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Abrahams SL, Nothup JK, Mansour TE, Gentleman S.
“Adenosine Cyclic 3',5'-Monophosphate in the Liver Fluke, fasciola hepatica. I. Activation of Adenylate Cyclase by 5-Hydroxytryptamine. II. Activation of Protein Kinase by 5- Hydroxytryptamine”.
Molec. Pharmacol.. 1976;12(1):49-58.
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Abstract
Fasciola hepatica were collected from fresh beef livers and particulate fractions of whole flukes, 'tails' or 'heads' (anterior ends) assayed for adenylate cyclase (AC) activity and cyclic AMP concentration using a protein binding assay. 5HT (Calbiochem) caused a marked concentration-dependent accumulation of cyclic AMP especially in 'heads', maximal activation of AC occurring at i mM, and an accompanying increase in motility of the isolated fluke head. The enhanced levels of cyclic AMP were preserved by both caffeine and papaverine, and NaF up to 5 mM caused a specific stimulation of AC. Maxi-mum accumulation of cyclic AMP occurred in 5-10 min. and decreases in levels was not accompanied by a decreasein motility. 5-Methoxytryptamine (1 mM, Calbiochem) caused a 4-fold increase in motility similar to 5HT but had a much diminished effect on cyclic AMP and AC acitivity, while tryptamine (Calbiochem), 5 - hydroxydimethyltryptamine (Sigma-Chem.), 5-hydroxytryptophan (Calbiochem) and dimethyl tryptamine (Sigma-Chem.), all at 1 mM, produced decreasing responses in both stimulation of motility and activation of AC. 0.1 mM LSD (Sandoz) and 1 mM amphetamine (Sigma-Chem.) both strongly stimulated motility but had no effect on cyclic AMP levels. In the presence of O.i mM 5HT, AC activity in the fluke particles was inhibited by 0.5 mcM LSD, and LSD also antagonized the 5HT stimulated increase in cyclic AMP levels, Adenine, adenosine, histamine (Calbiochem), adrenaline (Mann), tryptophan, dopamine, GTP (Sigma - Chem.), glucagon (Lilly) and noradrenaline (Winthrop) had no effect on motility or cyclic AMP levels. Protein kinase activity was 2-5 times greater in the 'head' than in the posterior end of the fluke and in both particulate and supernatant fractions half-maximal activation was obtained at 0.1-0.4 mcM cyclic AMP. The apparent Km for protamine was 0.1 mg/ml and for MgATP 20-50 mcM. Incubation of fluke heads for 5 min with 1 mM 5HT increased protein kinase activity of both fractions in the absence of added cyclic AMP, but with 5 mcM cyclic AMP protein kinase activity was increased inthe particulate and decreased in the super-natant fraction. The degree of activation by cyclic AMP was inversely correlated with endogenous cyclic AMP and 5HT concentration. Incubation of fluke heads with both 5HT and LSD reduced activation by 5HT, while LSD alone had no effect. Cyclic AMP increased phosphorylation of fluke protein in both fractions.
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