Cell surface ATP synthase-released H+ and ATP play key roles in cocoa butter intake-mediated regulation of gut immunity through releases of cytokines in rat.

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-1ß, 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-1ß, 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-1ß 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-1ß, 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.

Heatstroke risk informing system using wearable perspiration ratemeter on users undergoing physical exercise.

We constructed an informing system to users for the heatstroke risk using a wearable perspiration ratemeter and the users' thirst responses. The sweating ratemeter was constructed with a capacitive humidity sensor in the ventilated capsule. The timing point for informing heatstroke risk was decided to change from positive to negative on the second derivative of sweating curve. In addition, a wearable self-identification and -information system of thirst response was constructed with a smartphone. To evaluate the validity of wearable apparatus, we aimed to conduct human experiments of 16 healthy subjects with the step up and down physical exercises. The blood and urine samples of the subjects were collected before and after the 30-min physical exercise. The concentrations of TP, Alb, and RBC increased slightly with the exercise. In contrast, the concentrations of vasopressin in all subjects remarkably increased with the exercise. In almost subjects, they identified their thirst response until several min after the informing for heatstroke risk. In conclusion, the wearable ratemeter and self-information system of thirst response were suitable for informing system of heatstroke risk. The validity of timing point for informing heatstroke risk was confirmed with changes in the thirst response and concentrations of vasopressin in blood.

Physiological Roles of Lymph Flow-Mediated Nitric Oxide in Lymphatic System.

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.

Portal blood flow-dependent NO-mediated lymph formation in rat jejunum.

The higher permeability of the venules in jejunal microcirculation to albumin contributes to the increased mesenteric lymph formation. Recently, we demonstrated that water intake induced serotonin release from enterochromaffin cells in rat jejunum, serotonin of which circulated through the portal vein into blood circulation and then increased the mesenteric lymph formation. The mode of action of serotonin remains unclear. Therefore, we aimed to clarify the mechanisms involved in the regulation of the jejunal lymph formation with permeant albumin in in vivo rat experiments. We investigated the effects of intravenous administration of serotonin or water intake on the jejunal-originated lymph volume and the concentration of albumin in the lymph in the presence or absence of L-NAME. The effects of intravenous administration of L-NAME, nicardipine, A23187, and ML-7 on the lymph formation with permeant albumin were also evaluated. Serotonin or water intake significantly increased the mesenteric lymph volume with permeant albumin in the jejunal microcirculation. The serotonin- and water intake-mediated responses were significantly reduced by the pretreatment with intravenous administration of L-NAME. Intravenous administration of L-NAME itself also decreased significantly the jejunal lymph formation. Administration of A23187 and ML-7 significantly reduced the jejunal lymph formation with permeant albumin. In contrast, administration of nicardipine significantly increased the lymph formation. In conclusion, portal venous blood flow- or serotonin-mediated NO release from venular endothelial cells plays physiologically key roles in the lymph formation in rat jejunum via the extrusion of calcium ions and inactivation of MLCK in endothelial cells.

Estimation of the Lymph Flow Through Thoracic Duct in Human Subjects Using the Urine Osmolarity: Applicable for Evaluating the Effectiveness of Manual Lymph Drainage.

Currently, there are many methods to evaluate the effectiveness of manual lymph drainage in the treatment of lymphedema, that is, limb volume measurement, bio-electrical impedance measurement, computer tomography, and ultrasound imaging. However, it is difficult for these methods to accurately address the lymph flow generated by manual lymph drainage. Therefore, we aimed at developing a concise and accurate method to measure the lymph flow through the thoracic duct in human subjects, which is applicable for evaluating the effectiveness of manual lymph drainage. In the present mini-review, we demonstrate the developed method in detail and its scientific evidence for the effectiveness obtained with animal and human clinical experiments. In rat in vivo experiments, intragastric administration of distilled water significantly increased mesenteric flow, which was transported via the cisterna chyli and then the thoracic duct. The manual massage on the cisterna chyli in the anesthetized rabbit significantly accelerated the lymph flow through the thoracic duct, resulting in marked hemodilution. Abdominal respiration in the supine position in human subjects produced similar hemodilution, with a marked decrease in the concentration of vasopressin in the blood. On this basis, we developed a new method to accurately measure the lymph flow through the thoracic duct by using changes in the concentration of vasopressin in the blood. In addition, with changes in urine osmolarity depending on the concentration of vasopressin in the blood, we developed a more concise and noninvasive method for evaluating the lymph flow through the thoracic duct in human subjects. These methods may be applicable for evaluating the effectiveness for the manual lymph drainage in the patients with lymphedema.

From digestion and absorption to innate immunity and health care: water and food intake may contribute to IL-22 in ILC3-dependent mucosal immunity in the jejunum.

In this review, with our current studies we demonstrated medical evidence that water and food intake are useful for IL-22-related mucosal immunity-dependent maintenance of health care. The traditional Japanese health care practices recommend daily consumption of suitable volume of water. However, immunological mechanisms that support of the traditional practices are still unsolved. We focused on type 3 innate lymphoid cells (ILC3s), because the ILC3s are mainly housed in the lamina propria of the jejunum. IL-22 released from the ILC3 is transported through mesenteric lymph in collaboration with the albumin-mediated movement of consumed water. Thus, water intake-mediated upregulation of IL-22-dependent mucosal immunity contributes to the traditional Japanese health care practices. We also reviewed current studies that food intake-mediated increase in VIP-dependent neuronal activity in the small intestine and the food intake included with tryptophan-derived metabolites may accelerate the IL-22 in ILC3s-dependent mucosal immunity and then contribute in keeping health care.

Another route of CO2 gas excretion independent of red blood cells in human lungs.

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.

Water intake releases serotonin from enterochromaffin cells in rat jejunal villi.

The present study aims to investigate the roles of water intake in serotonin production and release in rat jejunum. We evaluated the changes in concentrations of serotonin in the portal vein and mesenteric lymph vessel induced by the intragastric administration of distilled water. The density of granules in enterochromaffin cells and the immunoreactivity of serotonin in the jejunal villi were investigated before and after water intake. The effects of intravenous administration of serotonin and/or ketanserin on mesenteric lymph flow and concentrations of albumin and IL-22 in the lymph were also addressed. Water intake increased serotonin concentration in the portal vein, but not in the mesenteric lymph vessel. The flux of serotonin through the portal vein was significantly larger than that through the mesenteric lymph vessel. Water intake decreased the density of granules in the enterochromaffin cells and increased the immunoreactivity of serotonin in the jejunal villi. The intravenous administration of serotonin increased significantly mesenteric lymph flow and the concentrations of albumin and IL-22; both were significantly reduced by the intravenous pretreatment with ketanserin. We showed that serotonin released from enterochromaffin cells by water intake was mainly transported through the portal vein. Additionally, serotonin in blood was found to increase mesenteric lymph formation with permeant albumin in the jejunal villi via the activation of 5-HT2 receptor.

Water intake accelerates ATP release from myofibroblast cells in rats: ATP-mediated podoplanin-dependent control for physiological function and immunity.

We previously demonstrated that water intake increased mesenteric lymph flow and the total flux of IL-22 in rat jejunum. The drained water and the higher permeability of albumin in the jejunal microcirculation contributed to increase the lymph flow and IL-22 transport via the activation of great bulk flow in the jejunal villi. To address the effects of water intake-mediated great bulk flow-dependent mechanical force on jejunal physiological function and immunological regulation of innate lymphoid cells (ILC)-3, we examined the effects of shear stress stimulation on cultured rat myofibroblast cells. Next, we investigated the effects of water intake on podoplanin and IL-22 expressions in cultured human intestinal epithelial cells and rat in vivo jejunal preparations, respectively. Shear stress stimulation of the myofibroblast cells induced ATP release via an activation of cell surface F1/F0 ATP synthase. ATP produced podoplanin expression in the intestinal epithelial cells. Water intake accelerated immunohistochemical expressions of podoplanin and IL-22 in the interepithelial layers and lamina propria of the jejunum. ATP dose-dependently increased IL-22 mRNA expression in ILC-3, which are housed in the lamina propria. Water intake also increased immunohistochemical and mRNA expressions of ecto-nucleoside triphosphate diphosphohydrolases 2 and 5 in jejunal villi. In conclusion, water intake-mediated shear stress stimulation-dependent ATP release from myofibroblast cells maintains higher tissue colloid osmotic pressure in the jejunal microcirculation through podoplanin upregulation in the interepithelial layers. ATP induces IL-22 mRNA expression in ILC-3 in jejunal villi, which may contribute to regulation of mucosal immunity in small intestine.NEW & NOTEWORTHY We investigated effects of shear stress stimulation on cultured myofibroblast cells and water intake on podoplanin and IL-22 expressions in rat jejunal villi. The stimulation induced ATP release from the cells. Water intake accelerated podoplanin and IL-22 expression levels. ATP increased IL-22 mRNA expression in innate lymphoid cells (ILC)-3. Hence, water intake maintains higher osmotic pressure in the jejunal villi through ATP release and podoplanin upregulation. Water intake may regulate the mucosal immunity.

Eyes Closing and Drowsiness in Human Subjects Decrease Baseline Galvanic Skin Response and Active Palmar Sweating: Relationship Between Galvanic Skin and Palmar Perspiration Responses.

We previously constructed a perspiration ratemeter for the measurement of palmar sweating in human subjects. Although galvanic skin response (GSR) has been used to evaluate emotional responses in human subjects, little is known about the relationships between the phasic and baseline components in GSR and active palmar sweating. From the aforementioned, we aimed to investigate the relationships in human subjects with handgrip exercise and eyes closing or opening. Fifteen healthy volunteers (mean age: 26.9 ± 8.7 years) participated in the present experiments. We investigated the effects of maximal handgrip exercise, eyes closing or opening, and self-awareness of drowsy on the GSR, active palmar sweating, R-R interval in electrocardiograph (ECG), and percentage of α wave in EEG. The faster phasic component in GSR completely agreed with the starting point of active palmar sweating. Handgrip exercise induced significantly faster spike in GSR, active palmar sweating, and decrease in R-R interval in ECG. Eyes closing produced significant decreases in baseline GSR and active palmar sweating in all human subjects. The percentage of α wave in electroencephalograph (EEG) also increased. In contrast, eyes opening increased significantly the baseline GSR and active palmar sweating. In the equivalent electrical model of human skin, the eyes closing-mediated time-dependent decrease in the baseline GSR completely agreed with the hypothesis that the palmar skin voltage only in the model decreased time dependently to 0.4 of the control during 6 min. The self-awareness of drowsy in mid-night working with computer produced similar decreases in baseline GSR and active palmar sweating to the responses with eyes closing in all human subjects. In conclusion, the faster spike in GSR completely agreed with the starting point of active palmar sweating. Eyes closing and opening or self-awareness of drowsy significantly produced changes in baseline GSR and active palmar sweating, which may become useful tools for evaluating clearness or drowsiness in human subjects.

Evaluating Lymph Flow Through the Thoracic Duct Using Urine Osmolarity in Human Participants.

Background: Previous animal studies have shown that intragastric administration of water can accelerate mesenteric lymph flow. Similarly, human studies have shown that abdominal breathing can induce thoracic lymph drainage. In these studies, lymph flow was measured by hemodilution and a corresponding reduction in blood anti-diuretic hormone (ADH) levels, the latter being linked to urine osmolarity. Hence, we questioned if induction of lymph flow through water administration and supine positioning could be measured by monitoring urine osmolarity. Methods and Results: Volunteers were given 250 mL of distilled water and then made to rest for either 10 or 30 minutes in a supine position. Blood samples were taken pre and postrest to monitor changes in plasma ADH, total protein, plasma albumin, red blood cell, and hemoglobin concentrations. Urine was collected to monitor [Na+], [Cl-], and osmolarity. Intake of 250 mL distilled water with 10-minute rest caused a significant reduction in plasma ADH concentration, with decreases in urine [Na+], [Cl-], and osmolarity. We found a linear relationship between the ratio of plasma ADH concentrations after/before rest (between 1.1 and 3.0 pg·mL) and the ratio of urine osmolarity after/before rest (between 180 and 601 mOsm·L). Conclusions: Intake of 250 mL distilled water with 10-minute rest in a supine position caused hemodilution and a reduction in urine osmolarity consistent with thoracic lymph drainage. Urine osmolarity is a simple, safe clinical measure for monitoring lymph flow that could be used to evaluate the technique of lymph edema therapists.

Water intake increases mesenteric lymph flow and the total flux of albumin, long-chain fatty acids, and IL-22 in rats: new concept of absorption in jejunum.

The traditional Japanese health care custom recommends that a suitable volume of water is consumed. However, physiological and immunological mechanisms in support of this practice are unknown. Therefore, we conducted rat and rabbit in vivo experiments to investigate the effects of intragastric administration of distilled water on the jejunal-originated lymph flow and the concentrations and total flux of cells, albumin, long-chain fatty acids, and innate lymphoid cell 3 (ILC-3)-secreted interleukin-22 (IL-22) through mesenteric lymph vessels. The distribution and activity of ILC-3 in rat small intestine by water intake were evaluated using flow cytometry and RT-PCR. The intragastric administration of distilled water caused significant increases in rat mesenteric lymph flow and in the total flux of cells, albumin, long-chain fatty acids, and IL-22 through the lymph vessels. Intravenously injected Evans blue dye was rapidly transported into rabbit mesenteric lymph vessel and cisterna chyli. The distribution of ILC-3 and the expression of IL-22 mRNA were maximal in the lamina propria cells of the rat jejunum. No significant presence of ILC-3 in the lymph was observed in the control and under water intake conditions. In conclusion, the absorbed water in the jejunum is transported through mesenteric lymph vessels. The higher permeability of albumin in the jejunal microcirculation may play key roles in the transport of consumed water and the reservoir and transporter of long-chain fatty acids. Water intake also accelerates the transfer of IL-22 to the mesenteric lymph, which may contribute, in part, to maintaining and promoting the innate immunity in the body. NEW & NOTEWORTHY The higher permeability of albumin-mediated transport of water-soluble substances in mesenteric lymph vessels of the jejunum may have a large impact on the classic concept suggesting that water-soluble small molecules travel to the liver via the portal vein. ILC-3 is mainly housed in the lamina propria of the jejunum, especially its upper part. IL-22 released from the ILC-3 is also transported through mesenteric lymph in collaboration with the albumin-mediated movement of consumed water.

Lymph Drainage from the Chylocyst-Induced Hemodilution in an In Vivo Rabbit Study.

To confirm our previous study that abdominal respiration has induced hemodilution in human subjects, we performed in-vivo experiments involving anesthetized rabbits. Fifteen 6- to 7-month-old male Japanese white rabbits were used in the animal experiments. Anesthesia was maintained with 2.5%-3.0% isoflurane under N2O + 100% O2 inhalation. Ventilation was maintained at 40 mL/breath for 20 breaths/min. Physiological saline solution was administered at rated 18 mL/h during the experiments. First, we attempted to evaluate lymph flow through the thoracic duct using Sonazoid-based contrast-enhanced ultrasound (CEUS)-guided method and then investigated the effects of manual lymph drainage of the chylocyst on the numbers of red blood cells (RBC), hematocrit (Ht) levels, and the blood concentrations of total protein (TP) and hemoglobin (Hb). In this study, we established surgical methods for identifying the left venous angle and chylocyst using Evans blue dye in anesthetized rabbits. We also confirmed that a Sonazoid-based CEUS-guided method was the most useful technique for producing real-time images of lymph flow through the thoracic duct in anesthetized rabbits. In addition, in present experiments involving anesthetized rabbits, we confirmed that manually massaging the chylocyst produced significant hemodilution. Thus, the procedure produced significant reductions of TP, RBC, Hb, and Ht level in the rabbits.

Abdominal Respiration Induces Hemodilution and Related Reduction in ADH Concentration of Blood.

To establish effective lymph drainage methods and develop concise and accurate clinical techniques for evaluating lymph drainage in healthy individuals and patients with cancer treatment-related lymph edema, we investigated the numbers of red (RBC) and white (WBC) blood cells, and platelets (PLT) in blood, hematocrit (Ht), and the blood concentrations of total protein (TP), albumin (Alb), and anti-diuretic hormone (ADH) before and after 5 min manual lymph drainage, followed by 30 min rest with or without abdominal respiration in the supine or sitting position in 48 healthy volunteers. The 5 min facial, upper and lower extremities lymph drainage, followed by 30 min rest in the supine position induced significant reductions of the TP and Alb in all subjects, and their RBC and Ht levels in some subjects. The 30 min rest only in the supine position without lymph drainage produced also significant reductions of blood TP and Alb. In addition, abdominal respiration in the supine position without manual lymph drainage caused more significant hemodilution, being significant reductions of TP, Alb, RBC, Ht, and ADH in all volunteers. These findings may be related to effective lymph drainage from the chylocyst. Furthermore, it also resulted in a significantly increased micturition desire. In conclusion, abdominal respiration during 30 min rest in the supine position is effective at inducing lymph drainage, and the associated induction of hemodilution and lowering of the blood ADH concentration (and increased micturition desire in some cases) can be used to accurately assess the extent of lymph drainage.

In vivo support for the new concept of pulmonary blood flow-mediated CO2 gas excretion in the lungs.

To further examine the validity of the proposed concept of pulmonary blood flow-dependent CO2 gas excretion in the lungs, we investigated the effects of intramediastinal balloon catheterization-, pulmonary artery catheterization-, or isoprenaline (ISP)-induced changes in pulmonary blood flow on the end-expiratory CO2 gas pressure (PeCO2 ), the maximal velocity of the pulmonary artery (Max Vp), systemic arterial pressure, and heart rate of anesthetized rabbits. We also evaluated the changes in the PeCO2 in clinical models of anemia or pulmonary embolism. An almost linear relationship was detected between the PeCO2 and Max Vp. In an experiment in which small pulmonary arteries were subjected to stenosis, the PeCO2 fell rapidly, and the speed of the reduction was dependent on the degree of stenosis. ISP produced significant increases in the PeCO2 of the anesthetized rabbits. Conversely, treatment with piceatannol or acetazolamide induced significant reductions in the PeCO2 . Treatment with a cell surface F1/FO ATP synthase antibody caused significant reductions in the PeCO2 itself and the ISP-induced increase in the PeCO2 . Neither the PeCO2 nor SAP was significantly influenced by marked anemia [%hematocrit (Ht), 70 ∼ 47%]. On the other hand, in the presence of less severe anemia (%Ht: 100 ∼ 70%) both the PeCO2 and SAP fell significantly when the rabbits' blood viscosity was decreased. The rabbits in which pulmonary embolisms were induced demonstrated significantly reduced PeCO2 values, which was compatible with the lowering of their Max Vp. In conclusion, we reaffirm the validity of the proposed concept of CO2 gas exchange in the lungs.

Proposed new lymphology combined with lymphatic physiology, innate immunology, and oncology.

As one of the lymphatic functions, it is well known that the transport and drainage of hydrophilic substances including plasma protein through the lymphatic system play pivotal roles in maintaining the homeostasis of the internal environment between the cells in tissues in collaboration with the exchange of the substances through the blood capillaries and venules. The physiological functions of the lymphatic system have been studied by many investigations of microcirculation, i.e., Yoffey and Courtice, Ruszunyak et al., Földie and Casley-Smigh et al., Roddie, Schmid-Schönbein et al., and Ohhashi et al. On the other hand, it is also well known that the initial clinical signs of primary diseases such as inflammation, tumors, and circulatory disorders including infarction and thrombosis appear as functional abnormalities of the internal environment in tissues. These abnormalities of the functions are strongly related to immunological defense reactions around the internal environment and abnormal actions of the transport and drainage of the lymphatic system. Taking into consideration the current inspired findings in lymphatic physiology, innate immunology, and oncology, we have proposed a new lymphology combined with new knowledge of the three above-mentioned academic fields from a defense mechanism points of view. In this review, we would like to demonstrate comprehensively our latest studies related to the possibility of establishing a new lymphology, hoping the readers will evaluate this possibility.

Pivotal roles of lymphatic endothelial cell layers in the permeability to hydrophilic substances through collecting lymph vessel walls: effects of inflammatory cytokines.

To address physiological and pathophysiological meanings of condensing effect of albumin in lymph through collecting lymph vessel walls, we established human lymphatic endothelial cells (LEC) and evaluated the size-dependent regulation of the permeability of such layers to hydrophilic substances. We also investigated the effects of tumor necrosis factor (TNF)-α or interleukin (IL)-1ß on the permeability and on the morphology of human LEC. Significant amounts of 4 kDa dextran, but not 12 or 66 kDa dextran, passed through the layers. TNF-α or IL-1ß induced significant increases in the permeability to 4 and 12 kDa dextrans. TNF-α or IL-1ß also produced significant redistribution of the cytoskeletal F-actin in the LEC, which resulted in changes in their shape. Pretreatment with Y-27632, a Rho kinase inhibitor, or PD98059, an extracellular signal-regulated kinase (ERK) phosphorylation inhibitor, significantly abolished the TNF-α- or IL-1ß-induced increases in the permeability of the layers to 4 and 12 kDa dextrans. Y-27632 and PD98059 significantly inhibited the changes in the F-actin distribution of the LEC produced by TNF-α or IL-1ß. TNF-α or IL-1ß caused significant increases in ERK 1/2 phosphorylation in the LEC, which were significantly inhibited by Y-27632 or PD98059. These findings suggest that the human LEC layer plays key roles in the transport of hydrophilic substances through collecting lymph vessel walls and that TNF-α or IL-1ß significantly increases the permeability of the layers to 4 and 12 kDa dextrans via Rho kinase activation and the ERK 1/2 phosphorylation-mediated reorganization of F-actin in the LEC.

Cell surface F1/FO ATP synthase contributes to interstitial flow-mediated development of the acidic microenvironment in tumor tissues.

To address pivotal roles of cell surface F1/FO ATP synthase in the development of acidic microenvironment in tumor tissues, we investigated effects of shear stress stimulation on the cultured human breast cancer cells, MDA-MB-231 and MDA-MB-157, or human melanoma cells, SK-Mel-1. Shear stress stimulation (0.5-5.0 dyn/cm(2)), the levels of which are similar to those produced by the interstitial flow, induced strength-dependent corelease of ATP and H(+) from the cells, which triggered CO2 gas excretion. In contrast, the same level of shear stress stimulation did not induce significant ATP release and CO2 gas excretion from the control human mammary epithelial cells (HMEC). Marked immunocytochemical and mRNA expression of cell surface F1/FO ATP synthase, vacuolar-ATPase (V-ATPase), carbonic anhydrase type IX, and ectonucleoside triphosphate diphosphohydrolase (ENTPDase) 3 were detected in MDA-MB-231 cells, but little or no expression on the HMEC. Pretreatment with cell surface F1/FO ATP synthase inhibitors, but not cell surface V-ATPase inhibitors, caused a significant reduction of the shear stress stimulation-mediated ATP release and CO2 gas excretion from MDA-MB-231 cells. The ENTPDase activity in the shear stress-loaded MDA-MB-231 cell culture medium supernatant increased significantly in a time-dependent manner. In addition, MDA-MB-231 cells displayed strong staining for purinergic 2Y1 (P2Y1) receptors on their surfaces, and the receptors partially colocalized with ENTPDase 3. These findings suggest that cell surface F1/FO ATP synthase, but not V-ATPase, may play key roles in the development of interstitial flow-mediated acidic microenvironment in tumor tissues through the shear stress stimulation-induced ATP and H(+) corelease and CO2 gas production.

Tumour PDGF-BB expression levels determine dual effects of anti-PDGF drugs on vascular remodelling and metastasis.

Anti-platelet-derived growth factor (PDGF) drugs are routinely used in front-line therapy for the treatment of various cancers, but the molecular mechanism underlying their dose-dependent impact on vascular remodelling remains poorly understood. Here we show that anti-PDGF drugs significantly inhibit tumour growth and metastasis in high PDGF-BB-producing tumours by preventing pericyte loss and vascular permeability, whereas they promote tumour cell dissemination and metastasis in PDGF-BB-low-producing or PDGF-BB-negative tumours by ablating pericytes from tumour vessels. We show that this opposing effect is due to PDGF-ß signalling in pericytes. Persistent exposure of pericytes to PDGF-BB markedly downregulates PDGF-ß and inactivation of the PDGF-ß signalling decreases integrin α1ß1 levels, which impairs pericyte adhesion to extracellular matrix components in blood vessels. Our data suggest that tumour PDGF-BB levels may serve as a biomarker for selection of tumour-bearing hosts for anti-PDGF therapy and unsupervised use of anti-PDGF drugs could potentially promote tumour invasion and metastasis.

Sphingosine 1-phosphate (S1P) induces S1P2 receptor-dependent tonic contraction in murine iliac lymph vessels.

OBJECTIVE: We studied the effects of S1P on the diameter and spontaneous contraction of murine iliac collecting lymph vessels. METHODS: The isolated lymph vessel was cannulated with two glass micropipettes and then pressurized to 4 cmH(2) O at the intraluminal pressure. The changes in lymph vessel diameter were measured using a custom-made diameter-detection device. Immunohistochemical studies were also performed to confirm S1P receptors on the lymph vessels. RESULTS: S1P (10(-7) M) had no significant effect on the frequency or amplitude of the lymph vessels' spontaneous contractions. In contrast, S1P (10(-8) -10(-6) M) produced a concentration-related reduction in lymph vessel diameter (tonic contraction). Pretreatment with 10(-4) M l-NAME or 10(-5) M aspirin had no significant effect on the S1P-induced tonic contraction of the lymph vessels. To evaluate the intracellular signal transduction pathway responsible for the S1P-induced tonic contractions and their Ca(2+) -dependence, we investigated the effects of JTE013, VPC23019, U-73122, xestospongin C, and nifedipine on the S1P-induced tonic contractions. All of these inhibitors except VPC23019 and nifedipine significantly reduced the S1P-induced tonic contractions. S1P (5x10(-7) M) also induced significant tonic contractions in the lymph vessels that had been superfused with high K(+) Krebs-bicarbonate solution or Ca(2+) -free high K(+) Krebs solution containing 1 mM EGTA. S1P2 receptors were immunohistochemically detected in the lymph vessels. CONCLUSION: These findings suggest that neither endogenous NO nor prostaglandins are involved in the S1P-induced tonic contraction of lymph vessels, which is mainly caused by Ca(2+) release from intracellular Ca(2+) stores through the activation of S1P2 and 1,4,5 IP(3) receptors.