Protein kinase C-mediated desensitization of the muscarinic response in rat lacrimal gland cells.

1. Desensitization of the Ca2+ release response evoked by acetylcholine in acinar cells from rat lacrimal glands was studied using the Ca(2+)-sensitive dye Fura-2. The evolution of the amplitude and half-maximal rise time (t1/2) of the Ca2+ response was followed as a function of trial number in a series of stimulations. 2. Under control conditions repetitive applications of acetylcholine (15 s long applications every minute) led to a linear decrease of the amplitude and to a linear increase of t1/2 with trial number. Both amplitude and t1/2 recovered their control values after 20 min of washing. 3. Staurosporine (0.2-1 microM), an inhibitor of protein kinase C, was found to decrease the slopes of the variation of amplitude and t1/2 with trial number. 4. Prolonged treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of protein kinase C (100-250 nM for 2-4 h), also led to a markedly decreased desensitization, presumably as a result of down-regulation of protein kinase C. On the other hand moderate pre-treatments with TPA (16-32 nM for 10 min) strongly inhibited the response, most probably as a result of protein kinase C activation. 5. Application of oleoylacetylglycerol (50 microM), a weaker activator of protein kinase C, inhibited the response and enhanced desensitization. These effects were, however, not obtained after down-regulation of protein kinase C with strong exposure to TPA. 6. We conclude that protein kinase C activation following the ACh-induced Ca2+ rise and the concomitant diacylglycerol production mediates desensitization of the response. 7. Arachidonic acid (100 microM) inhibited the ACh-induced response and enhanced desensitization. However, this effect did not appear to be mediated by protein kinase C since it was also obtained with docosahexaenoic acid, an analogue of arachidonic acid which does not activate protein kinase C.