Signaling in TRPV1-induced platelet activating factor (PAF) in human esophageal epithelial cells

Abstract

Transient receptor potential channel, vanilloid subfamily member 1 (TRPV1) receptors were identified in human esophageal squamous epithelial cell line HET-1A by RT-PCR and by Western blot. In fura-2 AM-loaded cells, the TRPV1 agonist capsaicin caused a fourfold cytosolic calcium increase, supporting a role of TRPV1 as a capsaicin-activated cation channel. Capsaicin increased production of platelet activating factor (PAF), an important inflammatory mediator that acts as a chemoattractant and activator of immune cells. The increase was reduced by the p38 MAP kinase (p38) inhibitor SB203580, by the cytosolic phospholipase A2 (cPLA(2)) inhibitor AACOCF3, and by the lyso-PAF acetyltransferase inhibitor sanguinarin, indicating that capsaicin-induced PAF production may be mediated by activation of cPLA(2), p38, and lyso-PAF acetyltransferase. To establish a sequential signaling pathway, we examined the phosphorylation of p38 and cPLA(2) by Western blot. Capsaicin induced phosphorylation of p38 and cPLA(2). Capsaicin-induced p38 phosphorylation was not affected by AACOCF3. Conversely, capsaicin-induced cPLA(2) phosphorylation was blocked by SB203580, indicating that capsaicin-induced PAF production depends on sequential activation of p38 and cPLA(2). To investigate how p38 phosphorylation may result from TRPV1-mediated calcium influx, we examined a possible role of calmodulin kinase (CaM-K). p38 phosphorylation was stimulated by the calcium ionophore A23187 and by capsaicin, and the response to both agonists was reduced by a CaM inhibitor and by CaM-KII inhibitors, indicating that calcium induced activation of CaM and CaM-KII results in P38 phosphorylation. Acetyl-CoA transferase activity increased in response to capsaicin and was inhibited by SB203580, indicating that p38 phosphorylation in turn causes activation of acetyl-CoA transferase to produce PAF. Thus epithelial cells produce PAF in response to TRPV1-mediated calcium elevation.

Fig. 1.

RT-PCR and Western blot data for transient receptor potential channel, vanilloid subfamily member 1 (TRPV1) receptors, in the cells of a human esophageal squamous epithelial cell line HET-1A. RT-PCR primers were derived from the conserved mRNA sequence of humans and other species. Primers for TRPV1 mRNA are shown. A band was recognized as expected (A). The PCR product was sequenced and found to be specific for TRPV1. To confirm the presence of TRPV1 protein in HET-1A cells, Western Blot analysis was performed. Cells were collected, and a 100-kDa band was immunoblotted with a TRPV1 antibody, confirming the presence of TRPV1 receptors in these esophageal epithelial cells. B: Western blots from 3 separate culture wells containing HET-1A cells.

Fig. 2.

Capsaicin-induced cytosolic Ca²⁺ increase in HET-1A cells. Cells were loaded with fura-2 AM 1.25 × 10⁻⁶ M (40 min) and placed in a 5-ml chamber mounted on the stage of an inverted microscope (Carl Zeiss, Germany). The bathing solution was HEPES-buffered solution. After obtaining the basal Ca²⁺ concentration, capsaicin was added to the bathing solution and the final concentration was 10⁻⁵ M. Ca²⁺ measurements were obtained using a modified dual-excitation wavelength imaging system (IonOptix, Milton, MA). The Ca²⁺ concentrations were measured from the ratios of fluorescence elicited by 340-nm excitation to 380-nm excitation. In fura-2 AM-loaded HET-1A cells, capsaicin caused an increase in cytosolic Ca²⁺ levels that occurred over several minutes, peaking at 4 min (*P < 0.05). Values are means ± SE, N = 8.

Fig. 3.

Capsaicin-induced production of platelet activating factor (PAF) in HET-1A cells. The cells were incubated for 24 h with capsaicin (10⁻⁷ M), alone or after 30-min exposure to the p38 MAPK inhibitor SB203580 (10⁻⁵ M), or the cytosolic phospholipase A2 (cPLA₂) inhibitor AACOCF3 (2 × 10⁻⁵ M) or the lyso-PAF acetyl-CoA transferase inhibitor sanguinarin (10⁻⁶ M). Because of the prolonged exposure to capsaicin required to measure PAF, a lower concentration was used than the one used for phosphorylation experiments that occur within 30 min. Capsaicin caused a significant (*P < 0.001) increase in PAF levels, that was significantly reduced by SB203580 (#P < 0.01), by AACOCF3 (#P < 0.01), and by sanguinarin (#P < 0.01), indicating that PAF production depends on activation of p38 MAP kinase, cPLA₂, and lyso-PAF acetyl-CoA transferase. Values are means ± SE for 4 experiments.

Fig. 4.

Capsaicin-induced p38 phosphorylation is mediated through TRPV1 receptors. The cells were incubated for 30 min with capsaicin (5 × 10⁻⁵ M), inducing elevated p38 phosphorylation (*P < 0.05). After 30-min pretreatment with the high-affinity TRPV1 antagonist iodoresiniferatoxin (IRTX) (10⁻⁶ M), p38 phosphorylation significantly decreased (#P < 0.05), confirming involvement of TRPV1 receptors in capsaicin-induced p38 phosphorylation. Values are means ± SE for 3 experiments.

Fig. 5.

Capsaicin-induced cPLA₂ phosphorylation. 30-min incubation with capsaicin (5 × 10⁻⁵ M) caused a significant increase in cPLA₂ phosphorylation (*P < 0.01). The increased cPLA₂ phosphorylation was significantly reduced (#P < 0.01) by 30-min preincubation with the p38 inhibitor SB203580 (10⁻⁵ M), indicating that cPLA₂ phosphorylation depends on activation of p38. Values are means ± SE for 3 experiments.

Fig. 6.

Capsaicin-induced p38 phosphorylation. 30-min incubation with capsaicin (5 × 10⁻⁵ M) caused a significant increase in p38 phosphorylation (*P < 0.01). The increased p38 phosphorylation was not reduced by 30-min preincubation with the cPLA₂ inhibitor AACOCF3 (2 × 10⁻⁵ M), indicating that p38 phosphorylation does not depend on activation of cPLA₂. Values are means ± SE for 3 experiments.

Fig. 7.

A23187-induced p38 phosphorylation. Cells were treated with the calcium ionophore A23187 (3 × 10⁻⁶ M, 30 min) alone or after 1-h pretreatment with the calmodulin kinase (CaM-K) inhibitors KN93 (5 × 10⁻⁶ M ) or STO-609 (10⁻⁵ M), the CaM inhibitor W-7 (10⁻⁵ M), or the calcium chelator BAPTA (4 × 10⁻⁴ M). The calcium ionophore A23187 induced a significant increase in p38 phosphorylation (*P < 0.001) that was abolished by the extracellular calcium chelator BAPTA (#P < 0.001), indicating that a cytosolic calcium elevation induces p38 phosphorylation. The A23187-induced increase in p38 phosphorylation was inhibited by W-7 (#P < 0.001) and by KN93 (#P < 0.001) but not by STO-609, suggesting that p38 phosphorylation results from calcium-mediated activation of CaM and CaM-K. Inhibition by KN93 but not by STO-609 suggests CaM-KII as the kinase responsible for p38 phosphorylation. Values are means ± SE for 3 experiments.

Fig. 8.

Capsaicin-induced p38 phosphorylation. Cells were treated with the capsaicin (5 × 10⁻⁵ M, 30 min ) alone or after 1-h pretreatment with the CaM-K inhibitors KN93 (5 × 10⁻⁶ M) or STO-609 (10⁻⁵ M), the CaM inhibitor W-7 (10⁻⁵ M), or the calcium chelator BAPTA (4 × 10⁻⁴ M) in combination with thapsigargin (10⁻⁷ M). Capsaicin induced a significant increase in p38 phosphorylation (*P < 0.001) that was reduced by the combination of BAPTA and thapsigargin (#P < 0.001), indicating that a cytosolic calcium elevation induces p38 phosphorylation. Capsaicin-induced p38 phosphorylation was inhibited by W-7 (#P < 0.001) and by KN93 (#P < 0.001) but not by STO-609, suggesting that p38 phosphorylation results from calcium-mediated activation of CaM and CaM-K. Inhibition by KN93 but not by STO-609 suggests CaM-KII as the kinase responsible for p38 phosphorylation. Values are means ± SE for 3 experiments.

Fig. 9.

Cells were treated for 45 min with 5 × 10⁻⁵ M capsaicin alone or after 30-min incubation with IRTX (10⁻⁶ M) or with SB203580 (10⁻⁵ M) or with AACOCF3 (2 × 10⁻⁵ M) or with the MAPK/ERK kinase (MEK) inhibitor PD98059 (2 × 10⁻⁵ M). Capsaicin caused activation of lyso-PAF acetyltransferase (*P < 0.001) that was inhibited by the TRPV1 antagonist IRTX (#P < 0.001) and by the p38 inhibitor SB203580 (#P < 0.001). In contrast, the MEK inhibitor PD98059 and the cPLA₂ inhibitor AACOCF3 had no effect on capsaicin-induced lyso-PAF acetyltransferase activation. The data indicate that P38 (but not ERK1/ERK2) is responsible for lyso-PAF acetyltransferase activation and that cPLA₂ does not contribute to activation of the lyso-PAF acetyltransferase enzyme. Values are means ± SE for 3 experiments.

Fig. 10.

Summary of the results of this investigation. Activation of TRPV1 causes influx of calcium (and possibly release from intracellular stores) into the cytoplasm, with binding to CaM and activation of CaM-KII. CaM-KII in turn phosphorylates p38, inducing activation of both cPLA₂ and acetyl-CoA:lyso-PAF acetyltransferase. cPLA₂ removes arachidonic acid (AA) from phospholipids, producing the PAF precursor lyso PAF-acether. Acetyl-CoA:lyso-PAF acetyltransferase then acetylates the lyso-PAF-acether to produce PAF.