Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, PC193
K+ channel identification in isolated smooth muscle cells of human placental chorionic plate arteries
M. F. Brereton1, S. L. Greenwood1, M. Wareing1
1. Maternal and Fetal Health Research Centre, The University of Manchester, Manchester, United Kingdom.
Appropriate control of fetoplacental blood flow is essential for adequate fetal growth and successful pregnancy. K+ channels expressed in smooth muscle cells (SMCs) regulate blood flow by modulating vascular tone. Pharmacological studies implicate voltage-gated (Kv), Ca2+-activated (KCa), and ATP-sensitive (KATP) channels in constriction and relaxation of human placental chorionic plate arteries [1-3] but the identity and localisation of these channels to SMCs is unknown. Here we use a model of freshly isolated chorionic plate artery SMCs to determine K+ channel expression and function. Arteries were dissected from placentas collected at term following normal pregnancy and SMCs isolated by enzyme digestion (papain 30mg/ml, DTT 200mg/ml, collagenase type 1A 20mg/ml). Expression of representative Kv, KCa, and KATP channel isoforms and markers of SMC phenotype was evaluated by immunocytochemistry. Whole-cell patch clamp was used to assess K+ currents, initially targeting Kv channels (5mM [K+]e, 140mM [K+]i, low [Ca2+]i, 0mM [ATP]i). Cells were clamped at -60mV and depolarised from -120mV to +90mV for 100ms to record current-voltage relationships. Kv channel function was assessed by extracellular application of 4-aminopyridine (4-AP; 5mM). SMCs showed positive staining for α-actin and myosin heavy chain-2. Variable expression was evident for h-caldesmon and non-muscle myosin heavy chain-B, markers of contractile and synthetic phenotype respectively. SMCs expressed Kv1.5, BKCa and KATP α-subunit proteins. Stable recordings (n=17 cells, N=8 placentas) displayed outward currents (IK) positive to -40mV. Normalised current-voltage relationships were qualitatively classified according to activation kinetics and current density (1) rapid activation, current density <100pA/pF (n=5) (2) rapid activation, current density >100pA/pF (n=2) (3) time-dependent activation, current density <100pA/pF (n=8) (4) time-dependent activation, current density >100pA/pF (n=2). 4-AP reversibly inhibited IK by 70±8% at +90mV (mean±SE; n=6, N=5); currents were significantly reduced at potentials positive to +50mV (p<0.05; two-way ANOVA, Bonferroni post-hoc test). Freshly isolated SMCs are a useful model to study K+ channel expression and function in chorionic plate arteries. The four K+ current profiles identified may relate to a range of SMC phenotypes given variable h-caldesmon and non-muscle myosin heavy chain-B expression observed in the present study and previous reports of SMCs with contractile and synthetic characteristics in native arteries . The contribution of 4-AP-sensitive channels to whole-cell current suggests the functional expression of Kv channel isoforms. Variable K+ currents and phenotypic profiles indicate a heterogeneous population of SMCs in chorionic plate arteries.
Where applicable, experiments conform with Society ethical requirements