Immunohistochemical characterization of elastic system fibers in rat molar periodontal ligament.

Among elastic system fibers, oxytalan fibers are known as a ubiquitous component of the periodontal ligament, but the localization and role of elastin-containing fibers, i.e., elastic and elaunin fibers, has yet to be clarified. In this study, we immunohistochemically investigated the localization of elastin and fibrillin, major proteins of elastin-containing fibers in the periodontal ligament of rat lower first molars. At the light microscope level, distribution of elastin-positive fibers was not uniform but often concentrated in the vicinity of blood vessels in the apical region of the ligament. In contrast, fibrillin-positive fibers were more widely distributed throughout the ligament, and the pattern of their distribution was comparable to the reported distribution of oxytalan fibers. At the ultrastructural level, assemblies or bundles of abundant fibrillin-containing microfibrils were intermingled with a small amount of elastin. This observation indicated that elastin-positive fibers observed under the light microscope were elaunin fibers. No mature elastic fibers, however, were found in the ligament. These results show that the major components of elastic system fibers in the periodontal ligament of the rat mandibular first molar were oxytalan and elaunin fibers, suggesting that the elastic system fibers play a role in the mechanical protection of the vascular system.     

Electron microscopy of the mucosal plexus of the rat colon.

The ultrastructure of neurons, glial cells and axons of the mucosal plexus of the rat colon descendens was studied. Serial semithin sections and a re-embedding technique were used in order to localize the ganglia. The ganglia are free of blood vessels and connective tissue. The ratio of neurons to glial cells is approximately 1. Ganglia and nerve strands are enclosed by a basement membrane, without a well-defined perineural connective tissue. The neurons show a structure similar to other enteric plexus. Synaptic contacts were observed frequently in the neuropil, where nerve endings and varicosities show a diverse outfit in vesicles. The glial cells, which contain immunocytochemically detectable glial fibrillary protein, possess the same ultrastructural attributes in the intra- and extraganglionic localizations. In the nerves, axonic profiles and varicosities appear in close relation with glial cells or their processes. The distance between the nerves and their target cells, i.e. the enterocytes, is 0.5 microns or more with interposed basement membranes and fibroblasts.     

Stress fibers in situ in proximal tubules of the rat kidney

Actin bundles in proximal tubules of the rat kidney were examined by immunofluorescence and confocal laser microscopy with special reference to their three-dimensional distribution and identification as stress fibers. Renal tubular segments were prepared from the fresh renal cortex by simple homogenization and centrifugation, and fixed in formaldehyde for staining with fluorescent dye-labeled phalloidin. Segments of the proximal tubules could be identified easily on the bases of their diameter, the height of epithelial cells and prominent brush borders. Confocal laser microscopy clearly demonstrated the overall distribution of actin bundles in the whole-mount proximal tubular segments. Actin bundles in the basal cytoplasm of epithelial cells were observed to run parallel to each other and at a right angle to the tubular axis. In the stereo views reconstructed from serial optical sections, the basal actin bundles appeared as straight rods with both ends tapered. They varied in length and width and extended rather short distances of not more than 10 microns. Often, two or more actin bundles were longitudinally aligned in tandem. Some bundles showed irregular bandings along their length. Each bundle was composed of tightly packed actin filaments which could be decorated with heavy meromyosin subfragment-1 to display a bi-directional arrangement within the bundle. Immunostaining of cryostat sections showed that actin bundles contained myosin and vinculin. Enzymatically isolated proximal tubules contracted upon addition of Mg-ATP. These observations collectively suggest that the actin bundles at the base of renal proximal tubule epithelial cells can be listed among the examples of stress fibers in situ.     

Intracellular pH regulation in colonocytes of rat proximal colon

The regulation of intracellular pH (pH(i)) in colonocytes of the rat proximal colon has been investigated using the pH-sensitive dye BCECF and compared with the regulation of pH(i) in the colonocytes of the distal colon. The proximal colonocytes in a HEPES-buffered solution had pH(i)=7.24+/-0.04 and removal of extracellular Na(+) lowered pH(i) by 0.24 pH units. Acid-loaded colonocytes by an NH(3)/NH(4)(+) prepulse exhibited a spontaneous recovery that was partially Na(+)-dependent and could be inhibited by ethylisopropylamiloride (EIPA). The Na(+)-dependent recovery rate was enhanced by increasing the extracellular Na(+) concentration and was further stimulated by aldosterone. In an Na(+)- and K(+)-free HEPES-buffered solution, the recovery rate from the acid load was significantly stimulated by addition of K(+) and this K(+)-dependent recovery was partially blocked by ouabain. The intrinsic buffer capacity of proximal colonocytes at physiological pH(i) exhibited a nearly 2-fold higher value than in distal colonocytes. Butyrate induced immediate colonocyte acidification that was smaller in proximal than in distal colonocytes. This acidification was followed by a recovery phase that was both EIPA-sensitive and -insensitive and was similar in both groups of colonocytes. In a HCO(3)(-)/CO(2)-containing solution, pH(i) of the proximal colonocytes was 7.20+/-0.04. Removal of external Cl(-) caused alkalinization that was inhibited by DIDS. The recovery from an alkaline load induced by removal of HCO(3)(-)/CO(2) from the medium was Cl(-)-dependent, Na(+)-independent and blocked by DIDS. Recovery from an acid load in EIPA-containing Na(+)-free HCO(3)(-)/CO(2)-containing solution was accelerated by addition of Na(+). Removal of Cl(-) inhibited the effect of Na(+). In summary, the freshly isolated proximal colonocytes of rats express Na(+)/H(+) exchanger, H(+)/K(+) exchanger ((H(+)-K(+))-ATPase) and Na(+)-dependent Cl(-)/HCO(3)(-) exchanger that contribute to acid extrusion and Na(+)-independent Cl(-)/HCO(3)(-) exchanger contributing to alkali extrusion. All of these are likely involved in the regulation of pH(i) in vivo. Proximal colonocytes are able to maintain a more stable pH(i) than distal cells, which seems to be facilitated by their higher intrinsic buffer capacity.     

Histo- and immunocytochemical characterization of the neurons of the mucosal plexus in the rat colon.

The mucosal plexus of the rat colon descendens is constituted of a network of nerves that, in contrast to most other segments of the digestive tract, contains also ganglia. The ganglia, consisting of neurons and glial cells, are located in the basal part of the lamina propria at distances between 100 and 1,200 microns. They are not vascularized. The neurons in these ganglia were characterized by means of: (1) the histochemical demonstration of acetylcholinesterase (AChE) activity, (2) the immunocytochemical identification of neurofilament proteins (NFP; 200 kD) and (3) their ultrastructure. The glial cells, which were AChE negative, could be distinguished from the neurons by differences in size and chromatin pattern. All neurons of the mucosal plexus reveal AChE activity in the perikaryon, but only parts of the axons are AChE positive. NFP-like immunoreactivity was detected in the perikarya but only in a minor part of the axons. These findings confirm previous light-microscopical observations and add new evidence for the existence of neurons (ganglia) in the mucosal plexus of the rat colon.     

Distribution of elastic system fibers in the peripheral nerves of mammals

Various nerves of 6 representative species of mammals (including the human) were studied by the comparative association of the selective staining methods of light microscopy with the ultrastructural observation after tannic acid-glutaraldehyde fixation, which provided a reliable means of characterizing the different elastic system fibers. Although mature elastic fibers are not present in nerves, elastic-related fibers are frequently observed: oxytalan fibers are found mainly in the endoneurium, whereas elaunin fibers predominate in the epineurium. These fibers are longitudinally disposed, in a parallel orientation to the axons. The fact that these findings were consistently observed in the nerves of all species studied argues strongly in favor of the existence of a uniform structural pattern of distribution of elastic system fibers in nerves as a general phenomenon in mammals.     

Essential role of nitric oxide in descending inhibition in the rat proximal colon

Possible mediators of descending inhibition in the rat proximal colon were studied. Localized distension with a small balloon caused relaxation of the circular muscle on the anal side of the distended region. This relaxation was still observed after the colonic segment had been desensitized to ATP, neurotensin and vasoactive intestinal peptide, so these compounds seem unlikely to mediate descending inhibition. Nitro-arginine inhibited the relaxation induced by the distension, and L-arginine counteracted the effect of nitro-arginine. Nitric oxide, isoamylnitrate and sodium nitroprusside caused relaxation. These results strongly suggest an essential role of nitric oxide in descending relaxation in the rat proximal colon.     

Epithelial and mucosal preparations of canine proximal colon in ussing chambers: comparison of responses

A novel epithelial preparation from proximal canine colon, devoid of its underlying muscularis mucosa, was studied in Ussing chambers and its responses to added agonists compared with those of a conventional full mucosal preparation. We assumed that the responses elicited from the former would reflect direct action of agonists on epithelial cells, and from the latter both direct and indirect effects mediated by functioning nerves in the attached submucosal plexus. Responses of the two preparations to tetrodotoxin, ouabain, scorpion venom, and field stimulation were different, the differences being explicable on the basis of functioning nerves in the mucosal preparation. Histamine stimulated both preparations; the increase in short-circuit current seen with the mucosal but not the epithelial preparation was inhibited by tetrodotoxin. Serotonin was a better stimulant of the epithelial preparation. Basal absorptions of both Na+ and Cl- were seen in both mucosa and epithelium, but net absorptions were significantly greater in the latter, suggesting tonic neuronal inhibition of absorption in the mucosa. These two preparations would prove useful in analysing direct and indirect effects of agonists on colonic transport.     

Multiple roles for elastic fibers in the skin.

Dermal elastic fibers are believed to have a primary role in providing elastic stretch and recoil to the skin. Here we compare the structural arrangement of dermal elastic fibers of chick skin and different animal species. Most elastic fibers in chick skin are derived from cells that line the feather follicle and/or smooth muscle that connects the pterial and apterial muscle bundles to feather follicles. Elastic fibers in the dermis of animals with single, primary hair follicles are derived from cells lining the hair follicle or from the ends of the pili muscle, which anchors the muscle to the matrix or to the hair follicle. Each follicle is interconnected with elastic fibers. Follicles of animals with primary and secondary (wool) hair follicles are also interconnected by elastic fibers, yet only the elastic fibers derived from the primary follicle are connected to each primary follicle. Only the primary hair follicles are connected to the pili muscle. Human skin, but not the skin of other primates, is significantly different from other animals with respect to elastic fiber organization and probably cell of origin. The data suggest that the primary role for elastic fibers in animals, with the possible exception of humans, is movement and/or placement of feathers or hair.     

Influence of VIP on the number of enterochromaffin and mucosal mast cells in the colon of the rat.

The possibility that VIP (Vasoactive intestinal peptide) could influence the enterochromaffin (EC) cell secretion of serotonin (5HT) and the action of VIP on the mast cell population of lamina propria were investigated in Wistar rat colon infused with a short chain fatty acid solution (sodium acetate), during a 1 h period. Under the action of an intravenous injection of synthetic porcine VIP, 14 micrograms/kg/h), the number of EC cells diminished significantly in the cecum and left colon, when compared to non-injected animals, both infused with a sodium acetate solution. At the same time, the number of mucosal mast cells in the crypts and lamina propria decreased significantly in the cecum. The postulate we put forward is that these VIP-induced changes are exerted through the stimulation of 5HT released from EC cells not only under normal physiological conditions but probably also under pathological conditions.     

Ultrastructural immunolocalization of elastic fibers in rat blood vessels using the protein A-gold technique

Identification of elastic fibers at the ultrastructural level is accomplished by a post-embedding immunohistochemical technique using the protein A-colloidal gold method. Antisera against elastins from human dermis and rat aorta have been characterized by radioimmunoassay and then applied to thin sections of rat blood vessels. Two fixative solutions and two embedding media have been tested. Both antibodies bind to elastic fibers of normal arteries and veins, indicating crossreactions among organs and species. The high sensitivity of this method is demonstrated by its application to the detection of neo-elastogenesis in the intimal thickening of aortic grafts.     

Endocrine cells in the rabbit colon mucosal epithelium

The epithelium of the rabbit colon was studied by light and electron microscopy. The highest number of endocrinocytes in colon are observed in terminal parts of colon, i.e. in a distal part of appendix (135 +/- 15 cells/mm2) and in rectum (142 +/- 20), to decrease in the ileocaeal region (caecum proxinmal part--39 +/- 9, colon proximal part--56 +/- 9), where the least number of cells was marked. Agrentaffin cells (EC) number the same way, however, with a weaker difference in the number of cells between terminal departments and ileocaeal region. An electron microscope study of mucosal epithelium of the colon enabled us to identify 5 types of endocrinocytes. I-III types: EC-, D- and L-cells. IV and V are seldom met types, the same way as the "mixed" cells have been indentified. Whose cytoplasm simultaneously contained both mucous and endocrine granules. The received data show a certain degree of similarity in the endocrine apparatus of the rabbit with that of humans, although essential differences exists in regards of the appendix pattern.     

The effect of different prostaglandins on gastric mucosal intracellular second messenger system in the rat.

After an oral administration of 100 micrograms/kg dose, the investigated prostaglandins: PGF2 alpha, PGE2 and a synthetic PGE2 derivative: FCE-20700, exerted a significant effect on cAMP and cGMP content of both parts (antral and fundic) of gastric mucosa, resulting in an elevated cAMP/cGMP ratio, while 6-keto-PGF1 alpha, the stable break-down product of prostacyclin, was inactive. Since the above-mentioned phenomenon seems to be proportionate to the cytoprotective (anti-ulcerogenic) property of the investigated prostaglandins, this cAMP/cGMP ratio "shift" is interpreted as a probable (molecular) sign of the reparative, (anti-ulcerogenic) processes.     

Effects of VIP and NO on the motor activity of vascularly perfused rat proximal colon

The effects of vasoactive intestinal polypeptide (VIP) and nitric oxide (NO) on the motor activity of the rat proximal colon were examined in an ex vivo model of vascularly perfused rat proximal colon. VIP reduced motor activity and this inhibitory effect was not altered by either atropine, hexamethonium, tetrodotoxin (TTX) nor TTX plus acetylcholine (ACh), but was completely antagonized by NO synthase inhibitor N(G)-nitro-L-arginine (L-NA) and by VIP receptor antagonist, VIP(10-28). These results suggest that VIP may exert a direct inhibitory effect on the motor activity of the rat proximal colon via a VIP receptor located on the smooth muscle and this effect is mediated by NO but not by cholinergic pathways. Atropine and hexamethonium reduced but ACh stimulated motor activity and the effect of ACh was not changed by TTX, suggesting that the cholinergic pathway may exert a direct stimulatory effect on motor activity. Single injection of TTX, VIP(10-28) or L-NA induced a marked increase in motor activity, suggesting that the motor activity of rat proximal colon is tonically suppressed by VIP and NO generating pathways, and elimination of inhibitory neurotransmission by TTX may induce an abnormal increase of the motor activity. The interaction between VIP and NO in regulation of motor activity was further examined by a measurement of NO release from vascularly perfused rat proximal colon. Results showed that NO release was significantly increased during infusion of VIP and this response was reversed by L-NA. These results suggest that VIP generating neurons may inhibit colonic motility by stimulating endogenous NO production in either smooth muscle cells or nerve terminals.     

Glycosaminoglycans in rat mucosal mast cells.

Rats were infected with the nematode Nippostrongylus brasiliensis, resulting in an approx. 5-fold increase in the number of mucosal mast cells and the histamine content of the intestinal (jejunum) wall. After injection of the infected animals with inorganic [35S]sulphate, a similar increase in the yield of labelled intestinal glycosaminoglycans was observed, compared with uninfected control rats. Autoradiography showed a highly selective labelling of the numerous mucosal mast cells and of the few connective-tissue mast cells in the subserosal region of the bowel. Analysis of the labelled polysaccharide from the infected animals showed that almost 60% of this material consisted of oversulphated galactosaminoglycan, whereas heparin-related polysaccharides accounted for only 13%. The galactosaminoglycan contained 4-monosulphated and 4,6-disulphated N-acetylgalactosamine residues in approx. 5:1 molar ratio, both being linked to D-glucuronic acid residues; the occurrence of L-iduronic acid units could not be excluded. No significant difference in structure was found between this polysaccharide and the corresponding component isolated from uninfected rats. It is concluded that the major polysaccharide produced by rat mucosal mast cells in vivo is an oversulphated galactosaminoglycan rather than heparin.     

Effect of age on NADPH-diaphorase-containing myenteric neurones of rat ileum and proximal colon

The effect of age on the distribution of NADPH-diaphorase-containing neurones was investigated in the myenteric plexus of ileum and proximal colon of embryonic day-19 rats, as well as in rats at postnatal day 4, 6 months and 26 months. The mean percentage of NADPH-diaphorase-stained neurones per ganglion was established using protein gene product 9.5 (protein found in most if not all neurones)-immunostained neurones as 100%. The results revealed that there was a significant relative increase in NADPH-diaphorase-positive neurones with increasing age in the myenteric plexus of proximal colon with nearly all protein gene product 9.5-immunoreactive neurones staining for NADPH-diaphorase in 26-month-old rats. This was in marked contrast with the ileum, where no significant relative increase in NADPH-diaphorase-positive neurones was seen in aged rats. The implications of these findings in relation to programmed cell survival and cell death are discussed.