Nitric oxide (NO) regulates flow and permeability. ACh and platelet-activating factor (PAF) lead to endothelial NO synthase (eNOS) phosphorylation and NO release. While ACh causes only vasodilation, PAF induces vasoconstriction and hyperpermeability. The key differential signaling mechanisms for discriminating between vasodilation and hyperpermeability are unknown. We tested the hypothesis that differential translocation may serve as a regulatory mechanism of eNOS to determine specific vascular responses. We used ECV-304 cells permanently transfected with eNOS-green fluorescent protein (ECVeNOS-GFP) and demonstrated that the agonists activate eNOS and reproduce their characteristic endothelial permeability effects in these cells. We evaluated eNOS localization by lipid raft analysis and immunofluorescence microscopy. After PAF and ACh, eNOS moves away from caveolae. eNOS distributes both in the plasma membrane and Golgi in control cells. ACh (10(-5) M, 10(-4) M) translocated eNOS preferentially to the trans-Golgi network (TGN) and PAF (10(-7) M) preferentially to the cytosol. We suggest that PAF-induced eNOS translocation preferentially to cytosol reflects a differential signaling mechanism related to changes in permeability, whereas ACh-induced eNOS translocation to the TGN is related to vasodilation.

Objective-To test the hypothesis that acupuncture on stomach 36 point (ST-36) reduces hypertension by activating nitric oxide synthase signaling mechanisms.Methods-The authors used the two-kidney, one-clip renal hypertension (2K1C) hamster model with electroacupuncture treatment.Results-Thirty-minute daily electroacupuncture treatment for 5 days reduced mean arterial pressure from 160.0 ± 7.6 to 128.0 ± 4.3 mmHg (mean ± SEM), compared to 115.0 ± 7.2 mmHg in sham-operated hamsters. Electroacupuncture increased periarteriolar NO concentration from 309.0 ± 21.7 nM to 417.9 ± 20.9 nM in the 2K1C hamster cheek pouch microcirculation when measured with NO-sensitive microelectrodes. Hypertension reduced endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) proteins relative to the sham-operated control, as measured by Western blotting. Electroacupuncture prevented the reduction of eNOS and nNOS associated with hypertension and showed even higher eNOS and nNOS expressions than shamoperated control in stomach and cheek pouch tissues, which are on the stomach meridian. Analysis of liver tissue, a non-stomach-meridian organ, indicated that electroacupuncture did not have a significant benefit in terms of enhanced expressions of eNOS and nNOS in the treated 2K1C hypertensive group.Conclusions-Activation of eNOS and nNOS is one of the mechanisms through which ST-36 electroacupuncture reduces blood pressure; this reduction works through the stomach meridian. Keywordsacupuncture; complementary and alternative medicine; hypertension; meridian theory; microcirculation; nitric oxide; nitric oxide synthase; Complementary and alternative medicine (CAM), which includes acupuncture and herbal medicine, is a current focus of interest for the general public and the medical profession. Acupuncture and herbal medicine have been used for a long time in the treatment of cardiovascular disease in Asia. In particular, acupuncture treatment has been used traditionally for the reduction of hypertension. However, this ancient practice has not yet been subjected systematically and thoroughly to the rigors of scientific testing.Hypertension affects millions of people in the United States and in the world. It causes loss of lives and carries significant economic cost. In addition, some medical therapeutic regimens and medications available for treating hypertension have significant side effects. Thus, it is

Endothelial nitric oxide (NO) synthase (eNOS) is thought to regulate microvascular permeability via NO production. We tested the hypotheses that the expression of eNOS and eNOS endocytosis by caveolae are fundamental for appropriate signaling mechanisms in inflammatory endothelial permeability to macromolecules. We used bovine coronary postcapillary venular endothelial cells (CVECs) because these cells are derived from the microvascular segment responsible for the transport of macromolecules in inflammation. We stimulated CVECs with platelet-activating factor (PAF) at 100 nM and measured eNOS phosphorylation, NO production, and CVEC monolayer permeability to FITC-dextran 70 KDa (Dx-70). PAF translocated eNOS from plasma membrane to cytosol, induced changes in the phosphorylation state of the enzyme, and increased NO production from 4.3+/-3.8 to 467+/-22.6 nM. PAF elevated CVEC monolayer permeability to FITC-Dx-70 from 3.4+/-0.3 x 10(-6) to 8.5+/-0.4 x 10(-6) cm/s. The depletion of endogenous eNOS with small interfering RNA abolished PAF-induced hyperpermeability, demonstrating that the expression of eNOS is required for inflammatory hyperpermeability responses. The inhibition of the caveolar internalization by blocking caveolar scission using transfection of dynamin dominant-negative mutant, dyn2K44A, inhibited PAF-induced hyperpermeability to FITC-Dx-70. We interpret these data as evidence that 1) eNOS is required for hyperpermeability to macromolecules and 2) the internalization of eNOS via caveolae is an important mechanism in the regulation of endothelial permeability. We advance the novel concept that eNOS internalization to cytosol is a signaling mechanism for the onset of microvascular hyperpermeability in inflammation.

Kim DD, Sánchez FA, Durán RG, Kanetaka T, Durán WN. Endothelial nitric oxide synthase is a molecular vascular target for the Chinese herb Danshen in hypertension. Am J Physiol Heart Circ Physiol 292: H2131-H2137, 2007. First published December 15, 2006; doi:10.1152 doi:10. /ajpheart.01027.2006 Chinese herb, reduces hypertension in Oriental medicine. We hypothesized that Danshen acts partially through endothelial nitric oxide synthase (eNOS) signaling mechanisms. We tested the hypothesis using tanshinone IIA, an active ingredient of Danshen, and the two-kidney, one-clip renovascular hypertension model in hamsters. Oral tanshinone (50 g/100 g body wt) reduced mean arterial pressure (MAP) from 161.2 Ϯ 6.9 to 130.0 Ϯ 7.8 mmHg (mean Ϯ SE; P Ͻ 0.05) in hypertensive hamsters. MAP in sham-operated hamsters was 114.3 Ϯ 9.2 mmHg. Topical tanshinone at 1 g/ml and 5 g/ml increased normalized arteriolar diameter from 1.00 to 1.25 Ϯ 0.08 and 1.57 Ϯ 0.11, respectively, and increased periarteriolar nitric oxide concentration from 87.1 Ϯ 11.3 to 146.9 Ϯ 23.1 nM (P Ͻ 0.05) at 5 g/ml in hamster cheek pouch. N G -monomethyl-L-arginine inhibited tanshinone-induced vasodilation. Hypertension reduced eNOS protein relative to sham-operated control. Tanshinone prevented the hypertension-induced reduction of eNOS and increased eNOS expression to levels higher than sham-operated control in hamster cheek pouch. Topical tanshinone increased normalized arteriolar diameter from 1.0 to 1.47 Ϯ 0.08 in the cremaster muscle of control mice and to 1.12 Ϯ 0.13 in cremasters of eNOS knockout mice. In ECV-304 cells transfected with eNOS-green fluorescent protein, tanshinone increased eNOS protein expression 1.35 Ϯ 0.05-and 1.85 Ϯ 0.07-fold above control after 5-min and 1-h application, respectively. Tanshinone also increased eNOS phosphorylation 1.19 Ϯ 0.07-and 1.72 Ϯ 0.20-fold relative to control after 5-min and 1-h application. Our data provide a basis to understand the action of a Chinese herb used in alternative medicine. We conclude that eNOS stimulation is one mechanism by which tanshinone induces vasodilation and reduces blood pressure.

Physiological changes in extracellular glucose, insulin, and leptin regulate glucose-excited (GE) and glucose-inhibited (GI) neurons in the ventromedial hypothalamus (VMH). Nitric oxide (NO) signaling, which is involved in the regulation of food intake and insulin signaling, is altered in obesity and diabetes. We previously showed that glucose and leptin inhibit NO production via the AMP-activated protein kinase (AMPK) pathway, while insulin stimulates NO production via the phosphatidylinositol-3-OH kinase (PI3K) pathway in VMH GI neurons. Hyperglycemia-induced inhibition of AMPK reduces PI3K signaling by activating the mammalian target of rapamycin (mTOR). We hypothesize that hyperglycemia impairs glucose and insulin-regulated NO production in VMH GI neurons. This hypothesis was tested in VMH neurons cultured in hyperglycemic conditions or from streptozotocin-induced type 1 diabetic rats using NO- and membrane potential-sensitive dyes. Neither decreased extracellular glucose from 2.5 to 0.5 mM, nor 5 nM insulin increased NO production in VMH neurons in either experimental condition. Glucose- and insulin-regulated NO production was restored in the presence of the AMPK activator, 5-aminoimidazole-4-carboxamide-1-b-4-ribofuranoside or the mTOR inhibitor rapamycin. Finally, decreased glucose and insulin did not alter membrane potential in VMH neurons cultured in hyperglycemic conditions or from streptozotocin-induced rats. These data suggest that hyperglycemia impairs glucose and insulin regulation of NO production through AMPK inhibition. Furthermore, glucose and insulin signaling pathways interact via the mTOR pathway.