James A. Snipes

High-Affinity Interaction between Gram-Negative Flagellin and a Cell Surface Polypeptide Results in Human Monocyte Activation

ABSTRACT Flagella from diverse gram-negative bacteria induce tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) synthesis by human monocytes (F. Ciacci-Woolwine, P. F. McDermott, and S. B. Mizel, Infect. Immun. 67:5176–5185, 1999). In this study, we establish that purified flagellin (FliC or FljB), the major filament protein from Salmonella enterica serovar Enteritidis,S. enterica serovar Typhimurium, and Pseudomonas aeruginosa, is an extremely potent inducer of TNF-α production by human monocytes and THP-1 myelomonocytic cells. Fifty percent of maximal TNF-α production (EC50) was obtained with 1.5 × 10−11 M flagellin (0.75 ng/ml). Mutagenesis studies revealed that the central hypervariable region of flagellin is essential for the TNF-α-inducing activity of the protein. Although less active than the wild-type protein, a Salmonellaflagellin mutant composed of only the central hypervariable region retained substantial TNF-α-inducing activity at nanomolar concentrations. In contrast, the conserved amino- and carboxy-terminal regions are inactive. Mutational analysis of the hypervariable region revealed that it contains two equally active TNF-α-inducing domains. The ability of THP-1 cells to respond to purified flagellins is dramatically reduced by mild trypsin treatment of the cells. Taken together, our results demonstrate that the cytokine-inducing activity of flagellins from gram-negative bacteria results from the interaction of these proteins with high-affinity cell surface polypeptide receptors on monocytes.

Diazoxide induces delayed pre-conditioning in cultured rat cortical neurons

We investigated the effect of diazoxide on neuronal survival in primary cultures of rat cortical neurons against oxygen–glucose deprivation (OGD). Diazoxide pre‐treatment induced delayed pre‐conditioning and almost entirely attenuated the OGD‐induced neuronal death. Diazoxide inhibited succinate dehydrogenase and induced mitochondrial depolarization, free radical production and protein kinase C activation. The putative mitochondrial ATP‐sensitive potassium channel blocker 5‐hydroxydecanoate abolished the protective effect of diazoxide while the non‐selective KATP channel blocker glibenclamide did not. The non‐selective KATP channel openers nicorandil and cromakalim did not improve viability. Superoxide dismutase mimetic, M40401, or protein kinase C inhibitor, chelerythrine, prevented the neuroprotective effect of diazoxide. Diazoxide did not increase reduced glutathione and manganese‐superoxide dismutase levels but we found significantly higher reduced glutathione levels in diazoxide‐pre‐conditioned neurons after OGD. In pre‐conditioned neurons free radical production was reduced upon glutamate stimulation. The succinate dehydrogenase inhibitor 3‐nitropropionic acid also induced pre‐conditioning and free radical production in neurons. Here, we provide the first evidence that diazoxide induces delayed pre‐conditioning in neurons via acute generation of superoxide anion and activation of protein kinases and subsequent attenuation of oxidant stress following OGD. The succinate dehydrogenase‐inhibiting effect of diazoxide is more likely to be involved in this neuroprotection than the opening of mitochondrial ATP‐sensitive potassium channels.

Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS.

Recent studies indicated that there is a distinct mitochondrial nitric oxide synthase (mtNOS) enzyme, which may be identical to the other known NOS isoforms. We investigated the possible involvement of the endothelial, the neuronal, and the inducible NOS isoforms (eNOS, nNOS, iNOS, respectively) in mitochondrial NO production. Mouse liver mitochondria were prepared by Percoll gradient purification from wild-type and NOS knockout animals. NOS activity was measured by the arginine conversion assay, NO production of live mitochondria was visualized by the fluorescent probe DAF-FM with confocal microscopy and measured with flow cytometry. Western blotting or immunoprecipitation was performed with 12 different anti-NOS antibodies. Mitochondrial NOS was purified by arginine, 2,5 ADP and calmodulin affinity columns. We observed NO production and NOS activity in mitochondria, which was not attenuated by classic NOS inhibitors. We also detected low amounts of eNOS protein in the mitochondria, however, NO production and NOS activity were intact in eNOS knockout animals. Neither nNOS nor iNOS were present in the mitochondria. Furthermore, we could not find mitochondrial targeting signals in the sequences of either NOS proteins. Taken together, the presented data do not support the hypothesis that any of the known NOS enzymes are present in the mitochondria in physiologically relevant levels.

Heart mitochondria contain functional ATP-dependent K+ channels

Recent observations challenged the functional importance or even the existence of mitochondrial ATP-dependent K+ (mitoK(ATP)) channels. In the present study, we determined the presence of K(ATP)-channel subunits in mouse heart mitochondria, and investigated whether known openers or blockers of the channel can alter mitochondrial membrane potential. Investigation of the channel composition was performed with antibodies against K(ATP)-channel subunits, namely the sulfonylurea receptor (SUR1 or SUR2) and the inwardly rectifying K+ channel (Kir6.1 or Kir6.2). Specific Kir6.1 and Kir6.2 proteins were found in the mitochondria by western blotting and immunogold electron microscopy. Neither SUR1 nor SUR2 was present in the mitochondria. In contrast, a mitochondrially enriched low molecular weight SUR2-like band was found at approximately 25 kDa. Mitochondrial-transport tags were identified in the sequences of Kir6.1 and Kir6.2, but not in SUR1 or SUR2. The fluorescent BODIPY-glibenclamide labeling of mitochondria indicated direct sulfonylurea binding. Pharmacological characterization of mitoK(ATP) was performed in isolated respiring heart mitochondria. Fluorescent confocal imaging with the membrane potential-sensitive dye MitoFluorRed showed that glibenclamide application changed membrane potential, while the specific mitoK(ATP)-channel openers, diazoxide or BMS-191095, reversed the effect. Mitochondrially formed peroxynitrite is a physiological opener of the channel. We conclude that a functional K(ATP) channel is present in heart mitochondria, which can be opened by diazoxide or BMS-191095. The channel can be composed of Kir6.1 and Kir6.2 subunits and does not contain either SUR1 or SUR2.

Investigation of the subunit composition and the pharmacology of the mitochondrial ATP-dependent K+ channel in the brain

Selective activation of mitoK(ATP) channels can protect the brain or cultured neurons against a variety of anoxic or metabolic challenges. However, little is known about the subunit composition or functional regulation of the channel itself. In the present study, we sought to characterize the mitoK(ATP) channel in the mouse brain using overlapping approaches. First, we determined that mitochondria contain the pore-forming Kir6.1 and Kir6.2 subunits with Western blotting, immunogold electron microscopy and the identification of mitochondrial transport sequences. In contrast, we found no evidence for the presence of either known sulfonylurea receptors (SUR1 or SUR2) in the mitochondria. However, the ATP-dependent K (K(ATP)) channel inhibitor glibenclamide specifically binds to mitochondria in both neurons and astrocytes, and small molecular weight SUR2-like proteins were concentrated in mitochondria. In addition to mice, similar results were found in rats and pigs. Second, live respiring mitochondria were stained with the membrane potential sensitive dye MitoFluorRed and visualized by confocal microscopy. We investigated the effects of pharmacological closing and opening of the channel with glibenclamide and the specific mitoK(ATP) openers diazoxide and BMS-191095. Closing of the channel inhibited the energization of the mitochondria, which was reversed by the application of the mitoK(ATP) openers. We also found that blocking mitochondrial peroxynitrite formation with FP15 has a similar effect to blocking the mitoK(ATP) channels. We conclude that brain mitochondria contain functional K(ATP) channels. The pore-forming subunit of the channel can be either Kir6.1 or Kir6.2, and the SUR subunit may be a SUR2 splice variant or a similar protein.

Putative Cyclooxygenase-3 expression in rat brain cells

Cyclooxygenase-3 (COX-3), a new acetaminophen-sensitive isoform of the COX family, has recently been cloned from canine tissues. Canine COX-3 apparently is identical to the full-length form of COX-1, with the exception that the COX-3 mRNA retains intron 1. Additionally, COX-3 mRNA expression is high in the brain. We investigated the expression of the putative rat COX-3 mRNA in primary cultures of neurons, astrocytes, endothelial cells, pericytes, and choroidal epithelial cells from the rat brain. Specific RT-PCR primers were designed to detect putative rat COX-3 mRNA, and the RT-PCR products were sequenced and compared to the known sequence of the rat COX-1 gene. Our results demonstrate that the mRNA of the putative COX-3 is expressed in all of the cell types except neurons. Cerebral endothelial cells showed the highest COX-3 expression. Whereas COX-2 expression increased several-fold after lipopolysaccharide (LPS) challenge, COX-1 and COX-3 expression did not change significantly, suggesting that cells constitutively express COX-3. In summary, we report, for the first time to our knowledge, that the putative COX-3 mRNA is detectable in rats and is differentially expressed in various cell types from rat brain, as well as that its expression is not stimulated by LPS.

Potassium Channel Dysfunction in Cerebral Arteries of Insulin-Resistant Rats Is Mediated by Reactive Oxygen Species

Background and Purpose— Insulin resistance (IR) increases the risk of stroke in humans. One possible underlying factor is cerebrovascular dysfunction resulting from altered K+ channel function. Thus, the goal of this study was to examine K+ channel–mediated relaxation in IR cerebral arteries. Methods— Experiments were performed on pressurized isolated middle cerebral arteries (MCAs) from fructose-fed IR and control rats. Results— Dilator responses to iloprost, which are BKCa channel mediated, were reduced in the IR compared with control arteries (19±2% versus 33±2% at 10−6 mol/L). Similarly, relaxation to the KATP opener pinacidil was diminished in the IR MCAs (17±2%) compared with controls (38±2% at 10−5 mol/L). IR also reduced the KATP channel–dependent component in calcitonin gene-related peptide–induced dilation; however, the magnitude of the relaxation remained unchanged in IR because of a nonspecified K+ channel–mediated compensatory mechanism. In contrast, Kir channel–mediated relaxation elicited by increases in extracellular [K+] (4 to 12 mmol/L) was similar in the control and IR arteries. Blockade of the Kir and Kv channels with Ba2+ and 4-aminopyridine, respectively, constricted the MCAs in both experimental groups with no significant difference. Pretreatment of arteries with superoxide dismutase (200 U/mL) plus catalase (150 U/mL) restored the dilatory responses to iloprost and pinacidil in the IR arteries. Immunoblots showed that the expressions of the pore-forming subunits of the examined K+ channels are not altered by IR. Conclusions— IR induces a type-specific K+ channel dysfunction mediated by reactive oxygen species. The alteration of KATP and BKCa channel–dependent vascular responses may be responsible for the increased risk of cerebrovascular events in IR.

Contribution of poly(ADP-ribose) polymerase to postischemic blood-brain barrier damage in rats.

The nuclear enzyme poly(ADP-ribose) polymerase (PARP) is activated by oxidative stress and plays a significant role in postischemic brain injury. We assessed the contribution of PARP activation to the blood–brain barrier (BBB) disruption and edema formation after ischemia–reperfusion. In male Wistar rats, global cerebral ischemia was achieved by occluding the carotid arteries and lowering arterial blood pressure for 20 mins. The animals were treated with saline or with the PARP inhibitor N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N, N-dimethylacetamide.HCl (PJ34); (10 mg/kg, i.v.) before ischemia. After 40 mins, 24, and 48 h of reperfusion, the permeability of the cortical BBB was determined after Evans Blue (EB) and Na-fluorescein (NaF) administration. The water content of the brain was also measured. The permeability of the BBB for EB increased after ischemia–reperfusion compared with the nonischemic animals after 24 and 48 h reperfusion but PARP inhibition attenuated this increase at 48 h (nonischemic: 170 ± 9, saline: 760 ± 95, PJ34: 472 ± 61 ng/mg tissue). The extravasation of NaF showed similar changes and PJ34 post-treatment attenuated the permeability increase even at 24 h. PARP inhibition decreased the brain edema seen at 48 h. Because PARP has proinflammatory properties, the neutrophil infiltration of the cortex was determined, which showed lower values after PJ34 treatment. Furthermore, PJ34 treatment decreased the loss of the tight junction protein occludin at 24 and 48 h. The inhibition of PARP activity accompanied by reduced post-ischemic BBB disturbance and decreased edema formation suggests a significant role of this enzyme in the development of cerebral vascular malfunction.

The mitochondrial KATP channel opener BMS-191095 induces neuronal preconditioning

BMS-191095, reportedly a selective mitoKATP channel opener which is free from the known side effects of the prototype mitoKATP channel opener diazoxide, induced acute and delayed preconditioning against glutamate excitotoxicity and delayed preconditioning against oxygen–glucose deprivation in primary cultures of rat cortical neurons. BMS-191095 dose dependently depolarized the mitochondria, increased the phosphorylation of PKC isoforms, but had no detectable effects on the activation of MAP kinases and did not influence the expressions of HSP70 and Mn-SOD. In BMS-191095-preconditioned neurons the glutamate-induced free-radical production was abolished. Our data give the first evidence that selective opening of mitoKATP channels with BMS-191095 leads to remarkable neuroprotection via mechanisms that involve mitochondrial depolarization, PKC activation and attenuated free radical production during neuronal stress.

Activation of mitochondrial ATP-sensitive potassium channels prevents neuronal cell death after ischemia in neonatal rats

Activation of mitochondrial ATP-sensitive potassium channels (mK(ATP)) has been shown to protect against cell death following ischemia/reperfusion in the heart but not in brain. We examined whether mK(ATP) activation with diazoxide (DIZ) prevents neuronal cell death following hypoxia-ischemia (HI) in 7-day-old rat pups. Rat pups were subjected to HI (left carotid ligation; 8% O(2); 2.5 h), following administration of vehicle, 1.9 mg/kg DIZ, 3.8 mg/kg DIZ or DIZ plus 10 mg/kg 5-hydroxydecanoic acid (mK(ATP) antagonist). Total infarct volume was reduced from 99.8+/-2.7% in vehicle animals to 80.6+/-4.2% in 3.8 mg/kg DIZ treated animals (n=85, P<0.05). Western blotting showed K(ATP) subunits concentrated in mitochondria. Fluorescent studies indicated DIZ directly depolarized the mitochondria. In conclusion, selective opening of mK(ATP) prior to HI results in neuroprotection in immature rats.