Tadashi Takewaki

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.

GTP‐binding protein involvement in membrane currents evoked by carbachol and histamine in guinea‐pig ileal muscle.

1. Single smooth muscle cells obtained by enzymic dispersion of the longitudinal muscle layer of guinea‐pig ileum were used for recording membrane currents under whole‐cell voltage clamp in response to carbachol (100 microM, unless otherwise stated) or histamine (100 microM) applied extracellularly. 2. At a holding potential of 0 mV, a transient outward current was evoked by carbachol and histamine. Responses to the two agonists were very similar in size and time course to the current response to caffeine (10 mM). The response to carbachol was virtually absent in the presence of histamine, and vice versa. Caffeine was without effect in the presence of either of these agonists. Inclusion of EGTA (10 or 20 mM) in the pipette abolished the responses to carbachol, histamine and caffeine. Thus, the outward current responses were considered to represent opening of Ca(2+)‐activated K+ channels in response to a massive release of Ca2+ from the same stores by these three agents. 3. An inward current was evoked by carbachol and histamine, but not by caffeine at a holding potential of ‐40 mV, which was considered to represent opening of cationic channels. The carbachol‐induced inward current was much longer in duration and larger in size than the histamine‐induced inward current. 4. Inclusion of GDP beta S (2 mM) in the pipette abolished the inward and outward current responses to histamine, but inhibited only part of those to carbachol. 5. When the holding potential was held at 0 mV with inclusion of GTP gamma S (0.1‐1 mM) in the pipette, spontaneous transient outward currents appeared immediately after break‐through but disappeared a few minutes later. Under these conditions, caffeine (10 mM) was almost without effect, suggesting that GTP gamma S had released Ca2+ stores. When the holding potential was held at ‐40 mV and GTP gamma S (0.1 or 0.2 mM) was present in the pipette, an inward current developed a few minutes after break‐through. During the GTP gamma S‐induced inward current, application of carbachol or histamine produced no further inward current. However, when 0.01 mM‐GTP gamma S was included in the pipette solution, carbachol‐ and histamine‐induced inward currents were potentiated. 6. Pretreated with 2‐5 micrograms/ml pertussis toxin (PTX) did not change noticeably the outward current responses to carbachol and histamine, but abolished or markedly reduced the inward current responses.(ABSTRACT TRUNCATED AT 400 WORDS)

Isulinotropic properties of Nigella sativa oil in Streptozotocin plus Nicotinamide diabetic hamster

The present study was designed to investigate the possible insulinotropic properties of Nigella sativa L. (N. sativa) oil in Streptozotocin plus Nicotinamide-induced diabetes mellitus in hamsters. Nicotinamide was injected intraperitoneally 15min before injection of Streptozotocin intravenously. Oral treatment with N. sativa oil began 4 weeks after induction of diabetes. Serum insulin was measured by enzymeimmunoassay. Islets insulin was stained using anti-insulin monoclonal antibody. Significant decrease in blood glucose level together with significant increase in serum insulin level were observed after treatment with N. sativa oil for 4 weeks. Big areas with positive immuno-reactivity for the presence of insulin were observed in the pancreases from N. sativa oil-treated group compared to non-treated one using immunohistochemical staining. Therefore, our data show that the hypoglycemic effect of N. sativa oil in Streptozotocin plus Nicotinamide diabetic hamsters resulted, at least partly, from a stimulatory effect on beta cell function with consequent increase in serum insulin level. These results indicate that N. sativa oil has insulinotropic properties in type 2-like model.

VIP‐ and PACAP‐mediated nonadrenergic, noncholinergic inhibition in longitudinal muscle of rat distal colon: involvement of activation of charybdotoxin‐ and apamin‐sensitive K+ channels

1 The mediators of nonadrenergic, noncholinergic (NANC) inhibitory responses in longitudinal muscle of rat distal colon were studied. 2 An antagonist of pituitary adenylate cyclase activating peptide (PACAP) receptors, PACAP6–38, concentration‐dependently inhibited the rapid relaxation of the longitudinal muscle induced by electrical field stimulation (EFS), resulting in a maximal inhibition of 47% at 3 μm. 3 PACAP6–38 inhibited the relaxation by 75% in the presence of the vasoactive intestinal peptide (VIP) receptor antagonist, VIP10–28 at μm, which inhibited the relaxation by 44%. 4 An antagonist of large conductance Ca2+‐activated K+ channels, charybdotoxin, concentration‐dependently inhibited the rapid relaxation of the longitudinal muscle, resulting in a maximal inhibition of 58% at 100 nM. 5 An antagonist of small conductance Ca2+‐activated K+ channels, apamin, concentration‐dependently inhibited the relaxation (58% at 1 μm). 6 Treatment with both K+ channel antagonists resulted in 84% inhibition of the EFS‐induced relaxation, which is comparable to the extent of inhibition induced by PACAP6–38 plus VIP10–28. 7 The inhibitory effect of VIP10–28 and of apamin, but not of charybdotoxin was additive: the same applied to PACAP6–38 and charybdotoxin, but not apamin. 8 Exogenously added VIP (100 nM‐1 μm) induced a slow gradual relaxation of the longitudinal muscle. Charybdotoxin, but not apamin significantly inhibited the VIP‐induced relaxation. VIP10–28, but not PACAP6–38 selectively inhibited the VIP‐induced relaxation. 9 Exogenously added PACAP (10–100 nM) also induced slow relaxation. Apamin and to a lesser extent, charybdotoxin, inhibited the PACAP‐induced relaxation. PACAP6–38, but not VIP10–28 selectively inhibited the PACAP‐induced relaxation. 10 Apamin at 100 nM inhibited inhibitory junction potentials (i.j.ps) induced by a single pulse of EFS. Apamin also inhibited a rapid phase, but not a delayed phase of i.j.ps induced by two pulses at 10 Hz. VIP10–28 did not inhibit i.j.ps induced by a single pulse, but significantly inhibited the delayed phase at two pulses. A combination of apamin and VIP10–28 abolished the i.j.ps induced by two pulses. 11 Both VIP and PACAP induced slow hyperpolarization of the cell membrane of the longitudinal muscle. Apamin inhibited the PACAP‐, but not VIP‐induced hyperpolarization. 12 From these findings it is suggested that VIP and PACAP are involved in NANC inhibitory responses of longitudinal muscle of the rat distal colon via activation of charybdotoxin‐ and apaminsensitive K+ channels, respectively.

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.

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.

Contrasting effects of ghrelin and des-acyl ghrelin on the lumbo-sacral defecation center and regulation of colorectal motility in rats

Background  We have previously demonstrated that a centrally penetrant ghrelin receptor agonist enhances colorectal motility, through activation of the lumbo‐sacral defecation center (L6‐S1 region of the spinal cord) in rats. In the present study, we examined the effects of the native peptide and its non‐acylated counterpart in eliciting this stimulatory effect on colorectal motility.

Nitric oxide‐mediated inhibitory response of rat proximal colon: independence from changes in membrane potential

1 We studied the relation of nitric oxide‐mediated relaxation of smooth muscle to changes in membrane potential of cells in the proximal colon of rats. 2 The resting membrane potential and electrical field stimulation (EFS)‐induced junction potentials were recorded from the circular and longitudinal muscle cells. 3 Localized distension with a small balloon caused relaxation of the circular muscle on the anal side of the distended region (descending relaxation). Relaxation of the longitudinal muscle was also induced by EFS. 4 Inhibitory junction potentials (i.j.ps) were recorded from all circular muscle cells tested, but rarely from the longitudinal muscle cells. 5 The i.j.ps were recorded only in the presence of atropine but relaxations of both muscles were induced even in the absence of atropine. 6 Apamin (100 nm) completely abolished the i.j.ps recorded in both circular and longitudinal muscle cells, but had no significant effect on the relaxations of either. 7 In contrast to apamin, NG nitro‐l‐arginine (10 μm) inhibited the relaxations of both muscles, but did not affect the i.j.ps. 8 Exogenously added nitric oxide (0.1–10 μm) induced relaxations of both muscles concentration‐dependently, but did not affect the membrane potentials at these concentrations. 9 These data strongly suggest that nitric oxide‐mediated relaxation of rat proximal colon is not associated with the i.j.ps of the cell membrane.

Inhibitory effects of zingerone, a pungent component of Zingiber officinale Roscoe, on colonic motility in rats

Ginger (rhizome of Zingiber officinale Roscoe) is an herbal medicine for the treatment of gastrointestinal disorders including constipation and diarrhea. Zingerone is a likely active constituent responsible for the antidiarrheal activity of ginger. The current study was designed to characterize pharmacological actions of zingerone on colonic motility. To evaluate pharmacological effects of zingerone on colonic motility, we used isolated colonic segments from rats, in which mechanical responses were recorded in the longitudinal direction. In addition, we evaluated the effects on colonic motility in vivo by measuring intraluminal pressure changes and expelled fluid volume from the colon in anesthetized rats. Zingerone was applied to the lumen of the colon to allow the drug to access from the mucosal side. Zingerone inhibited spontaneous contractile movements in the isolated colonic segments in a dose-dependent manner. The inhibitory effects of zingerone on colonic movements were not affected by pretreatment with capsazepine, a typical antagonist of transient receptor potential vanilloid 1. In addition, tetrodotoxin, a blocker of voltage-dependent sodium channels on neurons, did not affect the suppression of colonic movements by zingerone, suggesting that zingerone acts on the smooth muscles directly. Zingerone also attenuated colonic motility in vivo without affecting blood pressure and heart rate. The effects were reversible and reproducible. Our findings suggest that zingerone can inhibit colonic motility via direct action on smooth muscles. Zingerone might exert beneficial therapeutic effects on hypermotility-induced diarrhea by abrogating excessive gastrointestinal motility.

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.