. Nitric oxide donor induces temporal and dose-dependent reduction of gene expression in human endothelial cells. Am J Physiol Heart Circ Physiol 287: H1977-H1986, 2004. First published July 8, 2004 doi:10.1152/ ajpheart.00323.2004.-The present study tested the hypothesis that acute increases in nitric oxide (NO) exert substantial influences on gene transcription in endothelial cells (ECs) via guanylyl cyclase (GC). Human umbilical veins ECs (HUVECs) were exposed to 0.1, 1, and 10 mM of sodium nitroprusside (SNP) for 4 h and to 1 mM SNP or 250 M of (Z)-1[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]-diazen-1-ium-1,2-diolate (DETA-NONOate) for 2, 4, 8, and 24 h. Also, cells were exposed to DETA-NONOate in the presence and absence of the GC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 10 M) for 4 h. RNA was isolated, reverse transcribed, Cy3 and Cy5 labeled, and analyzed using cDNA microarrays. Increasing doses of SNP predominantly depressed gene expression in HUVECs. Gene function was related to growth, adhesion, and cell structure. DETA-NONOate evoked a wave of expression changes (maximum at 4 h), with a remarkable downregulation of the transcription factors MSX1, RELB, and Egr-1. Both SNP-and DETANONOate-induced gene expression had faded after 24 h, despite continued elevation of cGMP in the medium. Coadministration of ODQ decreased many, but not all, of the transcriptional responses to DETA-NONOate. NO pronouncedly depressed EC gene expression, in particular of transcription factors. The observation that many, but not all, transcriptional changes induced by NO could be inhibited by inhibition of GC indicates the presence of GC-independent NO actions on gene expression. Thus EC gene expression responds to NO; however, the transcriptional response fades during prolonged exposure. This could allow the EC to respond to increased shear, without vigorous changes in gene expression. microarray; nitrate tolerance; guanylate cyclase; Egr-1; V-Rel avian reticuloendotheliosis viral oncogene homolog B SUBSTANTIAL EVIDENCE is now available on the actions of nitric oxide (NO) in the vasculature (4). The, by now already classical, paradigm is that NO, released from endothelial cells (ECs) in response to shear stress, acts on vascular smooth muscle cells (VSMCs) by activating soluble guanylyl cyclase (GC), resulting in cGMP formation and vasodilation (4). In addition, NO affects multiple other processes in VSMCs, i.e., inhibits proliferation (41) and modulates expression of genes with vasoactive function (19). NO also affects EC function in an autocrine fashion and thereby inhibits adhesion, inflammation, and generation of proliferative stimuli. Indeed, exogenously applied NO can affect transcription of adhesion molecules, such as ICAM-1 and E-selectin (23) and VEGF (11) in ECs that are activated with IL-1␤ or TNF-␣. Interestingly, there is a large body of data available to support that NO actions in physiological settings are short lived, because of acute desensitization (5, 14) and slower adaptations of the GC ...

Single smooth muscle cells from bovine coronary artery were obtained by enzymatic digestion. 20.1 ± 3.2% (mean value of seven experiments) of the dispersed cells were in a relaxed, noncontracted state. These cells responded to electrically induced depolarization with a graded contraction to a maximum shortening of 47.8% of initial length; the corresponding maximum for K⁺-induced depolarization was 61.7% of initial length. Cells relaxed only a small percentage (≈ 20%) of the amount of shortening, but could be restimulated several times to contract to even shorter lengths. The decrease in mean cell length of dispersed cells exposed to serotonin, acetylcholine, and ergonovine maleate was 21.0, 23.0 and 25.5%, respectively (p < 0.05). No significant decrease in cell length was obtained with norepinephrine. The ultrastructure of the relaxed cells seemed to be unaltered, suggesting that the accuracy of the physiological results is not impaired by induced structural damage.