A key role of neurokinin 1 receptors in acute pancreatitis and associated lung injury

Earlier studies have shown that mice deficient in NK1 receptors or its ligand, substance P, are protected against acute pancreatitis and associated lung injury. In the current study, the protective effect of NK1 receptor blockage against acute pancreatitis and associated lung injury was investigated, using a specific receptor antagonist, CP-96345. Acute pancreatitis was induced in mice by intraperitoneal (i.p.) injections of caerulein. Substance P levels in plasma, pancreas, and lungs were found to be elevated in a caerulein dose-dependent manner. Mice treated with CP-96345, either prophylactically, or therapeutically, were protected against acute pancreatitis and associated lung injury as evident by attenuation in plasma amylase, pancreatic and pulmonary myeloperoxidase activities, and histological evidence of pancreatic and pulmonary injuries. Pulmonary microvascular permeability was also reduced as a result of CP-96345 treatment. These results point to a key role of NK1 receptors in acute pancreatitis and associated lung injury.     

Preprotachykinin-A gene products are key mediators of lung injury in polymicrobial sepsis

Preprotachykinin-A (PPT-A) gene products substance P and neurokinin-A have been shown to play an important role in neurogenic inflammation. To investigate the role of PPT-A gene products in lung injury in sepsis, polymicrobial sepsis was induced by cecal ligation and puncture in PPT-A gene-deficient mice (PPT-A(-/-)) and the wild-type control mice (PPT-A(+/+)). PPT-A gene deletion significantly protected against mortality, delayed the onset of lethality, and improved the long-term survival following cecal ligation and puncture-induced sepsis. PPT-A(-/-) mice also had significantly attenuated inflammation and damage in the lungs. The data suggest that deletion of the PPT-A gene may have contributed to the disruption in recruitment of inflammatory cells resulting in protection against tissue damage, as in these mice the sepsis-associated increase in chemokine levels is significantly attenuated.     

Role of substance P in hydrogen sulfide-induced pulmonary inflammation in mice

We have shown earlier that H(2)S acts as a mediator of inflammation. In this study, we have investigated the involvement of substance P and neurogenic inflammation in H(2)S-induced lung inflammation. Intraperitoneal administration of NaHS (1-10 mg/kg), an H(2)S donor, to mice caused a significant increase in circulating levels of substance P in a dose-dependent manner. H(2)S alone could also cause lung inflammation, as evidenced by a significant increase in lung myeloperoxidase activity and histological evidence of lung injury. The maximum effect of H(2)S on substance P levels and on lung inflammation was observed 1 h after NaHS administration. At this time, a significant increase in lung levels of TNF-alpha and IL-1beta was also observed. In substance P-deficient mice, the preprotachykinin-A knockout mice, H(2)S did not cause any lung inflammation. Furthermore, pretreatment of mice with CP-96345 (2.5 mg/kg ip), an antagonist of the neurokinin-1 (NK(1)) receptor, protected mice against lung inflammation caused by H(2)S. However, treatment with antagonists of NK(2), NK(3), and CGRP receptors did not have any effect on H(2)S-induced lung inflammation. Depleting neuropeptide from sensory neurons by capsaicin (50 mg/kg sc) significantly reduced the lung inflammation caused by H(2)S. In addition, pretreatment of mice with capsazepine (15 mg/kg sc), an antagonist of the transient receptor potential vanilloid-1, protected mice against H(2)S-induced lung inflammation. These results demonstrate a key role of substance P and neurogenic inflammation in H(2)S-induced lung injury in mice.     

Hydrogen sulfide up-regulates substance P in polymicrobial sepsis-associated lung injury

Hydrogen sulfide (H2S) has been shown to induce the activation of neurogenic inflammation especially in normal airways and urinary bladder. However, whether endogenous H2S would regulate sepsis-associated lung inflammation via substance P (SP) and its receptors remains unknown. Therefore, the aim of the study was to investigate the effect of H2S on the pulmonary level of SP in cecal ligation and puncture (CLP)-induced sepsis and its relevance to lung injury. Male Swiss mice or male preprotachykinin-A gene knockout (PPT-A-/-) mice and their wild-type (PPT-A+/+) mice were subjected to CLP-induced sepsis. DL-propargylglycine (50 mg/kg i.p.), an inhibitor of H2S formation was administered either 1 h before or 1 h after the induction of sepsis, while NaHS, an H2S donor, was given at the same time as CLP. L703606, an inhibitor of the neurokinin-1 receptor was given 30 min before CLP. DL-propargylglycine pretreatment or posttreatment significantly decreased the PPT-A gene expression and the production of SP in lung whereas administration of NaHS resulted in a further rise in the pulmonary level of SP in sepsis. PPT-A gene deletion and pretreatment with L703606 prevented H2S from aggravating lung inflammation. In addition, septic mice genetically deficient in PPT-A gene or pretreated with L703606 did not exhibit further increase in lung permeability after injection of NaHS. The present findings show for the first time that in sepsis, H2S up-regulates the generation of SP, which contributes to lung inflammation and lung injury mainly via activation of the neurokinin-1 receptor.     

Substance P and neurokinin-1 receptor expression by intrinsic airway neurons in the rat

Tachykinins and their receptors are involved in the amplification of inflammation in the airways. We analyzed the expression of preprotachykinin-A (PPT-A) and neurokinin-1 (NK-1) receptor genes by intrinsic airway neurons in the rat. We also tested the hypothesis that PPT-A-encoded peptides released by these neurons fulfill the requisite role of substance P in immune complex injury of the lungs. We found that ganglion neurons in intact and denervated airways or in primary culture coexpress PPT-A and NK-1 receptor mRNAs and their protein products. Denervated ganglia from tracheal xenografts (nu/nu mice) or syngeneic lung grafts had increased PPT-A mRNA contents, suggesting preganglionic regulation. Formation of immune complexes in the airways induced comparable inflammatory injuries in syngeneic lung grafts, which lack peptidergic sensory fibers, and control lungs. The injury was attenuated in both cases by pretreatment with the NK-1 receptor antagonist LY-306740. We conclude that tachykinins released by ganglia act as a paracrine or autocrine signal in the airways and may contribute to NK-1 receptor-mediated amplification of immune injury in the lungs.     

Substance P upregulates cyclooxygenase-2 and prostaglandin E metabolite by activating ERK1/2 and NF-kappaB in a mouse model of burn-induced remote acute lung injury

Acute lung injury (ALI) is a major cause of mortality in burn patients, even without direct inhalational injury. Identification of early mediators that instigate ALI after burn and of the molecular mechanisms by which they work are of high importance but remain poorly understood. We previously reported that an endogenous neuropeptide, substance P (SP), via binding neurokinin-1 receptor (NK1R), heightens remote ALI early after severe local burn. In this study, we examined the downstream signaling pathway following SP-NK1R coupling that leads to remote ALI after burn. A 30% total body surface area full-thickness burn was induced in male BALB/c wild-type (WT) mice, preprotachykinin-A (PPT-A) gene-deficient mice, which encode for SP, and PPT-A(-/-) mice challenged with exogenous SP. Local burn injury induced excessive SP-NK1R signaling, which activated ERK1/2 and NF-κB, leading to significant upregulation of cyclooxygenase (COX)-2, PGE metabolite, and remote ALI. Notably, lung COX-2 levels were abrogated in burn-injured WT mice by L703606, PD98059, and Bay 11-7082, which are specific NK1R, MEK-1, and NF-κB antagonists, respectively. Additionally, burn-injured PPT-A(-/-) mice showed suppressed lung COX-2 levels, whereas PPT-A(-/-) mice injected with SP showed augmented COX-2 levels postburn, and administration of PD98059 and Bay 11-7082 to burn-injured PPT-A(-/-) mice injected with SP abolished the COX-2 levels. Furthermore, treatment with parecoxib, a selective COX-2 inhibitor, attenuated proinflammatory cytokines, chemokines, and ALI in burn-injured WT mice and PPT-A(-/-) mice injected with SP. To our knowledge, we show for the first time that SP-NK1R signaling markedly elevates COX-2 activity via ERK1/2 and NF-κB, leading to remote ALI after burn.     

Expression of NK-1 and NK-3 tachykinin receptors in pancreatic acinar cells after acute experimental pancreatitis in rats

Activation of neurokinin (NK)-1 receptors but not of NK-3 stimulates amylase release from isolated pancreatic acini of the rat. Immunofluorescence studies show that NK-1 receptors are more strongly expressed than NK-3 receptors on pancreatic acinar cells under basal conditions. No studies have examined the expression of the two NK receptor populations in pancreatic acini during pancreatitis in rats. We therefore investigated the relationships between expression of these two tachykinin receptors and experimental acute pancreatitis induced by stimulating pancreatic amylase with caerulein (CK) in rats. Hyperstimulation of the pancreas by CK caused an increase in plasma amylase and pancreatic water content and resulted in morphological evidence of cytoplasmic vacuolization. Immunofluorescence analysis revealed a similar percentage of NK-1 receptor antibody immunoreactive acinar cells in rats with pancreatitis and in normal rat tissue but a larger percentage of NK-3 receptor immunoreactive cells in acute pancreatitis than in normal pancreas. Western blot analysis of NK-1 and NK-3 receptor protein levels after CK-induced pancreatitis showed no change in NK-1 receptors but a stronger increase in NK-3 receptor expression in pancreatic acini compared with normal rats thus confirming the immunofluorescence data. These new findings support previous evidence that substance P-mediated functions within the pancreas go beyond sensory signal transduction contributing to neurogenic inflammation, and they suggest that substance P plays a role in regulating pancreatic exocrine secretion via acinar NK-1 receptors. The significant increase in NK-3 receptors during pancreatic stimulation suggests that NK-3 receptors also intervene in the pathogenesis of mild acute pancreatitis in rats.     

Pro-inflammatory effects of hydrogen sulphide on substance P in caerulein-induced acute pancreatitis

Hydrogen sulphide (H(2)S), a novel gasotransmitter, has been recognized to play an important role in inflammation. Cystathionine-gamma-lyase (CSE) is a major H(2)S synthesizing enzyme in the cardiovascular system and DL-propargylglycine (PAG) is an irreversible inhibitor of CSE. Substance P (SP), a product of preprotachykinin-A (PPT-A) gene, is a well-known pro-inflammatory mediator which acts principally through the neurokinin-1 receptor (NK-1R). We have shown an association between H(2)S and SP in pulmonary inflammation as well as a pro-inflammatory role of H(2)S and SP in acute pancreatitis. The present study was aimed to investigate the interplay between pro-inflammatory effects of H(2)S and SP in a murine model of caerulein-induced acute pancreatitis. Acute pancreatitis was induced in mice by 10 hourly intraperitoneal injections of caerulein (50 (g/kg). PAG (100 mg/kg, i.p.) was administered either 1 hr before (prophylactic) or 1 hr after (therapeutic) the first caerulein injection. PAG, given prophylactically as well as therapeutically, significantly reduced plasma H(2)S levels and pancreatic H(2)S synthesizing activities as well as SP concentrations in plasma, pancreas and lung compared with caerulein-induced acute pancreatitis. Furthermore, prophylactic as well as therapeutic administration of PAG significantly reduced PPT-A mRNA expression and NK-1R mRNA expression in both pancreas and lung when compared with caerulein-induced acute pancreatitis. These results suggest that the pro-inflammatory effects of H(2)S may be mediated by SP-NK-1R pathway in acute pancreatitis.     

Treatment with antileukinate, a CXCR2 chemokine receptor antagonist, protects mice against acute pancreatitis and associated lung injury

Acute pancreatitis is a common, and as yet incurable, clinical condition, the incidence of which has been increasing over recent years. Chemokines are believed to play a key role in the pathogenesis of acute pancreatitis. We have earlier shown that treatment with a neutralizing antibody against CINC, a CXC chemokine, protects rats against acute pancreatitis-associated lung injury. The hexapeptide antileukinate (Ac-RRWWCR-NH2) is a potent inhibitor of binding of CXC chemokines to the receptors (CXCR2). This study aims to evaluate the effect of treatment with antileukinate on acute pancreatitis and the associated lung injury in mice. Acute pancreatitis was induced in adult male Swiss mice by hourly intra-peritoneal injections of caerulein (50 microg/kg/h) for 10 h. Antileukinate (52.63 mg/kg, s.c.) was administered to mice either 30 min before or 1 h after starting caerulein injections. Severity of acute pancreatitis was determined by measuring plasma amylase, pancreatic water content, pancreatic myeloperoxidase (MPO) activity, pancreatic macrophage inflammatory protein-2 (MIP-2) levels and histological examination of sections of pancreas. A rise in lung MPO activity and histological evidence of lung injury in lung sections was used as criteria for pancreatitis-associated lung injury. Treatment with antileukinate protected mice against acute pancreatitis and associated lung injury, showing thereby that anti-chemokine therapy may be of value in this condition.     

Hydrogen sulfide acts as a mediator of inflammation in acute pancreatitis: in vitro studies using isolated mouse pancreatic acinar cells

Hydrogen sulphide (H(2)S) is synthesized from L-cysteine via the action of cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS). We have earlier shown that H(2)S acts as a mediator of inflammation. However the mechanism remains unclear. In this study, we investigated the presence of H(2)S and the expression of H(2)S synthesizing enzymes, CSE and CBS, in isolated mouse pancreatic acini. Pancreatic acinar cells from mice were incubated with or without caerulein (10(-7) M for 30 and 60 min). Caerulein increased the levels of H(2)S and CSE mRNA expression while CBS mRNA expression was decreased. In addition, cells pre-treated with DL-propargylglycine (PAG, 3 mM), a CSE inhibitor, reduced the formation of H(2)S in caerulein treated cells, suggesting that CSE may be the main enzyme involved in H(2)S formation in mouse acinar cells. Furthermore, substance P (SP) concentration in the acini and expression of SP gene (preprotachykinin-A, PPT-A) and neurokinin-1 receptor (NK-1R), the primary receptor for SP, are increased in secretagogue caerulein-treated acinar cells. Inhibition of endogenous production of H(2)S by PAG significantly suppressed SP concentration, PPT-A expression and NK1-R expression in the acini. To determine whether H(2)S itself provoked inflammation in acinar cells, the cells were treated with H(2)S donor drug, sodium hydrosulphide (NaHS), (10, 50 and 100 muM), that resulted in a significant increase in SP concentration and expression of PPT-A and NK1-R in acinar cells. These results suggest that the pro-inflammatory effect of H(2)S may be mediated by SP-NK-1R related pathway in mouse pancreatic acinar cells.     

Prophylactic and therapeutic treatments with AT 1 and AT 2 receptor antagonists and their effects on changes in the severity of pancreatitis

Previous studies showed that a local pancreatic renin-angiotensin system (RAS) was upregulated in experimental acute pancreatitis. RAS inhibition could attenuate pancreatic inflammation and fibrosis, which casts a new light on the role of the pancreatic RAS in pancreatitis. The present study explores the prophylactic and therapeutic potentials, and possible molecular mechanism for the antagonism of angiotensin II receptors on the changes in the severity of pancreatic injury induced by acute pancreatitis. Experimental pancreatitis was induced by an intraperitoneal injection of supra-maximal dose of cerulein. The differential effects of angiotensin II receptors inhibitors losartan and PD123319 on the pancreatic injury were assessed by virtue of using the pancreatic water content, biochemical and histological analyses. Blockade of the AT(1) receptor by losartan at a dose of 200microg/kg could markedly ameliorate the pancreatic injury induced by cerulein, as evidenced by biochemical and histopathological studies. However, blockade of the AT(2) receptor by PD123319 appeared not to provide any beneficial role in cerulein-induced pancreatic injury. Both prophylactic and therapeutic treatments with losartan were effective against cerulein-induced pancreatic injury. The protective action of losartan was linked to an inhibition of NAD(P)H oxidase activity, thus consequential oxidative modification of pancreatic proteins in the pancreas. Inhibition of the AT(1) receptor, but not AT(2) receptor, may play a beneficial role in ameliorating the severity of acute pancreatitis. The differential effects of AT(1) and AT(2) inhibitors on cerulein-induced pancreatic injury might be due to the distinctive mechanism of the AT(1) and AT(2) receptors on the activation of NAD(P)H oxidase. Thus the protective role of AT(1) receptor antagonist, losartan, could be mediated by the inhibition of NAD(P)H oxidase-dependent generation of reactive oxygen species (ROS).     

Substance P acts via the neurokinin receptor 1 to elicit bronchoconstriction, oxidative stress, and upregulated ICAM-1 expression after oil smoke exposure

This study aimed to 1) assess whether substance P (SP) acts via neurokinin (NK)-1 and NK-2 receptors to stimulate neurogenic inflammation (indicated by formation of ICAM-1 expression and oxidative stress) following oil smoke exposure (OSE) in rats; and 2) determine if pretreatment with antioxidants ameliorates the deleterious effects of OSE. Rats were pretreated with NK-1 receptor antagonist CP-96345, NK-2 receptor antagonist SR-48968, vitamin C, or catechins. OSE was for 30-120 min. Rats were killed 0-8 h later. Total lung resistance (RL), airway smooth muscle activity (ASMA), lung ICAM-1 expression, neurogenic plasma extravasation (via India ink and Evans blue dye), bronchoalveolar lavage fluid SP concentrations, and reactive oxygen species formation [via lucigenin- and luminal-amplified chemiluminescence (CL)] were assessed. Lung histology was performed. SP concentrations increased significantly in nonpretreated rats following OSE in a dose-dependent manner. RL and total ASMA increased over time after OSE. Vitamin C and catechin pretreatments were associated with significantly reduced lucigenin CL 2 and 4 h after OSE. Pretreatment with catechins significantly reduced luminal CL counts 4 and 8 h after OSE. Evans blue levels were significantly reduced following 60 and 120 min of OSE in catechin- and CP-96345-pretreated rats. ICAM-1 protein expression was significantly decreased in all pretreatment groups after OSE. Thickening of the alveolar capillary membrane, focal hemorrhaging, interstitial pneumonitis, and peribronchiolar inflammation were apparent in OSE lungs. These findings suggest that SP acts via the NK-1 receptor to provoke neurogenic inflammation, oxidative stress, and ICAM-1 expression after OSE in rats.     

Characterisation of substance P-induced endocytosis of NK1 receptors on enteric neurons

Immunoreactivity for NK1 receptors is confined to specific nerve cell bodies in the guinea-pig ileum, including inhibitory motor neurons and secretomotor neurons. In the present work, endocytosis of NK1 receptors in these enteric neurons was studied following addition of substance P (SP) to isolated ileum. NK1 receptors were localised with antibodies against the C-terminus of this receptor. Some preparations were incubated with SP tagged with the fluorescent label, Cy3.18, so that the fate of SP bound to receptors could be followed. Preparations were analysed by confocal microcopy. In tissue that was incubated at 4° C in the absence of SP, most NK1 receptor immunoreactivity (IR) was confined to surface membranes of nerve cells. At 37° C in the presence of 10⁻⁷ M SP (plus 3×10⁻⁷M tetrodotoxin to prevent indirect activation via other neurons) the neuronal NK1 receptor was rapidly internalised. After 5 min, NK1 receptor IR was partially internalised, at 20 min NK1 receptor IR was throughout the cytoplasm and in perinuclear aggregates and at 30 min it was again at the cell surface. SP-induced NK1 receptor endocytosis was inhibited by the specific NK1 receptor antagonist, SR140333. Cy3-SP was colocalised with NK1 receptor IR and was internalised with the NK1 receptor. These results show that enteric neurons exhibit authentic NK1 receptors that are rapidly internalised when exposed to their preferred ligand.     

The neuropeptide substance P is a critical mediator of burn-induced acute lung injury

The classical tachykinin substance P (SP) has numerous potent neuroimmunomodulatory effects on all kinds of airway functions. Belonging to a class of neuromediators targeting not only residential cells but also inflammatory cells, studying SP provides important information on the bidirectional linkage between how neural function affects inflammatory events and, in turn, how inflammatory responses alter neural activity. Therefore, this study aimed to investigate the effect of local burn injury on inducing distant organ pulmonary SP release and its relevance to lung injury. Our results show that burn injury in male BALB/c mice subjected to 30% total body surface area full thickness burn augments significant production of SP, preprotachykinin-A gene expression, which encodes for SP, and biological activity of SP-neurokinin-1 receptor (NK1R) signaling. Furthermore, the enhanced SP-NK1R response correlates with exacerbated lung damage after burn as evidenced by increased microvascular permeability, edema, and neutrophil accumulation. The development of heightened inflammation and lung damage was observed along with increased proinflammatory IL-1beta, TNF-alpha, and IL-6 mRNA and protein production after injury in lung. Chemokines MIP-2 and MIP-1alpha were markedly increased, suggesting the active role of SP-induced chemoattractants production in trafficking inflammatory cells. More importantly, administration of L703606, a specific NK1R antagonist, 1 h before burn injury significantly disrupted the SP-NK1R signaling and reversed pulmonary inflammation and injury. The present findings show for the first time the role of SP in contributing to exaggerated pulmonary inflammatory damage after burn injury via activation of NK1R signaling.     

Substance P in the nucleus of the solitary tract augments bronchopulmonary C fiber reflex output

Bronchopulmonary C fibers defend the lungs against injury from inhaled agents by a central nervous system reflex consisting of apnea, cough, bronchoconstriction, hypotension, and bradycardia. Glutamate is the putative neurotransmitter at the first central synapses in the nucleus of the solitary tract (NTS), but substance P, also released in the NTS, may modulate the transmission. To test the hypothesis that substance P in the NTS augments bronchopulmonary C fiber input and hence reflex output, we stimulated the C fibers with left atrial capsaicin (LA CAP) injections and compared the changes in phrenic nerve discharge, tracheal pressure (TP), arterial blood pressure (ABP), and heart rate (HR) in guinea pigs before and after substance P injections (200 microM, 25 nl) in the NTS. Substance P significantly augmented LA CAP-evoked increases in expiratory time by 10-fold and increases in TP and decreases in ABP and HR by threefold, effects prevented by neurokinin-1 (NK1) receptor antagonism. Thus substance P acting at NTS NK1 receptors can exaggerate bronchopulmonary C fiber reflex output. Because substance P synthesis in vagal airway C fibers may be enhanced in pathological conditions such as allergic asthma, the findings may help explain some of the associated respiratory symptoms including cough and bronchoconstriction.     

Natural killer cells are involved in acute lung immune injury caused by respiratory syncytial virus infection

It is known that respiratory syncytial virus (RSV) is the main cause of bronchiolitis and pneumonia in young children. RSV infection often leads to severe acute lung immunopathology, but the underlying immune mechanisms are not yet fully elucidated. Here, we found that RSV infection induced severe acute lung immune injury and promoted the accumulation and activation of lung natural killer (NK) cells at the early stage of infection in BALB/c mice. Activated lung NK cells highly expressed activating receptors NKG2D and CD27 and became functional NK cells by producing a large amount of gamma interferon (IFN-γ), which was responsible for acute lung immune injury. NK cell depletion significantly attenuated lung immune injury and reduced infiltration of total inflammatory cells and production of IFN-γ in bronchoalveolar lavage fluid (BALF). These data show that NK cells are involved in exacerbating the lung immune injury at the early stage of RSV infection via IFN-γ secretion.     

Complement factor C5a exerts an anti-inflammatory effect in acute pancreatitis and associated lung injury

Complement factor C5a acting via C5a receptors (C5aR) is recognized as an anaphylotoxin and chemoattractant that exerts proinflammatory effects in many pathological states. The effects of C5a and C5aR in acute pancreatitis and in pancreatitis-associated lung injury were evaluated using genetically altered mice that either lack C5aR or do not express C5. Pancreatitis was induced by administration of 12 hourly injections of cerulein (50 microg/kg ip). The severity of pancreatitis was determined by measuring serum amylase, neutrophil sequestration in the pancreas, and acinar cell necrosis. The severity of lung injury was evaluated by measuring neutrophil sequestration in the lung and pulmonary microvascular permeability. In both strains of genetically altered mice, the severity of pancreatitis and pancreatitis-associated lung injury was greater than that noted in the comparison wild-type strains of C5aR- and C5-sufficient animals. This exacerbation of injury in the absence of C5a function indicates that, in pancreatitis, C5a exerts an anti-inflammatory effect. Potentially, C5a and its receptor are capable of both promoting and reducing the extent of acute inflammation.     

Functional expression of neurokinin 1 receptors on mast cells induced by IL-4 and stem cell factor

It is widely accepted that neurokinin 1 (NK(1)) receptors are not generally expressed on mast cells but little is known about their expression in inflammation. The present study shows expression of NK(1) receptors on bone marrow-derived mast cells (BMMC) under the influence of IL-4 or stem cell factor (SCF). Highest expression was found when both cytokines are present. Six days of coculture with the cytokines IL-4 and SCF showed significant expression of NK(1) receptors (NK(1) receptor(+)/c-kit(+) BMMC; control: 7%, IL-4/SCF: 16%), while 12 days of cytokine coculture increased this expression to 37% positive cells. A longer coculture with IL-4 and SCF did not give an additional effect. Increased expression in IL-4/SCF-treated BMMC was further confirmed using Western blot analysis. Next, we demonstrated the functional relevance of NK(1) receptor expression for mast cell activation, resulting in an enhanced degranulation upon stimulation by substance P. BMMC activation was significantly diminished by the NK(1) receptor antagonist RP67580 (10 micro M) when stimulated with low concentrations of substance P. The inactive enantiomer RP65681 had no effect. In addition, BMMC cultured from bone marrow of NK(1) receptor knockout mice showed significantly decreased exocytosis to low concentrations of substance P. The present study clearly shows that NK(1) receptor-induced activation contributes significantly at low physiological substance P concentrations (<100 micro M). In conclusion, BMMC were shown to express NK(1) receptors upon IL-4/SCF coculture. This expression of NK(1) receptors has been demonstrated to be of functional relevance and leads to an increase in the sensitivity of BMMC to substance P.