Contribution of phosphoinositide-dependent signalling to photomotility of Blepharisma ciliate

The effect of experimental procedures designed to modify an intracellular p hosphoinositide signalling pathway, which may be instrumental in the photop hobic response of the protozoan ciliate Blepharisma japonicum, has been inv estigated. To assess this issue, the latency time of the photophobic respon se and the cell photoresponsiveness have been assayed employing newly devel oped computerized videorecording and standard macro-photographic methods. C ell incubation with neomycin, heparin and Li+, drugs known to greatly imped e phosphoinositide turnover, causes evident dose-dependent changes in cell photomotile behaviour. The strongest effect on photoresponses is exerted by neomycin, a potent inhibitor of polyphosphoinositide hydrolysis. The prese nce of micromolar concentrations of neomycin in the cell medium causes both prolongation of response latency and decrease of cell photoresponsiveness. Neomycin at higher concentrations ( > 10 mu M) abolishes the cell response to light at the highest applied intensity. A slightly lower inhibition of cell responsiveness to light stimulation and prolongation of response laten cy are observed in cells incubated in the presence of heparin, an inositol trisphosphate receptor antagonist. Lithium ions, widely known to deplete th e intracellular phosphoinositide pathway intermediate, inositol trisphospha te, added to the cell medium at millimolar level, also cause a slowly devel oping inhibitory effect on cell photoresponses. Mastoparan, a specific G-pr otein activator, efficiently mimics the effect of light stimulation. In dar k-adapted ciliates, it elicits ciliary reversal with. the response latency typical for ciliary reversal during the photophobic response. Sustained tre atment of Blepharisma cells with mastoparan also suppresses the photorespon siveness, as in the case of cell adaptation to light during prolonged illum ination. The mastoparan-induced responses can be eliminated by pretreatment of the cells with neomycin. Moreover, using antibodies raised against bovi ne transducin, a cross-reacting protein with an apparent molecular mass of about 55 kDa in the Blepharisma cortex fraction is detected on immunoblots. The obtained results indirectly suggest that the changes in internal inosi tol trisphosphate level, possibly elicited by G-protein-coupled phospholipa se C, might play a role in the photophobic response of Blepharisma. However , further experiments are necessary to clarify the possible coupling betwee n the G-protein and the putative phospholipase C. (C) 2000 Elsevier Science S.A. All rights reserved.