Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury

Substance P, acting via the neurokinin 1 receptor (NK1R), plays an important role in mediating a variety of inflammatory processes. However, its role in acute pancreatitis has not been previously described. We have found that, in normal mice, substance P levels in the pancreas and pancreatic acinar cell expression of NK1R are both increased during secretagogue-induced experimental pancreatitis. To evaluate the role of substance P, pancreatitis was induced in mice that genetically lack NK1R by administration of 12 hourly injections of a supramaximally stimulating dose of the secretagogue caerulein. During pancreatitis, the magnitude of hyperamylasemia, hyperlipasemia, neutrophil sequestration in the pancreas, and pancreatic acinar cell necrosis were significantly reduced in NK1R−/− mice when compared with wild-type NK1R+/+ animals. Similarly, pancreatitis-associated lung injury, as characterized by intrapulmonary sequestration of neutrophils and increased pulmonary microvascular permeability, was reduced in NK1R−/− animals. These effects of NK1R deletion indicate that substance P, acting via NK1R, plays an important proinflammatory role in regulating the severity of acute pancreatitis and pancreatitis-associated lung injury.

A substance P (neurokinin-1) receptor mutant carboxyl-terminally truncated to resemble a naturally occurring receptor isoform displays enhanced responsiveness and resistance to desensitization

Two isoforms of the substance P (SP) receptor, differing in the length of the cytoplasmic carboxyl-terminus by ≈8 kDa, have been detected previously in rat salivary glands and other tissues. The binding and functional properties of these two isoforms have been investigated using full-length (407 amino acids) and carboxyl-terminally truncated (324 amino acids) rat SP receptors transfected stably into Chinese hamster ovary cells. Both the full-length and the truncated receptor bound radiolabeled SP with a similar Kd (≈0.1 nM). The average number of high affinity SP binding sites per cell was 1.0 × 105 and 0.3 × 105 for the full-length and the truncated SP receptor, respectively. In both cell lines, SP induced a rapid but transient increase in cytosolic calcium concentration ([Ca2+]i), which consisted of the release of Ca2+ from intracellular stores and the influx of extracellular Ca2+. Both components are dependent on phospholipase C activation. Although the full-length and the truncated receptor utilize the same calcium pathways, they differ in their EC50 values (0.28 nM for the full-length; 0.07 nM for the truncated). These differences in responsiveness may be related to the observed differences in receptor desensitization. The truncated receptor, in contrast to the full-length receptor, does not undergo rapid and long-lasting desensitization. Cells possessing the short isoform of the SP receptor would thus be expected to exhibit a prolonged responsiveness.

Preferential exclusion of sucrose from recombinant interleukin-1 receptor antagonist: Role in restricted conformational mobility and compaction of native state

Understanding the mechanism for sucrose-induced protein stabilization is important in many diverse fields, ranging from biochemistry and environmental physiology to pharmaceutical science. Timasheff and Lee [Lee, J. C. & Timasheff, S. N. (1981) J. Biol. Chem. 256, 7193–7201] have established that thermodynamic stabilization of proteins by sucrose is due to preferential exclusion of the sugar from the protein’s surface, which increases protein chemical potential. The current study measures the preferential exclusion of 1 M sucrose from a protein drug, recombinant interleukin 1 receptor antagonist (rhIL-1ra). It is proposed that the degree of preferential exclusion and increase in chemical potential are directly proportional to the protein surface area and that, hence, the system will favor the protein state with the smallest surface area. This mechanism explains the observed sucrose-induced restriction of rhIL-1ra conformational fluctuations, which were studied by hydrogen–deuterium exchange and cysteine reactivity measurements. Furthermore, infrared spectroscopy of rhlL-1ra suggested that a more ordered native conformation is induced by sucrose. Electron paramagnetic resonance spectroscopy demonstrated that in the presence of sucrose, spin-labeled cysteine 116 becomes more buried in the protein’s interior and that the hydrodynamic diameter of the protein is reduced. The preferential exclusion of sucrose from the protein and the resulting shift in the equilibrium between protein states toward the most compact conformation account for sucrose-induced effects on rhIL-1ra.

HOP-1, a Caenorhabditis elegans presenilin, appears to be functionally redundant with SEL-12 presenilin and to facilitate LIN-12 and GLP-1 signaling

Mutant presenilins have been found to cause Alzheimer disease. Here, we describe the identification and characterization of HOP-1, a Caenorhabditis elegans presenilin that displays much more lower sequence identity with human presenilins than does the other C. elegans presenilin, SEL-12. Despite considerable divergence, HOP-1 appears to be a bona fide presenilin, because HOP-1 can rescue the egg-laying defect caused by mutations in sel-12 when hop-1 is expressed under the control of sel-12 regulatory sequences. HOP-1 also has the essential topological characteristics of the other presenilins. Reducing hop-1 activity in a sel-12 mutant background causes synthetic lethality and terminal phenotypes associated with reducing the function of the C. elegans lin-12 and glp-1 genes. These observations suggest that hop-1 is functionally redundant with sel-12 and underscore the intimate connection between presenilin activity and LIN-12/Notch activity inferred from genetic studies in C. elegans and mammals.

Identification and analysis of the plant peroxisomal targeting signal 1 receptor NtPEX5

Protein translocation into peroxisomes takes place via recognition of a peroxisomal targeting signal present at either the extreme C termini (PTS1) or N termini (PTS2) of matrix proteins. In mammals and yeast, the peroxisomal targeting signal receptor, Pex5p, recognizes the PTS1 consisting of -SKL or variants thereof. Although many plant peroxisomal matrix proteins are transported through the PTS1 pathway, little is known about the PTS1 receptor or any other peroxisome assembly protein from plants. We cloned tobacco (Nicotiana tabacum) cDNAs encoding Pex5p (NtPEX5) based on the protein’s interaction with a PTS1-containing protein in the yeast two-hybrid system. Nucleotide sequence analysis revealed that the tobacco Pex5p contains seven tetratricopeptide repeats and that NtPEX5 shares greater sequence similarity with its homolog from humans than from yeast. Expression of NtPEX5 fusion proteins, consisting of the N-terminal part of yeast Pex5p and the C-terminal region of NtPEX5, in a Saccharomyces cerevisiae pex5 mutant restored protein translocation into peroxisomes. These experiments confirmed the identity of the tobacco protein as a PTS1 receptor and indicated that components of the peroxisomal translocation apparatus are conserved functionally. Two-hybrid assays showed that NtPEX5 interacts with a wide range of PTS1 variants that also interact with the human Pex5p. Interestingly, the C-terminal residues of some of these peptides deviated from the established plant PTS1 consensus sequence. We conclude that there are significant sequence and functional similarities between the plant and human Pex5ps.

Recruitment of IRAK to the interleukin 1 receptor complex requires interleukin 1 receptor accessory protein

The proinflammatory cytokine interleukin 1 (IL-1) activates the transcription of many genes encoding acute phase and proinflammatory proteins, a function mediated primarily by the transcription factor NF-κB. An early IL-1 signaling event is the recruitment of the Ser/Thr kinase IRAK to the type I IL-1 receptor (IL-1RI). Here we describe the function of a previously identified IL-1 receptor subunit designated IL-1 receptor accessory protein (IL-1RAcP). IL-1 treatment of cells induces the formation of a complex containing both IL-1RI and IL-1RAcP. IRAK is recruited to this complex through its association with IL-1RAcP. Overexpression of an IL-1RAcP mutant lacking its intracellular domain, the IRAK-binding domain, prevented the recruitment of IRAK to the receptor complex and blocked IL-1-induced NF-κB activation.

Correction of murine galactosialidosis by bone marrow-derived macrophages overexpressing human protective protein/cathepsin A under control of the colony-stimulating factor-1 receptor promoter

Galactosialidosis (GS) is a human neurodegenerative disease caused by a deficiency of lysosomal protective protein/cathepsin A (PPCA). The GS mouse model resembles the severe human condition, resulting in nephropathy, ataxia, and premature death. To rescue the disease phenotype, GS mice were transplanted with bone marrow from transgenic mice overexpressing human PPCA specifically in monocytes/macrophages under the control of the colony stimulating factor-1 receptor promoter. Transgenic macrophages infiltrated and resided in all organs and expressed PPCA at high levels. Correction occurred in hematopoietic tissues and nonhematopoietic organs, including the central nervous system. PPCA-expressing perivascular and leptomeningeal macrophages were detected throughout the brain of recipient mice, although some neuronal cells, such as Purkinje cells, continued to show storage and died. GS mice crossed into the transgenic background reflected the outcome of bone marrow-transplanted mice, but the course of neuronal degeneration was delayed in this model. These studies present definite evidence that macrophages alone can provide a source of corrective enzyme for visceral organs and may be beneficial for neuronal correction if expression levels are sufficient.

Desensitization of the Neurokinin-1 Receptor (NK1-R) in Neurons: Effects of Substance P on the Distribution of NK1-R, Gαq/11, G-Protein Receptor Kinase-2/3, and β-Arrestin-1/2

Observations in reconstituted systems and transfected cells indicate that G-protein receptor kinases (GRKs) and β-arrestins mediate desensitization and endocytosis of G-protein–coupled receptors. Little is known about receptor regulation in neurons. Therefore, we examined the effects of the neurotransmitter substance P (SP) on desensitization of the neurokinin-1 receptor (NK1-R) and on the subcellular distribution of NK1-R, Gαq/11, GRK-2 and -3, and β-arrestin-1 and -2 in cultured myenteric neurons. NK1-R was coexpressed with immunoreactive Gαq/11, GRK-2 and -3, and β-arrestin-1 and -2 in a subpopulation of neurons. SP caused 1) rapid NK1-R–mediated increase in [Ca2+]i, which was transient and desensitized to repeated stimulation; 2) internalization of the NK1-R into early endosomes containing SP; and 3) rapid and transient redistribution of β-arrestin-1 and -2 from the cytosol to the plasma membrane, followed by a striking redistribution of β-arrestin-1 and -2 to endosomes containing the NK1-R and SP. In SP-treated neurons Gαq/11 remained at the plasma membrane, and GRK-2 and -3 remained in centrally located and superficial vesicles. Thus, SP induces desensitization and endocytosis of the NK1-R in neurons that may be mediated by GRK-2 and -3 and β-arrestin-1 and -2. This regulation will determine whether NK1-R–expressing neurons participate in functionally important reflexes.

Molecular cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin

Pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 (PBSF/SDF-1) is a member of the CXC group of chemokines that is initially identified as a bone marrow stromal cell-derived factor and as a pre-B-cell stimulatory factor. Although most chemokines are thought to be inducible inflammatory mediators, PBSF/SDF-1 is essential for perinatal viability, B lymphopoiesis, bone marrow myelopoiesis, and cardiac ventricular septal formation, and it has chemotactic activities on resting lymphocytes and monocytes. In this paper, we have isolated a cDNA that encodes a seven transmembrane-spanning-domain receptor, designated pre-B-cell-derived chemokine receptor (PB-CKR) from a murine pre-B-cell clone, DW34. The deduced amino acid sequence has 90% identity with that of a HUMSTSR/fusin, a human immunodeficiency virus 1 (HIV-1) entry coreceptor. However, the second extracellular region has lower identity (67%) compared with HUMSTSR/fusin. PB-CKR is expressed during embryo genesis and in many organs and T cells of adult mice. Murine PBSF/SDF-1 induced an increase in intracellular free Ca2+ in DW34 cells and PB-CKR-transfected Chinese hamster ovary (CHO) cells, suggesting that PB-CKR is a functional receptor for murine PBSF/SDF-1. Murine PBSF/SDF-1 also induced Ca2+ influx in fusin-transfected CHO cells. On the other hand, considering previous results that HIV-1 does not enter murine T cells that expressed human CD4, PB-CKR may not support HIV-1 infection. Thus, PB-CKR will be an important tool for functional mapping of HIV-1 entry coreceptor fusin and for understanding the function of PBSF/SDF-1 further.

Preferential synaptic relationships between substance P-immunoreactive boutons and neurokinin 1 receptor sites in the rat spinal cord

Substance P plays an important role in the transmission of pain-related information in the dorsal horn of the spinal cord. Recent immunocytochemical studies have shown a mismatch between the distribution of substance P and its receptor in the superficial laminae of the dorsal horn. Because such a mismatch was not observed by using classical radioligand binding studies, we decided to investigate further the issue of the relationship between substance P and its receptor by using an antibody raised against a portion of the carboxyl terminal of the neurokinin 1 receptor and a bispecific monoclonal antibodies against substance P and horseradish peroxidase. Light microscopy revealed a good correlation between the distributions of substance P and the neurokinin 1 receptor, both being localized with highest densities in lamina I and outer lamina II of the spinal dorsal horn. An ultrastructural double-labeling study, combining preembedding immunogold with enzyme-based immunocytochemistry, showed that most neurokinin 1 receptor immunoreactive dendrites were apposed by substance P containing boutons. A detailed quantitative analysis revealed that neurokinin 1 receptor immunoreactive dendrites received more appositions and synapses from substance P immunoreactive terminals than those not expressing the neurokinin 1 receptor. Such preferential innervation by substance P occurred in all superficial dorsal horn laminae even though neurokinin 1 receptor immunoreactive dendrites were a minority of the total number of dendritic profiles in the above laminae. These results suggest that, contrary to the belief that neuropeptides act in a diffuse manner at a considerable distance from their sites of release, substance P should act on profiles expressing the neurokinin 1 receptor at a short distance from its site of release.

Genetic and pharmacological disruption of neurokinin 1 receptor function decreases anxiety-related behaviors and increases serotonergic function

Alterations in serotonin (5-hydroxytriptamine, 5-HT), norepinephrine, and γ-aminobutyric acid have been linked to the pathophysiology of anxiety and depression, and medications that modulate these neurotransmitters are widely used to treat mood disorders. Recently, the neuropeptide substance P (SP) and its receptor, the neurokinin 1 receptor (NK1R), have been proposed as possible targets for new antidepressant and anxiolytic therapies. However, animal and human studies have so far failed to provide a clear consensus on the role of SP in the modulation of emotional states. Here we show that both genetic disruption and acute pharmacological blockade of the NK1R in mice result in a marked reduction of anxiety and stress-related responses. These behavioral changes are paralleled by an increase in the firing rate of 5-HT neurons in the dorsal raphe nucleus, a major source of serotonergic input to the forebrain. NK1R disruption also results in a selective desensitization of 5-HT1A inhibitory autoreceptors, which resembles the effect of sustained antidepressant treatment. Together these results indicate that the SP system powerfully modulates anxiety and suggest that this effect is at least in part mediated by changes in the 5-HT system.

Identification in mice of two isoforms of the cysteinyl leukotriene 1 receptor that result from alternative splicing

Two classes of human G protein-coupled receptors, cysteinyl leukotriene 1 (CysLT1) and CysLT2 receptors, recently have been characterized and cloned. Because the CysLT1 receptor blockers are effective in treating human bronchial asthma and the mouse is often used to model human diseases, we isolated the mouse CysLT1 receptor from a mouse lung cDNA library and found two isoforms. A short isoform cDNA containing two exons encodes a polypeptide of 339 aa with 87.3% amino acid identity to the human CysLT1 receptor. A long isoform has two additional exons and an in-frame upstream start codon resulting in a 13-aa extension at the N terminus. Northern blot analysis revealed that the mouse CysLT1 receptor mRNA is expressed in lung and skin; and reverse transcription–PCR showed wide expression of the long isoform with the strongest presence in lung and skin. The gene for the mouse CysLT1 receptor was mapped to band XD. Leukotriene (LT) D4 induced intracellular calcium mobilization in Chinese hamster ovary cells stably expressing either isoform of the mouse CysLT1 receptor cDNA. This agonist effect of LTD4 was fully inhibited by the CysLT1 receptor antagonist, MK-571. Microsomal membranes from each transformant showed a single class of binding sites for [3H]LTD4; and the binding was blocked by unlabeled LTs, with the rank order of affinities being LTD4 >> LTE4 = LTC4 >> LTB4. Thus, the dominant mouse isoform with the N-terminal amino acid extension encoded by an additional exon has the same ligand response profile as the spliced form and the human receptor.

IL-1-induced NFκB and c-Jun N-terminal kinase (JNK) activation diverge at IL-1 receptor-associated kinase (IRAK)

Mutant I1A cells, lacking IL-1 receptor-associated kinase (IRAK) mRNA and protein, have been used to study the involvement of IRAK in NFκB and c-Jun N-terminal kinase (JNK) activation. A series of IRAK deletion constructs were expressed in I1A cells, which were then tested for their ability to respond to IL-1. Both the N-terminal death domain and the C-terminal region of IRAK are required for IL-1-induced NFκB and JNK activation, whereas the N-proximal undetermined domain is required for the activation of NFκB but not JNK. The phosphorylation and ubiquitination of IRAK deletion mutants correlate tightly with their ability to activate NFκB in response to IL-1, but IRAK can mediate IL-1-induced JNK activation without being phosphorylated. These studies reveal that the IL-1-induced signaling pathways leading to NFκB and JNK activation diverge either at IRAK or at a point nearer to the receptor.

Estradiol stimulates tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in the uterus.

The signaling pathways associated with estrogen-induced proliferation of epithelial cells in the reproductive tract have not been defined. To identify receptor tyrosine kinases that are activated in vivo by 17 beta-estradiol (E2), uteri from ovariectomized mice were examined for enhanced tyrosine phosphorylation of various receptors and a receptor substrate following treatment with this hormone. Within 4 hr after hormone exposure, extracts showed increased phosphotyrosine (P-Tyr) immunoreactivity at several bands, including 170- and 180-kDa; these bands were still apparent at 24 hr after E2. Analysis of immunoprecipitates from uterine extracts revealed that E2 enhanced tyrosine phosphorylation of the insulin-like growth factor-1 receptor (IGF-1R) and insulin receptor substrate-1 (IRS-1) by 6 hr. Comparison of supernatants from IRS-1 and control rabbit IgG immunoprecipitates indicated that the 170-kDa P-Tyr band in extracts was equivalent to IRS-1. The receptors for epidermal growth factor, platelet-derived growth factor, and basic fibroblast growth factor did not exhibit an E2-induced increase in P-Tyr content. The nonestrogenic steroid hormones examined did not stimulate the P-Tyr content of IGF-1R or IRS-1. Immunolocalization of P-Tyr and IRS-1 revealed strong reactivity in the epithelial layer of the uterus from E2-treated mice, suggesting that the majority of P-Tyr bands observed in immunoblots originate in the epithelium. Since hormonal activation of IRS-1 is epithelial, estrogen-specific, and initiated before maximal DNA synthesis occurs following treatment with hormone, this protein, as part of the IGF-1R pathway, may be important in mediating estrogen-stimulated proliferation in the uterus.

Functions of interleukin 1 receptor antagonist in gene knockout and overproducing mice.

Interleukin 1 receptor antagonist (IL-1ra) is a cytokine whose only known action is competitive inhibition of the binding of interleukin 1 (IL-1) to its receptor. To investigate the physiological roles of endogenously produced IL-1ra, we generated mice that either lack IL-1ra or overproduce it under control of the endogenous promoter. Mice lacking IL-1ra have decreased body mass compared with wild-type controls. They are more susceptible than controls to lethal endotoxemia but are less susceptible to infection with Listeria monocytogenes. Conversely, IL-1ra overproducers are protected from the lethal effects of endotoxin but are more susceptible to listeriosis. Serum levels of IL-1 following an endotoxin challenge are decreased in IL-1ra nulls and increased in IL-1ra overproducers in comparison to controls. These data demonstrate critical roles for endogenously produced IL-1ra in growth, responses to infection and inflammation, and regulation of cytokine expression.