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Effect of W07-toxin on gut physiological response in mice
Microbial Pathogenesis 37 (2004) 1–9 www.elsevier.com/locate/micpath
Effect of W07-toxin on gut physiological response in mice Shalmoli Bhattacharyyaa, Sujata Ghosha,*, Jasleen Shanta, Nirmal K. Gangulyb, Siddhartha Majumdara a
Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India b Indian Council of Medical Research, Government of India, New Delhi, India Received 20 May 2003; received in revised form 23 March 2004; accepted 29 March 2004
Abstract A number of unknown secretogenic factor(s) from Vibrio cholerae have been implicated to play a role in inducing cholera-like symptoms observed in patients. The present study has been carried out on the novel W07-toxin (pI 5.2) from V. cholerae W07, an epidemic cholera strain devoid of the ctx gene. The toxin showed maximum binding to GM1 and interacted with a 20 kDa glycoprotein present on the cell membrane of mice enterocytes in a GM1 specific manner. The analysis of biochemical parameters in enterocytes triggered with this toxin revealed a significant increase in intracellular calcium concentration and a massive secretion of Cl2. However, no absorption of Naþ was observed under the same condition. This toxin also elevated the level of cyclic adenosine 30 ,50 -monophosphate (cAMP) as well as protein kinase A (PKA). Thus, the novel toxin, although distinct from cholera-toxin, showed some functional homology to it and may be one of the key players inducing electrolyte imbalance within intestinal cells in the cholera-like symptoms associated with V. cholerae W07. q 2004 Elsevier Ltd. All rights reserved. Keywords: W07-toxin; Enterocytes; Protein kinase A; Electrolyte-imbalance
1. Introduction Cholera is an ancient disease that has taught many valuable lessons, from basic sanitation to molecular signaling. Although clinical management of cholera has advanced in the last 40 years, the disease continues to pose a serious problem in developing countries [1]. The horizontal gene transfer or recombination events raise the possibility of genesis of newer toxigenic strains of Vibrio cholerae with varied toxigenic potential. It has been previously shown that even the non-cholera toxin producing strains of V. cholerae might possess the ability to produce new secretogenic toxins [2]. This increases the importance of monitoring V. cholerae serogroups (including non-01 and non-0139) for their virulence gene content as a means of assessing their epidemic potential. The current study was carried out with a novel toxin from V. cholerae W07 isolated from a cholera epidemic in South India. Hybridization studies revealed the absence of ctx gene in the genome of this strain, although * Corresponding author. Tel.: þ91-172-2747585 ext. 5236; fax: þ 91172-2744401; þ 91-172-2705078. E-mail address: [email protected] (S. Ghosh). 0882-4010/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2004.03.002
the toxin produced by it could induce fluid accumulation in ligated rabbit ileal loop. This toxin also caused morphological change in CHO and Vero cells [3]. Cholera toxin (CT) is known as a classic secretory enterotoxin and its mechanism of action in the manifestation of diarrhoea has been elucidated in detail. The enzyme, adenylate cyclase is the intracellular target of CT, which catalyses the transformation of adenosine triphosphate (ATP) to cAMP. The adenylate cyclase, in turn, is regulated by guanine nucleotide binding proteins (G-proteins). It has been reported that the toxins (CT and other enterotoxins) act by the transfer of ADP-ribose moiety of NADþ to a specific arginine residue in the a subunit of Gs-protein leading to the inhibition of its intrinsic GTPase activity. Thus, the resulting activation of adenylate cyclase continues and the increased intracellular cAMP concentrations ultimately lead to electrolyte imbalance in the intestine. In this context, it would be interesting to know whether the mode of action of the W07-toxin from V. cholerae W07 is similar to that of CT or not. So, in the present study, an attempt has been made to investigate the status of gut physiological markers in response to the W07-toxin in mice enterocytes.
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2. Results The W07-toxin purified by the method of Walia et al. [3] was used in the present study. The homogeneity of the purified W07-toxin was confirmed by isoelectrofocussing. The isoelectric point of the toxin was found to be 5.2 (Fig. 1). The absence of CT in the W07-toxin preparation was confirmed in ELISA as well as in Western immunoblot. The IgG fraction (IgGWT) isolated from anti-W07-toxin sera could interact with the W07-toxin both in ELISA (OD 1.28 ^ 0.6) as well as in Western immunoblot (Fig. 2, lane1). However, the IgGWT could not show any reactivity with commercial CT (Sigma) in either case. Similarly, the IgG fraction isolated from anti-CT sera (IgGCT) reacted strongly with CT in ELISA (OD 1.7 ^ 0.12) and Western immunoblot (Fig. 2, lane 3) but could not show any reactivity to the W07-toxin. This toxin showed maximum binding specificity to GM1 (60.11 ^ 1.5%) followed by GM2 (36.08 ^ 0.5%), GM3 (30.16 ^ 0.5%) and asialoGM1 (17.94 ^ 0.7%). Further, the binding efficacy of the W07toxin and CT was better documented in GM1-ELISA using IgGWT and IgGCT, respectively. However, both these toxins could not show any crossreactivity. (Table 1). The interaction of the purified toxin with the membrane proteins of the enterocytes was visualized as darkly stained band of molecular weight ðMr Þ 20 kDa on the nitrocellulose paper in Western blot. This band was also highlighted in presence of the W07-toxin preincubated with the control antibody (preimmune serum). However, intensity of the band was reduced in presence of GM1 as well as IgGWT (Fig. 3). It was observed that 2 mg of the WO7-toxin induced an effective increase in the [Ca2þ]i at 5 min. In presence of 20 mM dantrolene (a drug known to trap calcium in intracellular stores), the WO7 toxin induced [Ca2þ]i in
Fig. 2. Western immunoblot showing the absence of CT in the W07toxin preparation. Lane 1: two subunits (40 and 58 kDa) of W07-toxin highlighted by IgG from anti-W07-toxin sera (IgGWT), lane 2: W07-toxin in presence of IgG from anti-CT sera (IgGCT), lane 3: A and B subunits of CT highlighted by IgGCT, lane 4: CT in presence of IgGWT.
the enterocytes was found to be reduced. Further, in enterocytes triggered with the WO7-toxin preincubated with IgGWT or GM1, the [Ca2þ]i was found to be decreased (Table 2). Fig. 4 reveals the [Ca2þ]i in the enterocytes triggered with different concentrations of CT for 5 min. It was found that [Ca2þ]i could be increased to similar extent in presence of 1 –4 mg of CT and 2 mg was used as control for the assays. No significant change in [Ca2þ]i was noticed in presence of the W07-toxin preincubated with IgGCT as well as in presence of CT preincubated with IgGWT. In the present study, a significant reduction in the fluorescence intensity was found at 5 min with the fluorescent indicator N-(6-methoxyquinolyl)acetoethyl ester (MQAE, Molecular Probe Co., Eugene, OR, USA) (Table 3). However, when the enterocytes were preincubated with 1 mg chlorotoxin, a chloride channel inhibitor and triggered with the WO7-toxin, a 2.5-fold increase in the fluorescence intensity was observed. Further, the WO7-toxin Table 1 GM1-ELISA showing binding of W07-toxin
Fig. 1. Homogeneity of the purified W07-toxin checked by isoelectrofocussing. (A) Lane 1: pI markers, Lane 2: Purified W07-toxin (pI 5.2). (B) Plot of pI vs. distance from the anode.
Group
OD
GM1 þ CT þ IgGCT GM1 þ W07-toxin þ IgG WT GM1 þ CT þ IgGWT GM1 þ W07-toxin þ IgG CT
0.334 ^ 0.01 0.412 ^ 0.07 ND ND
ND, non detectable (absorbance , 0.1). Values are expressed as mean ^ SD of triplicate experiments.
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Fig. 3. Identification of the W07-toxin binding proteins on the enterocyte membrane. (A) Lane 1: molecular weight ðMr Þ markers; lane 2: enterocyte membrane proteins (SDS-PAGE (10%)); Western blot showing interaction of the membrane protein(s) with lane 3: the W07-toxin; lane 4: W07-toxin preincubated with control antibody; lane 5: W07-toxin preincubated with IgGWT; lane 6: W07-toxin preincubated with GM1. (B) Plot of Mr of the marker proteins vs. the respective relative electrophoretic mobility on SDS-PAGE.
pretreated with IgGWT could also enhance the percentage of fluorescence intensity (26.02 ^ 1.12) of the MQAE labeled cells. The cytosolic free sodium levels in the enterocytes were assessed using sodium binding benzofuran isophtalate (SBFI, Molecular Probe Co., Eugene, OR, USA). Maximum fluorescence intensity (M1 region, taken as 100%) was
observed in control enterocytes. A minor decrease (2%) in the fluorescence intensity was noticed in enterocytes stimulated with the toxin for different time-period (5 s to 10 min) in either Naþ-HEPES buffer or Kþ-HEPES buffer. However, the enterocytes preincubated with amiloride ((1 mM), a Naþ Kþ ATPase inhibitor), followed by triggering with the WO7-toxin for 5 min could not reveal
Table 2 [Ca2þ]i in the Enterocytes triggered with the W07-toxin/CT in presence and absence of various inhibitors Groups
[Ca2þ]i (nM/106)
Enterocytes only (control) Enterocytes þ W07-toxin Enterocytes þ W07-toxin þ preimmune sera Enterocytes þ dantrolene þ W07-toxin Enterocytes þ W07-toxin þ IgGWT Enterocytes þ W07-toxin þ IgGCT Enterocytes þ CT Enterocytes þ CT þ preimmune sera Enterocytes þ CT þ IgGCT Enterocytes þ CT þ IgGWT
50.7 ^ 1.3 280.5 ^ 3.3*** 255.6 ^ 8.7*** 67.1 ^ 4.3 95.2 ^ 0.4 234.6 ^ 14.6*** 373 ^ 13.6*** 367.2 ^ 7.8*** 106 ^ 4.6 347 ^ 3.9***
Values are expressed mean ^ SD of triplicate experiments, ***p , 0:001:
Fig. 4. [Ca2þ]i in enterocytes (nmol/106 cells) triggered with different doses of CT. Values are expressed as mean ^ SD for triplicate experiments.
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Table 3 Level of cytosolic Cl2 in enterocytes triggered with W07-toxin (2 mg) for different time-period
Table 4 Adenylate cyclase dependent cAMP levels in presence of W07-toxin in mice enterocytes
Time
Fluorescence intensity of labeled cells (%)
Groups
Adenylate specific cAMP Levels (fmol/mg protein)
0s 5s 15 s 30 s 60 s 2.5 min 5 min 10 min 20 min
100.00 87.79 ^ 17.1 71.78 ^ 6.5 71.90 ^ 10.6 57.79 ^ 5.9 39.05 ^ 2.64 14.18 ^ 1.2** 3.45 ^ 0.34*** 3.44 ^ 0.67***
Enterocytes (control) Enterocytes þ WO7-toxin Enterocytes þ WO7-toxin þ IgGWT Enterocytes þ WO7-toxin þ GM1 Enterocytes þ WO7-toxin þ inhibitor Enterocytes þ cholera toxin (positive control)
ND 64.75 ^ 12.2 ND ND ND 85 ^ 12.4
Results were expressed in view of fluorescence intensity of labeled cells that could be directly correlated with cytosolic free Cl2. Values are expressed as mean ^ SD of triplicate experiments, **p , 0:01; ***p , 0:001 as compared to control at 0 s.
any significant change in the fluorescence intensity of the cells. Further, the WO7-toxin preincubated with IgGWT could also not induce any significant change. The GTPase activity (pmol/mg protein/min) in the membrane fraction of the WO7-toxin triggered enterocytes showed a statistically significant ðp , 0:01Þ decrease within 1 min (Fig. 5). Inhibition in the reduction of the GTPase activity was observed when the enterocytes were stimulated with the WO7-toxin preincubated with IgGWT (1:2500) or GM 1 (1 mg). The values were 25.59 ^ 0.76 and 28.87 ^ 1.75, respectively. The activity was found to be 26.73 ^ 1.99 in presence of 100 mM ouabain, a nonspecific inhibitor of GTPase activity. The enterocytes triggered with CT (2 mg) showed a statistically significant ðp , 0:01Þ decrease in the GTPase activity (15.75 ^ 0.29) at 1 min.
Fig. 5. GTPase activity in enterocytes (pmol/mg protein/min) stimulated with W07-toxin (2 mg) for different time-periods. CT was used as positive control. Values are expressed as mean ^ SD for triplicate experiments, **p , 0:01 as compared to control.
ND, non-detectable. Values are expressed as mean ^ SD of triplicate experiments.
The adenylate cyclase dependent cAMP levels (fmol/mg protein) in enterocytes triggered with the WO7-toxin or the WO7-toxin preincubated with IgGwT or GM1 are shown in Table 4. A significant increase in the level of cAMP was observed at 1 min in the WO7toxin as well as the CT (2 mg) stimulated enterocytes. Further, complete reduction in the level of cAMP was found in enterocytes treated with the WO7-toxin preincubated in presence of IgGWT or GM1. DDA (250 mM), the specific inhibitor of adenylate cyclase could also reduce the level of cAMP. The protein kinase A activity (units/106 cells) in enterocytes triggered with the WO7-toxin for different time-period was found to be maximum at 1 min (Fig. 6). However, in enterocytes treated with the WO7-toxin preincubated IgGWT or GM1, the protein kinase A (PKA) activity was found to be below detectable range. Further, in presence of a specific peptide inhibitor of PKA (TTYADFIASGRTGRRNAIHD, 10 mM), activity of the enzyme was also reduced to a non-detectable range. The CT (2 mg) could increase the PKA activity (2.5 ^ 0.3) significantly ðp , 0:001Þ as compared to that of control cells.
Fig. 6. PKA activity in enterocytes (units/ml) stimulated with W07-toxin (2 mg) for different time-periods. CT was used as positive control. Values are expressed as mean ^ SD for triplicate experiments, ***p , 0:001 as compared to control.
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3. Discussion and conclusion V. Cholerae W07 was found to be devoid of the ctx, ace or zot genes but caused a disease, which closely resembled cholera [3]. Data on the key pathogenicity genes of V. cholerae has shown that the structural genes ctxAB encoding cholera toxin might not serve as the only marker of epidemiologically dangerous strains [4]. Therefore, the ability of V. cholerae W07 to elaborate a novel toxin in absence of the known toxin genes is a matter of concern and needs better understanding. The reported yield of this toxin from in vitro culture was 1 mg from 2 l of the crude bacterial culture. It may be possible that in gut, the bacteria have ability to produce the toxin in excess thereby increasing the severity of the infection. Presence of a novel toxin in V. cholerae W07 was confirmed by N-terminal sequence of the subunits of purified W07-toxin as well as non-crossreactivity of CT with the antisera against W07-toxin in Western immunoblot [3]. In the present study, exclusion of contamination of CT in the W07-toxin preparation was further confirmed in ELISA and Western immunoblot in which the IgGCT could not reveal any crossreactivity to the W07-toxin (pI 5.2). The W07-toxin showed maximum binding efficacy for GM1 and we have identified a glycoprotein of Mr 20 kDa in the membrane fraction of mice enterocytes with which the W07-toxin could interact in a GM1 specific manner. It is still not clear whether this molecule is the receptor or not but since it could not be highlighted on the blot in presence of GM1, it could be assumed that the carbohydrate unit of this glycoprotein was somehow involved in binding of the WO7-toxin to the mice enterocyte membrane. In intestinal epithelium, the alteration in the level of cyclic nucleotides (cAMP, cGMP), Ca2þ, PKA and phospholipid metabolites was observed due to the action of enteric pathogens or their toxins [5]. Thus, in the present study, an attempt has been made to explore the mechanism by which this novel toxin from V. cholerae WO7 could exert its effect on the secretory pathway in the intestine. The increase in [Ca2þ]i observed in the W07-toxin stimulated enterocytes, is in good agreement with the previous finding in which a rise in the [Ca2þ]i was noticed in enterocytes isolated from V. cholerae 0139 (which is known to secrete CT) treated rabbit ileum [6]. Further, it was shown that the [Ca2þ]i in rat lymphocytes could be increased with CT [7,8]. Further, the [Ca2þ]i seems to have major involvement since significant decrease in [Ca2þ]i in presence of dantrolene in the WO7-toxin stimulated enterocytes has clearly indicated the involvement of intracellular Ca2þ for the release of Ca2þ within the cells. Our result is in supported by the observation of Hoque et al. [9] in which an increase in [Ca2þ]i was noticed in rat enterocytes in response to the heat stable enterotoxin of V. cholerae non-01 and this was found to be decreased in cells pretreated with dantrolene. We have also observed that
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the W07-toxin could induce an increase in [Ca2þ]i even after preincubation with IgGCT thus implying that the observed effect was due to the W07-toxin only. The net movement of electrolytes into the lumen results in a transepithelial osmotic gradient that causes water flow into the lumen and diarrheoa occurs when the massive volume of water is more than the absorptive capacity of the intestine [10]. It was reported that an increase in [Ca2þ]i caused a decrease in active Naþ and Cl2 absorption and/or stimulate active Cl2 secretion [11]. Therefore, in our study, we assessed the status of Naþ and Cl2 in enterocytes triggered with the WO7-toxin. The secretion of Cl2 under the effect of WO7-toxin was evident as almost 50% of cells showed decrease in Cl2 level within 1 min of toxin trigger and maximum reduction in Cl2 level was observed at 5 min. However, the W07-toxin could induce a minor decrease in the percentage of cells showing the Naþ specific label. This slight decrease might be due to passive diffusion of Naþ from the cells. In one experimental set up, sodium-citrate buffer was used to detect the level of Naþ absorption into the cells. In another assay, a sodium-free system was provided in order to assess the secretion of Naþ from the cells to the environment. Our result showed that in toxintriggered cells there was no net absorption of Naþ. The authenticity of these experiments was proved in presence of chlorotoxin, the Cl2 channel blocker and amiloride, the Naþ Kþ-ATPase inhibitor. Khurana et al. [12] have reported that the net intestinal secretion of Cl2 and Naþ might be due to an increase in serosal to mucosal fluxes and/or a decrease in mucosal to serosal fluxes of Cl2 and Naþ in Salmonella typhimurium enterotoxin induced diarrhoea in rat. The hemolysin from Vibrio parahaemolyticus was found to trigger a calcium dependent chloride secretion [13]. In the present study, it was observed that the WO7-toxin could reduce the GTPase activity in the mice enterocytes, thereby suggesting a major involvement of G-proteins in WO7-toxin induced transmembrane signaling. Further, an increase in the GTPase activity in enterocytes in presence of ouabain followed by the W07-toxin treatment confirmed the authenticity of previous observations. Binding of CT to GM1 on HeLa cell surface has been reported to be associated with an increase in cAMP accumulation in these cells [14]. Chang and Semrad [15] have shown that cAMP could stimulate the release of endogenous Ca2þ in enterocytes isolated from chicken small intestine. Thus, cAMP is thought to utilize the intracellular stores of calcium as also observed in the ileum and colon of both rabbit and rat. Shapiro et al. [16] showed that elevation in the levels of intracellular cyclic nucleotides could alter the structure of F-actin in intestinal epithelial cells and thus potentially contributing to intestinal secretion. In the present study, an increase in adenylate cyclase dependent cAMP can be correlated indirectly to the increased level of adenylate cyclase in the system. DDA is known to be a specific inhibitor of adenylate cyclase. Thus, in our study, a reduction in the level of cAMP in
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presence of DDA is in good agreement with previous observation. CT has been shown to increase intracellular cAMP levels and thus activate PKA in isolated intestinal epithelial cells. The activated kinase could phosphorylate numerous substrates in the cell [17]. In a study by Uhal et al. [18], it was observed that CT could elevate the cAMP level and increase the activity of PKA in adult rat type II pnenomocytes. Marshall et al. [19] have shown the involvement of Ca2þ, cAMP and PKA pathways in teleost intestine stimulated with ionomycin (Ca2þ ionophore) and dibutyryl-cAMP (cAMP-agonist). Subsequently, this could lead to activation of CFTR ion channels in the enterocytes, thereby leading to secretion of salt and fluid. Piacentino et al. [20] have given a ‘slip mode conductance’ hypothesis, which predicted that PKA could cause phosphorylation of Naþ channels, which could induce Ca2þ release from sarcoplasmic reticulum. The alteration in the levels of most of the intracellular parameters studied could be inhibited in presence of GM1 (1 mg) as well as IgGWT (diluted to 1:2500 in TBS). This indicates that both antibody-binding epitope as well as GM1-binding epitope of the WO7-toxin might have a precise role in transducing signals within the cell. The GM1-binding epitope might be involved in recognition of the cell surface receptor [21]. Hence, our data suggests that the W07-toxin might interact with the 20 kDa glycoprotein on mice enterocytemembrane and initiate a sequence of molecular events within the cells. It could enhance adenylate cyclase activity in a G-protein-dependent manner leading to the activation of cAMP/PKA mediated signal transduction pathway. This might further lead to phosphorylation of substrates involved in disturbance in ion transport which might then contribute to secretory response as has been reported for a number of enterotoxins [22]. It is also possible that the pathogenesis of secretion in response to this novel toxin from V. cholerae WO7 might also involve additional mechanisms as has been described in case of CT. Therefore, it can be said that the W07-toxin though unique from CT, might use common diarrheogenic pathways to cause the disease.
4. Materials and methods 4.1. Laboratory animals Inbred Balb/c mice (15 –20 g) and New Zealand White Rabbits (2 – 3 kg) were obtained from the Central Animal House of Postgraduate Institute of Medical Education and Research; Chandigarh. The use of animals was approved by the Institute’s Ethics Committee.
4.2. Bacterial strains Clinical isolate of V. cholerae W07 was a kind gift from Dr G.B. Nair, NICED, (Kolkata), India. The strain was maintained in trypticase soy broth. 4.3. Purification of toxin The novel toxin from V. cholerae W07was purified by the method of Walia et al. [3] and used for further assays. The purity of the toxin preparation was checked by isoelectrofocussing in phast system (Pharmacia, Sweden) using commercially available gels (Phast gel, TMI 3– 9) according to the manufacturers’ instructions. 4.4. Raising of antibodies Antibodies against W07-toxin as well as CT (Sigma) were raised in New Zealand White rabbits according to the method of Walia et al. [3]. The IgG from each antiserum was isolated using Protein A Sepharose CL-4B column according to the manufacturer’s instructions. The antibody titre was checked using ELISA [23]. The specificity of the IgG fraction to the respective toxin was checked on the Western immunoblot [24]. The crossreactivity between the two toxins was assessed by developing the blot containing W07-toxin with IgG from anti-CT immune sera (IgGCT) and the blot containing CT with IgG from antiW07-toxin immune sera (IgGWT) as primary antibody, respectively. 4.5. Binding efficacy of different gangliosides to W07-toxin The 96 well ELISA plates (Nunc, Denmark) were coated with the purified toxin (2.5 mg/well) in 50 mM carbonate/ bicarbonate buffer (pH 9.6) at 4 8C overnight in a humid chamber. The plates were washed once with TBS. Free binding sites of the coated wells were blocked with TBSBSA for 3 h at 37 8C. The wells were washed thrice in TBS and then incubated for 2 h at 25 8C in presence of various gangliosides (10 mg/ml) in twofold serial dilution. The control wells received only the toxin. After extensive washing with TBS containing 0.05% Tween 20 (TBST), the wells were treated with IgGWT (100 ml, diluted to 1:2500 with TBS-BSA) for 2 h at 37 8C as the primary antibody followed by standard ELISA procedure. The decrease in the absorbance at 492 nm for the ganglioside-treated wells compared to that of control wells (100%) was taken as the measure of ganglioside bound to the toxin. The binding was also checked in GM1-ELISA [25]. Briefly, the microtitre ELISA plates were coated with GM1 (1 mg/well) in 50 mM carbonate/bicarbonate buffer (pH 9.6) overnight at 4 8C. The W07-toxin was added to the GM1 coated ELISA plate previously blocked with TBS-BSA. The plates were washed and IgGWT/IgGCT, (each diluted to 1:2500) was added as the primary antibody. In a parallel set
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of experiment, CT was incubated in the GM1-coated plates and the IgGCT/IgGWT was used separately as primary antibody. The ELISA was processed using standard techniques. Sample giving absorbance $ 0.1 above the background were considered as positive. 4.6. Isolation of enterocytes Enterocytes were isolated from small intestine of mice as described by Pinkus [26] with modifications given by Toyoda et al. [27]. Briefly, the animals were sacrificed and the small intestine of each animal was quickly excised. The intestine was opened longitudinally, cut into small pieces and enterocytes were isolated by chelation-elution. The viability of cells was checked by the trypan blue exclusion test [12]. 4.7. Preparation of membrane fraction of enterocytes Mice enterocytes were extracted from the small intestine and suspended in saline. The cells were sonicated and the mixture was centrifuged (3000 £ g, 10 min) to remove the debris. The cell lysate containing a cocktail of protease inhibitors (Boehringer Mannheim) were further centrifuged (1,05,000 £ g, 1 h) at 4 8C and the pellet containing the membrane fraction of the enterocytes was suspended in 10 mM Tris/HCl (pH 7.2) containing 150 mM NaCl (TBS). 4.8. Identification of interacting molecule(s) for W07-toxin on enterocyte membrane The membrane preparation was subjected to SDS-PAGE (10%) under reducing condition [28] and the protein bands on the gel were electrophoretically transferred onto NCP strips [24]. The NCP strips were blocked with TBS-BSA and washed with TBS. These were then incubated separately with the purified W07-toxin (2 mg) preincubated with GM1 (1 mg/ml) or IgGWT (diluted to 1:2500). After washing successively with TBST and TBS, the strips were incubated with IgGWT at 37 8C for 2 h. This was followed by washing and incubation with HRP-labeled-goat anti-rabbit IgG (diluted to 1:1500 in TBS-BSA) at 37 8C for 1 h. Finally, the strips were developed with DAB using standard procedure. 4.9. Study on the mode of action of W07-toxin in mice enterocytes The viable enterocytes (106 cells/ml) were triggered with the required amount of toxin for different time. Further, to evaluate the role of intracellular mediators in the W07-toxin induced electrolyte imbalance in the enterocytes, the studies were carried out in presence and absence of various inhibitors/modulators.
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4.10. Estimation of intracellular free calcium concentration [Ca2þ]i [Ca2þ]i was estimated in enterocytes by the method of Pace and Galan [29]. Briefly, the isolated enterocytes (106 cells/ml) were taken in 20 mM HEPES buffer (pH 7.4) containing 145 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 1 mM CaCl2, 0.5 mM MgSO4 and 5 mM glucose. The cells were triggered with W07-toxin (2 mg) for 5 min and incubated in presence/absence in presence of control antiserum (diluted to 1:2500 in TBS) or GM1 (1 mg) or IgGWT (diluted to 1:2500 in TBS) or dantrolene (20 mM). Cells were incubated with 2 mM Fura-2/AM (Sigma, Chemicals, dissolved in DMSO) at 37 8C for 45 min. Finally, the enterocytes were suspended in the HEPES buffer and fluorescence measurements were performed at an excitation wavelength of 340 nm. The emission spectrum was recorded at 510 nm. The optimum dose of the toxin was then used to trigger the enterocytes for different time-period and the [Ca2þ]i was estimated. Further, the [Ca2þ]i in the enterocytes was estimated with different concentrations of CT (0.05, 0.1, 0.25, 0.50, 1, 2 and 4 mg) or CT (2 mg) preincubated with IgGWT(diluted to 1:2500). 4.11. Estimation of the intracellular Cl2 level The fluorescent indicator, MQAE was used for estimation of the level of Cl2 within the enterocytes [30]. In this assay, enterocytes were stimulated with the W07-toxin. The MQAE (5 mM) was loaded onto the cells by 3 min incubation in hypotonic solution (0.1% sodium citrate containing 0.1% Triton X-100) at 37 8C. The cells were centrifuged and resuspended in DMEM. The cells were acquired on a FACScan (Becton, Dickinson, USA) using the CELL QUEST program. The flowcytometric analysis was performed at an excitation wavelength of 360 nm and emission spectrum was recorded at 410 nm. The results were expressed in view of the mean fluorescence intensity of labeled enterocytes, which could be directly correlated to the level of cytosolic free Cl2. The level of cytosolic free Cl2 was also estimated in presence of GM1/IgGWT/chlorotoxin (1 mg). 4.12. Estimation of the cytosolic free Naþ level The fluorescent Naþ indicator, SBFI was used to determine the cytosolic free Naþ concentration in enterocytes [31]. For this assay, in one set of experiments the enterocytes were incubated with or without W07-toxin in sodium free Kþ-HEPES buffer (10 mM HEPES/5 mM KH2PO4 (pH 7.4) containing 150 mM KCl, 1 mM MgSO4 and 5 mM glucose). The cells were suspended in hypotonic solution containing a final concentration of 10 mM of SBFI-AM and incubated for
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3 min at 37 8C. Subsequently, the cells were centrifuged and resuspended in Kþ-HEPES/Naþ-HEPES buffer. The cells were acquired on FACScan (Becton, Dickinson, USA) using the CELL-QUEST programme and the cells were analyzed at an excitation wavelength of 385 nm. The emission spectrum was recorded at 490 nm. The results were expressed in view of the fluorescence intensity of labeled cells, which could be directly correlated to the level of cytosolic free Naþ. The level Naþ in enterocytes was also estimated in presence of GM1/IgGWT/amiloride (1 mM). 4.13. Measurement of GTPase activity The method of Sweeney [32] was followed to measure the GTPase activity in the membrane suspension (protein concentration 1 mg/ml) from the enterocytes. Briefly, the membrane suspension was taken in ADP-ribosylating buffer (25 mM Tris/HCl, pH 7.4, 1 mM EDTA, 1 mM DTT, 1 mM MgCl2, 1 mM ATP, 10 mM thymidine, 10 mM NADþ, 10 mM nicotinamide and 100 mM GTP), incubated with the W07-toxin for different time and finally resuspended in homogenizing buffer (10 mM Tris/HCl, pH 7.4, 1 mM EDTA, 5 mM DTT, 2 mM benzamidine, 50 mM chlorpromazine, 50 mM leupeptin, 0.25 TIU/ml aprotinin and 10 mM PMSF) for GTPase assay. The assay mixture contained 20 ml of this membrane suspension, 30 ml of water and 50 ml of GTPase reaction buffer (20 mM Tris/HCl (pH 7.4) containing 100 mM NaCl, 2 mM EDTA, 5 mM MgCl2, 2 mM DTT, 1 mM ATP, 1 mM creatine phosphate, 1 mM ouabain, 0.5 mM GTP and g32PGTP (final concentration 2.27 £ 104 mCi ml21)). Subsequently, each tube was centrifuged (1500 £ g, 10 min, 4 8C) after addition of icecold activated charcoal suspension (2%, 900 ml). The supernatant (200 ml) from each reaction mixture was removed and counted for 32P content in a liquid scintillation counter and the rate of GTP hydrolysis was expressed in pmol/mg protein/min. The activity of GTPase was also measured in the membrane fraction of enterocytes in presence of GM1/ IgGWT/ouabain (100 mM). 4.14. Measurement of adenylate cyclase dependent cAMP level The adenylate cyclase dependent cAMP level was measured in the enterocytes by using the Biotrake cAMP [125I] assay system (Amersham Pharmacia Biotech, Code RPA 509). In this assay, the enterocytes triggered with the purified W07-toxin were washed by centrifugation (1000 £ g, 5 min). Subsequently, cold 0.1N HCl (0.1 ml) was added to the cell pellet followed by incubation for 15 min at 37 8C. The cell debris was removed by centrifugation (500 £ g, 10 min). The supernatant was collected, titrated to pH 7.2 with 0.1N NaOH and used for estimation.
The activity of cAMP was also measured in presence of GM1/IgGWT/20 ,50 -dideoxy adenosine (DDA, a specific inhibitor of adenylate cyclase). 4.15. Measurement of protein kinase A levels The PKA activity in the enterocytes was measured by using a PepTagw non-radioactive cAMP-dependent protein kinase assay kit (Promega, USA, Cat. No. V5340). In this assay, the enterocytes triggered with the W07-toxin were centrifuged (1000 £ g, 10 min at 4 8C) to remove the excess toxin. Subsequently, the cells were resuspended in TBS (pH 7.2) and sonicated. The debris was removed by centrifugation (500 £ g, 10 min) and supernatant of each tube was used for the estimation of PKA. The activity of PKA was expressed as units 106/cells. The activity of PKA was also measured in presence of GM1/IgGWT/the peptide inhibitor (TTYADFIASGRTGRRNAIHD, 10 mM) specific for PKA. 4.16. Statistical analysis The data was analyzed by standard statistical methods (mean, SD, ANOVA and unpaired Student’s t-tests wherever applicable). In comparing groups p , 0:05 was taken as significant.
Acknowledgements The authors gratefully acknowledge the financial assistance provided by Council of Scientific and Industrial Research, Govt. of India to SB.
References [1] Heidelberg JF, Eisen JA, Nelson WC, Clayton RA, Gwinn ML, Dudson RJ, Haft DH, Hickey EK, Peterson JD, Umyam L, Gill SR, Nelson KE, et al. DNA sequence of both chromosomes of the cholera pathogen Vibrio cholerae. Nature 2000;406:477–83. [2] Singh DV, Matte MH, Matte GR, Jiang S, Sabeena F, Shukla BN, Sanyal SC, Colwell RR. Molecular analysis of V. cholerae 01, 0139, non-01 and non-0139 strains: clonal relationships between clinical and environmental isolates. Appl Environ Microbiol 2001;67: 910 –21. [3] Walia K, Ghosh S, Singh H, Nair GB, Ghosh A, Sahni G, Vohra H, Ganguly NK. Purification and characterization of novel toxin produced by Vibrio cholerae 01. Infect Immun 1999;67:5215–22. [4] Smirnova NI, Kirillina OA, Kutyrev VV. Genetic markers of epidemic strains of Vibrio cholerae. Zh Microbiol Epidemiol, Immunobiol 2000;5:87–91. [5] Chang EB, Brown DR, Wang NS, Field M. Secretagogue induced changes in membrane calcium permeability in chicken and chinchilla ileal mucosa. J Clin Invest 1986;78:281 –7. [6] Gorowara S, Sapru S, Ganguly NK. Role of intracellular second messengers and ROS in the pathophysiology of V. cholerae 0139 treated rabbit ileum. Biochem Biophys Acta 1998;1407:21–30.
S. Bhattacharyya et al. / Microbial Pathogenesis 37 (2004) 1–9 [7] Milani D, Minozzi MC, Petrelli L, Guidolin D, Skaper SD, Spoerri PE. Interaction of ganglioside GM1 with B-subunit of cholera-toxin modulates intracellular free calcium in sensory neurons. J Neurosci Res 1992;33:466–75. [8] Dixon SJ, Stewart D, Grisstein S, Spiegel S. Transmembrane signaling by B-subunit of CT: increased cytoplasmic calcium in rat lymphocytes. J Cell Biol 1987;105:1153–6. [9] Hoque KM, Pal A, Nair GB, Chattopadhyay S, Chakrabarty MK. Evidence of calcium influx across the plasma membranes depends upon the initial rise of cytosolic calcium with activation of IP3 in rat enterocytes by heat stable enterotoxin of V cholerae non 01. FEMS Microbiol Lett 2001;196:45–50. [10] Kaper JB, Fasano A, Trucksis M. Toxins of Vibrio cholerae. In: Wachsmuth IK, Blake PK, Olsvik O, editors. Vibrio cholerae and cholera. Washington, DC: American Society for microbiology; 1994. p. 145– 76. [11] Donowitz M. Ca2þ in the control of active intestinal Naþ and Cl2 transport: involvement in neurohumoral action. Am J Physiol 1983; 245:G165– 77. [12] Khurana S, Ganguly NK, Khullar M, Panigrahi D, Walia BNS. Studies on mechanisms of Salmonella typhimurium enterotoxin mediated diarrhoea. Biochim Biophys Acta 1991;1097: 171–6. [13] Raimondi F, Kao JP, Fiorentini C, Fabbri A, Donelli G, Gasparini N, Rubino A, Fasano A. Enterotoxocity and cytotoxicity of Vibrio parahemolyticus thermostable direct hemolysin in in-vitro systems. Infect Immun 2000;68:3180– 5. [14] Yamamoto K, Nagano T, Kunagai H, Okamoto Y, Otani S. Destruction of cholera toxin receptor on HeLa cell membrane using microbial endoglyco-ceraramidase. Arch Biochem Biophys 1996; 328:51–6. [15] Chang EB, Semrad CE. Calcium mediated cyclic-AMP inhibition of Naþ/Hþ exchange in chicken small intestine. Gastroenterology 1985; 88:1345. (Abstract). [16] Shapiro M, Matthews J, Hecht G, Delp C, Madara JL. Stabilization of F actin prevents cAMP elicited Cl2 secretion in T84 cells. J Clin Invest 1991;87:1903–9. [17] Chang EB, Rao MC. Intracellular mediators of intestinal electrolyte transport. In: Field M, editor. Diarrhoeal diseases. New York: Elsevier; 1991. p. 49–72. [18] Uhal BD, Papp M, Flynn K, Steek ME. Cholera toxin stimulates type II pneumocyte proliferation by a cAMP independent mechanism. Biochim Biophys Acta 1998;1405:99–109.
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[19] Marshall WS, Howard JA, Cozzi RR, Lynch EM. NaCl and fluid secretion by the intestine of the telost Fundulus heteroclitus: involvement of CFTR. J Exp Biol 2002;205:745 –58. [20] Piacentino V, Gaughan JP, Houser SR. L type Ca2þ currents overlapping threshold NaO currents: could they be responsible for the slip mode phenomenon in cardiac myocytes. Circ Res 2002;90: 435–42. [21] Minke WE, Roach C, Hol WGJ, Verlinde CLMJ. Structure based exploration of the ganglioside GM1 binding sites of E. coli heat labile enterotoxin and cholera toxin for the discovery of receptor. Biochemistry 1999;38:5684 –92. [22] Matthews JB, Awtrey CS, Madara JL. Microfilament dependent activation of Naþ/Kþ/2Cl2 cotransport by cAMP in intestinal epithelial monologues. J Clin Invest 1992;90:1608–13. [23] Voller A, Bidwell D, Bartlett A. Enzyme immunoassay in diagnostic medicine: theory and practice. Bull WHO 1976;53–5. [24] Towbin H, Stachelin T, Gordon J. Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc Natl Acad Sci USA 1979;76:4350–4. [25] Svennerholm A, Wiklund G. Rapid GM1-linked immunosorbent assay with visual reading for identification of E coli heat labile enterotoxin. J Clin Microbiol 1983;17:596–600. [26] Pinkus LM. Separation and use of enterocytes. Meth Enzymol 1981; 77:1154–62. [27] Toyoda S, Lee PC, Labenthal E. Physiological factors controlling release of enterokinase from rat enterocytes. Dig Dis Sci 1985;30: 1174– 80. [28] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 1970;227:680–5. [29] Pace JL, Galan JE. Measurement of free intracellular calcium levels in epithelial cells as consequence of bacterial invasion. Meth Enzymol 1994;236:482 –5. [30] Verkman AS, Sellers MC, Chao AC, Leung T, Ketcham R. Synthesis and characterization of improved chloride sensitive fluorescent indicators for biological applications. Anal Biochem 1989;178: 355–61. [31] Borin M, Siffert W. Stimulation by thrombin increases the cytosolic free Naþ concentration in human platelets. J Biol Chem 1990;265: 19543– 50. [32] Sweeney M. Measurement of the GTPase activity in signal transducing G-proteins in neuronal membranes. In: Kendall DA, Hill SJ, editors. Signal transduction protocols. Queens Medical Center, University of Nottingham: Humana Press; 1995. p. 51–62.
Effect of W07-toxin on gut physiological response in mice Shalmoli Bhattacharyyaa, Sujata Ghosha,*, Jasleen Shanta, Nirmal K. Gangulyb, Siddhartha Majumdara a
Department of Experimental Medicine and Biotechnology, Post Graduate Institute of Medical Education and Research, Chandigarh 160012, India b Indian Council of Medical Research, Government of India, New Delhi, India Received 20 May 2003; received in revised form 23 March 2004; accepted 29 March 2004
Abstract A number of unknown secretogenic factor(s) from Vibrio cholerae have been implicated to play a role in inducing cholera-like symptoms observed in patients. The present study has been carried out on the novel W07-toxin (pI 5.2) from V. cholerae W07, an epidemic cholera strain devoid of the ctx gene. The toxin showed maximum binding to GM1 and interacted with a 20 kDa glycoprotein present on the cell membrane of mice enterocytes in a GM1 specific manner. The analysis of biochemical parameters in enterocytes triggered with this toxin revealed a significant increase in intracellular calcium concentration and a massive secretion of Cl2. However, no absorption of Naþ was observed under the same condition. This toxin also elevated the level of cyclic adenosine 30 ,50 -monophosphate (cAMP) as well as protein kinase A (PKA). Thus, the novel toxin, although distinct from cholera-toxin, showed some functional homology to it and may be one of the key players inducing electrolyte imbalance within intestinal cells in the cholera-like symptoms associated with V. cholerae W07. q 2004 Elsevier Ltd. All rights reserved. Keywords: W07-toxin; Enterocytes; Protein kinase A; Electrolyte-imbalance
1. Introduction Cholera is an ancient disease that has taught many valuable lessons, from basic sanitation to molecular signaling. Although clinical management of cholera has advanced in the last 40 years, the disease continues to pose a serious problem in developing countries [1]. The horizontal gene transfer or recombination events raise the possibility of genesis of newer toxigenic strains of Vibrio cholerae with varied toxigenic potential. It has been previously shown that even the non-cholera toxin producing strains of V. cholerae might possess the ability to produce new secretogenic toxins [2]. This increases the importance of monitoring V. cholerae serogroups (including non-01 and non-0139) for their virulence gene content as a means of assessing their epidemic potential. The current study was carried out with a novel toxin from V. cholerae W07 isolated from a cholera epidemic in South India. Hybridization studies revealed the absence of ctx gene in the genome of this strain, although * Corresponding author. Tel.: þ91-172-2747585 ext. 5236; fax: þ 91172-2744401; þ 91-172-2705078. E-mail address: [email protected] (S. Ghosh). 0882-4010/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.micpath.2004.03.002
the toxin produced by it could induce fluid accumulation in ligated rabbit ileal loop. This toxin also caused morphological change in CHO and Vero cells [3]. Cholera toxin (CT) is known as a classic secretory enterotoxin and its mechanism of action in the manifestation of diarrhoea has been elucidated in detail. The enzyme, adenylate cyclase is the intracellular target of CT, which catalyses the transformation of adenosine triphosphate (ATP) to cAMP. The adenylate cyclase, in turn, is regulated by guanine nucleotide binding proteins (G-proteins). It has been reported that the toxins (CT and other enterotoxins) act by the transfer of ADP-ribose moiety of NADþ to a specific arginine residue in the a subunit of Gs-protein leading to the inhibition of its intrinsic GTPase activity. Thus, the resulting activation of adenylate cyclase continues and the increased intracellular cAMP concentrations ultimately lead to electrolyte imbalance in the intestine. In this context, it would be interesting to know whether the mode of action of the W07-toxin from V. cholerae W07 is similar to that of CT or not. So, in the present study, an attempt has been made to investigate the status of gut physiological markers in response to the W07-toxin in mice enterocytes.
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2. Results The W07-toxin purified by the method of Walia et al. [3] was used in the present study. The homogeneity of the purified W07-toxin was confirmed by isoelectrofocussing. The isoelectric point of the toxin was found to be 5.2 (Fig. 1). The absence of CT in the W07-toxin preparation was confirmed in ELISA as well as in Western immunoblot. The IgG fraction (IgGWT) isolated from anti-W07-toxin sera could interact with the W07-toxin both in ELISA (OD 1.28 ^ 0.6) as well as in Western immunoblot (Fig. 2, lane1). However, the IgGWT could not show any reactivity with commercial CT (Sigma) in either case. Similarly, the IgG fraction isolated from anti-CT sera (IgGCT) reacted strongly with CT in ELISA (OD 1.7 ^ 0.12) and Western immunoblot (Fig. 2, lane 3) but could not show any reactivity to the W07-toxin. This toxin showed maximum binding specificity to GM1 (60.11 ^ 1.5%) followed by GM2 (36.08 ^ 0.5%), GM3 (30.16 ^ 0.5%) and asialoGM1 (17.94 ^ 0.7%). Further, the binding efficacy of the W07toxin and CT was better documented in GM1-ELISA using IgGWT and IgGCT, respectively. However, both these toxins could not show any crossreactivity. (Table 1). The interaction of the purified toxin with the membrane proteins of the enterocytes was visualized as darkly stained band of molecular weight ðMr Þ 20 kDa on the nitrocellulose paper in Western blot. This band was also highlighted in presence of the W07-toxin preincubated with the control antibody (preimmune serum). However, intensity of the band was reduced in presence of GM1 as well as IgGWT (Fig. 3). It was observed that 2 mg of the WO7-toxin induced an effective increase in the [Ca2þ]i at 5 min. In presence of 20 mM dantrolene (a drug known to trap calcium in intracellular stores), the WO7 toxin induced [Ca2þ]i in
Fig. 2. Western immunoblot showing the absence of CT in the W07toxin preparation. Lane 1: two subunits (40 and 58 kDa) of W07-toxin highlighted by IgG from anti-W07-toxin sera (IgGWT), lane 2: W07-toxin in presence of IgG from anti-CT sera (IgGCT), lane 3: A and B subunits of CT highlighted by IgGCT, lane 4: CT in presence of IgGWT.
the enterocytes was found to be reduced. Further, in enterocytes triggered with the WO7-toxin preincubated with IgGWT or GM1, the [Ca2þ]i was found to be decreased (Table 2). Fig. 4 reveals the [Ca2þ]i in the enterocytes triggered with different concentrations of CT for 5 min. It was found that [Ca2þ]i could be increased to similar extent in presence of 1 –4 mg of CT and 2 mg was used as control for the assays. No significant change in [Ca2þ]i was noticed in presence of the W07-toxin preincubated with IgGCT as well as in presence of CT preincubated with IgGWT. In the present study, a significant reduction in the fluorescence intensity was found at 5 min with the fluorescent indicator N-(6-methoxyquinolyl)acetoethyl ester (MQAE, Molecular Probe Co., Eugene, OR, USA) (Table 3). However, when the enterocytes were preincubated with 1 mg chlorotoxin, a chloride channel inhibitor and triggered with the WO7-toxin, a 2.5-fold increase in the fluorescence intensity was observed. Further, the WO7-toxin Table 1 GM1-ELISA showing binding of W07-toxin
Fig. 1. Homogeneity of the purified W07-toxin checked by isoelectrofocussing. (A) Lane 1: pI markers, Lane 2: Purified W07-toxin (pI 5.2). (B) Plot of pI vs. distance from the anode.
Group
OD
GM1 þ CT þ IgGCT GM1 þ W07-toxin þ IgG WT GM1 þ CT þ IgGWT GM1 þ W07-toxin þ IgG CT
0.334 ^ 0.01 0.412 ^ 0.07 ND ND
ND, non detectable (absorbance , 0.1). Values are expressed as mean ^ SD of triplicate experiments.
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Fig. 3. Identification of the W07-toxin binding proteins on the enterocyte membrane. (A) Lane 1: molecular weight ðMr Þ markers; lane 2: enterocyte membrane proteins (SDS-PAGE (10%)); Western blot showing interaction of the membrane protein(s) with lane 3: the W07-toxin; lane 4: W07-toxin preincubated with control antibody; lane 5: W07-toxin preincubated with IgGWT; lane 6: W07-toxin preincubated with GM1. (B) Plot of Mr of the marker proteins vs. the respective relative electrophoretic mobility on SDS-PAGE.
pretreated with IgGWT could also enhance the percentage of fluorescence intensity (26.02 ^ 1.12) of the MQAE labeled cells. The cytosolic free sodium levels in the enterocytes were assessed using sodium binding benzofuran isophtalate (SBFI, Molecular Probe Co., Eugene, OR, USA). Maximum fluorescence intensity (M1 region, taken as 100%) was
observed in control enterocytes. A minor decrease (2%) in the fluorescence intensity was noticed in enterocytes stimulated with the toxin for different time-period (5 s to 10 min) in either Naþ-HEPES buffer or Kþ-HEPES buffer. However, the enterocytes preincubated with amiloride ((1 mM), a Naþ Kþ ATPase inhibitor), followed by triggering with the WO7-toxin for 5 min could not reveal
Table 2 [Ca2þ]i in the Enterocytes triggered with the W07-toxin/CT in presence and absence of various inhibitors Groups
[Ca2þ]i (nM/106)
Enterocytes only (control) Enterocytes þ W07-toxin Enterocytes þ W07-toxin þ preimmune sera Enterocytes þ dantrolene þ W07-toxin Enterocytes þ W07-toxin þ IgGWT Enterocytes þ W07-toxin þ IgGCT Enterocytes þ CT Enterocytes þ CT þ preimmune sera Enterocytes þ CT þ IgGCT Enterocytes þ CT þ IgGWT
50.7 ^ 1.3 280.5 ^ 3.3*** 255.6 ^ 8.7*** 67.1 ^ 4.3 95.2 ^ 0.4 234.6 ^ 14.6*** 373 ^ 13.6*** 367.2 ^ 7.8*** 106 ^ 4.6 347 ^ 3.9***
Values are expressed mean ^ SD of triplicate experiments, ***p , 0:001:
Fig. 4. [Ca2þ]i in enterocytes (nmol/106 cells) triggered with different doses of CT. Values are expressed as mean ^ SD for triplicate experiments.
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Table 3 Level of cytosolic Cl2 in enterocytes triggered with W07-toxin (2 mg) for different time-period
Table 4 Adenylate cyclase dependent cAMP levels in presence of W07-toxin in mice enterocytes
Time
Fluorescence intensity of labeled cells (%)
Groups
Adenylate specific cAMP Levels (fmol/mg protein)
0s 5s 15 s 30 s 60 s 2.5 min 5 min 10 min 20 min
100.00 87.79 ^ 17.1 71.78 ^ 6.5 71.90 ^ 10.6 57.79 ^ 5.9 39.05 ^ 2.64 14.18 ^ 1.2** 3.45 ^ 0.34*** 3.44 ^ 0.67***
Enterocytes (control) Enterocytes þ WO7-toxin Enterocytes þ WO7-toxin þ IgGWT Enterocytes þ WO7-toxin þ GM1 Enterocytes þ WO7-toxin þ inhibitor Enterocytes þ cholera toxin (positive control)
ND 64.75 ^ 12.2 ND ND ND 85 ^ 12.4
Results were expressed in view of fluorescence intensity of labeled cells that could be directly correlated with cytosolic free Cl2. Values are expressed as mean ^ SD of triplicate experiments, **p , 0:01; ***p , 0:001 as compared to control at 0 s.
any significant change in the fluorescence intensity of the cells. Further, the WO7-toxin preincubated with IgGWT could also not induce any significant change. The GTPase activity (pmol/mg protein/min) in the membrane fraction of the WO7-toxin triggered enterocytes showed a statistically significant ðp , 0:01Þ decrease within 1 min (Fig. 5). Inhibition in the reduction of the GTPase activity was observed when the enterocytes were stimulated with the WO7-toxin preincubated with IgGWT (1:2500) or GM 1 (1 mg). The values were 25.59 ^ 0.76 and 28.87 ^ 1.75, respectively. The activity was found to be 26.73 ^ 1.99 in presence of 100 mM ouabain, a nonspecific inhibitor of GTPase activity. The enterocytes triggered with CT (2 mg) showed a statistically significant ðp , 0:01Þ decrease in the GTPase activity (15.75 ^ 0.29) at 1 min.
Fig. 5. GTPase activity in enterocytes (pmol/mg protein/min) stimulated with W07-toxin (2 mg) for different time-periods. CT was used as positive control. Values are expressed as mean ^ SD for triplicate experiments, **p , 0:01 as compared to control.
ND, non-detectable. Values are expressed as mean ^ SD of triplicate experiments.
The adenylate cyclase dependent cAMP levels (fmol/mg protein) in enterocytes triggered with the WO7-toxin or the WO7-toxin preincubated with IgGwT or GM1 are shown in Table 4. A significant increase in the level of cAMP was observed at 1 min in the WO7toxin as well as the CT (2 mg) stimulated enterocytes. Further, complete reduction in the level of cAMP was found in enterocytes treated with the WO7-toxin preincubated in presence of IgGWT or GM1. DDA (250 mM), the specific inhibitor of adenylate cyclase could also reduce the level of cAMP. The protein kinase A activity (units/106 cells) in enterocytes triggered with the WO7-toxin for different time-period was found to be maximum at 1 min (Fig. 6). However, in enterocytes treated with the WO7-toxin preincubated IgGWT or GM1, the protein kinase A (PKA) activity was found to be below detectable range. Further, in presence of a specific peptide inhibitor of PKA (TTYADFIASGRTGRRNAIHD, 10 mM), activity of the enzyme was also reduced to a non-detectable range. The CT (2 mg) could increase the PKA activity (2.5 ^ 0.3) significantly ðp , 0:001Þ as compared to that of control cells.
Fig. 6. PKA activity in enterocytes (units/ml) stimulated with W07-toxin (2 mg) for different time-periods. CT was used as positive control. Values are expressed as mean ^ SD for triplicate experiments, ***p , 0:001 as compared to control.
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3. Discussion and conclusion V. Cholerae W07 was found to be devoid of the ctx, ace or zot genes but caused a disease, which closely resembled cholera [3]. Data on the key pathogenicity genes of V. cholerae has shown that the structural genes ctxAB encoding cholera toxin might not serve as the only marker of epidemiologically dangerous strains [4]. Therefore, the ability of V. cholerae W07 to elaborate a novel toxin in absence of the known toxin genes is a matter of concern and needs better understanding. The reported yield of this toxin from in vitro culture was 1 mg from 2 l of the crude bacterial culture. It may be possible that in gut, the bacteria have ability to produce the toxin in excess thereby increasing the severity of the infection. Presence of a novel toxin in V. cholerae W07 was confirmed by N-terminal sequence of the subunits of purified W07-toxin as well as non-crossreactivity of CT with the antisera against W07-toxin in Western immunoblot [3]. In the present study, exclusion of contamination of CT in the W07-toxin preparation was further confirmed in ELISA and Western immunoblot in which the IgGCT could not reveal any crossreactivity to the W07-toxin (pI 5.2). The W07-toxin showed maximum binding efficacy for GM1 and we have identified a glycoprotein of Mr 20 kDa in the membrane fraction of mice enterocytes with which the W07-toxin could interact in a GM1 specific manner. It is still not clear whether this molecule is the receptor or not but since it could not be highlighted on the blot in presence of GM1, it could be assumed that the carbohydrate unit of this glycoprotein was somehow involved in binding of the WO7-toxin to the mice enterocyte membrane. In intestinal epithelium, the alteration in the level of cyclic nucleotides (cAMP, cGMP), Ca2þ, PKA and phospholipid metabolites was observed due to the action of enteric pathogens or their toxins [5]. Thus, in the present study, an attempt has been made to explore the mechanism by which this novel toxin from V. cholerae WO7 could exert its effect on the secretory pathway in the intestine. The increase in [Ca2þ]i observed in the W07-toxin stimulated enterocytes, is in good agreement with the previous finding in which a rise in the [Ca2þ]i was noticed in enterocytes isolated from V. cholerae 0139 (which is known to secrete CT) treated rabbit ileum [6]. Further, it was shown that the [Ca2þ]i in rat lymphocytes could be increased with CT [7,8]. Further, the [Ca2þ]i seems to have major involvement since significant decrease in [Ca2þ]i in presence of dantrolene in the WO7-toxin stimulated enterocytes has clearly indicated the involvement of intracellular Ca2þ for the release of Ca2þ within the cells. Our result is in supported by the observation of Hoque et al. [9] in which an increase in [Ca2þ]i was noticed in rat enterocytes in response to the heat stable enterotoxin of V. cholerae non-01 and this was found to be decreased in cells pretreated with dantrolene. We have also observed that
5
the W07-toxin could induce an increase in [Ca2þ]i even after preincubation with IgGCT thus implying that the observed effect was due to the W07-toxin only. The net movement of electrolytes into the lumen results in a transepithelial osmotic gradient that causes water flow into the lumen and diarrheoa occurs when the massive volume of water is more than the absorptive capacity of the intestine [10]. It was reported that an increase in [Ca2þ]i caused a decrease in active Naþ and Cl2 absorption and/or stimulate active Cl2 secretion [11]. Therefore, in our study, we assessed the status of Naþ and Cl2 in enterocytes triggered with the WO7-toxin. The secretion of Cl2 under the effect of WO7-toxin was evident as almost 50% of cells showed decrease in Cl2 level within 1 min of toxin trigger and maximum reduction in Cl2 level was observed at 5 min. However, the W07-toxin could induce a minor decrease in the percentage of cells showing the Naþ specific label. This slight decrease might be due to passive diffusion of Naþ from the cells. In one experimental set up, sodium-citrate buffer was used to detect the level of Naþ absorption into the cells. In another assay, a sodium-free system was provided in order to assess the secretion of Naþ from the cells to the environment. Our result showed that in toxintriggered cells there was no net absorption of Naþ. The authenticity of these experiments was proved in presence of chlorotoxin, the Cl2 channel blocker and amiloride, the Naþ Kþ-ATPase inhibitor. Khurana et al. [12] have reported that the net intestinal secretion of Cl2 and Naþ might be due to an increase in serosal to mucosal fluxes and/or a decrease in mucosal to serosal fluxes of Cl2 and Naþ in Salmonella typhimurium enterotoxin induced diarrhoea in rat. The hemolysin from Vibrio parahaemolyticus was found to trigger a calcium dependent chloride secretion [13]. In the present study, it was observed that the WO7-toxin could reduce the GTPase activity in the mice enterocytes, thereby suggesting a major involvement of G-proteins in WO7-toxin induced transmembrane signaling. Further, an increase in the GTPase activity in enterocytes in presence of ouabain followed by the W07-toxin treatment confirmed the authenticity of previous observations. Binding of CT to GM1 on HeLa cell surface has been reported to be associated with an increase in cAMP accumulation in these cells [14]. Chang and Semrad [15] have shown that cAMP could stimulate the release of endogenous Ca2þ in enterocytes isolated from chicken small intestine. Thus, cAMP is thought to utilize the intracellular stores of calcium as also observed in the ileum and colon of both rabbit and rat. Shapiro et al. [16] showed that elevation in the levels of intracellular cyclic nucleotides could alter the structure of F-actin in intestinal epithelial cells and thus potentially contributing to intestinal secretion. In the present study, an increase in adenylate cyclase dependent cAMP can be correlated indirectly to the increased level of adenylate cyclase in the system. DDA is known to be a specific inhibitor of adenylate cyclase. Thus, in our study, a reduction in the level of cAMP in
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presence of DDA is in good agreement with previous observation. CT has been shown to increase intracellular cAMP levels and thus activate PKA in isolated intestinal epithelial cells. The activated kinase could phosphorylate numerous substrates in the cell [17]. In a study by Uhal et al. [18], it was observed that CT could elevate the cAMP level and increase the activity of PKA in adult rat type II pnenomocytes. Marshall et al. [19] have shown the involvement of Ca2þ, cAMP and PKA pathways in teleost intestine stimulated with ionomycin (Ca2þ ionophore) and dibutyryl-cAMP (cAMP-agonist). Subsequently, this could lead to activation of CFTR ion channels in the enterocytes, thereby leading to secretion of salt and fluid. Piacentino et al. [20] have given a ‘slip mode conductance’ hypothesis, which predicted that PKA could cause phosphorylation of Naþ channels, which could induce Ca2þ release from sarcoplasmic reticulum. The alteration in the levels of most of the intracellular parameters studied could be inhibited in presence of GM1 (1 mg) as well as IgGWT (diluted to 1:2500 in TBS). This indicates that both antibody-binding epitope as well as GM1-binding epitope of the WO7-toxin might have a precise role in transducing signals within the cell. The GM1-binding epitope might be involved in recognition of the cell surface receptor [21]. Hence, our data suggests that the W07-toxin might interact with the 20 kDa glycoprotein on mice enterocytemembrane and initiate a sequence of molecular events within the cells. It could enhance adenylate cyclase activity in a G-protein-dependent manner leading to the activation of cAMP/PKA mediated signal transduction pathway. This might further lead to phosphorylation of substrates involved in disturbance in ion transport which might then contribute to secretory response as has been reported for a number of enterotoxins [22]. It is also possible that the pathogenesis of secretion in response to this novel toxin from V. cholerae WO7 might also involve additional mechanisms as has been described in case of CT. Therefore, it can be said that the W07-toxin though unique from CT, might use common diarrheogenic pathways to cause the disease.
4. Materials and methods 4.1. Laboratory animals Inbred Balb/c mice (15 –20 g) and New Zealand White Rabbits (2 – 3 kg) were obtained from the Central Animal House of Postgraduate Institute of Medical Education and Research; Chandigarh. The use of animals was approved by the Institute’s Ethics Committee.
4.2. Bacterial strains Clinical isolate of V. cholerae W07 was a kind gift from Dr G.B. Nair, NICED, (Kolkata), India. The strain was maintained in trypticase soy broth. 4.3. Purification of toxin The novel toxin from V. cholerae W07was purified by the method of Walia et al. [3] and used for further assays. The purity of the toxin preparation was checked by isoelectrofocussing in phast system (Pharmacia, Sweden) using commercially available gels (Phast gel, TMI 3– 9) according to the manufacturers’ instructions. 4.4. Raising of antibodies Antibodies against W07-toxin as well as CT (Sigma) were raised in New Zealand White rabbits according to the method of Walia et al. [3]. The IgG from each antiserum was isolated using Protein A Sepharose CL-4B column according to the manufacturer’s instructions. The antibody titre was checked using ELISA [23]. The specificity of the IgG fraction to the respective toxin was checked on the Western immunoblot [24]. The crossreactivity between the two toxins was assessed by developing the blot containing W07-toxin with IgG from anti-CT immune sera (IgGCT) and the blot containing CT with IgG from antiW07-toxin immune sera (IgGWT) as primary antibody, respectively. 4.5. Binding efficacy of different gangliosides to W07-toxin The 96 well ELISA plates (Nunc, Denmark) were coated with the purified toxin (2.5 mg/well) in 50 mM carbonate/ bicarbonate buffer (pH 9.6) at 4 8C overnight in a humid chamber. The plates were washed once with TBS. Free binding sites of the coated wells were blocked with TBSBSA for 3 h at 37 8C. The wells were washed thrice in TBS and then incubated for 2 h at 25 8C in presence of various gangliosides (10 mg/ml) in twofold serial dilution. The control wells received only the toxin. After extensive washing with TBS containing 0.05% Tween 20 (TBST), the wells were treated with IgGWT (100 ml, diluted to 1:2500 with TBS-BSA) for 2 h at 37 8C as the primary antibody followed by standard ELISA procedure. The decrease in the absorbance at 492 nm for the ganglioside-treated wells compared to that of control wells (100%) was taken as the measure of ganglioside bound to the toxin. The binding was also checked in GM1-ELISA [25]. Briefly, the microtitre ELISA plates were coated with GM1 (1 mg/well) in 50 mM carbonate/bicarbonate buffer (pH 9.6) overnight at 4 8C. The W07-toxin was added to the GM1 coated ELISA plate previously blocked with TBS-BSA. The plates were washed and IgGWT/IgGCT, (each diluted to 1:2500) was added as the primary antibody. In a parallel set
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of experiment, CT was incubated in the GM1-coated plates and the IgGCT/IgGWT was used separately as primary antibody. The ELISA was processed using standard techniques. Sample giving absorbance $ 0.1 above the background were considered as positive. 4.6. Isolation of enterocytes Enterocytes were isolated from small intestine of mice as described by Pinkus [26] with modifications given by Toyoda et al. [27]. Briefly, the animals were sacrificed and the small intestine of each animal was quickly excised. The intestine was opened longitudinally, cut into small pieces and enterocytes were isolated by chelation-elution. The viability of cells was checked by the trypan blue exclusion test [12]. 4.7. Preparation of membrane fraction of enterocytes Mice enterocytes were extracted from the small intestine and suspended in saline. The cells were sonicated and the mixture was centrifuged (3000 £ g, 10 min) to remove the debris. The cell lysate containing a cocktail of protease inhibitors (Boehringer Mannheim) were further centrifuged (1,05,000 £ g, 1 h) at 4 8C and the pellet containing the membrane fraction of the enterocytes was suspended in 10 mM Tris/HCl (pH 7.2) containing 150 mM NaCl (TBS). 4.8. Identification of interacting molecule(s) for W07-toxin on enterocyte membrane The membrane preparation was subjected to SDS-PAGE (10%) under reducing condition [28] and the protein bands on the gel were electrophoretically transferred onto NCP strips [24]. The NCP strips were blocked with TBS-BSA and washed with TBS. These were then incubated separately with the purified W07-toxin (2 mg) preincubated with GM1 (1 mg/ml) or IgGWT (diluted to 1:2500). After washing successively with TBST and TBS, the strips were incubated with IgGWT at 37 8C for 2 h. This was followed by washing and incubation with HRP-labeled-goat anti-rabbit IgG (diluted to 1:1500 in TBS-BSA) at 37 8C for 1 h. Finally, the strips were developed with DAB using standard procedure. 4.9. Study on the mode of action of W07-toxin in mice enterocytes The viable enterocytes (106 cells/ml) were triggered with the required amount of toxin for different time. Further, to evaluate the role of intracellular mediators in the W07-toxin induced electrolyte imbalance in the enterocytes, the studies were carried out in presence and absence of various inhibitors/modulators.
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4.10. Estimation of intracellular free calcium concentration [Ca2þ]i [Ca2þ]i was estimated in enterocytes by the method of Pace and Galan [29]. Briefly, the isolated enterocytes (106 cells/ml) were taken in 20 mM HEPES buffer (pH 7.4) containing 145 mM NaCl, 5 mM KCl, 1 mM Na2HPO4, 1 mM CaCl2, 0.5 mM MgSO4 and 5 mM glucose. The cells were triggered with W07-toxin (2 mg) for 5 min and incubated in presence/absence in presence of control antiserum (diluted to 1:2500 in TBS) or GM1 (1 mg) or IgGWT (diluted to 1:2500 in TBS) or dantrolene (20 mM). Cells were incubated with 2 mM Fura-2/AM (Sigma, Chemicals, dissolved in DMSO) at 37 8C for 45 min. Finally, the enterocytes were suspended in the HEPES buffer and fluorescence measurements were performed at an excitation wavelength of 340 nm. The emission spectrum was recorded at 510 nm. The optimum dose of the toxin was then used to trigger the enterocytes for different time-period and the [Ca2þ]i was estimated. Further, the [Ca2þ]i in the enterocytes was estimated with different concentrations of CT (0.05, 0.1, 0.25, 0.50, 1, 2 and 4 mg) or CT (2 mg) preincubated with IgGWT(diluted to 1:2500). 4.11. Estimation of the intracellular Cl2 level The fluorescent indicator, MQAE was used for estimation of the level of Cl2 within the enterocytes [30]. In this assay, enterocytes were stimulated with the W07-toxin. The MQAE (5 mM) was loaded onto the cells by 3 min incubation in hypotonic solution (0.1% sodium citrate containing 0.1% Triton X-100) at 37 8C. The cells were centrifuged and resuspended in DMEM. The cells were acquired on a FACScan (Becton, Dickinson, USA) using the CELL QUEST program. The flowcytometric analysis was performed at an excitation wavelength of 360 nm and emission spectrum was recorded at 410 nm. The results were expressed in view of the mean fluorescence intensity of labeled enterocytes, which could be directly correlated to the level of cytosolic free Cl2. The level of cytosolic free Cl2 was also estimated in presence of GM1/IgGWT/chlorotoxin (1 mg). 4.12. Estimation of the cytosolic free Naþ level The fluorescent Naþ indicator, SBFI was used to determine the cytosolic free Naþ concentration in enterocytes [31]. For this assay, in one set of experiments the enterocytes were incubated with or without W07-toxin in sodium free Kþ-HEPES buffer (10 mM HEPES/5 mM KH2PO4 (pH 7.4) containing 150 mM KCl, 1 mM MgSO4 and 5 mM glucose). The cells were suspended in hypotonic solution containing a final concentration of 10 mM of SBFI-AM and incubated for
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3 min at 37 8C. Subsequently, the cells were centrifuged and resuspended in Kþ-HEPES/Naþ-HEPES buffer. The cells were acquired on FACScan (Becton, Dickinson, USA) using the CELL-QUEST programme and the cells were analyzed at an excitation wavelength of 385 nm. The emission spectrum was recorded at 490 nm. The results were expressed in view of the fluorescence intensity of labeled cells, which could be directly correlated to the level of cytosolic free Naþ. The level Naþ in enterocytes was also estimated in presence of GM1/IgGWT/amiloride (1 mM). 4.13. Measurement of GTPase activity The method of Sweeney [32] was followed to measure the GTPase activity in the membrane suspension (protein concentration 1 mg/ml) from the enterocytes. Briefly, the membrane suspension was taken in ADP-ribosylating buffer (25 mM Tris/HCl, pH 7.4, 1 mM EDTA, 1 mM DTT, 1 mM MgCl2, 1 mM ATP, 10 mM thymidine, 10 mM NADþ, 10 mM nicotinamide and 100 mM GTP), incubated with the W07-toxin for different time and finally resuspended in homogenizing buffer (10 mM Tris/HCl, pH 7.4, 1 mM EDTA, 5 mM DTT, 2 mM benzamidine, 50 mM chlorpromazine, 50 mM leupeptin, 0.25 TIU/ml aprotinin and 10 mM PMSF) for GTPase assay. The assay mixture contained 20 ml of this membrane suspension, 30 ml of water and 50 ml of GTPase reaction buffer (20 mM Tris/HCl (pH 7.4) containing 100 mM NaCl, 2 mM EDTA, 5 mM MgCl2, 2 mM DTT, 1 mM ATP, 1 mM creatine phosphate, 1 mM ouabain, 0.5 mM GTP and g32PGTP (final concentration 2.27 £ 104 mCi ml21)). Subsequently, each tube was centrifuged (1500 £ g, 10 min, 4 8C) after addition of icecold activated charcoal suspension (2%, 900 ml). The supernatant (200 ml) from each reaction mixture was removed and counted for 32P content in a liquid scintillation counter and the rate of GTP hydrolysis was expressed in pmol/mg protein/min. The activity of GTPase was also measured in the membrane fraction of enterocytes in presence of GM1/ IgGWT/ouabain (100 mM). 4.14. Measurement of adenylate cyclase dependent cAMP level The adenylate cyclase dependent cAMP level was measured in the enterocytes by using the Biotrake cAMP [125I] assay system (Amersham Pharmacia Biotech, Code RPA 509). In this assay, the enterocytes triggered with the purified W07-toxin were washed by centrifugation (1000 £ g, 5 min). Subsequently, cold 0.1N HCl (0.1 ml) was added to the cell pellet followed by incubation for 15 min at 37 8C. The cell debris was removed by centrifugation (500 £ g, 10 min). The supernatant was collected, titrated to pH 7.2 with 0.1N NaOH and used for estimation.
The activity of cAMP was also measured in presence of GM1/IgGWT/20 ,50 -dideoxy adenosine (DDA, a specific inhibitor of adenylate cyclase). 4.15. Measurement of protein kinase A levels The PKA activity in the enterocytes was measured by using a PepTagw non-radioactive cAMP-dependent protein kinase assay kit (Promega, USA, Cat. No. V5340). In this assay, the enterocytes triggered with the W07-toxin were centrifuged (1000 £ g, 10 min at 4 8C) to remove the excess toxin. Subsequently, the cells were resuspended in TBS (pH 7.2) and sonicated. The debris was removed by centrifugation (500 £ g, 10 min) and supernatant of each tube was used for the estimation of PKA. The activity of PKA was expressed as units 106/cells. The activity of PKA was also measured in presence of GM1/IgGWT/the peptide inhibitor (TTYADFIASGRTGRRNAIHD, 10 mM) specific for PKA. 4.16. Statistical analysis The data was analyzed by standard statistical methods (mean, SD, ANOVA and unpaired Student’s t-tests wherever applicable). In comparing groups p , 0:05 was taken as significant.
Acknowledgements The authors gratefully acknowledge the financial assistance provided by Council of Scientific and Industrial Research, Govt. of India to SB.
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