Marine snails of the genus Conus ("cone snails") use a particularly sophisticated strategy to capture their prey. They inject a venom containing a cocktail of bioactive peptides, which causes rapid immobilization by disrupting neurotransmission. The number of different peptides in the venom of a single species of cone snail may exceed 100. The isolation and characterization of these molecules, termed "conopeptides", has revealed that the venom possesses a considerable spectrum of pharmacological activities, targeting a variety of receptors and voltage-sensitive ion channels exposed on the cell's surface.
This thesis examines aspects of the pharmacology of three classes of conopeptides that act to disrupt sympathetic neurotransmission through the use of both tissue-based and molecular investigations. A key assay system employed isolated segments of the rat vas deferens, a tissue with dense sympathetic innervation. These responded to electrical field stimulation with a biphasic contraction, reflecting the involvement of two distinct neurotransmitters, adenosine 5'-triphosphate (ATP) and noradrenaline.
The ω-conopeptides target neuronal voltage-sensitive Ca2+ channels, and were used as pharmacological tools to investigate the role of these channels in supplying the Ca2+ required to trigger neurotransmitter release. The effects of three well characterized a)-conopeptides, GVIA, MVIIA and MVIIC, and four newly isolated ω-conopeptides, CVIA, CVIB, CVIC and CVID, were examined. Each acted to inhibit the purinergic and noradrenergic components of the electrically evoked contraction of the rat vas deferens, but displayed differences in terms of the potency, reversibihty, Hill slope parameter, and the extent of the inhibition produced. A dominant role for N-type Ca2+ channels in mediating the neuronal Ca2+ influx that triggers neurotransmitter release was established based on a strong correlation of the ω-conopeptides' potency with previously reported values for their binding potency at N-type Ca2+ channels. The involvement of P/Q-tpye Ca2+ channels was also demonstrated. While most of the differences in the pharmacology of the ω-conopeptides could be explained by variations in the Ca2+ channel subtype selectivity of the peptides, some of the results may also reflect that the ω-conopeptides vary in their ability to recognize the channel in different conformational states. The inhibitory action of the ω-conopeptides on the electrically evoked response was compared to the inhibition produced by the activation of presynaptic a2-adrenoceptor and µ-opioid receptors, and considered in terms of the concept of sympathetic co-transmission.
The second set of conopeptides consisted of TIA and a number of truncated analogues. These peptides resembled the a-conopeptides, which block neuronal or muscle nicotinic acetylcholine (ACh) receptors, structurally, but not pharmacologically. TIA reduced the noradrenergic component of the electrically evoked contraction of the rat vas deferens, but unlike the ω-conopeptides, spared the purinergic component. Concentration- response curves of the tissue to exogenously applied noradrenaline were shifted to the right in the presence of TIA, and the maximum response was depressed. These results are consistent with TIA acting as a non-competitive antagonist at the α1-adrenoceptor, a previously unrecognized target for the conopeptides. The new class of conopeptides defined by TIA was given the name, the "ρ-conopeptides". TIA was found not to block the a2-adrenoceptor in a functional assay using the rat vas deferens, and to target the three a1-adrenoceptor subtypes with approximately equal potency in binding studies. Other binding experiments confirmed the non-competitive nature of TIA's action at the a1-adrenoceptor. A comparison of the potency of TIA and its truncated analogues provided some information about the structure-activity relationship of the ρ-conopeptides.
MrIA and MrIB comprised the third set of conopeptides examined. These enhanced the noradrenergic component of the electrically evoked contraction of the rat vas deferens, without affecting the purinergic component. In the presence of MrIA, concentration-response curves to exogenously applied noradrenaline in the rat vas deferens were shifted leftward with no change in the maximum response, but the tissue's sensitivity to methoxamine was unaltered. While both noradrenaline and methoxamine are a1-adrenoceptor agonists, only noradrenaline is subject to removal by neuronal uptake, implicating the peptides as inhibitors of the neuronal noradrenaline transporter. This is the first demonstration that conopeptides target transporters, and the new class defined by MrIA and MrIB was named the "χ-conopeptides". Binding and functional experiments with cloned transporters indicated that MrIA does not act at the same site as noradrenaline or the classical noradrenaline transporter inhibitors, nor does it target the transporters for serotonin or dopamine.
These experiments have demonstrated three distinct ways that conopeptides can disrupt sympathetic neurotransmission: prevention of neurotransmitter release (ω-conopeptides), blockade of postsynaptic receptors (ρ-conopeptides), and inhibition of neurotransmitter clearance (χ-conopeptides). The three classes of conopeptides are useful pharmacological tools and may also have therapeutic applications.