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. 2019 Aug 8;14(8):e0220987.
doi: 10.1371/journal.pone.0220987. eCollection 2019.

The Epinephrine-Induced PGE2 Reduces Na+/K+ ATPase Activity in Caco-2 Cells via PKC, NF-κB and NO

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Free PMC article

The Epinephrine-Induced PGE2 Reduces Na+/K+ ATPase Activity in Caco-2 Cells via PKC, NF-κB and NO

Layla El Moussawi et al. PLoS One. .
Free PMC article

Abstract

We showed previously an epinephrine-induced inhibition of the Na+/K+ ATPase in Caco-2 cells mediated via PGE2. This work is an attempt to further elucidate mediators downstream of PGE2 and involved in the observed inhibitory effect. The activity of the Na+/K+ ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the enzyme. Changes in the protein expression of the Na+/K+ ATPase were investigated by western blot analysis which revealed a significant decrease in the abundance of the ATPase in plasma membranes. Treating the cells with epinephrine or PGE2 in presence of SC19220, a blocker of EP1 receptors abolished completely the effect of the hormone and the prostaglandin while the effect was maintained unaltered in presence of antagonists to all other receptors. Treatment with calphostin C, PTIO, ODQ or KT5823, respective inhibitors of PKC, NO, soluble guanylate cyclase and PKG, abrogated completely the effect of epinephrine and PGE2, suggesting an involvement of these mediators. A significant inhibition of the ATPase was observed when cells were treated with PMA, an activator of PKC or with 8-Br-cGMP, a cell permeable cGMP analogue. PMA did reduce the protein expression of IκB, as shown by western blot analysis, and its effect on the ATPase was not manifested in presence of an inhibitor of NF-κB while that of SNAP, a nitric oxide donor, was not affected. The results infer that NF-κB is downstream PKC and upstream NO. The data support a pathway in which epinephrine induces the production of PGE2 which binds to EP1 receptors and activates PKC and NF-κB leading to NO synthesis. The latter activates soluble guanylate cyclase resulting in cGMP production and activation of PKG which through direct or indirect phosphorylation inhibits the Na+/K+ ATPase by inducing its internalization.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Epinephrine acts via EP1 receptors.
The inhibitory effect of epinephrine (0.5mM, 20 min) disappeared in presence of (A) SC 19220, an EP1 antagonist (100 μM), but still appeared in presence of antagonists to (B) EP2(PF-7981106; 1μM), (C) EP3 (L-798106; 1μM) and (D) EP4 (GW 627368X; 10 μM). Values are means ± SEM of at least 3 observations. Bars not sharing a common letter are considered significantly different from each other at P<0.01.
Fig 2
Fig 2. PGE2 binds to EP1 receptors.
PGE2 (1 nM, 20 min) could not inhibit the Na+/K+ ATPase in presence of an antagonist to (A) EP1 receptors (SC 19220; 100 μM), while its effect appeared unaltered in presence of blocker of (B) EP2 (PF-7981106; 1μM), (C) EP3 (L-798106; 1μM) and (D) EP4 receptors (GW 627368X; 10 μM). Values are means ± SEM of at least 3 observations. Bars not sharing a common letter are considered significantly different from each other at P<0.01.
Fig 3
Fig 3. Epinephrine and PGE2 activate PKC.
The effect of the (A) hormone and the (B) prostaglandin disappeared in presence of calphostin (50 nM), an inhibitor of PKC while (C) PMA, an activator of the kinase, exerted a significant inhibitory effect on the ATPase that was maintained in presence of indomethacin, an inhibitor of COX enzymes. Values are means ± SEM of at least 3 observations. Bars not sharing a common letter are considered significantly different from each other at P<0.01.
Fig 4
Fig 4. Epinephrine and PGE2 induce NO production.
The effect of (A) epinephrine and (B) PGE2 did not appear in cells pre-treated for 20min with PTIO (30μM), a scavenger of NO. (C) SNAP (2μM, 20min) a NO donor caused a significant inhibition of the ATPase. Values are means ± SEM of at least 3 observations. Bars not sharing a common letter are considered significantly different from each other at P<0.01.
Fig 5
Fig 5. PKC activates NF-κB.
(A) PMA (100 nM) could not inhibit the ATPase in cells pre-treated with an inhibitor of NF-κB (15μM, 30 min) and (B) decreased significantly the protein expression of IκB. The effect of (C) SNAP (2μM, 20 min) was not affected by the inhibitor. Values are means ± SEM of at least 3 observations. Bars not sharing a common letter are considered significantly different from each other at P<0.01.
Fig 6
Fig 6. Epinephrine and PGE2 activate guanylate cyclase leading to cGMP production and PKG activation.
Pre-treating the cells with ODQ (3μM, 20 min) abolished the effect of (A) epinephrine and (B) PGE2. (C) 8-Br-cGMP (0.5 mM, 20 min) exerted a significant inhibitory effect on the Na+/K+ ATPase. (D) SNAP (2μM, 20 min) did not inhibit the ATPase when cells were pre-incubate with KT 5823 (1 μM) an inhibitor of PKG. Values are means ± SEM of at least 3 observations. Bars not sharing a common letter are considered significantly different from each other at P<0.01.
Fig 7
Fig 7. PGE2 reduces the protein expression of the Na+/K+ ATPase.
Cells treated with PGE2(1 nM, 20 min) were collected and fractionated by differential centrifugation to isolate plasma membranes. Eight microgram samples from the cell homogenate (A) and from the membrane fraction (B) were used for western blot analysis. PGE2 reduced significantly the expression of the ATPase as indicated by the student t test (P<0.01). The blots are representative of an experiment repeated three times.
Fig 8
Fig 8. Proposed signaling pathway.

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References

    1. Moussawi LE, Chakkour M, Kreydiyyeh SI. Epinephrine modulates Na /K ATPase activity in Caco-2 cells via Src, p38MAPK, ERK and PGE2. Plos One. 2018;13 - PMC - PubMed
    1. Fujii S, Suzuki K, Kawamoto A, Ishibashi F, Nakata T, Murano T, et al. PGE2 is a direct and robust mediator of anion/fluid secretion by human intestinal epithelial cells. Scientific Reports. 2016;6 - PMC - PubMed
    1. Esmann M. [10] ATPase and phosphatase activity of Na, K -ATPase: Molar and specific activity, protein determination. Methods in Enzymology Biomembranes Part P: ATP-Driven Pumps and Related Transport: The Na,K-Pump. 1988;105–15. - PubMed
    1. Taussky H, Shorr E. Microcolorimetric method for the determination of quinic acid and its contents in flue-cured tobacco. Microchemical Journal. 1959;3(4):587.
    1. Alam NA, Kreydiyyeh SI. Signaling pathway involved in the inhibitory effect of FTY720P on the Na /K ATPase in HepG2 cells. Journal of Cell Communication and Signaling. 2017;11(4):309–16. 10.1007/s12079-016-0369-z - DOI - PMC - PubMed

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This work was funded by SK University Research Board. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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