Pharmacologic characterization and autoradiographic distribution of binding sites for iodinated tachykinins in the rat central nervous system

P-type, E-type, and K-type tachykinin binding sites have been identified in the mammalian CNS. These sites may be tachykinin receptors for which the mammalian neuropeptides substance P, neuromedin K, and substance K are the preferred natural agonists, respectively. In the present investigation, we have compared the pharmacology and the autoradiographic distribution of CNS binding sites for the iodinated (¹²⁵I-Bolton-Hunter reagent) tachykinins substance P, eledoisin, neuromedin K, and substance K. Iodinated eledoisin and neuromedin K exhibited an E-type binding pattern in cortical membranes. Iodinated eledoisin, neuromedin K, and substance K each labeled sites that had a similar distribution but one that was considerably different from that of sites labeled by iodinated substance P. CNS regions where there were detectable densities of binding sites for iodinated eledoisin, neuromedin K, and substance K and few or no sites for iodinated substance P included cortical layers IV–VI, mediolateral septum, supraoptic and paraventricular nuclei, interpeduncular nucleus, ventral tegmental area, and substantia nigra pars compacta. Binding sites for SP were generally more widespread in the CNS. CNS regions where there was a substantial density of binding sites for iodinated substance P and few or no sites for iodinated eledoisin, neuromedin K, and substance K included cortical layers I and II, olfactory tubercle, nucleus accumbens, caudate-putamen, globus pallidus, medial and lateral septum, endopiriform nucleus, rostral thalamus, medial and lateral preoptic nuclei, arcuate nucleus, dorsal raphe nucleus, dorsal parabrachial nucleus, parabigeminal nucleus, cerebellum, inferior olive, nucleus ambiguus, retrofacial and reticular nuclei, and spinal cord autonomic and somatic motor nuclei. In the brainstem, iodinated substance P labeled sites in both sensory and motor nuclei whereas iodinated eledoisin, neuromedin K, and substance K labeled primarily sensory nuclei. Our results are consistent with either of two alternatives: (1) that iodinated eledoisin, neuromedin K, and substance K bind to the same receptor site in the rat CNS, or (2) that they bind to multiple types of receptor sites with very similar distribution.     

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.     

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).     

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.     

Pharmacological characterization of tachykinin septide-sensitive binding sites in the rat submaxillary gland

Binding studies have shown that [125I]NKA is a selective ligand of tachykinin septide-sensitive binding sites from membranes of the rat submaxillary gland. Indeed, this ligand bound with high affinity to a single population of sites. In addition, competition studies indicated that natural tachykinins and tachykinin-related compounds had a similar affinity for these sites than for those labeled with [3H]ALIE-124, a selective ligand of septide-sensitive binding sites. Moreover, selective tachykinin NK2, or NK3 agonists or antagonists exhibited weak or no affinity for [125I]NKA binding sites. As indicated by Ki values of several compounds, the pharmacological characteristics of the septide-sensitive binding sites (labeled with [125I]NKA) largely differ from those of classic NK1 binding sites, as determined on crude synaptosomes from the rat brain using [125I]Bolton-Hunter substance P (SP) as ligand. Indeed, several tachykinins including neurokinin A (NKA), neuropeptide K (NPK), neuropeptide gamma (NKgamma), and neurokinin B, as well as some SP and NKA analogues or C-terminal fragments such as septide, ALIE-124, SP(6-11), NKA(4-10), which have a weak affinity for classic tachykinin NK1 binding sites exhibited a high affinity for the septide-sensitive binding sites. In contrast, SP, classic selective NK1 agonists, and antagonists had a high affinity for both types of binding sites. The presence of a large population of tachykinin septide-sensitive binding sites in the rat submaxillary gland may thus explain why NPK and NPgamma induce salivary secretion and may potentiate the SP-evoked response in spite of the absence of tachykinin NK2 receptors in this tissue.     

Substance P and substance K receptor binding sites in the human gastrointestinal tract: Localization by autoradiography

Quantitative receptor autoradiography was used to localize and quantify the distribution of binding sites for ¹²⁵I-radiolabeled substance P (SP), substance K (SK) and neuromedin K (NK) in the human GI tract using histologically normal tissue obtained from uninvolved margins of resections for carcinoma. The distribution of SP and SK binding sites is different for each gastrointestinal (GI) segment examined. Specific SP binding sites are expressed by arterioles and venules, myenteric plexus, external circular muscle, external longitudinal muscle, muscularis mucosa, epithelial cells of the mucosa, and the germinal centers of lymph nodules. SK binding sites are distributed in a pattern distinct from SP binding sites and are localized to the external circular muscle, external longitudinal muscle, and the muscularis mucosa. Binding sites for NK were not detected in any part of the human GI tract. These results demonstrate that: 1) surgical specimens from the human GI tract can be effectively processed for quantitative receptor autoradiography; 2) of the three mammalian tachykinins tested, SP and SK, but not NK binding sites are expressed in detectable levels in the human GI tract; 3) whereas SK receptor binding sites are expressed almost exclusively by smooth muscle, SP binding sites are expressed by smooth muscle cells, arterioles, venules, epithelial cells of the mucosa and cells associated with lymph nodules; and 4) both SP and SK binding sites expressed by smooth muscle are more stable than SP binding sites expressed by blood vessels, lymph nodules, and mucosal cells.     

Action of the newly discovered mammalian tachykinins, substance K and neuromedin K, on gastroduodenal motility of anesthetized dogs

The present experiments examined the local effects of two new mammalian tachykinins isolated from porcine spinal cord, substance K and neuromedin K, on gastroduodenal motility of anesthetized dogs. Tachykinins were injected through the gastroepiploic and cranial pancreaticoduodenal arteries at concentrations ranging from 1 to 100 ng/ml. Substance K, neuromedin K and substance P increased gastroduodenal smooth muscle contractions in a dose-dependent manner. The contractile response of the gastric antrum to newly discovered tachykinins was not as long-lasting as that to substance P. The potencies of various tachykinins on contractile responses showed the following rank order of potencies: physalaemin = eledoisin = substance P greater than substance K = neuromedin K in gastric smooth muscle; physalaemin = substance P = eledoisin greater than substance K = neuromedin K in the duodenal smooth muscle. Administration of atropine (100-200 micrograms/kg) inhibited the effect of tachykinins both in the gastric antrum and in the proximal duodenum. These results indicate that substance K and neuromedin K could act as transmitters or as modulators of neuronal activity influencing gastroduodenal motility.     

Binding sites for tachykinin peptides in the brain and stomach of the dogfish, Scyliorhinus canicula.

In membranes of dogfish brain and stomach, two binding sites for tachykinins were identified. One site specifically bound [125I]-Bolton-Hunter substance P (BH-SP) and the rank potency of tachykinins to compete for BH-SP binding revealed similarities with the rank potency of an NK1 receptor. The pharmacology of the other site, which specifically bound [125I]-Bolton-Hunter scyliorhinin II (BH-Scy II), did not resemble any of the mammalian tachykinin receptors. The rank potency to inhibit BH-Scy II binding to this second site was: scyliorhinin II approximately scyliorhinin I greater than eledoisin approximately substance P approximately neurokinin A greater than phyllomedusin approximately physalaemin greater than [Sar9Met(O2)11]substance P. Neurokinin B and senktide did not displace BH-Scy II binding. In addition, nucleotide analogues inhibited BH-SP binding but not BH-Scy II binding. Our binding data suggest the existence of a mammalian-like NK1 receptor and of a nonmammalian tachykinin receptor in the dogfish.     

Distinct binding sites for substance P and neurokinin A (substance K): co-transmitters in rat brain

The distribution of binding sites in rat brain for iodinated neurokinin A and iodinated substance P were compared using autoradiography. Distinct patterns of binding for the two iodinated tachykinins were noted. Binding sites for iodinated neurokinin A were noted in the olfactory bulb, cortex, supraoptic n., paraventricular n., certain amygdaloid n., hippocampus, medial habenula, interpeduncular n., n. of the tractus solitarius, and dorsal horn of the spinal cord. This pattern was in contrast to low levels of binding of iodinated substance P to the cortex, supraoptic n., paraventricular n., and the interpeduncular n., but substantial density of binding sites in numerous other regions.     

Mapping of brain sites sensitive to the antidipsogenic effect of tachykinins

The present study investigated the sensitivity of 12 forebrain and midbrain structures to the antidipsogenic effect of eledoisin, physalaemin and substance P on angiotensin-induced drinking. The three tachykinins elicited the most potent effects when injected into the nucleus preopticus medialis, the nucleus anterior hypothalami and the subfornical organ. In other sites (nuclei lateralis, ventromedialis and posterior hypothalami, nucleus septi lateralis, nucleus interpeduncularis and substantia grisea periventricularis) the effect was lower, and most of these sites showed different sensitivity to the three tachykinins. Finally, the nucleus septi medialis, the nucleus preopticus lateralis and the substantia nigra were refractory to the three tachykinins. These results show that: (1) the antidipsogenic effect of tachykinins can be elicited not only in forebrain, but also in midbrain structures such as the substantia grisea periventricularis and the nucleus interpeduncularis; (2) the distribution of brain sites sensitive to the antidipsogenic effect of substance P and physalaemin is always overlapping, while this is not true for eledoisin. This probably reflects selective distribution and/or activation of distinct subtypes of tachykinin receptors.     

Tachykinin receptors in the rat brain bind alpha-bungarotoxin

alpha-Bungarotoxin was found to inhibit effectively the binding of 125I-labelled substance P and eledoisin to membrane and to solubilize preparations of the rat brain. Other postsynaptic neurotoxins exerted similar but less pronounced influence on the interaction of tachykinins with their receptors. The obtained results suggest that some alpha-bungarotoxin-binding polypeptides in brain are components of tachykinin receptors.     

Differential Effects of Tachykinins Injected Intranigrally on Striatal Dopamine Metabolism

The effects of intranigral injection of kassinin, eledoisin, and substance P on striatal dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) contents as well as circling behavior were studied in rats. Kassinin and eledoisin produced a marked dose-dependent increase of DOPAC concentrations in the ipsilateral striatum, as well as in the number of contralateral circlings. Substance P produced a similar but weaker effect. At the larger dose (5 nmol), the three tachykinins also induced an increase of DA concentrations in the ipsilateral striatum. The rank order of activity was kassinin greater than eledoisin greater than substance P. These results suggest that tachykinins stimulated the nigro-striatal dopaminergic system by accelerating the dopamine metabolism in striatum.     

Mammalian tachykinin-induced hydrolysis of inositol phospholipids in rat brain slices

The mammalian tachykinins substance K, neuromedin K and substance P stimulated inositol phospholipid hydrolysis in paired coronal sections through the rat brain. In contrast, none of these peptides had any effect on either basal or forskolin-stimulated cyclic AMP levels. The present results therefore implicate inositol phospholipid hydrolysis as a possible second messenger system mediating the effects of substance K and neuromedin K in addition to substance P.     

Binding of [125I]Bolton Hunter conjugated eledoisin to rat brain cortex membranes--evidence for two classes of tachykinin receptors in the mammalian central nervous system

[125I]Bolton Hunter conjugated eledoisin was prepared and purified by ion-paired reverse phase chromatography. The ligand binds to rat brain cortex membranes, and the binding is inhibited over 95% by unlabeled eledoisin (6.6 microM). The binding site appears to be distinct from the [125I]Bolton Hunter conjugated substance P receptor based on the relative potencies of substance P, eledoisin, kassinin, physalaemin and [pGlu]substance P (6-11) hexapeptide to displace the binding of these two ligands.     

Cyclic AMP Regulates the Expression of Neurokinin1 Receptors by Neonatal Rat Spinal Neurons in Culture

Neurokinin1 (NK1) receptors are up-regulated in the spinal cord during peripheral inflammation, but the biochemical mediators regulating this change have not been resolved. The promoter region of the gene encoding the NK1 receptor contains a cyclic AMP (cAMP)-responsive element. Therefore, we used primary cultures of neonatal rat spinal cord to test whether increasing intracellular cAMP can increase expression of NK1 receptors. Treatment with dibutyryl-cAMP (dbcAMP) resulted in a time-dependent increase in 125I-Bolton-Hunter-substance P (BHSP) binding in the cultures; treatment with dibutyryl-cyclic GMP did not. Treatment with forskolin plus 3-isobutyl-1-methylxanthine mimicked the increase in binding, providing further evidence for the involvement of cAMP in this effect. Scatchard analyses indicated that the increase in BHSP binding was due to an increase in binding capacity. The cAMP-induced increase in BHSP binding was preceded by an increase in levels of mRNA for NK1 receptor and was attenuated by pretreatment with cycloheximide. These data indicate that the cAMP-induced increase in binding was due to increased synthesis of NK1 receptors. Comparison of substance P (SP)-induced production of inositol phosphates between cultures pretreated with dbcAMP and controls suggested that increased expression of NK1 receptors did not result in increased generation of second messenger by NK1 receptor activation. Together, these data indicate that a persistent increase in intracellular cAMP increases expression of NK1 receptors. Because NK1 receptor activation contributes to increased excitability of spinal neurons, the increased expression of NK1 receptors may be important in maintaining responsiveness of spinal neurons to SP in central mechanisms underlying hyperalgesia.     

Characterization of the substance P receptor in rat brain cortex membranes and the inhibition of radioligand binding by guanine nucleotides

Rat brain cortex membranes bind to a conjugate of substance P and 125I-labeled Bolton-Hunter reagent, and this binding can be inhibited by a low concentration of substance P (Kd = 1.2 +/- 0.4 X 10(-8) M). This binding is reversible and saturable (0.5 +/- 0.1 pmol of binding sites/mg of protein). Fragments of substance P as small as the carboxyl-terminal hexapeptide can inhibit the binding although their potency decreases with the decrease in the length of the peptides. The binding affinities of smaller peptides or peptides in which the carboxyl-terminal amide or amino acids are removed are drastically reduced. Biologically active analogs of substance P, physalaemin, eledoisin, substance P methyl ester, [D-Ala0]hepta(5-11)substance P, kassinin, and the eledoisin-related hexapeptide also can inhibit the binding. However, the binding is not inhibited by polypeptides structurally unrelated to substance P or by amine hormones/neurotransmitters. The binding affinities of biologically active peptides to rat brain cortex membranes are almost identical with their affinities for rat parotid cells which we previously determined. Furthermore, the recently described substance P antagonist, [D-Pro, D-Trp]substance P, inhibits the binding of the 125I-labeled substance P derivative to brain cortex membranes and to parotid cells equally well. These results suggest that the substance P receptors in the brain cortex and the parotid gland are similar. The brain cortex membrane binding of the 125I-labeled substance P derivative can be inhibited by micromolar concentrations of GTP, GDP, and their analogs. ITP and IDP were less active. Adenine and pyridine nucleotides were inactive.     

Effect of chronic capsaicin treatment on tachykinin NK1 binding sites in the rat.

Binding sites for [125I]-Bolton-Hunter substance P (BHSP) were investigated in homogenates of rat submandibular gland, colon smooth muscle, and urinary bladder. In vehicle-treated animals, the equilibrium dissociation constant (KD) was similar for both submandibular gland (0.46 +/- 0.03 nM) and colon (0.57 +/- 0.04 nM), although the maximum density of binding sites (Bmax) was about six-fold higher in submandibular gland compared with colon. These binding parameters remained unchanged in capsaicin-pretreated animals (140 mg/kg IP). In contrast, capsaicin pretreatment reduced (p less than 0.05) the Bmax in urinary bladder by twenty-five percent (0.56 fmol/mg wet weight) when compared to vehicle-treated controls (0.73 fmol/mg wet weight), although the KD was unchanged (vehicle, 0.29 +/- 0.08 nM; capsaicin, 0.24 +/- 0.04 nM). These data demonstrate that the NK1 receptors in submandibular gland and colon smooth muscle are not associated with or dependent upon intact primary afferent sensory neurons. However, a minority of NK1 receptors in the urinary bladder were lost after capsaicin, indicating that these receptors are located on sensory terminals, or may be dependent on growth factors or other chemicals released from these nerves.     

Multiple tachykinins (neurokinin A, neuropeptide K and substance P) in capsaicin-sensitive sensory neurons in the guinea-pig

The occurrence of tachykinins in sensory neurons of the guinea-pig was studied by means of radioimmunoassay combined with ion-exchange and high-performance liquid chromatography as well as by immunohistochemistry. Antisera raised against kassinin (antiserum K12), neurokinin A (NKA) (antiserum NKA2) and substance P (SP) (antisera SP25 and SP2) were used. Antiserum K12 detected NKA, neuropeptide K (NPK) and a component eluting in the position of eledoisin (ELE) in extracts of the lung and ureter. Neurokinin B (NKB) was, however, not found. Neutral water extraction favored recovery of NKA and of the ELE-like component, while NPK was found only in acid extracts. The SP antisera detected two immunoreactive components of which the major form coeluted with synthetic SP. Capsaicin pretreatment depleted all these various forms of immunoreactivity in several peripheral organs including the ureter and lung. The immunoreactivity detected by antisera K12 or SP25 in radioimmunoassay had a similar regional distribution pattern in peripheral tissues. Immunohistochemical examination revealed that antiserum NKA2 stained the same spinal ganglion cells as the SP2 antiserum. The distribution of capsaicin-sensitive nerve fibers stained by these two antisera was also identical in peripheral organs such as the ureter, inferior mesenteric ganglion, heart and lung. It is concluded that multiple tachykinins, including SP, NKA, NPK and an ELE-like peptide, are present in capsaicin-sensitive sensory nerves in the guinea-pig. This finding can most likely be related to the origin of SP, NKA and NPK from the same precursor molecule, subsequent posttranslational tissue processing and axonal transport to terminal regions.     

3H-substance P binding to guinea-pig ileum longitudinal smooth muscle membranes

Specific binding of 3H-substance P was studied in guinea-pig ileum longitudinal smooth muscle membranes. A single population of non-interacting sites with an apparent dissociation constant of 1.8 nM was observed. The relative potencies of some structural analogues of substance P, notably eledoisin and substance P (5-11), in competing for 3H-substance P binding sites, however, had little resemblance to their potencies in contracting the intact muscle or in eliciting the breakdown of inositol phospholipids in this tissue. The results are discussed in the light of other binding studies for substance P.     

Substance P-like immunoreactivity in the stomatogastric nervous systems of the crab Cancer borealis and the lobsters Panulirus interruptus and Homarus americanus

Summary The distribution of substance P-like immunoreactivity in the stomatogastric nervous systems of three decapod crustacean species, Cancer borealis, Homarus americanus, and Panulirus interruptus, was studied. The stomatogastric ganglion showed dense staining in the neuropil, but none in the somata. A single neuron stained in the esophageal ganglion. Lucifer yellow backfills and intracellular injections followed by incubation with the substance P antibody showed that the axons of this neuron project into the inferior esophageal nerves towards the paired commissural ganglia. The commissural ganglia showed a pronounced projection from a large bundle of fibers in the anterior medial portion of the circumesophageal connective. Additionally, less dense neuropil and stained somata were seen in the commissural ganglia. Staining was completely blocked by preabsorption with authentic substance P, physalaemin, eledoisin, and substance K. These data suggest that in the nervous system of crustacean species a molecule with C-terminal homology to substance P and other tachykinins is released as a neuroregulator in the stomatogastric ganglion.