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Ischemic stroke is a devastating disease with limited therapeutic options. It is very urgent to find a new target for drug development. Here we found that the blood level of MIF in ischemic stroke patients is upregulated. To figure out the pathological role of MIF in ischemic stroke, both in vitro and in vivo studies were conducted. For in vitro studies, primary cortical neuron cultures and adult rat brain endothelial cells (ARBECs) were subjected to oxygen-glucose deprivation (OGD)/reoxygenation. Middle cerebral artery occlusion (MCAo) rodent models were used for in vivo studies. The results show that MIF exerts no direct neuronal toxicity in primary culture but disrupts tight junction in ARBECs. Furthermore, administration of MIF following MCAo shows the deleterious influence on stroke-induced injury by destroying the tight junction of blood-brain barrier and increasing the infarct size. In contrast, administration of MIF antagonist ISO-1 has the profound neuroprotective effect. Our results demonstrate that MIF might be a good drug target for the therapy of stroke.

Alzheimer’s disease (AD) is a major form of senile dementia, characterized by progressive memory and neuronal loss combined with cognitive impairment. AD is the most common neurodegenerative disease worldwide, affecting one-fifth of those aged over 85 years. Recent therapeutic approaches have been strongly influenced by five neuropathological hallmarks of AD: acetylcholine deficiency, glutamate excitotoxicity, extracellular deposition of amyloid-β (Aβ plague), formation of intraneuronal neurofibrillary tangles (NTFs), and neuroinflammation. The lowered concentrations of acetylcholine (ACh) in AD result in a progressive and significant loss of cognitive and behavioral function. Current AD medications, memantine and acetylcholinesterase inhibitors (AChEIs) alleviate some of these symptoms by enhancing cholinergic signaling, but they are not curative. Since 2003, no new drugs have been approved for the treatment of AD. This article focuses on the current research in clinical trials targeting the neuropathological findings of AD including acetylcholine response, glutamate transmission, Aβ clearance, tau protein deposits, and neuroinflammation. These investigations include acetylcholinesterase inhibitors, agonists and antagonists of neurotransmitter receptors, β-secretase (BACE) or γ-secretase inhibitors, vaccines or antibodies targeting Aβ clearance or tau protein, as well as anti-inflammation compounds. Ongoing Phase III clinical trials via passive immunotherapy against Aβ peptides (crenezumab, gantenerumab, and aducanumab) seem to be promising. Using small molecules blocking 5-HT6 serotonin receptor (intepirdine), inhibiting BACE activity (E2609, AZD3293, and verubecestat), or reducing tau aggregation (TRx0237) are also currently in Phase III clinical trials. We here systemically review the findings from recent clinical trials to provide a comprehensive review of novel therapeutic compounds in the treatment and prevention of AD.

Long-term opioid therapy for chronic pain may lead to analgesic tolerance, especially when administered intrathecally, thus preventing adequate pain relief. Discovering drug targets to treat opioid tolerance using a mechanism-based approach targeting opioid-induced neuroinflammation provides new therapeutic opportunities. In this study, we provide translational evidence that CXCL12/CXCR4 signaling contributes to the pathogenesis of opioid tolerance.The CXCL12 levels in the cerebrospinal fluid of opioid-tolerant patients were compared with those of opioid-naive subjects. For further investigation, a rodent translational study was designed using 2 clinically relevant opioid delivery paradigms: daily intraperitoneal morphine injections and continuous intrathecal morphine infusion. We measured rats' tail flick responses and calculated the percentage of maximum possible effects (%MPE) to demonstrate opioid acute antinociception and the development of analgesic tolerance. The effects of exogenous CXCL12, CXCL12 neutralizing antibody, and receptor antagonist AMD3100 were investigated by intrathecal administration. Data were presented as mean ± SEM.CXCL12 was significantly upregulated in the cerebrospinal fluid of opioid-tolerant patients for 892 ± 34 pg/mL (n = 27) versus 755 ± 33 pg/mL (n = 10) in naive control subjects (P = .03). Furthermore, after 2 and 5 days of intrathecal morphine infusion, rat lumbar spinal cord dorsal horn CXCL12 messenger RNA levels were significantly upregulated by 3.2 ± 0.7 (P = .016) and 3.4 ± 0.3 (P = .003) fold, respectively. Results from the daily intraperitoneal morphine injection experiments revealed that administering an intrathecal infusion of CXCL12 for 24 hours before the first morphine injection did not decrease antinociception efficacy on day 1 but accelerated tolerance after day 2 (%MPE 49.5% vs 88.1%, P = .0003). In the intrathecal morphine coinfusion experiments, CXCL12 accelerated tolerance development (%MPE 9.4% vs 43.4% on day 1, P.0001), whereas coadministration with CXCL12 neutralizing antibody attenuated tolerance (72.5% vs 43.4% on day 1, P.0001; 47.6% vs 17.5% on day 2, P.0001). Coadministration of receptor antagonist AMD 3100 can persistently preserve morphine analgesic effects throughout the study period (27.9% ± 4.1% vs 0.9% ± 1.6% on day 5, P = .03).The CXCL12/CXCR4 pathway contributes to the pathogenesis of opioid tolerance. Our study indicates that intervening with CXCL12/CXCR4 signaling has therapeutic potential for opioid tolerance.

Background: The pivotal role of glial activation and up-regulated inflammatory mediators in the opioid tolerance has been confirmed in rodents but not yet in humans. Here, the authors investigated the intraspinal cytokine and chemokine profiles of opioid-tolerant cancer patients; and to determine if up-regulated chemokines could modify opioid tolerance in rats. Methods: Cerebrospinal fluid samples from opioid-tolerant cancer patients and opioid-naive subjects were compared. The cerebrospinal fluid levels of tumor necrosis factor-alpha, CXCL1, CXCL10, CCL2, and CX3CL1 were assayed. The rat tail flick test was utilized to assess the effects of intrathecal CXCL1 on morphine-induced acute antinociception and analgesic tolerance. Results: CXCL1 level in cerebrospinal fluid was significantly up-regulated in the opioid-tolerant group (n = 30, 18.8 pg/ml vs. 13.2 pg/ml, P = 0.02) and was positively correlated (r2 = 0.49, P < 0.01) with opioid dosage. In rat experiment, after induction of tolerance by morphine infusion, the spinal cord CXCL1 messenger RNA was up-regulated to 32.5 ± 11.9-fold. Although CXCL1 infusion alone did not affect baseline tail-flick latency, the analgesic efficacy of a single intraperitoneal injection of morphine dropped significantly on day 1 to day 3 after intrathecal infusion of CXCL1. After establishing tolerance by intrathecal continuous infusion of morphine, its development was accelerated by coadministration of CXCL1 and attenuated by coadministration of CXCL1-neutralizing antibody or CXCR2 antagonist. Conclusions: CXCL1 is up-regulated in both opioid-tolerant patients and rodents. The onset and extent of opioid tolerance was affected by antagonizing intrathecal CXCL1/CXCR2 signaling. Therefore, the CXCL1/CXCR2 signal pathway may be a novel target for the treatment of opioid tolerance.

Neuroinflammation and oxidative stress are important factors that induce neurodegeneration in age-related neurological disorders. 5-Lipoxygenase (5-LOX) is the enzyme responsible for catalysing the synthesis of leukotriene or 5-HETE from arachidonic acid. 5-LOX is expressed in the central nervous system and may cause neurodegenerative disease. In this study, we investigated the effect of the pharmacological inhibition of 5-lipoxygenase on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/MPP(+)-induced dopaminergic neuronal death in midbrain neuron-glia co-cultures and in mice. It was found that 5-LOX was over-expressed in astrocytes after the injection of MPTP into C57BL6 mice. MK-886, a specific inhibitor of 5-LOX activating protein (FLAP), significantly increased [(3)H]-dopamine uptake, a functional indicator of the integrity of dopaminergic neurons, in midbrain cultures or the SH-SY5Y human dopaminergic cell line following MPP(+) treatment. In addition, LTB₄, one of 5-LOX's downstream products, was increased in the striatum and substantia nigra following MPTP injection in mice. LTB₄ but not LTD₄ and 5-HETE enhanced MPP(+)-induced neurotoxicity in primary midbrain cultures. MK-886 administration increased the number of tyrosine hydroxylase-positive neurons in the substantia nigra and the dopamine content in the striatum in MPTP-induced parkinsonian mice. Furthermore, the MPTP-induced upregulation of LTB₄ in the striatum and substantia nigra was antagonised by MK-886. These results suggest that 5-LOX inhibitors may be developed as novel neuroprotective agents and LTB₄ may play an important pathological role in Parkinson's disease.

Acetazolamide (AZ) is an carbonic anhydrase inhibitor, which has been used in the treatment of seizures, mountain sickness and glaucoma. Memory impairment by AZ has been reported in patient interviews; however, the related mechanism is unclear. We applied two fear conditioning paradigms, shuttle avoidance and passive avoidance, in both rats and mice to investigate this clinical anecdote. Adult Wistar rats receiving AZ 1 h before the shuttle avoidance test showed decreased avoidance rates, especially at high dosage. Adult ICR mice receiving AZ both before and after acquisition trials showed the decreased step-through latencies during the passive avoidance test. This impairment of fear memory was corroborated with decreased LTP by AZ in the amygdala. AZ only inhibited fear conditioning-induced ERK phosphorylation and had no effect on Akt phosphorylation. In conclusion, our study confirmed the adverse cognitive effect of AZ in animal and electrophysiological studies. In clinical practice, clinicians should be aware of this side effect in patients taking AZ. In addition, this inhibition of fear memory by AZ could potentially be applied to patients with posttraumatic stress disorder.

Osteoarthritis (OA) is a degenerative joint disease involving a combination of cartilage degradation and inflammation. EGb761, a standardized extract of Ginkgo biloba leaves, holds an anti-inflammatory potency. Here, we determined whether EGb761 could inhibit lipopolysaccharide (LPS)- and IL-1β-induced inflammatory responses in human articular chondrocytes and apply the chondroprotection in OA rats. We found that LPS markedly induced the productions of PGE2 and NO and the protein expressions of COX-2 and iNOS in human chondrocytes. LPS was also seen to up-regulate the expressions of toll-like receptor-4 (TLR4), its downstream signal TNF receptor-associated factor 6 (TRAF6), and nuclear factor (NF)-κB signaling. These LPS-induced inflammatory responses were efficaciously reversed by EGb761 and its active components quercetin and kampferol. The similar results could be observed by using IL-1β as an in vitro model to mimic an inflammatory response. In an OA rat model, PGE2 and NO levels in blood, the histological alterations, and COX-2 and nitrotyrosine expressions in cartilages were markedly increased, which were effectively reversed by EGb761. Our results suggested that EGb761 exerts the anti-inflammatory effects on human articular chondrocytes and OA rats.

Integrins are heterodimeric cell surface receptors that mediate cell-cell and cell-matrix interaction. The vitronectin and osteopontin receptor αvβ3 integrin has increased expression levels and is implicated in the adhesion, activation, and migration of osteoclasts on the bone surface as well as osteoclast polarization. αvβ3 integrin plays an important role in osteoclast differentiation and resorption. In addition, Arg-Gly-Asp (RGD)-containing peptides, small molecular inhibitors, and antibodies to αvβ3 integrin have been shown to inhibit bone resorption in vitro and in vivo. Here we examined the effects of a disintegrin HSA-ARLDDL a genetically modified mutant of rhodostomin conjugated with human serum albumin, which is highly selective of αvβ3, on RANKL-induced osteoclastogenesis and ovariectomy (OVX)-induced osteoporosis. In RANKL-induced osteoclastogenesis, HSA-ARLDDL significantly inhibited osteoclast formation, and IC50 was at nM range. Post-treatment HSA-ARLDDL also inhibits osteoclast formation. Furthermore, weekly administration of HSA-ARLDDL significantly inhibits the increase in serum bone resorption marker levels and decrease in cancellous bone loss in tibia and femur induced by OVX. On the other hand, HSA-ARLDDL did not affect the differentiation and calcium deposition of osteoblasts. These results indicate that the highly selective and long-acting αvβ3 integrin antagonists could be developed as effective drugs for postmenopausal osteoporosis.

Heme oxygenase-1 (HO-1) is up-regulated in response to oxidative stress and catalyzes the degradation of pro-oxidant heme to carbon monoxide (CO), iron and bilirubin. Bilirubin is a potent antioxidant and neuroprotectant. Neurotrophic factors of BDNF and GDNF also play important roles in survival and morphological differentiation of dopaminergic neurons. We have previously found that HO-1 induction by adenovirus containing human HO-1 gene (Ad-HO-1) in substantia nigra of rat increases BDNF and GDNF expression. We here further examined the possible mechanism of HO-1 action involved in the enhancement of neurotrophic factor expression. Treatment of anti-BDNF/GDNF antibody significantly enhanced dopaminergic neuronal death, whereas Ad-HO-1 co-treatment was able to antagonize the apoptosis-inducing effect of these antibodies. The confocal imaging shows that HO-1 induction appeared in dopaminergic neuron, astrocyte and microglia at 24 h after injecting Ad-HO-1. HO-1 induced-BDNF/GDNF mRNA expression in substantia nigra was 26/21 folds of that of the contralateral Ad-injected side. The downstream product bilirubin increased GDNF expression through ERK and PI3K-Akt pathways, and also enhanced NFkappaB (p65 and p50) nuclear translocation in glia-enriched cultures. In addition, bilirubin also enhanced BDNF expression through similar pathway in cortical neuron-enriched cultures. We also examined the effect of another HO-1 product, CO, by using CO donor. [Ru(CO)3Cl2]2 increased neurotrophic factor expression via sGC-PKG pathway in both neuron and glia. These results indicate that the downstream products of HO-1, i.e. bilirubin and CO, modulate BDNF and GDNF expression in neuron and astrocyte.

Stromal cell-derived factor-1 (SDF-1), also known as CXCL12, and its receptor CXC chemokine receptor 4 (CXCR4) express in various kinds of cells in central nervous system. The SDF-1/CXCR4 signaling pathway is regulated by diverse biological effects. SDF-1 is up-regulated in the ischemic penumbra following stroke and has been known to be associated with the homing of bone marrow cells to injury. However, the effect of SDF-1alpha/CXCR4 on cytokine production in microglia is mostly unknown. Here, we demonstrated that SDF-1alpha enhanced IL-6 production in both primary cultured microglia and BV-2 microglia. We further investigated the signaling pathway involved in IL-6 production stimulated by SDF-1alpha in microglia. SDF-1alpha increased IL-6 production in both protein and mRNA levels. These effects were attenuated by ERK, phosphatidylinositol 3-kinase (PI3K), NF-kappaB inhibitors, and IkappaB protease inhibitor. Stimulation of microglia with SDF-1alpha also increased Akt and ERK1/2 phosphorylation. In addition, SDF-1alpha treatment also increased IkappaB kinase alpha/beta (IKK alpha/beta) phosphorylation, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), translocation of p65 and p50 from cytosol to nucleus and kappaB-luciferase activity. Moreover, SDF-1alpha-mediated increase of kappaB-luciferase activity was inhibited by pre-transfection of DN-p85, DN-Akt or DN-ERK2. Increase of IKK alpha/beta phosphorylation and binding of p65 and p50 to the NF-kappaB element were both antagonized by PI3K and ERK inhibitors. Our results demonstrate a mechanism linking SDF-1alpha and IL-6, and provide additional support for the notion that SDF-1alpha plays a regulatory role in microglia activation.

Although much has been learned about the role of the amygdala in Pavlovian fear conditioning, relatively little is known about the signaling pathway involved in the acquisition of an active avoidance reaction. The aim of this study is to investigate the potentiating effects of the NO-guanylate cyclase activator YC-1 on learning and memory of shuttle avoidance test in rats. YC-1 enhanced the induction of long-term potentiation (LTP) in amygdala through NO-cGMP-PKG-ERK pathway and the increase of BDNF expression. The Western blot and PCR methods were used to examine the signaling pathways involved in fear memory. It was found that YC-1 increased the avoidance responses during learning period and the memory retention lasted longer than one week. The enhancement of learning behavior by YC-1 was antagonized by intracerebroventricular injection of NOS inhibitor l-NAME, PKG inhibitor Rp-8-Br-PET-cGMPS and MEK inhibitor PD98059, indicating that NO-cGMP-PKG and ERK pathways are involved in the learning potentiating action of YC-1. In addition, YC-1 increased the activation of ERK and Akt 30 min after Day-1 training in amygdala. YC-1 also potentiated the expression of BDNF and CREB in response to fear memory test. Taken together, these findings suggest that NO-cGMP-PKG-ERK signaling pathway is involved in the action of YC-1 in enhancing the fear memory.

Osteoporosis is a reduction in skeletal mass due to an imbalance between bone resorption and bone formation. Bone morphogenetic protein (BMP) plays important roles in osteoblastic differentiation and bone formation. Therefore, components involved in BMP activation are good targets for the development of anti-osteoporosis drugs. In this study, imperatorin and bergapten, two coumarin derivatives, were shown to enhance alkaline phosphatase (ALP) activity, type I collagen synthesis and bone nodule formation in primary cultured osteoblasts. Imperatorin and bergapten increased mRNA levels of BMP-2 using quantitative RT-PCR, whereas the BMP-2 antagonist noggin attenuated the increase of ALP activity induced by imperatorin and bergapten, indicating that BMP-2 expression is required for the action of imperatorin and bergapten in osteoblastic maturation. Both imperatorin and bergapten enhanced the phosphorylation of SMAD (transcription factors activated by TGF-beta) 1/5/8, p38 and extracellular signal-regulated protein (ERK). Pretreatment of osteoblasts with p38 inhibitor (SB203580) or mitogen-activated protein kinase inhibitor (PD98059) or transfected with dominant negative mutant of p38 or ERK antagonized the elevation of BMP-2 expression and ALP activity induced by imperatorin and bergapten. Local administration of imperatorin or bergapten into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the BMP-2 immunostaining and bone volume of secondary spongiosa in tibia. Taken together, our results provide evidence that coumarin derivatives increase BMP-2 expression and enhance bone formation in rat via the p38 and ERK-dependent signaling pathway.

The production of chemokine stromal cell-derived factor (SDF)-1 is significantly higher in synovial fluid of patients with osteoarthritis and rheumatoid arthritis. Matrix metalloproteinase (MMP)-13 may contribute to the breakdown of articular cartilage during arthritis. Here, we found that SDF-1alpha increased the secretion of MMP-13 in cultured human chondrocytes, as shown by reverse transcriptase-polymerase chain reaction, Western blot, and zymographic analysis. SDF-1alpha also increased the surface expression of CXCR4 receptor in human chondrocytes. CXCR4-neutralizing antibody, CXCR4-specific inhibitor [1-[[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane (AMD3100)], or small interfering RNA against CXCR4 inhibited the SDF-1alpha-induced increase of MMP-13 expression. The transcriptional regulation of MMP-13 by SDF-1alpha was mediated by phosphorylation of extracellular signal-regulated kinases (ERK) and activation of the activator protein (AP)-1 components of c-Fos and c-Jun. The binding of c-Fos and c-Jun to the activator protein (AP-1) element on the MMP-13 promoter and the increase in luciferase activity was enhanced by SDF-1alpha. Cotransfection with dominant-negative mutant of ERK2 or c-Fos and c-Jun antisense oligonucleotide inhibited the potentiating action of SDF-1alpha on MMP-13 promoter activity. Taken together, our results provide evidence that SDF-1alpha acts through CXCR4 to activate ERK and the downstream transcription factors (c-Fos and c-Jun), resulting in the activation of AP-1 on the MMP-13 promoter and contributing cartilage destruction during arthritis.

Perinatal insults and subsequent neuroinflammation are the major mechanisms of neonatal brain injury, but there have been only scarce reports on the associations between hypoxic preconditioning and glial activation. Here we use neonatal hypoxia-ischemia brain injury model in 7-day-old rats andin vitrohypoxia model with primary mixed glial culture and the BV-2 microglial cell line to assess the effects of hypoxia and hypoxic preconditioning on glial activation. Hypoxia-ischemia brain insult induced significant brain weight reduction, profound cell loss, and reactive gliosis in the damaged hemisphere. Hypoxic preconditioning significantly attenuated glial activation and resulted in robust neuroprotection. As early as 2 h after the hypoxia-ischemia insult, proinflammatory gene upregulation was suppressed in the hypoxic preconditioning group.In vitroexperiments showed that exposure to 0.5% oxygen for 4 h induced a glial inflammatory response. Exposure to brief hypoxia (0.5 h) 24 h before the hypoxic insult significantly ameliorated this response. In conclusion, hypoxic preconditioning confers strong neuroprotection, possibly through suppression of glial activation and subsequent inflammatory responses after hypoxia-ischemia insults in neonatal rats. This might therefore be a promising therapeutic approach for rescuing neonatal brain injury.

Exposure to hypoxia induced microglia activation and animal studies have shown that neuronal cell death is correlated with microglial activation following cerebral ischemia. Thus, it is likely that toxic inflammatory mediators produced by activated microglia under hypoxic conditions may exacerbate neuronal injury following cerebral ischemia. The hypoxia-inducible factor-1 (HIF-1) is primarily involved in the sensing and adapting of cells to changes in the O(2) level, which is regulated by many physiological functions. However, the role of HIF-1 in microglia activation under hypoxia has not yet been defined. In the current work, we investigate the signaling pathways of HIF-1alpha involved in the regulation of hypoxia-induced overexpression of inducible NO synthase (iNOS) in microglia. Exposure of primary rat microglial cultures as well as established microglial cell line BV-2 to hypoxia induced the expression of iNOS, indicating that hypoxia could lead to the inflammatory activation of microglia. iNOS induction was accompanied with NO production. Moreover, the molecular analysis of these events indicated that iNOS expression was regulated by the phosphatidylinositol 3-kinase (PI3-kinase)/AKT/ mammalian target of rapamycin (mTOR) signaling pathway and activation of hypoxia inducible factor-1alpha (HIF-1alpha). Thus, during cerebral ischemia, hypoxia may not only directly damage neurons, but also promote neuronal injury indirectly via microglia activation. In this study, we demonstrated that hypoxia induced iNOS expression by regulation of HIF-1alpha in microglia.

It has been shown that ultrasound (US) stimulation accelerates fracture healing in animal models and in clinical studies. Here we found that US stimulation transiently increased the surface expression of alpha2, alpha5, beta1, and beta3 integrins in cultured osteoblasts, as shown by flow cytometric analysis and immunofluorescence staining. US stimulation increased prostaglandin E(2) formation and the protein and mRNA levels of cyclooxygenase-2 (COX-2). At the mechanistic level, anti-integrin alpha5beta1 and alphavbeta3 antibodies or rhodostomin, a snake venom disintegrin, attenuated the US-induced COX-2 expression. Phosphatidylinositol 3-kinase (PI3K) inhibitors 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and wortmannin also inhibited the potentiating action of US. US stimulation increased the phosphorylation of focal adhesion kinase (FAK), extracellular signal-regulated kinases (ERK), p85 subunit of PI3K, and serine 473 of Akt. COX-2 promoter activity was enhanced by US stimulation in cells transfected with pCOX2-Luc. Cotransfection with dominant-negative mutant of FAK(Y397F), p85(Deltap85), Akt(K179A), or ERK2(K52R) inhibited the potentiating action of US on COX-2 promoter activity. Expression of mineralized nodule was lower in dominant-negative mutants of FAK, p85, and Akt-transfected clones than in vector-transfected control cells. Taken together, our results provide evidence that US stimulation increases COX-2 expression and promotes bone formation in osteoblasts via the integrin/FAK/PI3K/Akt and ERK signaling pathway.

Memory is one of the most fundamental mental processes, and various approaches have been used to understand the mechanisms underlying this process. Nitric oxide (NO), cGMP and protein kinase G (PKG) are involved in the modulation of synaptic plasticity in various brain regions. YC-1, which is a benzylindazole derivative, greatly potentiated the response of soluble guanylate cyclase to NO (up to several hundreds fold). We have previously shown that YC-1 markedly enhances long-term potentiation in hippocampal and amygdala slices via NO-cGMP-PKG-dependent pathway. We here further investigated whether YC-1 promotes learning behaviour in Morris water maze and avoidance tests. It was found that YC-1 shortened the escape latency in the task of water maze, increased and decreased the retention scores in passive and active avoidance task, respectively. Administration of YC-1 30 min after foot-shock stimulation did not significantly affect retention scores in response to passive avoidance test. Administration of scopolamine, a muscarinic antagonist, markedly impaired the memory acquisition. Pretreatment of YC-1 inhibited the scopolamine-induced learning deficit. The enhancement of learning behaviour by YC-1 was antagonized by intracerebroventricular injection of NOS inhibitor L-NAME and PKG inhibitors of KT5823 and Rp-8-Br-PET-cGMPS, indicating that NO-cGMP-PKG pathway is also involved in the learning enhancement action of YC-1. YC-1 is thus a good drug candidate for the improvement of learning and memory.

Although capsaicin analogs might be a potential strategy to manipulate inflammation, the mechanism is still unclear. In this study, the effects and action mechanisms of vanilloid analogs on iNOS and COX-2 expression were investigated in RAW264.7 macrophages. Capsaicin and resiniferatoxin (RTX) can inhibit LPS- and IFN-γ-mediated NO production, and iNOS protein and mRNA expression with similar IC50 values of around 10 μM. Capsaicin also transcriptionally inhibited LPS- and PMA-induced COX-2 expression and PGE2 production. However, this effect exhibited a higher potency (IC50: 0.2 μM), and RTX failed to elicit such responses at 10 μM. Interestingly, we found that capsazepine, a competitive TRPV1 antagonist, did not prevent the inhibition elicited by capsaicin or RTX. Nevertheless, it mimicked vanilloids in inhibiting iNOS/NO and COX-2/PGE2 induction with an IC50 value of 3 μM. RT–PCR and immunoblotting analysis excluded the expression of TRPV1 in RAW264.7 macrophages. The DNA binding assay demonstrated the abilities of vanilloids to inhibit LPS-elicited NF-κB and AP-1 activation and IFN-γ-elicited STAT1 activation. The reporter assay of AP-1 activity also supported this action. The kinase assay indicated that ERK, JNK, and IKK activation by LPS were inhibited by vanilloids. In conclusion, vanilloids can modulate the expression of inflammatory iNOS and COX-2 genes in macrophages through interference with upstream signalling events of LPS and IFN-γ. These findings provide new insights into the potential benefits of the active ingredient in hot chilli peppers in inflammatory conditions. British Journal of Pharmacology (2003) 140, 1077–1087. doi:10.1038/sj.bjp.0705533

Nitric oxide (NO) is known to affect synaptic plasticity in various regions of the brain via the cGMP-cGMP-dependent protein kinase (PKG) pathway. We found that a novel compound 3-(5-hydroxymethyl-2-furyl)-1-benzyl-indazole (YC-1), a drug known to modulate the response of soluble guanylyl cyclase to NO, greatly potentiates long-term potentiation (LTP). This compound markedly enhanced the induction of LTP in rat hippocampal and amygdala slices by weak tetanic stimulation. The potentiation of LTP by YC-1 was greatly reduced by NO synthase inhibitor Ng-nitro-l-arginine-methylester, guanylyl cyclase inhibitor 1 H-[1,2,4]-oxadiazolo(4,3-a)-quinoxalin-1-one, and PKG inhibitor (9S,10R,12R)-2,3,9,10,11,12, hexahydro-10-methoxy-2,9-dimethyl-1-ox0-9.12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT5823). In addition, mitogen-activated protein kinase kinase inhibitor 2'-amino-3'-methoxyflavone (PD98059) also markedly inhibited LTP potentiating action of YC-1. Intracellular increase of Ca2+ concentration derived from N-methyl-d-aspartate and glutamate metabotropic receptors contributes to the potentiating action of YC-1. Concurrent perfusion of YC-1 and NO donor sodium nitroprusside for a short time period resulted in the induction of LTP by stimuli at a frequency as low as 0.02 Hz. Incubation of unstimulated hippocampal slices with YC-1 plus nitroprusside increased the immunofluorescence of phospho-extracellular signal-regulated kinase (ERK) and phospho-cAMP response element binding protein (CREB). Furthermore, the Western blot shows that the phosphorylation of ERKs 1 and 2 and CREB of unstimulated hippocampal slices was increased by YC-1 plus nitroprusside, which was inhibited by KT5823. The NO-cGMP-PKG-ERK signaling pathway thus plays important role in the potentiation of LTP by YC-1.

Autophagy is an intracellular degradation pathway with dynamic interactions for eliminating damaged organelles and protein aggregates by lysosomal digestion. The EGFP-conjugated microtubule-associated protein 1 light chain 3 (EGFP-LC3) serves to monitor autophagic process. Extracellular β-amyloid peptide accumulation is reported as a major cause in Alzheimer's disease (AD) pathogenesis; large numbers of autophagic vacuoles accumulate in patients' brains. We previously demonstrated that extracellular Aβ (eAβ) induces strong autophagic response and α7nAChR acts as a carrier to bind with eAβ; which further inhibits Aβ-induced neurotoxicity via autophagic degradation. In the present study, we overexpressed LC3 in both neuroblastoma cells (SH-SY5Y/pEGFP-LC3) and mice (TgEGFP-LC3) to assess the effect of LC3 overexpression on Aβ neurotoxicity. SH-SY5Y/pEGFP-LC3 cells and primary cortical neuron cultures derived from E17 (embryonic day 17) TgEGFP-LC3 mice showed not only better resistance against Aβ neurotoxicity but also higher α7nAChR expression and autophagic activity than control. Administration of α-bungarotoxin (α-BTX) to block α7nAChR antagonized the neuroprotective action of SH-SY5Y/pECGF-LC3 cells, suggesting that eAβ binding with α7nAChR is an important step in Aβ detoxification. LC3 overexpression thus exerts neuroprotection through increasing α7nAChR expression for eAβ binding and further enhancing autophagic activity for Aβ clearance in vitro and in vivo.

Extracellular matrix proteins, such as fibronectin, laminin, and collagen, have been implicated in a wide variety of cellular properties, which include cell adhesion, migration, differentiation, and proliferation. In this study, we investigated the modulation of protein kinase A (PKA) activity by matrix proteins at developing motoneurons. The cultures of spinal neurons and myotomal cells were prepared from 1-day-old Xenopus laevis embryos. Spontaneous synaptic currents (SSC) were recorded from innervated myocytes of natural synapses by whole-cell voltage-clamped recordings (V(h) = -60 to approximately -65 mV). Bath application of agents, which directly or indirectly activate PKA, such as forskolin (20 microM), dibutyryl cAMP (DBcAMP) (1 mM), isoproterenol (10 microM), or albuterol (10 microM), significantly increased SSC frequency in cultures grown on fibronectin (FN)-coated substratum, but not on laminin- or collagen-coated glasses. The evoked synaptic currents increased in response to forskolin in neurons grown on FN substratum. Triflavin, an Arg-Gly-Asp-dependent disintegrin, inhibited potentiating action of isoproterenol in neurons grown on FN substratum, suggesting that integrin is involved in the potentiation of the PKA pathway in the regulation of acetylcholine (ACh) release. There is collaboration of neurotrophic factors and the FN matrix in regulating synaptic transmission in response to DBcAMP. Chronic treatment with neurotrophic factors, such as ciliary neurotrophic factor (150 ng/ml), glial cell line-derived neurotrophic factor (30 ng/ml), or neurotrophin-3 (50 ng/ml), enhanced the SSC-increasing action of DBcAMP in neurons grown on FN-coated glasses. These results suggest that the FN matrix potentiates synaptic transmission in response to PKA activation. Neurotrophic factors may collaborate with FN to regulate spontaneous ACh secretion at developing motoneurons, which may play an important role in the maturation of embryonic neuromuscular synapses.

The effects of boldine [(S)-2,9-dihydroxyl-1,10-dimethoxy-aporphine], a major alkaloid in the leaves and bark of Boldo (Peumus boldus Mol.), on neuromuscular transmission were studied using a muscle phrenic-nerve diaphragm preparation. Boldine at concentrations lower than 200 microM preferentially inhibited, after an initial period of twitch augmentation, the nerve-evoked twitches of the mouse diaphragm and left the muscle-evoked twitches unaffected. The twitch inhibition could be restored by neostigmine or washout with Krebs solution. The twitches evoked indirectly and directly were both augmented initially, suggesting that the twitch augmentation induced by boldine was myogenic. Boldine inhibited the acetylcholine-induced contraction of denervated diaphragm dose-dependently with an IC50 value of 13.5 microM. At 50 microM, boldine specifically inhibited the amplitude of the miniature end plate potential. In addition, boldine was similar to d-tubocurarine in its action to reverse the neuromuscular blocking action of alpha-bungarotoxin. These results showed that the neuromuscular blockade by boldine on isolated mouse phrenic-nerve diaphragm might be due to its direct interaction with the postsynaptic nicotinic acetylcholine receptor.

Erythropoietin (EPO) is a neuroprotective agent against cerebral ischemia/reperfusion (I/R)-induced brain injury. However, its crossing of blood-brain barrier is limited. Focused ultrasound (FUS) sonication with microbubbles (MBs) can effectively open blood-brain barrier to boost the vascular permeability. In this study, we investigated the effects of MBs/FUS on extending the therapeutic time window of EPO and its neuroprotective effects in both acute and chronic phases. Male Wistar rats were firstly subjected to two common carotid arteries and right middle cerebral artery occlusion (three vessels occlusion, 3VO) for 50 min, and then the rats were treated with hEPO (human recombinant EPO, 5000 IU/kg) with or without MBs/FUS at 5 h after occlusion/reperfusion. Acute phase investigation (I/R, I/R+MBs/FUS, I/R+hEPO, and I/R+hEPO+MBs/FUS) was performed 24 h after I/R; chronic tests including cylinder test and gait analysis were performed one month after I/R. The experimental results showed that MBs/FUS significantly increased the cerebral content of EPO by bettering vascular permeability. In acute phase, both significant improvement of neurological score and reduction of infarct volume were found in the I/R+hEPO+MBs/FUS group, as compared with I/R and I/R+hEPO groups. In chronic phase, long-term behavioral recovery and neuronal loss in brain cortex after I/R injury was significantly improved in the I/R+hEPO+MBs/FUS group. This study indicates that hEPO administration with MBs/FUS sonication even at 5 h after occlusion/reperfusion can produce a significant neuroprotection.

Multidrug resistance is a major cause of chemotherapy failure. Recent studies indicate that drug resistance can be rapidly induced by some soluble factors, such as cytokines, chemokines, growth factors, and cell adhesion factors in the tumor microenvironment. Osteopontin (OPN), an extracellular matrix protein, has a functional arginine-glycine-aspartic acid (RGD) domain for binding to integrin. Here we found OPN expression to be upregulated by hypoxic condition in PC-3 prostate tumor cells. OPN increased the mRNA and protein expression of p-glycoprotein (P-gp), a subfamily of ATP-binding cassette transporter in a concentration- and time-dependent manner. The increase in P-gp transporter by OPN was mediated by binding to αvβ3 integrin. Daunomycin (DUN), a chemotherapeutic agent with autofluorescence, was used to evaluate the pump activity, and OPN increased the drug pumping-out activity. OPN inhibited DUN-induced cell death, which was antagonized by αvβ3 monoclonal antibody. Long-term treatment with DUN further enhanced the expression of OPN. Knockdown of endogenous OPN potentiated the DUN-induced apoptosis of PC-3 cells. Furthermore, knockdown of OPN enhanced cell death caused by other drugs, including paclitaxel, doxorubicin, actinomycin-D, and rapamycin, which are also P-gp substrates. The animal studies also showed that OPN knockdown enhanced the cytotoxic action of DUN. These results indicate that OPN is a potential therapeutic target for cancer therapy to reduce drug resistance in sensitive tumors.

Metabolites from arachidonic acids play the pivotal roles in inflammatory arthritis. Arachidonic acid could be metabolized by cyclooxygenase (COX) and lipoxygenase (LOX) to produce the bioactive eicosanoids. Although the down-stream products of COX including prostaglandin E2 are well-known inflammatory stimulators, the role of LOX products in inflammatory arthritis is still unclear. Here we found that the downstream product of 15-LOX, 15-S-hydroxyeicosatetraenoic acid (15-(S)-HETE), can enhance the expression of placenta growth factor (PLGF), which is recently considered to play an important role in rheumatoid arthritis. 15-(S)-HETE increased the expression of PLGF in human rheumatoid arthritis synovial fibroblasts in a time-dependent and concentration-dependent manner. PI3K-Akt, NF-κB signaling pathways were involved in the potentiation effects of 15-(S)-HETE. In addition, COX-2 was up-regulated by the treatment of 15-(S)-HETE and the increase of COX-2 expression participated in 15-(S)-HETE-induced PLGF expression, which was confirmed by COX-2 shRNA or pharmacological COX-2 inhibitor. Moreover, it was found that treatment of prostaglandin E2 (PGE2), which was the main down-stream metabolite of COX-2, increased the expression of PLGF. EP1, EP2, EP3 and EP4 agonists could up-regulate PLGF as well. In animal studies, we found that the adjuvant-induced expression of PLGF and COX-2 was inhibited in 15-LOX knockout mice. These results indicated that PLGF up-regulation by 15-LOX downstream product may be involved in inflammatory arthritis.

15-Lipoxygenase (15-LOX) is involved in many pathological processes. The aim of this study is to examine the role of 15-LOX in the matrix metalloproteinase (MMP) expression and inflammatory arthritis. It was found that treatment of 15-LOX downstream product of 15-(S)-HETE (15-S-hydroxyeicosatetraenoic acid) increased the mRNA and protein levels of MMP-2 in rheumatoid arthritis synovial fibroblast (RASF) derived from rheumatoid arthritis patients. The enhancement effect of 15-(S)-HETE was antagonized by the addition of LY294002 (PI3K inhibitor) and PDTC (NF-κB inhibitor). Treatment of 15-(S)-HETE increased the phosphorylation of AKT, nuclear translocation of p65 and the breakdown of IκBα. TNF-α and IL-1β are the key cytokines involved in arthritis and also increase the activity of MMP-2 in RASF, which was antagonized by pretreatment with 15-LOX inhibitor PD146176 or knockdown of 15-LOX. It was also found that these two cytokines increased the expression of 15-LOX in RASF. Treatment of glucocorticoid but not NSAIDs inhibited 15-(S)-HETE-induced expression of MMP-2. In comparison with wild-type mice, adjuvant-induced arthritis and MMP-2 expression in synovial membrane were markedly inhibited in 15-LOX knockout (KO) mice. These results indicate that 15-LOX plays an important role in the disease progression of arthritis and may be involved in the inflammatory action induced by TNF-α and IL-1β. 15-LOX is thus a good target for developing drugs in the treatment of inflammatory arthritis.

Staurosporine, an inhibitor of protein kinases, induced outgrowth of cultured embryonic Xenopus myocytes. The outgrowing membrane elicited by staurosporine was stained uniformly with fluorescein isothiocyanate-phalloidin. Pretreatment with microfilament-disrupting agents but not microtubule inhibitors inhibited staurosporine-induced membrane outgrowth. Microfilament assembly is thus required for the action of staurosporine. Protein kinase C activators did not antagonize the membrane outgrowing effect of staurosporine. Furthermore, none of H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), H-8 N [2-(methylamino)ethyl]-5isoquinoline sulfonamide), sphingosine, phloretin, genistein or calmidazolium induced any significant morphological changes of embryonic myocytes, indicating that tyrosine kinases protein kinase C, protein kinase A or calmodulin-dependent protein kinases may not invloved in the membrane outgrowing action of staurosporine. Total pretein content of myocytes was not altered by staurosporine and protein or RNA synthesis inhibitors did not inhibit the membrane outgrowth induced by staurosporine. Furthermore, membrane outgrowth induced by staurosporine was less pronounced in older cultured myocytes or myocytes acutely isolated at later stages of tadpoles, indicating that there is different developmental susceptibility to the action of staurosporine.

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on nicotinic acetylcholine (ACh) receptor channels were studied in cultured myocytes of 1-day-old Xenopus embryos. The amplitude and decay time of iontophoretic ACh-induced currents were reduced by MPTP in a concentration-dependent manner. The inhibitory action of MPTP was frequency-dependent, being enhanced by higher frequencies of stimulation at 7 and 30 Hz. Neither pargyline nor tranylcypromine affected the inhibitory effects of MPTP on ACh-induced currents. Single channel recordings showed that MPTP decreased the opening frequency, mean open time as well as the conductance of both low- and high-conductance ACh channels. These results suggest that the inhibitory actions of MPTP on ACh receptor channels in skeletal muscle may contribute to the depression of the nerve-evoked contraction of the mouse diaphragm as previously reported.

1. Phorbol 12-myristate 13-acetate (TPA, 1 microM) and phorbol 12,13-dibutyrate (PDBu, 2 microM), activators of protein kinase C (PKC), increased the mean amplitude and decay time of the spontaneous synaptic currents of Xenopus nerve-muscle coculture, whereas, 4 alpha-phorbol (2 microM) which is an inactive phorbol analogue had no effect. 2. Staurosporine (0.5 microM) and H-7 (10 microM), inhibitors of PKC, inhibited the potentiation effects of TPA on the spontaneous synaptic currents. 3. Effects of TPA on the postsynaptic acetylcholine (ACh) sensitivity were examined by iontophoresis of ACh to the surface of embryonic muscle cells of 1-day-old Xenopus cultures. TPA increased both the amplitude and decay time of ACh-induced whole-cell currents in isolated myocytes. 4. TPA concentration-dependently increased the mean open time of low-conductance ACh channels but did not affect those of high-conductance ACh channels. PDBu but not 4 alpha-phorbol exhibited similar effects to TPA. Staurosporine and H-7 inhibited the increasing effects of TPA. 5. These results suggest that activation of PKC might be involved in synaptogenesis at developing neuromuscular synapses by the postsynaptic potentiation of ACh sensitivity.

1. The effects of 2,2',2''-tripyridine on the nicotinic acetylcholine (ACh) receptor channels were studied in the cultured myocytes of 1-day-old Xenopus embryos. 2. 2,2',2''-Tripyridine depressed the amplitude of iontophoretic ACh-induced current at a low frequency of 0.7 Hz stimulation and it not only decreased the initial responses but also enhanced the run-down of ACh-induced current at higher frequency stimulation of 7 Hz and 30 Hz. 3. Single ACh channel recordings showed that 2,2',2''-tripyridine decreased the channel conductance, the opening frequency and mean open time of both types of low- and high-conductance channels. 4. These results suggest that the blocking actions of 2,2',2''-tripyridine on ACh receptor channels in the skeletal muscle may contribute to the depression of the nerve-evoked contraction of the mouse diaphragm as reported previously.

Heme oxygenase-1 (HO-1) is up-regulated in response to oxidative stress and catalyzes the degradation of pro-oxidant heme to carbon monoxide (CO), iron, and bilirubin. Intense HO-1 immunostaining in the Parkinsonian brain is demonstrated, indicating that HO-1 may be involved in the pathogenesis of Parkinsonism. We here locally injected adenovirus containing human HO-1 gene (Ad-HO-1) into rat substantia nigra concomitantly with 1-methyl-4-phenylpyridinium (MPP(+)). Seven days after injection of MPP(+) and Ad-HO-1, the brain was isolated for immunostaining and for measurement of dopamine content and inflammatory cytokines. It was found that overexpression of HO-1 significantly increased the survival rate of dopaminergic neurons; reduced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in substantia nigra; antagonized the reduction of striatal dopamine content induced by MPP(+); and also up-regulated brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) expression in substantia nigra. Apomorphine-induced rotation after MPP(+) treatment was also inhibited by Ad-HO-1. On the other hand, inhibition of HO enzymatic activity by zinc protoporphyrin-IX facilitated the MPP(+)-induced rotatory behavior and enhanced the reduction of dopamine content. HO-1 overexpression also protected dopaminergic neurons against MPP(+)-induced neurotoxicity in midbrain neuron-glia cocultures. Overexpression of HO-1 increased the expression of BDNF and GDNF in astrocytes and BDNF in neurons. Our results indicate that HO-1 induction exerts neuroprotection both in vitro and in vivo. Pharmacological or genetic approaches targeting HO-1 may represent a promising and novel therapeutic strategy in treating Parkinsonism.

Sodium selenite was found to be toxic to chicks, with an LD50 of 8.5 micrograms/g, which was increased to 16.3 micrograms/g by NaCN. The major symptoms of chicks, treated with selenite, were sedation and then dyspnea and paralysis. The cause of death by selenite was apparently due to the respiratory failure. The possible mechanism of toxicity was explored in the isolated chick biventer cervicis nerve-muscle preparation. Selenite initially increased the amplitude of the twitch, reversed the suppression of the twitch caused by d-tubocurarine, Mg2+, Cd2+ or Mn2+ and significantly increased the quantal content and amplitude of endplate potentials. Subsequently, selenite depressed the amplitude of the twitch, blocked the axonal conduction and inhibited excitatory postsynaptic potentials. Both NH4+ and K+ enhanced the action of selenite in depressing the twitches. In addition, selenite induced a sustained contracture of the muscle, which was partially inhibited by removal of external Ca2+ and markedly blocked by EGTA. Entry of Ca2+ and release of the internal Ca2+ were considered to be responsible for inducing contracture by selenite. Pretreatment with trypsin, glutathione (GSH) and cyanide profoundly inhibited the effects of selenite, indicating that the site of action of selenite was on the outer membrane and the binding of selenite to the sulfhydryl groups of membrane proteins was proposed to be an essential step for selenite-induced contracture and neuromuscular action. These findings suggest that neuromuscular blockade and tetanic spasm, produced by selenite in chicks, may play a role in causing respiratory failure in vivo.

Glutamate, an important central excitatory neurotransmitter, is also secreted by osteoblasts and may be involved in the regulation of bone metabolism. Glutamate receptors for N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) are demonstrated in bone cells. Here we investigated the in vivo effects of glutamate by local injection of AMPA, NMDA, and their antagonists into tibia as well as their in vitro effects on the maturation of osteoblasts and formation of osteoclasts. AMPA receptor antagonist CNQX and NMDA receptor antagonist MK-801 significantly inhibited the maturation and mineralization of osteoblasts in high-glutamate alpha-MEM. On the other hand, AMPA and NMDA up-regulated the mineralized deposition and osteocalcin mRNA expression of primary osteoblasts cultured in glutamate-free DMEM. AMPA and NMDA induced the phosphorylation of extracellular signal-related kinases (ERK) in osteoblasts within 15 min. In addition, NMDA but not AMPA up-regulated the number of osteoclasts while MK801 antagonized this potentiating effect. To explore the action of glutamate agonists on bone formation in animal model, AMPA was locally injected into tibia and it was found that the bone volume in secondary spongiosa significantly increased and co-treatment of CNQX antagonized the enhancing effect of AMPA. These results suggest that glutamate may play a physiological role in regulating the maturation of osteoblasts and osteoclastogenesis. Activation of both AMPA and NMDA receptors regulates the maturation of osteoblasts. NMDA but not AMPA affects receptor for activation of NF-kappaB ligand (RANKL)-induced osteoclastogenesis.

Increased need for glycolysis and glucose uptake for ATP production is observed in tumor cells, particularly in cells lacking of oxygen supply. Because glucose is transported from blood to tumor, glucose molecules must be delivered across glucose transporters of the vascular endothelium and tumor cells. Here we found that glioma suffered from hypoxic insults can secrete factor(s) to regulate glucose transporter expression in brain endothelium. It was found that conditioned medium from rat C6 glioma cells under hypoxia up-regulated glucose transporter type 1 (GLUT1) expression in rat brain endothelial cells, whereas conditioned medium from C6 cells under normoxia caused no significant effect. We further investigated whether the observed potentiating effect was caused by vascular endothelial growth factor (VEGF) production from C6 cells, because secreted VEGF was markedly increased under hypoxic condition. By transfection of C6 cells with VEGF small interfering RNA, it was found that conditioned medium from transfected cells under hypoxia no longer up-regulated GLUT1 expression of endothelial cells. Moreover, the addition of VEGF-neutralizing antibody to the hypoxic conditioned medium could also exert similar inhibitory effects. Furthermore, it was found that the VEGF-induced increase of GLUT1 expression in endothelial cells was mediated by the phosphoinositide-3 kinase/Akt pathway. Our results indicate that hypoxic brain glioma may secrete VEGF to increase glucose transport across blood-brain barrier.

Cerebral microvascular endothelial cells form the anatomical basis of the blood-brain barrier (BBB), and the tight junctions of the BBB are critical for maintaining brain homeostasis and low permeability. Ischemia/reperfusion is known to damage the tight junctions of BBB and lead to permeability changes. Here we investigated the protective role of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), against chemical hypoxia and hypoxia/reoxygenation (H/R)-induced BBB hyperpermeability using adult rat brain endothelial cell culture (ARBEC). YC-1 significantly decreased CoCl2- and H/R-induced hyperpermeability of fluorescein isothiocyanate (FITC)-dextran in cell culture inserts. It was found that the decrease and disorganization of tight junction protein zonular occludens-1 (ZO-1) in response to CoCl2, and H/R was antagonized by YC-1. The protection of YC-1 may result from the inhibition of HIF-1alpha accumulation and production of its downstream target vascular endothelial growth factor (VEGF). VEGF alone significantly increased FITC-dextran permeability and down-regulated mRNA and protein levels of ZO-1 in ARBECs. We further used animal model to examine the effect of YC-1 on BBB permeability after cerebral ischemia/reperfusion. It was found that YC-1 significantly protected the BBB against ischemia/reperfusion-induced injury. Taken together, these results indicate that YC-1 may inhibit HIF-1alpha accumulation and VEGF production, which in turn protect BBB from injury caused by hypoxia.

Background and purpose: An inflammatory response in the central nervous system mediated by the activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. LPS has been reported to cause marked microglia activation. It is very important to develop drugs that can inhibit microglia activation and neuroinflammation. Here, we investigated the inhibitory effect of YC-1, a known activator of soluble guanylyl cyclase, against LPS-induced inflammatory responses in microglia. Experimental approach: To understand the inhibitory effects of YC-1 on LPS-induced neuroinflammation, primary cultures of rat microglia and the microglia cell line BV-2 were used. To examine the mechanism of action of YC-1, LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, iNOS, COX-2 and cytokine expression were analyzed by Griess reaction, ELISA, Western blotting and RT-PCR, respectively. The effect of YC-1 on LPS-induced activation of nuclear factor kappa B (NF-κB) was studied by NF-κB reporter assay and immunofluorocytochemistry. Key results: YC-1 inhibited LPS-induced production of NO and PGE2 in a concentration-dependent manner. The protein and mRNA expression of iNOS and COX-2 in response to LPS application were also decreased by YC-1. In addition, YC-1 effectively reduced LPS-induced expression of the mRNA for the proinflammatory cytokines, TNF-α and IL-1β. Furthermore, YC-1 inhibited LPS-induced NF-κB activation in microglia. Conclusions and implications: YC-1 was able to inhibit LPS-induced iNOS and COX-2 expression and NF-κB activation, indicating that YC-1 may be developed as an anti-inflammatory neuroprotective agent. British Journal of Pharmacology (2007) 151, 396–405; doi:10.1038/sj.bjp.0707187

Adipogenesis plays a central role in obesity development. The processes of adipogenesis include migration, adhesion, proliferation and survival of preadipocytes and differentiation to mature adipocytes. Many of these biological functions are related to integrins. Here, we found that snake venom-derived arginine-glycine-aspartic acid (RGD)-containing disintegrin inhibited adipogenesis. Rhodostomin but not rhodostomin RGD mutants (RGE-Rn and AKGDWN-Rn) caused the detachment of primary cultured preadipocyte. Furthermore, rhodostomin also inhibited focal adhesion of preadipocyte, including the inhibition of the expression of focal adhesion kinase (FAK) and FAK phosphorylation, assembly of vinculin and reorganization of actin cytoskeleton. Cell viability of preadipocytes was decreased after rhodostomin treatment in a concentration-dependent manner. The results of flow cytometric analysis showed that rhodostomin induced cell apoptosis. In addition, chromatin condensation was observed in DAPI staining. The increase of Bax expression and activation of capsase-3 was detected following rhodostomin treatment. Addition of dexamethasone, IBMX and insulin induced differentiation of preadipocytes into mature adipocytes and treatment of cells with rhodostomin during the initial 3 days showed less mature adipocytes following 9-10 days of differentiating period. The triglyceride content and gene expression of peroxisome proliferators-activated receptor gamma (PPARgamma) and leptin also decreased in response to the treatment of rhodostomin. These results suggest that disintegrin inhibits processes of adipogenesis and may be developed to treat obesity.

Injury to peripheral nerves may result in severe and intractable neuropathic pain. Many efforts have been focused on the elucidation of the mechanisms of neuropathic pain. It was found here that integrin plays an important role in the induction of neuropathic pain and treatment of disintegrin is able to attenuate neuropathic pain. The rats were induced hyperalgesia by tightly ligating the L5 spinal nerve and cut just distal to the ligature on one side. Mechanical and thermal stimuli were applied in the middle dermatome of the hind paw. Epidural administration of triflavin (TFV), an arginine-glycine-aspartic acid (RGD) containing disintegrin, inhibited hyperalgesia induced by either mechanical or thermal stimulation. Immunohistochemistry showed that the sprouting of sympathetic nerves into DRG by neuropathic surgery was markedly inhibited by TFV. Beta 1 integrin mRNA of L5 DRG increased immediately 1 day after tight ligation and cut of L5 spinal nerve. However, beta 1 integrin mRNA in uninjured L4 DRG increased later on Day 3 after surgery. On the other hand, alpha-CGRP precursor mRNA decreased in ipsilateral L5 DRG but increased in L4 DRG after neuropathic surgery. Immunohistochemistry shows that beta 3 integrins of L5 as well as L4 increased in response to neuropathic surgery and administration of triflavin antagonized the increasing action. These results suggest that there is interaction between injured and uninjured neurons and the induction of neuropathic pain is related to neuronal sprouting. Disintegrin is able to inhibit neuronal sprouting and the induction of hyperalgesia induced by peripheral nerve injury and may thus be a new category of drugs to be developed for the treatment of neuropathic pain.

Fibronectin (Fn) plays an important role in the regulation of adhesion, migration, and maturation of osteoblasts. Fn fibrillogenesis is involved in the process of bone mineralization. To elucidate the regulatory role of protein kinases in the formation of fibrillar Fn matrix, Fn synthesis and assembly were examined in cultured osteoblasts. Osteoblasts assembled the endogenously released soluble Fn into immobilized form on the substratum in a time-dependent manner. Both 12-O-tetradecanoylphorbol-13 acetate (TPA) and forskolin increased the synthesis of Fn. However, the extracellular assembly of Fn fibril from both endogenously released and exogenously applied soluble Fn was increased by TPA but decreased by forskolin. Protein kinase C (PKC) inhibitors, such as H7, Ro 318220, and Go 6976, inhibited Fn fibrillogenesis. These results suggest that the dynamic of Fn fibrillogenesis is differentially regulated by the activation of PKC and protein kinase A (PKA). Both classic and novel isoforms of PKC are involved in the action of TPA in osteoblasts. It has been reported that alpha5beta1 integrin is related to Fn fibrillogenesis. Immunocytochemistry and flow cytometry showed that TPA and forskolin increased and inhibited, respectively, the clustering and surface expression of alpha5 integrins. TPA and forskolin did not affect protein levels of alpha5 integrins. The Western blot and reverse transcriptase-polymerase chain reaction showed that protein and mRNA levels of beta1 integrins also were not affected by TPA and forskolin. These results suggest that TPA and forskolin may affect the surface expression of alpha5beta1 integrins. cAMP response element-binding protein phosphorylation is involved in the action of forskolin but not that of TPA. Our results suggest that PKC activation enhanced Fn fibrillogenesis, whereas PKA activation inhibited extracellular Fn fibrillogenesis in primary cultured osteoblasts. Cytosolic Fn synthesis and extracellular Fn assembly may be differentially regulated by the activation of PKA.

1. We have previously shown that the activation of either protein kinase A (PKA) or protein kinase C (PKC) enhanced the responses of muscle membrane to acetylcholine (ACh) by increasing the mean open time of embryonic-type ACh channels in Xenopus cultured myocytes. In the present study, we further investigated the interaction between these two kinases in the modulation of ACh channels by using the receptor ligands, adenosine diphosphate (ADP) and calcitonin gene-related peptide (CGRP) which selectively activate PKC and PKA, respectively. 2. ADP concentration-dependently increased the mean open time of embryonic-type ACh channels and 0.3 mM ADP is sufficient to achieve the maximal potentiating effect. alpha, beta-Methylene ATP and PMA (phorbol 12-myristate 13-acetate) but not adenosine, AMP, dibutyryl cyclic GMP have similar potentiating action. 3. Suramin (0.3 mM) pretreatment abolished the potentiating effect of ADP but left that of PMA unchanged. 4. CGRP increased the mean open time of embryonic-type ACh channels in a concentration-dependent manner and 1 microM CGRP produced the maximal effect. 5. The maximal effects of both ADP (0.3 mM) and CGRP (1 microM) in the prolongation of mean open time of ACh channels were additive. 6. These results suggest that the modulation of embryonic-type ACh channels by the endogenously released ligands via the activation of PKA and PKC is additive and possibly different sites of ACh channels may be involved in the potentiation effect of either PKC or PKA.

1. Previously we have shown that extracellular application of ATP, a substance co-stored and co-released with acetylcholine (ACh) in the peripheral nervous system, markedly potentiated the frequency of spontaneous synaptic currents (SSCs) produced by ACh. In the present study, we have further characterized the purinoceptor which mediates the potentiation effect of ATP and the role of endogenously released ATP. 2. Pretreatment with a P2-purinoceptor antagonist, suramin (0.3 mM), but not a P1-purinoceptor antagonist, 8-phenyltheophylline (0.1 mM), prevented the potentiating effect of ATP. 3. We studied the role of endogenously released ATP by examining the effect of a specific P2-purinoceptor antagonist on the frequency of spontaneous synaptic events at high-activity synapses (> or = 3 Hz) and found that suramin, but not 8-phenyltheophylline markedly reduced the frequency of SSCs at these high-activity synapses. In addition, desensitizing the P2-purinoceptor with alpha,beta-methylene ATP also produced similar effects to suramin. 4. Extracellular application of the L-type Ca2+ channel blockers, verapamil, nifedipine or diltiazem (10 microM each) reduced SSC frequency of high-activity synapses, while the N-type Ca2+ channel blocker, omega-conotoxin had no appreciable effect. The potentiating effect of ATP was further prevented by pretreatment with the L-type Ca2+ channel blockers. On the other hand, Bay K 8644, which is a depolarization-dependent L-type Ca2+ channel agonist, potentiated SSC frequency at these high-activity synapses. 5. These results suggest that endogenous release of ATP at developing neuromuscular synapses is responsible for the maintenance of high levels of spontaneous ACh release, which is known to play a crucial role in regulating postsynaptic differentiation.

The myotonic activity of mouse soleus and extensor digitorum longus muscles induced by either a combination of K+ channel blockers (4-aminopyridine) and a Cl- channel blocker (9-anthracene carboxylic acid) or a Cl- channel blocker in low Ca2+ (0.25 mM) Krebs or a Na+ channel activator (veratridine) was characterized in this paper. Myotonic activity was characterized by an increase in both the contraction amplitude and contraction duration accompanied by stimulus-related repeated action potentials. The slow soleus and fast extensor digitorum longus muscles appeared to differ in their responses to these ion channel modifiers. Nevertheless, chlorpromazine at a low concentration of 1 microM significantly inhibited all kinds of myotonic activity; it reduced the prolonged contraction duration and attenuated the stimulus-related repeated action potential firing. This depressant action of chlorpromazine was apparently not correlated with inhibition of either calmodulin or phospholipase A2 activity, since the myotonic depressant action of calmodulin inhibitors, such as dibucaine, flunarizine, chlorpromazine, trifluoperazine and diltiazem, was unrelated to their potency in inhibiting the activity of calmodulin or phospholipase A2. However, phosphatidylcholine was found to inhibit the myotonic depressant action of chlorpromazine. It is therefore, tentatively concluded that chlorpromazine interacted with membrane phospholipids, thereby changing membrane ion channel activity and depressing myotonic activity. These findings indicate that chlorpromazine might be useful in the management of clinical myotonia.

1. Extracellular application of ATP (1 mM), a substance co-stored and co-released with acetylcholine in peripheral nervous systems, potentiated the spontaneous secretion of acetylcholine (ACh) but had no effect on the amplitude and decay time constant of miniature endplate potentials (m.e.p.ps) at neuromuscular synapses in Xenopus tadpoles. 2. alpha,beta-Methylene ATP (0.3 mM) and GTP (1 mM) were also effective in increasing m.e.p.p. frequency. On the other hand, ADP, AMP and adenosine (all at 1 mM) decreased m.e.p.p. frequency. 3. Unlike the transient effect of ATP analogue and GTP on m.e.p.p. frequency, the phorbol ester TPA (2 microM) which is a protein kinase C activator, increased m.e.p.p. frequency consistently and the effects lasted as long as the presence of TPA. 4. Staurosporine (0.5 microM) and H-7 (10 microM), which are protein kinase C inhibitors, each decreased the basal level of m.e.p.p. frequency and markedly inhibited the effects of both ATP and TPA. 5. These results suggest that there is a basal activity of cytosolic protein kinases in the nerve terminals of Xenopus tadpoles and the effect of ATP is probably mediated by the binding of membrane surface purinoceptors which in turn activates cytosolic protein kinases and increases ACh release.

Effects of Cd2+ on the phrenic nerve-diaphragm preparation of the mouse varied markedly in media containing various Ca2+ concentrations. In normal 2.5 mM Ca2+ medium, Cd2+ inhibited acetylcholine release from nerve endings without appreciable effect on the muscle membrane. However, Cd2+ elicited stimulatory effects on the muscle membrane in low Ca2+ medium (10(-3)-1 mM). These stimulatory effects included the induction of spontaneous contractions, augmentation of twitch responses to direct electrical stimulation and potentiation of the muscle contracture induced by acetylcholine, carbachol and high K+. By contrast, caffeine contracture was not affected by Cd2+. Tetrodotoxin, procaine, cysteine and glycerol pretreatment abolished these stimulatory effects of Cd2+. Moreover, changing the ionic composition of the bathing medium to one containing low Na+, high K+, high Mg2+ or high Ca2+ also antagonized these effects of Cd2+. In contrast, low Mg2+ markedly potentiated the frequency of spontaneous contractions induced by Cd2+. (+)-Tubocurarine and beta-bungarotoxin had no effect on Cd2+-induced spontaneous contractions indicating that they may be myogenic rather than neurogenic in origin. By use of conventional microelectrodes, it was found that Cd2+ not only depolarized the muscle membrane but also induced spontaneous action potentials at a high frequency (173 +/- 17 Hz). It is concluded that increased Na+ permeability of the muscle membrane is the essential step bringing about spontaneous contractions. The binding of Cd2+ to -SH groups of the membrane is closely related to the induction of these effects.

In comparative studies on the effects of nine divalent cations (Mn 2+ , Co 2+ , Ni 2+ , Cd 2+ , Zn 2+ , Sr 2+ , Cu 2+ , Ba 2+ and UO 2 2+ on twitch responses to indirect and direct stimulations, on acetylcholine response and on Ca 2+ uptake, it was found that Cd 2+ was the most potent in inhibiting transmitter release from the motor nerve terminals but was without appreciable effect on the chick biventer cervicis muscle. Mn 2+ , Co 2+ , Ni 2+ and Zn 2+ were less potent than Cd 2+ in producing inhibition at presynaptic sites but were morep potent at postsynaptic sites. By contrast, UO 2 2+ potentiated twitch responses to indirect stimulation and induced a contracture of the muscle in the presence of physostigmine. Cu 2+ and Ba 2+ were particularly potent in inducing a contracture and at higher concentrations abolished twitch responses to indirect and direct stimulation. Sr 2+ at the very high concentration of 22 mM had only a week inhibitory action on twitch responses but was the only cation capable of substituting for Ca 2+ in inducing contraction. The studies on the effects of these cations on denervated muscle also led to the conclusion that Cd 2+ and UO 2 2+ act mainly on presynaptic nerve terminals and that Mn 2+ , Co 2+ , Ni 2+ and Zn 2+ act on presynaptic sites preferentially to postsynaptic sites while Cu 2+ and Ba 2+ act mainly on muscle. The effects of these cations can be antagonized by high Ca 2+ (10 mM) but those of Cu 2+ cannot. Cysteine (2.5p mM) antagonized the effects of all the cations except Mn 2+ , Ba 2+ and UO 2 2+ . d-Tubocurarine suppressed the contracture induced by UO 2 2+ but not that induced by Cu 2+ and Ba 2+ . These cations affected 45 Ca 2+ uptake and ionic contents of the muscle differently. 45 Ca 2+ uptake of the muscle was decreased by Mn 2+ , Co 2+ and Ni 2+ , not altered by UO 2 2+ but increased by Cd 2+ , Zn 2+ , Sr 2+ , Cu 2+ and Ba 2+ . In addition to greatly increasing 45 Ca 2+ uptake, Cu 2+ also increased calcium and sodium but decreased the potassium content of muscle. Zn 2+ and Sr 2+ increasedp the calcium content slightly but the other cations did not effect ionic contents appreciably. It is concluded that these cations exert their effects on nerve or on muscle depending on the ligands of the ligands of the membrane bindingp these cations. Some of the cations bi-nd with the—SH group of the membrane and thus alter the transmembrane movement of Ca 2+ bu the others do not.

The uranyl ion (UO2(2+)) produces rhythmic contractions of the longitudinal muscle of the ileum, similar to those induced by repetitive transmural stimulation. Hexamethonium inhibited the action of UO2(2+), indicating a preganglionic site of action of UO2(2+) and interneurons possibly being involved in the ACh-releasing effect of UO2(2+). In addition, the action of UO2(2+) was enhanced by physostigmine but antagonized by atropine, ATP, adrenaline and morphine suggesting multiple sites of action of UO2(2+). The effects of Ba2+ were studied simultaneously in order to compare them with those of UO2(2+). Atropine and hexamethonium had no effect on the rhythmic contractions with Ba2+ but physostigmine enhanced contractions induced by low concentration of Ba2+ indicating that Ba2+ induces contractions mainly by acting on the smooth muscle and releases ACh from the nerve to a lesser extent.

In this study, we found that sodium selenite was potent in inducing contracture of the mouse diaphragm. The possible mechanism of action of selenite was investigated. Contracture was induced by a direct action of selenite on the muscle membrane rather than that selenite enhanced transmitter release from the motor nerve terminals, since denervation, d-tubocurarine and tetrodotoxin did not inhibit the selenite-induced contracture. Although selenite decreased both the membrane potential and the amplitude of the muscle action potential, neither high K+ nor glycerol treatment, which closed the transverse tubule, reduced the selenite-induced contracture, suggesting that depolarization of the muscle membrane was not essential for the induction of the contracture. EGTA (1-50 mM) inhibited the selenite-induced contracture in a concentration-dependent manner. In contrast, varying the external Ca2+ concentrations from 10(-3) to 10 mM or raising Mg2+ concentration to 10 mM did not affect the contracture. Similarly, the contracture induced by caffeine was not affected by lowering the external Ca2+ concentration to 10(-3) mM but was completely inhibited by 30 mM EGTA. Selenite pretreatment markedly potentiated the caffeine contracture and prolonged treatment with caffeine inhibited the selenite contracture. All of these findings suggest that the selenite contracture was not dependent on external Ca2+ but was induced by the release of Ca2+ from internal membranes such as the sarcoplasmic reticulum. Pretreatment with trypsin, glutathione or cyanide blocked the selenite-evoked contracture. Therefore, we postulate that the selenite-induced contracture was induced by the initial binding of selenite to the sulfhydryl groups of the muscle membrane, which then triggered the release of Ca2+ from internal membranes such as the sarcoplasmic reticulum.

The effects of sodium selenite on the neuromuscular junction of the phrenic nerve-diaphragm of the mouse were studied. Nerve-evoked twitches of the diaphragm of the mouse, the frequency of miniature endplate potentials, the quantal content of endplate potentials and the compound action potentials of the axon were measured. Sodium selenite induced a slight increase of the amplitude of the twitch, followed by twitch depression. The amplitude of the twitch, increased by selenite, became more prominent after the suppression of the twitch induced by cadmium ions, d-tubocurarine or magnesium ions. It appeared that the increased amplitude of twitch was due to the facilitation of transmitter release, since selenite significantly increased the frequency of miniature endplate potentials, and the amplitude and quantal content of endplate potentials; the amplitude and half decay time of miniature endplate potentials were unaffected. Twitch depression induced by selenite was enhanced by ammonium ions, high potassium and low magnesium and attenuated by high calcium. During the period of gradual depression of the twitch, selenite decreased the amplitude of compound action potentials of the phrenic nerve axon and caused the disappearance of endplate potentials. Ammonium ions enhanced the blockade of axonal conduction induced by selenite. Moreover, the depolarizing agents, ammonium and high potassium also induced an initial increase of twitch amplitude followed by depression of the twitch. These findings indicate that selenite probably alters the release of the transmitter by depolarizing the nerve membrane. The effects of selenite were antagonized by glutathione and cyanide, suggesting that the binding of selenite to sulfhydryl groups of the membrane was essential for inducing its pharmacological actions.

The purpose of this investigation was to study the possible mechanism of the potentiation of the contractile response and myotonia caused by Cd2+ in the mouse diaphragm. Cd2+ increased both amplitude and duration of the contractile response to direct stimulation in either 0.25 mM Ca2+ Krebs or 2.5 mM Ca2+ Krebs containing the K+-channel blockers, 4-aminopyridine, uranyl nitrate or tetraethylammonium ion. High K+ and tetrodotoxin inhibited these effects of Cd2+. Electrophysiological studies revealed that only one or two action potentials were triggered by passing a short depolarizing current across the muscle fibre membrane in 0.25 mM Ca2+ Krebs, but in the presence of Cd2+, a train of action potentials (153 +/- 21 Hz) which lasted for 0.7 +/- 0.2 s was induced. Furthermore, Cd2+ triggered a train of action potentials evoked by a single extracellular direct stimulation on the muscle fibre in 2.5 mM Ca2+ Krebs solution containing either 4-aminopyridine or uranyl nitrate. The membrane depolarized during the repetitive firing and then repolarized immediately after the cessation of repetitive firing. Cd2+ (0.1 mM) increased the input resistance of the muscle fibre by 53 +/- 7% and this effect was inhibited in low [Cl-]o. These findings suggest that the contractile potentiation and myotonia induced by Cd2+ in the mouse diaphragm are mediated by lowering the Cl- conductance of the membrane.