Cytoskeletal toxicity of pectenotoxins in hepatic cells

Background and purpose.

Pectenotoxins are macrocyclic lactones found in dinoflagellates of the genus Dinophysis, which induce severe liver damage in mice after i.p. injection. Here, we have looked for the mechanism(s) underlying this hepatotoxicity.

Experimental approach.

Effects of pectenotoxin (PTX)-1, PTX-2, PTX-2 seco acid (PTX-2SA) and PTX-11 were measured in a hepatocyte cell line with cancer cell characteristics (Clone 9) and in primary cultures of rat hepatocytes. Cell morphology was assessed by confocal microscopy; F- and G-actin were selectively stained and cell viability measured by Alamar Blue fluorescence.

Key results.

Clone 9 cells and primary hepatocytes showed a marked depolymerization of F-actin with PTX-1, PTX-2 and PTX-11 (1–1000 nM) associated with an increase in G-actin level. However, morphology was only clearly altered in Clone 9 cells. PTX-2SA had no effect on the actin cytoskeleton. Despite the potent F-actin depolymerizing effect, PTX-1, PTX-2 or PTX-11 did not decrease the viability of Clone 9 cells after 24-h treatment. Only prolonged incubation (>48 h) with PTXs induced a fall in viability, and under these conditions, morphology of both Clone 9 and primary hepatocytes was drastically changed.

Conclusions and implications.

Although the actin cytoskeleton was clearly altered by PTX-1, PTX-2 and PTX-11 in the hepatocyte cell line and primary hepatocytes, morphological assessments indicated a higher sensitivity of the cancer-like cell line to these toxins. However, viability of both cell types was not altered.     

Tangeretin and its metabolite 4'-hydroxytetramethoxyflavone attenuate EGF-stimulated cell cycle progression in hepatocytes; role of inhibition at the level of mTOR/p70S6K

BACKGROUND AND PURPOSE

The mechanisms by which the dietary compound tangeretin has anticancer effects may include acting as a prodrug, forming an antiproliferative product in cancer cells. Here we show that tangeretin also inhibits cell cycle progression in hepatocytes and investigate the role of its primary metabolite 4′-hydroxy-5,6,7,8-tetramethoxyflavone (4′-OH-TMF) in this effect.

EXPERIMENTAL APPROACH

We used epidermal growth factor (EGF)-stimulated rat hepatocytes, with [³H]-thymidine incorporation into DNA as an index of progression to S-phase of the cell cycle, and Western blots for phospho-proteins involved in the cell signalling cascade.

KEY RESULTS

Incubation of tangeretin with microsomes expressing CYP1A, or with hepatocytes, generated a primary product we identified as 4′-OH-TMF. Low micromolar concentrations of tangeretin or 4′-OH-TMF gave a concentration-dependent inhibition of EGF-stimulated progression to S-phase while having little effect on cell viability. To determine whether time for conversion of tangeretin to an active metabolite would enhance the inhibitory effect we used long pre-incubations; this reduced the inhibitory effect, in parallel with a reduction in the concentration of tangeretin. The EGF-stimulation of hepatocyte cell cycle progression requires signalling through Akt/mTOR/p70S6K kinase cascades. The tangeretin metabolite 4′-OH-TMF selectively inhibited S6K phosphorylation in the absence of significant inhibition of upstream Akt activity, suggesting an effect at the level of mTOR.

CONCLUSIONS AND IMPLICATIONS

Tangeretin and 4′-OH-TMF both inhibit cell cycle progression in primary hepatocytes. The inhibition of p70S6K phosphorylation by 4′-OH-TMF raises the possibility that inhibition of the mTOR pathway may contribute to the anticancer influence of a flavonoid-rich diet.     

Okadaic acid induces matrix metalloproteinase-9 expression in fibroblasts: crosstalk between protein phosphatase inhibition and β-adrenoceptor signalling

BACKGROUND AND PURPOSE

Interactions between protein phosphatase inhibition and matrix metalloproteinase (MMP)-9 expression have implications for tissue remodelling after injury. Stimulation of β-adrenoceptors could affect such interactions as isoprenaline increases protein phosphatase 2A (PP2A) activity and MMP-9 abundance. We investigated the effect of okadaic acid (OA) on MMP-9 expression to assess interactions between phosphatase inhibition and β-adrenoceptor signalling in fibroblasts.

EXPERIMENTAL APPROACH

Fibroblasts were exposed to OA alone and in combination with isoprenaline. Effects on MMP-9 expression and intracellular signalling were studied using promoter assays, Western blot analysis and siRNA methodologies.

KEY RESULTS

Okadaic acid increased MMP-9 abundance in human cardiac ventricular fibroblasts, NIH3T3 fibroblasts and hepatic stellate cells. This effect was unaffected by PP2A knockdown in NIH3T3 cells. OA increased phosphorylation of NF-κB, but not NF-κB promoter activity, IκBα degradation, or nuclear translocation of p65-NF-κB. Exposure to SB202190 (p38 MAPK), U0126 (ERK1/2) and NF-κB III inhibitor revealed that OA induced MMP-9 activity through p38 MAPK. Isoprenaline inhibited OA-mediated MMP-9 expression in NIH3T3, in a β-arrestin 2- and PP2A-dependent manner. Mutation of the activator protein-1 (AP-1) and NF-κB binding sites demonstrated that OA-induced MMP-9 activity was mediated through the AP-1 but not NF-κB sites. The latter mediated the inhibitory effect of isoprenaline on OA-induced MMP-9 promoter activity.

CONCLUSION AND IMPLICATIONS

Okadaic acid induced MMP-9 activity through p38 MAPK and was inhibited by isoprenaline via a pathway involving β-arrestin 2, PP2A and an NF-κB binding motif. These findings elucidate how phosphoprotein phosphatases and adrenoceptors may modulate tissue remodelling by affecting fibroblast function.     

Decreased exposure of simvastatin and simvastatin acid in a rat model of type 2 diabetes

Aim:

Simvastatin is frequently administered to diabetic patients with hypercholesterolemia. The aim of the study was to investigate the pharmacokinetics of simvastatin and its hydrolysate simvastatin acid in a rat model of type 2 diabetes.

Methods:

Diabetes was induced in 4-week-old rats by a treatment of high-fat diet combined with streptozotocin. After the rats received a single dose of simvastatin (20 mg/kg, po, or 2 mg/kg, iv), the plasma concentrations of simvastatin and simvastatin acid were determined. Simvastatin metabolism and cytochrome P4503A (Cyp3a) activity were assessed in hepatic microsomes, and its uptake was studied in freshly isolated hepatocytes. The expression of Cyp3a1, organic anion transporting polypeptide 2 (Oatp2), multidrug resistance-associated protein 2 (Mrp2) and breast cancer resistance protein (Bcrp) in livers was measured using qRT-PCR.

Results:

After oral or intravenous administration, the plasma concentrations and areas under concentrations of simvastatin and simvastatin acid were markedly decreased in diabetic rats. Both simvastatin metabolism and Cyp3a activity were markedly increased in hepatocytes of diabetic rats, accompanied by increased expression of hepatic Cyp3a1 mRNA. Furthermore, the uptake of simvastatin by hepatocytes of diabetic rats was markedly increased, which was associated with increased expression of the influx transporter Oatp2, and decreased expression of the efflux transporters Mrp2 and Bcrp.

Conclusion:

Diabetes enhances the metabolism of simvastatin and simvastatin acid in rats via up-regulating hepatic Cyp3a activity and expression and increasing hepatic uptake.     

PPARy agonists inhibit TGF-β-PKA signaling in glomeru Iosclerosis

Aim:

To study the probable mechanisms of the anti-glomerulosclerosis effects induced by peroxisome proliferator-activated receptor gamma (PPARγ) agonists in rat intraglomerular mesangial cells (MCs).

Methods:

Cells were transfected with the pTAL-PPRE-tk-Luc⁺ plasmid and then treated with different concentrations of PPARγ agonist, either troglitazone or telmisartan, for the indicated times. Promega luciferase assays were subsequently used for the detection of PPARγ activation. Protein expression levels were assessed by Western blot, and PepTag^® assays were used for the non-radioactive detection of protein kinase A (PKA) activity. The deposition of α-smooth muscle actin (α-SMA) and p-cyclic AMP responsive element binding protein (pCREB) were analyzed by confocal laser scanning.

Results:

Both troglitazone and telmisartan remarkably inhibit the PKA activation and pCREB expression that is stimulated by TGF-β. The PPARγ agonists also inhibited α-SMA and collagen IV protein expression by blocking PKA activation.

Conclusion:

PPARγ ligands effectively suppress the activation of MCs and the accumulation of collagen IV stimulated by TGF-β in vitro. The renal protection provided by PPARγ agonists is partly mediated via their blockade of TGF-β/PKA signaling.     

Effect of prototypical inducers on ligand activated nuclear receptor regulated drug disposition genes in rodent hepatic and intestinal cells

Aim:

The aim of this study was to investigate the impact on expression of mRNA and protein by paradigm inducers/activators of nuclear receptors and their target genes in rat hepatic and intestinal cells. Furthermore, assess marked inter laboratory conflicting reports regarding species and tissue differences in expression to gain further insight and rationalise previously observed species differences between rodent and human based systems.

Methods:

Quantitative real time-polymerase chain reaction (QRT-PCR) and immunoblots were used to assess messenger RNA (mRNA) and protein expression for CYP2B2, CYP3A1, CYP3A2, CYP3A9, ABCB1a, ABCB1b, ABCC1, ABCC2, pregnane X receptor (PXR), farnesoid X receptor (FXR) and constituitive androstane receptor (CAR) in rat hepatoma cell line H411E, intestinal cells, Iec-6, and rat primary hepatocytes, in response to exposure for 18 h with prototypical inducers.

Results:

Dexamethasone (DEX) and pregnenolone 16α carbonitrile (PCN) significantly induced PXR, CYP3A9, ABCB1a and ABCB1b. However, when co-incubated, DEX appeared to restrict PCN-dependent induction. Chenodeoxycholic acid (CDCA) was the only ligand to induce FXR in all three cell types. Despite previously reported species differences between PCN and rifampicin (RIF), both compounds exhibited a similar profile of induction.

Conclusion:

Data presented herein may explain some of the discrepancies previously reported with respect to species differences from different laboratories and have important implications for study design.     

ROS generated by CYP450, especially CYP2E1, mediate mitochondrial dysfunction induced by tetrandrine in rat hepatocytes

Aim:

Tetrandrine, an alkaloid with a remarkable pharmacological profile, induces oxidative stress and mitochondrial dysfunction in hepatocytes; however, mitochondria are not the direct target of tetrandrine, which prompts us to elucidate the role of oxidative stress in tetrandrine-induced mitochondrial dysfunction and the sources of oxidative stress.

Methods:

Rat primary hepatocytes were isolated by two-step collagenase perfusion. Mitochondrial function was evaluated by analyzing ATP content, mitochondrial membrane potential (MMP) and the mitochondrial permeability transition. The oxidative stress was evaluated by examining changes in the levels of reactive oxygen species (ROS) and glutathione (GSH).

Results:

ROS scavengers largely attenuated the cytotoxicity induced by tetrandrine in rat hepatocytes, indicating the important role of ROS in the hepatotoxicity of tetrandrine. Of the multiple ROS inhibitors that were tested, only inhibitors of CYP450 (SKF-525A and others) reduced the ROS levels and ameliorated the depletion of GSH. Mitochondrial function assays showed that the mitochondrial permeability transition (MPT) induced by tetrandrine was inhibited by SKF-525A and vitamin C (VC), both of which also rescued the depletion of ATP levels and the mitochondrial membrane potential. Upon inhibiting specific CYP450 isoforms, we observed that the inhibitors of CYP2D, CYP2C, and CYP2E1 attenuated the ATP depletion that occurred following tetrandrine exposure, whereas the inhibitors of CYP2D and CYP2E1 reduced the ROS induced by tetrandrine. Overexpression of CYP2E1 enhanced the tetrandrine-induced cytotoxicity.

Conclusion:

We demonstrated that CYP450 plays an important role in the mitochondrial dysfunction induced by the administration of tetrandrine. ROS generated by CYP450, especially CYP2E1, may contribute to the mitochondrial dysfunction induced by tetrandrine.     

Indirect modulation of neuronal excitability and synaptic transmission in the hippocampus by activation of proteinase-activated receptor-2

BACKGROUND AND PURPOSE

Proteinase-activated receptor-2 (PAR2) is widely expressed in the CNS under normal physiological conditions. However, its potential role in modulating neuronal excitability and synaptic transmission remains to be determined. Here, we have investigated whether PAR2 activation modulates synaptic activity in the hippocampus.

EXPERIMENTAL APPROACH

PAR2 activation and its effect on the hippocampus were examined in rat primary cultures and acute slices using whole cell patch clamp and standard extracellular recordings, respectively.

KEY RESULTS

PAR2 activation leads to a depolarization of hippocampal neurones and a paradoxical reduction in the occurrence of synaptically driven spontaneous action potentials (APs). PAR2-induced neuronal depolarization was abolished following either the inhibition of astrocytic function or antagonism of ionotropic glutamate receptors whilst the PAR2-induced decrease in AP frequency was also reduced when astrocytic function was inhibited. Furthermore, when examined in acute hippocampal slices, PAR2 activation induced a profound long-term depression of synaptic transmission that was dependent on NMDA receptor activation and was sensitive to disruption of astrocytic function.

CONCLUSIONS AND IMPLICATIONS

These novel findings show that PAR2 activation indirectly inhibits hippocampal synaptic activity and indicate that these receptors may play an active role in modulating normal physiological CNS function, in addition to their role in pathophysiological disorders.     

Effects of atorvastatin metabolites on induction of drug-metabolizing enzymes and membrane transporters through human pregnane X receptor

BACKGROUND AND PURPOSE

Atorvastatin metabolites differ in their potential for drug interaction because of differential inhibition of drug-metabolizing enzymes and transporters. We here investigate whether they exert differential effects on the induction of these genes via activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR).

EXPERIMENTAL APPROACH

Ligand binding to PXR or CAR was analysed by mammalian two-hybrid assembly and promoter/reporter gene assays. Additionally, surface plasmon resonance was used to analyse ligand binding to CAR. Primary human hepatocytes were treated with atorvastatin metabolites, and mRNA and protein expression of PXR-regulated genes was measured. Two-hybrid co-activator interaction and co-repressor release assays were utilized to elucidate the molecular mechanism of PXR activation.

KEY RESULTS

All atorvastatin metabolites induced the assembly of PXR and activated CYP3A4 promoter activity. Ligand binding to CAR could not be proven. In primary human hepatocytes, the para-hydroxy metabolite markedly reduced or abolished induction of cytochrome P450 and transporter genes. While significant differences in co-activator recruitment were not observed, para-hydroxy atorvastatin demonstrated only 50% release of co-repressors.

CONCLUSIONS AND IMPLICATIONS

Atorvastatin metabolites are ligands of PXR but not of CAR. Atorvastatin metabolites demonstrate differential induction of PXR target genes, which results from impaired release of co-repressors. Consequently, the properties of drug metabolites have to be taken into account when analysing PXR-dependent induction of drug metabolism and transport. The drug interaction potential of the active metabolite, para-hydroxy atorvastatin, might be lower than that of the parent compound.     

Pharmacological profile of opicapone,a thirdgeneration nitrocatechol catechol-O-methyl transferase inhibitor,in the rat

Background and Purpose

Catechol-O-methyltransferase (COMT) is an important target in the levodopa treatment of Parkinson''s disease; however, the inhibitors available have problems, and not all patients benefit from their efficacy. Opicapone was developed to overcome those limitations. In this study, opicapone''s pharmacological properties were evaluated as well as its potential cytotoxic effects.

Experimental Approach

The pharmacodynamic effects of opicapone were explored by evaluating rat COMT activity and levodopa pharmacokinetics, in the periphery through microdialysis and in whole brain. The potential cytotoxicity risk of opicapone was explored in human hepatocytes by assessing cellular ATP content and mitochondrial membrane potential.

Key Results

Opicapone inhibited rat peripheral COMT with ED₅₀ values below 1.4 mg⋅kg⁻¹ up to 6 h post-administration. The effect was sustained over the first 8 h and by 24 h COMT had not returned to control values. A single administration of opicapone resulted in increased and sustained plasma levodopa levels with a concomitant reduction in 3-O-methyldopa from 2 h up to 24 h post-administration, while tolcapone produced significant effects only at 2 h post-administration. The effects of opicapone on brain catecholamines after levodopa administration were sustained up to 24 h post-administration. Opicapone was also the least potent compound in decreasing both the mitochondrial membrane potential and the ATP content in human primary hepatocytes after a 24 h incubation period.

Conclusions and Implications

Opicapone has a prolonged inhibitory effect on peripheral COMT, which extends the bioavailability of levodopa, without inducing toxicity. Thus, it exhibits some improved properties compared to the currently available COMT inhibitors.     

Pharmacokinetics, adverse effects and tolerability of a novel analogue of human pancreatic polypeptide, PP 1420

AIMS

The objectives of this phase 1 study were to confirm the tolerability of single ascending subcutaneous doses of PP 1420 in healthy subjects, to assess its adverse effects and to investigate the drug''s pharmacokinetics and dose proportionality.

METHODS

This was a double-blind, placebo-controlled, randomized study. There were three dosing periods. Each subject (n = 12) was randomized to receive one dose of placebo and two ascending doses of PP 1420, given as a subcutaneous injection. Blood samples were taken over 24 h to assess pharmacokinetics. Standard safety and laboratory data were collected. The primary endpoint was the tolerability of PP 1420. The secondary endpoint was exposure to PP 1420 as assessed by C_max and AUC(0,∞).

RESULTS

PP 1420 was well tolerated by all subjects with no serious adverse effects. Following single subcutaneous doses of PP 1420 at 2, 4 and 8 mg to male subjects, C_max was reached at a median t_max of approximately 1 h post dose (range 0.32–2.00 h). Thereafter, plasma concentrations of PP 1420 declined with geometric mean apparent terminal elimination t_1/2 ranging from 2.42–2.61 h (range 1.64–3.95 h) across all dose levels.

CONCLUSIONS

Subcutaneous PP 1420 was well tolerated in healthy human subjects at single doses between 2–8 mg, with no tolerability issues arising. Where observed, adverse events were not serious, and there was no evidence of a dose-relationship to frequency of adverse events. The results therefore support the conduct of clinical trials to investigate efficacy, tolerability and pharmacokinetics during repeated dosing.     

Benzamil sensitive ion channels contribute to volume regulation in canine chondrocytes

Background and Purpose

Chondrocytes exist within cartilage and serve to maintain the extracellular matrix. It has been postulated that osteoarthritic (OA) chondrocytes lose the ability to regulate their volume, affecting extracellular matrix production. In previous studies, we identified expression of epithelial sodium channels (ENaC) in human chondrocytes, but their function remained unknown. Although ENaC typically has Na⁺ transport roles, it is also involved in the cell volume regulation of rat hepatocytes. ENaC is a member of the degenerin (Deg) family, and ENaC/Deg-like channels have a low conductance and high sensitivity to benzamil. In this study, we investigated whether canine chondrocytes express functional ENaC/Deg-like ion channels and, if so, what their function may be.

Experimental Approach

Canine chondrocytes were harvested from dogs killed for unassociated welfare reasons. We used immunohistochemistry and patch-clamp electrophysiology to investigate ENaC expression and video microscopy to analyse the effects of pharmacological inhibition of ENaC/Deg on cell volume regulation.

Key Results

Immunofluorescence showed that canine chondrocytes expressed ENaC protein. Single-channel recordings demonstrated expression of a benzamil-sensitive Na⁺ conductance (9 pS), and whole-cell experiments show this to be approximately 1.5 nS per cell with high selectivity for Na⁺. Benzamil hyperpolarized chondrocytes by approximately 8 mV with a pD₂ 8.4. Chondrocyte regulatory volume decrease (RVI) was inhibited by benzamil (pD₂ 7.5) but persisted when extracellular Na⁺ ions were replaced by Li⁺.

Conclusion and Implications

Our data suggest that benzamil inhibits RVI by reducing the influx of Na⁺ ions through ENaC/Deg-like ion channels and present ENaC/Deg as a possible target for pharmacological modulation of chondrocyte volume.     

Inhibition of paracetamol glucuronidation by tyrosine kinase inhibitors

AIMS

We aimed to investigate the effects of tyrosine kinase inhibitors (TKIs) on paracetamol (acetaminophen) glucuronidation.

METHODS

The inhibition of nine small molecule TKIs on paracetamol glucuronidation was investigated in human liver microsomes (HLMs) and recombinant human UDP-glucuronosyltransferases (UGTs).

RESULTS

Sorafenib, dasatinib and imatinib exhibited mixed inhibition against paracetamol glucuronidation in pooled HLMs, and potent inhibition in UGT1A9 and UGT2B15. Dasatinib and imatinib also inhibited UGT1A1-mediated paracetamol glucuronidation. Axitinib, erlotinib, gefitinib, lapatinib, nilotinib and vandetanib exhibited weak inhibition of paracetamol glucuronidation activity in HLMs.

CONCLUSIONS

The inhibition of paracetamol glucuronidation by TKIs might be of particular concern when they are co-administered.     

The 5-lipoxygenase inhibitor, zileuton, suppresses prostaglandin biosynthesis by inhibition of arachidonic acid release in macrophages

BACKGROUND AND PURPOSE

Zileuton is the only 5-lipoxygenase (5-LOX) inhibitor marketed as a treatment for asthma, and is often utilized as a selective tool to evaluate the role of 5-LOX and leukotrienes. The aim of this study was to investigate the effect of zileuton on prostaglandin (PG) production in vitro and in vivo.

EXPERIMENTAL APPROACH

Peritoneal macrophages activated with lipopolysaccharide (LPS)/interferon γ (LPS/IFNγ), J774 macrophages and human whole blood stimulated with LPS were used as in vitro models and rat carrageenan-induced pleurisy as an in vivo model.

KEY RESULTS

Zileuton suppressed PG biosynthesis by interference with arachidonic acid (AA) release in macrophages. We found that zileuton significantly reduced PGE2 and 6-keto prostaglandin F1α (PGF1α) levels in activated mouse peritoneal macrophages and in J774 macrophages. This effect was not related to 5-LOX inhibition, because it was also observed in macrophages from 5-LOX knockout mice. Notably, zileuton inhibited PGE2 production in LPS-stimulated human whole blood and suppressed PGE2 and 6-keto PGF1α pleural levels in rat carrageenan-induced pleurisy. Interestingly, zileuton failed to inhibit the activity of microsomal PGE2 synthase1 and of cyclooxygenase (COX)-2 and did not affect COX-2 expression. However, zileuton significantly decreased AA release in macrophages accompanied by inhibition of phospholipase A2 translocation to cellular membranes.

CONCLUSIONS AND IMPLICATION

Zileuton inhibited PG production by interfering at the level of AA release. Its mechanism of action, as well as its use as a pharmacological tool, in experimental models of inflammation should be reassessed.     

Inhibition of the phosphatase PTEN protects mice against oleic acid-induced acute lung injury

Background and purpose

Injury to the lung parenchyma is a constitutional feature shared by many lung diseases. The protein, phosphatase and tensin homologue deleted on chromosome Ten (PTEN) is a major suppressor of phosphoinositide-3 kinase/Akt signalling, a vital survival pathway in lung parenchymal cells. Based on this, we hypothesized that PTEN inhibition in vivo would enhance cell tolerance to stress thereby preventing acute lung injury.

Experimental approach

We evaluated the ability of a PTEN inhibitor, potassium bisperoxo (1,10-phenanthroline) oxovanadate [bpV(phen)], to prevent acute lung injury induced by oleic acid (OA) in adult C57BL/6 mice. Lung assessments included bronchoalveolar lavage, tissue morphology, immunostaining for markers of cell death, cell identity, phospho-Akt and phospho-ERK levels and oximetry.

Key results

OA induced acute lung injury in a dose- and time-dependent manner. No injury was observed in the vehicle control or bpV(phen) treatment groups. PTEN inhibition by bpV(phen) increased lung tissue levels of phospho-Akt and ERK and but not focal adhesion kinase. This occurred in conjunction with a statistically significant reduction in protein content, lactate dehydrogenase, as well as tumour necrosis factor-α and chemokines in bronchoalveolar lavage fluid when compared with OA treatment alone. The incidence of alveolar lesions, consistent with acute lung injury, and terminal uridine deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive cells was also significantly reduced. Importantly, PTEN suppression maintained pulmonary function.

Conclusions and implications

Treatment with bpV(phen) significantly reduced the severity of acute lung injury in mice indicating that additional investigation is warranted to understand the important role that this phosphatase may play in the lung.     

The effect of inhibition of endoplasmic reticulum stress on lipolysis in white adipose tissue in a rat model of chronic kidney disease

Aim; Lipolysis in fat tissue plays an important role in the development of metabolic disturbances, a characteristic feature of chronic kidney disease （CKD）. In the present study, we tested the hypothesis that the inhibition of endoplasmic reticulum （ER） stress could alleviate lipolysis in white adipose tissue in a rat model of CKD. Methods： A rat model of CKD was established by a method of reduced renal mass （RRM）. Lipolysis was measured as the release of glycerol in ex vivo fat pads and cultured primary adipocytes. The activity of lipases and markers of ER stress were measured by Western blotting and immunoprecipitation. Results; Our data showed that lipolysis in visceral white adipose tissue was increased in RRM rats compared with control rats. In addition, increased phosphorylation of hormone-sensitive lipase （HSL） and binding of adipose triglyceride lipase （ATGL） to comparative gene identification-58 （CGI-58） protein were observed in the RRM rats. The phosphorylation of ER stress markers, including IREla, PERK, and eukaryotic initiation factor （elF） 2a, and the expression of ER stress marker 78 kDa glucose-regulated protein （GRP78） were significantly increased in RRM rats. Treatment with an inhibitor of ER stress partially but significantly alleviated lipolysis, and this alleviation was accompanied by reduced binding of ATGL to CGI-58. Conclusion： Our results showed that enhanced lipolysis and ER stress occurred in visceral white adipose tissue in a rat model of CKD. Moreover, inhibition of ER stress significantly alleviated lipolysis. These findings suggest that ER stress is a potential therapeutic target for the metabolic disturbances associated with CKD.     

Acute effects of short-chain alkylglycerols on blood-brain barrier properties of cultured brain endothelial cells

Background and Purpose

The blood-brain barrier (BBB) restricts drug penetration to the brain preventing effective treatment of patients suffering from brain tumours. Intra-arterial injection of short-chain alkylglycerols (AGs) opens the BBB and increases delivery of molecules to rodent brain parenchyma in vivo. The mechanism underlying AG-mediated modification of BBB permeability is still unknown. Here, we have tested the effects of AGs on barrier properties of cultured brain microvascular endothelial cells.

Experimental Approach

The effects of two AGs, 1-O-pentylglycerol and 2-O-hexyldiglycerol were examined using an in vitro BBB model consisting of primary cultures of rat brain endothelial cells, co-cultured with rat cerebral glial cells. Integrity of the paracellular, tight junction-based, permeation route was analysed by functional assays, immunostaining for junctional proteins, freeze-fracture electron microscopy, and analysis of claudin-claudin trans-interactions.

Key Results

AG treatment (5 min) reversibly reduced transendothelial electrical resistance and increased BBB permeability for fluorescein accompanied by changes in cell morphology and immunostaining for claudin-5 and β-catenin. These short-term changes were not accompanied by alterations of inter-endothelial tight junction strand complexity or the trans-interaction of claudin-5.

Conclusion and Implications

AG-mediated increase in brain endothelial paracellular permeability was short, reversible and did not affect tight junction strand complexity. Redistribution of junctional proteins and alterations in the cell shape indicate the involvement of the cytoskeleton in the action of AGs. These data confirm the results from in vivo studies in rodents characterizing AGs as adjuvants that transiently open the BBB.     

Involvement of protein kinase C and Src tyrosine kinase in acute tolerance to ethanol inhibition of spinal NMDA-induced pressor responses in rats

Background and purpose:

The present study was carried out to examine the role of protein kinases in the development of acute tolerance to the effects of ethanol on spinal N-methyl-D-aspartate (NMDA) receptor-mediated pressor responses during prolonged ethanol exposure.

Experimental approach:

Blood pressure responses induced by intrathecal injection of NMDA were recorded. The levels of several phosphorylated residues on NMDA receptor NR1 (GluN1) (NR1) and NMDA receptor NR2B (GluN2B) (NR2B) subunits were determined by immunohistochemistry and Western blot analysis.

Key results:

Ethanol inhibited spinal NMDA-induced pressor responses at 10 min, but the inhibition was significantly reduced at 40 min following continuous infusion. This effect was dose-dependently blocked by chelerythrine [a protein kinase C (PKC) inhibitor, 1–1000 pmol] or PP2 (a Src family tyrosine kinase inhibitor, 1–100 pmol) administered intrathecally 10 min following ethanol infusion. A significant increase in the immunoreactivity of phosphoserine 896 of NR1 subunits (pNR1-Ser896) and phosphotyrosine 1336 of NR2B subunits (pNR2B-Tyr1336) was found in neurons of intermediolateral cell column during the development of tolerance. Levels of pNR1-Ser896 and pNR2B-Tyr1336 were also significantly increased in lateral horn regions of the spinal cord slices incubated with ethanol for 40 min in vitro. The increases in pNR1-Ser896 and pNR2B-Tyr1336 levels were inhibited by post-treatment with chelerythrine and PP2, respectively, both in the in vivo and in vitro studies.

Conclusions and implications:

The results suggest that activation of PKC and Src tyrosine kinase during prolonged ethanol exposure leading to increases in the levels of pNR1-Ser896 and pNR2B-Tyr1336 may contribute to acute tolerance to inhibition by ethanol of NMDA receptor function.     

Timosaponin A3 induces hepatotoxicity in rats through inducing oxidative stress and down-regulating bile acid transporters

Aim:

To investigate the mechanisms underlying the hepatotoxicity of timosaponin A3 (TA3), a steroidal saponin from Anemarrhena asphodeloides, in rats.

Methods:

Male SD rats were administered TA3 (100 mg·kg⁻¹·d⁻¹, po) for 14 d, and the blood and bile samples were collected after the final administration. The viability of a sandwich configuration of cultured rat hepatocytes (SCRHs) was assessed using WST-1. Accumulation and biliary excretion index (BEI) of d8-TCA in SCRHs were determined with LC-MS/MS. RT-PCR and Western blot were used to analyze the expression of relevant genes and proteins. ROS and ATP levels, and mitochondrial membrane potential (MMP) were measured. F-actin cytoskeletal integrity was assessed under confocal microscopy.

Results:

TA3 administration in rats significantly elevated the total bile acid in serum, and decreased bile acid (BA) component concentrations in bile. TA3 inhibited the viability of the SCRHs with an IC₅₀ value of 15.21±1.73 μmol/L. Treatment of the SCRHs with TA3 (1–10 μmol/L) for 2 and 24 h dose-dependently decreased the accumulation and BEI of d8-TCA. The TA3 treatment dose-dependently decreased the expression of BA transporters Ntcp, Bsep and Mrp2, and BA biosynthesis related Cyp7a1 in hepatocytes. Furthermore, the TA3 treatment dose-dependently increased ROS generation and HO-1 expression, decreased the ATP level and MMP, and disrupted F-actin in the SCRHs. NAC (5 mmol/L) significantly ameliorated TA3-induced effects in the SCRHs, whereas mangiferin (10–200 μg/mL) almost blocked TA3-induced ROS generation.

Conclusion:

TA3 triggers liver injury through inducing ROS generation and suppressing the expression of BA transporters. Mangiferin, an active component in Anemarrhena, may protect hepatocytes from TA3-induced hepatotoxicity.