omega-agatoxin-iva and Brain-Ischemia

omega-Agatoxin IVA-sensitive Ca(2+) channels have been thought to be involved in physiological excitatory amino acid glutamate release and these channels may also contribute to the development of ischemic brain injury. Recently, we demonstrated that alpha-eudesmol from Juniperus virginiana Linn. (Cupressaceae) inhibits potently the presynaptic omega-agatoxin IVA-sensitive Ca(2+) channels. In the present study, we investigated the effects of alpha-eudesmol on brain edema formation and infarct size determined after 24 h of reperfusion following 1 h of middle cerebral artery occlusion in rats. We first found that alpha-eudesmol concentration-dependently inhibited glutamate release from rat brain synaptosomes and that its inhibitory effect was Ca(2+)-dependent. In the middle cerebral artery occlusion study, intracerebroventricular (i.c.v.) treatment with alpha-eudesmol significantly attenuated the post-ischemic increase in brain water content. alpha-Eudesmol also significantly reduced the size of the infarct area determined by triphenyltetrazolium chloride staining after 24 h of reperfusion. Using a microdialysis technique, we further demonstrated that alpha-eudesmol inhibits the elevation of the extracellular concentration of glutamate during ischemia. From these results, we suggest that alpha-eudesmol displays an ability to inhibit exocytotic glutamate release and to attenuate post-ischemic brain injury.

Topics: Animals; Brain Edema; Brain Ischemia; Calcium Channel Blockers; Cerebral Infarction; Dose-Response Relationship, Drug; Glutamic Acid; Male; Neuroprotective Agents; omega-Agatoxin IVA; Potassium; Rats; Rats, Sprague-Dawley; Rats, Wistar; Sesquiterpenes, Eudesmane; Terpenes

Calcium influx and transmitter efflux are central events in the neuropathological cascade that occurs during and following cerebral ischaemia. This study explored the role of voltage-gated calcium channels (VGCCs) in ischaemia-induced striatal dopamine (DA) release in vitro. Slices (350 microm thickness) of rat neostriatum were superfused (400 ml/h) with an artificial cerebrospinal fluid (aCSF) at 34 degrees C and subjected to episodes of 'ischaemia' by reduction of the glucose concentration from 4 to 2 mM and gassing with 95% N2/5% CO2. DA release was monitored with fast cyclic voltammetry at implanted carbon fibre microelectrodes. The time to onset, time to peak, rate and magnitude of DA release were measured. Non-selective blockade of VGCCs with a high concentration of Ni2+ (2.5 mM), markedly delayed (P < 0.01) and slowed (P < 0.05) DA release but preferential blockade of T-type VGCCs with a lower concentration (200 microM) had no effect. DA release was also unaffected by selective antagonism of L-type VGCCs with nimodipine and nicardipine (10 microM each). Selective blockade of N-type VGCCs with omega-conotoxin GVIA (100 nM) delayed DA release (P < 0.05) but did not affect its rate or magnitude. Blockade of P- and possibly Q-type VGCCs with omega-agatoxin IVA (up to 200 nM) both delayed (P < 0.05) and slowed (P < 0.05) DA release. Preferential blockade of P- type VGCCs with neomycin (500 microM) also delayed (P < 0.05) and slowed (P < 0.05) DA release. These findings suggest that N-, P- and possibly Q- but not L- or T-type VGCCs mediate ischaemia-induced DA release. Although it is not possible to say, on the basis of these results, that the effects are directly upon the dopamine terminals, these calcium channels nevertheless constitute promising targets for therapeutic intervention.

Topics: Animals; Brain Ischemia; Calcium Channel Blockers; Calcium Channels; Corpus Striatum; Dopamine; Dopamine Antagonists; Dose-Response Relationship, Drug; Electrophysiology; In Vitro Techniques; Ion Channel Gating; Male; Neomycin; Nickel; omega-Agatoxin IVA; omega-Conotoxin GVIA; Peptides; Rats; Rats, Wistar; Spider Venoms

The present study was undertaken to elucidate pathophysiological changes in the cortical presynaptic function, K(+)-stimulated calcium influx, noradrenaline release and noradrenaline uptake, on the 1st and 3rd days after microsphere embolism in rats. Voltage-dependent calcium channels were characterized pharmacologically using three types of calcium channel blockers, L-type (nifedipine and diltiazem), N-type (omega-conotoxin GVIA), and P-type channel (omega-agatoxin IVA) blockers. K(+)-stimulated calcium influx of the normal rat synaptosome was inhibited by 100 nM omega-agatoxin IVA, but not by 10 microM nifedipine, 10 microM diltiazem and 100 nM omega-conotoxin GVIA. Calcium influx of the cortical nerve terminals of the right hemisphere was decreased on the 1st and 3rd days after the embolism. Noradrenaline release and uptake were also decreased on the 1st and 3rd days after the embolism. However, the percent release of noradrenaline was not altered. The results suggest that P-type channels are predominant in the cerebrocortical nerve terminals in rats and that calcium influx, noradrenaline release and uptake in the cerebrocortical nerve terminals are decreased by microsphere embolism. The decrease in noradrenaline release may be mainly due to a reduction in the activity of noradrenaline uptake in cerebrocortical nerve terminals of the microsphere-embolized rat.

Topics: Animals; Brain Ischemia; Calcium; Calcium Channel Blockers; Calcium Channels; Cerebral Cortex; Diltiazem; Embolism; Ion Channel Gating; Male; Microspheres; Mollusk Venoms; Nifedipine; Norepinephrine; omega-Agatoxin IVA; omega-Conotoxin GVIA; Peptides; Potassium; Presynaptic Terminals; Rats; Rats, Wistar; Spider Venoms; Synaptosomes; Time Factors; Vasoconstrictor Agents

1. To examine the role of the phosphoinositide cascade triggered by disturbed Ca2+ homeostasis in ischaemic neurones, inositol 1,3,4,5-tetrakisphosphate (InsP4) was applied to the cytoplasmic face of membrane patches isolated from CA1 pyramidal neurones in the gerbil hippocampus. 2. In outside-out recordings, InsP4 induced an inward current which was increased by raising the extracellular [Ca2+]. In contrast, no clear channel openings could be observed in patches from neurones of sham-operated gerbils. 3. Open probabilities of InsP4-activated channels were significantly decreased upon application of omega-conotoxin but were not affected by omega-agatoxin or nifedipine. 4. In inside-out patches using high concentrations of Ca2+, Ba2+ or Sr2+ in the pipette solution, InsP4 enhanced inward currents. 5. Application of the isomers of InsP4 slightly enhanced the currents, but inositol 1,4,5-trisphosphate (InsP3) had no effect. 6. In the absence of InsP4 there was a single main Ba2+ current peak of 4.0 pA in amplitude, whereas upon its application two main peaks of 3.0 and 7.2 pA were present. 7. The open probabilities of these channels were apparently increased by InsP4. 8. These findings support the view that a disturbed phosphoinositide cascade occurs in the hippocampal pyramidal neurones after ischaemia and the InsP4 thus formed plays an important role in promoting the Ca2+ accumulation which results in neuronal death.

Topics: Animals; Brain Ischemia; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Membrane; Electrophysiology; Gerbillinae; Hippocampus; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Metals, Alkaline Earth; Neurons; Nifedipine; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Peptides; Spider Venoms

Excessive accumulation of Ca2+ in neurones and glutamate release are involved in neuropathological processes, including ischaemia. We investigated the neuroprotective effects of the Ca2+ channel antagonist, omega-Aga-IVA, in CA1 pyramidal neurones in rat hippocampal slices following an in vitro hypoxic-hypoglycaemic insult. Following this insult, evoked post-synaptic response amplitudes decreased from 3.7 +/- 0.5 mV to 0.6 +/- 0.2 mV and the CA1 neurones appeared dead using a live/dead fluorescence assay with confocal microscopy. Slices treated with 200 nM omega-Aga-IVA had evoked response amplitudes not significantly different from control (3.3 +/- 0.5 mV) and the CA1 neurones appeared viable using the live/dead fluorescence assay. The neuroprotective efficacy of omega-Aga-IVA suggests that omega-Aga-IVA-sensitive Ca2+ channels participate in ischaemic neuronal death and constitute a potential target of therapeutic intervention.

Topics: Animals; Brain Ischemia; Calcium Channel Blockers; Electrophysiology; Hippocampus; In Vitro Techniques; Male; Membrane Potentials; Microscopy, Electron; omega-Agatoxin IVA; Rats; Rats, Sprague-Dawley; Spider Venoms