Reactive oxygen species and small-conductance calcium-dependent potassium channels are key mediators of inflammation-induced hypotension and shock

Abstract

Septic shock is associated with life-threatening vasodilation and hypotension. To cause vasodilation, vascular endothelium may release nitric oxide (NO), prostacyclin (PGI2), and the elusive endothelium-derived hyperpolarizing factor (EDHF). Although NO is critical in controlling vascular tone, inhibiting NO in septic shock does not improve outcome, on the contrary, precipitating the search for alternative therapeutic targets. Using a hyperacute tumor necrosis factor (TNF)-induced shock model in mice, we found that shock can develop independently of the known vasodilators NO, cGMP, PGI2, or epoxyeicosatrienoic acids. However, the antioxidant tempol efficiently prevented hypotension, bradycardia, hypothermia, and mortality, indicating the decisive involvement of reactive oxygen species (ROS) in these phenomena. Also, in classical TNF or lipopolysaccharide-induced shock models, tempol protected significantly. Experiments with (cell-permeable) superoxide dismutase or catalase, N-acetylcysteine and apocynin suggest that the ROS-dependent shock depends on intracellular (*)OH radicals. Potassium channels activated by ATP (K(ATP)) or calcium (K(Ca)) are important mediators of vascular relaxation. While NO and PGI2-induced vasodilation involves K(ATP) and large-conductance BK(Ca) channels, small-conductance SK(Ca) channels mediate vasodilation induced by EDHF. Interestingly, also SK(Ca) inhibition completely prevented the ROS-dependent shock. Our data thus indicate that intracellular (*)OH and SK(Ca) channels represent interesting new therapeutic targets for inflammatory shock. Moreover, they may also explain why antioxidants other than tempol fail to provide survival benefit during shock.

Fig. 1

NO and cGMP in zVAD + TNF shock. a NO_x⁻ (NO₂⁻ + NO₃⁻) in serum collected 3 h after PBS or TNF (n above the bars). b cGMP in plasma collected 3 h after PBS or TNF (n above the bars). c cGMP in homogenates from kidneys, collected 3 h after PBS or TNF (n above the bars). ***P < 0.001, **P < 0.01, compared with PBS. d WT, iNOS^−/−, or double iNOS^−/− and eNOS^−/− mice (ixeNOS^{−−/−−}) were injected with TNF (T, open symbols) or zVAD + TNF (zT, filled symbols; n in the legend). ***P < 0.001, *P < 0.05, compared with TNF alone. e WT mice were injected with TNF (open circles, n = 9) or TNF + zVAD (closed circles, n = 9), and the effect of l-NAME 2 h (triangles, n = 5) or 30 min (triangles, n = 5) before TNF or together with TNF (diamonds, n = 8) was tested. *P < 0.05, compared with zVAD + TNF. f Mean arterial pressure in conscious free-moving catheterized mice injected through a catheter with TNF, with or without zVAD i.p. To analyze the role of NO, a group of zVAD + TNF mice was treated with l-NAME 45 min before TNF. n = 5 for each group, plotted is the median response

Fig. 2

ROS and EETs in zVAD + TNF toxicity. a Effect of catalase, PEG-catalase, or tempol on mortality induced by zVAD ± TNF. Plotted is the percent survival of all mice used in three independent experiments; the total number is indicated between brackets in the legend. Sensitization by zVAD corresponds to mortality within 6 h. Mice injected with TNF alone are also plotted for comparison. ***P < 0.0001 compared with zVAD + TNF (black bar). b Effect of the CYP inhibitors ABT, fluconazole, and SKF-525A. Plotted is the percent survival of all mice used in up to four independent experiments; the total number is indicated between brackets in the legend. Differences between zVAD + TNF and SKF-525A + zVAD + TNF are not significant (P > 0.07). c Blood pressure and (d) heart rate were monitored in conscious radiotelemetred mice injected with zVAD ± TNF. Two mice were treated with tempol 45 min before TNF. e, f Effect of tempol, SOD, or PEG-SOD on hypothermia (e) and mortality (f) induced by zVAD + TNF (n = 5), ***P < 0.001 compared with zVAD + TNF. g Tempol protects against TNF shock. Mice were injected i.v. with a lethal dose of TNF alone (n = 6), **P = 0.0012. h Tempol protects against LPS shock. Mice were injected i.v. with a lethal dose of LPS (n = 6), ***P = 0.0006

Fig. 3

Apocynin protects against acute zVAD + TNF shock. a Effect of apocynin on hypothermia induced by zVAD + TNF, *P < 0.05, **P < 0.01, ***P < 0.001 compared with zVAD ± TNF. b Effect of apocynin on zVAD ± TNF mortality, **P = 0.0013 compared with zVAD ± TNF. c, d Effect of apocynin on protection by tempol. *P < 0.05, **P < 0.01, ***P < 0.001 compared with zVAD ± TNF

Fig. 4

The role of K⁺ channels and H₂O₂ in ROS-dependent zVAD + TNF shock. a Effect of different K⁺ channel inhibitors. Plotted is the percent survival of all mice used in up to five independent experiments; total numbers are indicated between brackets in the legend. ***P < 0.0001 compared with zVAD + TNF (black bar). b, c Effect of apamin on hypothermia (b) and mortality (c) induced by zVAD ± TNF in a representative experiment (n in the legend), *P < 0.05, **P = 0.0049, ***P < 0.001 compared with zVAD ± TNF. d, e Mean arterial pressure and HR were monitored in conscious radiotelemetred mice injected with zVAD ± TNF. Three mice were treated with apamin 2 h before TNF, plotted are the non-survivor and one of the two survivors. f Effect of catalase and PEG-catalase on protection by tempol, ***P < 0.001 compared with tempol + zVAD + TNF (diamonds); data shown are from one individual representative experiment