Yasutake Shimizu

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.

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.

Evidence that stimulation of ghrelin receptors in the spinal cord initiates propulsive activity in the colon of the rat

Previous studies have failed to reveal an effect of the gastrointestinal peptide hormone ghrelin on colonic motility. In the present work, ghrelin was applied into the lumbo‐sacral spinal cord in the region of defecation control centres, and a synthetic ghrelin receptor agonist, CP464709, which crosses the blood–brain barrier, was applied intravenously or into the lumbo‐sacral cord. Both ghrelin and CP464709 elicited propulsive contractions and emptying of the colon in anaesthetized rats. In conscious rats, subcutaneous CP464709 caused fecal expulsion. The sites of action and nerve pathways involved in the stimulation of the colon by ghrelin receptor activation were investigated in anaesthetized rats. Intrathecal application of CP464709 at L6–S1, but not application at ponto‐medullary levels or to the thoracic spinal cord, elicited propulsive contractions. The stimulation evoked by intravenous CP464709 was prevented if the pelvic nerve outflows were severed, but not if the spinal cord was cut rostral to the defecation centre at L6–S3. The response was also blocked by hexamethonium. When ghrelin, applied intrathecally, was used to desensitize its receptors, the effect of intravenous CP464709 was blocked. CP464709 did not affect small intestine motility or the amplitudes of visceromotor reflexes caused by colorectal distension. It is concluded that activation of ghrelin receptors in the lumbo‐sacral spinal cord triggers co‐ordinated propulsive contractions that empty the colo‐rectum. The pathways through which these responses are generated pass out of the spinal cord via the pelvic nerves and cause propulsive contractions through activation of enteric neurons.

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.

Interleukin-1 increases norepinephrine turnover in the spleen and lung in rats

To clarify effects of interleukin-1 on sympathetic nerve activity, norepinephrine turnover in various organs was assessed in rats after intraperitoneal injection of recombinant human interleukin-1 beta. Interleukin-1 administration increased norepinephrine turnover in the spleen, lung and hypothalamus without appreciable effect in the heart, liver, submandibular gland, thymus, pancreas, brown adipose tissue and medulla oblongata. Similar changes in norepinephrine turnover were also found after the administration of bacterial endotoxin. It was concluded that interleukin-1 activates the sympathetic nerves specifically in the spleen and lung.

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.

The distribution of intermediate-conductance, calcium-activated, potassium (IK) channels in epithelial cells.

Intermediate‐conductance, calcium‐activated, potassium (IK) channels were first identified by their roles in cell volume regulation, and were later shown to be involved in control of proliferation of lymphocytes and to provide a K+ current for epithelial secretory activity. Until now, there has been no systematic investigation of IK channel localization within different epithelia. IK channel immunoreactivity was present in most epithelia, where it occurred in surface membranes of epithelial cells. It was found in all stratified epithelia, including skin, cornea, oral mucosa, vaginal mucosa, urothelium and the oesophageal lining. It occurred in the ducts of fluid‐secreting glands, the salivary glands, lacrimal glands and pancreas, and in the respiratory epithelium. A low level of expression was seen in serous acinar cells. It was also found in other epithelia with fluid‐exchange properties, the choroid plexus epithelium, the ependyma, visceral pleura and peritoneum, bile ducts and intestinal lining epithelium. However, there was little or no expression in vascular endothelial cells, kidney tubules or collecting ducts, lung alveoli, or in sebaceous glands. It is concluded that the channel is present in surface epithelia (e.g. skin) where it has a cell‐protective role against osmotic challenge, and in epithelia where there is anion secretion that is facilitated by a K+ current‐dependent hyperpolarization. It was also in some epithelial cells where its roles are as yet unknown.

Cold exposure increases glucose utilization and glucose transporter expression in brown adipose tissue

When rats were exposed to a cold environment (4 degrees C) for 10 days, tissue glucose utilization was increased in brown adipose tissue (BAT), a tissue specified for non-shivering thermogenesis, but not in skeletal muscle. Cold exposure also caused an increase in the amount of GLUT4, an isoform of glucose transporters expressed in insulin-sensitive tissues, in parallel with an increased cellular level of GLUT4 mRNA. In contrast to BAT, no significant effect of cold exposure was found in skeletal muscle. The results suggest the cold-induced increase in glucose utilization by BAT is attributable, at least in part, to the increased expression of GLUT4.

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.