vasoactive-intestinal-peptide and heliodermin

Mast cell degranulation is important in the pathogenesis of anaphylaxis and allergic disorders. Many animal venoms contain components that can induce mast cell degranulation, and this has been thought to contribute to the pathology and mortality caused by envenomation. However, we recently reported evidence that mast cells can enhance the resistance of mice to the venoms of certain snakes and that mouse mast cell-derived carboxypeptidase A3 (CPA3) can contribute to this effect. Here, we investigated whether mast cells can enhance resistance to the venom of the Gila monster, a toxic component of that venom (helodermin), and the structurally similar mammalian peptide, vasoactive intestinal polypeptide (VIP). Using 2 types of mast cell-deficient mice, as well as mice selectively lacking CPA3 activity or the chymase mouse mast cell protease-4 (MCPT4), we found that mast cells and MCPT4, which can degrade helodermin, can enhance host resistance to the toxicity of Gila monster venom. Mast cells and MCPT4 also can limit the toxicity associated with high concentrations of VIP and can reduce the morbidity and mortality induced by venoms from 2 species of scorpions. Our findings support the notion that mast cells can enhance innate defense by degradation of diverse animal toxins and that release of MCPT4, in addition to CPA3, can contribute to this mast cell function.

Topics: Amino Acid Sequence; Animals; Carboxypeptidases A; Intercellular Signaling Peptides and Proteins; Lizards; Mast Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Mutant Strains; Molecular Sequence Data; Peptides; Scorpion Venoms; Serine Endopeptidases; Vasoactive Intestinal Peptide; Venoms

An identification of PAC1- and VPAC-type receptors in a great number of neoplastic cells gave rise to intensive studies on the biochemical and physiological role of the mentioned peptides in cancers. Our earlier studies focused on effects of pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) in C6 glioma cells have shown their stimulatory receptor-mediated action on the cyclic adenosine monophosphate (cAMP)-generating system. In the present study, we demonstrated that truncated peptides, i.e., PACAP6-38 and VIP6-28, both produced a significant inhibition of the VIP-induced increase in cAMP production, whereas only PACAP6-38 did antagonize the PACAP-38 effect. In contrast to the well-expressed PACAP-38 and VIP effects on cAMP production in C6 cells, helodermin and secretin were poorly active as cAMP stimulators in this cell line, displaying some activity only at a high 5-microM dose. PACAP-38 and, to a lesser extent VIP stimulated the proliferation of C6 glioma cells, which was shown by an increased incorporation of 3H-thymidine into the cells, and the effects of these two peptides were antagonized by PACAP6-38. The truncated PACAP (10 microM) by itself significantly inhibited C6 cell proliferation. The study with the use of forskolin and dibutyryl-cAMP revealed that the growth effects of PACAP were cAMP independent. Our findings suggest that glioma C6 cells possess PAC1- and VPAC-type receptors, but the density of PAC1 seems to be much larger than VPAC receptors. Although the proliferative activity of PACAP and VIP is mediated via the PAC1-type receptor, the signaling cascade underlying this phenomenon does not seem to involve cAMP.

Topics: Animals; Bucladesine; Cell Line, Tumor; Cell Proliferation; Colforsin; Cyclic AMP; Enzyme Inhibitors; Intercellular Signaling Peptides and Proteins; Peptide Fragments; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Rats; Secretin; Vasoactive Intestinal Peptide

Three distinct complementary DNAs for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) receptors have been cloned and designated VIP-1 receptor (VIP-1R), VIP-2 receptor (VIP-2R), and PACAP receptor (PACAP-R). In the present study, we have characterized the binding sites on primary mouse calvarial osteoblasts for VIP and related peptides. By analyzing the cAMP response, the rank order of response observed was PACAP 38 > PACAP 27 > helodermin > VIP > helospectin > glucagon > PHI >>> secretin. The VIP-2R/PACAP-R antagonist, PACAP 6-38, inhibited both VIP- and PACAP-stimulated cAMP formation. Binding studies using an atomic force microscopy (AFM) technique showed high affinity binding for VIP and PACAP 38, but not for secretin. Radioligand binding studies using (125)I-VIP and (125)I-PACAP 38 demonstrated a more specific and higher affinity binding for PACAP 38 than for VIP. Secretin failed to inhibit both (125)I-VIP and (125)I-PACAP 38 binding. RT-PCR demonstrated that undifferentiated mouse calvarial osteoblasts express messenger RNA for VIP-2R, but not for VIP-1R or PACAP-R. When the osteoblasts were cultured for 20 days to induce bone noduli formation, VIP-1R, in addition to VIP-2R, were expressed when the nodules started to mineralize at 12 days. Taken together, these data demonstrate that mouse calvarial osteoblasts express functional VIP-2R with higher affinity binding for PACAP than for VIP and that the VIP-1R expression is induced during osteoblastic differentiation.

Topics: Animals; Cells, Cultured; Cyclic AMP; Glucagon; Intercellular Signaling Peptides and Proteins; Kinetics; Mice; Neuropeptides; Osteoblasts; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Polymerase Chain Reaction; Radioligand Assay; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Peptide, Type II; Secretin; Skull; Vasoactive Intestinal Peptide

Six forms of helospectin (a vasoactive intestinal peptide analogue) were purified from the venom of the Heloderma horridum lizard. Their identification was performed by combining sequencing by automated Edman degradation and electrospray mass spectrometry analysis on the complete peptides and their tryptic fragments. The products resulting from the action of an O-glycosidase were also analysed. Two forms were identified as the previously named Hs1 and Hs2 of 38 and 37 amino-acid residues, respectively. Two forms corresponded to Hs1 and Hs2 O-glycosylated by a N-acetylhexosamine-hexose motif attached to the Ser32 residue. Two other forms were not completely characterized but might correspond to the O-glycosylated forms bearing a phosphate or a sulfate group. The glycosylation did not affect the capacity of the helospectins to recognize and to activate the human and the rat VPAC1 and VPAC2 receptors.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Amphibian Venoms; Animals; Cell Membrane; CHO Cells; Cricetinae; Dose-Response Relationship, Drug; Enzyme Activation; Glycosylation; Humans; Inhibitory Concentration 50; Intercellular Signaling Peptides and Proteins; Lizards; Mass Spectrometry; Molecular Sequence Data; Peptides; Rats; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Peptide, Type II; Receptors, Vasoactive Intestinal Polypeptide, Type I; Recombinant Proteins; Sequence Homology, Amino Acid; Trypsin; Vasoactive Intestinal Peptide

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

Helodermin and exendin-4, two peptides isolated from the salivary gland of the Gila monster, Heloderma suspectum, are approximately 50% homologous to vasoactive intestinal peptide (VIP) and glucagon-like peptide-1 (GLP-1), respectively, and interact with the mammalian receptors for VIP and GLP-1 with equal or higher affinity and efficacy. Immunohistochemical studies suggested the presence of helodermin-like peptides in mammals. To determine whether helodermin and exendin-4 are present in mammals and their evolutionary relationship to VIP and GLP-1, their cDNAs were first cloned from Gila monster salivary gland. Northern blots and reverse transcription-polymerase chain reaction of multiple Gila monster tissues identified approximately 500-base pair transcripts only from salivary gland. Both helodermin and exendin-4 full-length cDNAs were approximately 500 base pairs long, and they encoded precursor proteins containing the entire amino acid sequence of helodermin and exendin-4, as well as a 44- or 45-amino acid N-terminal extension peptide, respectively, having approximately 60% homology. The size and structural organization of these cDNAs indicated that they were closely related to one another but markedly different from known cDNAs for the VIP/GLP-1 peptide family previously identified in both lower and higher evolved species. Cloning of the Gila monster VIP/peptide histidine isoleucine, pituitary adenylate cyclase activating polypeptide, and glucagon/GLP-1 cDNAs and Southern blotting of Gila monster DNA demonstrate the coexistence of separate genes for these peptides and suggests, along with the restricted salivary gland expression, that helodermin and exendin-4 coevolved to serve a separate specialized function. Probing of a variety of rat and human tissues on Northern blots, human and rat Southern blots, and genomic and cDNA libraries with either helodermin- or exendin-4-specific cDNAs failed to identify evidence for mammalian homologues. These data indicate that helodermin and exendin-4 are not the precursors to VIP and GLP-1 and that they belong to a separate peptide family encoded by separate genes. Furthermore, the existence of as yet undiscovered mammalian homologues to helodermin and exendin-4 seems unlikely.

Topics: Amino Acid Sequence; Animals; Base Sequence; Chickens; Cloning, Molecular; DNA Primers; DNA, Complementary; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Humans; Intercellular Signaling Peptides and Proteins; Lizards; Mammals; Molecular Sequence Data; Neuropeptides; Peptide Fragments; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Polymerase Chain Reaction; Protein Precursors; Rats; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Trout; Vasoactive Intestinal Peptide; Venoms

Effects of vasoactive intestinal peptide (VIP) on T cell migration are mediated by structurally distinct types I (VIPR1) and II (VIPR2) G protein-associated receptors. The two receptor types were proposed to transduce opposite effects on human T cells, since cytokine-induced chemotaxis of VIPR1-bearing HuT 78 human T cells, in contrast to T cells that express VIPR2, was inhibited by VIP. We studied chemotactic effects of VIP and related agonists with different affinities for VIP- and peptide histidine-isoleucine (PHI)-related receptors. All, VIP, secretin (SEC), a specific ligand for VIPR1, helodermin (HEL), an activator of helodermin-preferring VIPR2, as well as PHI, stimulated chemotaxis into micropore filters of both normal human peripheral blood T and B cells. Involvement of VIPRs was supported by inhibition of VIP-related agonist-induced migration of T and B cells with a VIPR antagonist. Peripheral blood lymphocyte (PBL) chemotaxis to VIP, SEC, HEL and PHI was reduced by inhibition of tyrosine kinase and pertussis or cholera toxin, whereas inhibition of protein kinase C only affected SEC-induced chemotaxis of PBL significantly. VIP-related agonists induced deactivation of migration at high concentrations. Findings in PBL suggest that VIPR1 activation can stimulate normal T and B cell chemotaxis. Different signaling mechanisms may be involved in mediating chemotactic activation of VIPRs and PHIRs, which may allow further exploration of receptor-dependent mechanisms and signaling pathways of VIP as mediator of PBL functions.

Topics: Cell Movement; Chemotaxis, Leukocyte; Cholera Toxin; Enzyme Inhibitors; Humans; Intercellular Signaling Peptides and Proteins; Lymphocytes; Peptides; Phosphotransferases; Receptors, Vasoactive Intestinal Peptide; Secretin; Vasoactive Intestinal Peptide; Virulence Factors, Bordetella

The effects of helodermin, a basic 35-amino acid peptide isolated from the venom of a lizard salivary gland, on arterial blood pressure and heart rate were examined in the rat, focusing on the possibility that activation of ATP sensitive K+ (K(ATP)) channels is involved in the responses. The results were also compared with those of vasoactive intestinal polypeptide (VIP). Helodermin produced hypotension in a dose-dependent manner with approximately similar potency and duration to VIP. Hypotension induced by both peptides was significantly attenuated by glibenclamide, which abolished a levcromakalim-produced decrease in arterial blood pressure. Oxyhemoglobin did not affect helodermin-induced hypotension, whereas it shortened the duration of acetylcholine (ACh)-produced hypotension. These findings suggest that helodermin-produced hypotension is partly attributable to the activation of glibenclamide-sensitive K+ channels (K(ATP) channels), which presumably exist on arterial smooth muscle cells. EDRF (endothelium-derived relaxing factor)/nitric oxide does not seem to play an important role in the peptide-produced hypotension.

Topics: Animals; Blood Pressure; Glyburide; Heart Rate; Hypotension; Intercellular Signaling Peptides and Proteins; Male; Oxyhemoglobins; Peptides; Potassium Channels; Rats; Rats, Wistar; Vasoactive Intestinal Peptide

Vasoactive intestinal polypeptide (VIP) acts through interaction with two subclasses of seven transmembrane G protein-coupled receptors named VIP1 and VIP2 receptors. These receptors have been cloned in different species, such as rat and human. Considering the different distribution of both receptor subclasses, there is considerable interest in the development of selective agonists and antagonists. The present study compares the binding properties of VIP, PACAP, GRF, secretin, and helodermin analogues on recombinant rat and human VIP1 and VIP2 receptors. On both rat and human receptors, secretin and GRF had a higher affinity for the VIP1 receptor subtypes. The amino-shortened VIP, and the carboxy terminal-shortened VIP and PACAP analogues also presented a higher affinity for the VIP1 receptor. PHI, PHV, helodermin, and helospectin were selective for the human VIP2 receptor subtypes. These results suggest that the helical structure of the carboxy terminal end is necessary for VIP2 recognition. The differences between species were the following: PHI, PHV, helodermin, and helospectin had a higher affinity for the rat VIP1 receptor than for the human VIP1 receptor. On both rat and human receptors, D-Ala4 VIP and D-Phe4 VIP had a high affinity for the VIP1 receptor and a low affinity for the VIP2 receptor. Thus, three domains of the ligand involved in VIP1/VIP2 receptor discrimination were identified: the amino acid residue in position 4 ([D-Ala4], [D-Phe4]VIP), in positions 8 and 9 (the effects of helodermin and helospectin), and the carboxy terminal end (the effects of the shortened VIP and pituitary adenylate cyclase activating polypeptide analogues).

Topics: Amino Acid Sequence; Animals; Binding, Competitive; CHO Cells; Cricetinae; Humans; Intercellular Signaling Peptides and Proteins; Molecular Sequence Data; Neuropeptides; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Rats; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Peptide, Type II; Receptors, Vasoactive Intestinal Polypeptide, Type I; Recombinant Proteins; Secretin; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Transfection; Vasoactive Intestinal Peptide

To determine the role of VIP-like peptides on neurotransmission of vagus nerve, we evaluated the effects of helodermin, helospectin, and vasoactive intestinal peptide (VIP) on the contraction of guinea pig tracheal smooth muscle evoked by electrical field stimulation (EFS) or the exogenous application of actylcholine (ACh). Isometric tension of tracheal strips was measured in the presence of indomethacin (10(-6) M) and of guanethidine (10(-6) M). VIP (10(-9) M to 10(-7) M) significantly suppressed the contraction evoked by EFS. VIP, at concentrations of 10(-9) M and 10(-8) M, did not affect the ACh-evoked contraction, but a concentration of 10(-7) M suppressed ACh-evoked contraction. Helospectin and helodermin (10(-8) M and 10(-7) M) significantly suppressed the EFS-evoked contraction, but 10(-9) M showed no effect. Helospectin and helodermin had no effect on the ACh sensitivity of smooth muscle up to 10(-8) M, but a concentration of 10(-7) M suppressed the ACh-evoked contraction. These results indicate that helodermin, helospectin, and VIP exert both pre- and post-junctional inhibitory effects on the airway smooth muscle of guinea pigs. These peptides, thus, inhibited tracheal smooth muscle contraction prejunctionally at low concentrations, and acted postjunctionally at higher concentrations.

Topics: Acetylcholine; Animals; Dose-Response Relationship, Drug; Electric Stimulation; Enzyme Inhibitors; Female; Guanethidine; Guinea Pigs; Indomethacin; Intercellular Signaling Peptides and Proteins; Male; Muscle Contraction; Muscle, Smooth; Neuromuscular Junction; Peptides; Sympatholytics; Synaptic Transmission; Trachea; Vagus Nerve; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide (VIP) stimulated adenylyl cyclase activity in rat and mouse peritoneal macrophage membranes. GTP potentiated the stimulatory effect of VIP so that it was routinely included at 10 microM GTP. Other agents like GTP, Gpp(NH)p, GTP-gamma-S, sodium fluoride, and forskolin, at a concentration of 0.1 mM, increased the basal activity of enzyme by 3.1, 5.7, 4.7, 3.6, and 7.8-fold, respectively. The stimulation of adenylyl cyclase by VIP was time, temperature, and membrane concentration dependent. Half-maximal enzyme activation (ED50) was very similar in rat and mouse peritoneal macrophage membranes (1.5 +/- 0.1 nM and 1.0 +/- 0.1 nM, respectively). However, VIP showed more efficacy in mouse macrophages membranes (about 3.1-fold basal values) than that in rat macrophage membranes (about 2.5-fold basal values). The relative potency of several peptides upon stimulation of adenylyl cyclase activity showed the following potency in both species: VIP = PACAP38 = PACAP27 > helodermin > PHI > secretin. On the other hand, a M(r)-45 kDa alpha s subunit of Gs protein was demonstrated by both ADP-ribosylation and immunoblot in mouse and rat peritoneal macrophage membranes. The present results, together other previous, strongly suggest that VIP play an important role in the regulation of macrophage function.

Topics: Adenosine Diphosphate Ribose; Adenylyl Cyclases; Animals; Cell Membrane; Cholera Toxin; Colforsin; Enzyme Activation; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylyl Imidodiphosphate; Intercellular Signaling Peptides and Proteins; Kinetics; Macrophages, Peritoneal; Mice; NAD; Neuropeptides; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Rats; Rats, Wistar; Secretin; Sodium Fluoride; Vasoactive Intestinal Peptide

We have examined the binding of radio-iodinated vasoactive intestinal peptide (VIP) to rat platelets. The binding was time- and temperature-dependent and was reversible, saturable and specific. Scatchard analysis of binding data suggested the presence of a single class of binding sites, with Kd = 2.49 +/- 0.76 nM and Bmax = 112.1 +/- 54.6 fmol/10(8) cells. Several VIP-related peptides inhibited 125I-VIP binding to rat platelets with the following order of potency: helodermin > or = VIP > peptide histidine isoleucine. Glucagon, secretin, growth hormone-releasing hormone (GHRH), and gastric inhibitory peptide (GIP) were ineffective. VIP and the other peptides increased cyclic AMP production with the same order of potency as the inhibition of binding, but the stimulation by VIP was less marked than that by prostacyclin (PGI2). We conclude that rat platelets have functional, adenylate cyclase-linked, receptors that bind preferentially to helodermin and VIP.

Topics: Animals; Binding Sites; Binding, Competitive; Blood Platelets; Cell Count; Cyclic AMP; Epoprostenol; Gastric Inhibitory Polypeptide; Glucagon; Growth Hormone-Releasing Hormone; Intercellular Signaling Peptides and Proteins; Kinetics; Peptides; Protein Binding; Rats; Rats, Sprague-Dawley; Receptors, Vasoactive Intestinal Peptide; Secretin; Temperature; Vasoactive Intestinal Peptide

Topics: Animals; Binding, Competitive; Cell Membrane; CHO Cells; Clone Cells; Cricetinae; Growth Hormone-Releasing Hormone; Humans; Intercellular Signaling Peptides and Proteins; Neuropeptides; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Polypeptide, Type I; Recombinant Proteins; Secretin; Transfection; Vasoactive Intestinal Peptide

Topics: Adenylyl Cyclases; Aged; Binding, Competitive; Cell Membrane; Humans; Intercellular Signaling Peptides and Proteins; Kinetics; Male; Middle Aged; Neuropeptides; Neurotransmitter Agents; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Prostate; Prostatectomy; Prostatic Hyperplasia; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Pituitary Hormone; Receptors, Vasoactive Intestinal Peptide; Secretin; Vasoactive Intestinal Peptide

Helodermin is a 35 amino acid-residue peptide of the vasoactive intestinal polypeptide (VIP) family, which was originally isolated from the venom of Heloderma suspectum on the basis of its capacity to stimulate adenylate cyclase in the rat pancreas. In the present study, using rat dispersed pancreatic acini, we examined the binding characteristics of helodermin, its action on amylase secretion, and the production of intracellular cyclic AMP (cAMP). Helodermin stimulated intracellular cAMP production dose dependently in a manner that was nearly identical to that of VIP. Helodermin stimulated amylase secretion dose dependently, showing an efficacy similar to that of VIP but with 100 times less potency than VIP. Adding 0.5 mM 3-isobutyl-1-methylxanthine increased the potency of helodermin's action on amylase secretion but did not change the efficacy. The binding study showed that the order of binding affinity to VIP receptors was VIP = helodermin > secretin, while the order of that to secretin receptors was secretin > helodermin > VIP. These results suggest that helodermin stimulated amylase secretion from rat dispersed pancreatic acini via VIP-preferring receptors that are coupled to the production of intracellular cAMP, but a part of the postreceptor mechanism for enzyme secretion is different from that of VIP.

Topics: Amylases; Animals; Binding, Competitive; Cyclic AMP; Dose-Response Relationship, Drug; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Intracellular Fluid; Male; Pancreas; Peptides; Radioligand Assay; Rats; Rats, Sprague-Dawley; Vasoactive Intestinal Peptide

Two chimeras were obtained by substituting the DNA sequence encoding the N-terminal extracellular domain of the VIP and secretin receptors by the homologous DNA sequence encoding the secretin (N-Sn/VIP.r) and VIP receptor (N-VIP/Sn.r), respectively. These chimeric receptors were transfected and stably expressed in CHO cells. Their pharmacological properties were then compared to the corresponding recombinant "wild type" receptors, expressed in the same cell line. Binding data were obtained for the wild types and the N-VIP/Sn.r but not for the N-Sn/VIP receptor. Functional data (adenylate cyclase activation) were obtained in all cases. In order to minimize the effects of an excess of receptors and thus, to compare validly binding and functional data, we determined agonists EC50 values after down regulation of the receptors (i.e. after a pretreatment of the cells for 24 h with VIP or secretin). The order of potency of the peptides for receptor occupancy and adenylate cyclase activation indicated that the N-terminal extracellular domain of each receptor was the key element for discrimination between secretin and VIP.

Topics: Adenylyl Cyclases; Animals; CHO Cells; Cricetinae; Enzyme Activation; Intercellular Signaling Peptides and Proteins; Ligands; Neuropeptides; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, G-Protein-Coupled; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Recombinant Fusion Proteins; Secretin; Structure-Activity Relationship; Vasoactive Intestinal Peptide

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

We have cloned and sequenced a cDNA isolated from a human SUP-T1 lymphoblast cell line library. It encoded a 457 amino acids protein having 87% identity with the rat PACAP type II, VIP2 receptor. Chinese hamster ovary (CHO) cells stably transfected with cloned cDNA expressed a specific binding of 125I[Acetyl-His1]PACAP-27. This binding was inhibited by GTP, and by the peptides helodermin, VIP, PACAP-27 and PACAP-38 that also stimulated adenylate cyclase activity. The order of potency was PACAP-38 > VIP > or = helodermin > or = PACAP-27. Comparison of the results in two cell lines expressing different receptor densities suggested that helodermin and PACAP-38 had a higher intrinsic activity than VIP and PACAP-27.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; CHO Cells; Cloning, Molecular; Cricetinae; DNA Primers; DNA, Complementary; Humans; Intercellular Signaling Peptides and Proteins; Lymphocytes; Molecular Sequence Data; Neuropeptides; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Rats; Receptors, Vasoactive Intestinal Peptide; Receptors, Vasoactive Intestinal Peptide, Type II; Sequence Homology, Amino Acid; Transfection; Vasoactive Intestinal Peptide

The present study was designed to study the localization and effects of some VIP-related peptides on the cerebral circulation in cats. A rich supply of nerve fibres containing vasoactive intestinal peptide- (VIP) was seen. Nerve fibres containing pituitary adenylate cyclase activating peptide and helospectin-like immunoreactivity (-IR) were moderate in numbers whereas only a sparse supply of fibres containing helodermin-IR was seen. Double immunostaining revealed that the majority of PACAP- and helospectin-IR nerve fibres contained VIP. Using a sensitive in vitro system prostaglandin F2 alpha-precontracted circular segments of the cat middle cerebral artery relaxed upon administration of VIP, PACAP, helospectin I, helospectin II and helodermin. These effects were non-endothelium dependent with pD2-values varying between 7.6 and 8.1. The maximum relaxation varied between 47% and 79% of precontraction. Local cerebral blood flow was studied in anaesthetised cats. Cortical injection of PACAP-38, helospectin or helodermin, 5 micrograms in a volume of 1 microliter, revealed moderate and consistent increases in flow. The increase in cerebral blood flow was rapid and concentration-dependent with maximum increases of 18 +/- 6% for PACAP, 21 +/- 5% for helodermin, 16 +/- 7% for helospectin I and 19 +/- 5% for helospectin II. The vehicle caused no significant response (2 +/- 4%).

Topics: Animals; Cats; Cerebral Arteries; Cerebrovascular Circulation; Dinoprost; Endothelium, Vascular; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Laser-Doppler Flowmetry; Neuropeptides; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; 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

Neuropeptides and neutrophils are postulated to be important immune effector molecules and cells, respectively, in a variety of lung inflammatory conditions.. We examined the effects of three lung neuropeptides, substance P, vasoactive intestinal peptide (VIP), and somatostatin and two relatively protease-resistant peptides, helodermin and sandostatin, on human neutrophil migration across 3 microns pore filters and human endothelial monolayers cultured on these filters.. At concentrations of 1 to 10 mumol/L, substance P induced significant neutrophil migration that was dose-responsive and equivalent through endothelium and filters. Neither VIP, helodermin, somatostatin, nor sandostatin induced significant neutrophil migration through either barrier at doses of 10(-14) to 10(-5) mol/L. Because VIP and somatostatin have been reported to inhibit some inflammatory cell functions, we also examined their effects on chemoattractant-induced neutrophil migration. Pretreatment of neutrophils and/or endothelium with VIP or somatostatin at either 10(-5) or 10(-10) mol/L did not significantly inhibit formyl-methionyl-leucyl-phenylalanine-, leukotriene B4-, or platelet activating factor-induced neutrophil migration through naked filters or endothelium. Thus only substance P had significant effects on neutrophil chemotaxis.. Substance P, but not VIP or somatostatin, may be important in directly influencing neutrophil migration across endothelium towards lung inflammatory sites. However, all three neuropeptides might still modulate neutrophil functions and lung inflammatory responses through effects on other lung cells.

Topics: Cell Line; Cell Migration Inhibition; Cell Separation; Chemotaxis, Leukocyte; Dose-Response Relationship, Drug; Endothelium, Vascular; Humans; Intercellular Signaling Peptides and Proteins; Neuropeptides; Neutrophils; Octreotide; Peptides; Somatostatin; Substance P; Vasoactive Intestinal Peptide

We earlier showed that the neuropeptide vasoactive intestinal peptide (VIP) reduces or prevents acute injury produced in rat lungs by xanthine and xanthine oxidase. We have now examined whether VIP can protect against lung injury induced by paraquat, a prooxidant pesticide. Isolated guinea pig lungs were perfused for 60 min with Krebs-4% albumin and mechanically ventilated with 95% O2-5% CO2. Infusion of paraquat (100 mg/kg) into the pulmonary artery (n = 9 observations) increased peak airway pressure from 10.1 +/- 0.6 to 54.7 +/- 6.5 cmH2O, perfusion pressure from 8.0 +/- 0.5 to 14.9 +/- 3.0 cmH2O, wet-to-dry lung weight ratio to 7.17 +/- 0.37, and bronchoalveolar lavage protein content to 2.70 +/- 0.83 mg/ml (P < 0.01). Pretreatment with 1-3 micrograms.kg-1 x min-1 VIP markedly attenuated or prevented all abnormalities. Of the related peptides tested, helodermin was as effective as VIP, but secretin and glucagon were ineffective. The results demonstrate that VIP and helodermin protect perfused guinea pig lungs against paraquat-induced injury and support the view that VIP has antioxidant activity.

Topics: Animals; Body Water; Guinea Pigs; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Lung; Lung Diseases; Male; Paraquat; Peptides; Perfusion; Permeability; Pressure; Prostaglandin-Endoperoxide Synthases; Proteins; Vasoactive Intestinal Peptide

We recently reported that the widely distributed neuropeptide vasoactive intestinal peptide (VIP) reduces inflammatory lung injury due to a variety of agents and inhibits the associated generation of cyclo-oxygenase and lipoxygenase products. We therefore investigated whether VIP may inhibit phospholipase A2 activity, thus reducing the release of arachidonic acid, the common precursor of all eicosanoids. VIP dose-dependently inhibited PLA2 of porcine pancreas and of Naja naja venom, as assessed by the release of free [3H]oleic acid from labeled Escherichia coli phospholipids. The potency of VIP was similar to that of mepacrine, with 50% inhibition at 400-500 microM. The closely related peptide helodermin produced 50% inhibition at 200 microM, but secretin and peptide histidine isoleucineamide produced little or no inhibition. The results suggest that VIP and helodermin selectively inhibit PLA2 in vitro. If this activity is exerted in vivo, it may contribute to the anti-inflammatory activity of these two peptides.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Calcium; Elapid Venoms; Escherichia coli; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Pancreas; Peptides; Phospholipases A; Phospholipases A2; Phospholipids; Quinacrine; Serum Albumin, Bovine; Vasoactive Intestinal Peptide

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

The aim of the present study was to investigate the action of pituitary adenylate cyclase-activating polypeptide (PACAP) on ion transport in the guinea pig distal colon. Submucosal/mucosal segments from distal colon were mounted in Ussing flux chambers, and increases in short-circuit current (Isc) were used as an index of secretion. Serosal addition of PACAP-38 and PACAP-27 produced concentration-dependent (10(-10)-10(-6) M) increases in Isc. Furosemide and chloride-free solutions significantly reduced the PACAP-evoked responses. Tetrodotoxin (TTX) completely blocked PACAP-evoked responses. Atropine significantly reduced the PACAP-evoked responses but did not abolish the responses. The results suggest that PACAP evokes chloride secretion through cholinergic and noncholinergic neural mechanism. Vasoactive intestinal polypeptide (VIP), peptide histidine-isoleucine amide, and helodermin evoked Isc in a concentration-dependent manner. Atropine reduced but did not abolish the VIP- and related peptides-evoked responses. TTX also significantly decreased the responses to higher concentrations of VIP and related peptides but did not abolish the responses. The results suggest that VIP and related peptides act on both submucosal neurons and the epithelial cell itself. VIP tachyphylaxis significantly decreased PACAP-38- and PACAP-27-evoked responses. These results provide evidence that PACAP recognizes, in some part, VIP receptors in the submucosal neurons to evoke chloride secretion.

Topics: Animals; Atropine; Biological Transport; Chlorides; Colon; Dose-Response Relationship, Drug; Evoked Potentials; Furosemide; Guinea Pigs; Intercellular Signaling Peptides and Proteins; Intestinal Mucosa; Ions; Male; Neurons; Neuropeptides; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Tetrodotoxin; Vasoactive Intestinal Peptide

Small-cell lung cancer (SCLC) is a common and highly fatal malignancy for which there is no satisfactory treatment. The amphibian peptide bombesin and its mammalian counterpart, gastrin-releasing peptide, serve as autocrine growth factors for the SCLC cells, but little is known about endogenous substances that inhibit the growth and proliferation of these tumor cells. We report that the neuropeptide vasoactive intestinal peptide (VIP) markedly inhibits the growth and multiplication of SCLC cell lines NCI-H345 and NCI-H69, and that the closely related peptide helodermin inhibits the proliferation of NCI-H345 cells with even higher efficacy. In the latter cells, the inhibition by VIP and isobutyl methyl xanthine paralleled their ability to stimulate cyclic adenosine monophosphate production within the cells. The peptide-induced suppression of SCLC proliferation is enhanced in the presence of an anti-bombesin monoclonal antibody. The antimitogenic activities of VIP and helodermin, and their enhancement by anti-bombesin antibody, offer the potential for a new approach to the pharmacologic control of SCLC.

Topics: Antibodies, Monoclonal; Bombesin; Carcinoma, Small Cell; Cell Division; Cell Line; Dose-Response Relationship, Drug; Humans; Intercellular Signaling Peptides and Proteins; Kinetics; Lung Neoplasms; Peptides; Time Factors; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Venoms

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

Vasoactive intestinal peptide (VIP, 10 nM) inhibited the release of cyclo-oxygenase products, detected by both bioassay and radioimmunoassay, induced by leukotriene (LT) D4 (3-30 pmol) and bradykinin (BK, 3-30 nmol) from guinea-pig isolated perfused lung. Helodermin (10 nM), a peptide that is structurally related to VIP, and salbutamol (10 nM), a beta 2-adrenoceptor agonist, evoked a similar inhibitory effect on LTD4-induced release of cyclo-oxygenase products. The generation of TxB2 and 6-keto-PGF1 alpha following stimulation with exogenously administered arachidonic acid (30-300 nmol) was not significantly attenuated in the presence of either VIP, helodermin or salbutamol. These results show that VIP, helodermin and salbutamol are potent inhibitors of the release of cyclo-oxygenase products induced by agonists known to activate endogenous arachidonic acid metabolism in guinea-pig lung. Since the metabolism of exogenously administered arachidonic acid was not inhibited these results suggest that the inhibitory effect may be exerted on events preceding the mobilisation of arachidonic acid and may involve cyclic AMP.

Topics: Albuterol; Animals; Bradykinin; Cyclic AMP; Guinea Pigs; Intercellular Signaling Peptides and Proteins; Lung; Male; Peptides; Perfusion; Prostaglandin-Endoperoxide Synthases; Prostaglandins; SRS-A; Thromboxane B2; Vasoactive Intestinal Peptide; Venoms

A number of regulatory peptides were investigated for their ability to elevate plasma cAMP. Pituitary adenylate cyclase activating peptide (PACAP)-27, PACAP-38, helodermin, helospectin I and II, vasoactive intestinal peptide (VIP), glucagon, parathyroid hormone (PTH), calcitonin and calcitonin gene-related peptide were among the peptides that were highly effective in raising plasma cAMP when given intravenously in equimolar doses to conscious mice. PACAP-27 and -38 were more effective than any of the other peptides. PACAP 16-38, secretin, gastrin-17, galanin, somatostatin, cholecystokinin-8s, pancreatic polypeptide, substance P, peptide YY and neuropeptide Y were inactive and also did not interfere with the PACAP-27-evoked rise in plasma cAMP levels. Repeated injections of PACAP-27 every 30 min caused a progressive reduction in the plasma cAMP response (measured 5 min after each injection). Forskolin, an activator of adenylate cyclase, dose-dependently raised the plasma concentration of cAMP and displayed a synergistic effect when given in a low dose concurrently with PTH or PACAP-38. The phosphodiesterase inhibitor rolipram dose-dependently raised the plasma concentration of cAMP. Combined treatment with PACAP-27 and a threshold dose of rolipram resulted in an exaggerated plasma cAMP response. Kidney hilus ligation suppressed the responses to PACAP-38, PTH, helodermin, helospectin, VIP, glucagon and calcitonin. Hepatectomy suppressed the response to glucagon but was without effect on the response to the other peptides. Pancreatectomy and spleenectomy reduced the response to VIP, but was without effect on the response to the other peptides. PACAP-27 stimulated cAMP efflux from the isolated rat tail vein. Hence, it cannot be excluded that blood vessels contribute to the peptide evoked plasma cAMP response in vivo.

Topics: Animals; Calcitonin; Calcitonin Gene-Related Peptide; Colforsin; Cyclic AMP; Drug Interactions; Glucagon; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Mice; Neuropeptides; Parathyroid Hormone; Peptides; Phosphodiesterase Inhibitors; Pyrrolidinones; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Rolipram; Tail; Vasoactive Intestinal Peptide

We have compared the effects of vasoactive intestinal peptide (VIP) and of the VIP-related peptides pituitary adenylate cyclase activating peptide (PACAP) 1-27 and 1-38, helodermin, helospectin I and helospectin II, on the electrically evoked twitches in the isolated vas deferens of the rat. While VIP was virtually without effect, PACAP 1-38 suppressed the electrically evoked twitches effectively and in a concentration-dependent manner (pIC50 value 7.5). The naturally occurring N-terminal fragment PACAP 1-27 was less effective than PACAP 1-38 (Imax values 37.2% suppression compared to 76.5%) and less potent. The C-terminal fragment PACAP 16-38 was virtually inactive. Also helodermin and helospectin I+II suppressed the electrically evoked twitches effectively and in a concentration-dependent manner (pIC50 values 6.9; 7.2; 6.8, respectively). The three peptides produced similar maximum reduction of the twitches (74-80%). The findings suggest that PACAP, helodermin and helospectin suppress the electrically evoked contractions in the rat vas deferens via receptors distinct from VIP receptors.

Topics: Amino Acid Sequence; Animals; Electric Stimulation; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Male; Molecular Sequence Data; Muscle Contraction; Muscle, Smooth; Neuropeptides; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Rats; Rats, Sprague-Dawley; Vas Deferens; Vasoactive Intestinal Peptide

Helodermin is an amidated peptide of 35 amino acid residues isolated from the lizard Heloderma suspectum. Homologous peptides, helospectins I and II, peptides of 38 and 37 amino acid residues, respectively, have been isolated from the lizard Heloderma horridum. This group of peptides stimulates the adenylate cyclase activity. Helodermin- and helospectin-like immunoreactivities were studied in the rat brain by using immunohistochemistry and radioimmunoassay in combination with high-performance liquid chromatography. The highest concentrations of helodermin-like immunoreactivity were found in the cerebellum and hypothalamus. The chromatographic analysis of rat brain extract revealed one main immunoreactive peak with elution properties similar to those of authentic lizard helodermin. Helodermin-immunoreactive neurons were located in the supraoptic nucleus, suprachiasmatic nucleus, periventricular nucleus, arcuate nucleus and central gray. Fibers and terminals of varying densities were observed in the bed nucleus of the stria terminalis, medial part of the central nucleus of amygdala, external layer of the median eminence, thalamus and central gray. The highest concentrations of helospectin-like immunoreactivity were found in the cerebral cortex, hypothalamus and medulla. The chromatographic analysis of brain extract revealed one major peak with elution properties similar to those of authentic helospectin I. Helospectin-immunoreactive neurons were located in the suprachiasmatic nucleus, central gray, cerebral cortex, dorsomedial hypothalamic nucleus and supramammillary nucleus. Helospectin-immunoreactive fibers and terminals were found in the bed nucleus of the stria terminalis, medial part of the central nucleus of amygdala, median eminence, lateral parabrachial nucleus, central gray, cerebral cortex, thalamus and nucleus of the solitary tract. The present study has revealed novel neuronal systems in the rat brain by using antisera against the lizard peptides helodermin and helospectin. The patterns of immunostaining suggest a role for the helodermin- and helospectin-like peptides in the hypothalamo-hypophyseal control of endocrine functions.

Topics: Animals; Antibody Specificity; Brain; Chromatography, High Pressure Liquid; Cross Reactions; Immunochemistry; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Lizards; Peptides; Radioimmunoassay; Rats; Rats, Inbred Strains; Vasoactive Intestinal Peptide

Competition binding curves, using [125I-acetyl-His1]PACAP-27 as radioligand and dose-effect curves of adenylate cyclase activation in human SUP-T1 lymphoblastic membranes showed that PACAP-27 and PACAP-38 stimulate the enzyme through a single class of helodermin-preferring VIP receptors with the following order of potency: helodermin = [acetyl-His1]PACAP-27 greater than PACAP-38 greater than PACAP-27 greater than VIP. PACAP (6-27) (Ki 0.5-0.8 microM) and [Des-His1, Asn3]PACAP-27 (Ki 1-2 microM) acted as competitive antagonists. Using a series of 13 PACAP-27 analogues and fragments and three VIP analogues, we identified positions 1, 2, 3, 9 and 13 in PACAP-27 as being of importance for high-affinity binding. Thus, we added further evidence for considering that the present helodermin-preferring VIP receptors, when compared to a majority of VIP receptors and PACAP receptors, exhibit an original specificity pattern.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Binding Sites; Binding, Competitive; Cell Line; Cell Membrane; Enzyme Activation; Humans; Intercellular Signaling Peptides and Proteins; Kinetics; Lymphocytes; Molecular Sequence Data; Neuropeptides; Peptides; Pituitary Adenylate Cyclase-Activating Polypeptide; Radioligand Assay; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Vasoactive Intestinal Peptide

Helodermin (HDM) belongs to the vasoactive intestinal polypeptide (VIP) family of polypeptides. Degradation of HDM in the tracheal tissue isolated from a guinea-pig and by an isolated enkephalinase was studied and compared with the degradation of VIP. The tracheal relaxing activity of VIP was potentiated by enkephalinase inhibitors, thiorphan and phosphoramidon, while the activity of HDM was not potentiated. On the other hand, bestatin, an aminopeptidase inhibitor, and captopril, an angiotensin converting enzyme inhibitor, did not influence the activity of VIP and HDM. The data suggests that the degradation of VIP but not HDM in the trachea was done by enkephalinase. Enkephalinase was then purified from the lung and the striatum membrane fraction through a DEAE-cellulose column, chromatofocusing column and hydroxyapatite column. The purified enkephalinase from the lung hydrolyzed VIP but not HDM. HDM and VIP were, however, hydrolyzed by the striatum enkephalinase. There was only a partial degradation of HDM by the striatum enkephalinase and the hydrolysis rate of HDM was slower than that of VIP. The degradation of VIP and HDM was inhibited by thiorphan. In conclusion, we found that VIP but not HDM was degraded by enkephalinase present in the respiratory system such as the trachea and the lung. Furthermore, enkephalinase, which hydrolyses HDM, was present in the brain.

Topics: Animals; Corpus Striatum; Guinea Pigs; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Lung; Male; Neprilysin; Peptides; Protease Inhibitors; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Trachea; Vasoactive Intestinal Peptide; Venoms

Cross-linking of [125I]helodermin to human SUP-T1 lymphoblasts with bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES) revealed a 63 K binding protein. This cross-linking was inhibited by helodermin and VIP. In cells submitted for 3-4 days to 0.2 microgram/ml tunicamycin, the Mr of an increasing proportion of helodermin-preferring receptors was reduced to 50 K and the total number of receptors was decreased by about 50%, without alteration in binding affinity and specificity. In parallel, the VIP-mediated adenylate cyclase stimulation was reduced by 30% with no change in NaF-, Gpp[NH]p-, and PGE1-stimulations. We conclude that a proper N-glycosylation of helodermin-preferring VIP receptors is required for normal receptor targeting and turnover but not for ligand binding and adenylate cyclase coupling.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Cell Line; Cross-Linking Reagents; Enzyme Activation; Glycosylation; Humans; Intercellular Signaling Peptides and Proteins; Lymphoma; Molecular Sequence Data; Peptides; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Succinimides; Tumor Cells, Cultured; Vasoactive Intestinal Peptide; Venoms

1. The effect of vasoactive intestinal peptide (VIP) was studied on the contractile response of guinea-pig lung parenchymal strips (GPP) induced by bronchoconstrictor agonists, such as leukotriene D4 (LTD4), histamine and acetylcholine (ACh). This effect of VIP was compared with helodermin, a peptide that is structurally related to VIP, and galanin, another neuropeptide that is thought to co-exist with VIP. 2. VIP (10 nM) induced a potent and reversible inhibition of the contractions of GPP induced by LTD4 (1-30 pmol) but did not affect those due to ACh (1-100 nmol) or histamine (1-30 nmol). A ten fold higher concentration of VIP (100 nM) did not further inhibit LTD4-induced responses or reduce those induced by histamine or ACh. 3. Helodermin (10 nM) had a similar inhibitory effect on contractions of GPP induced by LTD4 (3-30 pmol) but did not affect contractions induced by histamine (1-10 nmol). 4. Indomethacin (2.8 microM) and salbutamol (10 nM) significantly reduced responses elicited by LTD4 and histamine but not those due to ACh. A ten fold higher concentration of salbutamol (100 nM) further inhibited the contractions due to LTD4 and histamine and at this concentration responses induced by ACh were inhibited. 5. VIP (10 nM) and helodermin (10 nM) significantly reduced the LTD4-induced release of thromboxane A2 (TXA2), measured as TxB2 by radioimmunoassay, from GPP. The smaller release of TxA2 induced by histamine was not significantly reduced in the presence of VIP. 6. In comparative studies, galanin (10-100 nM) did not affect contractions of GPP induced by either LTD4, histamine or ACh. In contrast to VIP and helodermin, both at 0.1-3 nmol, which induced doserelated relaxations of guinea-pig trachea, galanin was inactive on this preparation in doses of up to 3 nmol.7. In conclusion, our results show that contractions of GPP induced by LTD4 are more sensitive to inhibition by VIP and helodermin than are contractions due to histamine or ACh. This inhibition appears to be associated with the different contribution of released TxA2 to contractions evoked by the agonists. VIP and helodermin inhibit the cyclo-oxygenase-dependent component of the LTD4-induced response, as in the case of indomethacin.

Topics: Acetylcholine; Albuterol; Animals; Galanin; Guinea Pigs; Histamine; Histamine Antagonists; In Vitro Techniques; Indomethacin; Intercellular Signaling Peptides and Proteins; Lung; Male; Muscle Contraction; Muscle, Smooth; Peptides; Prostaglandin Endoperoxides, Synthetic; Radioimmunoassay; SRS-A; Thromboxane B2; Trachea; Vasoactive Intestinal Peptide

Vasoactive intestinal peptide, at different concentrations, was tested on the migration of leucocytes by using the sealed capillary migration test. Vasoactive intestinal peptide, at 10(-7)-10(-9)M, inhibited, while at 10(-12)-10(-14)M, stimulated mononuclear leucocyte migration. The migration of polymorphonuclear leucocytes was inhibited by vasoactive intestinal peptide at 10(-6)-10(-9)M, a stimulation was found at 10(-13)-10(-14)M. The inhibiting effect of vasoactive intestinal peptide on leucocyte migration was abolished when vasoactive intestinal peptide was split into C- and N-terminal fragments, while a stimulating effect was retained in the N-terminal fragment, at 10(-14)M, for mononuclear cells. Helodermin and peptide T, as well as two other members of the secretin-glucagon family, secretin and gastric inhibitory peptide, had no effect on the migration. When VIP antiserum was tested, it had an inhibiting effect, which was not seen with control serum, supporting a physiological effect of the lower vasoactive intestinal peptide concentrations. Vasoactive intestinal peptide seems to have dual effects on mononuclear and polymorphonuclear leucocyte migration and, generally, intact vasoactive intestinal peptide seems to be needed for these effects.

Topics: Cell Migration Inhibition; Gastric Inhibitory Polypeptide; Humans; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Monocytes; Neutrophils; Peptide T; Peptides; Secretin; Vasoactive Intestinal Peptide

Helodermin and vasoactive intestinal polypeptide (VIP) are structurally related peptides. We have examined their effects on insulin and glucagon secretion in the mouse. Following intravenous injection, helodermin and VIP equipotently increased plasma glucagon levels with a maximal effect obtained at the dose level of 2 nmol/kg. The maximal response was not augmented by giving the two peptides together at maximal dose levels, showing that helodermin and VIP stimulate glucagon secretion by activating the same mechanisms. Furthermore, both peptides markedly potentiated glucagon secretion stimulated by the cholinergic agonist carbachol, showing that they sensitize glucagon secretion for muscarinic activation. This sensitizing action was abolished by methylatropine, whereas the direct glucagonotropic action of the peptides was insensitive to muscarinic antagonism. Plasma insulin levels were not affected by helodermin but slightly increased by VIP. The study suggests that helodermin and VIP (1) stimulate basal glucagon secretion by the same mechanism, which is insensitive to muscarinic antagonism, (2) sensitize the glucagon secretion for cholinergic activation, and (3) have no or only weak effect on insulin secretion.

Topics: Animals; Atropine Derivatives; Carbachol; Female; Glucagon; Insulin; Insulin Secretion; Intercellular Signaling Peptides and Proteins; Mice; Peptides; Receptors, Muscarinic; Secretory Rate; Stimulation, Chemical; Vasoactive Intestinal Peptide

We characterized a new type of vasoactive intestinal peptide (VIP) receptors in the CD4+ Stanford University Pediatric (SUP)-T1 lymphoma cell line, by comparing receptor occupancy [in the presence of (125I)helodermin and (125I)(acetyl-His1)VIP] and adenylate cyclase activation (in the presence of GTP). The order of potency of peptides on both parameters was: helodermin greater than (acetyl-His1)VIP greater than (Phe1)VIP = VIP greater than PHI while secretin was ineffective. In membranes, when Gs was permanently activated by Gpp(NH)p or by ADP-ribosylation (after pretreating intact lymphoblasts for 2 h with cholera toxin), there resulted a variably increased affinity of receptors for VIP-like peptides, suggesting reduced receptor selectivity. Preexposing intact lymphoblasts to the same peptides induced, within 5 min, homologous desensitization (i.e. reduced binding capacity and even more so impaired capability to activate adenylate cyclase), whose extent correlated with the Kd of each peptide at time 0. After prolonged (16 h) exposure to 30 nM VIP that resulted in marked (75%) downregulation, 60% of the adenylate cyclase responsiveness could recover within 30-120 min even in the presence of cycloheximide, but further resensitization was cycloheximide-sensitive. To conclude, VIP receptors coupled to adenylate cyclase showed distinct specificity in human SUP-T1 lymphoblasts. Their specificity decreased when Gs was permanently activated. In intact cells exposed to VIP-like peptides, the receptors were rapidly desensitized, then down-regulated, the resensitization mechanism being not immediately inhibited by cycloheximide.

Topics: Adenylyl Cyclases; CD4-Positive T-Lymphocytes; Cell Line; Down-Regulation; GTP-Binding Proteins; Humans; Intercellular Signaling Peptides and Proteins; Peptides; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Species Specificity; Vasoactive Intestinal Peptide

In the present study, we investigated whether peptides located within the thyroid gland, but not directly found in nerve fibers associated with blood vessels, might influence thyroid blood flow. Specifically, we evaluated the effects of helodermin, cholecystokinin (CCK), somatostatin (SRIF) and thyrotropin releasing hormone (TRH) given systemically on thyroid blood flow and circulating thyroid hormone levels. Blood flows in the thyroid and six other organs were measured in male rats using 141Ce-labeled microspheres. Circulating thyrotropin (TSH) and thyroid hormone levels were monitored by RIA. Helodermin (10(-10) mol/100 g BW, i.v. over 4 min) markedly elevated thyroid blood flow (52 +/- 6 vs. 10 +/- 2 ml/min.g in vehicle-infused rats; n = 5). Blood flows to the salivary gland, pancreas, lacrimal gland and stomach (but not adrenal and kidney) were also increased during helodermin infusions. CCK, SRIF, and TRH were without effect on blood flows to the thyroid and other organs even though these peptides were tested at higher molar doses than helodermin. Helodermin, CCK, or SRIF did not affect thyroid hormone or plasma calcium levels. As expected however, plasma TSH and T3 levels were increased at 20 min and 2 h, respectively, following TRH infusions. Since helodermin shares sequence homology with VIP, we next compared the relative effects of these two peptides on thyroid and other organ blood flows. VIP (10(-11) mol/100 g BW, i.v.) was more potent in increasing blood flows to the thyroid, salivary gland, and pancreas than an equimolar dose of helodermin. This study shows that while helodermin, like VIP, has the ability to increase thyroid and other organ blood flows, it appears to be a less potent vasodilator.

Topics: Animals; Calcium; Cholecystokinin; Intercellular Signaling Peptides and Proteins; Male; Pancreas; Peptides; Radioimmunoassay; Rats; Rats, Inbred Strains; Regional Blood Flow; Salivary Glands; Somatostatin; Thyroid Gland; Thyrotropin-Releasing Hormone; Vasoactive Intestinal Peptide

1. Helodermin, helospectin I and helospectin II, peptides recently isolated from the salivary gland venom of Heloderma suspectum, were compared to vasoactive intestinal peptide (VIP) with respect to effects on systemic blood pressure and on isolated femoral arteries in the rat. 2. They all reduced blood pressure in a dose-dependent manner; helodermin was less effective than VIP. However, at doses higher than 1 nmol kg-1 all four peptides reduced blood pressure to about the same extent. 3. The half-life of the hypotensive effect of VIP was longer than that of helodermin and the helospectins. 4. VIP and helodermin were equally potent in relaxing femoral arteries precontracted with phenylephrine or prostaglandin F2 alpha. 5. Helospectin I and II relaxed phenylephrine-contracted vessels to the same extent as VIP but with a lower potency. 6. Addition of VIP 1 microM to preparations exposed to helodermin 1 microM or to either of the helospectins did not produce a further relaxation. 7. The findings indicate that VIP, helodermin and helospectin I and II have a similar profile of action and therefore may act on a common receptor.

Topics: Amino Acid Sequence; Animals; Blood Pressure; Dinoprost; Female; Half-Life; Hemodynamics; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Male; Molecular Sequence Data; Peptides; Phenylephrine; Rats; Rats, Inbred Strains; Vasoactive Intestinal Peptide

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

The two peptides VIP (vasoactive intestinal peptide) and helodermin have both been shown to occur within the thyroid gland: VIP in intrathyroidal nerves and helodermin in the C-cells. Both peptides have previously been demonstrated to enhance the release of radioiodine from the prelabelled thyroid in vivo. Since a considerable amount of radioiodine released from the thyroid under these conditions may be non-hormonal, we reexamined the effects of VIP and helodermin on thyroid hormone secretion by the use of the specific technique of studying the release of radioiodine bound to specific T4 antiserum in mice. We thereby found that anti-T4-bound radioiodine in T3-pretreated animals increased after intravenous injection of VIP (1.5 nmol/animal) to 280 +/- 24% (P less than 0.001), and after intravenous injection of helodermin (1.5 nmol/-animal) to 186 +/- 26% (P less than 0.001) compared to 78 +/- 5% in controls. As a comparison, the corresponding figure after injection of TSH (70 microU/animal) was approximately 350% (P less than 0.001). In contrast, in animals not pretreated with T3, neither TSH, nor VIP helodermin significantly altered the plasma level of anti-T4-bound radioiodine. Also, VIP and helodermin did not change the plasma levels of free T4 in non-pretreated animals. In summary, the sensitive and specific technique of measuring the release of anti-T4-bound radioiodine in vivo after pretreatment with NA 125I and T3 detected a stimulation of the thyroid hormone secretion by VIP and helodermin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Female; Intercellular Signaling Peptides and Proteins; Mice; Peptides; Radioimmunoassay; Thyroxine; Triiodothyronine; 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

In fresh rat liver plasma membranes, high affinity VIP receptors were specifically labelled with [125I] helodermin and were well coupled to adenylate cyclase while low affinity VIP receptors were not. After freezing and thawing low affinity VIP receptors were also coupled to adenylate cyclase. This modification of adenylate cyclase activation was specific for the VIP response as freezing and thawing did not modify Gpp (NH)p, NaF and glucagon stimulations.

Topics: Adenylyl Cyclases; Animals; Binding, Competitive; Cell Membrane; Enzyme Activation; Freezing; Glucagon; Guanylyl Imidodiphosphate; Intercellular Signaling Peptides and Proteins; Liver; Peptides; Rats; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Sodium Fluoride; 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

Helodermin is a new peptide isolated from the venom of Heloderma suspectum. Its effects on rat pancreatic acini were compared with those of secretin and vasoactive intestinal peptide (VIP). Four classes of receptors with decreasing affinity for secretin (S1, S2, S3, and S4) were first delineated. Occupancy of S1 and S2 by secretin was responsible for a biphasic adenosine 3',5'-cyclic monophosphate (cAMP) response. S3 was VIP preferring so that the VIP-induced increase in cAMP could be inhibited by VIP-(10-28). S2 and S3 allowed cAMP elevation, protein phosphorylation, weak secretory effects, and potentiation of cholecystokinin octapeptide (CCK-8) when occupied by secretin and VIP, respectively. A more efficient exocytosis was observed with secretin interacting with low-affinity receptors S4. Helodermin increased cAMP levels 14-fold, this increase being inhibited by VIP-(10-28). Low concentrations of helodermin stimulated amylase secretion twofold and potentiated secretion by CCK-8. High concentrations of helodermin stimulated secretion another 2.6-fold. Helodermin bound to the four secretin receptors with a weak selectivity. At low concentration, helodermin stimulated cAMP elevation, protein phosphorylation, amylase release, and potentiation of CCK-8 through S3, whereas at high concentration it stimulated secretion via S4.

Topics: 1-Methyl-3-isobutylxanthine; Amylases; Animals; Cyclic AMP; Drug Synergism; Intercellular Signaling Peptides and Proteins; Pancreas; Peptides; Phosphoproteins; Rats; Receptors, G-Protein-Coupled; Receptors, Gastrointestinal Hormone; Secretin; Sincalide; Vasoactive Intestinal Peptide

The effect of helodermin on vascular physiology was studied in anesthetized dogs using a synthetic replicate of helodermin and helodermin related peptides. Intraarterial infusion of helodermin caused a dose-dependent increase in femoral blood flow. Helodermin was 16 times less potent than VIP and 5 times more potent than PHM (human PHI). The helodermin effect lasted significantly longer; the half-life of the helodermin effect was 6.5 times longer than VIP. Synthetic helodermin (Hd) N-terminal fragment Hd(1-27)NH2 retained substantial activity similar to the full helodermin molecule but the prolonged effect was lost. Hd(7-35) and Hd(22-35) were inactive in this system. Intravenous injection of synthetic helodermin produced prolonged systemic hypotension and tachycardia; and, similar to VIP, it increased the common carotid arterial blood flow while those of the superior mesenteric and femoral arteries were decreased. The results demonstrate the VIP-like vasodilating activity and cardiovascular effects of helodermin in anesthetized dogs.

Topics: Animals; Cardiovascular Physiological Phenomena; Cardiovascular System; Dogs; Female; Hemodynamics; Intercellular Signaling Peptides and Proteins; Male; Peptides; Regional Blood Flow; Vasoactive Intestinal Peptide; Vasodilation

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

Helodermin is a biologically active peptide isolated from the venom of the Gila monster lizard (Heloderma suspectum) whose structure is related to that of vasoactive intestinal peptide and secretin. Using a specific radioimmunoassay based on antisera prepared by immunizing rabbits with natural helodermin, we demonstrated the presence of helodermin-like material in mammalian salivary glands, including parotid, submaxillary and sublingual glands from rat and dog, and parotid and submaxillary glands from man. All helodermin-like materials had an apparent molecular mass of 4-12 kDa. Dog saliva, collected after pilocarpine stimulation, revealed similar immunoreactivity with a major component around 6 kDa.

Topics: Amino Acid Sequence; Animals; Chromatography, Gel; Dogs; Humans; Intercellular Signaling Peptides and Proteins; Peptides; Radioimmunoassay; Rats; Saliva; Salivary Glands; Secretin; Species Specificity; Vasoactive Intestinal Peptide

Helodermin, a newly isolated peptide from the venom of Gila monster (Heloderma suspectum) was shown to stimulate the adenylate cyclase activity of rat pancreatic membranes as efficiently as secretin and VIP. It also increased cyclic AMP levels and inhibited [125I]VIP binding in rat pancreatic acini. Finally, helodermin activated adenylate cyclase in membranes from rat heart, rat brain, and human heart, showing properties analogous yet distinct from those of secretin, VIP and PHI.

Topics: Adenylyl Cyclases; Animals; Biological Assay; Cell Membrane; Cyclic AMP; Enzyme Activation; Intercellular Signaling Peptides and Proteins; Kinetics; Lizards; Pancreas; Peptides; Rats; Secretin; Vasoactive Intestinal Peptide; Venoms

Helodermin, a newly isolated peptide from Gila Monster venom, is structurally related to VIP and secretin. When used as radioligand, [125I]helodermin bound rapidly and reversibly to crude rat liver membranes, the dissociation being accelerated by GTP. Competition binding curves of [125I]helodermin and [125I]VIP with unlabelled peptides showed the following order of decreasing affinity: VIP greater than helodermin greater than secretin greater than hpGRF(1-29)-NH2. The shape of binding curves and of concurrent adenylate cyclase activation is compatible with the specific labelling, by [125I]helodermin, of a class of high-affinity VIP receptors that is capable to stimulate adenylate cyclase.

Topics: Adenylyl Cyclases; Animals; Binding, Competitive; Cell Membrane; Enzyme Activation; Growth Hormone-Releasing Hormone; Guanosine Triphosphate; Intercellular Signaling Peptides and Proteins; Liver; Lizards; Male; Peptide Fragments; Peptides; Rats; Receptors, Cell Surface; Receptors, Vasoactive Intestinal Peptide; Secretin; Sermorelin; Time Factors; 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

A combination of three HPLC procedures applied to the venom of Gila monster (Heloderma suspectum) has led to the purification to homogeneity of two bioactive components: (i) a 17.5 kDa protein, isolated on the basis of its potent secretory effect on dispersed rat pancreatic acini, was accordingly designated PSF (pancreatic secretory factor); (ii) a 5.9-kDa peptide, designated helodermin, was purified on the basis of its ability to stimulate adenylate cyclase in rat pancreatic membranes. PSF was unable to activate adenylate cyclase and, conversely, helodermin was devoid of secretory action.

Topics: Adenylyl Cyclases; Animals; Biological Assay; Cell Membrane; Chromatography, High Pressure Liquid; Enzyme Activation; Intercellular Signaling Peptides and Proteins; Lizards; Molecular Weight; Pancreas; Pancreatic Juice; Peptides; Rats; Secretin; Vasoactive Intestinal Peptide; Venoms