Abstract 1438: Effect of C-type Natriuretic Peptide (CNP) on Endothelial Cell (EC) membrane Potential
CNP is a member of the natriuretic peptide family, expressed in many tissues, including endothelial cells. CNP has been found to promote relaxation in vascular smooth muscle cells (VSMC) and has been suggested to be endothelial derived hyperpolarizing factor (EDHF). EDHF has been shown to hyperpolarize both VSMC and ECs. However, the effect of CNP on EC membrane potential (Em) is unknown. We hypothesized that CNP modulates endothelial cell Em via the activation of K+ channels.
Methods: Rat microvascular EC were cultured on coverslips and loaded with a potentiometric bis-oxanol dye (DiBAC4) (1% change in fluorescent intensity= 1mV). The EC were subsequently exposed to vehicle or treatment in the presence or absence of channel blockers. Changes in fluorescence were measured using time-lapse fluorescence microscopy. In addition, currents were measured using nystatin perforated patch clamp recordings in voltage-clamp mode.
Results: CNP caused significant hyperpolarization within 5mins (control: −4±3 mV; CNP: −31±5 mV). To determine the role of cGMP in the CNP mediated hyperpolarization, 8-Br-cGMP was substituted for CNP. Similar to CNP, 8-Br-cGMP induced hyperpolarization within 5mins (−34±5 mV). Also, pretreatment with the PKG inhibitor, KT5823, completely blocked CNP mediated hyperpolarization. To identify the ion-channel involved, EC were exposed to CNP in the presence of a nonspecific K+ channel blocker, TEA, 2+, or a large-conductance Ca2+ activated K+ (BK) channel blocker, a Kir channel blocker, Ba iberiotoxin. Both TEA and iberiotoxin prevented the hyperpolarizing effects of CNP, while Ba2+ had no effect. Also, removal of extracellular Ca2+, and pretreatment with a nonspecific cation channel blocker, La3+, prevented CNP induced hyperpolarization. Perforated patch clamp studies confirmed an outwardly rectifying current induced by CNP that was blocked in part by iberiotoxin.
Conclusion: 1. CNP causes hyperpolarization of EC via BK channels. 2. CNP induced hyperpolarization is mediated by the NPR-B/particulate guanylyl cyclase pathway. 3. Extracellular Ca2+ entry via non-specific cation channels is required for the effect of CNP on Em.