Effects of cholecystokinin-58 on type 1 cholecystokinin receptor function and regulation

Cholecystokinin, like many peptide hormones, is present as multiple molecular forms. CCK-58 has been identified as the dominant form in the circulation, whereas most of the studies of CCK-receptor interactions have been performed with CCK-8. Despite both sharing the pharmacophoric region of CCK, representing its carboxy terminal heptapeptide amide, studies in vivo have demonstrated biological diversity of action of the two peptides, with CCK-58, but not CCK-8, stimulating pancreatic fluid secretion and lengthening the interval between meals. Here, we have directly studied the ability of these two CCK peptides to bind to the type 1 CCK receptor and to stimulate it to elicit an intracellular calcium response. The calcium response relative to receptor occupation was identical for CCK-58 and CCK-8, with the longer peptide binding with approximately fivefold lower affinity. We also examined the ability of the two peptides to elicit receptor internalization using morphological techniques and to disrupt the constitutive oligomerization of the CCK receptor using receptor bioluminescence resonance energy transfer. Here, both full agonist peptides had similar effects on these regulatory processes. These data suggest that both molecular forms of CCK act at the CCK1 receptor quite similarly and elicit similar regulatory processes for that receptor, suggesting that the differences in biological activity observed in vivo most likely reflect differences in the clearance and/or metabolism of these long and short forms of CCK peptides.     

VIP and PACAP display different vasodilatory effects in rabbit coronary and cerebral arteries

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) are closely related peptides with wide distribution in the nervous system. The aim of the present study was to investigate functional characteristics and the influence of sex steroids on the vasodilatory effects of these two peptides in cerebral and coronary vessels from female New Zealand White (NZW) rabbits.

The localization and concentration of VIP and PACAP in cardiovascular tissue was evaluated using immunohistochemistry and radioimmunoassays. The vasodilatory effects of VIP and PACAP were investigated using myographs, allowing isometric tension recordings. In order to evaluate the influence of steroid hormones, the rabbits were ovariectomized and randomized to treatment for 4 weeks with 17β-estradiol (E₂), Norethindrone Acetate (NETA), E₂+NETA or placebo. Ring segments of the posterior cerebral artery, the right proximal coronary artery and the distal left coronary artery were examined.

The highest concentrations of VIP/PACAP were observed in cerebral and coronary arteries: 5.0/5.7 and 2.8/3.5 pmol/g, respectively. The peptides were localized in nerve fibres innervating the arteries. Both peptides produced dose-dependent vasodilatory responses in all vessels investigated. While the effects of PACAP were identical in cerebral and coronary arterial segments, the effects of VIP displayed significant differences (E_max, pI₂, Hill-slope). Treatment with sex steroids induced no changes in the vascular effects of the two peptides.

These results indicate different mechanisms of action for the vasodilating effects of the two closely related peptides VIP and PACAP in different areas of the coronary and cerebrovascular tree. Treatment with female sex steroids does not seem to change these mechanisms.     

Helokinestatin-7 peptides from the venoms of Heloderma lizards

Helokinestatins 1-6 constitute a family of bradykinin antagonist peptides originally isolated from the venoms of the Gila Monster, Heloderma suspectum and the Mexican beaded lizard, Heloderma horridum. Here we report the identification, isolation and preliminary pharmacological characterization of two novel tridecapeptides, named helokinestatin-7S (FDDDSTELILEPR - 1550 Da) and helokinestatin-7H (FDDDSRKLILEPR - 1604 Da), whose primary structures were predicted from cDNAs cloned from venom libraries of respective Heloderma lizards. Computed molecular masses of putative helokinestatin-7 peptides were used as tools to locate these peptides in archived LC/MS fractions from respective venoms and sequences were confirmed by MS/MS fragmentation. A synthetic replicate of helokinestatin-7H was found to antagonize the relaxation effect of bradykinin on rat arterial smooth muscle but to have no measurable effects alone. In contrast, synthetic helokinestatin-7S was found to directly contract this preparation. Studies on related natural peptides with subtle differences in primary structure can provide the tools for structure/activity studies in pharmacological investigations directed toward unraveling the molecular basis of venom toxicity and for the evaluation of potential therapeutic leads.     

Mammalian regulatory peptide immunoreactivity in the trematode parasite Haplometra cylindracea and the lung of its frog host, Rana temporaria: comparative chromatographic characterisation using reverse-phase high-performance liquid chromatography.

1. Extracts of Haplometra cylindracea and lung tissues of its host, Rana temporaria, were subjected to radioimmunoassay using antisera to nine mammalian regulatory peptides. 2. In these extracts, immunoreactivity was measured to pancreatic polypeptide, substance P, neurokinin A, gastrin-releasing peptide and glucagon. The levels of each peptide varied considerably with some marked differences between those demonstrable in parasite and host (notably pancreatic polypeptide). 3. Reverse-phase HPLC fractionation of extracts revealed general chemical differences between parasite and host peptides, with some peptides present in more than one molecular form.     

Vasodilator action of guinea pig vasoactive intestinal polypeptide (VIP): comparison with common mammalian VIP.

The vascular activity of guinea pig (gp) and common mammalian (p) VIP were compared in anesthetized guinea pigs and dogs. In the guinea pig, intravenous injections of gpVIP and pVIP increased pancreatic blood flow and reduced the systemic arterial pressure and pancreatic vascular resistance in a dose-related manner. There were no significant differences in the vasodilator actions of these two VIPs, indicating that the overall cardiovascular actions of gpVIP and pVIP are similar in guinea pigs. In the dog, gpVIP, when given intra-arterially, was less potent (about 1/4) than pVIP in its action on femoral blood flow, suggesting that the blood vessels of the dog hind leg are more sensitive to its own VIP than to gpVIP. Oxidation of pVIP and gpVIP with H2O2 greatly reduced their vasodilator effects on the femoral arterial blood flow. The vascular effects were restored to control levels by reduction of the oxidized peptides with mercaptoethanol, which suggests that methionine residues of gpVIP and pVIP are important in the vasodilator effect on the femoral arterial bed in dogs.     

Glycosylated analogs of formaecin I and drosocin exhibit differential pattern of antibacterial activity

The synthetic glycopeptides are interesting model systems to study the effect of O-glycosylation in modulating their function and structure. A series of glycosylated analogs of two antibacterial peptides, formaecin I and drosocin, were synthesized by varying the nature of sugar and its linkage with bioactive peptides to understand the influence of structure variation of glycosylation on their antibacterial activities. Higher antibacterial activities of all glycopeptides compared to their respective non-glycosylated counterparts emphasize in part the importance of sugar moieties in functional implications of these peptides. The consequences of the unique differences among the analogs were apparent on their antibacterial activities but not evident structurally by circular dichroism studies. We have shown that differently glycosylated peptides exhibit differential effect among each other when tested against several Gram-negative bacterial strains. The change of monosaccharide moiety and/or its anomeric configuration in formaecin I and drosocin resulted into decrease in the antibacterial activity in comparison to that of the native glycopeptide, but the extent of decrease in antibacterial activity of glycosylated drosocin analogs was less. Probably, the variation in peptide conformation arising due to topological dissimilarities among different sugars in the same peptide resulting in possible modulation in binding properties appears to be responsible for differences in their antibacterial activities. Indeed, these effects of glycosylation are found to be sequence-specific and depend in the milieu of amino acid residues. Interestingly, none of the carbohydrate variants affected the basic property of these peptides, which is non-hemolytic and non-toxicity to eukaryotic cells.     

Block principle of organization and polyfunctional nature of regulatory peptides

Study on the primary structure of more than 100 regulatory peptides was carried out with the view of determining their structural similarity. Practically all regulatory peptides appeared to be related to each other. One regulatory peptide can bear similarity to several regulatory peptides (RPs) which is realized by different fragments. The structural similarity of RPs was in some cases unexpected, e. g., somatocrinin-thymosin, secretin-endorphin, etc. Gastro-intestinal peptides were related to most RPs. It seems likely that these peptides are the most ancient ones among RPs.     

O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry

O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (LWAC) and mass spectrometry. The effectiveness of this strategy on complex peptide mixtures was demonstrated through enrichment of 145 unique O-GlcNAc-modified peptides from a postsynaptic density preparation. 65 of these O-GlcNAc-modified peptides were sequenced and belonged to proteins with diverse functions in synaptic transmission. Beta-elimination/Michael addition, MS(3) on O-GlcNAc neutral loss ions, and electron capture dissociation were shown to facilitate analysis of O-GlcNAc-modified peptides/sites from lectin weak affinity chromatography enriched postsynaptic density samples. Bassoon and Piccolo, proteins critical to synapse assembly and vesicle docking, were extensively modified by O-GlcNAc. In some cases, O-GlcNAc was mapped to peptides previously identified as phosphorylated, indicating potential interplay between these modifications. Shared substrate amino acid context was apparent in subsets of O-GlcNAc-modified peptides, including "PVST" and a novel "TTA" motif (two hydroxyl-containing amino acids adjacent to an alanine). The results suggest specific roles for O-GlcNAc modification in synaptic transmission, establish a basis for site-specific regulatory studies, and provide methods that will facilitate O-GlcNAc proteome analysis across a wide variety of cells and tissues.     

Catecholamines increase urine formation in isolated toad (Bufo marinus) kidneys perfused at constant rate

Infusion of catecholamines into isolated kidneys of the toad (Bufo marinus) perfused at constant rate, produced increased arterial pressure accompanied by increased glomerular filtration rate, urine formation rate and sodium excretion. These parameters were all increased by arterial infusion of adrenaline or noradrenaline, or by infusion of adrenaline via the renal portal veins. Portal venous pressure increased slightly after arterial or portal infusion of adrenaline, but decreased after arterial infusion of noradrenaline. Estimation of segmental pressure gradients indicated that the efferent glomerular arterioles were selectively constricted by low concentrations of adrenaline or noradrenaline (3 X 10(-9), 3 X 10(-8) mol l(-1)). Higher concentrations of these amines constricted the preglomerular, as well as the postglomerular vasculature. These results demonstrate that the pericytes and/or endothelial cells which form the walls of the efferent arterioles in B. marinus are capable of active contraction.     

Hypotensive effect of novel chimeric peptides of met-enkephalin and FMRFa

Endogenous opioid peptides like endomorphins, met-enkephalin and NPFF/FMRFamide family of neuropeptides, besides playing a role in modulation of antinociception, also affect cardiovascular system. Based on MERF, which consists of overlapping sequences of FMRFa and met-enkephalin, two chimeric peptides YGGFMKKKFMRFamide (YFa) and [D-Ala2] YAGFMKKKFMRFamide ([D-Ala2] YFa) were designed and synthesized. In this study, effect of YFa and [D-Ala2] YFa on arterial blood pressure and heart rate was evaluated in anaesthetized rats. Both YFa and [D-Ala2] YFa showed a dose-dependent fall in mean arterial pressure in dose-range of 13-78 micromol/kg. After naloxone treatment (5 mg/kg), vasodepressor effect of [D-Ala2] YFa and YFa was only partially blocked as compared to met-enkephalin. Partial blockade of vasodepressive effect of YFa and [D-Ala2] YFa by naloxone may be attributed to interaction of these chimeric peptides with receptors other than naloxone-sensitive receptors such as anti-opioid receptors, adrenergic receptors and D-analogue receptors.     

Amino acid sequence pattern in the regulatory peptides.

The essential properties of the primary structure of regulatory peptides, i.e. amino acid residues and their combinations, which are characteristic of the whole population of regulatory peptides, have been revealed using statistical methodology. These properties are as follows: increased content of certain residues (Gly, Pro, Phe, Arg, Tyr, Met and Trp) as well as an increased rate of occurrence of certain pairs of residue as compared with proteins, a random sequence of residues and "nonregulatory" peptides. By representing regulatory peptides as a sequence of hydrophobic (2 types) and hydrophilic (3 types) segments, the pattern for alternation of these segments in regulatory peptides has been determined. The segments were classified into 5 types according to the peculiarities of mutual localization of hydrophobic and hydrophilic residues within the primary structure of regulatory peptides. As compared with proteins, "nonregulatory" peptides and a random sequence of segments, regulatory peptides were characterized by an increased frequency of 4 particular pairs of segments among 12 theoretically possible pairs. These 4 pairs are fragments of the periodic segment sequence with periods of 4 segments. The revealed pattern indicates that there exists a general principle of the regulatory peptide primary structure organization and possibly a common type of the regulatory peptides flexible peptide chain folding at the ligand-receptor complex formation.     

Ovarian jelly-peptides (OJPs), a new family of regulatory peptides identified in the cephalopod Sepia officinalis

In marine invertebrates, numerous water-borne peptides involved in reproductive behavior have been characterized. In this study, we focused on three ovarian water-borne peptides, released by full-grown oocytes (FGO) in the genital coelom and in the lumen of the oviduct in the cuttlefish Sepia officinalis. The first one (DQVKIVL), was characterized by the monitoring of HPLC purified fraction using a myotropic bioassay. Subsequently, a peptidomic approach consisting of a mass spectrometry comparative screening performed between the peptide content of FGO with that of FGO-conditioned medium, led to the identification of two additional water-borne peptides. The second peptide identified (DEVKIVL) was characterized by MS/MS and the primary structure of the third one (DEVKIVLD) was elucidated by a combination of Edman degradation, acid hydrolysis and MS/MS analysis. Sequence homology, tissue mapping and bioactivity demonstrate that these peptides belong to the same family. DQVKIVL-related-peptides strictly localized in the female genital tract modulate the whole female genital tract and the main nidamental gland contractions. Furthermore, these peptides form a jelly, when resuspended in water. This particular property could play an important role in the kinetics of peptide diffusion in the external medium. Thus, these regulatory peptides were named ovarian jelly-peptides (OJPs).     

Chemical coding of endocrine cells of the airways: presence of helodermin-like peptides

Summary The epithelium of the airways is rich in endocrine cells containing serotonin and/or a wide variety of regulatory peptides. These cells usually occur in clusters in the lungs but are also found scattered in the larynx and trachea. In the present study, endocrine cells in the airways of mouse, rat, hamster, guinea pig, pig, sheep and squirrel monkey were examined for the presence of serotonin, helodermin-like peptides and other regulatory peptides using immunocytochemistry and radioimmunoassay. In addition, we looked for the protein gene product 9.5 (PGP), which occurs in many peptide hormone-producing endocrine cells in the body. Both clustered and scattered endocrine cells in the airways were found to display coexistence of serotonin and peptides, such as a helodermin-like peptide, calcitonin and calcitonin gene-related peptide (CGRP). The PGP-immunoreactive cells were numerous and included elements containing serotonin and/or regulatory peptides. An additional PGP-immunoreactive endocrine cell population lacked serotonin and regulatory peptides. Helodermin-immunoreactive material was demonstrated in endocrine cells of the airways in the mouse and hamster but not in any of the other species studied. Serotonin was an endocrine cell constituent in all the species studied. Calcitonin and CGRP could be demonstrated by immunocytochemistry in the mouse, rat, and hamster, but not in the guinea pig, sheep, pig and monkey. In the hamster airways double immunostaining indicated that the helodermin-like peptide occurred in a subpopulation of the CGRP- and serotonin-containing cells. Most of the CGRP-containing cells stored serotonin; some of them also contained calcitonin. The chemical coding of these cells resembled that of the thyroid C cells.     

Differential effects of endothelin peptides in the systemic and pulmonary vascular beds of the cat

The cardiovascular and pulmonary responses to vasoactive intestinal contractor (VIC) were compared with those of endothelin (ET)-1, ET-2, ET-3 and sarafotoxin 6b (S6b) and the mechanism by which ET-1 alters vascular resistance was investigated in the hindquarters vascular bed of the cat. In a manner similar to ET-1 and ET-2, VIC at a dose of 0.3 nmol/kg i.v. produced increases in pulmonary arterial pressure (PAP) and biphasic changes in systemic arterial pressure (AP), systemic vascular resistance (SVR) and pulmonary vascular resistance (PVR). The biphasic changes were characterized by initial decreases followed by increases. In contrast, ET-3 and S6b at doses of 0.3 nmol/kg i.v. produced mainly decreases in AP and SVR, increases in PAP, and biphasic changes in PVR. A monocyclic ET-1 analog and the ET-1 C-terminal hexapeptide fragment produced no effect on AP, SVR, PAP and PVR at doses of 30–100 nmol/kg i.v. ET-1 at a dose of 0.3 nmol i.a. produced a biphasic change in hindquarters perfusion pressure. The initial vasodilation and secondary vasoconstriction were not modified by a variety of blocking agents, whereas the vasoconstrictor response was significantly reduced by infusion of nimodipine, a calcium entry blocking agent. Results of the present study indicate that VIC, a peptide specific to the mouse gastrointestinal tract, elicits cardiovascular responses that are similar to those elicited by ET-1 and ET-2. The present results indicate that responses to these novel peptides are complex and while the mechanism of action remains uncertain, these data indicate that structural differences among the peptides confer differences in biological activity.     

Effect of acidification of the duodenal contents on splanchnic circulation and metabolism

Portal vein (PF) and superior mesenteric artery blood flows (SMAF), arterial and portal venous oxygen concentrations were measured in dogs before and after duodenal introduction of 3 ml/kg 0.1 mol HCl. The duodenal acidification increased PF by 30% and SMAF by 35%. The change of mesenteric oxygen utilisation, from 0.80 +/- 0.10 to 0.81 ml/kg was not significant. It is concluded that the circulatory changes after acidification of the duodenal contents are not secondary to a metabolic effect.     

Chromogranin A-derived peptides: interaction with the rat posterior cerebral artery

Chromogranin A (CgA), an acidic granule protein of the regulated secretory pathway in the diffuse neuroendocrine system, is postulated to serve as a prohormone for regulatory peptides. Betagranin (rCgA(1-128)), the first N-terminal cleavage product of rat CgA, is 87% homologous to the bovine vasostatin I (bCgA(1-76)), previously shown to be vasoinhibitory in bovine resistance arteries. In this study the vasoactivity of homologous rat and bovine peptides was investigated in the rat posterior cerebral artery. Firstly, we examined the interaction of rhodamine (Rh)-labelled bCgA(7-40) and bCgA(47-70) with elements of the arterial wall by fluorescence microscopy. Secondly, rCgA(7-57), bCgA(1-40), bCgA(7-40) and bCgA(47-66) (chromofungin) were studied for effects on arterial tone and intracellular calcium as function of pressure in an arteriograph. Although without dilator or constrictor responses at 60-150 mm Hg, the rat peptide (rCgA(7-57)) evoked a significant delay in the onset of forced dilatation at 170 mm Hg, in contrast to the bovine peptides bCgA(1-40), bCgA(7-40) and bCgA(47-66) (chromofungin). Neither Rh-bCgA(7-40) nor Rh-bCgA(47-70) stained the endothelial layer, while Rh-bCgA(47-70) but not Rh-bCgA(7-40) stained the smooth muscle compartment. Analogously, bCgA(47-66) but not bCgA(7-40) reduced intracellular calcium, however without modifying the myogenic response. Thus, the betagranin peptide rCgA(7-57) and the two bovine chromofungin-containing peptides, highly homologous to the corresponding sequence (rCgA(47-66)), affected the rat cerebral artery without vasodilator effects, indicating significant species differences in vasoactivity of the N-terminal domain of CgA.     

Ca2+-dependent interaction of the inhibitory region of troponin I with acidic residues in the N-terminal domain of troponin C

Ca2+ regulation of vertebrate striated muscle contraction is initiated by conformational changes in the N-terminal, regulatory domain of the Ca2+-binding protein troponin C (TnC), altering the interaction of TnC with the other subunits of troponin complex, TnI and TnT. We have investigated the role of acidic amino acid residues in the N-terminal, regulatory domain of TnC in binding to the inhibitory region (residues 96-116) of TnI. We constructed three double mutants of TnC (E53A/E54A, E60A/E61A and E85A/D86A), in which pairs of acidic amino acid residues were replaced by neutral alanines, and measured their affinities for synthetic inhibitory peptides. These peptides had the same amino acid sequence as TnI segments 95-116, 95-119 or 95-124, except that the natural Phe-100 of TnI was replaced by a tryptophan residue. Significant Ca2+-dependent increases in the affinities of the two longer peptides, but not the shortest one, to TnC could be detected by changes in Trp fluorescence. In the presence of Ca2+, all the mutant TnCs showed about the same affinity as wild-type TnC for the inhibitory peptides. In the presence of Mg2+ and EGTA, the N-terminal, regulatory Ca2+-binding sites of TnC are unoccupied. Under these conditions, the affinity of TnC(E85A/D86A) for inhibitory peptides was about half that of wild-type TnC, while the other two mutants had about the same affinity. These results imply a Ca2+-dependent change in the interaction of TnC Glu-85 and/or Asp-86 with residues (117-124) on the C-terminal side of the inhibitory region of TnI. Since Glu-85 and/or Asp-86 of TnC have also been demonstrated to be involved in Ca2+-dependent regulation through interaction with TnT, this region of TnC must be critical for troponin function.     

The regulatory components of adenylate cyclase and transducin. A family of structurally homologous guanine nucleotide-binding proteins

G/F and transducin are guanine nucleotide-binding regulatory proteins that mediate activation of adenylate cyclase and of a rod outer segment cyclic GMP-specific phosphodiesterase, respectively. The substrate for islet-activating protein is a third guanine nucleotide-binding protein that is postulated to mediate inhibition of adenylate cyclase. The extent of structural homology among subunits of all three proteins was examined by analyses of amino acid compositions and electrophoretic patterns of proteolytic peptides. The lower molecular weight subunits (beta subunits; Mr = 35,000) of these proteins have identical amino acid compositions and yield similar peptides upon proteolysis with Staphylococcus aureus V8 protease and elastase. The higher molecular weight subunits (alpha subunits; Mr = 39,000, 41,000, and 45,000) are also similar to each other in these respects. Similarity between the subunits of transducin and the islet-activating protein (IAP) substrate is especially evident. Substantial differences do, however, exist between the lower and higher molecular weight subunits within each protein. In addition, evidence was obtained that the 41,000-Da subunit of the IAP substrate is not derived from the 45,000-Da subunit of G/F. It is concluded that transducin, the IAP substrate, and G/F represent a family of structurally homologous guanine nucleotide-binding regulatory proteins.