Glutathione and glutathione conjugate efflux from cultured liver cells

Efflux of glutathione (GSH) and GSH-conjugates from cultured rat liver epithelial cell lines; the non-tumorigenic ARL-15C₁ and the -glutamyl transpeptidase containing, tumorigenic ARL-16T₂, has been assessed under basal condition and during chronic treatment with 75 and 150 M ethacrynic acid (EA). The intracellular level of GSH increased in proportion to EA concentration during chronic exposure. The rates of GSH and GSH-EA conjugate efflux increased with intracellular GSH in both ARL cell lines.Glutathione-S-transferase activity measured with EA as substrate increased over the experimental time course after treatment with 150, but not 75 M EA. When intracellular GSH content was increased by treatment with the cysteine pro-drug, 2-L-oxothiazolidine 4-carboxylic acid, the rate of GSH efflux was increased, but not the rate of GS-EA conjugate export. Inhibition of -glutamyl transpeptidase by acivicin (AT-125) increased the GSH and GS-EA conjugate efflux rate in ARL-16T₂ cells by factors of approximately 2 and 15, respectively. Acivicin treatment of ARL-16T₂ cells chronically treated with EA elevated GSH efflux rate by 10-fold and GS-EA efflux by 40-fold versus control samples. These studies show that GSH and GSH conjugate efflux are accomplished as independently regulated processes. Efflux of GSH is enhanced by increased in racellular GSH, but increase in the conjugate transport rate requires the presence of the GSH conjugate. The response of the efflux process to treatment with a chronic GSH depleting agent was identical in two cell lines in which the metabolic fate of glutathione is known to differ fundamentally.Abbreviations GSH reduced glutathione - GSSG oxidized glutathione - GS-EA the glutathione conjugate of ethacrynic acid - EA ethacrynic acid - CDNB 1-chloro 2,4-dinitrobenzene - HBS HEPES buffered saline - OTC 2-L-oxothiazolidine 4-carboxylic acid - CYSSG cysteinyl-glutathione mixed disulfide - FDNB 1-fluoro-2,4-dinitrobenzene - GCS -glutamyl cysteine synthetase - GST glutathione-S-transferase - BCA bicinchoninic acid - SDS sodium dodecyl sulfate - PCA perchloric acid     

Mutation induction by γ and X-ray irradiation in tissue cultured lotus

Mutations of tissue cultured lotus were induced by treating plantlets with either acute -rays at doses of 0, 2, 3, 4, 5 or 6 krad or X-rays at doses of 0, 1, 2, 3, 4 or 5 krad. The 2-krad dose of either - or X-ray treatments resulted in a 50% survival rate. The use of - and X-rays to induce mutation in lotus resulted in 21 altered characteristics. Mutants from 1- and 2-krad of either or X-rays had long secondary roots and numerous adventitious roots. These mutants also exhibited good shoot growth and healthy rhizome development. Most plants treated with 3–5 krad of either - or X-rays exhibited abnormal characteristics including vitrification, chlorosis, deformed petioles and in addition had inhibited growth of lateral buds, secondary roots and rhizomes. All plants treated with 6 krad of -rays died within 4 weeks. Control plants had stoma lengths of 2.56 m and cytological analysis of the root tips confirmed the diploid chromosome number of 16. Two groups of aneuploid cells were achieved using irradiation at doses of 3 and 4 krad of either - or X-ray. Chromosome numbers were 2n=18 and 20 with associated stoma lengths of 3.43 and 4.34 m, respectively. Abnormal stomata (cyclocytic and deformity) were observed in plants treated with 4 krad of -ray.     

Secretion of recombinant human fibrinogen by the murine mammary gland

Transgenic animals secreting individual chains and assembled fibrinogen were produced to evaluate the capacity of the mammary gland for maximizing assembly, glycosylation and secretion of recombinant human fibrinogen (rhfib). Transgenes were constructed from the 4.1kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the A, B and fibrinogen chains. Transgenic mice secreted fully assembled fibrinogen into milk at concentrations between 10 and 200 g/ml, with total secretion of subunits approaching 700 g/ml in milk. Partially purified fibrinogen was shown to form a visible and stable clot after treatment with human thrombin and factor XIII. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the B and chains were rate limiting. Both the B and chains were glycosylated when co-expressed and the degree of saccharide maturation was dependent on expression level, with processing preferred for chains over B chains. Also, the subunit complexes ₂, A₂ and the individual subunits A, B and were found as secretion products. When the B was secreted individually, the glycosylation profile of the molecule was of a mature complex saccharide indicating recognition of the molecule by the glycosylation pathway without association with other fibrinogen chains. To date secretion of B chain has been not observed in any cell type, suggesting that the secretion pathway in mammary epithelia is less restrictive than that occurring in hepatocytes and other cells previously used to study fibrinogen assembly.     

Effect of lipid supplements on the production and glycosylation of recombinant interferon-γ expressed in CHO cells

The effects of lipids on the glycosylation of recombinant human interferon- expressed in a Chinese Hamster Ovary cell line were investigated in batch culture. Lipids form an essential part of the N-glycosylation pathway, and have been shown to improve cell viability. In control (serum-free) medium the proportion of fully-glycosylated interferon- deteriorated reproducibly with time in batch culture, but the lipoprotein supplement ExCyte was shown to minimise this trend. Partially substituting the bovine serum albumin content of the medium with a fatty-acid free preparation also improved interferon- glycosylation, possibly indicating that oxidised lipids carried on Cohn fraction V albumin may damage the glycosylation process.Abbreviations BSA bovine serum albumin - CHO chinese hamster ovary - DHFR dihydrofolate reductase - FCS foetal calf serum - IFN- human interferon-gamma - q _IFN specific interferon production rate - specific growth rate - 2N doubly-gycosylated - 1N singly-glycosylated - ON non-glycosylated     

The cryptoendolithic microbial environment in the Ross Desert of Antarctica: Satellite-transmitted continuous nanoclimate data, 1984 to 1986

Summary A satellite mediated station for monitoring nanoclimate (climate in the millimeter range) data, suitable for use in polar regions is described. The station, located in the Ross desert of Antarctica, has been in operation for more than 3 years, measuring rock temperatures, air temperature, light, snow, wind, and moisture. The data indicate that biological activity in the cryptoendolithic microbial ecosystem is limited to the period from mid November to mid February. The total number of hours of biological activity, based on assumptions of the minimum light, temperature and moisture requirements of the community, is less than 1000 h/year. The time above 0°C, representing more nearly optimal conditions, is between 50 and 550 h/year, depending on the orientation of the surface.Ross desert is an unofficial name for the ice-free region between 160° and 164°E and 76°30 and 78°30S. This area is more generally referred to as the dry valleys     

Rate of free-radical oxidation of C18 diene and triene fatty acids in aqueous micellar solutions and effectiveness of beta-carotene as an inhibitor of their oxidation

The rate of accumulation of conjugated dienes of polyunsaturated fatty acids was measured during free-radical oxidation of linoleic acid (18:2n-6, LA), -linolenic acid (18:3n-3, -LNA), and -linolenic acid (18:3n-6, -LNA) initiated by 2,2"-azo-bis-(2-amidinopropane) hydrochloride in aqueous micellar solutions of sodium dodecyl sulfate and sodium cholate. It was shown that, unlike homogeneous solutions, the oxidative stability of PUFAs in aqueous dispersions increased with an increase in the extent of unsaturation. The rate of LA oxidation was more than tenfold greater than that of - and -LNA. The antioxidant activity of -carotene, in contrast to homogeneous solutions, in both micellar systems studied depended on the degree of PUFA unsaturation. We found that 5 M -carotene effectively inhibited the LA oxidation (almost by 90%), whereas the oxidation of -LNA and -LNA was not inhibited by -carotene even at much greater concentration (30 M). The paradoxical discrepancy between the extent of unsaturation and the PUFA oxidation rate, as well as a decrease in the efficiency of -carotene-dependent inhibition of oxidation of more polyunsaturated fatty acids in reactions conducted in aqueous dispersions is consistent with the model according to which the peroxyl radicals of LA and fatty acids with the doublebond number greater than two exhibit different polarity.     

Diagnosing lactic acid fermentation based on specific rates of growth,substrate consumption and product formation

The on-line calculated specific rates of growth, substrate consumption and product formation were used to diagnose microbial activities during a lactic acid fermentation. The specific rates were calculated from on-line measured cell mass, and substrate and product concentrations. The specific rates were more sensitive indicators of slight changes in fermentation conditions than such monitored data as cell mass or product concentrations.List of Symbols 1/h specific rate of cell growth - 1/h specific rate of substrate consumption - 1/h specific rate of product formation - ^* dimensionless specific rate of cell growth - ^* dimensionless specific rate of substrate consumption - ^* dimensionless specific rate of product formation - _max 1/h maximum specific rate of cell growth - _max 1/h maximum specific rate of substrate consumption - _max 1/h maximum specific rate of product formation - X g/l cell mass concentration - S g/l substrate concentration - S ^* dimensionless substrate concentration - S ₀ g/l initial substrate concentration - P g/l product concentration     

Syntheses ofγ-fluoro-α-amino acids

Summary Methods for the synthesis of racemic and optically active title compounds are presented. Key step of these four-step procedures is the alkylation with 1-bromo-2-fluoroalkanes of glycine-ester-derived imines in anhydrous medium using lithium diisopropylamide as a base at low temperature or phase transfer catalyzed alkylation with 50% NaOH and triethylbenzylammoniumchloride as the phase transfer catalyst, respectively. Subsequent three-step deprotection gave the free acids in 13–33% overall yield. Deracemization of-fluoro--aminobutyric acid methyl and ethyl esters with-chymotrypsin was shown to give the (–)-enantiomers of the esters and (+)--fluoro--aminobutyric acid in >98% ee, while from thetert-butylester the opposite stereochemical result was observed giving the (–)-acid with 88% ee. Optically active-fluoro--amino acids were synthesized alternatively by phase transfer catalysis with N-benzyl-cinchonium chloride or using an auxiliary-directed asymmetric alkylation of the imine derived from (R)-(+)-camphor or (R)-(+)-2-hydroxypinan-3-one. These processes gave different enantiomers of-fluoro--aminobutyric acid via a monomeric lithium enolate in the first or a dimeric lithium enolate in the second case, respectively. The enantiomeric excess can be improved by lithium/magnesium exchange.     

Stimulation of non-specific resistance to tumors in the mouse using a poly(maleic-acid-styrene)-conjugated neocarzinostatin

Summary The development of non-specific resistance to tumors following stimulation with poly(maleic-acid-styrene)-conjugated neocarzinostatin (SMANCS), a polymer-conjugated derivative of neocarzinostatin, was investigated in mice. The growth of syngeneic solid tumors (Meth-A fibrosarcoma and RL 1 leukemia) inoculated into BALB/c mice was suppressed after one treatment with SMANCS at doses ranging from 0.14 mg/kg to 3.4 mg/kg i.v. 24 h before tumor implantation. Since previously observations concerning SMANCS have shown that it disappeared within 1.5 h after i.v. administration in mice and that it was inactivated quickly in plasma, SMANCS evidently inhibited tumor growth by mediating non-specific resistance. In addition, the non-specific resistance to tumors stimulated by SMANCS could be passively transferred to untreated mice by serum which was shown to contain interferon (IFN) from 12 h to 20 h after SMANCS administration. However, the resistance was not produced by serum prepared from mice at 8 h or 32 h after administration presumably because of the observation that the interferon activity was only demonstrated from 12 h to 28 h after SMANCS stimulation. When the serum specimens were treated with anti-IFN- antiserum, the antitumor activity of the sera was abrogated. However, no significant change was detected in the antitumor activity of the specimens following treatment with anti-IFN-/ antiserum. Treatment of mice with SMANCS and anti-IFN- antiserum together resulted in the elimination of the non-specific resistance to tumors. The IFN induced in the sera of mice by SMANCS was shown to be 57% IFN- and 41% IFN-/. Half of the interferon produced in SMANCS-stimulated mice could be eliminated by treatment with anti-IFN-, and treatment of SMANCS-stimulated mice with both anti-IFN- and anti-IFN-/ antisera resulted in a total absence of detectable interferon. These findings suggest that while the administration of SMANCS induces both IFN- and IFN-/ production, in this case, it is only the former which mediates the non-specific resistance to tumors.     

Interleukin 2-activated killer cells: generation in collaboration with interferonγ and its suppression in cancer patients

Summary The generation of lymphokine activated killer (LAK) cells by recombinant IL2 (rIL2) in collaboration with interferon (IFN) was examined in peripheral blood mononuclear cells (PBMC) from patients with malignant tumors of the digestive organs and breast cancer. LAK cytotoxicity could be induced by rIL2 at 10 units/ml in 10 of 12 patients and 20 of 37 using fresh autologous tumor cells and PK-1, an established solid tumor cell line as a target, respectively. Among 34 patients, in which titers of IFN produced were assayed, 12 showed no IFN production. All of these 12 patients had no or extremely low LAK activity, suggesting the correlation of LAK generation with the production of IFN in response to rIL2. LAK induction by rIL2 in PBMC of cancer patients was almost completely inhibited by addition of anti-IFN serum. Depressed LAK generation, which was accompanied by no or low levels of IFN production, was partially restored by addition of exogenous recombinant IFN. These results indicate that LAK induction by rIL2 in cancer patients involves the production of IFN and its interaction with rIL2.The results also suggested the presence of a factor(s) suppressing LAK induction by rIL2 in the serum of cancer patients. Based on these results, the cancer patients could be divided into the following three groups. Group 1, in which the serum suppressor activity was undetectable, had the same level of LAK cytotoxicity in PBMC as healthy controls. Group 2 showed the serum suppressor factor and had the lower level of cytotoxicity in PBMC when cultivated in autologous serum (AS) compared to healthy controls. The depressed LAK induction in AS medium was restored to a normal level in culture with fetal calf serum (FCS) plus rIL2, or by addition of rIFN, or high concentrations of rIL2 in AS medium. The last group (group 3), in which the serum suppressor factor was also found, had the lowest level of cytotoxicity compared to healthy controls. The LAK induction in these patients could not be restored to a normal level by culture in FCS medium, addition of exogenous rIFN or high concentrations of rIL2, suggesting the possibility that the deficit of LAK generation in this group might involve the dysfunction or the lack of IL2 responder cells, in addition to the presence of a serum suppressor factor(s).     

Substrate specificity and inhibition of UDP-GlcNAc:GlcNAcβ1-2Manα1-6R β1,6-N-acetylglucosaminyltransferase V using synthetic substrate analogues

UDP-GlcNAc:GlcNAc 1-2Man1-6R (GlcNAc to Man) 1,6-N-acetylglucosaminyltransferase V (GlcNAc-T V) adds a GlcNAc1-6 branch to bi- and triantennaryN-glycans. An increase in this activity has been associated with cellular transformation, metastasis and differentiation. We have used synthetic substrate analogues to study the substrate specificity and inhibition of the partially purified enzyme from hamster kidney and of extracts from hen oviduct membranes and acute myeloid leukaemia leukocytes. All compounds with the minimum structure GlcNAc1-2Man1-6Glc/Man-R were good substrates for GlcNAc-T V. The presence of structural elements other than the minimum trisaccharide structure affected GlcNAc-T V activity without being an absolute requirement for activity. Substrates with a biantennary structure were preferred over linear fragments of biantennary structures. Kinetic analysis showed that the 3-hydroxyl of the Man1-3 residue and the 4-hydroxyl of the Man- residue of the Man1-6(Man1-3)Man-RN-glycan core are not essential for catalysis but influence substrate binding. GlcNAc1-2(4,6-di-O-methyl-)Man1-6Glc-pnp was found to be an inhibitor of GlcNAc-T V from hamster kidney, hen oviduct microsomes and acute and chronic myeloid leukaemia leukocytes.Abbreviations all allyl - AML acute myeloid leukaemia - BSA bovine serum albumin - CML chronic myelogenous leukaemia - Gal G,d-galactose - Glc d-glucose - GlcNAc Gn,N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man M,d-mannose - mco 8-methoxycarbonyl-octyl, (CH₂)₈COOCH₃ - Me methyl - MES 2-(N-morpholino)ethanesulfonate - oct octyl - pnp p-nitrophenyl - T transferase     

Growth parameters (K s, μmax,Y s) of Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum was grown on a mineral salts medium in a fermenter gassed with H₂ and CO₂, which were the sole carbon and energy sources. Under the conditions used the bacterium grew exponentially. The dependence of the growth rate () on the concentration of H₂ and CO₂ in the incoming gas and the dependence of the growth yield ( ) on the growth rate were determined at pH 7 (the pH optimum) and 65° C (the temperature optimum).The curves relating growth rate to the H₂ and CO₂ concentration were hyperbolic. From reciprocal plots apparent K _s values for H₂ and CO₂ and _max were obtained: app. = 20%; app. = 11%; = 0.69 h^-1; t (max)=1 h. was 1.6 g mol^-1 and almost independent of the growth rate, when the rate of methane formation was not limited by the supply of either H₂ or CO₂. The yield increased to near 3 g mol^-1 when H₂ or CO₂ were limiting. These findings indicate that methane formation and growth are less tightly coupled at high concentrations of H₂ or CO₂ in the medium than at low concentrations. The physiological significance of these findings is discussed. K _s: H₂ and CO₂ concentration supporting 0.5 _max; _max: specific growth rate at infinite substrate concentration; Y _s:growth yield (g dry weight/mol substrate); t : doubling time     

Kinetics of growth and lactic acid production in continuous and batch culture

Summary In a lactic acid fermentation by Streptococcus faecalis, the specific consumption rate of glucose (v) and the specific production rate of lactic acid () were represented by the following simple equations as functions of the specific growth rate (): 1/=(1/) + 1/ = (1/) + By use of data from a batch culture, these two equations were derived from enzyme kinetics of the product inhibition. These equations were successfully applied to the results of batch culture and chemostat culture. In addition, calculation of ATP yield by these equations agreed with the experimental results better than the conventional Leudeking-Piret type equation, which includes two terms associated with growth and not with growth. Correspondence to: H. Ohara     

Expression of the alpha subunit of 7S nerve growth factor in the mouse submandibular gland

-NGF is an inactive serine protease that is associated in the mouse submandibular gland with a closely related serine protease, -NGF, and the neurotrophic factor, -NGF. The heterogeneity of purified -NGF has been examined by DEAE-cellulose chromatography and SDS polyacrylamide gel electrophoresis. A possible explanation for the observed heterogeneity is presented. Antibodies have been prepared against -NGF and purified by affinity chromatography so that they do not cross-react with -NGF. This antibody preparation recognizes two very similar proteins in male mouse submandibular gland RNA-directed cell-free translation mixtures. The expression of only one of these forms is regulated by testosterone. Oligonucleotide probes specific for each of the three NGF subunits have been prepared and used for Northern blot analysis of RNA from the mouse submandibular gland. The three subunits were found to be coordinately expressed and each were 30-fold more abundant in male than in female glands.Abbreviations used NGF nerve growth factor - -, -, and -NGF -, -, and -subunits of mouse 7S NGF - PBS phosphate buffered saline - DTT dithiothreitol - PPO 2,5-Diphenyloxazole - DMSO dimethylsulfoxide - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - SSC 0.15M NaCl, 15 mM sodium citrate Supported by USPHS research grant NS19964. This paper is respectfully dedicated to Profs. Eric M. Shooter and Silvio Varon in recognition of their many contributions to our understanding of the structure and function of nerve growth factor.     

Concurrent effect of visible light on γ-particles,chitin synthetase,and encystment capacity in zoospores ofBlastocladiella emersonii

Summary Zoospores derived from ordinary colorless plants ofBlastocladiella emersonii grown under 240 W/cm² of visible light contain an average of ca. 5 -particles and numerous aggregates of cytoplasmic granules which resemble the -matrix, while spores from dark-grown plants contain ca. 12 -particles but none of the granules. Correspondingly, the amount of chitin synthetase associated with -particles is approximately proportional to the number of -particles in the two spore types. The foregoing light effects, known to be accompanied by an increased capacity for encystment, probably take place before sporogenesis. Conversely, zoospores derived from dark-grown plants do not encyst appreciably over an 8 h period unless they are illuminated, but the number of -particles/spore decreases from 12 to ca. 6 whether or not light is present. Arguments are presented for considering the latter disappearance of -particles as an essential ark reaction which precedes a photochemical reaction, both being needed for light-induced encystment.     

Autoradiographic Characterisation of [35S]GTPγS Binding Sites in Rat Brain

The binding of [³⁵S]GTPS was characterised with autoradiography in rat brain. The binding was saturable, but the rate of dissociation was very slow. Analysis of binding isotherms revealed one class of binding sites with a Kd of 0.8 M. The specific binding was 98%. Different guanine nucleotides were all able to compete with [³⁵S]GTPS binding. However, no displacement was seen by the ATP-analogue App[NH]p, indicating that [³⁵S]GTPS does not bind to ATP-sites. Autoradiograms showed a highly homogenous distribution of [³⁵S]GTPS binding, in grey as well as in white matter. However, the pattern changed dramatically in the presence of GTP, which, unlike the non-hydrolysable GTP-analogues Gpp[NH]p and GTPS, did not displace [³⁵S]GTPS binding throughout the brain. In white matter areas the binding was potently displaced, while in many grey matter areas, e.g., the striatum, the binding was seen to increase. This GTP-induced increase in [³⁵S]GTPS binding was strongly Mg²⁺-dependent, with an optimum at 10 mM. This, together with the finding that the regional effects of GTP correspond well to previously reported distribution of low Km GTPase, suggest that the levels of binding of [³⁵S]GTPS in the presence of GTP may reflect functional G-protein activity.     

Characterization of specific calcitonin gene related peptide receptors present in hamster pancreatic β cells

Calcitonin gene-related peptide (CGRP) shares about 46% and 20% amino acid sequence homology with islet amyloid polypeptide (IAPP) and salmon calcitonin (sCT). We investigated whether these related peptides could cross-react with the specific binding of¹²⁵I-[His]hCGRP I to the CGRP receptor in hamster insulinoma cell membranes. A rapid dissociation of membrane bound¹²⁵I-[His]hCGRP I could be induced in the presence of 1 M chicken CGRP (cCGRP). The specific¹²⁵I-[His]hCGRP I binding was inhibited by the related peptides and their half-maximal inhibitory concentrations (IC₅₀) were: cCGRP (0.1 nM), rat CGRP I and human CGRP I and II (1.0–2.0 nM), fragment of hCGRP I (8-37) (150 nM), human IAPP (440 nM). The non-amidated form of hIAPP; human diabetes-associated peptide (hDAP) did not inhibit the binding of¹²⁵I-[His]hCGRP I and sCT was only effective at a high concentration (1 M). Binding of¹²⁵I-[His]hCGRP I was dose dependently inhibited by guanosine-5-O-(3-thiotriphosphate) or (GTPS) and a 70% reduction of binding was obtained with 0.1 mM GTPS. The IC₅₀ value of cCGRP (0.1 nM) was increased 100-fold in the presence of 0.1 mM GTPS. Human CGRP I and cCGRP at 2.5 M did not stimulate the activity of hamster insulinoma cell membranes adenylate cyclase, while glucagon (1 M) induced a 2-fold increase. Thus, specific CGRP receptors present in hamster cells are associated with G protein (s) and IAPP can interact with these receptors. These results and the observation that cCGRP and hCGRP I did not influence adenylate cyclase activity provide further evidence for CGRP receptor subtypes.Abbreviations CGRP calcitonin gene-related peptide - IAPP islet amyloid polypeptide - IC₅₀ half-maximal inhibitory concentration - GTPS guanosine-5-O-(3-thiotriphosphate) - ¹²⁵I [His]hCGRP I, (2[¹²⁵I]iodohistidyl¹⁰) human CGRP I     

Synthesis and secretion of caseins by the mouse mammary gland: Production and characterization of new polyclonal antibodies

Polyclonal antibodies to mouse - and /-caseins were raised in rabbits. These antibodies display tissue- and species specificity as shown by immunoblotting. Immunohistochemical analyses demonstrate that both - and /-caseins were synthesized and secreted from virtually all lactating mammary epithelial cells, in a pattern very similar to that of the mouse -lactalbumin. Residual amounts of caseins were located also in the apical surface of epithelial cells surrounding the ducal lumen of virgin mammary gland sections. In contrast to the significant level of -casein in the milk, the amount of this protein compared to - or -caseins was extremely low in medium conditioned for 24 h by mammary explants of mid-pregnant mice immediately after explantation or after 4 days.     

A role for G-proteins in the epidermal growth factor stimulation of phospholipase A2 in rat kidney mesangial cells

We have previously demonstrated phospholipase C (PLC) independent activation of phospholipase A₂(PLA₂) by epidermal growth factor (EGF) in glomerular mesangial cells in culture. In the current study using glass beads to permeabilize [³H]- or [¹⁴C]-arachidonate labelled mesangial cells we demonstrate that guanine nucleotides modulate the EGF-mediated stimulation of arachidonic acid release (75% inhibition with 100 M GDPS and 108% augmentation with 100 M GTPS). GTPS alone stimulated both the release of free arachidonic acid and production of diacylglycerol (DAG), while EGF itself neither stimulated DAG nor augmented the DAG response to GTPS. These findings suggest the intermediacy of a G-protein in PLC-independent stimulation of PLA₂ by a growth factor, and provide a model system for determining the relationship between G-protein intermediacy and the intrinsic tyrosine kinase activity of the growth factor receptor.Abbreviations EGF Epidermal Growth Factor - PLC phospholipase C - PLA₂ phospholipase A₂ - DAG Diacylglycerol - NEFA non-esterified fatty acid - GTPS guanosine-5-0-[3-thio]triphosphate - GDP\S guanosine-5-0-[2-thio]diphosphate