title: Zinc inhibits apoptosis and maintains NEP downregulation, induced by ropivacaine, in HaCaT cells.
abstract: Zinc (Zn), a cell-protective metal against various toxic compounds, is the key agent for neutral endopeptidase (NEP) functional structure. NEP is a zinc metalloenzyme which degrades endogenous opioids and is expressed in human keratinocytes (HaCaT). Ropivacaine, a widely used opiate local anaesthetic, exerts cell toxic and apoptotic effects against HaCaT cells. The aim of the present study is to investigate whether zinc modulates the effects of ropivacaine on proliferation, viability, apoptosis and NEP expression in HaCaT cells. To investigate the role of ropivacaine in NEP function, HaCaT cells overexpressing NEP were generated via cell transfection with plasmids carrying NEP cDNA. Ropivacaine's anti-proliferative effect was tested by Neubauer's chamber cell counting, and induction of cell death was demonstrated by trypan blue exclusion assay. Apoptosis due to ropivacaine was tested via DNA fragmentation and poly-ADP-ribose-polymerase (PARP) cleavage. NEP and PARP expression was performed by western blot analysis. Results showed that zinc (15 μΜ) inhibited proliferation and cell death induction by ropivacaine (0.5, 1 and 2 mM) (p < 0.05) as well as apoptosis induced by the drug (0.5 and 1 mM) in HaCaT cells. Ropivacaine (1.0, 2.0 and 5.0 mM) downregulated NEP expression in the presence of zinc (15 μΜ) while NEP overexpression enhanced ropivacaine's apoptotic effect. In conclusion, the abilities of zinc to inhibit the toxic and apoptotic effects of ropivacaine, to maintain NEP downregulation induced by the drug and, consequently, to enhance its anaesthetic result suggest that zinc may have a significant role in pain management and tissue protection.
authors.affiliation: Laboratory of Physiology, Medical School, University of Ioannina, 45110, Ioannina, Greece.
authors.forename: Evangelos
authors.initials: E
authors.lastname: Kontargiris
authors.forename: Athina
authors.initials: A
authors.lastname: Vadalouka
authors.forename: Vasilios
authors.initials: V
authors.lastname: Ragos
authors.forename: Vasiliki
authors.initials: V
authors.lastname: Kalfakakou
mesh_terms: D000577:Amides / Q000009:adverse effects / Q000494:pharmacology*; D000701:Analgesics, Opioid / Q000009:adverse effects / Q000494:pharmacology*; D000779:Anesthetics, Local / Q000009:adverse effects / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D019587:Dietary Supplements; D015536:Down-Regulation / Q000187:drug effects*; D004351:Drug Resistance; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D015260:Neprilysin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism; D009994:Osmolar Concentration; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D020011:Protective Agents / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism; D000077212:Ropivacaine; D015032:Zinc / Q000378:metabolism*; D017967:Zinc Compounds / Q000378:metabolism
chemical_list: D000577:Amides; D000701:Analgesics, Opioid; D000779:Anesthetics, Local; D009566:Nitrates; D020011:Protective Agents; D011994:Recombinant Proteins; D017967:Zinc Compounds; D000077212:Ropivacaine; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D015260:Neprilysin; C042103:zinc nitrate; D015032:Zinc