A non-inactivating potassium conductance determines the resting membrane potential of pulmonary artery smooth muscle cells (Evans et al. 1996). The two-pore domain potassium channel TASK-1 may encode this conductance, as it is similar in pharmacology and oxygen sensitivity (Gurney, 2002). In the present study RT-PCR was used to identify the presence of TASK-1 in rabbit pulmonary arterial myocytes.
Animals were humanely killed with pentobarbitone (80 mg kg -1, I.V.) and the main, branch and intrapulmonary arteries were excised. Smooth muscle cells were dissociated as previously described. A general primer pair which recognises multiple TASK channel isoforms identified transcripts in suspensions of pulmonary arteries (n = 3). TASK-1 transcripts were identified using eight different primer pairs (n = 3 each), recognising different parts of the sequence. PCR products were sequenced and confirmed to be TASK-1.
Immunohistochemistry was used to verify the presence of TASK-1 in mouse lung. Lungs were removed from mice which had been humanely killed by cervical dislocation, fixed in ice-cold 4 % paraformaldehyde in phosphate buffered saline for 15 min, dehydrated in sucrose under vacuum, and frozen in liquid N2. Antibodies raised against the C-terminus (Alomone Labs, n = 5) and N-terminus (Autogen Bioclear UK Ltd, n = 4) of human TASK-1 were used. Examination of cryosections by both epi-fluorescence and confocal microscopy revealed strong TASK-1 immunoreactivity in smooth muscle cells of pulmonary arteries. Immunoreactivity was also seen in alveolar cells and the epithelium of the bronchioles. Staining patterns were comparable with both antibodies. The antibody raised against the C-terminus of human TASK-1 also revealed TASK-1 immunoreactivity in smooth muscle cells dispersed from rabbit pulmonary artery.
In conclusion, we have demonstrated that smooth muscle cells from the pulmonary artery stain positively for TASK-1 protein and that they express transcripts for TASK, consistent with the proposed role for TASK-1 in regulating the membrane potential of pulmonary artery myocytes.
This work was supported by the British Heart Foundation and BBSRC.