Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC351

Poster Communications

Properties of a Na+ current in freshly dispersed smooth muscle cells from the rabbit airway

E. Bradley1, T. Webb1, G. P. Sergeant1, M. A. Hollywood1, N. G. McHale1, K. D. Thornbury1

1. Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Ireland.

A fast voltage-dependent Na+ current has been reported in cultured human bronchial smooth muscle cells, but not in freshly dispersed airway smooth muscle cells (ASMC)1,2. We investigated whether a similar current is present in freshly dispersed rabbit ASMC. Rabbits were humanely killed and their lungs removed. Secondary and tertiary airways were removed and cut into 1 mm3 pieces and ASMC isolated using a collagenase/protease mixture3. Cells were superfused with physiological saline at 37°C and studied under voltage clamp (whole cell configuration, CsCl containing pipette solution). Cells were held at -100 mV and depolarized through to +40 mV for 100 ms in 10 mV steps. Transient inward currents were evoked at potentials >--60 mV. These currents activated and inactivated within 5 ms at -40 mV. Changing external Na+ from 130 mM to 13 mM reduced the current at -40 mV from -489±90 pA to -28±18 pA (p<0.01, paired t-test, n=7). When activation curves were constructed, the voltage at half maximal activation (V1/2) was -41±2 mV (n=7). Steady state inactivation was studied by holding at conditioning potentials from -120 mV to -30 mV for 2s prior to test steps to -40 mV. The V1/2 of inactivation was -88±1 mV (n=8). To examine the effect of drugs, cells were hyperpolarized for 2s to -100 mV to remove inactivation and then depolarised to a test potential of -40 mV. Tetrodotoxin (TTX, 300 nM-10 μM) dose-dependently reduced the current (IC50 = 1.4±0.4 μM (n=8), showing that it was relatively insensitive to this toxin, compared to the Na+ current in freshly dispersed lymphatic smooth muscle cells3 and cultured human airway smooth muscle cells1,2. The current was also inhibited by the Na+/Ca2+ exchange blocker KBR-7943 (IC50, 2.5±1.3 μM, n=9) and lidocaine (IC50, 160±37 μM, n=8). The Na+ channel modulator, veratridine (50 μM), reduced the peak current from -535±96 pA to -362±77 pA (p<0.001, n=8), while enhancing the sustained current (at 25 ms) from -53±21 pA to -203 pA (p<0.001). The TTX-sensitivity and voltage-dependence of this current are similar to Na+ currents conducted by channels expressing the NaV1.5 α subunit4. RT-PCR was used to probe for transcripts of Na+ channels in both whole bronchi and groups of 10-20 isolated ASMC. Transcripts for NaV1.2 and NaV1.5 were found in ASMC (5 replications) and those for NaV1.2, NaV1.3, NaV1.4, NaV1.5, NaV1.8 & NaV1.9 in the whole bronchus. These data show that rabbit ASMC express a voltage-gated Na+ current with fast kinetics, which is relatively insensitive to TTX. The evidence suggests that most of this current is conduced by channels expressing the NaV1.5 isoform, although other subtypes may also contribute. More work will be necessary to determine the role of this current in health and disease.

Where applicable, experiments conform with Society ethical requirements