Tesi Dottorato (dott Ciro Coletta): Phospholipase A2: A New Target For Hydrogen Sulfide Activity

PHOSPHOLIPASE A2: A NEW TARGET FOR HYDROGEN SULFIDE ACTIVITY CHAPTER1 Hydrogen sulphide (H2S), a novel gaseous transmitter, is considered a physiological regulator of vascular homeostasis. Recent evidence suggests H2S is an endothelium hyperpolarizing factor (EDHF) candidate. To address this issue, we evaluated the vascular effect of sodium hydrogen sulphide (NaHS), an H2S donor on the rat mesenteric arterial bed. A NaHS concentration-response curve was performed on pre-constricted mesenteric arterial bed. In order to asses the contribute of EDHF, we performed a pharmacological dissection using indomethacin, NG-nitro-L-arginine methyl ester (L-NAME) or apamin and charibdotoxin, as cyclooxygenase, nitric oxide synthase and calcium-dependent potassium channel inhibitors, respectively. In another set of experiments we used 4-(4-Octadecylphenyl)-4-oxobutenoic acid, baicalein or proadifen as PLA2, lipoxygenase and P450 cytochrome inhibitors respectively. Finally, in an immunofluorescence study we evaluated the involvement of PLA2 in the NaHS-induced effect on the mesenteric artery. NaHS promoted a dual vascular effect, i.e., vasoconstriction and vasodilation. L-NAME administration did not affect NaHS-mediated vasodilation nor vasoconstriction, whereas apamin and charibdotoxin significantly inhibited NaHS-induced relaxation. Pre-treatment with PLA2 inhibitor abolished both the contracting and the relaxant effect, whereas P450 cytochrome blocker, significantly reduced NaHS-mediated relaxation. The immunofluorescence study showed that NaHS caused a migration of cPLA2 close to the nucleus, which implies activation of this enzyme. Our data indicate that H2S could activate PLA2 that in turn releases arachidonic acid leading, initially, to vasoconstriction followed by vasodilation mediated by cytochrome P450-derived metabolites. As EDHF has been presumed to be a cytochrome P450 derivative of the arachidonic acid, our results suggest H2S as a candidate for EDHF. CHAPTER 2 Background and purpose: Hydrogen sulphide (H2S), considered as a novel gas transmitter, is produced endogenously in mammalian tissue from L-cysteine by two enzymes cystathionine β- synthase (CBS) and cystathionine γ-lyase (CSE). Recently, it has been reported that H2S contributes to the local and systemic inflammation in several experimental animal models. We conducted this study to investigate on the signaling involved in H2S induced inflammation. Experimental approach: L-cysteine or sodium hydrogen sulphide (NaHS) were injected in the mouse hind paw and oedema formation was evaluated for 60 min. In order to investigate on the H2S–induced oedema formation, we used different pharmacological tools: serotonin and histamine receptor antagonists, KATP channels or arachidonic acid cascade inhibitors. Prostaglandin levels were determined in hind paw exudates by radioimmunoassay. Finally, the histological analysis was performed on L-cisteine injected hind paw. Key results: Both NaHS and L-cysteine caused oedema formation characterized by a fast onset which peaked at 30 minutes. This oedematogenic action was not associated with histamine, serotonin release or KATP channel activation. Conversely, the oedema formation was significantly inhibited by ciclooxygenase and phospholipase A2 selective inhibitors. Moreover, the prostaglandin levels were significantly increased in exudates of hind paw injected with NaHS or L-cysteine. The histological study clearly showed an inflammatory state with a loss of tissue organization. Conclusion and implication: We could assume that the phospholipase A2 and prostaglandins production are involved in H2S pro-inflammatory effect in the mouse hind paw. The present study could contribute to understand the role of L-cysteine/H2S pathway in inflammatory disease.