Summary
Functional responses to stimulation of rat 5-HT1C receptors expressed in A9 cells were studied using whole cell voltage clamp and calcium recording techniques. Stimulation of 5-HT1C receptors evoked outward currents clamped at −50 mV. The outward currents were reduced when GTP was excluded from the intracellular recording solution or when GDR-β-S was added.
8-Bromo cyclic AMP (5 mmol/l) neither produced an effect per se nor affected the 5-HT-induced outward current in A9 cells, thus excluding CAMP as a second messenger involved in 5-HT1C receptor activation. Phorbol myristic acetate (PMA; 10 μmol/l) did not affect the electrical activity of the transfected A9 cells but reduced the 5-HT-induced current amplitude to 71±9% of the control value (n = 12). This indicates that activation of protein kinase C does not play a direct role in the 5-HT-induced response in these cells.
The 5-HT induced currents mainly involved potassium ions, although a small contribution of chloride ions was also observed. The 5-HT-induced current was inhibited by the K+ channel blocking agents tetraethylammonium (1 mmol/l), apamin (0,5 μmol/l) and 4-aminopyridine (5 mmol/1). The 5-HT-induced currents recorded at −50 mV were unaffected by removal of extracellular calcium, but inclusion of the calcium chelator BAPTA (5 mmol/l) in the intracellular solutions abolished the current. Measurement with the calcium indicator Fluo-3 revealed a 5-HT-induced increase in intracellular calcium which was not affected by removal of extracellular calcium but declined after repeated stimulation.
Determinated of pD2 and KB values of several 5-HT ligands using Fura-2 calcium measurements confirmed the pharmacology of the 5-HT1C receptor.
The results show that cloned rat 5-HT1C C receptors expressed in A9 cells activate a calcium-dependent potassium conductance.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abercrombie RF, Masukawa LM, Sjodin RA, Livengood D (1981) Uptake and release of 45Ca bymyxicola axoplasm. J Gen Physiol 78:413–429
Blatz AL, Magleby KL (1986) Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle. Nature 232:718–720
Brooker G, Takashi S, Groll D, Wahlestedt C (1990) Calcium were evoked by activation of endogenous or exogenously expressed receptors in Xenopus oocytes. Proc Natl Acad Sci USA 87:1813–1817
Brown DA, Higashida H (1988) Inositol 1,4,5-triphosphate and diacylglycerol mimic bradykinin effects on mouse neuroblastomaxrat glioma hybrid cells. J Physiol 397:185–207
Conn PJ, Sanders-Bush E (1987) Relative efficacies of piperazines at the phosphoinositide hydrolysis-linked serotonergic (5-HT-2 and 5-HT-1C) receptors. J Pharm Exp Ther 242:-52–557
Evans MG, Marty A (1986) Potentiation of muscarinic and α-adrenergic responses by an analogue of guanosine 5′-triphosphate. Proc Natl Acad Sci USA 83:4099–4103
Furchgott RF (1972) The classification of adrenoceptors (adrenergic recpetors). An evaluation from the standpoint of receptor theory. In: Blaschko H, Muscholl E (eds) Catecholamines. Springer, Berlin Heidelberg New York Tokyo, pp 283–335
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3447
Hoyer D, Waeber C, Schoeffter P, Palacios JM, Dravid A (1989) 5-HT1C receptor-mediated stimulation of inositol phosphate production in pig choroid plexus, A pharmacological characterization. Naunyn-Schmiedeberg's Arch Pharmacol 339:252–258
Jones SVP, Barker JI, Bonner TI, Buckley NJ, Brann MR (1988) Electrophysiological characterization of cloned m1 muscarinic receptors expressed in A9 L cells. Proc Natl Acad Sci USA 85:4056–4060
Jones SVP, Barker JI, Goodman MB, Brann MR (1990) Inositol triphosphate mediates cloned muscarinic receptor-activated conductances in transfected mouse friboblast A9 L cells. J Physiol 421:499–519
Julius D, MacDermott AB, Axel R, Jessel TM (1988) Molecular characterization of a functional cDNA encoding the serotonin 1C receptor. Science 241:558–564
Lübbert H, Hoffmann BJ, Snutch TP, Van Dyke T, Levine AJ (1987) cDNA cloning of serotonin 5-HT1C receptor by electrophysiological assays of mRNA-injected Xenopus oocytes. Proc Natl Acad Sci USA 84:4332–4336
Minta A, Kao JPY, Tsien RY (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromofores. J Biol Chem 264:8171–8183
Morris AP, Gallagher DV, Irvine RF, Petersen OH (1987) Synergism of inositol triphosphate and tetrakisphosphate in activating calcium-dependent potassium channels. Nature 330:653–655
Oron Y, Dascal N, Nadler E, Lupu M (1985) Inositol-1,4,5-triphosphate mimics muscarinic response in Xenopus oocytes. Nature 313:141–143
Pazos A, Hoyer D, Palacios JM (1984) The binding of serotonergic ligand to the porcine choroid plexus: characterization of a new type of serotonin recognition site. Eur J Pharmacol 106:539–546
Pedigo NW, Yamamura HI, Nelson DL (1981) Discrimination of multiple [3H]5-hydroxtryptamine-binding sites by the neuroleptic spiperone in rat brain. J Neurochem 36:220–226
Sahin-Erdemli I, Schoeffter P, Hoyer D (1991) Competitive antagonism by recognised 5-HT2 receptor antagonists at 5-HT1C receptors in pig choroid plexus. Naunyn-Schmiedeberg's Arch Pharmacol 344:137–142
Sakuta H, Sekiguchi M, Okamoto K, Sakai Y (1991) Oscillatory muscarinic acetlycholine responses of Xenopus oocytes are desensitized by protein kinase C and sensitized by protein phosphatase 2B. Eur J Pharmacol Mol Sect 208:297–305
Schnellman RG, Waters SJ, Nelson DL (1984) [3H]5-hydroxytrypt-amine binding sites: species and tissue variation. J Neurochem 42:65–70
Schoeffter P, Hoyer D (1989) Interaction of arylpiperazines with 5-HT1a, 5-HT1b, 5-HT1C, and 5-HT1d receptors: do discriminatory 5-HT1b receptor ligands exist? Naunyn Schmiedeberg's Arch Pharmacol 339:675–683
Sheldon PW, Aghajanian GK (1991) Excitatory responses to serotonin (5-HT) in neurons of the rat piriform cortex: evidence for mediation by 5-HT1C receptors in pyramidal cells and 5-HT2 receptors in interneurons. Synpase 9:208–218
Teitler M, Leonhardt S, Weisberg EL, Hoffman BJ (1990) 4-[125I]Iodo-(2,5-dimethoxy)phenyhsopropylamine and [3H]ketanserin labeling of 5-hydroxytryptamine2 (5HT2) receptors in mammalian cells transfected with a rat 5HT2 cDNA: evidence for multiple states and not multiple 5HT2 receptor subtypes. Mol Pharmacol 38:594–598
Tsien RY, Pozzan T, Rink TJ (1982) Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol 94:325–331
Waeber C, Schoeffter P, Palacious JM, Hoyer D (1988) Molecular pharmacology of 5-HT1d recognition sites: radioligand binding studies in human, pig and calf brain membranes. Naunyn-Schmiedeberg's Arch Pharmacol 337:595–601
Author information
Authors and Affiliations
Additional information
Correspondence to H. W. G. M. Boddeke at the above address
Rights and permissions
About this article
Cite this article
Boddeke, H.W.G.M., Roffman, B.J., Palacios, J.M. et al. Characterization of functional responses in A9 cells transfected with cloned rat 5-HT1C receptors. Naunyn-Schmiedeberg's Arch Pharmacol 347, 119–124 (1993). https://doi.org/10.1007/BF00169255
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00169255