Modulation of fast sodium current in airway smooth muscle cells by Exchange Protein Directly Activated by cAMP (Epac)

Airway smooth muscle cells (ASM) from mouse bronchus express a fast sodium current mediated by NaV1.7. We present evidence that this current is regulated by cAMP. ASM were isolated by enzymatic dispersal and studied using the whole-cell patch clamp technique at room temperature. A fast sodium current, INa, was observed on holding cells under voltage clamp at -100 mV and stepping to -20 mV. This current was reduced in a concentration-dependent manner by denopamine (10 and 30 mM), a b-adrenergic agonist. Forskolin (1 mM), an activator of adenylate cyclase, reduced the current by 35%, but 6-MB-cAMP (300 mM), an activator of protein kinase A (PKA), had no effect. In contrast, 8-pCPT-2-O-Me-cAMP-AM (007-AM, 10 mM), an activator of Exchange Protein Directly Activated by cAMP (Epac), reduced the current by 48%. The inhibitory effect of 007-AM was still observed in the presence of dantrolene (10 mM), an inhibitor of ryanodine receptors, and when cytosolic [Ca2+] was buffered by inclusion of BAPTA (50 mM) in the pipette solution, suggesting that the inhibition of INa was not due to Ca2+-release from intracellular stores. When 007-AM was tested on the current-voltage relationship, it reduced the current at potentials from -30 - 0 mV, but had no effect on the steady state activation curve. However, the steady state inactivation V1/2, the voltage causing inactivation of 50% of the current, was shifted in the negative direction from -76.6 mV to -89.7 mV. These findings suggest that cAMP regulates INa in mouse ASM via Epac, but not PKA.