Calcium-dependent phosphorylation of proteins in rabbit ciliary processes.

Calcium-dependent phosphorylation of endogenous substrate proteins in albino rabbit ciliary processes was studied by SDS-polyacrylamide gel electrophoresis and autoradiography. In the soluble fraction, a modest augmentation of phosphorylation was observed by Ca2+ alone and together with the additional activators, calmodulin (CAM) or phorbol myristate acetate (PMA). However, there was a greater enhancement of protein phosphorylation by Ca2+ and activators in the particulate fraction. The degree of Ca2+-CAM-dependent protein phosphorylation was greater than that of Ca2+-PMA-dependent phosphorylation. Endogenous substrate proteins for Ca2+-CAM-dependent protein kinases had apparent molecular sizes of 205,170,150,130,77,58,40,32 and 18 kDa. Phosphorylation of the 58 kDa protein band was strongest. This protein was identified as vimentin on the basis of its behavior with Triton-X100 treatment, and by Western blotting using anti-vimentin antibody. Endogenous substrates of protein kinase C (Ca2+-PMA-dependent) were located at 87 kDa and possibly in the 56 and 54 kDa protein bands. A 50 kDa protein was found to be phosphorylated in the presence of Ca2+ alone, and was not affected by the presence of other activators (CAM or PMA). A Ca2+-dependent dephosphorylation of a 43 kDa protein was observed, and some proteins rapidly phosphorylated by Ca2+-CAM kinase were also relatively quickly dephosphorylated at incubation times greater than 1 min.

Role of phosphoinositides in the regulation of endothelial prostacyclin production.

To elucidate the role of the phosphoinositide signal transduction system in endothelial endothelial prostacyclin production, endothelial cells from human umbilical veins previously labelled with 3H-inositol were incubated with thrombin or histamine. Water-soluble inositol phosphates were separated on anion exchange columns. Both agonists evoked transient bursts of inositol phosphate production with inositol trisphosphate peaking at 15 seconds in histamine-stimulated cells and at 60 seconds in thrombin-stimulated cells. The inositol phosphate production was closely linked to prostacyclin production. After stimulation, there was concurrent desensitization to prostacyclin production and formation of inositol phosphates. Arachidonic acid and the Ca2+-ionophore A23187 did not affect inositol phosphate production in concentrations sufficient to increase prostacyclin production 20-fold, and they did not affect desensitization to a subsequent thrombin stimulation. The phorbol ester 12-o-tetradecanoyl phorbol 13-acetate, a stimulator of protein kinase C, inhibited thrombin-induced generation of inositol phosphates, enhanced A23187-mediated prostacyclin production, and had complex effects on thrombin-mediated prostacyclin production, but had no effect on its production from extrinsic arachidonic acid. The current data suggest that production of inositol phosphates is a link in receptor stimulation of endothelial cells to produce prostacyclin and that associated activation of protein kinase C affects both the generation of second messengers and the release of arachidonic acid.