Cocaine addiction is known to impair immune system function, but the effects of repeated treatment with cocaine in a self-administration model, its withdrawal as well as reinstatement of cocaine-seeking behavior on cell-mediated immunity are not well known. Cocaine self-administered for 18 days induced a significant increase in spleen weight, plasma corticosterone levels, interleukin (IL)-10, and tumor necrosis factor-α production, while concanavalin A–stimulated proliferation responses of peripheral blood T-lymphocytes and interferon-γ production by splenic lymphocytes were not altered. After 10 days withdrawal from cocaine, reinstatement of cocaine seeking behavior induced either by a priming dose of the drug (unconditioned stimulus), by cue previously associated with cocaine self-administration (conditioned stimuli), or by both these stimuli evoked similar changes in several immunological parameters, for example, a decrease in relative spleen weight, proliferative activity of splenocytes, and their ability to produce IL-10. The results showed that the cue previously associated with cocaine suppressed some parameters of cell-mediated immunity to the same degree as re-exposure to cocaine. The present study provides the first evidence that alterations of immune status can be conditioned by environmental stimuli paired with cocaine administration.

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Oridonin, a diterpenoid isolated from Rabdosia rubescences, could induce apoptosis through the generation of reactive oxygen species (ROS) in human hepatoma HepG2 cells. p53, a specific inhibitor of pifithrin α (PFT α), markedly inhibited ROS generation and apoptosis, showing that p53 was responsible for the cytotoxity of oridonin through mediation by ROS. Moreover, the ROS activated the p38 kinase, which in turn promoted the activation of p53, as verified by evidence showing that the ROS scavenger N-acetyl-cysteine (NAC) not only blocked the phosphorylation of p38 but also partially inhibited the activation of p53, and the p38 inhibitor SB203580 reduced the activation of p53 as well. Mitochondria were either the sources or the targets of ROS. This study showed that oridonin stimulated mitochondrial transmembrane permeabilization in a ROS-dependent manner because NAC almost thoroughly reversed the drop of mitochondrial transmembrane potential (Δψm) and the release of cytochrome c from the mitochondrial inter-membrane space into cytosol. Furthermore, as a result of mitochondrial permeability transition, procaspases-9 and -3 were cleaved into 37- and 17-kDa proteolytic products, respectively, which acted as executors of oridonin-induced apoptosis.

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The precise causative factors in neurodegenerative diseases such as Alzheimer’s (AD) and Parkinson’s disease remain elusive, but mechanisms implicated comprise excitotoxicity, mitochondrial dysfunction, and in the case of AD, the amyloid beta peptide (Aβ). Current therapeutic strategies for such disorders are very limited; thus, traditional herbal medicines currently receive increased attention. The seeds of Cassia obtisufolia have long been used in traditional eastern medicine and more recently the ethanolic fraction of the seeds (COE) has been shown to attenuate memory impairments in mice. In this study, we set out to determine the effect of COE (range: 0.1 – 10 μg/ml) on calcium dysregulation and cell death models in mouse primary hippocampal cultures implicated in general neurodegenerative processes and in the pathogenesis of AD: excitotoxicity, mitochondrial dysfunction, and Aβ toxicity. It was found that treatment with COE attenuated secondary Ca²⁺ dysregulation induced by NMDA (700 μM), while a pre-application of COE also reduced NMDA-induced cell death. Furthermore, COE was neuroprotective against the mitochondrial toxin 3-NP (1 mM), while having no significant effect on cell death induced by incubation with naturally-secreted oligomers of Aβ (8.2 pg/ml). Collectively, these results are important for the therapeutic use of COE in the treatment of neurodegenerative disorders.

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The β₂-agonist clenbuterol [4-amino-α(t-butyl-amino)methyl-3,5-dichlorobenzyl alcohol] is used as a non-steroidal anabolic drug for sports doping. The effects of clenbuterol on the transcriptional process and mRNA stability of β-adrenoceptor (β-AR) in skeletal and cardiac muscles are still unknown. Therefore, we investigated the effects of clenbuterol on β₁- and β₂-AR mRNA expressions of fast-twitch fiber–rich extensor digitorum longus (EDL), slow-twitch fiber–rich soleus (SOL), and left ventricle (LV) muscles by real-time RT-PCR. Adult male Sprague Dawley rats were divided into the clenbuterol-administered group and control group. The administration (dose = 1.0 mg/kg body weight/day, s.c.) of clenbuterol was maintained for 10 days. The administration of clenbuterol significantly increased the weight, RNA concentration, and total RNA content of EDL muscle. No effects of clenbuterol on those of SOL and LV muscles, however, were observed. The administration of clenbuterol significantly decreased β₁-AR mRNA expression of LV muscle. Furthermore, the administration of clenbuterol significantly decreased β₂-AR mRNA expression of EDL and LV muscles. No effect of clenbuterol on β₂-AR mRNA expression of SOL muscle, however, was observed. These results suggest that the effects of clenbuterol on β₁- and β₂-AR mRNA expressions and muscle hypertrophy depend on muscle fiber types.

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Circadian synchronization of cell proliferation is observed not only in normal healthy tissues but also in malignant solid tumors. However, the proliferation rhythm of tumor cells is often different from that of normal cells. We reported here that the peculiar rhythm of tumor cell proliferation was modulated by inhibition of platelet-derived growth factor (PDGF) signaling. DNA synthesis in tumor cells implanted in mice showed a 24-h oscillation apparently differing from that of normal bone marrow cells. Continuous administration of AG1295 (10 μg/h, s.c.), a PDGF receptor tyrosine kinase inhibitor, substantially suppressed DNA synthesis in the implanted tumor cells but not in the healthy bone marrow cells. During the administration of this drug, the rhythm of DNA synthesis in the tumor cells was synchronized with that in bone marrow cells. The present results suggest that the circadian rhythm of DNA synthesis in tumor cells is modulated by PDGF receptor signaling, which is activated following tumor progression. Because the rhythmic patterns of clock gene expression in tumor cells did not differ significantly from those in other healthy tissues, the enhanced signal transduction of PDGF receptor may cause an alteration in the rhythmicity of tumor cell proliferation without changing in the intracellular molecular clockwork.

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We examined the toxicity of cocatropine (cocaine/atropine mixture) and the therapeutic potential of diazepam on some behavioral and physiological parameters in rats. Atropine (20 and 60 mg/kg) or cocaine (40 mg/kg) alone did not induce any seizure or death, but the combination significantly increased both, after both acute and binge treatment. There was a significant increase of EEG mean total spectral power in cocatropine- in comparison with cocaine-treated animals. Hyperlocomotion was observed in non-seizuring rats treated with cocaine or cocatropine. Cocaine, atropine 60, and cocatropine (40 + 20 and 40 + 60) all induced hyperthermic effects in non-seizuring rats, while cocatropine (40 + 60)-seizuring animals had hypothermia. An initial hypertensive and tachycardiac effect within 15 min was followed by a secondary fall in the cocatropine (40 + 60) group. Cocatropine toxicity was partially or fully reversed by diazepam (5 mg/kg), given intraperitoneally after the first seizure. The present findings provide, for the first time, details of a synergistic toxic effect of the cocaine/atropine mixture and of the potential of diazepam for treating cocatropine-related hospital emergencies.

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Extracellular nucleotides are involved in the development of vascular inflammation. However, little is known about whether effects of nucleotides are modulated under inflammatory states. We investigated effects of interferon-γ (INF-γ) on ATP-induced responses in vascular endothelial cells. Treatment of human umbilical vein endothelial cells (HUVECs) with IFN-γ for 24 h resulted in an enhancement of the ATP-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) without affecting the UTP-induced one. The increased Ca²⁺ response to ATP in IFN-γ–treated cells was dependent on the extracellular Ca²⁺, and was not inhibited by the phospholipase C inhibitor U73122. RT-PCR and Western blotting revealed that HUVECs dominantly expressed P2X₄ receptor. IFN-γ increased P2X₄-receptor mRNA and protein, accompanied by an increase in ATP-triggered membrane current. IFN-γ did not affect P2X₄-receptor mRNA stability, but increased P2X₄-receptor gene transcription in a cycloheximide-insensitive manner. IFN-γ stimulated phosphorylation of signal transducer and activator of transcription-1 (STAT1). Epigallocatechin gallate (EGCG), an inhibitor of STAT1-mediated signaling, and AG490, a Janus kinase (JAK) inhibitor, impaired P2X₄-receptor mRNA up-regulation by IFN-γ. These results indicate that INF-γ selectively increases P2X₄-receptor gene expression, leading to an up-regulation of purinergic signaling in vascular endothelial cells.

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The present study was undertaken to clarify the epileptogenic activity induced by intracerebroventricular injection (i.c.v.) of methicillin-resistant Staphylococcus aureus (MRSA) antibiotics in mice. Teicoplanin (200 μg, i.c.v.) caused dose-related behavioral seizures such as head twitch and forelimb clonus. At the same time, the drug caused electroencephalographic (EEG) seizures characterized by spike-and-wave complex and a continuous spike with high amplitude. At a high dose (500 μg, i.c.v.), the drug caused a severe clonic convulsion followed by continuous spike and spike-and-wave complex on EEG. On the other hand, vancomycin caused no or almost no epileptogenic activity in both behavior and on EEG. Diazepam and sodium valproate dose-dependently antagonized epileptic seizures in behavior and on EEG induced by teicoplanin (500 μg, i.c.v.). In contrast, carbamazepine and ethosuximide caused no significant changes in both behavioral and EEG seizures induced by teicoplanin. From these findings, it can be concluded that teicoplanin may cause potent epileptogenic activity different from vancomycin when used clinically at extremely high doses. In addition, it may be that teicoplanin-induced seizure is closely related with the γ-amino butyric acid (GABA)-ergic mechanism.

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We characterized the effects of sphingosine-1-phosphate (S1P) on rat aortic myocytes with or without culture. Application of S1P induced a small Ca²⁺ response in 40% freshly dispersed aortic myocytes, whereas S1P caused a larger Ca²⁺ response in 90% myocytes cultured for 72 h. Concentration–response relationships of S1P in cultured myocytes were significantly different from that in non-cultured myocytes. Analysis of the expression of S1P-receptor mRNA transcripts revealed that S1P-receptor type 3 (S1P₃) was significantly increased when myocytes were cultured for 24 h. Neither the removal of serum from culture medium nor pretreatment with pharmacological agents, such as ERK, Rho, and PI3 kinase inhibitors, affected the progression of the S1P-induced Ca²⁺ response during culture. The sustained component of the Ca²⁺ response to S1P was sensitive to the removal of external Ca²⁺ and was effectively inhibited by inorganic Ca²⁺-channel blockers such as Gd³⁺, Cd²⁺, and Ni²⁺. However, application of S1P did not induce any contraction in organ-cultured as well as the intact aorta muscle strip. Aortic myocytes freshly dispersed from the organ-cultured muscle were also ineffective against S1P. Taken together, cell-culture changes the S1P₃-mediated Ca²⁺ response to S1P in rat aortic myocytes.

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Extracellular nucleotides are primary signals for tissue injury, acting together with various chemical mediators such as prostanoids at the inflammatory site. We investigated whether prostaglandin E₂ (PGE₂) affects purinergic signaling in murine J774 macrophages. J774 cells expressed four different purinoceptor mRNAs: the ionotropic P2X₄ and P2X₇ receptors and G-protein–coupled P2Y₂ and P2Y₆ receptors. Functional responses mediated by these purinoceptor subtypes were confirmed by measurement of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in fura-2–loaded cells. Thus, low concentrations (10 μM) of ATP (P2Y₂ agonist) and UDP (P2Y₆ agonist) evoked Ca²⁺ transient in a phospholipase C (PLC)-dependent manner, whereas the P2X₇ agonist benzoylbenzoyl-ATP (BzATP, 500 μM) caused a sustained rise in [Ca²⁺]_i. Furthermore, ivermectin, an activator of the P2X₄-receptor channel, enhanced the ATP-induced [Ca²⁺]_i elevation. PGE₂ inhibited ATP- and UDP-induced [Ca²⁺]_i elevation, without affecting the BzATP-induced sustained [Ca²⁺]_i elevation. Stimulation of J774 cells by UDP or BzATP increased the production of macrophage inflammatory peptide-α (MIP-α). PGE₂ abolished the UDP-induced MIP-α production, but not the BzATP-induced one. These results demonstrate that purinergic signalings in macrophages were regulated by PGE₂ in a subtype-specific manner. The different inhibitory effects on distinct purinoceptor functions may be related to the anti-inflammatory property of PGE₂.

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In this open, prospective study we assessed the prevalence of antiplatelet resistance among patients subjected to intracoronary stent implantation. In patients treated with aspirin + thienopyridine (N = 32), platelet reactivity index (PRI) significantly decreased after 2 and 7 days of dual antiplatelet treatment in comparison with the same patients on aspirin monotherapy (P<0.001, both). After 7 days of aspirin + thienopyridine treatment, insufficient antiplatelet response was observed in 28% (9/32) of the patients. High interindividual variability in response to aspirin + thienopyridine treatment emphasizes the significance of thienopyridine resistance, while the influence of statins on such a treatment should be reassessed.

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This study examines the functional role of Na⁺/H⁺ exchanger (NHE) in Ca²⁺ influx mediated by human endothelin type A receptor (ET_AR) expressed in Chinese hamster ovary (CHO) cells. Endothelin-1 (ET-1) increased extracellular acidification rate (ECAR), which was abolished by 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an NHE inhibitor. EIPA and KB-R7943, a Na⁺/Ca²⁺ exchanger (NCX) inhibitor, inhibited ET-1-induced sustained increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i), and EIPA had no effect on [Ca²⁺]_i after KB-R7943 treatment. ET-1-elicited sustained [Ca²⁺]_i increase was suppressed by reducing extracellular Na⁺ concentration. These results suggest that possible coupling of NHE with NCX via Na⁺ transport is involved in ET_AR-mediated sustained [Ca²⁺]_i increase.

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Effects of an injectable emulsion of propofol and its emulsifier on singlet oxygen (¹O₂) were examined. ¹O₂ released from activated human neutrophils was detected by chemiluminescence, and chemically generated ¹O₂ was detected by electron paramagnetic resonance (EPR). Both the propofol emulsion and the emulsifier suppressed ¹O₂ release from neutrophils. However, the emulsifier did not quench chemically generated ¹O₂, while the propofol emulsion quenched it. These results indicated that the emulsifier did not scavenge ¹O₂ released from neutrophils but inhibited ¹O₂ generation. The suppressive effects of propofol emulsion on ¹O₂ release from neutrophils consist of ¹O₂ scavenging and inhibition of ¹O₂ generation.

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Acute pretreatment with a single i.v. bolus injection of simvastatin (1 mg/kg) significantly protects rat kidney injured by ischemia-reperfusion (I/R) (45 min + 6 h). We aimed to determine the optimal timing of such a pretreatment. The effects of both injections of simvastatin before ischemia and reperfusion were similar regarding total histological score. However, simvastatin injected 30 min before ischemia was 30% – 75% more effective in reduction of serum creatinine levels and interstitial edema score, while its injections 5 and 30 min before reperfusion were 25% – 60% more effective in reduction of tubular necrosis score and fractional excretion of Na⁺. However, the observed differences do not seem to offer significant advantage in clinical settings.

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