Kuo-Shyan Lu

Mouse Taste Buds Use Serotonin as a Neurotransmitter

Synapses between gustatory receptor cells and primary sensory afferent fibers transmit the output signal from taste buds to the CNS. Several transmitter candidates have been proposed for these synapses, including serotonin (5-HT), glutamate, acetylcholine, ATP, peptides, and others, but, to date, none has been unambiguously identified. We used Chinese hamster ovary cells stably expressing 5-HT2C receptors as biodetectors to monitor 5-HT release from taste buds. When taste buds were depolarized with KCl or stimulated with bitter, sweet, or sour (acid) tastants, serotonin was released. KCl- and acid-induced 5-HT release, but not release attributable to sweet or bitter stimulation, required Ca2+ influx. In contrast, 5-HT release evoked by sweet and bitter stimulation seemed to be triggered by intracellular Ca2+ release. These experiments strongly implicate serotonin as a taste bud neurotransmitter and reveal unexpected transmitter release mechanisms.

Epithelial inducible nitric oxide synthase causes bacterial translocation by impairment of enterocytic tight junctions via intracellular signals of Rho-associated kinase and protein kinase C zeta.

Objective:Gut barrier dysfunction and bacterial translocation occur in various disorders, including intestinal obstruction. Overexpression of inducible nitric oxide synthase is implicated in the pathogenesis of bacterial translocation, of which the molecular mechanism remains unclear. Epithelial permeability is regulated by tight junction reorganization and myosin light chain phosphorylation. Our aim was to investigate the roles of Rho-associated kinase and protein kinase C &zgr; in epithelial nitric oxide synthase-mediated barrier damage. Design:Animal study and cell cultures. Setting:Research laboratory. Subjects:BALB/c mice. Interventions:Mouse distal small intestine was obstructed in vivo by a 10-cm loop ligation in which vehicle, L-Nil (a nitric oxide synthase inhibitor), or Y27632 (a Rho-associated kinase inhibitor) was luminally administered. After obstruction for 24 hrs, intestinal tissues were mounted on Ussing chambers for macromolecular flux. Liver and spleen tissues were assessed for bacterial counts. Caco-2 cells were exposed to 1 mM S-nitroso-N-acetylpenicillamine (a nitric oxide donor) for 24 hrs, and transepithelial resistance and permeability were evaluated. Measurements and Main Results:Mice with intestinal obstruction displayed epithelial barrier dysfunctions, such as permeability rise and bacterial translocation, associated with tight junction disruption and myosin light chain phosphorylation. Increased inducible nitric oxide synthase and phosphorylated protein kinase C &zgr; were observed in villus epithelium. Enteric instillation of L-Nil and Y27632 attenuated the functional and structural barrier damage caused by intestinal obstruction. L-Nil decreased intestinal obstruction-induced myosin light chain, myosin phosphatase target subunit 1, and protein kinase C &zgr; phosphorylation, suggesting that inducible nitric oxide synthase is upstream of Rho-associated kinase and protein kinase C &zgr; signaling. The intestinal phosphorylated myosin light chain level did not increase in inducible nitric oxide synthase(−/−) mice following intestinal obstruction. In vitro studies showed that S-nitroso-N-acetylpenicillamine-induced transepithelial resistance drop and permeability rise was independent of cell apoptosis. Y27632 inhibited S-nitroso-N-acetylpenicillamine-induced myosin light chain phosphorylation and permeability rise. S-nitroso-N-acetylpenicillamine also triggered phosphorylation and membrane translocation of protein kinase C &zgr;. Inhibitory protein kinase C &zgr; pseudosubstrate blocked S-nitroso-N-acetylpenicillamine-induced tight junction reorganization, but not myosin light chain phosphorylation. Conclusions:Epithelial inducible nitric oxide synthase activates two distinct signals, protein kinase C &zgr; and Rho-associated kinase, to disrupt tight junctions leading to bacterial influx.

Involvement of NO/cGMP signaling in the apoptotic and anti-angiogenic effects of β-lapachone on endothelial cells in vitro

Neovascularization is an essential process in tumor development, it is conceivable that anti‐angiogenic treatment may block tumor growth. In angiogenesis, nitric oxide (NO) is an important factor which mediates vascular endothelial cell growth and migration. β‐Lapachone (3,4‐dihydro‐2,2‐dimethyl‐2H‐naphtho‐[1,2‐b]pyran‐5,6‐dione), a natural product extracted from the lapacho tree (Tabebuia avellanedae), has been demonstrated to possess anti‐cancer and anti‐viral effects. Whether β‐lapachone can induce endothelial cell death or has an anti‐angiogenic effect is still an enigma. We investigated the in vitro effect of β‐lapachone on endothelial cells, including human vascular endothelial cell line, EAhy926, and human umbilical vascular endothelial cells (HUVEC). Our results revealed that (1) the intracellular cGMP levels and the mitochondria membrane potential (MMP) decreased, and calpain and caspases were activated, during β‐lapachone‐induced endothelial cell death; (2) co‐treatment with calpain inhibitors (ALLM or ALLN) or the intracellular calcium chelator, BAPTA, but not the general caspase inhibitor, zVAD‐fmk, provided significant protection against apoptosis by preventing the β‐lapachone‐induced MMP decrease and cytoplasmic calcium increase; (3) addition of NO downregulated the β‐lapachone‐induced cGMP depletion and protected the cells from apoptosis by blocking the MMP decrease and the calcium increase; and (4) exogenous NO protects endothelial cells against the cell death induced by β‐lapachone, but not the anti‐angiogenic effect. From all the data above, we demonstrated that NO can attenuate the apoptotic effect of β‐lapachone on human endothelial cells and suggest that β‐lapachone may have potential as an anti‐angiogenic drug. J. Cell. Physiol. 211: 522–532, 2007.

In vitro and in vivo wound healing-promoting activities of β-lapachone

Impaired wound healing is a serious problem for diabetic patients. Wound healing is a complex process that requires the cooperation of many cell types, including keratinocytes, fibroblasts, endothelial cells, and macrophages. beta-Lapachone, a natural compound extracted from the bark of the lapacho tree (Tabebuia avellanedae), is well known for its antitumor, antiinflammatory, and antineoplastic effects at different concentrations and conditions, but its effects on wound healing have not been studied. The purpose of the present study was to investigate the effects of beta-lapachone on wound healing and its underlying mechanism. In the present study, we demonstrated that a low dose of beta-lapachone enhanced the proliferation in several cells, facilitated the migration of mouse 3T3 fibroblasts and human endothelial EAhy926 cells through different MAPK signaling pathways, and accelerated scrape-wound healing in vitro. Application of ointment with or without beta-lapachone to a punched wound in normal and diabetic (db/db) mice showed that the healing process was faster in beta-lapachone-treated animals than in those treated with vehicle only. In addition, beta-lapachone induced macrophages to release VEGF and EGF, which are beneficial for growth of many cells. Our results showed that beta-lapachone can increase cell proliferation, including keratinocytes, fibroblasts, and endothelial cells, and migration of fibroblasts and endothelial cells and thus accelerate wound healing. Therefore, we suggest that beta-lapachone may have potential for therapeutic use for wound healing.

Investigation of the Blood-Ganglion Barrier Properties in Rat Sympathetic Ganglia by Using Lanthanum Ion and Horseradish Peroxidase as Tracers

Vascular permeability in various rat sympathetic ganglia, including superior cervical ganglia, thoracic ganglia and the celiac-mesenteric ganglia (CMG) complex, was investigated by using lanthanum and horseradish peroxidase (HRP) as tracers with special attention to the neuronal and small granule-containing (SGC) cell area. After lanthanum perfusion, lanthanum tracer was present within the lumen of blood vessels. No lanthanum depositions were found in the extravascular space surrounding neurons in the superior cervical and thoracic ganglia. By contrast, an accumulation of lanthanum was observed in both luminal, abluminal and subendothelial surface of blood vessels in neuronal and SGC cell areas of the CMG complex and surrounding SGC cells in superior cervical ganglia. Injecting HRP revealed that all blood vessels of various sympathetic ganglia, either in neuronal or in SGC cell areas, were impermeable to HRP. HRP reaction product was limited to the vascular lumen and macrophages. The escape of HRP was obstructed by the junctional complex at intercellular clefts of endothelia and also by the diaphragms of the fenestrated capillaries associated with SGC cells. We conclude that there are different properties in the blood-ganglion barriers among rat sympathetic ganglia: (1) continuous capillaries in superior cervical ganglia and thoracic ganglia provide an efficient blood-ganglion barrier that prevents the penetration of tracers, and (2) capillaries in the CMG complex and in regions of the superior cervical ganglia that contain SGC cells possess a selective blood-ganglion barrier that discriminates between tracers based on their molecular sizes.

Overexpression of neuronal intermediate filament protein α-internexin in PC12 cells

The neuronal intermediate filaments include not only the neurofilament triplet proteins but also peripherin and α‐internexin. To determine whether neurite outgrowth is enhanced by α‐internexin, the cDNA of rat α‐internexin tagged with enhanced green fluorescent protein (EGFP) was transfected into a rat adrenal pheochromocytoma cell line PC12 that responds to nerve growth factor (NGF) by induction of the neuronal phenotype. Selected stable clones were induced by NGF and examined for expression patterns of neuronal intermediate filaments by Western blot and immunocytochemistry. Differentiating neurons were also collected after NGF induction for RT‐PCR analysis. Overexpressed α‐internexin‐EGFPs were found mainly in cell bodies and the proximal part of neurites. It was also found that overexpression of α‐internexin‐EGFPs enhanced the neurite outgrowth of PC12 cells at the early stages of NGF induction. Meantime, NF‐L and NF‐M were upregulated by the overexpression of α‐internexin‐EGFPs. Interestingly, α‐internexin‐EGFP‐transfected cells obviously detached from culture plates at the later stages of NGF induction. Massive IF accumulations, swelling mitochondria, and degenerating neurites with numerous electron‐dense granules were observed ultrastructurally in the α‐internexin‐EGFP‐transfected cells. In addition, neuronal death was also characterized positively by the TUNEL assay. These observations may imply that cell death was occurring in α‐internexin‐EGFP‐transfected cells. From this study, it could be suggested that α‐internexin plays an important role in neurite outgrowth and regulates the expression of other neurofilaments during neuronal development. Apoptosis‐like cell death could also be induced by the overexpression of α‐internexin‐EGFP in PC12 cells after NGF induction.

The distribution of PGP9.5, BDNF and NGF in the vallate papilla of adult and developing mice

The development and innervation of vallate papillae and taste buds in mice were studied using antibodies against the neuronal marker, protein gene product 9.5 (PGP 9.5), and against nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). PGP 9.5 immunohistochemical studies revealed that the earliest sign of median vallate papilla formation was an epithelial bulge at embryonic day 13 (E13), and at E14, a dense nerve plexus was found within the connective tissue core of the papilla. Thin nerve fibers penetrated the apical and medial trench wall epithelium of the papilla at E16 and a few of these began to invade the lateral trench wall epithelium at E17. At postnatal day 1 (P1), the newly formed taste buds were recognizable and a small number of PGP 9.5-immunoreactive (IR) cells appeared on the medial trench wall epithelium. The number of PGP 9.5-IR taste bud cells then increased gradually and reached the adult level at postnatal week 2. PGP 9.5 immunoreactivity increased systematically with age. NGF and BDNF immunoreactivity was first seen at the boundary between the columnar cells in the apical epithelium of the developing vallate papilla at E13, then in the medial and lateral trench walls at E15 (BDNF) or E18 (NGF). At P1, BDNF immunoreactivity was exclusively present in the newly formed taste buds of the medial trench wall. The number of BDNF-IR taste bud cells then increased gradually, reaching the adult level at P7. Similar degrees of NGF and BDNF immunoreactivity were seen in the developing vallate papilla. In the present study, we found that the vallate papilla was formed prior to its innervation, and we propose that initiation of papilla formation does not require any direct influence from the specific gustatory nerve. We also suggest that neurotrophins in the early developing vallate papillae might act as local tropic factors for the embryonic growth of nerve fibers to induce differentiation of the taste buds.

Enteric dysbiosis promotes antibiotic-resistant bacterial infection: systemic dissemination of resistant and commensal bacteria through epithelial transcytosis.

Antibiotic usage promotes intestinal colonization of antibiotic-resistant bacteria. However, whether resistant bacteria gain dominance in enteric microflora or disseminate to extraintestinal viscera remains unclear. Our aim was to investigate temporal diversity changes in microbiota and transepithelial routes of bacterial translocation after antibiotic-resistant enterobacterial colonization. Mice drinking water with or without antibiotics were intragastrically gavaged with ampicillin-resistant (Amp-r) nonpathogenic Escherichia coli (E. coli) and given normal water afterward. The composition and spatial distribution of intestinal bacteria were evaluated using 16S rDNA sequencing and fluorescence in situ hybridization. Bacterial endocytosis in epithelial cells was examined using gentamicin resistance assay and transmission electromicroscopy. Paracellular permeability was assessed by tight junctional immunostaining and measured by tissue conductance and luminal-to-serosal dextran fluxes. Our results showed that antibiotic treatment enabled intestinal colonization and transient dominance of orally acquired Amp-r E. coli in mice. The colonized Amp-r E. coli peaked on day 3 postinoculation and was competed out after 1 wk, as evidenced by the recovery of commensals, such as Escherichia, Bacteroides, Lachnospiraceae, Clostridium, and Lactobacillus. Mucosal penetration and extraintestinal dissemination of exogenous and endogenous enterobacteria were correlated with abnormal epithelial transcytosis but uncoupled with paracellular tight junctional damage. In conclusion, antibiotic-induced enteric dysbiosis predisposes to exogenous infection and causes systemic dissemination of both antibiotic-resistant and commensal enterobacteria through transcytotic routes across epithelial layers. These results may help explain the susceptibility to sepsis in antibiotic-resistant enteric bacterial infection.

Commensal bacterial endocytosis in epithelial cells is dependent on myosin light chain kinase-activated brush border fanning by interferon-γ.

Abnormal bacterial adherence and internalization in enterocytes have been documented in Crohn disease, celiac disease, surgical stress, and intestinal obstruction and are associated with low-level interferon (IFN)-γ production. How commensals gain access to epithelial soma through densely packed microvilli rooted on the terminal web (TW) remains unclear. We investigated molecular and ultrastructural mechanisms of bacterial endocytosis, focusing on regulatory roles of IFN-γ and myosin light chain kinase (MLCK) in TW myosin phosphorylation and brush border fanning. Mouse intestines were sham operated on or obstructed for 6 hours by loop ligation with intraluminally administered ML-7 (a MLCK inhibitor) or Y27632 (a Rho-associated kinase inhibitor). After intestinal obstruction, epithelial endocytosis and extraintestinal translocation of bacteria were observed in the absence of tight junctional damage. Enhanced TW myosin light chain phosphorylation, arc formation, and brush border fanning coincided with intermicrovillous bacterial penetration, which were inhibited by ML-7 and neutralizing anti-IFN-γ but not Y27632. The phenomena were not seen in mice genetically deficient for long MLCK-210 or IFN-γ. Stimulation of human Caco-2BBe cells with IFN-γ caused MLCK-dependent TW arc formation and brush border fanning, which preceded caveolin-mediated bacterial internalization through cholesterol-rich lipid rafts. In conclusion, epithelial MLCK-activated brush border fanning by IFN-γ promotes adherence and internalization of normally noninvasive enteric bacteria. Transcytotic commensal penetration may contribute to initiation or relapse of chronic inflammation.

Morphological, immunohistochemical and quantitative studies of murine brain mast cells after mating

The mast cell is one of the immune cells, and can be triggered behaviorally to increase in the CNS of the sexually active dove. In the present study, we used ICR mice to investigate the number of brain mast cells in mated (one male with three female mice), non-mated (housed with female mice, but no mating) and control (four male mice housed together in one cage) male mice. We found that at least 40% of mated male mice had significant more mast cells than the maximum value seen in the controls, and that a significant correlation existed between the distribution index of mast cells and the postcoitum date. These mast cells were especially numerous in the thalamus and velum interpositum (VIP). Morphological observations showed that the increased mast cells were ultrastructurally similar to those in the controls, and displayed gonadotropin-releasing hormone (GnRH)-like immunoreactivity. Based on the facts that the number of brain mast cells in the male mice increased significantly after mating and that the change in the distribution of mast cells in the VIP and the thalamic parenchyma correlated well with time postcoitum, we speculate that, after mating, mast cells may migrate from the VIP to the thalamic parenchyma along the vascular tree of the brain. These results strongly suggest that mast cells are involved in the interaction among the immune, endocrine, and nervous systems in the mated male mouse brain.