We tested the hypothesis that vessel homeostasis is maintained through the cross talk of shear-induced production of prostacyclin and nitric oxide (NO). Confluent human umbilical vein endothelial cells (HUVEC) were exposed to fluid shear stress at 15 dyn/cm(2) using a cone-plate device, and the concentrations of 6-keto-PGF(1alpha) and NO metabolites (nitrate and nitrite) in the medium were measured with radioimmunoassay and the Greiss method, respectively. Compared with static control, shear stress increased cumulative prostacyclin production by twofold after 90 min of exposure. Inhibition of NO synthase enhanced flow-induced prostacyclin production by twofold without affecting the baseline production. Guanylyl cyclase inhibitor enhanced flow-induced prostacyclin production to the same degree. In contrast, a stable agonist of cGMP attenuated the rapid early phase of flow-dependent prostacyclin production. Shear-induced NO metabolite production was unaffected even after indomethacin inhibited prostacyclin production. We conclude that NO shows an inhibitory effect on prostacyclin production under shear stress and that vessel homeostasis may be maintained through an increase in prostacyclin production when NO synthesis is impaired in endothelial cells.

Background-We recently demonstrated that phospholipase C (PLC)-␦1 activity in cultured skin fibroblasts obtained from patients with coronary spastic angina (CSA) is enhanced. We tested the hypothesis that structural abnormality in PLC-␦1 isoform is a cause of the enhanced activity. Methods and Results-Sequence analysis of the cDNA coding for PLC-␦1 obtained from fibroblasts revealed that one conversion of guanine to adenine (A) was present at nucleotide position 864 in one CSA patient, resulting in the amino acid replacement of arginine 257 by histidine (R257H). The incidence of 864A/A in genomic DNA, analyzed by single-strand conformation polymorphism, was greater in patients with CSA than in male control subjects (6 of 57 patients with CSA versus 1 of 62 control subjects, PϽ0.05). The activity of the variant PLC-␦1 protein under free calcium concentration between 10 Ϫ8 and 10 Ϫ7 mol/L was 2-fold higher than that of the wild-type protein. Baseline intracellular calcium concentration ([Ca 2ϩ ] i ) in human embryonic kidney 293 cells transfected with the variant PLC-␦1 was higher than that in cells with the wild type. The peak increase in [Ca 2ϩ ] i in response to acetylcholine at 10 Ϫ6 and 10 Ϫ5 mol/L was greater in the cells with the variant PLC-␦1 than in those with the wild type. Conclusions-These findings indicate that the R257H variant in the PLC-␦1 gene detected in patients with CSA is associated with enhancement of enzyme activity, and they describe a novel mechanism for the enhanced coronary vasomotility in

We demonstrated recently that coupling factor 6, an essential component of the energy-transducing stalk of mitochondrial ATP synthase, suppresses the synthesis of prostacyclin in vascular endothelial cells. Here, we tested the hypothesis that coupling factor 6 is present on the cell surface and is involved in the regulation of systemic circulation. This peptide is present on the surface of CRL-2222 vascular endothelial cells and is released by these cells into the medium. In vivo, the peptide circulates in the vascular system of the rat, and its gene expression and plasma concentration are higher in spontaneously hypertensive rats (SHRs) than in normotensive controls. Elevation of blood pressure with norepinephrine did not affect the plasma concentration of coupling factor 6. Intravenous injection of recombinant peptide increased blood pressure, apparently by suppressing prostacyclin synthesis, whereas a specific Ab to coupling factor 6 decreased systemic blood pressure concomitantly with an increase in plasma prostacyclin. Interestingly, the antibody's hypotensive effect could be abolished by treating with the cyclooxygenase inhibitor indomethacin. These findings indicate that mitochondrial coupling factor 6 functions as a potent endogenous vasoconstrictor in the fashion of a circulating hormone and may suggest a new mechanism for hypertension.

Background-We showed that mitochondrial coupling factor 6 (CF6), an endogenous inhibitor of prostacyclin synthesis, is present in the systemic circulation as a pressor substance in rats. We investigated the possibility of vascular endothelial cells as a source of circulating CF6. Methods and Results-We used 2 cultured endothelial cell lines, human umbilical vein endothelial cells (HUVECs) and ECV 304 cells (transformed HUVECs), for this study. Immunofluorescence microscopy of both ECV 304 and HUVECs confirmed the surface-associated immunoreactivity of anti-CF6 antibody on the plasma membrane. The concentration of CF6 in the medium increased gradually with time in both ECV 304 and HUVECs in static conditions. Exposure of ECV 304 and HUVECs to a fluid shear stress enhanced the release of CF6: In ECV 304, the concentration of CF6 in the medium (ng · well Ϫ1 · 6 hours