Biochemical Pharmacology 93 (2015) 34–41

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Upregulation of FLJ10540, a PI3K-association protein, in rostral ventrolateral medulla impairs brain stem cardiovascular regulation during mevinphos intoxication Ching-Yi Tsai a,1, Chang-Han Chen a,1, Alice Y.W. Chang b, Julie Y.H. Chan a, Samuel H.H. Chan a,* a b

Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung 83301, Taiwan, Republic of China Institute of Physiology, National Cheng Kung University, Tainan, Taiwan, Republic of China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 28 October 2014 Accepted 29 October 2014 Available online 7 November 2014

FLJ10540, originally identified as a microtubule-associated protein, induces cell proliferation and migration during tumorigenesis via the formation of FLJ10540-PI3K complex and enhancement of PI3K kinase activity. Interestingly, activation of PI3K/Akt cascade, leading to upregulation of nitric oxide synthase II (NOS II)/peroxynitrite signaling in the rostral ventrolateral medulla (RVLM), the brain stem site that maintains blood pressure and sympathetic vasomotor tone, mediates the impairment of brain stem cardiovascular regulation induced by the pesticide mevinphos. We evaluated the hypothesis that upregulation of FLJ10540 in the RVLM is upstream to this repertoire of signaling cascade that underpins mevinphos-induced circulatory depression. Microinjection bilaterally of mevinphos (10 nmol) into the RVLM of anesthetized Sprague–Dawley rats induced a progressive hypotension that was accompanied by an increase (Phase I), followed by a decrease (Phase II) of an experimental index for baroreflexmediated sympathetic vasomotor tone. There was augmentation in FLJ10540 mRNA in the RVLM or FLJ10540 protein in RVLM neurons, both of which were causally and temporally related to an augmentation of binding between the catalytic subunit (p110) and regulatory subunit (p85) of PI3K, phosphorylation of Akt at Thr308 site, and NOS II, superoxide or peroxynitrite level in the RVLM. Immunoneutralization of FJL10540 in the RVLM significantly antagonized those biochemical changes, and blunted the progressive hypotension and the reduced baroreflex-mediated sympathetic vasomotor tone during mevinphos intoxication. We conclude that upregulation of FLJ10540 in the RVLM elicits impairment of brain stem cardiovascular regulation that underpins circulatory depression during mevinphos intoxication via activation of PI3K/Akt/NOS II/peroxynitrite signaling cascade in the RVLM. ß 2014 Elsevier Inc. All rights reserved.

Keywords: Organophosphate intoxication FLJ10540 PI3K/Akt signaling Rostral ventrolateral medulla Brain stem cardiovascular regulation

1. Introduction Abbreviations: aCSF, artificial cerebrospinal fluid; Akt, serine/threonine protein kinase; AP, arterial pressure; CP, 3-carboxy-proxyl radical; CPH, cyclic hydroxylamine 1-hydroxy-3-carboxyl-2,2,5,5-tetramethyl-pyrrolidine; ESCRT, endosomal sorting complex required for transport; ESR, electron spin resonance; HR, heart rate; BLF, low-frequency; MAP, mean arterial pressure; NeuN, neuron-specific nuclear protein; NF-kB, nuclear factor-kB; NO, nitric oxide; NOS, nitric oxide synthase; NRS, normal rabbit serum; PBS, phosphate buffer; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-triphosphate; PI3K, phosphoinositide 3-kinase; PKG, protein kinase G; RVLM, rostral ventrolateral medulla. * Corresponding author. Tel.: +886 7 7317123 x 8599; fax: +886 7 7317123 x 8569. E-mail address: [email protected] (Samuel H.H. Chan). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.bcp.2014.10.018 0006-2952/ß 2014 Elsevier Inc. All rights reserved.

FLJ10540, also designated Cep55 [1] or C10orf3 [2], was originally identified as a microtubule-association protein that is related to microtubule bundling [1]. During mitosis, FLJ10540 facilitates chromosome segregation by associating with the mitotic spindle and spindle midzone. FLJ10540 also regulates the terminal stage of cytokinesis via localization to the midbody [1,3]. During abscission, FLJ10540 recruits endosomal sorting complex required for transport (ESCRT) complex to execute membrane fission [4]. Based on depletion by siRNA, FLJ10540 has been shown to cause defects in cytokinesis or abscission and increase the number of multinucleated cells [3,5]. More recently, FLJ10540 is found to play a role in human cancer because it is overexpressed in many

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malignancies, including oral squamous cell carcinoma, hepatocellular carcinoma, nasopharyngeal carcinoma, colon cancer, and lung cancer [6–10]. Whether FLJ10540 exhibits other cellular functions is unknown. Mevinphos (3-[dimethoxyphosphosphinyl-oxyl]-2-butenoic acid methyl ester), a US Environmental Protection Agency Toxicity Category I organophosphate, is the most commonly used pesticide for suicidal purposes in Taiwan [11]. Our laboratory demonstrated previously [12] that mevinphos elicits cardiovascular depression by acting on the rostral ventrolateral medulla (RVLM), a brain stem site classically known to be responsible for the maintenance of arterial pressure (AP) and sympathetic vasomotor tone [13]. Specifically, we found that mevinphos acts on the RVLM to induce an increase (Phase I) followed by a decrease (Phase II) of the baroreflex-mediated sympathetic vasomotor tone [12,14]. We further established that nitric oxide (NO) produced by NO synthase I (NOS I) in the RVLM, followed by activation of the soluble guanyly cyclase/cGMP/protein kinase G (PKG) cascade, is responsible for sustaining brain stem cardiovascular regulation during Phase I mevinphos intoxication [14]. On the other hand, peroxynitrite formed by a reaction between superoxide anion and NOS IIproduced NO [14], which is upregulated transcriptionally by nuclear factor-kB (NF-kB), in the RVLM is responsible for the impairment of brain stem cardiovascular regulation that leads to hypotension during Phase II [14,15]. In our continuous efforts to delineate the cellular and molecular mechanisms that underlie the impairment of brain stem cardiovascular regulation during mevinphos intoxication, we reported very recently [16] that activation of phosphoinositide 3-kinase (PI3K)/serine/threonine protein kinase (Akt) signaling is upstream to the upregulation of NF-kB/NOS II/peroxynitrite cascade in the RVLM. Also found recently [7,10] is that one mechanism for FLJ10540 to induce cell proliferation and migration in hepatocellular carcinoma and lung cancer is via the formation of a FLJ10540PI3K complex that enhances PI3K kinase activity. It follows that upregulation of this PI3K-association protein in the RVLM, leading to activation of the PI3K/Akt/NOS II/peroxynitrite cascade, may mediate the impairment of brain stem cardiovascular regulation during mevinphos intoxication. The present study validated this hypothesis. 2. Materials and methods 2.1. Ethics statement All experimental procedures carried out in this study were approved by the Institutional Animal Care and Use Committee of the Kaohsiung Chang Gung Memorial Hospital, and were in compliance with the guidelines for animal care and use set forth by that committee. 2.2. Experimental animals Specific pathogen-free adult male Sprague–Dawley rats (235– 322 g; n = 177) purchased from the Experimental Animal Center of the National Science Council or BioLASCO, Taiwan, were used. Animas were housed in an AAALAC International-accredited Center for Laboratory Animals, with maintained room temperature (24  1 8C) and 12:12 light-dark control (lights on from 07:00). Standard laboratory rat chow and tap water were available ad libitum. 2.3. General preparation Preparatory surgery, including intubation of the trachea and cannulation of a femoral artery and a vein, was performed under an initial pentobarbital sodium anesthesia (50 mg kg1, i.p.). During

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the recording session, anesthesia was maintained by intravenous infusion of propofol (Zeneca, Macclesfield, UK) at 20– 25 mg kg1 h1. We have demonstrated [17] that this scheme provided satisfactory anesthetic maintenance while preserving the capacity of central cardiovascular regulation. 2.4. Mevinphos intoxication We demonstrated previously [12] that by acting on the RVLM, cardiovascular responses elicited by intravenous administration of mevinphos are comparable to those induced by microinjection of this organophosphate directly into the RVLM. The latter route of administration was therefore employed in the present study to produce site-specific cardiovascular actions of mevinphos. As in previous studies [14–16], we prepared our mevinphos solution so that for every 50 nL of the solution microinjected into the RVLM, the concentration of the insecticide (in gram molecular weight) delivered amounted to 10 nmol. AP recorded from the femoral artery was first digitized at a rate of 1000 Hz by an acquisition algorithm (Notocord, Croissy Sur Seine, France) and analyzed by an arterial blood pressure analyzer (APR31a, Notocord). Continuous, on-line, and real-time spectral analysis (SPA10a, Notocord) was applied to the thus obtained digitized AP signals to construct a power spectrum [12,14–16]. We were particularly interested in the low-frequency (BLF) (0.25– 0.8 Hz) band of the AP spectrum. This spectral component takes origin from the RVLM [18] and reflects the prevalence of baroreflex-mediated sympathetic vasomotor tone that emanates from this brain stem site [19]. Heart rate (HR) was derived instantaneously from the AP signals. Temporal changes in pulsatile AP, mean AP (MAP), HR and power density of the BLF component were displayed continuously before and after the administration of mevinphos and other test agents. During the recording session, animals were allowed to breathe spontaneously with room air, and the body temperature was maintained at 37 8C with a heating pad. 2.5. Characterization of anti-FLJ10540 antiserum Rabbit polyclonal anti-FLJ10540 antiserum was generated and characterized as reported previously [7] by one of the authors (C.H.C.), using purified recombinant Histagged FLJ10540 fusion protein. Western blot analysis using full-length gels in that study showed that this antiserum recognizes a single 54 kDa protein band in Huh7 cells, which corresponds with the estimated molecular mass of FLJ10540. Its specificity was further confirmed in competitive inhibition experiment, in which preabsorption of the antiserum with the immunizing antigen abolished FLJ10540 staining. 2.6. Microinjection of test agents into the RVLM The head of the animal was fixed to a stereotaxic head holder (Kopf, Tujunga, CA). Microinjection bilaterally of test agents into the RVLM, at a volume of 50 nL, was carried out with a stereotaxically positioned glass micropipette that was connected to a 0.5-mL Hamilton microsyringe (Reno, NV). The coordinates used were 4.5–5 mm posterior to the lambda, 1.8–2.1 mm lateral to midline, and 8.1–8.4 mm below the dorsal surface of cerebellum [14–16]. Test agents used in this study included mevinphos (kindly provided by Sinon Corporation, Taichung Country, Taiwan) or a rabbit polyclonal antiserum against FLJ10540 (prepared by C.H.C.). Possible volume effect of microinjection was controlled by injecting the same amount of artificial cerebrospinal fluid (aCSF) or normal rabbit serum (NRS) (Sigma–Aldrich, St. Louis, MO). The composition of aCSF was: NaCl 117 mM, KCl 4.7 mM, NaH2PO4 1.2 mM, MgCl2 1.2 mM, CaCl2 2.5 mM, NaHCO3 25 mM, and

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glucose 11 mM. As in our previous study [15,20], 0.02% Triton X-100 (Sigma–Aldrich) was added to facilitate transport of the antiserum or NRS across the cell membrane of RVLM neurons. AntiFLJ10540 antiserum or NRS was microinjected bilaterally into the RVLM 1 h before microinjection of mevinphos. To avoid the confounding effects of drug interactions, each animal received mevinphos or aCSF, plus FLJ10540 antiserum or NRS. 2.7. Collection of tissue samples from the RVLM We routinely collected tissue samples [14–16] during the peak of Phases I or II (mevinphos group), or 30 or 180 min after microinjection of aCSF or NRS. Medullary tissues collected from anesthetized animals but without treatment served as the shamcontrols. As a routine, microinjection sites were visually verified and recorded after the slice of medulla oblongata that contains the RVLM (0.5 to 1.5 mm rostral to the obex) was obtained. Tissues from both sides of the ventrolateral medulla were subsequently collected by micropunches made with a 1 mm (i.d.) stainless steel bore to cover the anatomical boundaries of the RVLM, and were co-extensive with the distribution of the microinjected test agents [15,20]. The samples were stored immediately in liquid nitrogen. 2.8. Isolation of RNA and real-time PCR Total RNA from the RVLM was isolated with a Total RNA Mini kit (Geneaid, Taipei, Taiwan) according to the manufacturer’s instructions. All RNA isolated was quantified by spectrophotometry and the optical density 260/280 nm ratio was determined. Reverse transcriptase reaction was performed using a Transcriptor First strand cDNA Synthesis kit (Roche, Mannheim, Germany). Real-time PCR analysis [16,21] was performed by amplification of cDNA using a LightCycler (Roche). Genes were quantified by SYBR Green real-time polymerase chain reaction with GAPDH as the endogenous control. The relative changes in mRNA expression were determined by the fold-change analysis, in which fold DD change = 2[ Ct], where DDCt = (Ctgene  CtGAPDH mevinphos or vehicle treatment  (Ctgene  CtGAPDH)sham control). Note that Ct value is the cycle number at which fluorescence signal crosses the threshold. Primers were designed by Roche LightCycler probe design software 2.0 using the sequence information of the NCBI database, and oligonucleotides were synthesized by Quality Systems (Taipei, Taiwan). The primer pairs used for amplification of target genes were: FLJ10540 (Genbank Accession: NM_001025646): Forward primer: 50 -TCCTTGAAGCAGCTACATGGATTTA-30 Reverse primer: 50 -CTGTACGCTGCACTTGG-30 GAPDH (Genbank Accession: NM_017008): Forward primer: 50 -CTTCTCTTGTGACAAAGTGGA-30 Reverse primer: 50 -TTAGCGGGATCTCGCTC-30

2.9. Double immunofluorescence staining and laser confocal microscopy As reported previously [15,22], double immunofluorescence staining was carried out using a rabbit polyclonal antiserum against FLJ10540 (prepared by C.H.C.) and a mouse monoclonal antiserum against a specific neuronal marker, neuron-specific nuclear protein (NeuN; Chemicon, Temecula, CA). The secondary antisera included a goat anti-rabbit IgG conjugated with Alexa Fluor 488 and a goat anti-mouse IgG conjugated with Alexa Fluor 568 (Molecular Probes, Eugene, OR). Viewed under a Fluoview

FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan), immunoreactivity for NeuN exhibited red fluorescence, and FLJ10540 exhibited green fluorescence. 2.10. Protein extraction As reported previously [15,16,20], tissues were homogenized on ice in a protein extraction buffer that contains protease and phosphatase inhibitors, and centrifuged at 10,000  g at 4 8C for 10 min. The supernatant was collected, and the concentration of total proteins was determined by the BCA protein assay (Pierce, Rockford, IL) by measuring absorbance at 562 nm. 2.11. Western blot analysis Western blot analysis [15,16,20] was carried out using a rabbit polyclonal antiserum against Akt (Cell Signaling Technology, Beverly, MA), phospho-Akt(Thr308) (Cell Signaling), NOS II (Santa Cruz Biotechnology, Santa Cruz, CA); or a mouse monoclonal antiserum against nitrotyrosine (Upstate, Lake Placid, NY) or bactin (Chemicon). This was followed by incubation with horseradish peroxidase-conjugated donkey anti-rabbit IgG (GE Healthcare, Little Chalfont, Buckinghamshire, UK), or sheep anti-mouse IgG (GE Healthcare). Specific antibody-antigen complex was detected using an enhanced chemiluminescence Western blot detection system. The amount of protein expression was quantified by the ChemiDoc XRS+ System (Bio-Rad, Hercules, CA), and was expressed as the ratio relative to b-actin protein. 2.12. Immunoprecipitation and immunoblot analysis Protein extracts from samples of the RVLM were immunoprecipitated with an affinity-purified antiserum against p110 that was conjugated with protein G-agarose beads (Santa Cruz). After dissociated from the beads, the immunoprecipitated proteins were subjected to immunblot analysis using an anti-rabbit antiserum against p85 (Santa Cruz). 2.13. Spin trapping and electron spin resonance (ESR) It has been shown that cyclic hydroxylamine 1-hydroxy-3carboxyl-2,2,5,5-tetramethyl-pyrrolidine (CPH) reacts with peroxynitrite and superoxide [23], and is transformed into a stable 3carboxy-proxyl radical (CP). Based on this principle, tissue samples were lysed with 50 mM sodium phosphate buffer (PBS; pH 7.4) treated with chelex (50 g/L, Sigma-Aldrich) for >3 h and added to 1 mM CPH (Enzo; Lausen, Switzerland) placed in 50-mL glass micropipettes. To inhibit iron-catalyzed reaction, 200 mM diethylenetriaminepentaacetic acid (Sigma–Aldrich) was added to all samples. ESR spectra were recorded at room temperature using an EMX-plus ESR spectrometer (BioSpin; Bruker, Ettingen, Germany). The ESR settings for CPH were: X-band, microwave frequency 9.88 GHz, microwave power 2 mW, modulation frequency 100 kHz, modulation amplitude 1 G, receiver gain 5.64  103, time constant 40.9 ms, sweep time 81.9 s. The average height of the triplet waveforms in the ESR spectrum was used for quantification of signal intensity. 2.14. Histology In some animals that were not used for biochemical analysis, the brain stem was removed at the end of the physiological experiment and fixed in 10% formaldehyde-saline solution that contains 30% sucrose for at least 72 h. Frozen 25-mm sections of the medulla oblongata stained with neural red were used for histological verification of the microinjection sites.

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2.15. Statistical analysis All values are expressed as mean  SEM. The averaged value of MAP or HR calculated every 20 min after administration of test agents, the sum of power density of BLF component in the AP spectrum over 20 min, ESR signal intensity, and protein expression level or the relative mRNA expression in the RVLM during each phase of mevinphos intoxication, was used for statistical analysis. One-way or two-way analysis of variance with repeated measures was used to assess group means, followed by the Scheffe´ multiple range test for post hoc assessment of individual means. P < 0.05 was considered statistically significant. 3. Results 3.1. FLJ10540 in the RVLM underlies impairment of brain stem cardiovascular regulation during mevinphos intoxication An a priori requirement for FLJ10540 to play a role in circulatory depression during mevinphos intoxication is that this PI3Kassociation protein in the RVLM is causally related to the mevinphos-elicited changes in brain stem cardiovascular regulation. Our first series of experiment established this fundamental requirement based on immunoneutralization of FLJ10540 because of the lack of appropriate pharmacological inhibitor. As reported previously [12,14,16], whereas microinjection bilaterally into the RVLM of aCSF and NRS (1:20) was ineffective, administration of Mev (10 nmol) and NRS (1:20) elicited a progressive hypotension that became significant 100 min after application (Fig. 1), accompanied by insignificant changes in HR. Concurrent alterations in the power density of the BLF component of the AP spectrum, an experimental index for baroreflex-mediated sympathetic vasomotor tone [19], revealed two distinct phases. Phase I entailed a significantly augmented BLF power that peaked at 20 min and endured 80 min (Fig. 1). Phase II exhibited further and significant reduction in the power density of the BLF component to below baseline, approaching zero towards the end of our 180-min observation period. On the other hand, microinjection of the same dose of mevinphos (10 nmol) and NRS (1:20) into sites adjacent to but outside of the confines of the RVLM, similar to aCSF and NRS (1:20), elicited minimal changes in MAP, HR and BLF power over 180 min (Fig. 1). Pretreatment with bilateral microinjection of a FLJ10540 antiserum (1:20) into the RVLM, 1 h before the administration of mevinphos, significantly blunted the induced hypotension and antagonized the reduced BLF power during Phase II mevinphos intoxication, accompanied by prolonged augmentation of the power density of the BLF component during Phase I (Fig. 1). However, pretreatment with NRS (1:20) was ineffective (Fig. 1).

Fig. 1. Temporal changes in mean arterial pressure (MAP), heart rate (HR) or power density of the low-frequency (BLF) component of AP spectrum after microinjection bilaterally into or outside (non-RVLM) the RVLM (at time 0) of mevinphos (Mev; 10 nmol) or artificial cerebrospinal fluid (aCSF) in animals that received pretreatment by application into the bilateral RVLM of an antiserum directed against FLJ10540 (FLJ10540 Ab; 1:20) or normal rabbit serum (NRS; 1:20). Values are mean  SEM of 6 to 7 animals per experimental group. *P < 0.05 versus NRS + aCSF group, and +P < 0.05 versus NRS + Mev group at corresponding time-points in the post hoc Scheffe´ multiple-range test. B = preinjection baseline.

3.2. Upregulation of FLJ10540 in the RVLM during mevinphos intoxication Our second series of experiments determined whether the FLJ10540-induced impairment of brain stem cardiovascular regulation during mevinphos intoxication entails its upregulation in the RVLM. Real-time PCR analysis revealed that, compared to sham-control and aCSF-controls, the FLJ10540 mRNA (Fig. 2) level in the RVLM was significantly elevated during both phases of mevinphos intoxication. Results from double-immunofluorescence staining further revealed the cellular distribution of the upregulated FLJ10540 protein in the RVLM. Viewed under a laser scanning confocal microscope (Fig. 3), cells stained positively with the neuronal marker, NeuN exhibited a clearly defined nucleus and nucleolus. Interestingly, the surge in FLJ10540 immunoreactivity during both phases of mevinphos intoxication was amply present

Fig. 2. Phasic fold-changes relative to sham-controls of FLJ10540 mRNA in the RVLM elicited by microinjection bilaterally into the RVLM of mevinphos (10 nmol) or aCSF. Tissue samples were collected during the peak of Phase I (MI) or Phase II (MII) in the mevinphos group; or 30 (AI) or 180 (AII) min in the aCSF group. Values are mean  SEM of triplicate analyses on individual samples obtained from 3 to 4 animals per experimental group. *P < 0.05 versus sham-control (S) group or corresponding aCSF group in the post hoc Scheffe´ multiple-range test.

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Fig. 3. Illustrative laser scanning confocal microscopic images showing cells in the RVLM that were immunoreactive to the neuronal marker, NeuN (red fluorescence) and additionally stained positively for FLJ10540 (green fluorescence) in sham controls (Basal) or during Phases I and II mevinphos intoxication. Symbol (*) denotes location of the nucleus in the corresponding RVLM neuron. Box in lowpower view of the medulla oblongata indicated the location of the FLJ10540immunoreactive neurons. These results are typical of 3 animals from each experimental group. Scale bar, 20 mm or 400 mm. NA = nucleus ambiguus.

in the cytoplasm and nucleus of these RVLM neurons (Fig. 3). We also noted the presence of residual FLJ10540 immunoreactivity that was not double-labelled with NeuN (Fig. 3). 3.3. FLJ10540 is upstream to PI3K/Akt activation in the RVLM We demonstrated recently [16] that activation of PI3K/Akt cascade in the RVLM underlies the impairment of brain stem cardiovascular regulation elicited by mevinphos. Furthermore, there are indications that during tumorigenesis, FLJ10540 forms a complex with PI3K and initiates the PI3K/Akt cascade [7,10]. Our third series of experiments established that this sequence of signaling events also takes place in the RVLM during mevinphos intoxication. Results from immunoprecipitation and immunoblot analysis (Fig. 4A) showed that pretreatment with microinjection of an anti-FLJ10540 antiserum bilaterally into the RVLM significantly antagonized the augmented formation of the heterodimer made up of the catalytic subunit (p110) and the regulatory subunit (p85) that signifies activation of PI3K in the RVLM during mevinphos intoxication. Results from Western blot analysis (Fig. 4B) also showed that pretreatment with an anti-FLJ10540 antiserum significantly antagonized the augmented phosphorylation of the kinase domain (Thr308) required for activation of Akt in the RVLM by mevinphos. 3.4. Activation of FLJ10540/PI3K/Akt signaling cascade upregulates NOS II or peroxynitrite in the RVLM during mevinphos intoxication We reported recently [16] that activation of the PI3K/Akt cascade preferentially upregulates NOS II or peroxynitrite in the RVLM during mevinphos intoxication. We next evaluated whether a causal relationship exists between activation of the FLJ10540/ PI3K/Akt cascade by mevinphos and the induced phasic changes in NOS II/peroxynitrite signaling in the RVLM. Western blot analysis

Fig. 4. Results from (A) immunoblot (IB) using an antiserum against p85 (PI3K) or IgG on proteins immunoprecipitated (IP) by an anti-p110 antiserum from the RVLM; or (B) Western blot analysis of expression of phosphorylated Akt at Thr308 site (p-Akt(T308) in the RVLM during Phase II mevinphos intoxication (MII) or 180 min after microinjection of aCSF (AII) into the bilateral RVLM in animals pretreated with an anti-FLJ10540 antiserum (FLJ10540 Ab; 1:20) or NRS (1:20). Values are mean  SEM from 4 to 5 animals per experimental group. *P < 0.05 versus NRS + aCSF group, and +P < 0.05 versus corresponding NRS + Mev group in the post hoc Scheffe´ multiple-range test.

(Fig. 5) showed that the level of NOS II or nitrotyrosine, an experimental marker for peroxynitrite [24], was progressively and significantly augmented over both phases. Microinjection of an anti-FLJ10540 antiserum bilaterally into the RVLM, at a dose that effectively blunted the activation of PI3K/Akt cascade in the RVLM (Fig. 4), significantly antagonized the augmented NOS II or nitrotyrosine in the RVLM during mevinphos intoxication. 3.5. Activation of FLJ10540 upregulates superoxide and peroxynitrite in the RVLM during mevinphos intoxication Our final series of experiments employed spin trapping and ESR to further ascertain a causal relationship between upregulation of FLJ10540 and the augmented production of superoxide and peroxynitrite in the RVLM during mevinphos intoxication. Fig. 6A illustrated the presence of the unique triplet waveform in the ESR spectrum that represents quantitatively the amount of superoxide and peroxynitrite trapped by CPH in the tissue samples [23,25] collected from the RVLM during mevinphos intoxication. Pretreatment with an anti-FLJ10540 antiserum significantly blunted the progressive augmentation of ESR signal intensity in the RVLM over both phases of mevinphos intoxication (Fig. 6B and C). However, those blunting actions were not observed when antiFLJ10540 antiserum was microinjected to sites outside the confines of the RVLM (Fig. 6B and C). 3.6. Microinjection sites Histological verifications of the tip of the microinjection pipettes (Fig. 7) confirmed that all observations were made from animals that received administration of mevinphos, aCSF, antiFLJ10540 antiserum or NRS within the confines of the RVLM. The only exception was when mevinphos (Fig. 1) or anti-FLJ10540 antiserum (Fig. 6) was applied to sites outside the RVLM. 4. Discussion As the most widely used pesticide in the world, it has been reported that organophosphate poisoning is the most commonly

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Fig. 5. Representative Western blots (insets), or percentage of NOS II or nitrotyrosine (NT), an experimental index for peroxynitrite, relative to to b-actin in the RVLM detected after microinjection bilaterally into the RVLM of mevinphos (Mev; 10 nmol) or aCSF in animals that received pretreatment by application into the bilateral RVLM of an antiFLJ10540 antiserum (FLJ10540 Ab; 1:20) or NRS (1:20). Values are mean  SEM of triplicate analyses on individual samples obtained from 5 to 6 animals per experimental group. * P < 0.05 versus NRS + aCSF group, and +P < 0.05 versus corresponding NRS + Mev group in the post hoc Scheffe´ multiple-range test.

used suicide methods in the world, accounting for approximately one-third of global suicides [26]. A generally accepted dogma stipulates that the clinical features of organophosphate poisons, which include salivation, seizure, hypotension, coma to death [27], result from acetylcholinesterase inhibition that leads to accumulation of acetylcholine in peripheral and central synapses [28]. Based on a mevinphos intoxication model [12,14–16], the present study provided the first demonstration that FLJ10540, a PI3K-association protein, plays a causal role in eliciting impaired brain stem cardiovascular regulation and hypotension induced by organophosphate poisoning via activating PI3K/Akt signaling in the RVLM, followed by upregulation of NOS II/peroxynitrite expression. The PI3K/Akt signaling, which is well-known to be involved in tumorigenesis [29], is now known to play a central role in the control of metabolism, cell growth, proliferation, survival or migration, and membrane transport or secretion [30]. PI3K generates phosphatidylinositol-3,4,5-triphosphate (PIP3) by phosphorylating phosphatidylinositol-4,5-bisphosphate (PIP2). The most common form of PI3K is a heterodimer composed of a catalytic subunit (p110) and a regulatory subunit (p85). An interaction between the catalytic and regulatory subunits is important for stability of the p110 catalytic subunit [31,32]. Tyrosine phosphorylation of p85 can relieve its inhibitory activity on PI3K [33,34], and gene deletion of p85 causes a decrease in the catalytic activity of PI3K [31,35]. FLJ10540 reportedly enhances PI3K activity and Akt phosphorylation by interacting with the endogenous p85-p110 heterodimeric complex [7,10]. The present study demonstrated an upregulation of FLJ10540 at the mRNA level in the RVLM during mevinphos intoxication. We further observed that immunoneutralization of FLJ10540 significantly antagonized the augmented level of the p85-p110 heterodimer in the RVLM during mevinphos intoxication. Together, these observations suggest that by interacting with the p85-p110 heterodimer, the upregulated FLJ10540 may stabilize the PI3K complex and enhance PI3K activity. As we demonstrated, this enhanced PI3K activity leads to the ligation of PIP3 and subsequent activation of Akt by phosphorylation of its kinase domain (Thr308). Our laboratory revealed recently [16] that one cellular consequence of activation of the PI3K/Akt cascade in the RVLM during mevinphos intoxication is the formation of peroxynitrite by a reaction between NOS II-derived NO and superoxide anion. The present study further indicated that upregulation of FLJ10540 is upstream to this repertoire of cellular signals, which elicits impairment of brain stem cardiovascular regulation that underpins

circulatory depression during mevinphos intoxication. In addition to results from Western blot, this notion was ascertained by employing ESR. Because of its short half-life and low steady-state concentration, superoxide or peroxynitrite cannot be directly detected in biological samples. CPH, a spin trapping reagent of cyclic hydroxylamines, can react with superoxide and peroxynitrite to form a stable nitroxide (CP) with a much longer half-time [23,25]. Based on ESR measurements of the trapped superoxide and peroxynitrite in the RVLM tissue samples, we demonstrated that upregulation of FLJ10540 in the RVLM is causally related to the significant and progressive augmentation of oxidative and nitrosative stress in the RVLM during mevinphos intoxication. Two observations from the present study suggest that this cascade of FLJ10540/PI3K/Akt/peroxynitrite in the RVLM contributes to the transition from Phase I to Phase II of mevinphos intoxication. First, pretreatment with anti-FLJ10540 antiserum preferentially antagonized the reduced BLF power during Phase II mevinphos intoxication. Second, whereas this pretreatment prolonged the augmented power density of the BLF component during Phase I, the peak effect was the same as in animals that received NRS pretreatment. Our results from double immunofluorescence coupled with confocal microscopy revealed that the upregulation of FLJ10540 took place preferentially in the RVLM neurons. We demonstrated previously that the BLF component of the AP spectrum takes origin from the RVLM [18] and reflects the prevalence of baroreflexmediated sympathetic vasomotor tone that emanates from this brain stem site [19]. It is well-known that direct projection from the RVLM to the preganglionic sympathetic neurons located in the intermediolateral cell column of the thoracic spinal cord exists [36], using glutamate as the major neurotransmitter [37,38]. It is therefore conceivable that RVLM neurons in which upregulation of FLJ10540 took place during mevinphos intoxication are glutamatergic in nature. We noted, however, the presence of residual FLJ10540 immunoreactivity that was not double-labelled with NeuN. We reported previously [39] that NOS II is also present in astrocytes and microglia at the RVLM. It is therefore conceivable that, in addition to neurons, our observed upregulation of NOS II by FLJ10540-activated PI3K/Akt signaling during mevinphos intoxication may also engage those glial cells in the RVLM. We are aware that our experiments were carried out in animals that were maintained under propofol anesthesia. We reasoned that this may not be a confounding factor since we have demonstrated previously [17] that his scheme of anesthetic management exerts minimal influence on the brain stem cardiovascular regulatory

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Fig. 7. Diagrammatic representation of the medulla oblongata showing the location of the tip of the microinjection pipette within the confines of the RVLM. NA = nucleus ambiguus; NOI = nucleus olivaris inferior; NP = nucleus prepositus hypoglossi; NR = nucleus raphe´; NTS = nucleus tractus solitarii; PY = tractus pyramidalis; RVLM = rostral ventrolateral medulla; V = nucleus and tractus trigemini spinalis. Scale denotes distance from lambda.

Fig. 6. (A) Typical triplet waveform in the electron spin resonance (ESR) spectrum of nitroxide (CP) formed by a reaction of the spin probe CPH with superoxide and peroxynitrite in RVLM tissue homogenate. (B) Illustrative example of the ESR spectrum during Phase II mevinphos intoxication; or (C) phasic changes of ESR signal intensity in arbitrary units (AU) after microinjection bilaterally into the RVLM of mevinphos (Mev; 10 nmol) or aCSF in animals that received pretreatment by application into or outside (non-RVLM) of the bilateral RVLM of NRS (1:20) or an anti-FLJ10540 antiserum (FLJ10540 Ab; 1:20). Values are mean  SEM from 4 to 5 animals per experimental group. *P < 0.05 versus NRS + aCSF group, and + P < 0.05 versus corresponding NRS + Mev group in the post hoc Scheffe´ multiplerange test.

machinery. Histological verifications confirmed that the test agents were microinjected into the RVLM. The specificity of the pharmacological effects thus induced is further ascertained by the lack of cardiovascular responses when mevinphos was microinjected to sites adjacent to but outside the confines of the RVLM (Fig. 2); or the lack of antagonism of the mevinphos-elicited responses when anti-FLJ10540 antiserum was microinjected

outside the RVLM (Fig. 7). Another confirmation arises from a unique pharmacological feature of mevinphos. Delivery of mevinphos, either by oral ingestion in human subjects [40] or by intravenous administration [12] or microinjection into the RVLM in animals [12,14–16], did not elicit significant alterations in HR. Anatomically, the RVLM is located immediately below the nucleus ambiguus (NA), the original of the vagus nerve that exerts an inhibitory action on the heart [13]. As such, the lack of induced changes in HR is another indicator that our observed responses were elicited by site-specific microinjection of mevinphos. In human cancer, patients with higher FLJ10540 expression in tumor tissues exhibit poorer survival rate [6–10]. It is therefore of clinical significance that our laboratory identified previously a unique phenotype from comatose patients who succumbed to organophosphate poisoning [40]; the power density of the BLF component (0.04–0.15 Hz in human) in the power spectrum of their AP signals invariably exhibits a dramatic reduction or loss before brain death ensues. Since this ‘‘life-and-death’’ signal takes origin from the RVLM [18], its disappearance before brain death greatly resembles the significant reduction in BLF power to below baseline during Phase II mevinphos intoxication. It follows that our present finding that FLJ10540-mediated activation of the PI3K/Akt cascade, leading to upregulation of NOS II/peroxynitrite signaling in the RVLM that result in impairment of brain stem cardiovascular regulation during mevinphos intoxication may offer a reasonable modus operandi for brain death. The realization that FLJ10540, which is overexpressed in tumor cell and plays an important role in tumor progression [2,7,41], also subserves organophosphate intoxication and brain death offers a new vista for future development of common therapeutic strategy against these fatal eventualities. Acknowledgements We thank Ms. Kuang-Yu Dai for technical assistance with the immunofluorescence staining experiments. This study was supported by the Chang Gung Medical Foundation, Taiwan (OMRPG8C0021 to S.H.H.C.).

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Upregulation of FLJ10540, a PI3K-association protein, in rostral ventrolateral medulla impairs brain stem cardiovascular regulation during mevinphos intoxication.

FLJ10540, originally identified as a microtubule-associated protein, induces cell proliferation and migration during tumorigenesis via the formation o...
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