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-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.

Regulation of store-operated Ca2+entry by CD38 in human airway smooth muscle

“…Six hours after initiation of transfection, DMEM containing 10% FBS was added to the transfection medium. After an additional 48 h, the medium was replaced with serum-free medium to elicit an ACh-responsive phenotype (19,32). For treatment with TNF␣ (20 ng/ml), the cytokine was introduced followed by 24 h of serum deprivation and continued for an additional 24 h. Nonspecific effects of CD38si were evaluated using Western blots for TRPC3 using previously described techniques (58).…”

β-Dystroglycan binds caveolin-1 in smooth muscle: a functional role in caveolae distribution and Ca2+ release

“…Real-time quantification of cytosolic Ca 2+ in cultured ASM cells was performed using the Ca 2+ -sensitive ratiometric fluorescent dye Fura-2 AM, as described previously (Gosens et al, 2007b;Mitchell et al, 2000). All measurements were carried out using myocytes grown on glass coverslips or chamber slides.…”

Airway Smooth Muscle Growth in Asthma: Proliferation, Hypertrophy, and Migration

“…With regard to studies in cells, stimulation of postconfluent cultures with either serum or growth factors has been shown to markedly repress the expression of contractile proteins such as a-smooth muscle actin and MLCK (36)(37)(38)(39). Conversely, serumdeprived cells demonstrate increased contractile protein expression.…”