065P Queen Elizabeth II Conference Centre London
BPS Winter Meeting 2010

 

 

Murine tracheal perfusion potential difference: carbachol-induced hyperpolarization in six inbred mouse strains.

Sarah Czarnecki, Henry Danahay, Kevin Coote, Derek Paisley. Novartis Institutes of Biomedical Research, RH12 5AB Horsham/West Sussex, United Kingdom.

 

The Calcium Activated Chloride Channel (CaCC) may provide a compensatory Cl- secretory pathway in CF lung disease. Brady et al (2001) used nasal potential difference (NPD) to demonstrate differences in CFTR-mediated chloride transport in six inbred mouse strains. Our aim was to establish the most suitable strain of mouse in which to assess tracheal epithelial CaCC responses in vivo.

Male mice were used at 20-25g. Following induction of surgical anaesthesia (16mg/ml ketamine/2mg/ml xylazine mixture i.p.), animals were secured supine and tilted nose down. Solutions (all containing 1mM amiloride) were perfused at 25µL/min onto the tracheal lumen via a six input manifold using a Pump 22 syringe pump (Harvard Apparatus, UK). Agar bridge exploring and reference electrodes were placed in the perfusate within the manifold on the trachea, and subcutaneously in the animals flank, respectively. Initially PBS was perfused to give a baseline reading, followed by Cl- free PBS, establishing a local Cl- gradient to promote Cl- secretion. The muscarinic agonist carbachol (10mM) was then perfused (in Cl- free PBS). Data are expressed as absolute or Δ mean TPD ± s.e.mean (mV), and tested for significance using ANOVA with Neuman-Kuells post-hoc test (unless stated otherwise). Tracheal potential difference measurements are summarised in Table 1.

Table 1. Summary of TPD measurements in 6 mouse strains

Mouse strain PBS Baseline TPD
mV ± s.e.mean		 Cl- free ΔTPD
mV ± s.e.mean		 10mM carbachol peak ΔTPD
mV ± s.e.mean		 n
Balb/c -2.4 ± 0.4 -0.1 ± 0.4 -6.7 ± 0.8 15
A/J -1.3 ± 0.7 0.8 ± 0.5 -5.6 ± 1.0 12
C3H -2.0 ± 0.6 -0.8 ± 0.4 -4.8 ± 0.6 13
DBA/2 -2.9 ± 0.3 -0.9 ± 0.5 -4.8 ± 0.7 15
129SV/S2 -1.5 ± 0.2 -0.5 ± 0.4 -4.3 ± 0.5 15
C57Bl/6Jax -2.8 ± 0.5 -0.3 ± 0.3 -3.5 ± 1.9 15

Carbachol induced a significant (P<0.05) decrease in TPD in all strains, however the only strains significantly different to one another were Balb/c and C57Bl/6jax mice (P<0.05). As Balb/c had the largest carbachol mediated increase in TPD, we tested this further to dissect the CaCC response. Perfusion of 20µM of the CFTR inhibitor CFTRinh-172 (Ma et al., 2002) depolarized the Cl- free response (+1.3 ± 0.4mV; n = 9), consistent with a tonic CFTR activation under basal conditions. The subsequent perfusion with carbachol (10mM) induced a significant hyperpolarisation of the TPD by -5.7 ± 0.5mV (P<0.05). The carbachol response was significantly attenuated by perfusion with DIDS (200μM), giving a depolarisation of +2.5 ± 0.5mV (P<0.05 student t-test).

We have shown that Cl- secretion can be measured in the mouse trachea. The detection of a carbachol response in the presence of CFTRinh-172 suggests that this is not CFTR mediated. Additionally, partial inhibition of the carbachol response by DIDS indicates CaCC involvement. These data are consistent with Balb/c being a suitable strain of mouse in which to begin to characterise pharmacological modulators of CaCC function.

 

Brady KG et al. Am J Physiol Lung cell Mol Physiol. 281: L1173-79 (2001)

Ma T et al. J Clin Invest. 110 (11): 1651-8 (2002).