Effects of a cardiotoxin from Naja naja kaouthia venom on skeletal muscle: Involvement of calcium-induced calcium release, sodium ion currents and phospholipases A2 and C

doi:10.1016/0041-0101(91)90005-C

Toxicon

Volume 29, Issue 12, 1991, Pages 1489-1500

https://doi.org/10.1016/0041-0101(91)90005-C Get rights and content

Abstract

Snake venom cardiotoxin (CTX) fractions induce contractures of skeletal muscle and hemolysis of red blood cells. The fractions also contain trace amounts of venom-derived phospholipase A₂ (PLA₂) contamination and activate tissue phospholipase C (PLC) activity. The present study examines the mechanisms of action of a CTX fraction from Naja naja kaouthia venom in skeletal muscle. Sphingosine competitively antagonized CTX-induced red blood cell hemolysis, but not skeletal muscle contractures. CTX rapidly lowered the threshold for Ca²⁺-induced Ca²⁺ release in heavy sarcoplasmic reticulum fractions, as monitored with arsenazo III. There was also a slower time-dependent reduction of Na⁺ currents, as assessed by whole cell patch-clamp techniques. The CTX fractions elevated levels of free fatty acids and diacylglycerol for 2 hr in primary cultures of human skeletal muscle by a combined action of venom-derived PLA₂ contamination in the fraction and activation of endogenous PLC activity. The activation of tissue PLC activity could be readily distinguished from the contribution of the venom PLA₂ by p-bromophenacyl bromide treatment of CTX fractions. The mechanism of action involved in contractures of skeletal muscle appears to be related to the immediate and specific effect of CTX (Ca²⁺ release by the sarcoplasmic reticulum), while the mechanisms involved in hemolysis of red blood cells and decreased Na⁺ currents in skeletal muscle most likely relate to long-term effects on lipid metabolism.

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These atypical toxins were less studied in depth compared to the classical S-NTX and L-NTX; nonetheless they have been shown to be able to interfere with neuromuscular transmission through interaction with nicotinic receptors, and perhaps other molecular targets yet to be investigated (Mordvintsev et al., 2009). At higher venom concentrations (3 and 5 μg/ml), the observable rapid neuromuscular blockade was accompanied by muscle contracture which reflected the action of cytotoxins (cobra cardiotoxins) in the venoms that caused the release of calcium ions from the sarcoplasmic reticula of skeletal muscle (Fletcher et al., 1991). While the lethal action of cobra venoms is mainly neurotoxic, local tissue necrosis occurs in almost all cobra bites and is attributed to the action of cytotoxins (Reid, 1964; WHO, 2010a).
The Southeast Asian monocled cobras (Naja kaouthia) exhibit geographical variations in their venom proteomes, especially on the composition of neurotoxins. This study compared the neuromuscular depressant activity of the venoms of N. kaouthia from Malaysia (NK-M), Thailand (NK-T) and Vietnam (NK-V), and the neutralization of neurotoxicity by a monospecific antivenom. On chick biventer cervicis nerve-muscle preparation, all venoms abolished the indirect twitches, with NK-T venom being the most potent (shortest t₉₀, time to 90% twitch inhibition), followed by NK-V and NK-M. Acetylcholine and carbachol failed to reverse the blockade, indicating irreversible/pseudo-irreversible post-synaptic neuromuscular blockade. KCl restored the twitches variably (NK-M preparation being the least responsive), consistent with different degree of muscle damage. The findings support that NK-T venom has the most abundant curarimimetic alpha-neurotoxins, while NK-M venom contains more tissue-damaging cytotoxins. Pre-incubation of tissue with N. kaouthia monovalent antivenom (NKMAV) prevented venom-induced twitch depression, with the NK-T preparation needing the largest antivenom dose. NKMAV added after the onset of neuromuscular depression could only halt the inhibitory progression but failed to restore full contraction. The findings highlight the urgency of early antivenom administration to sequester as much circulating neurotoxins as possible, thereby hastening toxin elimination from the circulation. In envenomed mice, NKMAV administered upon the first neurological sign neutralized the neurotoxic effect, with the slowest full recovery noticed in the NK-T group. This is consistent with the high abundance of neurotoxins in the NK-T venom, implying that a larger amount or repeated dosing of NKMAV may be required in NK-T envenomation.
Snake venomics of monocled cobra (Naja kaouthia) and investigation of human IgG response against venom toxins
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The most prominent component is the long α-neurotoxin α-cobratoxin (Richard et al., 2013), constituting about 25% of the venom (Kulkeaw et al., 2009) and belonging to the three-finger toxin family, of which N. kaouthia venom has several other members (Kulkeaw et al., 2007). Other proteins that have been studied include phospholipases A2 (PLA2s) (Joubert and Taljaard, 1980a; Reali et al., 2003; Doley and Mukherjee, 2003), l-amino acid oxidases (Tan and Swaminathan, 1992; Sakurai et al., 2001), cardiotoxins (Joubert and Taljaard, 1980b; Fletcher et al., 1991; Debnath et al., 2010; Jamunaa et al., 2012), and nerve growth factors (Kukhtina et al., 2001). However, a proteomic analysis of this venom that integrates an estimation of relative protein abundances together with a detailed screening of the toxicity of its various components, is pending.
The venom proteome of the monocled cobra, Naja kaouthia, from Thailand, was characterized by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses, yielding 38 different proteins that were either identified or assigned to families. Estimation of relative protein abundances revealed that venom is dominated by three-finger toxins (77.5%; including 24.3% cytotoxins and 53.2% neurotoxins) and phospholipases A₂ (13.5%). It also contains lower proportions of components belonging to nerve growth factor, ohanin/vespryn, cysteine-rich secretory protein, C-type lectin/lectin-like, nucleotidase, phosphodiesterase, metalloproteinase, l-amino acid oxidase, cobra venom factor, and cytidyltransferase protein families. Small amounts of three nucleosides were also evidenced: adenosine, guanosine, and inosine. The most relevant lethal components, categorized by means of a ‘toxicity score’, were α-neurotoxins, followed by cytotoxins/cardiotoxins. IgGs isolated from a person who had repeatedly self-immunized with a variety of snake venoms were immunoprofiled by ELISA against all venom fractions. Stronger responses against larger toxins, but lower against the most critical α-neurotoxins were obtained. As expected, no neutralization potential against N. kaouthia venom was therefore detected. Combined, our results display a high level of venom complexity, unveil the most relevant toxins to be neutralized, and provide prospects of discovering human IgGs with toxin neutralizing abilities through use of phage display screening.
Inhibition of Naja naja venom enzymes by the methanolic extract of Leucas aspera and its chemical profile by GC-MS
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L. aspera methanolic extract inhibited the hemolytic activity of the venom, at a ratio of 1:80 w/w (Fig. 4). Hemolytic activity is another distinct feature of cobra venoms greatly induced by multicomponents including metalloproteases, PLA2, and more specifically, cardiotoxins and cytotoxins of venom [26,27]. Though, L. aspera did not show inhibition on venom PLA2, it completely protected the hRBC from direct hemolytic activity of N. naja venom.
The present investigation was aimed at evaluating the anti-ophidian properties of ethnomedicinal herb Leucas aspera against Indian cobra, Naja naja venom enzymes.
Methanolic extract of Leucas aspera was evaluated, in vitro, for its ability to inhibit the major enzyme activities of Naja naja venom including protease, phospholipase A₂, hyaluronidase and hemolytic factors. The type of phytochemicals present in the extract was analyzed. Also, the major phytoconstituents in the extract was determined by gas chromatography–mass spectrometry (GC–MS).
Venom protease and hyaluronidase activities (two isoforms) were completely (100%) neutralized by the L. aspera methanolic extract at ratio of 1:50 w/w (venom: plant extract) and venom hemolytic activity was also completely neutralized at a ratio of 1:80 w/w by the plant extract. However, the extract failed to neutralize phospholipase A₂ activity even at the highest concentration used. Phytochemical analysis revealed the presence of alkaloids, acidic compounds, flavonoids, steroids and cardiac glycosides in the extract. GC–MS analysis indicated that a total of 14 compounds were present in the extract. The major bioactive constituents were found to be 6-octadecenoic acid (32.47%), n-hexadecanoic acid (25.97%), and 17-octadecen-14-yn-1-ol (14.22%) along with the minor constituents, sitosterol (2.45%) and stigmasterol (2%), which was previously reported to exhibit antivenom activity.
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Differential expression of choline kinase isoforms in skeletal muscle explains the phenotypic variability in the rostrocaudal muscular dystrophy mouse
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Choline kinase in mammals is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneous genomic deletion in murine Chkb results in neonatal forelimb bone deformity and hindlimb muscular dystrophy. Surprisingly, muscular dystrophy isn't significantly developed in the forelimb. We have investigated the mechanism by which a lack of choline kinase β, encoded by Chkb, results in minimal muscular dystrophy in forelimbs. We have found that choline kinase β is the major isoform in hindlimb muscle and contributes more to choline kinase activity, while choline kinase α is predominant in forelimb muscle and contributes more to choline kinase activity. Although choline kinase activity is decreased in forelimb muscles of Chkb^−/− mice, the activity of CTP:phosphocholine cytidylyltransferase is increased, resulting in enhanced phosphatidylcholine biosynthesis. The activity of phosphatidylcholine phospholipase C is up-regulated while the activity of phospholipase A₂ in forelimb muscle is not altered. Regeneration of forelimb muscles of Chkb^−/− mice is normal when challenged with cardiotoxin. In contrast to hindlimb muscle, mega-mitochondria are not significantly formed in forelimb muscle of Chkb^−/− mice. We conclude that the relative lack of muscle degeneration in forelimbs of Chkb^−/− mice is due to abundant choline kinase α and the stable homeostasis of phosphatidylcholine.
Understanding the muscular dystrophy caused by deletion of choline kinase beta in mice
2009, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Choline kinase in mice is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneously occurring genomic deletion in murine Chkb results in neonatal bone deformity and hindlimb muscular dystrophy. We have investigated the mechanism by which a lack of choline kinase β, encoded by Chkb, causes hindlimb muscular dystrophy. The biosynthesis of phosphatidylcholine (PC) is impaired in the hindlimbs of Chkb^−/− mice, with an accumulation of choline and decreased amount of phosphocholine. The activity of CTP:phosphocholine cytidylyltransferase is also decreased in the hindlimb muscle of mutant mice. Concomitantly, the activities of PC phospholipase C and phospholipase A₂ are increased. The mitochondria in Chkb^−/− mice are abnormally large and exhibit decreased inner membrane potential. Despite the muscular dystrophy in Chkb^−/− mice, we observed increased expression of insulin like growth factor 1 and proliferating cell nuclear antigen. However, regeneration of hindlimb muscles of Chkb^−/− mice was impaired when challenged with cardiotoxin. Injection of CDP-choline increased PC content of hindlimb muscle and decreased creatine kinase activity in plasma of Chkb^−/− mice. We conclude that the hindlimb muscular dystrophy in Chkb^−/− mice is due to attenuated PC biosynthesis and enhanced catabolism of PC.
Neurotoxic and myotoxic actions of Naja naja kaouthia venom on skeletal muscle in vitro
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The neuromuscular and skeletal muscle actions of Naja naja kaouthia snake venom were studied in mammalian (rat left hemidiaphragm) and avian (chick biventer cervicis) nerve–muscle preparations. The venom (5 and 10 μg/ml) produced neuromuscular blockade (85% in 36.8±2.0 min, mean±SEM, n=5, and 18±0.6 min, n=3, p<0.01, respectively) in the rat preparation. That the phospholipase A₂ (PLA₂) activity of the venom is involved in this effect was evaluated by inhibiting this enzyme with p-bromophenacyl bromide. This resulted in significantly (p<0.01) increasing the time required for 85% blockade with 5 and 10 μg/ml to 54±4.6 min (n=3) and 29±0.6 min (n=3), respectively. In chick preparations, the venom (5 μg/ml) produced neuromuscular blockade in 14.0±1.8 min (n=5). The contractures to exogenous acetylcholine were completely inhibited by the venom, whereas those to 134 μM KCl were partially blocked in chick preparations (n=4, n=3, respectively). The venom (5 μg/ml) produced a progressive decrease in the amplitude of miniature end-plate potentials (m.e.p.ps) in the rat hemidiaphragm, but did not alter the resting membrane potential at 5 μg/ml. Neostigmine (5.8 μM) immediate and partially reversed the 85% blockade produced by venom (61%, n=3) in rat preparations, as did 4-aminopyridine (53 μM) (∼59%, n=3). The 4-aminopyridine and neostigmine also restored the m.e.p.ps to pre-venom (control) values. In rat preparations, the venom damaged 47%±11% and 62.7±3.6% of the muscle fibers at concentrations of 5 and 10 μg/ml, respectively. For venom in which PLA₂ activity was inhibited, the corresponding values were 38±11.8% (5 μg/ml) and 67±9.6% (10 μg/ml). These findings suggest a post-synaptic neurotoxic action for N. n. kaouthia venom, and that inhibiting phospholipase activity of the venom reduces significantly the neuromuscular block but not the direct myotoxicity.

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