Related Experiment Videos
Dual cell cycle-specific mechanisms mediate the antimitogenic effects of nitric oxide in vascular smooth muscle cells
R Sarkar1, D Gordon, J C Stanley
1Department of Physiology, University of Michigan Medical Center, Ann Arbor 48109-0622, USA.
Objective:
To determine the cell cycle specificity and intracellular mechanisms involved in inhibition by nitric oxide (NO) of vascular smooth muscle cell mitogenesis.
Methods:
Cultured rat aortic smooth muscle cells were synchronized by serum withdrawal, treated with the NO donor S-nitroso-N-acetylpenicillamine and the cyclic GMP analog 8-Br-cGMP at various times during cell cycle progression, and DNA synthesis measured during the S phase. Two additional NO donors, 5-nitroso-glutathione and diethylamine NONOate, were used to confirm the inhibition of DNA synthesis by S-nitroso-N-acetylpenicillamine, and the ability of two antagonists of free NO to reverse the effects of NO donors was also evaluated. Bypass of ribonucleotide reductase by use of exogenous deoxynucleosides was attempted to determine whether inhibition of this S-phase enzyme was the mechanism by which NO inhibited DNA synthesis during the S phase.
Results:
Vascular smooth muscle cell mitogenesis was inhibited by cyclic GMP (cGMP) up to late G1 phase of the cell cycle, which corresponded to the point of greatest sensitivity to exogenous NO. In contrast to cGMP, three different NO donors inhibited DNA synthesis when added to cells synchronized in S phase, beyond the restriction point of cell cycle control in late G1 phase. This S-phase inhibition was reversible by removal of the NO donor or addition of two NO antagonists and was not observed with non-NO analogs of the donors. Inhibition by NO donors in S phase was neither reversed by the guanylate cyclase inhibitor methylene blue nor mimicked by exogenous cGMP. The S-phase inhibition by all three NO donors was reversed partially by bypass of ribonucleotide reductase, establishing this enzyme as an S-phase target of NO.
Conclusions:
These findings demonstrate that NO inhibits smooth muscle mitogenesis by cGMP-dependent and -independent mechanisms acting at distinct points in the cell cycle. NO is the first endogenous substance to have been shown to inhibit mitogenesis beyond the restriction point in late G1 phase, suggesting that it plays a role in regulation of cells that have lost normal mechanisms of G1 growth control, such as the hyperproliferative smooth muscle cells noted in hypertension and restenosis.
Insights
Nitric oxide (NO) inhibits vascular smooth muscle cell proliferation through both cyclic GMP (cGMP)-dependent and -independent pathways. NO uniquely inhibits DNA synthesis in the S phase, targeting ribonucleotide reductase, and plays a role in regulating hyperproliferative cells.
Area of Science:
- Vascular biology
- Cell cycle regulation
- Molecular signaling
Background:
- Vascular smooth muscle cell (VSMC) proliferation is a key process in cardiovascular health and disease.
- Nitric oxide (NO) is a critical signaling molecule in the vasculature.
- Understanding the cell cycle-specific mechanisms of NO action is crucial for developing targeted therapies.
Purpose of the Study:
- To elucidate the cell cycle specificity of nitric oxide (NO)-mediated inhibition of vascular smooth muscle cell (VSMC) mitogenesis.
- To identify the intracellular mechanisms underlying NO's inhibitory effects on VSMC proliferation.
Main Methods:
- Cultured rat aortic smooth muscle cells were synchronized and treated with NO donors and cyclic GMP analogs at different cell cycle stages.
- DNA synthesis was measured during the S phase.
- Ribonucleotide reductase inhibition was assessed by bypassing the enzyme with exogenous deoxynucleosides.
Main Results:
- Cyclic GMP (cGMP) inhibited VSMC mitogenesis up to late G1 phase.
- NO donors inhibited DNA synthesis specifically during the S phase, independent of cGMP.
- S-phase inhibition by NO was partially reversed by bypassing ribonucleotide reductase, identifying it as an NO target.
Conclusions:
- NO inhibits VSMC mitogenesis via distinct cGMP-dependent (G1 phase) and cGMP-independent (S phase) mechanisms.
- NO is the first endogenous substance shown to inhibit mitogenesis beyond the G1 restriction point.
- NO may regulate cells with dysregulated G1 control, relevant to hypertension and restenosis.