ARTICLE IN PRESS European Journal of
PROTISTOLOGY European Journal of Protistology 42 (2006) 201–208 www.elsevier.de/ejop
Detection of NADPH-diaphorase activity in Paramecium primaurelia Andrea Amarolia, Marzia Ognibenea,b, Francesca Triellia, Sonya Trombinoc, Carla Falugic, Maria Umberta Delmonte Corradoa, a
Dipartimento per lo Studio del Territorio e delle sue Risorse (DIP.TE.RIS.), University of Genoa, Genoa, Italy Laboratory of Molecular Biology, G. Gaslini Institute, Genoa, Italy c Department of Biology, University of Genoa, Genoa, Italy b
Received 17 November 2005; received in revised form 8 May 2006; accepted 13 May 2006
Abstract Recently, we showed that Paramecium primaurelia synthesizes molecules functionally related to the cholinergic system and involved in modulating cell–cell interactions leading to the sexual process of conjugation. It is known that nitric oxide (NO) plays a role in regulating the release of transmitter molecules, such as acetylcholine, and that the NO biosynthetic enzyme, nitric oxide synthase (NOS), shows nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity. In this work, we detected the presence of NADPH-d activity in P. primaurelia. We characterized this activity histochemically by examining its specificity for b-NADPH and a-NADH co-substrates, and sensitivity both to variations in chemico-physical parameters and to inhibitors of enzymes showing NADPH-d activity. Molecules immunologically related to NOS were recognized by the anti-rat brain NOS (bNOS) antibody. Moreover, bNOS immunoreactivity and NADPH-d activity sites were found to be co-localized. The non-denaturing electrophoresis, followed by exposure to b-NADPH or a-NADH co-substrates, revealed the presence of a band of apparent molecular mass of about 124 kDa or a band of apparent molecular mass of about 175 kDa, respectively. In immunoblot experiments, the bNOS antibody recognized a single band of apparent molecular mass of about 123 kDa. r 2006 Elsevier GmbH. All rights reserved. Keywords: NADPH-diaphorase activity; Nitric oxide synthase immunoreactivity; Paramecium; Ciliates
Introduction Recently, we showed that Paramecium primaurelia synthesizes molecules functionally related to the GABAergic (Delmonte Corrado et al. 2002) and cholinergic systems. The results of pharmacological experiments, carried out using cholinomimetic drugs and inhibitors of acetylcholinesterase (AChE) activity, Corresponding author. Tel.: +39 0103538031; fax: +39 0103538209. E-mail address:
[email protected] (M.U. Delmonte Corrado).
0932-4739/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejop.2006.05.002
indicated that the signal molecule acetylcholine (ACh) could be involved in modulating cell–cell interactions leading to the sexual process of conjugation (Delmonte Corrado et al. 1999, 2001, 2005; Trielli et al. 1997). It has been reported that nitric oxide (NO) plays a role, as a second messenger, in regulating cholinergic transmission in the central nervous system (Baccari et al. 1994), affecting choline acetyltransferase activity (Morot Gaudry-Talarmain et al. 1997) and exerting an inhibitory effect on ACh release (Belvisi et al. 1991; Knudsen and Tottrup 1992). NO, a very labile molecule, is synthesized by the enzyme nitric oxide synthase (NOS)
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that catalyzes the conversion of L-arginine to citrulline and NO (Alderton et al. 2001; Marletta 1993). Among the Protists, the ciliate P. caudatum (Malvin et al. 2003), the kinetoplastids Leishmania donovani (Basu et al., 1997), and Trypanosoma cruzi (Goldstein et al., 2000) express a protein that cross-reacts with mammalian brain NOS (bNOS) antibody on immunoblot. In addition, the presence of NOS activity has been reported in the erythrocyte stage of Plasmodium falciparum (Ghigo et al., 1995), in Entamoeba histolytica trophozoites (Hernandez-Campos et al., 2003), and in the plasmodial slime mold Physarum polycephalum (Golderer et al., 2001). It is known that neuronal NOS (nNOS) shows nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity, that is, the ability to transfer electrons from NADPH co-substrate to an electron acceptor substrate, a soluble tetrazolium salt, giving rise to a coloured insoluble compound (Dawson et al. 1991; Hope et al. 1991). According to some authors (Hope et al. 1991; Young et al. 1992), NADPH-d activity is synonymous with nNOS; therefore, it can be used as a histochemical marker for nNOS. However, NADPH-d activity can also be produced by enzymes other than nNOS (Matsumoto et al. 1993; Stoward et al. 1991; Tracey et al. 1993; Ward et al. 1992). The NADPH-d activity has long been studied in mammalian nervous systems (Grozdanovic et al. 1992; Hope et al. 1991). However, so far, there is no evidence for the presence of NADPH-d activity in Protists. In this work, we detected NADPH-d activity in P. primaurelia and characterized this activity histochemically by examining the co-substrate specificity, and the sensitivity to Triton X-100 concentration, to acidic or alkaline environment, and to inhibitors of enzymes showing diaphorase activity (Spessert and Claassen 1998; Wehby and Frank 1999). We then detected the presence of immunologically nNOS-related molecules, and of co-localized bNOS immunoreactivity and NADPH-d activity sites. Finally, we characterized NADPH-d activity and molecules immunologically related to nNOS by non-denaturing electrophoresis and immunoblot, respectively.
Materials and methods Cells and culturing methods P. primaurelia stock 90 cells, kindly supplied by Prof. Geoffrey H. Beale (Edinburgh) many years ago, were cultured at 25 1C in a lettuce medium, pH 6.8, inoculated with Enterobacter aerogenes and maintained in their logarithmic growth phase (Sonneborn 1970).
Chemicals The anti-rat bNOS antibody raised in mouse, antimouse fluorescein isothiocyanate (FITC)-labelled antibody, reduced a-nicotinamide adenine dinucleotide (a-NADH) and reduced b-nicotinamide adenine dinucleotide phosphate (b-NADPH) co-substrates, bovine serum albumin (BSA), dichlorophenolindophenol (DPIP), goat serum albumin (GSA), leupeptine, Nnitro-L-arginine-methylester (L-NAME), nitroblue tetrazolium (NBT) substrate, phenazine methosulphate (PMS), phenylmethylsulphonyl fluoride (PMSF), pyruvate, sodium azide and warfarin were purchased from Sigma (Milan, Italy). The anti-mouse immunoglobulins conjugated with alkaline phosphatase were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and the nitroblue tetrazolium salt/5-bromo-4-chloro-3indolyl phosphate (NBT/BCIP) Kit was purchased from Pierce (Milan, Italy).
Standard NADPH-diaphorase histochemistry Cells were fixed for 30 min in 2% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, and transferred onto the slides. After air-drying, the samples were processed for histochemistry and immunocytochemistry. The standard histochemical reaction for NADPH-d activity consisted of incubating the cells for 15 min at 37 1C in 50 mM Tris–HCl, pH 7.4, and then for 45 min at 37 1C in 1 mM b-NADPH co-substrate, 0.8 mM NBT substrate and 1% Triton X-100 in 50 mM Tris–HCl, pH 7.4. The histochemical reaction carried out in the absence of the b-NADPH served as a control (Spessert et al., 1994).
Modifications of NADPH-diaphorase reaction To characterize NADPH-d activity histochemically, the NADPH-d reaction was carried out under the following modified conditions. Co-substrate. Fixed cells were exposed to a-NADH substituted for b-NADPH at 1 mM concentration. Triton X-100 concentration of the incubation solution. The NADPH-d reaction was carried out in a Tris–HCl buffer containing 0.8 mM NBT, 1 mM b-NADPH or 1 mM a-NADH and 1%, 2%, 2.5% or 5% Triton X100. pH of the preincubation solution. The fixed cells were preincubated for 12 h at 4 1C with 50 mM Tris–HCl at various pH: 3.0, 5.0, 7.4, or 10.0. Afterwards, the samples were rinsed in 50 mM Tris–HCl, pH 7.4, and processed for the NADPH-d reaction in the presence of the b-NADPH or a-NADH co-substrate. Inhibitors of enzymes showing NADPH-diaphorase activity. Both preincubation, for 1 h at 25 1C, and
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incubation of the fixed cells were carried out in the presence of one of the following compounds: N-nitro-Larginine-methylester (L-NAME), 0.1 mM, an analogous compound of arginine, inhibiting NO synthesis (Spessert and Claassen 1998); dichlorophenolindophenol (DPIP), 0.1 mM, an electron acceptor and nNOS competitor (Spessert et al. 1994); pyruvate, 60 mM, a competitive inhibitor of lactate dehydrogenase activity (Wehby and Frank 1999); sodium azide, 5 mM, a competitive inhibitor of mitochondrial respiratory enzymes (Wehby and Frank 1999); warfarin, 0.5 mM, an inhibitor of NADPH-quinone oxidoreductase activity (Preusch and Suttie 1981); phenazine methosulphate (PMS), 1 mM, an electron coupler, transferring electrons from NAD(P)H to tetrazolium salts (Altman 1971). Incubation was performed according to the standard NADPH-d procedure, utilizing the b-NADPH or a-NADH co-substrate. The sample images were acquired and analyzed using ImageJ 1.33j software (NIH, USA). Student’s t-test was used to compare the mean optical densities, quantified by digital image analysis.
Immunocytochemistry To detect the presence of the molecules immunologically related to nNOS, fixed cells were incubated overnight with the anti-rat bNOS primary antibody, diluted 1:100, rinsed in 0.1 M PB, pH 7.4, and, lastly, incubated with an anti-mouse FITC-labelled secondary antibody, diluted 1:150, for 1 h at 37 1C. Unspecific reactions were blocked by adding 1% BSA and 5% GSA. The controls were carried out by omitting the primary antibody. The cells were rinsed and examined under a Leitz fluorescence microscope. To detect the presence of co-localized bNOS immunoreactivity and NADPH-d activity sites, samples of cells processed for immunocytochemistry were submitted to the standard NADPH-d reaction described above, after removing the coverslip (Hope et al., 1991).
Non-denaturing electrophoresis To characterize electrophoretically the NADPH-d activity, logarithmically growing P. primaurelia and E. aerogenes cells were centrifuged to concentrate the cells. After the addition of the protease inhibitors PMSF and leupeptine at the concentrations of 2 mM and 5 mg/ml, respectively, the samples were transferred to 80 1C for 10 min, homogenized in 0.3% Triton X-100, and centrifuged at 15,000g for 30 min. The total protein content was evaluated by BioRad Assay Kit, according to the manufacturer’s instructions. The supernatant was layered on 8% polyacrylamide gel. The molecules were electrophoretically separated for 2 h at 80 V. Afterwards,
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the gels were removed and agitated for 15 min at 25 1C in 50 mM Tris–HCl, pH 7.4. A staining bath containing 0.35% Triton X-100 in 50 mM Tris–HCl, pH 7.4, 0.25 mM NBT and 0.25 mM b-NADPH or a-NADH was added to cover the gels for 1 h at 25 1C. Enzyme activity was blocked in the gels by replacing the reaction mixture with a solution of 10% methanol and 7.5% acetic acid in distilled water (Kuonen et al. 1988). We used ImageJ 1.33j software (NIH, USA) to evaluate the apparent molecular mass of the experimental sample bands.
Immunoblot analysis A 30 mg total protein extract from P. primaurelia or E. aerogenes cells was run in 8% SDS-PAGE and then transferred to a nitrocellulose membrane. Non-specific binding sites were blocked with a blocking buffer containing Tris-buffered saline pH 7.4 and 0.1% Tween-20 with 3% non-fat milk powder for 45 min at 25 1C. The blot was incubated with a blocking buffer containing the anti-rat bNOS primary antibody, diluted 1:500, overnight at 4 1C. After washing, the blot was incubated with an anti-mouse immunoglobulins secondary antibody conjugated with alkaline phosphatase, diluted 1:500, for 3 h at 25 1C. After rinsing thoroughly, detection was performed directly on the membrane using the one-step NBT/BCIP Kit. The experiments were performed at least in triplicate. A 20 mg total protein mouse brain extract was used as a positive control for the bNOS antibody. We used ImageJ 1.33j software (NIH, USA) to evaluate the apparent molecular mass of the experimental sample bands.
Results Standard NADPH-diaphorase staining Logarithmically-growing cells of P. primaurelia showed intense diaphorase staining, mainly localized in the cytoplasm (Fig. 1A).
Effects of modifications of NADPH-diaphorase reaction To characterize histochemically the NADPH-d activity detected in P. primaurelia and to evaluate whether this activity was due to one or more enzymes, we modified the standard NADPH-d reaction by varying the co-substrate (Fig. 1), the Triton X-100 concentration of the incubation solution (Fig. 2), the pH of the preincubation solution (Fig. 3) and by exposing the cells to inhibitors of enzymes showing NADPH-d activity (Fig. 4).
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tion of the dark precipitate was less evident in the case of the b-NADPH co-substrate (Fig. 1A) than the a-NADH co-substrate (Fig. 1B).
Triton X-100 concentration of the incubation solution
Fig. 1. NADPH-d reaction. The reaction was carried out in the presence of the b-NADPH (A) (standard reaction) or a-NADH (B) co-substrate, and in the absence of the cosubstrate (C). Scale bars ¼ 10 mm.
The intensity of the diaphorase staining was affected differently by the Triton X-100 concentrations of the incubation solution, depending on the co-substrate utilized. In comparison with the standard reaction carried out at 1% Triton concentration in the presence of the b-NADPH co-substrate (Fig. 2A), the staining was not changed at 2.5% (Fig. 2B), but was nearly suppressed at 5% concentration (Fig. 2C). Conversely, in the presence of the a-NADH co-substrate, the diaphorase staining was found to be more intense at 5% Triton concentration (Fig. 2C0 ) than at 1% (Fig. 2A0 ) and 2.5% (Fig. 2B0 ) concentrations.
pH of the preincubation solution The preincubation of cells at increasing pH values of the preincubation solution affected the intensity of the diaphorase staining differently, depending on the cosubstrate used. In the presence of the b-NADPH cosubstrate, diaphorase staining was observed to be more intense at pH 5.0 (Fig. 3B) and pH 7.4 (Fig. 3C), than at pH 3.0 (Fig. 3A) and pH 10.0 (Fig. 3D). Using the aNADH co-substrate, the diaphorase staining peaked at pH 7.4 (Fig. 3C0 ) and pH 10.0 (Fig. 3D0 ). Faint staining was observed at pH 3.0 (Fig. 3A0 ) and intense staining appeared at pH 5.0 (Fig. 3B0 ).
Effects of inhibitors of enzymes showing NADPHdiaphorase activity on NADPH-d staining
Fig. 2. Effect of Triton X-100 concentration on NADPH-d reaction. NADPH-d reaction in the presence of the b-NADPH (A-C) or a-NADH (A0 -C0 ) co-substrates and at increasing Triton X-100 concentrations, 1% (A, A0 ), 2.5% (B, B0 ), 5% (C, C0 ), of the incubation solution. The controls (D, D0 ) were processed for the NADPH-d reaction in the absence of bNADPH or a-NADH. Scale bars ¼ 10 mm.
Co-substrate The intensity of the diaphorase reaction appeared to depend on the co-substrate used. Actually, the forma-
The results obtained by carrying out the NADPH-d reaction under modified conditions suggested that more than one enzyme showing NADPH-d activity is present in P. primaurelia. Therefore, the sensitivity of the NADPH-d staining to inhibitors of enzymes showing NADPH-d activity was evaluated. Fig. 4A reports the effects of inhibitors of enzymes showing NADPHdiaphorase activity on NADPH-d staining performed utilizing the b-NADPH co-substrate. Compared to the staining obtained with the standard NADPH-d reaction (white bar), the staining was unaffected by exposure to 0.1 mM L-NAME and 60 mM pyruvate (P40.05). Conversely, the staining decreased significantly after exposure to 5 mM sodium azide, 0.1 mM DPIP, an electron acceptor and nNOS competitor, and 0.5 mM warfarin (Po0.001). The staining was fully suppressed after exposure to 1 mM PMS, an electron coupler, transferring electrons from NAD(P)H to tetrazolium
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Fig. 3. Effect of different pH values on NADPH-d reaction. NADPH-d reaction in the presence of the b-NADPH (A-D) or a-NADH (A0 -D0 ) co-substrates and at increasing pH values, 3.0 (A, A0 ), 5.0 (B, B0 ), 7.4 (C, C0 ), 10.0 (D, D0 ), of the preincubation solution. The controls (E, E0 ) were processed for the NADPH-d reaction in the absence of b-NADPH or a-NADH. Scale bars ¼ 10 mm.
salts, and in the control, performed in the absence of the b-NADPH co-substrate. The effects of inhibitors of enzymes showing NADPHdiaphorase activity on NADPH-d staining performed utilizing the a-NADH co-substrate, are reported in Fig. 4B. Compared to the staining obtained with the NADPHd reaction in the absence of inhibitors (white bar), the staining appeared unaffected by exposure to 0.1 mM LNAME, 60 mM pyruvate, 5 mM sodium azide and 0.1 mM DPIP (P40.05). Contrarily, a significant decrease in staining intensity was found after exposure to 0.5 mM warfarin (Po0.001). The staining was fully suppressed after exposure to 1 mM PMS, and in the control, performed in the absence of the a-NADH co-substrate.
Immunocytochemical staining Immunocytochemical staining, performed using the anti-rat bNOS primary antibody and an anti-mouse
FITC-labelled secondary antibody, demonstrated the presence of immunoreactive sites, detected mainly in the cytoplasm (Fig. 5A). The presence of co-localized bNOS immunoreactivity (Fig. 6A) and NADPH-d activity (Fig. 6A0 ) sites was shown in samples of cells processed for immunocytochemistry followed by the standard NADPH-d reaction.
Non-denaturing electrophoresis to detect NADPHdiaphorase activity The electrophoretic analysis, carried out under nondenaturing conditions to detect NADPH-d activity, showed the presence of two bands in P. primaurelia. One band of about 124 kDa apparent molecular mass was found using the b-NADPH co-substrate (Fig. 7A), the other of about 175 kDa apparent molecular mass was found using the a-NADH co-substrate (Fig. 7B). The electrophoretic analysis carried out on E. aerogenes
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O.D.
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80 60
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*
0 120 B
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*
control
*
PMS
O.D.
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warfarin
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pyruvate
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Fig. 6. Co-localization of bNOS immunoreactivity and NADPH-d activity sites. A cell showing the immunological localization of nNOS-related molecules (A) and histochemical detection of NADPH-d activity (A0 ). Arrow-heads indicate the presence of co-localized positive sites. The control cell was exposed only to the FITC-conjugated secondary antibody (B) and processed for the NADPH-d reaction (B0 ). Scale bars ¼ 10 mm.
Fig. 4. Effects of exposure to inhibitors of enzymes showing NADPH-d activity on NADPH-d staining. NADPH-d reaction in the presence of the b-NADPH (A) or a-NADH (B) cosubstrate. NADPH-d staining (white bars), NADPH-d staining in the presence of inhibitors (black bars), NADPH-d staining in the absence of the co-substrate (grey bars). The inhibitors were: 0.1 mM L-NAME, 60 mM pyruvate, 5 mM sodium azide, 0.1 mM DPIP, 0.5 mM warfarin, and 1 mM PMS. On ordinate, optical density (O.D.) quantified by digital image analysis. Means of three experiments and standard deviation. Student’s t-test was used to compare the means. The symbol * indicates a significant difference (Po0.001) vs. NADPH-d staining (white bars). Sample size of each experiment ¼ 30 cells.
Fig. 7. Patterns of NADPH-d activity in Paramecium and Enterobacter compared by non-denaturing electrophoresis. The co-substrates used were b-NADPH (A) or a-NADH (B).
homogenates revealed the presence of two slight bands of about 124 and 47 kDa apparent molecular mass using the b-NADPH co-substrate (Fig. 7A), and no band using the a-NADH co-substrate (Fig. 7B). Fig. 5. Immunological localization of nNOS-related molecules. A cell exposed to the anti-rat bNOS primary antibody and an anti-mouse FITC-conjugated secondary antibody (A). The control cell (B) was exposed only to the FITC-conjugated secondary antibody. Scale bar ¼ 10 mm.
Immunoblot analysis of nNOS-related molecules In immunoblot experiments, the anti-rat bNOS antibody recognized a single immunoreactive band of about
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Fig. 8. Detection of nNOS-related molecules in Paramecium, mouse brain and Enterobacter by immunoblot. The anti-rat bNOS primary antibody raised in mice and an anti-mouse immunoglobulin secondary antibody conjugated with alkaline phosphatase were used.
123 kDa in P. primaurelia, no band in E. aerogenes, and two bands in mouse brain homogenate, used as a positive control (Fig. 8).
Discussion The main result of this study is that P. primaurelia cells show NADPH-d activity, revealed by histochemical and electrophoretic analyses. The different effects of the b-NADPH and a-NADH co-substrates on the staining intensity of the NADPH-d reaction, that appears lower or higher, respectively, suggests the presence of at least two enzyme activities with different co-substrate specificity. The histochemical characterization of the NADPH-d activities oxidizing the b-NADPH or a-NADH co-substrates reveals different sensitivity to both variations in the Triton X-100 concentration of the incubation solution and the pH of the preincubation solution. Moreover, exposure to specific inhibitors of enzymes showing diaphorase activity further supports the hypothesis that the NADPH-d activity is due to different enzymes in P. primaurelia. After incubation with DPIP, a nNOS competitor (Spessert et al. 1994), followed by the standard diaphorase reaction, the 50% decrease in staining intensity seems to indicate that the diaphorase activity revealed by the b-NADPH cosubstrate might be nNOS-related diaphorase activity. The 10% decrease in staining intensity caused by exposure to sodium azide and warfarin, a competitive inhibitor of mitochondrial respiratory enzymes and an inhibitor of NADPH-quinone oxidoreductase activity, respectively, suggests the presence of nNOS-unrelated
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diaphorase activity. Staining intensity appears to be unaffected by exposure to L-NAME, an analogous compound of arginine and inhibitor of NO synthesis, which is somewhat surprising since Malvin et al. (2003) found that 1 mM L-NAME reduced the metabolism of arginine to citrulline and reduced the production of nitrite by live P. caudatum. In our study on P. primaurelia, however, the reduction of the NBT substrate had occurred without conversion of L-arginine to citrulline and NO, according to the hypothesis that NO synthesis does not take place in fixed tissues (Spessert et al. 1994). The 70% decrease in diaphorase staining found after exposure to warfarin shows that nNOS-unrelated diaphorase activity oxidizes mainly the a-NADH co-substrate. The findings of the histochemical investigations are confirmed by the results of the non-denaturing electrophoretic analysis. Two distinct diaphorase activities are detected, one of apparent molecular mass of about 124 kDa that oxidizes the b-NADPH co-substrate, the other of apparent molecular mass of about 175 kDa that oxidizes the a-NADH co-substrate. Both activities clearly depend on P. primaurelia enzymes, as the electrophoretic patterns from the E. aerogenes homogenates reveal only the presence of two slight activities of apparent molecular mass of about 124 and 47 kDa using the b-NADPH co-substrate. The anti-rat bNOS antibody recognized nNOSrelated molecules in immunocytochemical experiments, and a single band of apparent molecular mass of about 123 kDa in immunoblot experiments. In conclusion, the results of this work show the detection of NADPH-d activity for the first time in Protists. Results from immunological tests suggest a possible link of this enzyme activity to NOS, but such a link is yet to be confirmed.
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