Smooth muscle cell expression of type I cyclic GMP-dependent protein kinase is suppressed by continuous exposure to nitrovasodilators, theophylline, cyclic GMP, and cyclic AMP.

A key component of the nitric oxide-cyclic guanosine monophosphate (cGMP) pathway in smooth muscle cells (SMC) is the type I GMP-dependent protein kinase (PK-G I). Activation of PK-G I mediates the reduction of cytoplasmic calcium concentrations and vasorelaxation. In this manuscript, we demonstrate that continuous exposure of SMC in culture to the nitrovasodilators S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) results in approximately 75% suppression of PK-G I mRNA by 48 h. PK-G I mRNA and protein were also suppressed by continuous exposure to cGMP analogues 8-bromo- and 8-(4-chlorophenylthio) guanosine-3,5-monophosphate or the cAMP analogue dibutyryl cAMP. These results suggest that activation of one or both of the cyclic nucleotide-dependent protein kinases mediates PK-G I mRNA suppression. Using isoform-specific cDNA probes, only the PK-G I alpha was detected in SMC, either at baseline or after suppression, while PK-G I beta was not detected, indicating that isoform switch was not contributing to the gene regulation. Using the transcription inhibitor actinomycin D, the PK-G I mRNA half-life in bovine SMC was observed to be 5 h. The half-life was not affected by the addition of SNAP to actinomycin D, indicating no effect on PK-G I mRNA stability. Nuclear runoff studies indicated a suppression of PK-G I gene transcription by SNAP. PK-G I suppression was also observed in vivo in rats given isosorbide dinitrate in the drinking water, with a dose-dependent suppression of PK-G I protein in the aorta. PK-G I antigen in whole rat lung extract was also suppressed by administration of isosorbide or theophylline in the drinking water. These data may contribute to our understanding of nitrovasodilator resistance, a phenomenon resulting from continuous exposure to nitroglycerin or other nitrovasodilators.

The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin.

Angiostatin, a potent naturally occurring inhibitor of angiogenesis and growth of tumor metastases, is generated by cancer-mediated proteolysis of plasminogen. Human prostate carcinoma cells (PC-3) release enzymatic activity that converts plasminogen to angiostatin. We have now identified two components released by PC-3 cells, urokinase (uPA) and free sulfhydryl donors (FSDs), that are sufficient for angiostatin generation. Furthermore, in a defined cell-free system, plasminogen activators [uPA, tissue-type plasminogen activator (tPA), or streptokinase], in combination with one of a series of FSDs (N-acetyl-L-cysteine, D-penicillamine, captopril, L-cysteine, or reduced glutathione] generate angiostatin from plasminogen. An essential role of plasmin catalytic activity for angiostatin generation was identified by using recombinant mutant plasminogens as substrates. The wild-type recombinant plasminogen was converted to angiostatin in the setting of uPA/FSD; however, a plasminogen activation site mutant and a catalytically inactive mutant failed to generate angiostatin. Cell-free derived angiostatin inhibited angiogenesis in vitro and in vivo and suppressed the growth of Lewis lung carcinoma metastases. These findings define a direct mechanism for cancer-cell-mediated angiostatin generation and permit large-scale production of bioactive angiostatin for investigation and potential therapeutic application.

Human prostate carcinoma cells express enzymatic activity that converts human plasminogen to the angiogenesis inhibitor, angiostatin.

Angiostatin is an inhibitor of angiogenesis and metastatic growth that is found in tumor-bearing animals and can be generated in vitro by the proteolytic cleavage of plasminogen. The mechanism by which angiostatin is produced in vivo has not been defined. We now demonstrate that human prostate carcinoma cell lines (PC-3, DU-145, and LN-CaP) express enzymatic activity that can generate bioactive angiostatin from purified human plasminogen or plasmin. Affinity purified PC-3-derived angiostatin inhibited human endothelial cell proliferation, basic fibroblast growth factor-induced migration, endothelial cell tube formation, and basic fibroblast growth factor-induced corneal angiogenesis. Studies with proteinase inhibitors demonstrated that a serine proteinase is necessary for angiostatin generation. These data indicate that bioactive angiostatin can be generated directly by human prostate cancer cells and that serine proteinase activity is necessary for angiostatin generation.

cAMP influence on transcription of thrombomodulin is dependent on de novo synthesis of a protein intermediate: evidence for cohesive regulation of myogenic proteins in vascular smooth muscle.

We have previously shown that cyclic adenosine monophosphate (cAMP) increases thrombomodulin (TM) mRNA and protein in vascular smooth muscle cells (VSMCs). The mechanism of that enhancement is now further defined. A time course evaluation of this effect by Northern blot analysis showed that exposure to the cAMP analog dibutyryl-cAMP and theophylline (CT) amplified TM mRNA sixfold by 3 hours. This effect was sustained through 9 hours and began to decline by 24 hours of CT exposure. In vitro exposure of VSMCs either to CT and actinomycin D or to actinomycin D alone showed equivalent half-lives for TM mRNA. This indicates that the increase in TM mRNA with CT supplementation was not the result of enhanced mRNA stability. Nuclear run-off analysis of VSMCs grown in the presence of control or CT-supplemented medium showed that the increase in TM mRNA in VSMCs with CT exposure was transcriptional. CT exposure was associated with an eightfold increase in measured TM transcription at 90 minutes. As previously reported, cAMP induced a decrease in tropomyosin and in alpha-actin mRNA species, a change that paralleled the enhancement of TM. Thus cAMP enhances transcription of this antithrombotic species while simultaneously causing diminished expression of these myogenic mRNA species. Addition of cycloheximide prevented the cAMP-mediated increase in TM mRNA and curtailed the down-regulation of myogenic mRNA species, alpha-actin, and tropomyosin. This suggests that the cAMP-mediated down-regulation of some smooth muscle-specific mRNA, including tropomyosin mRNA and alpha-actin mRNA, like the enhancement of TM transcription, is dependent on de novo protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)