NATIVE 5-HT1B RECEPTORS EXPRESSED IN OH CELLS DISPLAY DUAL COUPLING TO ELEVATION OF INTRACELLULAR CALCIUM CONCENTRATIONS AND INHIBITION OF ADENYLATE-CYCLASE

The opossum kidney (OK) cell line has been shown previously to express endogenous 5-HT1B receptors which negatively couple to adenylate cycl ase. Since other Gi-linked receptors have been shown to inhibit adenyl ate cyclase and to elevate intracellular calcium concentrations ([Ca2](i)), studies were initiated to determine whether native opossum 5-HT 1B receptors could also display dual coupling to these signal transduc tion mechanisms. Saturation studies using [I-125](-)-iodocyanopindolol ([I-125]CYP) to radiolabel the 5-HT1B receptor in OK cell membranes ( in the presence of 3 mu M (-)-isoproterenol to mask beta-adrenergic re ceptors) yielded an equilibrium dissociation constant (pK(d)) of 10.04 and binding site density (B-max) of 55 fmol/mg protein. Exposure of i ntact OK cells to 5-HT, CP 93,129, a selective rodent 5-MT1B receptor agonist, and (+/-)-cyanopindolol, a mixed 5-HT1A/1B receptor agonist/a ntagonist, produced concentration-dependent inhibitions of forskolin ( 3 mu M)-stimulated cAMP accumulation (FSCA; E-max=90-95%) and elevatio ns of [Ca2+](i) (E(max)similar to 200 nM increase above basal levels). Agonist potencies (pEC(50)) ranged from 9.7 to 8.1 and were comparabl e between the two second messenger assays, although slightly higher ag onist potencies (similar to threefold) were observed in the cAMP assay . GR 127,935, a selective 5-HT1B/1D receptor antagonist, behaved as a strong partial agonist in both the cAMP and calcium assays, with an in trinsic activity of 0.7 relative to 5-HT. Methiothepin, a nonselective 5-HT receptor antagonist, competitively antagonized the inhibitory cA MP response elicited by CP 93,129, yielding an apparent pK(b) value of 7.3. Methiothepin (10 mu M) completely antagonized the stimulatory ca lcium response evoked by a saturating concentration of CP 93,129 (100 nM). Pertussis toxin pretreatment blocked the CP 93,129-induced inhibi tion of FSCA and elevation of [Ca2+](i) in OK cells, indicating the in volvement of G(i/o) proteins in transducing these second messenger res ponses. The agonist properties of (+/-)-cyanopindolol and GR 127,935 o bserved in both second messenger assays suggests that a large degree o f receptor reserve may be present, even though 5-HT1B receptor express ion is low in OK cells. The OK cell line continues to serve as a model system to investigate 5-HT1B receptor-mediated signaling events.