Molecular pharmacological studies of the muscarinic receptors and the creatine transporter

Abstract

Murine fibroblast cell lines transfected with rat m1, m2, m3, m4 or m5 muscarinic receptor genes were used to characterize a novel muscarinic compound, YM796, and this drug was compared with other muscarinic receptor drugs. The cell lines were also used to study homologous down-regulation and desensitization of the muscarinic receptor subtypes. (-)YM796 (2,8-dimethyl-3-methylene-1-oxa-8-aza-spiro- (4,5) decane) showed minimal binding selectivity for the M1 muscarinic receptor with a Kᵢ value 16.4 μM, however, (-)YM796 appeared to be a functional selective M1 muscarinic receptor partial agonist. (-)YM796 stimulated [³H] IP₁ ([³H] inositol-1-phosphate) accumulation in LK3-3 cells without significant inhibition of the cAMP formation in M2LKB2-2 cells up to 1mM. (+)YM796 exhibited no significant efficacy for the M1 and M2 muscarinic receptors. Our study has demonstrated that the M1 muscarinic receptors were down-regulated without significant receptor internalization while the M2 muscarinic receptors were more sensitive to down-regulation than the M1 muscarinic receptors because of significant internalization of the M2 muscarinic receptors. The M1 muscarinic receptor partial agonist, (-)YM796 induced less down-regulation and no significant desensitization of the M1 muscarinic receptors and no substantial effect on the M2 muscarinic receptor density or function. Compared with the M1 and M2 muscarinic receptors, the M3 and M4 muscarinic receptors were relatively more resistant to down-regulation while the M5 muscarinic receptors were not regulated through receptor density and affinity after 24 hours exposure to the muscarinic receptor agonist, (+)-cismethyl-dioxolane (CD). While trying to isolate a cDNA encoding a human choline transporter, a human creatine transporter cDNA (hCRET) was isolated from a human brainstem/spinal cord and striatum cDNA library. This cDNA clone was comprised of an open reading frame of 1905 bp within a 2283 bp cDNA. The corresponding mRNAs were most prominently distributed in the skeletal muscle, heart and kidney. The predicted protein product revealed 12 putative transmembrane domains and a highly conserved amino acid identity with the other members ofthe sodium-dependent transporter family. Transient expression of the hCRET in COS-7 cells demonstrated sodium-dependent (¹⁴C) creatine uptake with a K(m) value of 14.9 μM, and the (¹⁴C) creatine uptake was attenuated by creatine and selective creatine transporter inhibitors.