title: Selective restoration of calcium coupling to muscarinic M(3) receptors in contractile cultured airway myocytes.
abstract: We previously demonstrated that after several days of serum deprivation about one-sixth of confluent cultured canine tracheal myocytes acquire an elongated, structurally and functionally contractile phenotype. These myocytes demonstrated significant shortening on ACh exposure. To evaluate the mechanism by which these myocytes acquire responsiveness to ACh, we assessed receptor-Ca(2+) coupling using fura 2-AM fluorescence imaging and muscarinic receptor expression using Western analysis. Cells were grown to confluence in 10% fetal bovine serum and then maintained for 7-13 days in serum-free medium. A fraction of serum-deprived cells exhibited reproducible intracellular Ca(2+) mobilization in response to ACh that was uniformly absent from airway myocytes before serum deprivation. The Ca(2+) response to 10(-4) M ACh was ablated by inositol 1,4,5-trisphosphate (IP(3)) receptor blockade using 10(-6) M xestospongin C but not by removal of extracellular Ca(2+). Also, 10(-7) M atropine or 10(-7) M 4-diphenylacetoxy-N-methylpiperidine completely blocked the response to ACh, but intracellular Ca(2+) mobilization was not ablated by 10(-6) M pirenzepine or 10(-6) M methoctramine. In contrast, 10(-5) M bradykinin (BK) was without effect in these ACh-responsive myocytes. Interestingly, myocytes that did not respond to ACh demonstrated robust increases in intracellular Ca(2+) on exposure to 10(-5) M BK that were blocked by removal of extracellular Ca(2+) and were only modestly affected by IP(3) receptor blockade. Serum deprivation increased the abundance of M(3) receptor protein and of BK(2) receptor protein by two- to threefold in whole cell lysates within 2 days of serum deprivation, whereas M(2) receptor protein fell by >75%. An increase in M(3) receptor abundance and restoration of M(3) receptor-mediated Ca(2+) mobilization occur concomitant with reacquisition of a contractile phenotype during prolonged serum deprivation. These data demonstrate plasticity in muscarinic surface receptor expression and function in a subpopulation of airway myocytes that show mutually exclusive physiological and pharmacological diversity with other cells in the same culture.
authors.affiliation: Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: R W
authors.initials: RW
authors.lastname: Mitchell
authors.forename: A J
authors.initials: AJ
authors.lastname: Halayko
authors.forename: S
authors.initials: S
authors.lastname: Kahraman
authors.forename: J
authors.initials: J
authors.lastname: Solway
authors.forename: M E
authors.initials: ME
authors.lastname: Wylam
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D001798:Blood Proteins / Q000494:pharmacology; D015153:Blotting, Western; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002478:Cells, Cultured; D003959:Diamines / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D016257:Fura-2; D053496:Inositol 1,4,5-Trisphosphate Receptors; D047028:Macrocyclic Compounds; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009130:Muscle, Smooth / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D010276:Parasympatholytics / Q000494:pharmacology; D010641:Phenotype; D010880:Piperidines / Q000494:pharmacology; D010890:Pirenzepine / Q000494:pharmacology; D043587:Receptor, Muscarinic M3; D018002:Receptors, Bradykinin / Q000032:analysis / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000032:analysis / Q000378:metabolism*; D014132:Trachea / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D014665:Vasodilator Agents / Q000494:pharmacology
chemical_list: D001798:Blood Proteins; D015220:Calcium Channels; D003959:Diamines; D005456:Fluorescent Dyes; D053496:Inositol 1,4,5-Trisphosphate Receptors; D047028:Macrocyclic Compounds; D018727:Muscarinic Antagonists; D010080:Oxazoles; D010276:Parasympatholytics; D010880:Piperidines; D043587:Receptor, Muscarinic M3; D018002:Receptors, Bradykinin; D018160:Receptors, Cytoplasmic and Nuclear; D011976:Receptors, Muscarinic; D014665:Vasodilator Agents; C108644:xestospongin A; D010890:Pirenzepine; D001285:Atropine; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; D000109:Acetylcholine; C054938:methoctramine; D001920:Bradykinin; D002118:Calcium; D016257:Fura-2