Native 5-HT1B receptors expressed in OK cells display dual coupling to elevation of intracellular calcium concentrations and inhibition of adenylate cyclase

Abstract

The opossum kidney (OK) cell line has been shown previously to express endogenous 5-HT_1B receptors which negatively couple to adenylate cyclase. Since other G_i-linked receptors have been shown to inhibit adenylate 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 transduction mechanisms. Saturation studies using [¹²⁵I](–)-iodocyanopindolol ([¹²⁵I]CYP) to radiolabel the 5-HT_1B receptor in OK cell membranes (in the presence of 3 µM (–)-isoproterenol to mask β-adrenergic receptors) yielded an equilibrium dissociation constant (pK _d) of 10.04 and binding site density (B _max) of 55 fmol/mg protein. Exposure of intact OK cells to 5-HT, CP 93,129, a selective rodent 5-HT_1B receptor agonist, and (±)-cyanopindolol, a mixed 5-HT_1A/1B receptor agonist/antagonist, produced concentration-dependent inhibitions of forskolin (3 µM)-stimulated cAMP accumulation (FSCA; E _max=90–95%) and elevations of [Ca²⁺]_i (E _max∼200 nM increase above basal levels). Agonist potencies (pEC₅₀) ranged from 9.7 to 8.1 and were comparable between the two second messenger assays, although slightly higher agonist potencies (∼three-fold) were observed in the cAMP assay. GR 127,935, a selective 5-HT_1B/1D receptor antagonist, behaved as a strong partial agonist in both the cAMP and calcium assays, with an intrinsic activity of 0.7 relative to 5-HT. Methiothepin, a nonselective 5-HT receptor antagonist, competitively antagonized the inhibitory cAMP response elicited by CP 93,129, yielding an apparent pK _b value of 7.3. Methiothepin (10 µM) completely antagonized the stimulatory calcium response evoked by a saturating concentration of CP 93,129 (100 nM). Pertussis toxin pretreatment blocked the CP 93,129-induced inhibition of FSCA and elevation of [Ca²⁺]_i in OK cells, indicating the involvement of G_i/o proteins in transducing these second messenger responses. The agonist properties of (±)-cyanopindolol and GR 127,935 observed in both second messenger assays suggests that a large degree of receptor reserve may be present, even though 5-HT_1B receptor expression is low in OK cells. The OK cell line continues to serve as a model system to investigate 5-HT_1B receptor-mediated signaling events.