Mitogen regulation of c-Raf-1 protein kinase activity toward mitogen-activated protein kinase-kinase.

The c-raf-1 protooncogene encodes a Ser/Thr protein kinase. A mitogen-activated protein kinase-kinase (MAPKK) purified from bovine brain is phosphorylated and activated 4-9-fold in vitro by c-Raf-1 from mitogen-treated cells. c-Raf-1 protein kinase activity, measured by the phosphorylation of brain MAPKK substrate, is detectably activated within 1 min after addition of platelet-derived growth factor (PDGF) to 3T3 cells, increasing more rapidly than the endogenous NIH 3T3 cell MAPKK activity.

Actin-binding protein contributes to cell volume regulatory ion channel activation in melanoma cells.

The cell volume regulatory response to a hypotonic stimulus is frequently initiated by activation of K+ and Cl- channels. We have characterized the hypotonic cell volume regulatory response of human melanoma cells devoid of actin-binding protein (ABP) and their genetically rescued counterpart transfected with the cDNA for ABP. ABP-deficient cells were unable to volume-regulate or activate K+ channels when exposed to a hypotonic stimulus.

Kid-1, a putative renal transcription factor: regulation during ontogeny and in response to ischemia and toxic injury.

We have identified a new putative transcription factor from the rat kidney, termed Kid-1 (for kidney, ischemia and developmentally regulated gene 1). Kid-1 belongs to the C2H2 class of zinc finger genes. Its mRNA accumulates with age in postnatal renal development and is detected predominantly in the kidney.

Phospholipase A2 (PLA2) activity in gerbil brain: characterization of cytosolic and membrane-associated forms and effects of ischemia and reperfusion on enzymatic activity.

Phospholipases A2 comprise a family of enzymes that hydrolyze the acyl bond at the sn-2 position of phospholipids to generate free fatty acids and lysophospholipids. In the central nervous system products of PLA2 regulate neurotransmission. In addition, the lysophospholipids, free fatty acids, eicosanoids, platelet activating factor and reactive oxygen species, generated by enhanced PLA2 activity and arachidonic acid metabolism, may be responsible for many destructive cellular processes in neuronal tissue.

Comparative studies of isolated CD3: CD8, CD3: CD3, and monovalent CD3 binding on CD8+ T-cell activation: model of progressive T-cell receptor aggregation synergism.

The TCR and CD8 complexes of CD8+ T cells bind to different regions of MHC class I molecules and both play important roles in the response of the CD8+ T cells to Ag/MHC on APCs. In this report, we mimicked common MHC binding with an anti-CD3:anti-CD8 (CD3,8) BSMAB to isolate the effect of CD3: CD8 pairing, compared this with the effect of CD3: CD3 pairing by the parental bivalent anti-CD3 MAB, and with monovalent anti-CD3 binding by an anti-CD3: anti-CD4 (CD3,4) BSMAB. CD3: CD8 pairing induced an increase in cytosolic free [Ca2+] 1.

Phospholipase A2 and signal transduction.

Phospholipases A2 (PLA2) comprise a family of enzymes that hydrolyze the acyl bond at the sn-2 position of phospholipids to generate free fatty acids and lysophospholipids. Different forms of PLA2 are involved in digestion, inflammation, and intercellular and intracellular signal transduction. The sn-2 position of phospholipids in mammalian cells is enriched in arachidonic acid, the precursor of eicosanoids, which have diverse physiologic and pathophysiologic effects on the kidney and other organs.

A cloned porcine renal calcitonin receptor couples to adenylyl cyclase and phospholipase C.

The signal transduction pathways of the recently cloned porcine kidney calcitonin (CT) receptor were evaluated. This receptor, when stably transfected into MC-3T3 cells, avidly bound salmon CT (SCT) [dissociation constant (Kd) = 4 nM]. Incubation with SCT resulted in a dose-dependent accumulation of adenosine 3',5'-cyclic monophosphate (cAMP) [50% effective concentration (EC50) = 0.

Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium.

Parathyroid hormone (PTH), a major regulator of mineral ion metabolism, and PTH-related peptide (PTHrP), which causes hypercalcemia in some cancer patients, stimulate multiple signals (cAMP, inositol phosphates, and calcium) probably by activating common receptors in bone and kidney. Using expression cloning, we have isolated a cDNA clone encoding rat bone PTH/PTHrP receptor from rat osteosarcoma (ROS 17/2.8) cells.

1 alpha,25-(OH)2 vitamin D3 enhances expression of the genes encoding Ca(2+)-binding proteins MRP-8 and MRP-14.

Two closely related Ca(2+)-binding proteins, migration inhibitory factor-related protein (MRP)-8 and MRP-14, are synthesized under specific conditions of myeloid cell differentiation. Because 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induces myeloid cell differentiation and expression of other S-100 class calcium-binding proteins, we examined the effects of 1,25-(OH)2D3 on MRP mRNA levels in human U-937 histiocytic lymphoma cells. 1,25-(OH)2D3 increased MRP-8 and MRP-14 mRNA levels in a time- and dose-dependent manner.

Kidney preservation ex vivo for transplantation.

Ischemic injury to the renal allograft, prior to implantation, is an important cause of delayed graft function. With improved understanding of the pathophysiological mechanisms involved, strategies have been devised to minimize ischemic injury during preservation ex vivo. It is clear that reducing the warm ischemic time, flushing the kidney with hypothermic solution containing cell-impermeant compounds, and maintaining the organ at low temperature ex vivo have increased the duration that the kidney can be preserved.

Heat shock protects cultured neurons from glutamate toxicity.

Expression of heat shock proteins (HSPs) occurs in brain after ischemia and status epilepticus. We report that induction of the heat shock response in cortical cultures protects neurons from glutamate-induced excitotoxicity. Cultures heated to 42.

Oscillations of intracellular calcium induced by vasopressin in individual fura-2-loaded mesangial cells. Frequency dependence on basal calcium concentration, agonist concentration, and temperature.

Intracellular free calcium concentration ([Ca2+]i) was measured in fura-2-loaded single rat mesangial cells by dual wavelength spectrofluorometry. Stimulation with arginine vasopressin (AVP) caused an initial sharp rise of [Ca2+]i followed by repetitive spikes. The frequency of the oscillations was dependent on the concentration of AVP.

Effects of dietary omega 3 fatty acids on vascular contractility in preanoxic and postanoxic aortic rings.

Background: Vasomotor reactivity may contribute to the pathophysiology of ischemic injury. The atherosclerotic vessel may be particularly susceptible to vasoconstriction because of the damaged endothelial layer with resultant loss of vasodilatory factors. While dietary omega 3 fatty acids have been proposed to protect against vascular occlusion, it is not clear to what extent this results from alterations in the function of platelets or from changes intrinsic to the blood vessel itself.

Characterization of phospholipase A2 (PLA2) activity in gerbil brain: enhanced activities of cytosolic, mitochondrial, and microsomal forms after ischemia and reperfusion.

Brain phospholipase A2 (PLA2) activity has not been well characterized. Given the importance of this enzymatic activity for a variety of cellular functions in the brain, we characterized the subcellular distribution of PLA2 activity in gerbil brain and evaluated how PLA2 activity was altered by ischemia and reperfusion. Cytosolic, mitochondrial, and microsomal fractions were prepared by differential centrifugation of forebrain homogenates.

Subcellular characteristics of phospholipase A2 activity in the rat kidney. Enhanced cytosolic, mitochondrial, and microsomal phospholipase A2 enzymatic activity after renal ischemia and reperfusion.

Phospholipase A2 (PLA2) activities in cytosolic, mitochondrial, and microsomal fractions of rat kidneys were characterized under control conditions, after ischemia, and subsequent to ischemia and reperfusion. Two forms of PLA2 activity were present in the cytosolic fraction: a high molecular weight form, active against phosphatidylcholine (PC), and phosphatidylethanolamine (PE), which upon purification has a molecular mass of 110 kD; and smaller form (Mr approximately 14 kD), active against PE. In mitochondrial and microsomal fractions a single form (Mr approximately 14 kD), active against both PC and PE, was dominant.

Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells.

Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane-associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine.

Noncyclooxygenase metabolites of arachidonic acid amplify the vasopressin-induced Ca2+ signal in glomerular mesangial cells by releasing Ca2+ from intracellular stores.

Noncyclooxygenase metabolites of arachidonic acid may be potent modulators of the mitogenic response of renal mesangial cells to the mitogenic vasoactive peptide arginine vasopressin (AVP). Since Ca2+ is a critical second messenger in the response of mesangial cells to AVP, and Ca2+ has been implicated in the regulation of growth, we determined whether noncyclooxygenase metabolites altered the phospholipase C-Ca2+ signalling cascade which is activated by AVP. Pretreatment of mesangial cells for 10 min with lipoxygenase and cytochrome P450 monooxygenase inhibitors, nordihydroguaiaretic acid (NDGA, 10(-5) M) or SKF-525A (2.

Localization of the protein product of the immediate early growth response gene, Egr-1, in the kidney after ischemia and reperfusion.

Egr-1 is an "immediate early" gene that is induced by growth factors and agents that induce differentiation and encodes a protein with a "zinc-finger" motif. This protein is believed to be involved in transcriptional regulation. Because the fate of the kidney, and hence the organism, after an ischemic insult is dependent upon cellular repair, differentiation, and proliferation, we examined whether there was expression of the Egr-1 protein after an ischemic insult to the rat kidney.

Endogenous non-cyclooxygenase metabolites of arachidonic acid modulate growth and mRNA levels of immediate-early response genes in rat mesangial cells.

The role of endogenous arachidonic acid and its metabolites as mediators of cell growth was studied in rat mesangial cells. Inhibitors of the cytochrome P450 monooxygenase and lipoxygenase systems (nordihydroguaiaretic acid (NDGA), SK&F 525A, and ketoconazole) significantly reduced serum-stimulated cell growth as determined by cell counts and incorporation of [3H]thymidine. Inhibition of cyclooxygenase or lipoxygenases alone had no effect on cell growth.

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.

Incorporation of marine lipids into mitochondrial membranes increases susceptibility to damage by calcium and reactive oxygen species: evidence for enhanced activation of phospholipase A2 in mitochondria enriched with n-3 fatty acids.

Experiments were designed to evaluate the susceptibility of mitochondrial membranes enriched with n-3 fatty acids to damage by Ca2+ and reactive oxygen species. Fatty acid content and respiratory function were assessed in renal cortical mitochondria isolated from fish-oil- and beef-tallow-fed rats. Dietary fish oils were readily incorporated into mitochondrial membranes.

Selection of T cell receptor expression mutants through the functionally linked Ly-6A.

Ly-6A is a glycosyl-phosphatidylinositol (GPI)-anchored molecule that participates in murine T cell activation. Activation of T cell hybridomas with anti-Ly-6A monoclonal antibody (mAb) leads to production of interleukin-2 (IL-2), but also to a paradoxical growth inhibition, which was used to select for signaling mutants. Fifteen subclones derived from two independent mutageneses and anti-Ly-6A selection were characterized.