Comparison of erythrocyte membrane fatty acid contents in renal transplant recipients and dialysis patients.

BACKGROUND: Alterations of erythrocyte membrane fatty acid (FA) composition play important roles in cellular function because they change the membrane microenvironment, including transmembrane receptors. The erythrocyte membrane oleic acid content is higher among patients with acute coronary syndrome and also in dialysis patients. However, available data are limited concerning erythrocyte membrane FA content in kidney transplant recipients (KTP). We sought to test the hypothesis that erythrocyte membrane FA content among KTP were different from those in dialysis patients. METHODS: In this cross-sectional study, we recruited 35 hemodialysis, 33 peritoneal dialysis 49 KTP, and 33 normal control subjects (CTL). Their erythrocyte membrane FA content were measured by gas chromatography. RESULTS: The mean ages of the enrolled dialysis patients, KTP, and CTL were 56.4 ± 10.1, 48.9 ± 10.4, and 49.5 ± 8.3 years, respectively. Mean kidney transplant duration was 89.8 ± 64.8 months and mean dialysis duration, 49.0 ± 32.6 months. The intakes of vegetable lipid and vegetable protein including total calories were significantly increased among KTP versus dialysis patients. Total cholesterol (P < .001) and high density lipoprotein cholesterol (HDL; P < .001) levels were significantly higher and C-reactive protein was significantly lower among KTP compared with dialysis patients. The erythrocyte membrane content of palmitoleic acid (P < .001) was significantly higher but oleic acid (P < .001) significantly lower in KTP compared with dialysis patients. The erythrocyte membrane contents of arachidonic acid and docosahexaenoic acid were significantly higher, and linoleic acid and the omega-6 FA to omega-3 FA ratio (P < .001) significantly lower in KTP compared with dialysis patients. The erythrocyte membrane content of oleic acid was independently associated with monounsaturated fatty acid (beta = 0.771, P < .001), eicosapentaeonic acid (beta = -0.244, P = .010), and HDL (beta = -0.139, P = .049) in KTP. CONCLUSIONS: FA contents of erythrocyte membranes were significantly different in KTP compared with dialysis patients. These differences may have been associated with improved dietary intake and immunosuppression after kidney transplantation.

Effect of omega-3 fatty acids on the modification of erythrocyte membrane fatty acid content including oleic acid in peritoneal dialysis patients.

Erythrocyte membrane fatty acids (FA), such as oleic acid, are related to acute coronary syndrome. There is no report about the effect of omega-3 FA on oleic acid in peritoneal dialysis (PD) patients. We hypothesized that omega-3 FA can modify erythrocyte membrane FA, including oleic acid, in PD patients. In a double-blind, randomized, placebo-controlled study, 18 patients who were treated with PD for at least 6 months were randomized to treatment for 12 weeks with omega-3 FA or placebo. Erythrocyte membrane FA content was measured by gas chromatography at baseline and after 12 weeks. The erythrocyte membrane content of eicosapentaenoic acid and docosahexaenoic acid was significantly increased and saturated FA and oleic acid were significantly decreased in the omega-3 FA supplementation group after 12 weeks compared to baseline. In conclusion, erythrocyte membrane FA content, including oleic acid, was significantly modified by omega-3 FA supplementation for 12 weeks in PD patients.

Association of adiponectin and leptin with serum lipids and erythrocyte omega-3 and omega-6 fatty acids in dialysis patients.

AIMS: Besides regulating energy metabolism, leptin promotes and adiponectin suppresses inflammation which is a common feature of end-stage renal disease (ESRD). Omega-3 fatty acids (n-3FA) exert anti-inflammatory actions by inhibiting pro-inflammatory signal transduction pathways whereas arachidonic acid (an n-6FA) facilitates inflammation by mediating inflammatory signals and serving as precursor of pro-inflammatory eicosanoids. Given the functional overlap between adipokines and n-3FA and n-6FA, we sought to explore their interrelationship in patients with ESRD. METHODS: 44 ESRD patients maintained on hemodialysis (HD), 29 patients receiving peritoneal dialysis (PD), and 10 healthy subjects were enrolled. Body mass index (BMI), plasma leptin, adiponectin, lipids and CRP and erythrocyte fatty acids were measured. RESULTS: Compared to controls adiponectin was elevated and leptin level was reduced in the ESRD group. Adiponectin levels were comparable among PD and HD patients, but leptin and BMI were higher in PD than in HD patients. Despite comparable BMIs, female patients had higher leptin than male patients. Leptin levels were positively associations with BMI, total and LDL cholesterol whereas adiponectin was inversely related with BMI, triglycerides and CRP and directly associated with HDL cholesterol in ESRD patients. Plasma adiponectin was directly associated with erythrocyte n-3 FA (r = 0.581, p = 0.023) and inversely associated with n-6FA (r = -0.640, p = 0.010) in the HD patients. CONCLUSION: A direct association was found between plasma levels of adiponectin and HDL and erythrocyte n-3FA in ESRD patients. Prospective trials are needed to explore the effect of n-3FA supplementation on plasma adipokines and markers of oxidative stress and inflammation in this population.

Protein tyrosine kinase inhibitors modulate radiosensitivity and radiation-induced apoptosis in K562 cells.

We studied the modulating effect of protein tyrosine kinase inhibitors on the response of cells of the human chronic myelogenous leukemia cell line K562 to radiation. The radiosensitivity of the cells was increased by treatment with herbimycin A and decreased by treatment with genistein. This modulating effect of protein tyrosine kinase inhibitors on radiation sensitivity was associated with the alteration of the mode of radiation-induced cell death. After X irradiation, the cells arrested in the G(2) phase of the cell cycle, but these TP53(-/-) cells were unable to sustain cell cycle arrest. This G(2)-phase checkpoint deficit caused cell death. The morphological pattern of cell death was characterized by swelling of the cytoplasmic compartments, cytosolic vacuolation, disruption of the plasma membrane, less evident nuclear condensation, and faint DNA fragmentation, all of which were consistent with oncosis or cytoplasmic apoptosis. The nonreceptor protein tyrosine kinase inhibitor herbimycin A accelerated the induction of typical apoptosis by X irradiation, which was demonstrated by morphological assessments using nuclear staining and electron microscopy as well as oligonucleosomal fragmentation and caspase 3 activity. Herbimycin A is known to be a selective antagonist of the BCR/ABL kinase of Philadelphia chromosome-positive K562 cells; this kinase blocks the induction of apoptosis after X irradiation. Our results showed that the inhibition of protein tyrosine kinase by herbimycin A enhanced radiation-induced apoptosis in K562 cells. This effect was associated with the activation of caspase 3 and rapid abrogation of the G(2)-phase checkpoint with progression out of G(2) into G(1) phase. In contrast, the receptor-type protein tyrosine kinase inhibitor genistein protected K562 cells from all types of radiation-induced cell death through the inhibition of caspase 3 activity and prolonged maintenance of G(2)-phase arrest. Further investigations using this model may give valuable information about the mechanisms of radiation-induced apoptosis and about the radiosensitivity and radioresistance of chronic myelogenous leukemia cells having the Philadelphia chromosome.

Promoted expression of mast cell-specific proteases in IgE-dependent passive cutaneous anaphylaxis responses.

BACKGROUND: Various factors can influence the protease expression phenotype of mast cells. METHODS: In an effort to understand the potential role of the mast cell proteases in the IgE-dependent passive cutaneous anaphylaxis (PCA) responses of murine tissues, we studied the changes of proteases expression. The expressions of proteases were examined by Northern blotting and immunohistochemistry. RESULTS: Promoted expression phenotypes of mouse mast cell protease (mMCP)-4, and rat mast cell protease I were accompanied by initiation of anti-dinitrophenyl (DNP) IgE-induced PCA responses, suggesting that the induction of these proteases expression are associated with IgE-mediated anaphylaxis responses. Elevated level of the L-histidine decarboxylase (HDC) mRNA expression was also observed in the PCA tissues and the activated mast cells, compared with that of the corresponding control tissue and cells, due to the activation of mast cells. CONCLUSIONS: Promoted protease expression phenotype appears to be linked with the induction of HDC expression.

Circulating levels of interleukin-8 and vascular endothelial growth factor in patients with carotid stenosis.

Interleukin (IL)-8 and vascular endothelial growth factor (VEGF) are important factors that induce the migration and proliferation of endothelial cells, increase the vascular permeability, and the modulate chemotaxis of monocytes. These molecules have been found in human atherosclerotic plaques. However, it is not clear whether the circulating levels of IL-8 and VEGF correlate with the extents of carotid stenosis. In this study, we investigated the relationship between circulating levels of IL-8 as well as VEGF and the extents of carotid stenosis. Sera from 41 patients with carotid stenosis were assessed for concentrations of IL-8 and VEGF by enzyme-linked immunosorbent assay. The degree of stenosis of extracranial carotid artery was calibrated by carotid B- mode ultrasonography. The serum concentration of IL-8 (r = -0.04733, p > 0.05) was not correlated with the degree of stenosis. However, the serum concentration of VEGF (r = 0.4974, p < 0.01) was significantly correlated with the degree of carotid stenosis. These findings suggest that increased serum level of VEGF might be a marker for higher degree of stenosis of extracranial carotid artery.

Age-related regional difference of interleukin-1 expression in rat brain after lipopolysaccharide treatment.

Aging is associated with altered immune responses including dysregulation of cytokine production. Of cytokines, interleukin-1 (IL-1) family has been primarily involved with central nervous system. To evaluate the age-related different response of IL-1 family following peripheral administration of lipopolysaccharide (LPS), immunohistochemical study of IL-1beta and IL-1 receptor expression was performed on Sprague-Dawley rat brain. Experimental animals were divided into four groups; saline-treated young (3-5 months) and old (over 24 months), and LPS-treated young and old groups. After intraperitoneal (i.p.) injection of LPS, three to five rats within each group were killed at 1, 2, 4, 8 and 16 hr. After fixation in 4% neutral buffered formalin, the brain slices were paraffin-embedded. Immunohistochemical staining using labelled streptavidin biotin was performed. The results showed that IL-1beta immunoreactivity was seen in the endothelial cell of pons in both LPS-treated young and old rats, with slightly longer persistency in old group. IL-1RI immunoreactivity appeared initially in the neurons of cerebral cortex in LPS-treated old group, compared with predominantly the cerebellum in LPS-treated young group. In conclusion, our study shows that there is age-related, different neuronal localization of IL-1RI expression at different points of time after LPS treatment.

Activated platelets induce secretion of interleukin-1beta, monocyte chemotactic protein-1, and macrophage inflammatory protein-1alpha and surface expression of intercellular adhesion molecule-1 on cultured endothelial cells.

Atherosclerosis is an inflammatory disease. Platelet-endothelium interaction plays an important role in the pathophysiology of atherogenesis. We investigated the role of activated platelets for secretion of interleukin (IL)-1beta, monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha and expression of intercellular adhesion molecule (ICAM)-1 on endothelial cells. Human umbilical vein endothelial cells (HUVEC) were incubated with non-stimulated or ADP-activated platelets for 6 hr. Secretion of interleukin (IL)-1beta, MCP-1 and MIP-1alpha and surface expression of ICAM-1 were measured by ELISA and flow cytometry. In the presence of activated platelets, the secretion of IL-1beta, MCP-1, and MIP-1alpha and surface expression of ICAM-1 were significantly increased compared with non-activated platelets. The present study shows that activated platelets may contribute to expression of various inflammatory mediators on endothelial cells.

Calmodulin-dependent activation of p38 and p42/44 mitogen-activated protein kinases contributes to c-fos expression by calcium in PC12 cells: modulation by nitric oxide.

Calcium and nitric oxide (NO) are important messengers for the activity-dependent immediate-early gene (IEG) expressions in neuronal cells. In the present study, we have investigated the roles of two mitogen-activated protein (MAP) kinases, extracellular signal-regulated protein kinase (ERK) and p38 MAP kinase (p38 kinase) in calcium- and NO-induced c-fos expression in PC12 cells. Membrane depolarization-induced calcium increases activated both ERK and p38 kinase within 5 min. The activation of both ERK and p38 kinase by calcium was a calmodulin-dependent process since the pretreatment of W13 or calmidazolium, specific calmodulin antagonists, blocked calcium-induced activation of both MAP kinases. Calcium-induced c-fos expression was significantly reduced by the pretreatment of either MEK inhibitor (PD98059) or p38 kinase inhibitor (SB203580). This finding indicates that the calmodulin-dependent activation of ERK and p38 kinase is involved in calcium-induced c-fos expression. However, sodium nitroprusside and SIN-1, known to release NO, dose-dependently activated only ERK. NO-induced c-fos expression was partially inhibited by the PD98059. We also observed that NO dose-dependently potentiates not only calcium-induced c-fos expression but also calcium-induced ERK activation. In the presence of PD98059, the amplification of calcium-induced c-fos expression by NO was not observed. This result suggests that calcium- and NO-signals converge into the MEK/ERK pathway, thereby enhance IEG expressions in neuronal cells.

Effect of t-butylhydroperoxide on chloride secretion in rat tracheal epithelia.

Oxidative stress has been known to play important roles in various inflammatory diseases of lung such as allergic bronchitis, dust particle-induced inflammatory diseases, or chronic bronchitis. However, the effects of oxidants on Cl- secretion in tracheal epithelia have not been determined. To examine the effects of oxidants on Cl- secretion of the airway epithelia rat tracheal epithelial cells were cultured on porous filters and short circuit current (Isc) was measured in an Ussing chamber system. t-Butylhydroperoxide, which was widely used as a model substance to study the mechanism of cell injury resulted from oxidative stress, induced a transient increase in Isc by dose-dependent manner. The response was not observed in Cl(-)-free medium, and inhibited by 100 microM bumetanide. N(-Diphenyl-1,4-phenylene-diamine (DPPD, 5 microM), an inhibitor of lipid peroxidation, blocked the t-butylhydroperoxide response. When t-butylhydroperoxide was added after the administration of forskolin or H-89, a protein kinase A inhibitor, the t-butylhydroperoxide-induce Isc increase was abolished. Pretreatment of indomethacin (10 microM) completely inhibited the t-butylhydroperoxide response, but pretreatment of thapsigargin (1 microM) did not, t-Butylhydroperoxide induced gradual increases in cytosolic Ca2+ level, and increased [3H]arachidonic acid release in the presence of thapsigargin. These results indicate that t-butylhydroperoxide stimulates Cl-secretion via activation of phospholipase A2 and subsequent production of cyclooxygenase metabolities by Ca(2+)-dependent and -independent mechanisms.

Seven novel mammalian SNARE proteins localize to distinct membrane compartments.

Soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins of the vesicle-associated membrane protein (VAMP) and syntaxin families play a central role in vesicular trafficking through the formation of complexes between proteins present on vesicle and target membranes. Formation of these complexes is proposed to mediate aspects of the specificity of vesicle trafficking and to promote fusion of the lipid bilayers. In order to further understand the molecular mechanisms that organize membrane compartments, we have characterized seven new mammalian proteins of the VAMP and syntaxin families. The proteins are broadly expressed; however, syntaxin 13 is enriched in brain and VAMP 8 in kidney. The seven novel SNAREs localize in distinct patterns overlapping with Golgi, endosomal, or lysosomal markers. Our studies support the hypothesis that evolutionary radiation of these two gene families gave rise to sets of proteins whose differential expression and combinatorial associations define and organize the membrane compartments of cells.

Stimulation of Cl- secretion by AlF4- and vanadate in T84 cells.

We investigated the mechanism of Cl- secretion by fluoroaluminate(AlF4-) and sodium orthovanadate(vanadate) using the human colonic T84 cell line. T84 cell monolayers grown on collagen-coated filters were mounted in Ussing chambers to measure short circuit current(ISC). Serosal addition of AlF4- or vanadate to T84 monolayers produced a sustained increase in ISC. Removal of Ca2+ from the serosal bathing solution partially inhibited AlF4-(-)and vanadate-induced ISC, and readministration of Ca2+ restored AlF4-(-)and vanadate-induced ISC. Carbachol application in the presence of forskolin, AlF4- or vanadate induced a synergistic increase of ISC. Forskolin and vanadate significantly increased cellular cAMP level, while carbachol and AlF4- did not. Carbachol, AlF4- and vanadate significantly increased [Ca2+]i. After Na+ in mucosal bathing solution was replaced with K+, and the mucosal membrane of T84 cell was permeabilized with amphotericin B, AlF4-, vanadate, and carbachol increased K+ conductance, but forskolin did not. After sodium chloride in serosal bathing solution was replaced with sodium gluconate and the serosal membrane was permeabilized with nystatin, forskolin, AlF4-, and vanadate increased Cl- conductance, but carbachol did not. AlF4-(-)induced ISC was remarkably inhibited by the pretreatment of pertussis toxin(2 micrograms/ml) for 2 hours. These results indicate that AlF4- and vanadate can increase Cl- secretion via simultaneous stimulation of Cl- channel and K+ channel in T84 cells. However, the AlF4- action is mostly attributed to stimulation of pertussis toxin-sensitive G-proteins, whereas the vanadate action mostly results from G protein-independent mechanisms.

Protein kinase A dependent membrane protein phosphorylation and chloride conductance in endosomal vesicles from kidney cortex.

Regulation of Cl conductance by protein kinase A may play a role in control of endosomal acidification [Bae, H.-R., & Verkman, A. S. (1990) Nature, 348, 637-639]. To investigate the mechanism of kinase A action, cell-free measurements of Cl transport and membrane protein phosphorylation were carried out in apical endocytic vesicles from rabbit kidney proximal tubule. Cl transport was measured by a stopped-flow quenching assay in endosomes labeled in vivo with the fluorescent Cl indicator 6-methoxy-N-(3-sulfopropyl)quinolinium. Phosphorylation was studied in a purified endosomal preparation by SDS-PAGE and autoradiography of membrane proteins labeled by [gamma-32P]ATP. Endosomes had a permeability (PCl) for conductive Cl transport of 3.1 x 10(-8) cm/s at 23 degrees C which was stilbene inhibitable. PCl was increased by 90 +/- 20% by a 10-min preincubation with the catalytic subunit of kinase A (PKA, 10 units/mL) and MgATP (0.5 mM) with anion selectivity Cl greater than I greater than Br. The increase in PCl was blocked by 100 microM N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) and was reversed by addition of alkaline phosphatase (AP, 40 units/mL) after incubation with PKA and MgATP; the increase in PCl was not blocked by pretreatment with AP.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity in ATP-dependent acidification in endocytic vesicles from kidney proximal tubule. Measurement of pH in individual endocytic vesicles in a cell-free system.

Measurement of membrane transport in suspensions of isolated membrane vesicles provides averaged information over a potentially very heterogeneous vesicle population. To examine the regulatory mechanisms for ATP-dependent acidification, methodology was developed to measure pH in individual endocytic vesicles. Endocytic vesicles from proximal tubule apical membrane of rat kidney were labeled in vivo by intravenous infusion of FITC-dextran (9 kD); a microsomal fraction was obtained from dissected renal cortex by homogenization and differential centrifugation. Vesicles were immobilized on a polylysine coated coverglass and imaged at high magnification by a silicon intensified target camera. ATP-dependent acidification was not influenced by endosome immobilization. Endosome pH was determined from the integrated fluorescence intensity of individual labeled vesicles after background subtraction. Calibration studies with high K and nigericin showed nearly identical fluorescence vs. pH curves for different endosomes with a standard deviation for a single pH measurement in a single endosome of approximately 0.2 pH units. In response to addition of 1 mM MgATP in the presence of K and valinomycin, endosome pH decreased from 7.2 to a mean of 6.4 with a unimodal distribution with width at half-maximum of approximately 1 pH unit. The drop in endosome pH increased and the shape of the distribution changed when the time between FITC-dextran infusion and kidney removal was increased from 5 to 20 min. Differences in ATP-dependent acidification could not be attributed to heterogeneity in passive proton conductance. These results establish a direct method to measure pH in single endocytic vesicles and demonstrate remarkable heterogeneity in ATP-dependent acidification which was interpreted in terms of heterogeneity in the number and/or activity of proton pumps at serial stages of endocytosis.

Protein kinase A regulates chloride conductance in endocytic vesicles from proximal tubule.

Regulation of ion transport by phosphorylation and G proteins occurs in several epithelial and non-epithelial cell plasma membranes1-5. It is not known whether transporters on intracellular membranes are target sites for second messengers. Here we present direct evidence that a chloride conductance in endocytic vesicles from rabbit proximal tubule is activated by phosphorylation through a cyclic AMP-dependent protein kinase. To measure chloride transport, endocytic vesicles were labelled in vivo with a Cl(-)-sensitive fluorescent indicator6-8. It was found that labelled endosomes contained an inward proton pump and a chloride conductance, but no ion-coupled chloride transport, and that the chloride conductance was regulated by protein kinase A. These results, taken together with measurements of chloride effects on ATP-dependent acidification, suggest that endosomal pH can be controlled by phosphorylation of a stilbene-sensitive conductive chloride transporter.