Neural anti-inflammatory action mediated by two types of acetylcholine receptors in the small intestine.

Gastrointestinal prokinetic agents function as serotonin-4 receptor (5-HT4R) agonists to activate myenteric plexus neurons to release acetylcholine (ACh), which then induce anti-inflammatory action. Details of this pathway, however, remain unknown. The aim of this study is to clarify the anti-inflammatory mechanism underlying the 5-HT4R agonist, mosapride citrate (MOS)-induced anti-inflammatory action on postoperative ileus (POI). POI models were generated from wild-type C57BL6/J (WT), 5-HT4R knock-out (S4R KO), α7 nicotinic AChR KO (α7 R KO), and M2 muscarinic ACh receptor KO (M2R KO) mice. MOS attenuated leukocyte infiltration in WT. MOS-induced anti-inflammatory action was completely abolished in both S4R KO and S4R KO mice upon wild-type bone marrow transplantation. MOS-induced anti-inflammatory action against macrophage infiltration, but not neutrophil infiltration, was attenuated in α7 R KO mice. Selective α7nAChR agonists (PNU-282987 and AR-R17779) also inhibited only macrophage infiltration in POI. MOS-mediated inhibition of neutrophil infiltration was diminished by atropine, M2AChR antagonist, methoctramine, and in M2R KO mice. Stimulation with 5-HT4R inhibits leukocyte infiltration in POI, possibly through myenteric plexus activation. Released ACh inhibited macrophage and neutrophil infiltration likely by activation of α7nAChR on macrophages and M2AChR. Thus, macrophage and neutrophil recruitment into inflamed sites is regulated by different types of AChR in the small intestine.

Electrical velocimetry for noninvasive cardiac output and stroke volume variation measurements in dogs undergoing cardiovascular surgery.

OBJECTIVE: To compare electrical velocimetry (EV) noninvasive measures of cardiac output (CO) and stroke volume variation (SVV) in dogs undergoing cardiovascular surgery with those obtained with the conventional thermodilution technique using a pulmonary artery catheter. STUDY DESIGN: Prospective experimental trial. ANIMALS: Seven adult Beagle dogs with a median weight of 13.6 kg. METHODS: Simultaneous, coupled cardiac index (CI; CO indexed to body surface area) measurements by EV (CIEV) and the reference pulmonary artery catheter thermodilution method (CIPAC) were obtained in seven sevoflurane-anaesthetized, mechanically ventilated dogs undergoing experimental open-chest cardiovascular surgery for isolated right ventricular failure. Relationships between SVV or central venous pressure (CVP) and stroke volume (SV) were analysed to estimate fluid responsiveness. Haemodynamic data were recorded intraoperatively and before and after fluid challenge. RESULTS: Bland-Altman analysis of 332 matched sets of CI data revealed an overall bias and precision of - 0.22 ± 0.52 L minute-1 m-2 for CIEV and CIPAC (percentage error: 30.4%). Trend analysis showed a concordance of 88% for CIEV. SVV showed a significant positive correlation (r2 = 0.442, p < 0.0001) with SV changes to a volume loading of 200 mL, but CVP did not (r2 = 0.0002, p = 0.94). Better prediction of SV responsiveness (rise of SV index of ≥ 10%) was observed for SVV (0.74 ± 0.09; p = 0.014) with a significant area under the receiver operating characteristic curve in comparison with CVP (0.53 ± 0.98; p = 0.78), with a cut-off value of 14.5% (60% specificity and 83% sensitivity). CONCLUSIONS AND CLINICAL RELEVANCE: In dogs undergoing cardiovascular surgery, EV provided accurate CO measurements compared with CIPAC, although its trending ability was poor. Further, SVV by EV, but not CVP, reliably predicted fluid responsiveness during mechanical ventilation in dogs.

Acute cardiac support with intravenous milrinone promotes recovery from early brain injury in a murine model of severe subarachnoid haemorrhage.

Early brain injury/ischaemia (EBI) is a serious complication early after subarachnoid haemorrhage (SAH) that contributes to development of delayed cerebral ischaemia (DCI). This study aimed to determine the role of inotropic cardiac support using milrinone (MIL) on restoring acute cerebral hypoperfusion attributable to EBI and improving outcomes after experimental SAH. Forty-three male C57BL/6 mice were assigned to either sham surgery (SAH-sham), SAH induced by endovascular perforation plus postconditioning with 2% isoflurane (Control), or SAH plus isoflurane combined with MIL with and without hypoxia-inducible factor inhibitor (HIF-I) pretreatment. Cardiac output (CO) during intravenous MIL infusion (0.25-0.75 µg/kg/min) between 1.5 and 2.5 hours after SAH induction was monitored with Doppler echocardiography. Magnetic resonance imaging (MRI)-continuous arterial spin labelling was used for quantitative cerebral blood flow (CBF) measurements. Neurobehavioral function was assessed daily by neurological score and open field test. DCI was analyzed 3 days later by determining infarction on MRI. Mild reduction of cardiac output (CO) and global cerebral blood flow (CBF) depression were notable early after SAH. MIL increased CO in a dose-dependent manner (P<.001), which was accompanied by improved hypoperfusion, incidence of DCI and functional recovery than Control (P<.05). The neuroprotective effects afforded by MIL or Control were attenuated by hypoxia-inducible factor (HIF) inhibition (P<.05). These results suggest that MIL improves acute hypoperfusion by its inotropic effect, leading to neurobehavioral improvement in mice after severe SAH, in which HIF may be acting as a critical mediator.

Review of studies that have used knockout mice to assess normal function of prion protein under immunological or pathophysiological stress.

Deletion of cellular isoform of prion protein (PrP(C)) increases neuronal predisposition to damage by modulating apoptosis and the negative consequences of oxidative stress. In vivo studies have demonstrated that PrP(C)-deficient mice are more prone to seizure, depression, and induction of epilepsy and experience extensive cerebral damage following ischemic challenge or viral infection. In addition, adenovirus-mediated overexpression of PrP(C) reduces brain damage in rat models of cerebral ischemia. In experimental autoimmune encephalomyelitis, PrP(C)-deficient mice reportedly have a more aggressive disease onset and less clinical improvement during the chronic phase than wild-type mice mice. In mice given oral dextran sulfate, PrP(C) has a potential protective role against inflammatory bowel disease. PrP(C)-deficient mice demonstrate significantly greater increases in blood glucose concentrations after intraperitoneal injection of glucose than wild-type mice. Further in vivo challenges to PrP gene-deficient models and conditional knockout models with siRNA and in vivo administration of PrP-ligating agents may assist in refining knowledge of the lymphoid function of PrP(C) and predicting the effects of anti-PrP treatment on the immune system. Together, these findings indicate that PrP(C) may have multiple neuroprotective and anti-inflammatory roles, which explains why this protein is so widely expressed.

Toxic effects of T-2 toxin and deoxynivalenol on the mitochondrial electron transport system of cardiomyocytes in rats.

The in vitro effects of 2 representative mycotoxins, T-2 toxin and deoxynivalenol (DON), of trichothecene group on the electron transport system (ETS) of mitochondria in rat cardiomyocytes were investigated by measuring oxygen consumption rates (OCR). The ATP-linked OCR and the reserve capacity (RC) of the mitochondria ETS were quantified by a "mitochondria stress test" which was estimated by the OCR responses to oligomycin and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, with an extracellular flux analyzer. The basal OCR was significantly inhibited by the application of T-2 toxin at concentrations of 6 × 10⁻¹ to 6 × 10⁻5 µM and DON at concentrations of 0.78 to 100 µM for 24 hr. The threshold of cardiomyocyte toxicity was estimated to be between 6.0 × 10⁻6 and 6.0 × 10⁻5 µM for T-2 toxicity on both ATP-linked OCR and RC and between 0.39 and 0.78 µM on ATP-linked OCR or between 1.56 and 3.13 µM on RC for DON. The decrease in OCR of cardiomyocytes exposed to T-2 toxin with a concentration of 6.0 × 10⁻³ and 6.0 × 10⁻4 µM was significantly inhibited by antioxidants, catalase and vitamin C. In conclusion, the present study demonstrated, through the direct and real-time measurement of respiratory function in mitochondria, that a marked inhibition of mitochondrial ETS function in cardiomyocytes was induced by T-2 toxin and DON and that the mitochondrial dysfunction by T-2 toxin was largely associated with oxidative stress.

Enhanced gastric retention of solid resin beads as a marker for emetic potential of agents in rats.

Whereas nausea and emesis are burdensome side effects that lead to poor treatment compliance especially in chemotherapy, it is difficult to predict the emetic potential of agents in rats and mice because rodents do not vomit. We examined the effect of emetics on gastric retention and role of serotonin (5-hydroxytryptamine, 5-HT)3 receptor in chemotherapeutic-induced enhancement of gastric retention in rats. The gastric retention of solid material was determined using resin beads, which were suitable to beads made with metals or glasses in size, hardness and weight. Each rat was orally given distilled water (0.5 ml/rat) containing 40 resin beads via a plastic feeding tube. The stomach was removed at 1 hr post-dose and cut along the greater curvature under carbon dioxide anesthesia. Beads were given immediately after administration of the drugs except with cisplatin, when there was a 1 hr delay. Cancer chemotherapeutics including cisplatin(0.1-3 mg/kg i.v.) and doxorubicin(0.3-10 mg/kg i.v.) and a nauseant, copper sulfate(1-30 mg/kg p.o.) enhanced gastric retention of beads. Ondansetron, a 5-HT3 receptor antagonist, dose-dependently antagonized the enhanced gastric retention by cisplatin and doxorubicin. The copper sulfate-induced enhancement was also reversed by ondansetron. Our results suggest that 5-HT3 receptors mediate the cancer chemotherapeutic-enhanced gastric retention of solid material in rats. This implicates that the gastric retention of solid material is a useful marker to predict the potential of compounds to induce nausea and/or emesis in non-vomiting rodents.

Nicotine treatment reduces LPS-induced sickness responses in telemetry monitoring rats.

Cholinergic anti-inflammatory pathway (CAP) inhibits unrestrained inflammatory response in a variety of experimental models. Limited research has been done yet to examine the mechanisms of activating CAP on bio-behavioral changes such as heart rate (HR), blood pressure (BP), body temperature (BT), locomotor activity (LA), and autonomic nervous activity (ANA). We observed these parameters using telemetry to clarify pathophysiological mechanisms of CAP. Nicotine significantly attenuated LPS-induced changes in HR, BP, LA, and ANA. These changes were accompanied by significant inhibition of TNF-α and IL-1ß syntheses. However the LPS-induced physiological responses persisted much longer than the cytokines production. These results indicate that systemic nicotine treatment inhibits LPS-induced cytokines production and attenuates the associated physiological and behavioral sickness responses.

Abundant expression of KCNE1 in the left ventricle of the miniature pig.

Delayed rectifier potassium currents such as I (Kr) and I (Ks) play an important role in the repolarization phase of the action potential in cardiac myocytes. Electrophysiological studies have shown that the pig is a useful animal not only for clinical use as a good candidate for humans, but also for basic research in heart function or arrhythmia. However, no studies concerning the potassium channels on a molecular level have been done. To elucidate the expression level and distribution of delayed rectifier potassium channels in pigs, we quantitatively investigated the I (Kr) and I (Ks) channel subunits using the real-time polymerase chain reaction (PCR) method. The hearts from Clawn miniature pigs were separated into the apical and basal regions, and subsequently excised into transmural trisections within each of the left ventricular walls, epicardium, midcardium, and endocardium. After RNA extraction from these sites, real-time PCR was executed with reverse transcriptional products for quantitative analysis. The expression level of KCNE1 was significantly higher than those of KCNQ1, KCNH2, and KCNE2, which were comparable in all sites. Transmural heterogeneity of these potassium channel subunits was not detected on the mRNA level. These results indicate that KCNE1 is a dominant subunit on the post-transcriptional level in the miniature pig.

Functional and molecular characterizations of chloride channels in rat pleural mesothelial cells.

The purposes of the present study were to clarify whether some Cl(-) channels exist in rat pleural mesothelial cells, and to investigate functional and molecular characteristics of these channels. Electrophysiological recordings were performed at room temperature using the whole-cell configuration of the patch-clamp technique. We could observe outwardly rectifying Cl(-) currents in rat pleural mesothelial cells under isotonic conditions. These currents exhibited time-dependent inactivation at potential over +60 mV and were inhibited by NPPB. It suggests the presence of voltage-dependent Cl(-) channels. Moreover, we observed the currents activated under hypotonic conditions. Their biophysical and pharmacological properties exhibited as follows; moderate outward rectification of whole-cell currents; time-dependent inactivation at large positive potential; anion selectivity with a type-I Eisenman's permeability sequence (I(-)>Br(-)>Cl(-)>F(-)>glutamate(-)); inhibited by NPPB. These properties are consistent with volume-regulated chloride channels (VRCCs), even though molecular identity of VRCCs could not have been determined, the molecular expressions of mRNA of the Cl(-) channels ClC-2, ClC-3, pI(Cln), MDR1 were confirmed. The properties of VRCCs in the pleural mesothelial cells were consistent with those of ClC-3 channels, and different from those of ClC-2. Therefore, these results suggest that ClC-3 might contribute to the modulation of VRCCs in rat pleural mesothelial cells.

Effects of cacao liquor polyphenols on cardiovascular and autonomic nervous functions in hypercholesterolaemic rabbits.

Many epidemiological studies have shown that polyphenols can reduce the risk of mortality from cardiovascular diseases. This study tested the hypothesis that cacao liquor polyphenols have the properties to restore the cardiovascular and autonomic nervous function in an animal model of familial hypercholesterolaemia. Male Kurosawa and Kusanagi-hypercholesterolaemic rabbits were housed in individual cages in a room where a 12-hr light:dark cycle (lights-on at 8:00 and lights-off at 20:00) was maintained. At 3 months of age, they were divided into two groups (standard diet and cacao liquor polyphenol) and the animals received 100 g of the respective diets per day and were provided with tap water ad libitum. Heart rate and blood pressure were measured by a telemetry system. To clarify the autonomic nervous function, power spectral analysis of heart rate variability, baroreflex sensitivity and autonomic nervous tone were measured. After 6 months of dietary administration of cacao liquor polyphenols, heart rate and blood pressure were lowered but plasma lipid concentrations were unchanged. The area of atherosclerotic lesions in the aorta in the cacao liquor polyphenol group was significantly smaller than that in the standard diet group. The high-frequency power of heart rate variability in the rabbits in the standard diet group was significantly decreased with ageing, but that in the cacao liquor polyphenol group was not different between short-term and long-term treatment. Moreover, cacao liquor polyphenols preserved parasympathetic nervous tone, although that in the standard diet group was significantly decreased with ageing. We conclude that cacao liquor polyphenols may play an important role to protect cardiovascular and autonomic nervous functions.

c-Fos suppresses systemic inflammatory response to endotoxin.

We explored the role of the transcription factor c-Fos in lipopolysaccharide (LPS)-induced cytokine response using mice lacking c-Fos (Fos-/- mice). Compared with wild-type controls, Fos-/- macrophages and mice showed significantly enhanced production of tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-12 p40, but reduced production of the anti-inflammatory cytokine IL-10. Bandshift analysis revealed that LPS-induced NF-kappaB binding activity to a functional site in the TNF-alpha promoter was significantly higher in Fos-/- than in wild-type macrophages. Using telemetry, we monitored body temperature and heart rate after LPS injection and found that Fos-/- mice undergo more severe hypothermia and bradycardia than wild-type mice. Such shock responses in Fos-/- mice were significantly reversed by neutralizing TNF-alpha. These data reveal a novel in vivo role for c-Fos as an anti-inflammatory transcription factor acting through suppression of NF-kappaB activity.

Statin protects endothelial nitric oxide synthase activity in hypoxia-induced pulmonary hypertension.

OBJECTIVE: We investigated the effects of fluvastatin on hypoxia-induced (1 to 3 weeks, 10% O2) pulmonary hypertension with focus on endothelial nitric oxide synthase (eNOS) activity. METHODS AND RESULTS: Oral fluvastatin treatment (1 mg/kg daily) prevented the causing and progression of pulmonary hypertension as determined by the right ventricular pressure, right ventricular hypertrophy, and muscularization of pulmonary artery. We also revealed that fluvastatin treatments prevented the hypoxia-induced decrease in cGMP production in the rat lung and restored the endothelium-dependent relaxation in the pulmonary artery. We revealed that this beneficial effect was not dependent on the increase in eNOS mRNA or protein expression, but was dependent on the inhibition of the eNOS-tight coupling with caveolin-1, the eNOS dissociation from heat shock protein 90, and the decrease in eNOS Ser1177-phosphorylation induced by hypoxia. Furthermore, in a whole-mount immunostaining the hypoxia-induced eNOS protein condensation with caveolin-1 of pulmonary endothelial cells was restored by the fluvastatin-treatment. CONCLUSIONS: These results suggest that the fluvastatin exerts beneficial effects on chronic hypoxia-induced pulmonary hypertension by protecting against the eNOS activity at the post-transcriptional level.

Suppressive effects of cacao liquor polyphenols (CLP) on LDL oxidation and the development of atherosclerosis in Kurosawa and Kusanagi-hypercholesterolemic rabbits.

We investigated the properties of cacao liquor polyphenols (CLP), which have an antioxidative effect on low-density lipoprotein (LDL) and an anti-atherosclerotic effect in the spontaneous familial hypercholesterolemic model, the Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbit. After 6 months of dietary administration of CLP at 1% (w/w) to the KHC rabbits, a higher total cholesterol concentration was observed in the treatment group compared to the control group. However, no other effects were noted in lipid profiles in plasma or lipoproteins. The plasma concentration of thiobarbituric acid reactive substances (TBARS), which is a lipid-peroxidation index, was significantly decreased 1 month after the start of CLP administration compared to that of the control group. The antioxidative effect of CLP on LDL was observed from 2 to 4 months of administration. The area of atherosclerotic lesions in the aorta in the CLP group (32.01+/-1.58%) was significantly smaller than that in the control group (47.05+/-3.29%), and the tissue cholesterol and TBARS concentrations were lower in the CLP group than in the control group. The anti-atherosclerotic effect of CLP was confirmed both rheologically and histopathologically. An in vitro study using KHC rabbit-derived LDL revealed that CLP significantly prolonged the lag time of LDL oxidation that was induced by a lipophilic azo-radical initiator, 2,2'-azobis(4-methoxy)-2,4-dimethylvaleronitrile (V-70), or Cu(2+) from a low concentration of 0.1 microg/mL. The antioxidative effect of CLP was superior to those of the well-known antioxidative substances, vitamin C, vitamin E and probucol. Therefore, CLP suppressed the generation of atherosclerosis, and its antioxidative effect appeared to have an important role in its anti-atherosclerotic activity.

Suppressive effect of cocoa powder on atherosclerosis in Kurosawa and Kusanagi-hypercholesterolemic rabbits.

We investigated the suppressive effect of cocoa powder (cacao polyphenol content: 7.8%) on atherosclerosis in a spontaneous familial hypercholesterolemic model, Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbits. Six-month dietary administration of cocoa powder had no effects on body weight, hematology or blood chemistry parameters or a lipid profile in KHC rabbits. Antioxidative activity of low-density lipoprotein (LDL) was observed in the 2nd month and 3rd month of administration. Thiobarbituric acid reactive substances (TBARS), the marker of lipid peroxidation, in plasma were decreased in the cocoa powder treated group from the 2nd month of administration during the study period compared to that in the control group. The area of atherosclerotic lesions in th aorta was significantly smaller in the cocoa powder group (30.87%) than in the control (52.39%). Tissue cholesterol content also tended to decrease. Distensibility of the aortic wall was improved significantly in the cocoa powder treated group due to decreases in fatty streaks and intimal thickening compared to that in the control group. These results suggest that cocoa powder has suppressive effect on development of atherosclerotic lesions. We consider that antioxidative activity of polyphenols rich in cocoa powder may be a key factor for the anti-atherosclerotic effect.

Ca2+-activated K+ channel blockers induce PKC modulated oscillatory contractions in guinea pig trachea.

Mechanisms underlying the Ca2+-activated K+ channel (K(Ca)) blockers-induced oscillatory contractions were investigated in guinea pig tracheal smooth muscle. The mean oscillatory frequencies induced by charybdotoxin (ChTX; 100 nM) and iberiotoxin (IbTX; 100 nM) were 9.8+/-0.8 (counts/h) and 8.0+/-1.3 (counts/h), respectively. Apamin (1 microM ), a blocker of SK(Ca), induced no contraction in guinea pig trachea and did not affect ChTX-induced oscillatory contractions. In Ca2+ free solution, no ChTX-induced contraction was observed. Nifedipine (100 nM), a blocker of voltage-dependent Ca2+ channels, and SK&F 96365 (10 microM), a blocker of capacitative Ca2+ entry, completely abolished ChTX-induced oscillatory contractions. Ryanodine (1 microM) decreased the amplitude, but increased the frequency of the oscillatory contractions. Thapsigargin (1 microM) changed contractions from the oscillatory type to the sustained type. Moreover, the protein kinase C (PKC) inhibitor, bisindolylamaleimide I (1 microM), decreased the amplitude and frequency, but PKC activator, phorbol 12-myristate 13-acetate (1 microM), increased the frequency of oscillatory contractions. These results suggest that K(Ca) inhibitors-induced oscillatory contractions are initiated by Ca2+ influx through L-type voltage-dependent Ca2+ channels. The ryanodine-sensitive calcium release channels in the sarcoplasmic reticulum may play an important role in maintaining the oscillatory contractions. Moreover, PKC activity modulates these oscillatory contractions.