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We describe the identification of a conopeptide sequence in venom duct mRNA from Conus victoriae that suppresses a vascular response to pain in the rat. PCR-RACE was used to screen venom duct cDNAs for those transcripts that encode specific antagonists of vertebrate neuronal nicotinic acetylcholine receptors (nAChRs). One of these peptides, Vc1.1, was active as an antagonist of neuronal nAChRs in receptor binding and functional studies in bovine chromaffin cells. It also suppressed the vascular responses to unmyelinated sensory nerve C-fiber activation in rats. Such vascular responses are involved in pain transmission. Furthermore, its ability to suppress C-fiber function was greater than that of MVIIA, an omega-conotoxin with known analgesic activity in rats and humans. Vc1.1 has a high degree of sequence similarity to the alpha-conotoxin family of peptides and has the 4,7 loop structure characteristic of the subfamily of peptides that act on neuronal-type nAChRs. The results suggest that neuronal alpha-conotoxins should be further investigated with respect to their potential to suppress pain.

Marine cone snails from the genus Conus are estimated to consist of up to 700 species. These predatory molluscs have devised an efficient venom apparatus that allows them to successfully capture polychaete worms, other molluscs or in some cases fish as their primary food sources. The toxic venom used by the cone shells contains up to 50 different peptides that selectively inhibit the function of ion channels involved in the transmission of nerve signals in animals. Each of the 700 Conus species contains a unique set of peptides in their venom. Across the genus Conus, the conotoxins represent an extensive array of ion channel blockers each showing a high degree of selectivity for particular types of channels. We have undertaken a study of the conotoxins from Australian species of Conus that have the capacity to inhibit specifically the nicotinic acetylcholine receptors in higher animals. These conotoxins have been identified by mass spectroscopy and their peptide sequences in some cases deduced by the application of modern molecular biology to the RNA extracted from venom ducts. The molecular biological approach has proven more powerful than earlier protein/peptide based technique tor the detection of novel conotoxins [1,2]. Novel conotoxins detected in this way have been further screened for their abilities to modify the responses of tissues to pain stimuli as a first step in describing their potential as lead compounds for novel drugs. This review describes the progress made by several research groups to characterise the properties of conopeptides and to use them as drug leads for the development of novel therapeutics for the treatment of a range of neurological conditions.

We have isolated and characterized ␣-conotoxin EpI, a novel sulfated peptide from the venom of the molluscivorous snail, Conus episcopatus. The peptide was classified as an ␣-conotoxin based on sequence, disulfide connectivity, and pharmacological target. EpI has homology to sequences of previously described ␣-conotoxins, particularly PnIA, PnIB, and ImI. However, EpI differs from previously reported conotoxins in that it has a sulfotyrosine residue, identified by amino acid analysis and mass spectrometry. Native EpI was shown to coelute with synthetic EpI. The peptide sequence is consistent with most, but not all, recognized criteria for predicting tyrosine sulfation sites in proteins and peptides.

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Single bovine adrenal medullary cells have been obtained by retrograde perfusion of adrenal medullae with a solution of 0.05% coUagenase in Ca §247 free Krebs Henseleit buffer. Chromaffin cells were obtained in high yield (5 x 106 cells/g medulla), and more than 95% of these were viable as shown by exclusion of trypan blue. The isolated cells were capable of respiring at a linear rate for a minimum of 120 min.Ultrastructural examination revealed that the cells were morphologically intact, and two distinct types of adrenal medullary cells were identified, on the basis of the morphology of their electron-dense vesicles, as (a) adrenalinecontaining and (b) noradrenaline-containing cells.Biochemical analysis showed that the cells contained catecholamines and dopamine-/3-hydroxylase (DBH). The cells released catecholamines and DBH in response to acetylcholine (ACh), and this release was accompanied by changes in the vesicular and surface membranes observed at the ultrastructural level. The time-course of ACh-stimulated catecholamine and DBH release, and the dependence of this release on the concentration of ACh and extracellular Ca + § have been investigated. The isolated cells were pharmacologically sensitive to the action of the cholinergic blocking agents, atropine and hexamethonium.

The opioid peptides Leu-enkephalin and Met-enkephalin are stored intraneuronally in the brain where they are thought to act as neurotransmitters and/or neuromodulators. Evidence for their release from nerve terminals has come from biochemical and pharmacological studies in vitro with brain tissue slices and synaptosomes. Enkephalins also exist in the peripheral nervous system in nerve cell bodies and axon terminals in the gastrointestinal tract, sympathetic ganglia and adrenal gland. In the adrenal gland, high levels of enkephalins are present both in axon terminals of the splanchnic nerve and in the adrenal medullary chromaffin cells where they are stored together with the catecholamines in the chromaffin granules. Stimulation of the adrenal gland in vivo or the perfused gland in vitro causes release of catecholamines and enkephalins into the adrenal vein. However, it is not clear whether the origin of the released enkephalins is the adrenal medullary chromaffin cells or the enkephalin-containing splanchnic nerve terminals that innervate the medulla. We now show that enkephalin and catecholamines are released together from primary cultures of bovine adrenal medullary chromaffin cells by nicotine in a Ca2+-dependent manner.