Substance P binding sites in the nucleus tractus solitarius of the cat

Substance P binding sites in the nucleus tractus solitarius were visualized with receptor autoradiography using Bolton-Hunter [¹²⁵I]substance P. Substance P binding sites were found to have distinct patterns within the cat nucleus tractus solitarius. The majority of substance P binding sites were present in the medial, intermediate and the peripheral rim of the parvocellular subdivisions. Lower amounts of substance P binding sites were present in the commissural, ventrolateral, interstitial and dorsolateral subdivisions. No substance P binding sites were present in the central region of the parvocellular subdivision or the solitary tract. The localization of substance P binding sites in the nucleus tractus solitarius is very similar to the patterns of substance P immunoreactive fibers previously described for this region. Results of this study add further support for a functional role of substance P in synaptic circuits of the nucleus tractus solitarius.     

Substance P-immunoreactive nerve fibres in the anterior segment of the rabbit eye

Summary Substance P-immunoreactive nerve terminals were found in several locations in the anterior segment of the rabbit eye. In the iris they occurred in the sphincter muscle and were randomly distributed in the iris stroma with some fibres running close to the dilator muscle. In the ciliary body these immunoreactive elements were few and occurred within bundles of nerve fibres, while in the ciliary processes they were more numerous with a predominantly subepithelial location. Blood vessels in the anterior uvea were often surrounded by substance P-immunoreactive fibres. No substance P-fibres were found in the cornea, while the sclera contained very few such elements.Using conventional in vitro techniques it was found that the sphincter pupillae muscle of the iris responded to electrical stimulation with a contraction that was resistant to cholinergic and adrenergic blockade, but was inhibited by the neuronal blocker tetrodotoxin. This indicates the existence of a non-cholinergic, non-adrenergic neuronal mediator of the contractile response. Exogenously applied substance P produced a long-lasting contraction of the spincter muscle, an observation compatible with the view that substance P is the noncholinergic, non-adrenergic neurotransmitter involved.     

Substance P-immunoreactive sensory nerves in the lower respiratory tract of various mammals including man

Summary The occurrence and origin of substance P (SP)-immunoreactive (IR) nerves in the lower respiratory tract was studied by means of immunohistochemistry in the guinea-pig, rat, cat and man. In addition, biopsies from human material were also analysed by radioimmunoassay. SP-IR nerves were seen in four principal locations: 1) under or within the lining epithelium, 2) around blood vessels, 3) within the bronchial smooth muscle layer, and 4) around local tracheobronchial ganglion cells. Ligation experiments combined with capsaicin pretreatments indicated that all SP-IR nerves in the respiratory tract are sensory. The trachea seems to be mainly supplied by the vagal nerves, while intrapulmonary bronchi and blood vessels receive SP-IR nerves of both vagal and non-vagal (spinal) origin. SP-IR nerves were also found in the human bronchi with principally similar location as in the guinea-pig. The levels of SP-IR in the trachea and peripheral bronchi of man were about 3–4 pmol/g, which is in the same range as the content of corresponding tissues from the guinea-pig.In conclusion, the present experimental findings of SP-IR nerves in the lower respiratory tract in both experimental animals and man support the functional evidence for the importance of SP in the vagal and non-vagal (spinal) control of bronchial smooth muscle tone and vascular permeability.     

Alteration of shock titration thresholds in the cat following intrathecal substance P administration

Substance P (SP) was injected intrathecally (10 or 100 micrograms) into cats previously implanted with nerve-stimulating electrodes and the effect on shock titration thresholds was evaluated. Elevated shock thresholds were observed in 5 of 8 cats following the 100 micrograms dose of SP. In addition, one cat exhibited a decreased threshold and two cats showed a triphasic (increase, decrease, increase) response. Overt behavioral effects of intrathecal SP were mild, but suggested that injection of the drug was aversive.     

Light- and electron-microscopic study of substance P-immunoreactive neurons in the guinea pig retina

Substance P (SP) immunoreactivity in the guinea pig retina was studied by light and electron microscopy. The morphology and distribution of SP-immunoreactive neurons was defined by light microscopy. The SP-immunoreactive neurons formed one population of amacrine cells whose cell bodies were located in the proximal row of the inner nuclear layer. A single dendrite emerged from each soma and descended through the inner plexiform layer toward the ganglion cell layer. SP-immunoreactive processes ramified mainly in strata 4 and 5 of the inner plexiform layer. SP-immunoreactive amacrine cells were present at a higher density in the central region around the optic nerve head and at a lower density in the peripheral region of the retina. The synaptic connectivity of SP-immunoreactive amacrine cells was identified by electron microscopy. SP-labeled amacrine cell processes received synaptic inputs from other amacrine cell processes in all strata of the inner plexiform layer and from bipolar cell axon terminals in sublamina b of the same layer. The most frequent postsynaptic targets of SP-immunoreactive amacrine cells were the somata of ganglion cells and their dendrites in sublamina b of the inner plexiform layer. Amacrine cell processes were also postsynaptic to SP-immunoreactive neurons in this sublamina. No synaptic outputs onto the bipolar cells were observed.     

鸡脊髓背角胶状质中calbindin-D28k阳性终末的超微结构及与substance P阳性终末之间的联系

目的 观察鸡脊髓背角胶状质中calbindin-D28k（CB）阳性终末的超微结构及其与含有substance P（SP）中央末梢之间的联系.方法 应用免疫电镜技术观察鸡脊髓背角胶状质中CB阳性终末的超微结构,并应用激光共聚焦显微镜观察鸡脊髓背角胶状质中CB和SP阳性突触小球中央末梢之间的关系.结果 电镜下观察：1）突触小球中含有心小泡的中央末梢呈CB免疫阳性;2）突触小球内或外的部分含小泡的树突呈CB免疫阳性;以及3）突触小球外的部分轴突呈CB免疫阳性.在突触结构内,CB免疫阳性反应物主要分布于突触后膜上.免疫荧光双标记法显示,SP阳性的含有心小泡的中央末梢呈CB阳性.结论 突触小球的中央末梢中CB与SP共存,提示CB可能通过其钙离子缓冲作用,参与脊髓的痛觉调制.     

Distribution and origin of bombesin,substance P and somatostatin in cat spinal cord

Bombesin (BN), substance P-(SP) and somatostatin (SRIF) were measured in individual laminae of the cervical, thoracic and lumbar (L) spinal cord of control cats, and in the L6 segment of cats receiving a spinal hemisection (L2) or deafferentation via dorsal rhizotomy at L6, 7, S1. The interlaminar distribution of BN, SP, and SRIF was remarkably similar. Highest concentrations were found in the superficial dorsal horn, and progressively less was found proceeding ventrally. Some intersegmental variations in peptide concentration within a single lamina were found. Dorsal rhizotomy caused a significant decline in BN, SP and SRIF in lamina I-III, therefore all three peptides appear to be contained in dorsal root ganglion cells. Evidence is presented for the existence of ascending BN and SP projections originating in lamina I-III and VII, for a descending SRIF pathway terminating in lamina VIII, and for an ascending BN path in lamina VIII. Dorsal root afferents to lamina VIII influence levels of BN, SP and SRIF.     

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.     

Immunohistochemical mapping of galanin-like neurons in the rat central nervous system

Using an antiserum generated in rabbits against synthetic galanin (GA) and the indirect immunofluorescence method, the distribution of GA-like immunoreactive cell bodies and nerve fibers was studied in the rat central nervous system (CNS) and a detailed stereotaxic atlas of GA-like neurons was prepared. GA-like immunoreactivity was widely distributed in the rat CNS. Appreciable numbers of GA-positive cell bodies were observed in the rostral cingulate and medial prefrontal cortex, the nucleus interstitialis striae terminalis, the caudate, medial preoptic, preoptic periventricular, and preoptic suprachiasmatic nuclei, the medial forebrain bundle, the supraoptic, the hypothalamic periventricular, the paraventricular, the arcuate, dorsomedial, perifornical, thalamic periventricular, anterior dorsal and lateral thalamic nuclei, medial and central amygdaloid nuclei, dorsal and ventral premamillary nuclei, at the base of the hypothalamus, in the central gray matter, the hippocampus, the dorsal and caudoventral raphe nuclei, the interpeduncular nucleus, the locus coeruleus, ventral parabrachial, solitarii and commissuralis nuclei, in the A1, C1 and A4 catechaolamine areas, the posterior area postrema and the trigeminal and dorsal root ganglia. Fibers were generally seen where cell bodies were observed. Very dense fiber bundles were noted in the septohypothalamic tract, the preoptic area, in the hypothalamus, the habenula and the thalamic periventricular nucleus, in the ventral hippocampus, parts of the reticular formation, in the locus coeruleus, the dorsal parabrachial area, the nucleus and tract of the spinal trigeminal area and the substantia gelatinosa, the superficial layers of the spinal cord and the posterior lobe of the pituitary. The localization of the GA-like immunoreactivity in the locus coeruleus suggests a partial coexistence with catecholaminergic neurons as well as a possible involvement of the GA-like peptide in a neuroregulatory role.     

Identification,biological characterization and pharmacophoric analysis of a new potent and selective NK1 receptor antagonist clinical candidate

The last two decades have provided a large weight of preclinical data implicating the neurokinin-1 receptor (NK₁) and its cognate ligand substance P (SP) in a broad range of both central and peripheral disease conditions. However, to date, only the NK₁ receptor antagonist aprepitant has been approved as a therapeutic and this is to prevent chemotherapy-induced nausea & vomiting (CINV). The belief remained that the full therapeutic potential of NK₁ receptor antagonists had yet to be realized; therefore clinical evidence that NK₁ receptor antagonists may be effective in major depression disorder, resulted in a significant further investment in discovering novel CNS penetrant druggable NK₁ receptor antagonists to address this condition. At GlaxoSmithKline after the discovery of casopitant, that went on to demonstrate efficacy as a novel antidepressant in the clinic, additional novel analogues of this NK₁ receptor antagonist were designed to further enhance its drug developability characteristics. Herein, we therefore describe the discovery process and the vivo pharmacological and pharmacokinetic profile of the new NK₁ receptor antagonist 3a (also called orvepitant), selected as clinical candidate and further progressed into clinical studies for major depressive disorder. Moreover, molecular modeling studies enabled us to improve the pharmacophore model of the NK₁ receptor antagonists with the identification of a region able to accommodate a variety of heterocycle moieties.     

Neurokinin A-like immunoreactivity in feline dental pulp: its distribution,origin and coexistence with substance P-like immunoreactivity

Summary The distribution and origin of neurokinin A (NKA)-like immunoreactivity were investigated in feline dental pulp by an indirect immunofluorescence method. NKA-containing nerve fibres with varicosities, which entered the dental pulp via apical foramen, were distributed throughout this tissue. Many NKA-containing nerve fibres were localized around blood vessels, but some were observed apart therefrom. At the odontoblastic layer, thin NKA-containing nerve fibres were observed running straight toward the pulp-predentinal border between odontoblasts. After inferior alveolar nerve section, all NKA-containing nerve fibres disappeared in the dental pulp, while the removal of the superior cervial ganglion resulted in no change in the distribution of these fibres. The correlation of NKA-like immunoreactivity and substance P (SP)-like immunoreactivity was also investigated by double-immunofluorescence technique. The distribution of NKA-containing nerve fibres was very similar to that of SP-containing nerve fibres; it appeared that all NKA-containing nerve fibres contained SP.     

Measurement of Substance P Metabolites in Rat CNS

A procedure based on ion-exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P (SP), the SP(1-7), and C-terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50- to 100-fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1-7) comigrated with the authentic standard whereas practically all activity isolated as C-terminal fragments comigrated with SP(5-11). The levels of C-terminal fragments in rat brain areas rich in SP and in spinal cord were 1-2% of those of parent compound. The levels of SP(1-7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1-7) levels fell more rapidly than those of SP or C-terminal fragments.     

Substance P-like-immunoreactive neurons in the photosensory pineal organ of the rainbow trout,Salmo gairdneri Richardson (Teleostei)

Summary Restricted numbers of substance P-like-immuno-reactive (SPL-IR) neurons were demonstrated in the photosensory pineal organ of the rainbow trout. The small parapineal organ of this teleost species receives a distinct SPL-IR innervation via the habenular nuclei, but displays no intrinsic SPL-IR neurons. Intrapineal SPL-IR neurons were located in the rostral portion of the pineal end-vesicle. Neuronal somata were found in a lateral position with smooth axonal processes extending mediad. Immunoreactive somata and axonal processes were observed intraparenchymally as well as in the pineal lumen. The pattern of immunoreactivity was not changed in excised pineal organs that had been incubated in tissue culture medium in the dark for 18 h. The possibility that the intrapineal SPL-IR neurons are not part of the neural circuitry involved in the transduction of photic information, but may have other functions, is discussed.Fellow of the Alexander von Humboldt-Stiftung, Bonn, Federal Republic of GermanySupported by research funds from the Deutsche Forschungsge-meinschaft (Ko 758/2-4)     

Comparative distribution of neuropeptide tyrosine-,vasoactive intestinal polypeptide-, substance P-immunoreactive,acetylcholinesterase-positive and noradrenergic nerves in the reproductive tract of the female rat

Summary Location, distribution and density of nerve fibers immunoreactive to neuropeptide tyrosine, vasoactive intestinal polypeptide and substance P were studied in the reproductive tract of the female rat and compared with acetylcholinesterase-positive (cholinergic) and noradrenergic nerves. Plexuses of all types of fibers were present in the vagina, uterine cervix, uterine horn and oviduct. In the tubular reproductive organs all of these types of nerve fibers appeared to innervate vascular and non-vascular smooth muscle and nearly all types of fibers formed plexuses subjacent to the epithelium lining the organs. Individual fibers of all classes appeared to innervate fascicles of smooth muscle in the mesometrium of the uterine horn. A few acetylcholinesterase-positive and substance P-immunoreactive fibers were present in the ovary but no vasoactive intestinal polypeptide-immunoreactive nerves were observed. Noradrenergic and neuropeptide tyrosine-immunoreactive nerves were numerous in the ovary where they were seen in the interstitial gland tissue and associated with follicles and blood vessels. It is suggested that these nerves may influence hemodynamic events and non-vascular smooth muscle in such functions as transport of sperm and ova and parturition. Substance P-immunoreactive nerve fibers are likely to be sensory fibers that could have roles in neurohormonal reflexes.     

Demonstration and Distribution of Kassinin-Like Material (Substance K) in the Rat Central Nervous System

Antiserum was raised against kassinin in rabbits. Cross-reactivity with other tachykinins was determined; these included substance K (100%) and substance P (0.1%). Peptides extracted from rat brain and synthetic tachykinins were chromatographed by reverse-phase HPLC. The major peak of kassinin-like material eluted at a time different from that of synthetic kassinin, eledoisin, physalaemin, neurokinin beta, and substance P but coeluted with substance K. Measurement of kassinin-like material in macrodissected and microdissected brain regions indicated that the distribution of kassinin-like material was similar to that of substance P.     

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.     

Substance P innervation of the rat thymus

Immunohistochemistry for substance P (SP) in the rat thymus revealed fine varicose neural profiles in specific regions of the thymus. Thymic SP innervation was abundant within the capsule and interlobular septa. The majority of SP+ nerve fibers within the septa were free of vascular association, although some fibers were associated with the vasculature deep within the septa. SP+ nerve fibers entered the thymic cortex from the septa and distributed among cortical thymocytes and mast cells. Along the corticomedullary junction, SP+ nerve fibers were found in association with the vasculature. The medullary region of the thymus received only a sparse innervation of SP+ fibers. In addition, SP+ nerve fibers coursed adjacent to OX-8+ cells and mast cells in the extrathymic connective tissue surrounding the thymus. The present study provides evidence that SP is present in nerve fibers in the thymus, and may be available to interact with thymocytes, mast calls, and other cells in the thymus, and affect their development and function.     

Distribution of neurotensin-like immunoreactive cell bodies and fibers in the brainstem of the adult male cat

We studied the distribution of cell bodies and fibers containing neurotensin in the brainstem of the cat using an indirect immunoperoxidase technique. A high or moderate density of immunoreactive perikarya was found in the interpeduncular nucleus, inferior colliculus, nucleus of the brachium of the inferior colliculus and in the lateral tegmental field. Moreover, a high density of neurotensin-immunoreactive fibers was observed in the periaqueductal gray, locus coeruleus and in the marginal nucleus of the brachium conjunctivum. The interpeduncular nucleus, nucleus of the solitary tract and the dorsal motor nucleus of the vagus contained a moderate density of immunoreactive fibers.     

Junctions in the central nervous system of the cat

Summary The leptomeningeal tissue of the choroid plexuses and of the brain surfaces have been studied by means of the freeze-etching technique. The pia-arachnoid membrane and the subdural neurothel represent the morphological barrier between the extracerebral tissue and the cerebrospinal compartment. The freeze-etch findings indicate that the arachnoid and neurothelial cells are coupled by extensive zonulae occludentes which seem to represent the structural basis of the barrier mechanism provided by these cell layers. Furthermore, it became evident that gap junctions of considerable structural heterogeneity occur on the pial and arachnoid cells of the interstitial choroidal compartment and of the free brain surfaces. The structural heterogeneity of the nexuses is taken as an indication of the plasticity of the leptomeningeal tissue. The different morphological characteristics of the nexal formations are discussed with respect to their probable functional meaning.This investigation was supported by the Deutsche Forschungsgemeinschaft SFB 114 (Bionach).     

Effect of Capsaicin Pretreatment on Substance P Binding to Synaptic Vesicles

Newborn or adult rats were pretreated with 50 mg kg-1 capsaicin. At the age of 2 to 4 months, binding of 125I-labelled Tyr8-substance P to synaptic vesicles prepared from different regions of the nervous system was examined. In both groups, capsaicin pretreatment led to a significant decrease in the number of binding sites in dorsal roots and spinal cord without having an effect on affinity. This decrease parallels the depletion of the substance P content (Gamse et al., 1980) and can be explained by degeneration of primary sensory neurons in newborn treated rats and by depletion of vesicles in adult treated rats.