Altered ion channel expression in response to prolonged hypoxia has been demonstrated in different cell types (e.g. Smirnov et al. 1994; Green & Peers, 2001), and probably contributes to remodelling of overall tissue responses to such insults. Here, we describe a preparation designed to examine the effects of chronic hypoxia on K+ channel expression in smooth muscle cells isolated from human internal mammary artery (IMA).
IMA samples were collected from patients undergoing coronary bypass grafting with local ethical permission, cleaned of adventitia, opened longitudinally and cut into segments ca 3-5 mm in length. These were then pinned, lumen uppermost onto Sylgard¿-coated 60 mm Petri dishes using A1 Minuten pins, and immersed in RPMI culture medium supplemented with 30 % fetal calf serum. Tissues were cultured at 37 °C in humidified incubators gassed with either air and 5 % CO2, or a gas mixture of 2.5 % O2, 87.5 % N2 and 5 % CO2. After 18 h, smooth muscle cells were dispersed as described previously (Kamishima et al. 2000), and allowed to adhere to poly-L-lysine-coated coverslips. Whole-cell patch-clamp recordings were made using pipette and extracellular solutions as previously described (Peers & Carpenter, 1998). Currents were evoked by step depolarizations (100 ms, 0.2 Hz) up to +60 mV from a holding potential of -70 mV.
Cell capacitance was significantly smaller (P < 0.05, Student’s unpaired t test) in normoxically cultured cells at 12.4 ± 1.1 pF (mean ± S.E.M., n = 14 cells from 8 patients) compared with 15.9 ± 1.3 pF in hypoxic cells (12 cells from the same 8 patients). K+ currents typically activated at ca -20 mV and increased in amplitude with increasing depolarization. Current density was significantly greater (P < 0.05, ANOVA) in cells cultured normoxically, compared with those maintained under hypoxic conditions. For example, at a test potential of +60 mV, current density was 55.3 ± 17.4 pA pF-1 in control cells, and 21.8 ± 5.8 pA pF-1 in hypoxic cells.
Our results indicate that this preparation allows isolation of viable smooth muscle cells from human tissue maintained in vitro for up to 48 h. Our preliminary results also suggest that K+ channel expression is sensitive to changes in O2 levels in vitro.
This work was supported by the British Heart Foundation, The Wellcome Trust and Pfizer Central Research.
All procedures accord with current local guidelines and the Declaration of Helsinki.