Phospholipase A2 mediates immediate early genes in cultured renal epithelial cells: possible role of lysophospholipid.

BACKGROUND: Exposure to high levels of oxalate induces oxidant stress in renal epithelial cells and produces diverse changes in cell function, ranging from cell death to cellular adaptation, as evidenced by increased DNA synthesis, cellular proliferation, and induction of genes associated with remodeling and repair. These studies focused on cellular adaptation to this oxidant stress, examining the manner by which oxalate exposure leads to increased expression of immediate early genes (IEGs). Specifically, our studies assessed the possibility that oxalate-induced changes in IEG expression are mediated by phospholipase A2 (PLA2), a common pathway in cellular stress responses. METHODS: Madin-Darby canine kidney (MDCK) cells were exposed to oxalate in the presence or absence of PLA2 inhibitors: mepacrine and arachidonyl trifluoromethyl ketone (AACOCF3). Expression of IEG (c-jun, egr-1, and c-myc) mRNA was assessed by Northern blot analysis. PLA2 activity was determined by measuring the release of [3H]arachidonic acid (AA) from prelabeled cells. RESULTS: Oxalate exposure (1 to 1.5 mmol/L) induced time- and concentration-dependent increases in IEG mRNA. Treatment with mepacrine resulted in a 75 to 113% reduction of oxalate-induced c-jun, egr-1, and c-myc mRNA, while AACOCF3 caused a 41 to 46% reduction of oxalate-induced c-jun and egr-1 mRNA. Of the two major byproducts of PLA2, only lysophosphatidylcholine (20 micromol/L) increased c-jun and egr-1 mRNA. In contrast, AA (25 micromol/L) attenuated the oxalate-induced increase in c-jun and egr-1 mRNA, presumably by inhibiting PLA2 activity. CONCLUSIONS: These findings suggest that PLA2 plays a major role in oxalate-induced IEG expression in renal epithelial cells and that lysophospholipids might be a possible lipid mediator in this pathway.

Oxalate-induced changes in the viability and growth of human renal epithelial cells.

Previous studies on the porcine renal epithelial LLC-PK1 cell line demonstrated that oxalate exposure produces concentration-dependent effects on renal cell growth and viability via process(es) involving free radicals. The present studies were conducted to determine whether these findings could be extended to a renal proximal tubular epithelial cell line derived from the human kidney. These studies examined oxalate-induced changes in membrane integrity after short-term exposure (4 h) and changes in cell survival after longer-term exposure (24 to 72 h). Oxalate-induced changes were also assessed in the expression of two genes: egr-1, a zinc-finger transcription factor, and osteopontin, a protein associated with tissue remodeling. The present studies also determined whether oxalate-induced changes in either cell viability or gene expression depended on free radicals. Oxalate at a concentration > or = 175 microM (free) produced membrane damage within 4 h. This effect was inhibited by Mn(III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP), a superoxide dismutase mimetic, but not by N-acetyl cysteine, a glutathione precursor, or by deferoxamine, an iron chelator. Acute oxalate-induced injury was followed by cell loss within 24 h, an effect maintained at 48 and 72 h at high concentrations of oxalate. Oxalate also promoted DNA synthesis. This mitogenic effect offset cell loss at lower oxalate concentrations (88 microM) leading to a small but significant increase in cell number at 72 h. Treatment with oxalate also increased expression of egr-1 mRNA within 1 h, a response that was attenuated by MnTMPyP; oxalate treatment for 8 h also increased abundance of osteopontin mRNA. These studies suggest that oxalate exposure produces changes in human renal cell growth and viability via a process(es) dependent on reactive oxygen intermediates. Such changes may play a role in the development and/or progression of renal disease via generation of reactive oxygen intermediates.

Role of phospholipase A2 in the cytotoxic effects of oxalate in cultured renal epithelial cells.

BACKGROUND: Oxalate, a common constituent of kidney stones, is cytotoxic for renal epithelial cells. Although the exact mechanism of oxalate-induced cell death remains unclear, studies in various cell types, including renal epithelial cells, have implicated phospholipase A2 (PLA2) as a prominent mediator of cellular injury. Thus, these studies examined the role of PLA2 in the cytotoxic effects of oxalate. METHODS: The release of [3H]-arachidonic acid (AA) or [3H]-oleic acid (OA) from prelabeled Madin-Darby canine kidney (MDCK) cells was measured as an index for PLA2 activity. The cell viability was assessed by the exclusion of ethidium homodimer-1. RESULTS: Oxalate exposure (175 to 550 microM free) increased the release of [3H]-AA in MDCK cells but had no effect on the release of [3H]-OA. Oxalate-induced [3H]-AA release was abolished by arachidonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of cytosolic PLA2 (cPLA2), but was not affected by selective inhibitors of secretory PLA2 and calcium-independent PLA2. The [3H]-AA release could be demonstrated within 15 minutes after exposure to oxalate, which is considerably earlier than the observed changes in cell viability. Furthermore, AACOCF3 significantly reduced oxalate toxicity in MDCK cells. CONCLUSIONS: Oxalate increases AA release from MDCK cells by a process involving cPLA2. In addition, based on the evidence obtained using a selective inhibitor of this isoform, it would appear that the activity of this enzyme is responsible, at least in part, for the cytotoxic effects of oxalate. The finding that oxalate can trigger a known lipid-signaling pathway may provide new insight into the initial events in the pathogenesis of nephrolithiasis.

Interaction of agonists and selective antagonists with gastric smooth muscle muscarinic receptors.

The interaction of cholinergic agonists and antagonists with smooth muscle muscarinic receptors has been investigated by measurement of displacement of the muscarinic antagonist [3H]QNB (quinuclidinyl benzilate) in membranes prepared from toad stomach. The binding of [3H]QNB was saturable, reversible and of high affinity (KD = 423 pM). The muscarinic receptor subtypes present in gastric smooth muscle were classified by determining the relative affinities for the selective antagonists pirenzepine (M1), AF-DX 116 (M2) and 4-DAMP (M3). The results from these studies indicate the presence of a heterogeneous population of muscarinic receptor subtypes, with a majority (88%) exhibiting characteristics of M3 receptors and a much smaller population (12%) exhibiting characteristics of M2 receptors. The binding curve for the displacement of [3H]QNB binding by the agonist oxotremorine was complex and was consistent with presence of two affinity states: 24% of the receptors had a high affinity (KD = 4.7 nM) for oxotremorine and 76% displayed nearly a 1,000-fold lower affinity (KD = 4.4 microM). When oxotremorine displacement of [3H]QNB binding was determined in the presence GTP gamma S, high affinity binding was abolished, indicating that high affinity agonist binding may represent receptors coupled to G proteins. Moreover, pertussis toxin pretreatment of membranes also abolished high affinity agonist binding, indicating that the muscarinic receptors are coupled to pertussis toxin-sensitive G proteins. Reaction of smooth muscle membranes with pertussis toxin in the presence [32P]NAD caused the [32P]-labelling of a 40 dD protein that may represent the alpha subunit(s) of G proteins that are known to by NAD-ribosylated by the toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the anti-calmodulin drugs calmidazolium and trifluoperazine on 45Ca transport in plasmalemmal vesicles from gastric smooth muscle.

The anti-calmodulin drugs calmidazolium (CMZ) and trifluoperazine (TFP) were shown to have a number of effects on 45Ca transport by plasmalemmal vesicles from gastric smooth muscle. Although these compounds produced the expected dose-dependent inhibition of the plasmalemmal ATP-dependent Ca2+ transport system, they also evoked a Ca2+ release comparable to that observed in the presence of the Ca2+ ionophore, ionomycin. This increased transmembrane Ca2+ flux was so large that it accounted for much of the apparent decrease in 45Ca uptake produced by these agents. Thus, direct effects of CMZ and TFP on ATP-dependent 45Ca uptake could only be reliably assessed for brief (less than or equal to 30 seconds) drug exposures. The explanation for the observed effects of CMZ and TFP on membrane Ca2+ permeability is unclear. The increased transmembrane Ca2+ flux may reflect nonspecific effects on membrane permeability or it may reflect a specific interaction of the anticalmodulin drugs with a Ca2+ release channel or with the Ca2+ transport ATPase. In any case, these results suggest the need for caution in the design and interpretation of studies using both CMZ and TFP as anticalmodulin agents.

Beta-adrenergic stimulation of 42K influx in isolated smooth muscle cells.

The present studies were designed to examine the effect of the beta-adrenergic agent isoproterenol (ISO) on 42K influx into isolated smooth muscle cells. Unidirectional 42K influx studies were carried out as described previously using suspensions of smooth muscle cells derived from the toad stomach. Results from these studies indicated that ISO (10(-4) M) produces an approximate threefold increase in 42K influx within 1 min after drug addition. This response is transient such that the flux rate returns to near basal levels within 5 min after drug addition. The stimulatory effect of ISO on 42K influx is blocked by pretreatment with ouabain (1-7.5 X 10(-4) M). ISO thus appears to increase 42K influx by stimulating the Na+-K+ pump in the smooth muscle cells. Other findings indicate that the stimulatory effect of ISO on Na+-K+ pumping is dose dependent, blocked by the beta-antagonist pindolol, and mimicked by dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP). These findings support our hypothesis that ISO elicits a beta-adrenergic, cAMP-dependent activation of the Na+-K+ pump in isolated smooth muscle cells.

Beta-adrenergic effects on transmembrane 45Ca fluxes in isolated smooth muscle cells.

The effect of the beta-adrenergic agent isoproterenol (ISO) on transmembrane fluxes of 45Ca was assessed in smooth muscle cells in suspension. Exposure of the cells to this smooth muscle relaxant provoked a large (13-fold) transient increase in 45Ca efflux from the isolated cells. This effect was blocked by the beta-adrenergic antagonist pindolol and mimicked by exposure of the cells to dibutyryl adenosine 3',5'-cyclic monophosphate. ISO had no significant effect on 45Ca binding or on 45Ca influx in those cells, thus the ISO-induced increase in 45Ca efflux appears to reflect increased Ca2+ extrusion from the smooth muscle cells. Because the ISO-induced increase in 45Ca efflux was abolished by pretreatment of the cells with the Na+-K+ pump inhibitor ouabain and by lowering extracellular Na+ levels, we propose that the increased Ca2+ extrusion in response to ISO is coupled to increased activity of the plasmalemmal Na+-K+ pump via increased Na+-Ca2+ exchange.

Phosphatidic acid and phosphatidylinositol labelling in adipose tissue. Relationship to the metabolic effects of insulin and insulin-like agents.

Exposure to phospholipase C increased the incorporation of [32P]Pi into phosphatidate, CMP-phosphatidate and phosphatidylinositol in rat adipose tissue and isolated adipocytes. A similar effect was observed in response to insulin and oxytocin. Theophylline, 3-isobutyl-1-methylxanthine and adenosine deaminase decreased [32P]Pi incorporation, and adenosine and N6-phenylisopropyladenosine reversed these effects. As with insulin, exposure of adipose tissue to phospholipase C stimulated oxidation of glucose, pyruvate and leucine and activated pyruvate dehydrogenase. Oxytocin and adenosine also mimicked the effects of insulin on leucine oxidation and pyruvate dehydrogenase. However, only insulin stimulated glycogen synthase activity, indicating that the regulation of synthase may be achieved by intracellular events distinct from those regulating changes in phospholipid metabolism, sugar transport and mitochondrial enzyme activities. It is postulated that exposure to phospholipase C forms diacylglycerol, which is phosphorylated to yield phosphatidate. The increased labelling of CMP-phosphatidate and phosphatidylinositol results from the conversion of phosphatidate into these lipids. The correlation between the effects of phospholipase C on phosphatidate synthesis and changes in adipose-tissue metabolism suggests the possibility that increased phosphatidate may directly or indirectly produce changes in membrane transport and enzyme activities. The pattern of phospholipid labelling produced by insulin, adenosine and oxytocin suggests that these stimuli may also increase phosphatidate synthesis, and, if so, changes in phospholipid metabolism could account for some of the metabolic actions of these stimuli.

Mechanism of beta-adrenergic relaxation of smooth muscle.

The mechanism of beta-adrenergic relaxation was investigated in isolated smooth muscle cells. Beta-adrenergic agents stimulate cyclic AMP-dependent phosphorylation, enhance Na+/K+ transport and induce relaxation. The stimulation of Na+/K+ transport is obligatory for relaxation, and we suggest that this stimulation induces relaxation through enhanced Na+/Ca2+ exchange.