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Receptor-operated and voltage-operated calcium entry mechansisms in agonist-evoked contraction of mouse small airways: potential role of TRPC6 In airway smooth muscle, extracellular Ca2+ influx through L-type voltage-operated Ca2+ channels (L-VOCCs) accounts for only part of the agonist-evoked extracellular Ca2+ entry in these cells, the remainder of which is mediated by receptor- and store-operated Ca2+ channels (Snetkov et al., 2001; Sweeney et al., 2002). TRPC6 is a receptor-operated cation channel (Boulay et al., 1997; Hofmann et al., 1999) that is present in human airway smooth muscle (Corteling et al., 2004) and highly expressed in mouse lung (Boulay et al., 1997), and is proposed to play a role in airway smooth muscle function (Cloutier et al., 2003). To assess the functional significance of TRPC6 in the regulation of airway smooth muscle tone, agonist-evoked contractions of mouse small airways were characterised in male wild-type and TRPC6- knockout mice (Deltagen Inc., San Carlos, CA) using wire myography. Mice were killed by exposure to a slowly rising concentration of CO2 before removal of the right lung. Third order airways (internal diameter 700 – 900 μm, length 1 – 2 mm) from wild-type and TRPC6-knockout mice (25 – 35 g) were dissected free of parenchyma and mounted in a Mulvany-Halpern wire myograph containing normal Krebs buffer gassed with 95% O2/5% CO2 and maintained at 37 °C (pH 7.4) . Following a 45 minute equilibration period, an active-tension length curve to the application of K+ (75 mM) was obtained for each airway segment and used to determine the degree of stretch that would be applied to study optimal responses to contractile agonists. Data are expressed as mean ± standard error of the mean. Statistical analysis was performed using Student’s unpaired t-test and a P value of < 0.05 was considered significant. The cumulative application of carbachol (10 nM – 10 μM) and the thromboxane A 2-mimetic, U46619 (0.1 nM – 1 μM) to isolated small airways from wild-type and TRPC6-knockout mice, evoked concentration-dependent contractions of these tissues. However, airways from the knockout mice demonstrated an augmented maximal contraction to carbachol (10 μM, 153.4 ± 10.5% and 189.3 ± 13.5% of contraction to 75 mM K+, wild type and knockout respectively, P < 0.05, n = 10) and a leftward shift of the concentration-response curve to U46619 compared with the wild-type (pD2 values 7.6 ± 0.1 and 8.3 ± 0.1, wild-type and knockout respectively, P < 0.05, n = 4 - 5). The magnitude of the response to 75 mM K+ did not differ between wild-type and knockout airways (3.0 and 3.0 mN respectively, P > 0.05, n = 3). In the presence of the selective L-VOCC blocker nifedipine (1 μM), the leftward shift of the concentration-response curve to U46619 was abolished. In contrast, the augmented maximal contraction to carbachol was unaffected by nifedipine. These data suggest that in small airways of TRPC6-deficient mice, there are agonist-specific increases in the functional contribution made by other Ca 2+ channels in contraction of these tissues. In particular, these findings suggest an increased functional contribution by L-VOCCs in U46619-evoked contraction.
Boulay et al., (1997) J. Biol. Chem. 272 : 47 29672 - 29680. |
