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J V Bonventre

Publications and source records attributed to J V Bonventre.

At least 19 recordsLinked to original sources
PubMed1992

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. The rat bone PTH/PTHrP receptor is 78% identical to the opossum kidney receptor; this identity indicates striking conservation of this receptor across distant mammalian species. Additionally, the rat bone PTH/PTHrP receptor has significant homology to the secretin and calcitonin receptors but not to any other G protein-linked receptor. When expressed in COS cells, a single cDNA clone, expressing either rat bone or opossum kidney PTH/PTHrP receptor, mediates PTH and PTHrP stimulation of both adenylate cyclase and phospholipase C. These properties could explain the diversity of PTH action without the need to postulate other receptor subtypes.

Amino Acid Sequence↗
PubMed1992

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+] 1.5 to 3.0-fold greater than the increase induced by CD3: CD3 pairing whereas monovalent CD3 binding induced only 20%-30% of the increase. Postbinding receptor migration studies suggested that microaggregation increased from monovalent CD3 binding to CD3: CD3 pairing to CD3: CD8 pairing. Further studies revealed that progressively higher concentrations of antibodies were needed from CD3,8 to CD3,3 to CD3,4 to initiate the same degree of DNA synthesis. These results demonstrated that Ti/CD3 and CD8 can indeed be bridged by a single molecule. A model of direct CD8: CD3 synergism was raised as a possible explanation for the enhanced activation induced by CD3: CD8 pairing. The observed parallel between all three parameters and the number of TCRs that can be directly linked by the Abs raised a nonmutually exclusive model whereby CD3 binding induces activated TCR intermediaries (aTCRi) that progressively synergize with other adjacent aTCRis. In this model, this dominant inter-aTCRi synergism may be enhanced by the di- and multimeric CD8 alpha chains serving as aTCRi-aggregation foci.

Antigen-Antibody Reactions↗
PubMed1992

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. The effectiveness of a number of other components of preservation solutions, as well as the relative merits of continuous perfusion of the organ ex vivo, is more controversial. In this chapter, we review the mechanistic features of ischemic acute renal failure and discuss various preservation strategies and their success in the context of these basic principles of ischemic pathophysiology.

Calcium↗
PubMed1992

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. MRP mRNA levels were maximal at 24 h and remained elevated for at least 96 h after exposure of the cells to 1,25-(OH)2D3. MRP-8 mRNA accumulation required 100- to 1,000-fold higher concentrations of 25-(OH)D3, which binds to the 1,25-(OH)2D3 intracellular receptor with 100- to 1,000-fold lower affinity. Other differentiating agents, dimethyl sulfoxide, retinoic acid, and dexamethasone, also increased levels of MRP-8 and MRP-14 mRNA. Phorbol myristate acetate enhanced MRP-14 mRNA levels to a greater extent than MRP-8 mRNA levels, suggesting differential regulation of MRP gene expression by protein kinase C. The 1,25-(OH)2D3-induced relative increase in MRP mRNA levels was not changed by a 1,000-fold reduction in extracellular [Ca2+]. Thus 1,25-(OH)2D3 is potentially a physiological modulator of MRP gene expression. Expression of the MRP-8 and MRP-14 genes may be important for differentiation of myeloid cells.

Calcitriol↗
PubMed1992

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) = 0.02 nM] in transfected cells (referred to as PC-1 cells). Binding kinetics and cAMP dose response relationships were similar to those of the native receptor in LLC-PK1 cells. PC-1 cells also responded to calcitonin gene-related peptide (CGRP), but the EC50 value for cAMP accumulation was more than three orders of magnitude higher than for SCT. Exposure of PC-1 cells to SCT (5 nM to 1 microM) produced a dose-dependent rise in cytosolic free Ca2+ concentration ([Ca2+]i), whereas CGRP did not. The initial rise in [Ca2+]i was not dependent on extracellular Ca2+, suggesting that SCT induced release of Ca2+ from intracellular stores. SCT also increased inositol trisphosphate production in PC-1 cells. In conclusion, the cloned, transfected porcine CT receptor functionally couples to and activates both adenylyl cyclase and phospholipase C. This dual coupling is also a characteristic of the parathyroid hormone receptor, which has significant homology in amino acid sequence with the CT receptor.

Adenylyl Cyclases↗
PubMed1992

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. Thus, the regulation of PLA2 activity has important implications for kidney function. PLA2 regulation involves: calcium, pH, protein kinases, GTP-binding proteins, inhibitory and activating proteins, metabolic product inhibition, and transcriptional control. The various roles of arachidonic acid and cyclooxygenase, lipoxygenase, and cytochrome P450 mono-oxygenase products of arachidonic acid metabolism, as intracellular messengers, in the regulation of membrane channel activities, intracellular enzyme activities, cellular calcium homeostasis, mitogenesis, differentiation, cytokine and early response gene expression are discussed.

Animals↗
PubMed1991

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. At 0.1, 1.0, 10.0, and 100.0 nM AVP, the frequencies of oscillations were 0.17 +/- 0.05 (n = 6), 0.32 +/- 0.05 (n = 6), 0.49 +/- 0.05 (n = 6), and 0.48 +/- 0.05 min-1 (n = 5), respectively. Reduction in extracellular [Ca2+] reduced the frequency of AVP-induced oscillations but did not abolish the oscillations. The frequency of calcium oscillations, upon stimulation with 1.0 nM AVP, was directly correlated with the basal [Ca2+]i prior to stimulation. Oscillation frequency increased with increasing temperature. An Arrhenius plot between 24 and 37 degrees C indicated a strong temperature dependency of the oscillations with a Q10 of 3.0. Protein kinase C stimulation by active phorbol esters inhibited AVP-induced calcium oscillations but not the initial [Ca2+] response to AVP. These observations are consistent with a model incorporating a feedback loop linking [Ca2+]i to the mechanism of [Ca2+]i increase. Ca(2+)-induced Ca2+ release may be involved, whereby inositol 1,4,5-trisphosphate (inositol 1,4,5-P3) formation releases Ca2+ from an inositol 1,4,5-P3-sensitive pool, with subsequent Ca2+ uptake and release from an inositol 1,4,5-P3-insensitive pool.

Animals↗
PubMed1991

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. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates.

Alkaloids↗
PubMed1991

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 (2.5 x 10(-5) M), but not the cyclooxygenase inhibitor indomethacin (2 x 10(-5) M), reduced the magnitude of the AVP (10(-8) and 10(-7) M)-induced increase in cytosolic free Ca2+ concentration ([Ca2+]i) without affecting inositol trisphosphate production. With 10(-8) M AVP, [Ca2+]i increased to 250 +/- 47 nM in NDGA-treated cells versus 401 +/- 59 nM in control cells (p less than 0.01). [Ca2+]i, measured 2 min after exposure to AVP, was also lower with NDGA (152 +/- 21 nM) when compared with AVP alone (220 +/- 22 nM, p less than 0.01). 14,15-epoxyeicosatrienoic acid (EET) (10(-8) M), which had no effect on inositol trisphosphate production, completely reversed the NDGA-induced inhibition of the [Ca2+]i transient, whereas 5-hydroperoxyeicosatetraenoic acid (HPETE) (5 x 10(-7) M) did not. Pretreatment with higher concentrations of 14,15-EET (10(-7)-10(-6) M) markedly potentiated the AVP-induced increase in [Ca2+]i. NDGA-induced inhibition of the AVP-generated [Ca2+]i transient was also observed when cells were incubated in low Ca2+ media ([Ca2+] less than 5 x 10(-8) M), suggesting that NDGA pretreatment impaired intracellular release of Ca2+. Since NDGA had no direct effect on inositol 1,4,5-trisphosphate-induced Ca2+ release, we postulated that NDGA blocked production of a metabolite that releases Ca2+ from intracellular stores. 14,15-EET and 15-HPETE, but not 15-hydroxyeicosatetraenoic acid (each at 3 x 10(-7) M), raised [Ca2+]i when added directly to cells in low Ca2+ media. In permeabilized cells 14,15-EET and 15-HPETE (10(-7) M) potently released Ca2+ from intracellular stores. In summary, noncyclooxygenase metabolites of arachidonic acid, and in particular P450 metabolites, are potent endogenous amplifiers of the AVP-induced [Ca2+]i signal by mechanisms not directly involving phospholipase C activation. This effect is mediated, at least in part, by enhanced release of Ca2+ from intracellular storage sites by an inositol 1,4,5-trisphosphate-independent mechanism.

8,11,14-Eicosatrienoic Acid↗
PubMed1991

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. Stimulation with arginine vasopressin, epidermal growth factor, or phorbol myristate acetate increased [3H]thymidine incorporation and mRNA levels of the immediate-early response genes c-fos and Egr-1. These increases in [3H]thymidine incorporation and mRNA levels were reduced by NDGA and ketoconazole. NDGA, SK&F 525A, and ketoconazole had no effect on cellular ATP levels. Indomethacin had no effect upon cell growth. 14,15-Epoxyeicosatrienoic acid potentiated the effect of arginine vasopressin to enhance [3H]thymidine incorporation. Reverse-phase high pressure liquid chromatography analysis of lipid extracts from cells prelabeled with [3H]arachidonic acid resulted in the detection of a radioactive peak which eluted with lipoxygenase and monooxygenase products, with the same retention time as vicinal dihydroxyeicosatrienoic acids. This peak increased after stimulation with arginine vasopressin or epidermal growth factor and was reduced by preincubation with NDGA. Furthermore, analysis of unlabeled cell extracts by gas chromatography-mass spectrometry revealed the presence of a compound with epoxyeicosatrienoic acid-like characteristics. These results indicate that mesangial cells in culture likely produce products of the cytochrome P450 monooxygenase system that are important endogenous mediators of the growth response to mitogenic agents.

Animals↗
PubMed1991

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 42.2 degrees C for 20 min showed an overall decrease in protein synthesis but an increase in the synthesis of approximately 72 and approximately 85 kd proteins and in the levels of HSP70 mRNA. Heat shock inhibited excitotoxicity in cells exposed to glutamate at 3 or 24 hr following heat exposure, but not when the interval between heat and glutamate exposure was shortened to 15 min or lengthened to 48 hr. Protection due to heat shock required new protein synthesis, since it did not occur when protein or RNA synthesis inhibitors were added. By ameliorating excitotoxic processes, HSPs may attenuate brain injury in certain pathologic conditions.

Animals↗
PubMed1991

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. We have previously reported that Egr-1 mRNA accumulates to high levels in mouse kidneys after 30 min of ischemia and 1 h of reperfusion. In the present study, performed in rats, we show that Egr-1 mRNA transiently accumulates to very high levels after 40 min of ischemia and 1 h of reperfusion, is decreased by 3 h, and is nondetectable by 24 h of reperfusion. Reperfusion is required for Egr-1 protein accumulation to occur. The Egr-1 protein was localized by immunohistochemical techniques primarily to the nuclei of the thick ascending limbs and principal cells of the collecting ducts in the cortex and medulla. The subcellular localization was exclusively nuclear. There was some staining of the glomerular tuft and staining was particularly prominent in the parietal epithelial cells. In parallel to the accumulation of Egr-1 mRNA, the expression of the protein was transient and was no longer apparent after 5 h of reperfusion. The Egr-1 protein may play an important role in regulation of the response to ischemia of those segments of the nephron that are highly susceptible to oxygen deprivation and have a high level of intrinsic plasticity. It is possible that this protein may modulate cellular processes important for the ultimate ability of these critical nephron segments to recover from an ischemic insult.

Animals↗
PubMed1991

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. METHODS AND RESULTS: The effects of dietary supplementation with fish oils on vascular contractility were examined in endothelialized and de-endothelialized aortic rings under pre- and postanoxic conditions. De-endothelialization was defined functionally by the loss of acetylcholine-induced vasodilation in norepinephrine-preconstricted aortic rings from rats fed normal rat chow. Three groups of rats were fed diets containing either 20% menhaden oil or 20% beef tallow, both supplemented with 3% corn oil or 23% corn oil for longer than 4 weeks. All animals received vitamin E. Under well-oxygenated conditions, de-endothelialized aortic rings from rats fed fish oil and corn oil contracted to similar extents with norepinephrine and vasopressin and less than rings from rats fed beef tallow. Endothelialized (intact) and de-endothelialized rings from rats fed fish oil relaxed more in response to acetylcholine than rings from rats fed beef tallow and corn oil. After anoxic exposure and reoxygenation, KCl-induced contraction of intact rings from rats fed fish oil and corn oil was similar and less than rings from rats fed beef tallow. Intact and de-endothelialized rings from rats fed fish oil relaxed more to acetylcholine than did rings from rats fed beef tallow and corn oil. CONCLUSIONS: Under preanoxic or postanoxic conditions, rings from rats fed fish oil and corn oil contracted less than rings from rats fed beef tallow. The relaxation response to acetylcholine, however, was greater in rings from rats fed fish oil than from rats fed either corn oil or beef tallow. These vascular effects of fish oil feeding may result in increased blood flow to ischemic and reperfused tissues in vivo.

Animals↗
PubMed1991

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. Activities in each fraction were optimal at pH 8.5-9.5. Cytosolic PLA2 activity was enhanced when Ca2+ concentration [( Ca2+]) was increased over the range of 10(-7) to 10(-6) M. Mitochondrial PLA2 activity required higher [Ca2+] for activation (greater than 10(-6) M). After 45 min of ischemia cytosolic PLA2 activity was decreased, whereas mitochondrial and microsomal activities were increased. When ischemia was followed by 1 h of reperfusion, cytosolic, mitochondrial, and microsomal activities were enhanced. Ischemia alone did not change the gel filtration chromatography patterns of PLA2 activity, but ischemia and reperfusion resulted in the appearance of a new peak of activity in cytosolic and mitochondrial fractions (Mr approximately 2-3 kD). Thus, the rat kidney has multiple forms of PLA2 activity, likely representing distinct enzymes, with Ca2+ dependencies suggesting regulation by Ca2+ in vivo. Ischemia and reperfusion result in stable increases of PLA2 activity in each subcellular fraction, perhaps related to covalent modifications of PLA2's, which likely account for membrane phospholipid degradation, and increased tissue levels of unsaturated free fatty acids.

Animals↗
PubMed1991

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. PLA2 activities of each fraction were assayed by measuring release of arachidonic acid (AA) from exogenous 14C-AA-phosphatidylcholine (PC), -phosphatidylethanolamine (PE), and -phosphatidylinositol (PI). Two forms of PLA2 were present in the cytosolic fraction: a high-molecular-weight form, active against PC and PE, and a smaller form with an Mr of approximately 14 kDa, active against PE. In the mitochondrial and microsomal fractions, a single form (Mr approximately 14 kDa) was dominant, active against both PC and PE. The role of PLA2 activation in ischemic brain injury remains controversial. PLA2 enzymatic activity was characterized in gerbil brain after 10 min of common carotid occlusion, followed by 10 min of reperfusion. Ischemic/reperfused brains had significantly higher PLA2 specific activities in each subcellular fraction. Ischemia and reperfusion did not change the gel-filtration elution patterns of PLA2 activity of the various forms of the enzyme. Cytosolic, mitochondrial, and microsomal activities were optimal at a pH of approximately 8.5. Cytosolic PLA2 activity was enhanced when Ca2+ concentration [( Ca2+]) was increased over the physiological range (10(-7) to 10(-6) M). Mitochondrial and microsomal PLA2 activities were also [Ca2+] dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

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. Thirteen subclones responded poorly or not at all to soluble anti-Ly-6A mAb. Although the selective pressure was exerted through Ly-6A, only one mutant did not express the Ly-6A antigen. Interestingly, 10 of the 15 subclones expressed either nondetectable or a very low level of T cell receptor/CD3 complex (TCR/CD3). Preferential expansion of TCR/CD3 expression mutants following anti-Ly-6A selection further established functional linkage between Ly-6A and TCR/CD3 complex. The mechanism of the functional coupling was investigated by analyzing the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), one of the early events in T cell activation. We showed that PIP2 was not hydrolyzed in response to anti-Ly-6A in TCR/CD3-negative mutants. Aluminum fluoride, which activates G protein directly, did induce PIP2 hydrolysis in these cells. These data suggest that activation signals originated from Ly-6A must be transmitted first to TCR/CD3 complex, which then couples to the G protein/phospholipase C system. A similar requirement also applies to the Thy-1 protein and lectin receptors. Thus, the TCR/CD3 complex plays a central role in the integration and transmission of activation signals that originated from several T cell surface molecules.

Aluminum↗