Functional expression of human 5-HT1A receptors and differential coupling to second messengers in CHO cells

Abstract

The signal transduction linkages of the cloned human 5-HT1A receptor as expressed stably in CHO cells were studied. A transfected clonal cell line which expresses 900 +/- 36 fmol 5-HT1A receptor/mg protein (designated CHO-5-HT1A/WT-27) responded to 5-HT and/or 8-OH-DPAT by coupling to several second messenger pathways. The 5-HT1A receptor inhibited, but did not stimulate, membrane adenylyl cyclase activity and whole cell cAMP accumulation in a dose-dependent manner (for 5-HT, IC50 = 146 +/- 27 and 55 +/- 12 nM, respectively). Activation of the receptor was associated with other signal transduction linkages: (i) a 40-50% increase in hydrolysis of inositol phosphates (for 5-HT, EC50 = 1.33 +/- 0.15 microM for 5-HT), (ii) a transient elevation of cytosolic Ca2+ levels (apparent at 1-100 microM 5-HT) which was not affected by chelation of extracellular Ca2+ by EGTA, and (iii) an augmentation of [3H]-arachidonic acid release pharmacologically with the calcium ionophore A23187 or by activation of endogenous thrombin or P2 purinergic receptors (for 5-HT, EC50 = 1.22 +/- 0.17 microM). This pathway may be an amplification mechanism for signaling in anatomic regions with high concentrations of several neuro-transmitters, hormones or autacoids, such as at neuronal junctions or near areas of platelet aggregation. All linkages were sensitive to pertussis toxin pre-treatment (IC50 approximately 0.5-0.6 ng/ml x 4.5 h for all pathways), suggesting the involvement of Gi protein(s) in these signal transduction pathways. Coupling to varied signal transduction pathways in a single cell system may be a common feature of receptors which classically inhibit adenylyl cyclase such as the 5-HT1A receptor.

Adenosine A1Receptor-Mediated Activation of Phospholipase C in Cultured Astrocytes Depends on the Level of Receptor Expression

“…In these cells the coupling of A 1 receptors to PLC was less efficient than the inhibitory coupling of A 1 receptors to adenylyl cyclase Fredholm, 1991, 1992a). Similarly, efficient inhibitory coupling to adenylyl cyclase and less efficient potentiation of PLC activity also was found for A 1 receptors transfected into CHO cells (Freund et al, 1994) and for other G i /o -protein receptors like ␣ 2 adrenergic receptors (Cotecchia et al, 1990), serotonin 5-HT 1A receptors (Raymond et al, 1992), and D 2 dopamine receptors (Vallar et al, 1992). In preliminary experiments we have shown that upregulation of adenosine A 1 receptors in cells with very low receptor levels (cerebellum) gives rise to the potentiating effect seen in other cultures.…”

Agonist-Promoted Down-Regulation and Functional Desensitization in Two Naturally Occurring Variants of the Human Serotonin1A Receptor

“…For this purpose, WT, Ser22, and Val28 5-HT 1A receptors were stably expressed in CHO-K1 cells. These cells do not ordinarily express any 5-HT receptor and, thus, are useful for pharmacological and functional studies of transfected 5-HT 1A receptors (Raymond 1991;Raymond et al 1992;Newman-Tancredi et al 1992). (Hoyer et al 1986;De Vivo and Maayani 1986;Dumuis et al 1988;Marazziti et al 1994).…”

Human Taste Thresholds Are Modulated by Serotonin and Noradrenaline

“…(4) We propose a serotonergic feedback from presynaptic to receptor cells where 5-HT acts via 5-HT 1A receptors (Delay et al, 1997;Herness and Chen, 1997;Herness and Chen, 2000;Kaya et al, 2004) to modulate bitter and sweet signaling. In some amphibian taste cells, activation of 5-HT 1A receptors is known to increase intracellular calcium levels (Delay et al, 1997), and 5-HT 1A receptors have been shown to affect intracellular calcium in other cell types (Raymond et al, 1992;Khan et al, 1995). 5-HT 1A receptors are expressed on a different cell population in the taste bud than 5-HT (Kaya et al, 2004), and 5-HT release occurs from presynaptic and not receptor cells (Y. J. .…”