PROMOTION OF CELL-GROWTH BY STIMULATION OF CLONED HUMAN 5-HT1D RECEPTOR-SITES IN TRANSFECTED C6-GLIAL CELLS IS HIGHLY SENSITIVE TO INTRINSIC ACTIVITY AT 5-HT1D RECEPTORS

5-Hydroxytryptamine (serotonin, 5-HT), essentially known as a neurotra nsmitter and vasoactive agent, also functions as a mitogen in various cell types through several different second messenger systems. Stimula tion of cloned human 5-HT1D receptor sites by sumatriptan in stably tr ansfected rat C6-glial/5-HT1D cells promotes cell growth (Pauwels et a l. (1996) Naunyn-Schmiedeberg's Arch Pharmacol 353: 144-156). In the p resent study, the pharmacology of this growth response was investigate d using a broad series of 5-HT receptor ligands. The data were compare d with the responses obtained by measuring inhibition of forskolin-sti mulated cAMP formation. 5-HT (EC(50): 25 nM) promoted cell growth of C 6-glial/5-HT1D cells, and this in contrast to the absence of any measu rable effect in pcDNA3-plasmid transfected and non-transfected CG-glia l cells. The 5-HT effect could be mimicked by the following compounds (EC(50) in nM): zolmitriptan (0.41), 2'-methyl-4'-(5-methyl[1,2,4] oxa diazol-3-yl)biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin -1-yl)phenyl] amide(CR 127,935; 0.86), naratriptan (0.92), metergoline (1.9), sumatriptan (2.9), 1,2,4-triazol-1-ylmethyl)-1H-indol-3-yl]eth ylamine (MK-462; 3.0), and R(+)-8-(hydroxy-2-(di-n-propylamino)tetrali n (R(+)-8-OH-DPAT; 30.7). These EC(50)-values correspond to the compou nds binding affinities at the human 5-HT1D receptor site and, with the exception of GR 127,935 and metergoline, also to the EC(50)-values fo und by measuring over 5 min inhibition of forskolin (100 mu M)-stimula ted cAMP formation. Prolonged exposure of GR 127,935 (3 h) and metergo line (30 min) to cells yielded EC(50) values in the cAMP assay more cl ose to those measured in the mitogenic response. The growth response t o sumatriptan, 5-HT, GR 127,935 and metergoline was blocked by the app arently silent antagonists methiothepin, ritanserin and ketanserin wit h potencies similar to blockade of inhibit-ion of stimulated cAMP form ation. The 8-OH-DPAT effect also is likely mediated by 5-HT1D receptor s; stereoselectivity was found with its enantiomers at this receptor s ite and the effect was blocked by ketanserin (1 mu M) but not by spipe rone (1 mu M). Micromolar concentrations of the 5-HT1B receptor agonis t -tetrahydro)-4-pyridil-5-pyrrolo[3,2-b]pyril-5-one (CP 93,129) and o f the 5-HT, receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopro pane (DOI) induced cell growth with a potency that accorded with the a ffinity of these compounds for the human 5-HT1D receptor site. These e ffects were sensitive to ketanserin (1 mu M) antagonism, but not to bl ockade by beta-adrenergic-blockers and the 5-HT2 receptor antagonist 2 -anilino-N-[2-(3-chlorophenoxy)-propyl] acetamidine hydroiodide (BW 50 1-C-67). The findings suggest that 5-HT1A, 5-HT1B and 5-HT2 receptors are not implicated in 5-HT-stimulated C6-glial/5-HT1D cell growth. In conclusion, human 5-HT1D receptors are involved in the growth of C6-gl ial/5-HT1D cells. This cellular response is highly sensitive to the in trinsic activity of compounds at 5-HT1D receptors.