Properties of highly purified lysyl oxidase from embryonic chick cartilage.

Lysyl oxidase the enzyme which oxidately deaminates lysine residues in collagen and elastin, was purified from embryonic chick cartialge by employing an affinity column of lathyritic rat skin collagen coupled to Sepharose, followed by separation on DEAE-cellulose. An enzyme preparation was obtained which was pure as shown by polyacrylamide gel electrophoresis. The specific activity was 1800-fold higher than that of the original extract. The pure enzyme utilized both collagen and elastin substrate. Furthermore, the ratios of enzyme activity with elastin substrate versus that with collagen substrate were the same at all stages of purity. Only one protein band was found after polyacrylamide gel electrophoresis of the pure lysyl oxidase in sodium dodecyl sulfate and mercaptoethanol. The molecular weight was estimated to be 28000. It was found that the enzyme contained a large number of cysteine and tyrosine residues. Evidence was obtained for molecular heterogeneity of lysyl oxidase. The enzyme eluted from DEAE-cellulsoe in at least four distinct regions. When the peaks were rechromatographed separately, they eluted at salt concentrations similar to those of the original chromatogram. However, the substrate specificity and the electrophoretic mobility on polyacrylamide gel were the same for all enzyme fractions.

Design, synthesis, and biological activity of a peptide mimic of vasopressin.

Our molecular modeling studies suggested that the conformational effects of the "cystine-line" residue Pmp1-Cys6 on the cyclohexapeptide ring of the vasopressin antagonist [Pmp1,D-Phe2,Val4]AVP might be mimicked by substitution of D-aminoadipic acid at position 6 and cyclization of its side-chain carboxyl to the alpha-amine of residue 2. The peptide was prepared with DL-aminoadipic acid, and following cyclization, the two diastereomeric peptides were separated and purified by preparative high-performance liquid chromatography. The structure of each was confirmed by amino acid analysis and fast atom bombardment mass spectrometry. The chirality of the aminoadipic acid residue of each peptide was determined by chiral gas chromatography. The circular dichroism spectrum of each peptide was run and compared with the appropriate agonist and antagonist peptide standards. These peptides demonstrated in vitro poor V2-receptor affinity and an inability to inhibit or stimulate vasopressin-induced adenylate cyclase formation, suggesting that they lack one or more key features of the agonist/antagonist pharmacophore.

Potent vasopressin antagonists modified at the carboxy-terminal tripeptide tail.

In a continuing effort to design more potent renal vasopressin (V2 receptor) antagonists, we have focused our attention on the carboxy-terminal tripeptide tail (Pro-Arg-Gly-NH2), a fragment common to both agonists and antagonists. Vasopressin antagonist analogues having a dibasic dipeptide tail, e.g., Arg-Arg-NH2 or Arg-Lys-NH2, attached directly to the cyclic hexapeptide ring are potent V2-receptor antagonists. Similar modification of a representative agonist drastically reduces its potency. We report the synthesis and pharmacological properties of a series of potent V2-receptor antagonists 3-9 where a combination of D or L dibasic dipeptide has been utilized to replace the common tripeptide fragment. Our results suggest a difference in the way agonists and antagonists bind to vasopressin receptor and further support the difference in the structure-activity relationships of agonists and antagonists. These results provide potentially useful insights for the design of novel V2-receptor antagonists.

Flushing and haemodynamic responses to vasopressin peptides in the rhesus monkey.

1. The mechanism of the flushing, hypotension and tachycardia associated with i.v. administration of desGlyd(CH2)5D-Tyr(Et)VAVP (SK&F 101926; 25 micrograms kg-1) and the selective V2 antidiuretic agonist, desamino-8-D-arginine vasopressin (dDAVP; 3 micrograms kg-1) was studied in ketamine-anaesthetized rhesus monkeys. 2. The flushing associated with SK&F 101926 was reduced by pretreatment with a mast cell stabilizer and by repeated administration of peptide (within 2-4 weeks). A similar desensitization to dDAVP-associated flushing was observed on repeated administration. 3. Treatment with dDAVP also resulted in reduced SK&F 101926-associated flushing. 4. The hypotension associated with SK&F 101926 was not affected by pretreatment with a mast cell stabilizer. A similar degree of hypotension was observed with repeated administration of either SK&F 101926 or dDAVP. 5. The tachycardia associated with SK&F 101926 was reduced by pretreatment with a mast cell stabilizer or repeated administration of SK&F 101926. Repeated administration of dDAVP, however, resulted in an enhanced tachycardia. 6. Indomethacin (5 mg kg-1 i.v.) did not alter the flushing or the hypotension associated with the administration of either SK&F 101926 or dDAVP, but resulted in an enhanced tachycardia to SK&F 101926. 7. Administration of a selective V1 vasopressor antagonist did not result in flushing, hypotension or tachycardia. 8. It was concluded that the flushing response to vasopressin-like peptides in rhesus monkeys may be due to an action on mast cells, whereas the haemodynamic responses are not, but probably involve direct vasodilator actions.

Induction of functional beta-adrenergic receptors in rat aortic smooth muscle cells by sodium butyrate.

Rat aortic smooth muscle cells in culture (A-10; ATCC CRL 1476) exhibited low levels of beta-adrenergic receptors as determined by specific binding of [125I]cyanopindolol ([125I]CYP) and marginal stimulation of adenylate cyclase in plasma membranes by (-)isoproterenol. When these cells were exposed to 5 mM sodium butyrate, the number of beta-adrenergic receptors and the beta-agonist-stimulated adenylate cyclase activity increased markedly. However, basal, GTP, Gpp(NH)p, and fluoride-stimulated activities did not change. The induction of beta-adrenergic receptors and beta-agonist stimulated adenylate cyclase activity was time- and dose-dependent, and was relatively specific for sodium butyrate. Propionate and valerate were less effective than butyrate, while isobutyrate, succinate, and malonate were ineffective. The induction involved RNA and protein synthesis because induction was prevented by treatment with cycloheximide, puromycin, and actinomycin D. Butyrate did not cause a general increase in cell surface receptors, because the number of vasopressin receptors did not change. The sustained presence of butyrate appeared to be necessary for the maintenance of the induced beta-receptors. When butyrate was removed, receptor number and beta-agonist-stimulated adenylate cyclase activity were decreased by 90% over 24 hr. We conclude that the poor response of rat aortic smooth muscle cell plasma membranes to beta-adrenergic agonists is due to the presence of a low number of beta-adrenergic receptors. Butyrate markedly increased the number of beta-receptors which resulted in a proportional increase in beta-agonist-stimulated adenylate cyclase activity. The increase in receptor number was dependent on RNA and protein synthesis. Butyrate treatment did not affect the activity of the cyclase unit and the efficiency of coupling between the receptors and the guanine nucleotide regulatory protein, Ns.

Desensitization of LLC-PK1 cells by vasopressin results in receptor down-regulation.

The molecular mechanism of desensitization of antidiuretic hormone receptors is not well understood. Preincubation of LLC-PK1 cells with lysine vasopressin (LVP) (10(-6) M, 5 h) decreased subsequent LVP-stimulated cAMP accumulation in cells by 83% and reduced the Vmax of LVP-stimulated adenylate cyclase by 81%. Such preincubation also reduced by 90% the binding of [3H]LVP to both intact cells and isolated plasma membranes, suggesting a loss of vasopressin receptors. Both the reduction in cAMP response and the apparent loss of receptors showed similar dose and time dependence. Monensin (33 microM) did not alter [3H]LVP binding or stimulation of cAMP by LVP, nor did it prevent desensitization. However, membranes prepared from cells preincubated with LVP in the presence of monensin did not show a decrease in [3H]LVP binding. Forskolin preincubation, at 0.1, 1, 10 and 100 microM, did not alter [3H]LVP binding or accumulation of cellular cAMP by LVP, nor did it induce desensitization to LVP. Cells desensitized with varying LVP concentrations in the presence of 10 microM forskolin displayed the same loss of [3H]LVP binding and LVP responsiveness as observed in the absence of forskolin. LVP-desensitized cells, upon removal from LVP-containing medium, recovered cAMP responsiveness to LVP and specific binding of [3H]LVP at the same rate, achieving control levels after 50 h. Recovery was prevented by cycloheximide (25 micrograms/ml). These findings are consistent with a desensitization process involving LVP-mediated receptor internalization, and a recovery process requiring protein synthesis.

Vasopressin receptor antagonism in rhesus monkey and man: stereochemical requirements.

The vasopressin antidiuretic (V2) antagonist activity of the position 6 stereoisomers of four vasopressin analogs were tested for water diuretic activity in the rhesus monkey and for activity to inhibit vasopressin-stimulated adenylate cyclase activity in rhesus monkey and human renomedullary tissue in vitro. Replacement of the mercapto groups of the cysteine residues with methylene groups resulted in compounds having similar in vitro potencies to their disulfide analogs; however, these 'dicarba' compounds demonstrated more potent aquaretic activity. Position 6 D enantiomers were associated with less vasopressin antagonist activity in vitro in both species. Based upon these studies, the most potent aquaretic structure identified was the dicarba analog SK & F 105494.

Vascular vasopressin receptors mediate phosphatidylinositol turnover and calcium efflux in an established smooth muscle cell line.

Vasopressin-induced phosphatidylinositol turnover and mobilization of intracellular Ca2+ was studied using an established smooth muscle cell line (A-10). The cells were subcloned to ensure a monoclonal cell population. The accumulation of inositol mono-, di-, and tris-phosphates (IP1, IP2, and IP3, respectively), and the mobilization of intracellular Ca2+ were dependent on the time of incubation and the concentration of arginine vasopressin (AVP). IP1, IP2, and IP3 were significantly elevated after 15 sec and remained elevated for up to 2 hr. The concentrations of AVP required for half-maximal stimulation of IP1, IP2, and IP3 formation were 2, 12, and 4 nM, respectively. LiCl was required to observe the accumulation of inositol phosphates in response to AVP. Significant 45Ca2+ efflux was observed within 15 sec after exposure to AVP. By employing the vasopressin receptor subtype selective antagonists [d(CH2)5Tyr(Me)AVP, V1; d(CH2)5D-Tyr(Et)VAVP,V1/V2; d(CH2) 5D-IleVAVP,V2] and agonists [AVP, V1/V2; dDAVP, V2; dVDAVP, V2], we found that the vasopressin-induced stimulation of phosphatidylinositol turnover and 45Ca2+ efflux were mediated by receptors of the vascular V1 subtype. Pertussis toxin pretreatment partially inhibited vasopressin-induced phosphatidylinositol turnover. These data demonstrate that activation of V1 receptors of vascular smooth muscle cells resulted in enhanced phosphatidylinositol turnover and mobilization of intracellular Ca2+.

Antibody activity and complement-mediated solubilization of precipitates during the immune response.

The avidity of antibodies against human serum albumin (HSA) and the complement-mediated solubilization of HSA anti-HSA precipitates has been investigated during the immune response in rats, mice, rabbits, and guinea-pigs. The avidity of the anti-HSA antibodies was quantitatively determined and expressed as the amount of HSA required to solubilize 50% of 10 ng HSA anti-HSA precipitate. In rats, from 25 to 100 days after primary immunization the avidity of the anti-HSA antibodies increased approximately 100-fold, whereas the affinity did not change significantly. The increase in avidity was accompanied by a thirty-six-fold decrease in the amount of immune precipitate that could be solubilized by complement. Booster injections of the rats did not prevent the increase in avidity (thirteen-fold) that occurred from 25 to 50 days after the first immunization; however, the subsequent eight-fold increase in avidity from 50 to 100 days could partially or completely be abolished by secondary immunizations. A close, inverse relationship between the avidity of the anti-HSA antibodies and the rate of complement-mediated solubilization was observed. In addition, an increase in avidity without a change in affinity of anti-HSA antibodies and a decrease in the rate of complement-mediated solubilization was found during the immune response against HSA not only in rats, but also in mice, rabbits, and guinea-pigs.

Complement-mediated solubilization of immune precipitates prepared with antibodies of different avidity.

Complement-mediated solubilization of immune precipitates prepared with HSA and rat IgG anti-HSA has been quantitatively analyzed. Early and late IgG anti-HSA antibodies were obtained 27 and 49 days after immunization, respectively. Immune precipitates prepared with early IgG anti-HSA were solubilized by rat serum to a larger extent than complexes prepared with late IgG anti-HSA. The affinities for HSA of the early and late antibodies were not significantly different. The quantitative differences in solubilization were neither due to differences in the Ab/Ag ratios of the immune precipitates, nor appeared to be brought about by changes in the distribution of the antibodies over the IgG sub-classes. The avidity of the late IgG anti-HSA antibodies was higher than the avidity of the early IgG antibodies. Presumably, the avidity of the antibodies greatly affected the complement-mediated solubilization of the immune precipitates. In addition, the solubilization was found to be dependent on the conditions employed to prepare the immune precipitates.

Antagonists of the antidiuretic activity of vasopressin.

Competitive antagonists of the antidiuretic (ADH) activity of vasopressin were first described some six years ago. When studied in vitro, ADH antagonists displace vasopressin from specific renal binding sites and antagonize, in a competitive fashion, vasopressin stimulation of adenylate cyclase and transepithelial water, salt, and urea fluxes. When studied in vivo, the ADH antagonists increase renal water excretion and antagonize, in a competitive fashion, the ADH activity of vasopressin. Marked species heterogeneity is apparent with ADH antagonists in vivo, and inconsistencies between in vitro and in vivo findings within the same species are reported. Other renal responses associated with administration of ADH antagonists include changes in renal hemodynamics and renal salt and urea excretion. The effects on salt excretion appear to be limited to those species in which vasopressin stimulation of epithelial salt reabsorption has been demonstrated. In summary, the role of vasopressin as the principal factor regulating renal water handling is supported by experience with ADH receptor antagonists. However, that experience also indicates the emerging significance of autocoids, and other synergistic factors, to affect ADH receptor/effector mechanisms and to modulate renal ADH responses.

Activation of prolyl hydroxylase in L-929 fibroblasts by ascorbic acid.

The addition of ascorbate to the culture medium of early log-phase mouse fibroblasts (L-929 cells) resulted in a 5-fold increase of prolyl hydroxylase activity. Maximal activity was reached within 2 hr after addition of 5 muM ascorbate. The total amount of enzyme-related antigen did not change on treatment with ascorbate and the activation was shown to be independent of RNA and protein synthesis. The increase in activity caused by ascorbate is therefore due to the activation of a preformed precursor. Enzyme (molecular weight 260,000-300,000) and putative precursor (molecular weight 85,000-105,000) were separated by chromatography on DEAE-Sephadex. Treatment of intact cells with dithiothreitol resulted in an almost quantitative conversion of the enzyme to the smaller inactive protein. When these cells were treated with ascorbate or incubated overnight in fresh medium the enzyme reappeared and precursor concentrations decreased proportionately. Ascorbate may act by bringing about aggregation of enzymatically inactive subunits.

Solubilization of a guanine nucleotide-sensitive form of vasopressin V2 receptors from porcine kidney.

Vasopressin (V2) receptors were solubilized from porcine kidney membranes with the detergent egg lysolecithin. Binding of [3H]vasopressin to the solubilized fraction was rapid, specific, and saturable. The agonist dissociation constants observed in membranes and solubilized fractions were 1.7 +/- 0.3 and 2.3 +/- 0.2 nM, respectively. In competition binding experiments, the solubilized fraction exhibited the same pharmacological profile as the membranes. Chemical crosslinking of [125I]vasopressin to the solubilized fraction followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated a 62-kDa band which was specifically labeled with [125I]vasopressin. Vasopressin binding sites from the solubilized fractions were resolved by gel filtration and ultracentrifugation on a sucrose gradient. In addition, agonist high affinity binding to V2 receptors and its sensitivity to guanine nucleotides were preserved even after solubilization in the absence of prebound agonist prior to solubilization. Addition of guanine nucleotides such as GTP gamma S decreased the specific binding of [3H]arginine vasopressin to these solubilized fractions in a dose-dependent manner, suggesting the solubilization of a V2 receptor-G protein complex. [32P]ADP ribosylation of the solubilized fraction by cholera and pertussis toxins revealed specifically labeled proteins with molecular weights of 42,000-43,000 and 39,000-41,000, respectively, on sodium dodecyl sulfate polyacrylamide gels. Furthermore [35S]GTP gamma S binding to these solubilized fractions was enhanced by vasopressin, confirming that a significant proportion of the vasopressin receptors must be closely coupled to G proteins even when these receptors are solubilized in the absence of agonist. These results are in contrast with those reported for beta, alpha 2 adrenergic and D2 dopaminergic receptor systems, but in agreement with D1 dopaminergic and A1 adenosine receptors. The molecular mechanism responsible for this difference remains to be determined.

Identification and characterization of vascular (V1) vasopressin receptors of an established smooth muscle cell line.

We report the identification and characterization of specific vasopressin-binding sites on intact cells and membranes of the established vascular smooth muscle cell line A-10, the fate of vasopressin associated with the cells, the role of guanine nucleotides in the regulation of the affinity of the vasopressin-binding sites, and the determination of the vasopressin receptor subtype. We have found specific vasopressin-binding sites on intact cells in monolayer (110,000 sites per cell during log growth and 60,000 sites per cell in stationary culture) with a KD of 6 nM at 37 degrees. After incubation of [3H]-8-arginine vasopressin ([3H]AVP) and cells for less than 20 min, cell-associated AVP was intact; with longer incubation times, AVP was progressively degraded. The major metabolites included phenylalanine and a fraction that eluted from a C18 reverse phase high performance liquid chromatography column between AVP and 8-arginine, 9-desglycinamide vasopressin. Extensive degradation also occurred when AVP was allowed to dissociate from the cells. With increased time of incubation, the amount of specifically bound AVP that could dissociate decreased, suggesting receptor-mediated endocytosis. In saturation equilibrium binding experiments with plasma membranes, two affinity states with KD of 0.7 nM and 379 nM were observed. The number of high affinity binding sites was similar to the number of receptors found on intact cells. Guanosine 5'-(beta,gamma-imido)triphosphate decreased vasopressin binding to the high affinity sites and did not significantly affect the low affinity sites. Competition binding experiments indicated that the vasopressin-binding sites of A-10 cells belong to the vascular V1 receptor subtype. We conclude that the established vascular smooth muscle cell line A-10 expressed vasopressin receptors of the vascular V1 subtype. Vasopressin bound to the receptors reversibly, but could also be degraded by the cells presumably after receptor-mediated endocytosis. The receptors might exist in different affinity states; guanosine 5'-(beta,gamma-imido)triphosphate decreased the affinity of the high affinity binding state.

Phorbol ester-mediated inhibition of vasopressin and beta-adrenergic responses in a vascular smooth muscle cell line.

We have reported previously that in the vascular smooth muscle cell line A-10 (ATCC CRL 1476), vasopressin stimulated phosphatidylinositol turnover Ca2+ efflux and inhibited isoproterenol-stimulated cAMP accumulation. Here we report that pretreatment of these cells with phorbol dibutyrate, an activator of protein kinase C, attenuated the responses to vasopressin and isoproterenol. This effect was concentration dependent and could be observed after pretreatment for 2 min. 4 alpha Phorbol 12,13-didecanoate, which does not activate protein kinase C, did not attenuate the responses. These data suggest that activation of protein kinase C by phorbol dibutyrate attenuates the responses of vascular smooth muscle cells to isoproterenol and vasopressin. Although phorbol ester did not affect [3H]-8-arginine vasopressin binding to intact cells, it appeared to uncouple vasopressin receptors from guanine nucleotide-binding protein.

Effects of N-ethylmaleimide on arginine vasopressin-induced responses in an established smooth muscle cell line.

We have previously reported that 8-arginine vasopressin (AVP) stimulates phosphatidylinositol turnover and Ca++ mobilization in rat aortic smooth muscle cells (A10). In the present study, N-ethylmaleimide (NEM) was used to further characterize the putative guanine nucleotide binding protein that transduces the V1 receptor effects on phosphatidylinositol turnover and Ca++ efflux in these cells. Pretreatment of the cells with low concentrations of NEM did not affect the basal levels of the inositol phosphates and Ca++ efflux but significantly inhibited the AVP-induced increases. NEM pretreatment did not significantly affect [3H]AVP binding to intact cells. Guanyl-5'-yl imidodiphosphate reduced the apparent binding affinity of AVP to cell membranes. NEM pretreatment abolished this guanyl-5'-yl imidodiphosphate effect. AVP stimulated a specific GTPase activity in cell membranes; this effect was also abolished by NEM pretreatment. The results suggest that in A10 cells a guanine nucleotide binding protein sensitive to NEM couples vasopressin receptors to phospholipase C.