Helodermin-like peptides.
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Amino Acid Sequence↗
Biomedical subjects
P De Neef
Publications and source records attributed to P De Neef.
Effects of d-tubocurarine on rat cardiac muscarinic receptors: a comparison with gallamine.
Gallamine and d-tubocurarine inhibited (3H)N-methyl-scopolamine [3H)NMS) binding to rat cardiac muscarinic receptors with I50 values of 0.7 microM and 22 microM, respectively. They decreased the association and dissociation rates of the two ligands (3H)NMS and (3H)Oxotremorine M [3H)Oxo-M). Gallamine interaction with muscarinic receptors was markedly inhibited by (3H)NMS and (3H)Oxo-M binding to the receptors. We were unable to demonstrate (3H)NMS or (3H)Oxo-M binding to the muscarinic receptor-gallamine complex. By contrast, d-tubocurarine interaction with rat cardiac muscarinic receptors was facilitated by (3H)Oxo-M binding and only slightly inhibited by (3H)NMS binding to muscarinic binding sites. Furthermore, (3H)NMS and (3H)Oxo-M bound to the receptor-d-tubocurarine complex, indicating that the latter drug interacted with an allosteric site on cardiac muscarinic receptors but did not recognize the muscarinic binding site (at concentrations below 1 mM).
Animals↗
[D-Phe4]peptide histidine-isoleucinamide ([D-Phe4]PHI), a highly selective vasoactive-intestinal-peptide (VIP) agonist, discriminates VIP-preferring from secretin-preferring receptors in rat pancreatic membranes.
The capacity of vasoactive intestinal peptide (VIP), peptide histidine-isoleucinamide (PHI), secretin, and a series of analogs to discriminate between VIP-preferring and secretin-preferring receptors that coexist in rat pancreatic plasma membranes was evaluated by their ability to inhibit [125I]iodo-VIP and [125I]iodo-secretin binding and to activate adenylate cyclase. VIP, the VIP analogs [D-His1]VIP, [D-Ser2]VIP, [D-Asp3]VIP and [D-Ala4]VIP, PHI, [D-Phe4]PHI, and secretin inhibited the binding of both ligands in a concentration range of 10(-11) M to 10(-5) M and with a selectivity factor varying from 18,000 to 0.1. The only exception was [D-Phe4]PHI that inhibited 125I-VIP binding only, with an IC50 of 7 nM, and with no inhibition of 125I-secretin binding at 10 microM. The peptides tested stimulated adenylate cyclase in the same membranes and the slope of the dose-effect curves indicated that all peptides, except [D-Phe4]PHI, interacted with at least two classes of receptors: VIP-preferring and secretin-preferring receptors. By contrast, the dose-effect curve of [D-Phe4]PHI activation of adenylate cyclase was monophasic and competitively modified by [D-Phe2]VIP (a VIP antagonist) but not by secretin(7-27) (a secretin antagonist), indicating an interaction with VIP-preferring receptors only. Thus, [D-Phe4]PHI appears to be a highly selective tool to characterize these receptors.
Adenylyl Cyclases↗
Chemical, immunological and biological properties of peptides like vasoactive-intestinal-peptide and peptide-histidine-isoleucinamide extracted from the venom of two lizards (Heloderma horridum and Heloderma suspectum).
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.
Adenylyl Cyclases↗
Interaction of vasoactive intestinal peptide (VIP) and N-terminally modified VIP analogs with rat pancreatic, hepatic and pituitary membranes.
Six vasoactive intestinal peptide (VIP) analogs inhibited [125I]iodo-VIP and [125I]iodo-helodermin binding to high-affinity VIP receptors in rat hepatic membranes. They also stimulated adenylate cyclase activity through these receptors, their decreasing order of potency being VIP greater than [D-Ala4]VIP greater than [D-Asp3]VIP greater than [D-Ser2]VIP greater than [D-His1]VIP greater than [D-Phe2]VIP greater than [D-Arg2]VIP, with the latter two peptides acting as partial agonists only. All VIP analogs tested on rat pancreatic membranes were able to stimulate adenylate cyclase, their order of potency being very similar to that observed on hepatic membranes. [D-Ser2]VIP, [D-His1]VIP, [D-Arg2]VIP and [D-Phe2]VIP were partial agonists with an intrinsic activity of, respectively, 0.8, 0.7, 0.35 and 0.09 as compared to that of VIP = 1.0. [D-Phe2]VIP competitively and selectively inhibited VIP-stimulated adenylate cyclase activity (Ki = 0.1 microM). On male rat anterior pituitary homogenates the order of potency of the peptides was VIP greater than [D-Ala4]VIP greater than [D-Asp3]VIP greater than [D-Ser2]VIP greater than [D-His1]VIP. [D-Ser2]VIP and [D-His1]VIP acted as partial agonists. Besides, [D-Phe2]VIP and [D-Arg2]VIP were inactive as well as unable to inhibit VIP-stimulated adenylate cyclase activity. These results indicated that (a) the efficacy of VIP receptor/effector coupling depended on the tissue tested; (b) the possibility exists to design a VIP antagonist by appropriate modification in the N-terminal moiety of the molecule.
Adenylyl Cyclases↗
Alterations of rat cardiac adenylate cyclase activity with age.
Basal, 5'-guanylimidodiphosphate, GTP-, NaF-, forskolin-, D,L-isoproterenol-, glucagon- and secretin-stimulated adenylate cyclase activities were investigated in cardiac membranes from young adult (6 month old), old (20 month old) and senescent (24 month old) Sprague Dawley rats. The only significant difference between old and young adult rats was a 43% decrease of the glucagon-stimulated enzyme activity. In senescent rats compared to young adult rats, we observed a 23% decrease in forskolin-stimulated enzyme activity, a more severe (-73%) decrease in glucagon-stimulated adenylate cyclase activity and a decrease (-38%) of the response to secretin. The response to the beta-adrenoreceptor agonist D,L-isoproterenol was unaffected. These results suggest an alteration with age in the vicinity of the catalytic unit of adenylate cyclase and a selective decrease of functional glucagon and secretin receptors.
Adenylyl Cyclases↗
Comparative structural requirements of thirty GRF analogs for interaction with GRF- and VIP receptors and coupling to adenylate cyclase in rat adenopituitary, liver and pancreas.
The ability of 30 synthetic GRF(1-29)-NH2 analogs to stimulate adenylate cyclase activity was investigated in membranes from rat adenopituitary, rat liver and rat pancreas. In adenopituitary membranes, GRF and GRF analogs interacted with specific GRF receptors, whereas in liver and pancreatic membranes, they interacted with VIP receptors. The C-terminal moiety of GRF was responsible for GRF receptor recognition as the hybrid analog (His1, D-Ala2)-GRF(1-9), VIP(10-28) stimulated pituitary adenylate cyclase through the occupancy of VIP receptors only. When GRF or VIP receptors were occupied by GRF analogs, the N-terminal part of the ligand appeared critical for adenylate cyclase activation. This was established by testing 30 GRF analogs mono-, bi- or tri-substituted in positions 1 to 10. Major observations included: (a) the characterization of (N-Ac-Tyr1, D-Arg2)-GRF(1-29)-NH2 as an antagonist of GRF-stimulated pituitary adenylate cyclase; (b) the discovery of the (N-Ac-Tyr1, D-Phe2)-, (His1, D-Ala2, D-Ser3, NLeu27)-, and (His1, D-Ala2, D-Thr7, NLeu27)-derivatives of GRF(1-29)-NH2 as specific antagonists of VIP receptors in rat pancreatic membranes; (c) the importance of the free NH2 function of amino acid residue 1 for pancreatic adenylate cyclase activation, and (d) the decreased efficiency of iodinated (Tyr1)-GRF(1-29)-NH2 as opposed to the non iodinated form, in all systems tested.
Adenylyl Cyclases↗
Interaction of synthetic N- and C-terminal fragments of helodermin with rat liver VIP receptors.
Synthetic helodermin was more potent than natural helodermin (purified from the venom of Gila monster) in activating adenylate cyclase in rat liver membranes. Two possible reasons for this discrepancy were discussed. Comparing adenylate cyclase activation to the binding of 125I-natural helodermin and 125I-VIP in the presence of six synthetic helodermin fragments (1-27, 7-35, 13-35, 17-35, 18-35 and 22-35), we conclude that the effects of both synthetic and natural helodermin were mediated through an interaction with VIP receptors. The C-terminal (28-35) extension of these peptides favored VIP receptor recognition. Their N-terminal extremity was not necessary for binding and for ensuing adenylate cyclase activation, illustrating again the atypical coupling of VIP receptors with the effector enzymatic system, in rat liver membranes.
Adenylyl Cyclases↗
Comparative efficacy of seven synthetic glucagon analogs, modified in position 1, 2 and/or 12, on liver and heart adenylate cyclase from rat.
Crude fresh membranes from rat liver and membranes from rat heart obtained according to Snyder and Drummond were tested for adenylate cyclase activation by glucagon (Gn) and seven glucagon analogs including (Ala2)-, (Arg12)-, (Des-His1, Arg12), (Phe1, Arg12)-, (N-Ac-His1, Arg12)-, (1-Me-His1, Arg12)-, and (3-Me-His1, Arg12)-glucagon. (Des-His1, Arg12)-glucagon acted as a competitive antagonist in heart membranes and as a partial agonist in liver membranes. Results obtained with analogs where His1 was modified suggest that the size of the imidazole ring and the charge of its nitrogen 1, but not the charge of the free amino group of histidine, played a major role in biological activity. When comparing functional glucagon receptors in liver and heart membranes, it appears that the first receptors were more sensitive to the hormone and more efficiently coupled to adenylate cyclase.
Adenylyl Cyclases↗
Propylbenzilylcholine mustard is selective for rat heart muscarinic receptors having a low affinity for agonists.
Propylbenzilylcholine mustard (PrBCM), an irreversible muscarinic antagonist, inactivated receptors with a low affinity for agonists faster than those with a high affinity in rat heart membranes. This result was obtained using either: (a) a low ionic strength buffer (allowing heterogeneity among antagonist binding sites, (b) the same buffer enriched with GTP, or (c) a high ionic strength buffer (where antagonists showed similar binding characteristics to all receptors). These data suggest either that PrBCM is a 'selective' antagonist which detects conformational differences between low and high affinity receptors, or that the agonist affinity of cardiac muscarinic receptors is determined, in part, by the relative concentrations of receptor and GTP binding protein.
Albumins↗
Specific labelling of high-affinity vasoactive intestinal peptide receptors in rat liver membranes by a growth hormone-releasing factor analog.
(125I-His1, D-Ala2, Nleu27)-growth hormone-releasing factor (GRF) (1-29)-NH2, initially developed as a possible radioligand for identifying GRF receptors in the anterior pituitary, was found to bind to rat hepatic membranes. The tracer was stable, bound rapidly and reversibly, and its dissociation was accelerated by GTP. Radioligand binding was enhanced by the divalent cations Mg2+, Ca2+ and Mn2+ and inhibited by the chelating agent EDTA. Vasoactive intestinal peptide (VIP), PHI, secretin, GRF(1-29)-NH2, (His1, D-Ala2, Nleu27)-GRF(1-29)-NH2, and (D-Ala2, Nleu27)-GRF(1-29)-NH2 dose-dependently inhibited tracer binding. The order of potency of the unlabelled peptides tested suggested that (125I-His1, D-Ala2, Nleu27)-GRF(1-29)-NH2 specifically identified a high-affinity subclass of VIP receptors in rat liver membranes.
Animals↗
Decreased stimulation of adenylate cyclase by growth hormone-releasing factor in the anterior pituitary of old rats.
Adenylate cyclase activity was studied in anterior pituitary homogenates from young adult (6 months) and old (20 and 24 months) male rats. Basal, NaF-, GTP-, 5'-guanylimidodiphosphate (Gpp(NH)p)- and forskolin-stimulated activities were similar in the three groups, implying that the stimulatory guanyl nucleotide regulatory binding site (NS) and the catalytical unit were unaffected by aging. Vasoactive intestinal peptide (VIP)-stimulated adenylate cyclase activity was also identical in the three groups. The efficacy (i.e., the maximum effect) of growth hormone-releasing factor [GRF(1-29)-NH2] on adenylate activity was reduced by 45-49% in old and senescent rats with no change in peptide potency (i.e., the concentration required for half-maximal enzyme stimulation). These results suggest that aging induced a selective loss of functional GRF receptors but influenced neither the coupling between receptors, NS and the catalytic unit nor the efficacy of the catalytic unit per se.
Adenylyl Cyclases↗
Effects of temperature and ethanol on agonist and antagonist binding to rat heart muscarinic receptors in the absence and presence of GTP.
The effect of temperature on the binding of four agonists and three antagonists to rat heart muscarinic receptors was studied in the absence and presence of GTP. The binding of agonists to two states (or classes) of receptors, in the absence of GTP, led to enthalpy and entropy changes that decreased sharply above 25 degrees C, suggesting that agonists induced 'isomerization' reactions (large conformational changes and/or receptor-effector association). Both temperature increase and ethanol decreased hydrophobic interactions, thereby hindering binding and/or agonist-induced 'isomerization' reactions. Addition of GTP to the incubation medium also appeared to reverse (or prevent) 'isomerization' reactions. For agonist binding to the low-affinity state, in the presence of GTP, and for antagonist binding, the thermodynamic parameters observed could be readily explained by simple receptor-ligand associations; large entropy increases and small enthalpy increases, provoked by hydrophobic and ionic interactions, were partly neutralized by entropy and enthalpy decreases, due to hydrogen bonds and van der Waals interactions. The muscarinic antagonists used (atropine, n-methylscopolamine and dexetimide), being more hydrophobic molecules than the agonists tested (carbamylcholine, oxotremorine and pilocarpine), induced larger entropy changes or more negative enthalpy changes.
Animals↗
Presence of helodermin-like peptides of the VIP-secretin family in mammalian salivary glands and saliva.
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.
Amino Acid Sequence↗
Immunoreactive helodermin-like peptides in rat: a new class of mammalian neuropeptides related to secretin and VIP.
Helodermin is a peptide from the venom of the lizard Heloderma suspectum (Gila Monster) showing a high degree of sequence similarity with VIP, PHI and secretin in its N-terminal moiety. The present data support the presence of peptide(s) closely related to helodermin in the brain, gut and salivary glands of rat. In our radioimmunoassays, we routinely used one of the three specific antisera obtained from rabbits that were immunized against lizard helodermin coupled to bovine serum albumin with carbodiimide. Heat- and acid-stable immunoreactive helodermin-like material was more abundant in striatum, hippocampus and anterior pituitary than in cerebral cortex and hypothalamus. High levels of helodermin-like material were also present in salivary glands, duodenum and jejunum. When submitted to gel permeation chromatography on a TSK-G 2000 SW column, the apparent molecular radius of most of the immunoreactive material ranged from 6 to 12 KDa.
Animals↗
Interaction of growth hormone-releasing factor (GRF) and 14 GRF analogs with vasoactive intestinal peptide (VIP) receptors of rat pancreas. Discovery of (N-Ac-Tyr1,D-Phe2)-GRF(1-29)-NH2 as a VIP antagonist.
Adenylate cyclase stimulation by GH-releasing factor (GRF) and 14 GRF analogs (modified in the N-terminal part) was compared to the capacity of the same peptides to inhibit [125I]iodo-vasoactive intestinal peptide (VIP) binding in rat pancreatic plasma membranes. These peptides interfered with VIP receptors as they inhibited [125I]iodo-VIP binding, and probably acted through VIP-preferring receptors as one of these peptides [(N-Ac-Tyr1,D-Phe2)-GRF(1-29)-NH2] selectively inhibited both VIP- and GRF-stimulated adenylate cyclase activities. In general, alterations in positions 6 and 7 (but not in positions 1-4) markedly reduced the affinity of the resulting GRF analog [based on Kact (concentration exerting half-maximal stimulation) values]. The intrinsic activity exerted by GRF analogs on adenylate cyclase was reduced by acetylation of the free NH2 group and by the replacement of Asp3, Ala4, Phe6, and Thr7 by the corresponding D-isomer. The presence of pCl-Phe6 and Trp6 also depressed this parameter. Substitution in GRF (or its N-acetylated derivative) by D-Phe2, D-Arg2, and D-Ala4 again reduced the intrinsic activity, whereas substitution of the natural L-amino acid residue by D-Ala2 and Phe4 gave superagonists.
Acetylation↗
Inhibitory effects of quinidine on rat heart muscarinic receptors.
Quinidine inhibited binding of the labelled agonist [3H]oxotremorine M [( 3H]Oxo-M) and the labelled antagonist [3H]N-methylscopolamine [( 3H]NMS) to rat heart muscarinic receptors. Kinetic studies demonstrated that quinidine decreased the association rates (I50: 4 and 7.5 microM) and dissociation rates (I50: 100 and 68 microM) of [3H]Oxo-M and [3H]NMS, with different potencies. These cooperative effects explained the low Hill coefficients and apparent selectivity of quinidine competition curves.
Animals↗
Importance of disulfide bonds in receptors for vasoactive intestinal peptide and secretin in rat pancreatic plasma membranes.
Vasoactive intestinal peptide (VIP), secretin, and C-terminal octapeptide of cholecystokinin (CCK-8) receptors were identified in rat pancreatic plasma membranes by the ability of these peptides to stimulate adenylate cyclase activity. The membrane preparation procedure was conducted through a series of steps including discontinuous sucrose density gradient fractionation. 5 mM beta-mercaptoethanol was added stepwise. Membrane preparations obtained stepwise were preincubated for 10 min at 25 degrees C in the presence of various concentrations of beta-mercaptoethanol or dithiothreitol before assaying adenylate cyclase. The use of the reducing agents exerted no effect on p[NH]ppG-, NaF-, and CCK-8- stimulated activities. By contrast, stimulation of adenylate cyclase by low VIP concentrations was specifically altered when beta-mercaptoethanol was used during tissue homogeneization at 5 degrees C. In addition, both VIP and secretin responses were highly sensitive towards a preincubation of 10 min at 25 degrees C in the presence of dithiothreitol. These results were likely to reflect alterations at the receptor level. 125I-VIP binding was, indeed, reduced after dithiothreitol preincubation, low concentrations of the thiol reagent decreasing the apparent number of high-affinity VIP receptors and higher dithiothreitol concentrations reducing the affinity of VIP receptors.
Adenylyl Cyclases↗