[3H]Neurokinin B and 125I-Bolton Hunter Eledoisin Label Identical Tachykinin Binding Sites in the Rat Brain

[3H]Neurokinin B ([3H]NKB) of high specific activity (75 Ci/mmol) was synthesized for study of its binding to crude synaptosomes from the rat cerebral cortex. The specific binding of [3H]NKB (75% of total binding) was temperature dependent, saturable, and reversible. Scatchard analyses and Hill plots showed the existence of a single population of noninteracting binding sites (KD = 4.3 nM; Bmax = 123 fmol/mg of protein). Competition studies indicated the following rank order of potencies among tachykinins: NKB greater than eledoisin (E) greater than kassinin greater than physalaemin greater than neurokinin A (NKA) greater than substance P (SP), a result suggesting that NKB might be the endogenous ligand for [3H]NKB binding sites. It is of interest that 127I-Bolton Hunter (BH) NKA (127I-BHNKA) was much more potent than NKA in inhibiting the specific binding of [3H]NKB, which raises certain questions concerning the use of 125I-BHNKA as a ligand for NKA binding sites in the brain. These results, as well as those obtained with different SP analogues, show a close similarity to those obtained previously with 125I-BHE binding to cortical synaptosomes. This suggested that the two ligands labeled identical binding sites. In addition, using either [3H]NKB or 125I-BHE as ligands, similar displacement curves were obtained with increasing concentrations of NKB and 127I-BHE. The similarity of the [3H]NKB and 125I-BHE binding sites was further confirmed by comparison of their localization on rat brain sections by autoradiography. The distribution of binding sites for [3H]NKB and 125I-BHE was identical throughout the brain, and the highest density of binding sites for the two ligands was found in layers IV and V of the cerebral cortex, the paraventricular nucleus of the hypothalamus (magnocellular part), and the ventral tegmental area.     

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.     

Marked Regional Heterogeniety of 125I-Bolton Hunter Substance P Binding and Substance P-Induced Activation of Phospholipase C in Astrocyte Cultures from the Embryonic or Newborn Rat

The specific binding of 125I-Bolton Hunter substance P (125I-BHSP) was estimated on 4- to 5-week-old primary cultures of astrocytes from several brain structures and the spinal cord of 16-day-old embryonic or newborn rats. In both cases, high levels of binding of 125I-BHSP were found on intact astrocytes from the brainstem, but this binding was low or negligible on cells from the cerebral cortex, striatum, hypothalamus, and mesencephalon. In addition, hippocampal astrocytes from newborn rats were also devoid of 125I-BHSP binding sites, while a binding of 125I-BHSP (half that of brainstem cells) was observed on astrocytes from the cerebellum and spinal cord. It was also shown that this regional heterogeneity in 125I-BHSP binding was not linked to differences in the inactivation of the ligand, cell plating density. or eventual cell contaminants. Five-day-old cultures from 16-day-old embryos were used to estimate 125I-BHSP binding on neuron-enriched cultures. Specific 125I-BHSP binding was found on cells from the brainstem, mesencephalon, and hypothalamus, but neurons from the cerebral cortex or the striatum contained low or negligible amounts of 125I-BHSP binding sites. Competition studies using tachykinins and SP analogues indicated that 125I-BHSP binding sites on brainstem astrocytes (16-day-old embryos) have the pharmacological profile expected for NK1 binding sites. SP (1 microM) stimulated phosphoinositide breakdown in cells rich in 125I-BHSP binding sites (brainstem) but not in those devoid of 125I-BHSP binding (striatum).(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of NK1 and NK3 receptors in guinea-pig brain

In this study the localizations of tachykinin neurokinin-1 (NK1) and neurokinin-3 (NK3) receptors in the guinea-pig brain are described. In agreement with studies in rat and human brain, the neurons that exhibited the most marked NK1 receptor immunoreactivity were found in the dorsomedial caudate putamen. NK1 receptors were also widely distributed in diencephalic structures and in the mid and hind brain. NK3 receptors were distributed in both superficial and deep layers of the cortex and many appeared to be located on cells with astrocyte-like morphology in the glia limitans. In several regions including the thalamus, hypothalamus, amygdala, periaqueductal gray, substantia nigra and area postrema, both NK1 and NK3 immunoreactivity were found. The present study revealed that tachykinin receptors are widely distributed in the guinea-pig central nervous system.     

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.     

Different subtypes of tachykinin NK(1) receptor binding sites are present in the rat brain

(2-[(125)I]iodohistidyl(1))Neurokinin A ([(125)I]NKA), which labels "septide-sensitive" but not classic NK(1) binding sites in peripheral tissues, was used to determine whether septide-sensitive binding sites are also present in the rat brain. Binding studies were performed in the presence of SR 48968 (NK(2) antagonist) and senktide (NK(3) agonist) because [(125)I]NKA also labels peripheral NK(2) binding sites and, as shown in this study, central NK(3) binding sites. [(125)I]NKA was found to label not only septide-sensitive binding sites but also a new subtype of NK(1) binding site distinct from classic NK(1) binding sites. Both subtypes of [(125)I]NKA binding sites were sensitive to tachykinin NK(1) antagonists and agonists but also to the endogenous tachykinins NKA, neuropeptide K (NPK), and neuropeptide gamma (NPgamma). However, compounds of the septide family such as substance P(6-11) [SP(6-11)] and propionyl-[Met(O(2))(11)]SP(7-11) and some NK(1) antagonists, GR 82334, RP 67580, and CP 96345, had a much lower affinity for the new NK(1)-sensitive sites than for the septide-sensitive sites. The hypothalamus and colliculi possess only this new subtype of NK(1) site, whereas both types of [(125)I]NKA binding sites were found in the amygdala and some other brain structures. These results not only explain the central effects of septide or SP(6-11), but also those of NKA, NPK, and NPgamma, which can be selectively blocked by NK(1) receptor antagonists.     

125I]-Bolton-Hunter scyliorhinin II: a novel, selective radioligand for the tachykinin NK3 receptor in rat brain

The cyclic tachykinin scyliorhinin II (SCYII) has high affinity for the [neurokinin B (NKB)-preferring] NK3 receptor. SCYII was iodinated using [125I]-Bolton-Hunter reagent and the product BHSCYII purified using reverse phase HPLC. In rat brain membranes, binding of BHSCYII and of the relatively unselective radioligand [125I]-Bolton-Hunter eledoisin (BHELE) was saturable, reversible and to an NK3 site. In competition studies, the rank order of potency in inhibiting binding of BHSCYII and BHELE was: SCYII greater than or equal to [MePhe7]-NKB approximately senktide greater than NKB greater than or equal to kassinin greater than or equal to eledoisin greater than [Pro7]-NKB greater than neurokinin A greater than neuropeptide K greater than or equal to substance P greater than [Sar9, Met(O2)11]-substance P. In "cold" saturation experiments, binding of BHELE occurred to a single class of high affinity sites (KD, 18.6 +/- 0.91 nM). Binding of BHSCYII was of greater affinity than for BHELE and could be resolved into a high (KD, 1.33 +/- 0.98 nM; 27% of sites) and low affinity (KD, 9.84 +/- 2.75; 73% of sites) component. The total number of binding sites was similar for both radioligands (BHSCYII, 8.27 +/- 0.98; BHELE, 7.94 +/- 0.32 fmol/mg wet weight). In vitro autoradiography in slide-mounted sections of rat brain showed identical binding patterns for both radioligands (100 pM), with dense binding localized predominantly to the cortex, Ammon's horn field 1, premammillary nuclei and interpeduncular nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of NK2 binding sites in the rat brain

Attempts were made to label tachykinin NK2 binding sites in the adult rat brain using [125I]neurokinin A (NKA) as ligand in the presence of NK1 and NK3 agonist or antagonist to avoid labelling of NK1 and NK3 binding sites, respectively. A high-affinity, specifically NK2-sensitive, [125I]NKA-binding, temperature-dependent, reversible, sensitive to GTPgammaS and correspondence to a single population of binding sites (K(D) and B(max) values: 2.2 nM and 7.3 fmol/mg protein) was demonstrated on hippocampal membranes. Competition studies performed with tachykinins and tachykinin-related compounds indicated that the pharmacological properties of these NK2-sensitive [125I]NKA binding sites were identical to those identified in the rat urinary bladder and duodenum. NKA, neuropeptide K, and neuropeptide gamma, as well as the potent and selective NK2 antagonists SR 144190, SR 48968 and MEN 10627, presented a nanomolar affinity for these sites. The regional distribution of these NK2-sensitive [125I]NKA binding sites differs markedly from those of NK1 and NK3 binding sites, with the largest labeling being found in the hippocampus, the thalamus and the septum. Binding in other brain structures was low or negligible. A preliminary autoradiographic analysis confirmed [125I]NKA selective binding in hippocampal CA1 and CA3 areas, particularly, and in several thalamic nuclei.     

Biochemical and pharmacological activities of SSR 146977, a new potent nonpeptide tachykinin NK3 receptor antagonist

SSR 146977 is a potent and selective antagonist of the tachykinin NK3 receptor. In Chinese hamster ovary cells expressing the human tachykinin NK3 receptor, SSR 146977 inhibited the binding of radioactive neurokinin B to NK3 receptors (Ki = 0.26 nM), senktide (10 nM) induced inositol monophosphate formation (IC50 = 7.8-13 nM), and intracellular calcium mobilization (IC50 = 10 nM). It antagonized [MePhe7]neurokinin B induced contractions of guinea pig ileum (pA2 = 9.07). Senktide (30 nM) induced firing rate increase of noradrenergic neurons in the guinea pig locus coeruleus and dopaminergic neurons in the guinea pig substantia nigra was also blocked by SSR 146977 (50 and 100 nM, respectively). In vivo, in the respiratory system, SSR 146977 inhibited bronchial hyperresponsiveness to acetylcholine, bronchial microvascular permeability hypersensitivity to histamine (doses of 0.1-1 mg/kg i.p.), and cough (doses of 0.03-1 mg/kg i.p.) provoked by citric acid in guinea pigs. In the central nervous system, SSR 146977 inhibited turning behaviour (ID50 = 0.2 mg/kg i.p. and 0.4 mg/kg p.o.) and prevented the decrease of locomotor activity (10 and 30 mg/kg i.p) mediated by the stimulation of NK3 receptors in gerbils. In guinea pigs, SSR 146977 antagonized senktide-induced acetylcholine release in the hippocampus (0.3 and 1 mg/kg i.p) and norepinephrine release in the prefrontal cortex (0.3 mg/kg i.p.). It also prevented haloperidol-induced increase of the number of spontaneously active dopamine A10 neurons (1 and 3 mg/kg i.p.).     

Stable expression of high affinity NK1 (substance P) and NK2 (neurokinin A) receptors but low affinity NK3 (neurokinin B) receptors in transfected CHO cells.

Stable CHO cell clones which selectively express all three rat tachykinin receptors were established by transfection. The binding of radiolabled substance P and neurokinin A (substance K) to CHO clones expressing the NK1 and NK2 receptors, respectively, were saturatable and of high affinity (Kd = 0.17 nM (NK1); 3.4 nM (NK2)). Scatchard analysis of the binding data indicated for both receptors binding to a single population of binding sites, and competition binding studies showed that the binding specificities of the receptors corresponded to those of classical NK1 and NK2 receptors. In contrast, the binding of eledoisin to the NK3 receptor expressed in the transfected CHO cells was of low affinity (IC50 = 240 nM) compared to the high affinity of the receptor found when it was transiently expressed in COS-7 cells (IC50 = 8 nM). However, in both cases the receptor exhibited the specificity of a classical NK3 receptor. The established cell clones may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of receptors for tachykinin peptides.     

NK1, NK2, NK3 and CGRP1 receptors identified in rat oral soft tissues,and in bone and dental hard tissue cells

The distribution of the tachykinin receptors neurokinin-1 (NK1), neurokinin-2 (NK2) and neurokinin-3 (NK3), and the calcitonin gene-related peptide-1 (CGRP1) receptor were examined in rat teeth and tooth-supporting tissues by immunohistochemical methods and light and confocal microscopy. Western blot analysis was performed to identify the NK1- and the CGRP1-receptor proteins in the dental pulp. The results showed that odontoblasts and ameloblasts, cementoblasts and cementocytes, osteoblasts and osteocytes are all supported with the tachykinin receptors NK1 and NK2, but a distinct, graded cellular labeling pattern was demonstrated. The ameloblasts were also positive for CGRP1 receptor. Blood vessels in oral tissues expressed the tachykinin receptors NK1, NK2 and NK3, and the CGRP1 receptor. Both gingival and Malassez epithelium were abundantly supplied by NK2 receptor. Pulpal and periodontal fibroblasts demonstrated NK1 and NK2 receptors. Western blot analysis identified both the NK1- and the CGRP1-receptor proteins in the dental pulp. These results clearly indicate that the neuropeptides substance P, neurokinin A, neurokinin B and CGRP, released from sensory axons upon stimulation, directly modulate the function of the different types of bone and dental hard tissue cells, and regulate functions of blood vessels, fibroblasts and epithelial cells in oral tissues.     

Interaction of Met297 in the seventh transmembrane segment of the tachykinin NK2 receptor with neurokinin A

We report the use of thiol chemistry to define specific and reversible disulfide interactions of Cys-substituted NK2 receptor mutants with analogues of neurokinin A (NKA) containing single cysteine substitutions. The NKA analogues were N-biotinylated to facilitate the rapid detection of covalent analogue-receptor interactions utilizing streptavidin reactivity. N-biotinyl-[Tyr1,Cys9]NKA, N-biotinyl-[Tyr1,Cys10]NKA were both found to reversibly disulfide bond to the NK2 receptor mutant Met297 --> Cys. This is consistent with the improved affinities of these particular analogues for the Met297 --> Cys receptor as compared with those for the wild-type and Met297 --> Leu receptors. In our three-dimensional model, Met297 occupies the equivalent position in helix 7 to the retinal binding Lys296 in rhodopsin. Binding of the NK2 receptor antagonist [3H]SR 48968 and of 125I-NKA was used to characterize additional receptor mutants. It seems that the aromatic residues Trp99 (helix 3), His198 (helix 5), Tyr266, His267, and Phe270 play an important role in NKA binding as structural determinants. The existence of overlapping SR 48968 and NKA binding sites is also evident. These data suggest that the peptide binding site of the NK2R is at least in part formed by residues buried deep within the transmembrane bundle and that this intramembranous binding domain may correspond to the binding sites for substantially smaller endogenous GPCR ligands.     

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.     

Characterization and visualization of neurotensin binding to receptor sites in human brain

The binding of monoiodo [125I-Tyr3]-neurotensin to human brain was characterized and visualized using radioreceptorassay and autoradiographic techniques. Specific binding to homogenates of human substantia nigra at 25 degrees C was maximal at 20 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two populations of binding sites with Kd values of 0.26 nM and 4.3 nM. Corresponding binding capacities were 26 and 89 fmol/mg of protein. Neurotensin analogs inhibited the binding of iodinated neurotensin with relative potencies that demonstrated the crucial role of the C-terminal hexapeptide portion of neurotensin for binding to its receptors. Autoradiography of human substantia nigra sections incubated with iodinated neurotensin revealed high levels of specific binding in the nucleus paranigralis and substantia nigra, pars compacta, and low levels in the substantia nigra, pars reticulata.     

Loss of high affinity neurotensin receptors in substantia nigra from parkinsonian subjects

Monoiodo [125I-Tyr3]-Neurotensin binding was studied in post mortem substantia nigra from 17 control and 15 parkinsonian subjects. Binding to individual homogenates was decreased by 58%, 49% and 26% at 0.36, 1.4, 5.5 M(-9) concentration of ligand, respectively. Saturation analysis using pooled substantia nigra demonstrated an almost complete loss of the high affinity component of the neurotensin receptor complex, yielding a 24% loss of the total binding capacity, with no alteration of the low affinity component. Similarly an important loss of binding was observed in monoiodo[125I-Tyr3]-Neurotensin autoradiograms of two substantia nigra from parkinsonian subjects. These results support the hypothesis of neurotensin receptors occurring on dopamine cell bodies and/or dendrites in human substantia nigra. Role of neurotensin may be of importance in the regulation of dopamine pathway involved in parkinsonism.     

RP 67580, a potent and selective substance P non-peptide antagonist]

The pharmacological properties of 7,7-Diphenyl-2 [1-imino-2 (2-methoxy-phenyl)-ethyl] perhydroisoindol-4-one (3 aR, 7 aR) or RP67580 are described. This compound, derived from a novel chemical family, is a potent and selective substance P (SP) antagonist, in vitro and in vivo. In vitro, it inhibited in a competitive manner (IC50 = 10 nM) 3H-SP binding in rat brain (NK1 receptors). It did not interact with the two other tachykinin receptor sites (NK2 and NK3) nor the other receptor sites tested. Moreover, RP67580 competitively antagonized the contractile activity of SP on guinea-pig ileum (pA2 = 7.16); in contrast, it was inactive in rabbit pulmonary artery and in rat portal vein tissues which contain NK2 and NK3 receptors, respectively. In vivo, in the rat, RP67580 inhibited the plasmatic extravasation induced by administration of SP (ED50 = 0.04 mg/kg i.v.) as well as that induced by antidromic stimulation of a peripheral sensory nerve (ED50 = 0.15 mg/kg i.v.). In mice and rats, RP67580, like morphine, potently blocked the nociceptive effects of phenylbenzoquinone and formalin; its antinociceptive effect does not involve opiate receptors since it was not reversed by naloxone. These results indicate that RP67580 is a particularly valuable tool for investigating the physiological and pathological role of SP.     

Leu9 psi(CH2NH)Leu10]-neurokinin A (4-10) (MDL 28,564) distinguishes tissue tachykinin peptide NK2 receptors

The neurokinin A analogue, MDL 28,564 (Asp-Ser-Phe-Val-Gly-Leu-CH2NH-Leu-NH2), inhibited 125I-NKA binding to hamster urinary bladder NK2 receptors with a KI of 130 nM. For rat submaxillary gland NK1 receptors and cerebral cortical NK3 receptors, the KI's for MDL 28,564 were greater than 250 microM and greater than 500 microM, respectively. MDL 28,564 did not relax dog carotid artery (NK1 tissue) or contract rat portal vein (NK3 tissue). In guinea-pig trachea tissues, MDL 28,564 stimulated phosphatidylinositol turnover and induced contraction with maximum effects similar to those of neurokinin A. In hamster urinary bladder tissue, MDL 28,564 stimulated phosphatidylinositol turnover with maximum effect only 10% of that of neurokinin A, did not produce sustained contraction itself and antagonized NKA-induced contraction. MDL 28,564 also produced full contraction in rabbit pulmonary artery (NK2 tissue) but was inactive in rat vas deferens (NK2 tissue). These data with MDL 28,564 are consistent with the NK2 receptors in guinea-pig trachea and rabbit pulmonary artery being different from those in hamster urinary bladder and rat vas deferens.     

Further evidence for the presence of specific binding sites for prolactin in the rabbit brain. Preferential distribution in the hypothalamus and substantia nigra

In the present research we have extended our work on the presence of binding sites for prolactin in the rabbit brain focusing our attention on the brain areas with high dopamine cell bodies density. Among these areas the hypothalamus showed the highest specific binding of labeled ovine prolactin (oPRL). Clearly detectible specific binding was observed also in substantia nigra, whereas in other brain regions the specific binding was very small, except for the striatum where a low but not negligible binding was found in female rabbits. The binding of 125I-oPRL showed a hormonal specificity and Scatchard analysis of the binding showed no clear difference in dissociation constant (Kd) between hypothalamus, nigra and striatum.     

Further evidence for the presence of "septide-sensitive" tachykinin binding sites in tissues possessing solely NK(1) tachykinin receptors

Binding experiments performed with [(125)I]-NKA allowed us to demonstrate the presence of "septide-sensitive" specific binding sites on membranes from rat CHO cells transfected with the NK(1) receptor cDNA (CHO-rat-NK1 cells), human astrocytoma U373 MG, or mouse cortical astrocytes, cells which express NK(1) but neither NK(2) nor NK(3) receptors. In all cases, [(125)I]-NKA was specifically bound with high affinity (2 to 5 nM) to a single population of sites. In the three preparations, pharmacological characteristics of [(125)I]-NKA binding sites were notably different from those of classical NK(1) binding sites selectively labelled with [(125)I]-BHSP. Indeed, the endogenous tachykinins NKA, NPK, and NKB and the septide-like compounds such as septide, SP(6-11), ALIE-124, [Apa(9-10)]SP, or [Lys(5)]NKA(4-10) had a much higher affinity for [(125)I]-NKA than [(125)I]-BHSP binding sites. Interestingly, differences were also found in the ratio of B(max) values for [(125)I]-NKA and [(125)I]-BHSP specific bindings from one tissue to another. These latter observations suggest that these two types of NK(1) binding sites are present on distinct NK(1) receptor isoforms (or conformers). Finally, while several tachykinins and tachykinin-related compounds stimulated cAMP formation or increased inositol phosphate accumulation in CHO-rat-NK1 cells, these compounds only increased the accumulation of inositol phosphates in the two other preparations.     

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.