Changes in the number of nitric oxide-synthesizing neurones on both sides of a chronic transection of the rat spinal cord.

Pain after chronic transection of the spinal cord is hypothesized to develop because of a hyperactivity in nociceptive neurones rostral to the lesion. One of the key substances in central nervous nociceptive processing is nitric oxide (NO). It has been demonstrated to tonically inhibit the background activity of dorsal horn neurones. Here, we show that in rats with chronic transection of the spinal cord there is a reduction of NO-synthesizing neurones on both sides of the lesion. This reduction is likely to be associated with a local lack of NO which could lead to an increased background activity of nociceptive dorsal horn neurones. The increased background activity of nociceptive neurones just rostral to the lesion might cause spontaneous pain that is perceived in segments close to the level of the lesion.

Protein kinase G expression in the small intestine and functional importance for smooth muscle relaxation.

In functional experiments, the nitric oxide (NO) donor N-morpholino-N-nitroso-aminoacetonitrile or the cGMP analog 8-(4-chlorophenylthio)-cGMP caused a concentration-dependent, tetrodotoxin-resistant relaxation of precontracted strips from rat small intestine. The inhibitory effect of both substances was completely blocked at lower concentrations and was significantly attenuated at higher concentrations by the selective cGMP-dependent protein kinase (cGK) antagonist KT-5823 (1 microM). cGK-I was identified by immunohistochemistry in circular and longitudinal muscle, lamina muscularis mucosae, and smooth muscle cells of the villi and in fibroblast-like cells of the small intestine. Additionally, there was staining of a subpopulation of myenteric and submucous plexus neurons. Double staining for neuronal NO synthase (nNOS) and cGK-I demonstrated a colocalization of these two enzymes. Western blot analysis of smooth muscle preparations and isolated nerve terminals demonstrated that these structures predominantly contain the cGK-Ibeta isoenzyme, whereas the cGK-Ialpha expression is about threefold less. The isoform cGK-II was entirely confined to mucosal epithelial cells. These results show that cGK-I is expressed in different muscular structures of the small intestine and participates in the NO-induced relaxation of gastrointestinal smooth muscle. The presence of cGK-I in NOS-positive enteric neurons further suggests a possible neuronal action site.

Regulation of VIP release from rat enteric nerve terminals: evidence for a stimulatory effect of NO.

The basal release of vasoactive intestinal polypeptide (VIP) from freshly prepared enriched synaptosomes was 159.1 +/- 17.3 fmol/mg protein (100%), which constituted 2.5% of the total VIP content. Basal VIP release was reduced by 65% by removal of external Ca2+. Release of VIP was stimulated by depolarization with KCl (65 mM, 143%) and in the presence of veratridine (10(-6) M, 184%), monensin (10(-5) M, 131%), and the Ca2+ ionophore A-23187 (10(-6) M, 160%). Stimulation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent mechanisms using isoproterenol (10(-6)-10(-4) M) and forskolin (10(-6) and 10(-5) M) had no stimulatory influence on VIP release. In contrast, sodium nitroprusside (10(-4) M, 198%), the nitric oxide (NO) donor 3-(morpholino)sydnonimine (10(-4) M, 155%), and the guanosine 3',5'-cyclic monophosphate (cGMP) analogue 8-bromo cGMP (10(-4) M, 196%) caused a significant release of VIP. L-Arginine (10(-3) M, 246%) also caused a significant increase of VIP release that was antagonized by the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (5 x 10(-4) M, 131%), which had no effect when given alone. The results demonstrate that VIP can be released from enriched synaptosomes by Ca(2+)-dependent mechanisms by NO agonists or NO-dependent mechanisms. It is speculated that this VIP release is induced by a presynaptic stimulatory mechanism of NO and this effect could enhance or contribute to the action of NO.

Locations of postganglionic nerve cells whose axons enter nerves originating from prevertebral ganglia.

Major nerve trunks that supply abdominal viscera contain axons of postganglionic neurons that originate in the coeliac ganglion, superior mesenteric ganglion, inferior mesenteric ganglia, hypogastric nerve ganglia and the sympathetic chain ganglia. Using the retrogradely transported neuronal marker Fast Blue, we mapped the distribution of labelled nerve cells after application of the dye to either the superior coeliac nerves, inferior coeliac nerves, superior mesenteric nerve, colonic nerves or hypogastric nerves. Distinctive patterns of nerve cell locations were associated with each nerve trunk. Within the coeliac ganglion, nerve cells that projected into the superior coeliac nerves were almost exclusively lateral, whilst neurons in the medial part of the coeliac ganglion and in the rostral pole of the superior mesenteric ganglion tended to project into the inferior coeliac nerves. Nerve cells located around the emerging superior mesenteric nerve in the superior mesenteric ganglion contributed the majority of axons to that nerve. Cells in both poles of the inferior mesenteric ganglia contributed the majority of postganglionic axons to the colonic nerves, but some cells in the caudal pole of the superior mesenteric ganglion also projected into the colonic nerves. Postganglionic axons in the hypogastric nerves originated from cells predominantly located in the caudal pole of the inferior mesenteric ganglion; however, cells in the rostral pole of the inferior mesenteric ganglia, the superior mesenteric, coeliac and hypogastric nerve ganglia also contributed some axons. Nerve cells of sympathetic chain ganglia projected into each of the nerve trunks; they were distributed rostro-caudally according to the nerve injected.

Locations and chemistries of sympathetic nerve cells that project to the gastrointestinal tract and spleen.

Retrograde tracing was used to determine the locations of sympathetic nerve cells whose axons project to the stomach, small intestine, caecum, proximal colon, distal colon and spleen of the guinea-pig. Projections from prevertebral ganglia were organotopically arranged within and between ganglia. The cranially located coeliac ganglion provided the major input to proximal gut regions; the distal gut received more caudal input, from superior and inferior mesenteric and the hypogastric nerve ganglia. Nevertheless, minor proportions of the innervation of some target organs arose from other than the closest ganglion and the caecum had input from each of the coeliac, superior mesenteric and inferior mesenteric ganglia. Topography within a ganglion was best defined in the coeliac, in which nerve cells whose axons projected to the spleen, stomach and duodenum were preferentially laterally located, whereas most of those projecting to the proximal colon were medial. Fewer neurons projected from paravertebral--compared with prevertebral--ganglia to abdominal viscera. Projections to the stomach came from all thoracic chain ganglia, those to the duodenum and spleen from lower thoracic ganglia and those to the large intestine from lumbar chain ganglia. It is suggested that the previously reported chemical topography of nerve cells in sympathetic ganglia might be secondary to their organotopic organization.

Direct tissue isoelectric focusing on mini ultrathin polyacrylamide gels followed by subsequent western blotting, enzyme detection, and lectin labeling as a tool for enzyme characterization in histochemistry.

Application of cryostat sections directly onto mini ultrathin polyacrylamide isoelectric focusing gels allows an elution of proteins out of the sections into the gels under conventional focusing conditions. Protein bands representing alkaline phosphatases can easily be identified on nitrocellulose after performing a modified Western blot procedure. Furthermore, carbohydrate residues of several isoforms of alkaline phosphatases separated by isoelectric focusing can be demonstrated in a single blot strip by subsequent incubation of this strip with substrates for alkaline phosphatase and peroxidase, the latter being employed as the enzyme to which the applied lectins are covalently linked. This simple and reproducible procedure is likely to enable histochemists to determine isoforms of enzymes from a single cryostat section.

Direct tissue isoelectric focusing on ultrathin polyacrylamide gels. Applications in enzyme, lectin and immunohistochemistry.

Application of cryostat sections directly onto ultrathin polyacrylamide gels and subsequent isoelectric focusing allows elution of proteins, glycoproteins and peptides out of the sections into the gels. The eluted compounds reveal clearly delineated band patterns in the polyacrylamide gels. The advantage of this method is that enzyme histochemical reactions can be directly performed in the gel and in the electroeluted tissue sections. Therefore, this method is suitable for specifying, in more detail, histochemical enzyme reactions and for detecting multiple forms of enzymes even from a single tissue section. Furthermore, the transfer of proteins, glycoproteins and peptides from the gel onto nitrocellulose by a modified Western blot procedure offers the possibility of checking findings obtained by lectin histochemistry and immunohistochemistry.

Lesion patterns of vasoactive intestinal polypeptide-containing neurons in the myenteric plexus induced by clamping or transsection of rat jejunum.

The acute reactions of vasoactive intestinal polypeptide (VIP)-containing neurons in the myenteric plexus of the rat jejunum in response to circumferential clamping or transection/reanastomosis were evaluated in a detailed time course of up to ten days. Whereas in sham-operated controls VIP immunostaining was confined exclusively to varicose fibers, either clamping or transection induced the appearance of strongly VIP-immunoreactive beaded neuronal processes and neuronal perikarya in the oral part of the lesion one day postoperatively. After five days strongly immunostained varicose fibers orientated in the longitudinal axis of the gut towards the lesion reappeared suggestive of the onset of regenerative processes.

Lectin-binding sites in the retina of the pig, baboon and cat.

There are notable interspecific differences in the lectin binding sites in the retina of mammals. In the porcine retina a distinct labeling of all the neurons of the stratum ganglionare is obtained by use of succinylated wheat-germ-agglutinin (WGA-S). In the baboon the stratum ganglionare does not label, though the entire inner plexiform layer can be demonstrated with WGA-S. In the cat WGA-S binds exclusively to the outer segments of the rods and cones. These specific differences will be compared with findings in the human retina [10] and discussed in connection with biochemical results obtained during our studies of the porcine retina.

Analysis of wheat germ agglutinin (WGA), Phaseolus vulgaris leucoagglutinin (PHA-L), and lens culinaris agglutinin (LCA) binding to isolated rat neocortical membrane glycoproteins and to brain tissue sections.

Different lectin transport directions--either anterograde or retrograde in tracing neuronal pathways--have been attributed to different sugar specificities of the lectins. To test this hypothesis, transmembrane neocortical glycoproteins were isolated to analyze their lectin-binding properties. The lectins tested exhibited a broad binding pattern to these glycoproteins. Arguing against the above-mentioned hypothesis, sugar residues of transmembranal glycoproteins probably are not exclusively responsible for the different transport directions observed in lectin tracing.