UNIPROT:P20366 - FACTA Search

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Query: UNIPROT:P20366 (substance P)
21,176 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Five unique, high affinity rabbit polyclonal antibodies against neuromedin B were characterized in a radioimmunoassay in terms of the following parameters: pH and type of buffer, ionic strength, and non-ionic detergents in order to optimize immunoglobulin-peptide interaction; specificity using peptides of the bombesin family, in addition to the tachykinin substance P; and affinity to neuromedin B. Optimum conditions included acidic pH (5.25), high ionic strength (greater than 0.1 M) and absence of non-ionic detergents, which inhibited the assay. Affinities for the 5 antibodies ranged from 10 to 48 fmol neuromedin B with titers from 1:1,000 to 1:10,000 and the sequence-specificity covered the entire peptide; cross-reactivity towards substance P was negligible. As a model tissue, rat spinal cord was homogenized with 5 different extraction solvents, including acetone, methanol, acid and alkaline conditions, and assayed by each polyclonal antiserum; neuromedin B immunoreactivity levels were highest in acid and alkaline extracts and reflected the specificity of the antibody used. Applying these antisera to rat brain extracts, the posterior pituitary gland contained the highest concentration of immunoreactive equivalents of neuromedin B followed by the anterior pituitary, hypothalamus, and hippocampus. The immunoreactive content in the pituitary and hypothalamus, however, depended on the particular antisera used with significant (P less than 0.01) differences existing between them. Further application of these polyclonal antibodies to a spinal cord extract analyzed by isocratic reverse-phase HPLC conditions also revealed differences in their cross-reactivity with the immunoreactive peptides. These antisera may now be used as molecular probes for the determination of extractable immunoreactive neuromedin B from neural tissue and in situ localization by immunohistochemical techniques.
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PMID:Assessment of neuromedin B polyclonal antibodies as molecular probes in neural tissue. 335 56

1. The pharmacological profile of GR71251, a new tachykinin receptor antagonist, and its effect on the responses evoked by stimulation of primary afferent fibres were studied in isolated spinal cord preparations of neonatal rats. Potential changes were recorded extracellularly from a lumbar ventral root (L3-L5). 2. Bath-application of substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) at 0.01-3 microM to the spinal cord induced depolarization of the ventral root in normal artificial cerebrospinal fluid (CSF). The NK1 agonist, acetyl-Arg6-septide, and the NK3 agonist, senktide, at 0.01-3 microM, also had potent depolarizing actions whereas two NK2 agonists, beta-Ala8NKA4-10 and Nle10NKA4-10, showed little depolarizing effects at 1 microM. 3. GR71251 (0.3-3 microM) caused a rightward shift of the concentration-response curves for SP, acetyl-Arg6-septide and NKA with pA2 values of 6.14, 6.75 or 6.70, respectively. The effects of GR71251 were readily reversible within 15-30 min after its removal. By contrast, GR71251 (1-5 microM) had little effect on the depolarizing responses to NKB and senktide. 4. GR71251 (1-3 microM) did not depress the depolarizing responses to bombesin, neuromedin B and gastrin-releasing peptide in normal artificial CSF. 5. Application of capsaicin to the spinal cord induced a depolarizing response, which was partially depressed by GR71251 (3-10 microM). 6. In the isolated spinal cord preparation, intense electrical stimulation of a dorsal root evoked a slow depolarizing response of the contralateral ventral root of the same segment. A similar slow ventral root depolarization was evoked by electrical stimulation of the ipsilateral saphenous nerve in an isolated spinal cord-saphenous nerve preparation. GR71251 (0.3-10 microM) dose-dependently depressed these slow ventral root potentials.7. In the spinal cord-peripheral nerve preparation, conditioning stimulation of the saphenous nerve evoked an inhibition of the muscle nerve-evoked monosynaptic reflex lasting about 20 s. The late part of the inhibition was markedly depressed by GR71251 (1-3 microM).8. The present results indicate that GR71251 is a potent and specific antagonist for tachykinin receptors in the spinal cord. The present study further provides evidence for the involvement of SP and NKA in the slow ventral root depolarization and the prolonged inhibition of monosynaptic reflex that are evoked by primary afferent stimulation.
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PMID:Depression of primary afferent-evoked responses by GR71251 in the isolated spinal cord of the neonatal rat. 750 77

Recently it has been established that both a gastrin-releasing peptide (GRP) receptor and a neuromedin B (NMB) receptor mediate the actions of bombesin-related peptides in mammals. Five different classes of peptides that function as GRP receptor antagonists have been identified; however, it is unknown whether similar strategies will yield antagonists for the closely related NMB receptor. In the present study we have used either native cells possessing only one bombesin (Bn) receptor subtype or cells stably transfected with one subtype to determine whether using the strategies that were used successfully for GRP receptors would allow NMB receptor antagonists to be identified. [DPhe12]Bn analogs; des Met14 amides, esters and alkylamides; psi 13-14 Bn pseudopeptides; and D-amino acid-substituted analogs of substance P (SP) or SP(4-11) were all synthesized and each functioned as a GRP receptor antagonist. All of these antagonists had low affinity for the NMB receptor. Application of similar strategies to NMB by formation of [DPhe8]NMB, [psi 9-10]NMB pseudopeptides, des-Met10 NMB amides, alkylamide or esters did not result in any potent NMB receptor antagonists. D-Amino acid SP and SP(4-11) analogs were weakly selective NMB receptor antagonists. No COOH-terminal fragments of NMB or GRP functioned as a GRP or NMB receptor antagonist. These results demonstrate that none of the known strategies used to prepare peptide GRP receptor antagonists are successful at the NMB receptor, suggesting that a different strategy will be needed for this peptide, such as the formation of somatostatin octapeptide or D-amino acid-substituted substance P analogs. These results suggest that even though there is a close homology between GRP and NMB and their receptors, their structure-function relations are markedly different. These results indicate that the development of receptor subtype-specific peptide agonists or peptide antagonists for newly discovered receptor subtypes of gastrointestinal hormones/neurotransmitters may be difficult because the strategies developed for one well-studied subtype may not apply to the other even though it is structurally closely related.
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PMID:Peptide structural requirements for antagonism differ between the two mammalian bombesin receptor subtypes. 756 61

Bombesin and 10 bombesin-related peptides were administered intracerebroventricularly to conscious and freely moving rats. All peptides tested were found to elicit excessive grooming, especially scratching behavior. Bombesin itself had the most potent and long-lasting activity in eliciting scratching behavior. Naturally occurring peptides such as neuromedin B and gastrin-releasing peptide (GRP)-(18-27) were short-acting compared with exogenous peptides such as bombesin and synthesized analogs. Two phyllolitorins, a new bombesin subfamily, were also examined in this study. [Leu8]phyllolitorin induced more scratching than [Phe8]phyllolitorin and proved to be virtually equipotent to bombesin. Corticotropin-releasing factor (CRF) and substance P induced considerable excessive grooming, but both peptides were strikingly weak in inducing scratching behavior. It is suggested that (1) scratching represents a specific behavior commonly induced by bombesin-related peptides and (2) the relative potency to induce scratching behavior reflects the metabolic stability of the peptide, e.g. endogenous versus exogenous, shorter versus longer sequences, or chemical protection of N-terminus.
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PMID:Scratching behavior induced by bombesin-related peptides. Comparison of bombesin, gastrin-releasing peptide and phyllolitorins. 769 21

BALB/c 3T3 cells do not normally express receptors for bombesin-like peptides [bombesin (Bn), gastrin-releasing peptide (GRP), and neuromedin B (NmB)]. Transfection of BALB/c 3T3 cells with complementary DNA-encoding GRP receptors or NmB receptors leads to stable expression of functional GRP receptors (GRP Rt) or NmB receptors (NmB Rt), respectively, which are coupled to cell membrane ion channels. Whole cell current analysis using patch electrodes shows that the activation of these newly expressed receptors induces cation conductance increases, most frequently a Ca(2+)-activated plasma membrane K+ conductance. The dose-response (peak-current) relations of both transfected receptor subtypes were sigmoidal and exhibited threshold activation concentration in the picomole range and the saturation of responses to higher concentrations than 10(-8) M. The GRP Rt responded about equally to GRP, NmB, and Bn when compared at equimolar levels, despite their known difference in binding affinity for the three peptides (GRP, Bn > NmB). In contrast, for the NmB Rt, the NmB was more potent than GRP or Bn. Among four GRP/Bn-receptor antagonists tested, the [D-Phe6]Bn(6-13) ethyl ester suppressed GRP Rt responses at low concentrations (10(-7) M). N-acetyl-GRP-(20-26) amide, [Leu13-psi(CH2NH)-Leu14]Bn, and [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P also blocked GRP Rt responses but at higher concentrations (10(-5) M). However, at these concentrations, these four antagonists had little effect on NmB Rt responses, thereby showing a specificity of these antagonists for the GRP receptors.
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PMID:Receptor-activated currents in mouse fibroblasts expressing transfected bombesin receptor subtype cDNAs. 823 11

The effects of neuromedin B (NMB) on C6 glioma cells were investigated. NMB bound with high affinity (IC50 = 1 nM) to C6 cells whereas BN and GRP were less potent (IC50 = 40 and 100 nM). NMB (1 nM) elevated cytosolic Ca2+ in individual C6 cells and the increase in cytosolic Ca2+ was reversed by 1 microM [D-Arg1, D-Pro2,D-Trp7.9,Leu11]substance P [APTTL]SP, a broad spectrum antagonist. NMB stimulated [3H]arachidonic acid release from C6 cells and the increase in [3H]arachidonic acid release was reversed [APTTL]SP. NMB increased transiently c-fos gene expression in C6 cells. NMB increased the number of C6 colonies in soft agar and the increase in growth caused by NMB was reversed by [APTTL]SP. These data suggest that NMB receptors may regulate the proliferation of C6 cells.
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PMID:Neuromedin B stimulates arachidonic acid release, c-fos gene expression, and the growth of C6 glioma cells. 853 98

We examined the effect of two des-Met-bombesin analogues, [(CH3)2CHCO-His-Trp-Ala-Val-D-Ala-His-Leu-NHCH3] (ICI 216140) and [D-Phe6,des-Met14]bombesin(6-14) ethylamide (DPDM-bombesin ethylamide), on neuromedin B-induced Ca2+ and [3H]arachidonate release in BALB 3T3 cells transfected with human neuromedin B receptors. ICI 216140 and DPDM-bombesin ethylamide both stimulated Ca2+ mobilisation in a concentration-dependent manner but were less potent and efficacious than neuromedin B. BIM 23042 [D-Nal-Cys-Tyr-D-Trp-Lys-Val-Cys-Nal-NH2], a selective neuromedin B antagonist and [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P, a broad-spectrum peptide receptor antagonist inhibited neuromedin B-, ICI 216140 and DPDM-bombesin ethylamide-induced Ca2+ release. Pretreatment of cells with either des-Met-bombesin analogue attenuated neuromedin B-induced Ca2+ elevations, suggesting similar agonist-sensitive Ca2+ pools. The pharmacological profiles revealed from the [3H]arachidonate assay were similar, although ICI 216140 was less potent and efficacious than DPDM-bombesin ethylamide. The data suggest that ICI 216140 and DPDM-bombesin ethylamide behave as agonists at the neuromedin B receptor, perhaps as a consequence of neuromedin B receptor overexpression.
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PMID:Pharmacological profiles of two bombesin analogues in cells transfected with human neuromedin B receptors. 881 45

DAB389 GRP is composed of the catalytic and transmembrane domains of diphtheria toxin fused to gastrin-releasing peptide (GRP). DAB389 GRP is selectively targeted to, and inhibits protein synthesis in, cell lines expressing GRP receptors. Protein synthesis in 5'ET4 cells (BALB/3T3 fibroblasts transfected with the gene encoding the GRP receptor) was inhibited by 50% in the presence of 20 pM DAB389 GRP (IC50, 20 pM). DAB389 GRP did not inhibit protein synthesis in untransfected BALB/3T3 cells. A second neuropeptide-conjugated toxin, DAB389 SP, directed to cells expressing substance P receptors, was not cytotoxic to 5'ET4 cells, nor was DAB389 GRP cytotoxic to substance P receptor-bearing cells. DAB389 GRP cytotoxic effects were receptor specific and were inhibited either by excess GRP or anti-GRP antibody. Cytotoxicity was mediated by passage through an acidic vesicle, because addition of 10 microM chloroquine to the reaction inhibited cytotoxicity. DAB389 GRP and DAB389 SP were tested on a number of tumor cell lines. DAB389 GRP inhibited protein synthesis in AR42J rat pancreatic acinar cells and HuTu 80 human duodenal adenocarcinoma cells with IC50s of 65 and 200 pM, respectively. DAB389 SP had an IC50 of 9.5 pM for the AR42J cells and 12 nM for the HuTu 80 cell line. A number of small cell lung cancer cell (SCLC) lines were tested, and the IC50 for DAB389 GRP ranged from 1.1 to 85 nM. Sensitivity to DAB389 GRP appeared to be based on receptor number and receptor type (i.e., GRP or neuromedin B preferring). SCLC cells were also sensitive to DAB389 SP, with IC50s ranging from 2.4 to 11.5 nM. These results suggest that a potential use exists for diphtheria-based fusion toxins as therapeutic agents for treatment of SCLC and other neuropeptide receptor-bearing cancers.
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PMID:Inhibition of protein synthesis in small cell lung cancer cells induced by the diphtheria toxin-related fusion protein DAB389 GRP. 900 May 70

The purpose of this study was to investigate the regulation of electrolyte transport across the porcine endometrium by gastrin-releasing peptide (GRP) and substance P (SP). Luminal addition of GRP, neuromedin B (NMB), SP, or neurokinin A(NKA) to mucosal tissues mounted in Ussing chambers produced a multiphasic change in short-circuit current (Isc) characterized by an initial rapid increase and subsequent decrease in current. A similar response was obtained after addition of ionomycin or thapsigargin to the tissues. The Isc response to the peptides or Ca ionophore was inhibited by pretreatment of the tissues with luminal amiloride or benzamil. GRP and SP were more potent [50% effective concentration (EC50) of 3 nM] than NMB or NKA (EC50 values of 46 and 26 nM, respectively) in producing the decrease in Isc. Pretreatment with the GRP receptor antagonist 3-Phe-His-Trp-Ala-Val-D-Ala-His-D-Pro-psi Phe-NH2 blocked the Isc response to GRP and NMB but not to SP or NKA, whereas the NMB receptor antagonist D-Nal-[Cys-Try-D-Trp-Orn-Val-Cys]-Nal-NH2 was ineffective in inhibiting the Isc response to any of the peptides. In contrast, pretreatment of the tissue with the nonpeptide SP receptor antagonist CP-99,994 blocked the Isc response to SP and NKA but not to GRP or NMB. Experiments with amphotericin B-permeabilized tissues showed that GRP, SP, ionomycin, and thapsigargin increased current through an outwardly rectifying K conductance located on the apical membrane of the cells. The K-to-Na selectivity ratio of this conductance was calculated to be 2.5:1. These experiments showed that GRP and SP, acting through different receptors, produced an increase in K efflux through a Ca-dependent K conductance present in the apical membrane of surface endometrial epithelial cells. In addition, immunohistochemistry data showed that GRP-like immunoreactivity was localized to surface and glandular epithelial cells, whereas GRP receptor antibody labeling was observed in both epithelial and stromal cells. These results suggest that GRP functions as both an autocrine and paracrine regulatory peptide in the endometrium.
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PMID:Mechanisms of electrolyte transport across the endometrium. II. Regulation by GRP and substance P. 925 43

Bombesin (Bn) receptor subtype 3 (BRS-3) is an orphan receptor that is a predicted member of the heptahelical G-protein receptor family and so named because it shares a 50% amino acid homology with receptors for the mammalian bombesin-like peptides neuromedin B (NMB) and gastrin-releasing peptide. In a recent targeted disruption study, in which BRS-3-deficient mice were generated, the mice developed obesity, diabetes, and hypertension. To date, BRS-3's natural ligand remains unknown, its pharmacology unclear, and cellular basis of action undetermined. Furthermore, there are few tissues or cell lines found that express sufficient levels of BRS-3 protein for study. To define the intracellular signaling properties of BRS-3, we examined the ability of [D-Phe6,beta-Ala11,Phe13, Nle14]Bn-(6-14), a newly discovered peptide with high affinity for BRS-3, and various Bn receptor agonists and antagonists to alter cellular function in hBRS-3-transfected BALB 3T3 cells and hBRS-3-transfected NCI-H1299 non-small cell lung cancer cells, which natively express very low levels of hBRS-3. This ligand stimulated a 4-9-fold increase in [3H]inositol phosphate formation in both cell lines under conditions where it caused no stimulation in untransfected cells and also stimulated an increase in [3H]IP1, [3H]IP2, and 3H]IP3. The elevation of [3H]IP was concentration-dependent, with an EC50 of 20-35 nM in both cell lines. [D-Phe6,beta-Ala11,Phe13,Nle14]Bn-(6-14) stimulated a 2-3-fold increase in [Ca2+]i, a 3-fold increase in tyrosine phosphorylation of p125(FAK) with an EC50 of 0.2-0.7 nM, but failed to either stimulate increases in cyclic AMP or inhibit forskolin-stimulated increases. None of nine naturally occurring Bn peptides or three synthetic Bn analogues reported to activate hBRS-3 did so with high affinity. No high affinity Bn receptor antagonists had high affinity for the hBRS-3 receptor, although two low affinity antagonists for gastrin-releasing peptide and NMB receptors, [D-Arg1,D-Trp7,9, Leu11]substance P and [D-Pro4,D-Trp7,9,10]substance P-(4-11), inhibited hBRS-3 receptor activation. The NMB receptor-specific antagonist D-Nal,Cys,Tyr,D-Trp,Lys,Val, Cys,Nal-NH2 inhibited hBRS-3 receptor activation in a competitive fashion (Ki = 0.5 microM). Stimulation of p125(FAK) tyrosine phosphorylation by hBRS-3 activation was not inhibited by the protein kinase C inhibitor, GF109203X, or thapsigargin, alone or in combination. These results show that hBRS-3 receptor activation increases phospholipase C activity, which causes generation of inositol phosphates and changes in [Ca2+]i and is also coupled to tyrosine kinase activation, but is not coupled to adenylate cyclase activation or inhibition. hBRS-3 receptor activation results in tyrosine phosphorylation of p125(FAK), and it is not dependent on activation of either limb of the phospholipase C cascade. Although the natural ligand is not a known bombesin-related peptide, the availability of [D-Phe6,beta-Ala11, Phe13,Nle14]Bn-(6-14), which functions as a high affinity agonist in conjunction with hBRS-3-transfected cell lines and the recognition of three classes of receptor antagonists including one with affinity of 0.5 microM, should provide important tools to assist in the identification of its natural ligand, the development of more potent selective receptor antagonists and agonists, and further exploration of the signaling properties of the hBRS-3 receptor.
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PMID:Ability of various bombesin receptor agonists and antagonists to alter intracellular signaling of the human orphan receptor BRS-3. 959 99

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