Methyl Ester HCl Research Articles

Veno-Arterial Extracorporeal Membrane Oxygenation (ECMO) Impairs Bradykinin-Induced Relaxation in Neonatal Porcine Coronary Arteries.

Extracorporeal membrane oxygenation (ECMO) is a lifesaving support for respiratory and cardiovascular failure. However, ECMO induces a systemic inflammatory response syndrome that can lead to various complications, including endothelial dysfunction in the cerebral circulation. We aimed to investigate whether ECMO-associated endothelial dysfunction also affected coronary circulation. Ten-day-old piglets were randomized to undergo either 8 h of veno-arterial ECMO (n = 5) or no treatment (Control, n = 5). Hearts were harvested and coronary arteries were dissected and mounted as 3 mm rings in organ baths for isometric force measurement. Following precontraction with the thromboxane prostanoid (TP) receptor agonist U46619, concentration–response curves to the endothelium-dependent vasodilator bradykinin (BK) and the nitric oxide (NO) donor (endothelium-independent vasodilator) sodium nitroprusside (SNP) were performed. Relaxation to BK was studied in the absence or presence of the NO synthase inhibitor Nω-nitro-L-arginine methyl ester HCl (L-NAME). U46619-induced contraction and SNP-induced relaxation were similar in control and ECMO coronary arteries. However, BK-induced relaxation was significantly impaired in the ECMO group (30.4 ± 2.2% vs. 59.2 ± 2.1%; p < 0.0001). When L-NAME was present, no differences in BK-mediated relaxation were observed between the control and ECMO groups. Taken together, our data suggest that ECMO exposure impairs endothelium-derived NO-mediated coronary relaxation. However, there is a NO-independent component in BK-induced relaxation that remains unaffected by ECMO. In addition, the smooth muscle cell response to exogenous NO is not altered by ECMO exposure.

Aldol Reactions of Conformationally Stable Axially Chiral Thiohydantoin Derivatives.

Two novel axially chiral ortho-trifluoromethylphenyl thiohydantoin derivatives have been prepared atroposelectively from the reaction of R and S alanine methyl ester HCl salts with ortho-trifluoromethylphenyl isothiocyanate in the presence of triethyl amine. It was found that after purification of the crude product by simple recrystallization, the R amino acid esters yielded thiohydantoins having solely M axial chirality whereas the S ones returned the P isomers only. This result prompted us to perform sterically controlled aldol reactions on M and P thiohydantoin atropisomers. It was found that during the aldol reaction of 3-o-trifluoromethyl-5-methylthiohydantoins, the o-trifluoromethyl group of the M isomers efficiently shielded the Si face of the intermediate and in this way, enabled the selective formation of only the R configured aldol products at C5 of the heterocyclic ring. The P thiohydantoins, on the other hand, yielded only the S C5 configured aldol products as a result of the Re face shielding of the ortho-trifluoromethyl group of intermediate enolates. A noteworthy face selectivity of the benzaldehyde molecule was not observed (anti/syn only 3/2) during the aldolization of trifluoromethylphenyl derivatives of thiohydantoins. Aldol reactions were also done using the previously synthesized axially chiral thiohydantoins with ortho-Cl, Br, and I phenyl substituents which had predominantly P conformations (P/M ratios > 95%), and the stereochemical outcomes were compared with those of the ortho-trifluoromethyl substituted ones. 80–90% face selectivity of the benzaldehyde molecule was observed for the axially chiral o-halophenyl substituted thiohydantoins. The syntheses done with axially chiral 3-ortho-trifluoromethylphenyl- and 3-ortho-iodophenyl-5-methyl thiohydantoins enabled stereoselective formation of quaternized chiral carbon centers at C5 of the thiohydantoin ring.

S-Ketamine Mediates Its Acute and Sustained Antidepressant-Like Activity through a 5-HT1B Receptor Dependent Mechanism in a Genetic Rat Model of Depression.

Rationale: The mechanisms responsible for the unique antidepressant properties of ketamine have only been partly resolved. Recent preclinical reports implicate the neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] in the antidepressant-like response of ketamine, and modulation of 5-HT1B receptors has been hypothesized to attain an important role.Objectives: To evaluate the role of endogenous stimulation of 5-HT1B heteroreceptors in the antidepressant-like activity of S-ketamine.Method: Flinders sensitive line (FSL) rats, a genetic model of depression, were depleted of endogenous 5-HT by 4-chloro-DL-phenylalanine methyl ester HCl administration (pCPA; 86 mg/kg/day for 3 days). In pCPA-pretreated and control FSL rats, the acute and sustained effects of a single dose of S-ketamine (15 mg/kg) and the selective 5-HT1B receptor agonist CP94253 (1–6 mg/kg) alone and in combination with S-ketamine were studied in the forced swim test (FST), a commonly used assay that detects antidepressant activity.Results: pCPA pretreatment decreased cortical 5-HT levels to ∼6% but did not affect the baseline behavioral phenotype of FSL rats. S-ketamine demonstrated acute and sustained antidepressant-like activity, both of which were abolished by 5-HT depletion. Combining S-ketamine with a sub-effective dose of CP94253 (1 mg/kg) rescued S-ketamine’s acute and sustained antidepressant-like effects, when CP94253 was administered 2 h prior to the FST. Co-administration of S-ketamine and CP94253 did not affect the plasma level of either compound, suggesting that the observed behavioral interaction could not be ascribed to a kinetic drug-drug interaction.Conclusion: 5-HT1B receptor activation during testing appears to be critical for S-ketamine’s antidepressant-like potentials in this model.

L-Citrulline acts as potential hypothermic agent to afford thermotolerance in chicks

Recently we demonstrated that L-citrulline (L-Cit) causes hypothermia in chicks. However, the question of how L-Cit mediates hypothermia remained elusive. Thus, the objective of this study was to examine some possible factors in the process of L-Cit-mediated hypothermia and to confirm whether L-Cit can also afford thermotolerance in young chicks. Chicks were subjected to oral administration of L-Cit along with intraperitoneal injection of a nitric oxide synthase (NOS) inhibitor, NG-nitro-l-arginine methyl ester HCl (L-NAME), to examine the involvement of NO in the process of hypothermia. Food intake and plasma metabolites were also analyzed after oral administration of L-Cit in chicks. To examine thermotolerance, chicks were orally administered with a single dose of L-Cit (15mmol/10ml/kg body weight) or the same dose twice within a short interval of 1h (dual oral administration) before the exposure to high ambient temperature (35 ± 1°C) for 180min. Although the rectal temperature was reduced following administration of L-Cit, L-NAME caused a greater reduction. L-NAME reduced total NO2 and NO3 (NOx) in plasma, which confirmed its inhibitory effect on NO. A single oral administration of L-Cit mediated a persistent state of hypothermia for the 300min of the study without affecting food intake. It was further found that plasma glucose was significantly lower in L-Cit-treated chicks. Dual oral administration of L-Cit, but not a single oral administration, afforded thermotolerance without a significant change in plasma NOx in chicks. In conclusion, our results suggest that L-Cit-mediated hypothermia and thermotolerance may not be involved in NO production. L-Cit-mediated thermotolerance further suggests that L-Cit may serve as an important nutritional supplement that could help in coping with summer heat.

Differential interaction with the serotonin system by S-ketamine, vortioxetine, and fluoxetine in a genetic rat model of depression.

The mechanisms mediating ketamine's antidepressant effect have only been partly resolved. Recent preclinical reports implicate serotonin (5-hydroxytryptamine; 5-HT) in the antidepressant-like action of ketamine. Vortioxetine is a multimodal-acting antidepressant that is hypothesized to exert its therapeutic activity through 5-HT reuptake inhibition and modulation of several 5-HT receptors. The objective of this study was to evaluate the therapeutic-like profiles of S-ketamine, vortioxetine, and the serotonin reuptake inhibitor fluoxetine in response to manipulation of 5-HT tone. Flinders Sensitive Line (FSL) rats, a genetic model of depression, were depleted of 5-HT by repeated administration of 4-chloro-DL-phenylalanine methyl ester HCl (pCPA). Using pCPA-pretreated and control FSL rats, we investigated the acute and sustained effects of S-ketamine (15mg/kg), fluoxetine (10mg/kg), or vortioxetine (10mg/kg) on recognition memory and depression-like behavior in the object recognition task (ORT) and forced swim test (FST), respectively. The behavioral phenotype of FSL rats was unaffected by 5-HT depletion. Vortioxetine, but not fluoxetine or S-ketamine, acutely ameliorated the memory deficits of FSL rats in the ORT irrespective of 5-HT tone. No sustained effects were observed in the ORT. In the FST, all three drugs demonstrated acute antidepressant-like activity but only S-ketamine had sustained effects. Unlike vortioxetine, the antidepressant-like responses of fluoxetine and S-ketamine were abolished by 5-HT depletion. These observations suggest that the acute and sustained antidepressant-like effects of S-ketamine depend on endogenous stimulation of 5-HT receptors. In contrast, the acute therapeutic-like effects of vortioxetine on memory and depression-like behavior may be mediated by direct activity at 5-HT receptors.

Arginine affects appetite via nitric oxide in ducks

The objective of the study was to investigate the mechanism by which arginine regulates feed intake in Pekin ducks. In experiment 1, one hundred forty-four 1-d-old male Pekin ducks were randomly allotted to 3 dietary treatments with 6 replicate pens of 8 birds per pen. Birds in each group were fed a corn-corn gluten meal diet containing 0.65, 0.95, and 1.45% arginine. Ducks fed the diet containing 0.65% arginine had lower feed intake and plasma nitric oxide level (P < 0.05) than the other 2 groups. In experiment 2, twenty 11-d-old ducks were allotted to 1 of 2 treatments. After 2 h fasting, birds in the 2 groups were intraperitoneally administrated saline andl-NG-nitro-arginine methyl ester HCl (L-NAME) for 3 d, respectively. Feed intake (P < 0.07) and plasma nitric oxide concentration (P < 0.05) 2 h postinjection in the L-NAME administered group were lower than those of the control group. In conclusion, the study implied that arginine modifies feeding behavior possibly through controlling endogenous synthesis of nitric oxide in Pekin ducks.

키랄 컬럼을 사용한 아미노산 에스테르의 니트로벤조옥사디아졸 유도체의 광학분리

A new convenient derivatization method of α-amino acid esters as nitrobenzoxadiazole (NBD) derivatives for chiral resolution was introduced and the enantiomer separation of α-amino acid esters as NBD derivatives was performed by normal HPLC using chiral columns based on polysaccharide derivatives. The NBD derivatives were readily prepared by stirring NBD-Cl and α-amino acid methyl ester HCl with NaHCO₃ in ethanol. The performance of Chiralpak IA was superior to the other chiral stationary phases for enantiomer resolution of NBD derivatives of several α-amino acid methyl esters. Owing to fluorescence detection as well as strong UV absorption, it is expected that the convenient analytical method developed in this study will be very useful for enantiomer separation of α-amino acid esters as NBD derivatives on polysaccharide-derived chiral columns.

Vortioxetine dose-dependently reverses 5-HT depletion-induced deficits in spatial working and object recognition memory: A potential role for 5-HT1A receptor agonism and 5-HT3 receptor antagonism

We previously reported that the investigational multimodal antidepressant, vortioxetine, reversed 5-HT depletion-induced memory deficits while escitalopram and duloxetine did not. The present report studied the effects of vortioxetine and the potential impact of its 5-HT1A receptor agonist and 5-HT3 receptor antagonist properties on 5-HT depletion-induced memory deficits. Recognition and spatial working memory were assessed in the object recognition (OR) and Y-maze spontaneous alternation (SA) tests, respectively. 5-HT depletion was induced in female Long-Evans rats using 4-cholro-DL-phenylalanine methyl ester HCl (PCPA) and receptor occupancies were determined by ex vivo autoradiography. Rats were acutely dosed with vortioxetine, ondansetron (5-HT3 receptor antagonist) or flesinoxan (5-HT1A receptor agonist). The effects of chronic vortioxetine administration on 5-HT depletion-induced memory deficits were also assessed. 5-HT depletion reliably impaired memory performance in both the tests. Vortioxetine reversed PCPA-induced memory deficits dose-dependently with a minimal effective dose (MED) ≤0.1mg/kg (∼80% 5-HT3 receptor occupancy; OR) and ≤3.0mg/kg (5-HT1A, 5-HT1B, 5-HT3 receptor occupancy: ∼15%, 60%, 95%) in SA. Ondansetron exhibited a MED ≤3.0μg/kg (∼25% 5-HT3 receptor occupancy; OR), but was inactive in the SA test. Flesinoxan had a MED ≤1.0mg/kg (∼25% 5-HT1A receptor occupancy; SA); only 1.0mg/kg ameliorated deficits in the NOR. Chronic p.o. vortioxetine administration significantly improved memory performance in OR and occupied 95%, 66%, and 9.5% of 5-HT3, 5-HT1B, and 5-HT1A receptors, respectively. Vortioxetine′s effects on SA performance may involve 5-HT1A receptor agonism, but not 5-HT3 receptor antagonism, whereas the effects on OR performance may involve 5-HT3 receptor antagonism and 5-HT1A receptor agonism.

Functional significance of synthesis of noradrenaline in adrenergic nerves of rat salivary glands.

Abstract The secretory response and the noradrenaline content of the submaxillary gland due to continuous electrical stimulation of the cervical sympathetic trunk with 5 Hz (5 V, 2 ms) were determined in five differently treated groups of rats. Stimulation during 5 h in untreated control rats reduced the noradrenaline content to about 40% while the secretory response remained unchanged. Another group received DL-α-methyl-m-tyrosine 400 mg kg−1 i.p. 26 and 18 h before the stimulation and 200 mg kg−1 2 h before. This treatment displaced 93% of the noradrenaline content of the glands, and the secretory response stabilized after an initial decrease, at a level of about two-thirds of the normal response, possibly indicating a reduced rate of transmitter synthesis compared to normal rats. Thus, a nerve with greatly reduced amounts of active transmitter in the store can satisfy rather high demands of transmitter release for long time periods, provided that the synthesis continues. A third group of rats received DL-α-methyl-m-tyrosine in the same doses as above but 50, 42 and 26 h before the experiments, respectively. After this treatment, the noradrenaline level was reduced to about 35% of normal, yet the secretory response was even higher than normal. Probably the decreased transmitter synthesis supposed to occur following treatment with α-methyl-m-tyrosine has been reversed at this time interval into an increased synthesis rate. To a fourth group of rats 500 mg kg−1 DL-α-methyl-p-tyrosine methylester HCl (H 44/68) was injected i.p. 15 min before stimulation, resulting in a virtually complete inhibition of the tyrosine hydroxylase. Three h of stimulation reduced noradrenaline to 15%, and the secretory response disappeared. When treatment with both drugs was given, a very rapid disappearance of an initially normal response was seen. In conclusion, synthesis of new transmitter appears to be essential for a normal function of the nerve.

Locomotor effects of nitrous oxide in mice: requirement of newly-synthesized and main intraneuronal storage pools of dopamine.

Nitrous oxide increases locomotor activity in mice. Other locomotor stimulants are thought to act via central dopaminergic mechanisms and can be divided into two groups as determined by their antagonism by tyrosine hydroxylase inhibitors or by reserpine pretreatment. The purpose of the present study was to determine if nitrous oxide fits one or the other of the groups. Mice were acclimatized for 1 h to exposure chambers (4 L filtration flasks), in air, delivered at 4 L min-1 and then exposed to N2O:O2 (50:50), also delivered at 4 L min-1. Locomotor activity was evaluated at 10 min intervals throughout the experiment. Racemic alpha-methyltyrosine methyl ester HCl (200 mg kg-1), administered at the beginning of acclimatization, almost totally eliminated the nitrous oxide effect but not that of methylphenidate HCl (20 mg kg-1). Reserpine pretreatment (5 mg kg-1, 18 h) totally eliminated the nitrous oxide effect but not that of amphetamine (5 mg kg-1). The results suggest that nitrous oxide requires both the newly synthesized and the main storage pools of dopamine and do not allow assignment of the agent, specifically, to either of the groups.

The Effect of an Acute Maternal Stress on β-Endorphin and Growth Hormone Releasing Factor in the Rat Fetus

Pregnant rats were injected with saline or L-tyrosine methylester HCl (200 mg/kg) and subjected to an acute forced immobilization stress on day 20 of gestation. At 10, 30, 60, and 120 minutes after the onset of stress, their fetuses were dissected out, and the contents of hypothalamic and pituitary immunoreactive beta-endorphin (IR-beta-EP) and hypothalamic immunoreactive growth hormone-releasing factor (IR-GRF) were determined by specific radioimmunoassays. The maternal stress arose a significant decrease of hypothalamic IR-beta-EP at 30 minutes, while pituitary IR-beta-EP slightly elevated at 30 minutes, then declined at 60 minutes. Hypothalamic IR-GRF showed a gradual increase during the maternal stress. Tyrosine supplementation tended to attenuate stress-induced changes in hypothalamic and pituitary IR-beta-EP, but the response of hypothalamic IR-GRF was less modified by tyrosine. These results showed the functional changes in fetal central beta-EP and GRF under maternal stress in the late gestational life, and suggested that catecholaminergic regulations participate, at least in part, in the fetal neuroendocrine response to maternal stress.

Intracerebroventricular injection of l-arginine induces sedative and hypnotic effects under an acute stress in neonatal chicks

L-arginine participates in many important and diverse biochemical reactions associated with the normal physiology of the organism. In the present study, we investigated the effect of central administration of L-arginine on the stress response and its mechanism in neonatal chicks. Intracerebroventricular (i.c.v.) injection of L-arginine clearly attenuated the stress response in a dose-dependent manner, and induced sleep-like behavior during 10 min. To clarify the mechanism by which L-arginine induces sedative and hypnotic effects in chicks, we investigated the effects of nitric oxide (NO) synthase (NOS) inhibitors on L-arginine-induced sedative and hypnotic effects, and as well as the effects of a NO donor. L-Arginine-induced (1.9 micromol) sedative and hypnotic effects were attenuated by i.c.v. co-injection with a non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester HCl (400 nmol). In addition, the effects of L-arginine were slightly attenuated by the inactive isomer of the NOS inhibitor N(G)-nitro-D-arginine methyl ester HCl (400 nmol). The i.c.v. injection of 3-morpholinosylnomine hydrochloride, a spontaneous NO donor, had little effect on postures. The i.c.v. injection of L-arginine had no effect on NOx concentration at various brain sites. These results suggested that the contribution of NO generation via NOS may be low in the sedative and hypnotic actions of L-arginine. Therefore, L-arginine and/or its metabolites, excluding NO, may be necessary for these actions.

Heparan sulfates mediate pressure-induced increase in lung endothelial hydraulic conductivity via nitric oxide/reactive oxygen species

We investigated the nonlinear dynamics of the pressure vs. hydraulic conductivity (L(p)) relationship in lung microvascular endothelial cells and demonstrate that heparan sulfates, an important component of the endothelial glycocalyx, participate in pressure-sensitive mechanotransduction that results in barrier dysfunction. The pressure vs. L(p) relationship was complex, possessing both time- and pressure-dependent components. Pretreatment of lung capillary endothelial cells with heparanase III completely abolished the pressure-induced increase in L(p). This extends our (7) previous observation regarding heparan sulfates as mechanotransducers for shear stress. Inhibition of nitric oxide (NO) synthase with L-NAME (N(G)-nitro-L-arginine methyl ester HCl) and intracellular scavenging of reactive oxygen species (ROS) by TBAP [tetrakis-(4-benzoic acid) porphorin] significantly attenuated the pressure-induced L(p) response. Intracellular NO/ROS were visualized using the fluorescent dye, 2'7'-dichlorofluorescein diacetate (DCFA), and cells demonstrated a pressure-induced increase in intracellular fluorescence. Heparanase pretreatment significantly reduced the pressure-induced increase in intracellular fluorescence, suggesting that cell-surface heparan sulfates directly participate in mechanotransduction that results in NO/ROS production and increased permeability. This is the first report to demonstrate a role for heparan sulfates in pressure-mediated mechanotransduction and barrier regulation. These observations may have important clinical implications during conditions where pulmonary microvascular pressure is elevated.

Pharmacological characterisation of capsaicin-induced relaxations in human and porcine isolated arteries

Capsaicin, a pungent constituent from red chilli peppers, activates sensory nerve fibres via transient receptor potential vanilloid receptors type 1 (TRPV1) to release neuropeptides like calcitonin gene-related peptide (CGRP) and substance P. Capsaicin-sensitive nerves are widely distributed in human and porcine vasculature. In this study, we examined the mechanism of capsaicin-induced relaxations, with special emphasis on the role of CGRP, using various pharmacological tools. Segments of human and porcine proximal and distal coronary arteries, as well as cranial arteries, were mounted in organ baths. Concentration response curves to capsaicin were constructed in the absence or presence of the CGRP receptor antagonist olcegepant (BIBN4096BS, 1 μM), the neurokinin NK1 receptor antagonist L-733060 (0.5 μM), the voltage-sensitive calcium channel blocker ruthenium red (100 μM), the TRPV1 receptor antagonist capsazepine (5 μM), the nitric oxide synthetase inhibitor Nω-nitro-l-arginine methyl ester HCl (l-NAME; 100 μM), the gap junction blocker 18α-glycyrrhetinic acid (10 μM), as well as the RhoA kinase inhibitor Y-27632 (1 μM). Further, we also used the K+ channel inhibitors 4-aminopyridine (1 mM), charybdotoxin (0.5 μM) + apamin (0.1 μM) and iberiotoxin (0.5 μM) + apamin (0.1 μM). The role of the endothelium was assessed by endothelial denudation in distal coronary artery segments. In distal coronary artery segments, we also measured levels of cyclic adenosine monophosphate (cAMP) after exposure to capsaicin, and in human segments, we also assessed the amount of CGRP released in the organ bath fluid after exposure to capsaicin. Capsaicin evoked concentration-dependent relaxant responses in precontracted arteries, but none of the above-mentioned inhibitors did affect these relaxations. There was no increase in the cAMP levels after exposure to capsaicin, unlike after (exogenously administered) α-CGRP. Interestingly, there were significant increases in CGRP levels after exposure to vehicle (ethanol) as well as capsaicin, although this did not induce relaxant responses. In conclusion, the capsaicin-induced relaxations of the human and porcine distal coronary arteries are not mediated by CGRP, NK1, NO, vanilloid receptors, voltage-sensitive calcium channels, K+ channels or cAMP-mediated mechanisms. Therefore, these relaxant responses to capsaicin are likely to be attributed to a non-specific, CGRP-independent mechanism.

A reevaluation of the effects of stimulation of the dorsal motor nucleus of the vagus on gastric motility in the rat

Our primary purpose was to characterize vagal pathways controlling gastric motility by microinjecting l-glutamate into the dorsal motor nucleus of the vagus (DMV) in the rat. An intragastric balloon was used to monitor motility. In 39 out of 43 experiments, microinjection of l-glutamate into different areas of the DMV rostral to calamus scriptorius (CS) resulted in vagally mediated excitatory effects on motility. We observed little evidence for inhibitory effects, even with intravenous atropine or with activation of gastric muscle muscarinic receptors by intravenous bethanechol. Inhibition of nitric oxide synthase with N(omega)-nitro-l-arginine methyl ester (l-NAME) HCl did not augment DMV-evoked excitatory effects on gastric motility. Microinjection of l-glutamate into the DMV caudal to CS produced vagally mediated gastric inhibition that was resistant to l-NAME. l-Glutamate microinjected into the medial subnucleus of the tractus solitarius (mNTS) also produced vagally mediated inhibition of gastric motility. Motility responses evoked from the DMV were always blocked by ipsilateral vagotomy, while responses evoked from the mNTS required bilateral vagotomy to be blocked. Microinjection of oxytocin into the DMV inhibited gastric motility, but the effect was never blocked by ipsilateral vagotomy, suggesting that the effect may have been due to diffusion of oxytocin to the mNTS. Microinjection of substance P and N-methyl-d-aspartate into the DMV also produced inhibitory effects attributable to excitation of nearby mNTS neurons. Our results do not support previous studies indicating parallel vagal excitatory and inhibitory pathways originating in the DMV rostral to CS. Our results do support previous findings of vagal inhibitory pathways originating in the DMV caudal to CS.

Satellite cell activation in stretched skeletal muscle and the role of nitric oxide and hepatocyte growth factor

In the present study, we examined the roles of hepatocyte growth factor (HGF) and nitric oxide (NO) in the activation of satellite cells in passively stretched rat skeletal muscle. A hindlimb suspension model was developed in which the vastus, adductor, and gracilis muscles were subjected to stretch for 1 h. Satellite cells were activated by stretch determined on the basis of 5-bromo-2'-deoxyuridine (BrdU) incorporation in vivo. Extracts from stretched muscles stimulated BrdU incorporation in freshly isolated control rat satellite cells in a concentration-dependent manner. Extracts from stretched muscles contained the active form of HGF, and the satellite cell-activating activity could be neutralized by incubation with anti-HGF antibody. The involvement of NO was investigated by administering nitro-L-arginine methyl ester (L-NAME) or the inactive enantiomer N(G)-nitro-D-arginine methyl ester HCl (D-NAME) before stretch treatment. In vivo activation of satellite cells in stretched muscle was not inhibited by D-NAME but was inhibited by L-NAME. The activity of stretched muscle extract was abolished by L-NAME treatment but could be restored by the addition of HGF, indicating that the extract was not inhibitory. Finally, NO synthase activity in stretched and unstretched muscles was assayed in muscle extracts immediately after 2-h stretch treatment and was found to be elevated in stretched muscle but not in stretched muscle from L-NAME-treated rats. The results of these experiments demonstrate that stretching muscle liberates HGF in a NO-dependent manner, which can activate satellite cells.

AT2 receptor‐mediated vasodilation in the mouse heart depends on AT1A receptor activation

Angiotensin (Ang) II type 2 (AT(2)) receptors are believed to counteract Ang II type 1 (AT(1)) receptor-mediated effects. Here, we investigated AT(2) receptor-mediated effects on coronary and cardiac contractility in C57BL/6 mice. Hearts were perfused according to Langendorff. Baseline coronary flow (CF) and left ventricular systolic pressure (LVSP) were 2.7 +/- 0.1 ml min(-1) and 111 +/- 3 mmHg (n = 50), respectively. Ang II (n = 14) concentration dependently decreased CF and LVSP, by maximally 41 +/- 4 and 25 +/- 3%, respectively (pEC(50)s 7.41 +/- 0.12 and 7.65 +/- 0.12). The AT(1) receptor antagonist irbesartan (n = 4) abolished all Ang II-induced changes, whereas the AT(2) receptor antagonist PD123319 (n = 6) enhanced (P < 0.05) the effect of Ang II on CF (to 59 +/- 1%) and LVSP (to 44 +/- 2%), without altering its potency. A similar enhancement was observed in the presence of nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine methyl ester HCl (L-NAME; n = 4). On top of L-NAME, PD123319 no longer affected the response to Ang II (n = 4). The AT(2) receptor agonist CGP42112A (n = 4) did not affect CF or LVSP, nor did CGP42112A (n = 4) alter the constrictor response to the alpha(1)-adrenoceptor agonist phenylephrine. Furthermore, Ang II exerted no effects in hearts of AT(1A)(-/-) mice (n = 5), whereas its effects in hearts of AT(1A)(+/+) wild-type control mice (n = 7) were indistinguishable from those in hearts of C57BL/6 mice. In conclusion, Ang II exerts opposite effects on coronary and cardiac contractility in the mouse heart via activation of AT(1A) and AT(2) receptors. AT(2) receptor-mediated effects depend on NO and occur only in conjunction with AT(1A) receptor activation.

Decrease in rat internal anal pressure with the use of a topical ointment containing a killed E. coli culture suspension

The present study aimed to clarify the mechanisms of a topical ointment containing an Escherichia coli culture suspension and hydrocortisone (Posterisan forte, BCS+HC) in lowering internal anal pressure in conscious rats. Internal anal pressure was measured using a water-filled balloon system for consecutive 10-min periods. The changes in pressure were evaluated by the number of peaks above 20 mmH2O between 1 and 8 min of recording. Topical intra-anal application of BCS+HC ointment (160 mg/kg) significantly decreased the internal anal pressure at 3 h after the application. Thereafter, this effect reached a maximum decrease at 4 h and lasted until 6 h. BCS+HC ointment (40, 80, and 160 mg/kg) lowered the internal anal pressure at 4-5 h in a dose-dependent manner. The maximum decrease ratios of the ointment and corresponding hydrocortisone-free ointment (Posterisan, BCS) were 32.6+/-12.7 and 25.7+/-9.0%, respectively, revealing significant pressure-lowering effects compared with a placebo (P<0.05). In contrast, the same ointment containing hydrocortisone alone and other ointments containing steroids or local anesthetics had no effects. Treatment with 1 mg/kg NG-nitro-L-arginine methyl ester HCl (L-NAME), a non-selective nitric oxide synthase inhibitor, significantly suppressed the effect of BCS+HC ointment (160 mg/kg) in lowering the internal anal pressure. Furthermore, BCS+HC ointment (160 mg/kg) significantly lowered capsaicin-induced high internal anal pressure compared to a placebo. These findings suggest that BCS+HC and BCS ointments containing an E. coli culture suspension significantly lowered the internal anal pressure due to endogenous nitric oxide production in conscious rats.

Nitric oxide involves in carnosine-induced hyperactivity in chicks

Carnosine has been characterized as a putative neurotransmitter and implicated as having a possible role in neuron–glia cell interactions. We previously confirmed that central administration of carnosine induced hyperactivity in chicks. In the present study, we investigated the effects of nitric oxide (NO) synthase (NOS) inhibitors on carnosine-induced hyperactivity in chicks. Carnosine-induced (3.2 μmol) hyperactivity was attenuated by intracerebroventricular (i.c.v.) co-administration with a non-selective NOS inhibitor N G-nitro- l-arginine methyl ester HCl (200 and 400 nmol) in a dose-dependent manner, while the hyperactivity was not attenuated by the inactive isomer of the NOS inhibitor N G-nitro- d-arginine methyl ester HCl (400 nmol). The i.c.v. injection of a selective inhibitor of inducible NOS (iNOS) l- N 6-(1-iminoethyl) lysine HCl (400 nmol) did not affect carnosine-induced hyperactivity. These results suggest that carnosine-induced hyperactivity may be linked to the constitutive NOS (cNOS), rather than iNOS, in the brain. Central carnosine may regulate brain function and/or behaviors by NO generation via cNOS in chicks.

INOS initiates and sustains metabolic arrest in hypoxic lung adenocarcinoma cells: mechanism of cell survival in solid tumor core

Nitric oxide (NO) modulates cellular metabolism by competitively inhibiting the reduction of O2 at respiratory complex IV. The aim of this study was to determine whether this effect could enhance cell survival in the hypoxic solid tumor core by inducing a state of metabolic arrest in cancer cells. Mitochondria from human alveolar type II-like adenocarcinoma (A549) cells showed a fourfold increase in NO-sensitive 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) fluorescence and sixfold increase in Ca2+-insensitive NO synthase (NOS) activity during equilibration from Po2s of 100-->23 mmHg, which was abolished by N(omega)-nitro-L-arginine methyl ester-HCl (L-NAME) and the inducible NOS (iNOS) inhibitor, N6-(1-iminoethyl)-L-lysine dihydrochloride (L-NIL). Similarly, cytosolic and compartmented DAF-FM fluorescence increased in intact cells during a transition between ambient Po2 and 23 mmHg and was abolished by transfection with iNOS antisense oligonucleotides (AS-ODN). In parallel, mitochondrial membrane potential (deltapsi(m)), measured using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), decreased to a lower steady state in hypoxia without change in glycolytic rate, adenylate energy charge, or cell viability. However, L-NAME or iNOS AS-ODN treatment maintained deltapsi(m) at normoxic levels irrespective of hypoxia and caused a marked activation of glycolysis, destabilization energy charge, and cell death. Comparison with other cancer-derived (H441) or native tissue-derived (human bronchial epithelial; alveolar type II) lung epithelial cells revealed that the hypoxic suppression of deltapsi(m) was common to cells that expressed iNOS. The controlled dissipation of deltapsi(m), absence of an overt glycolytic activation, and conservation of viability suggest that A549 cells enter a state of metabolic suppression in hypoxia, which inherently depends on the activation of iNOS as Po2 falls.