Selective restoration of calcium coupling to muscarinic M3 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-Ca2+ 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 Ca2+ mobilization in response to ACh that was uniformly absent from airway myocytes before serum deprivation. The Ca2+ response to 10− 4 M ACh was ablated by inositol 1,4,5-trisphosphate (IP3) receptor blockade using 10− 6 M xestospongin C but not by removal of extracellular Ca2+. Also, 10− 7 M atropine or 10− 7 M 4-diphenylacetoxy- N-methylpiperidine completely blocked the response to ACh, but intracellular Ca2+ 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 Ca2+ on exposure to 10− 5 M BK that were blocked by removal of extracellular Ca2+ and were only modestly affected by IP3 receptor blockade. Serum deprivation increased the abundance of M3 receptor protein and of BK2 receptor protein by two- to threefold in whole cell lysates within 2 days of serum deprivation, whereas M2receptor protein fell by >75%. An increase in M3receptor abundance and restoration of M3 receptor-mediated Ca2+ 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.