A NOVEL M(3) MUSCARINIC ACETYLCHOLINE-RECEPTOR IS EXPRESSED IN CHICK ATRIUM AND VENTRICLE

作者：GADBUT AP; GALPER JB

来源：Journal of Biological Chemistry, 1994, 269(41): 25823-25829.

Prior studies have suggested that heart expresses only the M(2) isoform of the muscarinic receptor (Peralta, E. G., Ashkenazi, A., Winslow, J. W., Smith, D. H., Ramachandran, J., and Capon, D, J. (1987) EMBO J. 6, 3923-3929). Tietje and Nathanson (Tietje, K. M., and Nathanson, N. M. (1991) J. Biol. Chem. 266, 17382-17387) have recently demonstrated that the chick heart may be unique since it expresses both the M(2) and M(4) isoforms of the muscarinic receptor. In this study, in order to determine whether other isoforms of the muscarinic receptor were present in the chick heart, a chick M(3) muscarinic recep tor was cloned, characterized, and its expression in chick tissues determined. Using a human M(3) muscarinic receptor cDNA as a probe, a 2.4-kilobase pair cDNA was isolated from a chick brain cDNA library which contained an open reading frame coding for a 639 amino acid protein. This protein demonstrated an 87 and 86% homology to the human and rat M(3) muscarinic receptor, respectively. Chinese hamster ovary (CHO-GRA) cells were stably transfected with the chick M(3) muscarinic receptor and one clone (CHO-CM3) expressed the M(3) receptor, as measured by the binding of quinuclidinly benzilate at 116 +/- 14 (+/-S.E., n = 3) fmol/mg protein with a K-d of 76 +/- 17 pM. This receptor demonstrated a rank order of potency for muscarinic antagonist binding characteristic for the M(3) receptor: with high affinity binding for hexahydrosiladifenidol, K-d: 16 +/- 2 nM (+/-S.E., n = 3); intermediate affinity for pirenzepine, K-d: 383 +/- 47 nM, and low affinity for methoctramine, K-d: 533 +/- 185 nM (+/-S.E., n = 3). Carbamylcholine stimulation of CHO-CM3 cells resulted in a 1.6-fold increase in cyclic AMP accumulation and a 3.5-fold increase in a pertussis toxin-insensitive inositol phosphate release. These data demonstrate that the chick M(3) muscarinic receptor has the properties characteristic of M(3) receptors from other species. RNase protection studies demonstrated the presence of M(3) muscarinic receptor mRNA in the brain, atria, and ventricle of chicks 17 days in ovo. Hence, the chick heart appears to have the unique capacity to express mRNAs coding not only for the M(2) and M(4) muscarinic receptors but also for the M(3) muscarinic receptor.

• 出版日期1994-10-14