Peptides (v.22, #7)
Structure-activity relationships of novel peptides related to the antiarrhythmic peptide AAP10 which reduce the dispersion 1 1 Abbreviations: DCC: Dicyclohexylcarbodiimide; DIPEA: Diisopropylethylamine; DMF: N,N-Dimethylformamide; DPPAD: iphenylphosphorazide; HOBt: 1-Hydroxybenzotriazole; NMM: N-Methylmorpholine; SAR: Structure activity relationship; TFA: Trifluoroaceticacid. of epicardial action potential duration by Rajiv Grover; Stefan Dhein (1011-1021).
We report the first study on short peptide structure-activity relationships (SAR) for the antiarrhythmic peptide AAP10 and its putative receptor. Synthetic improvements on the natural antiarrhythmic peptide AAPnat (H-Gly-Pro-Hyp-Gly-Ala-Gly) isolated from bovine atria led us to the synthesis of our lead molecule AAP10 (H-Gly-Ala-Gly-Hyp-Pro-Tyr-NH2) which reduces dispersion of epicardial potential duration and acts antiarrhythmically in isolated rabbit hearts. The aim of our study was to elucidate structure-activity relationships for AAP10 based on Langendorff experiments and molecular modeling. Mutation of the amino acid sequence led to 11 different peptides which were tested analogous to the lead molecule. Among these new synthetic peptides various including the cyclopeptide cAAP10RG, cyclo[CF3C(OH)-Gly-Ala-Gly-Hyp-Pro-Tyr] showed promising activities. (supported by the DFG and Köln-Fortune)
Keywords: Antiarrhythmic peptide; Gap Junction; Cardiac ischemia; Structure-activity relationships;
Effects of cod bradykinin and its analogs on vascular and intestinal smooth muscle of the Atlantic cod, Gadus morhua by Fatemeh Shahbazi; J. Michael Conlon; Susanne Holmgren; Jörgen Jensen (1023-1029).
The effects of [Arg0,Trp5,Leu8]-BK (cod [Arg0]BK) on vascular preparations from branches of the cod celiac artery and on longitudinal smooth muscle preparations from the cod intestine were investigated. Cod [Arg0]BK (3 × 10−8 M) caused a relaxation of the celiac artery precontracted with adrenaline. The relaxation was abolished by the cyclooxygenase inhibitor indomethacin, suggesting that the effect is mediated through the release of prostaglandins, but there was no evidence for the involvement of leukotrienes or nitric oxide in the response. In the intestinal preparations, cod [Arg0]BK produced concentration-dependent contractions (pD2 = 8.28 ± 0.16). Experiments with N-terminally and C-terminally truncated analogs and with alanine-substituted analogs of cod [Arg0]BK demonstrate that the central amino acid Gly4 and the C-terminal amino acids Leu8 and Arg9 are the most important in determining the conformation of the peptide that interacts with the receptor. The results indicate that the ligand binding properties of the cod BK receptor are considerably different from the receptor present in trout tissues and may resemble those of the mammalian B2 receptor more closely.
Keywords: Bradykinin; Vasorelaxation; Celiac artery; Cod intestine; Receptor;
Comparison of neurokinin SP with diazepam in effects on memory and fear parameters in the elevated T-maze free exploration paradigm by M.B. Echeverry; R.U. Hasenöhrl; J.P. Huston; C. Tomaz (1031-1036).
The elevated T-maze was combined with a free exploration protocol, which, in contrast to the conventional procedure, dispenses with handling of the animals during the experimental sessions. This allows measurement of fear indexes derived from the elevated plus-maze as well as assessment of acquisition of open arm avoidance and open arm escape in one continuous session. Retention of the different fear-responses is measured 72 h later without drug treatment. In order to assess the effects of two known anxiolytics in this paradigm, rats received an IP injection of diazepam (1 to 4 mg/kg), substance P (5 to 500 μg/kg) or vehicle (1 ml/kg) and were tested on the T-maze for 5 min. Diazepam elevated open arm activity, indicative of an anxiolytic effect. The drug also increased the latency to escape from the open arms, but did not significantly affect acquisition of open arm avoidance. During the retention trial, diazepam in higher doses impaired the performance of both fear-responses, suggestive of an anterograde amnesic effect. Substance P did not influence acquisition and retention of open arm avoidance and escape. However, in high doses, the peptide increased the sojourn time in the central arena of the maze, indicating reduced fear and, hence, a dissociation between anxiolytic and amnesic effects. The present findings demonstrate that the elevated T-maze free exploration paradigm is sensitive to anxiolytic and memory-modulating effects of drugs.
Keywords: Benzodiazepines; Neurokinin; Elevated T-maze; Conditioned fear; Unconditioned fear; Emotional reactivity;
Antidepressant-type effect of the NK3 tachykinin receptor agonist aminosenktide in mouse lines differing in endogenous opioid system activity by I Panocka; M Massi; I Lapo; T Swiderski; M Kowalczyk; B Sadowski (1037-1042).
The influence of the tachykinin NK3 receptor agonist, aminosenktide on the immobility in the forced swimming test was studied in mouse lines selectively bred for divergent magnitudes of stress-induced analgesia. The high analgesia (HA) line is known to display enhanced, and the low analgesia (LA) line displays reduced activity of the opioid system. Aminosenktide at doses of 125 μg/kg or 250 μg/kg intraperitoneally (IP) reduced, in naltrexone-reversible manner, the immobility more of opioid receptor-dense HA than of unselected mice, but was ineffective in the opioid receptor-deficient LA line. The effect of aminosenktide was quite similar to the antiimmobility action of desipramine (10 mg/kg IP), a prototypic antidepressant agent. None of the compounds increased animals’ locomotion as found with an open field test; therefore their antiimmobility effect cannot be attributed to a change in general motility. The results claim that aminosenktide causes an antidepressant effect, and endogenous opioids are involved in this process.
Keywords: Forced swimming test; NK3 receptors; Antidepressant action; Naltrexone; Mice;
Anxiogenesis induced by nitric oxide synthase inhibition and anxiolytic effect of melanin-concentrating hormone (MCH) in rat brain. by M.E Monzón; M.M Varas; S.R De Barioglio (1043-1047).
In this study, the involvement of nitric oxide (NO) in the mechanism of anxiety was investigated. The rats received an intraamygdaline or intrahippocampal injection of the nitric oxide synthase inhibitor, NG-nitro-l-arginine (L-NOARG), and were then tested in the plus-maze test. L-NOARG induced a decrease in the time spent by rats in the open arms. Conversely, the administration of the melanin-concentrating hormone (MCH) into these structures increased the number of entries into the open arms as well as the time spent on them. MCH injected in rats pretreated with L-NOARG also was able to revert the anxiogenic effects of L-NOARG in amygdala.
Asphyxia-induced release of α-melanocyte-stimulating hormone in newborn pigs by József Kovács; János Julesz; Mónika V. Mogyoróssy; Mária A. Deli; Csongor S. Ábrahám; Miklós Vecsernyés (1049-1053).
Asphyxia and reperfusion induced changes in the plasma and cerebrospinal fluid (CSF) concentrations of α-melanocyte-stimulating hormone (α-MSH) were studied in newborn pigs using a specific radioimmunoassay technique. Cardiovascular and metabolic failure induced by neonatal asphyxia resulted in a 3-fold, significant (P < 0.05) increase in plasma α-MSH levels, whereas the hormone concentration in CSF was significantly (P < 0.05) reduced by 50% during postasphyxial reperfusion. Our data indicate an asphyxia-induced release of α-MSH, and suggest a discordant regulation of plasma and CSF concentrations in newborn pigs.
Keywords: Asphyxia; Cerebrospinal fluid; α-melanocyte-stimulating hormone; Newborn; Plasma; Resuscitation;
An oxidation resistant radioligand for corticotropin-releasing factor receptors by Iman Q. Assil; Mansur E. Shomali; Abdul.B. Abou-Samra (1055-1061).
The methionine residues in Tyr-corticotropin-releasing factor (CRF) and Tyr-sauvagine radioligands are subject to oxidation, which renders them biologically inactive. Therefore [Tyr0, Gln1, Leu17]sauvagine (YQLS), in which the methionine was replaced with leucine was synthesized and labeled with 125Iodine using chloramine-T. Mass spectroscopy revealed that chloramine-T-treatment did not oxidize YQLS. 125I-YQLS bound with high affinity to cells expressing the murine CRF receptor 1 (CRFR1), CRF receptor 2 (CRFR2), and the mouse brain regions known to express both CRF receptors. 125I-YQLS chemically cross-linked to CRFR1. In conclusion, 125I-YQLS is oxidation-resistant, high affinity radioligand that can be chemically cross-linked to the CRF receptors.
Keywords: Corticotropin-releasing factor receptors; Radioligands; Sauvagine; Urotensin-I; Chemical crosslinking; Disuccinimidyl suberate;
Effects of forebrain microinjection of cholecystokinin on dopamine cell firing rate by Margaret E. Hamilton; F.Karl Weddige; Arthur S. Freeman (1063-1069).
In anesthetized rats, midbrain dopamine (DA) neuronal firing rate was differentially sensitive to focal brain microinjection of cholecystokinin peptides (CCK-4 and CCK-8) and N-methyl-D-aspartate (NMDA) into nucleus accumbens, amygdala and prefrontal cortex. Whereas changes in DA neuronal firing rate were frequently observed in response to intra-amygdalar microinjection of CCK peptides, NMDA was most effective in eliciting changes in DA neuronal activity following intra-accumbal microinjection. Thus, stimulation of amygdalar CCK receptors and accumbal excitatory amino acid receptors may participate in the afferent regulation of midbrain DA neuronal function.
Keywords: Dopamine electrophysiology; CCK receptors; N-methyl-D-aspartate; Excitatory amino acids;
Colocalization of GABA, enkephalin and neuropeptide Y in the tectum of the green frog Rana esculenta by Tamás Kozicz; Gyula Lázár (1071-1077).
The colocalization of GABA, enkephalin and neuropeptide Y immunoreactivity in neurons in the pretectal area and in the mesencephalic tectum of the green frog (Rana esculenta) was studied. Several Met-enkephalin immunoreactive perikarya were found in layer 6 of the tectum and every third of these neurons showed GABA-ir as well. Colocalization of GABA and NPY could also be shown in half of the neuropeptide Y immunopositive cells in the 6th layer of the tectum, but only a few cells were double stained in layers 9 and 4. In the pretectal area no colocalization of the investigated peptides and GABA was found.
Keywords: Amphibian visual system; Colocalization of peptides; Mesencephalic tectum; Pretectum; Immunohistochemistry;
Antinociceptive effect of somatostatin microinjected into caudate putamen by Roman Tashev; Stiliana Belcheva; Kiril Milenov; Iren Belcheva (1079-1083).
The effects of somatostatin microinjected bilaterally and unilaterally (left or right) at a dose of 10, 50 and 100 ng into the caudate putamen of male Wistar rats on nociception (analgesy-meter test) were studied. Somatostatin injected into caudate putamen resulted in analgesia. Bilateral microinjections of somatostatin significantly increased the pain threshold in a dose-dependent manner, i.e. somatostatin exerted antinociceptive effect. The pain threshold after left-side microinjections was significantly higher than that after injections into right-side. These findings suggest antinociceptive and asymmetric effects of somatostatin on pain in the caudate putamen.
Keywords: Somatostatin; Caudate putamen; Nociception; Analgesia; Asymmetry; Rat;
Identification of neuropeptide FF-related peptides in rodent spinal cord 1 1 Abbreviations: CNS: central nervous system; i.c.v.: intracerebroventricular; μHPLC/ESI MS: micro HPLC coupled on-line to electrospray mass spectrometry; RIA: radioimmunoassay; RP-HPLC: reverse phase high pressure liquid chromatography; SIM: single ion monitoring; SPC: subtilisin-like proprotein convertase. TPPII: tripeptidyl peptidase II. by Elisabeth Bonnard; Odile Burlet-Schiltz; Bernard Francés; Honoré Mazarguil; Bernard Monsarrat; Jean-Marie Zajac; Anne Roussin (1085-1092).
Peptides which should be generated from the neuropeptide FF (NPFF) precursor were identified in mouse and rat spinal cord, by using reverse phase high pressure liquid chromatography with radioimmunoassay and electrospray mass spectrometry detection. In both species, two octapeptides, NPFF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) and NPSF (Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe-amide) were identified but a longer peptide NPA-NPFF (Asn-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was present at the highest concentration in rat spinal cord. In mouse, the homologous peptide, SPA-NPFF (Ser-Pro-Ala-Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide) was not detected. Both peptides NPFF and NPSF reverse morphine-induced analgesia in the tail flick test. Our data reveal species differences in the maturation of NPFF precursor.
Keywords: Neuropeptide FF; Spinal cord; Mass spectrometry; Radioimmunoassay; Mouse; Rat;
MALDI-PSD-MS analysis of the phosphorylation sites of caseinomacropeptide by Gert H. Talbo; Detlev Suckau; Marina Malkoski; Eric C. Reynolds (1093-1098).
Caseinomacropeptide (CMP) is a 64 amino acid polypeptide corresponding to κ-casein 106–169. CMP naturally exists in several forms due to extensive posttranslational modifications including glycosylation and phosphorylation. The aglycosylated, phosphorylated form of CMP has been shown to exhibit antibacterial activity. The aim of this study was to use matrix assisted laser desorption/ionization post source decay mass spectrometry (MALDI-PSD-MS) to identify the phosphorylation sites in the CMP sequence. CMP was isolated from a chymosin digest of casein by HPLC and then digested with endoproteinase Glu-C to generate peptides suitable for MALDI-PSD-MS analysis. This analysis showed that CMP is fully phosphorylated at Ser149 and only partially phosphorylated at Ser127. Dehydroalanyl residues corresponding to the phosphoserines of CMP were detected upon MALDI-PSD-MS analysis suggesting that the phosphoryl bond in phosphoserine is very labile during PSD analysis such that the phosphoryl group may be lost before backbone fragmentation.
Keywords: Phosphopeptide; Caseinomacropeptide; Matrix assisted laser desorption; Ionization post source decay mass spectrometry;
Neuropeptides interact with glycolipid receptors A surface plasmon resonance study by Tania Valdes-Gonzalez; Junichi Inagawa; Tatsuo Ido (1099-1106).
Using Surface Plasmon Resonance (SPR) we investigated the interaction of seven neuropeptides with different characteristics and β-amyloid (Aβ42) peptide, with membranes containing gangliosides. A wide range of affinities characterized the bindings (KD = 10−3– 10−7 M), following the scheme: for GM1, Aβ42 > DYN > SP = GAL = SOM = BRD > OXY = ENK; for GD1a, Aβ42 = DYN = GAL > SP = SOM = BRD = OXY > ENK and for GT1b, Aβ42 > DYN > SP = GAL > SOM = BRD = OXY > ENK. The ganglioside sugar moiety, specifically the sialic acid, had an important role in the interactions. In general the affinities were higher with polysialo, than with monosialo gangliosides. The sensorgrams describing the interactions of Aβ42 and SP with gangliosides differed from the interactions of the other studied peptides. Ca2+ promoted changes in peptide-glycolipid interactions.
Keywords: neuropeptides; asialo-GM1; GM1; GT1b; GD1a; Surface Plasmon Resonance; Aβ42; Ca;
Circulating sensory peptide levels within 24 h of human bone fracture by Gracey N. Onuoha (1107-1110).
We designed this study to examine the circulatory levels of wound modulatory peptides [substance P (SP), calcitonin gene related peptide (CGRP] in patients with muscle injuries with bone fractures and within 24 h of the injury. The peripheral plasma levels of these sensory nerve peptides were measured on hospital admission (OA) and 24 h post-injury (PI), using ELISA technique. Mean (s.d) ng/liter of CGRP was higher in patients OA (270 ± 199), and PI (205 ± 176); than the controls (3 ± 81) P < 0.05. Substance P also increased in the patients OA: 101 ± 50; PI: 46 ± 3 than controls [8 ± 9] P < 0.001. Elastase (predictor of posttraumatic complication) was examined and there was no significant differences between patients and control samples (P = NS). This study shows that sensory nerve peptides are increased in bone fracture related injuries up to 24 h after injury. An intact nociceptor system of primary afferent sensory nerves is important for the initiation of the inflammatory process and successful tissue repair as dysfunction of this system could be a contributing factor for a delayed wound healing.
Keywords: Bone; Calcitonin gene-related peptide; Elastase; Substance P;
PACAP activates PKA, PKC and Ca2+ signaling cascades in rat neuroepithelial cells by Cheng-Ji Zhou; Toshihiko Yada; Daisuke Kohno; Sakae Kikuyama; Ryusuke Suzuki; Hidekatsu Mizushima; Seiji Shioda (1111-1117).
Several studies have reported that the PAC1 receptor (PAC1-R), the specific receptor for PACAP, is expressed at early developmental stages. Here, we describe that the cytosolic Ca2+ concentration ([Ca2+]i) was increased by PACAP, but not VIP, in a concentration range from 10−12 to 10−8 M via the PAC1-R in isolated single cells from the rat neural fold. This activation of the cells by PACAP was mimicked by agonists and inhibited by antagonists of the cAMP/PKA and PLC/PKC cascades. These data indicate that PACAP/PAC1-R is linked to [Ca2+]i signaling via two G-protein-coupled protein kinase pathways and may thereby play an important role in early neurodevelopment.
Keywords: PACAP; G-protein-coupled receptor; Alternative splicing variants; cAMP/protein kinase A cascades; Phospholipase C/protein kinase C cascade; Cytosolic Ca2+ concentration; Development; Neural fold; Primitive streak stage; Rat;
GRP-receptor-mediated signal transduction, gene expression and DNA synthesis in the human pancreatic adenocarcinoma cell line HPAF by Beáta Burghardt; Christoph Wenger; Kornélia Barabás; Gábor Rácz; Attila Oláh; Lajos Flautner; David H. Coy; Thomas M. Gress; Gábor Varga (1119-1128).
Bombesin-like peptides have been implicated as growth factors in various human cancers. Human adenocarcinoma cell lines (Capan-1, Capan-2, MiaPaCa-2 and HPAF) were tested to determine whether they express the gastrin-releasing peptide-preferring bombesin receptor (GRPR) and neuromedin B-preferring bombesin receptor (NMBR). Using RT-PCR the highest level of GRP receptor mRNA was found in HPAF cells. NMB receptor mRNA expression moderate in all cell lines investigated. We therefore selected the HPAF cell line to investigate whether bombesin treatment affects intracellular Ca2+ ([Ca2+]i), cAMP level, DNA synthesis as a measure of cell proliferation, and expression of three transcription factors: c-fos, c-myc and high mobility group protein IY (HMG-I(Y)).Bombesin administration led to an immediate increase in free intracellular Ca2+ concentration ([Ca2+]i) but did not change cAMP levels. The peptide also enhanced [3H]thymidine incorporation in HPAF cells (but not in the other cell lines), an effect that was concentration dependent, reaching 36 ± 5% stimulation over control values at 24 h with an EC50 of 2.27 × 10−12 M. Furthermore, bombesin stimulated c-fos, c-myc and HMG-I(Y) expression in a time-dependent manner: the c-fos mRNA level increased dramatically in the first 30 min of exposure, then returned to basal level within 2 h, while the c-myc and HMG-I(Y) mRNA levels peaked at 2 h and 4h, respectively. All actions of bombesin were blocked by BME (D-Phe6-bombesin-(6–13)-methylester), a selective GRP receptor antagonist, but not by the NMB receptor antagonist BIM-23127 (D-Nal-cyclo[Cys-Tyr-D-Trp-Orn-Val-Cys]-Nal-NH2).We conclude that HPAF cells express mRNA for GRP receptors and that functional receptors are present in the cell membrane. The occupation of these receptors leads to a sequence of intracellular events involving rapid mobilization of intracellular Ca2+, expression of c-fos, c-myc and HMG-I(Y) mRNA, and stimulation of cell proliferation. Conversely, although NMB receptor mRNA can be detected, its actual translation to functional receptors does not reach a detectable level.
Keywords: Bombesin; Antagonist; HPAF; Cell line; Cell proliferation;
Permeability of the peptidic GH secretagogues hexarelin and EP 51389, across rat jejunum by Marie Roumi; Elizabeth Kwong; Romano Deghenghi; Vittorio Locatelli; Sylvie Marleau; Patrick Du Souich; Richard Béliveau; Huy Ong (1129-1138).
The intestinal permeability of hexarelin and EP 51389, two growth hormone releasing hexa- and tri- peptide analogues, was assessed in vitro with side-by-side diffusion chambers in the apical-to-basolateral (AP-to-BL) and in the basolateral-to-apical (BL-to-AP) direction using excised rat jejunal segments. The effect of EP 51389 on P-glycoprotein (P-gp) was evaluated by rhodamine 123 accumulation on monolayers of CHRC5 cells with increasing concentrations of EP 51389. Hexarelin and EP 51389 permeability were found to be < 1%. Permeability coefficients (Papp) were 18.87 ± 2.86 (×10−7 cm/s) and 5.87 ± 0.45 (×10−7 cm/s) for hexarelin and EP 51389, respectively. Bidirectional studies revealed that hexarelin transport was similar in both directions. EDTA did not influence hexarelin permeability. Permeability was predominantly secretory for EP 51389 as Papp in the BL-to-AP direction [32.56 ± 6.11 (×10−7 cm/s)] was greater than AP-to-BL. Confirming involvement of a secretory transport system, chlorpromazine inhibited EP 51389 transport across the jejunum. EP 51389 inhibited P-gp in a dose dependent manner resulting in the intracellular accumulation of rhodamine in CHRC5 cells. These results suggest that: 1) the intestinal permeability of hexarelin and EP 51389 is poor; 2) the passage of hexarelin is mainly via a transcellular passive pathway since the contribution of paracellular permeability to the overall permeability is rather low; 3) P-gp may act as a potential barrier for the intestinal absorption of EP 51389.
Keywords: Hexarelin; EP 51389; Growth hormone releasing peptides; Intestinal permeability; P-glycoprotein; Rat intestinal segments; In vitro diffusion cell chambers;
GI side-effects of a possible therapeutic GRF analogue in monkeys are likely due to VIP receptor agonist activity by Tetsuhide Ito; Hisato Igarashi; Tapas K. Pradhan; Wei Hou; Samuel A. Mantey; John E. Taylor; William A. Murphy; David H. Coy; Robert T. Jensen (1139-1151).
Growth hormone (GH) is used or is being evaluated for efficacy in treatment of short stature, aspects of aging, cardiac disorders, Crohn’s disease, and short bowel syndrome. Therefore, we synthesized several stable growth hormone-releasing factor (GRF) analogues that could be therapeutically useful. One potent analog, [D-Ala2,Aib8, 18,Ala9, 15, 16, 22, 24–26,Gab27]hGRF(1–27)NH2 (GRF-6), with prolonged infusion caused severe diarrhea in monkeys; however, it had no side-effects in rats. Because GRF has similarity to VIP/PACAP and VIPomas cause diarrhea, this study investigated the ability of this and other GRF analogues to interact with the VIP/PACAP receptors. Rat VPAC1-R (rVPAC1-R), human VPAC1-R (hVPAC1-R), rVPAC2-R and hVPAC2-R stably transfected CHO and PANC 1 cells were made and T47D breast cancer cells containing native human VPAC1-R and AR4–2J cells containing PAC1-R were used. hGRF(1–29)NH2 had low affinity for both rVPAC1-R and rVPAC2-R while VIP had a high affinity for both receptors. GRF-6 had a low affinity for both rVPAC1-R and rVPAC2-R and very low affinity for the rPAC1-R. VIP had a high affinity, whereas hGRF(1–29)NH2 had a low affinity for both hVPAC1-R and hVPAC2-R. In contrast GRF-6, while having a low affinity for hVPAC2-R, had relatively higher affinity for the hVPAC1-R. In guinea pig pancreatic acini, all GRF analogues were full agonists at the VPAC1-R causing enzyme secretion. These results demonstrate that in contrast to native hGRF(1–29)NH2, GRF-6 has a relatively high affinity for the human VPAC1-R but not for the human VPAC2-R, rat VPAC1-R, rat VPAC2-R or rat PAC1-R. These results suggest that the substituted GRF analog, GRF-6, likely causes the diarrheal side-effects in monkeys by interacting with the VPAC1-R. Furthermore, they demonstrate significant species differences can exist for possible therapeutic peptide agonists of the VIP/PACAP/GRF receptor family and that it is essential that receptor affinity assessments be performed in human cells or from a closely related species.
Keywords: GRF; VIP; Receptor agonist; PAC1; Growth hormone release;
C-type natriuretic peptide system in rabbit oviduct by Suhn Hee Kim; Kyung Sun Lee; Sook Jeong Lee; Kyung Hwan Seul; Sung Zoo Kim; Kyung Woo Cho (1153-1159).
C-type natriuretic peptide (CNP), a third member of the natriuretic peptide family, is known to be distributed mainly in brain and vascular endothelium and is considered to act as a local regulator in many tissues. The purpose of this study was to determine the presence of CNP system and its biological function in rabbit oviduct. The serial dilution curve of tissue extracts was parallel to the standard curve of CNP(1–22) and a major peak of molecular profile of tissue extracts by HPLC was CNP(1–53). mRNA of CNP which was the same size as positive control was also detected by Southern blot analysis. CNP increased the production of 3′,5′-cyclic guanosine monophosphate (cGMP) in the purified membrane of oviduct, which was more in membranes derived from the isthmic portion than in the ampullar portion. The presence of mRNAs of natriuretic peptide receptor-A (NPR-A) and NPR-B was demonstrated by RT-PCR. Synthetic CNP(1–22) inhibited both frequency and amplitude of basal motility of oviduct in a dose-dependent manner. The inhibitory effect of CNP on the basal motility was more potent in the isthmic portion than in the ampullar portion. These results demonstrate the presence of CNP system in the oviduct and regional differences in motility inhibition by CNP between isthmic and ampullar portions. Therefore, these findings suggest the possible existence of a CNP system that may exert a local regulator of basal motility, either alone or in concert with other hormones.
Keywords: Oviduct; C-type natriuretic peptide; Motility; Natriuretic peptide receptor; cGMP;
Regulation of TRH and TRH-related peptides in rat brain by thyroid and steroid hormones by A.Eugene Pekary; Albert Sattin (1161-1173).
To investigate the possibility that TRH (pGlu-His-Pro-NH2) and EEP (pGlu-Glu-Pro-NH2) contribute to the behavioral and mood changes attending hypothyroidism, hyperthyroidism and hypogonadism, we have treated young, adult, male Sprague-Dawley rats (5/group, 250 g bw at time of sacrifice) for one week with either daily ip injections of saline, 5 μg T4, 3 mg PTU or castration. Immunoreactivity for TRH (TRH-IR), TRH-Gly (pGlu-His-Pro-Gly, a TRH precursor), EEP and Ps4 (prepro-TRH-derived TRH-enhancing peptide) was measured in 8 brain regions by RIA. Castration reduced the Ps4-IR levels in hippocampus by 80%. High pressure liquid chromatography revealed that in many brain regions EEP-IR and TRH-IR consisted of a mixture of TRH and other TRH-like peptides including EEP, Val2-TRH, Tyr2-TRH, Leu2-TRH and Phe2-TRH. Transition from the hyperthyroid to the hypothyroid state increased the Val2-TRH and Tyr2-TRH levels in the accumbens by 10-fold and 15-fold, respectively, and the corresponding ratios for the pyriform cortex increased 9-fold and 12-fold, respectively. Hypothyroidism and castration reduced the levels of TRH and the majority of other TRH-like peptides in the entorhinal cortex. This is the first report that thyroid and steroid hormones alter the levels of TRH, prepro-TRH-derived peptides, and a newly discovered array of TRH-like neuropeptides in limbic brain regions.
Keywords: Rat brain; TRH-like peptides; Depression; Thyroxine; Propylthiouracil; Castration; HPLC; Radioimmunoassay;
Expression of urotensin II and urotensin II receptor mRNAs in various human tumor cell lines and secretion of urotensin II-like immunoreactivity by SW-13 adrenocortical carcinoma cells by Kazuhiro Takahashi; Kazuhito Totsune; Osamu Murakami; Shigeki Shibahara (1175-1179).
Urotensin II is the most potent vasoconstrictor peptide identified so far. Expression of urotensin II and urotensin II receptor mRNAs was studied in various human tumor cell lines by reverse transcriptase polymerase chain reaction (PCR) method. Secretion of urotensin II by these tumor cells was studied by radioimmunoassay. The tumor cell lines studied were T98G glioblastoma cells, IMR-32 neuroblastoma cells, NB69 neuroblastoma cells, BeWo choriocarcinoma cells, SW-13 adrenocortical carcinoma cells, DLD-1 colorectal adenocarcinoma cells and HeLa cervical cancer cells. Urotensin II mRNA was expressed in 6 tumor cell lines except for NB69 neuroblastoma cells. Urotensin II receptor mRNA was expressed in all 7 tumor cell lines. A significant amount of urotensin II-like immunoreactivity was detected only in the culture medium of SW-13 adrenocortical carcinoma cells by radioimmunoassay. Sephadex G-50 column chromatography showed that the urotensin II-like immunoreactivity in the culture medium extract was eluted earlier than synthetic human urotensin II, suggesting that SW-13 cells secreted higher molecular weight materials, perhaps partially processed forms of the urotensin II precursor. Reverse phase high-performance liquid chromatography (HPLC) showed three immunoreactive peaks, one of which was eluted in the position of urotensin II. The present study has shown for the first time expression of urotensin II and urotensin II receptor mRNAs in various tumor cell lines and the secretion of urotensin II-like immunoreactivity by SW-13 adrenocortical carcinoma cells.
Keywords: Urotensin II; Tumor; Cancer; Adrenocortical;
Met-enkephalin levels during PTCA-induced myocardial ischemia by C. Parlapiano; M.C. Borgia; G. Tonnarini; G. Giancaspro; F. Pizzuto; E. Campana; T. Giovanniello; P. Pantone; G.M. Vincentelli; F. Alegiani; M. Negri (1181-1182).
Met-enkephalin (Met-enk) has been demonstrated to modulate myocardial-ischemia mechanisms via the opioid receptors, but no studies are now available on Met-enk levels in the coronary circulation.In this experience Met-enk levels were evaluated in aortic root and in coronary sinus at baseline (T0), during PTCA induced transient ischemia (T1) and during reperfusion (T2). No significant differences were found at any time. Thus, it appears that there is no Met-enk extraction from the coronary circulation during provoked myocardial ischemia and no Met-enk release from the ischemic heart.
Keywords: Met-enkephalin; PTCA; Coronary sinus; Aortic root;
Evolution of the insulin molecule: insights into structure-activity and phylogenetic relationships by J.Michael Conlon (1183-1193).
The conformation of insulin in the crystalline state has been known for more than 30 years but there remains uncertainty regarding the biologically active conformation and the structural features that constitute the receptor-binding domain. The primary structure of insulin has been determined for at least 100 vertebrate species. In addition to the invariant cysteines, only ten amino acids (GlyA1, IleA2, ValA3, TyrA19, LeuB6, GlyB8, LeuB11, ValB12, GlyB23 and PheB24) have been fully conserved during vertebrate evolution. This observation supports the hypothesis derived from alanine-scanning mutagenesis studies that five of these invariant residues (IleA2, ValA3, TyrA19, GlyB23, and Phe24) interact directly with the receptor and five additional conserved residues (LeuB6, GlyB8, LeuB11, GluB13 and PheB25) are important in maintaining the receptor-binding conformation. With the exception of the hagfish, only conservative substitutions are found at B13 (Glu → Asp) and B25(Phe → Tyr). In contrast, amino acid residues that were also considered to be important in receptor binding based upon the crystal structure of insulin (GluA4, GlnA5, AsnA21, TyrB16, TyrB26) have been much less well conserved and are probably not components of the receptor-binding domain. The hypothesis that LeuA13 and LeuB17 form part of a second receptor-binding site in the insulin molecule finds some support in terms of their conservation during vertebrate evolution, although the site is probably absent in some hystricomorph insulins. In general, the amino acid sequences of insulins are not useful in cladistic analyses especially when evolutionary distant taxa are compared but, among related species in a particular order or family, the presence of unusual structural features in the insulin molecule may permit a meaningful phylogenetic inference. For example, analysis of insulin sequences supports monophyletic status for Dipnoi, Elasmobranchii, Holocephali and Petromyzontiformes.
Keywords: Insulin; Receptor-binding; Structure-activity; Vertebrate evolution; Phylogeny;