Neurokinin A-like immunoreactivity in rat primary sensory neurons; coexistence with substance P

Rat spinal cord, dorsal root ganglia and skin were investigated employing immunohistochemical technique with specific antisera to neurokinin A and substance P. Neurokinin A-like immunoreactivity was detected in the spinal dorsal horn and skin with a similar distribution pattern as that of substance P-like immunoreactivity. After dorsal root transection a parallel decrease of neurokinin A and substance P-like immunoreactivity was observed in the dorsal horn. Using colchicine pretreatment a population of neurokinin A positive cell bodies was seen in the dorsal root ganglia, and by comparison of consecutive sections of the same cells stained for substance P it was revealed that these neurons also display substance P-like immunoreactivity. However, substance P-, but not neurokinin A-, immunoreactive cells were also observed. It is concluded that neurokinin A- and substance P-like immunoreactivity coexist in a population of rat primary sensory neurons.     

Neurokinin A-like immunoreactivity in rat primary sensory neurons; coexistence with substance P

Summary Rat spinal cord, dorsal root ganglia and skin were investigated employing immunohistochemical technique with specific antisera to neurokinin A and substance P. Neurokinin A-like immunoreactivity was detected in the spinal dorsal horn and skin with a similar distribution pattern as that of substance P-like immunoreactivity. After dorsal root transection a parallell decrease of neurokinin A and substance P-like immunoreactivity was observed in the dorsal horn. Using colchicine pretreatment a population of neurokinin A positive cell bodies was seen in the dorsal root ganglia, and by comparison of consecutive sections of the same cells stained for substance P it was revealed that these neurons also display substance P-like immunoreactivity. However, substance P-, but not neurokinin A-, immunoreactive cells were also observed. It is concluded that neurokinin A- and substance P-like immunoreactivity coexist in a population of rat primary sensory neurons.     

First identification and localization of a visual pigment in Hydra (Cnidaria, Hydrozoa)

The cnidarian Hydra does not possess identified photoreceptive structures or specialized cells for light detection; nevertheless, it shows a marked photosensitivity. So far no evidence has been previously reported about the localization of the proteins involved in the photoresponse. We used polyclonal antibodies and immunofluorescence microscopy on whole-mount Hydra to identify a putative rhodopsin-like protein. Our results show an immunoreactivity in the ectodermal layer of Hydra, which corresponds in position to the nervous epidermal sensory cells. These data provide the first identification of a rhodopsin-like protein in a phylogenetically old invertebrate and give a new insight into the Hydra photoreceptive response.     

Substance P-like immunoreactivity in the nervous system of hydra

Summary Using immunocytochemistry we find substance P-like material in nerve cells of hydra. These nerve cells are situated in the ectoderm of the basal disk and tentacles. Radioimmunoassay of hydra extracts gives dilution curves parallel to that of synthetic substance P, from which it can be calculated that one animal contains at least 0.6 fmol substance P-like immunoreactivity. After chromatography on Biogel P-100, the substance P-like immunoreactivity elutes as a peak in the void volume and a peak at the position of synthetic substance P.     

Transient appearance of substance P-like immunoreactivity in the granular convoluted tubules of the male mouse submandibular gland: light- and electron-microscopic studies

The time of appearance and distribution of substance P (SP)-like immunoreactivity in the granular convoluted tubule cells of the developing male mouse submandibular glands were examined, and the subcellular localization of SP-like immunoreactivity was investiagted by electron microscopy. At 25 days of age, SP-like immunoreactivity was first detected in the supranuclear cytoplasm of the granular convoluted tubule cells, which occurred either singly or in small clusters. At 30 and 35 days of age, granular convoluted tubule cells with SP-like immunoreactivity were more numerous than in the earlier stages, as the volume ratio of the cells increased. Not all granular convoluted tubule cells demonstrated SP-like immunoreactivity. The number of cells with SP-like immunoreactivity decreased at 60 days of age, and these cells had completely disappeared at 90 days of age. Most, but not all, secretory granules in the granular convoluted tubule cells were strongly labeled with gold particles, indicating that the subcellular site of SP-like substance is in the secretory granules within the cells. The findings suggest that the physiological role of the SP-like substance secreted from the GCT cells is restricted to the early postnatal stages, and that it may be involved in the development of the oral mucosa or digestive tract as a trophic factor.     

Substance P-like immunoreactivity in the nervous system of hydra

Using immunocytochemistry we find substance P-like material in nerve cells of hydra. These nerve cells are situated in the ectoderm of the basal disk and tentacles. Radioimmunoassay of hydra extracts gives dilution curves parallel to that of synthetic substance P, from which it can be calculated that one animal contains at least 0.6 fmol substance P-like immunoreactivity. After chromatography on Biogel P-100, the substance P-like immunoreactivity elutes as a peak in the void volume and a peak at the position of synthetic substance P.     

A comparative study of neuropeptides in the intestine of two stomachless teleosts (Poecilia reticulata,Leuciscus idus melanotus) under conditions of feeding and starvation

Summary Endocrine cells containing bombesin-, enkephalin-, gastrin/CCK-, 5-HT-, and substance P-like material were demonstrated in the alimentary tract of Poecilia reticulata and Leuciscus idus melanotus. Endocrine cells with neuropeptide-Y-like immunoreactivity were found only in P. reticulata, those with VIP-like immunoreactivity only in L. idus melanotus. Gut nerves showing bombesin-, G/CCK-5-HT-, neurotensin-, substance P-and VIP-like immunoreactivity were observed in both species investigated, enkephalin- and neuropeptide Y-like immunoreactivity in P. reticulata alone. The distribution and amount of endocrine cells and nerves along the gut as visualized with the appropriate antisera varied in both teleosts. Histologically, the intestinal tract of these stomachless fish can be divided into three regions. A large number of endocrine cells with VIP-like immunoreactivity was noted in the rectum of L. idus melanotus. Endocrine cells containing bombesin-, enkepha-lin- and substance P-like material were found only in intestinal parts I and II in L. idus melanotus. Neuropeptide Y-like immunoreactivity was absent from intestinal part I of P. reticulata. The influence of starvation on the immunoreactivity of nerves and enteroendocrine cells in the teleost intestine was examined. After a starvation period of more than 6 weeks, no alterations were observed either in the appearance or amount of nerve and endocrine cell immunoreactivity.     

Demonstration of enkephalin-, substance p- and glutamate decarboxylase-like immunoreactivity in cultured cells derived from newborn rat neostriatum

Summary The presence of cells exhibiting leucine-enkephalin-, substance P- and glutamate decarboxylase-like immunoreactivity was demonstrated in dissociated cultures from newborn rat neostriatum. The size and shape of the enkephalin-immunoreactive cells varied, but they were generally larger than substance P- and glutamate decarboxylase-immunoreactive cells, which formed relatively uniform cell populations. Cells of apparently non-neuronal origin did not show any immunoreactivity. It is unlikely that enkephalin is present in the same cells that contain substance P or glutamate decarboxylase because of norphological differences between these cells. The possible coexistence of substance P and glutamate decarboxylase in the same cells however, could not be excluded. The results of this study confirm that the cell bodies of neurons containing three possible neurotransmitters are located in the neostriatum.     

Demonstration of enkephalin-, substance P- and glutamate decarboxylase-like immunoreactivity in cultured cells derived from newborn rat neostriatum

The presence of cells exhibiting leucine-enkephalin-, substance P- and glutamate decarboxylase-like immunoreactivity was demonstrated in dissociated cultures from newborn rat neostriatum. The size and shape of the enkephalin-immunoreactive cells varied, but they were generally larger than substance P- and glutamate decarboxylase-immunoreactive cells, which formed relatively uniform cell populations. Cells of apparently non-neuronal origin did not show any immunoreactivity. It is unlikely that enkephalin is present in the same cells that contain substance P or glutamate decarboxylase because of morphological differences between these cells. The possible coexistence of substance P and glutamate decarboxylase in the same cells however, could not be excluded. The results of this study confirm that the cell bodies of neurons containing three possible neurotransmitters are located in the neostriatum.     

Substance P-like immunoreactivity in the trigeminal ganglion. A fluorescence, light and electron microscope study

The trigeminal ganglion of rat and guinea pig was studied for the presence of immunoreactive substance-P using fluorescence, light and electronmicroscopy. In untreated animals substance P containing cells, with a diameter of 15 to 50 micron, were distributed throughout the ganglion and comprised 10-30% of all ganglion cells. Colchicine, injected intraventricularly to inhibit intra-axonal transport, had no effect on the number of substance P cells; but when the drug was injected directly into the posterior root of the ganglion, the proportion of these cells increased to as much as 50%. In the electron microscope, immunoreactive substance-P was confined to ganglion cells classified as B type according to the arrangement of subcellular organelles, and to unmyelinated nerve fibers. Subcellularly the immunoreactivity appeared in cytoplasmic vesicles, as well as dispersed in the nerve fibers and the perikarya of neurons. The great number of substance P immunoreactive ganglion cells suggests that they do not comprise a well defined subpopulation of the B-cells. However, the immunoreactivity was restricted to a distinct ultrastructural type of neurons with unmyelinated nerve fibers, suggesting that they also may share some distinct functions.     

Identification and Characterization of Multiple Tachykinin Immunoreactivities in Bovine Retina: Evidence for the Presence of a Putative Oxidative Inactivation System for Substance P

Tachykinin immunoreactivity has been quantified and characterized in extracts of bovine retinae by combining radioimmunoassay, gel permeation chromatography, and reverse-phase HPLC. Using an antiserum specific for the C-terminal hexapeptide amide of substance P, levels of 3.43 +/- 0.33 ng g-1 and 12.45 +/- 0.76 ng g-1 (mean +/- SD, n = 5) were measured in extracts prepared by acidified ethanol and boiling 0.5 M acetic acid, respectively. Levels of neurokinin A immunoreactivity, assayed using an antiserum cross-reacting with neurokinin A (100%), neurokinin B (50%), neuropeptide K (85%), and substance P (less than 0.1%) were 12.46 +/- 0.47 ng g-1 and 7.20 +/- 0.37 ng g-1 in the same extracts. Gel permeation chromatography identified a single substance P immunoreactant eluting with substance P standard, whereas two neurokinin A immunoreactants were resolved eluting with neuropeptide K and neurokinin A standards. Reverse-phase HPLC analysis resolved immunoreactivity eluting with substance P, neurokinin A, neuropeptide K, and neurokinin B and their respective methionine sulphoxides. The amount of immunoreactive material co-eluting with the respective sulphoxides was higher in acidified ethanol extracts, and substance P was most susceptible to oxidative modification. Subsequent incubation of synthetic substance P with dispersed bovine retinal cells resulted in rapid conversion to three metabolites identified and isolated by reverse-phase HPLC. Each had an amino acid composition identical to that of substance P, and the major product had the same retention time as substance P sulphoxide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical identification of substance P in cutaneous sensory organs (electroreceptors) of gymnotid teleost fish

Summary The distribution of the neuropeptide substance P, which is considered to be a neurotransmitter or neuromodulator of the central nervous system, has been studied in the cutaneous electroreceptor organs (tuberous and ampullary organs) of 3 species of gymnotid fish: Apteronotus leptorhynchus, Eigenmannia virescens and Sternopygus sp. Immunohistochemical data have revealed that substance P is never present in the afferent fibers but is specifically localized in the electroreceptors of the three species examined. Substane P immunoreactivity is strictly localized in the sensory cells of the ampullary organs of all three species and in those of the tuberous organs of Apteronotus leptorhynchus and Sternopygus sp. In contrast, weak substance P immunoreactivity was observed only in certain tuberous sensory cells of Eigenmannia. Substance P immunoreactivity was also found in the accessory cells of certain organs: it was detected in the two types of accessory cells of the tuberous organs of Eigemmannia virescens, in the accessory cells type 2 of the tuberous organs of Sternopygus sp., and in all accessory cells of ampullary organs of Sternopygus sp. and Apteronotus leptorhynchus. In Sternopygus sp., positive staining was only evident if the substance P antibody was used at low concentration. Immunoreactivity for substance P in the sensory cells suggests that it has a transmitter or modulator function in these electroreceptors; the presence of substance P in the accessory cells remains to be explained.     

Calcitonin gene-related peptide-like immunoreactivity in nerve fibers in the human skin. Relation to fibers containing substance P-, somatostatin- and vasocactive intestinalpolypeptide-like immunoreactivity

Calcitonin gene-related peptide-like immunoreactivity was demonstrated in in sensory nerve fibers in the epidermis and dermis as free nerve endings and around blood vessels and hair follicles of the human finger pad and arm skin. The vast majority of the calcitonin gene-related immunoreactive fibers was shown to display also substance P-like immunoreactivity and a few fibers in the dermis were somatostatin positive. No fibers displaying both substance P and somatostatin-like immunoreactivity were found but a few substance P immunoreactive fibers in the dermis-epidermis region were found to contain also vasointestinal polypeptide-like immunoreactivity. In the sweat glands, abundant calcitonin gene-related peptide positive, but substance P negative, fibers were observed with a similar distribution pattern as the vasoactive intestinal polypeptide immunoreactive fibers and these fibers were suggested to be of sympathetic origin.     

Phylogenetic study of the oxytocin-like immunoreactive system in invertebrates

1. A phylogenetic study of oxytocin (OXT)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Oncidium verrucosum, Limax marginatus and Meretrix lamarckii of the Mollusca; and Baratha brassica of the Arthropoda. 3. No immunoreactive cells were found in Bipalium sp. of the Platyhelminthes; Pomacea canaliculata, Aplysia kurodai, Bradybaena similaris and Achatina fulica of the Mollusca; and Gnorimosphaeroma rayi, Procambarus clarkii, Hemigrapsus sanguineus, Helice tridens and Gryllus bimaculatus of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 4. These results demonstrate that an OXT-immunoreactive substance is widely present not only in vertebrates but also in invertebrates. 5. OXT seems to have been introduced into these invertebrates at an early stage of their phylogenetic history.     

Neurotransmitters and putative neuromodulators in the gut of Anguilla anguilla (L.). Localizations in the enteric nervous and endocrine systems

The gut of silver eels (Anguilla anguilla L.) was investigated in order to describe both the cholinergic and adrenergic intramural innervations, and the localization of possible accessory neuromediators. Histochemical reactions for the demonstration of nicotinamide adenine dinucleotide phosphate, reduced form-(NADPH-)diaphorase and acetylcholinesterase (AChEase) were performed, as well as the immunohistochemical testing of tyrosine hydroxylase, met-enkephalin, substance P, calcitonin gene-related peptide (CGRP), bombesin, vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), somatostatin, cholecystokinin-octapeptide (CCK-8), serotonin, cholineacetyl transferase. The results evidenced a different pattern in comparison with other vertebrates, namely mammals, and with other fish. Both NADPH-diaphorase and AChEase activities were histochemically detected all along the gut in the myenteric plexus, the inner musculature and the propria-submucosa. Tyrosine hydroxylase immunoreactivity was observed in the intestinal tract only, both in the myenteric plexus and in the inner musculature. Several neuropeptides (metenkephalin, CGRP, bombesin, substance P, VIP, NPY, somatostatin) were, in addition, detected in the intramural innervation; some of them also in epithelial cells of the diffuse endocrine system (met-enkephalin, substance P, NPY, somatostatin). Serotonin was only present in endocrine cells. Tyrosine hydroxylase immunoreactivity was present in localizations similar to those of NADPH-diaphorase-reactivity, and in the same nerve bundles in which substance P- and CGRP-like-immunoreactivities were detectable in the intestinal tract. In addition, NADPH-diaphorase-reactive neurons showed an anatomical relationship with AChEase-reactive nerve terminals, and a similar relationship existed between the latter and substance P-like immunoreactivity.     

Immunohistochemical and ultrastructural localisation of peptide-containing nerves and myocardial cells in the human atrial appendage

Summary The innervation and myocardial cells of the human atrial appendage were investigated by means of immunocytochemical and ultrastructural techniques using both tissue sections and whole mount preparations. A dense innervation of the myocardium, blood vessels and endocardium was revealed with antisera to general neuronal (protein gene product 9.5 and synaptophysin) and Schwann cell markers (S-100). The majority of nerve fibres possessed neuropeptide Y immunoreactivity and were found associated with myocardial cells, around small arteries and arterioles at the adventitial-medial border and forming a plexus in the endocardium. Subpopulations of nerve fibres displayed immunoreactivity for vasoactive intestinal polypeptide, somatostatin, substance P and calcitonin gene-related peptide. In whole-mount preparations of endocardium, substance P and calcitonin gene-related peptide immunoreactivities were found to coexist in the same varicose nerve terminals. Ultrastructural studies revealed the presence of numerous varicose terminals associated with myocardial, vascular smooth muscle and endothelial cells. Neuropeptide Y immunoreactivity was localised to large electron-dense secretory vesicles in nerve terminals which also contained numerous small vesicles. Atrial natriuretic peptide immunoreactivity occurred exclusively in myocardial cells where it was localised to large secretory vesicles. The human atrial appendage comprises a neuroendocrine complex of peptidecontaining nerves and myocardial cells producing ANP.     

Calcitonin gene-related peptide-like immunoreactivity in nerve fibers in the human skin

Summary Calcitonin gene-related peptide-like immunoreactivity was demonstrated in in sensory nerve fibers in the epidermis and dermis as free nerve endings and around blood vessels and hair follicles of the human finger pad and arm skin. The vast majority of the calcitonin generelated immunoreactive fibers was shown to display also substance P-like immunoreactivity and a few fibers in the dermis were somatostatin positive. No fibers displaying both substance P and somatostatin-like immunoreactivity were found but a few substance P immunoreactive fibers in the dermis-epidermis region were found to contain also vasointestinal polypeptide-like immunoreactivity. In the sweat glands, abundant calcitonin gene-related peptide positive, but substance P negative, fibers were observed with a similar distribution pattern as the vasoactive intestinal polypeptide immunoreactive fibers and these fibers were suggested to be of sympathetic origin.     

Immunohistochemical localisation of cholinergic markers in putative intrinsic primary afferent neurons of the guinea-pig small intestine

Antibodies against choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) were used to determine whether neurons that have previously been identified as intrinsic primary afferent neurons in the guinea-pig small intestine have a cholinergic phenotype. Cell bodies of primary afferent neurons in the myenteric plexus were identified by their calbindin immunoreactivity and those in the submucous plexus by immunoreactivity for substance P. High proportions of both were immunoreactive for ChAT, viz. 98% of myenteric calbindin neurons and 99% of submucosal substance P neurons. ChAT immunoreactivity also occurred in all nerve cell bodies immunoreactive for calretinin and substance P in the myenteric plexus, but in only 16% of nerve cells immunoreactive for nitric oxide synthase. VAChT immunoreactivity was in the majority of calbindin-immunoreactive varicosities in the myenteric ganglia, submucous ganglia and mucosa and also in the majority of the varicosities of neurons that were immunoreactive for calretinin and somatostatin and that had been previously established as being cholinergic. We conclude that the intrinsic primary afferent neurons are cholinergic and that they may release transmitter from their sensory endings in the mucosa.     

Localisation of substance P-like immunoreactivity in the ciliary ganglia of monkey (Macaca fascicularis) and cat: a light- and electron-microscopic study

The present study describes substance P-like immunoreactivity in the ciliary ganglia of monkey (Macaca fascicularis) and cat. About 60% of neurons in the monkey ciliary ganglion and 40% in the cat ciliary ganglion were substance P-like immunoreactive, ranging from faint to moderate staining. Substance P-like immunoreactivity was located in cell bodies, dendritic profiles and axons. In the monkey, substance P-like immunoreactive pericellular arborisations were associated with about 0.5%–3% of the ganglion cells, which were either negatively, faintly or moderately stained. An electron-microscopic study demonstrated the presence of either substance P-like immunoreactive positive or negative axon terminals synapsing or closely associated with positive dendritic profiles in both the monkey and cat ciliary ganglia. The results suggest that substance P plays an important role in the ciliary ganglion, perhaps as a modulator or transmitter.