In addition to nitric oxide (NO) and prostacyclin (PGI2), the endothelium generates the endothelium-derived hyperpolarizing factor (EDHF). We set out to determine whether an EDHF-like response can be detected in wild-type (WT) and endothelial NO synthase knockout mice (eNOS ؊͞؊) mice. Vasodilator responses to endothelium-dependent agonists were determined in vivo and in vitro. In vivo, bradykinin induced a pronounced, dose-dependent decrease in mean arterial pressure (MAP) which did not differ between WT and eNOS ؊͞؊ mice and was unaffected by treatment with N -nitro-L-arginine methyl ester and diclofenac. In the salineperfused hindlimb of WT and eNOS ؊͞؊ mice, marked N -nitro-L-arginine (L-NA, 300 mol͞liter)-and diclofenac-insensitive vasodilations in response to both bradykinin and acetylcholine (ACh) were observed, which were more pronounced than the agonistinduced vasodilation in the hindlimb of WT in the absence of L-NA. This endothelium-dependent, NO͞PGI2-independent vasodilatation was sensitive to KCl (40 mM) and to the combination of apamin and charybdotoxin. Gap junction inhibitors (18␣-glycyrrhetinic acid, octanol, heptanol) and CB-1 cannabinoid-receptor agonists (⌬ 9 -tetrahydrocannabinol, HU210) impaired EDHF-mediated vasodilation, whereas inhibition of cytochrome P450 enzymes, soluble guanylyl cyclase, or adenosine receptors had no effect on EDHFmediated responses. These results demonstrate that in murine resistance vessels the predominant agonist-induced endotheliumdependent vasodilation in vivo and in vitro is not mediated by NO, PGI 2 , or a cytochrome P450 metabolite, but by an EDHF-like principle that requires functional gap junctions. T he endothelium plays an important role in the control of vascular tone. At least three different vasodilating agents are synthesized by the endothelium upon exposure to mechanical forces, such as shear stress, or to receptor-dependent agonists, such as acetylcholine (ACh) and bradykinin. Whereas the properties of two of these vasodilators, nitric oxide (NO) and prostacyclin (PGI 2 ), have been extensively investigated, the nature and mechanism of action of the third vasodilator, the endothelium-derived hyperpolarizing factor (EDHF), is still controversial (1-7).The purpose of the present study was to characterize EDHFmediated responses in vivo, in isolated arteries, and in the perfused hindlimb of the mouse. To address the hypothesis that NO might be EDHF, we also determined endotheliumdependent relaxation in endothelial NO synthase (eNOS) knockout (eNOS Ϫ͞Ϫ) mice. Materials and MethodsMice. Homozygous eNOS Ϫ͞Ϫ mice were obtained from the colonies at Heinrich-Heine-Universität, Düsseldorf (genetic background C57BL͞b6) and Beth Israel

Abstract-Endogenously produced nitric oxide (NO) modulates nitrovasodilator-induced relaxation. We investigated the underlying mechanism in wild-type (WT) mice and endothelial NO synthase knockout (eNOS Ϫ/Ϫ ) mice to determine whether a chronic lack of endothelial NO alters the soluble guanylyl cyclase (sGC) pathway. In aortic segments from eNOS Ϫ/Ϫ mice, the vasodilator sensitivity to sodium nitroprusside (SNP) was significantly greater than that in WT mice. There was no difference in sensitivity to the G-kinase I activator 8-para-chlorophenylthio-cGMP or to cromakalim. N -Nitro-L-arginine had no effect on the SNP-induced relaxation in eNOS Ϫ/Ϫ but increased the sensitivity in WT mice so it was no longer different than that of eNOS Ϫ/Ϫ . Basal cGMP levels in aortic rings were significantly lower in eNOS Ϫ/Ϫ mice than in WT mice. SNP (300 nmol/L) induced a significantly greater cGMP accumulation in eNOSmice than in WT mice. The maximal SNP-induced (10 mol/L) increase in cGMP was similar in both strains. SNP-stimulated sGC activity was significantly greater in eNOS Ϫ/Ϫ mice than in WT mice. Incubation of aortic segments from WT mice with N -nitro-L-arginine increased sGC activity, an effect prevented by coincubation with SNP (10 mol/L). The aortic expressions of the sGC ␣1 and ␤1 subunits in WT and eNOS Ϫ/Ϫ mice were identical as determined with Western blot analysis. These data suggest that chronic exposure to endothelium-derived NO, as well as acute exposure to nitrovasodilator-derived NO, desensitizes sGC to activation by NO but does not alter sGC expression. Both the acute cessation of endothelial NO formation in WT mice and the chronic deficiency of NO in eNOS Ϫ/Ϫ mice restore the NO sensitivity of sGC and enhance vascular smooth muscle relaxation in response to nitrovasodilator agents. Key Words: nitric oxide Ⅲ mice Ⅲ genes Ⅲ vasodilator agents V ascular relaxation responses elicited by nitrovasodilator agents and by endothelium-derived nitric oxide (NO) are mainly mediated via the activation of soluble guanylyl cyclase (sGC) and a subsequent increase in intracellular cGMP levels. 1 Interactions between endogenous and exogenous NO have been reported to modulate vasodilatory responsiveness. 2-5 Different mechanisms may underlie this phenomenon, such as the downregulation of sGC, sGC desensitization, or inhibition of the cGMP-dependent signal transduction cascade. In the present study, we investigated the mechanism by which endothelium-derived NO affects the nitrovasodilator-induced relaxation in wild-type (WT) mice and in endothelial NO synthase knockout mice (eNOS Ϫ/Ϫ ). Methods Animals and Tissue PreparationsWT c57 black b6 mice were purchased from Charles River, and eNOS Ϫ/Ϫ mice and age-matched control animals were obtained from the Department of Physiology at Heinrich Heine Universität Düssel-dorf. 6 Mice were housed under conditions that conformed with the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication No. 85-23).Mice were killed by cervical dislocation. T...

L1 cell adhesion molecule (L1CAM) is a transmembrane cell adhesion molecule initially defined as a promigratory molecule in the developing nervous system that appears to be also expressed in some endothelial cells. However, little is known about the functional role of L1CAM on endothelial cells. We observed that L1CAM expression was selectively enhanced on endothelium associated with pancreatic adenocarcinoma in situ and on cultured pancreatic tumor-derived endothelial cells in vitro. L1CAM expression of endothelial cells could be augmented by incubation with immunomodulatory cytokines such as tumor necrosis factor alpha, interferon gamma, or transforming growth factor beta 1. Antibodies to L1CAM and the respective ligand neuropilin-1 blocked tube formation and stromal cell-derived factor 1beta induced transmigration of tumor endothelial cells in vitro. L1CAM expression on tumor-derived-endothelial cells enhanced Panc1 carcinoma cell adhesion to endothelial cell monolayers and transendothelial migration. Our data demonstrate a functional role of L1CAM expression on tumor endothelium that could favor metastasis and angiogenesis during tumor progression.