Capsaicinoids Regulate Airway Anion Transporters through Rho Kinase– and Cyclic AMP–Dependent Mechanisms

Abstract

To investigate the effects of capsaicinoids on airway anion transporters, we recorded and analyzed transepithelial currents in human airway epithelial Calu-3 cells. Application of capsaicin (100 μM) attenuated vectorial anion transport, estimated as short-circuit currents (I(SC)), before and after stimulation by forskolin (10 μM) with concomitant reduction of cytosolic cyclic AMP (cAMP) levels. The capsaicin-induced inhibition of I(SC) was also observed in the response to 8-bromo-cAMP (1 mM, a cell-permeable cAMP analog) and 3-isobutyl-1-methylxanthine (1 mM, an inhibitor of phosphodiesterases). The capsaicin-induced inhibition of I(SC) was attributed to suppression of bumetanide (an inhibitor of the basolateral Na(+)-K(+)-2 Cl(-) cotransporter 1)- and 4,4'-dinitrostilbene-2,2'-disulfonic acid (an inhibitor of basolateral HCO(3)(-)-dependent anion transporters)-sensitive components, which reflect anion uptake via basolateral cAMP-dependent anion transporters. In contrast, capsaicin potentiated apical Cl(-) conductance, which reflects conductivity through the cystic fibrosis transmembrane conductance regulator, a cAMP-regulated Cl(-) channel. All these paradoxical effects of capsaicin were mimicked by capsazepine. Forskolin application also increased phosphorylated myosin phosphatase target subunit 1, and the phosphorylation was prevented by capsaicin and capsazepine, suggesting that these capsaicinoids assume aspects of Rho kinase inhibitors. We also found that the increments in apical Cl(-) conductance were caused by conventional Rho kinase inhibitors, Y-27632 (20 μM) and HA-1077 (20 μM), with selective inhibition of basolateral Na(+)-K(+)-2 Cl(-) cotransporter 1. Collectively, capsaicinoids inhibit cAMP-mediated anion transport through down-regulation of basolateral anion uptake, paradoxically accompanied by up-regulation of apical cystic fibrosis transmembrane conductance regulator-mediated anion conductance. The latter is mediated by inhibition of Rho-kinase, which is believed to interact with actin cytoskeleton.

Capsaicin induces NKCC1 internalization and inhibits chloride secretion in colonic epithelial cells independently of TRPV1

“…Upon addition of nystatin to the basolateral compartment, the apical Cl Ϫ conductance was unmasked showing that capsaicin substantially stimulated apical Cl Ϫ conductance (Fig. 5C, P Ͻ 0.001 unpaired t-test) as reported by others (1,26). On the other hand, capsaicin did not enhance or block the forskolin-stimulated apical chloride current compared with untreated cells (P ϭ 0.4, unpaired t-test).…”

“…Similar behavior of capsazepine was described in Calu-3 cells. In this cell model capsaicin and capsazepine blocked the cAMP-stimulated anion secretion (26). Using TRPV1 agonists also suggests that TRPV1 is not involved in the inhibition of the FSK-I sc .…”

“…In Calu-3 cells, both capsaicin and capsazepine blocked the FSK-I sc via a Rho-kinase-dependent pathway (26). In guinea pig ileal longitudinal smooth muscle, capsaicin induced a capsazepine-insensitive relaxation that was 4-aminopyridine and high KCl-sensitive (17).…”

“…In the colon potassium conductance sustains chloride secretion (35,39), and in other preparations capsaicin was shown to inhibit potassium conductance (26,44). When tested in T84 cells, capsaicin did not block the basolateral potassium conductance.…”

“…Most studies related to TRPV1 focus on its neuronal function, but recent studies have shown the presence of TRPV1 in various epithelia including gastrointestinal epithelial cells, where its role remains elusive (2,6,7,24,30,51). In combination with the findings that capsaicin/TRPV1 affects chloride homeostasis (2,3,26,29), we hypothesized that capsaicin/TRPV1 may affect chloride secretion in the colon by a direct effect on epithelial cells responsible for chloride secretion independent of its effects on enteric neurons.…”