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).     

Contractile response to substance P in isolated smooth muscle strips from the intestinal bulb of the carp (Cyprinus carpio)

1. The effect of substance P on the mechanical activity of carp intestinal bulb smooth muscle was investigated in vitro. 2. Bath-applied substance P (1 nM-1 microM) caused concentration-dependent contraction of the smooth muscle. The EC50 value was 20 +/- 3 nM (N = 13). 3. Pretreatment with tetrodotoxin (780 nM) or atropine (500 nM) partially decreased the contractile response to substance P, while methysergide (3 microM) did not decrease the response. 4. The contractile response to substance P was not decreased by [D-Pro2, D-Trp7.9]-substance P or [D-Pro4, D-Trp7.9]-substance P (4-11) pretreatment (10 microM for 5 min). 5. Exposure of the intestinal bulb to substance P (100 nM and 1 microM for 15 min) decreased the response to subsequent application of substance P, physalaemin and eledoisin in a concentration dependent manner, while the contractile response to acetylcholine or methionine-enkephalin was not affected. 6. Exposure of the intestinal bulb to physalaemin and eledoisin (100 nM for 15 min) decreased the response to subsequent application of substance P. 7. The above results indicate that substance P causes the contraction of the carp intestinal bulb smooth muscle through its direct action on the smooth muscle and its indirect action through enteric cholinergic nerves. Long-term exposure to substance P causes desensitization of the preparation to substance P, physalaemin and eledoisin at the receptor level.     

The rat submaxillary gland contains predominantly P-type tachykinin binding sites

The specific binding of the ¹²⁵I-Bolton-Hunter labeled tachykinins substance K (BHSK), eledoisin (BHE), and substance P (BHSP) was examined in crude membrane suspensions and by autoradiography in rat submaxillary gland. All three ligands at 0.1 nM concentrations exhibited binding that was inhibited by tachykinins in a potency rank order of substance P > physalaemin > substance K > eledoisin > kassinin > neuromedin K with slope factors essentially equal to unity. All tachykinins were 5 to 10 times more potent in inhibiting BHSK and BHE binding compared to BHSP binding. Autoradiographic visualization of BHSK and BHSP binding sites in the gland revealed extensive labeling of mucous and serous acini. The intensity of labeling was much less for BHSK than for BHSP. The results indicate that the rat submaxillary gland contains predominantly P-type tachykinin binding sites.     

Tachykinin activation of muscarinic inhibition in canine small intestine is SPP in nature

Substance P when injected intraarterially into the small intestine of the anaesthetized dog during phasic activity produces three concentration dependent responses of the circular muscle. At lowest doses (approximately 10(-12) moles) inhibition occurs via release of acetylcholine to a muscarinic auto-receptor. At slightly higher doses (10(-10) moles) inhibition is preceeded by excitation via release of acetylcholine to muscarinic receptors on the smooth muscle. At still higher doses (10(-9) moles) substance P excites the smooth muscle directly. The present study demonstrates that other members of the tachykinin family also produce inhibition in vivo. The potency sequence was found to be physalaemin greater than or equal to substance P = neuromedin K greater than kassinin greater than alpha neurokinin = eledoisin. Such a sequence suggests that substance P is a natural stimulant of this pathway and that the receptor is SPP-like. The C-terminal fragment, substance P8-11, was a weak agonist at this receptor, while substance P1-9 was ineffective.     

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.     

A novel bioassay for the NK-2 neurokinin receptor: the guinea pig gallbladder

Although three neurokinin receptors (NK-1, NK-2, NK-3) have been identified by radioligand binding assays, only the NK-1 and NK-3 types have been found in smooth muscle bioassays. In this study, evidence is presented demonstrating functional NK-2 type receptors in the guinea pig gallbladder (GPGB). The potencies of the following neurokinins were determined in the GPGB and the guinea pig ileum (GPI): substance P (SP), physalaemin (PH), eledoisin (EL), substance K (SK) and kassinin (KA). ED50 values were determined by linear regression analysis of the dose-related increases in the force generated by each peptide. In the GPI, the rank order of potency was SP = PH = EL greater than SK = KA, indicating NK-1 selectivity. In the GPGB, the relative potencies were SK greater than KA greater than EL much greater than PH greater than SP, which is similar to that reported for the NK-2 receptor in radioligand binding assays. These findings demonstrate the NK-2 receptor tissue selectivity of the GPGB.     

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.     

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.     

Luminal tachykinin receptors on canine tracheal epithelium: functional subtyping

Luminal addition of tachykinins to the open-circuited canine tracheal epithelium produces a biphasic response in the transmucosal potential difference (PD). A rapid, transient decrease is followed by a subsequent rise, both phases being associated with changes in conductance. Concentration-response curves demonstrated the following orders of potency: substance P greater than physalaemin greater than eledoisin = kassinin for the tachykinins, and substance P greater than substance P-(4-11) greater than substance P-(6-11) using the C-terminal fragments. Both sequences are similar to those reported for the dog carotid artery. These observations were confirmed by cross-tachyphylaxis experiments. SP-O-methyl ester, a selective agonist for the SP-P (or NK-1) receptor, elicited identical responses, and exhibited cross-tachyphylaxis to substance P. Bradykinin produced similar luminal responses, though different receptors are involved, since no cross-tachyphylaxis was observed between bradykinin and the tachykinins.     

Regional distribution of neuromedin K and neuromedin L in rat brain and spinal cord

Neuromedin K and neuromedin L are novel mammalian tachykinins isolated from porcine spinal cord. We have developed a highly sensitive radioimmunoassay for neuromedin K. Since the radioimmunoassay for neuromedin K has significant crossreactivity with neuromedin L and substance P, we can simultaneously determine the tissue concentrations of neuromedin K, neuromedin L and substance P after separation of the tissue extracts by reverse phase high performance liquid chromatography. Substance P is found to be the most abundant mammalian tachykinin in every brain region. The ratio of the concentration of substance P to neuromedin K is small in cerebral cortex and large in medulla-pons, while that of substance P to neuromedin L is rather constant in a range of 2.0–2.5. In spinal cord, dorsal half contains more neuromedin K and L than ventral half as is the case with substance P. These results indicate that both neuromedin K and L are endogenous mammalian tachykinins with specific physiological functions.     

Possible influence of tachykinins on body fluid homeostasis in the rat

The effect on water intake, urine flow and vasopressin release of intracranial injections of substance P, physalaemin and eledoisin was studied in Wistar and Brattleboro, homozygous and heterozygous, rats. The tachykinins strongly inhibited water intake both in Wistar and in Brattleboro, homozygous and heterozygous, rats. Physalaemin and eledoisin reduced urine flow in Wistar and heterozygous, but not in homozygous, Brattleboro rats. Substance P never affected urine elimination. Physalaemin and eledoisin produced a dose-dependent, long lasting release of vasopressin in Wistar rats. Substance P did not affect the release of vasopressin. The results suggest that both substance P and physalaemin could influence brain mechanisms which control water intake, acting as thirst inhibitors, and that physalaemin could also participate in body fluid control by conserving water through vasopressin release.     

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.     

Effect of kassinin, neurokinin A and neurokinin B on drinking behaviour in the pigeon

Intracerebroventricular (i.c.v.) injection of kassinin produced a prompt and copious drinking response at doses of 10-1000 ng/pigeon, in the absence of other behavioural alterations or of changes in core temperature. Neurokinin A and B evoked drinking, but they were respectively 10 and 100 times less potent than kassinin. Intraperitoneal injection of kassinin elicited drinking, but at doses about 1000 X larger than the i.c.v. ones. The angiotensin antagonist [Sar1, Leu8]angiotensin II did not reduce drinking induced by i.c.v. kassinin, suggesting that its effect is not due to interaction with the central renin-angiotensin system. Moreover, the effect is apparently independent of the mechanisms controlling hypovolaemic and hyperosmotic thirst since exact additivity was found in the dipsogenic response when i.c.v. kassinin was administered in the presence of a hypovolaemic (subcutaneous (s.c.), polyethylene glycol) or hyperosmotic (s.c. hypertonic NaCl) dipsogenic stimulus. The present findings show that kassinin, neurokinin A and B share with the tachykinins already tested (eledoisin, physalaemin, substance P) a common dipsogenic action in pigeons. However, marked differences exist in their dipsogenic potency. This order of potency, eledoisin = kassinin = physalaemin greater than neurokinin A = substance P greater than neurokinin B, is not consistent with the tachykinin receptor subtypes so far proposed.     

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.     

Cloning and expression of a rat neuromedin K receptor cDNA

Functional cDNA clones for rat neuromedin K receptor were isolated from a rat brain cDNA library by cross-hybridization with the bovine substance K receptor cDNA. Injection of the mRNA synthesized in vitro from the cloned cDNA into Xenopus oocytes elicited electrophysiological responses to tachykinins, with the most potent sensitivity being to neuromedin K. Ligand-binding displacement in membranes of mammalian COS cells transfected with the cDNA indicated the rank order of affinity of the receptor to tachykinins: neuromedin K greater than substance K greater than substance P. The hybridization analysis showed that the neuromedin K receptor mRNA is expressed in both the brain and the peripheral tissues at different levels. The rat neuromedin K receptor consists of 452 amino acid residues and belongs to the family of G protein-coupled receptors, which are though to have seven transmembrane domains. The sequence comparison of the rat neuromedin K, substance P, and substance K receptors revealed that these receptors are highly conserved in the seven transmembrane domains and the cytoplasmic sides of the receptors. They also show some structural characteristics, including the common presence of histidine residues in transmembrane segments V and VI and the difference in the numbers and distributions of serine and threonine residues as possible phosphorylation sites in the cytoplasmic regions. This paper thus presents the first comprehensive analysis of the molecular nature of the multiple peptide receptors that exhibit similar but pharmacologically distinguishable activities.     

Presence of four tachykinins in an acid extract of the carp intestinal bulb (Cyprinus carpio)

1. The pharmacological and chemical properties of substance P-like peptides isolated from an acid extract of the carp intestinal bulb were examined using guinea-pig ileum longitudinal smooth muscle.2. On a Sephadex G25 column (3 × 96 cm), smooth muscle contracting material was eluted as two peaks (fraction-1 and fraction-2). The molecular weight of the fraction-1 was estimated to be 2300 and that of the fraction-2 to be 1530.3. The pharmacological properties of the contracting materials in fraction-1 and fraction-2 resembled those of substance P and neurokinin A.4. The susceptibility of the contracting activity of fraction-1 to proteolytic enzymes resembled that of physalaemin but, on the other hand, the susceptibility of that of fraction-2 resembled those of eledoisin and neurokinin A.5. Ion-exchange chromatography on sulfopropyl-Sephadex C25 indicated the presence of one contracting material in fraction-1 and three contracting materials in fraction-2. The elution positions of four materials were different from that of substance P.6. These results indicate that four tachykinins different from substance P are present in an acid extract of the carp intestinal bulb.     

The primary structures and myotropic activities of two tachykinins isolated from the African clawed frog,Xenopus laevis

Two peptides with limited structural similarity to mammalian substance P (SP) and neurokinin A (NKA) have been isolated from extracts of the intestine of the African clawed frog (Xenopus laevis). The primary structure of an SP-like peptide was established as: Lys-Pro-Arg-Pro-Asp-Gln-Phe-Tyr-Gly-Leu-Met.NH(2), which is identical to the previously characterized peptide, bufokinin isolated from the toad Bufo marinus. The primary structure of an NKA-related peptide was established as Thr-Leu-Thr-Thr-Gly-Lys-Asp-Phe-Val-Gly-Leu-Met.NH(2). Only the five amino acids at the C-terminal region of the peptide are identical to mammalian NKA whereas the N-terminal region shows no structural similarity to previously characterized tachykinins. Immunohistochemical investigations of the gut wall revealed a dense network of nerve fibres and nerve cell bodies containing SP/NKA-like substances. The myotropic effects of the Xenopus tachykinins were compared with the contractile effect of mammalian SP and NKA on isolated strips of circular smooth muscle from Xenopus stomach. No significant differences in potencies (-log EC(50)) or in intrinsic activities were observed between the Xenopus and mammalian peptides. The potencies for the Xenopus SP-like (8.49+/-0.15) and the NKA-like peptide (8.12+/-0.06) were similar suggesting that the amino acid sequence at the N-terminal region of the tachykinins is not important in activating the tachykinin receptors in Xenopus gastric smooth muscle. The maximum response to Xenopus SP (alpha=0.59+/-0.06) was significantly lower than to the NKA-like peptide (alpha=1.0) suggesting a more effective interaction of the NKA-like peptide with the tachykinin receptor(s) in Xenopus stomach.     

Presence of four tachykinins in an acid extract of the carp intestinal bulb (Cyprinus carpio).

1. The pharmacological and chemical properties of substance P-like peptides isolated from an acid extract of the carp intestinal bulb were examined using guinea-pig ileum longitudinal smooth muscle. 2. On a Sephadex G25 column (3 x 96 cm), smooth muscle contracting material was eluted as two peaks (fraction-1 and fraction-2). The molecular weight of the fraction-1 was estimated to be 2300 and that of the fraction-2 to be 1530. 3. The pharmacological properties of the contracting materials in fraction-1 and fraction-2 resembled those of substance P and neurokinin A. 4. The susceptibility of the contracting activity of fraction-1 to proteolytic enzymes resembled that of physalaemin but, on the other hand, the susceptibility of that of fraction-2 resembled those of eledoisin and neurokinin A. 5. Ion-exchange chromatography on sulfopropyl-Sephadex C25 indicated the presence of one contracting material in fraction-1 and three contracting materials in fraction-2. The elution positions of four materials were different from that of substance P. 6. These results indicate that four tachykinins different from substance P are present in an acid extract of the carp intestinal bulb.     

Effects of tachykinins on the electrical activity of isolated canine tracheal epithelium: an exploratory study

Substance P (SP) and other tachykinins altered the potential differences (P.D.) and resistances (omega) of open-circuited epithelial preparations. (1) The effects observed were critically dependent on the side to which the peptides were added. Luminal addition of SP (5 x 10(-7) M) produced within 8-20 s, a rapid decrease in P.D. (dip) followed by an increase that peaked transiently and declined. Serosal addition led to an increase in P.D. after a longer lag (40-90 s). In both cases, resistance decreased. (2) Low concentrations of SP (5 x 10(-12) M) elicited only an increase in P.D., the dip appearing at concentrations 50-100-fold higher, indicating perhaps receptors with different affinities. (3) Changes in P.D. and resistance were seen on luminal addition of physalaemin, eledoisin, kassinin, alpha-neurokinin, neuromedin K and C-terminal SP fragments larger than 5 amino acids. No responses were seen with SP tetrapeptide, SP 9-11, bombesin, litorin, neurotensin, dynorphin. The sequence Phe-X-Gly-Leu-Met-NH2 thus seems necessary to elicit changes in P.D. and resistance. (4) As with SP, low doses of physalaemin, eledoisin, kassinin elicited only an increase in PD, the dip appearing with higher concentrations.     

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.