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.     

Localization of tachykinin binding sites (NK1, NK2, NK3 ligands) in the rat brain

A comparative autoradiographic analysis of the distribution of tachykinin binding sites was made on brain serial sections using several ligands. (1) 3H-SP, 125I-BHSP and 3H-physalaemin labeled identical binding sites (NK1 type). (2) 3H-NKB, 125I-BHE and 3H-eledoisin also labeled identical sites (NK3 type). (3) 125I-BHNKA preferentially labeled NK3 binding sites, the distribution of 125I-BHNKA binding sites being identical to that of 3H-NKB or 125I-BHE binding sites. (4) The distributions of 3H-SP and 3H-NKB binding sites were markedly different. (5) A very low density of labeling was found with 3H-NKA or 125I-NKA, and these binding sites were distributed only in areas rich in either 3H-SP or 3H-NKB binding sites. (6) Particular efforts were made to look for the presence of tachykinin binding sites in the substantia nigra, since this structure is particularly rich in SP and NKA and contains functional tachykinin receptors of the NK1 and NK2 types as suggested by physiological studies. Confirming previous reports, low or very low labeling was observed in the substantia nigra with 3H-SP or 125I-BHSP and 3H-NKB or 125I-BHE. Similar results were found with 3H-NKA, 125I-NKA or 125I-BHNKA. In conclusion, our data do not provide evidence yet for the existence of NK2 binding sites in the rat brain.     

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.     

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.     

In vivo evidence for the activation of a septide-sensitive tachykinin receptor in guinea pig bronchoconstriction.

Intravenous administration of the undecapeptide [Sar⁹]substance P (SP) sulfone (1.5 nmol/kg) and the hexapeptide [Glp⁶,Pro⁹]SP(6ndash;11) (septide; 0.4 nmol/kg) produced a comparable (about 30–40 % of maximal effect) increase of insufflation pressure (bronchospasm) in anesthetized guinea-pigs. The non peptide NK-1 receptor antagonist, (±)CP 96,345 and the peptide NK-1 receptor antagonist, GR 82,334 antagonized dose-dependently the response to both agonists. Both antagonists were more potent against peptide than against [Sar⁹]SP sulfone (9 and 4 fold difference in ED50 for (±)CP 96,345 and GR 82,334, respectively). These findings indicate that a ‘septide-sensitive’ mechanism mediates bronchoconstriction in vivo and it influences the estimate of the potency of NK-1 receptor antagonists.     

Immunolocalization of full-length NK1 tachykinin receptors in human tumors.

Biological effects of substance P (SP) are mediated by the neurokinin-1 (NK1) receptor that exists as a full-length and as a carboxy-terminally truncated isoform in humans. Although NK1 receptor mRNA and binding sites have been detected in certain malignancies, little is known about the cellular and subcellular localization of NK1 receptor protein in human neoplastic tissues. We developed and characterized a novel anti-peptide antibody to the carboxy-terminal region of the human full-length NK1 receptor. Specificity of the antiserum was demonstrated by (1) detection of a broad band migrating at molecular mass 70,000-90,000 Da in Western blots of membranes from NK1-expressing tissues; (2) cell-surface staining of NK1-transfected cells; (3) translocation of NK1 receptor immunostaining after SP exposure; and (4) abolition of tissue immunostaining by preadsorption of the antibody with its immunizing peptide. Distribution of NK1 receptors was investigated in 72 formalin-fixed, paraffin-embedded human tumors showing that NK1 receptors were frequently expressed in glioblastomas and breast and pancreatic carcinomas. Immunoreactive NK1 receptors were clearly confined to the plasma membrane and uniformly present on nearly all tumor cells. Development of this novel NK1 receptor antibody allows the efficient localization of NK1 receptor protein in human formalin-fixed, paraffin-embedded tissues. NK1 receptor visualization with this simple and rapid immunohistochemical method will facilitate identification of tumors with a sufficient receptor overexpression for diagnostic or therapeutic intervention using SP analogs.     

Contribution of NK(2) tachykinin receptors to propulsion in the rabbit distal colon

The role of the tachykinin neurokinin (NK)(2) receptors on rabbit distal colon propulsion was investigated by using two selective NK(2)-receptor antagonists, MEN-10627 and SR-48968. Experiments on colonic circular muscle strips showed that contractile responses to [beta-Ala(8)]NKA-(4-10) (1 nM-1 microM), a selective NK(2)-receptor agonist, were competitively antagonized by MEN-10627 (1-100 nM), whereas SR-48968 (0.1-10 nM) caused an insurmountable antagonism, thus confirming the difference in the mode of action of the two compounds. Colonic propulsion was elicited by distending a mobile rubber balloon with 0.3 ml (submaximal stimulus) or 1.0 ml (maximal stimulus) of water. The velocity of anal displacement of the balloon (mm/s) was considered the main propulsion parameter. At low concentrations (1.0-100 nM and 0.1-10 nM, respectively), MEN-10627 and SR-48968 facilitated the velocity of propulsion, whereas at high concentrations (100 nM and 1 microM, respectively) they decelerated propulsion. The excitatory and inhibitory effects of both antagonists were observed only with submaximal stimulus. We focused on the hypothesis that the facilitatory effect on propulsion may result from blockade of neuronal NK(2) receptors and the inhibitory effect from suppression of the excitatory transmission mediated by NK(2) receptors on smooth muscle cells. In the presence of N(G)-nitro-L-arginine (300 microM), a nitric oxide synthase inhibitor, MEN-10627, at a concentration (10 nM) that was found to accelerate propulsion in control experiments inhibited the velocity of propulsion. In the presence of threshold (1-10 nM) or full (1 microM) concentration of atropine, which inhibited to a great extent the velocity of propulsion, the inhibitory effect of MEN-10627 (1 microM) was markedly increased. In conclusion, in the rabbit distal colon NK(2) receptors may decelerate propulsion by activating a nitric oxide-dependent neuronal mechanism and may accelerate it by a postjunctional synergistic interaction with cholinergic muscarinic receptors.     

Further delineation of the two binding sites (R*(n)) associated with tachykinin neurokinin-1 receptors using [3-Prolinomethionine(11)]SP analogues.

Two binding sites are associated with neurokinin-1 substance P receptors in both transfected cells and mammalian tissues. To further delineate the interactions between the crucial C-terminal methionine of substance P and these two binding sites, we have incorporated newly designed constrained methionines, i.e. (2S, 3S)- and (2S,3R)-prolinomethionines. The potencies of these C terminus-modified SP analogues to bind both sites and to activate phosphatidylinositol hydrolysis and cAMP formation have been measured, together with those of their corresponding sulfoxides and sulfones. The molecular nature of these two binding sites and their selective coupling to effector signaling pathways are discussed in the light of current models of receptor activation. The less abundant binding site is coupled to G(q/11) proteins, whereas the most abundant one interacts with G(s) proteins in Chinese hamster ovary cells transfected with human neurokinin-1 receptors. The specific orientation of the C-terminal methionine side chain imposed by these constraints shows that macroscopically chi(1) and chi(2) angles of this crucial C-terminal residue are similar in both binding sites. However, slight but significant variations in the rotation around the Cgamma-S bond yield different either stabilizing or destabilizing interactions in the two binding sites. These results highlight the need of such constrained amino acids to probe subtle interactions in ligand-receptor complexes.     

Peristalsis regulation by tachykinin NK1 receptors in the rabbit isolated distal colon

In the gastrointestinal tract, tachykinin NK1 receptors are widely distributed in a number of neuronal and nonneuronal cells involved in the control of gut motor activity. In particular, in the rabbit isolated distal colon, which is a suitable model system to investigate the contribution of tachykinins as noncholinergic excitatory transmitters, the influence of NK1 receptors in the regulation of peristalsis is not known. The selective NK1-receptor antagonists SR-140333 (0.3 and 1 nM) and MEN-10930 (0.3-10 nM) significantly enhanced the velocity of rabbit colonic propulsion to submaximal stimulation. The prokinetic effect of SR-140333 was prevented by N(omega)-nitro-L-arginine (L-NNA), a nitric oxide synthase inhibitor, indicating that NK1 receptors located on nitrergic innervation exert a functional inhibitory restraint on the circular muscle and probably on descending excitatory and inhibitory pathways during propulsion. Conversely, the selective NK1-receptor agonist septide (3-10 nM) significantly inhibited colonic propulsion. In the presence of L-NNA, the inhibitory effect of septide was reverted into a prokinetic effect, which is probably mediated by the activation of postjunctional excitatory NK1 receptors.     

Further evidence for the presence ofγ-aminobutyric acidA (GABAA) receptors on the cytoplasmic side of deiters' membrane

1. The permeation of labeled Cl- across single nerve membranes microdissected from rabbit Deiters' neurons was studied in a microchamber system. The in----out permeation of the ions was evaluated under control conditions and in the presence of either 10(-6) M GABA or 10(-6) M GABA plus 10(-5) M bicuculline methiodide (BMI) on the membrane cytoplasmic side. 2. In 32 experiments, involving one animal each, at least two membranes served as controls and at least two others were studied with the addition of GABA. Within each experiment all the membranes were obtained from the same animal. 3. In an additional 10 experiments, involving one animal each, at least two membranes served as controls and at least two others were studied in the presence of GABA plus bicuculline methiodide on the membrane cytoplasmic side. 4. The data show that 10(-6) M GABA on the Deiters' membrane cytoplasmic side stimulates Cl- permeation in----out by 42% (P = 0.0000001). When 10(-5) M BMI was present together with GABA, no stimulation of Cl- in----out permeation occurred.     

Involvement of tachykinin NK(1) and NK(2) receptors in changes in lung mechanics and airway microvascular leakage during the early phase of endotoxemia in Guinea pigs

We investigated the role of tachykinins in airway neurogenic responses occurring in the early phase of endotoxemia. Forty-eight anesthetized guinea pigs were evenly divided into six groups pretreated with either saline vehicle, CP-96,345 (a tachykinin NK(1) receptor antagonist), SR-48,968 (a tachykinin NK(2) receptor antagonist) or CP-96,345 and SR-48,968 in combination. Animals then received an intravenous injection of either saline (the vehicle for endotoxin) or endotoxin (30 mg/kg). Total lung resistance (R(L)) and dynamic lung compliance (C(dyn)) were continuously measured before and 30 min after administration of saline or endotoxin. Airway microvascular leakage was assessed at the end of the observation period. Endotoxin significantly increased R(L) and decreased C(dyn) 10 min after intravenous endotoxin injection. Plasma extravasation significantly increased in the trachea, main bronchi and intrapulmonary airways with endotoxin administration. These changes in lung mechanics were abolished by SR-48,968, but were unaffected by CP-96,345. The plasma extravasation was largely attenuated by CP-96,345 and/or SR-48,968. We conclude that (1) endogenous tachykinins play an important role in producing changes in lung mechanics and airway microvascular leakage during the early phase of endotoxemia and (2) activation of tachykinin NK(2) receptors is responsible for the former response, while activation of both tachykinin NK(1) and NK(2) receptors is involved in the latter response.     

Induction of steroid binding sites (receptors) and presence of steroid hormones in the unicellular Tetrahymena pyriformis

The unicellular Tetrahymena does not normally possess a steroid hormone (dehydroepiandrosterone, DHEA) or a glucocorticoid (dexamethasone) receptor, but both kinds of receptor can be induced in it by pretreatment (imprinting) with the adequate hormone. The specific receptors which arise are demonstrable experimentally. Examination of Tetrahymena cells for endogenous steroids by the radioimmunoassay (RIA) technique detected an appreciable concentration of DHEA and DHEA sulphate, and lesser concentrations of testosterone and estradiol in this unicellular organism.     

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.     

Neurons bearing NK3 tachykinin receptors in the guinea-pig ileum revealed by specific binding of fluorescently labelled agonists

The localisation of NK₃ tachykinin receptors in guinea-pig ileum was studied using the fluorescently labelled agonists, Cy3.5-neurokinin A and Cy3.5-kassinin. Binding to nerve cell bodies in the myenteric and submucosal plexuses was visualised using confocal microscopy. Binding to NK₁ receptors was blocked by the NK₁ receptor antagonist, CP-99994. NK₃ receptors, demonstrated by binding in the presence of CP-99994, occurred in 72% of myenteric and 38% of submucosal neurons. Colocalisation with other markers was examined to deduce the classes of neurons with NK₃ receptors. In myenteric ganglia, NK₃ receptors were present on the following: 73% of calbindin-immunoreactive (IR) intrinsic primary afferent neurons, 63% of calretinin-IR excitatory motor neurons and ascending interneurons, 63% of nitric oxide synthase-IR inhibitory motor neurons and descending interneurons, 79% of strongly neuropeptide Y (NPY)-IR secretomotor neurons, 67% of weakly NPY-IR descending interneurons and motor neurons, and 46% of NK₁ receptor-IR neurons. In submucosal ganglia, NK₃ receptors were on 65% of calretinin-IR secretomotor/vasodilator neurons, 81% of NPY-IR cholinergic secretomotor neurons, 2% of vasoactive intestinal peptide-IR non-cholinergic secretomotor neurons and were completely absent from substance P-IR intrinsic primary afferent neurons. The results support physiological studies suggesting that NK₃ receptors mediate tachykinin transmission between myenteric sensory neurons and to interneurons and/or motor neurons in descending inhibitory and ascending excitatory pathways. Accepted: 22 June 1999     

Direct evidence for the presence of selective binding sites for (3H) prostaglandin E2 on rat peritoneal macrophages

A method is presented which provides for a simple and rapid determination of PGE2 receptors on viable peritoneal macrophages. Incubation of the harvested cells with (3H)PGE2 revealed specific binding of (3H)PGE2 by use of the Millipore filter assay system. Maximum binding was attained in the presence of 1 mM EDTA. Specific binding was saturable at 65 fmol/mg protein with an equilibrium dissociation constant (Kd) of 3.2 X 10(-8)M. Inhibition of (3H)PGE2 binding with unlabelled prostaglandins revealed a potency series of PGE2 greater than PGE2 greater than PGI2. The PGE2 concentration which displaced 50% of the labelled ligand was 10(-7)M. Comparable kinetic data were obtained for adenylate cyclase stimulation, since the concentration which showed a halfmaximal stimulation of cAMP production was 2 X 10(-7)M of PGE2. Since PGE1 and PGI2 compete with (3H)PGE2 binding in a non-parallel manner compared to PGE2 itself, it is proposed that macrophages possess different types of PG receptors.     

Identification of the populations of enteric neurons that have NK1 tachykinin receptors in the guinea-pig small intestine

Simultaneous immunofluorescence labelling was used to investigate the patterns of colocalisation of the NK1 tachykinin receptor with other neuronal markers, and hence determine the functional classes of neuron that bear the NK1 receptor in the guinea-pig ileum. In the myenteric plexus, 85% of NK1 receptor-immunoreactive (NK1r-IR) nerve cells had nitric oxide synthase (NOS) immunoreactivity and the remaining 15% were immunoreactive for choline acetyltransferase (ChAT). Of the latter group, about 50% were immunoreactive for both neuropeptide Y (NPY) and somatostatin (SOM), and had the morphologies of secretomotor neurons. Many of the remaining ChAT neurons were immunoreactive for calbindin or tachykinins (TK), but not both. These calbindin immunoreactive neurons had Dogiel type II morphology. No NK1r-IR nerve cells in the myenteric plexus had serotonin or calretinin immunoreactivity. In the submucosal ganglia, 84% of NK1r-IR nerve cells had neuropeptide Y immunoreactivity and 16% were immunoreactive for TK. It is concluded that NK1r-IR occurs in five classes of neuron; namely, in the majority of NOS-immunoreactive inhibitory motor neurons, in ChAT/TK-immunoreactive excitatory neurons to the circular muscle, in all ChAT/NPY/SOM-immunoreactive secretomotor neurons, in a small proportion of ChAT/calbindin myenteric neurons, and in about 50% of ChAT/TK submucosal neurons.     

Lead-dependent deposits in diverse synaptic vesicles: suggestive evidence for the presence of anionic binding sites

We have observed electron dense deposits dependent on incubation of aldehyde-fixed tissues with lead ions within synaptic vesicles of several types of neurons that differ in the neurotransmitters utilized and in the secretory granules of the adrenal medulla. Evidently, vesicle components that can interact with lead ions are widespread. A plausible explanation for the occurrence of the deposits is the presence of anionic binding sites within the vesicles. This would agree well with other biochemical, cytochemical, and immunocytochemical evidence, such as that indicating the presence of sulfated macromolecules in certain synaptic vesicles. Anionic binding sites could play significant roles by participating in processes such as Ca2+ storage, stabilization of pH gradients, or the control of osmotic phenomena.     

Substance P stimulates Growth Hormone (GH) and GH-Releasing Hormone (GHRH) secretions through tachykinin NK2 receptors in sheep

Substance P is ubiquitous undecapeptide belonging to the tachykinins family. It has been found in the hypothalamus and is involved in the hypothalamo-hypophysial axis in several mammals, including human. Previous studies have shown that substance P increases GH secretions in rats and human. In this study, we have shown that intravenously infused substance P in sheep caused an increased level of Growth Hormone (GH) and GH-Releasing Hormone (GHRH), and decreased Somatotropin Release Inhibiting Hormone (SRIH) secretions. GH was obtained from peripheral blood. GHRH and SRIH were directly collected from hypophysial portal blood, using a trans-nasal surgery technique in a vigil sheep that allowed accessing to hypothalamo-hypophysial portal vessels. Hormones assays were performed by radioimmunoassay (RIA). Moreover, we showed that substance P-induced GH and GHRH secretion appears to be mediated by NK2 tachykinin receptors, since it is specifically blocked by a non peptidic tachykinin NK2 receptor antagonist (SR48968, Sanofi, Montpellier, France) whereas a non peptidic tachykinin NK1 antagonist (SR140333, Sanofi, Montpellier, France) failed to modify GH and GHRH hormones secretions.