Fumitaka Ikomi

Cilostazol, an Inhibitor of Type 3 Phosphodiesterase, Produces Endothelium-Independent Vasodilation in Pressurized Rabbit Cerebral Penetrating Arterioles

We investigated the effects of cilostazol, a potent inhibitor of cGMP-inhibited cAMP phosphodiesterase, on mechanical activity of isolated pressurized rabbit cerebral penetrating arterioles with special reference to the function of the endothelium. Both cilostazol and milrinone, another inhibitor of cAMP phosphodiesterase, produced vasodilation of the cerebral penetrating arterioles in a dose-dependent manner. Pretreatment with selective inhibitors of cyclooxygenase or nitric oxide synthase, or chemical denudation of the endothelial cells caused no significant effect on the cilostazol-mediated vasodilation of the cerebral arterioles. A selective large-conductance calcium-activated potassium channel inhibitor, iberiotoxin, and a selective protein kinase A inhibitor, H-89, caused no significant effect on the cilostazol-mediated vasodilation. In the cerebral arterioles, low concentration (10–6M) of cilostazol or milrinone caused a significant shift of the dose-vasodilatory response curve for adenosine to the left. These findings suggest that cilostazol produces vasodilation independent of the presence of the endothelium or activation of endogenous vasodilative prostaglandins, nitric oxide, calcium-activated potassium channel and protein kinase A. In conclusion, the vasodilator action of cilostazol may, in part, contribute to the beneficial effect of preventing lacunar cerebral infarction in patients with functional damage of the endothelium in cerebral penetrating arterioles.

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.

Effects of endothelin on spontaneous contractions in lymph vessels

A mode of action of endothelin (ET) on spontaneous contractions was investigated in ring preparations of isolated bovine mesenteric lymphatics. ET-1 at concentrations between 10-10 and 10-9 M caused a dose-dependent increase in the frequency of spontaneous contractions. The specific ETA-receptor antagonist BQ-123 (5 × 10-7 M) caused a significant inhibition of the ET-1-induced positive chronotropic effect in the ring preparations with and without the endothelium. Mechanical denudation of the lymphatic endothelial cells produced a significant potentiation of the ET-induced positive chronotropic effect. BQ-3020 (10-8-10-7M), a selective ETB-receptor agonist, induced dose dependently negative chronotropic and inotropic effects on the spontaneous contractions in the ring preparations with intact endothelium. Mechanical removal of the endothelium caused a significant reduction of the BQ-3020-induced negative chronotropic and inotropic effects. The ET-1-induced positive chronotropic effect was potentiated by pretreatment with N ω-nitro-l-arginine methyl ester (l-NAME) (10-5 M) but unaffected by aspirin (10-5 M). Additional treatment with l-arginine (10-4 M) completely reversed the l-NAME-mediated potentiation of the ET-induced chronotropic effect. These results suggest that stimulation of ETA receptors on the lymphatic smooth muscles causes a positive chronotropic effect on the spontaneous contractions, and stimulation of ETB receptors on the lymphatic endothelial cells induces a release of nitric oxide, which results in the chronotropic and inotropic effects on spontaneous contractions in isolated bovine mesenteric lymphatics.A mode of action of endothelin (ET) on spontaneous contractions was investigated in ring preparations of isolated bovine mesenteric lymphatics. ET-1 at concentrations between 10(-10) and 10(-9) M caused a dose-dependent increase in the frequency of spontaneous contractions. The specific ET(A)-receptor antagonist BQ-123 (5 x 10(-7) M) caused a significant inhibition of the ET-1-induced positive chronotropic effect in the ring preparations with and without the endothelium. Mechanical denudation of the lymphatic endothelial cells produced a significant potentiation of the ET-induced positive chronotropic effect. BQ-3020 (10(-8)-10(-7) M), a selective ET(B)-receptor agonist, induced dose dependently negative chronotropic and inotropic effects on the spontaneous contractions in the ring preparations with intact endothelium. Mechanical removal of the endothelium caused a significant reduction of the BQ-3020-induced negative chronotropic and inotropic effects. The ET-1-induced positive chronotropic effect was potentiated by pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME) (10(-5) M) but unaffected by aspirin (10(-5) M). Additional treatment with L-arginine (10(-4) M) completely reversed the L-NAME-mediated potentiation of the ET-induced chronotropic effect. These results suggest that stimulation of ET(A) receptors on the lymphatic smooth muscles causes a positive chronotropic effect on the spontaneous contractions, and stimulation of ET(B) receptors on the lymphatic endothelial cells induces a release of nitric oxide, which results in the chronotropic and inotropic effects on spontaneous contractions in isolated bovine mesenteric lymphatics.

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.

Recent Advance in Lymph Dynamic Analysis in Lymphatics and Lymph Nodes

Lymphatics are a unidirectional transport system that carries fluid from the interstitial space and back into the blood stream. Initial lymphatics take up not only fluid but also high-molecular-weight substances, such as plasma proteins and hyaluronan; immune cells, such as lymphocytes, macrophages, and dendritic cells; and colloidal particles, such as carbon particles, bacteria, and tattoo dye. Interstitially injected colloidal particles are known to accumulate in the regional lymph nodes. This phenomenon is applied to find sentinel lymph nodes in cancer patients. Lymph flow rate and composition are influenced by interstitial fluid, lymphatic pump activity, and intra-lymphatic pressure. Lymph composition is changed during its flow downstream. In this review, the main focus is on the mechanisms of lymph formation at the initial lymphatics and lymph transport through the collecting lymphatics and lymph nodes. (*English Translation of J Jpn Coll Angiol, 2008, 48: 113-123.).

Electrical stimulation-induced α1- and α2-adrenoceptors-mediated contraction in isolated dog thoracic ducts

Abstract The electrical stimulation-induced responses of isolated dog thoracic ducts were investigated using an organ bath technique. Electrical stimulation (0.7 ms in pulse width, 25 V in nominal voltage, 10 s in duration time, 1–32 Hz at frequency) produced frequency-related contractions in the lymphatic preparations. The contractions were abolished by pretreatment with tetrodotoxin (10 −7 M), guanethidine (10 −7 , 10 −6 M), and bretylium (10 −7 , 10 −6 M). Cocaine (10 −6 M) significantly potentiated the electrical stimulation−induced contractions. Phentolamine (10 −8 –10 −5 M), prazosin (10 −8 –10 −5 M), bunazosin (10 −6 , 10 −5 M), yohimbine (10 −8 –10 −6 M) and rauwolscine (10 −8 –10 −6 M) also dose−dependently reduced the contractions. On the other hand, propranolol (10 −8 –10 −6 M), atropine (10 −6 M), hexamethonium (10 −6 M), aspirin (3×10 −5 M), Nω -nitro- l -arginine methyl ester (L-NAME) (3×10 −5 M) and L-NAME (3×10 −5 M)+ l -arginine (10 −4 M) caused no significant effect on electrical stimulation-induced contractions. No significant difference in the electrical stimulation-induced responses was observed between the lymphatic preparations with and without an intact endothelium. The electrical stimulation caused only a small contraction with no relaxation in the thoracic duct preparation precontracted with 10 −8 M U46619. The small contraction was abolished by 10 −5 M phentolamine. These findings suggest that there exists α 1 - and α 2 -adrenoceptors-mediated excitatory innervation, but no NO-ergic inhibitory nerve fiber in dog thoracic ducts.

In Situ Lymph Dynamic Characterization through Lymph Nodes in Rabbit Hind Leg : Special Reference to Nodal Inflammation

In some lymph nodes, water and water-soluble substances of smaller molecular weight are known to be absorbed into blood vessels, and consequently the protein concentration of lymph within the nodes increases. In this study, we examined pressure-flow relationships of lymph nodes in situ and exchange properties of water and water-soluble substances through the nodes with special reference to inflamed lymph nodes. A lymph perfusion model through the lymph node in situ was constructed by cannulating one of the afferent lymphatics and an efferent lymphatic. Increasing infusion pressure (0 to 150 cmH(2)O) or decreasing outflow pressure (10 to -5 cmH(2)O) in the model caused a significant increase of the lymph outflow rate through the node. This rate was also increased significantly with increases in both intranodal venous pressure (range: control, 20, 30, and 40 mmHg) and prenodal lymph albumin concentration (range: 0%, 2.6%, and 10%). When formyl-Met-Leu-Phe-OH (fMLP)-mediated acute inflammation was produced in the lymph nodes, the lymph outflow rate through the node was significantly decreased. These results indicate that colloid osmotic pressure and hydrostatic pressure within the lymph node may play important roles in the transport of water and water-soluble substances through the node. Acute fMLP-mediated inflammation of lymph nodes also produced a significant decrease of the lymph flow rate through lymph nodes.

Electrical stimulation-induced α1- and α2-adrenoceptors-mediated contractions of isolated canine lymph nodes

Abstract The contractile effects of α-adrenoceptor agonists and field electrical stimulation were investigated pharmacologically in canine isolated tracheobronchial lymph nodes. Addition of noradrenaline (NA), phenylephrine (Phe) and UK14,304 caused dose-related contractions of the isolated lymph nodes. Pretreatment with prazosin (3×10 −9 , 3×10 −8 and 3×10 −7 M) shifted the concentration–response curve for phenylephrine to the right, whereas yohimbine (3×10 −7 M) did not affect the contractile responses to phenylephrine. On the other hand, the concentration–response curve for UK14,304 shifted to the right by treatment with yohimbine (3×10 −9 , 3×10 −8 and 3×10 −7 M) but not by prazosin (3×10 −7 M). Field electrical stimulation (25 V, 0.7 ms, 16 Hz) produced contractile responses of the lymph nodes. The electrical stimulation-induced contractions were completely reduced by pretreatment with tetrodotoxin (TTX) (3×10 −7 M) or bretylium (10 −6 M). Furthermore, phentolamine (10 −8 , 10 −7 and 10 −6 M), prazosin (3×10 −9 and 3×10 −8 M) and yohimbine (3×10 −8 and 3×10 −7 M) significantly reduced the electrical stimulation-induced contractions in the preparations. The present findings suggest that there are both α 1 - and α 2 -adrenoceptors located on the smooth muscle cells in canine tracheobronchial lymph nodes, and that the adrenoceptors-mediated excitatory mechanisms of adrenergic innervation exist in the lymph nodes.

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.