Transient receptor potential (TRP) channels are shown to modulate the expression of membrane proteins by selective endocytosis (Sidhaye et al., Proc Natl Acad Sci USA. 2006). This regulation is relevant to various patho-physiological conditions such as barrier function, edema, and mechanical stress. Therefore, we tested the effect of known TRP channel modulators on NCI-H441 cells, which commonly serve as a model for epithelial cells.

H441 cells were cultivated for two to three days and incubated with the Calcium sensing fluophore Fluo-4-AM ester for 30 min. After replacing the medium with control solution (C-sol in mM: NaCl 140, KCl 5, HEPES 10, MgCl₂ 1, CaCl₂ 2 and Glucose 5) containing 1 mg/ml brilliant black, the cells were placed into a plate reader for quantifying intracellular Ca²⁺ concentration, [Ca²⁺]_i by measuring the fluo-4 fluorescence intensity. Neither capsaicin (CPS) nor, 4a-phorbol 12,13-didecanoate (4a-PDD), both known as activators for TRPV1 and TRPV4 channels respectively, resulted in an increase of [Ca²⁺]_i. Another TRPV1 activator 2-Aminoethoxydiphenyl Borate (2-APB) elicited a transient increase in [Ca²⁺]_i. Surprisingly, the TRPV1 channel blocker capsazepine (CPZ) induced a biphasic increase of [Ca²⁺]_i in the presence of 2-APB. The biphasic response of 2-APB/CPZ consisted of transient and sustained components. Both components showed the same sensitivity to the TRP channel inhibitor SKF96365 but could be distinguished due to their different sensitivities to La³⁺, SB366791 and BCTC. Thus, 2-APB and CPZ activate two different Ca²⁺ entry pathways in H441 cells presumably, partially via activating TRP channels.

In order to address the question, if the above tested TRP channel modulators also affect endocytosis in H441 cells, we incubated H441 cells with the fluophore FM1-43 in the presence of TRP channel modulators. FM1-43 internalised by the cells, was fluorometrically quantified. Neither 2-APB nor CPZ alone induced any FM1-43 uptake. However, when both compounds were applied together, the FM1-43 uptake was approximately twofold increased compared to control cells. This increase was almost completely abolished by preloading cells with the Ca²⁺ chelator BAPTA, but not by thapsigargin. This suggests that the FM1-43 uptake, which is induced by 2-APB/CPZ, depends on Ca²⁺ influx. The 2-APB/CPZ induced FM1-43 uptake was also abolished by TRP channel blockers SKF96365, La³⁺ and SB366791, suggesting that the above identified Ca²⁺ influx triggers the FM1-43 uptake. The FM1-43 uptake was also blocked by Phenylarsine Oxide (PAO) but not by filipine, indomethacine and bafilomycin A1. Thus, the uptake of the fluophore depends on clathrine mediated endocytosis.

We conclude that clathrin mediated endocytosis in H441 cells might be modulated by TRP channel activity.

Supported by the DFG, grant D1402, the FWF, grant P15743, the 6th framework of the EU, Pulmo-Net and Boehringer Ingelheim.