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Contraction of pulmonary artery smooth muscle by KCNQ channel blockers Voltage-dependent K+ channels play an important role in regulating the tone in pulmonary artery smooth muscle, because their inhibition leads to membrane depolarization and activation of voltage-dependent Ca2+ channels, thereby causing Ca2+ influx. A class of voltage-dependent K+ channels encoded by KCNQ genes has gained increasing attention, due to mutations in family members being associated with specific diseases (Robbins 2001). The expression of KCNQ channels in murine portal vein myocytes was recently reported, suggesting a possible functional role in vasculature (Ohya et al., 2003 ). The aim of this study was to determine if KCNQ isoforms are expressed in pulmonary artery smooth muscle and if so whether they contribute to the regulation of vascular tone. Male BALB/c mice (4-5 weeks old), Sprague-Dawley rats (250-300g) and New Zealand white rabbits (2-3 kg) were killed in accordance with schedule1 of Animals (Scientific Procedures) Act 1986. The heart and lungs were removed en bloc, the intrapulmonary arteries were dissected from mice (100-200 µm), rats (300-400 µm) and rabbits (400 µm) and mounted on a wire myograph for isometric tension studies. An RNeasy Midi Kit was used to isolate RNA from rat pulmonary arteries, veins, airways, lungs, heart and brain, preserved with RNA later. RNA was reverse transcribed to cDNA, which was amplified using gene-specific primers designed against mouse KCNQ1, KCNQ2, KCNQ3, KCNQ4 and KCNQ5, following an established PCR protocol (Osipenko et al., 2000).Data are expressed as means ± S.E.M of n animals and values were considered statistically significant when p<0.05. In rat and mouse, but not rabbit pulmonary artery, KCNQ channel blockers produced constriction with EC50 values of 0.5 ± 0.3 µM (n=4) for linopirdine and 0.07 ± 0.07 µM (n=8) for XE991 in rat, and 0.3 ± 0.1 µM (n=9) for linopirdine and 0.3 ± 0.1 µM (n=9) for XE991 in mouse. These values are close to those reported for block of recombinant KCNQ channels (Robbins, 2001). In rat, endothelium removal did not significantly affect the contractile response to either drug. The maximum contraction, measured as % response to 50 mM KCl at 10 µM linopirdine or 1 µM XE991 was, 82 ± 12% and 69 ± 13 % (n=5), respectively, in rat, 54 ± 12 % and 69 ± 12 % (n=9), respectively, in mouse. Moreover, in rat, 1 µM nifedipine blocked the constrictor responses to linopirdine and XE991 by 99.8 ± 0.08 % and 98 ± 1 % respectively. They were blocked by 99.9 ± 0.07 % and 99.8 ± 0.7 % respectively in Ca2+-free medium. RT- PCR confirmed the presence of mRNAs for KCNQ subtypes in rat pulmonary arteries, namely KCNQ1, KCNQ4 and KCNQ5. Taken together these results suggest a functional role for KCNQ channels in regulating voltage-dependent Ca2+ influx and contraction in pulmonary artery smooth muscle cells, although species variation is apparent. Ohya S et al., (2003) Circ. Res. 1016-1023. |
