Hayato Matsuyama

Thymoquinone suppresses expression of inducible nitric oxide synthase in rat macrophages

The objective of the present study was to determine the immunomodulatory role of thymoquinone (TQ) regarding its effect on the production of nitric oxide (NO) by rat peritoneal macrophages. Under certain conditions, macrophagesand certain other cells can produce high concentrations of NO from its precursor L-arginine via inducible nitricoxide synthase (iNOS)pathway. TQ has been established as the major component of the oil extracted from Nigella saliva plant seeds, which is being used frequently in herbal medicine. TQ (IC50 1.4-2.76 microM) dose- and time-dependently reduced nitrite production, a parameter for NO synthesis, in supematants of lipopolysaccharide (LPS)-stimulated (5 microg/ml) macrophages without affecting the cell viability. The protein level of iNOS in peritoneal macrophages was also decreased by TQ in a concentration-dependent manner. In addition, TQ inhibited the increase in iNOS mRNA expression induced by LPS indicated by reverse transcription-polymerase chain reaction (RT-PCR). These inhibitory effects of TQ were confirmed by immunofluorescence staining of iNOS in macrophages which showed decreased immunoreactivity for iNOS after treatment with TQ if compared with the control LPS-stimulated cells. These results suggest that TQ suppresses the production of NO by macrophages; an effect which may be useful in ameliorating the inflammatory and autoimmune conditions.

Tachykinins and their functions in the gastrointestinal tract

Abstract.In the gastrointestinal tract, tachykinins are peptide neurotransmitters in nerve circuits that regulate intestinal motility, secretion, and vascular functions. Tachykinins also contribute to transmission from spinal afferents that innervate the gastrointestinal tract and have roles in the responses of the intestine to inflammation. Tachykinins coexist with acetylcholine, the primary transmitter of excitatory neurons innervating the muscle, and act as a co-neurotransmitter of excitatory neurons. Excitatory transmission is mediated through NK1 receptors (primarily on interstitial cells of Cajal) and NK2 receptors on the muscle. Tachykinins participate in slow excitatory transmission at neuro-neuronal synapses, through NK1 and NK3 receptors, in both ascending and descending pathways affecting motility. Activation of receptors (NK1 and NK2) on the epithelium causes fluid secretion. Tachykinin receptors on immune cells are activated during inflammation of the gut. Finally, tachykinins are released from the central terminals of gastrointestinal afferent neurons in the spinal cord, particularly in nociceptive pathways.

M2 and M3 muscarinic receptor-mediated contractions in longitudinal smooth muscle of the ileum studied with receptor knockout mice

Isometric contractile responses to carbachol were studied in ileal longitudinal smooth muscle strips from wild‐type mice and mice genetically lacking M2 or M3 muscarinic receptors, in order to characterize the mechanisms involved in M2 and M3 receptor‐mediated contractile responses. Single applications of carbachol (0.1–100 μM) produced concentration‐dependent contractions in preparations from M2‐knockout (KO) and M3‐KO mice, mediated via M3 and M2 receptors, respectively, as judged by the sensitivity of contractile responses to blockade by the M2‐preferring antagonist methoctramine (300 nM) or the M3‐preferring antagonist 4‐DAMP (30 nM). The M2‐mediated contractions were mimicked in shape by submaximal stimulation with high K+ concentrations (up to 35 mM), almost abolished by voltage‐dependent Ca2+ channel (VDCC) antagonists or depolarization with 140 mM K+ medium, and greatly reduced by pertussis toxin (PTX) treatment. The M3‐mediated contractions were only partially inhibited by VDCC antagonists or 140 mM K+‐depolarization medium, and remained unaffected by PTX treatment. The contractions observed during high K+ depolarization consisted of different components, either sensitive or insensitive to extracellular Ca2+. The carbachol contractions observed with wild‐type preparations consisted of PTX‐sensitive and ‐insensitive components. The PTX‐sensitive component was functionally significant only at low carbachol concentrations. The results suggest that the M2 receptor, through PTX‐sensitive mechanisms, induces ileal contractions that depend on voltage‐dependent Ca2+ entry, especially associated with action potential discharge, and that the M3 receptor, through PTX‐insensitive mechanisms, induces contractions that depend on voltage‐dependent and ‐independent Ca2+ entry and intracellular Ca2+ release. In intact tissues coexpressing M2 and M3 receptors, M2 receptor activity appears functionally relevant only when fractional receptor occupation is relatively small.

Phenotypic changes of morphologically identified guinea-pig myenteric neurons following intestinal inflammation

We investigated the responses of morphologically identified myenteric neurons of the guinea‐pig ileum to inflammation that was induced by the intraluminal injection of trinitrobenzene sulphonate, 6 or 7 days previously. Electrophysiological properties were examined with intracellular microelectrodes using in vitro preparations from the inflamed or control ileum. The neurons were injected with marker dyes during recording and later they were recovered for morphological examination. A proportion of neurons with Dogiel type I morphology, 45% (32/71), from the inflamed ileum had a changed phenotype. These neurons exhibited an action potential with a tetrodotoxin‐resistant component, and a prolonged after‐hyperpolarizing potential followed the action potential. Of the other 39 Dogiel type I neurons, no changes were observed in 36 and 3 had increased excitability. The afterhyperpolarizing potential (AHP) in Dogiel type I neurons was blocked by the intermediate conductance, Ca2+‐dependent K+ channel blocker TRAM‐34. Neurons which showed these phenotypic changes had anally directed axonal projections. Neither a tetrodotoxin‐resistant action potential nor an AHP was seen in Dogiel type I neurons from control preparations. Dogiel type II neurons retained their distinguishing AH phenotype, including an inflection on the falling phase of the action potential, an AHP and, in over 90% of neurons, an absence of fast excitatory transmission. However, they became hyperexcitable and exhibited anodal break action potentials, which, unlike control Dogiel type II neurons, were not all blocked by the h current (Ih) antagonist Cs+. It is concluded that inflammation selectively affects different classes of myenteric neurons and causes specific changes in their electrophysiological properties.

Macrophage-derived cytokine and nitric oxide profiles in type I and type II diabetes mellitus: effect of thymoquinone.

Comparing macrophage-derived cytokine and nitric oxide (NO) profiles in type I and type II diabetes mellitus (DM); and determining whether thymoquinone (TQ) has any modulatory effect were the main objectives of the present study. Peritoneal macrophages have been collected from Otsuka Long-Evans Tokushima Fatty (OLETF) as a model for type II DM and its control Long-Evans Tokushima Otsuka (LETO) rats, as well as from streptozotocin (STZ)-injected LETO ones as a model for type I DM. The cells were cultured and incubated with or without TQ (10 µM) in the absence or presence of lipopolysaccharide (LPS; 1 µg/ml). The same parameters have been also assessed in sera of the used animals with or without TQ treatment (3 mg/kg) under both LPS-stimulated (10 mg/kg) and unstimulated conditions. Nitrite, IL-1β and TNF-α were significantly higher in macrophage supernatants and sera of the acutely affected STZ-LETO rats either with or without LPS stimulation compared to corresponding controls. On the other hand, chronically diabetic OLETF rats’ macrophage supernatants showed significant decreases of IL-1β and TNF-α levels upon LPS stimulation or even without stimulation (IL-1β); and insignificant increase in nitrite concentration, which turned significant upon LPS stimulation. Sera of these animals, however, showed significant increase in TNF-α level. TQ normalised the elevated nitrite and cytokine profiles both in vitro and in vivo, yet had no significant effect on the already decreased parameters in chronically affected OLETF rats. These data suggest that there is a tendency for macrophage inflammatory products to increase in acute type I and to decrease in chronic type II DM; and that TQ has the potential to normalise the elevated levels of these macrophage-derived inflammatory mediators.

Role of intrinsic nitrergic neurones on vagally mediated striated muscle contractions in the hamster oesophagus

Oesophageal peristalsis is controlled by vagal motor neurones, and intrinsic neurones have been identified in the striated muscle oesophagus. However, the effect(s) of intrinsic neurones on vagally mediated contractions of oesophageal striated muscles has not been defined. The present study was designed to investigate the role of intrinsic neurones on vagally evoked contractions of oesophageal striated muscles, using hamster oesophageal strips maintained in an organ bath. Stimulation (30 μs, 20 V) of the vagus nerve trunk produced twitch contractions. Piperine inhibited vagally evoked contractions, while capsaicin and NG‐nitro‐L‐arginine methyl ester (L‐NAME) abolished the inhibitory effect of piperine. The effect of L‐NAME was reversed by subsequent addition of L‐arginine, but not by D‐arginine. L‐NAME did not have any effect on the vagally mediated contractions and presumed 3H‐ACh release. NONOate, a nitric oxide donor, and dibutyryl cyclic GMP inhibited twitch contractions. Inhibition of vagally evoked contractions by piperine and NONOate was fully reversed by ODQ, an inhibitor of guanylate cyclase. Immunohistochemical staining showed immunoreactivity for nitric oxide synthase (NOS) in nerve cell bodies and fibres in the myenteric plexus and the presence of choline acetyltransferase and NOS in the motor endplates. Only a few NOS‐immunoreactive portions in the myenteric plexus showed vanilloid receptor 1 (VR1) immunoreactivity. Our results suggest that there is a local neural reflex that involves capsaicin‐sensitive neurones, nitrergic myenteric neurones and vagal motor neurones.

Roles of M2 and M3 muscarinic receptors in cholinergic nerve‐induced contractions in mouse ileum studied with receptor knockout mice

The functional roles of M2 and M3 muscarinic receptors in neurogenic cholinergic contractions in gastrointestinal tracts remain to be elucidated. To address this issue, we studied cholinergic nerve‐induced contractions in the ileum using mutant mice lacking M2 or M3 receptor subtypes.

Receptor signaling mechanisms underlying muscarinic agonist-evoked contraction in guinea-pig ileal longitudinal smooth muscle

In guinea‐pig ileal longitudinal muscle, muscarinic partial agonists, 4‐(N‐[3‐chlorophenyl]‐carbomoyloxy)‐2‐butynyl‐trimethylammonium (McN‐A343) and pilocarpine, each produced parallel increases in tension and cytosolic Ca2+ concentration ([Ca2+]c) with a higher EC50 than that of the full agonist carbachol. The maximum response of [Ca2+]c or tension was not much different among the three agonists. The Ca2+ channel blocker nicardipine markedly inhibited the effects of all three agonists The contractile response to any agonist was antagonized in a competitive manner by M2 receptor selective antagonists (N,N′‐bis[6‐[[(2‐methoyphenyl)methyl]amino]hexyl]‐1,8‐octanediamine tetrahydrochloride and 11‐[[2‐[(diethlamino)methyl]‐1‐piperidinyl]acetyl]‐5,11‐dihydro‐6H‐pyrido[2,3‐b][1,4] benzodiazepine‐6‐one), and the apparent order of M2 antagonist sensitivity was McN‐A343>pilocarpine>carbachol. M3 receptor selective antagonists, 1,1‐dimethyl‐4‐diphenylacetoxypiperidinium iodide and darifenacin, both severely depressed the maximum response for McN‐A343, while darifenacin had a similar action in the case of pilocarpine. Both M3 antagonists behaved in a competitive manner in the case of the carbachol response. McN‐A343 failed to release Ca2+ from the intracellular stores, and the Ca2+‐releasing action of pilocarpine was very weak compared with that of carbachol. All three agonists were capable of increasing Ca2+ sensitivity of the contractile proteins. McN‐A343 rarely produced membrane depolarization, but always accelerated electrical spike discharge. Pilocarpine effect was more often accompanied by membrane depolarization, as was usually seen using carbachol. The results suggest that muscarinic agonist‐evoked contractions result primarily from the integration of Ca2+ entry associated with the increased spike discharge and myofilaments Ca2+ sensitization, and that Ca2+ store release may contribute to the contraction indirectly via potentiation of the electrical membrane responses. They may also support the idea that an interaction of M2 and M3 receptors plays a crucial role in mediating the contraction response.

ATP released from perivascular nerves hyperpolarizes smooth muscle cells by releasing an endothelium-derived factor in hamster mesenteric arteries

1 The interaction between perivascular nerves and endothelium was investigated by measuring the changes in smooth muscle membrane potentials using intracellular microelectrode techniques in hamster mesenteric thin (100–150 μm) and thick (300–350 μm) arteries. 2 In both arteries, nerve stimulation evoked excitatory junction potentials (EJPs) which were strongly inhibited by pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (0·5–5 μM). This result indicated that the EJPs were induced by the activation of P2X receptors. 3 Transient hyperpolarizations were evoked by trains of pulses at 20 Hz in PPADS (5 μM)‐pre‐treated thin arteries, but not in the thick arteries. ATP (100 μM) applied to adventitial surfaces mimicked the hyperpolarizations. Both the ATP‐ and nerve stimulation‐induced hyperpolarizations were blocked by cibacron blue F3GA (2–100 μM) and were also abolished after endothelium removal, indicating that the neurally released ATP evoked transient hyperpolarization through the activation of P2Y receptors located on the endothelium. 4 In endothelium‐intact preparations, intimal application of uridine 5′‐triphosphate (UTP 100 μM), a P2Y2‐like receptor agonist, but not 2‐methylthio ATP (7 μM), hyperpolarized the smooth muscle. The UTP‐induced hyperpolarization was significantly inhibited by cibacron blue F3GA and was abolished after endothelium removal. 5 These results suggest that ATP released from the perivascular nerves may reach the endothelium and activate P2Y2‐like receptors to induce the release of an endothelium‐derived hyperpolarizing factor in thin arteries.

Characterization of spontaneous depolarizations in smooth muscle cells of the guinea pig prostate

PURPOSE We characterized the electrical events recorded in small segments of the dorsal lobe of the prostate of immature male guinea pigs and examined some mechanisms underlying their generation. MATERIAL AND METHODS Membrane potential recordings were made in the stroma of the guinea pig prostate using conventional single microelectrode techniques. RESULTS Three distinct, spontaneously occurring electrical events were recorded in guinea pig prostate, namely slow waves, consisting of a depolarizing transient 14 mV in amplitude with 1 to 6 nifedipine sensitive spikes superimposed, pacemaker potentials, consisting of a larger depolarization 40 mV in amplitude, and STDs 1 to 10 mV in amplitude. Only spikes on slow waves were inhibited by nifedipine. The depolarizing transient of slow waves, pacemaker potentials and STDs were abolished by cyclopiazonic acid, a blocker of the SERCA pump, and the mitochondrial uncoupler cyanide m-chlorophenyl hydrazone as well as upon exposure to Ca(2+)-free saline or the Cl(-) channel blockers niflumic acid and anthracene-9-carboxylic acid (Sigma Chemical Co., St. Louis, Missouri). Examination of the stochastic properties of STDs revealed that they were not well modeled by Poisson statistics, but rather they occurred in a clustered manner, such they may well underlie pacemaker potential generation. CONCLUSIONS Guinea pig prostate shows STD and pacemaker potentials that arise from the release of Ca(2+) from intracellular stores and the activation of Ca(2+) activated Cl(-) channels. We speculate that the depolarizing transient of prostatic slow waves is the propagated response of pacemaker potentials evoked at sites electrically distant from the recording electrode.