Mechanisms underlying two different FMRF amide induced ionic currents in identified neurones of 'Helix aspersa'
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Application of the molluscan neuropeptide FMRFamide to two identified neurones in the cerebral ganglia of Helix aspersa induces quite different effects. In the Cl neurone, FMRFamide produces a slow hyperpolarizing current carried by K+ while with the C2 neurone it causes a fast depolarizing current carried by Na+. Possible mechanisms underlying the slow K+ response were examined and the fast response was characterized using voltage clamp techniques. Some patch clamp experiments were also used for the slow response. The slow response was shown to depend on a G protein, which was sensitive to inhibition by pertussis toxin, indicating that it was mediated by a Gi or Go protein. Second messengers such as cyclic AMP, cyclic GMP, IP3, arachidonic acid and Ca2+ along with the activation of protein kinase C were all found not to be directly involved in producing the FMRFamide response. These negative results with the second messengers gave rise to the view that the FMRFamide receptors and K+ ion channels may be linked directly through the activation of G proteins. 5-HT, probably acting through raised cyclic AMP levels, reduced the amplitude of the FMRFamide response which suggests that the channel opened by FMRFamide may be an "S" K+ type channel. Activation of protein kinase C by phorbol ester also reduced the FMRFamide response. A role for protein phosphorylation was indicated by the use of okadaic acid which inhibits protein phosphatases 1 and 2A. Its application reduced the amplitude of the FMRFamide response which suggested that increased protein phosphorylation levels lead to smaller responses. Thus, it seemed possible that protein phosphorylation levels controlled by cyclic AMP, protein kinase C activation and protein phosphatases 1 and 2A might modulate the activity of the receptor/ G protein/ ion channel complex. Alternatively, FMRFamide may operate through the activation of protein phosphatase(s) which reduce protein phosphorylation levels. Patch clamp studies in cell attached mode on the Cl neurone failed to reveal any channel openings induced by FMRFamide. This result also tends to rule out the direct involvement of a second messenger. The fast depolarizing FMRFamide response of the C2 neurone, which is due to the opening of a ligand gated channel, was found to be carried by Na+ and not Ca2+. Amiloride produced a reversible block of the current. Tetrodotoxin and lignocaine had no effect on the FMRFamide response while raised cyclic AMP levels potentiated the response. In the presence of okadaic acid and increased levels of cyclic AMP, the FMRFamide response is potentiated. This potentiation was not maintained in the presence of okadaic acid alone. The raised protein phosphorylation levels therefore did not cause potentiation, which suggested that cyclic AMP may have a direct effect on the receptor/channel complex.
Thesis, PhD Doctor of Philosophy
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