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F L Stassen

Publications and source records attributed to F L Stassen.

35 records · Page 2Linked to original sources
PubMed1986

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+.

Angiotensin II↗
PubMed1986

Vascular vasopressin receptors mediate inhibition of beta adrenergic receptor-induced cyclic AMP accumulation.

Beta adrenergic receptor agonists and forskolin stimulated cyclic AMP (cAMP) accumulation in cultured rat aortic smooth muscle cells (A-10). Furthermore, these cells display a high density of vasopressin receptors of the vascular (V1) subtype. Addition of vasopressin to these cells inhibited beta adrenergic agonist- and forskolin-stimulated cAMP accumulation by 30 to 40% and by 25 to 35%, respectively. The extent of inhibition was dependent on the concentration of vasopressin used. Half-maximal inhibition of cAMP accumulation by isoproterenol occurred at 8 X 10(-10) M vasopressin. Basal cAMP levels were not affected. The inhibition by arginine vasopressin was mediated by V1 receptors because the V2 renal receptor subtype selective agonists (1-deamino, 8-D-arginine)vasopressin and (1-deamino,4-valine,8-D-arginine)vasopressin were ineffective. Of the antagonists tested, the V1-selective antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]vasopressin was more potent than the mixed V1/V2 antagonist [1-beta-mercapto--beta, beta-cyclopentamethylenepropionic acid), 2-D-(O-ethyl)tyrosine,4-valine 8-arginine]vasopressin. The V2-selective antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-D-isoleucine,4-valine,8-arginine]vasopressin displayed minimal ability to block the vasopressin-mediated inhibitory effect. These data demonstrate that in rat aortic smooth muscle cells V1 receptors are negatively coupled to adenylate cyclase. The studies presented suggest that the vasoconstrictor activity of vasopressin might involve inhibition of beta adrenergic receptor-mediated vascular relaxation through inhibition of cAMP accumulation.

Animals↗
PubMed1985

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.

Adenylyl Cyclases↗
PubMed1985

Antidiuretic hormone antagonists and aquaresis in dogs: different vasopressin sensitivity and antagonist potency in renal cortex and papilla.

To understand the molecular mechanism of action of the novel class of diuretic agents, the antidiuretic hormone antagonists ["aquaretics" (specific water-losing activity as caused by vasopressin antagonists, as distinguished from the saluresis of conventional diuretics)], in the dog studies were made of the properties of the vasopressin-responsive adenylate cyclase system and the antagonist potencies of the vasopressin analogs [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-(O-ethyl)tyrosine,4-valine,8-arginine]vasopressin; [1-(beta-mercapto-beta-mercapto-beta, beta-cyclopentamethylenepropionic acid), 2-D-phenylalanine,4-valine,8-arginine]vasopressin; and [1-(beta-mercapto-beta, beta-cyclopenta-methylenepropionic acid), 2-D-(O-ethyl)tyrosine,4-valine,8-arginine]vasopressin (SK&F 100398, 101071 and 101498, respectively) using plasma membranes prepared from cortex, medulla and papilla of dog kidney. It was observed that the greatest sensitivity for vasopressin was in the papilla (concentration of 8-arginine vasopressin required for 50% activation of adenylate cyclase [Kact] was 2.0 X 10(-9)M, 1.1 X 10(-9)M and 5.1 X 10(-10) M in the cortex, medulla and papilla, respectively). The addition of 10(-5)M GTP did not alter the Kact of the cortex but enhanced 10-fold the vasopressin sensitivity of the papilla to 5.2 X 10(-11) M. The vasopressin analogs were competitive antagonists of vasopressin-stimulated adenylate cyclase of cortex and papilla with the greatest potency for the papillary enzyme (Ki in papilla was 3.6 X 10(-9)M, 4.6 X 10(-9)M and 1.0 X 10(-9)M for SK&F 100398, 101071 and 101498, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗
PubMed1982

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.

Animals↗
PubMed1982

Molecular mechanisms of novel antidiuretic antagonists: analysis of the effects on vasopressin binding and adenylate cyclase activation in animal and human kidney.

These studies describe the molecular mechanisms of a potential new class of diuretic agents, vasopressin antagonists. The inhibition of the antidiuretic response to antidiuretic hormone (ADH) by the novel vasopressin analogs d(CH2)5Tyr(Me)VAVP, d(CH2)5Tyr(Et)VAVP, d(CH2)5Tyr(Et)VDAVP and d(CH2)5D-TyrVAVP was studied using medullary membranes of pig kidney. These analogs were competitive inhibitors of vasopressin binding and adenylate cyclase activation by vasopressin with potencies that were 5- to 7-fold higher than those of d(CH2)5VDAVP (Kbind was 6.7 x 10(-7) M; Ki was 2.3 x 10(-7) M), an analog with no in vivo anti-ADH activity. The antagonists were judged selective for vasopressin receptors because the activation of renal adenylate cyclase by beta adrenergic agonists and prostaglandins E1, E2 and I2 was not affected by d(CH2)5D-TyrVAVP. Furthermore, blockade of the vasopressin receptors with this analog did not impair the other components of the adenylate cyclase system since basal enzyme activity and activity stimulated by guanyl-5'-yl imidodiphosphate and NaF were not diminished. In addition, d(CH2)5D-TyrVAVP was a potent inhibitor of vasopressin activation of adenylate cyclase in pig, rat and dog kidney, and also in human kidney (Ki was 1.9 x 10(-8) M). The possibility that these or similar agents could be useful therapeutic agents in man as novel diuretics must now be considered.

Adenylyl Cyclases↗
PubMed1979

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.

Animals↗
PubMed1976

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.

Amino Acid Oxidoreductases↗
PubMed1973

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.

Animals↗
PubMed1973

Cell-free synthesis of procollagen: L-929 fibroblasts as a cellular model for dermatosparaxis.

A cell-free system that actively synthesizes collagen was prepared from L-929 fibroblasts. Chromatographic and electrophoretic techniques were used to demonstrate that the only collagenous products are pro alpha(1) and pro alpha(2) chains. The collagen synthesized by the cell-free system was also compared to the collagen extracted from the cells. The cellular collagen was composed of aggregates of pro-alpha chains, while no alpha chains were found. Procollagen peptidase activity could not be detected in the cells, and the activity present in the medium was low, comparable to that in dermatosparaxic cell cultures. These properties indicate that L-929 cells may be a model system for dermatosparaxis.

Animals↗