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Department of Medical Education & Research, Kaohsiung Veterans General Hospital (J.Y.H.C., L.-L.W., C.-C.O.), and Center for Neuroscience, National Sun Yat-sen University (A.Y.W.C, S.H.H.C.), Taiwan, Republic of China
Received September 25, 2006; accepted January 16, 2007
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Abstract |
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), atherosclerosis (Dickhout et al., 2005), ischemia/reperfusion injury (Eefting et al., 2004), and septic shock (Sharahar et al., 2003). Apoptosis is a form of programmed cell death whose initiation and execution are orchestrated by activation of a family of aspartate-specific cysteine proteases called caspases (Takahashi and Earnshaw, 1996). The mitochondrial (caspase 9/caspase 3) pathway, death receptor (caspase 8/caspase 3) pathway, and endoplasmic reticulum (caspase 12/caspase 3) pathway represent three distinct routes via which the caspase cascades are activated (Takahashi and Earnshaw, 1996). Of note is that all three pathways converge to caspase 3 activation, leading to DNA fragmentation as the final step to apoptosis.
The rostral ventrolateral medulla (RVLM) is a brainstem site in which sympathetic premotor neurons that are responsible for maintaining basal vasomotor tone are located (Ross et al., 1984). We reported previously that by reducing the sympathetic vasomotor outflow (Chan et al., 2001b), overproduction of nitric oxide (NO) by the inducible NO synthase (iNOS) in the RVLM plays a pivotal role in fatal cardiovascular depression in an animal model of endotoxemia induced by Escherichia coli lipopolysaccharide (LPS) (Chan et al., 2001a). We demonstrated subsequently (Chan et al., 2005) that iNOS-derived NO causes the release of cytochrome c from the mitochondria to the cytosol, resulting in caspase 3-dependent apoptotic cell death in the RVLM.
The release of apoptosis-initiation factors, including cytochrome c, from the intermediate space via mitochondrial permeability transition pore (mPTP) (Martinou and Green, 2001) plays a key role in the subsequent apoptosome formation and caspase 3 activation (Liu et al., 1996). Composed of adenine nucleotide translocase (ANT) and voltage-dependent anion protein (VDAC) (Halestrap and Brennerb, 2003), opening of the mPTP is reciprocally regulated by various Bcl-2 family proteins. Thus, antiapoptotic proteins such as Bcl-2 and Bcl-xL reduce, and proapoptotic proteins such as Bax and Bak enhance mitochondrial cytochrome c release (Kroemer, 1997). On activation of death signals, Bax translocates from the cytosol to the mitochondria and increases mitochondrial membrane permeabilization via interactions with ANT and/or VDAC (Shimizu et al., 1999). As such, Bax may serve as an intracellular trigger for mitochondrion-dependent apoptosis.
We identified recently (Chang et al., 2006) an interplay between Bax and caspase 3-dependent apoptotic cell death in the RVLM during experimental endotoxemia. The intracellular signals that interpose between generation of NO, activation and translocation of Bax, and cytosolic release of cytochrome c in this apoptotic cascade are unknown. One potential candidate is protein kinase C (PKC). LPS mediates its cellular effects via Toll-like receptor 4-associated second-messenger pathways, including PKC (Comalada et al., 2003), and mitochondrial translocation of activated Bax after its dissociation from the PKC/Bax complex is engaged in hypoxia/reperfusion-induced apoptosis in endothelial cell (Wang et al., 2005). We hypothesize that generation of NO by up-regulation of iNOS in the RVLM leads to PKC activation, which triggers activation and translocation of Bax that opens mPTP by interacting with ANT or VDAC in the mitochondria, followed by activation of the cytochrome c-caspase 3 apoptotic cascade, which underlies cardiovascular depression during endotoxemia. The present study validated this hypothesis. We demonstrated that iNOS activation in the RVLM after LPS treatment resulted in membrane-bound translocation of PKC from the cytosol and the dissociation of PKC/Bax complex. After mitochondrial translocation, the activated Bax formed a heterodimeric complex with ANT and VDAC, leading to cytochrome c release, activation of caspase 3, and DNA fragmentation in the RVLM. It is important to note that these cellular events play a crucial role in the manifestation of endotoxin-induced cardiovascular depression.
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Materials and Methods |
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Animals. Experiments were carried out in adult male Sprague-Dawley rats (200–270 g, n = 268) purchased from the Experimental Animal Center of the National Applied Research Laboratories (Taipei, Taiwan). They were housed in an animal room under temperature control (24 ± 0.5°C) and 12-h light/dark (8 AM to 8 PM) cycle. Standard laboratory rat chow (PMI Nutrition International, Brentwood, MO) and tap water were available ad libitum. All animals were allowed to acclimatize for at least 7 days before experimental manipulations.
General Preparation. Rats were anesthetized initially with pentobarbital sodium (50 mg/kg i.p.) to perform preparatory surgery (Chan et al., 2001a,b, 2002, 2005), which routinely included intubation of the trachea and cannulation of the femoral artery and vein. Thereafter, animals received continuous intravenous infusion of propofol (30 mg/kg/h), which provided satisfactory anesthetic maintenance while preserving the capacity of central cardiovascular regulation (Yang et al., 1995). Animals were mechanically ventilated to maintain end-tidal CO2 to be within 4 to 5%, as monitored by a capnograph (Datex Normocap, Helsinki, Finland). All data were collected from animals with a maintained rectal temperature of 37 ± 0.5°C. At the end of each experiment, rats were killed with intravenous injection of an overdose of pentobarbital sodium (100 mg/kg).
Power Spectral Analysis of SAP Signals. Pulsatile and mean systemic arterial pressure (MSAP) and heart rate (HR) were recorded on a polygraph (Gould, Valley View, OH), along with simultaneous online and real-time power spectral analysis of the SAP signals (Chan et al., 2001a,b; 2002). We were particularly interested in the very low-frequency (0–0.25 Hz) and low-frequency (0.25–0.8 Hz) components in the SAP spectrum. Our laboratory demonstrated previously (Kuo et al., 1997) that these spectral components of SAP signals take origin from the RVLM, and their power density reflects the prevailing neurogenic sympathetic vasomotor tone.
Induction of Experimental Endotoxemia. Experimental endotoxemia was induced by intravenous injection of Escherichia coli LPS (15 mg/kg, serotype 0111:B4; Sigma-Aldrich, St. Louis, MO) (Chan et al., 2001a, 2005; Chang et al., 2006). Injection of the same amount of 0.9% saline served as the vehicle and volume control. The temporal changes in mean SAP, HR, and power density of the vasomotor components of the SAP signals were routinely followed for 6 h.
Microinjection of Test Agents into the RVLM. Test agents were microinjected bilaterally and sequentially at a volume of 50 nl into the RVLM (Chan et al., 2001a,b, 2002, 2005; Chang et al., 2006). The coordinates for RVLM were 4.5 to 5 mm posterior to lambda, 1.8 to 2.1 mm lateral to the midline, and 8.0 to 8.5 mm below the dorsal surface of cerebellum. Test agents used included an iNOS inhibitor, S-methylisothiourea (SMT; Tocris Cookson, Bristol, UK); an NO trapping agent, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-L-oxyl-3-oxide (carboxy-PTIO; Tocris Cookson); selective PKC inhibitors, calphostin C, or Gö6983 (Calbiochem, San Diego, CA); a chloride-channel inhibitor that blocks mitochondrial translocation of Bax, furosemide (Sigma-Aldrich); cell membrane-permeable Bax inhibitor peptides, Pro-Met-Leu-Lys-Glu (P5; Tocris Cookson) or Val-Pro-Met-Leu-Lys (V5; Tocris Cookson); a Bax channel inhibitor (BCI), 3,6-dibromo-
-(1-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride (Tocris Cookson); an inhibitory ligand of the mitochondrial ANT, bongkrekic acid (BA; Sigma-Aldrich); or a cell-permeable caspase 3 inhibitor, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (z-DEVD-fmk; Calbiochem). The dose and treatment scheme were adopted from previous reports (Chan et al., 2001b, 2005; Wu et al., 2003) that used the same test agents for the same purpose as in this study or established during the initial pilot studies. With the exception of Gö6983, BCI, or z-DEVD-fmk, which used 1% DMSO as the solvent, all test agents were prepared with artificial cerebrospinal fluid (aCSF). Microinjection of these two solvents served as the vehicle and volume control.
Collection of Ventrolateral Medullary Samples. Rats were killed with an overdose of pentobarbital sodium and perfused intracardiacally with 150 ml of warm (37°C) saline containing heparin (100 U/ml). The brain was rapidly removed and placed on dry ice. Both sides of the ventrolateral medulla, at the level of the RVLM (0.5–1.5 mm rostral to the obex), were collected by micropunches made with a stainless steel bore (1 mm internal diameter). Medullary tissues collected from animals under anesthesia but without treatment served as the sham control.
Isolation of Membranous, Cytosolic, or Mitochondrial Fractions. We isolated membranous, cytosolic, or mitochondrial fraction by discontinuous Percoll gradient centrifugation according to procedures described previously (Chuang et al., 2002; Chan et al., 2005). This procedure yields 10 to 15% of the total mitochondria and enriches the mitochondrial fraction by at least 10-fold compared with tissue homogenates (Kantrow et al., 1997). The amount of protein in each fraction was determined by the method of Bradford (1976) with a protein assay kit (Bio-Rad Laboratories, Hercules, CA).
Western Blot Analysis. Western blot analysis (Chan et al., 2002, 2005; Chang et al., 2006) was carried out on proteins extracted from the membranous, cytosolic, or mitochondrial fraction. The primary antiserum used in this study included rabbit polyclonal anti-PKC (1:1000), anti-PKC- (1:500), anti-PKC-
I (1:2000), anti-PKC-II (1: 2000), anti-PKC-
(1:2000), anti-PKC-
(1:2000), anti-PKC-
(1: 1000), anti-PKC-
(1:1000), anti-PKC-
(1:2000), anti-PKC-
(1:2000; all from Calbiochem), anti-cytochrome c (1:1000 and 1:5000 for cytosolic and mitochondrial fractions, respectively; Calbiochem), anti-caspase 3 that recognizes the inactive procaspase 3 (36 kDa) and the active cleaved fragment (20 KDa) of caspase 3 (1:1000); anti-Bcl-xL (1:1000), anti-Bcl-2 (1:1000; all from Calbiochem), anti-ANT (1:1000), anti-VDAC (1:1000; both from Santa Cruz Biotechnology, Santa Cruz, CA), anti-HSP70 (1:2000; StressGen, Victoria, BC, Canada) or anti--tubulin (1:5000; Sigma-Aldrich) antiserum. The secondary antisera used included horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:5000; Jackson ImmunoResearch Laboratories, West Grove, PA). Specific antibody-antigen complex was detected by an enhanced chemiluminescence Western blot detection system (PerkinElmer Life and Analytical Sciences, Boston, MA). The amount of protein was quantified by Photo-Print Plus software (ETS Vilber-Lourmat, Marne La Vallee, France), and was expressed as the ratio (percentage) to -tubulin protein, which served as the internal control to demonstrate equal loading of the proteins.
Immunoprecipitation. For immunoprecipitation assay (Chang et al., 2006), protein A- or G-agarose beads were added to each protein fraction. Immunoprecipitation with either a mouse monoclonal anti-Bax 6A7 antiserum that recognizes specifically the conformational change in Bax protein associated with its activation (Yethon et al., 2003) or a rabbit polyclonal anti-Bcl-2, anti-Bcl-xL, anti-ANT, anti-VDAC, or anti-PKC antiseum was performed at 4°C overnight, and the precipitated beads were washed with an ice-cold lysis buffer followed by a kinase buffer (25 mM HEPES, pH 7.4, 20 mM MgCl2, 0.1 mM Na3VO4, and 2 mM dithiothreitol). Western blot analysis of Bax (1:1000; Calbiochem) was carried out as described above.
Sandwich Enzyme-Linked Immunosorbent Assay for Histone-Associated DNA Fragmentation. To quantify apoptosis-related DNA fragmentation, a cell death enzyme-linked immunosorbent assay (Roche Molecular Biochemicals, Mannheim, Germany) that detects apoptotic but not necrotic cell death (Bonfoco et al., 1995) was used to assay the level of histone-associated DNA fragments in the cytoplasm (Saito et al., 2004). In brief, protein from the cytosolic fraction of the ventrolateral medullary samples was used as the antigen source, together with primary anti-histone antibody and secondary anti-DNA antibody coupled to peroxidase. The amount of nucleosomes in cytoplasm was quantitatively determined using 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate] as the substrate. Absorbance was measured at 405 nm and referenced at 490 nm using a microtiter plate reader (Hitachi, Tokyo, Japan).
Histology. For verification of microinjection sites, the brain stem, except for those dissected by micropunches for extraction of protein, was removed from animals after the physiological experiments and fixed in 30% sucrose in 10% formaldehyde-saline solution for 
72 h. Frozen 25-µm sections of the medulla oblongata were stained with 1% neural red for histological verification of the location of microinjection sites. One percent Evans blue dye was added to the microinjection solution to facilitate this process.
Statistical Analysis. All values are expressed as mean ± S.E. One-way or two-way analysis of variance with repeated measures was used to assess group means, as appropriate, to be followed by the Scheffé multiple-range test for post hoc assessment of individual means. p < 0.05 was considered statistically significant.
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). Western blot analysis revealed an increased expression of PKC-I, PKC-, PKC-, or PKC- isoform in the membranous fraction of samples from ventrolateral medulla during experimental endotoxemia, accompanied by a concomitant decrease of those PKC isoforms in the cytosolic fraction (Fig. 1). This membrane translocation of PKC-I, PKC-, PKC-, or PKC- occurred 15 min after LPS administration, lasting at least 60 min. The expression of PKC- or PKC- isoforms in the cytosolic or membranous fraction, on the other hand, remained unchanged. In addition, expression of PKC-II, PKC-, or PKC- in both fractions was lower than our detection limit (data not shown).
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I, PKC-, PKC-, or PKC- expression in the membranous fraction of samples from ventrolateral medulla (Fig. 2). Similar observations were found in animals that received the NO trapping agent carboxy-PTIO (50 nmol) in the bilateral RVLM (data not shown). SMT or carboxy-PTIO, on the other hand, had no effect on basal cytosolic or membranous expression of those PKC isoforms (data not shown).
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Mitochondrial Translocation of Activated Bax in Ventrolateral Medulla During Experimental Endotoxemia. We next investigated whether mitochondrial translocation of activated Bax from the cytosol took place in the RVLM during experimental endotoxemia. Coimmunoprecipitation experiments pairing an anti-6A7 antiserum that specifically recognizes the conformational change in Bax protein associated with its activation with an anti-Bax antiserum or vice versa (Fig. 3B) also revealed a progressive increase in the expression of activated Bax in ventrolateral medulla measured 60, 120, or 240 min after LPS administration. Whereas SMT treatment (250 pmol) again suppressed this endotoxin-promoted Bax activation, the total Bax expression in ventrolateral medulla was unaffected by LPS, given alone or with SMT. We further demonstrated in samples obtained from ventrolateral medulla 120 min after LPS treatment that the appreciable increase in activated Bax was present in both the cytosolic and mitochondrial fraction (Fig. 3, C and D). Although this augmentation of cytosolic and mitochondrial Bax was blunted by the PKC inhibitors calphostin C (100 pmol) or Gö6983 (100 pmol) (Fig. 3C), only the increase in mitochondrial Bax was attenuated by inhibition of Bax translocation with furosemide (1, 5, or 10 nmol) (Fig. 3D). Furosemide, at the same time, caused further increases in activated Bax expression in the cytosol (Fig. 3D).
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Bax-Dependent Release of Mitochondrial Cytochrome c, Activation of Caspase 3, or DNA Fragmentation in Ventrolateral Medulla During Experimental Endotoxemia. Similar to our previous observations (Chan et al., 2005; Chang et al., 2006), experimental endotoxemia was accompanied by the release of cytochrome c from the mitochondria to the cytosol, followed by activation of the proapoptotic caspase 3 in ventrolateral medulla (Fig. 5A). Up-regulation of cytosolic cytochrome c was detected at 120 min, and activated caspase 3 was expressed 180 min after LPS treatment. Microinjection bilaterally into the RVLM of BCI (0.5 nmol) (Fig. 5A) or BA (1 µ mol) (data not shown) significantly antagonized this LPS-induced cytochrome c release or caspase 3 activation. The same treatments, along with furosemide (10 nmol) or the caspase 3 inhibitor, z-DEVD-fmk (100 nmol), also significantly suppressed DNA fragmentation induced in ventrolateral medulla during experimental endotoxemia (Fig. 5B).
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; Chang et al., 2006), LPS treatment resulted in an initial followed by a delayed decline in MSAP and HR, along with a reduction in the power density of the vasomotor components of SAP signals during the 360-min observation period (Figs. 6 and 7). Compared with vehicle controls, microinjection immediately after LPS administration into the bilateral RVLM of calphostin C (50 or 100 pmol) (Figs. 6A and 8A), furosemide (5 or 10 nmol) (Fig. 8A), Bax inhibitor peptides V5 (10 or 20 pmol) or P5 (10 or 20 pmol) (Fig. 8A), BCI (0.5 or 1 nmol) (Figs. 7A and 8A), BA (5 or 10 nmol) (Fig. 8A), or z-DEVD-fmk, (50 or 100 nmol) (Fig. 8A) significantly antagonized the cardiovascular depression during experimental endotoxemia. Of note is that, given 120 min after LPS treatment, furosemide (Fig. 8B), V5 or P5 (Fig. 8B), BCI (Figs. 7B and 8B), BA (Fig. 8B), or z-DEVD-fmk (Fig. 8B), but not calphostin C (Figs. 6B or 8B), significantly attenuated the LPS-promoted decrease in MSAP, HR, or sympathetic neurogenic vasomotor tone.
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) that iNOS-derived NO triggers the cytosolic release of mitochondrial cytochrome c during experimental endotoxemia, resulting in caspase 3-dependent apoptotic cell death in the RVLM. We further showed (Chang et al., 2006) that this process involves the participation of the proapoptotic protein Bax. The present study provided the novel identification that activated PKC represents the crucial interposing intracellular signal that triggers mitochondrial translocation of Bax after iNOS activation. We demonstrated that the temporal sequence of signaling events after activation of iNOS in the RVLM during experimental endotoxemia includes membrane-bound translocation of cytosolic PKC, dissociation of the cytosolic PKC/Bax complex, mitochondrial translocation of Bax, heterodimerization between activated Bax and ANT or VDAC, cytosolic release of cytochrome c, activation of caspase 3, and DNA fragmentation. Most importantly, we demonstrated that the formation of mitochondrial Bax/ANT or Bax/VDAC complex, which initiates apoptosis in the RVLM as a result of PKC-dependent mitochondrial translocation of activated Bax, plays a pivotal role in the manifestation of endotoxin-induced cardiovascular depression.
Membrane-bound translocation of PKC from the cytosol is a hallmark of PKC activation (Buchner et al., 1999). Accordingly, the present study revealed an early activation of conventional PKCIor , novel PKC, and atypical PKC isoforms in ventrolateral medulla after LPS treatment. Ischemia/reperfusion injury results in activation of PKC in the hippocampus (Bright and Mochly-Rosen, 2005), and an early activation of PKC is involved in the N-methyl-D-aspartate-induced apoptosis in cortical neurons (Crisanti et al., 2005). The reversal by SMT of membrane-bound translocation of all of those PKC isoforms further supports a causal relationship between the iNOS-derived NO and PKC activation. NO may activate PKC via at least two mechanisms. One is by reacting with superoxide anion to form peroxynitrite that activates PKC (Lochner et al., 2000), and the other is by direct nitration of tyrosine residues in PKC (Balafanova et al., 2002). It is therefore of interest that we reported previously (Chan et al., 2002) that formation of peroxynitrite by a reaction between iNOS-derived NO and superoxide anion in the RVLM contributes primarily to cardiovascular depression during experimental endotoxemia.
Our data showed that PKC forms a complex with Bax under basal condition in ventrolateral medulla. It is intriguing that PKC activation on LPS treatment was accompanied by the dissociation of this PKC/Bax complex, concurrent with elevated levels of activated Bax that was inhibited by SMT or PKC inhibitors. These results suggest, for the first time, that membrane-bound translocation of PKC after iNOS activation leads to dissociation of PKC from the complex, which triggers conformational changes that transform Bax to an active form that in turn translocates to the mitochondria. Of note is that activation and translocation of Bax, which was readily detectable in the mitochondrial fraction 60 min after LPS treatment, occurred without a decrease in overall Bax expression in the cytosol. This observation suggests that de novo synthesis of Bax may contribute to the increase in Bax translocation during endotoxemia. A conformational change in Bax as a result of changes in the ionic composition of the cytosolic milieu underlies intracellular translocation of Bax (Roucou and Martinou, 2001). It is therefore intriguing to note that furosemide reduced the augmented expression of the activated Bax in the mitochondria in a dose-related fashion, alongside further increases in cytosolic Bax expression. These results are interpreted to suggest that by inhibiting the chloride channels, furosemide may alter the cytosolic pH and/or ionic strength (Cooper and Hunter, 1997) and effectively prevent the conformational change in Bax that is required for mitochondrial translocation.
Conflicting reports exist on whether PKC inhibits apoptosis (Pierchala et al., 2004; Weinreb et al., 2004) or mediates apoptotic processes (Bright et al., 2004; Bright and Mochly-Rosen, 2005). Bax moves from the cytosol to mitochondria under conditions that induce cell death by apoptosis (Wolter et al., 1997). The present study provided novel demonstration that a key determinant of this Bax-dependent apoptotic activity in the RVLM during experimental endotoxemia is the extent of association between PKC and Bax. We further observed that the temporal profile of Bax translocation to the mitochondria after LPS treatment coincided with that of cytochrome c release to the cytosol and was followed by activation of caspase 3 and apoptotic cell death in ventrolateral medulla. More importantly, that furosemide prevented all those LPS-induced apoptotic signaling indicated that mitochondrial translocation of Bax on dissociation from the activated PKC is a prerequisite for triggering the cytochrome c-caspase 3 cascade of mitochondrial apoptotic event in the RVLM during endotoxemia.
The importance of Bax in the execution of mitochondrial death pathway was reported in a recent study in which denervation-induced cytochrome c release, increase in caspase 3 activity, and apoptotic cell death in the muscle are absent in Bax-knockout mice (Siu and Always, 2006). There are two mechanisms by which Bax may target mitochondria to trigger apoptotic cell death (Adams and Cory, 1998). One is for Bax to interact with antiapoptotic members of Bcl-2 family such as Bcl-2 and Bcl-xL to block their actions, and the other is to directly enhance the mitochondrial outer-membrane permeability. The present study provided novel demonstration that enhanced Bax/ANT or Bax/VDAC interactions in mitochondria, but not association of Bax with Bcl-2 or Bcl-xL, contribute to the LPS-promoted apoptotic cell death in the RVLM during experimental endotoxemia. Heterodimerization between Bax and ANT or VDAC directly changes the conformation of mPTP and increases membrane permeability (Shimizu et al., 1999; Cao et al., 2001), and the release of cytochrome c from mPTP plays a key role in triggering caspase-3 activation and the associated mitochondrial apoptotic pathway (Liu et al., 1996; Halestrap and Brennerb, 2003). Furthermore, ectopic expression of Bax-induced apoptosis occurs in wild-type but not in ANT- or VDAC-deficient cells (Shimizu et al., 1999). Our observations that Bax channel or ANT inhibitor significantly reduced the formation of Bax/ANT or Bax/VDAC complex, suppressed release of cytochrome c into the cytosol, and attenuated LPS-induced apoptosis in the RVLM are therefore consistent with the stipulated enhancement of mitochondrial outer-membrane permeability by Bax in the initiation of the apoptotic pathway. A reduction in LPS-promoted formation of mitochondrial Bax/ANT or Bax/VDAC complex in the presence of furosemide further suggests that only activated Bax that translocates to the mitochondria is associated with this complex.
NO derived from iNOS in the RVLM suppresses sympathetic neurogenic vasomotor activity and induces severe hypotension and bradycardia after LPS treatment (Chan et al., 2001a, 2002). Perhaps the most intriguing finding of the present study is the demonstration, for the first time, that our identified PKC-dependent mitochondrial translocation of activated Bax, formation of mitochondrial Bax/ANT and Bax/VDAC complexes, cytosolic release of cytochrome c, and caspase 3-dependent apoptosis in the RVLM represent a crucial cascade of intracellular events that are triggered by iNOS-derived NO and play a pivotal role in the manifestation of cardiovascular depression during endotoxemia. Treatments with the same PKC, Bax translocation, or Bax channel inhibitors at doses that sufficiently blunted the relevant cellular events also significantly attenuated the LPS-promoted decrease in MSAP, HR, or sympathetic neurogenic vasomotor tone, providing ample support for this notion. We noted that although pharmacological blockade of mitochondrial Bax translocation or Bax-associated mitochondrial apoptotic signaling delivered 120 min after LPS treatment was effective against the late-stage cardiovascular depression, coadministration of PKC inhibitors into the RVLM at this time point exerted minimal effects. These observations are again complementary to our biochemical results and reinforced the contention that iNOS-dependent activation of PKC constitutes an early step of cellular events that mediate LPS-induced cardiovascular depression. The contribution of PKCI, PKC, PKC, or PKC isoforms to apoptosis in the RVLM and cardiovascular depression during experimental endotoxemia, however, requires further delineation.
Human and animal studies have shown that cardiovascular depression is a crucial and often fatal event that may lead to multiple organ failure during endotoxemia. Whereas endotoxemia is a well-defined clinical phenomenon, the pathogenic basis and the molecular mediators that trigger circulatory depression, particularly in brain areas that are involved in cardiovascular regulation, remain poorly understood. Based on results from the present study, we propose that iNOS-derived NO triggered during endotoxemia induces membrane-bound translocation of PKC, resulting in dissociation of cytosolic PKC/Bax complex, to be followed by mitochondrial translocation of the activated Bax, formation of mitochondrial Bax/ANT or Bax/VDAC complex, leading to cytosolic release of cytochrome c and execution of caspase 3-associated apoptotic cell death in the RVLM. Most importantly, this cascade of intracellular events plays a pivotal role in the elicitation of cardiovascular depression during endotoxemia. This detailed dissection of the intracellular signaling mechanisms in the RVLM after iNOS activation opens a new therapeutic vista in our search for new and more effective agents against cardiovascular depression during endotoxemia. It is of significance that we found that the effectiveness of pharmacological blockade of PKC, mitochondrial Bax translocation, or Bax-associated mitochondrial apoptotic signaling against cardiovascular depression was dependent on the time of administration after LPS treatment. These novel observations pointed to the importance of therapeutic windows when targeting different cellular events in the design of management strategies against endotoxin-induced cardiovascular depression.
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Acknowledgements |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: RVLM, rostral ventrolateral medulla; aCSF, artificial cerebrospinal fluid; ANT, adenine nucleotide translocase; BA, bongkrekic acid; BCI, 3,6-dibromo--(1-piperazinylmethyl)-9H-carbazole-9-ethanol dihydrochloride; carboxy-PTIO, carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-L-oxyl-3-oxide; HR, heart rate; iNOS, inducible nitric-oxide synthase; LPS, lipopolysaccharide; mPTP, mitochondrial permeability transition pore; MSAP, mean systemic arterial pressure; P5, Pro-Met-Leu-Lys-Glu; PKC, protein kinase C; SAP, systemic arterial pressure; SMT, S-methylisothiourea; V5, Val-Pro-Met-Leu-Lys; VDAC, voltage-dependent anion protein; z-DEVD-fmk, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone; DMSO, dimethyl sulfoxide; Gö6983, 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl) maleimide.
Address correspondence to: Dr. Samuel H. H. Chan, Center for Neuroscience, National Sun Yat-sen University, Kaohsiung 80424, Taiwan, Republic of China. E-mail: schan{at}mail.nsysu.edu.tw
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