PHARMACOLOGY OF CLONED HUMAN 5-HT1D RECEPTOR-MEDIATED FUNCTIONAL-RESPONSES IN STABLY TRANSFECTED RAT C6-GLIAL CELL-LINES - FURTHER EVIDENCEDIFFERENTIATING HUMAN 5-HT1D AND 5-HT1B RECEPTORS

This study was undertaken to investigate the pharmacology of human ser otonin (5-HT)(1D) receptor sites by measuring two functional cellular responses, inhibition of forskolin-stimulated cAMP formation and promo tion of cell growth, using transfected rat C6-glial cell lines and a b road series of 5-HT receptor agonists. Stable and separate transfectio n of a pcDNA3 or pRcRSV plasmid, each containing a cloned human 5-HT1D receptor gene, in rat C6-glial cells was confirmed with RT-PCR of 5-H T1D receptor mRNA and radioligand binding with [H-3] 5-carboxamidotryp tamine (5-CT) and [H-3] sumatriptan. The 5-HT1D receptor density was 3 50 and 1050 fmol/mg protein for the C6-glial/pcDNA3/5-HT1D and C6-glia l/pRcRSV/S-HT1D cell line, and forskolin (100 mu M)-induced cAMP forma tion was inhibited by 45 and 78% in the presence of 1 mu M 5-HT, respe ctively. A comparison of the intrinsic agonist activities for sixteen 5-HT receptor ligands with their corresponding binding affinities for the human 5-HT1D receptor site showed similar results for both cell li nes with the exception of the partial agonist m-trifluoro-phenyl-piper azine (TFMPP). Three classes of compounds were observed: 1) efficaciou s agonists, such as 5-CT, 5-methoxytryptamine, 5-HT, sumatriptan, bufo tenine, 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl) 1H-indole (RU 24,9 69), tryptamine and 8-hydroxy-2(di-n-propilamino)tetralin (8-OH-DPAT), with agonist potency close to their binding affinity; 2) the partial agonists metergoline, oromethyl-4(4-methyl-1-piperazinyl)-pyrolo-(1,2- a) quinoxaline (CGS 12066B), 1-naphthylpiperazine and 2'-methyl-4-(5-m ethyl-[1,2,4]oxadiazol-3 -yl)-biphenyl-4-carboxylic acid [4-methoxy-3- (4-methylpiperazin-1 -yl)-phenyl]-amide (GR 127,935) with marked intri nsic agonist activity but at concentrations higher than their binding affinity; and 3) the silent antagonists ritanserin, ketanserin and met hiothepin, apparently free of intrinsic agonist activity, with antagon ist potency close to their binding affinity. The cAMP data were furthe r supported by the observed promotion of cell growth by stimulation of both transfected cell lines with sumatriptan under serum-free conditi ons; half-maximal stimulation was obtained at 4.4 nM (C6-glial/pcDNA3/ 5-HT1D) fully in agreement with its EC(50)-value (5.7 nM) for inhibiti on of cAMP formation. This growth promoting effect was antagonised by 1 mu M methiothepin and not observed in pcDNA3-plasmid-transfected and non-transfected C6-glial cells. A comparative study with a C6-glial/p cDNA3/5-HT1B cell line expressing a similar amount of cloned human 5-H T1B receptors (B-max: 360 fmol/mg protein) showed almost no intrinsic agonist activity for metergoline, 1-naphtylpiperazine and GR 127,935. Together with the 5-HT1D receptor binding selectivity and antagonist a ctivity of ketanserin and ritanserin, the findings define important ph armacological differences between cloned human 5-HT1D and 5-HT1B recep tor sites.