title: Spilanthol from Acmella Oleracea Lowers the Intracellular Levels of cAMP Impairing NKCC2 Phosphorylation and Water Channel AQP2 Membrane Expression in Mouse Kidney.
abstract: Acmella oleracea is well recognized in Brazilian traditional medicine as diuretic, although few scientific data have been published to support this effect. Aim of this study was to determine the molecular effect of Acmella oleracea extract and its main alkylamide spilanthol on two major processes involved in the urine concentrating mechanism: Na-K-2Cl symporter (NKCC2) activity in the thick ascending limb and water channel aquaporin 2 accumulation at the apical plasma membrane of collecting duct cells. Phosphorylation of NKCC2 was evaluated as index of its activation by Western blotting. Rate of aquaporin 2 apical expression was analyzed by confocal laser microscopy. Spilanthol-induced intracellular signalling events were dissected by video-imaging experiments. Exposure to spilanthol reduced the basal phosphorylation level of NKCC2 both in freshly isolated mouse kidney slices and in NKCC2-expresing HEK293 cells. In addition, exposure to spilanthol strongly reduced both desmopressin and low Cl--dependent increase in NKCC2 phosphorylation in mouse kidney slices and NKCC2-expressing HEK293 cells, respectively. Similarly, spilanthol reduced both desmopressin- and forskolin-stimulated aquaporin 2 accumulation at the apical plasma membrane of collecting duct in mouse kidney slice and MCD4 cells, respectively. Of note, when orally administered, spilanthol induced a significant increase in both urine output and salt urinary excretion associated with a markedly reduced urine osmolality compared with control mice. Finally, at cellular level, spilanthol rapidly reduced or reversed basal and agonist-increased cAMP levels through a mechanism involving increases in intracellular [Ca2+]. In conclusion, spilanthol-induced inhibition of cAMP production negatively modulates urine-concentrating mechanisms thus holding great promise for its use as diuretic.
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari "Aldo Moro", 70126, Bari, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Gerbino
authors.affiliation: Department of Sciences, University of Basilicata, Viale dell'Ateneo Lucano 10, 85100, Potenza, Italy.
authors.forename: Giorgia
authors.initials: G
authors.lastname: Schena
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari "Aldo Moro", 70126, Bari, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Milano
authors.affiliation: Department of Sciences, University of Basilicata, Viale dell'Ateneo Lucano 10, 85100, Potenza, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Milella
authors.affiliation: Department of Food Technology, Federal Rural University of Rio de Janeiro, BR 465 Km 07, Seropédica, Rio de Janeiro, Brazil.
authors.forename: Alan Franco
authors.initials: AF
authors.lastname: Barbosa
authors.affiliation: Department of Sciences, University of Basilicata, Viale dell'Ateneo Lucano 10, 85100, Potenza, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Armentano
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari "Aldo Moro", 70126, Bari, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Procino
authors.affiliation: Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari "Aldo Moro", 70126, Bari, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Svelto
authors.affiliation: Department of Sciences, University of Basilicata, Viale dell'Ateneo Lucano 10, 85100, Potenza, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Carmosino
mesh_terms: D000577:Amides / Q000302:isolation & purification / Q000494:pharmacology*; D000818:Animals; D051399:Aquaporin 2 / Q000378:metabolism*; D019659:Asteraceae / Q000737:chemistry; D001938:Brazil; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D004232:Diuretics; D015536:Down-Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008519:Medicine, Traditional; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation / Q000187:drug effects; D028321:Plant Preparations / Q000302:isolation & purification / Q000494:pharmacology; D053284:Polyunsaturated Alkamides; D064466:Solute Carrier Family 12, Member 1 / Q000378:metabolism*
chemical_list: D000577:Amides; D051399:Aquaporin 2; D004232:Diuretics; C489347:N-isobutyl-2E-decenamide; D028321:Plant Preparations; D053284:Polyunsaturated Alkamides; C578715:Slc12a1 protein, mouse; D064466:Solute Carrier Family 12, Member 1; D000242:Cyclic AMP