Modulation of fast sodium current in airway smooth muscle cells by exchange protein directly activated by cAMP

Airway smooth muscle (ASM) cells from mouse bronchus express a fast sodium current mediated by Na(V)1.7. We present evidence that this current is regulated by cAMP. ASM cells were isolated by enzymatic dispersal and studied using the whole cell patch clamp technique at room temperature. A fast sodium current, I-Na, 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 mu M), a beta-adrenergic agonist. Forskolin (1 mu M), an activator of adenylate cyclase, reduced the current by 35%, but 6-MB-cAMP (300 mu M), an activator of protein kinase A (PKA), had no effect. In contrast, 8-pCPT-2-O-Me-cAMP-AM (007-AM, 10 mu M), 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 mu M), an inhibitor of ryanodine receptors, and when cytosolic [Ca2+] was buffered by inclusion of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, Sigma (BAPTA) (50 mu M) in the pipette solution, suggesting that the inhibition of I-Na 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 to 0 mV, but had no effect on the steady-state activation curve. However, the steady-state inactivation V-1/2, 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 I-Na in mouse ASM via Epac, but not PKA