scholarly journals Stimulation of phospholipase D in rabbit platelet membranes by nucleoside triphosphates and by phosphocreatine: roles of membrane-bound GDP, nucleoside diphosphate kinase and creatine kinase

1994 ◽  
Vol 299 (3) ◽  
pp. 701-709 ◽  
Author(s):  
X T Fan ◽  
J L Sherwood ◽  
R J Haslam
Keyword(s):  
Creatine Kinase ◽  
Phospholipase D ◽  
Phorbol Esters ◽  
Nucleoside Diphosphate Kinase ◽  
Rabbit Platelet ◽  
Platelet Membranes ◽  
Nucleoside Triphosphates ◽  
Membrane Bound ◽  
Fold Stimulation ◽  
Stimulation Of

Previous work has shown that guanosine 5′-[gamma-thio]triphosphate (GTP[S]) and GTP stimulate phospholipase D (PLD) in rabbit platelet membranes and that these effects are greatly enhanced by pretreatment of platelets with phorbol esters that activate protein kinase C [Van der Meulen and Haslam (1990), Biochem. J. 271, 693-700]. In the present study, the effects of Mg2+, various nucleoside triphosphates and phosphocreatine (PCr) were investigated. Platelet membranes containing phospholipids labelled with [3H]glycerol were assayed for PLD in the presence of an optimal Mg2+ concentration (10 mM) by measuring [3H]phosphatidylethanol formation in incubations that included 300 mM ethanol. In membranes from phorbolester-treated platelets, the same maximal increases in PLD activity (5-fold) were seen with 1 microM GTP[S]), and 100 microM GTP. Addition of adenosine 5′-[gamma-thio]triphosphate (ATP[S]), ITP, XTP, UTP and CTP had similar stimulatory effects, but only at > or = 1 mM. In contrast, ATP had a biphasic action, causing a maximal (2-fold) stimulation at 10 microM and smaller effects at higher concentrations; the inhibitory component of the action of ATP was blocked by 2 microM staurosporine. Guanosine 5′-[beta-thio]diphosphate decreased the stimulatory effects of ATP and ATP[S]. UDP, which can inhibit nucleoside diphosphate kinase (NDPK), decreased the activation of PLD by ATP[S], ATP, XTP, CTP and to a lesser extent ITP, but had no effect on the actions of GTP[S] and GTP. Rabbit platelet membranes contained NDPK and addition of [gamma-32P]ATP led to the formation of [32P]GTP in amounts sufficient to explain most or all of the activation of PLD; UDP prevented GTP formation. PCr (0.04-1 mM) also stimulated membrane PLD activity, an effect that was dependent on endogenous membrane-bound creatine kinase (CK). UDP and guanosine 5′-[beta-thio]diphosphate each inhibited this effect of PCr. The results show that in rabbit platelet membranes, CK, NDPK and the GTP-binding protein that activates PLD can be functionally coupled. However, assay of membrane preparations at increasing dilutions showed that stimulation of PLD by the compounds studied, with the partial exception of ATP[S], involved diffusible rather than protein-bound intermediates.

Insulin increases the turnover rate of Na+-K+-ATPase in human fibroblasts

2001 ◽  
Vol 280 (4) ◽  
pp. C912-C919 ◽  
Author(s):  
Nicola Longo ◽  
Fernando Scaglia ◽  
Yuhuan Wang
Keyword(s):  
Plasma Membrane ◽  
Turnover Rate ◽  
Phorbol Esters ◽  
Human Fibroblasts ◽  
Insulin Stimulation ◽  
Automatic Response ◽  
Intracellular Content ◽  
Membrane Bound ◽  
Measurable Change ◽  
Stimulation Of

Insulin stimulates K+ transport by the Na+-K+-ATPase in human fibroblasts. In other cell systems, this action represents an automatic response to increased intracellular [Na+] or results from translocation of transporters from an intracellular site to the plasma membrane. Here we evaluate whether these mechanisms are operative in human fibroblasts. Human fibroblasts expressed the α1 but not the α2 and α3 isoforms of Na+-K+-ATPase. Insulin increased the influx of Rb+, used to trace K+ entry, but did not modify the total intracellular content of K+, Rb+, and Na+ over a 3-h incubation period. Ouabain increased intracellular Na+ more rapidly in cells incubated with insulin, but this increase followed insulin stimulation of Rb+ transport. Bumetanide did not prevent the increased Na+ influx or stimulation of Na+-K+-ATPase. Stimulation of the Na+-K+- ATPase by insulin did not produce any measurable change in membrane potential. Insulin did not affect the affinity of the pump toward internal Na+ or the number of membrane-bound Na+-K+-ATPases, as assessed by ouabain binding. By contrast, insulin slightly increased the affinity of Na+-K+-ATPase toward ouabain. Phorbol esters did not mimic insulin action on Na+-K+-ATPase and inhibited, rather than stimulated, Rb+ transport. These results indicate that insulin increases the turnover rate of Na+-K+-ATPases of human fibroblasts without affecting their number on the plasma membrane or modifying their dependence on intracellular [Na+].

scholarly journals Sulphation of proteochondroitin and 4-methylumbelliferyl β-d-xyloside-chondroitin formed by mouse mastocytoma cells cultured in sulphate-deficient medium

1993 ◽  
Vol 296 (1) ◽  
pp. 119-126 ◽  
Author(s):  
J E Silbert ◽  
G Sugumaran ◽  
J N Cogburn
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Deae Cellulose ◽  
Size Distributions ◽  
Sulphate Concentration ◽  
Deficient Medium ◽  
Mastocytoma Cells ◽  
Membrane Bound ◽  
Fold Stimulation ◽  
Stimulation Of

Mouse mastocytoma cells were cultured in medium containing [3H]GlcN and concentrations of [35S]sulphate varying from 0.01 to 0.5 mM. Intracellular [35S]sulphate incorporation increased severalfold from the lowest concentrations, reaching a maximum at 0.1-0.2 mM, whereas incorporation of [3H]hexosamine remained constant at all sulphate concentrations. Proteo[3H]-chondroitin [35S]sulphate was isolated and incubated with chondroitin ABC lyase, yielding 35S-labelled and/or 3H-labelled delta Di-0S and delta Di-4S disaccharide products. The increasing percentage of delta Di-4S was consistent with the increasing sulphate incorporation at each higher [35S]sulphate concentration. Examination of proteochondroitin [35S]sulphate size by Sepharose CL-6B chromatography indicated a range consistent with various numbers of glycosaminoglycan chains on the protease-resistant serglycin core protein. Alkali-cleaved chondroitin [35S]sulphate products indicated similar size distributions at all sulphate concentrations with no indication of preferential sulphation being related to smaller or larger size. DEAE-cellulose chromatography of [3H]chondroitin [35S]sulphate glycosaminoglycans indicated a random undersulphation as [35S]sulphate concentration was lowered. Addition of 4-methylumbelliferyl beta-D-xyloside to the cultures resulted in a 2-2.5-fold stimulation of [3H]chondroitin [35S]sulphate synthesis with formation of beta-xyloside-[3H]chondroitin [35S]sulphate which was much smaller, as estimated by Sepharose CL-6B chromatography, than the decreased amount of [3H]chondroitin [35S]sulphate derived from proteo[3H]chondroitin [35S]sulphate. Much higher concentrations of sulphate were necessary to produce sulphation of the beta-xyloside-[3H]chondroitin comparable with that of proteo[3H]-chondroitin, as indicated by chondroitin ABC lyase products and DEAE-cellulose chromatography. The specific radioactivities of the [3H]GalN in the proteo[3H]chondroitin [35S]sulphate and beta-xyloside-[3H]chondroitin [35S]sulphate were calculated from the 3H and 35S c.p.m. of isolated dual-labelled delta Di-4S from each, and indicated that the presence of the beta-xyloside resulted in a dilution of the [3H]GlcN by endogenous GlcN that was 4 times higher than that of cultures lacking the beta-xyloside. The higher sulphate concentrations needed for sulphation of beta-xyloside-chondroitin suggests that the membrane-bound nature of the proteochondroitin acceptor in juxtaposition to a chondroitin sulphate-synthesizing enzyme complex effectively reduces the apparent Km for adenosine 3′-phosphate 5′-phosphosulphate.

Interaction of Vasopressin with Human Blood Platelets: Dependency on Mg2+

1986 ◽  
Vol 56 (03) ◽  
pp. 260-262 ◽  
Author(s):  
Isabella Roos ◽  
Fabrizia Ferracin ◽  
Alfred Pletscher
Keyword(s):  
Phosphatidic Acid ◽  
Human Blood ◽  
Arginine Vasopressin ◽  
Specific Binding ◽  
Blood Platelets ◽  
Bivalent Cations ◽  
Platelet Membranes ◽  
Maximal Rise ◽  
Human Blood Platelets ◽  
Stimulation Of

SummaryArginine-vasopressin (AVP) in the presence of Mg2+ but not in the absence of bivalent cations led to accumulation of [32P]-phosphatidic acid ([32P]-PA) in human blood platelets. Mg2+ also enhanced the specific binding of [3H]-AVP to intact platelets. The concentrations of the cation which enabled AVP to cause half maximal rise of [32P]-PA and those inducing half maximal [3H]-AVP-binding were of the same order. It is concluded that the stimulation of phosphatidyl inositide breakdown by AVP in presence of Mg2+ is at least partially due to a Mg2+-induced enhancement of specific AVP-binding to the platelet membranes.

Kinetin-Mediated Stimulation of Accumulation of Buckwheat Flavonoids in the Dark

1983 ◽  
Vol 38 (9-10) ◽  
pp. 711-718 ◽  
Author(s):  
U. Margna ◽  
T. Vainjärv
Keyword(s):  
Anthocyanin Biosynthesis ◽  
Flavonoid Biosynthesis ◽  
Accumulation Rates ◽  
Sharp Rise ◽  
Short Treatment ◽  
Exogenous Substrate ◽  
Percent Increase ◽  
High Level ◽  
Fold Stimulation ◽  
Stimulation Of

A short treatment of excised buckwheat cotyledons with a solution of kinetin lead to an up to 9-fold stimulation of anthocyanin biosynthesis, to an about 50 percent increase in the accumula­tion of rutin, and to an about 30 percent increase, on the average, in the accumulation of C-glycosylflavones in the treated material during its posttreatment incubation in the dark. When the treated cotyledons were incubated in a solution of ʟ--phenylalanine anthocyanin accumulation in the dark practically attained the same high level as it was observed in the illuminated cotyledons fed with exogenous ʟ--phenylalanine. In experiments with l4C-labelled L-phenylalanine kinetin induced a sharp rise in the labelling (resp. in the utilization of exogenous substrate for biosynthesis) of anthocyanins and rutin in the dark and a slight increase in the radioactivity of C-glycosylflavones. Similar labelling changes occurred in the illuminated cotyledons. However, both kinetin and light still more effectively promoted biosynthetic use of the endogenous sub­strate. As a result the relative portion of flavonoids formed from exogenous L-phenylalanine under these conditions showed a decrease as compared with the ratio of precursor use in the un­treated cotyledons. The results show that low accumulation rates of anthocyanins and other flavo­noids in the dark are conditioned by the limited access of substrate (ʟ--phenylalanine) molecules to the flavonoid enzymes lending further support to the idea that flavonoid biosynthesis is normally controlled at the substrate rather than at the enzymic level.

Phorbol Esters Stimulate Phospholipase D Activity in C-6 Glioma Cells

1989 ◽  
Vol 16 (3) ◽  
pp. 257-262
Author(s):  
Lena Gustavsson ◽  
Christofer Lundqvist ◽  
Christer Ailing
Keyword(s):  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Culture Medium ◽  
Phorbol Esters ◽  
Glioma Cells

The effects of phorbol esters on phospholipase D activity were studied in C-6 glioma cells. The cell lipids were prelabelled with [3H]-glycerol or [14C]-arachidonic acid. Phosphatidylethanol was formed during stimulation with 100nM 12-0-tetradecanoylphorbol-13-acetate (TPA), when ethanol was present in the culture medium. After 30 minutes of stimulation, phosphatidylethanol constituted 2.6% of the [3H]-glycerol-labelled lipids. Stimulating the cells with TPA in the absence of ethanol caused a significant increase in labelled phosphatidic acid. This increase was inhibited by ethanol. The present findings demonstrate that TPA stimulates phospholipase D activity in cultured C-6 glioma cells.

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