Limits...
Signaling function of Na,K-ATPase induced by ouabain against LPS as an inflammation model in hippocampus.

Kinoshita PF, Yshii LM, Vasconcelos AR, Orellana AM, Lima Lde S, Davel AP, Rossoni LV, Kawamoto EM, Scavone C - J Neuroinflammation (2014)

Bottom Line: The involvement of the inflammatory transcription factor NF-κB in the OUA effect was indicated by its prevention of LPS-induced nuclear translocation of the NF-κB subunit, RELA (p65), as well as the decreased cytosol levels of the NF-κB inhibitor, IKB, in the hippocampus.Our results suggest that a low dose of OUA has an important anti-inflammatory effect in the rat hippocampus.This effect was associated with decreased GFAP induction by LPS in the dentate gyrus, a brain area linked to adult neurogenesis.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neuropharmacology Laboratory, Department of Pharmacology, Institute of Biomedical Science, University of São Paulo, 05508-900, São Paulo, Brazil. paula.f.kinoshita@gmail.com.

ABSTRACT

Background: Ouabain (OUA) is a newly recognized hormone that is synthesized in the adrenal cortex and hypothalamus. Low doses of OUA can activate a signaling pathway by interaction with Na,K-ATPase, which is protective against a number of insults. OUA has central and peripheral anti-inflammatory effects. Lipopolysaccharide (LPS), via toll-like receptor 4 activation, is a widely used model to induce systemic inflammation. This study used a low OUA dose to evaluate its effects on inflammation induced by LPS injection in rats.

Methods: Adult male Wistar rats received acute intraperitoneal (ip) OUA (1.8 μg/kg) or saline 20 minutes before LPS (200 μg/kg, ip) or saline injection. Some of the animals had their femoral artery catheterized in order to assess arterial blood pressure values before and after OUA administration. Na,K-ATPase activity, cytokine mRNA levels, apoptosis-related proteins, NF-κB activation brain-derived neurotrophic factor BDNF, corticosterone and TNF-α levels were measured.

Results: OUA pretreatment decreased mRNA levels of the pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS) and IL-1β, which are activated by LPS in the hippocampus, but with no effect on serum measures of these factors. None of these OUA effects were linked to Na,K-ATPase activity. The involvement of the inflammatory transcription factor NF-κB in the OUA effect was indicated by its prevention of LPS-induced nuclear translocation of the NF-κB subunit, RELA (p65), as well as the decreased cytosol levels of the NF-κB inhibitor, IKB, in the hippocampus. OUA pretreatment reversed the LPS-induced glial fibrillary acidic protein (GFAP) activation and associated inflammation in the dentate gyrus. OUA also prevented LPS-induced increases in the hippocampal Bax/Bcl2 ratio suggesting an anti-apoptotic action in the brain.

Conclusion: Our results suggest that a low dose of OUA has an important anti-inflammatory effect in the rat hippocampus. This effect was associated with decreased GFAP induction by LPS in the dentate gyrus, a brain area linked to adult neurogenesis.

Show MeSH
Pretreatment of ouabain (OUA) (1.8 μg/kg) in lipopolysaccharide (LPS) (200 μg/kg) inflammation model induced no changes on Na,K-ATPase (NKA) activity. The total NKA activity was tested by adding 10 μg of the particulate fraction from the rat hippocampus obtained 2 hours after OUA (1.8 μg/kg, ip) or saline pretreatment + LPS (200 μg/kg, ip). α2/3-NKA activity was measured by subtracting the activity obtained with 3 μM OUA (it inhibits just these 2 isoforms) from total NKA activity. To determine the α1 subunit-associated NKA activity, the total NKA activity was subtracted from the activity obtained with 3 mM OUA (Mg-ATPase). Results are expressed as nmol/mg.min protein (mean + SEM) from five individual experiments. One-way ANOVA followed by Newman-Keuls post-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4307894&req=5

Fig1: Pretreatment of ouabain (OUA) (1.8 μg/kg) in lipopolysaccharide (LPS) (200 μg/kg) inflammation model induced no changes on Na,K-ATPase (NKA) activity. The total NKA activity was tested by adding 10 μg of the particulate fraction from the rat hippocampus obtained 2 hours after OUA (1.8 μg/kg, ip) or saline pretreatment + LPS (200 μg/kg, ip). α2/3-NKA activity was measured by subtracting the activity obtained with 3 μM OUA (it inhibits just these 2 isoforms) from total NKA activity. To determine the α1 subunit-associated NKA activity, the total NKA activity was subtracted from the activity obtained with 3 mM OUA (Mg-ATPase). Results are expressed as nmol/mg.min protein (mean + SEM) from five individual experiments. One-way ANOVA followed by Newman-Keuls post-test.

Mentions: One-hour OUA administration (1.8 μg/kg; ip) did not change mean arterial pressure (Before: 113 ± 3.16 versus After OUA: 112 ± 4.71 mmHg; P > 0.05) or heart rate (Before: 323 ± 21.35 versus After OUA: 331 ± 15.46 bpm; P > 0.05). In addition, this OUA dose does not change total ATPase, Mg-ATPase, α1−NKA and α2/3−NKA activities (Figure 1) in the rat hippocampus, suggesting the absence of OUA effects on blood pressure and central NKA activity.Figure 1


Signaling function of Na,K-ATPase induced by ouabain against LPS as an inflammation model in hippocampus.

Kinoshita PF, Yshii LM, Vasconcelos AR, Orellana AM, Lima Lde S, Davel AP, Rossoni LV, Kawamoto EM, Scavone C - J Neuroinflammation (2014)

Pretreatment of ouabain (OUA) (1.8 μg/kg) in lipopolysaccharide (LPS) (200 μg/kg) inflammation model induced no changes on Na,K-ATPase (NKA) activity. The total NKA activity was tested by adding 10 μg of the particulate fraction from the rat hippocampus obtained 2 hours after OUA (1.8 μg/kg, ip) or saline pretreatment + LPS (200 μg/kg, ip). α2/3-NKA activity was measured by subtracting the activity obtained with 3 μM OUA (it inhibits just these 2 isoforms) from total NKA activity. To determine the α1 subunit-associated NKA activity, the total NKA activity was subtracted from the activity obtained with 3 mM OUA (Mg-ATPase). Results are expressed as nmol/mg.min protein (mean + SEM) from five individual experiments. One-way ANOVA followed by Newman-Keuls post-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4307894&req=5

Fig1: Pretreatment of ouabain (OUA) (1.8 μg/kg) in lipopolysaccharide (LPS) (200 μg/kg) inflammation model induced no changes on Na,K-ATPase (NKA) activity. The total NKA activity was tested by adding 10 μg of the particulate fraction from the rat hippocampus obtained 2 hours after OUA (1.8 μg/kg, ip) or saline pretreatment + LPS (200 μg/kg, ip). α2/3-NKA activity was measured by subtracting the activity obtained with 3 μM OUA (it inhibits just these 2 isoforms) from total NKA activity. To determine the α1 subunit-associated NKA activity, the total NKA activity was subtracted from the activity obtained with 3 mM OUA (Mg-ATPase). Results are expressed as nmol/mg.min protein (mean + SEM) from five individual experiments. One-way ANOVA followed by Newman-Keuls post-test.
Mentions: One-hour OUA administration (1.8 μg/kg; ip) did not change mean arterial pressure (Before: 113 ± 3.16 versus After OUA: 112 ± 4.71 mmHg; P > 0.05) or heart rate (Before: 323 ± 21.35 versus After OUA: 331 ± 15.46 bpm; P > 0.05). In addition, this OUA dose does not change total ATPase, Mg-ATPase, α1−NKA and α2/3−NKA activities (Figure 1) in the rat hippocampus, suggesting the absence of OUA effects on blood pressure and central NKA activity.Figure 1

Bottom Line: The involvement of the inflammatory transcription factor NF-κB in the OUA effect was indicated by its prevention of LPS-induced nuclear translocation of the NF-κB subunit, RELA (p65), as well as the decreased cytosol levels of the NF-κB inhibitor, IKB, in the hippocampus.Our results suggest that a low dose of OUA has an important anti-inflammatory effect in the rat hippocampus.This effect was associated with decreased GFAP induction by LPS in the dentate gyrus, a brain area linked to adult neurogenesis.

View Article: PubMed Central - PubMed

Affiliation: Molecular Neuropharmacology Laboratory, Department of Pharmacology, Institute of Biomedical Science, University of São Paulo, 05508-900, São Paulo, Brazil. paula.f.kinoshita@gmail.com.

ABSTRACT

Background: Ouabain (OUA) is a newly recognized hormone that is synthesized in the adrenal cortex and hypothalamus. Low doses of OUA can activate a signaling pathway by interaction with Na,K-ATPase, which is protective against a number of insults. OUA has central and peripheral anti-inflammatory effects. Lipopolysaccharide (LPS), via toll-like receptor 4 activation, is a widely used model to induce systemic inflammation. This study used a low OUA dose to evaluate its effects on inflammation induced by LPS injection in rats.

Methods: Adult male Wistar rats received acute intraperitoneal (ip) OUA (1.8 μg/kg) or saline 20 minutes before LPS (200 μg/kg, ip) or saline injection. Some of the animals had their femoral artery catheterized in order to assess arterial blood pressure values before and after OUA administration. Na,K-ATPase activity, cytokine mRNA levels, apoptosis-related proteins, NF-κB activation brain-derived neurotrophic factor BDNF, corticosterone and TNF-α levels were measured.

Results: OUA pretreatment decreased mRNA levels of the pro-inflammatory cytokines, inducible nitric oxide synthase (iNOS) and IL-1β, which are activated by LPS in the hippocampus, but with no effect on serum measures of these factors. None of these OUA effects were linked to Na,K-ATPase activity. The involvement of the inflammatory transcription factor NF-κB in the OUA effect was indicated by its prevention of LPS-induced nuclear translocation of the NF-κB subunit, RELA (p65), as well as the decreased cytosol levels of the NF-κB inhibitor, IKB, in the hippocampus. OUA pretreatment reversed the LPS-induced glial fibrillary acidic protein (GFAP) activation and associated inflammation in the dentate gyrus. OUA also prevented LPS-induced increases in the hippocampal Bax/Bcl2 ratio suggesting an anti-apoptotic action in the brain.

Conclusion: Our results suggest that a low dose of OUA has an important anti-inflammatory effect in the rat hippocampus. This effect was associated with decreased GFAP induction by LPS in the dentate gyrus, a brain area linked to adult neurogenesis.

Show MeSH