Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells

Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25–30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10⁻⁶M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R.     

Effects of substance P on osteoblastic differentiation and heme oxygenase-1 in human periodontal ligament cells

Although substance P (SP) is associated with osteoclast differentiation and bone resorption, little is known about the osteogenic differentiation-inducing effects of SP in periodontal ligament (PDL) cells. This study investigated whether PDL cells could differentiate into osteoblastic-like cells by SP. The expression of osteoblastic differentiation markers such as osteopontin (OPN), osteonectin (ON), osteocalcin (OCN) and bone sialoprotein (BSP) were evaulated by Western blotting. Additionally, SP-mediated heme oxygenase-1 (HO-1) pathways were further clarified.SP increased HO-1 and osteogenic differentiation in concentration- and time-dependent manners, as determined by OPN, ON, OCN and BSP expression. Furthermore, treatment with inhibitors of p38, ERK MAPK, and NF-κB abolished SP-induced osteogenic differentiation and HO-1 expression. SP-induced translocation of Nrf-2 was also observed. The combined results suggest that SP activates the stress-response enzymes HO-1 and Nrf-2, subsequently leading to upregulation of osteogenic differentiation in human PDL cells.     

Functional and immunological responses of Jurkat lymphocytes transfected with the substance P receptor

1. We have transfected the rat substance P receptor (SPR) cDNA into the leukemic T-lymphocyte cell line Jurkat (J-wt) in order to study the effects of substance P (SP) on lymphocyte signaling mechanisms and the resultant neuropeptide-induced immunological changes. 2. The SPR cDNA was transfected into J-wt by the method of electroporation. Clones expressing SPRs were selected using a functional assay that measured SP-induced mobilization of intracellular Ca2+ ([Ca2+]i) in a fluorescence activated cell sorter (FACS) and by their expression of specific 125I-SP binding. 3. One clone, J-SPR, was identified and shown by Northern blot and 125I-SP saturation binding techniques to express the 2.2-kb SPR message and approximately 50,000 SPRs/cell with a Kd of 0.3 nM, respectively. Stimulation of J-SPR by SP resulted in the rapid mobilization of [Ca2+]i. This response was dose dependent in the range 10(-11)-10(-6) M SP and was maximal at 10(-7) M SP, with an EC50 of 0.3-0.5 nM SP. We further demonstrated that the SPR is rapidly desensitized following SP stimulation and by activation of the cell's T-cell receptor (TCR). Whole-cell patch-clamp experiments on J-SPR show that SP stimulation induces a Cl- current by a Ca2+ mediated process dependent on Ca2+/calmodulin-dependent protein kinase (CaMK). 4. Stimulation of J-SPR by SP results in changes in the cell surface expression of a number of molecules that play important roles in cell adhesion and activation: the expression of LFA-1 is decreased, and CD2 and IL-2 receptors are increased by 30 min, 6 hr, and 24 hr, respectively, following stimulation, as assessed by antibody staining in a FACS. 5. The expression of functional SPRs in Jurkat lymphocytes will not permit a detailed examination of how the activation of SPRs result in altered immune responses and further elucidate the role this neuropeptide receptor plays in inflammation.     

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.     

Correlation of substance P-induced desensitization with substance P amino terminal metabolites in the mouse spinal cord.

Intrathecal injection of mice with substance P (SP) or its C-terminal fragments results in a behavioral syndrome characterized by reciprocal caudally directed biting and scratching. Repeated injection of SP, but not SP C-terminal fragments, results in a decrease in the intensity of, or desensitization to, these SP-induced behaviors. Peptidase inhibitors, phosphoramidon (PH), bacitracin (BAC), diprotin A (DPA) and angiotensin converting enzyme inhibitor (ACEI OR SQ20881), together with [3H]SP, were used to investigate the possible accumulation of tritiated N-terminal metabolites in the mouse spinal cord in vivo during the development of desensitization to SP. SP N-terminal metabolites in the spinal cord were quantified by reverse-phase HPLC. The magnitude of SP-induced desensitization correlated well (r = .95) with total SP N-terminal metabolites recovered from the spinal cords of the same mice studied in vivo. The magnitude of SP-induced desensitization was also found to be negatively correlated (r = .95) with total recovered intact [3H]SP. The rank order of potency of the peptidase inhibitors in decreasing the magnitude of SP-induced desensitization was BAC = PH much greater than ACEI greater than DPA. The order of potency for in vitro inhibition of SP metabolism using synaptic membrane-derived peptidases was BAC greater than PH much greater than ACEI. These results support the hypothesis that desensitization to SP-induced behaviors depends, at least in part, on the concentration of SP N-terminal metabolites in the spinal cord.     

The mood-stabilizing agent valproate inhibits the activity of glycogen synthase kinase-3

Valproic acid (VPA) is a potent broad-spectrum anti-epileptic with demonstrated efficacy in the treatment of bipolar affective disorder. It has previously been demonstrated that both VPA and lithium increase activator protein-1 (AP-1) DNA binding activity, but the mechanisms underlying these effects have not been elucidated. However, it is known that phosphorylation of c-jun by glycogen synthase kinase (GSK)-3beta inhibits AP-1 DNA binding activity, and lithium has recently been demonstrated to inhibit GSK-3beta. These results suggest that lithium may increase AP-1 DNA binding activity by inhibiting GSK-3beta. In the present study, we sought to determine if VPA, like lithium, regulates GSK-3. We have found that VPA concentration-dependently inhibits both GSK-3alpha and -3beta, with significant effects observed at concentrations of VPA similar to those attained clinically. Incubation of intact human neuroblastoma SH-SY5Y cells with VPA results in an increase in the subsequent in vitro recombinant GSK-3beta-mediated 32P incorporation into two putative GSK-3 substrates (approximately 85 and 200 kDa), compatible with inhibition of endogenous GSK-3beta by VPA. Consistent with GSK-3beta inhibition, incubation of SH-SY5Y cells with VPA results in a significant time-dependent increase in both cytosolic and nuclear beta-catenin levels. GSK-3beta plays a critical role in the CNS by regulating various cytoskeletal processes as well as long-term nuclear events and is a common target for both lithium and VPA; inhibition of GSK-3beta in the CNS may thus underlie some of the long-term therapeutic effects of mood-stabilizing agents.     

Cardioprotection by ischemic postconditioning is lost in isolated perfused heart from diabetic rats: Involvement of transient receptor potential vanilloid 1, calcitonin gene-related peptide and substance P

We previously found that the expression of transient receptor potential vanilloid 1 (TRPV1) and contents of calcitonin gene-related peptide (CGRP) and substance P (SP), two main neuropeptides released from TRPV1, were decreased in diabetic hearts. This study aimed to test whether decreased TRPV1, CGRP and SP levels were responsible for the loss of cardioprotection by ischemic postconditioning (IPostC) in isolated perfused heart from streptozotocin-induced diabetic rats. IPostC effectively protected non-diabetic hearts against ischemia/reperfusion injury by improving cardiac function and lowering creatine kinase (CK) and cardiac troponin I (cTnI) release, which could be abolished by inhibiting TRPV1, CGRP receptor or SP receptor. However, IPostC had no effect on cardiac function and the release of CK and cTnI in diabetic hearts regardless of whether TRPV1, CGRP receptor or SP receptor were inhibited. CGRP or SP-induced postconditioning significantly prevented both non-diabetic and diabetic hearts from ischemia/reperfusion injury by improving cardiac function and lowering CK and cTnI release. Additionally, IPostC markedly increased CGRP and SP release in non-diabetic hearts, which could be reversed with TRPV1 inhibition, but not CGRP receptor or SP receptor inhibition. However, IPostC failed to affect CGRP and SP release in diabetic hearts in the presence or absence of TRPV1, CGRP receptor or SP receptor inhibition. These results indicate that the loss of cardioprotection by IPostC during diabetes is partly associated with a failure to increase CGRP and SP release, likely due to decreased TRPV1 expression and CGRP and SP contents in diabetic hearts.     

Effect of regulatory polypeptides on the substance P stimulated lower esophageal sphincter pressure in pigs

The effect of graded doses of vasoactive intestinal polypeptide (VIP), enkephalin, neuropeptide Y (NPY), gastrin-17, pentagastrin, cholecystokinin (CCK)-4, CCK-8, neurotensin, somatostatin, and thyrotropin-releasing hormone (TRH) on the substance P (SP)-stimulated lower esophageal sphincter pressure (LESP) in anaesthetized pigs was studied by direct infusion of the peptides into the arterial supply of the lower esophageal sphincter (LES). Infusion of SP in a dose of 20 pmol/kg per min for 3 min significantly increased the LESP (P less than 0.01). Simultaneous VIP infusion at 5--40 pmol/kg per min showed a dose-dependent inhibition of the effect of SP on the LESP. None of the other peptides had any effect on the LESP during simultaneous infusion of SP. Pharmacological blockade by atropine (250 mu/kg) or guanethidine (1 mg/kg) had no effect on the SP-stimulated LESP. In conclusion, the SP-induced stimulation of the LESP is abolished by VIP, and both peptides seem to play a role in the complex regulation of the LESP.     

Regulation of apoptosis by somatostatin and substance P in peritoneal macrophages

Recent studies have shown that somatostatin (SOM) inhibits interleukin 6 (IL-6) and interferon gamma (IFNgamma) production by lymphocytes and peritoneal macrophages, whereas substance P (SP) enhances these cytokines production. To define the mechanism of the cytokine production enhancements and inhibitions by SOM and SP, we examined the expression of apoptosis modulator, p53, Bcl-2, Bax, inducible nitric oxide synthase (iNOS), Fas, caspase-8 and nitric oxide (NO) in thioglycolate-elicited peritoneal macrophages. SOM caused up-regulation of p53, Bcl-2, Fas and caspase-8 activities, and down-regulation of iNOS expression and NO production. On the other hand, SP slightly induces p53 and highly induces Bcl-2, iNOS expression and NO production. These data suggest that apoptosis by SOM may occur by a Bax- and NO-independent p53 accumulation, and through Fas and caspase-8 activation pathways, and that the inducible expression of Bcl-2 and NO production by SP may contribute to prevent the signals of apoptosis by Bax, and via Fas and caspase-8 activation.     

Modulation of peripheral inflammation by the substance P N-terminal metabolite substance P1-7

The N-terminal metabolite of the undecapeptide substance P (SP), substance P1-7 (SP1-7), is known to modulate nociception in the central nervous system (CNS) and often has opposite effects from SP. This study investigated the ability of SP(1-7) to modulate the vasodilatation response to SP in anaesthetized rats under different injury conditions using a blister model of inflammation on the hind footpad. The results indicated that SP1-7 inhibited the vascular response to SP in a dose-dependent manner. The putative antagonists naloxone and D-Pro2-D-Phe7-SP1-7 (D-SP1-7) reversed the effect of SP1-7. D-SP1-7 improved the responsiveness to SP under chronic nerve injury, which suggests a role for endogenous SP1-7 in this model. SP1-7 did not inhibit the response to electrical stimulation of the sciatic nerve, which indicates that the heptapeptide interacts at a post-terminal binding site. The current results suggest that SP1-7 may have inhibitory properties in inflammation, analogous to its antinociceptive role in the central nervous system.     

Effect of the NEP inhibitor SCH32615 on airway responses to intravenous substance P in guinea pigs.

We examined the effects of the selective neutral endopeptidase (NEP) inhibitor SCH32615 on airway responses to rapid intravenous infusions of substance P (SP) and neurokinin A (NKA) and on recovery of administered tachykinins from arterial blood in anesthetized mechanically ventilated guinea pigs. SCH32615, in doses that cause a marked increase in the magnitude of bronchoconstriction induced by infused NKA, had little effect on the changes in pulmonary conductance (GL) or dynamic compliance induced by SP. In animals in which SCH32615 (1 mg/kg) was administered in combination with the angiotensin-converting enzyme (ACE) inhibitor captopril (5.7 mg/kg), the dose of SP required to decrease GL by 50% was fourfold less than in animals that received captopril alone (P < 0.005). SP measured in arterial blood withdrawn within 45 s of intravenous administration of this tachykinin was not different in control and SCH32615-treated animals, whereas captopril caused an approximately threefold increase in SP concentrations (P < 0.005). When SCH32615 and captopril were administered together, significantly more SP was recovered than when captopril or SCH32615 was administered alone (P < 0.0005). Our results are consistent with the hypothesis that both NEP and ACE contribute to the degradation of intravenously infused SP. ACE degradation of SP is sufficient to limit SP-induced bronchoconstriction even in the presence of specific NEP inhibition.     

Diverse effects of intrathecal pituitary adenylate cyclase-activating polypeptide on nociceptive transmission in mice spinal cord

Pituitary adenylate cyclase-activating polypeptide (PACAP) immunoreactive neural elements have been detected in the mouse spinal cord. The discrepancy of PACAP actions in the role of sensory transmission has been proposed to have potentiation and inhibition on nociceptive responses after intrathecal application of PACAP. The aim of the present study was to assess nociceptive transmission of PACAP in the mouse spinal cord by comparison with that of substance P (SP). The intrathecal injection of PACAP induced licking or scratching behavior similar to that of SP. These PACAP-induced aversive behaviors showed different manner from SP-induced responses in point of time course. SP-induced aversive responses quickly increased and suddenly disappeared almost within 1 min. Meanwhile, following a long latency after the injection, PACAP-induced aversive responses gradually appeared, and then persisted more than 60 min. In the early phase, PACAP produced an increase of tail flick latency. Pretreatment with 6-hydroxydopamine (6-OHDA) which destroys noradrenaline neuron of descending pain inhibitory systems in the spinal cord markedly abridged the latency and augmented the duration of PACAP-induced aversive responses. In this way, PACAP exhibits diverse effects on nociception, such as an analgesic role in early phase of the injection and subsequently lasting algesia. These results suggest that PACAP as a neurotransmitter or neuromodulator might have crucial role in nociceptive transmission system.     

Effect of galanin on substance P- and vasoactive intestinal polypeptide-induced nociceptive trigemino-hypoglossal reflex in rats.

Substance P (SP), vasoactive intestinal polypeptide (VIP) and galanin (GAL), present in primary sensory neurons, are involved in transmission of nociceptive signaling from the peripheral to central nervous system. In this study we investigated the effect of GAL on SP-induced or VIP-induced evoked tongue jerks (ETJ) in response to noxious tooth pulp stimulation during perfusion of the cerebral ventricles with SP or VIP solutions. The experiments were carried out on rats under chloralose anesthesia. It was shown that both, SP and VIP, perfused through the cerebral ventricles enhanced the ETJ amplitude as compared with control, but the effect produced by SP was stronger. The intracerebroventricular perfusion of GAL 5 minutes before SP caused a dose-dependent inhibition of SP-induced ETJ, whereas GAL perfused through the cerebral ventricles 5 minutes before VIP did not reduce the excitatory effect of VIP on ETJ. These results indicate that the antinociceptive effect of GAL perfused through the cerebral ventricles, tested on the trigemino-hypoglossal reflex in rats, is specifically mediated by the SP-ergic system.     

Involvement of substance P and the NK-1 receptor in cancer progression

Many data suggest the deep involvement of the substance P (SP)/neurokinin (NK)-1 receptor system in cancer: (1) Tumor cells express SP, NK-1 receptors and mRNA for the tachykinin NK-1 receptor; (2) Several isoforms of the NK-1 receptor are expressed in tumor cells; (3) the NK-1 receptor is involved in the viability of tumor cells; (4) NK-1 receptors are overexpressed in tumor cells in comparison with normal ones and malignant tissues express more NK-1 receptors than benign tissues; (5) Tumor cells expressing the most malignant phenotypes show an increased percentage of NK-1 receptor expression; (6) The expression of preprotachykinin A is increased in tumor cells in comparison with the levels found in normal cells; (7) SP induces the proliferation and migration of tumor cells and stimulates angiogenesis by increasing the proliferation of endothelial cells; (8) NK-1 receptor antagonists elicit the inhibition of tumor cell growth; (9) The specific antitumor action of NK-1 receptor antagonists on tumor cells occurs through the NK-1 receptor; (10) Tumor cell death is due to apoptosis; (11) NK-1 receptor antagonists inhibit the migration of tumor cells and neoangiogenesis. The NK-1 receptor is a therapeutic target in cancer and NK-1 receptor antagonists could be considered as broad-spectrum antitumor drugs for the treatment of cancer. It seems that a common mechanism for cancer cell proliferation mediated by SP and the NK-1 receptor is triggered, as well as a common mechanism exerted by NK-1 receptor antagonists on tumor cells, i.e. apoptosis.     

Reduction induced by substance P of the initial accumulation of myo(3H)inositol in acinar cells of the parotid gland in rats

The stimulation of rat parotid acinar cells by substance P(SP) resulted in a significant reduction in the initial accumulation of cytosolic myo[3H]inositol and in the initial labelling of phosphoinositides. The SP-induced reduction was concentration-dependent, the EC50 of SP was 5.8 +/- 2.5 nM. Spantide, [D-Arg1, D-Trp7,9, Leu11]SP, a SP antagonist, used at a concentration of 10(-5) M, competitively shifted the dose-response curve of SP. The pharmacological analysis of the effects of several tachykinins and analogues, suggests the implication of NK1 receptors (specific receptor of SP).