Abstract
Characterization of muscarinic acetylcholine receptors in acinar cells from rat pancreas and lacrimal and parotid glands was achieved by binding of the reversible muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) and the specific alkylating reagent [3H]propylbenzilylcholine mustard (PrBCM) to intact acini or dispersed acinar cells. Binding studies with [3H]QNB showed that acinar cells from pancreas contain 26,400, from parotid 21,400, and from lacrimal gland 25,700 binding sites/cell. To assess molecular size of the receptor in each gland, acini were prepared by digestion with purified collagenase and singly dispersed acinar cells were prepared by a combination of digestion with crude collagenase, hyaluronidase, and alpha-chymotrypsin and divalent cation chelation using EDTA. Muscarinic receptors on acini or dispersed cells were covalently labeled with 5 nM [3H]PrBCM, solubilized directly in hot sodium dodecyl sulfate buffer, and resolved by polyacrylamide gel electrophoresis. When solubilized acini were electrophoresed, a major labeled peak was observed on gels along with a smaller peak of lower apparent molecular weight. For pancreatic acini, the apparent molecular weights of these peaks were 117,600 and 85,700; for parotid acini, 104,800 and 74,500; and for lacrimal acini, 87,200 and 63,100. Addition of muscarinic antagonists to the labeling medium abolished both peaks. When dispersed acinar cells were labeled, the larger peak was eliminated, and all radioactivity was concentrated in a single peak: 87,600 for pancreas, 78,000 for parotid gland, and 62,800 for lacrimal gland. Digestion of prelabeled acini with the mixture of enzymes used to produce dispersed acinar cells similarly shifted all radioactivity into this second peak. Limited digestion of acini or dispersed cells with 1 mg/ml of papain resulted in the disappearance of these higher molecular weight peaks and the appearance of a broad peak at Mr = 40,000. Cells of nonepithelial origin, IM-9 lymphocytes and NG108 neuroblastoma X glioma hybrids, also were labeled with [3H]PrBCM and electrophoresed.(ABSTRACT TRUNCATED AT 250 WORDS)
Highlights
Results of the studies presented here indicate that the molecular size of muscarinic acetylcholine receptors as deduced from covalent antagonist labeling of intact cells varies considerably from one cell type to another, even among parenchymal cells of exocrine glands from the same animal
That the [3H]PrBCM-labeled peaks resolvedon polyacrylamide gels do represent muscarinic receptors or fragments thereof is confirmed by their disappearance from gels when cells were incubated with [3H]PrBCMtogether with an excess of a competing muscarinic antagonist, atropine,QNB, or scopolamine
Another observation of interest in these studies was the presence of atropine-inhibitable [3H]PrBCMbinding to acinar cells abovethe level that could beaccounted for by binding of the radioligand to the receptor
Summary
Characterization of muscarinic acetylcholine recep- Muscarinic acetylcholine receptors are important mediators tors in acinacrells from rat pancreas and lacrimal and of secretory events in mammaliaenxocrine organs such as the parotid glands was achieved by binding of the revers- pancreasand lacrimal and salivaryglands.Occupation of ible muscarinic antagonist [3H]quinuclidinylbenzilate these receptors in these organs by acetylcholine or its syn(QNB)and the specific alkylating reagent [3H]propyl- thetic analoguesleads tocharacteristicalterationsinthe tcbdscwbforheriieoinsurlontlpmddhwszieieniwnrelppcsydguaeeeoltrrrcsadlhoeiilhcftaatahoieigtecdpsledig/irncnnael2ceeaacap1olnsiralm,nled.4lrc,Taa,eu0ehrgcods0labyeeltc,asyalan.silrlaanssaudBnsifercerdsiwoon(osaPnemdmmfnirrridBdebnooaigmpCplsnseriaeMascnen,ttlupguiac)olaladcrayntrnerrioeeiadodsmdisasfiniz-sadwtcepgaclihbeiogclotyayrftehnsnmatsethddct[adoi3eiiiog2tnnHarrne5iyec]sw,Qcpi27tneisi6N0ooptia,nh0nrB-r400pcatifm(,rnu3elniaetgn)pdrs.mgtcamfoHetclbirureaoorseilawmnddlgnuaisebevillemnayescrptstbrreoaseerrrtlticatayusoienceopsrpteehnittssooooofnrsr(pZfetsohoo)hme,frceocacsairabniueencnootpedosltseatahriynacrytlgirchcmdlyeaehyelonpoysingilenutioevhnc(nvIsleeek)uanl,niwldinteaoesrinlfwtetlhlciaunenenxaa.cildnsltTrsrseeda.oaumtdIosealineftesvilditmmaatuCairlcrbagaaenrlrte2noeoae+etslnvvisxfyeeoretocrnnepremnosterlsftl-otein and divalentcation chelation using EDTA Muscarinic this resultsfrom a lackof knowledge concerning themolecular receptors on acini or dispersed cells were covalently characteristics of epithelial muscarinic receptors and of the labeled with 5 nM [‘HIPrBCM, solubilized directly in assemblages of enzymes and ion channels with which they are hotsodiumdodecyl sulfatebuffer,and resolved by functionally associatedin the secretory cell plasmamempolyacrylamide gel electrophoresis. In each of of muscarinic receptors in both epithelial and non- these studies, reducing agents such as mercaptoethanol and epithelial cell types and indicate that the preparation dithiothreitol had noeffect on themolecular weight of labeled utilized can affect the size of the receptor
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