Calcium/calmodulin transduces thrombin-stimulated secretion: studies in intact and minimally permeabilized human umbilical vein endothelial cells.

Thrombin stimulates cultured endothelial cells (EC) to secrete stored von Willebrand factor (vWF), but the signal transduction pathways are poorly defined. Thrombin is known to elevate the concentration of intracellular calcium ([Ca2+]i) and to activate protein kinase C (PKC) in EC. Since both calcium ionophores and phorbol esters release vWF, both second messenger pathways have been postulated to participate in vWF secretion in response to naturally occurring agonists. We find that in intact human EC, vWF secretion stimulated by either thrombin or by a thrombin receptor activating peptide, TR(42-55), can be correlated with agonist-induced elevations of [Ca2+]i. Further evidence implicating calcium in the signal transduction pathway is suggested by the finding that MAPTAM, a cell-permeant calcium chelator, in combination with the extracellular calcium chelator EGTA, can inhibit thrombin-stimulated secretion. In contrast, the observation that staurosporine (a pharmacological inhibitor of PKC) blocks phorbol ester- but not thrombin-stimulated secretion provides evidence against PKC-mediated signal transduction. To examine further the signal transduction pathway initiated by thrombin, we developed novel conditions for minimal permeabilization of EC with saponin (4-8 micrograms/ml for 5-15 min at 37 degrees C) which allow the introduction of small extracellular molecules without the loss of large intracellular proteins and which retain thrombin-stimulated secretion. These minimally permeabilized cells secrete vWF in response to exogenous calcium, and EGTA blocks thrombin-induced secretion. Moreover, in these cells, thrombin-stimulated secretion is blocked by a calmodulin-binding inhibitory peptide but not by a PKC inhibitory peptide. Taken together, these findings demonstrate that thrombin-stimulated vWF secretion is transduced by a rise in [Ca2+]i and provide the first evidence for the role of calmodulin in this process.

LY290181, an inhibitor of diabetes-induced vascular dysfunction, blocks protein kinase C-stimulated transcriptional activation through inhibition of transcription factor binding to a phorbol response element.

Previous studies have shown that high glucose levels and diabetes induce an elevation in protein kinase C (PKC) activity in vascular cells and tissues susceptible to diabetic complications. In addition, PKC activation has been shown to modulate vascular cell growth, permeability, and gene expression, processes thought to be involved in the development of vascular complications. Using two in vivo model systems, we have identified a novel inhibitor of diabetic vascular dysfunction, LY290181. LY290181 prevented glucose-induced increases in blood flow and permeability in rat granulation tissue and corresponding vascular changes in the retina, sciatic nerve, and aorta of diabetic rats. Tested for its ability to inhibit PKC-regulated processes, LY290181 inhibited phorbol ester-stimulated plasminogen activator activity in a dose-dependent manner in bovine retinal endothelial cells and in human dermal fibroblasts. In addition, LY290181 inhibited phorbol ester-stimulated activation of the porcine urokinase plasminogen activator (uPA) promoter (-4600/+398) linked to the chloramphenicol acetyltransferase (CAT) reporter gene (p4660CAT). More detailed analysis of the uPA promoter revealed that LY290181 inhibited phorbol ester-stimulated activation of the uPA phorbol response element (-2458/-2349) located upstream of the thymidine kinase promoter (puPATKCAT). LY290181 appears to inhibit uPA promoter activation by blocking phorbol ester-stimulated binding of nuclear proteins to the uPA PEA3/12-0-tetradecanoylphorbol 13-acetate responsive element (TRE). These results suggest that LY290181 may inhibit diabetes-induced vascular dysfunction by inhibiting transcription factor binding to specific PKC-regulated genes involved in vascular function.

Prolonged peak elevations in cytoplasmic free calcium ions, derived from intracellular stores, correlate with the extent of thrombin-stimulated exocytosis in single human umbilical vein endothelial cells.

We have used indo-1-loaded human endothelial cells (EC) in monolayer culture and quantitative laser scanning fluorescence microscopy techniques to investigate the magnitude and duration of the change in cytoplasmic free calcium ([Ca2+]i) required for thrombin-stimulated von Willebrand factor (vWF) secretion in individual EC. Both alpha-thrombin and a 14 amino acid thrombin receptor activating peptide stimulate an increase in EC [Ca2+]i that is agonist dose dependent. Low-dose agonist treatment generates asynchronous oscillations (i.e., repetitive spikes < 80 sec duration) in [Ca2+]i. Stimulation with higher agonist concentrations generates a prolonged single peak elevation in [Ca2+]i. Both the number of cells displaying prolonged [Ca2+]i peaks and the mean amplitude of the peaks increase as a function of agonist concentration. Higher doses of agonist also cause sustained elevations in [Ca2+]i that depend upon extracellular Ca2+. Oscillations in [Ca2+]i are not sufficient to stimulate significant vWF secretion, and sustained elevations in [Ca2+]i are not required for maximal secretion. Both the number of cells displaying prolonged peaks and the mean peak amplitude correlate with increasing levels of vWF secretion from the culture. We have used the expression of P-selectin, a secretory granule membrane protein, as a marker for measuring thrombin-induced exocytosis in individual EC. Both the number of secreting cells and the amount of secretion per cell increase as a function of thrombin concentration. The graded responses in [Ca2+]i amplitudes and the graded exocytotic response may be causally related.

Protein kinase C isozymes differentially regulate promoters containing PEA-3/12-O-tetradecanoylphorbol-13-acetate response element motifs.

To investigate the regulation of promoters containing classical phorbol ester response sequences (PEA-3/12-O-tetradecanoylphorbol-13-acetate response element motifs) by protein kinase C (PKC) isozymes, co-transfections were performed in human dermal fibroblasts with a plasmid containing either the human collagenase promoter or the porcine urokinase plasminogen activator (uPA) promoter linked to the chloramphenicol acetyltransferase gene and a plasmid expressing an individual PKC isozyme. Using this experimental design, seven PKC isozymes were analyzed for their ability to trans-activate the collagenase and uPA promoters. Our results demonstrate that only PKC delta, epsilon, and eta trans-activated the collagenase promoter and that binding of Ap-1 family members to the collagenase 12-O-tetradecanoylphorbol-13-acetate response element (TRE) was not responsible for the isozyme-specific trans-activation. In contrast, the uPA promoter was stimulated by all of the PKC isozymes examined (PKC alpha, betaII, gamma, delta, epsilon, zeta, and eta). These results indicate that PKC isozymes differentially regulate promoters containing PEA-3/TRE motifs and suggest that individual isozymes play unique roles within the cell.