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The participation of protein kinase C (PKC) in the regeneration of tentacles ofHydra vulgaris was studied. Regeneration was induced by 1,2-sn-dioctanoyl-glycerol (diC8) and the novel diterpenoidic diacylglycerol verrucosin B (VB), a potent PKC activator extracted from marine sources. VB substantially increasedHydra average tentacle number (ATN) at concentrations 10,000 times lower than those needed for diC8 to exert an analogous effect. When both synthetic and natural VB analogues were tested, the structure/activity relationship found inHydra tentacle regeneration was identical to that known for DAG-induced activation of PKC in vitro. VB-induced increase of ATN was strongly counteracted by the PKC inhibitors sphingosine and A3, but was not synergic with a tenfold increase of extracellular Ca2+ concentration or with an increase of intracellular Ca2+ concentration obtained either with the ionophore A23187 or with thapsigargin. This suggested the involvement of a non-Ca2+-dependent PKC in VB-triggeredHydra tentacle regeneration. The involvement of phospholipase A2 (PLA2) activation inHydra regenerative processes was studied using the novel site-specific inhibitor of the enzyme, oleyloxyethylphosphorylcholine (OOPC), which brought about a striking inhibition of ATN in the low μmolar range. This effect was reversed by arachidonic acid (AA), while an enhancement of ATN was also observed with an inhibitor of AA uptake from membrane phospholipids, thus suggesting that PLA2-catalysed liberation of AA is involved inHydra tentacle regeneration. OOPC also blocked verrucosin B-induced PKC-mediated enhancement of ATN, thus suggesting that this effect is also mediated by PLA2 activation. ATN was increased also by compound 48/80, a direct activator of pertussis toxin-sensitive GTP-binding proteins, and this effect was counteracted by pertussis toxin pretreatment. None of the known AA cascade inhibitors exhibited an effect on ATN comparable to that exerted by OOPC, but, surprisingly, the cycloxygenase inhibitor indomethacin strongly enhanced ATN, thus suggesting that prostanoids might effect a negative control onHydra regenerative processes. This represents the first attempt so far reported to study the implication of more than one biochemical pathway as a signalling event in the hydroid regenerative processes.

Previous studies conducted in cytosolic extracts of the freshwater hydrozoanHydra vulgarisled to the finding of an abundant 11(R)-lipoxygenase catalyzing the peroxidation of polyunsaturated fatty acid (PUFAs) on the tenth carbon atom from the aliphatic end (ω10 peroxidation). Here we describe experiments aimed at identifying the actual metabolites generated in vivo by such enzymic activity. Homogenates ofH. vulgarispolyps were analyzed by HPLC. This showed the presence of three major components chromatographically identical to three metabolites obtained when incubating the homogenates with exogenous α-linolenic acid (α-LA). The presence, in extracts of polyps prelabelled with [14C]-α-linolenic acid, of radioactive metabolites displaying the same chromatographic properties, substantiated the hypothesis that the natural products isolated in vivo are derived from α-LA. Gas chromatographic analyses revealed that this was the most abundant PUFA in both free and phosphoglyceride-bound fatty acid pools. [1H]-NMR analysis of the endogenous substances, carried out in comparison with products obtained from exogenously incubated α-LA, indicated that their structures were those of 9-hydroxy-, 9-hydroperoxy- and 9-keto-octadeca-10E-12Z-15Z-trienoic acids (9-α-HOTrE,-HPOTrE and-KOTrE).Hydrahomogenates transformed 9-α-HPOTrE partly into 9-α-HOTrE and partly into 9-α-KOTrE. Chiral phase HPLC conducted on 9-α-HOTrE established that this metabolite was composed mostly of theRanantiomer. These observations, and the finding that the presence of exogenous arachidonic acid in incubated homogenates significantly reduces the production of α-LA metabolites, provide strong evidence that these compounds are produced by an enzymic activity identical to the previously-describedH. vulgaris (R)-ω10-lipoxygenase. Further experiments suggested that α-LA, acting as the native substrate for this enzyme, is mainly esterified on the 2 position ofHydraphosphoglycerides, and that the production of the α-LA metabolites described here for the first time from natural sources, can be potentially enhanced in vivo by stimuli activating phospholipase A2.