Endogenous Neurokinin Research Articles

Endocytic recycling prevents copper accumulation in astrocytoma cells stimulated with copper-bound neurokinin B

Neurokinin B (NKB) is a key neuropeptide in reproductive endocrinology where it contributes to the generation of pulses of gonadotropin-releasing hormone. NKB is a copper-binding peptide; in the absence of metal NKB rapidly adopts an amyloid structure, but copper binding inhibits amyloid formation and generates a structure that can activate the neurokinin 3 receptor. The fate of copper once it binds NKB and activates the neurokinin 3 receptor is not understood, but endocytosis of NKB occurs even when the peptide is coordinated to copper. Using astrocytoma cells that express endogenous neurokinin 3 receptor, this work shows that endocytosis of apo- and copper-bound NKB occurs in concert with the receptor via a trafficking pathway that includes the early endosome. When cells are stimulated with copper-bound NKB the cellular copper concentration does not significantly increase, however when the cells are pre-treated with the recycling inhibitor, brefeldin A, they are capable of accumulating copper. This data shows that copper-bound NKB can activate the neurokinin 3 receptor then endocytosis abstracts metal, peptide and receptor from the cell surface. The cell does not accumulate the copper but instead it enters recycling pathways that ultimately leads to metal release from the cell. The work reveals a novel receptor-mediated copper trafficking pathway that retains metal in membrane bound organelles until it is exported from the cell.

Similar effects of substance P on learning and memory function between hippocampus and striatal marginal division.

Substance P is an endogenous neurokinin that is present in the central and peripheral nervous systems. The neuropeptide substance P and its high-affinity receptor neurokinin 1 receptor are known to play an important role in the central nervous system in inflammation, blood pressure, motor behavior and anxiety. The effects of substance P in the hippocampus and the marginal division of the striatum on memory remain poorly understood. Compared with the hippocampus as a control, immunofluorescence showed high expression of the substance P receptor, neurokinin 1, in the marginal division of the striatum of normal rats. Unilateral or bilateral injection of an antisense oligonucleotide against neurokinin 1 receptor mRNA in the rat hippocampus or marginal division of the striatum effectively reduced neurokinin 1 receptor expression. Independent of injection site, rats that received this antisense oligonucleotide showed obviously increased footshock times in a Y-maze test. These results indicate that the marginal division of the striatum plays a similar function in learning and memory to the hippocampus, which is a valuable addition to our mechanistic understanding of the learning and memory functions of the marginal division of the striatum.

Antagonist profile of ibodutant at the tachykinin NK2 receptor in guinea pig isolated bronchi

In this study we have characterized the pharmacological profile of the non-peptide tachykinin NK2 receptor antagonist ibodutant (MEN15596) in guinea pig isolated main bronchi contractility. The antagonist potency of ibodutant was evaluated using the selective NK2 receptor agonist [βAla8]NKA(4-10)-mediated contractions of guinea pig isolated main bronchi. In this assay ibodutant (30, 100 and 300nM) induced a concentration-dependent rightward shift of the [βAla8]NKA(4-10) concentration-response curves without affecting the maximal contractile effect. The analysis of the results yielded a Schild-plot linear regression with a slope not different from unity (0.95, 95% c.l. 0.65–1.25), thus, indicating a surmountable behavior. The calculated apparent antagonist potency as pKB value was 8.31±0.05. Ibodutant (0.3–100nM) produced a concentration-dependent inhibition of the nonadrenergic–noncholinergic (NANC) contractile response induced by electrical field stimulation (EFS) of intrinsic airway nerves in guinea pig isolated main bronchi. At the highest concentration tested (100nM) ibodutant almost abolished the EFS-induced bronchoconstriction (95±4% inhibition), the calculated IC50 value was 2.98nM (95% c.l. 1.73–5.16nM). In bronchi from ovalbumin (OVA) sensitized guinea pigs ibodutant (100nM) did not affect the maximal contractile response to OVA, but completely prevented the slowing in the fading of the motor response induced by phosphoramidon pretreatment linked to the endogenous neurokinin A release. Altogether, the present study demonstrates that ibodutant is a potent NK2 receptor antagonist in guinea pig airways.

Esmolol activates endogenous neurokinin activity inhibiting infarction-induced arrhythmias in rats: Novel mechanisms of anti-arrhythmia

Endogenous neurokinin and adrenergic mechanisms might co-participate in the pathology of acute myocardial infarction (MI). This study sought to investigate the role of endogenous neurokinin and its relationship with β1-adrenergic mechanism in the infarction induced arrhythmias.In 60min of MI in rats, the contents of substance P (SP), a native agonist of neurokinin 1 receptor (NK1-R), norepinephrine (NE), NK1-R and β1-adrenergic receptor in the myocardium at risk of ischemia were examined and the ventricular arrhythmias were analyzed. The effects of pretreatment with D-SP (152ng/kg), a specific antagonist of NK1-R, esmolol (10mg/kg), a specific blocker of β1-adrenergic receptor, and a combination of the two blockers were studied. The results showed that the overlaps of up-regulation of NE, SP and the increase of ventricular arrhythmias were observed. D-SP exacerbated the episodes and duration of VT & VF by 54% and 104%, respectively (all P<0.05). Esmolol inhibited the morbidity rate, the episodes and the duration of VT & VF by 66%, 92% and 95%, respectively. Surprisingly, esmolol significantly attenuated the arrhythmogenic effect of D-SP throughout the MI, beyond the time span of esmolol action, during which a significant up-regulation of the NK1-R (by 19%, P<0.05) was detected.In conclusion, the findings of this study may indicate an anti-arrhythmic effect of endogenous neurokinin mechanism, through the activation of which, via up-regulation of NK1 receptor, esmolol may exert its anti-arrhythmic action at the early time of acute myocardial infarction.

Degeneration of sensory afferent nerves enhances pulmonary inflammatory alterations in acute myocardial infarction in rats

Evidence suggests proinflammatory changes in the lungs during acute myocardial infarction and a participation of neural mechanisms and substance P in the pathology. This study was undertaken to investigate the role and the mechanisms by which sensory afferent degeneration at neonatal stages exacerbates the pulmonary inflammatory responses to acute myocardial infarction in the adult rats. The degeneration of capsaicin-sensitive afferent nerves was induced by administration of capsaicin to neonatal rats. The pulmonary inflammatory changes following coronary artery occlusion (CAO) were assessed by the analysis of the infiltration of neutrophils and tissue morphology in the lungs. Significant increases in the pulmonary infiltration of neutrophils, up to 240% and 218% of the sham controls at 3 and 6 h, respectively, after CAO (P<.05) and marked pulmonary edema were observed. Degeneration of capsaicin-sensitive afferent nerves or antagonism of endogenous neurokinin (NK)-1 receptor exacerbated the pulmonary infiltration of neutrophils (up to 214% and 254% of the controls, respectively) and pulmonary tissue edema following the CAO. The findings indicate that degeneration of sensory afferent nerves enhances the pulmonary inflammatory changes in acute myocardial infarction, in which the endogenous NK may play a role.

Degeneration of capsaicin sensitive sensory nerves enhances myocardial injury in acute myocardial infarction in rats

BackgroundEvidence indicated an involvement of afferent nerves in the pathology of acute myocardial infarction. This study was undertaken to clarify the role and mechanisms by which the sensory afferent degeneration exacerbates the myocardial injury in acute myocardial infarction in rats. MethodsThe myocardial injury was assessed by analysis of 1) the differences in the infarct size, myocyte apoptosis, the caspase activity in the myocardium and cardiac troponin I in serum between the denervated and non-denervated rats; 2) the differences in the size of infarctiom with and without antagonisms of endogenous neurokinin 1 receptor or calcitonin gene related peptide receptor in acute myocardial infarction. ResultsDegeneration of the afferent nerves resulted in marked increase in the pain threshold and decrease in substance P and calcitonin gene related peptide in dorsal root ganglia, spinal dorsal horn and myocardium. Increases of the infarction size (39%±4% vs. 26%±4%,), troponin-I (28.4±8.89ng/ml, vs. 14.6±9.75ng/ml), apoptosis of myocytes (by 1.8±0.2 folds) and caspase-3 activity (1.6±0.3 vs. 1.05±0.18) were observed in the denervated animals at 6h of myocardial infarction, compared with the non-denervated rats. Antagonisms of the endogenous neurokinin 1 receptor or calcitonin gene related peptide receptor caused increase of the size of infarction in the animals. ConclusionDegeneration of capsaicin sensitive afferent nerves enhances the myocardial injury of acute myocardial infarction, possibly due to reduction of endogenous calcitonin gene related peptide and substance P.

Knockout of the Neurokinin-1 Receptor Reduces Cholangiocyte Proliferation in Bile Duct Ligated Mice

Background and aims . Cholangiocytes, the cells that line biliary ducts, have the capacity to respond to a variety of hormones and neuropeptides, regulating their surrounding cell functions and proliferative activity1. There is no evidence that the sensory neuropeptide substance P (SP) regulates cholangiocyte hyperplasia interacting with its endogenous neurokinin 1 receptor (NK1-R)2. Methods . To shed light on this interaction, Wild-type (WT, +/+) and NK-1 receptor (NK-1R) knock-out (NK-1R -/-) mice underwent sham or BDL for 1 week. Then, we evaluated: (i) NK-1R expression by immunohistochemistry, (ii) necrosis, apoptosis, steatosis and the number of cholangiocytes positive for citokeratin-19 (CK-19) in liver sections; (iii) PCNA expression and PKA phosphorylation by western blots. In vitro, we determined the effects of SP on growth by MTS proliferation assay, cAMP intracellular levels and PKA phosphorylation by western blots. Results . Cholangiocytes express NK-1R with upregulation following BDL. In normal NK-1R (-/-) mice, there was higher apoptosis and scattered steatosis compared to controls. In NK-1R -/- BDL mice, there was a decrease in the number of CK-19-positive cholangiocytes and enhanced biliary apoptosis compared to controls. In cholangiocytes from BDL NK-1R -/- mice there was decreased PCNA expression and PKA phosphorylation. In vitro, SP increased cAMP levels, proliferation and PKA phosphorylation of cholangiocytes. Conclusion . In the present study we have demonstrated that modulation of SP, by NK-1R, such as other neuroendocrine factors, may offer a new target in the inhibition and in the regulation of biliary functions during cholangiopathies.

Novel triple neurokinin receptor antagonist CS-003 strongly inhibits neurokinin related responses

Neurokinins are known to induce neurogenic inflammation related to respiratory diseases, though there is little information on triple neurokinin receptor antagonists. The pharmacological properties of the novel triple neurokinin 1, 2 and 3 receptor antagonist [1-{2-[(2 R)-(3,4-dichlorophenyl)-4-(3,4,5-trimethoxybenzoyl)morpholin-2-yl]ethyl}spiro[benzo[ c]thiophene-1(3 H),4′-piperidine]-(2 S)-oxide hydrochloride] (CS-003) were evaluated in this study. The binding affinities of CS-003 were evaluated with human and guinea pig neurokinin receptors. As well, the in vivo antagonism of CS-003 against exogenous neurokinins and effects on capsaicin-induced and citric acid-induced responses were investigated in guinea pigs. CS-003 exhibited high affinities for human neurokinin 1, neurokinin 2 and neurokinin 3 receptors with K i values (mean ± S.E.M.) of 2.3 ± 0.52, 0.54 ± 0.11 and 0.74 ± 0.17 nM, respectively, and for the guinea pig receptors with K i values of 5.2 ± 1.4, 0.47 ± 0.075 and 0.71 ± 0.27 nM, respectively. Competitive antagonism was indicated in a Schild analysis of substance P-, neurokinin A- and neurokinin B-induced inositol phosphate formation with pA 2 values of 8.7, 9.4 and 9.5, respectively. CS-003 inhibited substance P-induced tracheal vascular hyperpermeability, neurokinin A- and neurokinin B-induced bronchoconstriction with ID 50 values of 0.13, 0.040 and 0.063 mg/kg (i.v.), respectively. CS-003 also inhibited capsaicin-induced bronchoconstriction (ID 50: 0.27 mg/kg, i.v.), which is caused by endogenous neurokinins. CS-003 significantly inhibited citric acid-induced coughs and the effect was greater than those of other selective neurokinin receptor antagonists. CS-003 is a potent antagonist of triple neurokinin receptors and may achieve the best therapeutic efficacy on respiratory diseases associated with neurokinins compared to selective neurokinin receptor antagonists.

Solution structure of amphibian tachykinin Uperolein bound to DPC micelles

Uperolein, a physalaemin-like endecapeptide, has been shown to be selective for Neurokinin 1 receptor. As a first step towards understanding the structure–activity relationship, we report the membrane-induced structure of Uperolein with the aid of circular dichroism and 2D 1H NMR spectroscopy. Sequence-specific resonance assignments of protons have been made using correlation spectroscopy (TOCSY, DQF–COSY) and NOESY spectroscopy. The interproton distance constraints and dihedral angle constraints have been utilized to generate a family of structures using torsion angle molecular dynamics within program DYANA. The conformational range of the peptide revealed by NMR and CD studies has been analysed in terms of characteristic secondary features. Analysis of NMR data indicates that the global fold of Uperolein can be explained in terms of equilibrium between 3 10-helix and α-helix from residues 5 to 11. An extended highly flexible N-terminus displays some degree of order and a possible turn structure. A comparison between the structures of Uperolein and Substance P, a prototype and endogenous Neurokinin 1 receptor agonist, indicates several common features in the distribution of hydrophobic and hydrophilic residues. Both the peptides show an amphiphilic character towards the middle region. The similarities suggest that the molecules interact with the receptor in an analogous manner.

Neurokinin release in the rat nucleus of the solitary tract via NMDA and AMPA receptors

Neurokinins (substance P, neurokinin A and neurokinin B) and the neurokinin receptors, the NK1 and NK3 receptors, are largely expressed in the nucleus of the solitary tract (NST) where they are involved in the central regulation of visceral function. Studying the mechanisms that control neurokinin release can provide valuable information concerning the control of autonomic functions subserved by the NST. Glutamate is the principal excitatory neurotransmitter in the NST and the main neurotransmitter of afferent vagal fibers. Neurokinins and glutamate may interact within the NST. In the present study, we have examined the contribution of the N-methyl- D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtypes of glutamate receptors on the release of the endogenous neurokinins in the NST. We used internalization of the NK1 or NK3 receptor as an index of endogenous neurokinin release assessed by immunocytochemical visualization of the NK1 or NK3 receptor endocytosis. Experiments were performed in vitro using rat brainstem slices. A first series of experiments were done in order to validate our in vitro preparation. Application of substance P, neurokinin A or neurokinin B induced dose-dependent internalization of NK1 and NK3 receptor. This was blocked by the endocytosis inhibitor, phenylarzine oxide. The NK1 receptor antagonist SR140333 blocked internalization of NK1 receptor induced by the three neurokinins. In addition, the internalization NK1 or NK3 receptor was reversible. These results demonstrate that internalization and recycling mechanisms of NK1 or NK3 receptor were preserved in in vitro brainstem slices. Application of NMDA or AMPA induced internalization of NK1 receptor. This was blocked by the application of SR140333 suggesting that NK1 receptor internalization is due to the binding of endogenous neurokinin released under the effects of NMDA and AMPA. Application of NMDA or AMPA had no effect on NK3 receptor. Application of tetrodotoxin blocked NK1 receptor internalization induced by NMDA, demonstrating that the release of neurokinins is dependent of axon potential propagation. This result excludes the hypothesis of a release on neurokinins via pre-synaptic NMDA receptors located on neurokinin-containing axon terminals. NMDA or AMPA may directly induce neurokinin release in the NST by acting on receptors located on the cell bodies and dendrites of neurokinin-containing neurons. Release of neurokinins may also be the result of a general activation of neuron networks of the NST by NMDA or AMPA. To conclude, our results suggest that glutamate, through activation of post-synaptic NMDA and AMPA receptors, contributes to neurokinin signaling in the NST.

Endogenous neurokinins facilitate synaptic transmission in guinea pig airway parasympathetic ganglia.

Neurokinin-containing nerve fibers were localized to guinea pig airway parasympathetic ganglia in control tissues but not in tissues pretreated with capsaicin. The purpose of the present study was to determine whether neurokinins, released during axonal reflexes or after antidromic afferent nerve stimulation, modulate ganglionic synaptic neurotransmission. The neurokinin type 3 (NK(3)) receptor antagonists SB-223412 and SR-142801 inhibited vagally mediated cholinergic contractions of bronchi in vitro at stimulation voltages threshold for preganglionic nerve activation but had no effect on vagally mediated contractions evoked at optimal voltage or field stimulation-induced contractions. Intracellular recordings from the ganglia neurons revealed that capsaicin-sensitive nerve stimulation potentiated subsequent preganglionic nerve-evoked fast excitatory postsynaptic potentials. This effect was mimicked by the NK(3) receptor agonist senktide analog and blocked by SB-223412. In situ, senktide analog markedly increased baseline tracheal cholinergic tone, an effect that was reversed by atropine and prevented by vagotomy or SB-223412. Comparable effects of intravenous senktide analog on pulmonary insufflation pressure were observed. These data highlight the important integrative role played by parasympathetic ganglia and indicate that activation of NK(3) receptors in airway ganglia by endogenous neurokinins facilitates synaptic neurotransmission.

Interaction between interleukin 1β and endogenous neurokinin 1 receptor agonists in mediating plasma extravasation and neutrophil accumulation in the cutaneous microvasculature of the rat

Interleukin 1β (IL-1β) is a potent mediator of neutrophil accumulation. Antidromic stimulation of the rat saphenous nerve leads to neurogenic oedema formation mediated by endogenous tachykinins. Here, we have investigated links between IL-1β and the tachykinin 1 (NK 1) receptors in microvascular events in rat skin. Saphenous nerve-induced plasma extravasation was not modulated by skin pretreatment with IL-1β (3 pmol/site intradermally). In addition, the long-lasting antidromic electrical stimulation did not induce significant neutrophil accumulation in naive rat skin. By comparison, the effect of IL-1β-induced neutrophil accumulation was significantly potentiated by co-stimulation of the ipsilateral saphenous nerve; an effect prevented by an NK 1 receptor antagonist (SR140333, 480 nmol/kg, i.v.). We conclude that IL-1β-induced neutrophil accumulation can be influenced in a pro-inflammatory manner by ongoing neurogenic inflammation, of relevance to the sensory nerve input that occurs during ongoing inflammatory disease.

Neuroanatomical localisation of Substance P in the CNS and sensory neurons

The anatomical distribution of Substance P (SP) has been investigated since the development of antibodies against it in the 1970s. Although initial studies were performed with antibodies that also recognised the other endogenous neurokinins, most of the initial descriptions are surprisingly still valid today. In this review, we provide an integrated overview of the pathways containing SP in the central and peripheral nervous systems. The highest densities of SP immunoreactivity occur in the superficial dorsal horn of the spinal cord, in the substantia nigra and in the medial amygdaloid nucleus. In the peripheral nervous system, SP occurs in high concentrations in small diameter primary sensory fibres and in the enteric nervous system. SP is extensively co-localised with classical transmitters and other neuropeptides. In the spinal cord, SP immunoreactive axonal boutons are preferentially presynaptic to neurons expressing the SP receptor, suggesting that the neurokinin acts at a short distance from the release site. In contrast, in the periphery, the situation probably differs in the autonomic ganglia, where the targets are directly innervated by SP, and in other peripheral territories, where SP has to diffuse through the connective tissue to reach the structures expressing the receptor.

NK 3 receptor blockade prevents hyperalgesia and the associated spinal cord substance P release in monoarthritic rats

Previous studies in vitro have shown that NK 3 receptors exist on primary afferent terminals in rat spinal cord and mediate potentiation of the depolarisation-evoked substance P (SP) release. In the present study we have investigated the role of the NK 3 receptor-mediated SP release system in a model of inflammatory pain. Monoarthritis was induced in rats by unilateral injection of complete Freund's adjuvant (CFA); withdrawal latencies to a thermal stimulus were subsequently measured at various times following CFA. The CFA-treated paw displayed hyperalgesia as early as 4 h after CFA injection and hyperalgesia was maintained until day 4 but had disappeared by day 21. The thermal hyperalgesia was associated with an increase in basal SP release from spinal cord synaptosomes. The possible involvement of endogenous neurokinin B acting at NK 3 receptors was tested by using SB 223412-A [(S)-(−)- N-(α-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carboxamide hydrochloride], a novel, potent ( K i=30 nM) and selective ( K i>10 000 nM for NK 1 and NK 2 receptors), non-peptidic NK 3 receptor antagonist. In vitro SB 223412-A antagonised the potentiation of SP release produced by senktide in spinal cord synaptosomes. Administered systemically to monoarthritic rats (50 mg/kg, p.o., b.i.d., for 4 days), the NK 3 receptor antagonist SB 223412-A significantly reduced thermal hyperalgesia and normalised the basal release of SP from spinal cord synaptosomes. The data suggest that neurokinin B acting at NK 3 receptors that mediate SP release within the spinal cord play a role in inflammation. These NK 3 receptors may represent, therefore, appropriate targets in the therapy of inflammatory pain.

An open assessment of the safety and efficacy of venlafaxine in mild to moderate and in severe depression in usual care settings in Hungary

The locomotor activity (LMA) response induced after infusion of selective neurokinin (NK) agonists into the cell body (A10) and a terminal region of the mesolimbic pathway of the rat was investigated. Infusion of the NK1 receptor-selective agonist, GR73632, into the ventral tegmental area (VTA: A10) or the nucleus accumbens (NAS) significantly and dose-dependently increased basal LMA. Agonists selective for the NK2 and NK3 receptors, GR64349 and senktide respectively, had no effect on LMA after intra-NAS infusion. The LMA induced by GR73632 is mediated via dopamine (DA) since the response was abolished by haloperidol. From these studies it would appear that the elevated LMA reported previously after VTA or NAS administration of substance P probably occurs via NK1 receptors. Such data supports the notion that endogenous NKs are likely to be important in modulating the mesolimbic DA pathway and, as a consequence, compounds which antagonise their effects could be useful for the treatment of disorders associated with this system. However, simultaneous infusion of the NK1 agonists, ±CP-96, 345 and its analogue CPQ, into the VTA did not attenuate the LMA induced after intra-VTA infusion of GR73632. Co-infusion of the NK1 antagonist CPQ, but not±CP-96,345, attenuated the LMA response induced by GR73632 in the NAS. The apparent poor susceptibility of these responses to blockade by the recently developed non-peptide NK1 antagonists was unexpected but may reflect their poor affinity for the rat variant of the NK1 receptor. Further advances in the chemistry of NK1 antagonists are now required to confirm the apparent importance of NK1 receptor activation in the control of the mesolimbic DA pathway.

Spinal NK 2 receptors contribute to the increased excitability of the nociceptive flexor reflex during persistent peripheral inflammation

The role of endogenous neurokinin A in changes in the excitability of spinal neurons during adjuvant-induced, peripheral inflammation was examined by determining the effect of a selective NK 2 receptor antagonist, GR103537, on the nociceptive flexor reflex in rats. Intrathecal administration of GR103537 (1.4–14 nmol) dose-dependently attenuated the increased activity of the flexor reflex ipsilateral to the inflamed paw. The activity of GR103537 at NK 2 receptors was confirmed by blockade of the facilitation of the reflex by neurokinin A but not substance P in normal rats. These results indicate that endogenous neurokinin A increases the excitability of spinal neurons during persistent peripheral inflammation.

Further Identification of Neurokinin Receptor Types and Mechanisms of Calcium Signaling Evoked by Neurokinins in the Murine Neuroblastoma C1300 Cell Line

It has been suggested that murine neuroblastoma C1300 cells express endogenous neurokinin NK2 receptors with features that differ from those of NK2 receptors characterized in other systems. In this study, we have further characterized the neurokinin receptor types present in this cell line. RNA blots showed that mRNAs of NK2 and NK3 receptors, but not of NK1 receptors, were expressed in C1300 cells. The increase in the cytosolic calcium concentration ([Ca2+]i) induced by 0.33 microM neurokinin A was completely inhibited by SR 48968, an NK2 receptor antagonist, whereas the partial response to 0.33 microM neurokinin B was unaffected, and the response was completely inhibited by SR 142801, and NK3 receptor antagonist. In addition, the [Ca2+]i increase by 0.33 microM senktide, an NK3 receptor agonist, was inhibited by SR 142801 but not by SR 48968. These findings indicated that C1300 cells endogenously express functional NK2 and NK3 receptors. It was also demonstrated that NK2 and NK3 receptors can be activated independently by 3.3 microM neurokinin A in the presence of 1.0 microM SR 142801 or 1.0 microM senktide, respectively. Therefore, the mechanisms of Ca2+ signaling mediated by endogenous NK2 and NK3 receptors were investigated. The independent activation of NK2 or NK3 receptors induced not only the [Ca2+]i increase, but also stimulated the formation of inositol trisphosphates; both these responses were inhibited by U73122, a phospholipase C (PLC) inhibitor. In addition, NK2 and NK3 receptor-mediated [Ca2+]i increase was partially attenuated in the absence of extracellular Ca2+ or in the presence of nickel, an inorganic Ca2+ influx blocker, but was unaffected by nifedipine and omega-conotoxin, L- and N-type voltage-dependent Ca2+ channel blockers, respectively. Furthermore, the depolarization by 60 mM K+ did not affect the [Ca2+]i. These findings suggested that the NK2 and NK3 receptor-mediated [Ca2+]i increase was due to the activation of PLC and was dependent on the mobilization of internal Ca2+ and the entry of extracellular Ca2+ through voltage-independent channels. This study showed that the C1300 cell line is a useful system with which to investigate pharmacological functions and signaling pathways of endogenous NK2 and NK3 receptors.

SR 48968 specifically depresses neurokinin A- vs. substance P-induced hyperalgesia in a nociceptive withdrawal reflex

To determine the role of neurokinin A and tachykinin NK 2 receptors in processing of nociceptive information at the spinal level, the selective NK 2 receptor antagonist, SR 48968 ( S)-N-methyl- N[4-(4-acetylamino-4-[phenyl piperidino)-2-(3,4-dichlorophenyl)-butyl]benzamide, was tested for its effects on the hyperalgesia produced in the tail flick reflex by intrathecal administration of neurokinin A and of substance P. SR 48968 was also tested in a model in which noxious peripheral stimulation has been shown to produce hyperalgesia via a substance P mechanism. SR 48968 given intrathecally had a dose-dependent inhibitory effect on both the behaviour and the hyperalgesia induced by neurokinin A but not on either of these effects produced by substance P. In addition, systemic administration of SR 48968 depressed the hyperalgesic effect of intrathecal administration of neurokinin A. First, this evidence indicates a unique role for neurokinin A in the spinal cord as distinct from that of its homologue, substance P, and confirms that neurokinin A acts via the tachykinin NK 2 receptor, rather than non-specifically via the NK 1 receptor. Second, the data indicate that in this model substance P does not express any of its effects non-selectively via activation of NK 2 receptors. Third, SR 48968 appears to have access to the spinal cord upon systemic administration. Fourth, intrathecal administration of the NK 1 receptor antagonist, CP-96,345 [(2 S,3 S)-cis-2-(diphenylmethyl)- N-[(2-methoxy-phenyl)-methyl]-1-azabicyclo[2.2.2]-octan-3-amine], had no effect on the responses to intrathecal administration of neurokinin A. Finally, the hyperalgesia produced by sustained noxious thermal stimulation of the tip of the tail was unaffected by intrathecal administration of SR 48968; thus, it remains to find a physiological response in which endogenous neurokinin A and NK 2 receptors at the spinal level are involved in the rat in vivo.

Substance P released endogenously by high-intensity sensory stimulation potentiates purinergic inhibition of nociceptive dorsal horn neurons induced by peripheral vibration

To investigate the interaction at the spinal level of endogenously released substance P with the effects of endogenously released adenosine, extracellular single-unit activity was recorded from dorsal horn neurons in the anesthetized cat. Vibration to the skin inhibited on-going activity of nociceptive neurons; 20 mg/kg caffeine reversibly blocked this inhibition, indicating mediation via adenosine receptors. In half of the cases, this inhibition was potentiated by iontophoretic application of substance P. High-intensity electrical stimulation to a sensory nerve produced excitation which was blocked by an NK-1 (substance P) receptor antagonist, implicating an endogenous neurokinin. When electrical stimulation preceded the vibrational stimulus, the inhibitory effect of vibration was potentiated. Thus, we suggest that endogenous substance P may potentiate the inhibitory response to endogenous adenosine. The results have important implications for integration of inputs from different sensory modalities, especially as they relate to nociception and pain.

SR 48968 selectively prevents faecal excretion following activation of tachykinin NK2 receptors in rats.

We tested the ability of SR 48968, (S)-N-methyl-N(4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichloropheny l)butyl ) benzamide, a non-peptide antagonist highly selective for tachykinin NK2 receptors, to prevent defecation induced in rats by several agents. The tachykinin agonists substance P, [MePhe7]neurokinin B and [beta-Ala8]neurokinin A (4-10) all promoted defecation and increased faecal water content, the last compound being over ten times more potent than the other two (intraperitoneal dose inducing the excretion of 1 g faeces dry weight = 6.7 micrograms kg-1). SR 48968 given either orally (p.o.) or subcutaneously (s.c.) was similarly potent in dose-dependently inhibiting faecal output stimulated by the selective NK2-agonist [beta-Ala8]neurokinin A (4-10) (doses causing 50% inhibition 0.4 microgram kg-1, p.o. and 0.3 microgram kg-1, s.c.). This inhibition was long-lasting (more than 18 h after 1 microgram kg-1 SR 48968 either s.c. or p.o.). At the higher doses tested, SR 48968 also significantly prevented the increase in faecal water content produced by [beta-Ala8]neurokinin A (4-10). In rats treated with SR 48968, stimulation of faecal output by the alpha 2-adrenergic antagonist idazoxan and by salmonella endotoxin (LPS), but not by the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin, 5-HT, carbachol or platelet-activating factor, was partially prevented. The present results suggest that activation of intestinal NK2 receptors, either directly by the selective agonist [beta-Ala8]neurokinin A (4-10) or indirectly through the release of endogenous neurokinin A (by idazoxan or LPS), promotes defecation, presumably as a consequence of increased gut motility or secretion, or both. SR 48968 should therefore be useful for studying the role of neurokinin A-dependent mechanisms in health and disease, including those of the gastrointestinal system, and possibly for developing new therapeutic agents.