Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P20366 (substance P)
 21,176 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate substance P (SP) receptors on an established human astrocytoma cell line (U-87 MG), [3H][Sar9,Met(O2)11]-SP, a selective SP receptor agonist, was used to identify and characterize the cell membrane binding sites for SP. SP receptor mRNA was examined by solution hybridization analysis, and the existence of SP binding protein on the surface of membranes was evaluated by flow cytometry using an anti-SP binding protein antibody. In U-87 MG and U-373 MG RNA preparations, transcripts were identified that corresponded to both mature and partially spliced receptor forms. In U-87 MG cell membrane-enriched preparations, the binding of [3H][Sar9,Met(O2)11]-SP was found to be time and cell number dependent, specific, saturable, and of high affinity. Equilibrium binding analysis revealed a single class of binding sites with an apparent KD of 1.15 +/- 0.15 nM and a Bmax of 108 +/- 9.8 fmol/mg of protein. [3H][Sar9, Met(O2)11]-SP binding was basically not influenced by addition of mono (Na+, Li+) or divalent (Mg2+, Mn2+, Ca2+) cations; only high doses of divalent cations decreased the binding. GTP and guanylyl-5'-imidodiphosphate, but not GDP and GMP, reduced the Bmax without changing the affinity of [3H][Sar9,Met(O2)11]-SP. We also examined the effects of pretreatment with three lectins [concanavalin A (con A), wheat germ agglutinin (WGA), and Lens culinaris agglutinin (LCA)] to determine the nature of carbohydrate chains on the U-87 MG cell. Of three lectins analyzed for effects on agonist binding, WGA and LCA had an inhibitory effect, whereas con A was ineffective. These results suggest that SP receptors on the human astrocytoma cell line U-87 MG have either a biantennary complex-type or a high mannose-type of carbohydrate chain and may be regulated by GTP-binding protein(s).
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PMID:Human astrocytoma cells (U-87 MG) exhibit a specific substance P binding site with the characteristics of an NK-1 receptor. 886 85

We investigated the effect of Mn2+ on the mechanical responses evoked by high K+ (60 mM) or low Na+ (25 mM) solutions, oxytocin and neurokinin A in the oestrogen-primed rat uterus. In a Ca2+-free, Mn2+ (0.54 mM)-containing solution, high K+ or low Na+ solutions produced contractions of smaller amplitude than those observed in a normal Ca2+ (0.54 mM) solution, which were abolished by nifedipine (1 microM). Oxytocin (1 microM) and neurokinin A (1 microM, in the presence of phosphoramidon 1 microM) evoked nifedipine-insensitive contractile responses similar to (oxytocin) or smaller (neurokinin A) in amplitude than those observed in Ca2+ (0.54 mM)-containing solution. In strips loaded with Ca2+ (2.16 mM) for 10 min and then exposed to a Ca2+- and Mn2+-free, EGTA (3 mM)-containing medium for 4 min, both oxytocin and neurokinin A induced transient contraction followed by a small sustained response. The transient component of the response was abolished by cyclopiazonic acid (10 microM). When preparations were loaded with Mn2+ (2.16 mM) for 10 min, only the small, tonic contraction was observed. In Ca2+-containing solution, Mn2+ (0.01-10 mM) inhibited in a concentration-dependent manner the rhythmic contractions developed either spontaneously or by electrical stimulation as well as high K+- and neurokinin A-induced contractions. Mn2+ also abolished the rhythmic, but not the tonic component of the response to oxytocin, and the preparation remained maximally contracted. These data suggest that in the oestrogen-primed rat uterus, Mn2+ acts as an antagonist of Ca2+ influx through L-type voltage-operated Ca2+ channels. In addition, Mn2+ enters the cell mainly through nifedipine-insensitive receptor-operated channels and, to a lesser degree, through L-type Ca2+ channels to produce contraction by directly activating the contractile machinery.
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PMID:Effects of Mn2+ on the responses induced by different spasmogens in the oestrogen-primed rat uterus. 919 74

1. In human U373 MG astrocytoma cells agonist-induced increases in intracellular Ca2+ ([Ca2+]i) are rapidly returned towards prestimulated levels. Examination of the effect of histamine and substance P on [Ca2+]i in thapsigargin-treated cells has allowed a mechanism contributing to this effect to be characterized. 2. Histamine and substance P stimulated [3H]-inositol monophosphate ([3H]-IP1) accumulation in U373 MG cells. Concentration-response curves of [3H]-IP1 accumulation in suspensions of U373 MG cells in HEPES buffer containing 30 mM Li+ yielded best-fit EC50 values of 19.1+/-1.5 microM for histamine and 5.7+/-1.3 nM for substance P. 3. In confluent monolayers of fura-2 loaded U373 MG cells perfusion with 100 microM histamine resulted in a transient 597+/-50 nM increase in [Ca2+]i. The best-fit EC50 for histamine was 4.6+/-2.2 microM. The initial, transient, histamine response was often followed by further small transient increases in [Ca2+]i. 4. Treatment of U373 MG cells with 5 microM thapsigargin, followed by the readdition of 1.8 mM Ca2+ to the perfusion buffer, resulted in a steady-state level of [Ca2+]i 97+/-5 nM above pretreated levels (measured 400 s after readdition of Ca2+). Perfusion of histamine (100 microM, 100 s) caused a rapid decline in the thapsigargin-induced steady state level of [Ca2+]i. This effect of histamine was normally reversible upon washout. The best-fit EC50, for the histamine response was 0.8+/-0.2 microM. Substance P (10 nM, 100s) also caused a reduction in thapsigargin-induced steady-state levels of [Ca2+]i. 5. Neither 100 microM histamine nor 10 nM substance P inhibited the rate of quench of fura-2 fluorescence by Mn2+ in U373 MG cells pretreated with 5 microM thapsigargin, indicating that the depressant effect on steady-state raised [Ca2+]i was probably not due to a block of Ca2+ entry. 6. The depressant effect of histamine on [Ca2+]i was blocked by 1 microM mepyramine, and was partially reduced by pre-incubation with 1 microM staurosporine (61+/-7% reduction) and with Ro 31-8220 (24+/-10% and 50+/-6% reduction by 1 and 10 microM Ro 31-8220, respectively). Pre-incubation with H-89 did not alter the depressant effect of histamine. 7. Neither 1 microM staurosporine nor 10 microM KN-62 inhibited the binding of [3H]-mepyramine to guinea-pig cerebellar membranes, whereas it was reduced by 17+/-1% and 55+/-2% by 1 and 10 microM Ro 31-8220, respectively. However, [3H]-IP1 accumulation stimulated by histamine in U373 MG cells was not inhibited by 1 or 10 microM Ro 31-8220 and in 2 out of 3 experiments there was a significant potentiation of the response to histamine with both concentrations of Ro 31-8220. Staurosporine, 1 microM, similarly potentiated the response to 100 microM histamine in 3 out of 4 experiments. KN-62 (10 microM) did not stimulate histamine-induced [3H]-IP1 accumulation. 8. In HEPES buffer to which no Ca2+ had been added, histamine stimulated a transient 451+/-107 nM increase in [Ca2+]i. Pretreatment with 1 microM and 10 microM Ro 31-8220 did not significantly alter the initial peak response to histamine, but slowed the rate at which histamine-induced increases in [Ca2+]i were returned to prestimulated levels. Pretreatment with KN-62 had no significant effect on the response to histamine, but consistently inhibited the secondary slower phase of the decline in [Ca2+]i. H-89 did not alter the histamine response. 9. The effect of histamine in stimulating Ca2+ extrusion was not confined to U373 MG cells, since 100 microM histamine also caused a rapid decrease in steady-state levels of [Ca2+]i in thapsigargin-treated human HeLa cells. 10. The results indicate that agonists which increase [Ca2+]i via activation of phosphoinositide metabolism can also stimulate a homeostatic mechanism which acts to reduce [Ca2+]i. The balance of the evidence indicates that in U373 MG cells the latter effect most likely involves a PKC-mediated stimulation of a Ca2+-extrusion pump.
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PMID:Dual effects of histamine and substance P on intracellular calcium levels in human U373 MG astrocytoma cells: role of protein kinase C. 950 96

The nine Leucophaea Tachykinin-Related Peptides (LemTRP 1-9) isolated from the midgut and brain of the cockroach, Leucophaea maderae, all induced increases in spontaneous contractions of the L. maderae hindgut. Synthetic LemTRP 1 and 3-9, were equally potent in inducing contractions of the hindgut. More than seven of the nine C-terminal residues of the closely related locust peptide locustatachykinin I (LomTK I) are required for full activity of the peptide on the L. maderae hindgut. Proctolin, a well characterized myostimulatory neuropeptide, was shown to be more potent than LemTRPs. LemTRP 1 and proctolin did not have synergistic actions in potentiating the amplitude and tonus of contractions of the L. maderae hindgut. Several differences could be seen in actions of LemTRP 1 and proctolin. In contrast to proctolin, LemTRP 1 could not override the inhibitory action of 10(-9) M of the myoinhibitory peptide leucomyosuppressin. Spantide I, an antagonist of the mammalian tachykinin receptors, at a concentration of 5 microM, blocked the response to LemTRP 1, but not to proctolin. The competitive proctolin receptor antagonist [alpha-methyl-L-tyrosine2]-proctolin blocked the action of both proctolin and LemTRP 1 when applied at 1 microM, whereas cycloproctolin had no antagonist action on either peptide. Verapamil, a blocker of voltage gated Ca2+-channels, and the less specific Ca2+-channel blocker Mn2+, abolished the action of LemTRP 1, but not of proctolin. The results obtained indicate that LemTRPs act on receptors distinct from those of proctolin. Double label immunocytochemistry revealed that all LomTK-like immunoreactive fibers impinge on the proctolinergic fibers in the hindgut. This finding and the inhibitory actions of Ca2+-channel blockers on TRP responses and of the proctolin receptor antagonist on both peptides, may suggest that the LemTRP receptors are not on the hindgut muscle fibers but on the terminals of the proctolinergic neurons. Thus, LemTRPs may induce release of proctolin on the hindgut. An alternative is that LemTRPs act by mechanisms clearly distinct from those of proctolin.
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PMID:Characterization of actions of Leucophaea tachykinin-related peptides (LemTRPs) and proctolin on cockroach hindgut contractions. 953 32

The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E. C. 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo.
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PMID:Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme. 1110 90

A cDNA (LeAPP2) was cloned from tomato coding for a 654 amino acid protein of 72.7 kDa. The deduced amino acid sequence was >40% identical with that of mammalian aminopeptidase P, a metalloexopeptidase. All amino acids reported to be important for binding of the active site metals and catalytic activity, respectively, were conserved between LeAPP2 and its mammalian homologues. LeAPP2 was expressed in Escherichia coli in N-terminal fusion with glutathione S-transferase and was purified from bacterial extracts. LeAPP2 was verified as an aminopeptidase P, hydrolyzing the amino-terminal Xaa-Pro bonds of bradykinin and substance P. LeAPP2 also exhibited endoproteolytic activity cleaving, albeit at a reduced rate, the internal -Phe-Gly bond of substance P. Apparent K(m) (15.2 +/- 2.4 microm) and K(m)/k(cat) (0.94 +/- 0.11 mm(-1) x s(-1)) values were obtained for H-Lys(Abz)-Pro-Pro-pNA as the substrate. LeAPP2 activity was maximally stimulated by addition of 4 mm MnCl(2) and to some extent also by Mg(2+), Ca(2+), and Co(2+), whereas other divalent metal ions (Cu(2+), Zn(2+)) were inhibitory. Chelating agents and thiol-modifying reagents inhibited the enzyme. The data are consistent with LeAPP2 being a Mn(II)-dependent metalloprotease. This is the first characterization of a plant aminopeptidase P.
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PMID:Cloning, expression, and characterization of tomato (Lycopersicon esculentum) aminopeptidase P. 1142 53

Using a functional genomic approach, we have identified and characterized a cytosolic form of aminopeptidase P from Drosophila melanogaster. This study represents the first characterization of an insect aminopeptidase P. The complete sequence of a 12.5 kbp genomic clone from D. melanogaster showed the presence of a 1,839 bp ORF, encoding a protein of 613 amino acids with a calculated molecular mass of 68.5 kDa. The deduced amino acid sequence was 48% identical and 66% similar to rat and human cytosolic aminopeptidase P. Amino acids important for catalytic activity and the metal binding ligands were found to be conserved between Drosophila AP-P and its mammalian homologues. The recombinant enzyme expressed in Escherichia coli hydrolyzed the amino terminal Xaa-Pro bond of substance P and bradykinin, revealing its functional identity. Further enzyme characterization showed the enzyme to be a manganese-dependent metallopeptidase. Immunoblot analysis showed that DAP-P is located exclusively in the cytosol and is temporally regulated during Drosophila development. In the adult fly, the protein could be detected in gut, testis and ovary, with a high level of expression in brain.
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PMID:A cytosolic form of aminopeptidase P from Drosophila melanogaster: molecular cloning and characterization. 1187 74

Carotid body glomus cells produce and release acetylcholine (ACh), catecholamines, and neuropeptides, and there is biochemical evidence that these cells possess receptors for these substances. Thus, we studied the effects of cholinergics [ACh, nicotine (Nic), bethanechol (BN)] and peptides [met-enkephalin (ME), substance P (SP)] on the membrane potential (Em), voltage noise (Erms), and input resistance (Ro) of glomus cells. Sliced carotid bodies (for cell visualization) of cats, rabbits, and mice were used. The mean Em and Ro of rabbit glomus cells were lower than those of cat and mouse. Ro of mouse cells was the largest, whereas Erms was similar in all species. The various agents had qualitatively similar effects on the cells of the three species although some quantitative differences were sometimes observed. But, for simplicity, results were pooled. ACh depolarized most cells (effect depressed by zero [Ca2+]o and Mn2+), reduced their resistance, and induced variable changes in Erms. Different ACh doses produced non-linear effects on DeltaEm. Nic and BN also depolarized most cells, reducing Ro and Erms. Atropine depressed the cell responses to BN; alpha-bungarotoxin the depolarizing response to Nic. ME and SP depolarized most cells, but only ME significantly reduced Ro. Neither peptide significantly changed voltage noise. Comparing the effects of all drugs showed that BN was the most effective depolarizing agent, producing the largest reductions in Ro. There were negative correlations between DeltaEm and DeltaRo with the cholinergics and SP; correlations between DeltaErms and DeltaRo were significant and positive only with the cholinergics. These results confirm the presence of nicotinic, muscarinic, and peptidergic receptors in glomus cells. The similar effects of cholinergics and peptides and those of flow interruption and anoxia suggest that the latter may partly act via autoreceptors for the released transmitters.
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PMID:Effects of Putative Neurotransmitters of the Carotid Body on its Own Glomus Cells. 1210 5

We investigated whether substance P modulates pacemaker currents generated in cultured interstitial cells of Cajal of murine small intestine using whole cell patch-clamp techniques at 30 degrees C. Interstitial cells of Cajal generated spontaneous inward currents (pacemaker currents) at a holding potential of -70 mV. Tetrodotoxin, nifedipine, tetraethylammonium, 4-aminopyridine, or glibenclamide did not change the frequency and amplitude of pacemaker currents. However, divalent cations (Ni2+, Mn2+, Cd2+, and Co2+), nonselective cationic channel blockers (gadolinium and flufenamic acid), and a reduction of external Na+ from normal to 1 mM inhibited pacemaker currents indicating that nonselective cation channels are involved in their generation. Substance P depolarized the membrane potential in current clamp mode and produced tonic inward pacemaker currents with reduced frequency and amplitude in voltage clamp mode. [D-Arg1, D-Trp7,9, Leu11] substance P, a tachykinin NK1 receptor antagonist, blocked these substance P-induced responses. Furthermore, [Sar9, Met(O2)11] substance P, a specific tachykinin NK1 receptor agonist, depolarized the membrane and tonic inward currents mimicked those of substance P. Substance P continued to produce tonic inward currents in external Ca2+-free solution or in the presence of chelerythrine, a protein kinase C inhibitor. However, substance P-induced tonic inward currents were blocked by thapsigargin, a Ca2+-ATPase inhibitor in the endoplasmic reticulum or by an external 1 mM Na+ solution. Our results demonstrate that substance P may modulate intestinal motility by acting on the interstitial cells of Cajal by activating nonselective cation channels via the release of intracellular Ca2+ induced by tachykinin NK1 receptor stimulation.
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PMID:Substance P induces inward current and regulates pacemaker currents through tachykinin NK1 receptor in cultured interstitial cells of Cajal of murine small intestine. 1521 18

Electron capture dissociation (ECD) of the peptide Substance P (SubP) complexed with divalent metals has been investigated. ECD of [SubP + H + M]3+ (M2+ = Mg2+ -Ba2+ and Mn2+ -Zn2+) allowed observation of a larger number of product ions than previous investigations of doubly charged metal-containing peptides. ECD of Mg-Ba, Mn, Fe, and Zn-containing complexes resulted in product ions with and without the metal from cleavage of backbone amine bonds (c' and z* -type ions). By contrast, ECD of Co and Ni-containing complexes yielded major bond cleavages within the C-terminal methionine residue (likely to be the metal ion binding site). Cu-containing complexes displayed yet another behavior: amide bond cleavage (b and y'-type ions). We believe some results can be rationalized both within the hot hydrogen atom mechanism and mechanisms involving electron capture into excited states, such as the recently proposed amide superbase mechanism. However, some behavior, including formation of (cn 'M - H)+ ions for Ca-Ba, is best explained within the latter mechanisms with initial electron capture at the metal. In addition, the ECD behavior appears to correlate with the metal second ionization energy (IE2). Co and Ni (displaying sequestered fragmentation) have IE2s of 17.1 and 18.2 eV, respectively, whereas IE2s for Mg-Ba, Mn, and Fe (yielding random cleavage) are 10.0 to 16.2 eV. This behavior is difficult to explain within the hot hydrogen atom mechanism because hydrogen transfer should not be influenced by IE2s. However, the drastically different fragmentation patterns for Co, Ni, and Cu compared to the other metals can also be explained by their higher propensity for nitrogen (as opposed to oxygen) binding. Nevertheless, these results imply that directed fragmentation can be accomplished via careful selection of the cationizing agent.
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PMID:Divalent metal ion-peptide interactions probed by electron capture dissociation of trications. 1695 59


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