Endogenous Tachykinins Research Articles

Characterization of neuropeptide K processing in rat spinal cord S9 fractions using high-resolution quadrupole-Orbitrap mass spectrometry.

Tachykinins are a family of pronociceptive neuropeptides with a specific role in pain and inflammation. Several mechanisms regulate endogenous tachykinins levels, including the differential expression of protachykinin mRNA and the controlled secretion of tachykinin peptides from neurons. We suspect that proteolysis regulates extracellular neuropeptide K (NPK) and neurokinin A (NKA) concentrations and NPK is a precursor of NKA. Here, we provide evidence that proteolysis controls NPK and NKA levels in the spinal cord, leading to the formation of active C-terminal peptide fragments. Using high-resolution mass spectrometry, specific tachykinin fragments were identified and characterized. The metabolic stability in rat spinal cord fractions of NPK and NKA was very short, resulting in half-lives of 1.9 and 2.2 min respectively. Following the degradation of NPK, several C-terminal fragments were identified, including NPK1-26 , NKA, NKA2-10 , NKA3-10 , NKA5-10 and NKA6-10 , which conserve affinity for the neurokinin 2 receptor but also for the neurokinin 1 receptor. Interestingly, the same fragments were identified following the degradation of NKA. A specific proprotein convertases inhibitor was used and showed a significant reduction in the rate of formation of NKA, providing strong evidence that proprotein convertase is involved in C-terminal processing of NPK in the spinal cord, leading to the formation of NKA.

Targeted high-resolution quadrupole-Orbitrap mass spectrometry analyses reveal a significant reduction of tachykinin and opioid neuropeptides level in PC1 and PC2 mutant mouse spinal cords

Tachykinin and opioid neuropeptides play a fundamental role in pain transmission, modulation and inhibition. The proteolysis control of endogenous tachykinin and opioid neuropeptides has a significant impact on pain perception. The role of proprotein convertases (PCs) in the proteolysis of proneuropeptides was previously established but very few studies have shown the direct impact of PCs on the regulation of specific tachykinin and opioid peptides in the central nervous system. There is an increasing interest in the therapeutic targeting of PCs for the treatment of pain but it is imperative to assess the impact of PCs on the pronociceptive and the endogenous opioid systems. The objective of this study was to determine the relative concentration of targeted neuropeptides in the spinal cord of WT, PC1−/+ and PC2−/+ animals to establish the impact of a restricted PCs activity on the regulation of specific neuropeptides. The analysis of tachykinin and opioid neuropeptides were performed on a HPLC-MS/MS (High-Resolution Quadrupole-Orbitrap Mass Spectrometer). The results revealed a significant decrease of Dyn A (p<0.01), Leu-Enk (p<0.001), Met-Enk (p<0.001), Tach58–71 (p<0.05), SP (p<0.01) and NKA (p<0.001) concentrations in both, PC1−/+ and PC2−/+ animals. Therefore, the modulation of PCs activity has an important impact on specific pronociceptive peptides (SP and NKA), but the results also shown that endogenous opioid system is hindered and consequently it will affect significantly the pain modulatory pathways. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies.

Kinetic binding and activation profiles of endogenous tachykinins targeting the NK1 receptor

Ligand–receptor binding kinetics (i.e. association and dissociation rates) are emerging as important parameters for drug efficacy in vivo. Awareness of the kinetic behavior of endogenous ligands is pivotal, as drugs often have to compete with those. The binding kinetics of neurokinin 1 (NK1) receptor antagonists have been widely investigated while binding kinetics of endogenous tachykinins have hardly been reported, if at all. Therefore, the aim of this research was to investigate the binding kinetics of endogenous tachykinins and derivatives thereof and their role in the activation of the NK1 receptor.We determined the binding kinetics of seven tachykinins targeting the NK1 receptor. Dissociation rate constants (koff) ranged from 0.026±0.0029min−1 (Sar9,Met(O2)11-SP) to 0.21±0.015min−1 (septide). Association rate constants (kon) were more diverse: substance P (SP) associated the fastest with a kon value of 0.24±0.046nM−1min−1 while neurokinin A (NKA) had the slowest association rate constant of 0.001±0.0002nM−1min−1. Kinetic binding parameters were highly correlated with potency and maximal response values determined in label-free impedance-based experiments on U-251 MG cells.Our research demonstrates large variations in binding kinetics of tachykinins which correlate to receptor activation. These findings provide new insights into the ligand–receptor interactions of tachykinins and underline the importance of measuring binding kinetics of both drug candidates and competing endogenous ligands.

Characterization of Substance P processing in mouse spinal cord S9 fractions using high-resolution Quadrupole-Orbitrap mass spectrometry

Tachykinins are a family of pronociceptive neuropeptides with a specific role in pain and inflammation. Several mechanisms regulate endogenous tachykinins and Substance P (SP) levels, including the differential expression of protachykinin mRNA and the controlled secretion of tachykinins from neurons. Proteolysis is suspected to regulate extracellular SP concentrations but few studies were conducted on the metabolism of proneuropeptides and neuropeptides. Here, we provide evidence that proteolysis controls SP levels in the spinal cord leading to the formation of active C-terminal fragments. Using high-resolution mass spectrometry, specific tachykinins fragments were characterized and quantified. The metabolic stability of β-Tachykinin58–71 and SP were very short resulting in half-life of 5.7 and 3.5min respectively. Several C-terminal fragments were identified, including SP3–11, SP5–11 and SP8–11, which conserve affinity for the Neurokinin 1 receptor. Interestingly, the metabolic stability of C-terminal fragments was significantly superior. Two specific Prolyl endopeptidase inhibitors were used and showed a significant reduction in the rate of formation of SP3–11 and SP5–11 providing strong evidence that Prolyl endopeptidase is involved into N-terminal processing of SP in the spinal cord.

An Endogenous Tachykinergic NK2/NK3 Receptor Cascade System Controlling the Release of Serotonin from Colonic Mucosa.

5-Hydroxytryptamine (5-HT) released from colonic mucosal enterochromaffin (EC) cells is a major signaling molecule, which participates in the pathophysiological regulation of colonic functions in gut disorder including irritable bowel syndrome (IBS), but the endogenous modulator system for the 5-HT release is not yet well elucidated. Our in vitro studies in guinea-pig colon have indicated that the cascade pathway of neuronal tachykinergic NK3 receptors and NK2 receptors on peptide YY (PYY)-containing endocrine L cells represents an endogenous modulator system for 5-HT release from EC cells and that melatonin, endogenous tachykinins and PYY play important roles in modulation of the release of 5-HT from EC cells via the endogenous NK2/NK3 receptor cascade system. This review aims at examining the potential role of the endogenous tachykinergic NK2/NK3 receptor cascade system controlling the release of 5-HT from EC cells, with special attention being paid to the pathophysiology of gut disorders including IBS.

In vitro pharmacological evaluation of the radiolabeled C-terminal substance P analogue Lys-Phe-Phe-Gly-Leu-Met-NH2: Does a specific binding site exist?

In the present paper, we report the synthesis, radiolabeling and comprehensive pharmacological evaluation of a C-terminally truncated tachykinin derivative, 3H-KFFGLM-NH2. The C-terminal fragments of endogenous tachykinins are pharmacophores responsible for interaction with the tachykinin receptors NK1, NK2 and NK3. The N-terminal fragments are responsible for modulation of receptor selectivity and interactions with other receptor systems. To evaluate and separate the function of an NK-pharmacophore from the activity of its parent neurokinin, KFFGLM-NH2 was synthesized in both tritiated and unlabeled forms. It has been proposed that the obtained NK-binding profiles of specific reference ligands and KFFGLM-NH2 differentiate monomeric and dimeric forms of NK receptors. We hypothesize that dimers of NK receptors could be specific receptor(s) for C-terminal fragments of all neurokinins as well as their C-terminal fragments, including H-KFFGLM-NH2. Dissociation of dimers into monomers opens access to additional allosteric binding sites. Fully elongated undecapeptide substance P interacts with both the "tachykinin pocket" and the "allosteric pocket" on the monomeric NK1 receptor, resulting in high and selective activation. However, C-terminal hexapeptide fragment analogues, recognizing only the "tachykinin pocket", may have less specific interactions with all tachykinin receptors in both monomeric and dimeric forms.

Ovarian steroids regulate tachykinin and tachykinin receptor gene expression in the mouse uterus

BackgroundIn the mouse uterus, pregnancy is accompanied by changes in tachykinin and tachykinin receptor gene expression and in the uterotonic effects of endogenous tachykinins. In this study we have investigated whether changes in tachykinin expression and responses are a result of changes in ovarian steroid levels.MethodsWe quantified the mRNAs of tachykinins and tachykinin receptors in uteri from ovariectomized mice and studied their regulation in response to estrogen and progesterone using real-time quantitative RT-PCR. Early (3 h) and late (24 h) responses to estrogen were evaluated and the participation of the estrogen receptors (ER), ERalpha and ERbeta, was analyzed by treating mice with propylpyrazole triol, a selective ERalpha agonist, or diarylpropionitrile, a selective agonist of ERbeta.ResultsAll genes encoding tachykinins (Tac1, Tac2 and Tac4) and tachykinin receptors (Tacr1, Tacr2 and Tacr3) were expressed in uteri from ovariectomized mice. Estrogen increased Tac1 and Tacr1 mRNA after 3 h and decreased Tac1 and Tac4 expression after 24 h. Tac2 and Tacr3 mRNA levels were decreased by estrogen at both 3 and 24 h. Most effects of estrogen were also observed in animals treated with propylpyrazole triol. Progesterone treatment increased the levels of Tac2.ConclusionThese results show that the expression of tachykinins and their receptors in the mouse uterus is tightly and differentially regulated by ovarian steroids. Estrogen effects are mainly mediated by ERalpha supporting an essential role for this estrogen receptor in the regulation of the tachykinergic system in the mouse uterus.

Endogenous tachykinins and neprilysin modulate human sperm motility

OBJECTIVE: The purpose of this work was to examine the function and expression of the tachykinin substance P (SP) and the enzyme neprilysin in capacitated human spermatozoa. DESIGN: To examine the function and expression of the tachykinin substance P (SP) and the enzyme neprilysin in capacitated human spermatozoa we have used molecular biology techniques. MATERIALS AND METHODS: Freshly ejaculated semen was collected from 30 healthy human donors. Liquefied semen samples were capacitated on a discontinuous Percoll density gradient followed by a swim-up. Sperm motility was measured using WHO procedures. Reverse transcriptase polymerase chain reaction and immunofluorescence assays were used to detect the mRNAs and proteins of interest. RESULTS: The mRNAs of TAC1 and MME, the genes that encode SP and neprilysin, respectively, were expressed in human sperm cDNA. Immunocytochemistry studies showed the presence of SP and neprilysin proteins in sperm cells. Phosphoramidon, a high selective inhibitor of neprilysin, caused an increase in sperm progressive motility and its effects were reduced in the presence of a combination of the tachykinin receptor-selective antagonists SR 140333 (NK1 receptor-selective), SR 48968 (NK2 receptor-selective) and SR 142801 (NK3 receptor-selective). CONCLUSIONS: These findings demonstrate that neprilysin plays a role in the regulation of human sperm motility. The effects of neprilysin are due, at least in part, to a modulation of metabolism of endogenous tachykinins.

Capsaicin-evoked bradycardia in anesthetized guinea pigs is mediated by endogenous tachykinins

The present study was done to characterize the effects of endogenous tachykinins on heart rate in urethane-anesthetized guinea pigs. Intravenous injection of capsaicin (32 nmol/kg) was used to evoke release of tachykinins and calcitonin gene-related peptide (CGRP) from cardiac sensory nerve fibers. Such injections caused a brief decrease in heart rate (−37±7 beats/min, n=6) that was followed by a more prolonged increase (+44±10 beats/min). Blood pressure was lowered by −11±2 mmHg. Bilateral vagotomy did not affect the chronotropic or depressor responses to capsaicin, but atropine (1 µmol/kg) nearly abolished the bradycardic response (−8±3 beats/min, n=7). Combined blockade of NK2 and NK3 receptors, with SR48968 and SR14801 respectively, also caused a significant reduction of capsaicin-evoked bradycardia (−14±3 beats/min, n=4) but did not affect bradycardia evoked by vagal nerve stimulation. Blockade of CGRP receptors eliminated capsaicin-evoked tachycardia and prolonged the capsaicin-evoked bradycardia. These findings suggest that capsaicin-evoked bradycardia in the anesthetized guinea pig is mediated by tachykinins that stimulate cardiac cholinergic neurons. This effect appears to be truncated by the positive chronotropic action of CGRP that is also released from cardiac afferents by capsaicin.

Ventilatory and cardiovascular actions of centrally administered trout tachykinins in the unanesthetized trout

The brains of teleost fish contain members of the tachykinin family that are the products of orthologous genes expressed in mammalian nervous tissues, but little is known regarding the physiological effects of these peptides in their species of origin. The present study compares the central actions of trout neuropeptide gamma (NPgamma), substance P (SP) and neurokinin A (NKA) (5-250 pmol) on ventilatory and cardiovascular parameters in the unanesthetized rainbow trout Oncorhynchus mykiss. Intracerebroventricular (ICV) injection of NPgamma evoked a dose-dependent elevation of the ventilation rate (f(V)) but a reduction of the ventilation amplitude (V(AMP)) that was caused by a reduction of the magnitude of the adduction phase of the ventilatory signal. The net effect of NPgamma was to produce an hypoventilatory response since the total ventilation (V(TOT)) was significantly reduced. The minimum effective dose for a significant effect of NPgamma on f(V) and V(AMP) was 50 pmol. SP evoked a significant elevation of f(V), a concomitant depression of V(AMP), and a resultant decrease in V(TOT) but only at the highest dose (250 pmol). NKA was without action on f(V) but significantly decreased V(AMP) at only the highest dose tested. In this case also, the net effect of NKA was to reduce V(TOT). When injected centrally, none of the three peptides, at any dose tested, produced changes in heart rate or mean dorsal aortic blood pressure (P(DA)). Intra-arterial injection of the three tachykinins (250 pmol) produced a significant (P<0.05) increase in P(DA), but only SP and NKA induced concomitant bradycardia. None of the three peptides produced any change in f(V) or V(AMP). In conclusion, our results demonstrate that centrally injected tachykinins, particularly NPgamma, produce a strong hypoventilatory response in a teleost fish and so suggest that endogenous tachykinins may be differentially implicated in neuroregulatory control of ventilation.

Tachykinin regulation of cholinergic transmission in the limbic/prefrontal territory of the rat dorsal striatum: implication of new neurokinine 1‐sensitive receptor binding site and interaction with enkephalin/mu opioid receptor transmission

The tachykinin neurokinin 1 receptors (NK(1)Rs) regulation of acetylcholine release and its interaction with the enkephalin/mu opioid receptors (MORs) transmission was investigated in the limbic/prefrontal (PF) territory of the dorsal striatum. Using double immunohistochemistry, we first showed that in this territory, cholinergic interneurons contain tachykinin NK(1)Rs and co-express MORs in the last part of the light period (afternoon). In slices of the striatal limbic/PF territory, following suppression of the dopaminergic inhibitory control of acetylcholine release, application of the tachykinin NK(1)R antagonist, SSR240600, markedly reduced the NMDA-induced acetylcholine release in the morning but not in the afternoon when the enkephalin/MOR regulation is operational. In the afternoon, the NK(1)R antagonist response required the suppression of the enkephalin/MOR inhibitory control of acetylcholine release by betafunaltrexamine. The pharmacological profile of the tachykinin NK(1)R regulation tested by application of the receptor agonists [[Pro(9)]substance P, neurokinin A, neuropeptide K, and substance P(6-11)] and antagonists (SSR240600, GR205171, GR82334, and RP67580) indicated that the subtype of tachykinin NK(1)R implicated are the new NK(1)-sensitive receptor binding site. Therefore, in the limbic/PF territory of the dorsal striatum, endogenous tachykinin facilitates acetylcholine release via a tachykinin NK(1)R subtype. In the afternoon, the tachykinin/NK(1)R and the enkephalin/MOR transmissions interact to control cholinergic transmission.

Role for NK 1 and NK 2 receptors in the motor activity in mouse colon

The present study examined the effects induced by endogenous and exogenous activation of NK 1 and NK 2 receptors on the mechanical activity of mouse proximal colon. Experiments were performed in vitro recording the changes in intraluminal pressure from isolated colonic segments. Electrical field stimulation in the presence of atropine and guanethidine produced a small relaxation, followed by nonadrenergic noncholinergic (NANC) contraction. SR140333, NK 1 receptor antagonist, or SR48968, NK 2 receptor antagonist, significantly reduced the contraction, although SR48968 appeared more efficacious. The co-administration of SR140333 and SR48968 virtually abolished the NANC contraction. [Sar 9, Met(O 2) 11]-substance P, selective NK 1 receptor agonist, induced a concentration-dependent biphasic effect, contraction followed by reduction of the mechanical spontaneous activity. Both effects were antagonized by SR140333, but not by SR48968. [β-Ala 8]-neurokinin A (4-10), selective NK 2 receptor agonist, evoked concentration-dependent contraction, which was antagonized by SR48968, but not by SR140333. The contraction induced by [Sar 9, Met(O 2) 11]-substance P, but not by [β-Ala 8]-neurokinin A (4-10), was reduced by tetrodotoxin or atropine, and increased by N ω-nitro-L-arginine methyl ester (L-NAME), inhibitor of nitric oxide synthase. The inhibitory effects induced by [Sar 9, Met(O 2) 11]-substance P were abolished by tetrodotoxin or L-NAME. The results of the present study suggest that in mouse colon both NK 1 and NK 2 receptors are junctionally activated by endogenous tachykinins to cause an additive response. NK 1 receptors appear to be located on cholinergic and on nitrergic neurons as well as on smooth muscle cells, whereas NK 2 receptors seem to be present exclusively on smooth muscle cells.

Endogenous Tachykinin Release Contributes to the Locomotor Activity in Lamprey

Tachykinins are present in lamprey spinal cord. The goal of this study was to investigate whether an endogenous release of tachykinins contributes to the activity of the spinal network generating locomotor activity. The locomotor network of the isolated lamprey spinal cord was activated by bath-applied N-methyl-D-aspartate (NMDA) and the efferent activity recorded from the ventral roots. When spantide II, a tachykinin receptor antagonist, was bath-applied after reaching a steady-state burst frequency (>2 h), it significantly lowered the burst rate compared with control pieces from the same animal. In addition, the time to reach the steady-state burst frequency (>2 h) was lengthened in spantide II. These data indicate that an endogenous tachykinin release contributes to the ongoing activity of the locomotor network by modulating the glutamate-glycine neuronal network responsible for the locomotor pattern. We also explored the effects of a 10-min exogenous application of substance P (1 microM), a tachykinin, and showed that its effect on the burst rate depended on the initial NMDA induced burst frequency. At low initial burst rates (approximately 0.5 Hz), tachykinins caused a marked further slowing to 0.1 Hz, whereas at higher initial burst rates, it instead caused an enhanced burst rate as previously reported, and in addition, a slower modulation (0.1 Hz) of the amplitude of the motor activity. These effects occurred during an initial period of approximately 1 h, whereas a modest long-lasting increase of the burst rate remained after >2 h.

Ontogeny of excitatory and inhibitory control of gastrointestinal motility in the African clawed frog,Xenopus laevis

The transparent body wall of Xenopus laevis larvae during the first developmental stages allows in vivo studies of gastrointestinal tract activity. The purpose of this study was to chart the ontogeny of gut motility in Xenopus larvae and to identify the most important control systems during the first developmental stages. Coordinated descending contraction waves first occurred in the gut at Nieuwkoop and Faber stage 43 [0.8 +/- 0.1 contractions/min (cpm)] and increased to 4.9 +/- 0.1 cpm at stage 47. The cholinergic receptor agonist carbachol (5-10 microM) increased contraction frequency already at stage 43, as did neurokinin A (NKA, 0.3-1 microM). The muscarinic antagonist atropine (100 microM) first affected contraction frequency at stage 45, which coincides with the onset of feeding. The tachykinin antagonist MEN-10,376 (6 microM) blocked NKA-induced contractions but not spontaneous motility. Both sodium nitroprusside [nitric oxide (NO) donor, 1-10 microM] and vasoactive intestinal peptide (VIP, 0.1-1 microM) inhibited contractions from the earliest stage onward. Blocking NO synthesis using NG-nitro-L-arginine methyl ester (100 microM) had no effect per se, but antagonized VIP evoked inhibition at stage 47. We conclude that gastrointestinal motility is well developed in the Xenopus laevis larvae before the onset of feeding. Functional muscarinic and tachykinin receptors are present already at the onset of motility, whereas a cholinergic tone develops around the onset of feeding. No endogenous tachykinin tone was found. Functional VIP receptors mediate inhibition at the onset of motility. NO seems to mediate the VIP effect at later stages.

Pivotal role of endogenous tachykinins and the NK 1 receptor in mediating leukocyte accumulation, in the absence of oedema formation, in response to TNFα in the cutaneous microvasculature

Tachykinins including substance P (SP) are well known to play a role in influencing oedema formation and leukocyte accumulation during tissue insult and inflammation. Cutaneous inflammatory models to characterize a TNFα-dependent mechanism where endogenous SP act via the NK 1 receptor to promote leukocyte accumulation in the absence of oedema formation were used. We found that TNFα induced dose-dependent leukocyte accumulation at 4 h, which returned towards basal levels at 8 h in NK 1 +/+ mice. This response was absent in both the NK 1 +/+ mice treated with an NK 1 receptor antagonist and NK 1 −/− mice. At the highest dose IL-6 induced a significant accumulation in NK 1 +/+ and NK 1 −/− mice but IL-12 was ineffective. SP induced skin oedema but none of the cytokines did. Either co-injection of SP with low dose of TNFα (0.3 pmol/site) or SP previously injected (30 min) to TNFα evoked a significant increase in MPO activity when compared with that induced by the cytokine alone. In contrast, SP injected i.d. 3.5 h after TNFα failed to produce additive response. Control, but not capsaicin-pretreated rats (to deplete sensory nerves), exhibited a marked increase in MPO activity in response to TNFα. Histological analysis showed that TNFα caused tissue infiltrate of leukocytes in NK 1 +/+ mice, whilst leukocytes accumulated at intravascular sites in NK 1 −/− mice, but did not appear to emigrate, suggesting a defect in trans-endothelial migration. Interestingly, monocytes in addition to neutrophils accumulated 4 h post TNFα injection. In conclusion, the NK 1 receptor plays a functional role in mediating leukocyte accumulation independently of the historically important NK 1 mediated oedema formation. It seems that TNFα directly activates sensory nerve in addition to its chemoattractant activity. The NK 1 receptor agonist influences the accumulation of monocytes in addition to that of PMN by 4 h, thus revealing an important influence of the NK 1 receptor on TNFα mediated events in mouse skin.

Cigarette smoke increases mucosal permeability in guinea pig trachea via tachykinin NK 2 receptor activation

We investigated whether exposure to cigarette smoke increases the mucosal permeability in guinea pig trachea and if this effect could be mediated by tachykinin NK 2 receptor activation. Guinea pigs were exposed to either three different doses of cigarette smoke or room air. Mucosal permeability was measured by monitoring the rate of appearance in the circulation of horseradish peroxidase (HRP) that had been instilled into the isolated tracheal segment. Exposure to 20 and 30 puffs but not 10 puffs of cigarette smoke increased the tracheal mucosal permeability. Pretreatment with the tachykinin NK 2 receptor antagonist SR48,968 [( S)- N-methyl- N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide] completely inhibited the increase in the permeability of the tracheal mucosa induced by exposure to cigarette smoke, whereas the tachykinin NK 1 receptor antagonist SSR240,600 [( R)-2-(1-{2-[4-{2-[3,5-bis(trifluoromethyl)phenyl]acetyl}-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl}-4-piperidinyl)-2-methylpropanamide] and the tachykinin NK 3 receptor antagonist SR142,801 [( S)-( N)-(1-[3-(1-benzoyl-3(3,4-dichlorophenyl)piperidine-3-yl)propyl]-4-phenylpiperidin-4-yl)- N-methyl-acetamide] had no effect. It is concluded that endogenous tachykinins via NK 2 receptor activation mediate the increase in the permeability of the tracheal mucosa induced by exposure to cigarette smoke in guinea pigs.

Role of Neurokinin 3 Receptors in Supraoptic Vasopressin and Oxytocin Neurons

Neurokinin 3 receptors (NK3-Rs) are expressed in the supraoptic nucleus (SON), and SON is innervated by substance P (SP)-expressing A1 neurons in the medulla. Because SP stimulates vasopressin (VP) and oxytocin release from explants of the hypothalamo-neurohypophyseal system (HNS), two hypotheses were tested: (1) SP-stimulated VP release is mediated by NK3-Rs, and (2) stimulation of the A1 pathway by hypotension activates SON NK3-Rs. Senktide, an NK3-R agonist, stimulated VP release from HNS explants, but neither a neurokinin 1 receptor antagonist [L732,138 (N-acetyl-L-tryptophan 3,5-bis(tri-fluoromethyl)benzyl ester)] nor two NK3-R antagonists (SB222200 and SB235375) prevented SP-stimulated VP release. Because the affinity of these antagonists for rat NK-Rs may limit their efficacy, NK3-R internalization was used to assess the ability of SP to activate SON NK3-Rs. Senktide, SP, or vehicle was microinjected above SON. The brain was perfused 5 min after injection and stained for NK3-R immunoreactivity. Using confocal microscopy, the number of NK3-R-immunoreactive (-IR) endosomes was counted in a 5.6(2) mu region of cytoplasm in SON neurons. Senktide, but not SP or vehicle, significantly increased the number of NK3-R-IR endosomes in the cytoplasm. When hypotension was induced with hydralazine, NK3-R internalization was observed within 5 min (p < 0.005). A decrease in cytoplasmic NK3-R immunoreactivity was observed within 15 min of hypotension. Unexpectedly, both senktide and hypotension resulted in translocation of NK3-R-IR immunoreactivity to the nucleus. Thus, although these studies do not identify SP as the NK3-R ligand, they do provide evidence for hypotension-induced release of an endogenous tachykinin in SON and evidence suggesting a role for NK3-Rs in transcription regulation.

A novel role for tachykinin neurokinin-3 receptors in regulation of human bronchial Ganglia neurons.

The neuropeptide tachykinins and their receptors have been implicated in the pathogenesis of lung disease, although the role of the tachykinin neurokinin-3 receptor has not been elucidated. Using confocal microscopy, we identified tachykinin neurokinin-3 receptors on human bronchial parasympathetic ganglion neurons. Electrophysiologic recordings demonstrated that activation of sensory nerve fibers, either by antidromic stimulation or capsaicin, depolarized these neurons. This response was mimicked by exogenously applied tachykinin neurokinin-3 receptor-selective agonist, senktide analogue, but not significantly by tachykinin neurokinin-1 or neurokinin-2 receptor-selective agonists. Responses to endogenous tachykinins or exogenous selective tachykinin neurokinin-3 receptor activation with senktide analogue were inhibited by the selective tachykinin neurokinin-3 receptor antagonists, SB 223412 or SB 235375. We provide the first evidence that tachykinin neurokinin-3 receptors regulate human bronchial parasympathetic ganglion neurotransmission by activation of a peripheral reflex. This pathway may play a significant role in controlling bronchomotor tone and air flow to the lung.

Neprilysin 2: A Novel Messenger Peptide-Inactivating Metalloprotease

Neprilysin 2 is a recently identified glycoprotein displaying the highest degree of sequence identity with neprilysin (EC 3.4.24.11), the prototypical member of the M13 family of zinc-dependent metalloproteases. Whereas neprilysin has been shown to be involved in the inactivation of endogenous messenger peptides, like enkephalins and tachykinins, the true physiological functions of neprilysin 2 remain unknown.

The new neurokinin 1-sensitive receptor mediates the facilitation by endogenous tachykinins of the NMDA-evoked release of acetylcholine after suppression of dopaminergic transmission in the matrix of the rat striatum.

Using an in vitro microsuperfusion procedure, the NMDA-evoked release of [3H]ACh was studied after suppression of dopamine (DA) transmission (alpha-methyl-p-tyrosine) in striatal compartments of the rat. The effects of tachykinin neurokinin 1 (NK1) receptor antagonists and the ability of appropriate agonists to counteract the antagonist responses were investigated to determine whether tachykinin NK1 classic, septide-sensitive and/or new NK1-sensitive receptors mediate these regulations. The NK1 antagonists, SR140333, SSR240600, GR205171 but not GR82334 and RP67580 (0.1 and 1 microM) markedly reduced the NMDA (1 mm + D-serine 10 microM)-evoked release of [3H]ACh only in the matrix. These responses unchanged by coapplication with NMDA of NK2 or NK3 agonists, [Lys5,MeLeu9,Nle10]NKA(4-10) or senktide, respectively, were completely counteracted by the selective NK1 agonist, [Pro9]substance P but also by neurokinin A and neuropeptide K (1 nM each). According to the rank order of potency of agonists for counteracting the antagonist responses ([Pro9]substance P, 0.013 nM > neurokinin A, 0.15 nM >> substance P(6-11) 7.7 nM = septide 8.7 nM), the new NK1-sensitive receptors mediate the facilitation by endogenous tachykinins of the NMDA-evoked release of ACh in the matrix, after suppression of DA transmission. Solely the NK1 antagonists having a high affinity for these receptors could be used as indirect anti-cholinergic agents.