Transporters, Ion Channels, and Junctional Proteins in Choroid Plexus Epithelial Cells.

The choroid plexus (CP) plays significant roles in secreting cerebrospinal fluid (CSF) and forming circadian rhythms. A monolayer of epithelial cells with tight and adherens junctions of CP forms the blood-CSF barrier to control the movement of substances between the blood and ventricles, as microvessels in the stroma of CP have fenestrations in endothelial cells. CP epithelial cells are equipped with several kinds of transporters and ion channels to transport nutrient substances and secrete CSF. In addition, junctional components also contribute to CSF production as well as blood-CSF barrier formation. However, it remains unclear how junctional components as well as transporters and ion channels contribute to the pathogenesis of neurodegenerative disorders. In this manuscript, recent findings regarding the distribution and significance of transporters, ion channels, and junctional proteins in CP epithelial cells are introduced, and how changes in expression of their epithelial proteins contribute to the pathophysiology of brain disorders are reviewed.

Subcellular distribution of α2-adrenoceptor subtypes in the rodent kidney.

Renal α2-adrenoceptors have been reported to play a role in the regulation of urinary output, renin secretion, and water and sodium excretion in the kidneys. However, the distribution of α2-adrenoceptor subtypes in the kidneys remains unclear. In this study, we aimed to investigate the localization of α2-adrenoceptor subtypes in rat kidneys using 8-week-old Sprague-Dawley rats. Immunofluorescence imaging revealed that both α2A- and α2B-adrenoceptors were expressed in the basolateral, but not apical, membrane of the epithelial cells of the proximal tubules. We also found that α2A- and α2B-adrenoceptors were not expressed in the glomeruli, collecting ducts, or the descending limb of the loop of Henle and vasa recta. In contrast, α2C-adrenoceptors were found to be localized in the glomeruli and lumen of the cortical and medullary collecting ducts. These results suggest that noradrenaline may act on the basement membrane of the proximal tubules through α2A- and α2B-adrenoceptors. Moreover, noradrenaline may be involved in the regulation of glomerular filtration and proteinuria through the induction of morphological changes in mesangial cells and podocytes via α2C-adrenoceptors. In the collecting ducts, urinary noradrenaline may regulate morphological changes of the microvilli through α2C-adrenoceptors. Our findings provide an immunohistochemical basis for understanding the cellular targets of α2-adrenergic regulation in the kidneys. This may be used to devise therapeutic strategies targeting α2-adrenoceptors.

Effects of intravesicular loading of a Ca2+ chelator and depolymerization of actin fibers on neurotransmitter release in frog motor nerve terminals.

Ca2+ -induced Ca2+ release (CICR) via type-3 ryanodine receptor enhances neurotransmitter release in frog motor nerve terminals. To test a possible role of synaptic vesicle in CICR, we examined the effects of loading of EGTA, a Ca2+ chelator, into synaptic vesicles and depolymerization of actin fibers. Intravesicular EGTA loading via endocytosis inhibited the ryanodine sensitive enhancement of transmitter release induced by tetanic stimulation and the associated rises in intracellular-free Ca2+ ([Ca2+ ]i : Ca2+ transients). Latrunculin A, a depolymerizer of actin fibers, enhanced both spontaneous and stimulation-induced transmitter release, but inhibited the enhancement of transmitter release elicited by successive tetanic stimulation. The results suggest a possibility that the activation of CICR from mobilized synaptic vesicles caused the enhancement of neurotransmitter release.

Histomorphometry of ectopic mineralization using undecalcified frozen bone sections.

To investigate the correlation between mineral formation and enhanced expressions of some proteins using undecalcified frozen bone sections. Histological studies have revealed that some proteins, such as BMP2, BMPR1A, and Connexin 43, are expressed in and around sites of ectopic ossification. However, the relationship between the expressed proteins considered to be associated with the ossification and mineral formation in vivo is not clear. Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1)-mutant spinal hyperostotic TWY mice and ICR mice as controls were euthanized after calcein labeling, and undecalcified frozen sections were obtained from the middle thoracic spine. Intervertebral disc areas were examined histologically and by measuring calcein-labeled areas and areas showing immunoreactivity for BMP2, BMPR1A, and Connexin 43. Calcein-labeled areas, indicating mineralization in the ectopic mineralization sites, were significantly larger in the mutant mice than in controls. The expression of Connexin 43 was elevated in the annulus fibrosus. Increases in the calcein-labeled areas was not correlated with increases in the areas showing immunoreactivity for Connexin 43 in the annulus fibrosus. There was no statistical correlation between enhanced immunohistochemical expression and elevated calcein-labeled areas. By applying the morphometrical analysis method using undecalcified frozen sections to ENPP1-mutant mice, quantitative evaluation of the mineralization and proteins expressed in the surrounding area in the same animal became possible.

Global metabolic reprogramming of colorectal cancer occurs at adenoma stage and is induced by MYC.

Cancer cells alter their metabolism for the production of precursors of macromolecules. However, the control mechanisms underlying this reprogramming are poorly understood. Here we show that metabolic reprogramming of colorectal cancer is caused chiefly by aberrant MYC expression. Multiomics-based analyses of paired normal and tumor tissues from 275 patients with colorectal cancer revealed that metabolic alterations occur at the adenoma stage of carcinogenesis, in a manner not associated with specific gene mutations involved in colorectal carcinogenesis. MYC expression induced at least 215 metabolic reactions by changing the expression levels of 121 metabolic genes and 39 transporter genes. Further, MYC negatively regulated the expression of genes involved in mitochondrial biogenesis and maintenance but positively regulated genes involved in DNA and histone methylation. Knockdown of MYC in colorectal cancer cells reset the altered metabolism and suppressed cell growth. Moreover, inhibition of MYC target pyrimidine synthesis genes such as CAD, UMPS, and CTPS blocked cell growth, and thus are potential targets for colorectal cancer therapy.

Hypoxia-Inducible Factor-1α in Smooth Muscle Cells Protects Against Aortic Aneurysms-Brief Report.

OBJECTIVE: The purpose of this study was to determine the role of smooth muscle cell-derived hypoxia-inducible factor-1α (Hif-1α) in the pathogenesis of aortic aneurysms. APPROACH AND RESULTS: Control mice and smooth muscle cell-specific hypoxia-inducible factor-1α-deficient mice were infused with ß-aminopropionitrile for 2 weeks and angiotensin II for 6 weeks to induce aortic aneurysm formation. Mutant mice experienced increased levels of aneurysm formation of the thoracic or abdominal aorta with more severe elastin disruption, compared with control mice. Smooth muscle cell-specific hypoxia-inducible factor-1α deficiency did not affect matrix metalloproteinase-2 activity; however, the activity of lysyl oxidase and the levels of tropoelastin mRNA in the angiotensin II- and ß-aminopropionitrile-treated aortae, associated with elastin fiber formation, were suppressed. Furthermore, we observed reduced volumes of mature cross-linked elastin in the thoracoabdominal aorta after treatment with angiotensin II and ß-aminopropionitrile. CONCLUSIONS: Deficiency of smooth muscle cell-derived hypoxia-inducible factor-1α augments aortic aneurysms, accompanied by disruption of elastin fiber formation, but not changes of elastin fiber degradation.

Albumin microvascular leakage in brains with diabetes mellitus.

Their aim was to examine whether microvascular leakage of endogenous albumin, a representative marker for blood-brain barrier (BBB) damage, was induced in the periventricular area of diabetic db/db mice because periventricular white matter hyperintensity formation in magnetic resonance images was accelerating in elderly patients with diabetes mellitus. Using light and electron microscopes, and semi-quantitative analysis techniques, immunoreactivity of endogenous albumin, indicating vascular permeability, was examined in the periventricular area and spinal cord of db/db mice and db/+m control mice. Greater immunoreactivity of albumin was observed in the vessel wall of the periventricular area of db/db mice than in controls. Additionally, weak immunoreactivity was observed in the spinal cord of both db/db mice and controls. The number of gold particles, indicating immunoreactivity of albumin, in the perivascular area of db/db mice was significantly higher than that of control mice, but there was no significant difference in the number of particles in the spinal cord between db/db mice and controls. These findings suggest that albumin microvascular leakage, or BBB breakdown, is induced in the periventricular area of diabetic mice. Microsc. Res. Tech. 79:833-837, 2016. © 2016 Wiley Periodicals, Inc.

Blood-brain barrier damage in vascular dementia.

New findings on flow or drainage pathways of brain interstitial fluid and cerebrospinal fluid have been made. The interstitial fluid flow has an effect on the passage of blood-borne substances in the brain parenchyma, especially in areas near blood-brain barrier (BBB)-free regions. Actually, blood-borne substances can be transferred in areas with intact BBB function, such as the hippocampus, the corpus callosum, periventricular areas, and medial portions of the amygdala, presumably through leaky vessels in the subfornical organs or the choroid plexus. Increasing evidence indicates that dysfunction of the BBB function may play a significant role in the pathogenesis of vascular dementia. Accordingly, we have examined which insults seen in patients suffering from vascular dementia have an effect on the BBB using experimental animal models exhibiting some phenotypes of vascular dementia. The BBB in the hippocampus was clearly deteriorated in Mongolian gerbils exposed to acute ischemia followed by reperfusion and also in stroke-prone spontaneously hypertensive rats (SHRSP) showing hypertension. The BBB in the corpus callosum was clearly deteriorated in Wistar rats with permanent ligation of the bilateral common carotid arteries showing chronic hypoperfusion. The BBB in the hippocampus and the olfactory bulb was mildly deteriorated in aged senescence accelerated prone mice (SAMP8) showing cognitive dysfunction. The BBB in the hippocampus was mildly deteriorated in aged animals with hydrocephalus. Mild endothelial damage was seen in hyperglycemic db/db mice. In addition, mRNA expression of osteopontin, matrix metalloproteinase-13 (MMP-13), and CD36 was increased in vessels showing BBB damage in hypertensive SHRSP. As osteopontin, MMP-13 and CD36 are known to be related to brain injury and amyloid ß accumulation or clearance, BBB damage followed by increased gene expression of these molecules not only contributes to the pathogenesis of vascular dementia, but also bridges the gap between vascular dementia and Alzheimer's disease.

Long-Term Chronic Toxicity and Mesothelial Cell Reactions Induced by Potassium Octatitanate Fibers (TISMO) in the Left Thoracic Cavity in A/J Female Mice.

The present study was conducted to examine the chronic effects of potassium octatitanate fibers (trade name TISMO; chemical formula K2O·6TiO2) on the mouse lung and thoracic cavity. This method of infusion was employed to examine the direct effects of the fibers to the pleura. In the present study, 52- and 65-week experiments were employed to examine the long-term chronic effects after infusion of fiber-shaped TISMO into the thoracic cavities of A/J mice. Following this infusion, TISMO fibers were observed in the alveoli, indicating penetration through the visceral pleura. The additional histopathological detection of TISMO fibers in the liver, spleen, kidneys, ovary, heart, bone marrow, and brain of TISMO-infused mice indicated migration of the fibers out from the thoracic cavity. Atypical mesothelial cells with severe pleural proliferation were observed, but malignant mesotheliomas were not detected. This study demonstrated that intrathoracic infusion of TISMO fiber did not cause malignant mesothelioma but did cause severe chronic inflammation and proliferation of pleural mesothelial cells.

Clearance of beta-amyloid in the brain.

Intravascular substances invade extracellular spaces in the brain via endothelial cells in the sites without bloodbrain barrier (BBB) and move not only in the cerebrospinal fluid (CSF) but also in the interstitial fluid (ISF) of brain parenchyma adjacent to non-BBB sites. It is likely that CSF drains directly into the blood via arachnoid villi and granulations and also to lymph nodes via subarachnoid spaces in the brain and nasal lymphatics, whereas ISF drains to cervical lymph nodes through pathways along vascular wall of capillaries and arteries. As the supposed pathways of fluids seem to be critical for the maintenance of normal brain function, it is reasonable to suspect that an obstacle to the passage of fluids through these pathways likely induces some kinds of brain dysfunction such as Alzheimer's disease. According to assumed pathways for the elimination of amyloid-ß (Aß) from the brain, Aß peptides produced mainly in neurons are degraded by peptidases, flow out of the brain parenchyma into the blood through efflux transporters located in cerebral vessels, drain through perivascular pathways into the cervical lymph nodes, or are taken up by some kinds of cells in the brain. As for the perivascular pathways, ISF including Aß peptides diffuses in the extracellular spaces of the brain parenchyma, enters basement membranes of capillaries, passes into the tunica media of arteries, and drains out of the brain. In this review, these pathways for the clearance of fluids including Aß from the brain into the blood are briefly reviewed and the relationship between dysfunction of these pathways and brain diseases is discussed.

An immunohistochemical study of human platelets using a rabbit antibody against H18-K24 of apolipoprotein CIII (HATKTAK).

H18-K24 of human apolipopotein CIII (Apo CIII) (HATKTAK) is an activator of the macromolecular activators of phagocytosis from platelets (MAPPs). Using a rabbit antibody against HATKTAK, we performed an immunohistochemical study of human platelets. Indirect ELISA showed that this antibody reacts with Apo CIII-derived peptides with a C-terminal of HATKTAK, but not with Apo CIII. Immunoelectron microscopy revealed that reaction of anti-HATKTAK antibody occurred in the pseudopods of activated platelets. In blood coagula produced from the peripheral blood and formalin-fixed after various incubation periods, reaction of this antibody with platelets appeared rapidly with a peak at 3 to 6 h of incubation, and then diminished gradually. Leukocytes in the blood coagula were stained strongly positive. In tissue sections, fresh thrombi and hemorrhages with slight fibrin formation revealed a positive response of platelets to anti-HATKTAK antibody, whereas older ones with leukocytic infiltration, fibrin formation and organization did not. In addition to platelets, endothelial cells and leukocytes were stained positive by anti-HATKTAK antibody. All of the positive reactions by anti-HATKTAK antibody disappeared or diminished by co-incubation with HATKTAK. In conclusion, the anti-HATKTAK antibody reveals platelets during the early phase of activation.

CD36 expression in the brains of SAMP8.

SAMP8, senescence accelerated mice with age-related deficits in memory and learning, are known to show age-related increases of amyloid precursor protein (APP) and immunopositivity for amyloid-ß (Aß) proteins, and moreover to be under elevated oxidative stress. The elevated expression of class B scavenger receptor CD36, which is the receptor of oxidized LDL and also one of efflux transporters of Aß proteins in the cerebral vessels, is thought to mediate free radical production in cerebral ischemia and induce oxidative stress. Accordingly, by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemical techniques, we examined whether the expression of CD36 was increased in the brains of 10-12-week-old SAMP8 with elevated oxidative stress. Ten to 12-week-old SAMR1 mice were used as controls without the features. The gene and protein expression of CD36 was significantly higher in the brains of SAMP8 than those of SAMR1. Confocal microscopic examination revealed that the CD36 immunoreactivity was seen in the cytoplasm of endothelial cells and F4/80-positive perivascular cells of the brains. These findings indicate that the expression of CD36 in the brains of SAMP8 is increased compared with that of SAMR1.

Presence of FXYD6 in the endolymphatic sac epithelia.

A homeostasis of the electrochemical properties and volume of the endolymph in the inner ear is essential for hearing and equilibrium sensing and is maintained by ion-transport across an epithelial tissue, the endolymphatic sac. One of the key proteins in the maintenance is Na(+), K(+)-ATPase. Although we previously found that the Na(+), K(+)-ATPase in the sac plays a pivotal role in the control of the endolymphatic volume, the mechanism remains unclear. Therefore, in this study, we examined the expression of FXYD6, a functional modulator of the Na(+), K(+)-ATPase, in the epithelial cells of the endolymphatic sac using various approaches. Laser capture microdissection RT-PCR was used to identify FXYD6 mRNA in the endolymphatic sac. Immunolabeling with the specific antibody showed that FXYD6 was predominantly expressed in the intermediate portion of the endolymphatic sac, and it was colocalized with the Na(+), K(+)-ATPase. Because the Na(+), K(+)-ATPase in this region is known to exhibit a high level of activity, an interaction of FXYD6 with this transporter may be critically involved in the regulation of the characteristics of the endolymph.

Interaction of the profilaggrin N-terminal domain with loricrin in human cultured keratinocytes and epidermis.

The relationship between the two coexpressed differentiation markers, profilaggrin and loricrin, is not clear right now. In this study, we explored the interaction of profilaggrin N-terminal domain (PND) with loricrin in keratinocytes and epidermis. Confocal immunofluorescence microscopic analysis of human epidermis showed that PND colocalized with loricrin. Loricrin nucleofected into HaCaT cells colocalized with PND in the nucleus and cytoplasm. The PND localizes to both the nucleus and cytoplasm of epidermal granular layer cells. Nucleofected PND also colocalized with keratin 10 (K10) in the nucleus and cytoplasm. Immunoelectron microscopic analysis of human epidermis confirmed the findings in nucleofected keratinocytes. Yeast two-hybrid assays showed that the B domain of human and mouse PND interacted with loricrin. The glutathione S-transferase (GST) pull-down analysis using recombinant GST-PND revealed that PND interacted with loricrin and K10. Knockdown of PND in an organotypic skin culture model caused loss of filaggrin expression and a reduction in both the size and number of keratohyalin granules, as well as markedly reduced expression of loricrin. Considering that expression of PND is closely linked to keratinocyte terminal differentiation, we conclude that PND interacts with loricrin and K10 in vivo and that these interactions are likely to be relevant for cornified envelope assembly and subsequent epidermal barrier formation.

The expression of LDL receptor in vessels with blood-brain barrier impairment in a stroke-prone hypertensive model.

We previously reported that the blood-brain barrier (BBB) function was deteriorated in vessels located in the hippocampus in stroke-prone spontaneously hypertensive rats (SHRSP). In order to assess whether substances with oxidative stress such as amyloid-beta (Abeta) can be scavenged in the BBB-damaged vessels, we examined the gene expression of representative efflux and influx transporters of Abeta, such as low-density lipoprotein receptor (LDLR), LDL-related protein 1 (LRP1), and the receptor for advanced glycation end product (RAGE) in the hippocampus of SHRSP with the BBB impairment and Wistar Kyoto rats (WKY) without the impairment. Real-time quantitative reverse transcriptase-polymerase chain reaction analysis revealed that LDLR gene expression was increased in the samples of SHRSP compared with those of WKY, while there was no significant difference in LRP1 or RAGE gene expression between SHRSP and WKY. Western blot analysis revealed that the protein expression of LDLR was increased in the samples of SHRSP compared with those of WKY. Immunoelectron microscopic examination revealed that the LDLR expression was seen in the luminal and abluminal cytoplasmic membranes and vesicular structures of the endothelial cells and the cytoplasm of perivascular cells, especially in vessels with immunoreactivity of albumin showing increased vascular permeability. These findings suggest that the expression of LDLR was increased in the hippocampus of SHRSP compared with that of WKY and was seen in the luminal and abluminal cytoplasmic membranes and vesicular structures of endothelial cells, suggesting a role of LDLR in the vessels with BBB impairment.

Mineralocorticoid receptor blockade enhances the antiproteinuric effect of an angiotensin II blocker through inhibiting podocyte injury in type 2 diabetic rats.

Treatment with angiotensin II type 1 receptor blockers (ARBs) is the first-line therapy for hypertensive patients with diabetic nephropathy. However, emerging clinical evidence indicates that mineralocorticoid receptor (MR) blockers have blood pressure-independent antiproteinuric effects. We sought to determine whether treatment with an MR blocker, eplerenone, enhances the effects of an ARB, telmisartan, on podocyte injury and proteinuria in type 2 diabetic Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats. From 20 to 50 weeks old, diabetic OLETF rats showed higher systolic blood pressure (SBP) and urinary protein excretion (U(protein)V) than nondiabetic control Long-Evans-Tokushima-Otsuka rats. At 50 weeks old, OLETF rats also showed glomerular sclerosis and podocyte injury, whereas nephrin and podocin mRNA levels in isolated glomeruli were significantly decreased. Treatment with telmisartan (3 mg/kg/day p.o.) decreased SBP and U(protein)V, increased nephrin and podocin mRNA levels, and attenuated glomerular sclerosis and podocyte injury. Eplerenone (100 mg/kg/day p.o.) did not alter SBP but elicited similar changes in renal parameters. However, greater reductions in U(protein)V and podocyte injury and greater increases in nephrin and podocin mRNA levels were observed in the combination treatment group. Hydralazine (25 mg/kg/day p.o.) decreased SBP but did not alter any renal parameters. These data indicate that MR blockade enhances the SBP-independent antiproteinuric effect of an ARB through inhibiting podocyte injury in type 2 diabetic rats.

The expression of matrix metalloproteinase-13 is increased in vessels with blood-brain barrier impairment in a stroke-prone hypertensive model.

We previously reported that the blood-brain barrier (BBB) function was deteriorated in vessels located in the hippocampus, but not the cerebral cortex, in 3-month-old stroke-prone spontaneously hypertensive rats (SHRSP). Recently published data suggest that matrix metalloproteinase (MMP)-2 and MMP-9 play a critical role in the BBB disruption in stroke or cerebral ischemia. In this study, we examined gene and protein expressions of MMPs in the BBB-damaged hippocampal vessels of 3-month-old SHRSP, in the cerebral cortical vessels without BBB damage of SHRSP, and in the hippocampal and cerebral cortical ones without BBB damage of 3-month-old Wistar Kyoto (WKY) rats. The expressions of MMPs were examined by real-time quantitative reverse transcriptase-PCR (RT-PCR), western blotting and immunohistochemical techniques. The gene and protein expressions of MMP-13 were significantly increased in the hippocampal samples of SHRSP compared with samples without BBB damage, such as cerebral cortical samples of SHRSP or hippocampal samples of WKY. Immunostaining of MMP-13 was seen in the cytoplasm of ED-1-positive perivascular cells in both rats and was colocalized with those of type IV collagen or osteopontin. The type IV collagen was also localized in the basement membrane. These findings indicate that the expression of MMP-13 is increased in BBB-damaged hippocampal vessels in hypertensive SHRSP compared with vessels without BBB impairment in normotensive WKY rats and may be involved in vascular remodeling.

Strict angiotensin blockade prevents the augmentation of intrarenal angiotensin II and podocyte abnormalities in type 2 diabetic rats with microalbuminuria.

OBJECTIVES: Beneficial effects of angiotensin II type 1 receptor blockers have been indicated for patients with diabetic nephropathy. We investigated the effects of an angiotensin II type 1 receptor blocker, telmisartan, on intrarenal angiotensin II levels and the progression of albuminuria or glomerular injury in type 2 diabetic Otsuka Long-Evans Tokushima Fatty rats with microalbuminuria. METHODS AND RESULTS: Otsuka Long-Evans Tokushima Fatty rats were randomly treated with telmisartan (10 mg/kg/day, orally), hydralazine (25 mg/kg/day in drinking water) or vehicle from the initiation of albuminuria (13 weeks old). At this age, Otsuka Long-Evans Tokushima Fatty rats showed low but detectable albuminuria (1.0 +/- 0.1 mg/day) and higher systolic blood pressure, postprandial blood glucose and kidney angiotensin II levels than age-matched nondiabetic Long-Evans Tokushima Otsuka rats. At 35 weeks of age, vehicle-treated Otsuka Long-Evans Tokushima Fatty rats did not show apparent glomerular injury or tubulointerstitial fibrosis but did exhibit severe albuminuria (72.6 +/- 5.9 mg/day) and accumulation of cytoplasmic granules containing albumin in podocytes. Otsuka Long-Evans Tokushima Fatty rats also showed higher systolic blood pressure, postprandial blood glucose, collagen gene expression, desmin staining (a marker of podocyte injury) and angiotensin II levels than Long-Evans Tokushima Otsuka rats. Treatment with telmisartan did not affect postprandial blood glucose but decreased systolic blood pressure, collagen gene expression, desmin staining and angiotensin II levels. Telmisartan also prevented the development of albuminuria (0.6 +/- 0.1 mg/day at 35 weeks old) and accumulation of cytoplasmic granules. Hydralazine treatment resulted in a similar reduction in systolic blood pressure and partially attenuated the albuminuria (35.4 +/- 1.8 mg/day at 35 weeks old) but did not affect the other parameters. CONCLUSION: The present results suggest the contribution of augmented intrarenal angiotensin II levels to the initiation and progression of albuminuria as well as podocyte abnormalities in type 2 diabetic rats. Angiotensin II blockade may inhibit the transition from microalbuminuria to overt nephropathy through prevention of intrarenal angiotensin II augmentation, independently of changes in blood pressure and glucose levels.

Involvement of endogenous nitric oxide in angiotensin II-induced activation of vascular mitogen-activated protein kinases.

Angiotensin II (ANG II) is a powerful activator of mitogen-activated protein (MAP) kinase cascades in cardiovascular tissues through a redox-sensitive mechanism. Nitric oxide (NO) is considered to antagonize the vasoconstrictive and proarteriosclerotic actions of ANG II. However, the role of endogenous NO in ANG II-induced redox-sensitive signal transduction is not yet clear. In this study using catheterized, conscious rats, we found that acute intravenous administration of N(G)-nitro-L-arginine methyl ester (L-NAME; 5 mg/kg) enhanced phosphorylation of aortic MAP kinases extracellular signal regulated kinase (ERK) 1/2 and p38, which were suppressed only partially by a superoxide dismutase mimetic (Tempol), whereas ANG II-induced MAP kinase phosphorylation was markedly suppressed by Tempol. FK409, a NO donor, had little effect on vascular MAP kinase phosphorylation. On the other hand, acute exposure to a vasoconstrictor dose of ANG II (200 ng x kg(-1) x min(-1) iv) failed to enhance phosphorylation of aortic MAP kinases in the chronically L-NAME-treated rats, whereas the vasoconstrictor effect of ANG II was not affected by L-NAME treatment. Furthermore, three different inhibitors of NO synthase suppressed, in a dose-dependent manner, ANG II-induced MAP kinase phosphorylation in rat vascular smooth muscle cells, which was closely linked to superoxide generation in cells. These results indicate the involvement of endogenous NO synthase in ANG II-induced signaling pathways, leading to activation of MAP kinase, and that NO may have dual effects on the vascular MAP kinase activation associated with redox sensitivity.

Effects of aging and HIF-1alpha deficiency on permeability of hippocampal vessels.

We examined age-related changes in the blood-brain barrier (BBB) of neural cell-specific hypoxia inducible factor-1alpha (HIF-1alpha) deficient mice, which showed hydrocephalus with neuronal cell loss, to investigate an effect of neural cell-specific HIF-1alpha deficiency or hydrocephalus on vascular function. Vascular permeability of horseradish peroxidase (HRP) and binding of cationized ferritin (CF) particles to the endothelial cell luminal surface, as a marker of glycocalyx, were investigated. The thickness of CF-labeled glycocalyx was significantly decreased in the cortex in mutant mice compared with that of control mice, although it was not paralleled by increased vascular permeability. In addition, strong staining for HRP was seen around vessels located along the hippocampal fissure in 24-month-old mutant mice. The reaction product of HRP appeared in an increasing number of the endothelial cell abluminal vesicles and within the thickened basal lamina of arterioles in the hippocampus, showing increased vascular permeability. There were no leaky vessels in 10-week-old mutant mice or 10-week-old and 24-month-old control mice. These findings suggest the necessity of two factors, aging and hydrocephalus, for BBB dysfunction in HIF-1alpha deficient mice.