ABSTRACT
The effects of four peptides of the endothelin/sarafotoxin (ET/SRTX) family on the motility of the rat uterus were examined during the different stages of the estrous cycle. ET-1, ET-3, SRTX-b and SRTX-c showed similar effects on the contraction of the uterus: a slight increase in the maximum tension of the spontaneous rhythmic contractions, a suppression of the relaxation phase of these contractions and an increase in their rate. All three effects were concentration dependent. Of the four peptides, ET-1 and SRTX-b showed the highest potency and efficacy, suggesting that among the various peptides of this family so far studied, ET-1 and SRTX-b are the two full agonists. The rank order of susceptibility of the different stages was, in most cases: proestrus greater than estrus greater than metestrus. Freshly excised diestrus uteri showed no spontaneous contractions and did not respond to any of the peptides. The binding potency of ET-1 and SRTX-b to uterine membranes was similar at the various estrous stages, but their maximal binding decreased gradually from proestrus to diestrus. All four peptides induced phosphoinositide (PI) hydrolysis in uterine slices at all four different stages, with ET-1 and SRTX-b again being more potent than ET-3 or SRTX-c. The maximal PI hydrolysis correlated with the increased rate of the rhythmic contractions. It is suggested that the reaction of the rat uterus to the ET/SRTX peptides depends on its hormonal status and that ET may act in concert with steroid hormones in the modulation of the estrous cycle.
Subject(s)
Endothelins/pharmacology , Phosphatidylinositols/metabolism , Uterine Contraction/drug effects , Uterus/drug effects , Vasoconstrictor Agents/pharmacology , Viper Venoms/pharmacology , Animals , Binding Sites/drug effects , Dose-Response Relationship, Drug , Endothelins/metabolism , Estrus/drug effects , Female , Hydrolysis , Rats , Uterus/metabolism , Vasoconstriction/drug effects , Vasoconstrictor Agents/metabolism , Viper Venoms/metabolismABSTRACT
Neuraminidase was used in an attempt to determine whether the endothelin (ET)/sarafotoxin (SRTX) receptor subtypes are glycoproteins and, if so, to determine the role of the carbohydrate moiety in the binding of ligands to the receptor. Incubation of rat cerebellar membranes with neuraminidase was accompanied by a decrease in the capacity of the receptors to bind ET-1 and SRTX-b. In contrast, treatment of the rat caudate putamen and strium or of guinea pig ileum with the enzyme did not affect the binding properties of these receptors. Following exposure of [125I]-ET-1 affinity-labeled receptor to neuraminidase, gel electrophoresis and autoradiography revealed a decrease in molecular mass in the cerebellar and atrial preparations of about 2.5-2.8 kDa. These data indicate that some of the ET/SRTX receptors are glycoproteins and that the sugar moiety is important for ligand binding. Thus, glycosylation might be responsible for the observed heterogeneity among ET/SRTX receptors.
Subject(s)
Caudate Nucleus/metabolism , Cerebellum/metabolism , Glycoproteins/metabolism , Ileum/metabolism , Myocardium/metabolism , Putamen/metabolism , Receptors, Cell Surface/metabolism , Receptors, Cholinergic/metabolism , Receptors, Peptide , Animals , Autoradiography , Cell Membrane/metabolism , Electrophoresis, Polyacrylamide Gel , Endothelins/metabolism , Glycoproteins/isolation & purification , Glycosylation , Guinea Pigs , Iodine Radioisotopes , Kinetics , Molecular Weight , Neuraminidase/pharmacology , Organ Specificity , Rats , Receptors, Cell Surface/isolation & purification , Receptors, Cholinergic/isolation & purification , Receptors, EndothelinABSTRACT
A highly specific and sensitive radioimmunoassay (RIA) was developed for the potent vasoconstrictor peptides, sarafotoxin-b and human endothelin. The antigenic determinants of the antibodies employed in studies with these assays were found to be localized within the amino acid sequence at positions 4-7. This was confirmed by CNBr cleavage of the methionyl residue at position 6 in the sarafotoxin and at position 7 in the endothelin. The chemically characterized modified peptides showed very low cross reactivity in the RIAs. On the other hand, the binding properties as well as the ability to induce phosphoinositide hydrolysis were very similar in the modified and native peptides, indicating that despite cleavage of the peptide bond the biologically active conformation responsible for either binding or phosphoinositide hydrolysis is retained, probably because of the disulfide bonds. Thus, structural alteration might be a valuable means of curtailing some of the various activities induced by the sarafotoxin/endothelin family of peptides.
Subject(s)
Peptides/immunology , Viper Venoms/immunology , Animals , Binding, Competitive , Chromatography, High Pressure Liquid , Cyanogen Bromide , Endothelins , Epitopes , Peptide Fragments/immunology , Protein Conformation , Rabbits , RadioimmunoassayABSTRACT
Functional receptors for the peptides of the endothelin (ET) and sarafotoxin (SRTX) families were detected in the rat uterus. These receptors specifically bind 125I-SRTX-b (Bmax = 220 fmol/mg protein), as well as ET-1, ET-3 and SRTX-c (IC50's 10, 5, 300 and 780 nM, respectively). Activation of the uterine ET/SRTX receptors induced dose-dependent phosphoinositide (PI) hydrolysis and three typical contractile responses: 1) increase in the muscle tonic tension; 2) increase in frequency of the spontaneous rhythmic contractions; 3) decrease of relaxation in each spontaneous rhythmic cycle. All three effects appeared at doses as low as 0.5-1 nM. Dose responses yield ED50 values of 5.5, 30 and 680 nM for ET-1, SRTX-b and ET-3, respectively. SRTX-c was the least effective peptide in achieving decrease in relaxation. In view of these results, and since the uterine responses to the peptides were almost immediate and reversible, we suggest that the functional ET/SRTX receptor of the rat uterus that is coupled to PI hydrolysis may be of physiological significance.
