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-P 3), 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-P 3 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-P 3 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-P 3 caused a dose-dependent, long-lasting inhibition of subsequent Ins1,4,5-P 3 and 5HT responses. Desensitization induced by Ins1,4,5-P 3 affected both 5HT1 and 5HT2. Thus, Ins1,4,5-P 3 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-P 3 response was reversed by PhoOBt2 suggesting a role for protein kinase C in recovery from desensitization.
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References
Alkon DL, Rasmussen H (1988) A spatial-temporal model of cell activation. Science 239: 998–1005
Bader CR, Bertrand D, Schlichter R (1987) Calcium-activated chloride current in cultured sensory and parasympathetic quail neurons. J Physiol (Lond) 394: 125–148
Bazzi MD, Nelsestuen GL (1988) Properties of membrane-inserted protein kinase C. Biochemistry 27: 7589–7593
Berridge MJ (1987) Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem 56: 159–193
Berridge MJ (1988) Inositol trisphosphate-induced membrane potential oscillations in Xenopus oocytes. J Physiol (Lond) 403: 589–599
Berridge MJ, Irvine RF (1984) A novel second messenger in cellular signal transduction. Nature 312: 315–321
Berridge MJ, Irvine RF (1989) Inositol phosphates and cell signaling. Nature 341: 197–205
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: 511–534
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: 1–6
Burgoyne RD (1989) A role for membrane-inserted protein kinase C in cellular memory? Trends Biochem Sci 14: 87–88
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: 677–6826
Dascal N (1987) The use of Xenopus oocytes for the study of ion channels. CRC Crit Rev Biochem 22: 317–387
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: 552–574
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: 299–313
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: 201–209
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: 1411–1415
Garland LG, Bonser RW, Thompson NT (1987) Protein kinase C inhibitors are not selective. Trends Pharmacol Sci 8: 33
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: 349–361
Gundersen CB, Miledi R, Parker I (1984) Messenger RNA from human brain induced drug and voltage-operated channels in Xenopus oocytes. Nature 308: 421–424
Hill TD, Dean NM, Boynton AL (1988) Inositol 1,3,4,5-tetrakisphosphate induces Ca2+ sequestration in rat liver cells. Science 242: 1176–1178
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: 523–535
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: 318–321
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: 155–160
Julius D, MacDermont AB, Axel R, Jessel T (1988) Molecular characterization of a functional cDNA encoding the serotonin 1c receptor. Science 241: 558–564
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: 11–19
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: 766–773
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: 411–416
Lester HA (1988) Heterologous expression of excitability proteins: route to more specific drugs? Science 241: 1057–1063
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: 239–248
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: 1159–1165
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: 4332–4336
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: 83–87
Lupu-Meiri M, Shapira H, Oron Y (1989) Dual regulation by protein kinase C of the muscarinic respone in Xenopus oocytes. Pflügers Arch 413:498–504
Malinow R, Madison DV, Tsien RW (1988) Persistent protein kinase activity underlying long-term potentiation. Nature 335: 820–824
Maniatis T, Fritch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Marty A, Tan YP, Trautman A (1984) Three types of calcium-dependent channels in rat lacrimal gland. J Physiol (Lond) 357: 293–325
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: 836–838
Moran O, Dascal N (1989) Protein kinase C modulates neurotransmitter responses in Xenopus oocytes injected with rat brain RNA. Mol Brain Res 5: 193–202
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: 6977–7000
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: 113–123
Oron Y, Dascal N, Nadler E, Lupu M (1985) Inositol 1,4,5-trisphosphate mimics muscarinic response in Xenopus oocytes. Nature 313: 141–143
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: 918–925
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: 307–315
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: 491–496
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: 37–45
Shears SB 61989) Inositol phosphate metabolism: further problems and some solutions. Cell Signaling 2: 125–133
Sibley DR, Lefkowitz RJL (1985) Molecular mechanisms of receptor desensitization using the beta-adrenergic receptor-coupled adenylate cyclase system as a model. Nature 317: 124–129
Singer D, Boton R, Dascal N (1988) Serotonin (5HT) response inRNA-injected Xenopus oocytes: short-and long-lasting desensitization. Biophys J 53: 518a
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: 1048–11051
Sugiyama H, Hisanaga Y, Hirono C (1985) Induction of muscarinic cholinergic responsiveness in Xenopus oocytes by mRNA isolated from rat brain. Brain Res 338: 346–350
Sugiyama H, Ito I, Hirono C (1987) A new type of glutamate receptor linked to inositol phospholipid metabolism. Nature 325: 531–533
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: 5063–5067
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: 641–646
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: 497–500
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D. Singer and R. Boton are to be considered as equal first authors of this paper
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Singer, D., Boton, R., Moran, O. et al. 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. Pflügers Arch 416, 7–16 (1990). https://doi.org/10.1007/BF00370215
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DOI: https://doi.org/10.1007/BF00370215