Elsevier

Toxicon

Volume 40, Issue 4, April 2002, Pages 335-393
Toxicon

Review
Ophidian envenomation strategies and the role of purines

https://doi.org/10.1016/S0041-0101(01)00232-XGet rights and content

Abstract

Snake envenomation employs three well integrated strategies: prey immobilization via hypotension, prey immobilization via paralysis, and prey digestion. Purines (adenosine, guanosine and inosine) evidently play a central role in the envenomation strategies of most advanced snakes. Purines constitute the perfect multifunctional toxins, participating simultaneously in all three envenomation strategies. Because they are endogenous regulatory compounds in all vertebrates, it is impossible for any prey organism to develop resistance to them. Purine generation from endogenous precursors in the prey explains the presence of many hitherto unexplained enzyme activities in snake venoms: 5′-nucleotidase, endonucleases (including ribonuclease), phosphodiesterase, ATPase, ADPase, phosphomonoesterase, and NADase. Phospholipases A2, cytotoxins, myotoxins, and heparinase also participate in purine liberation, in addition to their better known functions. Adenosine contributes to prey immobilization by activation of neuronal adenosine A1 receptors, suppressing acetylcholine release from motor neurons and excitatory neurotransmitters from central sites. It also exacerbates venom-induced hypotension by activating A2 receptors in the vasculature. Adenosine and inosine both activate mast cell A3 receptors, liberating vasoactive substances and increasing vascular permeability. Guanosine probably contributes to hypotension, by augmenting vascular endothelial cGMP levels via an unknown mechanism. Novel functions are suggested for toxins that act upon blood coagulation factors, including nitric oxide production, using the prey's carboxypeptidases. Leucine aminopeptidase may link venom hemorrhagic metalloproteases and endogenous chymotrypsin-like proteases with venom L-amino acid oxidase (LAO), accelerating the latter. The primary function of LAO is probably to promote prey hypotension by activating soluble guanylate cyclase in the presence of superoxide dismutase. LAO's apoptotic activity, too slow to be relevant to prey capture, is undoubtedly secondary and probably serves principally a digestive function. It is concluded that the principal function of L-type Ca2+ channel antagonists and muscarinic toxins, in Dendroaspis venoms, and acetylcholinesterase in other elapid venoms, is to promote hypotension. Venom dipeptidyl peptidase IV-like enzymes probably also contribute to hypotension by destroying vasoconstrictive peptides such as Peptide YY, neuropeptide Y and substance P. Purines apparently bind to other toxins which then serve as molecular chaperones to deposit the bound purines at specific subsets of purine receptors. The assignment of pharmacological activities such as transient neurotransmitter suppression, histamine release and antinociception, to a variety of proteinaceous toxins, is probably erroneous. Such effects are probably due instead to purines bound to these toxins, and/or to free venom purines.

Introduction

Recently, in the course of analyzing enzymatic compositional patterns relative to prey preference among coral snake taxa (Jorge da Silva and Aird, 2001) it was difficult to explain the ubiquitous nature of some relatively non-toxic venom constituents. The nearly universal occurrence of some enzymes suggested a central role in envenomation, but no satisfactory explanation has ever been given for their presence in venoms. As recently as 1999, Cousin and Bon (1999) noted that, ‘snake venoms are rich in proteins and enzymes whose functions are unknown’, citing nerve growth factor (NGF), L-amino acid oxidase (LAO) and phosphodiesterase (PDE) as examples. Numerous other enzymes and non-enzymatic toxins could also have been added to this short list.

At the same time, research in the Laboratório de Toxinas Naturais confirmed the earlier observation of Francis et al. (1997) that venom of the Brazilian coral snake, Micrurus frontalis, contains significant quantities of guanosine. Preliminary evidence suggested that a significant portion of the nucleoside is not free in solution, but that it is apparently tightly bound to postsynaptic neurotoxins and perhaps to phospholipases and other compounds as well. Intrigued by these findings I set out in search of an explanation for the role of purines in venoms.

Micrurus frontalis venom is far from being unique with regard to its purine content. The first such report appeared nearly 50 years ago when Fischer and Dörfel (1954) found adenosine in venoms of the puff adder (Bitis arietans), a large viperid, and of the eastern green mamba (Dendroaspis angusticeps), an elapid, and suggested that this compound might contribute to the hypotension promoted by these venoms. Doery (1956) reported the presence of adenosine, adenosine 3′-phosphate and ‘guanine compounds’ in venoms of several dissimilar elapids, Acanthophis antarcticus, Notechis scutatus, Dendroaspis angusticeps and Denisonia superba (Austrelaps superbus), and also in that of Bitis arietans. Doery (1957) later isolated guanosine, inosine and hypoxanthine from Notechis scutatus venom. Eight years passed before Wei and Lee (1965) reported that guanosine constitutes slightly over 1% of crude Bungarus multicinctus venom, but found no evidence that it influenced blood pressure in cats. Lo and Chen (1966) found adenosine, guanosine and inosine in the venom of the Chinese cobra, Naja atra. Lin and Lee (1971) suggested that nucleoside contamination might explain the detection of carbohydrate (ribose or deoxyribose) in many apparently pure elapid neurotoxins. Eterovic et al. (1975) reported that Bungarus multicinctus venom contains large amounts of free guanosine which accounted for 10% of the 280 nm absorbance of the crude venom. This material did not bind to a CM Sephadex column equilibrated in 50 mM ammonium acetate (pH 7.0). Takasaki et al. (1991) also identified adenosine, guanosine, and inosine in venom of the long-glanded coral snake (Maticora bivirgata).

In the present paper I outline three fundamental ophidian envenomation strategies which appear to be employed by all advanced venomous snakes. I suggest that purines act as multifunctional toxins, exerting synchronous effects upon virtually all cell types, and I believe that exogenous and released endogenous purines play a central role in all three envenomation strategies, which previously has been almost entirely overlooked. I offer hypothetical explanations for the presence of many venom enzymes which has hitherto been unexplained. Direct experimental evidence from venom studies is still lacking for most of the hypotheses presented herein, but pharmacological research in other fields provides ample evidence to suggest that they are probably correct and that they are worthy of experimental verification.

Section snippets

Venom functions

Snake venoms are extremely complex mixtures of proteins, peptides, carbohydrates, lipids, metal ions and organic compounds, with proteins and peptides accounting for approximately 90% of the dry weight (Bieber, 1979). While snake venoms have an obvious role in self-defense, this is of relatively little importance with regard to venom composition. Their primary function is to immobilize and kill prey organisms (Karlsson, 1979). Venoms simultaneously commence digestion of the prey from within,

Nomenclature

Two groups of nitrogenous bases are of immense importance in biological systems. The structurally more simple pyrimidines are six-sided, heterocyclic, aromatic rings, containing two nitrogen and four carbon atoms. The most important naturally occurring pyrimidines include cytosine, uracil and thymine (Fig. 1). Purines, with which the present paper is primarily concerned, comprise a fused ring system consisting of the pyrimidine ring linked to a five-sided imidazole ring. Five purines are well

Known synergistic actions of snake venom constituents

Historically snake venom research has necessarily been quite reductionist because only by isolating and characterizing individual components has it been possible to begin to understand the complex pathological state induced by envenomation. Perhaps because the majority of venom research has been done by biochemists and pharmacologists, there has also been a tendency to ignore the fact that venoms are optimized to function in specific prey organisms as integrated assemblages of compounds, and

Hypotensive strategy

Most snake venoms employ a variety of means to induce rapid and profound hypotension, leading to circulatory shock, prey immobilization and death (Bjarnason et al., 1983). The hypotensive peptides and kininogenases discussed above are only two of the many mechanisms employed for this purpose (Fig. 5). Several other well known hypotensive mechanisms will be briefly summarized before a variety of previously unrecognized pharmacological strategies are discussed.

Paralytic strategy

As with the hypotensive strategy, there are many paralytic strategy elements that are well known, and it is not the intent of this paper to review these; however, an incomplete picture would be presented if some of the better known examples were not mentioned briefly.

Digestive strategy

Prey immobilization is the first priority in all envenomation strategies and all snake venom components depend upon the prey's circulatory system for distribution throughout prey tissues (Fig. 5, Fig. 7). Nonetheless prey digestion also commences at the instant of envenomation and undoubtedly continues beyond the prey's death until venom constituents are inactivated by prey protease inhibitors or proteases, or by the snake's digestive enzymes. Some venom constituents have digestive functions

Role of venom purines other than adenosine

As previously mentioned, venoms have also been reported to contain guanosine, inosine and hypoxanthine. What possible contributions do these make to the envenomation strategies of the snakes that employ them?

Purine interactions with other toxins and miscellaneous roles

Lin and Lee (1971) suggested that nucleoside contamination might explain the detection of carbohydrate (ribose or deoxyribose) in many apparently pure toxins. I suggest that many toxins bind adenosine, guanosine and possibly inosine and that these bound purines also play an essential role in envenomation. If this hypothesis proves correct, much of the pharmacological data gathered with such toxins to date is probably in error. Specifically, toxin research involving neurotransmitter release,

Supporting evidence from arthropod venoms

Snake venoms are not the only toxic animal secretions to employ purines. Nucleotides and nucleosides are also important components of various spider venoms and at least one ant venom. Chan et al. (1975) discovered ATP, ADP and AMP in venoms of the tarantulas (Theraphosidae), Dugesiella and Aphonopelma. ATP, the principal nucleotide constituent, was present at a concentration of approximately 55 mM in Dugesiella, and 100 mM in Aphonopelma. ATP acted synergistically with the necrotoxin, the major

Conclusions

  • Snake envenomation employs three well integrated strategies: prey immobilization via hypotension, prey immobilization via paralysis, and prey digestion.

  • Purines (especially adenosine, inosine and guanosine) are concluded to play a central role in envenomation by most advanced venomous snakes.

  • Purines constitute the perfect multifunctional toxins, participating simultaneously in all three envenomation strategies. Because they are endogenous regulatory and homeostatic compounds in all vertebrates,

Acknowledgements

I express my gratitude to my wife, Yayoi, whose love and support made this research possible, and also to my parents, John S. and Laurel J. Aird, for photocopying several hundred publications at the National Library of Medicine and the National Institutes of Health Library, without which this study could not have been done in Brazil. I gratefully acknowledge the assistance of Dr Saad Lahlou, not only for extensive editorial criticisms of the manuscript, but for his expertise in interpreting the

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