Venoms have historically proven to be a rich source of powerful neuroactive agents. Recent discovery of SNX-325, a selective blocker of class B (N-type) calcium channels, has provided clinicians with a novel agent for the treatment of intractable pain. Currently available calcium channel drugs, including the dihydropyridines, target channels are functionally and pharmacologically distinct from the peripheral L-type channels and the exploration of their therapeutic modulation is limited by the present lack of specific agonists and antagonists. Drugs targeted toward these channels would be useful, for example, in minimizing neuronal damage caused by excess calcium entry during periods of ischemia. Recent findings from the applicant's laboratory demonstrate that the most powerful of the Hawaiian water coelenterate venoms, Carybdea alata venom, contains at least three distinct activities. The general goal of the collaborative research proposed is to augment the ability of the applicant to screen for novel neuroactive agents using ion flux measurements utilizing imaging techniques for various ions including Ca2+ and Na+. Potential identification of novel calcium channel blockers may ultimately result in elucidation of therapeutic interventions to accomplish neuroprotection which may be applied in the treatment of ischemia and other types of brain injury. Studies are proposed to test the working hypothesis that this venom, as well as other blockers. To test this, the effects of isolated constituent toxins will be tested on various model preparations including crayfish nerve chord (electrophysiological measurement) and single neuronal cells (ion flux and cAMP measurements utilizing fluorescence imaging techniques). The project will be conducted as an integrated and coordinated collaboration between investigators at the University of Hawaii and Johns Hopkins University. The applicant's component of the project will focus on biochemical separation of unique constituent toxins and their respective electrophysiological and morphological effects upon target tissues, while the collaborating component will focus on the evaluation of ion flux and secondary messenger system effects, with particular emphasis on calcium and cAMP flux measurements. The hypotheses to be tested in both components are closely interrelated as are the experimental designs, and will allow the complementary expertise of the investigators to be applied in a maximally productive and mutually beneficial way. The collaboration will also provide ample opportunities for students and fellows at the University of Hawaii to obtain training in the basic neuroscientific techniques to be implemented in the conduct of this research effort.
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