Abstract
Prior studies have suggested that heart expresses only the M2 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 M2 and M4 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 M3 muscarinic receptor receptor was cloned, characterized, and its expression in chick tissues determined. Using a human M3 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 M3 muscarinic receptor, respectively. Chinese hamster ovary (CHO-GRA) cells were stably transfected with the chick M3 muscarinic receptor and one clone (CHO-CM3) expressed the M3 receptor, as measured by the binding of quinuclidinly benzilate at 116 +/- 14 (+/- S.E., n = 3) fmol/mg protein with a Kd of 76 +/- 17 pM. This receptor demonstrated a rank order of potency for muscarinic antagonist binding characteristic for the M3 receptor: with high affinity binding for hexahydrosiladifenidol, Kd: 16 +/- 2 nM (+/- S.E., n = 3); intermediate affinity for pirenzepine, Kd: 383 +/- 47 nM, and low affinity for methoctramine, Kd: 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 M3 muscarinic receptor has the properties characteristic of M3 receptors from other species. RNase protection studies demonstrated the presence of M3 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 M2 and M4 muscarinic receptors but also for the M3 muscarinic receptor.
Highlights
Usinga human M, 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
Using RNase protection analysis we further demonstrated that mRNA coding for this chick M, receptor was expressed in the mal essential medium supplemented by dialyzed fetal calf serum in the absence of nucleosides.Ten positive clones wereisolated, amplified,and assayed for [,H]QNB binding
41-kDa band (Fig. 6c).The addition of Lubrol (0.1%) to homogenates of cells which were pretreated with pertussitsoxin had no effect on the level of incorporation of [”‘PINAD, suggesting that the absence of pertussis toxin substrates in these homotussis toxin-insensitive.CHO-CM3 cells were grown to 80% conflu- Tissue Distribution of Muscarinic Receptor mRNA by RNase ence and incubated for h with 1.25 pCUm1 [DHlmyoinositol.a,cells were incubated with the indicated concentrations of carbamylcholine for min at 37 “C and total inositol phosphate release determineads described under “Materials and Methods.”
Summary
Scribed (Galper and Smith, 1980)in 250-pl aliquots of cell homogenate containing 100-150 pg of membrane protein and the indicated concen-. Two clones coded for forskolin, lo-, M isobutylmethylxanthine, and various concentrations of the same unique full-length receptor protein. This clone was completely carbamylcholine.Cellswerelysed and CAMP assayed as described sequenced on bothstrands using a combination of subcloned restriction (Marsh and Roberts, 1987)using a radioimmunoassay kit (Biomedical fragments and synthetic oligonucleotideprimers, by the dideoxy chain Technologies Inc.). Termination method (Sanger et al, 1977).Other clones isolated included Inositol Phosphate Assay-The production of inositol phosphate was cDNAscodingfor full-length chick M, and M, receptors which were assayed as described previously (Barnett et al, 1990). RNase ProtectionAssays-Total RNA was isolated from 17-dayin ouo chick tissues using guanidium CsCl, centrifugation For the M, RNase protection probea 636-bptemplate was generated
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