Forskolin, phosphodiesterase inhibitors, and cyclic AMP analogs inhibit proliferation of cultured bovine aortic endothelial cells

Abstract

The role of cyclic AMP on endothelial cell proliferation was investigated, since these cells can be exposed to high concentrations of physiological and pharmacological agents that alter cyclic AMP metabolism. Cloned bovine aortic endothelial cells were plated at 25,000 cells/35mm dish and grown for 5 days in the presence of phosphodiesterase (PDE) inhibitors, forskolin, or cyclic AMP analogs. The PDE inhibitors dipyridamole, ZK 62 711, isobutylmethylxanthine (IBMX) and theophylline inhibited cell growth in a concentration-dependent manner. Dipyridamole produced a 30% and a 50% inhibition at 5 microM and 12.5 microM, while higher concentrations were cytotoxic. At its therapeutic plasma concentration range (50-100 microM) theophylline inhibited cell proliferation by 15-25%, while IBMX and the highly specific cyclic AMP phosphodiesterase inhibitor, ZK 62 711 inhibited growth by 60-80% and 40-50%, respectively. Forskolin (5 microM) increased cyclic AMP levels and cyclic AMP-kinase activity ratios by 2.5-fold and 2-fold. In the absence of PDE inhibitors forskolin produced a 20% growth inhibition at 0.5 microM and a 60% inhibition at 10 microM. The forskolin dose-response curve was not altered by theophylline, but was shifted to the left by approximately 10-fold with dipyridamole and ZK 62 711 and 5-fold with IBMX. Forskolin (5 microM), by itself produced a 1.8-fold increase in cyclic AMP. In the presence of 5 microM theophylline, dipyridamole, IBMX, and ZK 62 711, cyclic AMP was increased by forskolin 2.0, 2.6, 3.5, and 6.6-fold, respectively. 8-Bromo cyclic AMP and dibutyryl cyclic AMP produced a 55% and 60% growth inhibition at 100 microM. The cyclic GMP analogs were less effective inhibitors of growth (15-30%). Our results demonstrate that cyclic AMP analogs and pharmacological agents that elevate intracellular cyclic AMP levels inhibit cell growth and suggest that cyclic AMP may be an important endogenous regulator of endothelial cell proliferation.

Cyclic Nucleotide Hydrolysis in Bovine Aortic Endothelial Cells in Culture: Differential Regulation in Cobblestone and Spindle Phenotypes

“…The long-term effect of PDE2 and PDE4 inhibition in the cobblestone phenotype suggests that the increase in cAMP due to PDE inhibition contributes to maintaining the cobblestone phenotype, and to preventing the appearance of proliferating cell types which is required for angiogenesis. This is in agreement with the antiproliferative effect reported for cAMP in endothelial cells [9]. …”

“…A cross-talk between PDEs mediated by cGMP may participate in intracellular signalling [1, 7, 8], and furthermore, cyclic nucleotides have been shown to be important endogeneous regulators of cell proliferation. Endothelial cell growth inhibition is associated with an elevation of intracellular cAMP [9], while either a decrease [10]or an increase [11]in intracellular cGMP has been reported to inhibit growth.…”

Mitogenic response of rat schwann cells to fibroblast growth factors is potentiated by increased intracellular cyclic AMP levels

“…Furthermore, fibronectin was shown to stimulate Schwann cell growth ' isobutylmethylxanthine (IBMX) is partially active in enhancing the cellular response to FGFs. Elevated intracellular cAMP levels by forskolin or phosphodiesterase inhibitor treatment were shown to be growth inhibitory in bovine aortic endothelial cells (Leitman et al, 1986) and human foreskin fibroblasts (Heldin et al, 1989). In this report, we showed that the growth response of fibroblasts isolated from connective tissues surrounding the sciatic nerves to bFGF was also slightly inhibited by forskolin at a concentration that was optimally stimulatory for Schwann cells.…”

Opposing Effects on Modulation of Angiogenesis by Protein Kinase C and cAMP-Mediated Pathways

“…cAMP has been shown to enhance or inhib it cell proliferation depending on the cell type used. Inhi bition of proliferation was observed with bovine aortic EC [31], pig aortic EC [32], and rat brain microvascular EC [33], whereas stimulation was observed in a fetal bovine aortic EC line [34], Furthermore, cAMP levels are increased by a number of growth factors [14] and eleva tion of cAMP can lead to reversal of the transformed phe notype [15]. The antitumor activity of the cAMP analog 8-Cl-cAMP may be due to its ability to restore normal gene transcription in tumor cells through an action on type II PKA [10].…”