THESIS: D. J.West submitted this thesis for the degree of Doctor of Philosophy in Neuroscience and was examined at the School of Biological Sciences, Royal Holloway, University of London, U.K., in 1997.
Advisors: Professor M.C. Thorndyke, Dr. A.R. McVean and Dr E.B. Andrews.
ABSTRACT. Some marine snails produce toxic secretions that are used to overcome their prey. Such animals have proved to be a rich source of toxins which have enabled neuroscientists to study key components of both vertebrate and invertebrate nervous systems.
The accessory salivary glands of some marine snails have been proposed to function as venom glands. This study has found that the accessory salivary glands of Nucella lapillus produce a pharmacologically active secretion. A neuroactive substance was detected that relaxed molluscan smooth muscle and contracted vertebrate smooth muscle. Its pharmacological action could be blocked by the serotonin receptor antagonists mersalyl acid (500 µM) and cyproheptadine (100 µM), respectively. The active component was isolated by high performance liquid chromatography (HPLC) and identified as serotonin by thermospray mass spectrometry (TS-MS), thin layer chromatography, HPLC elution time and capillary electrophoresis. High levels of serotonin were detected in gland extracts (0.25 mg/g of gland). Immunocytochemical investigation of the accessory salivary glands demonstrated that the serotonin originated not from intrinsic neurons but from large numbers of secretory cells previously proposed as toxin-producing cells. Additional pharmacological activity was found in gland extracts. Extracts were cardioinhibitory in molluscs and inhibited neuromuscular transmission by 78% (±9%) in rat phrenic nerve-hemidiaphragm preparations, effects that were not due to the presence of serotonin. Fluorimetric measurements of intracellular calcium using fura-2, showed that gland extracts contain at least 2 components that inhibit voltage-activated calcium channels (55 ± 2% and 14 ± 5% maximum inhibition, respectively) in rat cortical synaptosomes.
A second species of marine snail, Neptunea antiqua, has also been studied. Extracts of the salivary glands (which are distinct from the accessory salivary glands) were found to contain a component that contracted molluscan smooth and cardiac muscle, an effect which was blocked by the cholinergic antagonist, d-tubocurarine. Extracts also completely blocked neuromuscular transmission in rat phrenic nerve-hemidiaphragm preparations. The active component was isolated and identified by HPLC, thin layer chromatography and its pharmacological properties as the marine toxin tetramine (tetramethylammonium chloride). Large amounts of tetramine were isolated from the salivary glands (5.7 mg/g of gland). Electrophysiological analysis of rat neuromuscular junction demonstrated that tetramine induced a depolarisation of the postsynaptic membrane and reduced the mean amplitude of miniature end-plate potentials (MEPPs) by inducing a depolarising block of postsynaptic acetylcholine receptors. At least 3 additional components were found to inhibit voltage-activated calcium channels in rat cortical synaptosomes. The 3 fractions gave rise to maximum inhibitions of 44 ± 7%, 43 ± 5% and 35 ± 4%, respectively.
It is clear from this study that glands from both these species contain pharmacological agents that could be used in the paralysis of prey. The salivary glands of Neptunea are true salivary glands and any venomous function is incidental to their primary function, the lubrication and partial digestion of food. However, we present strong evidence to support the notion that the accessory salivary glands of Nucella are suited to a venomous function. Unlike the salivary glands, they empty their contents externally and their thick muscular walls allow them to propel their pharmacologically active secretion with some force (c.f. the venom apparatus of the Conus snails). Therefore, we propose that the accessory salivary glands produce a venom that is used to overcome prey. Since no salivary or digestive function can be attributed to these glands we suggest that they are renamed "venom glands". This work was carried out in collaboration with Lilly Research Centre Ltd., Eli Lilly & Co., U.K.
REFERENCES
01 WEST DJ., ANDREWS EB., BOWMAN D., McVEAN AR., THORNDYKE MC. Toxins from some poisonous and venomous marine snails. Comp. Biochem. Physiol., 1996, 113C, 1-10.
02 WEST DJ., ANDREWS EB., McVEAN AR., OSBORNE D., THORNDYKE MC. Isolation of serotonin from the accessory salivary glands of the marine snails Nucella lapillus. Toxicon, 1994, 32, 1261-4.
CORRESPONDENCE TO:
D. J. WEST - Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, U.K. E-mail: djw36@cam.ac.uk
Publication Dates
-
Publication in this collection
26 Nov 1998 -
Date of issue
1998
