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Proper food intake is important for maintaining good health in humans. Chocolate is known to exert anti-inflammatory effects; however, the mechanisms remain unclear. In this study, we aimed to investigate the effects of cocoa butter intake on gut immunity in rats and rabbits. Cocoa butter intake increased the lymph flow, cell density, and IL-1β, IL-6 and IL-10 levels in mesenteric lymph. Clodronate, a macrophage depletion compound, significantly enhanced the release of all cytokines. The immunoreactivities of macrophage markers CD68 and F4/80 in the jejunal villi were significantly decreased with clodronate. Piceatannol, a selective cell surface ATP synthase inhibitor significantly reduced the cocoa butter intake-mediated releases of IL-1β, IL-6 and IL-10. The immunoreactivities of cell surface ATP synthase were observed in rat jejunal villi. Shear stress stimulation on the myofibroblast cells isolated from rat jejunum released ATP and carbon dioxide depended with H+ release. In rabbit in vivo experiments, cocoa butter intake increased the concentrations of ATP and H+ in the portal vein. The in vitro experiments with isolated cells of rat jejunal lamina propria the pH of 3.0 and 5.0 in the medium released significantly IL-1β and IL-6. ATP selectively released IL-10. These findings suggest that cocoa butter intake regulates the gut immunity through the release and transport of IL-1β, IL-6, and IL-10 into mesenteric lymph vessels in a negative feedback system. In addition, the H+ and ATP released from cell surface ATP synthase in jejunal villi play key roles in the cocoa butter intake–mediated regulation of gut immunity. [ABSTRACT FROM AUTHOR]

It is known that nitric oxide (NO) is a gas and synthesized from l-arginine by the NO synthase (NOS) in vascular endothelial cells. The diffused NO activates the guanosine monophosphate, which initiates a series of intracellular events, leading to physiological response such as vasodilation. There are three different types of NOS, namely endothelial constitutive NOS (ecNOS), neuronal NOS (nNOS), and cytokine-inducible NOS (iNOS). The ecNOS and nNOS are expressed constitutively at low levels and can be activated rapidly by an increase in cytoplasmic calcium ions. In contrast, the iNOS is induced when macrophages are activated by cytokine, resulting in the induction of pathophysiological effects. Lymph flow is known to stimulate the release of NO from lymphatic endothelial cells (LEC) and then produce the relaxation of lymphatic smooth muscle cells. The NO also plays a key role in the control of lymphatic pump activity in vivo. Many studies have shown the NO-mediated findings in various kinds of lymph vessels. However, there is no or little study to demonstrate the effects of lymph flow on the molecular expression of ecNOS mRNA and the protein. In addition, little study is available for clarifying the relationship between NO and sympathetic nerve fibers in the regulation of lymph transport and production. Therefore, in this review, the experimental findings of lymph flow-mediated increases in the ecNOS mRNA and the protein in LEC are demonstrated in detail. In addition, the roles of NO and aminergic nerve fibers in the physiological control system of lymph transport and production are discussed. [ABSTRACT FROM AUTHOR]

To clarify the roles of lymphatic endothelial cells (LEC) in the regulation of endothelial constitutive nitric oxide synthase (ecNOS) expression, we examined the effects of shear stress on ecNOS immunohistochemical staining and mRNA and protein expression in human LEC as well as on ATP release from these cells. Shear stress at 0.5 or 1.0 dyn/[cm.sup.2] increased ecNOS immunohistochemical staining and ecNOS mRNA and protein expression in cultured LEC. The same strength of shear stress produced a significant release of ATP from the LEC. Exogenous ATP ranging in concentration from [10.sup.-9] to [10.sup.-6] M produced a significant increase in ecNOS immunohistochemical expression in a dose-dependent manner. The increase in ecNOS expression mediated by [10.sup.-6]M ATP was significantly reduced by [10.sup.-5] M suramin. Suramin ([10.sup.-5] M) caused a significant reduction in the shear stress-mediated increases in ecNOS immunohistochemical staining and mRNA expression. The shear stress-mediated increases in ecNOS expression were significantly reduced by 3 mM tetraethylammonium, [10.sup.-4] M apamin, [10.sup.-9] M iberiotoxin, [10.sup.-5] M 2-aminoethoxydephenyl borate, or [10.sup.-5]M xestospongin C, but not [10.sup.-5] M glybenclamide or [10.sup.-5] M nifedipine. The shear stress-mediated increases in ecNOS expression were significantly potentiated by pinacidil or NS1619 in a dose-dependent manner. The immunohistochemical expression of small--([SK.sub.Ca]) and big-conductance ([BK.sub.Ca]) [Ca.sup.2+]-activated [K.sup.+] channels was confirmed on the surfaces of human LEC. These findings suggest that shear stress produces a significant release of ATP from LEC, which activates the purinergic P2X/2Y receptor, thereby facilitating ecNOS mRNA and protein expression through inositol 1,4,5-trisphosphatemediated release of intracellular [Ca.sup.2+] ions and the activation of [Ca.sup.2+]-activated [K.sup.+] channels in LEC. lymph flow rate; [Ca.sup.2+]-activated [K.sup.+] channel; mRNA; 2-aminoethoxydephenyl borate; xestospongin C; apamin; iberiotoxin; pinacidil doi:10.1152/ajpcell.00249.2009

We examined the effects of supernatants of culture media of MDA-MB-231 and MCF-7 cells on the expression of adhesion molecules on human lymphatic endothelial cells (LECs) and evaluated whether the overexpression of adhesion molecules facilitated the attachment of carcinoma cells to LECs. The 48-h stimulation of MDA-MB-231, but not MCF-7, supernatant produced a significant expression of ICAM-1 on human LECs but little or no expression of E-selectin. Chemical treatment with dialyzed substances of intercellular adhesion molecule-1; sentinel lymph node

Microcirculation of the sheath of the rat sciatic nerve fiber was investigated by using an intravital microscope, and changes in the diameter of the epineurial arterioles in response to highly oxygenated Krebs-bicarbonate solution were evaluated. Superfusion of low-oxygen (0%) Krebs-bicarbonate solution (LKS) onto rat sciatic nerves did not affect changes in the diameter of the arterioles. Nifedipine, a [Ca.sup.2+]-channel blocker, caused a dose-dependent dilation of the epineurial arterioles in LKS. In contrast, superfusion of high-oxygen (21%) Krebs-bicarbonate solution (HKS) onto rat sciatic nerves significantly constricted the epineurial arterioles in a time-dependent manner. The HKS-induced constriction of the epineurial arterioles was significantly reduced by treatment with 120 U/ml superoxide dismutase (SOD) alone or 5,000 U/ml catalase alone. In the presence of 120 U/ml SOD plus 5,000 U/ml catalase, [10.sup.-4] M tempol, [10.sup.-6] M diphenyleneiodium, 2 x [10.sup.-4] M apocynin, or [10.sup.-6] M allopurinol, the HKS-induced constriction of the epineurial arterioles completely disappeared. These results suggest that superfusion of highly oxygenated solution onto rat sciatic nerves constricts the epineurial arterioles through reactive oxygen species (ROS), including superoxide and hydrogen peroxide, and that production of superoxide involves a NADPH oxidase- or xanthine oxidase-dependent pathway. In conclusion, ROS play significant roles in the regulation of microcirculation of rat sciatic nerves in vivo. peripheral nerves; microcirculation; superoxide; hydrogen peroxide

The wall effective permeability of hydrophilic substances labeled with fluorescent dyes was evaluated in an isolated cannulated rat single lymph vessel through a videomicroscope system. Sodium fluorescein (NaFl; 332 mol wt) and FITC-dextrans (4,400, 12,000, and 71,200 mol wt) were administered into the intraluminal space of the lymph vessels and then excited by a Xenon lamp. Changes in the fluorescence intensity of the dyes were continuously measured by a silicon-intensified target camera through appropriate filters. The net flux of each dye in the wall of the lymph vessels was calculated by the relationship between the fluorescence intensity and the concentration of the dyes. NaFl and FITC-dextran 4,400 in the intraluminal space of isolated rat lymph vessels significantly penetrated the wall of the lymph vessels. FITC-dextran 12,000 in the intraluminal space of isolated rat lymph vessels slightly passed through the lymphatic wall, whereas FITC-dextran 71,200 did not penetrate the wall. Intraluminal pressures ranging from 4 to 8 cm[H.sub.2]0 did not significantly affect the net flux of dyes used in the present study. After administration of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate into the lymph vessels, the net flux of FITC-dextran 4,400 and 12,000 but not 71,200 was augmented significantly. These results suggest that small molecular hydrophilic substances ([less than or equal to]4,400) are permeable from the intraluminal to extraluminal space of isolated lymph vessels and that the endothelial cell surface structure may play a barrier role in the effective permeability of large molecular hydrophilic substances (4,400 to 12,000) through the wall of the lymph vessels. endothelium; dextran; fluorescence

We examined the effects of ATP on intrinsic pump activity in lymph vessels isolated from the rat. ATP caused significant dilation with a cessation of lymphatic pump activity. Removal of the endothelium or pretreatment with [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) significantly reduced ATP-induced inhibitory responses of lymphatic pump activity, whereas reduction was not suppressed completely by [10.sup.-6] M ATP. L-Arginine significantly restored ATP-induced inhibitory responses in the presence of L-NAME. ATP-induced inhibitory responses in lymph vessels with endothelium were also significantly, but not completely, suppressed by pretreatment with glibenclamide. 8-Cyclopentyl-1,3-dipropylxanthine (a selective adenosine [A.sub.1] receptor antagonist), but not suramine (a P2X and P2Y receptor antagonist) or 3,7-dimethyl-1-proparglyxanthine (a selective adenosine [A.sub.2] receptor antagonist), significantly decreased ATP-induced inhibitory responses. [alpha],[beta]-Methylene ATP (a selective P2X and P2Y receptor agonist) had no significant effect on lymphatic pump activity. In some lymph vessels with endothelium (24 of 30 preparations), adenosine also caused dose-dependent dilation with a cessation of lymphatic pump activity. L-NAME significantly reduced the inhibitory responses induced by the lower (3 x [10.sup.-8]-3 x [l0.sup.-7] M) concentrations of adenosine. Glibenclamide or 8-cyclopentyl-1,3-dipropylxanthine also significantly suppressed adenosine-induced inhibitory responses. These findings suggest that ATP-induced dilation and inhibition of pump activity of isolated rat lymph vessels are endothelium-dependent and -independent responses. ATP-mediated inhibitory responses may be, in part, related to production of endogenous nitric oxide, involvement of ATP-sensitive [K.sup.+] channels, or activation of adenosine [A.sub.1] receptors in lymphatic smooth muscle and endothelium. rat; endothelium; smooth muscle; nitric oxide

To evaluate whether or not Rho-Rho kinase pathway is involved in the regulation of mechanical activity of lymph vessels, effects of Y-27632 and okadaic acid on lymph pump activity and myogenic, pressure- and agonist-induced tone were examined in isolated rat lymph vessels. Y-27632 caused a significant dilation with a cessation of the lymph pump activity. Y-27632 also produced a dose-related dilation of the lymph vessels precontracted by norepinephrine (NE)-, U-46619- or 80 mM KCl. Okadaic acid significantly constricted the lymph vessels and reduced the frequency of the lymph pump activity. Okadaic acid also produced a dose-related constriction of the lymph vessels precontracted by NE or U-46619. The Y-27632-induced decrease of the frequency of lymph pump activity was significantly reversed by the pretreatment with okadaic acid. In the presence of Y-27632, the pressure-mediated tone of the lymph vessel was significantly decreased. On the other hand, okadaic acid significantly increased the pressure-mediated tone. These findings suggest that Rho kinase and myosin phosphatase activity in lymphatic smooth muscles may contribute to the regulation of lymph pump activity and may be also involved in the control of myogenic pressure- and agonist-induced tone. rat; Y-27632; okadaic acid; lymphatic smooth muscle

Nakaya, Kei, Risuke Mizuno, and Toshio Ohhashi. B16-BL6 melanoma cells release inhibitory factor(s) of active pump activity in isolated lymph vessels. Am J Physiol Cell Physiol 281: C1812-C1818, 2001.--We investigated whether supernatant cultured with melanoma cell lines B16-BL6 and K1735 or the Lewis lung carcinoma cell line (LLC) can regulate lymphatic pump activity with bioassay preparations isolated from murine iliac lymph vessels. B16-BL6 and LLC supernatants caused significant dilation of lymph microvessels with cessation of pump activity. B16-BL6 supernatant produced dose-related cessation of lymphatic pump activity. There was no significant tachyphylaxis in the supernatant-mediated inhibitory response of lymphatic pump activity. Pretreatment with 3 x [10.sup.-5] M [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) or [10.sup-7] M or [10.sup.-6] M glibenclamide and 5 x [10.sup.-4] M 5-hydroxydecanoic acid caused significant reduction of supernatant-mediated inhibitory responses. Simultaneous treatment with [10.sup.-3] M L-arginine and 3 x [10.sup.-5] M L-NAME significantly lessened L-NAME-induced inhibition of the supernatant-mediated response, suggesting that endogenous nitric oxide (NO) plays important roles in supernatant-mediated inhibitory responses. Chemical treatment dialyzed substances of malignant melanoma; active lymph transport; nitric oxide; mitochondrial adenosine 5'-triphosphate-sensitive potassium channel Received 28 February 2001; accepted in final form 23 July 2001

An investigation was conducted on the mode of action of endothelin (ET) on spontaneous contractions in ring preparations of isolated bovine mesenteric lymphatics. A significant inhibition of the ET-1-induced positive chromotropic effect in the ring preparations with and without the endothelium which was caused by the specific ET(sub A)-receptor antagonist BQ-123. The results showed that stimulation of ET(sub A) receptors on the lymphatic endothelial cells induced a release of nitric oxide.

The role of prostaglandins in the flow- or acetylcholine-induced vasodilation in the spinal resistance-sized small arteries was examined in rabbits. Treatment with aspirin produced a slightly significant reduction in the flow-mediated vasodilation. Prostanoids such as indomethacin played a major role in the flow- or acetylcholine-induced vasodilation in the rabbit spinal resistance-sized small arteries.

The stimulation of acetylcholine (ACh) or an increase in flow produces and releases less nitric oxide (NO) in the venous endothelial cells of canine large vein than in the arteries. The flow-mediated responses in arteries and veins are partly due to the release of NO from the endothelial cells. N(super G)-nitro-l-arginine methyl ester (l-NAME) produces a decrease in the ACh- and flow-induced vasodilation. The treatment with l-arginine reverses the l-NAME-induced suppression of ACh-induced vasodilation, but has no effect on flow-induced vasodilation.

Receptors of 5-hydroxytryptamine (5-HT) are present in the popliteal lymph nodes of monkeys whose stimulation activates cytosolic guanylate cyclase in the cells and a nitric oxide (NO) dependent relaxation is seen. However, the basal tones of the lymph nodes are unaffected. This 5-HT-induced relaxation is inhibited by pretreatment with methiothepin, methylsergide and methylene blue. Aspirin and ketanserin did not affect the relaxation. The release of NO by 5-HT probably helps control the microvascular permeability of fluids and proteins in the lymph nodes.

Changes in the spontaneous contractions of isolated bovine messenteric lymph vessels due to acetylcholine (ACh) were examined. The mechanical activity of 82 lymphatics was measured after the direct application of ACh into the bathing medium by a microsyringe. Negative chronotropic and inotropic effects on spontaneous contractions in bovine mesenteric lymphatics were derived after the addition of low concentrations of ACh.

We demonstrated pulmonary arteriolar blood flow-mediated CO2 gas excretion in rabbit lungs. The shear stress stimulation produced CO2 gas in cultured human endothelial cells of pulmonary arterioles via the activation of F1/Fo ATP synthase. To confirm the findings in human subjects undergoing the operation with heart–lung machines, we aimed to evaluate the effects of a stepwise switch, from a partial to a complete cardiopulmonary bypass, of the circulatory blood volume (BV, 100% = 2.4 × cardiac index), on the end-expiratory CO2 pressure (PetCO2), maximal flow velocity in the pulmonary artery (Max Vp), the inner diameter (ID) of pulmonary artery, pulmonary arterial CO2 pressure (P mix v CO2), pulmonary arterial O2 pressure (P mix v O2), hematocrit (Hct), pH, the concentration of HCO3−, and base excess (BE) in mixed venous blood in 9 patients with a mean age of 72.3 ± 3.4 years. In addition, the effects of the decrease in Hct infused with physiological saline solution (PSS) on PetCO2 were investigated in the human subjects. An approximately linear relationship between the PetCO2 and Max Vp was observed. The pumping out of 100% BV produced little or no change in the Hct, pH, P mix v CO2, and P mix v O2, respectively. The hemodilution produced by intravenous infusion of PSS caused a significant decrease in the Hct, but not in the PetCO2. In conclusion, another route of CO2 gas excretion, independent of red blood cells, may be involved in human lungs. [ABSTRACT FROM AUTHOR]