Inhibition of pacemaker activity in interstitial cells of Cajal by LPS via NF-κB and MAP kinase

Figure 1 Effects of AH6809 or NG-Nitro-L-arginine Methyl Ester on lipopolysaccharide-induced action in interstitial cells of Cajal. A: Pacemaker currents of interstitial cells of Cajal (ICCs) at a holding potential of -70 mV in control condition; B: Pacemaker currents of ICCs incubated at 37 °C with 200 μg/mL of lipopolysaccharide (LPS) for 12 h; C: Pacemaker currents in ICCs pretreated with AH6809 (10 μmol/L) for 2 h prior to LPS incubation; D: Pacemaker currents in ICCs pretreated with NG-Nitro-L-arginine Methyl Ester (10 μmol/L) for 2 h prior to LPS incubation. LPS: Lipopolysaccharide; L-NAME: NG-Nitro-L-arginine Methyl Ester.

Figure 2 Expression of TLR4, iNOS and COX-2 in c-Kit positive cells. A: Double labeling of TLR4- and c-Kit-like immunoreactivity in cultured interstitial cells of Cajal (ICCs). Green (c-Kit) and red (TLR4) result in the mixed color yellow, indicating the colocalization of both peptides; B: Double labeling of iNOS and c-Kit-like immunoreactivity in ICCs. Green (c-Kit) and red (iNOS) result in the mixed color yellow, indicating the colocalization of both peptides; C: Double labeling of COX-2 and c-Kit-like immunoreactivity in ICCs. Green (c-Kit) and red (COX-2) result in the mixed color yellow, indicating the colocalization of both peptides (bar = 20 μm).

Figure 3 Effects of catalase on lipopolysaccharide-induced action in interstitial cells of Cajal. A: Pacemaker currents of interstitial cells of Cajal (ICCs) incubated at 37 °C with 200 μg/mL of lipopolysaccharide (LPS) for 12 h; B: Pacemaker currents in ICCs pretreated with catalase (3000 unit/mL) for 2 h prior to LPS incubation. The effects of catalase on the LPS-induced action in ICCs are summarized in C and D. Bars represent mean ± SE values (n = 4 per group).

Figure 4 Effects of SN-50 or actinomycin D on lipopolysaccharide-induced action in interstitial cells of Cajal. A: Pacemaker currents of interstitial cells of Cajal (ICCs) incubated at 37 °C with 200 μg/mL of lipopolysaccharide (LPS) for 12 h at a holding potential of -70 mV; B: Pacemaker currents in ICCs pretreated with SN-50 (10 μmol/L) for 2 h prior to LPS incubation; C: Pacemaker currents in ICCs pretreated with actinomycin D (10 μmol/L) for 2 h prior to LPS incubation. The effects of SN-50 or actinomycin D on the LPS-induced action in ICCs are summarized in D and E. Bars represent mean ± SE values (n = 5 per group). ^aP < 0.05 vs LPS alone treatment. SN: SN-50, AD: Actinomycin D.

Figure 5 Effects of Mitogen-activated protein kinases inhibitors on lipopolysaccharide-induced action in interstitial cells of Cajal. A: Pacemaker currents of interstitial cells of Cajal (ICCs) incubated at 37  °C with 200 μg/mL of lipopolysaccharide (LPS) for 12 h at a holding potential of -70 mV; B: Pacemaker currents after pretreatment with PD 98059 (10 μmol/L) for 2 h prior to LPS incubation; C: Pacemaker currents after pretreatment with SB 203580 (10 μmol/L) for 2 h prior to LPS incubation; D: Pacemaker currents after pretreatment with c-Jun N-terminal kinase (JNK) inhibitor II (10 μmol/L) for 2 h prior to LPS incubation. The effects of mitogen-activated protein kinases inhibitors on the LPS-induced action are summarized in E and F. Bars represent mean ± SE values (n = 7 per group). ^aP < 0.05 vs LPS.

Figure 6 Effects of mitogen-activated protein kinases inhibitors on prostaglandin E2-induced action in interstitial cells of Cajal. A: Pacemaker currents of interstitial cells of Cajal (ICCs) treated with 5 μmol/L prostaglandin E2 (PGE₂) at a holding potential of -70 mV; B: Pacemaker currents after pretreatment with PD 98059 (10 μmol/L) prior to PGE₂ treatment; C: Pacemaker currents after pretreatment with SB 203580 (10 μmol/L) prior to PGE₂ treatment. The effects of mitogen-activated protein kinases inhibitors on the PGE₂-induced action are summarized in D and E. Bars represent mean ± SE values (n = 4 per group). ^aP < 0.05 vs PGE₂. Con: Control, PD: PD 98059, SB: SB203580.

Figure 7 Effects of mitogen-activated protein kinases inhibitors on (±)-S-nitroso-N-acetylpenicillamine-induced action in interstitial cells of Cajal. A: Pacemaker currents of interstitial cells of Cajal (ICCs) treated with 100 μmol/L SNAP at a holding potential of -70 mV; B: Pacemaker currents after pretreatment with PD 98059 (10 μmol/L) prior to SNAP treatment; C: Pacemaker currents after pretreatment with SB 203580 (10 μmol/L) prior to SNAP treatment; D: Pacemaker currents after pretreatment with L-NAME (10 μmol/L) prior to prostaglandin E2 (PGE₂) (5 μmol/L) treatment. The effects of mitogen-activated protein kinases inhibitors on the (±)-S-nitroso-N-acetylpenicillamine-induced action are summarized in E and F. Bars represent mean ± SE values (n = 5 per group). Con: Control, PD: PD 98059, SB: SB203580; SNAP: S-nitroso-N-acetylpenicillamine; L-NAME: NG-Nitro-L-arginine Methyl Ester.