The importance of being outer: consequences of the distinction between the outer and inner surfaces of flavivirus glycoprotein E

Glycoprotein E of West Nile, dengue and other flaviviruses is the principal stimulus for the development of neutralizing antibodies and contains a fusion peptide responsible for inserting the virus into the host cell membrane. This glycoprotein lies flat on the surface of the virion and therefore only epitopes on the outer or lateral surface are important immunogens. Changes in antigen recognition after exposure of the virus to low pH have yielded clues to the fusion process.     

External adrenergic innervation of the three neuron types of dogiel in the plexus myentericus and the plexus submucosus externus of the porcine small intestine

Summary For the simultaneous demonstration of intramural enteric ganglion cells and the adrenergic nerve fibres in the porcine small intestine a combined histochemical method was developed using a hypertonic solution, the main chemicals of which were glyoxylic acid, Nitro-BT^* and NADH. By means of the enzymatic histochemical method reaction for the NADH-dependent dehydrogenase activity with Nitro-BT as an electron acceptor, the identification of the three neuron types of Dogiel (i.e. type I, type II, type III) was for the first time realized in relation with the glyoxylic acid induced fluorescence (GIF) of the plexus myentericus (Auerbach) and the plexus submucosus externus (Schabadasch). Besides the close topographic relationship between the adrenergic varicose axons on the one hand and the perikarya and dendrites of the multidendritic uniaxonal type I cells characterized by radially oriented short and lamellar dendrites and the multidendritic uniaxonal type III cells, characterized by radially oriented long and tapering dendrites on the other hand, it is striking that for the adendritic multiaxonal type II cells the fluorescent varicose fibres adhere closer to the cell bodies and their processes. In principle, the relation between adrenergic varicose axons and neuron types is identical in plexus myentericus (Auerbach) and plexus submucosus externus (Schabadasch), yet with the exception that in the latter no type I neurons are observed.2,2-Di-p-nitrophenyl-5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-diphenylene) ditetrazolium chloride     

Stimulation of adenosine A1 and A2A receptors by AMP in the submucosal plexus of guinea pig small intestine

Actions of adenosine 5'-monophosphate (AMP) on electrical and synaptic behavior of submucosal neurons in guinea pig small intestine were studied with "sharp" intracellular microelectrodes. Application of AMP (0.3-100 microM) evoked slowly activating depolarizing responses associated with increased excitability in 80.5% of the neurons. The responses were concentration dependent with an EC(50) of 3.5 +/- 0.5 microM. They were abolished by the adenosine A(2A) receptor antagonist ZM-241385 but not by pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid, trinitrophenyl-ATP, 8-cyclopentyl-1,3-dimethylxanthine, suramin, or MRS-12201220. The AMP-evoked responses were insensitive to AACOCF3 or ryanodine. They were reduced significantly by 1) U-73122, which is a phospholipase C inhibitor; 2) cyclopiazonic acid, which blocks the Ca(2+) pump in intraneuronal membranes; and 3) 2-aminoethoxy-diphenylborane, which is an inositol (1,4,5)-trisphosphate receptor antagonist. Inhibitors of PKC or calmodulin-dependent protein kinase also suppressed the AMP-evoked excitatory responses. Exposure to AMP suppressed fast nicotinic ionotropic postsynaptic potentials, slow metabotropic excitatory postsynaptic potentials, and slow noradrenergic inhibitory postsynaptic potentials in the submucosal plexus. Inhibition of each form of synaptic transmission reflected action at presynaptic inhibitory adenosine A(1) receptors. Slow excitatory postsynaptic potentials, which were mediated by the release of ATP and stimulation of P2Y(1) purinergic receptors in the submucosal plexus, were not suppressed by AMP. The results suggest an excitatory action of AMP at adenosine A(2A) receptors on neuronal cell bodies and presynaptic inhibitory actions mediated by adenosine A(1) receptors for most forms of neurotransmission in the submucosal plexus, with the exception of slow excitatory purinergic transmission mediated by the P2Y(1) receptor subtype.     

Fine structural study of interstitial cells associated with the deep muscular plexus of the rat small intestine,with special reference to the intestinal pacemaker cells

Two types of interstitial cells have been demonstrated in close association in the deep muscular plexus of rat small intestine, by electron microscopy. Cells of the first type are characterized by a fibroblastic ultrastructure, i.e. a well-developed granular endoplasmic reticulum, Golgi apparatus and absence of the basal lamina. They form a few small gap junctions with the circular muscle cells and show close contact with axon terminals containing many synaptic vesicles. They may play a role in conducting electrical signals in the muscle tissue. Cells of the second type are characterized by many large gap junctions that interconnect with each other and with the circular muscle cells. Their cytoplasm is rich in cell organells, including mitochondria, granular endoplasmic reticulum and Golgi apparatus. They show some resemblance to the smooth muscle cells and have an incomplete basal lamina, caveolae and subsurface cisterns. However, they do not contain an organized contractile apparatus, although many intermediate filaments are present in their processes. They also show close contacts with axon terminals containing synaptic vesicles. These gap-junction-rich cells may be regular components of the intestinal tract and may be involved in the pacemaking activity of intestinal movement.     

Character of Auerbach's plexus receptors in the stomach and small intestine]

The motility of the stomach and jejunum in 8 fed dogs with the intact vagus nerves was registered by the balloon method. Subcutaneous injection of benzohexonium (0.125--0.5 ml of 2.5% soltuion) and atropine (0.12--0.25 ml of 0.1% solution) or metacine (0.125--0.25 ml of 0.1% solution) to 6 dogs proved to induce a transition from digestive motility to the periodic form after a transient depression of the digestive motility. The same effect followed injection of 0.5--1.0 ml of 0.1% atropine only in 2 dogs and 1.0 ml of 0.1% metacine in 1 dog. Since retention of periodic motility following food consumption was inherent for vagotomized dogs, a conclusion was drawn that the experimental dog had "pharmacologic vagotomy". It was suggested that the muscarine receptors on the Auerbach's plexus cells exceeded the nicotine receptor in number.     

Functional and structural differences between cultured outer and inner layer cells of bovine adrenal cortex

Summary Outer and inner layer cells of bovine adrenal cortex were cultured separately to compare cellular structural characteristics and functional differences. Outer layer cells were polygonal in shape with radially distributed lipid droplets in the cytoplasm, and produced mainly aldosterone and cortisol. The aldosterone production increased upon stimulation with angiotensin II or dibutyryl-cAMP. In contrast, inner layer cells were spindle-shaped and had fine diffused lipid droplets. They produced four times as much cortisol as outer layer cells but no aldosterone. Cortisol production increased with ACTH or dibutyryl-cAMP stimulation.When stimulated by ACTH or by dibutyryl-cAMP, both types of adrenocortical cells showed cellular retraction whereby the number of cytoplasmic lipid droplets decreased and microvilli on the cellular surface increased. At the same time, the transverse distribution of actin fibers disappeared and the microtubules changed their distribution pattern from circular to radial. Stimulation by angiotensin II, on the other hand, brought no marked structural changes.These results indicate that, in functional terms, the outer layer cells and the inner layer cells in this culture system reflect zona glomerulosa and zona fasciculata-reticularis, respectively.     

Topography, architecture and structure of the plexus submucosus externus (Schabadasch) of the porcine small intestine in scanning electron microscopy

Whole-mount preparations of the porcine small intestine, consisting of the tela submucosa and the adjacent lamina muscularis mucosae, were used for scanning electron-microscopic investigation of the plexus submucosus externus (Schabadasch) after enzymatic digestion, fixation and HCI hydrolysis. The present results confirm previous light-microscopic data and provide irrefutable proof that within the submucosal plexus, considered by most authors as one ganglionated nerve plexus situated in the entirety of the tela submucosa, two distinct nerve meshworks can be distinguished, one lying close to the lamina muscularis mucosae, i.e., the plexus submucosus internus (Meissner), and the other, i.e., the plexus submucosus externus (Schabadasch), situated in the outer region of the tela submucosa against the circular smooth muscle layer. In addition to the distinct location of both plexuses, they are quite different with regard to the pattern and diameter of their nerve strands and the number and appearance of their ganglia.     

Topography, architecture and structure of the plexus submucosus internus (Meissner) of the porcine small intestine in scanning electron microscopy

Scanning electron microscopy of whole-mount preparations of the tela submucosa in the porcine small intestine, examined after trypsin digestion, fixation and HCl hydrolysis, visualized a clear differentiation of the submucosal plexuses, i.e., the plexus submucosus internus (Meissner) and the plexus submucosus externus (Schabadasch). The distinctive features refer to the topography, number, size and shape of the ganglia and the number and diameter of the nerve strands. The plexus of Meissner is closely apposed to the external surface of the lamina muscularis mucosae by the enveloping connective tissue and by connecting strands penetrating the lamina muscularis mucosae. Three distinctive subdivisions of connecting strands can be identified. Since the glial cells covering the ganglia and connecting strands have been preserved, neither individual neuronal cells nor axons can be observed.     

Vascularization of the myenteric plexus in the small intestine of pigeons]

By means of light and electron microscopy vascularization of the myenteric plexus has been studied in the pigeon small intestine. Ganglia of the plexus, their cell composition, ultrastructure of neurons have been described. Links of the microcirculatory bed of the intramural ganglia are characterized, interrelations of capillaries with neurons are described, quantitative estimation of microhemovessels, surrounding the microcirculatory bed of the myenteric plexus in the intestinal wall in birds and mammalia.     

Expression of differentiated functions in the developing porcine small intestine

Heterologous cDNA clones were used as hybridization probes to define the temporal expression of intestinal functions during fetal and postnatal development in the pig. Northern hybridization analysis revealed the presence of the mRNAs for the cellular retinol binding protein CRBP II, for the digestive enzyme aminopeptidase N, and for the microvillar proteins villin and ezrin in the small intestine of both weaned and 40-day fetal pigs. The presence of these mRNAs suggests that at the end of the first third of gestation the pig fetal intestine is already exhibiting some characteristics of a differentiated epithelium. The mRNAs for the two fatty acid-binding proteins I-FABP and L-FAPB, both involved in the metabolism of long chain fatty acids, were detected only in the intestinal mRNA extracted from weaned animals, while that for the cellular retinol-binding protein CRBP I was expressed only in the fetal tissue. The temporal limits of expression of intestinal genes in the pig epithelium seem therefore more easily defined than in other experimental animals with shorter times of fetal development. To isolate pig genes expressed at different developmental stages during intestinal epithelial cell differentiation, a cDNA library was constructed from poly(A) + RNA extracted from mature pig intestine. This library was employed in the isolation of clones encoding CRBP II and L-FABP. The nucleotide sequence of the two pig cDNA clones was determined, and the sequences of the deduced proteins compared with their homologues from other species. The results of this analysis showed that the two pig clones share a high level of homology with human and rat homologues both at the DNA and at the protein level.     

Histochemical characterization of myenteric plexus in domestic fowl small intestine

The myenteric plexus of the domestic fowl (Gallus domesticus) small intestine was studied by means of silver staining, glyoxylic acid-induced fluorescence, the modified Koelle-Friedenwald method for the detection of acetylcholinesterase, NADH-diaphorase techniques and the unlabelled antibody method involving the use of an antiserum raised against GABA conjugated by glutaraldehyde to bovine serum albumin. The majority of the perikarya were in the ganglia, with an average density of 3370 +/- 942 nerve cells/cm2. Cholinesterase-positive and a few GABA-immunoreactive nerve cell bodies were seen in the myenteric ganglia, while fluorescent ganglion cells were not observed. In addition to AChE and GABA-positive nerve fibres, a rich fluorescent network of varicose and nonvaricose nerve fibres was detected, pointing to the presence of an extrinsic aminergic system in the domestic fowl myenteric plexus. Electron microscopic observations on nerve cells, axon profiles and varicosites with various vesicle populations were in good agreement with the histochemical findings.     

Total numbers of neurons in myenteric ganglia of the guinea-pig small intestine

Two techniques that are thought to stain all of the neurons in the myenteric ganglia of the intestine are NADH diaphorase histochemistry and immunhistochemistry using a nerve cell body antiserum. However, this assumption has never been directly verified. In the present study myenteric ganglia of the guinea-pig ileum were prepared as whole-mounts and stained with either of these techniques. All nerve cells that could be identified in the whole-mounts were counted. The whole-mounts were then embedded flat in resin and serially sectioned at 1 m. Nerve cells were identified and counted from the serial sections, and the data compared to those obtained from the whole-mounts. NADH diaphorase histochemistry did not reveal all the neurons at incubation times that gave selective staining. In contrast, nerve cell body antiserum stained the entire neuronal population. To determine the total number of nerve cell bodies/ganglion and the proportion of nerve cell bodies with calbindin immunoreactivity, whole-mounts that had been processed for calbindin immunohistochemistry were serially sectioned and reconstructed. The total number of neurons per myenteric ganglion was 105±10 (SE). Calbindin-immunoreactive neurons comprised about 20% of the myenteric neurons, which is considerably less than previous estimates, because previously the total population has been underestimated. The spatial density of myenteric neurons in the undistended ileum of the guinea-pig is 17300 nerve cells/cm².     

Shapes and projections of tertiary plexus neurons of the guinea-pig small intestine

The axons of neurons that innervate the longitudinal muscle of the small intestine in small mammals such as rabbit, rat, guinea pig and mouse form a network, the tertiary plexus, against the inner surface of the muscle. In general, because of their substantial overlap, it has not been possible to follow the ramifications of individual axons in the tertiary plexus. In the present work, the longitudinal muscle motor neurons were filled with marker dyes through an intracellular microelectrode, and their morphologies and projections were examined in whole-mount preparations of longitudinal muscle and myenteric plexus. Most neurons that were examined were in the small intestine (ileum and duodenum), but a few were examined in the distal colon. Neurons in all regions had similar morphologies and projections. The cell bodies were amongst the smallest in myenteric ganglia, with major and minor axes of 14 microns and 25 microns (mean, n = 40) in the plane of the myenteric plexus. Each neuron had a single axon that branched extensively in the tertiary plexus, most had multiple lamellar dendrites and a few had filamentous dendrites or a mixture of filamentous and lamellar dendrites. The mean area of muscle covered by an axon and its branches extended 1.6 mm orally to anally and 1.7 mm circumferentially. The area covered was 2.8 +/- 1.9 mm2 (mean +/- SD, n = 23). From the density of occurrence of cell bodies, it can be calculated that each point in the longitudinal muscle is innervated by the processes of about 100 motor neurons and is influenced by electrotonic conduction of signals through the muscle by about 300 motor neurons.     

ATP-induced currents in submucous plexus neurons of the guinea pig small intestine

ATP-induced membrane durrents in the submucous neurons of the guinea pig small intestine were studied using the whole-cell patch-clamp recording technique. Being applied at –50 mV. ATP activated an inward non-selective cationic current in 68.3% of the investigated neurons. An increase in ATP concentration within the 1–1,000 µM range resulted in the s-like increase in the amplitude of ATP-induced current. The EC₅₀ was 150.0±18.5 µM, while the Hill number was 1.6. The current was selectively activated by ATP and was not blocked by P2 purinoreceptor antagonist suramin (50–300 µM).,-Methylene-ATP (100–200 µM) and,-methylene-ATP (100–200µM), which are P2-purinoreceptor agonists, as well as adenosine (100–300 µM), exerted no effects. Reactive blue 2, if applied up to 4 min, enhanced ATP-induced current, while its longer application partially suppressed this current. In most submucous neurons, acetylcholine (ACh) likewise activated an inward cationic current. The amplitude of ACh-induced current was lower if ACh was applied during a long-lasting application of ATP than if ACh only was applied. Hexamethonium (50 µM), d-tubocurarine (20–40 µM), and trimethaphan (30 µM) completely and reversibly blocked ACh-induced currents, regardless of the presence of ATP, and did not affect ATP-induced currents. The results suggest that ATP-induced currents in submucous neurons are due to activation of a unique type of P2 purinoreceptors, which function in connection with nicotinic ACh receptors.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 100–110, March–June, 1996.     

Catabolism of neurotensin in the epithelial layer of porcine small intestine

The mammalian small intestine is both a source and a site of degradation of neurotensin. Metabolites produced by incubation of the peptide with dispersed enterocytes from porcine small intestine were isolated by high-performance liquid chromatography and identified by amino-acid analysis. The principal sites of cleavage were at the Tyr-11-Ile-12 bond, generating neurotensin-(1-11), and at the Pro-10-Tyr-11 bond, generating neurotensin-(1-10). The corresponding COOH-terminal fragments, neurotensin-(11-13) and -(12-13) were metabolized further. Formation of neurotensin-(1-11) and -(1-10) was completely inhibited by phosphoramidon (Ki = 6 nM), an inhibitor of endopeptidase 24.11, but not by captopril, an inhibitor of peptidyl dipeptidase A. Incubation of neurotensin with purified endopeptidase 24.11 from pig stomach also resulted in cleavage of the Tyr-11-Ile-12 and Pro-10-Tyr-11 bonds. A minor pathway of cell-surface-mediated degradation was the phosphoramidon-insensitive cleavage of the Tyr-3-Glu-4 bond, generating neurotensin-(1-3) and neurotensin-(4-13). No evidence for specific binding sites (putative receptors) for neurotensin was found either on the intact enterocyte or on vesicles prepared from the basolateral membranes of the cells. Neurotensin-(1-8), the major circulating metabolite, was not formed when neurotensin(1-13) was incubated with cells, but represented a major metabolite (together with neurotensin-(1-10] when neurotensin-(1-11) was used as substrate. The study has shown that degradation of neurotensin in the epithelial layer of the small intestine is mediated principally through the action of endopeptidase 24.11, but this enzyme is probably not responsible for the production of the neurotensin fragments detected in the circulation.