetylcholine responses in Xenopus oocytes injected with rat brain messenger RNA. Mol Brain Res 1: 201–209Google Scholar
  16. Dierks P, Van Ooyen A, Mantei N, Weissmann C (1981) DNA sequences preceding the rabbit β-globin RNA gene are required for formation in mouse L-cells of β-globin RNA with correct 5-terminus. Proc Natl Acad Sci USA 78: 1411–1415Google Scholar
  17. Garland LG, Bonser RW, Thompson NT (1987) Protein kinase C inhibitors are not selective. Trends Pharmacol Sci 8: 33Google Scholar
  18. Gillo B, Lass Y, Nadler E, Oron Y (1987) The involvement of inositol 1,4,5-trisphosphate and calcium in the two-component response to acetylcholine in Xenopus oocytes. J Physiol (Lond) 342: 349–361Google Scholar
  19. Gundersen CB, Miledi R, Parker I (1984) Messenger RNA from human brain induced drug and voltage-operated channels in Xenopus oocytes. Nature 308: 421–424Google Scholar
  20. Hill TD, Dean NM, Boynton AL (1988) Inositol 1,3,4,5-tetrakisphosphate induces Ca2+ sequestration in rat liver cells. Science 242: 1176–1178Google Scholar
  21. Hirono C, Ito I, Sugiyama H (1987) Neurotensin and acetylcholine evoke common responses in frog oocytes injected with rat brain messenger ribonucleic acid. J Physiol (Lond) 382: 523–535Google Scholar
  22. Houamed KM, Bilbe G, Smart TG, Constanti A, Brown DA, Barnard EA, Richards BM (1984) Expression of functional GABA, glycine and glutamate receptors in Xenopus oocytes injected with rat brain mRNA. Nature 310: 318–321Google Scholar
  23. Ito I, Hirono C, Yamagishi S, Nomura Y, Kaneko S, Sugiyama H (1988) Roles of protein kinases in neurotransmitter responses in Xenopus oocytes injected with rat brain RNA. J Cell Physiol 134: 155–160Google Scholar
  24. Julius D, MacDermont AB, Axel R, Jessel T (1988) Molecular characterization of a functional cDNA encoding the serotonin 1c receptor. Science 241: 558–564Google Scholar
  25. Kaneko S, Kato K-I, Yamagishi S-I, Sugiyama H, Nomura Y (1987) GTP-binding proteins Gi and Go transplanted onto Xenopus oocytes by rat brain messenger RNA. Mol Brain Res 3: 11–19Google Scholar
  26. Kato K, Kaneko S, Nomura Y (1988) Phorbol ester inhibition of current responses and simultaneous protein phosphorylation in Xenopus oocyte injected with brain mRNA. J Neurochem 50: 766–773Google Scholar
  27. Kubo T, Fukuda K, Mikami A, Maeda A, Takahashi H, Mishina M, Haga T, Haga K, Ichiyama A, Kangawa K, Kojima M, Matsuo H, Hirose T, Numa S (1986) Cloning, sequencing and expression of complementary DNA encoding the muscarinic acetylcholine receptor. Nature 323: 411–416Google Scholar
  28. Lester HA (1988) Heterologous expression of excitability proteins: route to more specific drugs? Science 241: 1057–1063Google Scholar
  29. Llano I, Marty A (1987) Protein kinase C activators inhibit the inositol trisphosphate-mediated muscarinic current responses in rat lacrimal cells. J Physiol (Lond) 394: 239–248Google Scholar
  30. Lubbert H, Snutch TP, Dascal N, Lester HA, Davidson N (1987a) Rat brain 5HT1c receptors are encoded by a 5–6 kb mRNA size class and functionally expressed in injected Xenopus oocytes. J Neurosci 7: 1159–1165Google Scholar
  31. Lubbert H, Hoffman BJ, Snutch TP, van Dyke T, Hartig PR, Lester HA, Davidson N (1987b) cDNA cloning of a serotonin 5-HT1c receptor by electrophysiological assays of mRNA-injected Xenopus oocytes. Proc Natl Acad Sci USA 84: 4332–4336Google Scholar
  32. Lupu-Meiri M, Shapira H, Oron Y (1988) Hemispheric asymmetry of rapid chloride responses to inositol trisphosphate and calcium in Xenopus oocytes. FEBS Lett 240: 83–87Google Scholar
  33. Lupu-Meiri M, Shapira H, Oron Y (1989) Dual regulation by protein kinase C of the muscarinic respone in Xenopus oocytes. Pflügers Arch 413:498–504Google Scholar
  34. Malinow R, Madison DV, Tsien RW (1988) Persistent protein kinase activity underlying long-term potentiation. Nature 335: 820–824Google Scholar
  35. Maniatis T, Fritch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.Google Scholar
  36. Marty A, Tan YP, Trautman A (1984) Three types of calcium-dependent channels in rat lacrimal gland. J Physiol (Lond) 357: 293–325Google Scholar
  37. Masu Y, Nakayama K, Tamaki H, Harada Y, Kuno M, Nakanishi S (1987) cDNA cloning of bovine substance-K receptor through oocyte expression system. Nature 329: 836–838Google Scholar
  38. Moran O, Dascal N (1989) Protein kinase C modulates neurotransmitter responses in Xenopus oocytes injected with rat brain RNA. Mol Brain Res 5: 193–202Google Scholar
  39. Muller D, Turnbull J, Baudry M, Lynch G (1988) Phorbol ester-induced synaptic facilitation is different than long-term potentiation. Proc Natl Acad Sci USA 85: 6977–7000Google Scholar
  40. Nomura Y, Kaneko S, Kato K-I, Yamagishi SI, Sugiyama H (1987) Inositol phosphate formation and chloride current responses induced by acetylcholine and serotonin through GTP-binding proteins in Xenopus oocyte after injection of rat brain messenger RNA. Mol Brain Res 2: 113–123Google Scholar
  41. Oron Y, Dascal N, Nadler E, Lupu M (1985) Inositol 1,4,5-trisphosphate mimics muscarinic response in Xenopus oocytes. Nature 313: 141–143Google Scholar
  42. Oron Y, Gillo B, Straub RE, Gershengorn MC (1987) Mechanism of membrane electrical response to thyrotropin-releasing hormone in Xenopus oocytes injected with GH3 pituitary cell messenger ribonucleic acid. Mol Endocrinol 1: 918–925Google Scholar
  43. Parker I, Miledi R (1986) Changes in intracellular calcium and in membrane currents evoked by injection of inositol trisphosphate into Xenopus oocytes. Proc R Soc Lond [Biol] 228: 307–315Google Scholar
  44. Parker I, Gundersen CB, Miledi R (1985) Intracellular Ca2+ -dependent and Ca2+ -independent responses of rat brain serotonin receptors transplanted to Xenopus oocytes. Neurosci Res 2: 491–496Google Scholar
  45. Parker I, Sumikawa K, Miledi R (1987) Activation of a common effector system by different brain neurotransmitter receptors in Xenopus oocytes. Proc R. Soc Lond [Biol] 231: 37–45Google Scholar
  46. Shears SB 61989) Inositol phosphate metabolism: further problems and some solutions. Cell Signaling 2: 125–133Google Scholar
  47. Sibley DR, Lefkowitz RJL (1985) Molecular mechanisms of receptor desensitization using the beta-adrenergic receptor-coupled adenylate cyclase system as a model. Nature 317: 124–129Google Scholar
  48. Singer D, Boton R, Dascal N (1988) Serotonin (5HT) response inRNA-injected Xenopus oocytes: short-and long-lasting desensitization. Biophys J 53: 518aGoogle Scholar
  49. Snyder PM, Krause KH, Welsh MJ (1988) Inositol trisphosphate isomers, but not inositol 1,3,4,5-tetrakisphosphate, induce calcium influx in Xenopus laevis oocytes. J Biol Chem 263: 1048–11051Google Scholar
  50. Sugiyama H, Hisanaga Y, Hirono C (1985) Induction of muscarinic cholinergic responsiveness in Xenopus oocytes by mRNA isolated from rat brain. Brain Res 338: 346–350Google Scholar
  51. Sugiyama H, Ito I, Hirono C (1987) A new type of glutamate receptor linked to inositol phospholipid metabolism. Nature 325: 531–533Google Scholar
  52. Takahashi T, Neher E, Sakmann B (1987) Rat brain serotonin receptors in Xenopus oocytes are coupled by intracellular calcium to endogenous channels. Proc Natl Acad Sci USA 84: 5063–5067Google Scholar
  53. Taleb O, Felth P, Bossu J-L, Feltz A (1988) Small-conductance chloride channels activated by calcium on cultured endocrine cells from mammalian pars intermedia. Pflügers Arch 412: 641–646Google Scholar
  54. Van Wezenbeek LACM, Tonnaer JADM, Ruigt GSF (1988) The endogenous muscarinic acetylcholine receptor in Xenopus oocytes is of the M3 subtype. Eur J Pharmacol 151: 497–500Google Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • Dafna Singer
    • 1
  • Rony Boton
    • 1
  • Ofira Moran
    • 1
  • Nathan Dascal
    • 1
  1. 1.Department of Physiology and Pharmacology, Sackler Faculty of MedicineTel Aviv UniversityRamat AvivIsrael

Personalised recommendations