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1.
Nature ; 415(6871): 497-502, 2002 Jan 31.
Article in English | MEDLINE | ID: mdl-11823852

ABSTRACT

Ralstonia solanacearum is a devastating, soil-borne plant pathogen with a global distribution and an unusually wide host range. It is a model system for the dissection of molecular determinants governing pathogenicity. We present here the complete genome sequence and its analysis of strain GMI1000. The 5.8-megabase (Mb) genome is organized into two replicons: a 3.7-Mb chromosome and a 2.1-Mb megaplasmid. Both replicons have a mosaic structure providing evidence for the acquisition of genes through horizontal gene transfer. Regions containing genetically mobile elements associated with the percentage of G+C bias may have an important function in genome evolution. The genome encodes many proteins potentially associated with a role in pathogenicity. In particular, many putative attachment factors were identified. The complete repertoire of type III secreted effector proteins can be studied. Over 40 candidates were identified. Comparison with other genomes suggests that bacterial plant pathogens and animal pathogens harbour distinct arrays of specialized type III-dependent effectors.


Subject(s)
Gram-Negative Aerobic Rods and Cocci/genetics , Bacterial Proteins/metabolism , Biological Evolution , Genome, Bacterial , Genomics , Gram-Negative Aerobic Rods and Cocci/pathogenicity , Solanum lycopersicum/virology , Molecular Sequence Data , Sequence Analysis, DNA , Virulence/genetics
2.
Mol Microbiol ; 36(2): 249-60, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10792714

ABSTRACT

As in many other Gram-negative plant pathogenic bacteria, the Ralstonia solanacearum hrp genes are involved in the production of a type III secretion apparatus that allows the translocation of PopA protein to the external medium. Here, we show that hrp genes are also involved in the biogenesis of pili that are mainly composed of the HrpY protein. These pili are produced at one pole of the bacterium and are also released into the external medium where they can form very long straight bundles. An hrpY mutant is defective in pilus production, impaired in interactions with plants and in secretion of the PopA protein but not in attachment to plant cells.


Subject(s)
Bacterial Proteins/physiology , Betaproteobacteria/physiology , Fimbriae, Bacterial/metabolism , Plant Diseases/microbiology , Plants/microbiology , Amino Acid Sequence , Bacterial Adhesion , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Betaproteobacteria/genetics , Betaproteobacteria/metabolism , Fimbriae, Bacterial/chemistry , Fimbriae, Bacterial/genetics , Fluorescent Antibody Technique, Indirect , Gene Expression Regulation, Bacterial , Microscopy, Electron , Microscopy, Interference , Molecular Sequence Data , Multigene Family , Sequence Analysis, Protein , Time Factors
3.
Peptides ; 9(2): 339-45, 1988.
Article in English | MEDLINE | ID: mdl-2836826

ABSTRACT

The ability of VIP, PHI, secretin, helodermin, and seven N-terminally D-amino monosubstituted VIP and PHI analogs to occupy (125I)iodo-VIP labeled receptors and to activate adenylate cyclase was tested on human lung membranes purified by the method of Schachter et al. Best fitted Kd, Kact and % of max. values suggested the coexistence, in near equal proportions, of two classes of VIP-preferring binding sites coupled to adenylate cyclase that showed similar decreasing affinity for: VIP greater than (D-Ala4)-VIP greater than (D-Asp3)-VIP = (D-Ser2)-VIP greater than (D-His1)-VIP greater than PHI greater than (D-Phe2)-VIP greater than (D-Phe4)-VIP. (D-Arg2)-VIP was a non-selective agonist. A third receptor type, coupled to adenylate cyclase and showing high affinity for secretin and helodermin but not for VIP, was also detected.


Subject(s)
Lung/metabolism , Receptors, Gastrointestinal Hormone/metabolism , Vasoactive Intestinal Peptide/metabolism , Adult , Binding, Competitive , Cell Membrane/metabolism , Humans , Kinetics , Middle Aged , Receptors, Vasoactive Intestinal Peptide
4.
Pancreas ; 3(5): 529-35, 1988.
Article in English | MEDLINE | ID: mdl-3186683

ABSTRACT

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.


Subject(s)
Pancreas/analysis , Receptors, Gastrointestinal Hormone/analysis , Adenylyl Cyclases/analysis , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Humans , Intercellular Signaling Peptides and Proteins , Peptide Fragments/pharmacology , Peptide PHI/pharmacology , Peptides/pharmacology , Receptors, G-Protein-Coupled , Receptors, Gastrointestinal Hormone/drug effects , Vasoactive Intestinal Peptide/pharmacology
5.
Life Sci ; 42(5): 505-10, 1988.
Article in English | MEDLINE | ID: mdl-2828794

ABSTRACT

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.


Subject(s)
Adenylyl Cyclases/metabolism , Freezing , Liver/metabolism , Receptors, Gastrointestinal Hormone/metabolism , Animals , Binding, Competitive , Cell Membrane/metabolism , Enzyme Activation/drug effects , Glucagon/pharmacology , Guanylyl Imidodiphosphate/pharmacology , Intercellular Signaling Peptides and Proteins , Peptides/metabolism , Rats , Receptors, Vasoactive Intestinal Peptide , Sodium Fluoride/pharmacology , Vasoactive Intestinal Peptide/metabolism , Vasoactive Intestinal Peptide/pharmacology
6.
Eur J Biochem ; 165(2): 243-9, 1987 Jun 01.
Article in English | MEDLINE | ID: mdl-3036504

ABSTRACT

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.


Subject(s)
Pancreas/metabolism , Peptide PHI/analogs & derivatives , Secretin/metabolism , Vasoactive Intestinal Peptide/metabolism , Adenylyl Cyclases/metabolism , Animals , Binding, Competitive , Cell Membrane/metabolism , Female , In Vitro Techniques , Male , Peptide PHI/metabolism , Rats , Rats, Inbred Strains , Receptors, G-Protein-Coupled , Receptors, Gastrointestinal Hormone/metabolism , Receptors, Vasoactive Intestinal Peptide , Stereoisomerism , Structure-Activity Relationship
7.
Eur J Biochem ; 159(1): 45-9, 1986 Aug 15.
Article in English | MEDLINE | ID: mdl-3017717

ABSTRACT

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.


Subject(s)
Liver/metabolism , Pancreas/metabolism , Pituitary Gland, Anterior/metabolism , Receptors, Cell Surface/metabolism , Vasoactive Intestinal Peptide/analogs & derivatives , Vasoactive Intestinal Peptide/metabolism , Adenylyl Cyclases/metabolism , Animals , Enzyme Activation/drug effects , Female , Liver/enzymology , Male , Pancreas/enzymology , Pituitary Gland, Anterior/enzymology , Rats , Rats, Inbred Strains , Receptors, Vasoactive Intestinal Peptide , Vasoactive Intestinal Peptide/pharmacology
8.
Eur J Pharmacol ; 126(1-2): 91-5, 1986 Jul 15.
Article in English | MEDLINE | ID: mdl-3758167

ABSTRACT

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.


Subject(s)
Adenylyl Cyclases/metabolism , Myocardium/enzymology , Aging , Animals , Colforsin/pharmacology , Glucagon/pharmacology , Guanylyl Imidodiphosphate/pharmacology , Isoproterenol/pharmacology , Male , Rats , Rats, Inbred Strains , Secretin/pharmacology , Sodium Fluoride/pharmacology
9.
Neuroendocrinology ; 44(1): 108-11, 1986.
Article in English | MEDLINE | ID: mdl-3024053

ABSTRACT

(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.


Subject(s)
Growth Hormone-Releasing Hormone/analogs & derivatives , Liver/metabolism , Receptors, Gastrointestinal Hormone/metabolism , Animals , Binding, Competitive , Cations, Divalent/pharmacology , Edetic Acid/pharmacology , Growth Hormone-Releasing Hormone/metabolism , In Vitro Techniques , Intercellular Signaling Peptides and Proteins , Kinetics , Ligands , Membranes/metabolism , Peptides/metabolism , Rats , Receptors, Vasoactive Intestinal Peptide
10.
J Membr Biol ; 93(1): 23-32, 1986.
Article in English | MEDLINE | ID: mdl-3795260

ABSTRACT

n-Alkanols (from methanol to decanol) have a biphasic effect on rat cardiac adenylate cyclase either basal or stimulated by GTP, GppNHp, NaF or hormones (isoproterenol, glucagon, secretin) in the presence of GTP. At high concentration, all the enzyme activities are inhibited. At low concentration, adenylate cyclase activity is either unchanged or potentiated depending on both the stimulus and the alkanols involved. Potentiation is due to an increase of maximum velocity with no change in the activation constant of the enzyme. Basal activity is unchanged as well as the isoproterenol- and glucagon-stimulated enzyme. The secretin-stimulated enzyme is potentiated. It is the guanyl nucleotide regulatory protein-mediated stimulation of adenylate cyclase which is mainly affected. An attempt was made to relate these effects on adenylate cyclase with physical parameters of the alkanols (partition coefficient). From the data obtained as a function of the alkanol chain-length and of temperature on the adenylate cyclase stimulated by GTP, GppNHp, NaF and permanently activated, it is concluded that the increase in efficacy observed in the presence of alkanol is due to an interaction with the protein moeity particularly with the guanyl nucleotide regulatory protein.


Subject(s)
Adenylyl Cyclases/metabolism , Alcohols/pharmacology , Myocardium/enzymology , Animals , Cell Membrane/enzymology , Glucagon/pharmacology , Guanosine Triphosphate/pharmacology , Guanylyl Imidodiphosphate/pharmacology , Isoproterenol/pharmacology , Kinetics , Rats , Secretin/pharmacology , Sodium Fluoride/pharmacology , Structure-Activity Relationship
11.
Neuroendocrinology ; 44(4): 429-32, 1986.
Article in English | MEDLINE | ID: mdl-3102990

ABSTRACT

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.


Subject(s)
Adenylyl Cyclases/metabolism , Aging/metabolism , Growth Hormone-Releasing Hormone/pharmacology , Pituitary Gland, Anterior/enzymology , Animals , Colforsin/pharmacology , Guanosine Triphosphate/pharmacology , Guanylyl Imidodiphosphate/pharmacology , Male , Rats , Rats, Inbred Strains , Sodium Fluoride/pharmacology , Vasoactive Intestinal Peptide/pharmacology
12.
Peptides ; 7 Suppl 1: 109-12, 1986.
Article in English | MEDLINE | ID: mdl-3018688

ABSTRACT

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.


Subject(s)
Adenylyl Cyclases/metabolism , Glucagon/analogs & derivatives , Amino Acid Sequence , Animals , Enzyme Activation/drug effects , Glucagon/pharmacology , In Vitro Techniques , Liver/enzymology , Myocardium/enzymology , Rats , Receptors, Cell Surface/drug effects , Receptors, Glucagon , Structure-Activity Relationship
13.
Peptides ; 7 Suppl 1: 53-9, 1986.
Article in English | MEDLINE | ID: mdl-3018703

ABSTRACT

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.


Subject(s)
Adenylyl Cyclases/metabolism , Growth Hormone-Releasing Hormone/analogs & derivatives , Receptors, Cell Surface/metabolism , Receptors, Neuropeptide , Receptors, Pituitary Hormone-Regulating Hormone , Animals , Enzyme Activation/drug effects , Growth Hormone-Releasing Hormone/metabolism , Growth Hormone-Releasing Hormone/pharmacology , In Vitro Techniques , Liver/metabolism , Male , Pancreas/metabolism , Pituitary Gland, Anterior/metabolism , Rats , Receptors, Vasoactive Intestinal Peptide , Structure-Activity Relationship
14.
Peptides ; 7 Suppl 1: 79-82, 1986.
Article in English | MEDLINE | ID: mdl-3018706

ABSTRACT

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.


Subject(s)
Peptide Fragments/metabolism , Peptides/metabolism , Receptors, Cell Surface/metabolism , Adenylyl Cyclases/metabolism , Animals , Enzyme Activation/drug effects , In Vitro Techniques , Intercellular Signaling Peptides and Proteins , Liver/drug effects , Liver/metabolism , Peptide Fragments/chemical synthesis , Peptide Fragments/pharmacology , Peptides/pharmacology , Rats , Receptors, Cell Surface/drug effects , Receptors, Vasoactive Intestinal Peptide
15.
Endocrinology ; 117(5): 1759-64, 1985 Nov.
Article in English | MEDLINE | ID: mdl-2994998

ABSTRACT

The efficacy and potency of 14 GH-releasing factor (GRF) analogs, substituted in position 1 to 7, on adenylate cyclase activation in crude homogenates from rat anterior pituitary were related to those of human pancreatic GRF(1-29)-amide and vasoactive intestinal peptide. Among several D-amino acid substitutions, that in position 2 was the only one to yield a super-agonist [with a Kact (concentration required for half-maximal adenylate cyclase activation) 2 times lower than that of GRF(1-29)-NH2]. By contrast, D-isomer substitution in position 1 and 3 was without effect and D-isomer substitution in position 4, 6, or 7 decreased the affinity of the analog. The N-acetylated analog of GRF was as potent and active as the parent peptide, and the identity of the amino acid in position 2 of (N-Ac-Tyr1)-GRF(1-29)-NH2 proved to be determining for enzyme activation, with D-Phe2 and D-Trp2 derivatives acting as partial agonists and the (N-Ac-Tyr1,D-Arg2) analog being an efficient competitive antagonist of GRF(1-29)-NH2. With use of this antagonist, it was possible to demonstrate that GRF and vasoactive intestinal peptide receptors represent distinct entities in the rat anterior pituitary.


Subject(s)
Adenylyl Cyclases/metabolism , Growth Hormone-Releasing Hormone/pharmacology , Pituitary Gland, Anterior/physiology , Receptors, Cell Surface/drug effects , Receptors, Neuropeptide , Receptors, Pituitary Hormone-Regulating Hormone , Animals , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Growth Hormone-Releasing Hormone/analogs & derivatives , Growth Hormone-Releasing Hormone/antagonists & inhibitors , Male , Peptide Fragments/pharmacology , Rats , Receptors, Vasoactive Intestinal Peptide , Structure-Activity Relationship , Vasoactive Intestinal Peptide/pharmacology
16.
Naunyn Schmiedebergs Arch Pharmacol ; 331(1): 71-5, 1985 Oct.
Article in English | MEDLINE | ID: mdl-2999617

ABSTRACT

We evaluated the effects of ischemic injury on the myocardial adenylate cyclase system, 5 h after ligation of the left anterior descending coronary in 5 anesthetized dogs. Crude cardiac membrane preparations were isolated from control and ischemic areas of ventricular myocardium and tested for: 1. L-(125I)iodocyanopindolol binding, in the absence and presence of +/- -isoprenaline and GTP, and 2. adenylate cyclase activity. The density of beta-adrenoceptors increased by 35% in membranes from ischemic areas while the proportion of receptors in a high affinity state for +/- -isoprenaline decreased from 43% to 20%. Adenylate cyclase activities in the basal state and under stimulation with NaF, forskolin, Gpp(NH)p, +/- -isoprenaline and VIP were all markedly and similarly reduced, being only about 30% of comparable activities in membranes from control areas. The +/- -isoprenaline subsensitivity of cardiac adenylate cyclase can, thus, be attributed to a defective enzymatic system and not to a reduction in the number of beta-adrenoceptors implying that the internal components of the system were more sensitive to acute ischemia than the outward oriented hormone receptors. It is tempting to ascribe this uncoupling to a functional depletion in the guanine nucleotide-binding regulatory protein Ns that might reflect a loss of high energy phosphate stores including GTP.


Subject(s)
Adenylyl Cyclases/metabolism , Coronary Disease/metabolism , Myocardium/metabolism , Receptors, Adrenergic, beta/metabolism , Animals , Binding, Competitive/drug effects , Colforsin/pharmacology , Coronary Disease/enzymology , Dogs , Guanosine Triphosphate/pharmacology , Guanylyl Imidodiphosphate/pharmacology , In Vitro Techniques , Isoproterenol/pharmacology , Male , Membranes/metabolism , Myocardium/enzymology , Receptors, Adrenergic, beta/drug effects , Sodium Fluoride/pharmacology , Vasoactive Intestinal Peptide/pharmacology
17.
Endocrinology ; 116(6): 2643-9, 1985 Jun.
Article in English | MEDLINE | ID: mdl-2859987

ABSTRACT

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.


Subject(s)
Growth Hormone-Releasing Hormone/analogs & derivatives , Growth Hormone-Releasing Hormone/pharmacology , Pancreas/drug effects , Receptors, Cell Surface/drug effects , Vasoactive Intestinal Peptide/antagonists & inhibitors , Acetylation , Adenylyl Cyclases/analysis , Animals , Dose-Response Relationship, Drug , In Vitro Techniques , Peptide Fragments/pharmacology , Rats , Receptors, Vasoactive Intestinal Peptide , Sermorelin , Structure-Activity Relationship
18.
Peptides ; 5(5): 877-81, 1984.
Article in English | MEDLINE | ID: mdl-6504721

ABSTRACT

The importance of the N-terminal His residue of VIP for stimulating adenylate cyclase was appreciated by estimating the intrinsic activity and EC50 of four VIP analogues on membranes from rat lung, liver, brain, anterior pituitary, and pancreas, and on human heart membranes. In all tissue preparations tested except one, the order of efficacy (and often potency) was: VIP greater than (Ac-His1)VIP greater than (Phe1)VIP = (3-Me-His1)VIP greater than (D-His1)VIP. In rat heart membranes, the order of efficacy was somewhat different: VIP greater than (Ac-His1)VIP = (Phe1)VIP greater than (D-His1)VIP greater than (3-Me-His1)VIP. These data demonstrated the key role of His1 in VIP in activating adenylate cyclase. They suggest that a given VIP analogue might act as full agonist in tightly coupled adenylate cyclase systems (such as those of rat lung and liver membranes) whereas the same analogue could not promote full activity in poorly coupled systems (such as that present in rat brain synaptic membranes).


Subject(s)
Adenylyl Cyclases/metabolism , Histidine , Vasoactive Intestinal Peptide/pharmacology , Animals , Brain/enzymology , Humans , Liver/enzymology , Lung/enzymology , Myocardium/enzymology , Pancreas/enzymology , Pituitary Gland, Anterior/enzymology , Rats , Structure-Activity Relationship , Synaptic Membranes/enzymology , Vasoactive Intestinal Peptide/analogs & derivatives , Vasoactive Intestinal Peptide/analysis
19.
FEBS Lett ; 172(1): 55-8, 1984 Jun 25.
Article in English | MEDLINE | ID: mdl-6329822

ABSTRACT

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.


Subject(s)
Liver/metabolism , Peptides/metabolism , Receptors, Cell Surface/metabolism , Venoms/metabolism , Adenylyl Cyclases/metabolism , Animals , Binding, Competitive , Cell Membrane/metabolism , Enzyme Activation , Growth Hormone-Releasing Hormone/metabolism , Guanosine Triphosphate/pharmacology , Intercellular Signaling Peptides and Proteins , Lizards , Male , Peptide Fragments/metabolism , Rats , Receptors, Vasoactive Intestinal Peptide , Secretin/metabolism , Sermorelin , Time Factors , Vasoactive Intestinal Peptide/metabolism
20.
Biochim Biophys Acta ; 773(2): 271-8, 1984 Jun 27.
Article in English | MEDLINE | ID: mdl-6329286

ABSTRACT

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.


Subject(s)
Cell Membrane/metabolism , Pancreas/metabolism , Receptors, Cell Surface/metabolism , Receptors, Gastrointestinal Hormone , Secretin/metabolism , Sincalide/metabolism , Vasoactive Intestinal Peptide/metabolism , Adenylyl Cyclases/metabolism , Animals , Disulfides/analysis , Enzyme Activation , Kinetics , Mercaptoethanol/pharmacology , Rats , Receptors, Cholecystokinin , Receptors, G-Protein-Coupled , Receptors, Vasoactive Intestinal Peptide , Secretin/pharmacology , Vasoactive Intestinal Peptide/pharmacology
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