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Mechanism of inhibition by cyclic AMP of protein kinase C-triggered respiratory burst in Ehrlich ascites tumour cells
Cellular Signalling Vol. 4, No. 6, pp. 651~563, 1992.
0898-6568/92 $5.00 + 0.00 © 1992 Pergamon P~'~s Ltd
Printed in Great Britain,
MECHANISM OF INHIBITION BY CYCLIC AMP OF PROTEIN KINASE C - T R I G G E R E D RESPIRATORY BURST IN EHRLICH ASCITES T U M O U R CELLS BHARATHI P. SALIMATHand G. SAVITHA Department of Biochemistry, University of Mysore, Manasagangothri, Mysore 570 006, India (Received 19 March 1992; and accepted 11 June 1992)
Abstract--The superoxide anion generation in Ehrlich aseites tumour (EAT) cells increased more than two-fold in the presence of the tumour promoter, tetradecanoyl phorbol myristate acetate (TPA). Epinephrine and dibutryl cAMP (Bt2 cAMP) inhibited in a dose-dependent manner, both basal and TPA-triggered superoxide generation in EAT cells. The kinetics of inhibition of superoxide generation showed a maximum inhibition between 30 and 40 min of preincubation with epinephrine or Bt2 cAMP of EAT cells and coincided with an increase in activity of a phosphoprotein phosphatase. In TPA-treated EAT cells, epinephrine or Bt2 cAMP increased the phosphatase activity in a dose-dependent manner. In vitro EGTA, EDTA and sodium fluoride inhibited phosphatase activity. Superoxide generation in response to TPA in Triton-permeabilized EAT cells was inhibited by inclusion of the phosphatase in the assay. Taken together, these results clearly suggest that the phosphatase activity in EAT cells develops as a result of protein kinase A (PKA) and protein kinase C (PKC)mediated phosphorylation of the phosphatase which then mediates dephosphorylation of the PKC-triggered phosphorylation of proteins to inhibit respiratory burst. A cross-talk between PKA and PKC pathways negatively modulates superoxide generation in EAT cells. Key words: Respiratory burst, protein kinase C, cAMP, phosphoprotein phosphatase, EAT cells.
INTRODUCTION Tim REACTIVEoxygen species generating respiratory burst oxidase activity of professional phagocytes (neutrophils, macrophages, monocytes and eosinophils) is of prime importance for the microbicidal, tumouricidal and antigen processing functions of the phagocytes [1]. The phagocytes from patients with chronic granulomatous disease (CGD), an inherent disorder which is characterized by recurrent infections, and a granulomatous tissue reaction, fail to mount an effective respiratory burst activity. On another extreme is the increased or overproduction of reactive oxygen species by Abbreviations: BSA--bovine serum albumin; CGD--chronic granulomatous disease; DMSO----dimethylsulphoxide; EAT--Ehrlich ascites tumour; HBSS----Hanks' balanced salt solution; NBT--Nitroblue tetrazoliurn; OD---optieal density;Pi--inorganie phosphate; PKA--proWin kinas¢ A; PKC--protein kinase C; SOD-----supcroxide dismutase; TPA--tetradeeanoylphorbol myristateacetate. 651
N A D P H oxidase of macrophages, fibroblasts and eosinophils which are implicated in inflammation and damaging of the joints, in rheumatoid arthritis, and alterations in basement membranes of blood vessels and the surrounding connective tissues, respectively [2,3]. Therefore, the regulation of N A D P H oxidase, the respiratory burst enzyme, is central in maintenance of normal well-being as any modulations in regulation have deleterious effects. The property of respiratory burst activity is not restricted to phagocytes and is also observed in tumour cells and several other cell types. To mention a few, human leukemia cells (HL 60) differentiated into neutrophil-like cells, AK-5 cells, a macrophage-like cell line [4], lymphocytes from burkitt cell lines and tonsillar B lymphocytes are shown to generate reactive oxygen species [5]. An intricate network of several signalling pathways is implicated in the regulation of
652
B.P. SALIMATHand G. SAVITHA
N A D P H oxidase [6]. Protein phosphorylation is a major regulatory mechanism governing key cellular events in m a n y cells and tissues. This is also true with phagocytes where protein phosphorylation by protein kinase C (PKC) is a major mechanism regulating the activity of N A D P H oxidase [7,8]. Studies have shown that cytosolic proteins (p48 and p67) which are deficient in variants of C G D are essential for activation of NADPH oxidase [9]. Phosphorylation and translocation of these cytosolic proteins to phagocyte plasma membrane is obligatory in activation of respiratory burst [10,11]. While several studies have been oriented towards phosphorylation and activation of respiratory burst or identification and characterization of substrates for PKC, the reversal of the event by dephosphorylation and inactivating the active phagocyte to normal resting state are not undertaken. Several responses of phagocytes, including respiratory burst, are inhibited by fl-adrenergic agonists or agents which increase intracellular c A M P concentration [12-14]. However, the mechanism of inhibition has not been delineated. Intensive work on phosphoprotein phosphatases has suggested that phosphorylation of specific phosphatase by A-kinase and thereby its activation, results in dephosphorylation of phosphorylated proteins and inactivation of agonist-induced activation [15]. This logic was applied to delineate the mechanism of inhibition by c A M P of tetradecanoyl phorbol myristate acetate (TPA)-triggered respiratory burst of Ehrlich ascites tumour (EAT) cells.
MATERIALS AND METHODS TPA, phenyl methyl sulphonyl fluoride, superoxide dismutase (SOD), alkaline phosphatase, bovine serum albumin (BSA) and dibutryl cAMP (Bt2 cAMP) were obtained from Sigma, St Louis, MO, U.S.A. Nitroblue tetrazolium (NBT), dioxan and malachite green were from Sisco Research Labs, India. Epinephrine was purchased from Weyth Laboratories, India. Mice bearing EAT cells were obtained from the Cancer Research Centre, Bombay, India. All other chemicals and reagents used were of analytical grade.
Culture of EAT cells EAT cells were grown in the peritoneal cavity of 6-8-week-old Swiss mice by serial transplantation. Cells were harvested 8-10 days after transplantation and the viability of the cells was determined by trypan blue exclusion. More than 95% of the freshly harvested cells were viable. These cells were suspended in Hanks' balanced salt solution (HBSS) containing 0.4% BSA.
Assay of superoxide generation The SOD-inhibitable superoxide generation by NADPH oxidase of the EAT cells was assayed by NBT-reduction assay, as described earlier [16]. Briefly, EAT cells (5 x 104) were incubated with or without TPA (160nM) at 37°C for 8 min. NBT 60 nmol was added and the incubation was con: tinued for 20 more rain at 37°C. Whenever epinephrine or Bt2 cAMP was used in the assay, the EAT cells were preincubated with it for either 20 rain or for different time intervals prior to exposure to TPA (160 nM). The blue formazan formed as a result of NBT reduction due to generation of reactive oxygen species was extracted using dioxan and absorbance was read at 540 nm. A standard curve for NBT (0-80 nmoi) was done in parallel.
Assay of phosphoprotein phosphatase Preparation of the cytosol and membrane fractions Freshly harvested EAT cells were taken as six aliquots containing equal numbers of cells. Cells were either treated with TPA (160nM) or with epinephrine (100#M) or with Bh cAMP (500#M), or with both TPA and epinephrine (100#M or various concentrations), or TPA and Bh cAMP (500/zM or various concentrations). Cells without TPA or epinephrine or Bt2 cAMP served as a control. Cells with epinephrine or Bt 2 cAMP were preincubated for 20 min or for various time intervals prior to addition of TPA. After 10 rain of incubation with TPA at 37°C, the cells were collected by centrifugation (400g, 8 min 4°C) and resuspended in cold 10 mM Tris-HC1 buffer (pH 7-5) containing 0.1 mM PMSF. The cells were disrupted by sonication (10 s, 20 W, on ice). After removing the cell debris and nuclei by low-speed centrifugation (400 g, 10 rain), the supernatant was subjected to centrifugation at 100,000g, for 1 h. While the high-speed supernatant(s) was used as a source of crude phosphatase activity, the high-speed pellet of TPA-treated ceils served as PKC, phosphorylated endogenous membrane protein substrates.
Inhibition of respiratory burst by phosphoprotein phosphatase
Phosphatase assay A sensitive non-radioactive method for the assay of phosphoprotein phosphatase as described by Geladopoulos et al. [17] was adopted for the assay of phosphatase from EAT cells. The total reaction mixture of 2ml consisted of 10mM Tris-HCl buffer (pH 8.0), 200 #g of substrate, 100/~g of enzyme source and + sodium fluoride. The enzyme was added to the prewarmed reaction mixture to initiate the reaction. After incubation for 10 min, the reaction was stopped by chilling. The solution was centrifuged at 1000g for 10 min. An aliquot of the supernatant was added to 0.5ml malachite green solution and was incubated for 10 min at 37°C. The absorbance was read at 620 nm. Suitable controls of either enzyme or substrate alone of six different groups of cells were included. The corrected values of optical density (OD) at 620 nm were obtained after subtracting the additive OD of enzyme alone and substrate alone from the OD obtained with both enzyme and substrate together. A standard curve of absorbance at 620nm for phosphate (0-12 nmol) was determined in parallel. Preparation of labelled membrane substrate EAT cells (1 x 107 cells) in prewarmed HBSS buffer (pH 7.4) were incubated at 37°C for 60 min in the presence of 200-250/~Ci of 32p-pi. After 60 min the cells were placed on ice and used in subsequent experiments [18]. In experiments to check the effect of epinephrine on TPA-triggered phosphorylation, labelled EAT cells were first incubated for 20 min at 37°C with epinephrine (100 #M). TPA (160 nM) was added and incubation was continued for a further 10 min. Labelled EAT cells without any TPA or epinephrine or with TPA alone or with epinephrine alone served as appropriate controls. The cells were collected by centrifugation (400g, 8 min, 4°C) and resuspended in ice-cold 10mM Tris-HC1 buffer (pH 7.4) containing 0.25% sucrose, disrupted in cold using a glass-Teflon homogenizer. The lysate was centrifuged at 100,000g, for 1 h, and the pellet redissolved in the electrophoresis sample buffer. After immersion of the samples in a boiling water bath for 2 min, the samples were subjected to SDS--PAGE, on 1-mm-thick slab gels, consisting of 4.5% (w/v) acrylamide stacking gel and 12.5% (w/v) separating gel and gels were run at a constant voltage of 180V according to the method of Laemmli [19]. After staining and destaining, gels were dried and subjected to autoradiography at -70°C for 2-4 days using Kodak X-Omat X-ray films with intensifying screens. An aliquot of phosphorylated membrane from the TPA-treated labelled EAT cells, and the cold phosphatase (100 #g cytosol) from the TPA- and epinephrine-treated cells (prepared as described above), was incubated for 10 rain at 37°C with or without sodium fluoride. The reaction was terminated by the addition of electrophoresis sample buffer. SDS--PAGE and autoradiography were done as described above.
653
Protein was estimated according to Lowry et al.
[20] using BSA as standard. TPA and Bt 2 cAMP were prepared as a stock solution in dimethyl sulphoxide (DMSO I mg/ml) and the final concentration of DMSO in the assay system was less than 0.1% throughout.
RESULTS
Effect of TPA epinephrine and Bt 2 c A M P on respiratory burst The preliminary data demonstrating that E A T cells show a preactivated respiratory burst, when compared to resting phagocytes, are shown in Fig. I. With T P A as an activator, a further two-fold increase of respiratory burst was observed. Epinephrine (50#M) and Bt 2 c A M P (500#M) inhibited respiratory burst both in control and TPA-treated E A T cells.
Phosphatase activity in cells receiving dual signals In cytosol from either control cells or from cells treated with TPA there is a basal level of phosphatase activity (Table 1A). In the presence of epinephrine (50 #M) there is a slight increase in phosphatase activity. However, dual signalling with both T P A and epinephrine or TPA and Bt2 cAMP, dramatically activates the phosphatase by approximately four- to sevenfold. The phosphatase activity obtained from T P A and epinephrine or T P A and Bt 2 cAMPtreated E A T cell cytosol released inorganic phosphate (Pi) from both control E A T cell membrane or TPA-activated EAT cell membrane. The phosphatase(s) had no effect on the membrane as substrate obtained either from TPA plus epinephrine- or T P A plus Bt: cAMPtreated cells or on epinephrine alone or Bt 2 cAMP-treated cells (Table I B). Membranes from control cells were not such good substrates when compared to that from TPA-activated cells, where there was a two-fold increased release of Pi from TPA-activated cell membranes by the dual signal-induced phosphatase activity. Alkaline phosphatase had no
654
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FIG. I. Inhibition by epinephrine and Bt~ cAMP of basal and TPA-triglFred respiratory burst in EAT cells. I-q, EAT cells; [], normal phagocytes. The data represent the mean+S.D, of the results obtained from three experiments performed in duplicate.
effect whatsoever on any of the substrate sources.
Kinetics of inhibition of respiratory burst with simultaneous activation of phosphatase by epinephrine or Bt2 cAMP Kinetics of inhibition of respiratory burst by epinephrine or Bt2 cAMP in both control and TPA-activated cells are shown in Fig. 2a and b. Half-maximal inhibition of respiratory burst in control cells was seen at 30 min while the same effect was seen within 5 min in TPA-activated cells, by either epinephrine or Bt: cAMP. Within 30 min, epinephrine and Btz cAMP caused 100% inhibition. Because of the effectiveness of epinephrine or Bt: cAMP inhibition of TPA-activated cells, the cytosol from such cells was assayed for kinetics of phosphatase activity with TPA-treated EAT cell membrane
as substrate. With an increase in the time of incubation with epinephrine or Bh cAMP, a simultaneous increase in phosphatase activity was seen. By 30 min, maximum phosphatase activity could be detected in both cases.
Effect of increasing concentration of epinephrine and Bt2 cAMP on respiratory burst and phosphatase activity The maximum respiratory burst seen in the presence o f TPA was inhibited in a dose-dependent manner by epinephrine or Bt2 cAMP with I¢ 50 values of 20 and 80/~M, respectively. As is shown in Fig. 3a and b, in the presence of TPA and in the absence of epinephrine or Bh cAMP, little or no phosphatase activity could be detected. With the inclusion o f various concentrations o f epinephrine or Bh cAMP, there was a rise in the phosphatase activity in the cytosol.
655
Inhibition of respiratory burst by phosphoprotein phosphatase TABLE 1A. DUAL SIGNALS,TPA AND EPINEPHRINE-OR TPA
A N D 13t 2 CYTOSOL EXHIBIT PHOSPHATASE ACTIVITY
Substrate for phosphatase (200/~g)
Specific activity (nmol Pi/mg protein/10 min)
TPA activated cell 100,000 g Pellet TPA activated cell 100,000 g Pellet TPA activated cell 100,000 g Pellet TPA activated cell 100,000 g Pellet TPA activated cell 100,000 g Pellet TPA activated cell 100,000 g Pellet
4.3 +_1
Source of phospbatase 1.
Control cell cytosol
2.
TPA-activated cell cytosol
3.
TPA + epinephrine-treated cell cytosol
4.
TPA + Bt2 cAMP-treated cell cytosoi
5.
Epinephrine-treated cell cytosol
6.
Bt2 cAMP-treated cell cytosol
cAMP-TP.EATm)EAT CELL
4.9 +- 1.2 28 -I-3 37 + 4 7 +0.9
The crude phosphatase (100 #g) or the alkaline phosphatase (2.5 U) was incubated with the substrate (200/zg) for 10 min at 37°C. The Pi in the supernatants was estimated by the malachite green method. The data represent the mean +-S.D. of the three experiments performed in duplicate. TABLE 1 B . T P A - P H O S P H O R Y L A T E D MEMBRANE ACTS AS THE SUBSTRATE
Source of cellular phosphatase (nmol Pi/mg protein/10 min at 37°C) Substrate for phosphatase (200 #g) 1. 2. 3. 4. 5. 6.
Control cell--100,000g pellet TPA-activated cell--100,000g pellet TPA +epinephrine-treated cell-100,000 g pellet Epinephrine-treated cell--100,000g pellet TPA+Bt2 cAMP-treated ceU--100,000 g pellet Bt2 cAMP-treated cell--100,000g pellet
TPA and epinephrinetreated cytosol
TPA and Bt2 cAMPtreated cytosol
16.0+_2.5 26.0+_2.8 0
20+_2 33 + 1.9 ND
0
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phosphatase (2.5 U)
B
The crude phosphatase (100 #g) or the alkaline phosphatase (2.5 U) was incubated with the substrate (200 #g) for 10 min at 37°C. The Pi in the supernatants was estimated by the malachite green method. The data represent the mean-t-S.D, of the three experiments performed in duplicate.
M a x i m u m phosphatase activity was seen in 100#M epinephrine- or 500#M Bt 2 cAMPtreated E A T (5.6 x 106) cells, with 160 nM TPA.
At this concentration (100#M) o f epinephrine or 500pM Bt 2 cAMP, respiratory burst was inhibited by 100%.
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FIG. 2. (a, b) Inhibition of superoxide anion production coincides with the development of phosphatase activity by the EAT cells, as a function of time in the presence of agents which increase intracellular cAMP concentration. NBT reduction and Pi release were assayed as described in Materials and Methods. Cells were incubated with 100 pM epinephrine (a) or 500/~M Bt 2 cAMP (b) for the times indicated. A, control; C), TPA-triggered cells; 0 , phosphatase activity. The values are the mean+ S.D. of three experiments performed in duplicate.
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FIG. 3. Dose-dependent inhibition by epinephrine or Bt2 cAMP of TPA-triggered respiratory burst and activation of phosphatase activity. Superoxide anion release was measured after exposure of EAT cells (5 x 106 cells) to different concentrations of epinephrine or Bt2 cAMP for 20 min at 37°C, in the absence ( A ) or presence of TPA (160 riM; ©). The Pi release ( O ) was measured by the malachite green method from the cytosol of cells treated with TPA and epinephrine or Bt~ cAMP using TPA-treated EAT cell membrane as substrate. The values are the mean + S.D. of three experiments performed in duplicate.
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FIG. 4. Dose-dependent inhibition of phosphatase activity by sodium fluoride. The crude phosphatase (100 #g) was incubated with the substrate (200 #g), with or without different concentrations of sodium fluoride for 10 rain at 37°C. The Pi release was measured as described in Materials and Methods. C), TPA-epinephrine-treated cytosol; O, TPA-Bh cAMP-treated cytosol. The data represent the mean_+S.D, of the results obtained from three experiments performed in duplicate.
Effect of sodium fluoride on phosphatase activity The phosphatase activity from cells treated with both TPA and epinephrine or TPA and Bt2 cAMP could effectively be inhibited in a dosedependent manner by sodium fluoride, with a half-maximal inhibition at 250 #M and 200/~M of sodium fluoride, respectively (Fig. 4).
Effect of divalent cations on phosphatase activity Addition of the divalent cations to the incubation mixture did not affect the Pi release from the phosphorylated substrate by the phosphatase. However, addition of EGTA or EDTA to the assay mixture inhibited the Pi release by 100 and 45% in epinephrine--TPA-treated cytosol and 96 and 95% in Bh cAMP TPA-treated cytosol, respectively (Table 2).
Effect of phosphatase on respiratory burst EAT cells were permeabilized using low levels of Triton X-100. When such cells were activated for respiratory burst with TPA (160nM), inclusion of crude phosphatase that is the cytosol from TPA and epinephrine- or TPA and Bt2 cAMP-treated cells, the TPA-triggered respiratory burst was inhibited in a dose-dependent manner (Fig. 5).
Effect of epinephrine and phosphatase on protein phosphorylation Previously, TPA has been shown to induce phosphorylation of up to six proteins in rabbit neutrophils. Although it is difficult to resolve all individual proteins in one dimensional gel there is a definite TPA-induced increase in four to five bands of molecular weight similar to those reported earlier plus a general increased density
Inhibition of respiratory burst by phosphoprotein phosphatase
659
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FIG. 5. Effect of phosphatase on TPA-triggered respiratory burst in Triton-permeabilized EAT cells. (a) 25, (b) 50, (c) 100, (d) 200 #g phosphatases; values are the average of three experiments performed in duplicate.
o f phosphorylation in the entire lane. Figure 6 (photograph o f an autoradiogram) shows the profile of the phosphorylated E A T cell membranes in the presence of T P A (lane c) and
TABLE 2. EFFECT OF METAL ION,
the dephosphorylation effect of epinephrine (100 #M, lane b; 200 #M, lane a). The concentration of epinephrine which inhibits the respiratory burst in control or T P A activation, blocks
EGTA AND E D T A ON PHOSPHATASE ACTIVITY
Phosphatase activity (% of control)
System Control + 10 mM MgCI2 or MnCI 2 + 100 mM MgCI2 or MnC12 +50 and 100 #M C a C i 2 + I mM EGTA +1 mM EDTA
TPA + epinephrinetreated cytosol (a)
TPA + Bt2 cAMPtreated cytosol (b)
1O0 100 100 100 -4.5
1O0 I00 100 100 5.6 5.0
*Control activity was 26 and 29 nmol Pi/mg protein/10 min for a and b, respectively. Phosphatase was assayed as described in Materials and Methods except that the assay mixtures were supplemented with salts at the concentration indicated. Values are the average of three experiments performed in duplicate.
660
B.P. SALIMATHand G. SAVITHA
the phosphorylation of both high- and lowmolecular weight proteins. Further, treatment of TPA-phosphorylated membrane with the phosphatase from TPA and epinephrine-treated EAT cell cytosol (100 and 200#g) induced dephosphorylation of the membrane proteins (lanes d, e). Inclusion of NaF (200#M) protected phosphorylation to a certain extent (lane f). DISCUSSION The respiratory burst in EAT cells is higher when compared to that exhibited by normal resting phagocytes. PKC-triggered phosphorylation of protein(s) is known to activate respiratory burst in neutrophils [21]. The same appears to be the case with EAT cells, where the two-fold increase in respiratory burst was obtained on addition of TPA. Cyclic nucleotides, being among the negative modulators of respiratory burst, when assessed with EAT cells inhibited the respiratory burst. The inhibition of respiratory burst by Bt: cAMP further confirmed the negative modulation by cAMP. The possibility of dephosphorylation and inactivation of respiratory burst seems more likely than multiple-site phosphorylation of a protein by different signals operating as negative modulation. Our results proved this hypothesis where EAT cells expressed phosphoprotein phosphatase activity in the cytosol after treatment with epinephrine or Bt2 cAMP and the activity was further increased in cells treated with TPA and epinephrine or Bt2 cAMP. From these results one can infer that phosphorylation by both PKC and protein kinase A (PKA) is required to activate a phosphatase activity which then dephosphorylates the phosphoproteins responsible for the respiratory burst activity. As EAT cells without TPA signal exhibit respiratory burst, it can be assumed that membrane proteins are in a phosphorylated state. Further, treatment with TPA activates respiratory burst and results in further increase of phosphorylated membrane proteins. This was proved by our results which show that a
membrane preparation from TPA-treated EAT cells was a better substrate for the dual-signal phosphatase than that from EAT cells alone. Further evidence for the existence of the phosphatase activity and its regulation by both PKC and PKA came from our results on kinetic studies which showed that in cells treated with both TPA and epinephrine or TPA and Bt2 cAMP, maximum inhibition of respiratory burst was obtained at 30 min of incubation during which time the maximum phosphatase activity was obtained. This view was strengthened by our results on dose-dependent inhibition of TPA-treated EAT cell respiratory burst by either epinephrine or Bt 2 cAMP, where maximum inhibition of respiratory burst at 100 and 500#M, respectively, was accompanied with increased phosphatase activity. Our results on the effect of sodium fluoride, a commonly used phosphatase inhibitor [22], clearly demonstrate the existence of the phosphatase in cells treated with dual signals. Direct evidence for inactivation of respiratory burst by dephosphorylation was obtained from results on the effect of phosphatase on TPA-triggered respiratory burst in permeabilized EAT cells. Labelling studies revealed that TPA-triggered phosphorylation of EAT membrane could be inhibited in the cells treated with epinephrine and the treatment of the dual-signal triggered phosphatase could dephosphorylate the TPA-phosphorylated membrane proteins. In conclusion, the results presented in this paper clearly demonstrate that negative modulation of respiratory burst in EAT cells by cAMP involves dephosphorylation by a phosphoprotein phosphatase. A cross-talk between PKC and PKA occurs in many cell types [23]. So also in EAT cells one can speculate that the phosphatase is regulated by phosphorylation by both PKA and PKC. This is a first preliminary report to show dual-signal regulated phosphatase activity. However, purification of this phosphatase and use of specific kinase-phosphorylated 47,000 or 67,000 Mr as a substrate in vitro will give better evidence for the specificity of the phosphatase. These studies are currently being undertaken.
FIG. 6. Effect of epinephrine, phosphatase and NaF on the phosphorylation of the protein of EAT cells. Protein(s) phosphorylated in EAT cell membrane by TPA (lane c) are dephosphorylated by incubation of cells with epinephrine (lanes a, b) or treatment of membrane with phosphatase (lanes d, e). NaF (lane f) protects phosphorylation.
661
Inhibition of respiratory burst by phosphoprotein phosphatase Acknowledgements--S.G. thanks the council of Scientific Industrial Research for a Senior Research Fellowship. The authors thank PRov. P. S. SASTRY I.I.SC., Bangalore, and DR C. SMARATHKUMAR,City X-ray & Scanning Research Centre, Mysore, for their help in radio labelling techniques.
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0898-6568/92 $5.00 + 0.00 © 1992 Pergamon P~'~s Ltd
Printed in Great Britain,
MECHANISM OF INHIBITION BY CYCLIC AMP OF PROTEIN KINASE C - T R I G G E R E D RESPIRATORY BURST IN EHRLICH ASCITES T U M O U R CELLS BHARATHI P. SALIMATHand G. SAVITHA Department of Biochemistry, University of Mysore, Manasagangothri, Mysore 570 006, India (Received 19 March 1992; and accepted 11 June 1992)
Abstract--The superoxide anion generation in Ehrlich aseites tumour (EAT) cells increased more than two-fold in the presence of the tumour promoter, tetradecanoyl phorbol myristate acetate (TPA). Epinephrine and dibutryl cAMP (Bt2 cAMP) inhibited in a dose-dependent manner, both basal and TPA-triggered superoxide generation in EAT cells. The kinetics of inhibition of superoxide generation showed a maximum inhibition between 30 and 40 min of preincubation with epinephrine or Bt2 cAMP of EAT cells and coincided with an increase in activity of a phosphoprotein phosphatase. In TPA-treated EAT cells, epinephrine or Bt2 cAMP increased the phosphatase activity in a dose-dependent manner. In vitro EGTA, EDTA and sodium fluoride inhibited phosphatase activity. Superoxide generation in response to TPA in Triton-permeabilized EAT cells was inhibited by inclusion of the phosphatase in the assay. Taken together, these results clearly suggest that the phosphatase activity in EAT cells develops as a result of protein kinase A (PKA) and protein kinase C (PKC)mediated phosphorylation of the phosphatase which then mediates dephosphorylation of the PKC-triggered phosphorylation of proteins to inhibit respiratory burst. A cross-talk between PKA and PKC pathways negatively modulates superoxide generation in EAT cells. Key words: Respiratory burst, protein kinase C, cAMP, phosphoprotein phosphatase, EAT cells.
INTRODUCTION Tim REACTIVEoxygen species generating respiratory burst oxidase activity of professional phagocytes (neutrophils, macrophages, monocytes and eosinophils) is of prime importance for the microbicidal, tumouricidal and antigen processing functions of the phagocytes [1]. The phagocytes from patients with chronic granulomatous disease (CGD), an inherent disorder which is characterized by recurrent infections, and a granulomatous tissue reaction, fail to mount an effective respiratory burst activity. On another extreme is the increased or overproduction of reactive oxygen species by Abbreviations: BSA--bovine serum albumin; CGD--chronic granulomatous disease; DMSO----dimethylsulphoxide; EAT--Ehrlich ascites tumour; HBSS----Hanks' balanced salt solution; NBT--Nitroblue tetrazoliurn; OD---optieal density;Pi--inorganie phosphate; PKA--proWin kinas¢ A; PKC--protein kinase C; SOD-----supcroxide dismutase; TPA--tetradeeanoylphorbol myristateacetate. 651
N A D P H oxidase of macrophages, fibroblasts and eosinophils which are implicated in inflammation and damaging of the joints, in rheumatoid arthritis, and alterations in basement membranes of blood vessels and the surrounding connective tissues, respectively [2,3]. Therefore, the regulation of N A D P H oxidase, the respiratory burst enzyme, is central in maintenance of normal well-being as any modulations in regulation have deleterious effects. The property of respiratory burst activity is not restricted to phagocytes and is also observed in tumour cells and several other cell types. To mention a few, human leukemia cells (HL 60) differentiated into neutrophil-like cells, AK-5 cells, a macrophage-like cell line [4], lymphocytes from burkitt cell lines and tonsillar B lymphocytes are shown to generate reactive oxygen species [5]. An intricate network of several signalling pathways is implicated in the regulation of
652
B.P. SALIMATHand G. SAVITHA
N A D P H oxidase [6]. Protein phosphorylation is a major regulatory mechanism governing key cellular events in m a n y cells and tissues. This is also true with phagocytes where protein phosphorylation by protein kinase C (PKC) is a major mechanism regulating the activity of N A D P H oxidase [7,8]. Studies have shown that cytosolic proteins (p48 and p67) which are deficient in variants of C G D are essential for activation of NADPH oxidase [9]. Phosphorylation and translocation of these cytosolic proteins to phagocyte plasma membrane is obligatory in activation of respiratory burst [10,11]. While several studies have been oriented towards phosphorylation and activation of respiratory burst or identification and characterization of substrates for PKC, the reversal of the event by dephosphorylation and inactivating the active phagocyte to normal resting state are not undertaken. Several responses of phagocytes, including respiratory burst, are inhibited by fl-adrenergic agonists or agents which increase intracellular c A M P concentration [12-14]. However, the mechanism of inhibition has not been delineated. Intensive work on phosphoprotein phosphatases has suggested that phosphorylation of specific phosphatase by A-kinase and thereby its activation, results in dephosphorylation of phosphorylated proteins and inactivation of agonist-induced activation [15]. This logic was applied to delineate the mechanism of inhibition by c A M P of tetradecanoyl phorbol myristate acetate (TPA)-triggered respiratory burst of Ehrlich ascites tumour (EAT) cells.
MATERIALS AND METHODS TPA, phenyl methyl sulphonyl fluoride, superoxide dismutase (SOD), alkaline phosphatase, bovine serum albumin (BSA) and dibutryl cAMP (Bt2 cAMP) were obtained from Sigma, St Louis, MO, U.S.A. Nitroblue tetrazolium (NBT), dioxan and malachite green were from Sisco Research Labs, India. Epinephrine was purchased from Weyth Laboratories, India. Mice bearing EAT cells were obtained from the Cancer Research Centre, Bombay, India. All other chemicals and reagents used were of analytical grade.
Culture of EAT cells EAT cells were grown in the peritoneal cavity of 6-8-week-old Swiss mice by serial transplantation. Cells were harvested 8-10 days after transplantation and the viability of the cells was determined by trypan blue exclusion. More than 95% of the freshly harvested cells were viable. These cells were suspended in Hanks' balanced salt solution (HBSS) containing 0.4% BSA.
Assay of superoxide generation The SOD-inhibitable superoxide generation by NADPH oxidase of the EAT cells was assayed by NBT-reduction assay, as described earlier [16]. Briefly, EAT cells (5 x 104) were incubated with or without TPA (160nM) at 37°C for 8 min. NBT 60 nmol was added and the incubation was con: tinued for 20 more rain at 37°C. Whenever epinephrine or Bt2 cAMP was used in the assay, the EAT cells were preincubated with it for either 20 rain or for different time intervals prior to exposure to TPA (160 nM). The blue formazan formed as a result of NBT reduction due to generation of reactive oxygen species was extracted using dioxan and absorbance was read at 540 nm. A standard curve for NBT (0-80 nmoi) was done in parallel.
Assay of phosphoprotein phosphatase Preparation of the cytosol and membrane fractions Freshly harvested EAT cells were taken as six aliquots containing equal numbers of cells. Cells were either treated with TPA (160nM) or with epinephrine (100#M) or with Bh cAMP (500#M), or with both TPA and epinephrine (100#M or various concentrations), or TPA and Bh cAMP (500/zM or various concentrations). Cells without TPA or epinephrine or Bt2 cAMP served as a control. Cells with epinephrine or Bt 2 cAMP were preincubated for 20 min or for various time intervals prior to addition of TPA. After 10 rain of incubation with TPA at 37°C, the cells were collected by centrifugation (400g, 8 min 4°C) and resuspended in cold 10 mM Tris-HC1 buffer (pH 7-5) containing 0.1 mM PMSF. The cells were disrupted by sonication (10 s, 20 W, on ice). After removing the cell debris and nuclei by low-speed centrifugation (400 g, 10 rain), the supernatant was subjected to centrifugation at 100,000g, for 1 h. While the high-speed supernatant(s) was used as a source of crude phosphatase activity, the high-speed pellet of TPA-treated ceils served as PKC, phosphorylated endogenous membrane protein substrates.
Inhibition of respiratory burst by phosphoprotein phosphatase
Phosphatase assay A sensitive non-radioactive method for the assay of phosphoprotein phosphatase as described by Geladopoulos et al. [17] was adopted for the assay of phosphatase from EAT cells. The total reaction mixture of 2ml consisted of 10mM Tris-HCl buffer (pH 8.0), 200 #g of substrate, 100/~g of enzyme source and + sodium fluoride. The enzyme was added to the prewarmed reaction mixture to initiate the reaction. After incubation for 10 min, the reaction was stopped by chilling. The solution was centrifuged at 1000g for 10 min. An aliquot of the supernatant was added to 0.5ml malachite green solution and was incubated for 10 min at 37°C. The absorbance was read at 620 nm. Suitable controls of either enzyme or substrate alone of six different groups of cells were included. The corrected values of optical density (OD) at 620 nm were obtained after subtracting the additive OD of enzyme alone and substrate alone from the OD obtained with both enzyme and substrate together. A standard curve of absorbance at 620nm for phosphate (0-12 nmol) was determined in parallel. Preparation of labelled membrane substrate EAT cells (1 x 107 cells) in prewarmed HBSS buffer (pH 7.4) were incubated at 37°C for 60 min in the presence of 200-250/~Ci of 32p-pi. After 60 min the cells were placed on ice and used in subsequent experiments [18]. In experiments to check the effect of epinephrine on TPA-triggered phosphorylation, labelled EAT cells were first incubated for 20 min at 37°C with epinephrine (100 #M). TPA (160 nM) was added and incubation was continued for a further 10 min. Labelled EAT cells without any TPA or epinephrine or with TPA alone or with epinephrine alone served as appropriate controls. The cells were collected by centrifugation (400g, 8 min, 4°C) and resuspended in ice-cold 10mM Tris-HC1 buffer (pH 7.4) containing 0.25% sucrose, disrupted in cold using a glass-Teflon homogenizer. The lysate was centrifuged at 100,000g, for 1 h, and the pellet redissolved in the electrophoresis sample buffer. After immersion of the samples in a boiling water bath for 2 min, the samples were subjected to SDS--PAGE, on 1-mm-thick slab gels, consisting of 4.5% (w/v) acrylamide stacking gel and 12.5% (w/v) separating gel and gels were run at a constant voltage of 180V according to the method of Laemmli [19]. After staining and destaining, gels were dried and subjected to autoradiography at -70°C for 2-4 days using Kodak X-Omat X-ray films with intensifying screens. An aliquot of phosphorylated membrane from the TPA-treated labelled EAT cells, and the cold phosphatase (100 #g cytosol) from the TPA- and epinephrine-treated cells (prepared as described above), was incubated for 10 rain at 37°C with or without sodium fluoride. The reaction was terminated by the addition of electrophoresis sample buffer. SDS--PAGE and autoradiography were done as described above.
653
Protein was estimated according to Lowry et al.
[20] using BSA as standard. TPA and Bt 2 cAMP were prepared as a stock solution in dimethyl sulphoxide (DMSO I mg/ml) and the final concentration of DMSO in the assay system was less than 0.1% throughout.
RESULTS
Effect of TPA epinephrine and Bt 2 c A M P on respiratory burst The preliminary data demonstrating that E A T cells show a preactivated respiratory burst, when compared to resting phagocytes, are shown in Fig. I. With T P A as an activator, a further two-fold increase of respiratory burst was observed. Epinephrine (50#M) and Bt 2 c A M P (500#M) inhibited respiratory burst both in control and TPA-treated E A T cells.
Phosphatase activity in cells receiving dual signals In cytosol from either control cells or from cells treated with TPA there is a basal level of phosphatase activity (Table 1A). In the presence of epinephrine (50 #M) there is a slight increase in phosphatase activity. However, dual signalling with both T P A and epinephrine or TPA and Bt2 cAMP, dramatically activates the phosphatase by approximately four- to sevenfold. The phosphatase activity obtained from T P A and epinephrine or T P A and Bt 2 cAMPtreated E A T cell cytosol released inorganic phosphate (Pi) from both control E A T cell membrane or TPA-activated EAT cell membrane. The phosphatase(s) had no effect on the membrane as substrate obtained either from TPA plus epinephrine- or T P A plus Bt: cAMPtreated cells or on epinephrine alone or Bt 2 cAMP-treated cells (Table I B). Membranes from control cells were not such good substrates when compared to that from TPA-activated cells, where there was a two-fold increased release of Pi from TPA-activated cell membranes by the dual signal-induced phosphatase activity. Alkaline phosphatase had no
654
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FIG. I. Inhibition by epinephrine and Bt~ cAMP of basal and TPA-triglFred respiratory burst in EAT cells. I-q, EAT cells; [], normal phagocytes. The data represent the mean+S.D, of the results obtained from three experiments performed in duplicate.
effect whatsoever on any of the substrate sources.
Kinetics of inhibition of respiratory burst with simultaneous activation of phosphatase by epinephrine or Bt2 cAMP Kinetics of inhibition of respiratory burst by epinephrine or Bt2 cAMP in both control and TPA-activated cells are shown in Fig. 2a and b. Half-maximal inhibition of respiratory burst in control cells was seen at 30 min while the same effect was seen within 5 min in TPA-activated cells, by either epinephrine or Bt: cAMP. Within 30 min, epinephrine and Btz cAMP caused 100% inhibition. Because of the effectiveness of epinephrine or Bt: cAMP inhibition of TPA-activated cells, the cytosol from such cells was assayed for kinetics of phosphatase activity with TPA-treated EAT cell membrane
as substrate. With an increase in the time of incubation with epinephrine or Bh cAMP, a simultaneous increase in phosphatase activity was seen. By 30 min, maximum phosphatase activity could be detected in both cases.
Effect of increasing concentration of epinephrine and Bt2 cAMP on respiratory burst and phosphatase activity The maximum respiratory burst seen in the presence o f TPA was inhibited in a dose-dependent manner by epinephrine or Bt2 cAMP with I¢ 50 values of 20 and 80/~M, respectively. As is shown in Fig. 3a and b, in the presence of TPA and in the absence of epinephrine or Bh cAMP, little or no phosphatase activity could be detected. With the inclusion o f various concentrations o f epinephrine or Bh cAMP, there was a rise in the phosphatase activity in the cytosol.
655
Inhibition of respiratory burst by phosphoprotein phosphatase TABLE 1A. DUAL SIGNALS,TPA AND EPINEPHRINE-OR TPA
A N D 13t 2 CYTOSOL EXHIBIT PHOSPHATASE ACTIVITY
Substrate for phosphatase (200/~g)
Specific activity (nmol Pi/mg protein/10 min)
TPA activated cell 100,000 g Pellet TPA activated cell 100,000 g Pellet TPA activated cell 100,000 g Pellet TPA activated cell 100,000 g Pellet TPA activated cell 100,000 g Pellet TPA activated cell 100,000 g Pellet
4.3 +_1
Source of phospbatase 1.
Control cell cytosol
2.
TPA-activated cell cytosol
3.
TPA + epinephrine-treated cell cytosol
4.
TPA + Bt2 cAMP-treated cell cytosoi
5.
Epinephrine-treated cell cytosol
6.
Bt2 cAMP-treated cell cytosol
cAMP-TP.EATm)EAT CELL
4.9 +- 1.2 28 -I-3 37 + 4 7 +0.9
The crude phosphatase (100 #g) or the alkaline phosphatase (2.5 U) was incubated with the substrate (200/zg) for 10 min at 37°C. The Pi in the supernatants was estimated by the malachite green method. The data represent the mean +-S.D. of the three experiments performed in duplicate. TABLE 1 B . T P A - P H O S P H O R Y L A T E D MEMBRANE ACTS AS THE SUBSTRATE
Source of cellular phosphatase (nmol Pi/mg protein/10 min at 37°C) Substrate for phosphatase (200 #g) 1. 2. 3. 4. 5. 6.
Control cell--100,000g pellet TPA-activated cell--100,000g pellet TPA +epinephrine-treated cell-100,000 g pellet Epinephrine-treated cell--100,000g pellet TPA+Bt2 cAMP-treated ceU--100,000 g pellet Bt2 cAMP-treated cell--100,000g pellet
TPA and epinephrinetreated cytosol
TPA and Bt2 cAMPtreated cytosol
16.0+_2.5 26.0+_2.8 0
20+_2 33 + 1.9 ND
0
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phosphatase (2.5 U)
B
The crude phosphatase (100 #g) or the alkaline phosphatase (2.5 U) was incubated with the substrate (200 #g) for 10 min at 37°C. The Pi in the supernatants was estimated by the malachite green method. The data represent the mean-t-S.D, of the three experiments performed in duplicate.
M a x i m u m phosphatase activity was seen in 100#M epinephrine- or 500#M Bt 2 cAMPtreated E A T (5.6 x 106) cells, with 160 nM TPA.
At this concentration (100#M) o f epinephrine or 500pM Bt 2 cAMP, respiratory burst was inhibited by 100%.
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FIG. 2. (a, b) Inhibition of superoxide anion production coincides with the development of phosphatase activity by the EAT cells, as a function of time in the presence of agents which increase intracellular cAMP concentration. NBT reduction and Pi release were assayed as described in Materials and Methods. Cells were incubated with 100 pM epinephrine (a) or 500/~M Bt 2 cAMP (b) for the times indicated. A, control; C), TPA-triggered cells; 0 , phosphatase activity. The values are the mean+ S.D. of three experiments performed in duplicate.
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FIG. 3. Dose-dependent inhibition by epinephrine or Bt2 cAMP of TPA-triggered respiratory burst and activation of phosphatase activity. Superoxide anion release was measured after exposure of EAT cells (5 x 106 cells) to different concentrations of epinephrine or Bt2 cAMP for 20 min at 37°C, in the absence ( A ) or presence of TPA (160 riM; ©). The Pi release ( O ) was measured by the malachite green method from the cytosol of cells treated with TPA and epinephrine or Bt~ cAMP using TPA-treated EAT cell membrane as substrate. The values are the mean + S.D. of three experiments performed in duplicate.
658
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FIG. 4. Dose-dependent inhibition of phosphatase activity by sodium fluoride. The crude phosphatase (100 #g) was incubated with the substrate (200 #g), with or without different concentrations of sodium fluoride for 10 rain at 37°C. The Pi release was measured as described in Materials and Methods. C), TPA-epinephrine-treated cytosol; O, TPA-Bh cAMP-treated cytosol. The data represent the mean_+S.D, of the results obtained from three experiments performed in duplicate.
Effect of sodium fluoride on phosphatase activity The phosphatase activity from cells treated with both TPA and epinephrine or TPA and Bt2 cAMP could effectively be inhibited in a dosedependent manner by sodium fluoride, with a half-maximal inhibition at 250 #M and 200/~M of sodium fluoride, respectively (Fig. 4).
Effect of divalent cations on phosphatase activity Addition of the divalent cations to the incubation mixture did not affect the Pi release from the phosphorylated substrate by the phosphatase. However, addition of EGTA or EDTA to the assay mixture inhibited the Pi release by 100 and 45% in epinephrine--TPA-treated cytosol and 96 and 95% in Bh cAMP TPA-treated cytosol, respectively (Table 2).
Effect of phosphatase on respiratory burst EAT cells were permeabilized using low levels of Triton X-100. When such cells were activated for respiratory burst with TPA (160nM), inclusion of crude phosphatase that is the cytosol from TPA and epinephrine- or TPA and Bt2 cAMP-treated cells, the TPA-triggered respiratory burst was inhibited in a dose-dependent manner (Fig. 5).
Effect of epinephrine and phosphatase on protein phosphorylation Previously, TPA has been shown to induce phosphorylation of up to six proteins in rabbit neutrophils. Although it is difficult to resolve all individual proteins in one dimensional gel there is a definite TPA-induced increase in four to five bands of molecular weight similar to those reported earlier plus a general increased density
Inhibition of respiratory burst by phosphoprotein phosphatase
659
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FIG. 5. Effect of phosphatase on TPA-triggered respiratory burst in Triton-permeabilized EAT cells. (a) 25, (b) 50, (c) 100, (d) 200 #g phosphatases; values are the average of three experiments performed in duplicate.
o f phosphorylation in the entire lane. Figure 6 (photograph o f an autoradiogram) shows the profile of the phosphorylated E A T cell membranes in the presence of T P A (lane c) and
TABLE 2. EFFECT OF METAL ION,
the dephosphorylation effect of epinephrine (100 #M, lane b; 200 #M, lane a). The concentration of epinephrine which inhibits the respiratory burst in control or T P A activation, blocks
EGTA AND E D T A ON PHOSPHATASE ACTIVITY
Phosphatase activity (% of control)
System Control + 10 mM MgCI2 or MnCI 2 + 100 mM MgCI2 or MnC12 +50 and 100 #M C a C i 2 + I mM EGTA +1 mM EDTA
TPA + epinephrinetreated cytosol (a)
TPA + Bt2 cAMPtreated cytosol (b)
1O0 100 100 100 -4.5
1O0 I00 100 100 5.6 5.0
*Control activity was 26 and 29 nmol Pi/mg protein/10 min for a and b, respectively. Phosphatase was assayed as described in Materials and Methods except that the assay mixtures were supplemented with salts at the concentration indicated. Values are the average of three experiments performed in duplicate.
660
B.P. SALIMATHand G. SAVITHA
the phosphorylation of both high- and lowmolecular weight proteins. Further, treatment of TPA-phosphorylated membrane with the phosphatase from TPA and epinephrine-treated EAT cell cytosol (100 and 200#g) induced dephosphorylation of the membrane proteins (lanes d, e). Inclusion of NaF (200#M) protected phosphorylation to a certain extent (lane f). DISCUSSION The respiratory burst in EAT cells is higher when compared to that exhibited by normal resting phagocytes. PKC-triggered phosphorylation of protein(s) is known to activate respiratory burst in neutrophils [21]. The same appears to be the case with EAT cells, where the two-fold increase in respiratory burst was obtained on addition of TPA. Cyclic nucleotides, being among the negative modulators of respiratory burst, when assessed with EAT cells inhibited the respiratory burst. The inhibition of respiratory burst by Bt: cAMP further confirmed the negative modulation by cAMP. The possibility of dephosphorylation and inactivation of respiratory burst seems more likely than multiple-site phosphorylation of a protein by different signals operating as negative modulation. Our results proved this hypothesis where EAT cells expressed phosphoprotein phosphatase activity in the cytosol after treatment with epinephrine or Bt2 cAMP and the activity was further increased in cells treated with TPA and epinephrine or Bt2 cAMP. From these results one can infer that phosphorylation by both PKC and protein kinase A (PKA) is required to activate a phosphatase activity which then dephosphorylates the phosphoproteins responsible for the respiratory burst activity. As EAT cells without TPA signal exhibit respiratory burst, it can be assumed that membrane proteins are in a phosphorylated state. Further, treatment with TPA activates respiratory burst and results in further increase of phosphorylated membrane proteins. This was proved by our results which show that a
membrane preparation from TPA-treated EAT cells was a better substrate for the dual-signal phosphatase than that from EAT cells alone. Further evidence for the existence of the phosphatase activity and its regulation by both PKC and PKA came from our results on kinetic studies which showed that in cells treated with both TPA and epinephrine or TPA and Bt2 cAMP, maximum inhibition of respiratory burst was obtained at 30 min of incubation during which time the maximum phosphatase activity was obtained. This view was strengthened by our results on dose-dependent inhibition of TPA-treated EAT cell respiratory burst by either epinephrine or Bt 2 cAMP, where maximum inhibition of respiratory burst at 100 and 500#M, respectively, was accompanied with increased phosphatase activity. Our results on the effect of sodium fluoride, a commonly used phosphatase inhibitor [22], clearly demonstrate the existence of the phosphatase in cells treated with dual signals. Direct evidence for inactivation of respiratory burst by dephosphorylation was obtained from results on the effect of phosphatase on TPA-triggered respiratory burst in permeabilized EAT cells. Labelling studies revealed that TPA-triggered phosphorylation of EAT membrane could be inhibited in the cells treated with epinephrine and the treatment of the dual-signal triggered phosphatase could dephosphorylate the TPA-phosphorylated membrane proteins. In conclusion, the results presented in this paper clearly demonstrate that negative modulation of respiratory burst in EAT cells by cAMP involves dephosphorylation by a phosphoprotein phosphatase. A cross-talk between PKC and PKA occurs in many cell types [23]. So also in EAT cells one can speculate that the phosphatase is regulated by phosphorylation by both PKA and PKC. This is a first preliminary report to show dual-signal regulated phosphatase activity. However, purification of this phosphatase and use of specific kinase-phosphorylated 47,000 or 67,000 Mr as a substrate in vitro will give better evidence for the specificity of the phosphatase. These studies are currently being undertaken.
FIG. 6. Effect of epinephrine, phosphatase and NaF on the phosphorylation of the protein of EAT cells. Protein(s) phosphorylated in EAT cell membrane by TPA (lane c) are dephosphorylated by incubation of cells with epinephrine (lanes a, b) or treatment of membrane with phosphatase (lanes d, e). NaF (lane f) protects phosphorylation.
661
Inhibition of respiratory burst by phosphoprotein phosphatase Acknowledgements--S.G. thanks the council of Scientific Industrial Research for a Senior Research Fellowship. The authors thank PRov. P. S. SASTRY I.I.SC., Bangalore, and DR C. SMARATHKUMAR,City X-ray & Scanning Research Centre, Mysore, for their help in radio labelling techniques.
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