Effect of Nitric Oxide on DNA Replication Induced by Angiotensin II in Rat Cardiac Fibroblasts

Abstract

Our previous in vivo studies (Hou et al. J Clin Invest. 1995;96:2469-2477.) demonstrated that chronic inhibition of nitric oxide synthase led to an exaggerated response to relatively low doses of angiotensin II, resulting in a rapid and marked cardiac fibrosis. To examine further the importance of angiotensin II in inducing cardiac fibrosis and the possibility that nitric oxide serves as a modulator of the proliferative effects of angiotensin II, we used cultured rat cardiac fibroblasts to study the interrelationships between these substances. Angiotensin II induced a delayed DNA synthetic response in quiescent cells that occurred 30 hours after exposure to the hormone. The most pronounced effect of angiotensin II on thymidine uptake occurred 36 to 42 hours after the addition to cells. This response was inhibited in a dose-dependent manner by the addition of either S-nitroso-N-acetylpenicillamine or sodium nitroprusside, each a source of nitric oxide. The nitric oxide donor was most effective in reducing thymidine incorporation when added 12 hours after angiotensin II, whereas the metabolite N-acetylpenicillamine had no effect at any time. The inhibitory effect of S-nitroso-N-acetylpenicillamine was mimicked by 8-bromoguanosine 3':5'-cyclic monophosphate but not by 8-bromoadenosine 3':5'-cyclic monophosphate. Nitric oxide donors did not appear to inhibit the induction of c-fos, Egr-1, or other immediate-early genes in response to angiotensin II. The results suggest that nitric oxide affects the cell cycle following the transition into G, and modulates the proliferation of fibroblasts during cardiac fibrosis induced by angiotensin II.

Nitric Oxide–Induced Increase in p21 Sdi1/Cip1/Waf1 Expression During the Cell Cycle in Aortic Adventitial Fibroblasts

“…In an earlier study, 17 we had shown that SNAP would inhibit angiotensin-induced thymidine uptake by cardiac fibroblasts when added at quiescence, yet the NO donor did not prevent cell changes characteristic of transition from G 0 to G 1 , suggesting an effect within the cell cycle. SNAP addition was actually more effective in reducing thymidine incorporation into DNA when added after the G 0 to G 1 transition than when added at quiescence.…”

“…At the end of the incubation period, incorporation of labeled thymidine was determined essentially as described previously by us. 17 All experiments were performed in quadruplicate and are representative of 2 or 3 separate experiments with different preparations of cells.…”

“…18 Northern blot analysis was carried out as previously described. 17,19 The cDNA probe for rat p21 containing a 0.7-kb insert from the open reading frame was kindly provided by Dr B. Schreiber (Boston University School of Medicine). The p53 cDNA probe was a 1-kb insert from mouse p53 cDNA obtained by treatment with SacII and XhoI of a plasmid obtained from Dr K. Ravid (Boston University School of Medicine).…”

Regulation of Gene Expression by Cyclic GMP

“…In VSMCs, mesangial cells, and various fibroblasts, cGMP inhibits proliferation, and the effect is mostly mediated by PKG, although it may involve PKA cross-activation under some conditions. 4,[45][46][47][48][49][50][51][52][53][54] The antiproliferative effect correlates with cGMP inhibition of growth factor-induced extracellular signal-regulated kinase (Erk-1/2) activity (online Table, available in the online data supplement at http://www.circresaha.org; also discussed later), increased expression of MAP kinase phosphatase-1 (MKP-1), 46,47,53 modulation of cell cycle-associated genes, and reduction of ET-1 synthesis. 52,55 In contrast to the antiproliferative effects in VSMCs and fibroblasts, cGMP increases proliferation of endothelial cells.…”

The renin-angiotensin system in the year 2000

“…A somewhat separate, but very pertinent report, especially in relation to the aforementioned effect of angiotensin II in facilitating cardiac remodelling and causing myocardial lesions, was published by Takizawa et al 252 These workers found that nitric oxide modulated the proliferation of fibroblasts with angiotensin II-induced cardiac fibrosis.…”