Monoclonal antibodies against different epitopes of peptide hormones. Use of photoreactive analogues in studies on vasopressin.

In order to produce monoclonal antibodies directed against different epitopes of the neurohypophyseal hormone vasopressin, the hormone was coupled to carrier proteins via photoreactive groups at different positions in the vasopressin sequence: [2-(4-azidophenylalanine), 8-arginine]vasopressin (peptide P1, photoreactive group at position 2) and desamino-[8-N6-(4-azidophenylamidino)lysine]vasopressin (peptide P2, photoreactive group at position 8) were conjugated to thyroglobulin by flash photolysis. Monoclonal antibodies against these conjugates bound ([3H]8-arginine]vasopressin with dissociation constants ranging over 40-400 nM. Epitope analysis by means of competitive ELISA showed that the monoclonal antibody obtained with peptide P1 as hapten was directed against the C-terminal acyclic tripeptide when its conformation was stabilized by interaction with the disulphide-linked cyclic hexapeptide. In contrast, the epitope analysis of three monoclonal anti-(peptide P2) antibodies demonstrated that they recognized antigenic determinants in the cyclic hexapeptide ring, mainly the hydrophobic surface formed by Tyr2 and Phe3. Our results suggest that monoclonal antibodies against different epitopes in small peptide hormones can be generated selectively by using photoreactive peptides in such a way that different antigenic sites are exposed in the hapten-carrier conjugate.     

The central mechanism of the immunomodulating action of vasopressin and its structural analogs]

It is shown that the effect of neuropeptides depends on the dose and their structure. Considerable immunostimulation is observed after administration of [8-arginine]vasopressin and des-9-glucine-[8-arginine]vasopressin as against the control. This stimulation is prevented by the preliminary heloperidol blockade of dopaminergic receptors, i.e. it is dopamine-dependent. At the same time, injection of tetrapeptide vesopressin-(2-5) induces deep immunosuppression in all the applied doses. The peptide immunosuppression is shown to be a result of interaction with another neuromediator (serotoninergic); it has no effect under preliminary cuproheptadine blockade of C-2 serotonin postsynaptic receptors. The immunomodulating effect of the neuropeptides studied is central one and is realized via the hypothalamus-hypophysis complex.     

Suppression of natriferic activity of the vasopressin molecule by modifications in positions 1, 2 and 4

The capacity of five synthetic analogs of [8-arginine] vasopressin (AVP) to stimulate frog skin sodium transport (natriferic activity) was characterized electrophysiologically using the method of short-circuit current, and compared to that of synthetic AVP. The analogs used were [8-arginine] vasopressins modified in positions 1 and 2: [1-(1-mercapto-4-tert-butylcyclohexaneacetic acid)] AVP (I); [1-(1-mercapto-4-methylcyclohexaneacetic acid)] AVP (II); [1-(1-mercapto-4-methylcyclohexaneacetic acid)-2-O-methyltyrosine] AVP (III); and in position 4: [1-(1-mercaptocyclohexaneacetic acid)-4-arginine] AVP (IV); [1-(2-mercaptopropionic acid)-4-arginine] AVP (V). The addition of synthetic vasopressins I, II and V to the frog skin resulted in a weaker stimulation of the skin sodium transport, measured as the level of the short-circuit current (Isc), as compared to that induced by synthetic AVP. In relation to natriferic activity, analogs III and IV did not change the electrical parameters of the skin. It is concluded that introduction of cyclic structure at the beta-carbon in position 1 of the vasopressin molecule decreased its natriferic activity by about 70%. The same reduction of the activity was caused by the replacement of the glutamine residue in position 4 with arginine, and deamination in position 1. Cyclic structure bound in position 1 together with methylation of tyrosine in position 2 resulted in a full suppression of natriferic activity. Similarly, introduction of cyclic group in position 1 in combination with substitution of glutamine in position 4 with arginine totally abolished natriferic activity.     

Hydroosmotic activities of arginine-vasopressins modified either in positions 1, 2 and 4 or at N-terminal extensions.

Vasopressin and its synthetic analogs were studied for their effect on transepithelial water flux in frog urinary bladder. As compared with AVP, 1-deamino-8-D-arginine vasopressin (dDAVP) was about 40 times less effective in stimulating osmotic water flow. The vasopressin analogs obtained by modification in positions 1 and 2 were: [1-(1-mercapto-4-tert-butylcyclohexaneacetic acid)] AVP (I); [1-(1-mercapto-4-methylcyclohexaneacetic acid)]AVP (II); [1-(1-mercapto-4-methylcyclohexaneacetic acid)-2-O-methyltyrosine]AVP (III); and those modified in position 4 were: [1-(1-mercaptocyclohexaneacetic acid)-4-arginine] AVP (IV); [1-(2-mercaptopropionic acid)-4-arginine]AVP (V). Any of the above analogs did not influence basal, but antagonized vasopressin-stimulated water flux. N-terminally extended analogs of AVP: Ala-AVP (VI); Ser-Ala-AVP (VII) and Thr-Ser-Ala-AVP (VIII) stimulated osmotic water flux to the same extent in concentration 200 times higher as that of AVP. We conclude from these studies that vasopressin analogs (I-V) competitively antagonize vasopressin-stimulated hydroosmotic activity in frog urinary bladder probably at the epithelial vasotocin V1 and/or V2 receptor site. N-terminal extension of the vasopressin molecule did not influence the capacity of AVP to induce V2 receptor-mediated action, even when used at higher concentrations.     

Neural regulation of parvalbumin expression in mammalian skeletal muscle.

The receptor mechanisms underlying vasopressin-induced human platelet activation were investigated with respect to stimulation of phosphoinositide metabolism and changes in the cytosolic free Ca2+ concentration ([Ca2+]i). Vasopressin stimulated phosphoinositide metabolism, as indicated by the early formation of [32P]phosphatidic acid ([32P]PtdA) and later accumulation of [32P]phosphatidylinositol ([32P]PtdIns). In addition, vasopressin elicited a transient depletion of [glycerol-3H]PtdIns and accumulation of [glycerol-3H]PtdA. The effects of vasopressin on phosphoinositide metabolism were concentration-dependent, with half maximal [32P]PtdA formation occurring at 30 +/- 15 nM-vasopressin. In the presence of 1 mM extracellular free Ca2+, vasopressin induced a rapid, concentration-dependent elevation of [Ca2+]i in quin2-loaded platelets: half-maximal stimulation was observed at 53 +/- 20 nM-vasopressin. The V1-receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]-vasopressin selectively inhibited vasopressin (100 nM)-induced [32P]PtdA formation [I50 (concn. giving 50% inhibition) = 5.7 +/- 2.4 nM] and elevation of [Ca2+]i (I50 = 3 +/- 1.5 nM). Prior exposure of platelets to vasopressin rendered them unresponsive, in terms of [32P]PtdA formation and elevation of [Ca2+]i, to a subsequent challenge with vasopressin, but responsive to a subsequent challenge with U44069, a thromboxane-A2 mimetic. These results indicate that vasopressin-induced human platelet activation is initiated by combination with specific V1 receptors on the platelet, and that the sequelae of receptor occupancy (stimulation of phosphoinositide metabolism and elevation of [Ca2+]i) are equally susceptible to inhibition by receptor antagonists and by receptor desensitization.     

Stimulation of inorganic-phosphate incorporation into phosphatidylinositol in rat thoracic aorta mediated through V1-vasopressin receptors.

Vasopressin stimulates the incorporation of [32P]Pi into phosphatidylinositol but not into other phospholipids in rat thoracic aorta strips. The relative abilities of three vasopressin analogues to stimulate phosphatidylinositol labelling in rat aorta are similar to their relative pressor potencies in vivo and to their relative potencies in stimulating the metabolism of rat hepatocytes, but very different from their relative antidiuretic potencies. The vasopressor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin competitively inhibits [Arg8]vasopressin-stimulated phosphatidylinositol labelling in rat aorta with a pA2 of 8.1. It is concluded that the Ca2+-mobilizing vasopressin receptors (V1-receptors) of the rat aorta stimulate phosphatidylinositol metabolism, probably by enhancing phosphatidylinositol breakdown.     

The composition of crystalline complexes of neurophysin-M with [8-arginine]-vasopressin and oxytocin

Neurophysin-M, a methionine-containing protein that is the major constituent of neurophysin, has been crystallized as complexes with [8-arginine]-vasopressin. Three moles of vasopressin alone or 2 moles of vasopressin together with 1 mole of oxytocin are bound/mole of protein. An amorphous complex of the protein with oxytocin alone contains 2 moles of the hormone/mole of protein. Deamino-[8-arginine]-vasopressin, a highly active basic analogue of vasopressin, is not bound by neurophysin. The primary amino group of both vasopressin and oxytocin is necessary for binding with neurophysin.     

Resistance to hepatic action of vasopressin in genetically obese (ob/ob) mice.

1. Fatty acid synthesis, measured in the perfused liver of genetically obese (ob/ob) mice with 3H2O or [14C]actate, did not show the inhibition by [8-arginine]vasopressin (antidiuretic hormone) that is observed in livers from normal mice. 2. Hepatic glycogen breakdown in obese mice was stimuulated by vasopressin, but not as extensively as in lean mice. 3. If obese mice received a restricted amount of food, then fatty acid synthesis still did not respond to vasopressin, but glycogen breakdown was fully stimulated. 4. Cholesterol synthesis was not inhibited by vasopressin in livers from obese mice. 5. Vasopressin inhibited fatty acid synthesis in intact lean mice, but not in obese animals. 6. These results suggest that genetic obesity could be due to an inborn error within the mechanisms (other than adenylate cyclase) which mediate responses to extracellular effectors.     

Proteolytic conversion of oxytocin by brain synaptic membranes: role of aminopeptidases and endopeptidases.

The proteolytic conversion of oxytocin and vasopressin by purified rat brain synaptic membranes was studied at 37 degrees C and physiological pH 7.4. The formed peptide fragments were isolated by high performance liquid chromatography and characterized by amino acid analysis. When oxytocin was incubated with synaptic membranes, either C- or N-terminal fragments were found. The most abundant were [Cyt6]oxytocin(4-9), [Cyt6]oxytocin(3-9), [Cyt6]oxytocin(2-9), oxytocin(1-8) and oxytocin(1-7). In contrast, only C-terminal fragments, [Cyt6-Arg8]vasopressin(4-9), [Cyt6-Arg8]vasopressin(3-9) and [Cyt6-Arg8]vasopressin(2-9), were found by incubating [Arg8]vasopressin. The formation of C-terminal oxytocin and vasopressin fragments was inhibited by the aminopeptidase inhibitors amastatin and bestatin, while the formation of oxytocin(1-7) and (1-8) was inhibited by the divalent cations Hg(2+) and Zn(2+). The formation of oxytocin(1-7) was also partially prevented by the endopeptidase inhibitor phosphoramidon. The formation of both C- and N-terminal fragments was inhibited by o-phenanthroline. The results suggest that, while [Arg8]vasopressin is metabolized only by membrane-bound aminopeptidases, oxytocin is also metabolized by membrane-bound endopeptidases.     

Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin

A photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N6-4-azidophenylamidino)lysine]-vasopressin, was compared to salmon calcitonin and [8-arginine]-vasopressin with respect to stimulation of cAMP synthesis in the LLC-PK1 pig kidney epithelial cell line. Without photoactivation, the vasopressin analogue-elicited responses were identical to those induced by vasopressin, in that cAMP synthesis returned to the basal, unstimulated level about 4 h after hormonal treatment. In contrast, the levels of activation of cAMP-dependent protein kinase induced by salmon calcitonin returned to basal approx. 12 h after hormone addition. When activated by ultraviolet irradiation, the vasopressin analogue induced 'permanent' stimulation of adenylate cyclase, whereby cAMP production could be detected even 12.5 h after treatment. Both salmon calcitonin and the photoactivated vasopressin analogue inhibited growth of LLC-PK1 cells, in contrast to vasopressin or the nonactivated analogue. Growth inhibition appeared to be a consequence of the prolonged stimulation of adenylate cyclase. This conclusion was supported by the fact that a LLC-PK1 cell mutant in cAMP-dependent protein kinase was resistant to growth inhibition by salmon calcitonin and activated vasopressin analogue. The results imply that the cAMP-dependent protein kinase is the mediator of the hormone-stimulated growth inhibition.     

Isolation and structure elucidation of bovine pineal arginine vasopressin: arginine vasotocin not identified

A large number of reports have demonstrated the presence of neurohypophysial hormone-like peptides in mammalian pineal glands and an antigonadotropic function has been ascribed to pineal arginine vasotocin (AVT). We have undertaken large scale purification of bovine pineal neurohypophysial hormone-like substances which demonstrate mouse mammary milk-ejection activity (ME-activity) in vitro. Peptides with ME-activity were extracted from more than 5 kg of bovine pineal glands. ME-activity containing peptides were found in both high (Mr approximately 10,000-15,000) and low (Mr approximately 500-1000) Mr species from Sephadex G-25 chromatography of 0.2 N acetic acid extracts. After ultrafiltration in 5% formic acid, the neurohypophysial hormone-like peptides were localized to an ultrafiltration Mr 500-1000 retentate. A homogeneous peptide, which shared an identical retention time (RT) and amino acid sequence with synthetic 8-arginine vasopressin (AVP), was isolated by serial semipreparative high performance liquid chromatography. On the other hand, the non-mammalian nonapeptide AVT was not identified.     

Vasopressin receptor subtypes in dorsal hindbrain and renal medulla

We have investigated the ability of a series of synthetic vasopressin analogues and related peptides to compete with (3H)-arginine8 vasopressin for binding sites in rat renal medulla and dorsal hindbrain. In renal medulla, arginine8 vasopressin and deamino arginine8 vasopressin, a selective antidiuretic, were equipotent while two antagonists of the pressor action of arginine vasopressin were less potent. In the dorsal hindbrain, arginine8 vasopressin and the pressor antagonists were more potent than the synthetic antidiuretic. Potency profiles of these and other analogues suggest that the renal medulla and dorsal hindbrain vasopressin receptors represent different subtypes.     

Nature and properties of human platelet vasopressin receptors.

The binding of 3H-labelled [8-arginine]vasopressin to human platelets or crude platelet membranes was examined. Both preparations specifically bound [8-arginine]vasopressin. The binding increased linearly with protein concentration, it was temperature- and time-dependent, saturable and could be reversed to a large extent by EDTA (10 mM). In this latter case, addition of an excess of MgCl2 (20 mM) restored the initial level of binding. Intact platelets and membranes derived from these platelets presented a single population of binding sites with a dissociation constant (Kd) of 1.3 +/- 0.2 and 1.8 +/- 0.3 nM and a maximal binding capacity of 142 +/- 48 and 270 +/- 17 fmol/mg of protein, respectively. The Kd values of various analogues correlated well with those determined on rat liver membrane V1 vasopressin receptors but not with those determined on rat kidney membrane V2 receptors.     

The synthesis and biological activity of four novel fluorescent vasopressin analogs

We synthesized and tested the biological properties of four fluorescent vasopressin analogs: [1-(2-mercapto)propionic acid]-8-lysine-N6-5-dimethylamino-naphthalene-1-sulfonyl vasopressin (D-MLVP), [1-(2-mercapto)propionic acid]-8-lysine-N6-carboxyfluorescein vasopressin (F-MLVP), [1-(2-mercapto)propionic acid]-8-lysine-N6-2-N-methylanthranilamide vasopressin (MA-MLVP), and [1-(2-mercapto)propionic acid]-8-lysine-N6-carboxytetramethylrhodamine vasopressin (R-MLVP). All fluorescent analogs were prepared by coupling the appropriate fluorochrome to the 6-amino group of the lysine residue in [1-(2-mercapto)propionic acid]-8-lysine vasopressin (MLVP) which was synthesized by the Merrifield solid-phase method. The structures of high performance liquid chromatography-purified MLVP and the fluorescent analogs were confirmed by fast atom bombardment mass spectrometry. F-MLVP, MA-MLVP, and R-MLVP effectively competed for 8-arginine vasopressin (AVP)-binding sites in canine renal plasma membranes and on the surface of porcine kidney cells (LLC-PK1, ATCC CL101). Dissociation constants for F-MLVP, MA-MLVP, and R-MLVP of 32, 8.8, and 26 nM, respectively, were calculated from the results of competition binding assays conducted with membranes. D-MLVP did not bind to plasma membranes. Dissociation constants for F-MLVP, MA-MLVP, and R-MLVP of 390, 38, and 160 nM, respectively, were calculated from the results of competition binding assays conducted with cells. F-MLVP, MA-MLVP, and R-MLVP at a concentration of 10(-6) M increased adenylate cyclase activity in canine renal plasma membranes to values 2.4, 2.9, and 2.6 times that of basal activity, respectively. A maximally active concentration of AVP (1 microM) increased adenylate cyclase activity in canine renal plasma membranes to a value 2.7 times that of basal activity. D-MLVP did not stimulate adenylate cyclase activity. F-MLVP, MA-MLVP, and R-MLVP at a concentration of 10(-6) M increased the cAMP content of porcine kidney cells from a basal level of 43 to 267, 160, and 469 pmol/mg of cell protein, respectively. Specific binding of these fluorescent analogs to receptors on the surface of LLC-PK1 cells was observed by fluorescence microscopy. These observations indicate that F-MLVP, MA-MLVP, and R-MLVP are biologically active fluorescent vasopressin analogs which are well-suited to the study of renal vasopressin receptors by fluorescence microscopy.     

纤维蛋白单体D区与E区聚合后E区结构的变化

应用纤维蛋白单克隆抗体IF 5 3,观察当纤维蛋白的“A”位点与另一纤维蛋白D区域的“a”位点结合后纤维蛋白E区的变化 .纤维蛋白原Aα链经赖氨酰肽链内切酶消化后 ,应用反相HPLC分离纯化 ;通过ELISA法检测单克隆抗体IF 5 3与纤维蛋白原及其衍生物的反应情况 ;应用放射免疫法检测RGD合成肽抑制纤维蛋白单体与IF 5 3反应的情况 .发现IF 5 3能与纤维蛋白原Aα链的一个片段反应 ,该片段经氨基酸序列分析显示为纤维蛋白原Aα链氨基末端 (1～ 2 9) .该抗体能与酸溶解的纤维蛋白单体和可溶性纤维蛋白及XDP反应 ,但不能与酸化纤维蛋白原或GPRP反应 ,因此IF 5 3的抗原决定簇在Aα 2 0～ 2 9,与凝血酶作用于纤维蛋白肽A ,暴露出的聚合位点“A”(Aα17～19)紧邻 .当GPRP存在于纤维蛋白原溶液时 ,经凝血酶作用产生这种纤维蛋白单体不能与IF 5 3反应 .Aα(93～ 99) (ILRGDFS)合成肽部分抑制纤维蛋白单体与IF 5 3的反应 .实验结果提示 ,当纤维蛋白单体相互聚合 ,或纤维蛋白单体与纤维蛋白原聚合时 ,纤维蛋白单体结构会发生变化 ,其中Aα2 0～ 2 9片段成为新抗原暴露于E区表面 ,并且Aα2 0～ 2 9与纤维蛋白原细胞粘附区域RGD1片段邻近     

Circular-dichroic spectra of vasopressin analogues and their cyclic fragments.

The circular dichroic spectra of [Arg8]vasopressin, [Mpr1, Arg8]vasopressin, [Mpr1, D-Arg8]-vasopressin, pressinamide, deaminopressinamide, tocinamide, deaminotocinamide, [Leu4, D-Arg8]-vasotocin, [Mpr1, Leu4, D-Arg8]vasotocin and [Phe2, Lys8]vasopressin have been studied. All these substances showed a characteristic positive dichroic band at about 225 nm due to the presence of tyrosine in sequence position 2. The intensity of this band was affected by interactions between the tyrosine side-chain and other structural elements in the molecule, such as the Na-amino group, the side-chain of phenylalanine in position 3 and the linear C-terminal peptide. Analysis of the response of this band to structural modifications of the molecule and change in the solvent (particularly comparing neutral aqueous solutions with hexafluoroacetone solutions) allowed some conformational conclusions. The linear C-terminal tripeptide is probably situated over the cyclic portion of the molecule both in vasopressin and oxytocin substances. Its steric interaction with the tyrosine side-chain seems to be particularly efficient in molecules containing D-arginine in position 8. In the vasopressin series the stacking interaction of neighbouring aromatic amino acid residues furthermore limits the conformational freedom of the tyrosine side-chain and also probably distorts the dihedral angles of residues 1-3 in comparison with oxytocin. The interactions of phenylalanine and arginine with tyrosine relatively decrease the conformational effects of the primary amino group. Consequently the local conformation of vasopressin in the region of the tyrosine residue is more rigid and less sensitive to changes in medium than that of oxytocin. The circular dichroic spectra did not show any basic conformational differences in the backbone peptide chain of oxytocin and vasopressin substances. A weak negative disulphide band at about 290 nm could be observed in the spectra of both series of substances.     

Conformation of [8-arginine]vasopressin and V1 antagonists in dimethyl sulfoxide solution derived from two-dimensional NMR spectroscopy and molecular dynamics simulation

Structural and dynamic properties of [8-arginine]vasopressin and a class of highly potent vasopressin V1 antagonists which contain 3-mercapto-3,3-cyclopentamethylene propionic acid (Mca) in position 1 of the vasopressin sequence have been determined. On the basis of two-dimensional NMR experiments in dimethyl sulfoxide solution, interproton distances were derived according to which model conformations were built and refined using molecular dynamics simulations. The conformation of vasopressin and the V1 antagonists differ mainly in the region of the mutated residue. The antagonistic property was found to be related to an inversed chirality of the disulfide bridge. In all investigated molecules, characteristic beta-turn structure elements were found for the backbone conformation of the endocyclic residues Tyr2-Asn5. For this portion of the peptide sequence, various conformational equilibria were detected which matched different time scales. For [Arg8]vasopressin, averaged NMR parameters were obtained which could be explained by rapid interconversion between different beta-turn geometries, whereas multiple slowly exchanging conformations were observed for the V1 antagonists. V1 antagonists containing sarcosine in position 7 exhibited multiple spectral patterns for the exocyclic part attributed to cis/trans isomerization. The X-ray structure of deamino-oxytocin [Wood, S. P., Tickle, I. J., Treharne, A. M., Pitts, J. E., Mascarenhas, Y., Li, J. Y., Husain, J., Cooper, S., Blundell, T. L., Hruby, V. J., Buku, A., Fischman, A. J. & Wyssbrod, H. R. (1986) Science 232, 633-636] was found to represent one sample of the conformational space covered by the multiple conformations found for [Mca1, Arg8]vasopressin.     

Biotinyl analogues of vasopressin as biologically active probes for vasopressin receptor expression in cultured cells

Biotinyl analogues of [Arg8]vasopressin were synthesized with the biotinyl moiety at position 4. This involved the substitution of 2, 4-diaminobutyric acid (Dab) for Gln4 in [1-deamino-Arg8]vasopressin to give the parent peptide des-[Dab4,Arg8]vasopressin. Two biotinyl analogues with different spacers between the side chain of Dab4 and the biotinyl residue were then prepared and characterized in detail. The analogues retained high binding affinities for the V2-receptor in both bovine kidney membranes and LLC-PK1 renal epithelial cells and for the V1-receptor in rat liver membranes. Both analogues were as potent as [Arg8] vasopressin in stimulating the cAMP-dependent protein kinase and the production of urokinase-type plasminogen activator in LLC-PK1 cells, with concentration dependence consistent with receptor binding affinities. Avidin or streptavidin did not appear to reduce receptor binding or biological activity of the biotinyl analogues. The use of the biotinylated vasopressin analogue des-[Dab-(biotinylamido)hexanoyl4, Arg8]vasopressin together with fluorescein-labeled streptavidin as a fluorescent probe for the V2-receptor in LLC-PK1 cells demonstrated the following: 1) Specific binding of the biotinyl analogue shown by quantitative single-cell fluorescence measurements using the technique of fluorescence microphotolysis; 2) the V2-receptor visualized by fluorescence microscopy; and 3) the expression of the V2-receptor detected by flow cytometry.     

Mechanism of the inhibition of protein synthesis by vasopressin in rat liver

A recent study reported that protein synthesis was inhibited in rat livers perfused with medium containing vasopressin (Chin, K. -V., Cade, C., Brostrom, M. A., and Brostrom, C. O. (1988) Int. J. Biochem. 20, 1313-1319). The inhibition of protein synthesis caused by vasopressin was associated with a disaggregation of polysomes, suggesting that peptide chain initiation was slowed relative to elongation. In contrast, Redpath and Proud (Redpath, N. T., and Proud, C. G. (1989) Biochem. J. 262, 69-75) recently reported an inhibition of peptide chain elongation by a calcium/calmodulin-dependent mechanism. Therefore, the question remained whether only peptide chain initiation was inhibited or both initiation and elongation were affected by vasopressin. In the present study, vasopressin was found to inhibit protein synthesis in both perfused rat livers and isolated rat hepatocytes. Ribosomal half-transit times in isolated hepatocytes averaged 1.9 +/- 0.1 min with or without vasopressin present in the media, demonstrating that the rate of peptide chain elongation was unaffected by vasopressin. Instead, the inhibition of protein synthesis induced by vasopressin was manifested at the level of peptide chain initiation. Vasopressin treatment resulted in both a 2-fold increase in the number of free ribosomal particles and a greater than 50% decrease in the amount of [35S]methionine bound to 43 S preinitiation complexes. In addition, the activity of eukaryotic initiation factor (eIF) 2B in crude extracts from perfused livers was reduced to 53% of the control value in response to vasopressin. The inhibition of eIF-2B activity was associated with an increase in the proportion of the alpha-subunit of eIF-2 in the phosphorylated form from 9.6% in control livers to 30.7% in livers perfused with medium containing vasopressin. The results demonstrate the novel finding that the inhibition of protein synthesis in vasopressin-treated livers is caused by a reduction in eIF-2B activity due to an increase in phosphorylation of eIF-2 alpha.     

Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin

A photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N⁶-4-azidophenylamidino)lysine]-vasopressin, was compared to salmon calcitonin and [8-arginine]-vasopressin with respect to stimulation of cAMP synthesis in the LLC-PK₁ pig kidney epithelial cell line. Without photoactivation, the vasopressin analogue-elicited responses were identical to those induced by vasopressin, in that cAMP synthesis returned to the basal, unstimulated level about 4 h after hormonal treatment. In contrast, the levels of activation of cAMP-dependent protein kinase induced by salmon calcitonin returned to basal approx. 12 h after hormone addition. When activated by ultraviolet irradiation, the vasopressin analogue induced ‘permanent’ stimulation of adenylate cyclase, whereby cAMP production could be detected even 12.5 h after treatment. Both salmon calcitonin and the photoactivated vasopressin analogue inhibited growth of LLC-PK₁ cells, in contrast to vasopressin or the nonactivated analogue. Growth inhibition appeared to be a consequence of the prolonged stimulation of adenylate cyclase. This conclusion was supported by the fact that a LLC-PK₁ cell mutant in cAMP-dependent protein kinase was resistant to growth inhibition by salmon calcitonin and activated vasopressin analogue. The results imply that the cAMP-dependent protein kinase is the mediator of the hormone-stimulated growth inhibition.