The effects of cardiac myocytes on interstitial fibroblasts in toxic iron overload.

Iron deposits preferentially in myocytes in mixed cultures of cardiac myocytes and nonmyocytic fibroblasts. In vivo, iron overload is associated with cardiac fibrosis. Therefore, we examined whether iron loading of cardiac myocytes in culture could trigger a response in nonmyocytes characteristic of a fibrogenic phenotype. We found that the nonmyocytes adopted a myofibroblast phenotype in culture. The rate of DNA synthesis (measured by [3H]thymidine incorporation) by the nonmyocytes was decreased by the myocyte-conditioned medium, compared to that of the unconditioned medium, and this activity was retained in <10-kDa fractions. The rate was partially restored when the medium was obtained from iron-loaded myocytes, and in this medium, the <10-kDa fraction was even more effective in reversing the suppression of proliferation. This suppression suggests a decreased secretion of a growth inhibitory substance in the iron-loaded myocytes, and this effect was partially reversed when the iron-loaded cells were treated with the iron chelator, deferoxamine. This indicates that cardiac myocytes may play a paracrine role in suppressing the proliferation of myofibroblasts that is partially overcome when the myocytes are iron overloaded. The myocyte-conditioned medium also affects the myofibroblast phenotype, increasing the cells' fibronectin mRNA content and decreasing alpha-smooth-muscle actin mRNA. The myocyte-conditioned medium increases transforming growth factor-beta (TGF-beta) secretion by myofibroblasts, but the TGF-beta content of the conditioned medium was found to play, at most, a minor role in determining the response of the myofibroblast.

Stress-activated protein kinase-dependent induction of c-fos by Cd(2+) is mediated by MKK7.

Exposure of mesangial cells to ionic Cd(2+) induces the proto-oncogene c-fos, while activating both Erk and stress-activated protein kinase (SAPK) MAP kinase pathways. While we have previously used a pharmacological inhibitor of Erk activation to implicate involvement of this pathway in the induction of c-fos by Cd(2+), the consequences of SAPK activation remained unknown. Here we use dominant negative inhibitors of the SAPK kinases, SEK1 and MKK7, to show that Cd(2+) activates SAPK through MKK7, but that partial inhibition of SAPK alone is insufficient to significantly affect the magnitude of the Cd(2+)-dependent increase in c-fos mRNA. However, inhibition of Erk and SAPK pathways together abrogates the increase, suggesting that these pathways act in concert in the induction of c-fos by this toxic metal.

Heterogeneity in the response of vascular smooth muscle to heparin: altered signaling in heparin-resistant cells.

OBJECTIVE: Vascular smooth muscle cells show phenotypic heterogeneity in vivo that affects the extent to which they respond to the antimitogenic effects of heparin. In vitro, heparin-resistant cells are readily selected. This study was undertaken to determine whether differences in the antiproliferative response to heparin involve differences in activity of heparin-sensitive signal transduction pathways. METHODS: Rat thoracic aorta smooth muscle cells (ASMC) at early passage together with two established vascular smooth muscle lines, PAC-1 and A10, were examined before and after selection for growth in the presence of heparin (10 micrograms/ml). Cells were rendered quiescent and then stimulated with serum. RESULTS: The three cell types showed different sensitivities to the antimitogenic effects of heparin. With respect to [3H]thymidine incorporation, A10 cells were insensitive to 1 microgram/ml heparin whereas PAC-1 cells responded down to 0.05 microgram/ml and ASMC were of intermediate sensitivity. ASMC and PAC-1 cells but not A10 showed a decrease in c-fos mRNA in response to 1 microgram/ml heparin, and a decrease in the c-Fos content of AP-1 DNA binding activity. None of the cells had decreased c-jun mRNA in the presence of heparin. Although induction of c-fos by serum is thought to signal through the Erk mitogen activated protein kinase family, Erk activity was decreased more by 1 microgram/ml heparin in A10 cells than in PAC-1 or ASMC. When cells were selected by growth in the presence of 10 micrograms/ml heparin, A10 cells were unaffected but PAC-1 and ASMC showed a blunted effect of heparin on serum stimulation. In contrast to A10 and their controls not exposed to continuous heparin, heparin-selected PAC-1 and ASMC showed a diminished ability to induce c-fos in response to serum. CONCLUSIONS: Smooth muscle cell lines show different responses to the antimitogenic effects of heparin that correlate with the heparin sensitivity of c-Fos/c-Jun expression. Although Erk is implicated in c-fos induction, cells comparatively resistant to heparin still show heparin-dependent inhibition of Erk activation, suggesting that other pathways may be more important for heparin resistance. Furthermore, cells selected for heparin resistance may develop c-fos-independent pathways for proliferation.

Changes in gene expression with iron loading and chelation in cardiac myocytes and non-myocytic fibroblasts.

Iron overload is associated with long-term cardiac iron accumulation and tissue changes such as fibrosis. To determine short-term iron-dependent changes in expression of genes associated with iron homeostasis and fibrosis we measured mRNA on Northern blots prepared from cultured rat neonatal cardiomyocytes and non-myocytes (fibroblasts) as a function of iron loading and chelation. Transferrin receptor mRNA was reduced in myocytes exposed to various concentrations of iron for 3 days and this decline was associated with a 63% decline in iron-response element (IRE) binding of iron regulatory protein-1, indicating that myocytes utilize IRE-dependent mechanisms to modulate gene expression. In myocytes iron caused a dose-dependent decline in mRNAs coding for transforming growth factor- beta(1)(TGF- beta(1)), biglycan, and collagen type I while plasminogen activator inhibitor-1 mRNA was unaffected by iron loading and decorin mRNA doubled. Total TGF- beta bioactivity was also decreased by iron loading. Thus, the effects of iron loading on genes related to cardiac fibrosis are gene-specific. Addition of deferoxamine for 1 day did not have any significant effect on any of these genes. Parallel changes in gene expression were exhibited by non-myocytes (fibroblasts), where chelation also decreased TGF- beta(1)mRNA and activity, and mRNA for collagen type I and biglycan, and collagen synthesis. In addition to these changes in transcripts associated with matrix formation the mRNA of the metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase was unaffected by iron loading but doubled in both cell types upon treatment with deferoxamine. These findings suggest that in both cardiac myocytes and non-myocyte fibroblasts gene expression is coupled to intracellular iron pools by gene-specific and IRE-dependent and idependent mechanisms. This linkage may influence matrix deposition, a significant component of cardiac injury.

Activation of parallel mitogen-activated protein kinase cascades and induction of c-fos by cadmium.

Cadmium is a toxic divalent cation that can initiate either mitogenic signals or apoptosis, possibly as a consequence of inducing different patterns of oncogene expression in different cells. We previously showed that Cd(2+) caused transcriptional activation of the c-fos protooncogene in mesangial cells (Wang and Templeton, J. Biol. Chem. 273, 73-79, 1998). The present study was undertaken to identify the signaling pathways that might be involved. Exposure to 10 microM CdCl(2) for 8 h caused a prolonged activation of Erk kinase and accumulation of c-fos mRNA. Inhibition of Erk activation with PD98059 only partially inhibited c-fos induction, indicating that additional pathways are involved. The c-Jun kinase/stress-activated protein kinase (SAPK) was also activated by Cd(2+). All three signals, i.e., Erk activity, SAPK activity, and c-fos mRNA levels in response to Cd(2+) showed a similar biphasic time course with an initial increase at 15-30 min and then a larger and more prolonged increase several hours later. Each signal also showed a similar concentration dependence, with less than 1 microM Cd(2+) causing the initial increase but values above 3 microM being required for the prolonged phase. These events showed high specificity for Cd(2+); other divalent metals tested under the same conditions (Mg(2+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), and Hg(2+)) were without significant effects. We conclude that Cd(2+) is a specific inducer of c-fos in mesangial cells, probably through activation of both Erk kinase and SAPK pathways. The similar time and concentration dependence of the response of both pathways to Cd(2+) suggests a common basis for activation.

Heparan sulfate chains with antimitogenic properties arise from mesangial cell-surface proteoglycans.

Heparan sulfate (HS) chains accumulate in both the medium and the cell layer of mesangial cell cultures. When given in fresh medium to quiescent cultures at naturally occurring concentrations, they suppress entry into the cell cycle and progression to DNA synthesis. We have attempted to identify the proteoglycan (PG) source of the antimitogenic HS chains from mesangial cell layers (HS(c)) and medium (HS(c)). When cells were labeled for 16 hours with [35S]sulfate, 25% of the label was found in intracellular HS chains and 5% in extracellular HSPGs. Cell-surface HSPGs accounted for the remaining 70% of the label associated with cell-layer HS and were released by either trypsin or 2% Triton X-100. About 20% of this cell-surface fraction was released by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), and probably represents glypican-like PG; glypican mRNA was present in the cells. The remainder of this fraction could be incorporated into liposomes, indicating the presence of hydrophobic transmembrane regions suggestive of syndecans. Upon purification and deglycosylation, an antiserum to rat liver HSPGs that reacts primarily with syndecan-2 showed a strong signal corresponding to this protein and three weaker bands that may represent additional syndecans. mRNAs for syndecan-1, -2, and -4 were present in the cultures. Syndecan-1 and -2 mRNAs were increased 30 minutes after stimulation of quiescent rat mesangial cells (RMCs) with serum. Heparin, HS(c), and HS(m) all prevented this increase. Syndecan-4 mRNA was not affected by serum, heparin, or HS. In pulse-chase experiments, the amount of 35S appearing in the cellular protein-free HS fraction was accounted for almost entirely by cell-surface PGs, as matrix-associated label was a minor contribution at the end of the pulse-labeling. The appearance of [35S]HS in cell extracts was unaffected by phospholipase C treatment, indicating that turnover of the newly labeled syndecan fraction is the source of the antimitogenic HS chains.

Absorption and retention of nickel from drinking water in relation to food intake and nickel sensitivity.

Two studies were performed to examine the influence of fasting and food intake on the absorption and retention of nickel added to drinking water and to determine if nickel sensitization played any role in this regard. First, eight nonallergic male volunteers fasted overnight before being given nickel in drinking water (12 micrograms Ni/kg) and, at different time intervals, standardized 1400-kJ portions of scrambled eggs. When nickel was ingested in water 30 min or 1 h prior to the meal, peak nickel concentrations in serum occurred 1 h after the water intake, and the peak was 13-fold higher than the one seen 1 h after simultaneous intake of nickel-containing water and scrambled eggs. In the latter case, a smaller, delayed peak occurred 3 h after the meal. Median urinary nickel excretion half-times varied between 19.9 and 26.7 h. Within 3 days, the amount of nickel excreted corresponded to 2.5% of the nickel ingested when it was mixed into the scrambled eggs. Increasing amounts were excreted as the interval between the water and the meal increased, with 25.8% of the administered dose being excreted when the eggs were served 4 h prior to the nickel-containing drinking water. In the second experiment, a stable nickel isotope, 61Ni, was given in drinking water to 20 nickel-sensitized women and 20 age-matched controls, both groups having vesicular hand eczema of the pompholyx type. Nine of 20 nickel allergic eczema patients experienced aggravation of hand eczema after nickel administration, and three also developed a maculopapular exanthema. No exacerbation was seen in the control group. The course of nickel absorption and excretion in the allergic groups did not differ and was similar to the pattern seen in the first study, although the absorption in the women was less. A sex-related difference in gastric emptying rates may play a role. Thus, food intake and gastric emptying are of substantial significance for the bioavailability of nickel from aqueous solutions.

Inactivation of kinase cascades in mesangial cells grown on collagen type I.

Growth on collagen type I gels is known to suppress the mitogenic responsiveness of mesangial cells. Because these cells proliferate in some renal diseases and themselves synthesize collagen type I, we examined the influence of growth on collagen upon several kinase signaling cascades involved in mesangial cell proliferation. Quiescent mesangial cells grown on collagen type I and then stimulated with serum showed a markedly diminished induction of the protooncogene c-fos, compared with their counterparts on plastic or fibronectin. This effect was accompanied by decreased activation of mitogen-activated (Erk family) and Ca2+/calmodulin-dependent protein kinases. Cells on collagen showed lower basal protein kinase C (PKC) activity and diminished levels of PKC-alpha and -zeta isoforms. Global phosphorylation of tyrosine residues was diminished on collagen, and tyrosine phosphorylation of Erk and focal adhesion kinase in response to serum was not detected, in contrast to cells on plastic. We conclude that attachment of mesangial cells to collagen type I results in a broad suppression of protein phosphorylation that is reflected in diminished induction of the c-fos gene and probably underlies the conversion of cultured mesangial cells to a nonproliferative phenotype.

Long-term safety and effectiveness of iron-chelation therapy with deferiprone for thalassemia major.

BACKGROUND: Deferiprone is an orally active iron-chelating agent that is being evaluated as a treatment for iron overload in thalassemia major. Studies in an animal model showed that prolonged treatment is associated with a decline in the effectiveness of deferiprone and exacerbation of hepatic fibrosis. METHODS: Hepatic iron stores were determined yearly by chemical analysis of liver-biopsy specimens, magnetic susceptometry, or both. Three hepatopathologists who were unaware of the patients' clinical status, the time at which the specimens were obtained, and the iron content of the specimens examined 72 biopsy specimens from 19 patients treated with deferiprone for more than one year. For comparison, 48 liver-biopsy specimens obtained from 20 patients treated with parenteral deferoxamine for more than one year were similarly reviewed. RESULTS: Of the 19 patients treated with deferiprone, 18 had received the drug continuously for a mean (+/-SE) of 4.6+/-0.3 years. At the final analysis, 7 of the 18 had hepatic iron concentrations of at least 80 micromol per gram of liver, wet weight (the value above which there is an increased risk of cardiac disease and early death in patients with thalassemia major). Of 19 patients in whom multiple biopsies were performed over a period of more than one year, 14 could be evaluated for progression of hepatic fibrosis; of the 20 deferoxamine-treated patients, 12 could be evaluated for progression. Five deferiprone-treated patients had progression of fibrosis, as compared with none of those given deferoxamine (P=0.04). By the life-table method, we estimated that the median time to progression of fibrosis was 3.2 years in deferiprone-treated patients. After adjustment for the initial hepatic iron concentration, the estimated odds of progression of fibrosis increased by a factor of 5.8 (95 percent confidence interval, 1.1 to 29.6) with each additional year of deferiprone treatment. CONCLUSIONS: Deferiprone does not adequately control body iron burden in patients with thalassemia and may worsen hepatic fibrosis.

Cadmium and calcium-dependent c-fos expression in mesangial cells.

Cadmium is a carcinogenic metal known to increase the expression of several protooncogenes in a variety of cells. although the underlying mechanisms are unknown. Renal mesangial cells are smooth muscle cells in which Ca2+ signaling pathways regulate the induction of c-fos through both cAMP-dependent and mitogen-activated protein kinase- (MAPK-) dependent pathways. We report that c-fos is induced in these cells by both protein kinase C- (PKC-) dependent (phorbol ester, platelet-derived growth factor), and independent (serum, ionomycin) mechanisms. In all cases, prevention of an increase in cytosolic [Ca2+] with the chelator BAPTA prevented this induction. CdCl2 (10 microM) caused an accumulation of c-fos mRNA over 30 min that was sustained for at least 8 h. Cycloheximide inhibits turnover of c-fos mRNA and shows a synergistic effect with Cd2+ on transcript levels. Together with a similar half life of the transcript whether accumulated in response Cd2+ or induced by phorbol ester, this suggests induction of c-fos by Cd2+ rather than an effect of Cd2+ on transcript stability. Cadmium increased MAPK activity by 5 min; this was sustained for at least 8 h, consistent with the time course of c-fos mRNA accumulation. The MAPK kinase inhibitor PD98059 caused a marked decrease in the induction of c-fos by Cd2+, but did not eliminate the phenomenon completely. Although Cd2+ has been reported to activate PKC in vitro, no effect was found on PKC activity in Cd2+ -treated cells, indicating the activation of MAPK by Cd2+ is through an unidentified PKC-independent pathway. We conclude that Cd2+ can cause a sustained induction of c-fos in part through sustained activation of MAPK, that contrasts with the transient activation of these species in response to physiological mitogenic stimuli.

Heparin inhibits Ca2+/calmodulin-dependent kinase II activation and c-fos induction in mesangial cells.

Like vascular smooth-muscle cells, rat mesangial cells (RMCs) display an anti-mitogenic response to heparin. In particular, heparin partially suppresses the ability of quiescent RMCs to enter the cell cycle and induce c-fos expression. When the mitogenic stimulus is serum, phorbol ester or platelet-derived growth factor, this response appears to result from the ability of heparin to suppress activation of the extracellular-signal-regulated kinase family of mitogen-activated protein kinases. However, we have also shown that heparin suppresses c-fos expression in response to ionophores such as ionomycin, an event independent of mitogen-activated protein kinase [Miralem, Wang, Whiteside and Templeton (1996) J. Biol. Chem. 271, 17100-17106]. Here we identify this second heparin-sensitive pathway as involving Ca2+/calmodulin-dependent kinase (CaMK) II. Ionomycin (100 nM) caused a transient rise in intracellular Ca2+ concentration ([Ca2+]i) in quiescent RMCs to 386+/-55 nM, with an increase in CaMK II activity that peaked 30 s later. The accumulation of c-fos mRNA that ensued 30 min later was prevented when the increase in [Ca2+]i was prevented with the intracellular Ca2+ chelator, 1,2-bis-(2-aminophenyoxy)ethane-N,N,N',N'-tetra-acetic acid. The broad-specificity CaMK inhibitor, KT 5926, inhibited ionomycin-dependent c-fos induction at a concentration at which it was without effect on induction by serum or phorbol ester. The CaMK II-specific inhibitor, KN-93, likewise inhibited c-fos induction by ionomycin, but not by serum or phorbol ester. ML-7, an inhibitor of the CaMK-related myosin light-chain kinase (MLCK), was without effect. Heparin (1 microg/ml) suppressed ionomycin-dependent c-fos induction. It was without effect on [Ca2+]i, but inhibited the development of autonomous CaMK II activity. However, when heparin was added to the CaMK II assay solution in vitro, it was without effect on autonomous activity. Furthermore, heparin did not prevent full activation of CaMK II by the Ca2+-calmodulin complex in vitro. Heparin did not affect myosin light-chain phosphorylation or RMC contraction, processes mediated by MLCK. We conclude that ionomycin induces c-fos in RMCs through the CaMK II pathway, and that heparin prevents CaMK II activation by an indirect process mediated by other cell components. Heparin does not affect activation of the closely related CaMK, MLCK.

Induction of c-fos proto-oncogene in mesangial cells by cadmium.

Cadmium is mitogenic under some circumstances and has been shown to cause accumulation of transcripts for several proto-oncogenes in a variety of cells, but the mechanism(s) remain to be delineated. Here we show that CdCl2 causes an increase in c-fos mRNA within 30 min of exposure of mesangial cells. At 10 microM Cd2+, this increase persists for at least 8 h in both rat and human cells. The half-life of c-fos mRNA is the same whether it accumulates following 4 h of treatment with Cd2+ or is induced transiently by phorbol ester. Cycloheximide, which stabilizes the transcript, causes a synergistic increase when administered with CdCl2. Nuclear run-on analysis confirms that Cd2+ causes transcriptional activation of the c-fos gene. Calmodulin and Ca2+/calmodulin-dependent kinase, and classical protein kinase C (PKC) isoforms represent two Ca2+-dependent signaling pathways that can lead to induction of c-fos, and Cd2+ has been shown to activate both calmodulin and PKC in vitro, possibly by virtue of the similar ionic radii of Cd2+ and Ca2+. Therefore, we investigated the effect of Cd2+ on these pathways in vivo. 10 microM CdCl2 did not increase total PKC activity or Ca2+/calmodulin-dependent kinase II activity and inhibited the latter at higher concentrations, ruling out either pathway in the Cd2+-dependent induction of c-fos. However, Cd2+ did lead to a sustained activation of the Erk family mitogen-activated protein kinases (MAPK) that correlated with induction of c-fos. A specific inhibitor of the MAPK kinases, PD98059, partially inhibited the induction of c-fos by Cd2+. We conclude that Cd2+ induces c-fos at least in part by causing a sustained activation of MAPK independent of its ability to activate PKC and calmodulin in vitro.

Chelation of intracellular calcium prevents mesangial cell proliferative responsiveness.

Mesangial cell transformation into a proliferative phenotype, observed in many glomerular diseases, occurs in response to growth factors and cytokines. This study tests the hypothesis that intracellular calcium is necessary for stimulation of mesangial cell proliferative responsiveness to a variety of growth factors. Furthermore, these experiments tested whether nonspecific calcium entry via a calcium ionophore was sufficient to elicit the same response. Rat primary mesangial cells (passages 5 to 10) were growth-arrested for 48 h in 0.5% fetal bovine serum (FBS), then stimulated with 0.1 microM endothelin-1, 1.9 microM platelet-derived growth factor (PDGF)-BB, 0.5% FBS, or 0.1 microM ionomycin, with or without the intracellular calcium chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA). Calcium signaling was measured in Fura-2-loaded cells on coverslips by dual-wavelength spectrofluorometry and in Fluo-3-loaded cells by confocal fluorescence laser microscopy. [3H]-Thymidine incorporation was measured after 12 to 24 h of stimulation with each test agent. Expression of c-fos mRNA was analyzed by Northern blot. All test agents stimulated a significant increase in cytosolic and nuclear calcium, which were both effectively inhibited with BAPTA. All agents stimulated a significant increase in [3H]-thymidine incorporation and enhanced c-fos mRNA expression (no detectable c-fos mRNA was observed in quiescent cells). BAPTA prevented the enhanced [3H]-thymidine incorporation stimulated by endothelin-1 and PDGF, and partial inhibition of FBS-stimulated incorporation with BAPTA was observed. BAPTA inhibited c-fos expression observed in response to these agents. Phorbol ester induction of c-fos mRNA in the absence of raised cytosolic or nuclear calcium was also suppressed by BAPTA. Cell viability as measured by thiazolyl blue and trypan blue was not altered by BAPTA. It is concluded that normal regulation of intracellular calcium is necessary for mesangial cell proliferative responsiveness.

Characterization of Fe2+ and Fe3+ transport by iron-loaded cardiac myocytes.

Plasma iron overload causes cardiac iron accumulation leading to toxicity and organ failure. In order to understand the basis of iron acquisition, we examined mechanisms of Fe3+ and Fe2+ uptake in control and iron-loaded cardiomyocyte cultures. Iron loading increased rates of Fe3+ and Fe2+ uptake, primarily by increasing Vmax. Inhibition of Fe3+ transport by impermeable Fe2+ chelators and the presence of a cell surface ferricyanide reductase activity are consistent with a role for redox cycling in Fe3+ uptake. However, flavoproteins and copper-dependent oxidases known to be required for redox-active iron transport in yeast do not appear to be involved in iron uptake by cardiac myocytes, nor do the abundant cardiac L-type Ca2+ channels. The data suggest that both redox states of iron contribute to cardiac iron accumulation in iron overload.

Posttranscriptional effects of glucose on proteoglycan expression in mesangial cells.

Hyperglycemic conditions are known to increase mRNA and protein levels of several extracellular matrix molecules in cultured mesangial cells, but accompanying increases in proteoglycan mRNA have not been found, and there are discrepant reports of normal or decreased proteoglycan synthesis with or without undersulfation in diabetic kidneys and hyperglycemic cultures. We examined the effects in proliferating cells of glucose on [35S]Sulfate incorporation into heparan and dermatan sulfates and on mRNA levels of decorin, biglycan, and basement membrane perlecan. In both mesangial cells and vascular smooth muscle cells, 30 mmol/L glucose caused a decrease of 15% to 25% in the amount of sulfate incorporated into each proteoglycan in cultures confluent for 1 to 4 days, compared with 10 mmol/L glucose. The effect showed no specificity for the class of proteoglycan and was not a consequence of changes in total protein synthesis, which increased, or cell proliferation, which was unaffected. No decrease in charge density of any of the proteoglycan fractions was observed by ion-exchange chromatography. Therefore, the decrease in labeling was due to a decrease in synthesis and not undersulfation. mRNA levels for biglycan and perlecan increased slightly and transiently, and these changes cannot account for the decreased synthesis. Decorin mRNA was detected only in smooth muscle cells, where it and biglycan were differentially affected by glucose, apparently at the transcriptional level; stabilities of the two messages were unaffected by glucose. Although transforming growth factor-beta 1 (TGF-beta 1) mRNA levels increased in response to glucose, the cytokine did not appear to regulate proteoglycan synthesis, because structural changes in proteoglycans elicited by addition of TGF-beta 1 to the culture medium did not occur in the hyperglycemic cultures. On the other hand, inhibition and downregulation of protein kinase C (PKC), while decreasing net sulfate incorporation into mesangial cell proteoglycans, prevented the effect of high glucose. We conclude that a high glucose concentration causes a general decrease in the synthesis of all classes of proteoglycans at a posttranscriptional level, and can do so without affecting the charge density of individual proteoglycan molecules.

Cellular factors mediate cadmium-dependent actin depolymerization.

Cadmium exposure produces depolymerization of the actin cytoskeleton in several cultured cell lines, and we have previously shown that in renal mesangial cells this effect is caused by picomolar concentrations of cytosolic Cd2+. The present study was undertaken to explore further the basis of this action of Cd2+. Skeletal muscle actin was labeled with pyrene and rates of polymerization and depolymerization were followed in vitro by changes in fluorescence. Concentrations of 100 microM or higher Cd2+ increased the rate of polymerization while lower concentrations stabilized actin filaments. Because this is in contrast to the observation that Cd2+ favors depolymerization in cultured mesangial cells, we examined the role of cellular factors in modifying the actions of Cd2+ on the polymerization/depolymerization equilibrium. Inclusion in the in vitro polymerization assay of cytosol from mesangial cells pre-treated with 5 microM CdCl2 caused a decrease in the rate of polymerization and an increase in the rate of depolymerization, relative to cytosol from control cells. EGTA normalized these rates, indicating involvement of Ca2+-dependent factors. Likewise, cytoskeleton from Cd2+-treated cells caused a Ca2+-dependent decrease in actin polymerization. Ligand blotting with [125I]actin revealed loss of binding of a 93-kDa protein from the cytosol and an increase in binding of a 49-kDa protein in the cytoskeletal fraction upon treatment of mesangial cells with Cd2+. Based on their electrophoretic mobility and Ca2+ dependence of actin binding these are tentatively identified as members of the gelsolin and severin families, respectively. Thus, Cd2+ may exert its effect on the mesangial cell cytoskeleton by altering the levels or actin-binding activity of actin-associated proteins.

Heparin inhibits mitogen-activated protein kinase-dependent and -independent c-fos induction in mesangial cells.

Heparin suppresses mitogenic responses in renal mesangial cells, and when quiescent mesangial cells are stimulated with serum, heparin blocks the induction of c-fos seen at 15 min. Because heparin is taken up by cells over a much longer time course, we addressed mechanisms whereby extracellular heparin might suppress c-fos induction at such early times. Quiescent cells were treated with serum, 12-O-tetradecanoylphorbol-13-acetate, or low concentrations of Ca2+ ionophores that produced increases in intracellular Ca2+ concentration ([Ca2+]i) in the physiological range. Each treatment caused an increase in c-fos mRNA, but they did so by different mechanisms. Serum activated mitogen-activated protein kinase (MAPK) and increased [Ca2+]i without affecting protein kinase C. Activation of protein kinase C with phorbol ester activated MAPK without much effect on [Ca2+]i. Ionophores increased [Ca2+]i without affecting basal levels of protein kinase C or MAPK. Heparin (1 microg/ml) suppressed the induction of c-fos initiated by all three treatments. It did not affect the activity of protein kinase C, but inhibited activation of MAPK by either serum or phorbol ester, suggesting a common site of action at or below the probable convergence of the induced signals at Ras/Raf-1 activation. Heparin also inhibited the serum-stimulated entry of extracellular Ca2+ to the same extent as verapamil, consistent with the ability of verapamil to block L-type Ca2+ channels and the known presence of these channels in mesangial cells. However, this effect does not appear to be related to heparin's ability to inhibit induction of c-fos. First, verapamil had no effect on induction of c-fos by serum. Second, heparin had no effect on changes in [Ca2+]i achieved by ionophores. We conclude that heparin suppresses induction of c-fos in mesangial cells by blocking at least two different points in signal transduction cascades, one upstream of MAPK and the other independent of MAPK, but dependent on intracellular Ca2+.

Sample collection guidelines for trace elements in blood and urine. IUPAC Commission of Toxicology.

This paper presents an organized system for element-specific sample collection and handling of human blood (whole blood, serum or plasma, packed cells or erythrocytes) and urine also indicating a proper definition of the subject and sample. Harmonized procedures for collection, preparation, analysis and quality control are suggested. The aim is to assist scientists worldwide to produce comparable data which will be useful on a regional, national and international scale. The guidelines are directed to the elements aluminium, arsenic, cadmium, chromium, cobalt, copper, lead, lithium, manganese, mercury, nickel, selenium and zinc. These include the most important elements measured for their occupational or clinical significance, and serve as examples of principles that will guide development of methods for other elements in the future.

Collagen type I enhances endothelin-mediated contraction and induces nonproliferating phenotype in mesangial cells.

Accumulation of glomerular extracellular matrix is a characteristic accompaniment of mesangial cell proliferation in progressive renal disease. We examined how growth on several matrices affected the proliferative phenotype of cultured rat mesangial cells. Compared with growth on plastic, Matrigel, or mesangial matrix, collagen type I caused a decreased cell number at 72 h, decreased total DNA per culture, and a decrease in the incorporation of [3H]thymidine during S phase in cells released from quiescence. These antiproliferative and antimitogenic effects of collagen type I required growth on a collagen gel; soluble collagen or collagen fragments were without effect. Because a number of agents elicit both proliferative and contractile responses in mesangial cells, we examined the effect of growth on collagen on contractility. Compared with plastic, cells grown on collagen type I were more contractile, showed a higher Ca2+ signal in response to endothelin, and responded to endothelin with a more rapid myosin light-chain kinase-dependent phosphorylation of myosin light chain. We conclude that growth on a collagen type I gel uncouples contractility from a proliferative response in mesangial cells, suppressing proliferation while enhancing contraction and Ca2+ signaling in response to endothelin.