peptide-phi and heliodermin

Vasoactive intestinal polypeptide (VIP) is known as an important regulator of airway function. It has been suggested that VIP is involved in the pathogenesis of asthma due to its relaxant effects on smooth muscles. The present study was designed to characterize the effects of the peptides of the VIP family on airway mucus secretion. The peptides VIP, PHI, PACAP-27, PACAP-38, GLP-I, exendin-4, helodermin, helospectin I and helospectin II were investigated using isolated rat trachea. Data show that PACAP-27 is the most potent stimulator of airway mucus secretion (225% stimulation). The rank order of potency was PACAP-27 > VIP > helospectin II > PHI > exendin-4 = helodermin = helospectin I = PACAP-38. The addition of the protease inhibitor thiorphan enhanced the effects of PHI and helodermin, but not of the other peptides. These data show that the peptides of the VIP family stimulate airway mucus secretion differently.

Topics: Animals; Exenatide; Glucagon; Glucagon-Like Peptide 1; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Male; Mucus; Neuropeptides; Peptide Fragments; Peptide PHI; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Protease Inhibitors; Protein Precursors; Rats; Rats, Sprague-Dawley; Thiorphan; Trachea; Vasoactive Intestinal Peptide; Venoms

In the present study, we examined the effect of pretreatment with VIP and various peptides structurally related to VIP such us PHI, helodermin, and secretin on VIP receptor number and affinity, as well as VIP-stimulated cyclic AMP production in rat peritoneal macrophages. Short-term (5-30 min) exposures of rat peritoneal macrophages to 0.1 microM VIP induced a rapid reduction in specific binding. Pretreatment for 15 and 30 min caused 26% (SEM = 6) and 48% (SEM = 4) reduction in specific binding, respectively. The maximal effect was observed at 120 min, causing a decrease of 67% (SEM = 6) in specific binding. Pretreatment with 0.1 microM VIP for 15, 30, and 120 min caused 23% (SEM = 9), 52% (SEM = 4), and 76% (SEM = 4) reduction in cyclic AMP production, respectively. Only VIP concentrations at the nanomolar level and higher were shown to be effective. The potency of VIP and related peptides to desensitize was similar to their potency to occupy receptors and to activate cyclic AMP production. The internalization of radioiodinated VIP was also studied. It was shown that receptor-bound ligand is internalized during the downregulation process. However, the diminution in VIP binding to macrophages was not completely explained by internalization.

Topics: Animals; Cells, Cultured; Cyclic AMP; Dose-Response Relationship, Drug; Down-Regulation; Insulin; Intercellular Signaling Peptides and Proteins; Kinetics; Macrophages, Peritoneal; Peptide PHI; Peptides; Rats; Rats, Wistar; Receptors, Vasoactive Intestinal Peptide; Secretin; Vasoactive Intestinal Peptide

In dispersed acini from rat pancreas, verapamil (a phenylalkylamine calcium channel blocker) potentiated amylase secretion stimulated by vasoactive intestinal peptide (VIP), secretin, peptide histidine isoleucine, helodermin, forskolin, and 8-bromocyclic AMP. The action of verapamil on VIP-stimulated amylase secretion was detectable at 10 microM verapamil and maximal at 100 microM verapamil. Verapamil did not alter binding of 125I-VIP, basal cAMP, the increase in cAMP caused by VIP, or the increase in cAMP-dependent protein kinase caused by VIP. The effects of verapamil on stimulated amylase secretion were fully reversible and could be reproduced by nicardipine (a 1,4-dihydropyridine calcium channel blocker) and diltiazem (a benzothiazepine calcium channel blocker), but not by cinnarizine (a piperazine calcium channel blocker). Although 300 microM verapamil increased outflux of 45Ca, 100 microM verapamil, the concentration that produced maximal potentiation of VIP-stimulated amylase secretion, did not alter 45Ca outflux. Our results indicate that the action of verapamil to potentiate amylase secretion stimulated by secretagogues that activate the cAMP pathway occurs at a step that is distal to the activation of cAMP-dependent protein kinase.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Amylases; Animals; Colforsin; Cyclic AMP; Intercellular Signaling Peptides and Proteins; Male; Pancreas; Peptide PHI; Peptides; Rats; Rats, Sprague-Dawley; Secretin; Vasoactive Intestinal Peptide; Verapamil

The lower airways of guinea pigs were analysed for helospectin and helodermin using immunocytochemistry. A moderate supply of helospectin/helodermin-like immunoreactive nerve fibers and few nerve fibers displaying helodermin immunoreactivity was seen in the smooth muscle, around seromucous glands and small blood vessels in the trachea and around bronchi and pulmonary blood vessels. Helospectin I-, helospectin II- and helodermin-induced suppression of smooth muscle responses were analysed using isolated circular segments of trachea and pulmonary arteries of guinea pigs. In both airways and arteries the peptides caused a concentration-dependent relaxation of precontracted segments. The maximal relaxant activity observed was more pronounced in the airways than in the arteries. The effects of the helospectins and helodermin were compared to those of vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), pituitary adenylate cyclase activating peptide (PACAP) and acetylcholine (ACh). All peptides, with the exception of PACAP, caused a total or nearly total relaxation of the precontracted tracheal segments. In the trachea PACAP was significantly more potent than the other five peptides whereas only small potency differences were seen in the pulmonary artery. The relaxant responses to helospectin I, helospectin II and helodermin in the trachea and the intrapulmonary arteries were unaffected by pretreatment with atropine, prazosin, yohimbine, propranolol, mepyramine and cimetidine. Conceivably, nerve fibers containing helospectin and helodermin may play a role in the regulation of airway resistance and in the regulation of local pulmonary blood flow.

Topics: Acetylcholine; Animals; Guinea Pigs; Immunohistochemistry; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Lung; Male; Neuropeptides; Peptide PHI; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Pulmonary Artery; Tissue Distribution; Trachea; Vasoactive Intestinal Peptide; Vasodilation

Vasoactive intestinal peptide (VIP) has been shown to stimulate adenylyl cyclase activity in human endometrial membranes. The effect was dependent on the time and temperature of incubation as well as on the concentration of endometrial membrane proteins in the medium. In the presence of 1 microM GTP, half-maximal stimulation of adenylyl cyclase activity was observed at 25.0 +/- 7.0 nM VIP, whereas the maximal activity (at 1 microM VIP) corresponded to an increase of about 140% with respect to basal values (7.5 +/- 0.6 pmol cyclic AMP/min/mg of protein). However, the maximal stimulation of adenylyl cyclase activity was obtained with helodermin (1 microM) that increased the activity by 170% over the basal. The relative potency of VIP-related peptides upon the adenylyl cyclase activity was: helodermin (ED50 = 1.8 +/- 1.4 nM) > VIP (ED50 = 25.0 +/- 7.0 nM) > PHI (ED50 = 725.0 +/- 127.2 nM). Secretin had a faint effect upon the adenylyl cyclase activity and glucagon was completely inefficient at this level. The presence of alpha s and alpha i subunits of G proteins in human endometrium was detected by immunoblot. Preliminary results showed the presence of two classes of 125I-VIP receptors in human endometrial membranes with the following stoichoimetric parameters: high affinity receptor (Kd = 2.0 nM, binding capacity 0.1 pmol VIP/mg protein) and low affinity receptor (Kd = 0.43 microM, binding capacity 13.1 pmol VIP/mg protein). The present results together with the known presence of VIP in human uterus and the actions of this neuropeptide in the adjacent myometrial tissue support the idea that VIP and related peptides may have a role in human endometrium.

Topics: Adenylyl Cyclases; Adult; Endometrium; Female; GTP-Binding Proteins; Humans; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Kinetics; Membranes; Middle Aged; Peptide PHI; Peptides; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Two out of three rabbit anti-helodermin antisera previously shown to be useful for radioimmunoassay failed to detect up 100 ng of the peptide helodermin spotted directly on a nitrocellulose membrane. A lesser shortcoming of dot immunoassay was encountered with porcine PHI (peptide histidine-isoleucinamide), a member of the same peptide family, and an anti-PHI antiserum. To improve antigen-antibody interactions in the solid phase, we compared five methods of prior peptide immobilization. The best result was obtained when the nitrocellulose membrane was pretreated for 1 min in 4% ovalbumin, followed by 10 min activation with 2.5% glutaraldehyde, before peptide spotting. After cross-linking, the peptide was immunodetected with a F(ab')2 fragment of an anti-rabbit IgG coupled to alkaline phosphatase. The peptide cross-linking method was capable of increasing the sensitivity of ensuing immunodetection by more than 1000-fold, i.e., made feasible the detection of 0.1 ng peptide/dot in cases when the direct spotting method was inefficient. The sensitivity of this new, reliable and simple dot immunoassay for peptides was comparable to the conventional dot assay of directly immobilized large proteins.

Topics: Amino Acid Sequence; Animals; Collodion; Cross-Linking Reagents; Immunoblotting; Immunoenzyme Techniques; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Peptide PHI; Peptides; Protein Binding; Rabbits

1. Based on radioligand binding and adenylate cyclase activation, functional receptors to vasoactive intestinal peptide(VIP)/helodermin, were shown to coexist with beta 2-adrenoceptors and prostaglandin receptors in membranes from a cultured cloned BL/VL3 cell line of murine T-cell lymphoma induced by a radiation leukemia virus. 2. The relative potency of VIP-related peptides to stimulate adenylate cyclase activity was: helodermin greater than VIP greater than peptide histidine isoleucinamide. Five VIP analogs inhibited 125I-iodo-VIP binding and stimulated adenylate cyclase activity, their decreasing order of potency being: VIP greater than [D-Asp3]VIP greater than [D-Ser2]VIP greater than [D-Ala4]VIP = [D-His1]VIP = [D-Phe2]VIP. [D-Phe2]VIP acted as a partial agonist (with an intrinsic activity of 0.1 as compared to that of VIP = 1.0) and competitively inhibited helodermin- and VIP-stimulated adenylate cyclase activity with a similar Ki (0.07-0.10 microM). These data suggest the existence, in this murine T-cell lymphoma, of VIP receptors of the 'helodermin-preferring' subtype that are coupled to adenylate cyclase.

Topics: Adenylyl Cyclases; Alprostadil; Animals; Catecholamines; Cell Membrane; Enzyme Activation; Intercellular Signaling Peptides and Proteins; Leukemia, Radiation-Induced; Lymphoma; Mice; Peptide PHI; Peptides; Receptors, Adrenergic, beta; Receptors, Gastrointestinal Hormone; Receptors, Prostaglandin; Receptors, Vasoactive Intestinal Peptide; Retroviridae; T-Lymphocytes; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

1. Functional vasoactive intestinal peptide (VIP)/helodermin receptors and beta 2-adrenoceptors coexist in membranes from a cultured cloned BL/VL3 cell line of murine T-cell lymphoma induced by a radiation leukemia virus (see preceding paper in this journal). 2. Short-term (5-30 min) exposures of BL/VL3 cells to VIP or isoproterenol induced both homologous and heterologous desensitization. The potency of VIP and isoproterenol to desensitize was similar to their potency to occupy receptors and activate adenylate cyclase. 3. Long-term (16-h) exposure of BL/VL3 cells to VIP induced homologous down regulation only, whereas isoproterenol induced both homologous and heterologous down regulation. The potency of VIP, peptide histidine isoleucinamide, helodermin, helospectin, and [D-Phe2]VIP on the one hand, and of isoproterenol on the other hand, to decrease homologous responses was comparable to their potency for receptor occupancy and adenylate cyclase activation.

Topics: Adenylyl Cyclases; Animals; Cell Membrane; Drug Tolerance; Enzyme Activation; Guanylyl Imidodiphosphate; Intercellular Signaling Peptides and Proteins; Isoproterenol; Leukemia, Radiation-Induced; Lymphoma; Mice; Peptide PHI; Peptides; Receptors, Adrenergic, beta; Retroviridae; Sodium Fluoride; T-Lymphocytes; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Helodermin, VIP and PHI, which share a high degree of homology with secretin, have been identified in the gut but their physiological role is unknown. In this study 3 series of tests were carried out to determine the actions of helodermin, VIP and PHI on pancreatic secretion in 6 conscious dogs and amylase release from the dispersed canine pancreatic acini and to correlate the alterations in pancreatic secretory and circulatory effects in 24 anesthetized dogs. Helodermin, VIP and PHI infused i.v. in graded doses (12.5-200 pmol/kg.h) resulted in a dose-dependent increase in pancreatic HCO3 secretion reaching, respectively, 100%, 7% and 2% of secretin maximum. When combined with constant dose infusion of CCK-8 (100 pmol/kg.h), helodermin but not VIP or PHI augmented dose-dependently the HCO3 secretion. When added in various concentrations (10(-10)-10(-5)M) to the incubation medium of dispersed pancreatic acini only helodermin but not VIP or PHI increased dose-dependently amylase release reaching about 50% of CCK-8 maximum. In anesthetized dogs, the pancreatic blood flow (PBF) measured by electromagnetic blood flowmetry showed an immediate and dose-dependent increase following the injections of various doses of helodermin, VIP, PHI and secretin, the peak blood flow preceding by about 1 min the peak secretory stimulation. This study shows that helodermin resembles secretin in its potent pancreatic HCO3 stimulation but differs from VIP or PHI which are poor secretagogues but potent vasodilators. We conclude that if tested peptides are released in the gut, helodermin, like secretin, may be involved in the hormonal stimulation of exocrine pancreas, whereas VIP and PHI may serve mainly as vasodilators in the pancreatic circulation.

Topics: Animals; Cholecystokinin; Dogs; Gastrins; Intercellular Signaling Peptides and Proteins; Pancreas; Peptide PHI; Peptides; Proteins; Secretin; Vasoactive Intestinal Peptide

A new type of VIP receptor was characterized in human SUP-T1 lymphoblasts. The order of potency of unlabeled peptides, in the presence of [125I]helodermin, was: helodermin(1-35)-NH2 = helodermin(1-27)-NH2 greater than helospectin greater than VIP = PHI greater than [D-Ser2]VIP greater than [D-Asp3]VIP greater than [D-His1]VIP greater than or equal to [D-Ala4]VIP greater than or equal to secretin = GRF. This specificity was distinct from that of all VIP receptors described so far in that: (i) the affinity for helodermin (Kd = 3 nM) was higher than that of VIP (Kd = 15 nM) and PHI (Kd = 20 nM); and (ii) position 4 played an important role in ligand binding. The labeled sites were likely to be functional receptors as adenylate cyclase in crude lymphoblastic membranes (200-10,000 x g pellets) was stimulated by peptides, in the presence of GTP, with the following order of potency: helodermin(1-35)-NH2 greater than helodermin(1-27)-NH2 greater than helospectin = VIP = PHI.

Topics: Adenylyl Cyclases; Cell Membrane; Guanosine Triphosphate; Humans; Intercellular Signaling Peptides and Proteins; Kinetics; Lymphoma; Peptide Fragments; Peptide PHI; Peptides; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; T-Lymphocytes; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Crude membranes (27,000 g pellets) from five normal human pancreases were prepared. In the presence of GTP, the peptides of the secretin family stimulated adenylate cyclase activity, their order of potency being: secretin greater than helodermin greater than peptide histidine isoleucinamide (PHI) greater than or equal to vasoactive intestinal peptide (VIP) greater than growth hormone releasing factor (GRF) (1-29)-NH2. In addition, helodermin and PHI were more efficient than secretin. Secretin (3-27) inhibited fully the secretin stimulation and partially only the helodermin and PHI stimulation of the enzyme. Secretin receptors were investigated by the ability of secretin and related peptides to inhibit tracer binding. [125I]Secretin binding was fully inhibited by secretin (Kd 0.8 nM), helodermin (Kd 200 nM), and PHI (Kd 250 nM). VIP and GRF(1-29)-NH2 induced partial (20%) inhibition at a high 10 microM concentration. The fragments secretin (2-27), (3-27), (4-27), and (7-27) showed the same low potency and efficacy based on their ability to stimulate adenylate cyclase and to occupy secretin receptors. The analogues [Val5]secretin and [Ala2]secretin had a higher potency than secretin. Based on this comparison of adenylate cyclase stimulation and [125I]secretin binding inhibition, it is tempting to conclude that the human pancreas: (a) possesses highly specific secretin receptors and (b) such receptors could not fully account for the whole pattern of adenylate cyclase activation by related peptides, so that the presence of an added type of "helodermin-PHI-preferring" receptors is suggested.

Topics: Adenylyl Cyclases; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Intercellular Signaling Peptides and Proteins; Pancreas; Peptide Fragments; Peptide PHI; Peptides; Receptors, G-Protein-Coupled; Receptors, Gastrointestinal Hormone; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) receptors on guinea pig pancreatic acini differ from those on all other tissues in containing a high-affinity VIP receptor and a low-affinity VIP receptor that has a high affinity for secretin. To characterize the molecular components of these receptors, 125I-VIP was covalently cross-linked to these receptors by four different cross-linking agents: disuccinimidyl suberate, ethylene glycol bis (succinimidyl succinate), dithiobis (succinimidylpropionate), and m-maleimidobenzoyl N-hydroxysuccinimide ester. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a single major polypeptide band of Mr 45,000 and a minor polypeptide band of Mr 30,000 were cross-linked to 125I-VIP. Covalent cross-linking only occurred when a cross-linking agent was added, was inhibited by GTP, was inhibited by VIP receptor agonists or antagonists that interact with VIP receptors, and not by other pancreatic secretagogues that interact with different receptors. For inhibiting both cross-linking and binding of 125I-VIP to the major polypeptide Mr 45,000 and the minor polypeptide Mr 30,000 components, the relative potencies were VIP greater than helodermin greater than rat growth hormone releasing factor greater than peptide histidine isoleucine greater than secretin. The apparent molecular weight of the cross-linked polypeptides were unchanged by dithiothreitol. Thus the high-affinity VIP receptor on pancreatic acinar cell membranes consists of a single major polypeptide of Mr 45,000, and this polypeptide is not a subunit of a larger disulfide-linked structure. Furthermore, either the low-affinity VIP/secretin-preferring receptor was not covalently cross-linked under the experimental conditions or it consists of a major polypeptide with the same molecular weight as the high-affinity VIP receptor.

Topics: Animals; Bombesin; Carbachol; Cell Membrane; Cross-Linking Reagents; Growth Hormone-Releasing Hormone; Guanosine Triphosphate; Guinea Pigs; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Molecular Weight; Pancreas; Peptide PHI; Peptides; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Secretin; Sincalide; Substance P; Vasoactive Intestinal Peptide

Having previously isolated helodermin, the major peptide like vasoactive-intestinal-peptide and peptide-histidine-isoleucinamide, from the venom of the lizard Heloderma suspectum, we decided on a systematic exploration of all (VIP-PHI)-like peptides present in the venom of another lizard of the Helodermatidae family: Heloderma horridum. Six (VIP-PHI)-like peptides (PHH1 to 6) were purified to homogeneity from the venom of the lizard H. horridum with PHH3 and PHH4 representing two minor forms. All peptides cross-reacted in radioimmunoassays for helodermin and PHI but not for VIP. They yielded four fragments (T1 to T4) after trypsin digestion. T1, T2 and T3 showed the same retention time by reverse-phase HPLC and the same amino acid composition; the differences were confined to T4, the C-terminal sequence. PHH5 and PHH6 were found to be identical to synthetic helospectins I and II respectively. PHH1 and PHH3 probably resulted from a secondary modification of PHH5, while PHH2 and PHH4 derived from PHH6. Thus, the VIP-like peptides, previously called helospectins, are in fact typical of H. horridum venom. We confirmed that helodermin is the major (VIP-PHI)-like peptide of the venom of H. suspectum and observed its absence in H. horridum venom. Also, we found that positions 8 and 9 of helodermin are occupied by two Glu residues instead of two Gln as previously published. Helospectin-like material was also present in H. suspectum venom but in very small amount. In both venoms all VIP-like peptides were equally potent and efficient when tested for (a) their ability to occupy VIP as well as secretin receptors in rat pancreatic membranes and VIP receptors in rat liver membranes, and (b) the ensuing activation of adenylate cyclase in both membrane preparations.

Topics: Adenylyl Cyclases; Animals; Enzyme Activation; Immunochemistry; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Lizards; Peptide Fragments; Peptide PHI; Peptides; Species Specificity; Vasoactive Intestinal Peptide; Venoms

The effect of helodermin, a member of the secretin/vasoactive intestinal polypeptide (VIP)/peptide histidine isoleucine (PHI) family of peptides, on pituitary prolactin (PRL) secretion was examined in the rat. Either i.c.v. or i.v. injection of helodermin resulted in a dose-related increase in plasma PRL levels in urethane-anesthetized male rats. At the doses tested the potency of helodermin to raise plasma PRL levels was greater than that of rat PHI and porcine PHI, and as great as that of VIP.

Topics: Animals; Injections, Intravenous; Injections, Intraventricular; Intercellular Signaling Peptides and Proteins; Male; Peptide PHI; Peptides; Prolactin; Rats; Rats, Inbred Strains; Vasoactive Intestinal Peptide

The pharmacological effects of peptide histidine isoleucine (PHI), glucagon and secretin were compared with vasoactive intestinal polypeptide (VIP) on rabbit urethra and anococcygeus muscle. VIP and PHI dose-dependently inhibited induced contractions of both smooth muscle preparations. Cross-tachyphylaxis between VIP and PHI was demonstrated in the urethra preparation, suggesting that their activity is mediated via a common receptor or second messenger. Glucagon and secretin were without effect on either preparation. Radioimmunoassays demonstrated substantial concentrations of VIP and PHI in both urethra and anococcygeus tissue extracts. These observations suggest that PHI is an additional candidate together with VIP to mediate relaxation of rabbit urethra and anococcygeus muscle. When compared with VIP, Gila monster venom was found to inhibit both smooth muscle preparations, producing concentration-response curves parallel to those produced by VIP.

Topics: Animals; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Lizards; Male; Muscle, Smooth; Peptide PHI; Peptides; Rabbits; Radioimmunoassay; Tissue Extracts; Urethra; Vasoactive Intestinal Peptide; Venoms

The complete amino acid sequence of helodermin isolated from the venom of Gila monster was elucidated. The peptide was shown to be a basic pentatriacontapeptide amide: His-Ser-Asp-Ala-Ile-Phe-Thr-Gln-Gln-Tyr-Ser-Lys-Leu-Leu-Ala-Lys-Leu-Ala- Leu-Gln-Lys- Tyr-Leu-Ala-Ser-Ile-Leu-Gly-Ser-Arg-Thr-Ser-Pro-Pro-Pro-NH2. A high degree of sequence similarities to secretin/VIP/PHI/(PHM)/GRF from mammal and bird was observed over the entire N-terminal 1-27 sequence. In particular, the amino acid residues in positions 3, 6 and 7 were found to be common to 9 peptides of the family. Another interesting feature of the structure of helodermin was its C-terminal -Pro-Pro-Pro-NH2 sequence. Isolation of helodermin was the first demonstration of the existence of a secretin/VIP-related peptide in an animal that is neither mammal nor bird.

Topics: Amino Acid Sequence; Animals; Biological Evolution; Chymotrypsin; Growth Hormone-Releasing Hormone; Humans; Intercellular Signaling Peptides and Proteins; Lizards; Peptide Fragments; Peptide PHI; Peptides; Secretin; Trypsin; Vasoactive Intestinal Peptide; Venoms