Tumor Necrosis Factor Alpha Inhibits L-Type Ca(2+) Channels in Sensitized Guinea Pig Airway Smooth Muscle through ERK 1/2 Pathway

Abstract

Tumor necrosis factor alpha (TNF-α) is a potent proinflammatory cytokine that plays a significant role in the pathogenesis of asthma by inducing hyperresponsiveness and airway remodeling. TNF-α diminishes the L-type voltage dependent Ca(2+) channel (L-VDCC) current in cardiac myocytes, an observation that seems paradoxical. In guinea pig sensitized tracheas KCl responses were lower than in control tissues. Serum from sensitized animals (Ser-S) induced the same phenomenon. In tracheal myocytes from nonsensitized (NS) and sensitized (S) guinea pigs, an L-VDCC current (ICa) was observed and diminished by Ser-S. The same decrease was detected in NS myocytes incubated with TNF-α, pointing out that this cytokine might be present in Ser-S. We observed that a small-molecule inhibitor of TNF-α (SMI-TNF) and a TNF-α receptor 1 (TNFR1) antagonist (WP9QY) reversed ICa decrease induced by Ser-S in NS myocytes, confirming the former hypothesis. U0126 (a blocker of ERK 1/2 kinase) also reverted the decrease in ICa. Neither cycloheximide (a protein synthesis inhibitor) nor actinomycin D (a transcription inhibitor) showed any effect on the TNF-α-induced ICa reduction. We found that CaV1.2 and CaV1.3 mRNA and proteins were expressed in tracheal myocytes and that sensitization did not modify them. In cardiac myocytes, ERK 1/2 phosphorylates two sites of the L-VDCC, augmenting or decreasing ICa; we postulate that, in guinea pig tracheal smooth muscle, TNF-α diminishes ICa probably by phosphorylating the L-VDCC site that reduces its activity through the ERK1/2 MAP kinase pathway.

Figure 1

Sensitization diminished KCl-induced contraction in guinea pig tracheas. Responses to 3 consecutive stimulations of 60 mM KCl were higher in tracheal rings from nonsensitized guinea pigs (NS, n = 14) than in tissues from sensitized (S, n = 17) animals (inset). (a) The third KCl response from NS tissues was considered as 100%, and statistical difference was found when compared with the S group. (b) Nonsensitized tracheas, incubated with different percentage (V/V) concentrations of sensitized guinea pigs serum (Ser-S, n = 5, each), showed decreased responses to KCl (20–60 mM), reaching statistical significance only with the highest Ser-S concentration tested. ^∗ p < 0.05, ^∗∗ p < 0.01 compared with control group. Bars and symbols represent mean ± SEM.

Figure 2

Voltage dependent L-type Ca²⁺ currents were diminished by sensitized guinea pig serum and by TNF-α in guinea pig tracheal myocytes. (a) Similar Ba²⁺ currents, equivalent to Ca²⁺ currents, induced by 10 mV increments were observed in myocytes from nonsensitized (NS) animals grown with fetal bovine serum (FBS) or serum from nonsensitized guinea pigs (Ser-NS). These currents were significantly diminished when myocytes from NS and sensitized (S) animals were grown with serum from sensitized guinea pigs (Ser-S, 10%). This phenomenon was not seen when myocytes from S were grown with FBS (n = 9 for each group). (b) Ba²⁺ currents in myocytes from NS grown with FBS showed a decrease when TNF-α was added. These responses were concentration dependent and only the highest (1000 μg/L) produced a significant reduction of this current (n = 5 for each group). Insets in each figure represent original recordings. ^∗ p < 0.05, ^∗∗ p < 0.01, and ^† p < 0.01 when compared with NS + FBS group. Symbols represent mean ± SEM.

Figure 3

TNF-α diminishes the L-type Ca²⁺ currents through activation of the TNF-α receptor 1 (TNFR1) and MAP kinase signaling pathway in guinea pig tracheal myocytes. (a) Myocytes from nonsensitized animals (NS) grown with fetal bovine serum (FBS) showed an inward Ba²⁺ current. This current was reduced when NS myocytes were grown with serum from sensitized guinea pig (Ser-S, 10%). This decrease was abolished when the small-molecule inhibitor of TNF-α (SMI-TNF, n = 7), which impedes this cytokine from binding to its receptor, was incubated during cell growth. (b) The Ba²⁺ current diminution induced by Ser-S incubation in NS myocytes was also reversed, in a concentration dependent manner, by the TNFR1 antagonist (WP9QY, n = 9). (c) The Ba²⁺ current decrease induced by Ser-S was abolished when myocytes were incubated with an inhibitor of ERK 1/2 kinase (U0126, n = 6). Insets in each figure represent original recordings. ^∗ p < 0.05, ^∗∗ p < 0.01, ^† p < 0.01 when compared with NS + Ser-S group. Symbols represent mean ± SEM.

Figure 4

The Ba²⁺ current reduction induced by TNF-α is not mediated by a synthetic pathway activation in guinea pig tracheal myocytes. Ba²⁺ current evoked by step depolarization from −60 to 50 mV in tracheal cells from nonsensitized guinea pigs added with fetal bovine serum (NS + FBS, n = 7) were significantly diminished when myocytes were grown with TNF-α (n = 7). Neither actinomycin D (Act D, n = 6) nor cycloheximide (Cyclohex, n = 9) addition during myocyte growth altered TNF-α induced effect on the Ba²⁺ current. Inset represents original recordings. ^∗ p < 0.05, ^∗∗ p < 0.01, when compared with TNF-α group. Symbols represent mean ± SEM.

Figure 5

Detection of mRNA for L-VDCC subunits in guinea pig tracheal smooth muscle, as revealed by RT-PCR. (a) In airway smooth muscle, the PCR products at 470 and 459 bp length correspond to Ca_V1.2 and Ca_V1.3 cDNA, respectively. In this tissue, Ca_V1.1 and Ca_V1.4 were not found. Positive controls for these subunits were skeletal muscle (SKM, ~500 bp) and retina (~200 bp). Lane at the left corresponds to 1 Kb Plus DNA Ladder. (b) Representative PCR blots for Ca_V1.2 and Ca_V1.3 from nonsensitized (NS, n = 3) and sensitized (S, n = 4) smooth muscles. The first column in each blot corresponds to a negative control without template. The lower panel displays constitutive cDNA of GAPDH. (c) Densitometry data analysis for mRNA from Ca_V1.2 and Ca_V1.3 subunits showing no statistical significance between NS and S. Bars correspond to mean ± SEM.

Figure 6

Immunofluorescence for Ca_V1.2 in nonsensitized and sensitized guinea pig tracheal smooth muscle. The first column shows immunoreactivity for Ca_V1.2 (stained green) in nonsensitized (a) and sensitized tissues (e); notice that Ca_V1.2 is located in the airway smooth muscle (SM) and epithelium (EPI, pointed by arrow); blocking peptide completely eliminated the fluorescence (i). The second and the third columns illustrate smooth muscle α-actin (stained red; (b), (f), (j)) and cell nuclei (DAPI, stained blue; (c), (g), (k)). The last column depicts merged images of the former three columns ((d), (h), (l)). In these merged images, Ca_V1.2 is seen to be colocalized with α-actin (stained yellow) on the smooth muscle.

Figure 7

Immunofluorescence for Ca_V1.3 in nonsensitized and sensitized guinea pig tracheal smooth muscle. The first column shows immunoreactivity for Ca_V1.3 (stained green) in nonsensitized (a) and sensitized tissues (e); notice that Ca_V1.3 is located in the airway smooth muscle (SM) and epithelium (EPI, pointed by arrows); blocking peptide completely eliminated the fluorescence (i). The second and the third columns illustrate smooth muscle α-actin (stained red; (b), (f), (j)) and cell nuclei (DAPI, stained blue; (c), (g), (k)). The last column depicts merged images of the former three columns ((d), (h), (l)). In these merged images, Ca_V1.3 is seen to be colocalized with α-actin (stained yellow) on the smooth muscle.

Figure 8

Schematic representation of the proposed mode of action of tumor necrosis factor α (TNF-α) on the L-type voltage dependent calcium channel (L-VDCC) of the guinea pig airway smooth muscle. TNF-α whether exogenous or present in sensitized guinea pig serum (Ser-S) activates its receptor 1 (TNFR1). Afterwards, it promotes synthetic signaling pathways: it activates receptor interacting protein (RIP), NF-κB-inducing kinase (NIK), and IκB kinases (IκKs) that phosphorylate NFκB inhibitors (IκBs) activating nuclear factor κB (NFκB) and through TNF receptor-associated factor (TRAF), map kinase kinase (MEK) and extracellular signal-regulated kinase (ERK 1/2), p38 MAPK, or c-Jun N-terminal kinase (JNK). We demonstrated that a synthetic pathway was not responsible for the diminution in the Ca²⁺ current (ICa, see Figure 4). Nevertheless, ERK 1/2 might be directly phosphorylating serine⁴⁹⁶  (S⁴⁹⁶) on the β ₂ subunit of the L-VDCC, favoring a downregulation of the ICa. Inset illustrates ICa in tracheal myocytes and contraction in tracheal rings from guinea pigs. NS implies nonsensitized tissues or cells and this indicates the absence of exogenous TNF-α or serum from Ser-S. S illustrates original recordings from sensitized tissues or cells in the presence of Ser-S; notice that both ICa and contraction are diminished. Other subunits of the L-VDCC: α ₁, α ₂, γ, δ. SR: sarcoplasmic reticulum.