Bothrops snake myotoxins induce a large efflux of ATP and potassium with spreading of cell damage and pain

Abstract

Myotoxins play a major role in the pathogenesis of the envenomations caused by snake bites in large parts of the world where this is a very relevant public health problem. We show here that two myotoxins that are major constituents of the venom of Bothrops asper, a deadly snake present in Latin America, induce the release of large amounts of K + and ATP from skeletal muscle. We also show that the released ATP amplifies the effect of the myotoxins, acting as a "danger signal," which spreads and causes further damage by acting on purinergic receptors. In addition, the release of ATP and K + well accounts for the pain reaction characteristic of these envenomations. As Bothrops asper myotoxins are representative of a large family of snake myotoxins with phospholipase A 2 structure, these findings are expected to be of general significance for snake bite envenomation. Moreover, they suggest potential therapeutic approaches for limiting the extent of muscle tissue damage based on antipurinergic drugs. muscle damage | phospholipase A2 | C2C12 cells

The adjuvant MF59 induces ATP release from muscle that potentiates response to vaccination

“…PO 4 , and 11 mM glucose at pH 7.4) at 37°C. The ATP released in the buffer was determined with the ATPlite luciferase assay (Perkin-Elmer) as previously described (8).…”

“…injection, we posited that a high local concentration of adjuvant is generated in a confined portion of the muscle and that the first cell membrane they come in contact with is the sarcolemma. Because we found that the muscle injection of membrane-interacting snake phospholipase A2 myotoxin induces the release of ATP, which is contained in large amounts inside muscle fibers (7,8), we decided to evaluate the possibility that other putative membrane-interacting agents such as the major adjuvants mentioned earlier might similarly induce ATP release. This possibility would be particularly relevant in the context of adjuvanticity, as ATP is a "danger signal" acting on a variety of purinergic P2 receptors and, as such, is a strong modulator of immune responses (9)(10)(11).…”

Lys49 myotoxin from the Brazilian lancehead pit viper elicits pain through regulated ATP release

“…BomoTx belongs to a group of secreted phospholipase A2 (sPLA2)-like proteins that have the conserved PLA2 fold but lack enzymatic activity, and is most closely related to so-called Lys49 myotoxins found in snake species within the Crotalinae subfamily (9). These toxins are devoid of phospholipase activity due to key enzymatic site mutation of Asp49 to Lys49, but promote release of ATP from myotubes through an as-yet uncharacterized mechanism (10)(11)(12). Similarly, we show that BomoTx lacks phospholipase activity and excites a cohort of sensory neurons through a mechanism involving ATP release and activation of P2X 2 and P2X 3 purinergic receptors.…”

Cellular Mechanisms of Action of Snake Phospholipase A2 Toxins

“…Although the precise binding sites and/or targets of myotoxins have not been identified so far, the common belief is that myotoxic PLA2s affect the plasma membrane integrity, with consequent rapid influx of calcium ions that triggers a series of degenerative events (Montecucco et al 2008). In addition, different molecules released by damaged cells may act as danger signals, thus contributing to extend the damage to the surrounding tissues (Cintra-Francischinelli et al 2010;Zornetta et al 2012). The putative internalization of group II PLA2s and their possible interaction with intracellular targets have not been yet explored.…”

“…In both kinds of cells, these toxins induce a calcium release from intracellular stores, followed by massive calcium entry from the extracellular medium only in myotubes and by a rapid cell lysis (Cintra-Francischinelli et al 2009). These toxins also cause the release of K + and ATP (Cintra-Francischinelli et al 2010). ATP release has been studied in details also in RAW274.7 and J744 macrophage cell lines intoxicated with the K49 PLA2-like myotoxin II of Bothrops asper (Mt-II).…”