Specific substance P binding sites in rat thymus and spleen: in vitro autoradiographic study

Substance P binding sites were localized in rat thymus and spleen by incubation of tissue sections with [125I]Bolton-Hunter substance P, [3H]Ultrofilm autoradiography with image analysis coupled to computerized microdensitometry and comparison with 125I standards. The tissue localization of the binding sites was determined with emulsion autoradiography. A single type of specific, saturable, high affinity binding sites was found associated with the vasculature in the medulla of the thymus and the marginal sinus of the spleen, with a Kd of 0.10 and 0.14 nM, respectively. Of all the unlabeled tachykinins tested (substance P, physalaemin, substance K, eledoisin, kassinin, and neuromedin K) substance P was the most potent inhibitor of [125I]Bolton-Hunter substance P binding, with an IC50 of approximately 0.5 nM, indicating the presence of substance P-P binding sites. Our results support the hypothesis of a role for substance P in the modulation of the immune system.     

Retinal laser burn-induced neuropathy leads to substance P-dependent loss of ocular immune privilege

Inflammation in the eye is tightly regulated by multiple mechanisms that together contribute to ocular immune privilege. Many studies have shown that it is very difficult to abrogate the immune privileged mechanism called anterior chamber-associated immune deviation (ACAID). Previously, we showed that retinal laser burn (RLB) to one eye abrogated immune privilege (ACAID) bilaterally for an extended period of time. In an effort to explain the inflammation in the nonburned eye, we postulated that neuronal signals initiated inflammation in the contralateral eye. In this study, we test the role of substance P, a neuroinflamatory peptide, in RLB-induced loss of ACAID. Histological examination of the retina with and without RLB revealed an increase of the substance P-inducible neurokinin 1 receptor (NK1-R) in the retina of first, the burned eye, and then the contralateral eye. Specific antagonists for NK1-R, given locally with Ag within 24 h, but not 3, 5, or 7 d post-RLB treatment, prevented the bilateral loss of ACAID. Substance P knockout (KO) mice retained their ability to develop ACAID post-RLB. These data support the postulate that substance P transmits early inflammatory signals from the RLB eye to the contralateral eye to induce changes to ocular immune privilege and has a central role in the bilateral loss of ACAID. The possibility is raised that blocking of the substance P pathway with NK1-R antagonists postocular trauma may prevent unwanted and perhaps extended consequences of trauma-induced inflammation in the eye.     

Determination of ligand binding affinities for endogenous seven-transmembrane receptors using fluorometric microvolume assay technology.

We have developed a fluorescence-based mix and read method for the quantitative determination of receptor-ligand binding interactions. This method was used to determine IC(50) values for peptide ligands of two endogenous seven-transmembrane receptors that are expressed in cultured human cancer cells. Substance P, neurokinin A, and galanin were labeled with Cy5 and were shown to retain their native binding affinities. The cell-associated fluorescence was quantified using a fluorometric microvolume assay technology (FMAT) scanner that was designed to perform high-throughput screening assays in multiwell plates with no wash steps. The binding of fluorescently labeled substance P and neurokinin A was tested on the human astrocytoma cell line UC11 that expresses endogenous NK(1) receptor. Galanin binding was measured on endogenous galanin type 1 receptors in the Bowes neuroblastoma cell line. IC(50) values were determined for substance P, neurokinin A, and galanin and were found to correspond well with reported values from radioligand binding determinations. To demonstrate FMAT as instrumentation for high-throughput screening, it was utilized to successfully identify individual wells in a 96-well plate in which Cy5-substance P binding in UC11 cells was competed with unlabeled substance P. In addition, we developed a two-color multiplex assay in which cells individually expressing neuropeptide Y and substance P receptors were mixed in the same well. In this assay, the fluorescent ligands substance P and neuropeptide Y bound only to their respective cell types and binding was specifically competed. Therefore, two different seven-transmembrane receptor targets can be tested in one screen to minimize reagent consumption and increase throughput.     

Characterization of a neurokinin B receptor site in rat brain using a highly selective radioligand

We have recently characterized a tachykinin receptor subtype (SP-N) whose preferred ligand is the mammalian neuropeptide, neurokinin B (Laufer, R., Wormser, U., Friedman, Z. Y., Gilon, C., Chorev, M., and Selinger, Z. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7444-7448). To investigate this novel tachykinin receptor, we have now prepared a radiolabeled peptide, N alpha-[( 125I]desamino-3-iodotyrosyl)-[Asp5,6, N-methyl-Phe8]substance P (5-11) heptapeptide (125I-BH-NH-Senktide), which selectively interacts with the SP-N receptor subtype. The binding of 125I-BH-NH-Senktide to rat cerebral cortex membranes was studied under conditions that minimized nonspecific binding. Unlike other tachykinin receptor probes, this radioligand is not degraded during the binding experiment. Binding of 125I-BH-NH-Senktide is reversible, saturable, and of high affinity (KD = 0.9 nM). The radioligand labels a single class of binding site (122 fmol binding sites/mg of protein), as indicated by a linear Scatchard plot and a Hill coefficient close to unity (nH = 1.05). The pharmacological specificity of this binding site corresponds to that of the neuronal SP-N receptor in guinea pig ileum myenteric plexus, which was determined by a functional bioassay. Among various rat brain regions, the highest binding was observed in the cerebral cortex, olfactory bulb, hypothalamus, and hippocampus. These results suggest the existence and specific distribution of a neurokinin B receptor site of the SP-N type in rat brain. 125I-BH-NH-Senktide is the first selective and potent probe for this receptor and is thus an important tool for further studies of its distribution, regulation, and functional role.     

Ranakinin: A Novel NK1 Tachykinin Receptor Agonist Isolated with Neurokinin B from the Brain of the Frog Rana ridibunda

An extract of the whole brain of the frog Rana ridibunda contained high concentrations of substance P-like immunoreactivity, measured with an antiserum directed against the COOH-terminal region of mammalian substance P and neurokinin B-like immunoreactivity, measured with an antiserum directed against the NH2-terminus of neurokinin B. The primary structure of the substance P-related peptide (ranakinin) was established as: Lys-Pro-Asn-Pro-Glu-Arg-Phe-Tyr-Gly-Leu-Met-NH2. Mammalian substance P was not present in the extract. The primary structure of the neurokinin B-related peptide was established as: Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2. This amino acid sequence is the same as that of mammalian neurokinin B. Ranakinin was equipotent with substance P and [Sar9,Met(O2)11]substance P in inhibiting the binding of 125I-Bolton-Hunter-[Sar9,Met(O2)11]substance P, a selective radioligand for the NK1 receptor, to binding sites in rat submandibular gland membranes (IC50 1.6 +/- 0.3 nM; n = 5). It is concluded that ranakinin is a preferred agonist for the mammalian NK1 tachykinin receptor subtype.     

Tachykinin receptors and the airways.

The tachykinins, substance P, neurokinin A and neurokinin B, belong to a structural family of peptides. In mammalian airways, substance P and neurokinin A are colocalized to afferent C-fibres. Substance P-containing fibres are close to bronchial epithelium, smooth muscle, mucus glands and blood vessels. Sensory neuropeptides may be released locally, possibly as a result of a local reflex, and produce bronchial obstruction through activation of specific receptors on these various tissues. Three types of tachykinin receptors, namely NK-1, NK-2 and NK-3 receptors, have been characterized by preferential activation by substance P, neurokinin A and neurokinin B respectively. NK-1 and NK-2 receptors were recently cloned. The determination of receptor types involved in the effects of tachykinins in the airways has been done with synthetic agonists and antagonists binding specifically to NK-1, NK-2 and NK-3 receptors. Although the existence of species differences, the conclusion that bronchial smooth muscle contraction is mainly related to activation of NK-2 receptors on bronchial smooth muscle cell has been drawn. The hypothesis of a NK-2 receptor subclassification has been proposed with NK-2A receptor subtype in the guinea-pig airways. Other effects in the airways are related to stimulation of NK-1 receptors on mucus cells, vessels, epithelium and inflammatory cells. A non-receptor-mediated mechanism is also involved in the effect of substance P on inflammatory cells and mast cells.     

Demonstration of two distinct tachykinin receptors in rat brain cortex

Eledoisin and substance P are members of a class of peptides termed tachykinins. They share a similar spectrum of biological activities but their relative potencies in various pharmacological assays differ. We have investigated whether there is more than one receptor for these tachykinins in rat brain cortex membranes. 125I-Bolton Hunter-conjugated eledoisin specifically binds to rat brain cortex membranes with high affinity. The binding is inhibited over 95% by unlabeled eledoisin (6.6 microM). Scatchard analysis of the binding of this ligand is curvilinear suggesting that there are two binding sites with KD values of 0.9 +/- 0.7 nM and 20 +/- 10 nM. We tested various analogs and fragments of substance P and eledoisin for their ability to inhibit the binding of 125I-Bolton Hunter-conjugated eledoisin and 125I-Bolton Hunter-conjugated substance P to these membranes. The following peptides are more potent as inhibitors of the 125I-Bolton Hunter-conjugated eledoisin binding site than of the 125I-Bolton Hunter-conjugated substance P binding site: nonradioactive Bolton Hunter-conjugated eledoisin (greater than 100-fold), eledoisin (12-fold), kassinin (22-fold), neuromedin K (greater than 58-fold), and pyroglutamyl substance P(6-11)hexapeptide (4-fold). In contrast, substance P (21-fold), physalaemin (8-fold), and substance P methyl ester (1200-fold) were more potent as inhibitors of 125I-Bolton Hunter-conjugated substance P binding. These results suggest that these two ligands may bind to distinct receptors. 125I-Bolton Hunter-conjugated substance P binds specifically to rat parotid cell receptors, but 125I-Bolton Hunter-conjugated eledoisin does not, indicating that parotid cells contain only one of the receptor subtypes. The cortex membrane binding of both ligands is stimulated by low concentrations of MnCl2 (ED50 = 0.05 mM) and is inhibited by guanylyl-5'-(beta, gamma-imido)diphosphate (IC50 = 0.5 microM).     

Structural Motifs Encoded by Individual Exons of the Human Neurokinin-1 Receptor Gene Interact Differentially with Selective Agonists and Antagonists

Abstract: Three chimeric receptors were constructed by exchanging exons between human neurokinin NK₁ and NK₃ receptor genes. The N-terminal sequences of these chimeric receptors are encoded by exon 1, exon 1–2, or exon 1–3 of the NK₁ receptor gene, whereas the remaining C-terminal sequences of these chimeric receptors are encoded by corresponding exons of the human NK₃ receptor gene. Substance P bound with high affinities to all three chimeric receptors, suggesting that in addition to the common structures composed of conserved amino acid residues among neurokinin receptors, structural elements encoded by the first exon of the human NK₁ receptor gene may also play an important role for substance P binding. On the contrary, potent NK₁ antagonists L703,606 and SR140,333 did not show any detectable binding to these chimeric receptors. In accordance, sequences encoded by exon 4, and possibly exon 5, are likely to contain important structural motifs that may directly or indirectly influence the binding of these antagonists. Further comparison of the binding affinities of highly selective NK₁ agonists, [Sar⁹,Met(O₂)¹¹]substance P, substance P methyl ester, and septide, revealed that each agonist may interact differently with the human NK₁ receptor. These results show that the exon-exchanging technique can be a useful tool for studying structure-function relationships of receptors in which exon-intron junctions are fully conserved among receptor subtypes.     

Existence of atrial natriuretic peptide in choroid, retina and ciliary body in rabbits]

The presence of atrial natriuretic factor (ANF) in choroid, retina and ciliary body of rabbit eyes has been studied. The peptide was extracted by reverse phase chromatography using Sep-Pak C18 cartridges. Immunoreactive ANF (Ir-ANF) was measured by a specific RIA. In the choroid, retina and ciliary body the ir-ANF concentrations were 444 +/- 94, 67 +/- 12 and 223 +/- 75 pg/g tissue weight (n = 16), respectively. The biological activity of the acid extract ANF was evaluated by a 125I-ANF binding assay to a retinal particulate preparation. Results demonstrate the existence of biological active ANF in choroid, retina and ciliary body of rabbit eyes. The presence of ANF and its specific receptors in the ocular tissues suggests that this hormone might play a physiological role in the intraocular fluid homeostasis.     

The inhibitory effect of [D-Arg1,D-Phe,D-Try7,9,Leu11] substance P on endothelin-1 binding sites in rat cardiac membranes.

The specific binding of [125I]ET-1 to rat cardiac membrane fragments was inhibited by [D-Arg1,D-Phe, D-Try7,9,Leu11] substance P [substance P(D)], a potent bombesin antagonist. This inhibitory effect required high concentrations (greater than 3X10(-6)M) of substance P(D) and was accompanied by a steep increase in non-specific binding, and not a decrease in total binding. Such results indicate that substance P(D) does not competitively inhibit the specific binding of [125I]ET-1 to rat cardiac membrane fragments.     

The two NK-1 binding sites correspond to distinct, independent, and non-interconvertible receptor conformational states as confirmed by plasmon-waveguide resonance spectroscopy

Two nonstoichiometric ligand binding sites have been previously reported for the NK-1 receptor, with the use of classical methods (radioligand binding and second messenger assays). The most populated (major, NK-1M) binding site binds substance P (SP) and is related to the adenylyl cyclase pathway. The less populated (minor, NK-1m) binding site binds substance P, C-terminal hexa- and heptapeptide analogues of SP, and the NK-2 endogenous ligand, neurokinin A, and is coupled to the phospholipase C pathway. Here, we have examined these two binding sites with plasmon-waveguide resonance (PWR) spectroscopy that allows the thermodynamics and kinetics of ligand-receptor binding processes and the accompanying structural changes of the receptor to be monitored, through measurements of the anisotropic optical properties of lipid bilayers into which the receptor is incorporated. The binding of the three peptides, substance P, neurokinin A, and propionyl[Met(O(2))(11)]SP(7-11), to the partially purified NK-1 receptor has been analyzed by this method. Substance P and neurokinin A bind to the reconstituted receptor in a biphasic manner with two affinities (K(d1) = 0.14 +/- 0.02 nM and K(d2) = 1.4 +/- 0.18 nM, and K(d1) = 5.5 +/- 0.7 nM and K(d2) = 620 +/- 117 nM, respectively), whereas only one binding affinity (K(d) = 5.5 +/- 0.4 nM) could be observed for propionyl[Met(O(2))(11)]SP(7-11). Moreover, binding experiments in which one ligand was added after another one has been bound to the receptor have shown that the binding of these ligands to each binding site was unaffected by the fact that the other site was already occupied. These data strongly suggest that these two binding sites are independent and non-interconvertible on the time scale of these experiments (1-2 h).     

An analogue of substance P with broad receptor antagonist activity

[DPro4,DTrp7,9,10]Substance P-4-11 functions as a substance P receptor antagonist in several different systems. Because some analogues of substance P can function as receptor antagonists for bombesin as well as substance P, we tested [DPro4,DTrp7,9,10]substance P-4-11 for its ability to modify the interaction of various pancreatic secretagogues with their receptors in dispersed acini from guinea pig pancreas. [DPro4,DTrp7,9,19]Substance P-4-11 did not stimulate amylase secretion and did not alter the stimulation of amylase secretion caused by secretin, vasoactive intestinal peptide, calcitonin gene-related peptide or carbachol, but did inhibit the stimulation of amylase secretion caused by substance P, bombesin or cholecystokinin. With substance P, bombesin and cholecystokinin, [DPro4,DTrp7,9,10]substance P-4-11 caused a parallel rightward shift in the dose-response curve for stimulation of amylase secretion with no change in the maximal response. Schild plots of these results gave straight lines with slopes that were not significantly different from unity. [DPro4,DTrp7,9,10]Substance P-4-11 inhibited binding of 125I-labeled substance P, 125I-[Tyr4]bombesin and 125I-cholecystokinin octapeptide over the same range of concentrations as that in which it inhibited biologic activity of each of these peptides. Half-maximal inhibition of binding of 125I-substance P occurred with 4 microM, of 125I-[Tyr4]bombesin with 17 microM and of 125I-cholecystokinin octapeptide with 5 microM. With each radiolabeled peptide the value of Ki for inhibition of binding by [DPro4,DTrp7,9,10]substance P-4-11 was not significantly different from the corresponding value of Ki calculated from the appropriate Schild plot. The present results indicate that [DPro4,DTrp7,9,10]substance P-4-11 is a competitive antagonist at receptors for substance P, for bombesin and for cholecystokinin. Thus, these receptors must share a common peptide recognition mechanism even though they interact with agonists that have no obvious structural similarity.     

Synthesis and biological activities of neurokinin alpha and beta

Two novel neuropeptides, neurokinin alpha and beta isolated from porcine spinal cord, were announced. We have synthesized neurokinin alpha as Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2, which were 98-99% pure by HPLC. Assays on the isolated guinea pig ilium showed neurokinin alpha to have 81% and neurokinin beta to have 65% of the activity of Substance P. Knowledge of these three related peptides having a common activity opens new considerations of their intrinsic physiological roles in neurotransmission versus pharmacological activities, and reappraisal of the diverse activities of Substance P including that in the inflammatory response of the eye.     

Inhibition of substance P degradation in rat brain preparations by peptide hydroxamic acids

A peptidase activity of rat diencephalon membranes, which acts on the C-terminal hexapeptide sequence of substance P, was characterized using the radiolabeled substrate N alpha-[( 125I]iododesaminotyrosyl)-substance P (6-11)-hexapeptide. This activity presents certain characteristics similar to those of the substance-P-degrading enzyme purified from human brain by Lee et al. [Eur. J. Biochem. 114, 315-327 (1981)]. It is inhibited by metal chelators and some thiol reagents, but is insensitive to inhibitors of serine proteases and aminopeptidases. The activity is different from angiotensin-converting enzyme and enkephalinase, since it is not affected by specific inhibitors of these enzymes. Substance P and substance P C-terminal fragments longer than the pentapeptide inhibited the degradation of the radiolabeled substrate with inhibition constants around 200 microM. Short fragments of the substance P sequence, such as Boc-Phe-Phe-OMe and Boc-Phe-Phe-Gly-OEt, were also found to inhibit the degradation of the substrate. When the metal-chelating hydroxamic acid moiety was attached to the carboxyl terminus of these short peptides, potent inhibitors of the substance-P-degrading activity were obtained, with inhibition constants in the micromolar range. The most potent of these compounds, iododesaminotyrosyl-Phe-Phe-Gly-NHOH (IBH-Phe-Phe-Gly-NHOH), is a competitive inhibitor, with a Ki value of 1.9 microM. The degradation of substance P by rat diencephalon slices was inhibited to the same extent (40-50%) by IBH-Phe-Phe-Gly-NHOH (20 microM) and by phosphoramidon (1 microM). A combination of both reagents reduced the degradation rate by 75-80%, suggesting that both enkephalinase and the substance-P-degrading activity are involved in the metabolism of substance P in this preparation. IBH-Phe-Phe-Gly-NHOH seems to be quite specific for the latter enzyme, since at a high concentration (0.1 mM) it did not affect the degradation of the radiolabeled substrate by alpha-chymotrypsin, papain, or thermolysin.     

Tachykinin Receptors of the NK1 Type (Substance P) Coupled Positively to Phospholipase C on Cortical Astrocytes from the Newborn Mouse in Primary Culture

Specific 125I-Bolton-Hunter substance P (125I-BHSP) binding sites are present on intact cortical astrocytes of the newborn mouse in primary culture. Therefore, these cells were used to ascertain the existence of functional substance P (SP) receptors coupled positively to phospholipase C. SP stimulated phosphoinositide breakdown with an EC50 value (4.5 x 10(-10) M) similar to its IC50 value (3.8 x 10(-10) M) for inhibiting 125I-BHSP binding. The maximal response to (10(-6) M SP for 60 min) obtained was approximately 500% of control values. The rank order of potency of tachykinins was SP greater than neurokinin (NK) A greater than NKB. Long SP C-terminal fragments were more potent than shorter ones in stimulating the accumulation of 3H-inositol phosphates. SP free acid and SP N-terminal fragments were without effect. [L-Pro9]SP and SP methyl ester, two selective agonists of NK1 receptors, were almost as potent as SP. An excellent correlation was found when the abilities of tachykinins and their analogs for stimulating phosphoinositide breakdown and for inhibiting 125I-BHSP binding were compared. Finally, when used at a concentration of 3 x 10(-6) M, spantide [( D-Arg1, D-Trp7,9, Leu11]SP), an SP antagonist, competitively reduced the stimulatory effect of SP on accumulation of 3H-inositol phosphates. These results demonstrate the presence of functional SP receptors (NK1) on cortical astrocytes from the newborn mouse in primary culture.     

In vivo autoradiographic analysis of prolactin binding in brain and choroid plexus of the domestic ring dove

Summary The binding of intravenously administered prolactin to choroid plexus and brain tissue was determined radioautographically in the ring dove, a species that exhibits prolactin-induced alterations in brain function. An intense autoradiographic reaction was detected over the epithelial cells of the choroid plexus 5 min after the intravenous injection of ¹²⁵I-ovine prolactin. A significant reaction was also observed over the infundibulum but no significant uptake of prolactin occurred in other brain areas. The binding of radiolabelled prolactin to infundibulum appeared to be non-specific, since excess unlabelled hormone did not reduce silver grain density. In contrast, ¹²⁵I-ovine prolactin binding in choroid plexus was significantly reduced by excess unlabelled ovine prolactin or human growth hormone, but not by ovine luteinizing hormone. Specific binding to choroid plexus was also detected in vitro. The lack of significant brain uptake of prolactin in vivo is discussed in relation to recent in vitro evidence for specific binding sites for prolactin in several dove brain regions. Similarities between the binding results obtained in this avian species and those reported previously in mammals suggest that the two vertebrate groups exhibit similar patterns of prolactin interaction with neural target tissues.     

Localization of 2-[125I]Iodomelatonin Binding Sites in Mammalian Retina

Specific melatonin binding sites were localized in the mammalian retina using the selective radioligand 2-[125I]iodomelatonin. Frozen sections obtained from both pigmented and albino rabbit eyes and albino mouse eyes were incubated with 2-[125I]iodomelatonin in the absence and presence of competing agents. In eyecups from albino rabbits, the highest density of specific 2-[125I]iodomelatonin binding sites was localized over the inner plexiform layer. Approximately 40-60% of the binding was specific, as determined with both the agonist 6-chloromelatonin and the antagonist luzindole. A high density of binding sites was observed over the choroid and retinal pigmented epithelium, but no statistical difference between total and nonspecific binding was detected. Results were similar with eyecups from pigmented rabbits. Albino mice showed a significant extent of 2-[125I]iodomelatonin binding in both the inner plexiform and the outer and inner segment layers. The specific binding of 2-[125I]iodomelatonin in retinas from albino rabbits maintained in the light for 24 h before decapitation was increased in the inner retina compared with the control. The distribution of 2-[125I]iodomelatonin binding sites in the various layers of the mammalian retina is consistent with the described functions for this hormone in retinal physiology.     

The NK1 receptor is involved in the neurokinin-induced shape change of rabbit platelets.

Substance P and selective neurokinin receptor agonists have been tested for their ability to induce shape change in rabbit platelets. Substance P and the NK1 receptor agonist Ac [Arg6,Sar9,Met(O2)11]-substance P (6-11) induced shape change (EC50 = 3 and 6 nM, respectively), whereas the selective NK2 agonist [Nle10]-Neurokinin A (4-10) and the selective NK3 agonist [MePhe7]-Neurokinin B did not show any effect. Moreover, the specific NK1 receptor antagonist CP-96,345 selectively and dose-dependently counteracted the effect of substance P or of the NK1 receptor agonist (IC50 = 2 and 0.8 nM, respectively), whereas the selective NK2 receptor antagonist, SR 48968, had no effect. Unlike for serotonin or low doses of ADP, epinephrine did not allow substance P or the NK1 receptor agonist to become a proaggregating substance. These data therefore show that the NK1 receptor is solely involved in the neurokinin-induced shape change of rabbit platelets.     

Mapping, quantitative distribution and origin of substance p- and VIP-containing nerves in the uvea of guinea pig eye

VIP- and substance P-like immunoreactivities were found in considerable concentrations (VIP: 17.3 +/- 4.8 pmol/g, mean +/- SEM; substance P:11.1 +/- 1.8 pmol/g) in the uveal portion of the guinea pig eye. Immunocytochemistry localised these two regulatory peptides to nerve fibres found principally in a plexus in the iris (substance P) and in an extensive network surrounding the blood vessels of the choroid (VIP). A remarkable anatomical demarcation of the two types of peptide-containing nerves was established by the staining of substance P-containing nerves, which stops at the level of the ciliary body. This uveal area is known to be involved in the ocular responses to nociceptive stimuli. At the ultrastructural level, immunoreactivity for both peptides was localised to distinct subpopulations of p-type nerves, distinguishable by the size of their large dense-cored vesicles. Those immunoreactive for VIP were significantly larger (p less than 0.0005) than those immunoreactive for substance P (95 +/- 7 nm and 82 +/- 9 nm respectively; mean +/- SD). Interruption of the trigeminal pathway produced a remarkable decrease of substance P immunoreactivity in the anterior portion of the uvea (9.1 +/- 1.5 pmol/g, mean +/- SEM, control; 5.3 +/- 1.3 pmol/g, denervated), but not of VIP immunoreactivity in the choroid. Following colchicine treatment, VIP-immunoreactive neuronal cell bodies were localised in the choroid. The separate anatomical localisations and distributions of the two uveal peptides appear to be related to their different origins and functional roles in the response of the eye to noxious stimuli.     

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.