Thomas W. Honeyman

Annexin II is the membrane receptor that mediates the rapid actions of 1α,25-dihydroxyvitamin D3†

1α,25‐Dihydroxyvitamin D3 has been shown to exert its effects by both genomic (minutes to hours) and rapid (seconds to minutes) mechanisms. The genomic effects are mediated by interaction with the nuclear vitamin D receptor. We show that the vitamin D analog, [14C]‐1α,25‐dihydroxyvitamin D3 bromoacetate, is specifically bound to a protein (molecular weight 36 kDa) in the plasma membrane of rat osteoblastlike cells (ROS 24/1). The plasma membrane protein labeled with the bromoacetate analog was identified as annexin II by sequence determination and Western blot. Partially purified plasma membrane proteins (PI 6.9–7.4) and purified annexin II exhibited specific and saturable binding for [3H]‐1α,25‐dihydroxyvitamin D3. Antibodies to annexin II inhibited [14C]‐1α,25‐dihydroxyvitamin D3 bromoacetate binding to ROS 24/1 plasma membranes, immunoprecipitated the ligand–protein complex, and inhibited 1α,25‐dihydroxyvitamin D3–induced increases in intracellular calcium in ROS 24/1 cells. The results indicate that annexin II may serve as a receptor for rapid actions of 1α,25‐dihydroxyvitamin D3. J. Cell. Biochem. 78:34–46, 2000.

Oxalate toxicity in LLC-PK1 cells, a line of renal epithelial cells

PURPOSE The present studies assessed the possibility that high concentrations of oxalate may be toxic to renal epithelial cells. MATERIALS AND METHODS Subconfluent cultures of LLC-PK1 cells were exposed to oxalate, and the effects on cell morphology, membrane permeability to vital dyes, DNA integrity and cell density were assessed. RESULTS Oxalate exposure produced time- and concentration-dependent changes in the light microscopic appearance of LLC-PK1 cells with higher concentrations ( > 140 microM.) inducing marked cytosolic vacuolization and nuclear pyknosis. Exposure to oxalate also increased membrane permeability to vital dyes, promoted DNA fragmentation and, at high concentrations (350 microM. free oxalate), induced a net loss of LLC-PK1 cells. CONCLUSIONS Since high concentrations of oxalate can be toxic to renal epithelial cells, hyperoxaluria may contribute to several forms of renal disease including both calcium stone disease and end-stage renal disease.

Phosphoinositide hydrolysis is correlated with agonist-induced calcium flux and contraction in the rabbit aorta

In the present study changes in the extent of 32P labelling of membrane phospholipids were correlated with the alpha 1-adrenoceptor-induced events of increased 45Ca influx, 45Ca release and contraction in the rabbit aorta. Under basal conditions 32P incorporation into all phospholipids proceeded without saturation through 80 min of labelling. During a 5 min exposure to 10(-5) M norepinephrine (NE) after 25 min of prelabelling the incorporation of 32P into certain phospholipids was substantially increased. Phosphatidic acid (PA) labelling was increased above basal levels by 4.1 fold, phosphatidylinositol (PI) 2.5 fold and phosphatidylcholine (PC) 1.8 fold. Half maximal stimulation of 32P labelling of PA occurred at 2.0 microM, which was similar to the EC50 value for stimulation of 45Ca influx (2.5 microM) and 45Ca release (2.1 microM) but slightly higher than the value for contractile response (0.9 microM). Antagonist sensitivity studies reinforced the alpha 1 receptor subtype character of the rabbit aorta. Prazosin (10(-7) M) reduced agonist-induced events by 63-82% while yohimbine (10(-7) M) was without influence. Phenoxybenzamine (10(-8) M) reduced agonist-induced events by 56-76%. A temporal comparison showed that agonist stimulation of PA labelling was slower than 45Ca release, but similar to the time course of 45Ca influx. Hydrolysis of 32P-labelled phosphatidylinositol diphosphate (PIP2) was more rapid and paralleled 45Ca release. These findings suggest that PIP2 hydrolysis may account for the rapid phase of norepinephrine-induced contraction in rabbit aorta while PA or its immediate precursor diacylglycerol may account for receptor-induced Ca2+ influx.

Adenosine A1 receptor-mediated antiadrenergic effects are modulated by A2a receptor activation in rat heart.

Presently, the physiological significance of myocardial adenosine A2a receptor stimulation is unclear. In this study, the influence of adenosine A2a receptor activation on A1 receptor-mediated antiadrenergic actions was studied using constant-flow perfused rat hearts and isolated rat ventricular myocytes. In isolated perfused hearts, the selective A2a receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385) potentiated adenosine-mediated decreases in isoproterenol (Iso; 10-8 M)-elicited contractile responses (+dP/d t max) in a dose-dependent manner. The effect of ZM-241385 on adenosine-induced antiadrenergic actions was abolished by the selective A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (10-7 M), but not the selective A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-1191, 10-7 M). The A2a receptor agonist carboxyethylphenethyl-aminoethyl-carboxyamido-adenosine (CGS-21680) at 10-5 M attenuated the antiadrenergic effect of the selective A1 receptor agonist 2-chloro- N 6-cyclopentyladenosine (CCPA), whereas CSC did not influence the antiadrenergic action of this agonist. In isolated ventricular myocytes, CSC potentiated the inhibitory action of adenosine on Iso (2 × 10-7 M)-elicited increases in intracellular Ca2+concentration ([Ca2+]i) transients but did not influence Iso-induced changes in [Ca2+]itransients in the absence of exogenous adenosine. These results indicate that adenosine A2areceptor antagonists enhance A1-receptor-induced antiadrenergic responses and that A2a receptor agonists attenuate (albeit to a modest degree) the antiadrenergic actions of A1 receptor activation. In conclusion, the data in this study support the notion that an important physiological role of A2a receptors in the normal mammalian myocardium is to reduce A1 receptor-mediated antiadrenergic actions.Presently, the physiological significance of myocardial adenosine A2a receptor stimulation is unclear. In this study, the influence of adenosine A2a receptor activation on A1 receptor-mediated antiadrenergic actions was studied using constant-flow perfused rat hearts and isolated rat ventricular myocytes. In isolated perfused hearts, the selective A2a receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2, 4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385) potentiated adenosine-mediated decreases in isoproterenol (Iso; 10(-8) M)-elicited contractile responses (+dP/dtmax) in a dose-dependent manner. The effect of ZM-241385 on adenosine-induced antiadrenergic actions was abolished by the selective A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (10(-7) M), but not the selective A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191, 10(-7) M). The A2a receptor agonist carboxyethylphenethyl-aminoethyl-carboxyamido-adenosine (CGS-21680) at 10(-5) M attenuated the antiadrenergic effect of the selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), whereas CSC did not influence the antiadrenergic action of this agonist. In isolated ventricular myocytes, CSC potentiated the inhibitory action of adenosine on Iso (2 x 10(-7) M)-elicited increases in intracellular Ca2+ concentration ([Ca2+]i) transients but did not influence Iso-induced changes in [Ca2+]i transients in the absence of exogenous adenosine. These results indicate that adenosine A2a receptor antagonists enhance A1-receptor-induced antiadrenergic responses and that A2a receptor agonists attenuate (albeit to a modest degree) the antiadrenergic actions of A1 receptor activation. In conclusion, the data in this study support the notion that an important physiological role of A2a receptors in the normal mammalian myocardium is to reduce A1 receptor-mediated antiadrenergic actions.

Oxalate-induced redistribution of phosphatidylserine in renal epithelial cells: implications for kidney stone disease.

Aims: The present studies assessed the possibility that exposure to oxalate leads to alterations in membrane structure that promote crystal binding to renal epithelial cells. Specifically, we determined whether oxalate exposure produces a redistribution of membrane phosphatidylserine (PS) and an increase in the binding of 14C-oxalate crystals to renal epithelial cells. Methods: PS distribution was monitored in MDCK cells and in phospholipid-containing vesicles using NBD-PS, a fluorescent derivative of PS. Superfical PS was also detected by monitoring the binding of annexin V to MDCK cells. Results: Oxalate exposure rapidly increased the abundance of superficial NBD-PS and increased the binding of annexin V to MDCK cells. Oxalate exposure also increased PS at the surface of phospholipid vesicles, suggesting that oxalate may interact directly with PS. The oxalate concentrations that increased superficial PS also increased binding of 14C-oxalate crystals to MDCK cells, and the increased crystal binding was blocked by annexin V. Conclusions: These findings provide direct evidence that oxalate exposure promotes both a redistribution of PS and an increase in crystal binding in renal epithelial cells and support the notion that oxalate toxicity may contribute to the development of stone disease by altering the properties of the renal epithelial cell membrane.

HOW ELEVATED OXALATE CAN PROMOTE KIDNEY STONE DISEASE: CHANGES AT THE SURFACE AND IN THE CYTOSOL OF RENAL CELLS THAT PROMOTE CRYSTAL ADHERENCE AND GROWTH

The present review assesses the mechanisms by which oxalate-induced alterations in renal cell function may promote stone disease focusing on 1) changes in membrane surface properties that promote the attachment of nascent crystals and 2) changes in the expression/secretion of urinary macromolecules that alter the kinetics of crystal nucleation, agglomeration and growth. The general role of renal cellular injury in promoting these responses and the specific role of urinary oxalate in producing injury is emphasized, and the signaling pathways that lead to the observed changes in cell surface properties and in the viability and growth of renal cells are discussed. Particular attention is paid to evidence linking oxalate-induced activation of cytosolic phospholipase A2 to changes in gene expression and to the activation of a second signaling pathway involving ceramide. The effects of the lipid signals, arachidonic acid, lysophosphatidylcholine and ceramide, on mitochondrial function are considered in some detail since many of the actions of oxalate appear to be secondary to increased production of reactive oxygen molecules within these organelles. Data from these studies and from a variety of other studies in vitro and in vivo were used to construct a model that illustrates possible mechanisms by which an increase in urinary oxalate levels leads to an increase in kidney stone formation. Further studies will be required to assess the validity of various aspects of this proposed model and to determine effective strategies for countering these responses in stone-forming individuals.

Intracellular Events in the Initiation of Calcium Oxalate Stones

This review summarizes our current understanding of intracellular events in the initiation of kidney stone formation, focusing on results from studies using renal epithelial cells in vitro. Such studies have shown that oxalate – either in crystalline or in soluble form – triggers a spectrum of responses in renal cells that favor stone formation, including alterations in membrane surface properties that promote crystal attachment and alterations in cell viability that provide debris for crystal nucleation. Activation of cytosolic PLA2 appears to play an important role in oxalate actions, triggering a signaling cascade that generates several lipid mediators (arachidonic acid, AA; lysophosphatidylcholine, Lyso-PC; ceramide) that act on key intracellular targets (mitochondria, nucleus). The net effect is increased production of reactive oxygen molecules (that in turn affect other cellular processes), an increase in cell death and an induction of a number of genes in surviving cells, some of which may promote proliferation for replacement of damaged cells, or may promote secretion of urinary macromolecules that serve to modulate crystal formation. A scheme is provided that explains how such oxalate-induced alterations could initiate stone formation in vivo.

TGF-? signaling in A549 lung carcinoma cells: lipid second messengers

Transforming growth factor‐β (TGF‐β) is a potent inducer of numerous extracellular matrix components, largely through a transcriptional mechanism. To define the postreceptor signaling pathways used by TGF‐β in the induction of extracellular matrix gene expression, we have utilized the human lung carcinoma cell line, A549, in transfection experiments with the TGF‐β inducible reporter construct, p3TP‐Lux. Previous work from this laboratory using pharmacologic agents suggested that a phosphatidylcholine‐specific phospholipase C and protein kinase C may be involved in early aspects of TGF‐β signaling. Here we provide evidence that TGF‐β induces a rapid and transient increase in diacylglycerol (DAG) production. When cells transfected with the p3TP‐Lux reporter plasmid are simultaneously treated with TGF‐β and a DAG kinase inhibitor, we observed a higher level of luciferase than with TGF‐β alone. We also find elevated levels of phosphocholine in cells following TGF‐β treatment. Further, exogenously added bacterial phosphatidylcholine phospholipase C (PC‐PLC) is capable of inducing expression of the p3TP‐Lux reporter to the same extent as TGF‐β indicating that the bacterial PC‐PLC can mimic the TGF‐β effect. In contrast, neither hexanoyl sphingosine (a ceramide analogue) nor arachadonic acid induce expression of the p3TP‐Lux reporter. Measurements with the fluorescent, calcium‐sensitive dye, FURA2, indicated that there was no change in intracellular calcium in response to TGF‐β. Furthermore, buffering intracellular calcium with the calcium chelating agent BAPTA/AM failed to block TGF‐β induction of the p3TP‐Lux reporter. Thus the TGF‐β signaling pathway appears to involve the production of diacylglycerol but is independent of calcium. J. Cell. Biochem. 78:588–594, 2000.

Oxalate-Induced Changes in Intracellular Calcium Levels in Renal Papillary Cells

Alterations in renal tubular and cellular function have been reported in clinical and experimental nephrolithiasis1-3, and it has been suggested that the presence of calcium oxalate stones within the kidney elicits these changes as a result of mechanical injury to the tubular cells. Evidence in support of this possibility was provided by studies in experimental animals where the induction of calcium oxalate crystal formation led to renal tubular damage (enzymuria, proteinuria, etc,4, 5). The present studies assessed the possibility that oxalate produces alterations in cell function in the absence of overt crystal formation. Specifically we determined whether or not exposure to physiological levels of oxalate would elicit changes in intracellular calcium levels in isolated papillary cells from normal rat kidneys.

Independent modulation of hepatic protein kinase activities.

The effects of hormonal status on protein kinase activity was examined in homogenates of rat liver. Protein kinase activity was evaluated from incorporation of 32P from [gamma-32P]ATP into protamine or histone as receptor substrates. Protamine phosphorylation in the presence or absence of cyclic AMP exceeded histone phosphorylation by at least a factor or two. Hypophysectomy markedly increased protamine phosphorylation in the presence or absence of saturating amounts of cyclic AMP. In contrast, hypophysectomy only slightly increased cyclic AMP independent phosphorylation of histone. These results could not be amounted for by differences in ATPase or protein phosphase activities. Cortisone (2 mg/day x 3) decreased total protein kinase activity in livers of hypophysectomized rats when protamine was substrate, but had no effect on the total activity toward histone. Growth hormone (100 mug/day x 3) significantly increased histone, but not protamine phosphorylation in livers of hypophysectomized rats. Administration of 5 mug of triiodothyonine/day to hypophysectomized rats also markedly increased the phosphorylation of histone, but not protamine when saturating amounts of cyclic AMP were present. These results support the hypothesis that liver may contain more than one type of protein kinase activity and that the different protein kinase activities can be separately affected by hormones. Such control distal to cyclic AMP might allow selective modulation of cyclic AMP-dependent processes in cells which carry out more than one such process.