Risuke Mizuno

Establishment of rat lymphatic endothelial cell line.

Objective: The objective of the present study was to establish a rat lymphatic endothelial cell line and then to investigate the morphological and immunohistochemical properties of the cells.

Involvement of ATP-sensitive K+ channels in spontaneous activity of isolated lymph microvessels in rats

Physiological roles of ATP-sensitive K+ channels for spontaneous activity in isolated rat mesenteric lymph microvessels (maximum diameter ∼80-150 μm) were investigated. The lymph microvessels were cannulated with glass micropipettes and pressurized at a perfusion pressure of 6 cmH2O. Changes in the diameter and frequency of spontaneous contractions in the lymphatics were measured with videomicroscopy. Pinacidil (K+-channel opener) inhibited the spontaneous activity. In the presence of glibenclamide (selective ATP-sensitive K+-channel blocker; 10-7 and 10-6 M) and tetraethylammonium (TEA; nonselective K+-channel blocker; 10-4 and 10-3 M), the pinacidil-induced inhibition of the spontaneous contractions in lymph microvessels was significantly reversed. Glibenclamide and TEA themselves, however, did not affect the frequency of spontaneous activity in the lymph microvessels. These results suggest that ATP-sensitive K+ channels are involved in the regulation of spontaneous activity in the smooth muscles of isolated lymph microvessels of rat mesenteries.

Involvement of NO and EDHF in Flow-Induced Vasodilation in Isolated Hamster Cremasteric Arterioles

Flow-induced vasodilation in hamster cremasteric arterioles was investigated with special reference to the roles of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). Arterioles (∼60 µm resting diameter) were cannulated, and suffused with MOPS solution at 37°C (mean intraluminal pressure: 80 cm H2O). Step increases in the perfusate flow elicited a dose-dependent vasodilation, which was almost proportional to the increases in calculated wall shear stress (WSS). Nω-nitro L-arginine methyl ester (L-NAME, 100 µM) reduced the flow-induced vasodilation by ∼50%, whereas indomethacin (10 µM) produced no significant effect. In the presence of L-NAME, the residual vasodilation was eliminated by treatment with the cytochrome P-450 monooxygenase inhibitor 17-octadecynoic acid (17-ODYA, 50 µM), sulfaphenazol (10 µM), tetraethylammonium (TEA, 3 mM; a nonselective Ca2+-activated K+ channel inhibitor), or charybdotoxin (ChTX, 0.1 µM; intermediate or large conductance Ca2+-activated K+ channel inhibitor). In the absence of L-NAME, the dilation was also reduced by ∼50% by treatment with 17-ODYA, TEA, or ChTX. The residual vasodilation was eliminated by additional treatment with L-NAME. The inhibitor of ATP-sensitive K+ channels (KATP), glibenclamide, also caused a significant, but partial, reduction of the flow-induced vasodilation. The residual vasodilation was completely reduced by additional treatment with 17-ODYA, but not L-NAME. These findings suggest that in hamster cremaster, higher flow rate produces NO, KATP, and EDHF vasodilation of the arterioles under physiological conditions.

Critical roles of VEGF-C-VEGF receptor 3 in reconnection of the collecting lymph vessels in mice

Molecular mechanisms of reconnection of collecting lymph vessels were analyzed by using murine popliteal prenodal lymph vessels. At 1 and 2 weeks after being divided by cutting the lymph vessel, lymphatic reconnection was frequently observed accompanied by mesh‐like lymphatic channels. Electron microscopic study also showed a monolayer of endothelial cells in the newly developed lymph vessels. Smooth muscle markers were immunofluorescently demonstrated in the wall of the new vessels. At 1 week after the procedure of cutting, augmented expressions of VEGF receptors 1, 2 and 3 were found immunohistochemically at the site of the reconnected lymph vessels. The expression of mRNA for VEGF receptor 3 was enhanced at 5 days and 1 week in small pieces of the tissues containing the reconnected lymph vessels, compared with that in the corresponding tissues obtained with sham operated ones. The administration of VEGF‐C at the cutting site of the collecting lymph vessel significantly increased the rate of the reconnected lymph vessels, whereas additional treatment with Flt4/Fc chimera protein significantly reduced the rate of the reconnected ones. These results suggest that activation of VEGF‐C‐VEGF receptor 3 has critical roles in reconnection of the collecting lymph vessels in adult mice.

Recanalization of the Collecting Lymphatics in Rabbit Hind Leg

Objective: This study was designed to examine whether mature collecting lymphatics can regenerate in the adult tissue or not.

Glucose and Glucose Transporters Regulate Lymphatic Pump Activity through Activation of the Mitochondrial ATP-Sensitive K+ Channel

We investigated the pivotal roles of glucose and its transporter in the regulation of mechanical activity of isolated rat thoracic ducts and then examined whether mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)) are involved in those responses. In the absence of extracellular glucose, the thoracic ducts showed pump activity during 120 min. Extracellular glucose caused a dose-dependent increase in the frequency of pump activity and a constriction in the thoracic ducts. Pump activity of the thoracic ducts in 0 mm glucose was completely inhibited in the presence of chlorogenic acid (an inhibitor of glucose-6-phosphatase). Cytochalasin B, an inhibitor of facilitative glucose transporter (GLUT), or phlorizin, an inhibitor of sodium-dependent glucose cotransporter (SGLT), significantly reduced the frequency of pump activity and dilated the thoracic ducts. A decrease in the frequency of pump activity induced by 5-hydroxydecanoate (5-HD, a selective blocker of mitoK(ATP)) was completely reversed by ruthenium red (an inhibitor of Ca(2+) uniporter in mitochondria). Diazoxide (a selective opener of mitoK(ATP)) significantly increased the frequency of pump activity. Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP, a protonophore of mitochondrial proton pump action) significantly reduced the frequency of pump activity and dilated the thoracic ducts. Collectively, these findings suggest that glucose derived from intracellular glycogen and/or through GLUT/SGLT in lymphatic smooth muscles contributes to the regulation of the pump activity of isolated rat thoracic ducts, and that mitoK(ATP) in the cells may partially serve as a modulator of the mechanical functions associated with mitochondrial Ca(2+) uptake.

NT-702, a Selective Phosphodiesterase 3 Inhibitor, Dilates Rabbit Spinal Arterioles via Endothelium-Dependent and Endothelium-Independent Mechanisms

We investigated the effects of NT-702, a selective phosphodiesterase (PDE) 3 inhibitor, on arterioles isolated from rabbit lumbar spinal cords. NT-702 caused a dose-dependent dilation of the isolated spinal arterioles. The disruption of endothelium produced a significant reduction of higher concentrations (10(-7) and 10(-6) M), but not lower concentrations (less than 10(-8) M), of NT-702-induced vasodilation. The NT-702-induced vasodilation of the arterioles with endothelium was not affected by pretreatment with an inhibitor of nitric oxide, cyclooxygenase, or cytochrome P-450 monooxygenase. In contrast, catalase reduced significantly the higher concentrations of NT-702-induced vasodilation only. Tetraethylammonium (TEA) completely reduced the lower concentrations of NT-702-induced vasodilation, but decreased only partially the higher concentrations of NT-702-induced vasodilation of the arterioles with endothelium. Hydrogen peroxide dilated significantly the isolated arterioles with endothelium, the response of which was reduced significantly by TEA. KT5720 (a selective protein kinase inhibitor) significantly decreased both the lower and higher concentrations of NT-702-induced vasodilation of the arterioles with endothelium. The findings suggest that NT-702 dose-dependently dilated the isolated spinal arterioles of rabbits via endothelium-dependent and endothelium-independent mechanisms. Protein kinase A (PKA)- and TEA-sensitive K(+) channels may be involved in the NT-702-induced vasodilation. Moreover, hydrogen peroxide may contribute in part to the endothelium-dependent higher concentrations of NT-702-induced vasodilation.

Effects of exercise intensity, posture, pressure on the back and ambient temperature on palmar sweating responses due to handgrip exercises in humans

We have, by using newly developed ratemeters, attempted to examine the effects of exercise intensity, posture, pressure on the skin of the back, and ambient hyperthermic conditions (approximately 30 degrees C) on the 5-s handgrip exercise-mediated responses of active palmar sweating in humans. Thirty-five right-handed male (n=5) and female (n=30) volunteer students (20.2+/-1.3 years old) participated in the present study. Oral explanation of only the isometric handgrip exercise (IHG) caused a rapid and oscillatory response (pre-operational) of active palmar sweating in almost all subjects (10 of 14 subjects). Performing the IHG for 5-s caused a significant increase in active sweating rate (operation-mediated response) in both ipsi- and contra-lateral palmar surfaces of the thumbs of all subjects. The operation-mediated responses of active palmar sweating to the IHG were reproducible, resulting in no habituation. The increase of operation-mediated responses to the IHG was dependent upon exercise intensity (100-25% maximal voluntary contractions). The IHG-mediated ipsi- and contra-lateral responses of active palmar sweating were significantly decreased by changing the body posture from a seated to a supine position or by pressing the skin of the back. Ambient hyperthermic conditions (approximately 30 degrees C) for 60 min also resulted in a significant decrease in the back-pressure-dependent reduction of the operation-mediated responses of active palmar sweating to the IHG. In conclusion, in order to optimize the precision and reproducibility of clinical tests involving palmar sweating responses, it is important that subjects maintain a constant handgrip force and posture and that ambient temperature be kept under normothermic conditions.

Head-down tilt posture elicits transient lymphocyte mobilization from the iliac, but not mesenteric, lymph nodes of rats.

The effects of short-term simulated microgravity on the lymph dynamics of rat lymph nodes were investigated using a combination of Bollmans cage and head-down tilt (HDT). Efferent lymphatics of the iliac and mesenteric lymph nodes were cannulated for the collection of lymph. There was no significant difference in lymph flow rate from the iliac lymph nodes between non-HDT (control) and HDT rats. Lymph flow rate from the mesenteric lymph nodes in HDT rats was slightly higher than that obtained with the control. The cell count obtained from the iliac lymph nodes in HDT rats was significantly larger than those of the controls, while no significant difference in the number of cells from the mesenteric lymph nodes was observed between the control and HDT groups. The cells from the iliac lymph nodes in the control and HDT rats were mostly lymphocytes. The distribution of subsets of lymphocytes (CD3+, CD4+, CD8a+, and CD45R+) from the iliac lymph nodes in HDT rats was not significantly different from the subsets of lymphocytes in the control. Immunization did not affect the distribution of lymphocyte subsets from the iliac lymph nodes in the control and HDT groups. There was no significant difference in the concentrations of lymph albumin in iliac afferent or efferent lymphatics between the control and HDT groups. These findings suggest that HDT posture in Bollmans cage induces transient output of lymphocytes from the iliac lymph nodes of rats in vivo without changing the flow rate, lymphocyte subsets, or concentration of albumin.