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Biomedical subjects

A V Cybulsky

Publications and source records attributed to A V Cybulsky.

At least 19 recordsLinked to original sources
PubMed1995

Phospholipase A2 activity is increased in guinea pig uterine cervix in late pregnancy and at parturition.

Release of arachidonic acid from phospholipids by phospholipases A2 (PLA2) is the rate-limiting step in prostaglandin (PG) synthesis. PGE2 and PGF2 alpha are essential intermediates in interstitial collagenase-mediated degradation of type I collagen, a key step in cervical dilatation at parturition. We demonstrate that PLA2 is present in cytosolic fractions of guinea pig cervix and PLA2 activity is increased during cervical dilatation at parturition. In the cervix of nonpregnant animals, PLA2 activity against phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was 13 +/- 6 and 49 +/- 27 pmol.min-1.mg protein-1, respectively. Levels were similar at day 25 of pregnancy. At day 50, PLA2-PC increased to 190 +/- 54 pmol.min-1.mg-1, and PLA2-PE rose to 592 +/- 127 pmol.min-1.mg-1. At parturition (68 +/- 2 days) there were further increases of 49-67%. PLA2 activity declined toward basal levels 2 days postpartum. Almost 50% of the enhanced cytosolic PLA2 (cPLA2) activity at day 50 or at parturition was of high molecular mass and was identified as the "85-kDa cPLA2." Increases in cPLA2 activity at these time points were not, however, associated with increases in cPLA2 protein or phosphorylation of cPLA2, compared with nonpregnant animals. This study suggests that multiple PLA2 enzymes, including cPLA2, are involved in cervical dilatation at parturition.

Animals↗
PubMed1995

Complement C5b-9 activates cytosolic phospholipase A2 in glomerular epithelial cells.

In rat membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury and proteinuria, which, in some models, is partially mediated by eicosanoids. By analogy, sublytic C5b-9 injures plasma membranes and releases arachidonic acid (AA) and eicosanoids in cultured rat GEC. In this study, we demonstrate that, in GEC, sublytic C5b-9 stably increased the activity of a high-molecular-mass cytosolic phospholipase A2 (PLA2), which we identified as "cPLA2." This increase was abolished with inhibitors of protein kinase C. C5b-9 did not affect low-molecular-mass membrane-associated or secretory PLA2 activities. In GEC that stably overexpress cPLA2 activity and protein (produced by transfection of cPLA2 cDNA), immunoblot analysis showed that sublytic C5b-9 induced a decreased mobility of cPLA2, consistent with cPLA2 phosphorylation. Incubation of cPLA2-transfected GEC with sublytic C5b-9 significantly increased production of free AA and prostaglandin E2, whereas, in control GEC, the C5b-9-induced changes in free AA and prostaglandin E2 were small. Furthermore, both C5b-9-dependent sublytic cytotoxicity and cytolysis were enhanced in GEC overexpressing cPLA2, compared with control cells. Thus C5b-9 increased cPLA2 activity, probably via phosphorylation involving a protein kinase C-dependent pathway. Phospholipid hydrolysis by cPLA2 resulted in release of substrate for eicosanoid synthesis and in enhancement of C5b-9-dependent GEC injury. Both processes may facilitate glomerular damage in membranous nephropathy.

Animals↗
PubMed1995

Insulin-like growth factor-1 enhances epidermal growth factor receptor activation and renal tubular cell regeneration in postischemic acute renal failure.

Growth factors such as insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and hepatocyte growth factor have been shown to accelerate the recovery from postischemic acute renal failure (ARF) with a concomitant increase in DNA synthesis. Interactions between growth factors have been demonstrated in a number of in vitro studies. This study examined the effect of exogenous IGF-1 on the DNA synthesis and EGF receptor (EGF-R) activation in postischemic rat kidneys. Thirty minutes after the relief of 30-minute total occlusion of the left renal artery in anesthetized 225 to 300 gm Sprague-Dawley rats, either IGF-1 (75 micrograms/kg) or normal saline solution (NS, 0.2 ml) was given by intravenous bolus, followed by twice daily subcutaneous injections of IGF-1 (50 micrograms/kg) or 0.2 ml NS for 4 days, respectively, in IGF-1-Tx) and NS treated (NS-Tx) groups (n = 8 each). On the day after the completion of treatment, inulin clearance (ml/kg/min) of the postischemic kidneys in the IGF-1-Tx group was significantly higher (p < 0.01) than inulin clearance of kidneys in the NS-Tx group. This was associated with improved kidney morphology. IGF-1 treatment also enhanced the labeling index of 5-bromo-2'-deoxyuridine (percent of stained tubule cells), a marker for active DNA synthesis, in the outer medulla of postischemic kidneys at 1 day and 2 days after the injury. EGF-R tyrosine phosphorylation (which reflects receptor activation) increased in postischemic kidneys in both NS-Tx (n = 5) and IGF-1-Tx (n = 3) groups 1 day after the injury as compared with nonischemic contralateral kidneys. In the IGF-1-Tx group there was also increased iodine 125-labeled EGF binding and EGF-R protein. Our results demonstrate a beneficial effect of IGF-1 on postischemic ARF. Furthermore, they suggest that EGF-R activation is involved in tubular regeneration and that IGF-1 may enhance EGF-R activation by increasing EGF-R expression.

Acute Kidney Injury↗
PubMed1992

Adhesion of rat glomerular epithelial cells to extracellular matrices: role of beta 1 integrins.

Glomerular epithelial cells (GEC) maintain glomerular permselectivity and are a target of immunological glomerular injury, which may lead to proliferation or detachment from extracellular matrix (ECM). We studied adhesion mechanisms in rat GEC in culture, focusing on adhesion molecules of the beta 1 integrin family. At early time points (1 hr after plating of cells into culture wells that had been pre-incubated with purified ECM proteins), adhesion of GEC to collagen I, collagen IV, laminin, and fibronectin was inhibited with anti-beta 1 integrin antibody. The peptide RGDS inhibited adhesion to fibronectin and laminin. Immunoprecipitation studies demonstrated the presence of alpha 2, alpha 3, and beta 1 integrins; the alpha 1, alpha 4, alpha 5, alpha 6, alpha v, and beta 3 subunits were undetectable. Adhesion to all ECM proteins was dependent on divalent cations, but the effects of individual cations varied among substrata. In rat GEC, alpha 2 beta 1 and/or alpha 3 beta 1 integrins appear to mediate adhesion to collagen I, collagen IV, and laminin. The alpha 3 beta 1 integrin is also the likely receptor for fibronectin, interacting through an RGD binding site. Furthermore, single integrins or combinations of integrins appear to have distinct ligand-binding functions that are differentially regulated by divalent cations. Characterization of GEC adhesion molecules may facilitate the understanding of mechanisms of glomerular development, and cell detachment or proliferation in immune glomerular injury.

Amino Acid Sequence↗
PubMed1992

Eicosanoids enhance epidermal growth factor receptor activation and proliferation in glomerular epithelial cells.

Proliferation of glomerular epithelial cells (GEC) and release of prostaglandins (PG) and thromboxane (Tx) A2 may occur in glomerular injury. We studied the relationship of eicosanoids to epidermal growth factor (EGF)-induced proliferation of rat GEC in culture. After 48 h of serum-deprivation, EGF stimulated [3H]thymidine incorporation ninefold above serum-deprived cells. Inhibition of cyclooxygenase with indomethacin or of Txsynthase with OKY-046 decreased the proliferative effect of EGF by 50 and 38%, respectively. The effect of indomethacin was reversed by addition of PGE2. Synthesis of PGE2, PGF2 alpha, and TxA2 by serum-deprived GEC was not enhanced by EGF. Scatchard analysis of 125I-EGF binding to GEC demonstrated two populations of EGF receptors; the high-affinity site had a dissociation constant (Kd) of 444 pM and 24,864 receptors/cell. EGF receptor autophosphorylation (reflecting receptor activation) was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of GEC membrane proteins with anti-phosphotyrosine antibody. EGF increased phosphorylation of a protein of approximately 170 kDa, which comigrated with proteins immunoprecipitated from [35S]methionine-labeled GEC with antibodies to EGF receptor. Indomethacin and OKY-046 decreased the EGF-dependent phosphorylation of the 170-kDa protein, and this decrease was overcome by addition of PGE2. Indomethacin and OKY-046 did not, however, reduce 125I-EGF binding. Thus, in GEC, the basal synthesis of eicosanoids enhanced EGF-induced proliferation. This effect appears to be due to enhancement of EGF receptor activation.

Animals↗
PubMed1991

Effect of nephritogenic antibody on complement regulation in cultured rat glomerular epithelial cells.

In passive Heymann nephritis, a rat model of membranous nephropathy, antibody (anti-Fx1A) activates C on the surface of the glomerular epithelial cell (GEC), leading to GEC injury and proteinuria. In this study, we examined C activation by anti-Fx1A in cultured rat GEC. In addition to anti-Fx1A IgG, anti-Fx1A F(ab')2 and Fab' led to GEC injury in the presence of rat or human sera as sources of C. Cytotoxicity was Mg2+ and factor B dependent, but Ca2+ independent, indicating that anti-Fx1A activated the C alternative pathway (AP). Furthermore, in the presence of Mg2+ and factor B, anti-Fx1A enhanced 125I-C3b deposition on GEC in the absence of classical pathway activation. AP C3 and C5 convertases formed on GEC (GEC-C3bBbP) were inactivated over time, probably due to binding of GEC C regulatory proteins. This inactivation was prevented when GEC-C3bBbP were incubated with anti-Fx1A IgG. An antibody raised against cultured GEC that binds to GEC in vitro and in vivo had no effect on C3 and C5 convertases, suggesting that stabilization of C3bBbP is unique to anti-Fx1A. Anti-Fx1A Fab' also stabilized GEC-C3bBbP, indicating that cross-linking of membrane Ag was not required. C3bBbP on E were not affected by anti-Fx1A, excluding direct stabilization of convertases by anti-Fx1A. Therefore, anti-Fx1A inhibits C regulation on GEC, which can account for its ability to activate the AP. This represents a potentially powerful mechanism of producing disease in vivo.

Animals↗
PubMed1991

Release of arachidonic acid by complement C5b-9 complex in glomerular epithelial cells.

In experimental membranous nephropathy, C5b-9 induces noncytolytic glomerular epithelial cell (GEC) injury and proteinuria, which in some models is partially mediated by metabolites of arachidonic acid. In cultured GEC, sublytic C5b-9 increases cytosolic Ca2+ concentration ([Ca2+]i), activates phospholipase C (PLC), and releases arachidonic acid and eicosanoids. This study examined mechanisms of arachidonic acid production by C5b-9. In GEC labeled with [3H]arachidonate C5b-9 increased free [3H]arachidonic acid and 1,2-[3H]-arachidonoyl-diacylglycerol (DAG), an endogenous activator of protein kinase C (PKC). Elevated [Ca2+]i was not sufficient to account for increased free arachidonic acid. Moreover, in GEC that had been depleted of PKC by preincubation for 18 h with 2 microM phorbol myristate acetate, the C5b-9-induced arachidonate release was inhibited by greater than 75%. Reacylation of phospholipids was not decreased by C5b-9. Homogenates of GEC that had been stimulated with C5b-9 released more [14C]arachidonate from exogenously added 2-[14C]arachidonoyl-phosphatidyl-ethanolamine or 2-[14C]arachidonoyl-phosphatidylcholine than homogenates of unstimulated cells (assayed at a Ca2+ concentration of 2 mM). These experiments demonstrate directly that C5b-9 increased phospholipase A2 (PLA2) activity. PLA2 appeared to be stimulated as a result of PKC activation (probably secondary to increased DAG) in association with elevated [Ca2+]i. The C5b-9-induced activation of PLA2 may lead to release of eicosanoids, which may contribute toward impaired glomerular capillary wall permselectivity in experimental membranous nephropathy.

Animals↗
PubMed1990

Cytosolic calcium and protein kinase C reduce complement-mediated glomerular epithelial injury.

In rat membranous nephropathy, protein-uria is due to formation of the C5b-9 membrane attack complex of complement (C), and is associated with morphological evidence of glomerular epithelial cell (GEC) injury. Analogous morphological changes are induced by C5b-9 in cultured GEC. In addition, in cultured GEC C5b-9 induces Ca2+ influx, as well as Ca2+ mobilization and increased 1,2-diacylglycerol due to the activation of phospholipase C. In this study we investigated how this GEC activation pattern might influence C-mediated GEC injury. We demonstrate that the C5b-9-induced increase in cytosolic Ca2+ concentration ([Ca2+]i) did not impair ATP generation by mitochondria, suggesting that it does not contribute to cytotoxicity. Moreover, this increase in [Ca2+]i protected GEC from C-mediated cytolysis. However, a large increase in [Ca2+]i (produced by the Ca2+ ionophore A23187) impaired ATP generation and aggravated C-mediated cytotoxicity, suggesting that intact mitochondrial activity is necessary for GEC to withstand C attack. Activation of protein kinase C (PKC) by phorbol myristate acetate (PMA) also decreased C-mediated cytolysis. Conversely, C lysis was enhanced in GEC that had been pretreated for 18 hours with a high dose of PMA to deplete PKC, and following PKC inhibition with H-7. Therefore, PKC activation, possibly resulting from C5b-9-induced increase in 1,2-diacylglycerol, triggered mechanisms that protected GEC from C-mediated injury. Thus, as a consequence of C5b-9-induced phospholipase activation, the amount of C-induced GEC injury is diminished.

Adenosine Triphosphate↗
PubMed1990

Extracellular matrix regulates proliferation and phospholipid turnover in glomerular epithelial cells.

To understand how glomerular epithelial cell (GEC) growth might be regulated in health and disease, we studied the effects of growth factors and extracellular matrix on proliferation and membrane phospholipid turnover in cultured rat GECs. In GECs adherent to type I collagen matrix, epidermal growth factor (EGF), insulin, and serum stimulated DNA synthesis and increased cell number. In addition, GECs proliferated when adherent to type IV collagen, but not to laminin or plastic substrata. Attachment of GECs to the substrata that facilitated proliferation (types I or IV collagen) produced increases in 1,2-diacylglycerol (DAG), an activator of protein kinase C (PKC). Increased DAG was associated with hydrolysis of inositol phospholipids and an increase in inositol trisphosphate and was not dependent on the presence of growth factors. After PKC downregulation (by preincubation with a high dose of phorbol myristate acetate), DNA synthesis was enhanced in GECs adherent to collagen. Thus contact of GECs with collagen matrices is required for serum, EGF, or insulin to induce proliferation. Collagen matrix also activates phospholipase C. As a result, the DAG-PKC signaling pathway desensitizes GECs to the mitogenic effects of growth factors and might promote cell differentiation. Understanding the interaction between GECs, growth factors, and extracellular matrix may elucidate the mechanisms of proliferation during glomerular injury.

Animals↗
PubMed1990

Effects of tumor necrosis factor on glomerular mesangial and epithelial cells in culture.

We have previously documented the importance of tumor necrosis factor (TNF) alpha in the pathogenesis of nephrotoxic-serum nephritis in rats. In this study, we evaluated the possible relevance of the well-established cytocidal TNF activity to the mechanism of glomerular injury by assessing sensitivity of rat mesangial and epithelial cell populations to recombinant murine TNF alpha (rMuTNF). Radiolabelled confluent mesangial cell cultures that were incubated with rMuTNF released significantly more 3H-thymidine than control monolayers (maximum specific release was 11.4 +/- 4.9% at 1,000 pg/ml of rMuTNF). rMuTNF was, however, approximately 1,000-fold less cytolytic in mesangial cells than in the lymphotoxin-sensitive L929 cell line. Conversely, glomerular epithelial cells were not affected by exposure to rMuTNF under the same conditions. These results suggest that the cytolytic effect of TNF may contribute to glomerular injury in nephrotoxic-serum nephritis.

Animals↗
PubMed1989

Decay accelerating factor regulates complement activation on glomerular epithelial cells.

Epithelial cells of the glomerular capillary are the site of C5b-9 mediated injury in rat membranous nephropathy. We investigated the regulation of C activation by cultured glomerular epithelial cells (GEC). Rat and human GEC were more resistant to C injury by homologous C than heterologous C. In human GEC homologous C cytotoxicity was enhanced by antiserum to decay accelerating factor (DAF) indicating that homologous C activation was, at least in part, restricted by membrane DAF. Anti-DAF immunoprecipitated a 67-kDa protein from human glomeruli. In rat GEC, pronase and phosphatidylinositol-specific phospholipase C (which are known to inactivate human DAF) enhanced cytotoxicity by homologous C. Thus, DAF is present on human GEC in culture and in human kidney glomeruli, and a DAF-like protein is present on cultured rat GEC. These proteins regulate C activation in vitro and may play a role in controlling C activation on GEC in vivo.

Animals↗
PubMed1989

Complement C5b-9 complex activates phospholipases in glomerular epithelial cells.

In rat membranous nephropathy, formation of the C5b-9 membrane attack complex (MAC) leads to proteinuria in association with glomerular visceral epithelial cell (GEC) injury. These alterations in GEC function and morphology might result from changes in intracellular free Ca2+ concentration [( Ca2+]i) and activation of phospholipases. We demonstrate that in cultured rat GEC, antibody-directed formation of noncytolytic amounts of the MAC induced a rapid and sustained increase in [Ca2+]i that was partly inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The MAC elevated levels of inositol bis- (IP2) and trisphosphate (IP3), as well as 1,2-diacylglycerol (DAG) and phosphatidic acid (PA). In permeabilized GEC, IP3 released Ca2+ from intracellular stores. Cellular 45Ca2+ uptake was also increased by the MAC. Thus, in GEC, the MAC induced Ca2+ mobilization from intracellular stores secondary to activation of phospholipase C and production of IP3, as well as enhanced Ca2+ influx. In addition, C5b-9 stimulated release of arachidonic acid (AA), prostaglandin F2 alpha, and thromboxane A2. Indomethacin partially inhibited the increase in DAG levels observed with the MAC, whereas the prostaglandin H2/thromboxane A2 analogue U46619 elevated DAG, suggesting that an eicosanoid product of MAC-induced AA release may enhance the activation of phospholipase C. Activation of phospholipases by the MAC may lead to altered GEC function and thereby contribute to the pathophysiological changes that characterize complement-dependent rat membranous nephropathy.

Animals↗
PubMed1989

Studies with antibodies to cultured rat glomerular epithelial cells. Subepithelial immune deposit formation after in vivo injection.

To investigate the role of glomerular epithelial cell (GEC) membrane proteins in the in situ formation of subepithelial immune deposits, the authors raised a rabbit antiserum against GEC that had been grown in culture (anti-GEC). By indirect immunofluorescence (IF) on normal rat kidney, anti-GEC stained proximal tubular brush border (BB). After intravenous injection into animals, granular glomerular capillary wall staining for IgG was present by IE and subepithelial immune deposits were identified by standard transmission and immunoelectron microscopy. Using the latter technique, injected anti-GEC IgG was identified beneath slit diaphragms and in endocytic-coated pits and intracellular vesicles of podocytes. Anti-GEC immunoprecipitated gp330 and two other proteins from radiolabeled BB. These proteins also were identified by sheep anti-rat Fx1A, the antiserum responsible for passive Heymann nephritis. Anti-GEC and anti-Fx1A also immunoprecipitated five identical proteins from surface-labeled GEC. Biosynthetically-labeled but not surface-labeled GEC contained immunoprecipitable gp330. Thus, injection into rats of antibodies raised against cultured GEC can produce subepithelial immune deposits, a disease process classically induced by antibodies to BB or its purified components. In addition to gp330, GEC and BB share other antigenic determinants that may contribute to the formation of these immune deposits.

Animals↗
PubMed1988

Anti-Fx1A produces complement-dependent cytotoxicity of glomerular epithelial cells.

Glomerular injury in passive Heymann nephritis (PHN) in rats is mediated by the C5b-9 membrane attack complex (MAC) and is associated with morphologic changes in glomerular visceral epithelial cells (GEC). We determined if the nephritogenic antibody of PHN (gamma 1 sheep anti-Fx1A IgG) directs insertion of the MAC into GEC plasma membranes with consequent cytotoxicity. Antibody-sensitized GEC were exposed to various sera serving as sources of complement. Loss of cell viability was determined by trypan blue uptake and/or by release of cellular lactate dehydrogenase (LDH). Incubation of antibody-sensitized primary and passaged GEC in fresh human serum (FHS) resulted in sigmoidal relationships between cytotoxicity and complement dose (r = 0.97 and 0.94, respectively) such that cytolysis approached 100% with FHS (10% vol/vol). Cytotoxicity was not evident if C8-deficient (C8D) plasma was substituted for FHS, but was restored in a dose-dependent manner by reconstitution with purified rat C8. Sublytic injury was demonstrated by wide separation between simultaneous release curves of cell-incorporated biscarboxyethyl carboxyfluorescein (BCECF; mol wt approximately equal to 520) and LDH at limiting doses of complement (at 2% FHS, BCECF release was 51.1 +/- 0.6% of maximum vs. 3.2 +/- 1.3% for LDH; N = 3) and by blebbing of the plasma membrane on electron microscopy. Thus, the pathogenic antibody of PHN produces complement-mediated sublytic as well as lytic cytotoxicity of GEC.

Animals↗
PubMed1987

A role for thromboxane in complement-mediated glomerular injury.

The membrane attack complex (MAC) of complement (C) has been shown to stimulate prostaglandin (PG) and thromboxane (Tx) synthesis in nucleated cells. Because glomerular epithelial cell injury and altered permeability in rat membranous nephropathy are mediated by the MAC, the authors examined whether MAC-induced proteinuria is linked to glomerular prostanoid synthesis. In kidneys containing non-nephritogenic, non-C-fixing gamma 2 sheep anti-Fx1A (planted antigen) that were perfused in vitro with C-fixing guinea pig anti-sheep IgG and a C source (fresh human plasma, 50% vol/vol in buffered bovine albumin), heavy proteinuria developed, reaching 4.27 +/- 1.20 mg/min/g at 100-120 minutes (n = 8). Cyclooxygenase blockade with 10(-4) M indomethacin (n = 6) inhibited urinary PGE2 excretion (569 +/- 47 to 124 +/- 18 pg/min/g, P less than 0.001) and lowered proteinuria (1.06 +/- 0.42 mg/min/g, P less than 0.001). Reduced protein excretion (0.88 +/- 0.12 mg/min/g, n = 6, P less than 0.001) also occurred with inhibition of Tx synthetase by OKY-046, 10(-4) M, a dose that was shown in separate perfusions to inhibit urinary TxB2 excretion by greater than 85%. Control kidneys, without planted antigen and perfused with anti-sheep IgG and plasma, excreted 0.30 +/- 0.05 mg protein/min/g (n = 6). Because inulin clearance was reduced by indomethacin, renal hemodynamic factors may have contributed to the reduction in proteinuria observed with this drug. However, insulin clearance was not significantly affected by OKY-046, implying that inhibition of Tx synthetase reduced proteinuria independently of changes in renal hemodynamics. Thus, proteinuria in rat membranous nephropathy is due to MAC-dependent glomerular epithelial injury and is mediated, in part, by Tx.

Animals↗