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.     

Substance P and hydra: an immunohistochemical and physiological study

1. The distribution of substance P-like immunoreactivity was studied in Hydra attenuata using the peroxidase-antiperoxidase technique. 2. Positive immunoreactivity was observed in ectodermal nerve cells and fibers as well as in nematoblasts at various stages of differentiation. 3. Administration of synthetic substance P to regenerating hydra did not affect regeneration rates. Exogenous substance P administration stimulated tentacle contraction and nematocyst displacement within battery cells. 4. It is suggested that substance P acts on the contractile apparatus of Hydra tissues.     

Striatal substance P-like immunoreactivity: characterization of high-performance liquid chromatography and aspects of subcellular distribution and in vitro release by potassium

Substance P-like immunoreactivity (SPLI) in acetic acid extracts of striatum elutes in a single peak that corresponds to that for synthetic substance P, using reverse-phase high-performance liquid chromatography. This finding suggests that striatal SPLI, as detected with the radioimmunoassay used here, largely represents a single molecular species. Striatal SPLI is highly concentrated in the crude mitochondrial fraction (P2); over 70% of this P2-SPLI is recovered in the synaptosomal subfraction. The potassium-induced release of SPLI from striatal tissue in vitro occurs only when enough extracellular calcium is available. These data suggest that substance P in striatal neurons may participate in neural transmission locally, exclusive of its probable role in maintaining the substance P supply to nerve terminals in the substantia nigra.     

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.     

Immunochemical evidence for substance P-like peptide in tissues of the earthworm Lumbricus terrestris: action on intestinal contraction

The presence of a substance P-like peptide in intestinal and body wall tissues, ventral nerve fiber and seminal vesicles of the earthworm Lumbricus terrestris has been demonstrated by means of a radioimmunoassay technique. The greatest substance P-like immunoreactivity was measured in intestinal tissues where it stimulates the rate of spontaneous contraction. This effect is inhibited by the substance P antagonist (D-pro2, D-trp7,9)-SP suggesting a possible involvement of receptor mechanisms. Dual localization of substance P-like immunoreactivity in earthworm intestinal and nerve tissues follows the pattern observed of peptidal hormones in vertebrates which are common to both endocrine and non-endocrine tissues.     

Studies on the distribution of calcitonin gene-related peptide-like and substance P-like immunoreactivities in rat hind limb muscles

Summary The tibialis anterior, extensor digitorum longus and soleus muscles in the rat were examined with respect to the presence of calcitonin gene-related peptide-like as well as substance P-like immunoreactivity. In some of the motor endplates in these muscles, identified by staining for acetylcholinesterase activity, calcitonin gene-related peptide-like immunoreactivity was detected, but in others it was not. Calcitonin gene-related peptide-like immunoreactivity was found to coexist with substance-P-like immunoreactivity in nerve fibres located outside and inside the capsule of the muscle spindles, as well as in nerve fibres located in nerve fascicles. These fibres presumably represent sensory nerve fibres. Calcitonin gene-related peptide-like immunoreactivity, but not substance P-like immunoreactivity, was also detected, in cap-like structures located on the surface of the intrafusal muscle fibres in the polar regions of the spindles, structures which are likely to correspond to motor plate endings. The observations suggest that calcitonin gene-related peptide is heterogeneously present in the endplates of rat hind limb muscles, and gives for the first time immunohistochemical evidence for the presence of calcitonin gene-related peptide and substance P in the innervation of muscle spindles.     

Characterization of substance P-like immunoreactivity in peripheral sensory nerves and enteric nerves by high pressure liquid chromatography and radioimmunoassay

Material exhibiting immunoreactivity for substance P in enteric nerves, obtained from the myenteric plexus of the guinea pig small intestine, and in the peripheral ends of sensory nerves of the ureter, atrium and superior mesenteric artery, was characterized by separation by high pressure liquid chromatography, and quantified by radioimmunoassay of fractions collected from the chromatograph. Capsaicin, which depletes substance P-like immunoreactivity from sensory, but not from other substance P-containing nerves, reduced the content of substance P-like immunoreactivity in ureter, atrium and superior mesenteric artery by more than 99.5%, whereas the reduction in immunoreactive material in the myenteric plexus was less than 10%. Separation of extracts of myenteric plexus, ureter and atrium on a reversed-phase column gave major peaks corresponding to authentic substance P and minor peaks that coeluted with oxidized substance P. If the extracts were oxidized with hydrogen peroxide before chromatography, all the immunoreactivity was found in the peak corresponding to oxidized substance P. In the superior mesenteric artery extracts, in addition to the components corresponding to substance P and its oxidized derivative, there was a small intermediate peak that has yet to be identified. Physalaemin, which has been suggested to be present in mammalian nerves, was not detectable in any of the extracts. It is concluded that both enteric nerves and the peripheral processes of sensory nerves which show immunoreactivity for substance P in this species contain the authentic peptide.     

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.     

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.     

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.     

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.     

Nerve growth factor receptor-like immunoreactivity in primary and permanent canine tooth pulps of the cat

Summary The distribution of nerve growth factor receptor (NGF receptor)-like immunoreactivity in pulps of developing primary and mature permanent cat canine teeth was examined, by use of a monoclonal antibody against NGF receptor detected by fluorescence immunohistochemistry and pre-embedding immunocytochemical light- and electron microscopy. Both primary and permanent pulps contained a vast number of NGF receptor-like immunoreactive nerves. Immunolabelling appeared to be localized both to axons and Schwann cells. In addition, many blood vessel walls in immature primary tooth pulps showed NGF receptor-like immunoreactivity, in contrast to permanent pulps where blood vessels rarely were NGF receptor-immunoreactive. Double-labelling immunofluorescence experiments revealed that in the permanent pulp a majority of the NGF receptor-positive nerves also showed calcitonin gene-related peptide (CGRP)-like immunoreactivity, and many showed substance P-like immunoreactivity. However, nerve fibers with neuropeptide Y-like immunoreactivity lacked NGF receptor-like immunoreactivity. In developing primary tooth pulps fewer NGF receptor-positive nerves were CGRP-like immunoreactive or substance P-like immunoreactive, as compared to the permanent pulp. Neuropeptide Y-like immunoreactive nerve fibers were not detected in the primary tooth pulp. The results suggest a role for nerve growth factor in both developing and mature sensory nerves of the tooth pulp.     

Substance P-like immunoreactivity in sympathetic ganglion from toad

Substance P-like immunoreactivity cellular in toad sympathetic ganglia was studied in normal and capsaicin-treated ganglia. In the eighth sympathetic ganglion substance P-like immunoreactive are found in mast cells and SIF cells. The effect of substance P (0.001-0.003 mM) caused increase of compound action potential during tetanical stimulation (50 Hz by 40 sec.) and post-tetanic potentiation (0.1 Hz). Our results show that substance P facilitates synaptic transmission in the sympathetic ganglia from Caudiverbera caudiverbera.     

Intimate relationship between interstitial cells of Cajal and enteric nerves in the guinea-pig small intestine

Recent studies have suggested that enteric inhibitory neurotransmission is mediated via interstitial cells of Cajal in some gastrointestinal tissues. This study describes the physical relationships between enteric neurons and interstitial cells of Cajal in the deep muscular plexus (IC-DMP) of the guinea-pig small intestine. c-Kit and vimentin were colocalized in the cell bodies and fine cellular processes of interstitial cells of the deep muscular plexus. Anti-vimentin antibodies were subsequently used to examine the relationships of interstitial cells with inhibitory motor neurons (as identified by nitric oxide synthase-like immunoreactivity) and excitatory motor neurons (using substance P-like immunoreactivity). Neurons with nitric oxide synthase- and substance P-like immunoreactivities were closely associated with the cell bodies of interstitial cells and ramified along their processes for distances greater than 300 7m. With transmission electron microscopy, we noted close relationships between interstitial cells and the nitric oxide synthase- and substance P-like immunoreactive axonal varicosities. Varicosities of nitric oxide synthase and substance P neurons were found as close as 20 and 25 nm from interstitial cells, respectively. Specialized junctions with increased electron density of pre- and postsynaptic membranes were observed at close contact points between nitric oxide synthase- and substance P-like immunoreactive neurons and interstitial cells. Close structural relationships (approximately 25 nm) were also occasionally observed between either nitric oxide synthase- and substance P-like immunoreactive varicosities and smooth muscle cells of the outer circular muscle layer. The data suggest that interstitial cells in the deep muscle plexus are heavily innervated by excitatory and inhibitory enteric motor neurons. Thus, these interstitial cells may provide an important, but probably not exclusive, pathway for nerve-muscle communication in the small intestine.     

Bombesin-like immunoreactivity in the nervous system of hydra

Summary With immunocytochemical methods, nerve cells have been detected in Hydra attenuata containing bombesin-like immunoreactivity. These nerve cells are located in the ectoderm of all body regions of the animal and are especially abundant in basal disk and tentacles. Radioimmunoassay of extracts of hydra demonstrated at least 0.2 pmol/g wet weight of bombesinlike immunoreactivity. The immunoreactive material elutes from Sephadex G-50 in a similar position to synthetic bombesin. The data show that bombesin-like peptides are among the phylogenetically oldest neuropeptides found so far.     

Distribution of substance p-like immunoreactivity in nerves of the gastrointestinal tract of the frog Rana esculenta L.

Summary Substance P-like immunoreactivity in the alimentary canal of the frogRana esculenta L. was studied by means of the indirect immunoperoxidase method. In all segments of the gastrointestinal tract, immunoreactivity was revealed in both the myenteric and the submucosa plexus. Stained nerve cells were observed in the myenteric plexus but not in the submucous plexus. The proximal part of the oesophagus and hindgut were free of immunoreactive perkarya. The stained nerve cells were of the Dogiel type I category in the foregut, and type II in the midgut. Both the musculature and gastrointestinal glands were supplied with immune-positive fibres. These results indicate that substance P may play similar roles in the frog gut, as described previously in mammals and fish.     

Substance P-like immunoreactive nerve fibers in the pars distalis of the anterior pituitary in the dog

Summary The pars distalis of the anterior pituitary is known to be regulated by hypothalamic hormones. Recently, we have discovered the presence of substance P-like immunoreactive nerve fibers in the pars distalis of the monkeys. Substance P-like immunoreactivity in the pars distalis of the dog was investigated in this study. A substantial amount of substance P-like immunoreactive nerve fibers with a large amount of varicosities were found. They were widely distributed in the gland, more abundant along its periphery. Most of them were closely related to the glandular tissue, some were located on vascular walls. Substance P-like immunoreactive nerve fibers were also found in the meningeal sheath of the anterior pituitary. They could be followed into the parenchyma of the gland.     

Distribution of serotonin-, Gamma-Aminobutyric Acid- and substance P-like immunoreactivity in the central and peripheral nervous system of Mytilus galloprovincialis

The distribution of serotonin-, GABA- and substance P-like immunoreactivity has been studied in the cerebral and visceral ganglia and in some peripheral tissues of Mytilus galloprovincialis (Moleusca, Bivalvia). Cerebral ganglia contain a developed serotonin-immunoreactive neuronal subpopulation and numerous GABA-immunoreactive neurons, whereas neurons positive for substance P are sparse. In peripheral tissues innervated by the cerebral ganglia (labial palps and oesophagus) only serotonin-immunoreactive nerve fibers were found. In the visceral ganglia, serotonin- and GABA-immunoreactive neurons are far less numerous than in the cerebral ganglia, whereas several neurons positive for substance P are scattered in all cortical zones. Serotonin-immunoreactive plexuses innervate the posterior adductor muscle and the gill filaments which contain also a developed nerve network positive for substance P. The distribution pattern of the immunoreactive elements in the ganglia and in peripheral territories indicates that GABA should exert only a central action, whereas serotonin and a substance P-like peptide are involved both in central and peripheral neurotransmission.     

Development of an Antiserum to the Midportion of Substance P: Applications for Biochemical and Anatomical Studies of Substance P-Related Peptide Species in CNS Tissues

This report describes the generation and biochemical characterization of a high-affinity antiserum that recognizes an epitope contained in the midportion sequence of substance P, i.e., substance P4-10. Designated A47, this reagent bound a variety of related peptide species containing the substance P4-10 sequence with apparent equipotency. A double radioimmunoassay procedure was developed that utilized A47, in combination with a traditional high-affinity COOH-terminally directed anti-substance P serum, to provide quantification of mature and immature forms of substance P in CNS tissues. Across most rat CNS areas, levels of substance P-like immunoreactivity were consistently 15% higher when monitored by analyses using A47 versus anti-substance P serum. In the dorsal root ganglia, an apparent enhancement in levels of substance P-like immunoreactivity of approximately 40%, when quantified by analyses using A47 versus anti-substance P serum, was observed; this most likely reflected the presence of an active biosynthetic pool of intermediate processing forms of substance P in this tissue. Coordinated HPLC/radioimmunoassay analyses of extracted dorsal root ganglia tissues demonstrated multiple peaks of immunoreactivity corresponding to mature substance P and to several of its precursor forms found in the normal biosynthetic pathway. Of the total recovered HPLC-fractionated immunoreactivities, that corresponding to the putative immediate precursor to substance P, i.e., substance P-glycine, was the predominant peak. In an additional series of HPLC/radioimmunoassay analyses, selective decreases in immunoreactive peaks corresponding to precursor forms of substance P were observed in dorsal root ganglia tissues from rats treated with the neurotoxic agent capsaicin. These results indicated decreased turnover of substance P as a consequence of drug treatment. Finally, initial immunohistochemical analyses employing affinity-purified A47 produced an unusual pattern of labeling characterized by well defined punctate terminal elements within the superficial aspects of the dorsal horn of the spinal cord.