Mechanisms of Motility Change on Trinitrobenzenesulfonic Acid-Induced Colonic Inflammation in Mice

Ulcerative colitis is an inflammatory bowel disease (IBD) characterized by recurrent episodes of colonic inflammation and tissue degeneration in human or animal models. The contractile force generated by the smooth muscle is significantly attenuated, resulting in altered motility leading to diarrhea or constipation in IBD. The aim of this study is to clarify the altered contractility of circular and longitudinal smooth muscle layers in proximal colon of trinitrobenzen sulfonic acid (TNBS)-induced colitis mouse. Colitis was induced by direct injection of TNBS (120 mg/kg, 50% ethanol) in proximal colon of ICR mouse using a 30 G needle anesthetized with ketamin (50 mg/kg), whereas animals in the control group were injected of 50% ethanol alone. In TNBS-induced colitis, the wall of the proximal colon is diffusely thickened with loss of haustration, and showed mucosal and mucular edema with inflammatory infiltration. The colonic inflammation is significantly induced the reduction of colonic contractile activity including spontaneous contractile activity, depolarization-induced contractility, and muscarinic acetylcholine receptor-mediated contractile response in circular muscle layer compared to the longitudinal muscle layer. The inward rectification of currents, especially, important to Ca2+ and Na+ influx-induced depolarization and contraction, was markedly reduced in the TNBS-induced colitis compared to the control. The muscarinic acetylcholine-mediated contractile responses were significantly attenuated in the circular and longitudinal smooth muscle strips induced by the reduction of membrane expression of canonical transient receptor potential (TRPC) channel isoforms from the proximal colon of the TNBS-induced colitis mouse than the control.

Forskolin Changes the Relationship between Cytosolic Ca2+ and Contraction in Guinea Pig Ileum

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic Ca2+ level ([Ca2+]i), and Ca2+ sensitivity in guinea pig ileum. Forskolin (0.1 nM~10 micrometer) inhibited high K+ (25 mM and 40 mM)- or histamine (3 micrometer)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high K+-evoked contractions. Spontaneous changes in [Ca2+]i and contractions were inhibited by forskolin (1 micrometer) without changing the resting [Ca2+]i. Forskoln (10 micrometer) inhibited muscle tension more strongly than [Ca2+]i stimulated by high K+, and thus shifted the [Ca2+]i-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 micrometer) inhibited both [Ca2+]i and muscle tension without changing the [Ca2+]i-tension relationship. In alpha-toxin-permeabilized tissues, forskolin (10 micrometer) inhibited the 0.3 micrometer Ca2+-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the Ca2+-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in Ca2+ sensitivity of contractile elements in high K+-stimulated muscle and a decrease in [Ca2+]i in histamine-stimulated muscle.

Differentiation of neuroepithelial progenitor cells implanted into newborn rat brain striatum

It has been demonstrated that multipotent neuronal progenitor cells can be isolated from the developing or adult CNS and proliferated in vitro in response to epidermal growth factor. The present study was undertaken to investigate the differentiation of neuronal progenitor cells after transplantation into the neonatal rat forebrain striatum. Primary cultured progenitor cells were labeled with 3,3'-dioctadecycloxacarbonyl- amine perchlorate (DiO). DiO labeled progenitor cells were implanted into neonatal rat striatum. Implanted DiO labeled progenitor cells were differentiated into astrocytes and GABAergic neurons. These results suggest that implanted progenitor cells can be differentiated into neurons in host forebrain striatum. In addition, our data show that DiO labeling is a useful technique for tracing implanted progenitor cells.

Adrenergic sensitivity of uninjured C-fiber nociceptors in neuropathic rats

We investigated the adrenergic sensitivity of afferent fibers in the L4 dorsal roots of rats with a unilateral ligation of the L5-L6 spinal nerves. About 12% of nociceptive fibers on the affected side were excited by sympathetic stimulation or by intra-arterial injection of norepinephrine which did not affect A beta-fiber activity. Sympathetic excitation of nociceptive fibers was suppressed by alpha 1-antagonist prazosin, while it was unaffected by alpha 2-antagonist yohimbine. Most of these fibers were excited by intra-arterial injection of alpha 1-agonist phenylephrine, without being affected by an injection of alpha 2-agonist clonidine. Sympathetic excitation was blocked by lidocaine applied near the receptive fields of recorded fibers. The results suggested that some nociceptors remaining intact after partial nerve injury become sensitive to sympathetic activity by the mediation of alpha 1-adrenoceptors in the peripheral endings.

Mechanism of L-NAME-resistant endothelium-dependent relaxation induced by acetylcholine in rabbit renal artery

In the rabbit renal artery, acetylcholine (ACh, 1 nM ~ 10 micrometer) induced endothelium-dependent relaxation of arterial rings precontracted with norepinephrine (NE, 1 micrometer) in a dose-dependent manner. NG-nitro-L-arginine (L-NAME, 0.1 mM), an inhibitor of NO synthase, or ODQ (1 micrometer), a soluble guanylate cyclase inhibitor, partially inhibited the ACh-induced endothelium-dependent relaxation. The ACh-induced relaxation was abolished in the presence of 25 mM KCl and L-NAME. The cytochrome P450 inhibitors, 7-ethoxyresorufin (7-ER, 10 micrometer), miconazole (10 micrometer), or 17-octadecynoic acid (17-ODYA, 10 micrometer), failed to inhibit the ACh-induced relaxation in the presence of L-NAME. 11,12-epoxyeicosatrienoic acid (11,12-EET, 10 micrometer) had no relaxant effect. The ACh-induced relaxation observed in the presence of L-NAME was significantly reduced by a combination of iberiotoxin (0.3 micrometer) and apamin (1 micrometer), and almost completely blocked by 4-aminopyridine (5 mM). The ACh-induced relaxation was antagonized by P2Y receptor antagonist, cibacron blue (10 and 100 micrometer), in a dose-dependent manner. Furthermore, 2-methylthio-ATP (2MeSATP), a potent P2Y agonist, induced the endothelium-dependent relaxation, and this relaxation was markedly reduced by either the combination of iberiotoxin and apamin or by cibacron blue. In conclusion, in renal arteries isolated from rabbit, ACh produced non-NO relaxation that is mediated by an EDHF. The results also suggest that ACh may activate the release of ATP from endothelial cells, which in turn activates P2Y receptor on the endothelial cells. Activation of endothelial P2Y receptors induces a release of EDHF resulting in a vasorelaxation via a mechanism that involves activation of both the voltage-gated K+ channels and the Ca2+-activated K+ channels. The results further suggest that EDHF does not appear to be a cytochrome P450 metabolite.

The effects of laminin on the characteristics and differentiation of neuronal cells from epidermal growth factor-responsive neuroepithelial cells

Many extracellular matrix molecules are expressed in the embryonic nervous system and there is some evidence that they are important regulators of neural development. Of these molecules, laminin appears to be the most potent, affecting virtually all neurons of the peripheral and central nervous system. This study was undertaken to investigate the effects of laminin on the proliferation and differentiation of cultured neuroepithelial cells taken from fetal rat forebrains (embryonic day 17-19). The results are summarized as follows. 1) Neuroepithelial cells cultivated in epidermal growth factors containing serum-free medium subsequently differentiated into neurons, astrocytes, and oligodendrocytes. 2) Neuronal cells derived from neuroepithelial cells were immunoreactive for gamma-aminobutyric acid (GABA) or substance P, but were not for serotonin and tyrosine hydroxylase. 3) In western blot analysis, the phosphorylated neurofilament content in neuronal cells was higher in culture on laminin than in culture on poly-L-lysine (PLL). 4) The proliferation rate of GABAergic neurons was higher in culture on laminin than in culture on PLL. These results suggest that GABAergic and substance P-ergic neurons can be differentiated from neuroepithelial cells and that laminin promotes the differentiation of neuronal cells from neuroepithelial cells and the increased proliferation rate of GABAergic cells.

Effects of ketamine on contractile responses in vascular smooth muscle

This study was designed to determine the effects of ketamine on contractions induced by norepinephrine (NE), K+ or histamine (Hist) and on agonist-induced calcium mobilization, in rabbit thoracic aorta with or without endothelium. Contractile responses to NE, K+ or Hist were markedly attenuated by prior exposure to ketamine. Subsequent addition of ketamine to the rabbit aorta undergoing an isometric contraction induced by NE, K+ or Hist also decreased the contractile responses in a calcium ion concentration-dependent manner. Preincubation with ketamine produced a concentration-dependent inhibition of contractile responses elicited by the addition of calcium ion (1.6 mM) to a Ca(++)-free depolarizing solution. However, the phasic contraction produced by NE with 2mM lanthanum pretreatment, which is release of intracellular calcium, was also inhibited by ketamine. Moreover, the tonic contraction produced by NE after depletion of the agonist-releasable pool of intracellular calcium, which is thought to be due to calcium influx, was depressed by ketamine. These data suggest that ketamine relaxes NE-contracted rings of rabbit thoracic aorta by decreasing calcium entry and by producing an extracellular calcium-independent relaxant effect.