In vitro and in vivo pharmacology of S 16474, a novel dual tachykinin NK1 and NK2 receptor antagonist

Since tachykinins released from lung sensory nerve endings are thought to play a role in inflammatory diseases of airways via NK1 and NK2 receptors, dual tachykinin NK1 and NK2 receptor antagonists may have a great therapeutic potential. In vitro, the cyclopeptide S 16474 (cyclo-[Abo-Asp(D-Trp(Suc0Na)-Phe-N-(Me)Bzl)]) bound to both human tachykinin NK1 and NK2 receptors expressed in two lines of transfected Chinese hamster ovary cells (IC50 values 85 nM and 129 nM, respectively), while showing a poor affinity for the rat tachykinin NK1 receptor. S 16474 inhibited the contractions induced by substance P in isolated rabbit vena cava (pA2 7.0) and by neurokinin A in rabbit pulmonary artery (pA2 5.6). In vivo in anaesthetized guinea-pigs, S 16474 was found to dose dependently inhibit the bronchoconstrictions induced by intravenously administered substance P, neurokinin A and capsaicin. Plasma extravasation evoked in bronchi by endogenously released tachykinins under vagus nerve stimulation was abolished by S 16474 (10 mu mol/kg i.v.). These results demonstrate clearly that S 16474 is a tachykinin receptor antagonist exhibiting, in vitro and in vivo, a dual inhibitory effect on NK1 and NK2 receptors.     

The unpredicted high affinities of a large number of naturally occurring tachykinins for chimeric NK1/NK3 receptors suggest a role for an inhibitory domain in determining receptor specificity

Three chimeric receptors were constructed by exchanging exon sequences between human NK1 and NK3 receptor genes. The resulting chimeric receptors not only retained high affinities for their natural ligands substance P and neurokinin B but also exhibited surprisingly high affinities for other naturally occurring tachykinins including neurokinin A, neuropeptide K, neuropeptide gamma, eledoisin, kassinin, physalaemin, and phyllomedusin. In contrast, these chimeric receptors displayed a wide range of variability in their affinities for non-naturally occurring ligands including selective agonists and antagonists of NK1, NK2, and NK3 receptors. Since the only common feature among these naturally occurring neurokinin peptides is the conserved C-terminal sequences, our data suggest that these conserved sequences must play the major role in conferring high affinity binding to the chimeric receptors. To explain the apparently "improved" affinities of these naturally occurring ligands for the chimeric receptors as compared with their affinities for the parent NK1 and NK3 receptors, we are proposing that certain inhibitory domains that are present in the NK1 and/or NK3 receptors are compromised in these chimeric receptors. Upon disruption of these inhibitory domains during the formation of chimeras, the naturally occurring ligands can interact more favorably with chimeric receptors through their conserved C-terminal sequences. Based on this hypothesis, the binding affinities of natural tachykinin ligands may be largely determined by their conserved C-terminal sequences, whereas receptor selectivities of these ligands are influenced more by the presence or absence of inhibitory domains rather than specific binding domains on their target receptors.     

Tachykinins alter inositol phosphate formation, but not cyclic AMP levels, in primary cultures of neonatal rat spinal neurons through activation of neurokinin receptors

The naturally occurring tachykinins, substance P, neurokinin A and neurokinin B, induce the formation of inositol phosphates or cAMP in a variety of tissues but their effects on neurons have not been resolved. We used primary cultures of neonatal rat spinal cord to determine whether neurokinin receptors mediate changes in these second messengers in spinal neurons. We found that substance P, neurokinin A and neurokinin B induced the formation of inositol phosphates in a concentration-dependent manner with similar potencies (EC50S: 3.6, 5.7 and 21.3 nM, respectively), but at concentrations tested (0.1-1.0 microM) these peptides had no effect on cAMP levels. All three tachykinins induced the formation of inositol phosphates predominately by activation of neurokinin1 receptors. CP-96,345 and WIN 51,708, neurokinin1 receptor antagonists, attenuated the response to substance P, neurokinin A and neurokinin B. GR 103,537, a neurokinin2 receptor antagonist, had no effect on the responses induced by any of the tachykinins. Furthermore, the selective neurokinin1 receptor agonist, GR-73632, induced the formation of inositol phosphates in a concentration-dependent manner, whereas the selective neurokinin2 receptor agonist, GR-64349, generated inositol phosphates only at the highest concentration tested (10 microM). Senktide, a neurokinin3 receptor agonist, did not induce the formation of inositol phosphates at any of the concentrations tested (0.01-10 microM). Inositol phosphate formation appeared to be due to a direct effect of the tachykinins on neuronal neurokinin1 receptors. These results suggest that biological responses in spinal neurons following activation of neurokinin1 receptors are mediated mainly by the hydrolysis of phosphoinositol 4,5-bisphosphate to form inositol 1,4,5-trisphosphate and diacylglycerol. It remains to be determined which of these second messengers mediates the increased neuronal excitability and depolarization that occurs in response to substance P.     

Potencies of agonists acting at tachykinin receptors in the oestrogen-primed rat uterus: effects of peptidase inhibitors

The uterotonic potencies of the naturally occurring mammalian tachykinins and the synthetic subtype-selective agonist analogues of these agents [Lys5,MeLeu9,Nlel0]neurokinin A-(4-10) and [Nle10]neurokinin A-(4-10) (tachykinin NK2 receptor-selective), [Sar9,Met(O2)11]substance P (tachykinin NK1 receptor-selective) and senktide (tachykinin NK3 receptor-selective) were determined using preparations from oestradiol-treated rats. The endopeptidase 24.11 inhibitor, N-[N-[1-(S)-carboxyl-3-phenylpropyl]-(S)-phenyl-alanyl-(S)-isoserine+ ++ (SCH 39370), potentiated responses to neurokinin A, neurokinin B and substance P, but not to [Lys5,MeLeu9,Nle10)]neurokinin A-(4-10) or senktide. [Nle10]neurokinin A-(4-10) effects were potentiated by SCH 39370 with amastatin and those to [Sar9,Met(O2)11]substance P were potentiated by SCH 39370 and captopril in combination. In the presence of optimal concentrations of peptidase inhibitors the relative order of agonist potency was: neurokinin A > substance P > neurokinin B for the naturally occurring mammalian tachykinins and [Lys5,MeLeu9,Nle10]neurokinin A-(4-10) > [Nle10]neurokinin A-(4-10) > [Sar9,Met(O2)11]substance P > senktide for the synthetic tachykinin analogues. Thus, while a tachykinin NK2 receptor predominates in the oestrogen-primed uterus, a tachykinin NK1 receptor may also be present. The non-peptide tachykinin NK3 receptor antagonist, SR 142801, did not antagonise the effects of senktide suggesting that tachykinin NK3 receptors do not mediate its relatively minor effect on the uterus of the oestrogen-primed rat.     

The tachykinin NK1 receptor. Part I: ligands and mechanisms of cellular activation

The tachykinin NK1 receptor is widely expressed in the mammalian central and peripheral nervous system. Powerful pharmacological tools (agonists and antagonists) are now available to elucidate the physiological role of NK1 receptors at these levels, as well as to understand their role in diseases and establish the possible therapeutic usefulness of NK1 receptor antagonists for treatment of human diseases. The structure-activity studies that have led to the development of potent peptide and non-peptide ligands for the tachykinin NK1 receptor are here reviewed. Among the peptide agonists and antagonists, linear and cyclic sequences have been developed. The non peptide antagonists belong to different chemical classes, i.e. steroids, perhydroisoindolones, quinuclidines, piperidines and tryptophane derivatives. The first non peptide antagonists for NK1 receptors have been obtained by random screening of chemical compounds large collections. The resulting leads were optimized with 'classic' structure activity approaches, aiming at identifying 'common' motifs for interaction with the receptor by ligands of different chemical classes. The results derived from the recent application of molecular biology techniques were useful to drive the design of new ligands toward a precise structural definition of ligand-receptor bi-molecular interactions. Studies on mutant receptors have established that the sites of interaction of peptide agonists and non peptide antagonists with the tachykinin NK1 receptor are largely non overlapping. Moreover, data obtained from mutagenesis of the NK1 receptor further indicate that some amino acid residues in the NK1 receptor sequence are critical for determining the binding affinity of some but not all ligands. Therefore, different antagonists discovered from random screening may not possess common points of interaction or common structural and conformational characteristics for their interaction with the tachykinin NK1 receptor. The tachykinin NK1 receptor couples with G-proteins to determine its biological effects in target cells. Several G-proteins both sensitive (Go, Gi) and insensitive (Gq, G11) to pertussis toxin can mediate the action of NK1 receptors. Moreover, several second messanger signalling systems (elevation of intracellular calcium, stimulation of phosphoinositol turnover, arachidonic acid mobilization, cAMP accumulation) have to be activated following NK1 receptor signalling. Also a direct modulation of certain ion channels at membrane level has been proposed. The NK1 receptor undergoes prompt and significant tachyphylaxis upon exposure to the agonist: this has been shown to be linked with receptor internalization which also occurs physiologically when the NK1 receptor is stimulated by endogenous tachykinins.     

Mechanisms of desensitization and resensitization of G protein-coupled neurokinin1 and neurokinin2 receptors

We compared the desensitization of neurokinin1 and neurokinin2 (NK1 and NK2) receptors expressed in Chinese hamster ovary cells to substance P and neurokinin A, respectively. Substance P and neurokinin A stimulated a rapid increase in intracellular Ca2+ concentration ([Ca2+]i) for both receptors, which was due to release of Ca2+ from intracellular stores. This was followed by a plateau in [Ca2+]i, which was due to influx of extracellular Ca2+, and was more sustained for the NK2 receptor. When Ca2+ was present in the extracellular solution, the Ca2+ response of the NK1 receptor, but not the NK2 receptor, rapidly desensitized and slowly resensitized to two exposures to agonist. In contrast, the [Ca2+]i response, measured in Ca2+-free solution, and inositol triphosphate generation desensitized and resensitized similarly for the NK1 and NK2 receptors. Thus, differences in desensitization between the NK1 receptor and the NK2 receptor may be related to differences in entry of extracellular Ca2+. We compared endocytosis of the NK1 and NK2 receptors to determine whether disparities could account for differences in desensitization. Fluorescent and radiolabeled substance P and neurokinin A were internalized similarly by cells expressing NK1 and NK2 receptors. Thus, disparities in internalization cannot account for differences in desensitization. We used inhibitors to examine the contribution of endocytosis, recycling, and phosphatases to desensitization and resensitization of the NK1 receptor. Desensitization did not require endocytosis. However, resensitization required endocytosis, recycling, and phosphatase activity. This suggests that the NK1 receptor desensitizes by phosphorylation and resensitizes by dephosphorylation in endosomes and recycling.     

Gly166 in the NK1 receptor regulates tachykinin selectivity and receptor conformation

We have studied the pharmacological properties of genetically engineered human NK1 tachykinin receptors in which residues at the extracellular surface of the fourth transmembranal domain were substituted with the corresponding amino acids from the NK2 receptor. We show that substitution of G166C:Y167F in the human NK1 receptor induces high affinity binding of a group of tachykinin ligands, known as 'septides' (i.e. neurokinin A, neurokinin B, [pGlu6,Pro9]-substance P6-11 and substance P-methylester). In contrast, binding of substance P and non-peptide antagonists is unaffected by these mutations. This effect parallels that found on the rat receptor and is therefore species specific. Second, we demonstrate that mutation of Gly166 to Cys alone is both necessary and sufficient to create this pan-reactive tachykinin receptor, whereas replacement of Tyr167 by Phe has no detectable effect on the pharmacological properties of the receptor. Furthermore, analysis of the effect of N-ethylmaleimide and dithiothreitol on binding of radiolabelled substance P documents differences in the mode in which this ligand interacts with wild-type and mutant receptors and supports the existence of a mutational induced change in the conformational status of the NK1 receptor.     

Are inner or outer hair cells the source of summating potentials recorded from the round window?

Previous studies indicated that antidromic stimulation of capsaicin-sensitive vagal afferent fibers activated, via peripheral release of tachykinins, nonadrenergic, noncholinergic parasympathetic ganglion neurons that mediate relaxations of guinea pig trachealis. On the basis of the effects of selective agonists and inhibition with a nonselective receptor antagonist (SR 48968), we speculated that tachykinin-mediated activation of neurokinin3 (NK3) receptors might be involved. Using the recently developed NK3-selective receptor antagonist SR 142801, we further assessed the role of NK3 receptors in these relaxant responses. Relaxations of the guinea pig trachea elicited by antidromic stimulation of capsaicin-sensitive vagal afferent nerves were markedly inhibited by 0.3 microM SR 142801 and were abolished by a combination of SR 142801 and either of the NK1-selective receptor antagonists SR 140333 and CP 99994 (0.3 microM each). The NK3 receptor antagonist had similar effects on the relaxant responses elicited by capsaicin and substance P, but it had no effect on relaxations of the trachealis elicited by electrical field stimulation of the postganglionic nerves that innervate the trachealis or by stimulation of the preganglionic parasympathetic vagal nerves that innervate the trachea. These results and the observation that the ganglion neurons that mediate these responses are densely innervated by substance P-containing nerve fibers lead us conclude that stimulation of capsaicin-sensitive visceral afferent fibers activates, upon peripheral release of tachykinins, nonadrenergic, noncholinergic inhibitory neurons innervating guinea pig trachealis via activation of both NK3 and NK1 receptors.     

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.     

Characterization of tachykinin NK2 receptor on dog proximal colon. Antagonism by MEN 10,627 and SR 48,968

The nature of the tachykinin receptors involved in the contraction of the circular muscle of dog colon has been investigated. The following rank order of potency for agonists was obtained: [Sar9,Met(O2)11]substance P > or = neurokinin A > [beta-Ala8]neurokinin A-(4-10) > [MePhe7]neurokinin B. The efficacy of the tachykinin NK2 receptor agonists was significantly greater than that of the tachykinin NK1 receptor agonists and of carbachol. A concentration-dependent rightward shift of the motor response to neurokinin A (obtained in the presence of (+/-)-CP 96,345) was induced by peptide and non-peptide tachykinin NK2 receptor antagonists with this rank order: MEN 10,627 = SR 48,968 > L 659,877 > MEN 10,376 > MDL 28,564. MEN 10,627 and SR 48,968 affinities were similar to those measured in human tissues. In conclusion, the tachykinin NK2 receptor plays a predominant role in tachykinin-induced contraction of the canine colonic circular muscle and this tissue could be useful to predict the pharmacological actions of MEN 10,627 and SR 48,968 in human colon.     

Nutritional assessment of folate and cyanocobalamin status in a Spanish elderly group

The mammalian tachykinins, neurokinin A (NKA) and NKA(4-10), along with the tachykinin NK2 receptor-selective antagonist MEN 10,376, were compared to their C-terminal free acid derivatives, NKA-OH, NKA(4-10)-OH and MEN 10,456, respectively, on several in vitro bioassays for NK1, NK2 and NK3 tachykinin receptors. NKA-OH and NKA(4-10)-OH were much weaker agonists than NKA or NKA(4-10) in the endothelium-deprived rabbit pulmonary artery (endowed with NK2A receptors) and in the guinea pig isolated bronchus (endowed with NK2A and NK1 receptors), where they produced submaximal contractile responses, and were inactive in the hamster isolated trachea (endowed with NK2B receptors) and in the rat isolated portal vein (endowed with NK3 receptors). At NK1 receptors of the guinea pig isolated ileum, NKA-OH produced weak agonist responses, whereas NKA(4-10)-OH was ineffective. In sharp contrast, MEN 10,456, while maintaining the same antagonist potency of the parent compound MEN 10,376 in the rabbit pulmonary artery and hamster isolated trachea, developed a clear-cut agonist character in the rat isolated portal vein, guinea pig isolated ileum and guinea pig isolated bronchus. The agonist responses produced by MEN 10,456 (10 microM) were reduced by MEN 10,376 in the guinea pig isolated bronchus and by the NK1 receptor antagonist GR 82,334 in the guinea pig isolated ileum. These results, although indicating the importance of C-terminal amidation for the agonist activity of natural tachykinins, suggest that the C-terminal amide group may not be directly involved in stimulation of the tachykinin receptors, but could induce agonist activity through a conformation effect.     

Intercostal pedicle flap for thoracic oesophageal perforations

Since exogenously applied tachykinins (substance P and neurokinin A) prevent the neurogenic hyperaemia which is elicited by acid back-diffusion in the rat stomach, we investigated whether endogenous tachykinins would act in a similar manner. Acid back-diffusion, induced by perfusing the stomach with 15% ethanol in the presence of 0.05 M HCI, increased gastric mucosal blood flow (GMBF) by 60-100% as determined by hydrogen clearance in urethane-anaesthetized rats. This response remained unchanged after pretreatment with the tachykinin NK1 receptor antagonist SR 140,333 (300 nmol/kg) but tended to be enhanced by the NK2 receptor antagonist MEN 10,627 (200 nmol/kg). When given during ongoing acid back-diffusion, MEN 10,627 significantly enhanced the acid-evoked vasodilatation as compared with vehicle or SR 140,333. We conclude that endogenously released tachykinins, acting via NK2 receptors, limit the gastric hyperaemic response to acid.     

Heterogeneity of tachykinin receptors in the rabbit lung

The tachykinins, substance P (SP) and neurokinin A (NKA), are agonists for the NK(1) and NK(2) receptors, respectively. Tachykinins have various respiratory effects, including bronchoconstriction. This study characterizes tachykinin binding sites in the rabbit lung. We hypothesize that (2-[(125)I]iodohistidyl(1))Neurokinin A ([(125)I]NKA) interacts with NK1 and NK2 binding sites in the rabbit lung. The K d determined from saturation isotherms was 0.69 times/divided by 1.14 nM (geometric mean times/divided by SEM) and the B max was 4.15 + or - 0.22 femtomole/mg protein (arithmetic mean + or - SEM). Competitive inhibition studies with NKA, SP and various selective tachykinin agonists showed the rank order of potency; [beta-Ala(8)]-Neurokinin A 4-10 = SP > NKA > [Sar(9),Met(02)11]-Substance P. [beta-Ala(8)]-Neurokinin A 4-10, a selective NK(2) agonist, and SP inhibition of [(125)I]NKA binding were best described using a two-site model. Competitive inhibition studies using the selective nonpeptide NK(2) antagonist (SR 48968) and the selective nonpeptide NK(1) antagonist (CP 96,345) revealed Ki's of 5.5 nM and 8.1 nM, respectively. Our data therefore suggest that [(125)I]NKA binds to both the NK(1) and NK(2) receptors in the lung.     

Pharmacological characterization of tachykinin-induced intracellular calcium rise in a human NK2 receptor transfected cell line

We have examined the effect of various natural and synthetic tachykinins on the steady state Ca(++)-rise ([Ca++]i) in transfected chinese hamster ovary cells expressing recombinant human Neurokinin 2 (NK2) receptors. The rank order of potency with natural tachykinins was NeurokininA > Neurokinin B > Eledoisin > Physaelamin > substance P. The selective NK2 agonist, [beta-Ala8]NKA(4-10) was very potent, with an EC50 value of 4.83 x 10(-9) M whereas Senktide, MePhe7NKB and Sar9, (MetO2)11 substance P, selective NK3 and NK1 agonists, respectively, did not have any effect on [Ca++]i in hrNK2CHO cells, suggesting a selective and preferential recognition and activation of NK2 receptors in these cells. (+/-) SR 48968, a selective NK2 antagonist, abolished the beta-AlaNKA-induced [Ca++]i with an IC50 value of 0.7 nM. Two other peptidic NK2 antagonists, MEN 10376 and L-658977, were less active with IC50 values of 49 nM and 5.29 microM, respectively. In contrast, (+/-) CP-96,345 and (+/-)CP-99,994 and RP 67580, all selective NK1 antagonists, did not have any effect on the beta-AlaNKA-induced [Ca++]i in hrNK2CHO cells (+/-) SR 140333, a potent and selective NK1 antagonist, had a 35% inhibition under similar conditions. These data demonstrate a high selectivity and sensitivity to NK2 receptor mediated [Ca++]i in rhNK2R-CHO cells and may be of value as a rapid, selective test of drug action at the human NK2 receptors in vitro.     

Characterization of the tachykinin receptors involved in spinal and supraspinal cardiovascular regulation

The pharmacological characterization of the tachykinin receptors involved in spinal and supraspinal cardiovascular regulation is reviewed in this report. In conscious rats, substance P (SP), neurokinin A (NKA), neurokinin B (NKB), neuropeptide K (NPK), and neuropeptide gamma (NP gamma) were injected either intrathecally (i.t.) or intracerebroventricularly (i.c.v.), and their effects were assessed on mean arterial blood pressure (MAP) and heart rate (HR). Moreover, selective antagonists for NK1 ((+/-)-CP-96045 and RP-67580), NK2 (SR-48968), and NK3 (R-486) receptors were tested against the agonists. I.t. tachykinins elicited dose-dependent increases in MAP and HR (NPK > NP gamma > SP > NKA > NKB). The cardiovascular response to i.t. SP, NPK, and NP gamma was significantly attenuated by the prior i.t. administration of (+/-)-CP-96345 and RP-67580 but not by SR-48968 and R-486. By the i.c.v. route, tachykinins also elicited pressor and tachycardiac responses dose dependently (NPK > NP gamma > SP > NKA > NKB). Senktide and [MePhe7]NKB, two NK3-selective agonists, were slightly more potent than NKB on both parameters. Whereas the cardiovascular response to NPK was largely blocked by (+/-)-CP-96345 and RP-67580, that to SP was reduced by 40-50%. This treatment had no effect on the cardiovascular response to NKA and [MePhe7]NKB. Conversely, SR-48968 reduced by 40-50% the NKA-induced cardiovascular changes without affecting the central mediated effects of NPK, SP, and [MePhe7]NKB. However, when coadministered, RP-67580 and SR-48968 abolished the effects to SP and NKA while leaving untouched those induced by [MePhe7]NKB. Finally, the central effects mediated by [MePhe7]NKB, senktide, and NKB were blocked by R-486. These findings suggest that the i.t. action of tachykinins on the rat cardiovascular system is mediated by a NK1 receptor in the spinal cord, while NK1, NK2, and NK3 receptors are likely involved in the supraspinal (hypothalamus) effects of these neuropeptides. It is also concluded that NPK is a pure and powerful NK1 agonist, in contrast to SP and NKA, which are not selective for NK1 and NK2 receptors, respectively.     

Assessing cognitive functions in tree shrews: visuo-spatial and spatial learning in the home cage

In the wild-type tachykinin NK3A receptor histidyl residues are present at two positions in TM-V, V:01 and V:05, at which Zn2+ functions as an antagonist in NK1 and kappa-opioid receptors with engineered metal-ion sites. Surprisingly, in the NK3A receptor Zn2+ instead increased the binding of the agonist 125I-[MePhe7]neurokinin B to 150%. [MePhe7]neurokinin B bound to the NK3A receptor in a two-component mode of which Zn2+ eliminated the subnanomolar binding mode but induced a higher binding capacity of the nanomolar binding mode. Signal transduction was not induced by ZnCl2 but 10 microM ZnCl2 enhanced the effect of neurokinin B. Ala-substitution of HisV:01 eliminated the enhancing effect of Zn2+ on peptide binding. It is concluded that physiological concentrations of Zn2+ have a positive modulatory effect on the binding and function of neurokinin B on the NK3A receptor through a bis-His site in TM-V.     

Tachykinin NK1 receptor antagonists enhance stress-induced c-fos in rat locus coeruleus

These experiments tested the hypothesis that substance P neurotransmission at tachykinin NK1 receptors in the locus coeruleus is involved in stress-induced activation of the locus coeruleus, using c-fos as an index of activation. Selective tachykinin NK1 receptor antagonists administered systemically did not result in substantial locus coeruleus c-fos expression. Restraint stress resulted in a large number of locus coeruleus c-fos expressing cells. Administration of two selective tachykinin NK1 receptor antagonists prior to restraint resulted in an increase in the number of locus coeruleus c-fos expressing cells, compared to restraint alone. These results suggest that the enhanced c-fos expression observed in response to tachykinin NK1 receptor antagonists combined with stress, could be due to the blockade of tachykinin NK1 receptor-mediated activity at sites other than the locus coeruleus, resulting in an overall activation of the locus coeruleus.     

Inhibition of nitric oxide synthesis reveals non-cholinergic excitatory neurotransmission in the canine proximal colon

1. Neuromuscular transmission in the circular muscle of the canine proximal colon was examined, in the presence and absence of nitric oxide synthase inhibitors, by use of mechanical and intracellular microelectrode recording techniques. 2. Electrical field stimulation (EFS; 0.1-20 HZ) produced frequency-dependent contractions of circular muscle strips which reached a maximum at 15 Hz. These responses were enhanced by NG-monomethyl-L-arginine (L-NMMA; 300 microM) and reduced by atropine (1 microM). The effects of L-NMMA were reversed by L-arginine (3 mM). All responses to EFS were abolished by tetrodotoxin (1 microM). 3. In the presence of atropine, phentolamine and propranolol (all at 1 microM; 'non-adrenergic, non-cholingergic (NANC) conditions'), EFS evoked frequency-dependent inhibition of phasic contractions which reached a maximum at 5 Hz. At higher frequencies of EFS, inhibition diminished, and these responses were followed by post-stimulus excitation. 4. Under NANC conditions and in the presence of L-NG-nitroarginine methyl ester (L-NAME; 200 microM), EFS evoked contractions at frequencies of 5 Hz or greater. These contractions were reduced by co-incubation with L-arginine (2 mM) and abolished by tetrodotoxin (1 microM). 5. In the presence of atropine (1 microM), EFS (5-20 Hz) caused frequency-dependent inhibition of electrical slow waves. In the presence of L-NAME (100 microM) and atropine, the inhibitory response to EFS was abolished and an increase in slow wave duration was seen at stimulation frequencies greater than 5 Hz. The effects of EFS on slow wave duration were abolished by tetrodotoxin (1 microM). 6. Atropine-resistant contractions to EFS were enhanced by indomethacin (10 microM) and reduced or abolished by the non-selective NK1/NK2 tachykinin receptor antagonist D-Pro2, D-Trp7,9 SP, and by the selective NK2 receptor antagonist MEN 10,376 (10 microM).7. Exogenous tachykinins mimicked non-cholinergic excitatory electrical and mechanical responses. The rank order of potency for contraction was neurokinin A>neurokinin B>substance P, suggesting a predominance of the NK2 sub-type of tachykinin receptors on colonic smooth muscle cells. Low concentrations of neurokinin A also increased the amplitude and duration of electrical slow waves.8. These results suggest that: (i) in previous studies, non-cholinergic excitatory responses were masked by the simultaneous release of NO; (ii) non-cholinergic excitatory responses occur throughout the period of stimulation and are not manifest only as 'rebound' excitation; (iii) one or more tachykinins, possibly,acting via NK2 receptors, may mediate non-cholinergic excitatory responses.     

Molecular characterization and functional expression of a substance P receptor from the sympathetic ganglion of Rana catesbeiana

Substance P is an important neuropeptide neurotransmitter in the central, autonomic and enteric nervous systems. In sympathetic ganglia, substance P is thought to play a role in modulating synaptic transmission. Release of substance P by neuronal stimulation or direct application of substance P to ganglionic neurons increases neuronal excitability. An amphibian substance P receptor complementary DNA has been cloned and characterized from bullfrog, Rana catesbeiana, sympathetic ganglion complementary DNA libraries. The deduced primary structure contains features indicative of a seven transmembrane domain G-protein-coupled receptor. The deduced protein sequence shows 69% identity to previously cloned mammalian substance P receptors. In situ hybridization analysis performed on bullfrog sympathetic ganglia using digoxigenin-labelled complementary RNA probe demonstrated that approximately 75% of the principal neurons displayed reaction product above background levels. Radioligand binding studies were performed on stably transfected cells with [(125)I]Tyr-1-substance P as the ligand. Substance P had an IC50 of 16 nM and the agonist potency profile was substance P>neurokinin A > neurokinin B. The order of potency for three tachykinins to increase intracellular calcium when applied to a stably transfected clonal cell line was substance P>neurokinin A > neurokinin B. This order of agonist potency also held for inhibition of the M-type potassium current in intact bullfrog sympathetic neurons. The non-peptide substance P antagonists CP-96345 and RP-67580 at concentrations that block mammalian substance P receptors had little or no effect on the responses to substance P at the bullfrog receptor. Overall, these results demonstrate that the cloned sequence has the features consistent with and characteristic of a substance P receptor. The results are discussed with reference to the established pharmacology of the bullfrog substance P receptor and known structure activity relationships of mammalian tachykinin receptors.