The effect of substance P (SP) and SP partial sequences on nerve fiber growth in tissue culture

Explants of the ganglion trigeminale (PNS) and of the telencephalon (CNS) from chick embryos were cultivated in MAXIMOW chambers in semisynthetic media in the presence of dipeptide fragments (Lys(Z)-Pro . HCl, Lys-Pro-2HBr, Arg-Pro-2HCl) and the heptapeptide (SP5-11) of substance P as well as the complete substance P (SP1-11). 1. Histological examination of the dipeptide-treated CNS explants indicates that the structure of outgrowth in vitro is changed. Fascicel were observed. A stimulation of nerve fibre extension did not take place. 2.1. In dipeptide-treated PNS cultures the index of areas covered by the explants increased. 2.2. The index of nerve fibre growth increased significantly. The stimulation was caused in multiplication of fibres. Only Lys(Z)-Pro . HCl presents a prolongation of neurites. 2.3. SP5-11 effects in no case the growth of nerve fibres. SP1-11 stimulated significantly the fibre regeneration. 3. The possible role of SP1-11 with different effects under in vitro conditions is discussed. Only the N-terminal dipeptides stimulate the growth of nerve fibres. The C-terminal SP5-11 is without effect. Finally it is stated that the best results in neuritic enlargement and neurogenesis can only be obtained by cultivation with SP1-11.     

The influence of substance P and its fragments on endogenous phosphorylation of synaptosomal membrane protein (synapsin) from cerebral cortex of rat brain.

1. The effects of substance P and its fragments and analogue of a C-terminal fragment on cyclic AMP-dependent phosphorylation of synapsin I in synaptosomal membranes (SM) from cerebral cortex were investigated. 2. SP(I-II) and SP(1-4) at 10(-3) M caused a marked stimulation of synapsin I phosphorylation. 3. A C-terminal fragment of SP (SP6-11) had no effect on phosphorylation of synapsin 1. 4. Analogue of C-terminal fragment [(Tyr8)SP6-11] at 10(-3) M distinctly inhibits phosphorylation of synapsin I. 5. These data suggest that SPI-II and its C- and N-terminal fragments have a modulator function against the phosphorylation of some rat brain proteins.     

Effect of substance P on nerve fiber regeneration in tissue culture

Explants of the ganglion trigeminale from chick embryos (PNS) and of the hippocampus from fetal rats (CNS) were cultivated in maximow chambers with growth medium or maintanance medium. Varied concentrations of substance P (SP . 3 CH3COOH . 4 H2O) were added. 1. The effect of substance P (SP) is related to concentration. In the presence of 10(-7)M SP in the growth medium and of 10(-4)M SP in the maintanance medium the cultivation of PNS cultures indicates positive results. These doses are suitable. 2. Within the first 24 hours in vitro SP stimulates the index of area in PNS cultures. The index of characterizes the relation of the outgrowth zone to the explant. In CNS cultures a significant difference of this effect was not observed. 3. The index of growth of nerve fibers may compare the test cultures with the control cultures. SP significantly increases the index of fiber growth in PNS cultures. A stimulation of CNS cultures was observed, significance was not found. 4. From the beginning of the cultivation with SP up to 48 hours in vitro the growth of nerve fibers significantly increases in the treated cultures in comparison with the control cultures. After this time the growth of nerve fibers decreased and a morphological conformity of test cultures and controls was observed. 5. The role of SP is discussed in specific activity on PNS tissue in vitro. The reactive neurons may be from the medio dorsal group of cells of the sensible ganglion.     

Modulation of isolation-induced fighting by N- and C-terminal analogs of substance P: evidence for multiple recognition sites

Substance P (SP) significantly reduced fighting in mice made aggressive by prolonged isolation. The N-terminal heptapeptide fragment SP (1-7) also reduced fighting. The C-terminal fragment SP(4-11) was without activity, while the shorter C-terminal fragment analog less than E-SP(7-11) significantly increased isolation-induced fighting. The aggression-enhancing effect of less than E-SP(7-11) was antagonized by naloxone, which by itself had no significant effect. The aggression-reducing effect of SP(1-11) was significantly enhanced by naloxone, while the effect of SP(1-7) was unchanged. These results demonstrate that a behavioral effect of SP may be duplicated by an N-terminal fragment of the SP molecule, and that peptide fragments or analogs of the N- and C-terminal portions of the SP molecule can exert opposing effects on a specific behavior. These findings represent a structure/activity relationship that is strikingly different from any previously described for SP. The differing effects of naloxone on N- and C-terminal fragment analogs suggest that these two effects may be mediated by different mechanisms.     

Measurement of Substance P Metabolites in Rat CNS

A procedure based on ion-exchange chromatography for chemical separation and radioimmunoassays for quantitation of substance P (SP), the SP(1-7), and C-terminal fragments, respectively, has been developed. The procedure allows the determination of these fragments in the presence of large (i.e., 50- to 100-fold) excess of parent compound. The chemical identity of isolated SP and fragments was studied with preparative electrophoresis on dilute agarose gel and with HPLC. The activity identified as SP(1-7) comigrated with the authentic standard whereas practically all activity isolated as C-terminal fragments comigrated with SP(5-11). The levels of C-terminal fragments in rat brain areas rich in SP and in spinal cord were 1-2% of those of parent compound. The levels of SP(1-7) were always higher, in the spinal cord markedly higher (three to five times). Postmortem storage of samples from brain and spinal cord indicated that SP(1-7) levels fell more rapidly than those of SP or C-terminal fragments.     

Effect of substance P on the ganglion trigeminale in tissue culture (author's transl)

Explants of the ganglion trigeminale from chick embryos were cultivated in Maximow chambers in the presence of 10-6...10-8 M substance P (SP . 3CH3COOH.4H2O). 1. In SP-treated cultures the index of areas covered by the explants was increased in shorttime tests. 2. The density of cells was related to the type of medio-dorsal (MD) and ventro-lateral (VL) neuroblasts. The density of SP-treated VL cells was not altered. The density of MD cells decreased. 3. The percentage of dark neuroblasts was decreased under the influence of SP. 4. A stimulation of VL neuroblasts did not take place. 5. The diameters of MD pericarya and the areas of MD cell nuclei and the areas of nuclei from nonneuronal cells increased. 6. The possible role of SP as a factor controlling In-vitro-processes is discussed.     

Alternative proteolytic processing of the arterivirus replicase ORF1a polyprotein: evidence that NSP2 acts as a cofactor for the NSP4 serine protease.

The C-terminal half of the replicase ORF1a polyprotein of the arterivirus equine arteritis virus is processed by a chymotrypsinlike serine protease (SP) (E. J. Snijder et al., J. Biol. Chem. 271:4864-4871, 1996) located in nonstructural protein 4 (nsp4). Three probable SP cleavage sites had previously been identified in the ORF1a protein. Their proteolysis explained the main processing products generated from the C-terminal part of the ORF1a protein in infected cells (E. J. Snijder et al., J. Virol. 68:5755-5764, 1994). By using sequence comparison, ORF1a expression systems, and site-directed mutagenesis, we have now identified two additional SP cleavage sites: Glu-1430 / Gly and Glu-1452 / Ser. This means that the ORF1a protein can be cleaved into eight processing end products: nsp1 to nsp8. By microsequence analysis of the nsp5 and nsp7 N termini, we have now formally confirmed the specificity of the SP for Glu / (Gly/Ser) substrates. Importantly, our studies revealed that the C-terminal half of the ORF1a protein (nsp3-8) can be processed by the SP following two alternative pathways, which appear to be mutually exclusive. In the majority of the nsp3-8 precursors the SP cleaves the nsp4/5 site, yielding nsp3-4 and nsp5-8. Subsequently, the latter product is cleaved at the nsp7/8 site only, whereas the newly identified nsp5/6 and nsp6/7 sites appear to be inaccessible to the protease. In the alternative proteolytic cascade, which is used at a low but significant level in infected cells, it is the nsp4/5 site which remains uncleaved, while the nsp5/6 and nsp6/7 sites are processed to yield a set of previously unnoticed processing products. Coexpression studies revealed that nsp3-8 has to interact with cleaved nsp2 to allow processing of the nsp4/5 junction, the first step of the major processing pathway. When the nsp2 cofactor is absent, the nsp4/5 site cannot be processed and nsp3-8 is processed following the alternative, minor pathway.     

A comparison of the effects of substance P and shorter analogues on the synaptosomal ATPases activities in the rat brain

The influence in vitro of SP and C-terminal fragments of analogues SP(5-11) (pyroGlu5, Tyr8); SP(6-11) (pyroGlu6, Tyr8); SP(6-11) (pyroGlu6, D-Phe7); SP(6-11) (pyroGlu6, D-Phe8) on the (Ca, Mg) and (Na, K) ATPases activities from synaptosomal membranes of cerebral cortex and hippocampus of rat brain were compared. The data obtained in this study indicate the following: 1. Substance P stimulates the activities of (Na, K) and (Ca, Mg) ATPases more effectively in synaptosomal membranes from hippocampus than cerebral cortex. 2. Heptapeptide SP(5-11) (pyroGlu5, Tyr8) causes a more distinct increase of (Ca, Mg) ATPase activity in cortical synaptosomal membranes than SP does. 3. The change of L-Phe conformation to D in position 7 in hexapeptide induces reduction of enzymes activities in hippocampus. 4. Especially important for the maintenance of biological activity of drugs is the replacement of Gln5 with pyroGlu6 and conformation of Phe residues. 5. SP and shorter analogues of fragments SP C-terminal SP regulate the active cation transport in synaptosomal membranes of cerebral cortex and hippocampus.     

Morphological studies of the effect of substance P partial sequences on nerve tissue culture

Explants of the telencephalon and of the ganglion trigeminale from chick embryos were cultivated in the presence of 10(-7) M of substance P partial sequences SP 1-2 (Arg-Pro . 22HCl) and SP3-4 (Lys(Z)-Pro . HCl or Lys-Pro . 2HBr). The morphology of the living outgrowth in fact the growth of nerve fibers, cell migration and proliferation was observed. SP1-2 and SP3-4 influenced particularly the morphology of pns cultures.     

Hydrolysis of substance p and neurotensin by converting enzyme and neutral endopeptidase

Angiotensin I converting enzyme (ACE) and neutral endopeptidase ("enkephalinase"; NEP), were purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln6-Phe7,-Phe8, and Gly9-Leu10 and neurotensin (NT) at Pro10-Tyr11 and Tyr11-Ile12. NEP hydrolyzed 0.1 mM SP, NT and their C-terminal fragments at the following rates (mumol/min/mg): SP1-11 = 7.8, SP4-11 = 11.7, SP5-11 = 15.4, SP6-11 = 15.6, SP8-11 = 6.7, NT1-13 = 2.9, and NT8-13 = 4.0. Purified ACE rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe8-Gly9 and Gly9-Leu10 to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl- dependent and inhibited by captopril. ACE released mainly C-terminal tripeptide from SP methyl ester, but only dipeptide from SP free acid. Modification of arginine residues in ACE with cyclohexanedione or butanedione similarly inhibited hydrolysis of SP, bradykinin and Bz-Gly-Phe-Arg (80-93%) indicating an active site arginine is required for hydrolysis of SP. ACE hydrolyzed NT at Tyr11-Ile12 to release Ile12-Leu13. SP, NT and their derivatives (0.1 mM) were cleaved by ACE at the following rates (mumol/min/mg): SP1-11 = 1.2, SP methyl ester = 0.7, SP free acid = 8.5, SP4-11 = 2.4, SP5-11 = 0.9, SP6-11 = 1.4, SP8-11 = 0, NT1-13 = 0.2, and NT8-13 = 1.3. Peptide substrates were used as inhibitors of ACE (substrate = FA-Phe-Gly-Gly) and NEP (substrate = Leu5-enkephalin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional Studies with Substance P Analogues: Effects of N-Terminal, C-Terminal, and C-Terminus-Extended Analogues of Substance P on Nicotine-Induced Secretion and Desensitization in Cultured Bovine Adrenal Chromaffin Cells

Abstract: Substance P (SP) and SP analogues, including C-terminal, N-terminal, and C-terminus-extended analogues, have been investigated for their ability to modulate nicotine-induced secretion from bovine adrenal chromaffin cells in culture. Secretion was monitored by measuring the release of endogenous catecholamines by electrochemical detection following separation on HPLC and the release of endogenous ATP with an on-line luciferin-luciferase bioluminescence technique. SP is known to have the following two effects on nicotine-induced secretion of catecholamines (see Livett and Zhou, 1991): inhibition of the nicotinic response and protection against nicotinic desensitization. Secretion induced by 10^-5M nicotine was inhibited 70-80% by SP, SP-methyl ester, and the C-terminus-extended analogue SP-Tyr¹²-NH₂, 65% by (Ala³)SP-NH₂, 45% by the C-terminal analogue SP(4-11), and 20 and 5% by the N-terminal analogues SP(1-7) and SP(1-5), respectively, when these peptides were present at 3 ×; 10^-5M concentrations. The order of potency was SP = SP-methyl ester = SP-Tyr¹²-NH₂ > (Ala³)SP-NH₂ > SP(4-11) > SP(1-7) > SP(1-5). SP, SP-methyl ester, and (Ala³)SP-NH₂ protected against nicotinic desensitization by 40-55%, and SP(4-11) protected by 20% (all at 3 ×; 10^-5M). In contrast, the N-terminal analogues SP(1-7) and SP(1-5) and the C-terminus-extended analogue SP-Tyr¹²-NH₂ at 3 × 10^-5M did not protect against nicotinic desensitization. Cyclo-SP(3-9), Ac-SP(3-9)-NH₂, SP(3-9), and SP(3-6) had neither inhibitory nor facilitatory effects on secretion. Of the 20 SP analogues extended at the C terminus by one amino acid, there were only three that protected against nicotinic desensitization, whereas the majority inhibited nicotine-evoked catecholamine secretion. The present work indicates that for inhibition of nicotine-evoked secretion, both the C terminus and N terminus of SP are necessary. For the protection against nicotine-induced desensitization, the C terminus of SP is important. This suggests that the two mechanisms, inhibition of nicotine-evoked secretion and protection against nicotinic desensitization, are regulated independently.     

Common functional elements of Drosophila melanogaster seminal peptides involved in reproduction of Drosophila melanogaster and Helicoverpa armigera females

Sex peptide (SP) and Ductus ejaculatorius peptide (Dup) 99B are synthesized in the retrogonadal complex of adult male Drosophila melanogaster, and are transferred in the male seminal fluid to the female genital tract during mating. They have been sequenced and shown to exhibit a high degree of homology in the C-terminal region. Both affect subsequent mating and oviposition by female D. melanogaster. SP also increases in vitro juvenile hormone (JH) biosynthesis in excised corpora allata (CA) of D. melanogaster and Helicoverpa armigera. We herein report that the partial C-terminal peptides SP(8-36) and SP(21-36) of D. melanogaster, and the truncated N-terminal SP(6-20) do not stimulate JH biosynthesis in vitro in CA of both species. Both of these C-terminal peptides reduce JH-III biosynthesis significantly. Dup99B, with no appreciable homology to SP in the N-terminal region, similarly lacks an effect on JH production by H. armigera CA. In contrast, the N-terminal peptides - SP(1-11) and SP(1-22) - do significantly activate JH biosynthesis of both species in vitro. We conclude that the first five N-terminal amino acid residues at the least, are essential for allatal stimulation in these disparate insect species. We have previously shown that the full-length SP(1-36) depresses pheromone biosynthesis in H. armigera in vivo and in vitro. We now show that full-length Dup99B and the C-terminal partial sequence SP(8-36) at low concentrations strongly depress (in the range of 90% inhibition) PBAN-stimulated pheromone biosynthesis of H. armigera. In addition, the N-terminal peptide SP(1-22), the shorter N-terminal peptide SP(1-11) and the truncated N-terminal SP(6-20) strongly inhibit pheromone biosynthesis at higher concentrations.     

Isolation of cDNA for a Xenopus sperm-specific basic nuclear protein (SP4) and evidence for expression of SP4 mRNA in primary spermatocytes

A cDNA library was prepared in lambda gt 11 from poly(A)+ mRNA isolated from a pure population of Xenopus round spermatids and screened with an antibody against SP3-5 (sperm-specific proteins) of Xenopus sperm. Positive clones were sequenced and an arginine-rich clone, designated pXSP531, was obtained. The 473-nucleotide sequence of pXSP531 contained an open reading frame of 237 nucleotides which was preceded by a 5' untranslated region of 67 nucleotides. The 3' untranslated region contained 149 nucleotides, including a consensus polyadenylation signal (AAATAAAA). Twenty nucleotides of a poly(A) tail was contained in the pXSP531. SP3-5 were separated from each other by reverse-phase chromatography and sequenced. The amino acid sequence of the peptide fragments which were obtained by digestion of SP4 with V8 protease and separated by reverse-phase chromatography was identical to the sequence of the N-terminal 43 and C-terminal 15 amino acids deduced from the nucleotide sequence of pXSP531. This result demonstrates that pXSP531 encodes SP4. Northern hybridization of RNA extracted from primary spermatocytes and round spermatids on Days 0 and 6 with SP4 cDNA probe (pXSP531) showed that SP4 mRNA is present both in primary spermatocytes and in round spermatids as is protamine mRNA in the rainbow trout. The size of the SP4 mRNA in round spermatids on Day 0 was longer by 60 nucleotides compared to that in primary spermatocytes and that in spermatids on Day 6 was shorter by 30 nucleotides compared to that on Day 0. These size differences were due to differences in the length of the poly(A) tracts because digestion of poly(A) with ribonuclease H resulted in the shortening of mRNA to the same size for three stages.     

A cAMP-responsive element regulates expression of the mouse steroid 11 beta-hydroxylase gene

In Y1 mouse adrenocortical tumor cells, expression of steroid 11 beta-hydroxylase (11 beta-OHase) is stimulated by cAMP following a delay of 4-6 h. Our results demonstrate that a cAMP-responsive element (CRE) within the 11 beta-OHase promoter region is a major determinant of this induction. The 5'-flanking sequences from the mouse 11 beta-OHase gene were placed in front of a human growth hormone reporter gene and transfected into Y1 cells. Treatment of transfected cells with 8-bromo-cAMP increased expression directed by the 11 beta-OHase 5'-flanking region by 3.8-fold. In 5'-deletion analyses, 123 base pairs of 5'-flanking sequences were sufficient for cAMP induction, whereas cAMP treatment did not affect expression of a plasmid with only 40 base pairs of 5'-flanking sequence. Within these 123 base pairs, a region from -56 to -49 matched 7 of 8 bases comprising the consensus sequence for the CRE. 11 beta-OHase 5'-flanking sequences from -65 to -42, including the CRE-like sequence, conferred cAMP inducibility to promoters from the thymidine kinase and chorionic gonadotropin alpha-subunit genes. DNase I footprinting and Southwestern blotting analyses demonstrated that the protein which interacted with the CRE in the 11 beta-OHase promoter region was similar to the CRE-binding protein associated with other cAMP-regulated genes. Together, these results suggest that an interaction between the 11 beta-OHase CRE and CRE-binding protein mediates cAMP induction of the 11 beta-OHase gene.     

Effects of tachykinins on the electrical activity of isolated canine tracheal epithelium: an exploratory study

Substance P (SP) and other tachykinins altered the potential differences (P.D.) and resistances (omega) of open-circuited epithelial preparations. (1) The effects observed were critically dependent on the side to which the peptides were added. Luminal addition of SP (5 x 10(-7) M) produced within 8-20 s, a rapid decrease in P.D. (dip) followed by an increase that peaked transiently and declined. Serosal addition led to an increase in P.D. after a longer lag (40-90 s). In both cases, resistance decreased. (2) Low concentrations of SP (5 x 10(-12) M) elicited only an increase in P.D., the dip appearing at concentrations 50-100-fold higher, indicating perhaps receptors with different affinities. (3) Changes in P.D. and resistance were seen on luminal addition of physalaemin, eledoisin, kassinin, alpha-neurokinin, neuromedin K and C-terminal SP fragments larger than 5 amino acids. No responses were seen with SP tetrapeptide, SP 9-11, bombesin, litorin, neurotensin, dynorphin. The sequence Phe-X-Gly-Leu-Met-NH2 thus seems necessary to elicit changes in P.D. and resistance. (4) As with SP, low doses of physalaemin, eledoisin, kassinin elicited only an increase in PD, the dip appearing with higher concentrations.     

The effects of the main body cations on the peptidase(s) activity against pyroGlu6[125I-Tyr8]SP6-11 in the nuclear and synaptosomal fraction of the cortex and hippocampus of rat brain

1. Peptidase(s) activity of nuclear and synaptosomal fraction from cortex and hippocampus of rat brain against pyroGlu6[125I-Tyr8]SP6-11 was evaluated in different concentration of Ca2+, Mg2+, K+ and Na+ in about "isotonic" conditions. 2. The effects of studied ions on the peptidase activities forming N-terminal and C-terminal fragments are different especially in synaptosomes of both areas. 3. The differences of ionic requirements for N- and C-forming activities are particularly relevant for Ca2+ at the cortex and K+ at the hippocampus. 4. Ca2+ activate forming of N-terminal fragments in the nuclear fraction whereas inhibit it in synaptosomes from both areas. 5. The ionic requirements for C-terminal fragments' formation in synaptosomes of both areas are contradictory.     

Primary structure of mannuronate lyases SP1 and SP2 fromTurbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability

The complete amino acid sequences of two isoforms, SP1 and SP2, of mannuronate lyase from a wreath shell,Turbo cornutus, were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.     

Primary structure of mannuronate lyases SP1 and SP2 fromTurbo cornutus and involvement of the hydrophobic C-terminal residues in the protein stability

The complete amino acid sequences of two isoforms, SP1 and SP2, of mannuronate lyase from a wreath shell,Turbo cornutus, were determined to elucidate amino acid residues responsible for causing the more stable protein conformation of SP2. The sequences of the two isoforms were identical except for two hydrophobic C-terminal amino acid residues of SP2, Ile and Leu, which were additionally attached to Thr of the C-terminal residue of SP1 (253 residues in total). The molecular weight of SP2 was calculated to be 28,912 from the amino acid sequence data. Two disulfide bond cross-linkages were found to be between 106 and 115 and between 145 and 150, and a partially buried single SH group was located at 236. A carbohydrate chain that consisted of 3 GlcNAc, 3 Fuc, and 1 Man was anchored on Asn-105 in a typical carbohydrate-binding motif of Asn-X-Ser. This is the first evidence of the primary structure of mannuronate lyase, and no significant homology of the amino acid sequence among other proteins was found. The C-terminal truncated SP2, which was produced by digestion with carboxypeptidase Y and corresponded structurally to SP1, showed a thermal stability identical to that of SP1. These results indicate that the higher stability of SP2 than SP1 arises from the presence of the C-terminal two hydrophobic amino acid residues.