A "septide-sensitive" receptor is not involved in tachykinin-mediated secretory and inositol phosphate responses in rat parotid gland: are several transduction pathways involved after the stimulation of the NK1 receptor?

In the rat parotid gland, the neuropeptide substance P (SP), as well as SP(4-11), and septide elicited inositol phosphate production (EC50 values 0.44, 2, and 20 nM, respectively). No additivity of the maximal response to the three agonists was observed. SP, SP(4-11), and septide also stimulated protein secretion; for SP, two EC50 were determined (0.5 and 160 nM), whereas a single one could be determined for SP(4-11) and septide (EC50 values 15 and 20 nM, respectively). The selective tachykinin NK1 receptor antagonist RP67580 acted as a competitive inhibitor of both SP- and SP(4-11)-induced inositol phosphate production. Its effect on septide-induced inositol phosphate production was noncompetitive. RP67580 is apparently as potent at antagonizing septide, SP, or SP(4-11) (in all cases KB = 3 nM). These results show that in parotid gland, only NK1 receptors are activated by SP, SP(4-11), and septide. We also showed that the protein secretion stimulated by SP was inhibited competitively by RP67580, whereas the effect of RP67580 was noncompetitive on protein secretion when SP(4-11) or septide was used. Our data indicate that in rat parotid gland, the existence of a specific "septide-sensitive" receptor can be ruled out and that only the NK1 receptor is present and mediates cellular responses. Taken together, these results show that in this tissue the NK1 receptor would present at least two different binding sites that could be coupled to different transduction pathways and that would regulate protein secretion.     

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.     

Tachykinin peptides affect differently the second messenger pathways after binding to CHO-expressed human NK-1 receptors

The human NK-1 receptor transfected in Chinese hamster ovary (CHO) cells was studied with use of different tachykinin analogs: Substance P, [Pro9]SP, [Sar9, Met(O2)11]SP, [Gly9 psi (CH2CH2) Leu10]SP, Ac-Arg-septide, septide, [Gly9 psi (CH2CH2) Gly10]SP, NKA, [pGlu6]SP(6-11) and [Lys5]NKA(4-10). Binding experiments with [3H][Pro9]SP discriminated two classes of peptides with either high affinity (K iota in the nanomolar range) for the human NK-1 receptor or with low affinity (K iota in the micromolar range); this second group of peptides included NKA and [pGlu6]SP(6-11). In spite of these differences, both peptide families evoked potent stimulation of phosphatidylinositol hydrolysis (EC50 in the nanomolar range). In contrast, only NK-1 agonists, with high affinity, stimulated with great potency cyclic AMP formation (EC50 from 8 to 50 nM), whereas the second family of peptides were only weak agonists (EC50 in the micromolar range). RP 67580, CP 96345 and GR 94800, a NK-2 antagonist, were either competitive or uncompetitive inhibitors of inositol phosphates or cyclic AMP formations induced by [Pro9]SP, septide or NKA, independently of the agonist or the response studied. Thus, NKA, the presumed NK-2 endogenous peptide that may be co-released with SP, and the enzymatically produced C-terminal fragment of SP, [pGlu6]SP(6-11), may trigger specific pharmacological responses via the NK-1 receptor, at nanomolar concentrations, and thus regulate the action of SP at the NK-1 receptor.     

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.     

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.     

Effect of early cyclosporine levels on kidney allograft rejection

Experiments were performed on strips of mouse stomach and urinary bladder to characterize the receptors involved in the contractile responses of these tissues to neurokinins (substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and neuropeptide gamma (NP gamma). The neurokinin receptors were characterized by using assays with selective agonists as well as peptide and nonpeptide antagonists and by applying the two Schild criteria for receptor classification, namely, the order of potency of agonists and the apparent affinity of competitive antagonists. The mouse stomach contains primarily NK1 and NK2 functional sites and possibly some NK3 receptors, whereas the urinary bladder possesses only the NK2 receptor. The rank order of potency of agonists in the stomach is Ac[Arg6,Sar9,Met(O2)11]SP-(6-11) > NKA > SP > [beta-Ala8]NKA-(4-10) > NKB > [MePhe7]NKB. Among the selective agonists, Ac[Arg6,Sar9,Met(O2)11]SP-(6-11) is more active than SP and [Sar9,Met(O2)11]SP on the NK1 receptor, whereas the order of potency on the NK2 receptor is NKA > NP gamma > or = [beta-Ala8]NKA-(4-10) > [Nle10]NKA-(4-10). The order of potency of agonists in the bladder is NP gamma > NKA > [beta-Ala8]NKA-(4-10). The myotropic responses mediated by NK1 selective agonists are blocked by SR 140333 (pA2 8.57) and those mediated by the NK2 selective agonists are inhibited by SR 48968 (pA2 9.05). RP 67580 (pA2 8.41) is more active than CP 99994 (pA2 6.06) on the mouse NK1 receptor. The NK1 receptor of the mouse shows, therefore, a pharmacological profile similar to that of the NK1 receptor of the rat. Similarly, MEN 10627 (pA2 9.20) is more active than R 396 (pA2 6.21), suggesting that the mouse NK2 receptor is similar to that of the rabbit. The mouse NK2 receptor of the urinary bladder shows similarity with that of the stomach, but is less sensitive to [beta-Ala8]NKA-(4-10).     

Excitatory motor and electrical effects produced by tachykinins in the human and guinea-pig isolated ureter and guinea-pig renal pelvis

1. In isolated tissue experiments, neurokinin A (NKA) produced concentration-dependent contraction of human and guinea-pig ureter (pD2 = 6.7 and 7.2, respectively); an effect greatly reduced (>80% inhibition) by the tachykinin NK2 receptor-selective antagonist MEN 11420 (0.1 microM). The tachykinin NK1 and NK3 receptor agonists septide and senktide, respectively, were ineffective. 2. Electrical field stimulation (EFS) of the guinea-pig isolated renal pelvis produced an inotropic response blocked by MEN 11420 (0.01-1 microM). In the same preparation MEN 11420 (0.1 microM) blocked (apparent pK(B) = 8.2) the potentiation of spontaneous motor activity produced by the NK2 receptor-selective agonist [betaAla8]NKA(4-10). 3. In sucrose-gap experiments, EFS evoked action potentials (APs) accompanied by phasic contractions of human and guinea-pig ureter, which were unaffected by tetrodotoxin or MEN 11420 (3 microM), but were blocked by nifedipine (1-10 microM). NKA (1-3 microM) produced a slow membrane depolarization with superimposed APs and a tonic contraction with superimposed phasic contractions. NKA prolonged the duration of EFS-evoked APs and potentiated the accompanying contractions. MEN 11420 completely prevented the responses to NKA in both the human and guinea-pig ureter. 4. Nifedipine (1-10 microM) suppressed the NKA-evoked APs and phasic contractions in both human and guinea-pig ureter, and slightly reduced the membrane depolarization induced by NKA. A tonic-type contraction of the human ureter in response to NKA persisted in the presence of nifedipine. 5. In conclusion, tachykinins produce smooth muscle excitation in both human and guinea-pig ureter by stimulating receptors of the NK2 type only. NK2 receptor activation depolarizes the membrane to trigger the firing of APs from latent pacemakers.     

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.     

Genetic basis of disease: studies of eicosanoids, and some complex disease traits

The three main tachykinins, substance P, neurokinin A (NKA), and neurokinin B, are believed to be selective ligands for respectively the NK-1, NK-2 and NK-3 receptors. However, NKA also has actions which cannot be mediated through its normal NK-2 receptor and the synthetic peptide [pGlu6,Pro9]-Substance P9-11--called septide--is known to have tachykinin-like actions despite its apparent lack of binding to any known tachykinin receptor. In the cloned NK-1 receptor expressed in COS-7 cells NKA and septide as expected were poor competitors for radiolabeled substance P. However, by using radiolabeled NKA and septide directly, it was found that both peptides in homologous binding assays as well as in competition against each other in fact bound to the NK-1 receptor with high affinity: Kd values of 0.51 +/- 0.15 nM (NKA) and 0.55 +/- 0.03 nM (septide). It is concluded that NKA and septide are high-affinity ligands for the NK-1 receptor but that they are poor competitors for substance P, which in contrast competes very well for binding with both NKA and septide.     

Bronchoconstrictor and respiratory effects of neurokinin A in dogs

Neurokinin A (NKA) is the primary bronchoconstrictor tachykinin in the lungs of several species, including humans and has been implicated as an important mediator of inflammatory lung disorders, such as asthma. In this study, we investigated the effect of NKA on airway mechanics (lung resistance, dynamic lung compliance) and respiration (tidal volume, respiratory rate) in anesthetized, spontaneously breathing, male beagle dogs. The dogs were challenged with aerosolized NKA that was delivered from a jet nebulizer to the airways through an endotracheal tube. The challenge consisted of five separate inflations of 600 ml of air/inflation over a 1-min period. Challenge with aerosolized NKA (0.1-1%) produced a dose-dependent increase in lung resistance and a decrease in dynamic lung compliance. The bronchoconstriction induced by 1% NKA peaked at 0.5 min after challenge and had a duration of approximately 5 min. Challenge with 1% NKA also reduced tidal volume and increased respiratory rate. Pretreatment of dogs with the NK-2 receptor antagonist, SR 48968 dose-dependently (1-10 mg/kg, p.o.) blocked the bronchoconstriction and respiratory responses to NKA challenge. Pretreatment with the NK1-receptor antagonist, CP 99994 (1 mg/kg, i. v.) had no effect on the increase in lung resistance and the decrease in dynamic lung compliance due to NKA challenge, but blunted the respiratory response to NKA. Pretreatment of dogs with inhaled ipratropium bromide (0.01%) slightly, but significantly reduced the increase in lung resistance due to NKA challenge but had no effect on the decrease of dynamic lung compliance or on the respiratory responses to NKA. As expected, the bronchoconstrictor response to inhaled methacholine was completely blocked by inhaled ipratropium bromide (0.01%). In conclusion, we have identified an NK2-receptor mediated bronchoconstrictor effect of NKA in dogs. Cholinergic reflexes play a small, but significant role in this response. Furthermore, both NK1 and NK2-receptors appear to be involved with the development of the rapid, shallow breathing response to NKA challenge. These results demonstrate an effect of tachykinins on airway mechanics and ventilatory reflexes in dogs.     

Membrane-induced secondary structures of neuropeptides: a comparison of the solution conformations adopted by agonists and antagonists of the mammalian tachykinin NK1 receptor

We present what we believe to be the first documented example of an inducement of distinctly different secondary structure types onto agonists and antagonists selective for the same G-coupled protein receptor using the same membrane-model matrix wherein the induced structures are consistent with those suggested to be biologically active by extensive analogue studies and conventional binding assays. 1H NMR chemical shift assignments for the mammalian NK1 receptor-selective agonists alpha-neurokinin (NKA) and beta-neurokinin (NKB) as well as the mammalian NK1 receptor-selective antagonists [d-Pro2,d-Phe7,d-Trp9]SP and [d-Arg1, d-Pro2,d-Phe7,d-His9]SP have been determined at 600 MHz in sodium dodecyl sulfate (SDS) micelles. The SDS micelle system simulates the membrane-interface environment the peptide experiences when in the proximity of the membrane-embedded receptor, allowing for conformational studies that are a rough approximation of in vivo conditions. Two-dimensional NMR techniques were used to assign proton resonances, and interproton distances were estimated from the observed nuclear Overhauser effects (NOEs). The experimental distances were used as constraints in a molecular dynamics and simulated annealing protocol using the modeling package DISCOVER to generate three-dimensional structures of the two agonists and two antagonists when present in a membrane-model environment to determine possible prebinding ligand conformations. It was determined that (1) NKA is helical from residues 6 to 9, with an extended N-terminus; (2) NKB is helical from residues 4 to 10, with an extended N-terminus; (3) [d-Pro2,d-Phe7,d-Trp9]SP has poorly defined helical properties in the midregion and a beta-turn structure in the C-terminus (residues 6-9); and (4) [d-Arg1,d-Pro2, d-Phe7,d-His9]SP has a helical structure in the midregion (residues 4-6) and a well-defined beta-turn structure in the C-terminus (residues 6-10). Attempts have been made to correlate the observed conformational differences between the agonists and antagonists to their binding potencies and biological activity.     

The arterial blood supply of the human patella. Its clinical importance for the operating technique in vascularized knee joint transplantations

1 This study aimed to assess the effect of cyclopiazonic acid (CPA), an inhibitor of sarcoplasmic reticulum calcium (Ca) pump, against contractile responses produced by selective tachykinin NK1 and NK2 receptor agonists, [Sar9]substance P (SP) sulfone and [beta Ala8]neurokinin A (NKA) (4-10), respectively, on the circular muscle of guinea-pig colon. All experiments were performed in the presence of atropine (1 microM) and indomethacin (10 microM). 2 In organ bath experiments, a submaximally equieffective concentration of the two agonists (10 nM) was selected: [Sar9]SP sulfone (10 nM) produced a biphasic contraction, the two amplitudes averaging 75 +/- 2 and 43 +/- 3% of the maximal response to KCl (80 mM) at 1 and 15 min from application of the agonist, respectively. CPA (3 microM for 60 min) slightly reduced the phasic response to [Sar9]SP sulfone (16 +/- 4% inhibition) and markedly suppressed the tonic component (89 +/- 3% inhibition). 3 The contraction produced by [beta Ala8]NKA (4-10) (10 nM) was more sustained than that induced by the NK1 receptor agonist: it averaged 69 +/- 5 and 73 +/- 4% of the response to KCl at 1 and 15 min from application of the agonist, respectively. CPA slightly and evenly depressed the response to [beta Ala8]NKA (4-10) (18 +/- 7 and 21 +/- 5% inhibition at 1 and 15 min). 4 In the presence of tachykinin NK1 and NK2 receptor antagonists (SR 140333 and MEN 10627, respectively, 1 microM each) and of L-nitroarginine (100 microM), KCl (40 mM) produced a distinct phasic and tonic contraction which was suppressed by 1 mM nifedipine. CPA (3 microM) did not affect the phasic contraction to KCl but abolished the tonic component of the response. 5 In the presence of 1 microM nifedipine, the response to [beta Ala8]NKA (4-10) was slightly depressed (32 +/- 6% inhibition) in its early component only, while the response to [Sar9]SP sulfone was abolished. CPA produced a slight inhibition (15 +/- 9 and 33 +/- 10% at 1 and 15 min, respectively) of the nifedipine-resistant response to [beta Ala8]NKA (4-10), an effect similar to that observed in the absence of nifedipine. Therefore, a large part of the response to [beta Ala8]NKA (4-10) persisted in the presence of both CPA and nifedipine. 6 In the sucrose gap, a prolonged superfusion with [Sar9]SP sulfone (0.1 microM for 5 min) produced sustained depolarization with superimposed spikes and contraction. CPA (3 microM) produced transient depolarization and contraction. In the presence of CPA, the initial responses (depolarization, spikes and contraction) to [Sar9]SP sulfone were unaffected but the sustained component of contraction was absent; the latter effect was accompanied by a suppression of spikes while the sustained depolarization was present. 7 We conclude that, during sustained depolarization produced by the NK1 receptor agonist, blockade of the sarcoplasmic reticulum Ca pump by CPA produces a faster Ca-dependent inactivation of Ca channels, thereby eliminating spikes and abolishing the tonic component of contraction. Ca mobilization/reuptake from a CPA-sensitive store seems to be of minor importance for regulating the NK2 receptor-mediated contractile responses.     

Tachykinin NK1 but not NK2 receptors mediate non-cholinergic excitatory junction potentials in the circular muscle of guinea-pig colon

1. The effect of tachykinin NK1 and NK2 receptor antagonists on noncholinergic excitatory junction potentials (e.j.ps) evoked by electric field stimulation (EFS) in the circular muscle of the guinea-pig proximal colon was investigated by means of a sucrose-gap technique. 2. In the presence of 1 microM atropine, submaximal EFS (10 Hz, 20-30 V, 0.5 ms pulse width, 1 s train duration) evoked an inhibitory junction potential (i.j.p.) followed by e.j.p. with superimposed action potentials (APs) and contraction. Addition of either NG-nitro-L-arginine (L-NOARG, 0.1 mM) or apamin (0.1 microM) inhibited the evoked i.j.p. and the combined administration of the two agents almost abolished it. In the presence of both L-NOARG and apamin, an atropine-resistant e.j.p. was the only electrical response evoked by EFS in 50% of cases and a small i.j.p. (10% of original amplitude) followed by e.j.p. was evident in the remainder. 3. In the presence of L-NOARG and apamin, the tachykinin NK1 receptor antagonists, (+/-)-CP 96,345 and GR 82,334 (10 nM-3 microM) concentration-dependently inhibited the atropine-resistant e.j.p. and accompanying contraction evoked by EFS. EC50 values were: 0.77 microM (e.j.p. inhibition) and 0.22 microM (inhibition of contraction) for (+/-)-CP 96,345; 0.61 microM (e.j.p. inhibition) and 0.20 microM (inhibition of contraction) for GR 82,334. The tachykinin NK2 receptor antagonists, MEN 10,376 (up to 3 microM) and SR 48,968 (up to 1 microM) had no effect on the atropine-resistant e.j.p. MEN 10,376 (3 microM) but not SR 48,968 produced a slight inhibition of the evoked contraction. 4. (+/- )-CP 96,345 (3 microM) and GR 82,334 (3 microM) markedly reduced (81 and 89% inhibition, respectively)the atropine-resistant ej.p. in the absence of L-NOARG and apamin, without affecting the ij.p. MEN 10,376 (3 microM) and SR 48,968 (1 microM) had no significant effect on noncholinergic ij.p. and ej.p. evoked in the absence of apamin and L-NOARG.5. The electrical and mechanical responses to the NK, receptor agonist [Sar9]substance P (SP) sulfone were blocked by (+/-)-CP 96,345 (3 1M) or GR 82,334 (3 microM) which, at the same concentration, failed to affect the responses to the NK2 receptor agonist [PAla8] neurokinin A (NKA) (4-10). In contrast, MEN10,376 (3 microM) or SR 48,968 (1 microM) blocked the response to [beta Ala8]NKA(4-10) without affecting the response to [Sar9]SP sulfone.6. In the presence of L-NOARG and apamin, and in the absence of atropine, EFS of low pulse width(0.02-0.03 ms, other parameters as above) produced cholinergic ej.ps and contraction which were unaffected by GR 82,334 (3 microM). (+/-)-CP 96,345 (3 JAM) produced 24% reduction in the area of the atropine-sensitive ej.p. without affecting the peak amplitude of ej.p. or contraction.7. These findings demonstrate that the noncholinergic ej.ps and accompanying contraction of the circular muscle of the guinea-pig colon are produced through activation of intramural tachykininergic nerves and that the resultant smooth muscle response is almost entirely mediated through NK1 receptors.     

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.     

Tachykinins mediate noncholinergic excitatory neural responses in the circular muscle of rat proximal colon

Using the sucrose-gap technique, we attempted to assess a role for tachykinins (TKs) in mediating noncholinergic excitatory junction potential (EJP) and contraction, in the circular muscle of rat proximal colon. Excitatory responses were evoked by submaximal electrical field stimulation (EFS) in the presence of atropine (1 microM), guanethidine (1 microM), indomethacin (10 microM), and N(omega)-nitro-L-arginine methyl ester (L-NAME) (100 microM). The NK1 receptor antagonist, SR 140,333 (up to 3 microM) or the NK2 receptor antagonists, SR 48,968 and MEN 10,627 (up to 5 microM) produced a partial inhibition of the excitatory responses to EFS. The co-administration of the selective NK1 and NK2 receptor antagonists produced additive effects on the responses to EFS. Selective NK1 receptor agonist, [Sar9, Met (O2)11]-substance P, induced depolarization and contraction, antagonized by SR 140,333, but not by NK2 receptor antagonists. NK2 receptor agonist, [betaAla8]-neurokinin A (4-10), also produced electrical and mechanical excitatory effects that were antagonized by SR 48,968 or MEN 10,627, but not by the NK1 receptor antagonist. Our results provide evidence that, in circular muscle of rat colon, endogenous tachykinins are the main excitatory transmitters for nonadrenergic, noncholinergic (NANC) excitation and their action is mediated by both NK1 and NK2 receptors.     

Antinociceptive activity of the tachykinin NK1 receptor antagonist, CP-99,994, in conscious gerbils

1. The ability of CP-99,994, and its less active enantiomer, CP-100,263, to inhibit spontaneous behaviours and hyperalgesia induced by central infusion of the NK1 receptor agonist, GR73632 or intraplantar injection of formalin was investigated in rats and gerbils. 2. GR73632 (3 pmol, i.c.v.)-induced foot tapping in gerbils was dose-dependently inhibited by CP-99,994 (0.1-1 mg kg-1, s.c.), but not by CP-100,263 (10 mg kg-1, s.c.) using pretreatment times up to 60 min. The centrally active dose-range for CP-99,994 was increased to 1-10 mg kg-1 s.c. with a higher challenge dose of GR73632 (30 pmol, i.c.v.). 3. In gerbils, intrathecal (i.t.) injection of GR73632 (30 pmol) elicited behaviours (licking, foot tapping or flinching and face washing) which closely resembled, but which was less specifically localized than, behaviours seen in animals injected with formalin (0.1-5%) into one hindpaw. 4. In rats, CP-100,263, but not CP-99,994 (up to 30 mg kg-1), inhibited the early phase response to intraplantar injection of 5% formalin (ID50 = 13.9 mg kg-1). The late phase was inhibited by both compounds (ID50 values 36.3 and 20.9 mg kg-1, respectively). In gerbils, there was marginal evidence for enantioselective inhibition of the early phase induced by formalin (2%). The ID50 values were 6.2 mg kg-1 for CP-99,994 and 13.4 mg kg-1 for CP-100,263. 5. Intrathecal injection of GR73632 (30 pmol) caused thermal hyperalgesia in igerbils which was inhibited enantioselectively by s.c. administration of CP-99,994 (ID50= 2.46 mg kg-1), but not by CP-100,263 (30 mg kg-1).6. In gerbils, intraplantar injection of formalin (0.1%) caused thermal hyperalgesia which was inhibited by CP-99,994 (ID50= 1.1 mg kg-1, s.c.). There was a nonsignificant trend for an anti-algesic effect of CP-100,236 (estimated ID50 = 8.2 mg kg-1, s.c.).7 These findings support the proposal that NK1 receptor antagonists may be useful in the clinical management of pain and reinforce the need to dissociate specific and nonspecific antinociceptive effects of available compounds.     

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.     

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.     

Activation of the cloned human NK3 receptor in Chinese Hamster Ovary cells characterized by the cellular acidification response using the Cytosensor microphysiometer

1. The aim of the present study was to validate the Cytosensor microphysiometer, a novel system that measures the extracellular acidification rate as a reliable index of the integrated functional response to receptor activation, as a method for studying NK3 receptor pharmacology, and then to use this system to assess the functional activity of novel compounds at this receptor. 2. The selective NK3 agonist senktide caused reproducible, concentration-related increases in acidification ratein CHO-NK3 cells, with a pEC50 value of 8.72+/-0.11 (n=15). [Beta-Ala8]NKA(4-10), the selective NK2 agonist, elicited a much weaker response (pEC50=6.68+/-0.08, n=4), while the NK1-selective agonist substance P methylester only caused a very weak response at concentrations > or =3 microM (n=2). The rank order of potency for the endogenous tachykinins NKB>NKA>substance P (n=3) confirmed the response was mediated by the NK3 receptor. Moreover, the actual potencies obtained were consistent with affinities measured in radioligand binding studies. 3. The novel compounds PD156319-121 (0.3-1 microM), PD161182 (10-300 nM), PD168001 (10-100 nM) and PD168073 (10-100 nM) all acted as surmountable antagonists of the senktide-induced acidification response, with pA2 values of 7.49, 8.67, 9.17 and 9.25 respectively (n=3-5). In comparison the known NK3 antagonist SR142801 (10-100 nM) had a pA2 value of 8.83 (n=8) for the interaction with senktide. Again, these values are consistent with the radioligand binding data. 4. Amiloride (1 mM) inhibited the senktide-induced acidification response by 68.3+/-3.3 (n=4), indicating that the Na+/H+ antiporter plays an important role in this response, and this is consistent with the importance of this antiporter in other acidification responses. 5. Inhibition of protein kinase C with staurosporine (0.1 microM), or depletion of the intracellular Ca2+ stores with thapsigargin (1 microM), both resulted in a reduction in the maximum response to senktide (63.3+/-1.7 and 68.9+/-3.2% respectively, n=3-5), and co-application of these inhibitors abolished the response (n=3). This strongly suggested that the NK3 receptor was coupling via phospholipase C (PLC), as would be expected, although this could not be confirmed by the use of the putative PLC/PLA2 inhibitor U73122. 6. In conclusion, we have demonstrated the utility of the Cytosensor in the characterization of functional responses to agonists, and assessment of the affinities of antagonists in CHO cells expressing the human NK3, and have shown that our series of novel compounds are non-peptide NK3 antagonists of high affinity, as exemplified by PD168073.     

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.