Bolton-Hunter Substance Research Articles

Amide-based atropisomers in tachykinin NK 1-receptor antagonists: synthesis and antagonistic activity of axially chiral N-benzylcarboxamide derivatives of 2,3,4,5-tetrahydro-6 H-pyrido[2,3- b][1,5]oxazocin-6-one

A series of novel N-benzylcarboxamide derivatives of bicyclic compounds, 3,4-dihydropyrido[3,2- f][1,4]oxazepin-5(2 H)-one and 2,3,4,5-tetrahydro-6 H-pyrido[2,3- b][1,5]oxazocin-6-one, were synthesized by cyclization of N-benzyl-2-chloro- N-(2-hydroxyethyl)- [and -(3-hydroxypropyl)-] nicotinamides, respectively. Atropisomerism was observed in 5-[3,5-bis(trifluoromethyl)benzyl]-7-phenyl-2,3,4,5-tetrahydro-6 H-pyrido[2,3- b][1,5]oxazocin-6-ones due to steric hindrance of the carboxamide moiety and restriction of its rotation. Cyclization of N-[3,5-bis(trifluoromethyl)benzyl]-2-chloro- N-[(2 S)-3-hydroxy-2-methylpropyl]-5-methyl-4-phenylnicotinamide gave (3 S)-5-[3,5-bis(trifluoromethyl)benzyl]-3,8-dimethyl-7-phenyl-2,3,4,5-tetrahydro-6 H-pyrido[2,3 b][1,5]oxazocin-6-one, which exists predominantly in the thermodynamically stable aR-conformer in CDCl 3. This compound showed excellent NK 1-antagonistic activity with IC 50 value (in vitro inhibition of [ 125I]-Bolton–Hunter-substance P binding in human IM-9 cells) of 0.47 nM, which is ca. 200-fold more potent than that of its enantiomer, indicating that the atropisomer chirality affects NK 1-receptor recognition.

Tachykinin receptor (NK 1, NK 2, NK 3) binding sites in the rat caudal brainstem following neonatal capsaicin administration

Binding of [ 125I]-Bolton–Hunter substance P ([ 125I]-BHSP), [ 125I]-neurokinin A and [ 3H]-senktide to tachykinin NK 1, NK 2 and NK 3 receptors, respectively, was examined in caudal brainstem sections of 10-week-old rats pretreated as neonates (P2) with capsaicin (50 mg/kg, s.c.) or vehicle. [ 125I]-BHSP binding was localised to the nucleus of the solitary tract (NTS), hypoglossal nucleus and inferior olivary complex, whereas [ 125I]-neurokinin A and [ 3H]-senktide binding were confined to the NTS. The distribution and density of binding sites were similar in vehicle- and capsaicin-pretreated rats, suggesting that sensory neuron ablation by neonatal capsaicin does not affect tachykinin receptor numbers in the rat caudal brainstem.

Radioligand binding and functional characterisation of tachykinin receptors in chicken small intestine.

Tachykinin receptors in chicken intestine were studied using radioligand binding and functional techniques. Mechanisms of tachykinin-induced contraction were also investigated. Binding of [125I]Bolton-Hunter substance P ([125I]BH-SP) to chicken ileal membranes was rapid, saturable, of high affinity and to a single population of binding sites with Kd 0.72 nM and Bmax 0.48 fmol/ wet weight tissue. The rank order of agonists competing for [125I]BH-SP binding sites was [Sar9]SP > [Arg3]SP (natural tachykinin in chickens) > SP > [Pro9]SP > or = NKA > eledoisin > [Sar9,Met(O2)11]SP >> [Lys5,MeLeu9,Nle10]-NKA(4-10) >> senktide, suggesting similarities to the mammalian NK1 receptor. The NK1 receptor antagonist CP 99994, and NK2 receptor antagonist SR 48968 were weak competitors while spantide, RP 67580, GR 82334, GR 94800 and MEN 11420 were ineffective. The radioligand [125I]NKA showed no specific binding to ileal membranes. The potency order of most tachykinins in contacting isolated ileal longitudinal segments was in good agreement with that obtained from competition binding studies. Contractions to [Arg3]SP, NKA and senktide were greatly reduced by tetrodotoxin, suggesting that neurally-mediated responses were primarily involved. [Arg3]SP and NKA acted mainly by increasing release of acetylcholine, prostaglandins and probably tachykinins. Responses to [Arg3]SP were virtually abolished by nifedipine but were unaffected by NK1 receptor antagonists. Senktide-induced contraction was inhibited by the NK3 receptor antagonist, SR 142801, but was unaffected by atropine or L-NAME. The study provides evidence for a tachykinin receptor with similarities to the NK1 receptor in the chicken small intestine. In addition, senktide may act on a receptor similar to the mammalian NK3 receptor.

Effects of age and clustered hypoxia on [ 125I]substance P binding to neurotachykinin-1 receptors in brainstem of developing swine

This work focused on the postnatal development of substance P-bound neurotachykinin-1 (NK-1) receptors in the porcine brainstem using 2–3-, 6–11-, 16–18-, and 21–28-day-old piglets versus adult, and on alterations in these receptors after single and six-daily repeated clustered hypoxia using 6–11- and 21–28-day-old piglets. NK-1 receptor localization and densities were determined by quantitative autoradiography using mono-iodinated Bolton-Hunter substance P ([ 125I]BHSP). Slide-mounted brainstem sections, incubated in [ 125I]BHSP and then exposed to film, have shown [ 125I]BHSP binding throughout many brainstem nuclei and tracts, including the ambigual/periambigual (nAmb), dorsal motor vagal (dmnv), gigantocellular (nGC), hypoglossal (nHyp), medial parabrachial (nPBM), lateral reticular (nRL), raphe magnus (nRMg), raphe obscurus (nROb) and solitary tract (nTS) nuclei. NK-1 receptor densities decreased with age. As compared to normoxia, NK-1 receptor densities increased significantly after the six-daily hypoxia protocol in nAmb, dmnv, nHyp, nRL, nRMg, nROb, and nTS of both the young and older age groups. This increase may represent receptor upregulation as an adaptation to repeated hypoxia.

Electroconvulsive shock increases tachykinin NK 1 receptors, but not the encoding mRNA, in rat cortex

Recent studies have suggested that the substance P (tachykinin NK 1) receptor may be a pharmacological target for the treatment of mood disorders. Here, the effects of electroconvulsive shock on tachykinin NK 1 receptor gene expression in the rat brain was investigated. Rats received either a single electroconvulsive shock or five shocks on alternate days. Quantitative autoradiography with [ 125I]Bolton Hunter-substance P, and in situ hybridisation histochemistry, were used to measure tachykinin NK 1 receptor-binding site densities and mRNA abundance, respectively. Densities of tachykinin NK 1 receptor-binding sites were significantly increased in the cerebral cortex following repeated electroconvulsive shock compared to sham treated animals. Densities remained unchanged in the hippocampus, striatum and amygdala. Neither single nor repeated electroconvulsive shock altered tachykinin NK 1 receptor mRNA in the brain regions examined. Hence, repeated electroconvulsive shock increases tachykinin NK 1 receptors in the rat brain in a regionally specific way. Upregulation of receptor-binding sites without a change in mRNA indicates that translational or post-translational mechanisms underlie this process.

Effect of non-peptide tachykinin NK 1 receptor antagonists on non-adrenergic, non-cholinergic neurogenic mucus secretion in ferret trachea

We investigated, in ferret trachea in vitro, the binding characteristics and the inhibition of non-adrenergic, non-cholinergic (NANC) neural mucus secretion of four tachykinin receptor antagonists: the non-peptide tachykinin NK 1 receptor antagonists CGP 49823 ((2 R,4 S)-2-benzyl-1-(3,5-dimethylbenzoyl)-4-(quinolin-μ-ylmethyl amino) piperidine), CGP 55000 ((2 R,4 S)-2-benzyl-1-(3,5-bistrifluoromethyl-benzoyl)-4-(quinolinyl-methylamino)piperidine) and CP 99,994 ((+)-(2 S,3 S)-3-methoxybenzyl amino)-2-phenylpiperidine), and the peptide tachykinin NK 2 receptor antagonist MEN 10,627 (cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2β–5β)). CGP 49823, CGP 55000 and CP 99,994 concentration-dependently displaced [ 125I]Bolton–Hunter substance P binding in tracheal membranes with Hill coefficients not different from unity and IC 50 values of 1.4, 1.7 and 1.3 nM, respectively. In contrast, MEN 10,627 displaced binding according to a two-site model, with IC 50s of 0.2 nM and 1.3 μM. Electrical stimulation of tracheal segments with adrenoceptor and cholinoceptor blockade increased output of the mucus marker 35SO 4 by 59% above baseline (representing the NANC neural secretory response). CGP 49823, CGP 55000 or CP 99,994 concentration-dependently inhibited NANC neural secretion with IC 50 values of 30, 8 and 120 nM, respectively. In contrast, MEN 10,627 (3 μM) did not inhibit secretion. The NK 1 antagonists, but not the NK 2 antagonist, inhibited [Sar 9]substance P-induced secretion, while none of the antagonists affected acetylcholine-induced secretion. We conclude that NANC neural secretion in ferret trachea in vitro is a useful test system for tachykinin NK 1 receptor antagonists with therapeutic potential in conditions of the airways in which tachykininergic mechanisms and mucus hypersecretion are implicated in pathophysiology, for example asthma and chronic bronchitis.

Ontogeny of neurokinin-1 receptors in the porcine respiratory system

We characterized the ontogeny of NK-1 receptor agonist affinity ( K d) and density ( B max) in membranes from tracheal epithelium, smooth muscle, and lung of pigs aged 1–7 days, 8–21 days, and adult in comparison to contractile responses in vitro. Affinity of [ 125I] Bolton-Hunter substance P ([ 125I]BH-SP) in epithelium and smooth muscle was three- to fourfold lower in young piglets than in adults. The B max of NK-1 sites in epithelium was elevated by more than twofold at 8–21 days relative to 1–7 days piglets and adults. In the lung, NK-1 density as well as affinity was lower than in trachea, regardless of age. In all three groups, [ 125I]BH-SP binding was potently inhibited by Gpp(NH)p, in both trachea and lung, implying coupling to G-proteins. Inhibition by Gpp(NH)p was most potent in the adult relative to younger animals, in both tracheal epithelium and smooth muscle. Functional sensitivity to the NK-1 agonists substance P and septide was reduced in neonates, as shown by the higher concentration of agonist required to elicit contractile responses. We conclude that the reduced sensitivity of newborn piglet airways to substance P reflects immaturity of G-protein coupling to NK-1, independent of receptor density.

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 NK 2 or NK 3 agonists or antagonists exhibited weak or no affinity for [ 125I]NKA binding sites. As indicated by K i values of several compounds, the pharmacological characteristics of the septide-sensitive binding sites (labeled with [ 125I]NKA) largely differ from those of classic NK 1 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 γ (NKγ), 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 NK 1 binding sites exhibited a high affinity for the septide-sensitive binding sites. In contrast, SP, classic selective NK 1 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 NPγ induce salivary secretion and may potentiate the SP-evoked response in spite of the absence of tachykinin NK 2 receptors in this tissue.

Tachykinin receptors in the small intestine of the cane toad (Bufo marinus): a radioligand binding and functional study.

In this study, we have used radioligand binding and functional techniques to investigate tachykinin receptors in the small intestine of the cane toad Bufo marinus. The radioligand [125I]Bolton-Hunter [Sar9,Met(O2)11]substance P (selective at mammalian NK-1 receptors) showed no specific binding. Specific binding of [125I]Bolton-Hunter substance P ([125I]BHSP) was saturable, of high affinity (Kd 0.3 nM) and was inhibited by SP (IC50 0.64 nM) > ranakinin approximately neurokinin A (NKA) > or = SP(5-11) > or = neuropeptide gamma > or = scyliorhinin II > scyliorhinin I > or = [Sar9]-SP > or = neurokinin B approximately physalaemin approximately carassin >> SP(7-11) approximately eledoisin > or = SP(4-11) approximately SP(6-11). Binding was also inhibited by Gpp[NH]p > or = GTPgammaS > App[NH]p, indicating a G-protein coupled receptor. The order of potency of tachykinins and analogues in contracting the isolated lower small intestine was carassin (EC50 1.4 nM) > eledoisin approximately SP > or = physalaemin > or = ranakinin > SP(6-11) > scyliorhinin II > or = neuropeptide gamma > neurokinin B approximately NKA approximately scyliorhinin I > or = SP(4-11) > or = SP(5-11) > [Sar9]SP > SP(7-11). In both studies, the selective mammalian NK-1, NK-2 and NK-3 receptor agonists [Sar9,Met(O2)11]SP, [Lys5,Me-Leu9,Nle10]NKA(4-10) and senktide were weak or ineffective. There was a strong positive correlation between the pD2 and pIC50 values for mammalian tachykinins and analogues (r = 0.907), but not for the non-mammalian tachykinins, which were all full agonists but variable binding competitors. [Sar9,Met(O2)11]-SP(pD2 5.7) was approximately 25-fold less potent as an agonist than [Sar9]SP, which was itself 25-fold weaker than SP. Responses to SP were significantly reduced (n = 8, P<0.001) by the antagonist [D-Arg1,D-Trp7,9,Leu11]-SP (spantide; 1 microM). Highly selective NK-1 receptor antagonists including CP 99994 and GR 82334 (both 1 microM) were ineffective in both functional and binding studies. Tetrodotoxin (1 microM) did not inhibit contractile responses to SP, NKA and senktide. In summary, this study has shown the presence of one or more tachykinin receptor in the toad intestine. The binding site recognised by [125I]BHSP prefers SP and ranakinin. This toad "NK-1-like receptor" differs from the mammalian NK-1 receptor in having a low affinity for all mammalian NK-1 selective ligands, including antagonists. For some non-mammalian peptides, their high potency as contractile agonists relative to their poor binding affinity suggests the existence of other tachykinin receptors in the toad small intestine.

Presence of NK1receptors on a mucosal-like mast cell line, RBL-2H3 cells

Reverse transcription--polymerase chain reaction of mRNA from rat RBL-2H3 cells yielded a 316 base pair band consistent with that predicted for the neurokinin-1 (NK1) receptor. Saturation and competition binding with 125I-labeled Bolton-Hunter substance P, substance P fragments, and a series of selective tachykinin receptor agonists and antagonists demonstrated that RBL-2H3 cells express high affinity binding sites for substance P on their surfaces with the kinetic and pharmacological properties of NK1 receptors. The pharmacology of these 125I-labeled substance P binding sites was (from most to least potent) [Sar9,Met(O2)11]substance P > substance P 4-11 >> GR82334 << MEN 10,376. However, substance P 1-4, substance P 8-11, substance P 9-11, and [Trp7, beta-Ala8]neurokinin A 4-10 failed to compete for binding. The metabolically stable NK1 receptor agonist, [Sar9,Met(O2)11] substance P, caused a 49% increase in 5-hydroxytryptamine release above basal levels. The results demonstrate the presence of functional NK1 receptors on RBL-2H3 cells, a mucosal-like mast cell line.

A quantitative structure-activity relationship study of some substance P-related peptides. A multivariate approach using PLS and variable selection.

Nine new analogues of substance P (SP) were designed using quantitative sequence-activity models based on the amino acid z-scales with PLS as the statistical method and the GOLPE procedure for variable selection. The nine SP analogues were synthesised by solid-phase peptide synthesis and tested for affinity to the NK-1 receptor from rat brain with radio receptor assay using [125I]-Bolton-Hunter substance P as labelled ligand. All of the new substance P analogues showed high affinities, with IC50 values of less than 0.8 nM. One analog, Lys-Arg-Ala-Lys-Phe-Met-Met-Phe-Phe-Gly-Leu-Let-NH2, showed a exceptional high affinity for the NK1 receptor, with IC50 = 5 pM.

Substance‐P receptors in human primary neoplasms: Tumoral and vascular localization

Primary human neoplasms were examined for the presence of substance-P receptors by receptor autoradiography with 125I-labelled Bolton-Hunter substance P. Substance-P receptors were localized and characterized in the neoplastic cells of 9/12 astrocytomas, 10/10 glioblastomas, 10/12 medullary thyroid carcinomas, 8/16 breast carcinomas and 4/5 ganglioneuroblastomas. Conversely, substance-P receptors were not or only rarely identified on non-small-cell carcinomas of the lung (1/16), neuroblastomas (0/8), adenocarcinomas of the colon (1/21) or the pancreas (1/9), or on malignant lymphomas (3/18). However, in the great majority of the investigated tumours, substance-P receptors were found on intra- and peritumoral blood vessels. All substance-P receptors detected had the pharmacological characteristics of the neurokinin-I receptor sub-type. In addition, the expression of somatostatin receptors was examined in all the neoplastic tissues mentioned above. Both substance-P and somatostatin receptors were present in astrocytomas and in ganglioneuroblastomas, whereas little or no receptor was found in pancreatic and non-small-cell lung carcinomas. The extent of somatostatin-receptor expression was inversely correlated to that of the substance-P receptors in glioblastomas, neuroblastomas and non-Hodgkin's lymphomas. The tumoral and vascular localization of substance-P receptors in tumours may have clinical implications. The use of radiolabelled substance P for in vivo scintigraphy may supplement the current set of diagnostic tools. Substance-P antagonists might be used in the treatment of tumours, as their binding to vascular receptors may decrease tumoral blood supply and drainage.

Autoradiographic localization of NK1 and NK3 tachykinin receptors in rat kidney.

The distribution of neurokinin receptors in rat kidney, renal artery, renal vein, and proximal ureter was evaluated by autoradiography after in vitro labeling of NK1 sites with [125I]Bolton-Hunter substance P (BHSP) or NK3 sites with [125I][MePhe7]neurokinin B ([MePhe7]NKB). Film autoradiography using [125I][MePhe7]NKB revealed specific binding sites associated with the renal vein and its large branches, the renal pelvis, the inner strip of outer renal medulla, and the proximal ureter. High-resolution autoradiograms demonstrated that these sites were localized to the smooth muscle layer in the veins, pelvis, and ureter. Neither the renal arterial system nor the renal cortex contained specific [125I][MePhe7]NKB binding sites although a high level of nonspecific binding was associated with the renal artery. Specific binding of [125I]BHSP was associated with the renal artery and renal pelvis but not the renal veins. Arterial NK1 receptors appeared to be localized to the adventitia. The results indicate that at least two types of tachykinin receptor are present in the rat kidney. The distinct localization observed for most of the NK1 and NK3 receptors suggests that they have different functions.

Opioid and substance p receptor adaptations in the rat spinal cord following sub-chronic intrathecal treatment with morphine and naloxone

The effect of continuous intrathecal infusion with morphine (5 μ/h) or naloxone (2 μ/h) was investigated with regard to analgesia and the apparent density of μ- and δ-opioid and neurokinin-I/substance P receptors in the rat spinal cord. Morphine infusion increased tail-flick and paw-pressure responses until day 4 after the mini-osmotic pump implant. A decline in antinociception, reflecting tolerance to morphine, was then apparent in both tests. Quantitative in vitro receptor autoradiography of [ 125I]FK-33824, [ 125I][ d-Ala 2]deltorphin-I and [ 125I] Bolton-Hunter substance P binding sites, as ligands of μ, δ and neurokinin-I/substance P receptors, respectively, was performed on lumbosacral spinal cord sections of seven-days tolerant animals. Treatments with morphine and naloxone induced a similar increase (37%) in the number of δ binding sites in the superficial laminae of the dorsal horn. In contrast, the density of μ-opioid receptors was only affected by naloxone (50% increase). Neurokinin-I/substance P binding parameters were not altered by these treatments. Thus, it appears that δ-opioid binding sites may be of special relevance with regard to the development of tolerance to opiates in the spinal cord.

Receptor-Mediated Specific Biological Activity of a β-Amyloid Protein Fragment for NK-1 Substance P Receptors

A β-amyloid protein fragment AP21-35 (tetradeapeptide with amino acid residues 21-35) was found to be a highly selective agonist of substance P receptors (NK-1) among three tachykinin receptor subtypes. This peptide fragment contracted the smooth muscle preparations of guinea pig ileum in a dose dependent manner (EC50 = 22 μM). This activity was completely reversed by CP-96,345-1, a specific antagonist of NK-1 receptors, whereas atropine for NK-3 had no effect. The peptide was inactive in rat vas deferens which contains predominantly NK-2 receptors. These results indicated that AP21-35 is a highly selective agonist for NK-1 receptors. In the radio-ligand receptor binding assay using [125I]-Bolton-Hunter substance P to membranes from guinea pig ileum, the fragment exhibited a distinct dose-response curve (IC50 = 11 μM). All the present data strongly suggest that β-amyloid protein function as a peptide ligand of substance P receptors with some pathophysiologic activities.

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 (K D) 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 (B max) 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 < 0.05) the B max 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 K D 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.

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 NK 1 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 ≈ scyliorhinin I > eledoisin ≈ substance P ≈ neurokinin A > phyllomedusin ≈ physalaemin > [Sar 9Met(O 2) 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 NK 1 receptor and of a nonmammalian tachykinin receptor in the dogfish.

Complex allosteric interaction of heparin with neurokinin-1 receptors

The effects of several heparin preparations on rat striatal neurokinin-1 receptor labelling were investigated. A low concentration of heparin dose-dependently stimulated [ 125I]Bolton-Hunter substance P ([ 125I]BH-SP) binding to rat striatal membranes. This effect was mainly due to an increase of the association velocity reflected by a decrease of the equilibrium dissociation rate constant. A higher heparin concentration inhibited specific [ 125I]-BH-SP labelling by a negative allosteric mechanism as a result of an increase of the dissociation velocity.

A novel substance P binding site in bovine adrenal medulla

Radioligand binding techniques were used to characterize the substance P (SP) binding site on membranes prepared from bovine adrenal medullae. 125I-labelled Bolton-Hunter substance P (BHSP), which recognises the C-terminally directed, SP-preferring NK1 receptor, showed no specific binding. In contrast, binding of [ 3H]SP was saturable (at 6 nM) and reversible, with an equilibrium dissociation constant ( K d) 1.46 ± 0.73 nM, B max 0.73 ± 0.06 pmol/g wet weight and Hill coefficient 0.98 ± 0.01. Specific binding of [ 3H]SP was displaced by SP > neurokinin A (NKA) > SP(3–11) ≈ SP(1–9) > SP(1–7) ≈ SP(1–4) ≈ SP(1–6), with neurokinin B (NKB) and SP(1–3) very weak competitors and SP(5–11), SP(7–11) and SP(9–11) causing negligible inhibition (up to 10 μM). This potency order is quite distinct from that seen with binding to an NK1 site, a conclusion confirmed by the lack of BHSP binding. It appears that Lys 3 and/or Pro 4 are critical for binding, suggesting an anionic binding site. These data suggest the existence of an unusual binding site which may represent a novel SP receptor. This site appears to require the entire sequence of the SP molecule for full recognition.

Characterization of tachykinin receptors in endothelial cells of porcine artery

The binding of iodine-labeled Bolton-Hunter substance P ( 125I-BHSP) to porcine endothelial cell membranes was examined. The endothelial cells had a single high-affinity binding site with a dissociation constant of 0.10 nM, and a maximum number of binding sites of 52.2 fmol/mg protein. The relative potencies of various tachykinins to displace the binding of 47 pM 125I-BHSP suggested that endothelial cells of porcine aorta contain the NK-1 subtype of tachykinin receptor. A GTP analogue, guanyl-5′-yl imidodiphosphate, induced marked reduction in the number of 125I-BHSP binding sites suggesting that these binding sites are coupled with GTP-binding protein.