Morphological assessment of the luminal surface of olfactory epithelium in mice deficient in tissue plasminogen activator following bulbectomy.

OBJECTIVE: This study aimed to investigate the function of tissue plasminogen activator in the olfactory epithelium of mice following neural injury. METHOD: Transmission electron microscopy was used to study the changes in the morphology of the olfactory epithelium 1-7 days after surgical ablation of the olfactory bulb (bulbectomy). RESULTS: Prior to bulbectomy, a uniformly fine material was observed within some regions of the olfactory epithelium of mice deficient in tissue plasminogen activator. At 2-3 days after bulbectomy, there were degenerative changes in the olfactory epithelium. At 5-7 days after bulbectomy, we noted drastic differences in olfactory epithelium morphology between mice deficient in tissue plasminogen activator and wild-type mice (comparisons were made using findings from a previous study). The microvilli seemed to be normal and olfactory vesicles and receptor neuron dendrites were largely intact in the olfactory epithelium of mice deficient in tissue plasminogen activator. CONCLUSION: The tissue plasminogen activator plasmin system may inhibit the regeneration of the olfactory epithelium in the early stages following neural injury.

Microwave irradiation for fixation and immunostaining of endothelial cells in situ.

Using microwave irradiation during tissue fixation and immunostaining reduces sample preparation time and facilitates penetration of fixatives and antibody solutions into the tissues. This results in improved fixation and reduction of non-specific binding of antibodies, respectively. Experimental analyses of endothelial cells in blood vessels in situ have been limited because of the difficulty of tissue preparation. We report here a technique using intermittent microwave irradiation for blood vessel fixation and immunostaining the fixed tissues. Intermittent microwave irradiation during fixation reduced blood vessel contraction and resulted in well preserved morphology of blood vessels, especially the endothelial cells. Microwave irradiation also reduced non-specific binding of fluorescein-labeled antibodies. These microwave irradiation-assisted techniques are useful for analysis of endothelial cell function and for pathological study of blood vessels in situ.

Systemic delivery of IL-10 by an AAV vector prevents vascular remodeling and end-organ damage in stroke-prone spontaneously hypertensive rat.

Interleukin-10 (IL-10) ameliorates various T-helper type 1 cell-mediated chronic inflammatory diseases. Although the therapeutic benefits of IL-10 include antiatherosclerotic effects, pathophysiological effects of IL-10 on vascular remodeling in hypertension have not yet been elucidated. These studies were designed to determine whether sustained IL-10 expression, mediated by an adeno-associated virus (AAV) vector, prevents vascular remodeling and target-organ damage in the stroke-prone spontaneously hypertensive rat (SHR-SP)-an animal model of malignant hypertension. A single intramuscular injection of an AAV1 vector encoding rat IL-10 introduced long-term IL-10 expression. These IL-10-transduced rats had decreased stroke episodes and proteinuria, resulting in improved survival. Histological examination revealed a reduced level of deleterious vascular remodeling of resistance vessels in the brain and kidney of these rats. Immunohistochemical analysis indicated that IL-10 inhibited the enhanced renal transforming growth factor-beta expression and perivascular infiltration of monocytes/macrophages and nuclear factor-kappaB-positive cells normally observed in the SHR-SP. Four weeks after IL-10 vector injection, systolic blood pressure significantly decreased and this effect persisted for several months. Overall, AAV vector-mediated systemic IL-10 expression prevented vascular remodeling and inflammatory lesions of target organs in the SHR-SP. This approach provides significant insights into the prevention strategy of disease onset with unknown genetic predisposition or intractable polygenic disorders.

Adeno-associated virus vector-mediated interleukin-10 gene transfer inhibits atherosclerosis in apolipoprotein E-deficient mice.

Inflammation is a major contributor to atherosclerosis by its effects on arterial wall biology and lipoprotein metabolism. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that may modulate the atherosclerotic disease process. We investigated the effects of adeno-associated virus (AAV) vector-mediated gene transfer of IL-10 on atherogenesis in apolipoprotein E (ApoE)-deficient mice. A murine myoblast cell line, C2C12, transduced with AAV encoding murine IL-10 (AAV2-mIL10) secreted substantial amounts of IL-10 into conditioned medium. The production of monocyte chemoattractant protein-1 (MCP-1) by the murine macrophage cell line, J774, was significantly inhibited by conditioned medium from AAV2-mIL10-transduced C2C12 cells. ApoE-deficient mice were injected with AAV5-mIL10 into their anterior tibial muscle at 8 weeks of age. The expression of MCP-1 in the vascular wall of the ascending aorta and serum MCP-1 concentration were decreased in AAV5-mIL10-transduced mice compared with AAV5-LacZ-transduced mice. Oil red-O staining of the ascending aorta revealed that IL-10 gene transfer resulted in a 31% reduction in plaque surface area. Serum cholesterol concentrations were also significantly reduced in AAV5-mIL10-transduced mice. To understand the cholesterol-lowering mechanism of IL-10, we measured the cellular cholesterol level in HepG2 cells, resulting in its significant decrease by the addition of IL-10 in a dose-dependent manner. Furthermore, IL-10 suppressed HMG-CoA reductase expression in the HepG2 cells. These observations suggest that intramuscular injection of AAV5-mIL10 into ApoE-deficient mice inhibits atherogenesis through anti-inflammatory and cholesterol-lowering effects.

Changes of perivascular macrophages in the process of brain edema induced by cold injury.

Perivascular macrophages are considered as cerebral scavenger cells under physical and pathological conditions. In this study, we tried to examine changes of perivascular macrophages, especially changes of the characteristic lysosomal inclusion bodies that are rich in hydrolytic enzymes, in the process of brain edema induced by cold injury. Wistar male rats aged 4 months were treated with dry ice for 20 minutes through a drilled hole at the parietal bone. At different time points after the cold injury, cerebral cortex was excised and the immunoreaction for cathepsin D, one kind of lysosomal protease, was examined by post-embedding immuno-electron microscopy. The reactions of cathepsin D were located in the inclusion bodies of perivascular macrophages. At 5 and 10 hours after cold injury, the reactions increased dramatically. Then the reactions inclined to decrease, and reached the minimum at 1 week after cold injury. The reactions seemed to recover at 2 weeks after cold injury. The changes of cathepsin D reactions suggest that the function of perivascular macrophages as scavenger cells were activated in the early phase of the process of brain edema, their later declines might be caused by severe pathological conditions.

Effect of postural change on blood volume in long-term hemodialysis patients.

In healthy subjects, the blood volume (BV) increases rapidly after postural change from standing to the supine position. However, little is known about the effect of postural change on BV in long-term hemodialysis (HD) patients. Therefore, we have examined the BV change caused by adopting the supine position from standing by continuous hematocrit monitoring, using the CRIT-LINE instrument, in 8 anuric HD patients. The hematocrit was monitored for 25 min with the patient in the supine position just before HD. The percentage change in the BV (% Delta BV) was calculated from the hematocrit and approximated using the equation: % Delta BV = b - [1 - exp(-c x time (min)] -a x time (min). Coefficient a was the slope of the linear part in the % Delta BV, b was the magnitude of BV increase and c was the rate of BV increase. Then we examined the relationship between the coefficients (a, b and c) and clinical parameters. In all patients, % Delta BV increased quickly after adopting the supine position. The mean increases were 2.8 +/- 0.6% after 5 min and 4.8 +/- 0.5% after 25 min. There was a significant correlation between the value of % Delta BV calculated from the hematocrit and the value calculated using above equation (0.92 < r < 0.99, p < 0.001). Although coefficient a did not correlate with a clinical parameter, coefficient b showed a significant positive linear correlation with the serum albumin level (r = 0.816, p < 0.05) and coefficient c showed a significant positive linear correlation with the percentage change in interdialytic weight gain (r = 0.736, p < 0.05). Furthermore, based on the % Delta BV, we calculated the change in total BV, which had increased by 181.5 +/- 21.9 ml after 25 min in the supine position. In conclusion, the change in the BV with time by continuous hematocrit monitoring using the CRIT-LINE instrument can be approximated by a modified monoexponential equation. BV increased quickly in HD patients after postural change from standing to the supine position.

[Ultrastructural study on intracerebral small blood vessels and fluorescent granular perithelial (FGP) cells in experimental cerebral ischemia].

In order to clarify the sequential changes of the morphology of vascular cells and FGP cells under cerebral ischemia, 32 male Wistar rats were employed. The FGP cells in the present paper are distributed along cerebral microvessels, and markedly potent in the uptake capacity for endo- and exogenous substances under the physiological and pathological conditions. Under the anesthesia of pentobarbital, experimental animals suffered from cerebral ischemia were produced by (1) occlusion of bilateral vertebral arteries, (2) unilateral ligation of common carotid artery accompanied with occlusion of vertebral arteries and (3) temporary clipping of bilateral common carotid arteries accompanied with occlusion of vertebral arteries. On 1 to 14 days after the treatments mentioned above, the cerebral cortices of animals were examined with the electron microscope with paying special attention to morphological changes of FGP cells. From the observation, it is confirmed that: (1) after occlusion of vertebral arteries (first group of experimental animals), the FGP cells become edematous without any severe damage of cerebral neurons through 2 weeks.: (2) In case of the unilateral ligation of common carotid artery (second group of experimental animals), the FGP cells and neurons tend to degenerate at the ligated side on 14 days, but at the opposite side, the considerable vacuolation and swelling of the FGP cells are evident, without accompanying with any degeneration of neurons and FGP cells, and: (3) In the reflow experiment (third group of experimental animals), the neurons are not affective and the FGP cells show some degenerative changes on 7 days, but most of them recovered on 14 days. From these observations, it may be concluded that the morphological changes of FGP cells run parallel with the change of microenvironment surrounding neurons, and the FGP cells, in addition to astrocytes, are reliable morphological markers of cerebral edema.

Regional difference of lipid distribution in brain of apolipoprotein E deficient mice.

According to recent knowledge, apolipoprotein E (apo E) plays a significant role in the homeostasis of intracellular cholesterol level in various tissues. Apo E deficient mice develop hyperlipidemia, and suffer from atherosclerosis in extracerebral blood vessels and neurodegeneration in the central nervous system. Furthermore, Walker et al. (Am. J. Path., 1997;151:1371-1377) demonstrated cerebral xanthomas of various sizes in the brain of apo E deficient mice. In the present study, it is illustrated that in the homozygous apo E deficient mice of advancing age, a great number of foamy macrophages extravasate from microvessels in thalamus and fimbria hippocampi, and scatter in the perivascular regions and migrate toward the ependyma, fimbria hippocampi, hippocampus, and thalamus. Here, it must be pointed out that under hyperlipidemia, although foamy macrophages made clusters in the perivascular region, the cerebral microvessels did not develop atherosclerosis. On the other hand, in the other cerebral regions such as cerebral cortex, caudoputamen, globus pallidus, and substantia nigra, macrophages did not appear and microvessels retained normal shapes, but the fluorescent granular perithelial (in short, FGP) cells accompanied by these vessels contained a certain amount of lipids. That is, in the cerebral cortex and caudoputamen, lipid components are detected in FGP cells and microglia, while in the globus pallidus and substantia nigra, they are mainly localized in astrocytes. The reason why the astrocytes in such defined regions contain, specifically, a high quantity of lipid components remains unsettled. Axonal degenerations are often represented in thalamus, globus pallidus, and substantia nigra. On the other hand, in the specimens of Wild-type mice, lipid components were observed only in FGP cells, and the vascular architecture took a normal profile. Any lipid laden macrophages and the axonal degenerations could not be detected through the cerebral parenchyma. Furthermore, it is also a noticeable finding that immunohistochemically, the FGP cells express a positive reaction against the antibody of apo E in the Wild-type mice, but those of homozygous apo E deficient mice are immunonegative. FGP cells are not only provided with the scavenger receptor, but also contribute to the lipid metabolism in the brain.

The effect of EGF on electrolyte transport is mediated by tyrosine kinases in the rabbit cortical collecting duct.

Epidermal growth factor (EGF) inhibits amiloride-sensitive Na(+) conductance in the apical membrane of the isolated rabbit cortical collecting duct. However, there is no information on the relationship between electrolyte transport and tyrosine kinase. We examined the effect of EGF on transport of potassium and chloride as well as sodium and the roles of tyrosine kinases in the rabbit cortical collecting duct using in vitro isolated tubular microperfusion. Basolateral EGF depolarized the transepithelial voltage in a dose-dependent manner within a concentration range of 10(-10) in 10(-8) M. Basolateral ouabain and luminal amiloride completely abolished EGF-induced depolarization. However, luminal BaCl(2) did not abolish its depolarization. To confirm the mechanism, sodium, potassium, and chloride fluxes were measured in the presence of 10(-10) M EGF. EGF significantly decreased the lumen-to-bath isotope flux of sodium and chloride from 93.6+/-12.5 to 61.1+/-9.6 pmol/mm/min (n = 5, p<0.05) and from 86.6+/-10.0 to 54. 8+/-9.7 pmol/mm/min (n = 10, p<0.01), respectively. EGF also decreased net potassium secretion from -27.7+/-5.9 to -7.8+/-1.5 pmol/mm/min (n = 6, p<0.01). To examine whether EGF-induced depolarization is mediated by tyrosine kinase, tyrosine kinase inhibitors were applied from the basolateral side. Pretreatment with 1 microg/ml herbimycin A for 120 min completely abolished EGF-induced depolarization (90.9+/-5.4%, n = 4; NS). Herbimycin A itself also did not change the lumen-to-bath isotope flux of sodium and completely abolished the inhibition of Na(+) absorption on EGF action (control 65.4+/-6.8, herbimycin A 61.8+/-6.3, EGF with herbimycin A 60.0+/-4.4 pmol/min/mm, n = 5; NS). In conclusion, EGF depolarizes transepithelial voltage by inhibiting sodium transport primarily and potassium and chloride transport secondarily. These effects were blocked by nonspecific tyrosine kinase inhibitors.

Ultrastructural and immunohistochemical changes of fluorescent granular perithelial cells and the interaction of FGP cells to microglia after lipopolysaccharide administration.

Fluorescent granular perithelial (FGP) cells introduced by Mato et al. (1979, 1980, 1981) are situated in the Virchow-Robin space of cerebral microvessels and exhibit a marked uptake capacity for the endo- and exogenous materials in physiological conditions. That is, the FGP cells are the perivascular macrophage lineage enclosed by a basal lamina and glia limitans and are distributed between blood vessels and cerebral parenchyma. As reported previously, the FGP cells were distinguishable from microglia and pericytes in their morphology, location, and epitopes on their cytoplasmic membrane. On the other hand, it is established that microglia are upregulated by lipopolysaccaride (LPS) administration, but it remains unsettled whether the FGP cells, the perivascular macrophagic cells, are activated by LPS or not and what role the FGP cells play in the upregulation of microglia. Thus far, the morphological relationship between FGP cells and microglia in the process of upregulation has not been clarified. Thirty-six 7-month-old Wistar male rats were employed. The animals were sacrificed at 2, 5, 10, 24, and 72 h after the intravenous administration of 500 microg/kg of LPS. The FGP cells and microglia in their cerebral cortex were studied with immunohistochemical and electron microscopical techniques. The findings of the present study indicated that the majority of FGP cells were upregulated by LPS from the results of immunohistochemical and morphological data, but some of them tended to degenerate. Furthermore, in the time course after LPS administration, microglia were also upregulated and approached the microvessels and occasionally contacted the FGP cells directly. From these findings, it is hypothesized that FGP cells may significantly influence the mobilization and upregulation of microglia caused by LPS administration.

Immunohistochemical evidence for a macrophage scavenger receptor in Mato cells and reactive microglia of ischemia and Alzheimer's disease.

Macrophage scavenger receptors (MSR) are implicated in the development of atherosclerosis and amyloid b-protein deposition in Alzheimer's disease. However, histopathological studies of MSR expression in human tissues have been hampered by a lack of specific antibodies. Using MSR-deficient mice, we successfully raised a novel monoclonal antibody against human MSR together with high-titer antisera. These antibodies specifically recognized human tissue macrophages and human MSR protein purified from differentiated THP1 cells. In normal brain, MSR staining was mainly distributed to the perivascular cells, which correspond to Mato's fluorescent granular perithelial cells (Mato cells). In the lesions of ischemia and Alzheimer's disease, a subset of microglia stained positive for MSR. These novel antibodies are useful tools for analysis of MSR expression in human tissues.

[Study on Mato cells (Mato's FGP cells) under various conditions].

Mato cells are unique macrophagic cells locating in the Virchow-Robin space of cerebral microvessels. They play a significant role in blood brain barrier, and uptake and digest proteinous and lipoidal materials derived from surroundings. They are provided with epitopes of macrophage lineages such as MHC Class II and scavenger receptor. Under pathological conditions, -cerebral injury, hypercholesteremia, hypertension and congenital dysfunction of nerve and some enzymes-induce heavy damages of Mato cells in shape and contents, and some of Mato cells are going into degeneration. Subsequently, the architecture of microvessels is also modified and result in the narrowing of vascular luminae. Finally, the authors discusses these findings referring to the earlier reports of Alzheimer (1913).

Growth retardation of Mato's fluorescent granular perithelial (FGP) cells in scavenger receptor knockout (SRKO) mice.

BACKGROUND: Mato's fluorescent granular perithelial (FGP) cells are situated between the basal lamina of cerebral microvessels and the glia limitans covering astrocytic processes. Because they are potent enough to take up endo- and exogenous substances in a steady state, they are regarded as scavenger cells in the central nervous system. The scavenger receptor for oxidized low-density lipoprotein (LDL) is also expressed on their cytoplasmic membrane. We studied the differentiation and growth of FGP cells in wild-type and scavenger receptor knockout (SRKO) mice. METHODS: Wild-type (129 strain) and SRKO mice at 1, 3, 5, 8, 10, 14, and 28 days after birth were employed in this study. Stretch specimens of their cerebral cortices were used for immunohistochemical investigations, and Epon-embedded specimens of the same region were studied with an electron microscope. RESULTS: In wild-type mice, perivascular cells derived from the pial tissue were transformed at approximately 8 days after birth into the FGP cells, which, provided with several epitopes common to macrophage lineages, developed over a period of 14 days. The FGP cells possessed several inclusions and were surrounded by two defined sheets of basal lamina and continuous glia limitans. At 4 weeks, their morphology, enzymatic activity, and epitopes advanced with the aging of the mice. However, in SRKO mice, at 8 days, the perivascular cells took fibroblastoid forms with scarce lysosomal inclusions, and, even at 14 days, glia limitans remained discontinuous and most of FGP cells remained immature in their cytoplasmic organelles. Different from the case with the wild-type, the intensity of the labeling of epitopes remained at a low level. At 4 weeks, some of the FGP cells fell into degeneration, and the others were differentiated to a certain degree. CONCLUSIONS: The development of Mato's FGP cells in SRKO mice was retarded in the morphology, enzyme activity, and epitopes. The scavenger receptor, which reacted with the antibody 2F8, seems to be closely associated with the development of Mato's FGP cells.

Alteration in water channel AQP-2 by removal of AVP stimulation in collecting duct cells of dehydrated rats.

Dehydration increased the expression of aquaporin of collecting duct (AQP-2) and translocated AQP-2 to the apical plasma membranes from cytoplasmic vesicles of collecting duct cells. We determined whether the abrupt decrease in circulating arginine vasopressin (AVP) by giving excess water affects the expression of AQP-2 mRNA and subcellular localization of AQP-2 in collecting duct cells of the dehydrated rats. The 72-h water deprivation increased plasma AVP levels to 3.1 pg/ml and the expression of AQP-2 mRNA by 336% in rats, which were concomitantly abolished by the 40 ml/kg oral water load. A 50% inhibition ofAQP-2 mRNA expression was obtained with 20 min after the forced water load. In immunohistochemistry and electron microscopy, the AQP-2 was manifestly present around the apical edge of collecting duct cells in the 72-h dehydrated rats. The AQP-2 was diffusely translocated into the cytoplasm 1 h after the forced water administration. These results indicate that AVP plays the on-off regulation of AQP-2 mRNA expression and that a majority of AQP-2 is regulated by the shuttle recycling in the collecting duct cells.

Involvement of specific macrophage-lineage cells surrounding arterioles in barrier and scavenger function in brain cortex.

The transport of solutes between blood and brain is regulated by a specific barrier. Capillary endothelial cells of brain are known to mediate barrier function and facilitate transport. Here we report that specific cells surrounding arterioles, known as Mato's fluorescent granular perithelial (FGP) cells or perivascular microglial cells, contribute to the barrier function. Immunohistochemical and in situ hybridization studies indicate that, in normal brain cortex, type I and type II macrophage scavenger receptors are expressed only in FGP/perivascular microglial cells, and surface markers of macrophage lineage are also detected on them. These cells mediate the uptake of macromolecules, including modified low density lipoprotein, horseradish peroxidase, and ferritin injected either into the blood or into the cerebral ventricles. Accumulation of scavenged materials with aging or after the administration of a high-fat diet results in the formation of honeycomb-like foam cells and the narrowing of the lumen of arterioles in the brain cortex. These results indicate involvement of FGP/perivascular microglial cells in the barrier and scavenger functions in the central nervous system.

Study on distribution of pericyte and fluorescent granular perithelial (FGP) cell in the transitional region between arteriole and capillary in rat cerebral cortex.

BACKGROUND: The FGP (fluorescent granular perithelial) cell nominated by M. Mato were distributed around small cerebral vessels and potent in the uptake capacity. They are provided with surface antigens related to macrophage lineage. The aim of this report is to show the regional distribution of pericytes and novel perivascular cells (FGP cell) in relation to the glia limitans and basal lamina. METHODS: The perietal cortex of rat cerebrum were fixed with a mixture containing 2.5% glutaraldehyde and 2% paraformaldehyde with or without 1% tannic acid and 1% osmium tetroxide buffered with 0.1 M phosphate. The specimens were embedded in Epon 812 and cut serially and observed with the light microscope. After selecting the appropriate region of blood vessels in the sections, they were reembedded in Epon 812 and were prepared for electron microscopic observation. RESULTS: Fixatives containing tannic acid gave good results for the discrimination between the glia limitans and the basal lamina at an electron microscopical level. The FGP cells were situated in the interstices between thick basal lamina of blood vessel and thin glia limitans of astrocyte (Virchow Robin space). In the terminal part of cerebral arteriole, the vascular basal lamina abutted against the glia limitans and both laminae were fused. The basal lamina enclosing pericytes was common to endothelium and the basal lamina of capillary was assumed to be the composite of proper basal lamina and glia limitans. Subsequently, the FGP cells surround only arteriole and venule, whereas the pericytes belong to capillaries. At the boundary of arterioles and capillaries, FGP cells coexisted with pericytes and were localized at the external site of pericytes bordering with basal lamina. CONCLUSIONS: Based on these findings, the FGP cells were different from pericytes in their localization and distribution. Some roles of FGP cells in cerebral metabolism are discussed.

[A case of hemolytic uremic syndrome accompanied by massive proteinuria and ascites].

A 50-year-old male was admitted to our hospital because of proteinuria, thrombocytopenia and moderate renal dysfunction. On admission, he had massive ascites, which was transudatory. During his clinical course, renal function deteriorated and the urine volume was decreased. Hemolytic uremic syndrome with massive ascites was diagnosed based on the finding of thrombocytopenia, acute renal failure and hemolytic anemia. Methylpredonisolone pulse therapy was not effective, but plasma exchange given 22 times in total combined with vincristine sulfate, PGI2 analogue and vitamin E administration was very effective for thrombocytopenia, renal dysfunction and hemolytic anemia. Massive ascites disappeared at the same time. After complete recovery of renal function, renal biopsy was performed, revealing the reticulation of mesangial matrix and mesangiolysis, which correspond to hemolytic uremic syndrome.

Additional mechanisms of nafamostat mesilate-associated hyperkalaemia.

OBJECTIVE: Nafamostat mesilate, a potent protease inhibitor, is widely used for the treatment of pancreatitis, disseminated intravascular coagulation and as an anticoagulant in haemodialysis. However, hyperkalaemia associated with nafamostat mesilate has been reported. It is thought to be due to decreased urinary potassium excretion, of the drug suppression of aldosterone secretion, and a direct inhibitory action on the apical Na+ conductance in collecting ducts. We have seen two cases of nafamostat mesilate associated-hyperkalaemia, which indicated that extrarenal potassium imbalance might play a role in inducing hyperkalaemia. METHODS: To examine the effect of nafamostat mesilate on potassium transport in erythrocytes in vitro, 86RbCl uptake was measured in red blood cells from eight healthy volunteers. RESULTS: Nafamostat mesilate and a metabolite, 6-amidino-2-naphthol, at concentrations of 10(-4) and 10(-3) M, respectively, significantly, suppressed potassium influx whilst another metabolite, p-guanidino-benzoic acid, had no effect. The inhibitory action of nafamostat mesilate was not affected by various inhibitors. CONCLUSION: Nafamostat mesilate and its metabolite, 6-amidino-2-naphthol, suppressed potassium influx in erythrocytes by inhibition of a Na-K ATPase dependent pathway, which was not inhibited by amiloride, barium, nor by frusemide (furosemide).

The distribution of TM-316-associated surface antigen on polymorphonuclear leucocytes: an immunoelectron microscopic study.

It has been established that MoAb TM-316 recognizes an epitope on leucocytes and specifically inhibits the chemotactic behavior of leucocytes. In the present paper, the distribution of this epitope on the cell surface and in intracellular organelles was studied by immunoelectron microscopy. Leucocytes separated from the blood of healthy men and from synovial fluid from patients suffering from rheumatoid arthritis were used. They were fixed with a mixture containing paraformaldehyde, glutaraldehyde and picric acid. As the second antibody, goat anti-mouse IgM conjugated to 10 nm gold colloids was employed. In normal specimens, the epitope was found to some extent on the cytoplasmic membrane of neutrophilic leucocytes, but it was only sparsely distributed on eosinophilic and basophilic leucocytes. On activated neutrophilic leucocytes, obtained from the synovial fluid of rheumatoid arthritis patients, the immunolabeling was markedly increased. The number of sites where the epitope occurs on the surface of leucocytes is thus associated with the cell type, and also with the level of activation of the leucocytes. In order to investigate the processing of the antigen, the intracellular localization of the epitope in the neutrophilic leucocytes was also studied. The epitope recognized by TM-316 was also detected in/on the characteristic granules and Golgi stacks.