Toshio Ohhashi

The response of lymphatic smooth muscles to vasoactive substances

A study was made of the isotonic response of bovine mesenteric lymphatics to several physiological vasoactive substances. Contractions of lymphatic smooth muscles were induced by serotonin (5-HT), prostaglandin F2α (PGF2α), noradrenaline (NA), histamine, dopamine and acetylcholine (ACh). The smooth muscles were particularly sensitive to 5-HT. Excepting PGF2α no other substances could equal 5-HT in the magnitude of the maximum response. The majority of 5-HT receptors seemed to be the D receptors. The decreasing order of the contractile responses was as follows: 5-HT>PGF2α>NA>histamine>dopamine>ACh. The contractile response to ACh was observed only in specimens involving valvular region. It was very likely that, in the lymphatics, there were 2 kinds of receptors for catecholamines, i.e. α and β receptors, and the stimulation of the former induced smooth muscle contraction and that of the latter relaxation. A difference was noticed between the responses of valvular and intervalvular segments to NA. Relaxations of lymphatic smooth muscles were induced not only by isoproterenol but also by adenosine and adenine nucleotides. The decreasing order of the relaxant responses was as follows: ISP>adenosine >ATP>ADP>cyclic AMP≧AMP. The relaxant responses to adenine nucleotides tended to reduce with decrease in the number of high energy phosphates.

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-β signalling in pericytes. Persistent exposure of pericytes to PDGF-BB markedly downregulates PDGF-β and inactivation of the PDGF-β signalling decreases integrin α1β1 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.

Human perspiration measurement

We review various methods developed for human perspiration measurement and their physiological applications, with special reference to the performance and application of a new home-made ratemeter and instrumentation with a microscope. Many kinds of humidity sensor based on humidity-sensitive electrical properties have been investigated and placed on the market. Recently a capacitive thin-film humidity sensor was constructed and confirmed to be one of the best humidity sensors for accurately and quickly detecting changes in the relative humidity of gas-flow perfused through a ventilated chamber for human perspiration measurement. In this paper we also introduce a new home-made ratemeter with a capacitive humidity sensor, the electrical output of which is not disturbed by changes in ambient temperature, and new instrumentation for directly observing drops of sweat secreted from eccrine glands in human skin and simultaneously measuring the change in amount of perspiration at the same area of skin. Finally, we review physiological applications of the methods for measuring human palmar perspiration including emotional sweating.

Critical Role for GATA3 in Mediating Tie2 Expression and Function in Large Vessel Endothelial Cells

Endothelial phenotypes are highly regulated in space and time by both transcriptional and post-transcriptional mechanisms. There is increasing evidence that the GATA family of transcription factors function as signal transducers, coupling changes in the extracellular environment to changes in downstream target gene expression. Here we show that human primary endothelial cells derived from large blood vessels express GATA2, -3, and -6. Of these factors, GATA3 was expressed at the highest levels. In DNA microarrays of human umbilical vein endothelial cells (HUVEC), small interfering RNA-mediated knockdown of GATA3 resulted in reduced expression of genes associated with angiogenesis, including Tie2. At a functional level, GATA3 knockdown inhibited angiopoietin (Ang)-1-mediated but not vascular endothelial cell growth factor (VEGF)-mediated AKT signaling, cell migration, survival, and tube formation. In electrophoretic gel mobility shift assays and chromatin immunoprecipitation, GATA3 was shown to bind to regulatory regions within the 5′-untranslated region of the Tie2 gene. In co-immunoprecipitation and co-transfection assays, GATA3 and the Ets transcription factor, ELF1, physically interacted and synergized to transactivate the Tie2 promoter. GATA3 knockdown blocked the ability of Ang-1 to attenuate vascular endothelial cell growth factor stimulation of vascular cell adhesion molecule-1 expression and monocytic cell adhesion. Moreover, exposure of human umbilical vein endothelial cells to tumor necrosis factor-α resulted in marked down-regulation of GATA3 expression and reduction in Tie2 expression. Together, these findings suggest that GATA3 is indispensable for Ang-1-Tie2-mediated signaling in large vessel endothelial cells.

Shear stress-induced ATP-mediated endothelial constitutive nitric oxide synthase expression in human lymphatic endothelial cells

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(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(-9) to 10(-6) M produced a significant increase in ecNOS immunohistochemical expression in a dose-dependent manner. The increase in ecNOS expression mediated by 10(-6)M ATP was significantly reduced by 10(-5) M suramin. Suramin (10(-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(-4) M apamin, 10(-9) M iberiotoxin, 10(-5) M 2-aminoethoxydephenyl borate, or 10(-5)M xestospongin C, but not 10(-5) M glybenclamide or 10(-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(Ca)) and big-conductance (BK(Ca)) Ca(2+)-activated K(+) 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-trisphosphate-mediated release of intracellular Ca(2+) ions and the activation of Ca(2+)-activated K(+) channels in LEC.

Effects of calcitonin gene-related peptide on neuromuscular transmission in the isolated rat diaphragm

The present study was undertaken to investigate a possible interaction between the cholinergic nerve neurotransmitter and CGRP on neuromuscular transmission in the isolated rat diaphragm. Electrical stimulation of the isolated phrenic nerve resulted in twitch contractions which were dose-dependently potentiated by CGRP in concentrations ranging from 1.2 x 10(-9) M to 3 x 10(-7) M. The potentiating action of CGRP (3 x 10(-7) M) disappeared in about 25 min. The same dose of CGRP 40 min later produced an augmentation of contraction amplitude similar to that observed prior to the administration of CGRP. The action of CGRP was dependent upon the stimulation pulse width ranging from 0.2 to 1.0 msec. Rat calcitonin (4.5 x 10(-7) M) caused a minimal change in the amplitude of twitch contractions. CGRP had no effect on the quiescent striated muscle. Twitch responses to direct electrical stimulation were also enhanced by CGRP (6 x 10(-8) M-6 x 10(-7) M) in the absence and presence of 10(-5) M d-tubocurarine. These results suggest that CGRP modulates the action of acetylcholine at the motor end plates of striated muscle.

Innervation of bovine mesenteric lymphatics: From the histochemical point of view

Abstract Histochemical studies were carried out on the distribution of adrenergic and cholinergic nerves in bovine mesenteric lymphatics. Most of the catecholamine-specific fluorescent nerve fibers demonstrated by the glyoxylic acid method encircled these vessels, although a few of them ran spirally or longitudinally. The fluorescent nerve fibers were present not only in the adventitia but also in the smooth muscle layers. The peak wavelengths of excitation and emission spectra of the fluorescence analyzed by means of a microepifluorescence spectrophotometer were about 415 and 465 nm, respectively. Both the values agreed with those of norepinephrine. Most of the cholinesterase-positive fibers stained by the copper thiocholine method were longitudinal in direction. The pattern of their distribution was similar to that of the catecholamine-containing nerve fibers, though the distribution density of the former fibers was less than the latter. Labeling was not affected by 10 −4 M iso-OMPA in the presence of acetylthiocholine iodide as a substrate—an indication that the staining of these fibers is specific for cholinesterase activity. These results suggest that the adrenergic and cholinergic nerve fibers are distributed in the smooth muscle layers as well as in the adventitia of bovine mesenteric lymphatics.

MDA-MB-231 produces ATP-mediated ICAM-1-dependent facilitation of the attachment of carcinoma cells to human lymphatic endothelial cells

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 <1,000 molecular weight (MW) caused a complete reduction of the supernatant-mediated response. In contrast, pretreatment with heating, digestion with protease, or chemical treatment with dialyzed substances of <500 MW produced no significant effect on the supernatant-mediated response. ATP (10(-7) M) caused overexpression of ICAM-1 on human LECs similar to that produced by the supernatant of MDA-MB-231. The ATP- and MDA-MB-231 supernatant-mediated responses were significantly reduced by treatment with 10(-6) M suramin (a purinergic P2X and P2Y receptor antagonist). In attachment assays, 10(-7) M ATP or MDA-MB-231 supernatant produced a significant increase in the attachment of carcinoma cells to human LECs. The treatment with 10(-6) M suramin caused a significant reduction of ATP- and supernatant-mediated facilitation of the attachment responses. Additional treatment with anti-ICAM-1 antibody also caused a significant reduction of ATP- and supernatant-mediated facilitation of the attachment responses. The experimental findings suggest that MDA-MB-231 may release or leak ATP, which produces the overexpression of ICAM-1 on human LECs through activation of purinergic P2X and/or P2Y receptors and then facilitates ICAM-1-mediated attachment of carcinoma cells to LECs.

Vasa vasorum within the media of bovine mesenteric lymphatics.

Summary Smooth muscles in bovine mesenteric lymphatics were well-developed and arranged in three layers, i.e., the internal longitudinal, intermediate circumferential, and external longitudinal. The outermost one was much thicker than the other two. Blood capillaries were found within the smooth muscle layers as well as in the ad-ventitia. These blood capillaries may be essential for maintaining vigorous rhythmic contractions of the lymphatic smooth muscles which act as a driving force for the propulsion of lymph. The presence of numerous mitochondria and glycogen granules seemed to reflect a high metabolic activity of the smooth muscle cells. The authors are indebted to Dr. T. Suganuma and Mr. R. Ichikawa for their technical advice and assistance.

Vasoactive intestinal peptide inhibitory innervation in bovine mesenteric lymphatics. A histochemical and pharmacological study.

The localization of vasoactive intestinal peptide-immunoreactive nerves innervating bovine lymphatic vessels was studied by an immunohistochemical technique. Nerve fibers containing vasoactive intestinal peptide immunoreactivity were present in the smooth muscle layers as well as in the adventitia of all mesenteric lymphatics that were examined. The effect of vasoactive intestinal peptide on isolated lymphatic vessels in vitro was studied. Vasoactive intestinal peptide caused a concentration-dependent relaxation of bradykinin-induced contractions of lymphatic vessels. The threshold and maximum relaxations were achieved with vasoactive intestinal peptide at concentrations less than 6 X 10(-9) M and 3 X 10(-7) M, respectively. The relaxant response to vasoactive intestinal peptide was not modified by atropine, propranolol, bretylium, or tetrodotoxin. These results suggest that vasoactive intestinal peptide may be a possible inhibitory neurotransmitter that causes relaxation of lymphatic vessels.