Biomedical subjects

F L Stassen

Publications and source records attributed to F L Stassen.

At least 19 recordsLinked to original sources

PubMed1991

Vasopressin antidiuretic agonist and antagonist activity in dogs: structural and stereochemical relationship between bridge and carboxyl terminus.

A series of vasopressin analogs with various amino acid tail modifications were tested for antidiuretic agonist and antagonist (water diuretic) activity in the water-loaded indomethacin-treated and hydropenic dogs, respectively. Changing the carboxy terminus from Cys6-Pro7-Arg8-NH2 in SK&F 101926 to Cys6-Arg7-NH2 or to D-Cys6-Pro7-Arg8-NH2 or to D- or L-Cys6-Arg7-D-Arg8-NH2 reduced antidiuretic agonist and increased water diuretic activity. Replacement of the sulfur atoms in the cysteine residues with methylene groups further reduced the antidiuretic agonist activity of all carboxy terminus-modified compounds which possessed full agonist activity. It also increased the water diuretic activity of those disulfide analogs with both weak agonist and antagonist activity. These results indicate that alterations in the geometry of the hexapeptide ring and in the stereochemical relationship between the ring and the carboxy terminus of the molecule substantially modify the in vivo agonist and antagonist activity of vasopressin analogs.

Amino Acid Sequence↗

PubMed1989

Prolonged incubation with phorbol esters enhanced vasopressin-induced calcium mobilization and polyphosphatidylinositol hydrolysis of vascular smooth muscle cells.

Arginine vasopressin (AVP)-induced formation of inositol phosphates and increased calcium efflux in smooth muscle cells (A-10) were inhibited by short term treatment with phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C (Ca2+/phospholipid-dependent protein kinase) (Aiyar, N., Nambi, P., Whitman, M., Stassen, F. L., and Crooke, S. T. (1987) Mol. Pharmacol. 31, 180-184). Here we report that prolonged treatment of A-10 cells (48 h) with PDBu markedly enhanced AVP-induced calcium mobilization but inhibited ATP- and thrombin-induced calcium mobilization. PDBu (400 nM) doubled [Ca2+]i induced with 3 nM AVP, while the basal calcium concentrations before and after AVP were not different from those of untreated cells. The EC50 for a 24-h exposure was 2.3 nM PDBu. Phorbol 12-myristate 13-acetate was also effective, while 4-alpha-phorbol 12,13-didecanoate (48 h at 400 nM) was without effect. 4-alpha-phorbol 12,13-didecanoate also did not affect inositol phosphate formation. PDBu markedly enhanced inositol phosphate formation induced by AVP but not by NaF. PDBu did not affect basal inositol phosphate and polyphosphoinositide levels, and cytosolic and membrane-associated phospholipase C activity. PDBu treatment (48 h, 400 nM) decreased membrane-associated and cytosolic protein kinase C activity by 80 and 90%, respectively. However, the dose response and time course of changes in protein kinase C activity did not correlate with the same curves for PDBu enhancement of AVP-induced calcium mobilization. We conclude that prolonged PDBu treatment selectively enhanced AVP-induced calcium mobilization and polyphosphoinositide hydrolysis. These effects were not caused by an increase in vasopressin receptor number and apparent affinity, an increase in phospholipase C activity, G-protein-phospholipase C coupling, formation of polyphosphoinositide, or inhibition of inositol phosphate metabolizing enzymes. Enhancement of the AVP responses did not correlate with desensitization or activation of protein kinase C. We suggest that prolonged PDBu treatment might sensitize a putative V1 receptor-G-protein-phospholipase C complex.

Animals↗

PubMed1989

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.

Animals↗

PubMed1989

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.

Adenylyl Cyclase Inhibitors↗

PubMed1989

Cyclooxygenase inhibition unmasks the full antidiuretic agonist activity of the vasopressin antagonist, SK&F 101926, in dogs.

The vasopressin (AVP) antagonist, SK&F 101926 (beta-mercapto-beta beta-cyclopentamethylene propionic acid1, D-Tyr(Et)2, Phe3, Val4, Asn5, Cys6, Pro7, Arg8-NH2), is an antidiuretic antagonist in rats and squirrel monkeys in vivo. In rat, dog, pig, squirrel monkey and human in vitro studies, SK&F 101926 is an AVP antagonist with no discernable agonist activity. Not predicted by these studies was the discovery that SK&F 101926 is an antidiuretic agonist in humans. The purpose of these studies was to show that indomethacin, which potentiates the antidiuretic activity of AVP in hydrated dogs, also potentiates the antidiuretic agonist activity of SK&F 101926, and to determine what structural modifications of this AVP antagonist would confer diminished agonist activity. Treatment of dogs with i.v. indomethacin (2 mg/kg bolus + 3 mg/kg/hr infusion, a dose which inhibited 80-90% of the urinary prostaglandin E2 excretion) unmasked the full antidiuretic activity of SK&F 101926. SK&F 104146 (Arg7-D-Arg8-NH2 peptide tail modification of SK&F 101926) in the presence of indomethacin caused a partial antidiuretic response. Replacement of the 1 to 6 disulfide bridge of SK&F 104146 with methylene groups to form a "dicarba bridge" resulted in SK&F 105494, a compound lacking antidiuretic agonist activity. Using the indomethacin-treated dog model, we have unmasked the full antidiuretic agonist activity of SK&F 101926 in nonhumans. In addition, both SK&F 104146 and 105494 blocked the antidiuretic agonist activity of SK&F 101926, suggesting that the agonist effect seen in the presence of indomethacin treatment is receptor mediated.

Animals↗

PubMed1988

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.

Adenylyl Cyclase Inhibitors↗

PubMed1988

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.

Animals↗

PubMed1988

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.

Animals↗

PubMed1988

Oxytocin induces a transient increase in cytosolic free [Ca2+] in renal tubular epithelial cells: evidence for oxytocin receptors on LLC-PK1 cells.

We examined the effects of oxytocin on renal tubular epithelial LLC-PK1 cells. In cells loaded with Fura 2, we found that 1 microM oxytocin induced a rapid increase in cytosolic free [Ca2+]i from 120 nM to 250 nM within 12 sec. [Ca2+]i then decreased and leveled at 148 nM. Calcium was mobilized from intra- and extra-cellular sources. Oxytocin-induced calcium mobilization was dose dependent (EC50 between 5 and 30 nM). Oxytocin also stimulated calcium efflux which was blocked by the selective oxytocin antagonist KB-5-21. Calcium mobilization was a likely consequence of enhanced phosphatidylinositol turnover, because oxytocin rapidly increased the formation of inositol phosphates including Ins1,4,5P3. Calcium transients were induced by oxytocin and the oxytocin selective analog AM-2-40 and blocked by the oxytocin-selective antagonist KB-5-21. Lysine vasopressin, the selective V2 agonist dDAVP, and the V1-selective agonist SK&F 105349 were at least 10- to 100-fold less potent than oxytocin and exhibited only partial agonist activity. Using peptide analogs, a poor correlation was found between antagonism of oxytocin-induced calcium transients of LLC-PK1 cells and pig kidney V2 and rat liver V1 receptor affinity. These data indicate that oxytocin-induced calcium transients in LLC-PK1 cells were not mediated by V1 or V2 vasopressin receptors, but by oxytocin receptors. However, the poor correlation between antagonism at the LLC-PK1 receptors and the rat uterus oxytocin receptors suggests marked differences in antagonist recognition. We have also identified specific, saturable, high affinity oxytocin-binding sites of low density on intact LLC-PK1 cells (KD = 1.9 nM; Bmax = 3.2 fmol/10(6) cells). The relative analog affinities for these binding sites correlated well with their effects on oxytocin-induced calcium transients. We conclude that in LLC-PK1 cells, oxytocin stimulates a transient rise in cytosolic free [Ca2+]i and the formation of inositol phosphates, including Ins1,4,5P3. The effects on [Ca2+]i probably are not mediated by V1 and V2 vasopressin receptors, but by putative oxytocin receptors.

Animals↗

PubMed1988

SK&F 105494: a potent antidiuretic hormone antagonist devoid of partial agonist activity in dogs.

Previous studies from our laboratory have shown that in vivo cyclooxygenase blockade in dogs unmasks the antidiuretic agonist activity associated with the vasopressin antagonist, SK&F 101926, and have revealed two new vasopressin analogs, SK&F 104146 and 105494, with greatly reduced antidiuretic agonist activity. The purpose of the present study was to characterize SK&F 104146 and SK&F 105494 for water diuretic activity (aquaretic activity) in hydropenic dogs and for antagonism of vasopressin-stimulated antidiuresis in hydrated dogs. The vasopressin receptor affinity and inhibition of vasopressin-stimulated adenylate cyclase activity in renal membranes were also studied. When administered to hydropenic dogs, SK&F 101926 (3 or 30 micrograms/kg) did not cause a water diuresis. Substitution of the dipeptide tail of SK&F 101926 with Arg7D-Arg8NH2 (SK&F 104146; 30 micrograms/kg) was associated with a reduction of urine osmolality from 1876 +/- 182 to 349 +/- 94 mOsm/kg of H2O, and an increase in free water clearance (from -0.32 +/- 0.09 to 0.06 +/- 0.09 ml/min). Replacement of the 1 to 6 disulfide bridge of SK&F 104146 with a 1 to 6 dicarba bridge (SK&F 105494; 3 micrograms/kg) was associated with a further reduction of urine osmolality (1709 +/- 281 to 210 +/- 79 mOsm/kg of H2O) and a net positive free water clearance (from -0.56 +/- 0.02 to 0.6 +/- 0.35 ml/min). In water diuretic dogs, SK&F 104146 and 105494 shifted the vasopressin dose-response for antidiuresis to the right. SK&F 105494 appeared to be 3 times more potent than SK&F 104146.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

SK&F 105494 is a potent antidiuretic hormone antagonist in the rhesus monkey (Macaca mulatta).

The vasopressin analog desGly(CH2)5D-Tyr(Et)VAVP (SK&F 101926) is a potent antagonist of the antidiuretic action of vasopressin in rats, dogs, and squirrel monkeys, demonstrating minimal agonist activity in these species. In humans, however, SK&F 101926 was a potent full antidiuretic agonist. This agonist response was not predicted by in vitro studies with human tissue. The purpose of the present study was to determine if the agonist activity of SK&F 101926 could be modeled in an old-world primate, the rhesus monkey, and, if agonist activity was demonstrated, to determine how SK&F 101926 could be modified to reduce agonist activity and permit the elaboration of antagonist activity in that species. We observed that i.v. administration of SK&F 101926 to the hydrated rhesus monkey resulted in a full antidiuretic agonist response, as observed in humans, and did not reduce urine osmolality or increase urine flow when administered to hydropenic rhesus monkeys. Changing the terminal amino acids from Pro7, Arg8 to Arg7, D-Arg8 produced a compound (SK&F 104146) that was also an agonist, although less potent than SK&F 101926. Further substitution in SK&F 104146 of the sulfide groups of cysteine residues with methyl groups in the peptide ring to produce a "dicarba bridge" resulted in a compound (SK&F 105494) that was associated with minimal agonist activity in hydrated monkeys. Administration of SK&F 105494 (30 micrograms/kg i.v.) reduced urine osmolality from 629 +/- 20 to 91 +/- 7 mosmol/kg H2O (P less than .01) and increased free water clearance to positive levels (from -0.16 +/- 0.03 to 0.73 +/- 0.18 ml/min; P less than .05) in anesthetized hydropenic monkeys.

Adenylyl Cyclases↗

PubMed1987

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.

Animals↗

PubMed1987

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.

Animals↗

PubMed1987

A novel radiolabeled vasopressin antagonist: [3H-Phe]-desGlyd(CH2)5D-Tyr(Et)VAVP, [3H]-SK&F 101926.

We report the vasopressin receptor-binding properties of [3H-Phe]-desGlyd(CH2)5D-Tyr(Et)VAVP, [3H]-SK&F 101926, the first radiolabeled vasopressin receptor antagonist. We chose to radiolabel SK&F 101926 because this vasopressin analog is a potent antagonist of vascular V1 and renal V2 vasopressin receptors in all species studied. [3H]-SK&F 101926 bound with a single high affinity to intact vascular smooth muscle cells (A-10; KD = 0.5 nM), and plasma membranes A-10 cells (KD = 0.4 nM) and rat liver (KD = 0.2 nM). In competition experiments with [3H]-SK&F 101926 and [3H]arginine vasopressin ([3H]AVP) using cell and liver membranes, the affinity rank orders of vasopressin analogs were the same and were typical for the V1 receptor subtype. In competition binding experiments with [3H]-SK&F 101926 using cell and liver membranes, guanosine 5'-(beta,gamma-imido)triphosphate did not significantly alter the affinity of the V1 antagonist d(CH2)5Tyr(Me)AVP, but the affinity of AVP was decreased. These data indicate that the V1 receptor can exist in at least two affinity states that are modulated by guanine nucleotides. [3H]-SK&F 101926 also bound specifically and with high affinity to V2 receptors of MDCK cells. We conclude that [3H]-SK&F 101926 binds with high affinity to V1 and V2 vasopressin receptors and is a powerful new tool for the identification of vasopressin receptors and the study of molecular mechanisms involved in the interaction of vasopressin with its receptors.

Animals↗

PubMed1987

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.

Animals↗

PubMed1987

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.

Animals↗

PubMed1987

Vasopressin antagonism in the squirrel monkey (Saimiri sciureus).

Vasopressin antagonism and water diuresis (aquaresis) is demonstrated after i.p. or i.v. administration of vasopressin antagonists in a primate species, the squirrel monkey (Saimiri sciureus). Antagonism of vasopressin-stimulated renal medullary adenylate cyclase activity was evaluated in vitro; the most potent antagonists were those with D-tyrosine (alkyl) substitutions at position 2. Aquaresis was evaluated in vivo; the most potent aquaretic agents were again those with D-tyrosine (alkyl) substitutions at position 2. Correlation of in vitro vasopressin antagonist and in vivo aquaretic potencies for a series of antagonists was r = 0.7880 (P less than .05). Renal excretion of electrolytes, creatinine and urea tended to increase slightly as a function of vasopressin antagonist dose; the rates of solute excretion approached but seldom exceeded those rates associated with water diuresis in squirrel monkeys. The vasopressin antagonists antagonized the antidiuretic activity of exogenous vasopressin in vivo. Onset of the aquaretic response to i.v. administration of desGlyd(CH2)5D-Tyr(Et)VAVP was within 30 min; duration was greater than 120 min. These studies establish vasopressin antagonism and aquaresis associated with administration of vasopressin antagonists in a primate species.

Adenylyl Cyclases↗

PubMed1986

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.

Adenylyl Cyclases↗