The participation of protein kinase C (PKC) in the regeneration of tentacles of Hydra vulgaris was studied. Regeneration was induced by 1,2-sn-dioctanoyl-glycerol (diC-8) and the novel diterpenoidic diacylglycerol verrucosin B (VB), a potent PKC activator extracted from marine sources. VB substantially increased Hydra average tentacle number (ATN) at concentrations 10,000 times lower than those needed for diC-8 to exert an analogous effect. When both synthetic and natural VB analogues were tested, the structure/activity relationship found in Hydra 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-triggered Hydra tentacle regeneration. The involvement of phospholipase A2 (PLA2) activation in Hydra 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 mumolar 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 in Hydra 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 on Hydra 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.
THE POSSIBLE INVOLVEMENT OF PROTEIN-KINASE-C AND PHOSPHOLIPASE-A2 IN HYDRA TENTACLE REGENERATION
De Petrocellis L;Di Marzo V;
1993
Abstract
The participation of protein kinase C (PKC) in the regeneration of tentacles of Hydra vulgaris was studied. Regeneration was induced by 1,2-sn-dioctanoyl-glycerol (diC-8) and the novel diterpenoidic diacylglycerol verrucosin B (VB), a potent PKC activator extracted from marine sources. VB substantially increased Hydra average tentacle number (ATN) at concentrations 10,000 times lower than those needed for diC-8 to exert an analogous effect. When both synthetic and natural VB analogues were tested, the structure/activity relationship found in Hydra 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-triggered Hydra tentacle regeneration. The involvement of phospholipase A2 (PLA2) activation in Hydra 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 mumolar 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 in Hydra 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 on Hydra 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.