Nerve-mediated action of forskolin on guinea pig ileal mucosa

Summary The effects of forskolin on myenteric neuronal activity and mucosal function were examined in guinea pig ileum. Forskolin increased the excitability of myenteric neurons, and increased mucosal chloride secretion by stimulating enteric neurons as well as by acting directly on enterocytes.Acknowledgment. The authors thank E. Martens, S. Bowen for technical assistance and N. Schaad for secretarial assistance. This work was supported by National Institutes of Health Grants, F32-AM07301, K-104-AM1004, RO1-AM29699, RO1-AM26742 and an Eloise Gerry Fellowship.     

Effects of methylene blue on electrical behavior of myenteric neurons

Summary Intracellular recording methods were used to investigate the action of methylene blue on electrical behavior of myenteric neurons in guinea pig small intestine. The neurophysiological studies were done in parallel with studies on contractile activity of the intestinal musculature. Methylene blue depolarized the membranes, increased the input resistance, augmented excitability and reduced postspike hyperpolarizing potentials in AH/Type 2 myenteric neurons. These effects, with the exception of suppression of postspike hyperpolarization, were reversed by exposure to elevated calcium. The mechanism of action of methylene blue appeared to be suppression of calcium-dependent potassium conductance in the neuronal membranes. The neuronal action of methylene blue was manifest as a release of excitatory neurontransmitter substances with evoked contraction of the small intestinal longitudinal muscle.     

Effects of methylene blue on electrical behavior of myenteric neurons

Intracellular recording methods were used to investigate the action of methylene blue on electrical behavior of myenteric neurons in guinea pig small intestine. The neurophysiological studies were done in parallel with studies on contractile activity of the intestinal musculature. Methylene blue depolarized the membranes, increased the input resistance, augmented excitability and reduced postspike hyperpolarizing potentials in AH/Type 2 myenteric neurons. These effects, with the exception of suppression of postspike hyperpolarization, were reversed by exposure to elevated calcium. The mechanism of action of methylene blue appeared to be suppression of calcium-dependent potassium conductance in the neuronal membranes. The neuronal action of methylene blue was manifest as a release of excitatory neurontransmitter substances which evoked contraction of the small intestinal longitudinal muscle.     

The enteric neural receptor for 5-hydroxytryptamine

An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using 3H-5-HT as a radioligand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of 3H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these 3H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of 3H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of 3H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of 3H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

The enteric neural receptor for 5-hydroxytryptamine

Summary An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using³H-5-HT as a radiologand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of³H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these³H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of³H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of³H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of³H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

Neuropeptide Y in the guinea-pig biliary tract

High concentrations of neuropeptide Y (NPY) have been demonstrated in the gall bladder (16.7 +/- 5.4 pmol/g), cystic duct (25.4 +/- 9.2 pmol/g) and common bile duct (54.7 +/- 11.5 pmol/g) of the guinea-pig using a recently developed radioimmunoassay. Immunoreactive NPY containing nerves were demonstrated in all layers of the biliary tree using immunocytochemistry, being particularly dense in the myenteric and mucosal plexuses.     

Increase in neuronal nitric oxide synthase content of the gastroduodenal tract of diabetic rats

This study examined the changes occurring in the pattern of distribution and expression of neuronal nitric oxide synthase (nNOS)-positive nerves in the gastroduodenal tract of streptozotocin-induced diabetic rats. The ganglion cells of the myenteric plexus of the gastric antrum of normal rats contain nNOS. We also observed nNOS-positive neurons and fibres in the myenteric plexus of the duodenum of normal rats. After the onset of diabetes, the number and intensity of staining of nNOS-positive nerve profiles in the gastric antrum and duodenum did not change significantly. However, Western blotting showed a significant increase in the expression of nNOS after the onset of diabetes. In conclusion, diabetes of 4 and 32 weeks duration induced an increase in the tissue content of nNOS in the gastroduodenum of rat. The increase in the level of nNOS in the gastroduodenum of diabetic rats may explain why impaired gastric emptying is common in patients with diabetes.     

Neuropeptide Y in the guinea-pig biliary tract

Summary High concentrations of neuropeptide Y (NPY) have been demonstrated in the gall bladder (16.7±5.4 pmol/g), cystic duct (25.4±9.2 pmol/g) and common bile duct (54.7±11.5 pmol/g) of the guinea-pig using a recently developed radio-immunoassay. Immunoreactive NPY containing nerves were demonstrated in all layers of the biliary tree using immunocytochemistry, being particularly dense in the myenteric and mucosal plexuses.Acknowledgments. JMA is a recipient of a Wellcome Trust Training Fellowship. JG is a visiting scholar from the Department of Pathology, Peking Medical College, Peking, China.     

Histamine H1- and H2-receptor-mediated gastric microcirculatory effects in the aetiology of stress ulceration in the rat stomach

Stress produced severe mucosal ulcers, increased mucosal microcirculation and lowered mast cell counts in the glandular wall of rat stomachs. Mepyramine i.m. or metiamide i.p. effectively prevented both ulceration and microcirculatory changes but not stress-reduced mast cell counts.     

Local and target-derived actions of neurotrophins during peripheral nervous system development

Neurotrophic factors are present in limiting quantities, and neurons that obtain an adequate supply of the required neurotrophic factor survive whereas those that compete unsuccessfully die. Analysis of null mutant mice for neurotrophins and Trk receptors as well as in vivo experiments in ovo in the chick applying exogenous neurotrophins or neutralising antisera have significantly increased knowledge of the roles they play during development. This review focuses on recent advances in understanding the various roles of neurotrophins in dorsal root ganglion sensory neuron development at different times in embryonic development - an early local role for differentiation of the sensory precursor cells and a later survival-promoting target-derived role for the mature neurons. Neurotrophic factors are present in limiting quantities, and neurons that obtain an adequate supply of the required neurotrophic factor survive whereas those that compete unsuccessfully die. Analysis of null mutant mice for neurotrophins and Trk receptors as well as in vivo experiments in ovo in the chick applying exogenous neurotrophins or neutralising antisera have significantly increased knowledge of the roles they play during development. This review focuses on recent advances in understanding the various roles of neurotrophins in dorsal root ganglion sensory neuron development at different times in embryonic development - an early local role for differentiation of the sensory precursor cells and a later survival-promoting target-derived role for the mature neurons.     

The topography of posttetanic potentiation in guinea-pig ileum

The myenteric plexus-longitudinal muscle preparation of the guinea-pig ileum offers, by its anatomical arrangement, the possibility of studying a new aspect of posttetanic potentiation (PTP); its topography. Evidence was sought and obtained that during PTP more distal junctional sites of cholinergic nerve terminals may be recruited into the transmitter secretion process.     

Further support for the postsynaptic action of substance P and its blockade with baclofen in neurons of the guinea-pig hypothalamus in vitro

Effects of substance P on neurons of the guinea-pig hypothalamus in vitro and antagonism between substance P and baclofen were investigated. Substance P increased the firing rate of neurons in the medium containing 0 mM Ca2+ and 12 mM Mg2+. The excitatory action of substance P was antagonized by a low dose of baclofen whereas that of acetylcholine was not antagonized even by much higher doses of baclofen.     

The right neuron at the wrong place: biology of heterotopic neurons in cortical neuronal migration disorders, with special reference to associated pathologies

During the development of the neocortex, neurogenesis and neuronal differentiation occur in two separate locations. Thus neurons have to migrate through the future white matter. Arrested or excessive migration leads neurons to differentiate in a heterotopic position. Such neuronal migration disorders (NMDs) occur sporadically in normal development but are markedly increased as a consequence of genetic defects or after exposure to toxic drugs during the period of migration. Anatomofunctional studies in rodents with NMDs have revealed that heterotopic neurons form essentially normal afferent and efferentconnections, which has been interpreted as evidence that the connectionpattern of cortical neurons is specified prior to migration. In addition, recent data show that heterotopic neurons can be contacted by environmental, that is local, fibres that normally never innervate the neocortex. This dual connectivity leads heterotopias to form bridges between their environmental and original network. Such an abnormal pattern of connectivity could contribute to the pathophysiology of disorders associated with NMDs such as epilepsy. Received 16 December 1998; received after revision 5 February 1999; accepted 9 February 1999     

Failure of coffee to inhibit the pharmacodynamic activity of morphine in vivo

Summary High doses of caffeine-containing as well as decaffeinated instant coffee neither inhibited morphine-induced analgesia in mice nor the morphine-induced fall of blood pressure, heart rate and respiratory rate in rats. On the contrary, caffeine-containing coffee even enhanced the analgesic effects of morphine in mice. Coffee thus does not exhibit opiate-antagonizing activity in the whole organism in vivo. The very weak morphine-antagonistic efficacy of coffee powder in the myenteric plexus-longitudinal muscle preparation from the guinea pig ileum is of no practical importance.     

Ethanol impairs insulin-stimulated mitochondrial function in cerebellar granule neurons

Ethanol impairs insulin-stimulated survival and mitochondrial function in immature proliferating neuronal cells due to marked inhibition of downstream signaling through P13 kinase. The present study demonstrates that, in contrast to immature neuronal cells, the major adverse effect of chronic ethanol exposure (50 mM) in post-mitotic rat cerebellar granule neurons is to inhibit insulin-stimulated mitochondrial function (MTT activity, MitoTracker Red fluorescence, and cytochrome oxidase immunoreactivity). Ethanol-impaired mitochondrial function was associated with increased expression of the p53 and CD95 pro-apoptosis genes, reduced Calcein AM retention (a measure of membrane integrity), increased SYTOX Green and propidium iodide uptake (indices of membrane permeability), and increased oxidant production (dihydrorosamine fluorescence and H2O2 generation). The findings of reduced membrane integrity and mitochondrial function in short-term (24 h) ethanol-exposed neurons indicate that these adverse effects of ethanol can develop rapidly and do not require chronic neurotoxic injury. A role for caspase activation as a mediator of impaired mitochondrial function was demonstrated by the partial rescue observed in cells that were pre-treated with broad-spectrum caspase inhibitors. Finally, we obtained evidence that the inhibitory effects of ethanol on mitochondrial function and membrane integrity were greater in insulin-stimulated compared with nerve growth factor-stimulated cultures. These observations suggest that activation of insulin-independent signaling pathways, or the use of insulin sensitizer agents that enhance insulin signaling may help preserve viability and function in neurons injured by gestational exposure to ethanol.     

慢病毒介导的CBS基因过表达对原代皮质神经元β淀粉样蛋白分泌的影响

目的通过过表达手段上调胱硫醚β-合成酶(Cystathionine-β-synthase,CBS)在原代神经元细胞中的表达,观察其对原代神经元β淀粉样蛋白(Aβ)分泌的影响。方法使用过表达CBS慢病毒载体感染体外培养的原代皮质神经元,设定为慢病毒介导阳性(CBS)组、另设阴性对照慢病毒感染(NC)组、未经慢病毒感染(CON)组。采用实时荧光定量PCR检测CBS基因mRNA的表达,Western blot检测CBS蛋白的表达,采用Elisa检测Aβ40的表达水平。结果使用过表达CBS的慢病毒载体感染体外培养的神经元,实时荧光定量PCR检测其mRNA的表达上调明显升高,与NC组和CON组相比差异有统计学意义(P0.01),Western blot结果显示蛋白表达升高与定量PCR一致,Elisa检测感染72小时后CBS组、NC组、CON组Aβ40的分泌分别为(182.19±24.52)ng/L、(411.90±12.43)ng/L、(390.41±54.53)ng/L,CBS组与NC组和CON组相比差异有统计学意义(P0.05)。结论慢病毒介导的CBS基因过表达可降低原代皮质神经元Aβ40的分泌。     

Failure of coffee to inhibit the pharmacodynamic activity of morphine in vivo

High doses of caffeine-containing as well as decaffeinated instant coffee neither inhibited morphine-induced analgesia in mice nor the morphine-induced fall of blood pressure, heart rate and respiratory rate in rats. On the contrary, caffeine-containing coffee even enhanced the analgesic effects of morphine in mice. Coffee thus does not exhibit opiate-antagonizing activity in the whole organism in vivo. The very weak morphine-antagonistic efficacy of coffee powder in the myenteric plexus-longitudinal muscle preparation from the guinea pig ileum is of no practical importance.     

Histamine H1- and H2-receptor-mediated gastric microcirculatory effects in the aetiology of stress ulceration in the rat stomach

Summary Stress produced severe mucosal ulcers, increased mucosal microcirculation and lowered mast cell counts in the glandular wall of rat stomachs. Mepyramine i.m. or metiamide i.p. effectively prevented both ulceration and microcirculatory changes but not stress-reduced mast cell counts.Acknowledgment. The authors thank Dr W.A.M. Duncan (Smith, Kline and French Labs Ltd, England) for the generous gift of metiamide.     

Intestinal epithelial barrier and mucosal immunity

The mucosal immune system maintains a delicate balance between providing robust defense against infectious pathogens and, at the same time, regulating responses toward innocuous environmental and food antigens and commensal microbes. The Peyer's patch (PP) has been studied in detail as a major inductive site for mucosal immunity within the small intestine. While the mechanisms responsible for the induction of mucosal immunity versus tolerance are not yet fully understood, recent studies have highlighted mucosal dendritic cells (DCs) as regulators of the immune responses to orally administered antigens. Here we discuss recent studies that describe the role of PP DCs in immune induction and speculate on the mechanism by which the resident DCs regulate T cell and immunoglobulin A (IgA) responses in the gastrointestinal mucosa.     

Intestinal epithelial barrier and mucosal immunity

The mucosal immune system acts as a first line of defense against bacterial and viral infections while also playing a crucial role in the establishment and maintenance of mucosal homeostasis between the host and the outside environment. In addition to epithelial cells and antigen-presenting cells (dendritic cells and macrophages), B and T lymphocytes form a dynamic mucosal network for the induction and regulation of secretory IgA (S-IgA) and cytotoxic T lymphocyte (CTL) responses. This review seeks to shed light on the pathways of induction and regulation of these responses and to elucidate the role they simultaneously play in fending off pathogen invasion and maintaining mucosal homeostasis.