Intraspecific variations in Conus geographus defence-evoked venom and estimation of the human lethal dose.

Conus geographus is the most dangerous cone snail species known, with reported human fatality rates as high as 65%. Crude venom gland extracts have been used to determine animal LD50 and to aid the isolation of several potent paralytic toxins. However, not only is the composition of injected venoms known to differ significantly from that in dissected venom glands, but also to vary according to predatory or defensive stimuli. Therefore, to study the venom that is directly relevant to human envenomation, the defense-evoked venom of several specimens of C. geographus was collected and analyzed by standard LC-MS methods. The molecular composition of individual defense-evoked venom showed significant intraspecific variations, but a core of paralytic conotoxins including α-GI, α-GII, µ-GIIIA, ω-GVIA and ω-GVIIA was always present in large amounts, consistent with the symptomology and high fatality rate in humans. Differences between injected and dissected venoms obtained from the same specimen were also evident. Interestingly, an apparent linear correlation between the dry weight/volume of injected venom and the size of the shell allowed extrapolation to a human lethal dose (0.038-0.029 mg/kg) from an historic fatal case of C. geographus envenomation, which may help in the management of future victims.

Voltage-gated calcium channels involved in the inhibitory motor responses and vasoactive intestinal polypeptide release in the rat gastric fundus.

Ca(2+) inflow responsible for neurotransmitter release at most peripheral junctions is mainly mediated by activation of Ca(V)2.2 and Ca(V)2.1 channels. The aim of the present study was to characterize the voltage-gated Ca(2+) channels (VGCCs) responsible for the non-adrenergic non-cholinergic (NANC) relaxation and vasoactive intestinal polypeptide (VIP)-like immunoreactivity release in the rat gastric fundus. Precontracted longitudinal muscle strips of the rat gastric fundus were subjected to electrical field stimulation (EFS) under NANC conditions to evoke the relaxation and VIP-like immunoreactivity release. Nifedipine (1microM) completely relaxed the preparations, so that its effects on EFS-induced NANC relaxations could not be investigated. omega-Conotoxin GVIA (0.3-100nM) concentration-dependently reduced the amplitude of low frequency and the area under the curve (AUC) of high-frequency EFS-evoked relaxations (maximal reductions: approximately 55% and 42% of controls, respectively). The omega-conotoxin GVIA-resistant component of relaxation was not affected by omega-agatoxin IVA (300nM), omega-conotoxin MVIIC (100nM), SNX-482 (100nM) or flunarizine (1microM). omega-Conotoxin GVIA (30nM), omega-agatoxin IVA (30nM) and omega-conotoxin MVIIC (100nM) reduced high-frequency EFS-evoked VIP-like immunoreactivity release by approximately 70%, 27% and 35% of controls, respectively. omega-Conotoxin GVIA (30nM) plus omega-conotoxin MVIIC (100nM) almost abolished the EFS-induced VIP-like immunoreactivity outflow. In the rat gastric fundus, the activation of Ca(V)2.2 and P-type of Ca(V)2.1 channels is responsible for the EFS-induced VIP-like immunoreactivity release. In contrast, Ca(V)1 channels, novel VGCCs and/or molecular variants of VGCCs cloned to date may mediate a substantial component of the NANC relaxation.

Pyrethroid action on calcium channels: neurotoxicological implications.

Actions of cismethrin versus deltamethrin were compared using two functional attributes of rat brain synaptosomes. Both pyrethroids increased calcium influx but only deltamethrin increased Ca(2+)-dependent neurotransmitter release following K(+)-stimulated depolarization. The action of deltamethrin was stereospecific, concentration-dependent, and blocked by omega-conotoxin GVIA. These findings delineate a separate action for deltamethrin and implicate N-type rat brain Ca(v)2.2 voltage-sensitive calcium channels (VSCC) as target sites that are consistent with the in vivo release of neurotransmitter caused by deltamethrin. Deltamethrin (10(-7) M) reduced the peak current (approx. -47%) of heterologously expressed wild type Ca(v)2.2 in a stereospecific manner. Mutation of threonine 422 to glutamic acid (T422E) in the alpha(1)-subunit results in a channel that functions as if it were permanently phosphorylated. Deltamethrin now increased peak current (approx. +49%) of T422E Ca(v)2.2 in a stereospecific manner. Collectively, these results substantiate that Ca(v)2.2 is directly modified by deltamethrin but the resulting perturbation is dependent upon the phosphorylation state of Ca(v)2.2. Our findings may provide a partial explanation for the different toxic syndromes produced by these structurally-distinct pyrethroids.