Characterization of central and peripheral effects of septide with the use of five tachykinin NK1 receptor antagonists in the rat.
(9/471)
1. Effects of two tachykinin NK1 receptor selective agonists (septide and [Sar9, Met(O2)11]SP) were compared on the increases in mean arterial pressure (MAP), heart rate (HR) and motor behaviour following intracerebroventricular (i.c.v.) administration in unanaesthetized rat, and on the vascular permeability increases to intradermal (i.d.) injection in the anaesthetized rat. Moreover, five tachykinin NK1 receptor selective antagonists (LY303870, LY306740, LY303241, SR140333 and RP67580) were tested against the two agonists to compare their pharmacological profile. 2. [Sar9, Met(O2)11]SP and septide (10-100 pmol per rat, i.c.v.) were equipotent in increasing MAP and HR, yet they had dissimilar time-course. Both agonists increased dose-dependently face washing and sniffing while [Sar9, Met(O2)11]SP was the sole to produce grooming, septide was more potent than [Sar9, Met(O2)11]SP (6.5-650 pmol) in increasing vascular permeability. 3. For most centrally mediated responses, LY303870 and RP67580 were significantly more potent in inhibiting septide than [Sar9, Met(O2)11]SP. In some parameters, greater blockade was achieved when antagonists (particularly LY306740) were given 1 h instead of 10 min prior to i.c.v. septide. 4. All antagonists except LY303241 blocked dose-dependently the increases in vascular permeability to equipotent doses of [Sar9, Met(O2)11]SP and septide. LY303870 and LY306740 were more potent against septide. 5. The antagonism afforded by LY303870, LY306740 and LY303241 was stereoselective and only SR140333 was found to cause central and peripheral non specific effects. 6. The data confirm a distinct pharmacological profile for septide in vivo. RP67580 and LY306740 are currently the most valuable tachykinin NK1 receptor antagonists for in vivo studies in rat. (+info)
Post-translational modifications of the insect sulfakinins: sulfation, pyroglutamate-formation and O-methylation of glutamic acid.
(10/471)
We identified and chemically characterized the two major forms of sulfakinins from an extract of 800 corpora cardiaca/corpora allata complexes of the American cockroach, Periplaneta americana. Bioactivity during the purification was monitored by measuring heart beat frequency in a preparation in situ. By Edman degradation analysis and MS, these main forms were identified as having the primary structures Pea-SK [EQFDDY(SO(3)H)GHMRFamide] and Lem-SK-2 [pQSDDY(SO(3)H)GHMRFamide]. The sulfation was confirmed by UV, MS and peptide synthesis. In addition, post-translationally modified sulfakinins of both major forms were isolated and identified. Firstly, nonsulfated forms of these peptides are present in considerable amounts in the corpora cardiaca/allata. Secondly, the N-terminally blocked Pea-SK and the nonblocked Lem-SK-2 occur naturally in neurohaemal release sites. Thirdly, modified Pea-SK with O-methylated glutamic acid occurs which is not an artefact of peptide purification. The major forms of the sulfakinins were shown to be highly active on both the heart and hindgut with threshold concentrations of approximately 5 x 10(-10) M (heart) and 2 x 10(-9) M (hindgut). (+info)
A head-activator binding protein is present in hydra in a soluble and a membrane-anchored form.
(11/471)
The neuropeptide head activator plays an important role for proliferation and determination of stem cells in hydra. By affinity chromatography a 200 kDa head-activator binding protein, HAB, was isolated from the multiheaded mutant of Chlorohydra viridissima. Partial amino acid sequences were used to clone the HAB cDNA which coded for a receptor with a unique alignment of extracellular modules, a transmembrane domain, and a short carboxy-terminal cytoplasmic tail. A mammalian HAB homologue with identical alignment of these modules is expressed early in brain development. Specific antibodies revealed the presence of HAB in hydra as a transmembrane receptor, but also as secreted protein, both capable of binding head activator. Secretion of HAB during regeneration and expression in regions of high determination potential hint at a role for HAB in regulating the concentration and range of action of head activator. (+info)
Post-translational regulation of AP-1 transcription factor DNA-binding activity in the rat conceptus.
(12/471)
Activator protein-1 (AP-1) transcription factor DNA binding is induced during transient oxidative stress in the midorganogenesis rat conceptus in culture. L-2-Oxothiazolidine-4-carboxylate (OTC), a cysteine prodrug, prevented oxidative stress and the induction of AP-1 binding activity in the embryo but not in the yolk sac. Because AP-1 activity may be a significant determinant of developmental outcome after insult, we investigated the regulation of AP-1 activity in the conceptus. Supershift assays indicated that basal AP-1 binding in the embryo was due primarily to JunD, whereas in the yolk sac c-Jun and JunD were important. Under oxidative stress, c-Fos and c-Jun contributed to the AP-1 binding in the embryo; in the yolk sac, a c-Fos-shifted complex emerged. OTC protection from oxidative stress did not change the AP-1 composition, suggesting that increased AP-1 activity was due to post-translational modifications. Changes in AP-1 activity in embryos under oxidative stress or with OTC protection were not the result of alterations in the net phosphorylation state of Fos or Jun proteins or of changes in activities of the extracellular signal-regulated kinases 1 and 2 or stress-activated protein kinases. However, immunodepletion of redox factor 1 (Ref-1), a nuclear factor that promotes AP-1 binding, eliminated AP-1 activity from embryonic nuclear extracts under both basal and oxidative stress conditions. Therefore, Ref-1 plays a critical role in regulating AP-1 activity in the conceptus; it is plausible that Ref-1-mediated modulation of the AP-1 stress response is a determinant of embryonic fate. (+info)
The effects of the phyllolitorin analogue [desTrp(3), Leu(8)]phyllolitorin on scratching induced by bombesin and related peptides in rats.
(13/471)
Bombesin along with several closely related neuropeptides elicit scratching behavior when administered centrally. The first part of the study was designed to determine the antagonistic effects of a novel phyllolitorin analogue [desTrp(3),Leu(8)]phyllolitorin (DTP) on scratching induced by three peptides (bombesin, neuromedin-C, and [Leu(8)]phyllolitorin). In addition, the binding affinity of each peptide for the bombesin receptor site was determined. DTP (30 microg) inhibited scratching induced by these peptides, but unlike the peptides, DTP had no affinity for the bombesin site, thereby suggesting that DTP is displaying physiological antagonism through an unknown mechanism. (+info)
Glutamate transport asymmetry and metabolism in the functioning kidney.
(14/471)
Renal glutamate extraction in vivo shows a preference for the uptake of D-glutamate on the antiluminal and L-glutamate on the luminal tubule surface. To characterize this functional asymmetry, we isolated rat kidneys and perfused them with an artificial plasma solution containing either D- or L-glutamate alone or in combination with the system X-AG specific transport inhibitor, D-aspartate. To confirm that removal of glutamate represented transport into tubule cells, we monitored products formed as the result of intracellular metabolism and related these to the uptake process. Perfusion with D-glutamate alone resulted in a removal rate that equaled or exceeded the L-glutamate removal rate, with uptake predominantly across the antiluminal surface; L-glutamate uptake occurred nearly equally across both luminal and antiluminal surfaces. Thus the preferential uptake of D-glutamate at the antiluminal and L-glutamate at the luminal surface confirms the transport asymmetry observed in vivo. Equimolar D-aspartate concentration blocked most of the antiluminal D-glutamate uptake and a significant portion of the luminal L-glutamate uptake, consistent with system X-AG activity at both sites. D-Glutamate uptake was associated with 5-oxo-D-proline production, whereas L-glutamate uptake supported both glutamine and 5-oxo-L-proline formation; D-aspartate reduced production of both 5-oxoproline and glutamine. The presence of system X-AG activity on both the luminal and antiluminal tubule surfaces, exhibiting different reactivity toward L- and D-glutamate suggests that functional asymmetry may reflect two different X-AG transporter subtypes. (+info)
Intracisternal PYY increases gastric mucosal resistance: role of cholinergic, CGRP, and NO pathways.
(15/471)
The influence of intracisternal injection of peptide YY (PYY) on gastric lesions induced by ethanol was studied in urethan-anesthetized rats. Gastric lesions covered 15-22% of the corpus as monitored 1 h after intragastric administration of 45% ethanol (5 ml/kg) in intracisternal vehicle control groups. PYY, at doses of 23, 47, or 117 pmol 30 min before ethanol, decreased gastric lesions by 27%, 63%, and 59%, respectively. Thyrotropin-releasing hormone (TRH) receptor antisense oligodeoxynucleotide pretreatment (intracisternally, 48 and 24 h before intracisternal PYY) did not influence the gastroprotective effect of intracisternal PYY (47 pmol) but abolished that of intracisternal TRH analog RX-77368 (4 pmol). RX-77368 (2.6 pmol) and PYY (6 pmol) were ineffective when injected intracisternally alone but reduced ethanol lesions by 44% when injected simultaneously. Atropine (subcutaneously), the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37) (intravenously), or the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, intravenously) completely abolished the gastroprotective effect of intracisternal PYY (47 pmol), whereas indomethacin (intraperitoneally) had no effect. The L-NAME action was reversed by L-arginine but not by D-arginine (intravenously). These results suggest that intracisternal PYY acts independently of medullary TRH to decrease ethanol-induced gastric lesions. The PYY action involves vagal cholinergic-mediated CGRP/NO protective mechanisms. (+info)
Human TRH-degrading ectoenzyme cDNA cloning, functional expression, genomic structure and chromosomal assignment.
(16/471)
Thyrotropin-Releasing Hormone (TRH) is an important extracellular signal substance that acts as a stimulator of hormone secretion from adenohypophyseal target cells and fulfills many criteria for the function of a neuromodulator/neurotransmitter within the central and peripheral nervous systems. The inactivation of TRH-signals is catalysed by a highly specific ectoenzyme. Here, we characterize the human TRH-degrading ectoenzyme (TRH-DE) by primary sequence, functional expression, genomic structure and chromosomal assignment. By screening a cDNA-library constructed from human lung, 5.7 kb of cDNA were identified. The longest open reading frame predicts a type II integral membrane protein of 117 kDa. The extracellular domain contains the HEXXH + E motif that is characteristic of a certain family of Zn-dependent aminopeptidases. Within this family, the sequences of human and rat TRH-DE reveal an unusual high degree of conservation (96% identical residues). Specific enzymatic activity was observed after transfecting COS-7 cells with human TRH-DE cDNA yielding a Km for TRH hydrolysis of 29.7 microM. Northern blot analysis demonstrated a restricted tissue distribution with highest transcript levels in the brain. Using fluorescent in situ hybridization with the cDNA and a genomic lambda clone, respectively, we localized the TRH-DE gene to the long arm of human chromosome 12. Five independent P1 artificial chromosome clones were required to span the complete cDNA sequence and revealed that it is distributed on 19 exons. Interspecies Southern analysis suggests that the gene is present as a single copy in human, monkey, rat, mouse, dog, bovine, rabbit and chicken DNA. All of these data further the notion that the TRH-DE is not an ordinary enzyme but a specific neuropeptidase that has been highly conserved among species. (+info)