Faculty Bibliography

Exposure of the gastrin-releasing peptide (GRP) receptor to agonists causes a rapid desensitization of the receptor-stimulated mobilization of intracellular calcium. Homologous desensitization occurs by uncoupling the G-proteins from the receptor and by ligand induced internalization. The molecular determinants of desensitization of the GRP receptor are not well known. The importance of tyrosine 324 which is located in a highly conserved NPX2-3 Y motif of the GRP receptor was investigated. Kirsten murine sarcoma virus-transformed rat kidney (KNRK) cells were transfected with expression vectors encoding either the wild type or the mutant (tyrosine 324 to alanine 324) rat GRP receptor. The wild type and mutant GRP receptors were expressed at a high level in the KNRK cells, 2.0 x 10(6) and 0.5 x 10(6) receptors per cell, respectively. The wild type and mutant GRP receptors bound GRP with the same affinity (Kd = 6-7 nM). KNRK cells expressing the wild type or mutant GRP receptor had similar [Ca2+]i, dose response to GRP. KNRK cells expressing the GRP receptor rapidly internalized bound 125I-GRP at 37 degree C. Internalization was inhibited at 4 degrees C and by 0.45 m sucrose. The internalization of bound 125I-GRP by the mutant GRP receptor was identical to the wild type receptor. Fluorescent microscopy was used to directly observe the GRP receptor expressed on the surface of the KNRK cells and to visualize its ligand induced internalization. There was no difference in the pattern of internalization between the wild type and mutant GRP receptors expressed in KNRK cells. Therefore, the highly conserved tyrosine 324 does not have a role in GRP binding, receptor-G-protein interaction, or initial events of ligand induced receptor internalization. The NPXnY motif is not a general sequestration sequence for seven transmembrane G-protein linked receptors.

Interactions between neutral endopeptidase-24.11 (NEP) and the substance P receptor (SPR; NK1) were investigated by examining substance P (SP) degradation, SP binding and SP-induced Ca2+ mobilization in epithelial cells transfected with cDNA encoding the rat SPR and rat NEP. Expression of NEP accelerated the degradation of SP by intact epithelial cells and by membrane preparations, and degradation was reduced by the NEP inhibitor thiorphan. In cells expressing SPR alone, specific 125I-SP binding after 20 min incubation at 37 degrees C was 92.2 +/- 3.1% of maximal binding and was unaffected by thiorphan. Coexpression of NEP in the same cells as the SPR markedly reduced SP binding to 13.9 +/- 0.5% of maximal, and binding was increased to 82.7 +/- 2.4% of maximal with thiorphan. Coexpression of NEP in the same cells as the SPR also reduced to undetectable the increase in intracellular Ca2+ in response to low concentrations of SP (0.3 and 0.5 nM), and significantly reduced the response to higher concentrations (1 and 3 nM). The Ca2+ response was restored to control values by inhibition of NEP with thiorphan. In contrast, SP binding and SP-induced Ca2+ mobilization were only slightly reduced when cells expressing SPR alone were mixed with a 3- to 24-fold excess of cells expressing NEP alone. Therefore, in this system, NEP markedly down-regulates SP binding and SP-induced Ca2+ mobilization only when coexpressed in the same cells as the SPR.

Antibodies to neuropeptide receptors can be used to localize and characterize the receptors in tissues and cell lines. Two strategies were used to study the rat substance P receptor (SPR, NK-1) by immunological methods. First, a polyclonal antiserum was raised by immunizing rabbits with a peptide corresponding to the 15 amino acid residues (KTMTESSSFYSNMLA, SPR393-407) at the intracellular C-terminus of the rat SPR coupled to bovine thyroglobulin. An antiserum was obtained with a titer for half-maximal binding of 125I-SPR393-407 of 1:70,000. Nonradioactive SPR393-407 inhibited 50% of binding at a concentration of 10 pM. Binding of 125I-SPR393-407 to the antiserum was also displaced in a parallel manner by membrane proteins from tissues expressing high levels of the SPR (brain and submaxillary gland). Second, a chimeric SPR construct of a hydrophilic Flag peptide (DYKDDDDK) genetically engineered in sequence with the extracellular N-terminus of rat SPR was generated by polymerase chain reaction. The Flag-SPR chimera was expressed in rat kidney epithelial cells (KNRK) and judged to be fully functional, assessed by binding of 125I-substance P (apparent Kd of 5.63 nM) and calcium mobilization in response to substance P (EC50 of 0.66 nM). Antibodies to SPR393-407 and the Flag peptide stained the plasma membrane of KNRK cells expressing the native SPR or the Flag-SPR chimera. Staining was abolished by preincubation with SPR393-407 or the Flag peptide. Cells transfected with vector alone were unstained. The SPR antiserum recognized a broad protein band on Western blots of membranes prepared from cells expressing SPR but not from cells transfected with vector alone. The signal was quenched by preincubation of the antiserum with SPR393-407. By immunohistochemistry, the SPR antiserum was found to bind to neurons in the dorsal horn of the rat spinal cord and to ganglion cells in the myenteric plexus of the rat ileum near substance P-immunoreactive nerve fibers. Staining was abolished by preabsorption of the antiserum with SPR393-407. These antibodies can be used to localize the SPR in tissues and cells and to examine the function of the receptor in cell lines.

OBJECTIVE:The goal of this investigation was to determine the role of calcitonin gene-related peptide (CGRP) in gastric mucosal resistance to ulceration. SUMMARY BACKGROUND DATA/BACKGROUND:CGRP is a 37-amino acid peptide found in the peripheral ends of afferent gastric neurons. CGRP is known to inhibit acid secretion, stimulate mucosal blood flow, and stimulate release of somatostatin. METHODS:The release of CGRP in response to intragastric and intra-arterial administration of capsaicin in the isolated, vascularly perfused rat stomach was measured by radioimmunoassay. The molecular forms of CGRP released were analyzed by gel filtration chromatography. The effect of intravenous CGRP or intragastric capsaicin on gastric ulceration induced by 100 mmol/L HCl and indomethacin was studied in intact and endogenous CGRP-depleted rats. RESULTS:Intra-arterial capsaicin (concentration range, 10(-7) to 10(-5) mol/L) stimulated a prompt and sustained release of immunoreactive CGRP, of which 84% coeluted with rat 1-37 CGRP I by gel filtration. Intragastric capsaicin (range, 10(-5) to 10(-4) mol/L) failed to release CGRP into the vascular perfusate. In intact rats, intragastric capsaicin (10(-6) mol/L) or intravenous CGRP I (10 micrograms/kg/hr) reduced the number and area of mucosal lesions caused by HCl and indomethacin compared with the findings in control rats. Rats depleted of endogenous CGRP were more susceptible to gastric ulceration than were normal rats. Intragastric capsaicin failed to protect the mucosa of CGRP-depleted rats, whereas exogenous intravenous CGRP was effective. CONCLUSIONS:These data support the hypothesis that CGRP released from gastric enteric neurons mediates gastric mucosal resistance to ulceration by noxious agents.

BACKGROUND:The action of tachykinins in the circular muscle of the human esophageal body is not known. The present study aimed to determine the response to tachykinins and the receptor type mediating this response. METHODS:Specimen were obtained from organ donors or patients undergoing esophagectomy for cancer, and isometric tension in response to tachykinins was measured. RESULTS:Substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) evoked a concentration-dependent contraction with the following order of potency: NKA > NKB > SP. The neutral endopeptidase inhibitor, phosphoramidon, increased only the response to SP. [beta Ala8]NKA(4-10), a selective agonist of the NK2 receptor, produced a concentration-dependent contraction, whereas [Sar9,Met(O2)11]SP and [MePhe7]NKB, selective agonists of NK1 and NK3 receptors, respectively, had no effect. Contraction evoked by NKA was inhibited by the nonpeptide NK2 antagonist SR 48968 but not by the nonpeptide NK1 receptor antagonist CP-96,345, tetrodotoxin, or atropine. SR 48968 did not affect the response to carbachol. CONCLUSIONS:Tachykinins contract the circular muscle of human esophageal body by activation of NK2 receptors without involvement of neural mechanisms. Response to SP is modulated by a phosphoramidon-sensitive enzymatic activity.

The contractile response to natural tachykinins and selective peptide agonists for tachykinin receptors was studied in strips of circular smooth muscle of human lower esophageal sphincter in vitro. The effects of phosphoramidon, which inhibits neutral endopeptidase (EC.3.4.24.11) and of the non-peptide compounds, SR 48968 and CP-96,345, which selectively block NK1 and NK2 receptors, respectively, were also investigated. Substance P, neurokinin A and neurokinin B produced a concentration-dependent contractile response. The rank order of potency was neurokinin A > neurokinin B > substance P. Phosphoramidon (1 microM) potentiated the response to substance P without changing the order of potency of natural tachykinins. The NK2-selective agonist, ([ beta Ala8]neurokinin A-(4-10)), produced a concentration-dependent contraction. The NK1 ([Sar9,Met(O2)11]substance P, 1 microM) and NK3 ([MePhe7]neurokinin B, 1 microM) selective agonists, however, did not exert any contractile effect. The selective NK2 antagonist, SR 48968, potently inhibited in a concentration-dependent (10 nM-1 microM) manner the response to neurokinin A, without affecting the response to carbachol. The selective NK1 antagonist, CP-96,345 (1 microM), did not affect the response to neurokinin A. These results indicate that tachykinins contract the circular muscle of human lower esophageal sphincter, and that this effect is mediated by NK2 receptor stimulation. Moreover, a phosphoramidon-sensitive mechanism plays a role in the regulation of the response to substance P.

Intestinal inflammation induced by the nematode Trichinella spiralis is accompanied by increased intestinal concentrations of substance P, a mediator of inflammation and a stimulant of smooth muscle contraction, and by intestinal hypermotility. The expression of neutral endopeptidase (NEP), a cell surface enzyme that degrades substance P in the extracellular fluid, was examined in the inflamed intestine. NEP enzymatic activity, measured by a fluorometric assay, was reduced by 84-fold in jejunal mucosa-circular muscle and by 12-fold in jejunal longitudinal muscle-myenteric plexus within 6 days after infection with T. spiralis. The downregulation was unaffected by treatment with betamethasone and was still observed in athymic animals. NEP protein levels, examined by Western blotting, confirmed the loss of NEP from inflamed tissue. The specific activity for degradation of substance P was reduced by sixfold in jejunal mucosa-circular muscle and by twofold in jejunal longitudinal muscle-myenteric plexus of rats infected with T. spiralis compared with uninfected controls. Thus the downregulation of NEP resulted in reduced substance P degradation, which may contribute to functional abnormalities of the inflamed intestine.

The distribution of neutral endopeptidase (NEP; EC 3.4.24.11) was examined in the alimentary tract of the rat. Immunoreactive NEP and NEP mRNA were localized to epithelial cells of the small intestine and to muscle cells in the stomach, small intestine, and colon by immunohistochemistry and in situ hybridization histochemistry. NEP antisera recognized a protein on Western blots of membranes from gastric, jejunal, and colonic mucosa and gastric muscle with an electrophoretic mobility identical to that of recombinant human NEP (approximately 95 kDa). An antisense cRNA probe to NEP hybridized to RNA of approximately 3.5 kb and approximately 6.5 kb, corresponding to the primary transcripts of rat NEP, on Northern blots of total RNA from the jejunal mucosa. NEP message was detected in mRNA from jejunal and colonic mucosa and gastric, jejunal, and colonic muscle using a ribonuclease protection assay. NEP enzymatic activity, assessed by DL-thiorphan-inhibitable degradation of glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine, was highest in homogenates of jejunal mucosa (868 +/- 98 pmol.h-1 x micrograms protein-1) and was between 49- and 413-fold lower in other gastrointestinal tissues. The cellular origin of NEP in the gastric and colonic mucosa could not be determined.

The objectives of this investigation were to characterize neuropeptide-degrading enzymes on the surface of gastric muscle cells and to determine their physiological function. Neutral endopeptidase (NEP, EC 3.4.24.11) activity was measured using the fluorogenic substrate glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine. The NEP inhibitors phosphoramidon and DL-thiorphan (1 microM) inhibited degradation of the substrate by gastric muscle membranes by 100% and by freshly dispersed gastric muscle cells by 55-60%. The phosphoramidon or DL-thiorphan-inhibitable activity, attributed to NEP, of membranes was 112 +/- 4.0 pmol h-1 (micrograms protein)-1 and of cells was 4.2 +/- 0.8 nmol h-1 (10(6) cells)-1. This activity was associated with membranes prepared from cells and was not detected in the cytoplasm or in the cell incubation solution. Gastric muscle membranes were fractionated by electrophoresis and analysed by Western blotting using two NEP antisera. Both antisera recognized a protein in membranes with an electrophoretic mobility identical to that of recombinant human NEP and an apparent molecular mass of approximately 95 kDa. Neuropeptides were degraded by membranes with specific activities in the order of [Leu5]enkephalin > [Met5]enkephalin > gastrin-releasing peptide-10 (GRP-10) > [D-Ala2][Leu5]enkephalin > somatostatin-14. Phosphoramidon and DL-thiorphan similarly inhibited the degradation of GRP-10 (mean of 35% inhibition), somatostatin-14 (57%) and the aminopeptidase-resistant analogue, [D-Ala2][Leu5]enkephalin (75%). When aminopeptidases were inhibited with amastatin (10 microM) phosphoramidon inhibited degradation of [Leu5]enkephalin (54%) and [Met5]enkephalin (100%). Phosphoramidon increased the potency of the contractile effects of neuropeptides on muscle cells by > 280-fold for somatostatin-14, 17-fold for GRP-10, 18-fold for [Met5]enkephalin and 14-fold for [Leu5]enkephalin. The results show that an NEP-like enzyme on the surface of gastric muscle cells degrades and inactivates enkephalins, GRP-10 and somatostatin-14 and acts in a manner analogous to that of acetylcholinesterase in the neuromuscular junction of skeletal muscle.

1. A protein A-rat substance P receptor (SPR) fusion protein was genetically engineered and used as an immunogen to raise a polyclonal antiserum to the SPR. The fusion protein was expressed in Escherichia coli driven by the heat-inducible lambda promoter (lambda Pr). 2. The fusion protein was purified using an IgG-Sepharose column, which specifically binds proteins containing the protein A moiety. The IgG fraction obtained after the immunization was cleaved to produce Fab fragments, which were subsequently purified using a fusion protein affinity column. The serum (anti-SPR Fab serum) was analyzed by fluorescence-activated cell sorting (FACS) and immunohistochemistry on both a constitutive cell line for the SPR (AR42J) and a cell line transfected with the SPR (KNRKSPR). 3. Specificity of the antiserum for SPR was confirmed by immunohistochemistry on cells using antiserum that had been preincubated with the protein A fusion protein (blocked). 4. The Ca2+ signal normally observed on stimulation of SPR with SP in AR42J cells and SP binding to KNRKSPR cells was shown to be diminished in the presence of anti-SPR Fab serum. SPR from both cell lines was immunoprecipitated using the anti-SPR Fab serum. The antiserum itself did not induce intracellular Ca2+ mobilization normally observed when cells were incubated with SP. 5. This specific SPR antiserum will be a useful tool to investigate further the mechanisms of SP/SPR interactions.