Most regions of the gastrointestinal tract generate spontaneous electrical and mechanical activity in the absence of stimulation. When electrical recordings are made from slow muscle cells lying in the gastrointestinal tract, a regular discharge of long lasting waves of depolarization, slow waves, is detected. It has recently become apparent that slow waves are generated by a specialized population of smooth muscle cells, known as interstitial cells of Cajal (ICC). ICC can be subdivided into at least two separate groups. In most regions of the gastrointestinal tract, one group of ICC form a network that generates pacemaker potentials, so producing rhythmical membrane potential changes in the adjacent muscle layers. The second group of ICC are distributed amongst the smooth muscle cells and are tightly electrically coupled to them. In some regions of the gut, the second group of ICC augment the waves of pacemaker depolarization, so ensuring that voltage-dependent calcium channels in the smooth muscles are activated during each slow wave cycle. In addition, the second group of ICC are densely innervated by inhibitory and excitatory nerve terminals. Thus intrinsic nerve terminals, rather than communicating directly with smooth muscle cells, selectively innervate ICC and release transmitters directly onto them. The signals that are generated in the ICC, by the neurally released transmitters, then alter the activity of surrounding smooth muscle cells.
Five isoforms of Na+/Ca2+ exchanger have been identified: NCX1, NCX2, NCX3, NCX-SQ1, and CALX. In all of the exchangers, the Na+/Ca2+ exchange current, which was recorded in inside-out membrane patches, was regulated by cytoplasmic Ca2+. However, the mode of regulation is different among the exchangers. NCX1, NCX2, and NCX-SQ1 are positively regulated by cytoplasmic Ca2+, but CALX is negatively regulated. NCX3 apparently has both positive and negative regulation mechanisms. In this review, I briefly summarize the Ca2+-dependent regulation mechanisms of the exchangers.
The stoichiometry of the Na+/Ca2+ exchanger (NCX) had been generally accepted as 3 Na+:1 Ca2+. However, recently a challenging stoichiometry of 4:1 was proposed. Therefore, using guinea pig ventricular cells, we re-examined the stoichiometry by measuring the reversal potential of the NCX current and intracellular Ca2+ concentrations under the whole-cell voltage clamp. We confirmed that the stoichiometry of NCX is 3:1 not 4:1. In addition, we explored the possible reasons for obtaining erroneous results of a 4:1 stoichiometry.
Sensitivities of the reverse-mode Na+/Ca2+ exchange activity measured as the Na+i-dependent Ca2+ uptake to extracellular monovalent cations K+, Li+, and Na+ were compared between the K+-dependent (NCKX2) and the K+-independent Na+/Ca2+ exchanger (NCX1) overexpressed in a fibroblast cell. Interestingly, the exchange activity of NCKX2 was not influenced by Li+ while it was increased by K+. On the contrary, the activity of NCX1 was increased by Li+. Thus, the cation sensitivities to K+ and Li+ markedly differed between NCKX2 and NCX1. In addition, Na+ exerted a significantly smaller inhibitory effect on the activity in NCKX2 than in NCX1. The Na+/Ca2+ exchange activities of NCKX2 and NCX1 are considered to be regulated differentially via the respective binding site domains that have distinct sensitivities to the external monovalent cations.
We used Na+/Ca2+ exchanger (NCX) knockout mice to evaluate the effects of NCX in cardiac function and the infarct size after ischemia/reperfusion injury. The contractile function in NCX KO mice hearts was significantly better than that in wild type (WT) mouse hearts after ischemia/reperfusion and the infracted size was significantly smaller in NCX KO mice hearts compared with that in WT mice hearts. NCX is critically involved in the development of ischemia/reperfusion-induced myocardial injury, and therefore the inhibition of NCX function may contribute to cardioprotection against ischemia/reperfusion injury.
The Na+/Ca2+ exchanger (NCX) is an ion transporter that exchanges Na+ and Ca2+ in either Ca2+ efflux or Ca2+ influx mode, depending on membrane potential and transmembrane ion gradients. In myocytes, neurons, and nephron cells, NCX is thought to play an important role in the regulation of intracellular Ca2+ concentration. Recently, the benzyloxyphenyl derivatives KB-R7943, SEA0400, and SN-6 have been developed as selective NCX inhibitors. Currently, SEA0400 is the most potent and selective inhibitor. These inhibitors possess different isoform-selectivities, although they have similar properties, such as Ca2+ influx mode-selectivity and I1 inactivation-dependence. Recent site-directed mutagenesis has revealed that these inhibitors possess some molecular determinants (Phe-213, Val-227, Tyr-228, Gly-833, and Asn-839) for interaction with NCX1. These benzyloxyphenyl derivatives are expected to be useful tools to study the physiological roles of NCX. Moreover, such inhibitors may have therapeutic potential as a new remedy for ischemic disease, arrhythmias, heart failure, and hypertension.
The improving effects of various components of Toki-Shakuyaku-San (TSS) and fractions isolated from Angelica acutiloba Radix (Toki) on scopolamine-induced spatial memory impairment were investigated in eight-armed radial maze. The scopolamine-induced memory impairment was characterized by prominent increase of error choices in addition to decreased correct choices. Toki, Cnidium officinale Rhizoma (Senkyu), Poria cocos Hoelen (Bukuryo), Alisma orientale Rhizoma (Takusha), and Atractylodes lancea Rhizoma (Sojutsu) increased the correct choices, while only the Toki, Sojutsu, and Takusha decreased the error choices. No effect was produced by Paeonia lactiflora Radix (Shakuyaku). Investigation of effects of fractions isolated from Toki revealed that its activity mainly resided in the butanol layer and its contents of N-methyl-β-carboline-3-carboxamide and amines. Moreover, the alkaloid, internal and external solutions (containing poly-, di-, and monosaccharides) obtained by dialysis with Visking cellophane tubing also improved the memory. However, no improving properties were detected for methanol and hexanol layers, L-(−)-tryptophan, L-arginine, L-(−)-lysine, and choline chloride. The results showed that the TSS components could improve the reference and working memory impaired by scopolamine. The improving effect of TSS is produced greatly by the Toki component, the activity of which was greatly produced by the fraction extracted by butanol.
Adenosine A1 receptors in the brain are believed to play an important role in brain functioning. We have discovered a novel adenosine A1 receptor antagonist, FR194921 (2-(1-methyl-4-piperidinyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone), and characterized the pharmacological activity in the present study. FR194921 showed potent and selective affinity for the adenosine A1 receptor without affinity for A2A and A3 receptors and did not show any species differences in binding affinity profile among human, rat, and mouse. Pharmacokinetic study in rats revealed that FR194921 was orally active and highly brain penetrable. Oral administration of FR194921 dose-dependently ameliorated the hypolocomotion induced by the A1 receptor agonist N6-cyclopentyladenosine in rats, indicating this compound exerts A1-antagonistic action in vivo. In the passive avoidance test, scopolamine (1 mg/kg)-induced memory deficits were significantly ameliorated by FR194921 (0.32, 1 mg/kg). In two animal models of anxiety, the social interaction test and elevated plus maze, FR194921 showed specific anxiolytic activity without significantly influencing general behavior. In contrast, FR194921 did not show antidepressant activity even at a dose of 32 mg/kg in the rat forced swimming test. These results indicate that the novel, potent, and selective adenosine A1 receptor antagonist FR194921 exerts both cognitive-enhancing and anxiolytic activity, suggesting the therapeutic potential of this compound for dementia and anxiety disorders.
Embryonic stem (ES) cells have many of the characteristics of an optimal cell source for cell-replacement therapy. Although the usefulness of the in vitro generation of dopamine (DA)-neural precursors from ES cells has been widely discussed, functional recovery in animal models of Parkinson’s disease is not fully understood. In 6-hydroxydopamine-lesioned rats, apomorphine markedly induced contralateral rotation. Apomorphine-induced rotation was significantly reduced by transplantation of neuron-like cells that had differentiated from mouse ES cells using nicotinamide, but not L-lysine. In addition, methamphetamine-induced ipsilateral rotation was significantly reduced. On the other hand, picrotoxin did not inhibit apomorphine-induced rotational asymmetry. Fluoxetine alone and fenfluramine alone induced slight contralateral rotation and rotation in both directions, respectively, and these effects were similar in transplanted rats. Although immunoreactivity for tyrosine hydroxylase (TH) was almost completely lost in the ipsilateral striatum in hemiparkinsonian rats, TH immunoreactivity was detected in transplanted cells and sprouting fibers. In contrast, immunoreactivities for γ-aminobutyric acid (GABA) and serotonin (5-HT) neurons were not changed. These results suggest that improvement of rotational behavior may be induced predominantly by transplantation of nicotinamide-treated ES cell-derived DA neurons, rather than by changes in the activities of GABA or 5-HT neural systems, in hemiparkinsonian rats.
The novel calmodulin (CaM) antagonist DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) with an apparent neuroprotective effect in vivo preferentially inhibits neuronal nitric oxide synthase (nNOS), Ca2+/CaM-dependent protein kinase IIα (CaMKIIα), and calcineurin in vitro. In the present study, we investigated the molecular mechanism underlying its neuroprotective effect with the gerbil transient forebrain ischemia model, by focusing on its inhibition of these Ca2+/CaM-dependent enzymes. Post-ischemic DY-9760e treatment (5 mg/kg, i.p.) immediately after 5-min ischemia significantly reduced the delayed neuronal death in the hippocampal CA1 region. CaMKIIα was transiently autophosphorylated immediately after reperfusion with concomitant sustained decrease in its total amounts in the Triton X-100-soluble fractions. Calcineurin activity, accessed by the phosphorylation state of dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) at Thr34, was elevated at 6 h after reperfusion. Post-treatment of DY-9760e had no effects on both CaMKIIα and DARPP-32 phosphorylation at 6 h after reperfusion. However, DY-9760e significantly inhibited nitrotyrosine formation, as a biomarker of NO, and in turn, peroxynitrite (ONOO−) production. These results suggest that DY-9760e primarily inhibits Ca2+/CaM-dependent neuronal NOS, without any effects on CaMKII and calcineurin, and the inhibition of NO production possibly accounts for its neuroprotective action in brain ischemic injury.
Carbachol (CCh) caused a dose-dependent release of β-hexosaminidase and an increase in the production of inositol 1,4,5-trisphosphate (IP3) in RBL-2H3 cells transfected with m2 mAChR cDNA (RBL-m2 cells). The secretion was completely inhibited by LaCl3 and pertussis toxin. The secretion was dependent on extracellular Ca2+ and mediated through the pertussis toxin-sensitive G protein. Exposing RBL-m2 cells to 100 μM CCh for 30 min in Ca2+-free medium (desensitizing treatment) inhibited the secretion induced by the subsequent addition of 10 μM CCh plus Ca2+, but not by stimulating the high affinity IgE receptor (FcεRI). Desensitizing treatment of RBL-m2 cells reduced the affinity of the lipophilic ligand [3H]quinuclidinyl benzilate to m2 mAChR without a reduction of the total m2 mAChR number. The treatment also decreased the cell surface mAChR number to 14% with a slight reduction in its affinity. Desensitizing treatment of RBL-m2 cells inhibited the CCh-induced transient increase in levels of IP3 and intracellular Ca2+ concentration. The results suggested that the CCh-induced desensitization of m2 mAChR-mediated secretion is due to the receptor sequestration followed by blocking the increase in [Ca2+]i and that this desensitizing mechanism is receptor-subtype-specific.
Yogurt containing Lactobacillus gasseri OLL2716 (LG21 yogurt) is reported to improve Helicobacter pylori-induced gastric mucosal inflammation in clinical studies. However, other beneficial effects of LG21 yogurt have not been clarified. Therefore, we examined whether LG21 yogurt exhibits a gastroprotective action against acute gastric lesion or antral ulcer in rats. Moreover, the mechanism of gastroprotective action was also evaluated. After fasting, acute gastric lesions were induced by 0.6 M HCl. Gastric mucosal folds were stained by oral administration of methyl violet. Antral ulcers were induced by the combined administration of diethyldithiocarbamate and HCl in refed rats after fasting. LG21 yogurt was orally administered before HCl treatment or staining the mucosal folds. LG21 yogurt significantly and dose-dependently inhibited the formation of acute gastric lesions, and this gastroprotective action was attenuated by pretreatment with indomethacin. LG21 yogurt also significantly increased prostaglandin E2 generation in the gastric mucosa. Stained length of gastric mucosal fold was reduced by LG21 yogurt. Antral ulcer formation was also significantly inhibited by LG21 yogurt. From the above results, it was found that the ingestion of LG21 yogurt is useful for the prevention of gastric ulcer. Moreover, endogenous prostaglandin was suggested to be one of the gastroprotective mechanisms of LG21 yogurt.
We analyzed gene expression in rat anti-Thy1 antibody-induced glomerulonephritis by using the cDNA microarray method. Ninety-seven genes that differed by more than 1.5-fold intensity in comparison with the controls were selected. Cluster analysis showed that the expression of genes associated with inflammation reached maximum levels at 24 h, while genes involved in the development of fibrosis increased at 7 days after injection. Microarray analysis of animal disease models may be a powerful approach for understanding the gene expression programs that underlie these disorders.