Biomedical subjects

D Stübner

Publications and source records attributed to D Stübner.

9 recordsLinked to original sources

PubMed1990

Inhibition of adenylate cyclase by polyadenylate.

The effects of ribo- and deoxyribonucleic acids on the activity of detergent-dispersed adenylate cyclases from rat and bovine brain were examined. Mn2+ (10 mM)-activated adenylate cyclase was inhibited by micromolar concentrations of poly(A) (IC50 congruent to 0.45 microM). This inhibition was directly due to poly(A) and was not mediated by: (a) protein contamination of the poly(A) preparation, (b) metal chelation, (c) formation of an acid-soluble inhibitor of adenylate cyclase, (d) effects on the specific activity of [alpha-32P]ATP, (e) competition with MnATP for binding to adenylate cyclase, or (f) diversion of substrate to an alternate polymerase reaction. Inhibition of adenylate cyclase by poly(A) was on the enzyme's catalytic unit, as purified preparations of the enzyme from bovine brain were inhibited by poly(A). This inhibition by poly(A) was not likely mediated via the enzyme's "P"-site, through which activated forms of the enzyme are selectively inhibited by specific adenosine phosphates. In contrast with inhibition by the "P"-site agonist 3' AMP, inhibition of adenylate cyclase by poly(A) was slow in onset and was not reversible by dilution and showed a different metal-dependence. Inhibition of adenylate cyclase was relatively specific for poly(A) as poly(U) caused less than 50% inhibition and deoxyribonucleic acids had no effect. The potency and specificity of the inhibition of adenylate cyclase by poly(A) imply a biochemically interesting interaction that is possibly also of physiological significance.

Adenylyl Cyclase Inhibitors↗

PubMed1990

Growth regulation of porcine thyroid follicles in-vitro by growth factors.

Primarily intact thyroid follicles were isolated from explanted thyroid glands of hogs immediately after sacrifice. These intact thyroid follicles had a preserved polarity, still contained thyroglobulin, could be liberated from contaminating mesenchymal single cells and could be kept in culture for at least 14 days. They respond to bovine TSH (1 mU/ml) with a 10-fold increase in cAMP production within 15 min, trapp and organify iodine and secrete thyroid hormones. In contrast to thyroid monolayers, prepared from these thyroid follicles, intact thyroid follicles did not proliferate in response to bTSH (1-1000 uU/ml) stimulation. When thyroid follicles, however, were stimulated with EGF (1-5 ng/ml) a dose dependent increase in cell number as well as [3H]-thymidine incorporation into acid precipitable DNA was seen. The growth response of thyroid follicles to EGF (5 ng/ml) in comparison to thyroid monolayer cells was 275% versus 145% increase in cell number within 6 days. With IGF I, a proliferative response of thyroid follicles was found, and this was synergistic with simultaneous incubation with EGF. When thyroid follicles were incubated with EGF together with TSH, a dose dependent inhibition of [3H]-thymidine incorporation as well as cell number with increasing amounts of TSH (1-1000 mU/ml) occurred. The comparable effect was found, when thyroid follicles were stimulated to growth with IGF I (100 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Forskolin decreases sensitivity of brain adenylate cyclase to inhibition by 2',5'-dideoxyadenosine.

The effects of forskolin on the sensitivity of adenylate cyclase to 'P'-site-mediated inhibition were studied. Stimulation of crude and purified preparations of adenylate cyclase by forskolin led to decreased sensitivity to inhibition by 2',5' dideoxyadenosine with enzyme from rat and bovine brain. This is in contrast with the enhancement of P-site sensitivity induced by calmodulin, divalent cations, and stable GTP analogs and is in contrast with behavior seen with enzyme from liver and S49 cyc membranes. The effect of forskolin on P-site sensitivity of the brain adenylate cyclase was not dependent on the presence of G-proteins or calmodulin. It was not the consequence of proteolysis nor was it due to an obvious artifact in the assay procedures. This distinct behavior of the brain enzyme is most likely due to a structural difference in the catalytic subunit.

Adenylyl Cyclase Inhibitors↗

PubMed1989

Ammonium ions enhance proteolytic activation of adenylate cyclase and decrease its sensitivity to inhibition by "P"-site agonists.

A detergent-dispersed adenylate cyclase from rat brain was used to study the effects of ammonium salts and polyamines on the proteolytic activation of the enzyme by a sperm protease and on the sensitivity of adenylate cyclase to inhibition via its "P"-site. A purified preparation of a trypsin-like, serine protease from bovine sperm was used to activate solubilized adenylate cyclase in the presence of guanosine 5'-O-(3-thiotriphosphate (GTP gamma S). The proteolytically activated form of adenylate cyclase was found to be particularly sensitive to further activation by ammonium bicarbonate. The activation by NH4HCO3 was found to be due to the NH+4 cation and was characterized by an increased Vmax and by a decreased sensitivity of adenylate cyclase to inactivation by elevated concentrations of the sperm protease or by trypsin. NH4Cl and (NH4)2SO4 also caused biphasic effects on adenylate cyclase, which mimicked but were less effective than those caused by NH4HCO3. Consistent with observations of others, adenylate cyclase activity was enhanced by ammonium ions whether in the presence of reversible (Mn2+) or irreversible (GTP gamma S) activators. Mn2+- and GTP gamma S-stimulated activities were similarly optimally enhanced by 30 mM (NH4)2SO4 and by 30 to 150 mM NH4Cl or NH4HCO3. Ammonium ions did not increase the activity of the purified catalytic unit. Moreover, the effect of ammonium ions was not accompanied by an increased rate of activation by GTP gamma S, suggesting that the activation of Gs (guanine nucleotide-dependent stimulatory component) may not be the primary cause of stimulation by ammonium salts. Several polyamines at millimolar concentrations blocked the stimulatory effect of NH+4. This was observed when adenylate cyclase was activated by Mn2+, but not when it was activated by GTP gamma S or by the sperm protease + GTP gamma S. The inhibitory effect of polyamines was not due to the formation of a complex with ATP. Both the increase in Vmax of the Mn2+-stimulated enzyme by NH+4 and the decrease in Vmax caused by spermine were accompanied by an increase in the enzyme's Km MnATP app. Spermine increased the IC50 for inhibition of Mn2+-activated adenylate cyclase by 2',5'-dideoxyadenosine (2',5'-ddAdo) from 0.75 to 4.6 microM, consistent with the idea that increased sensitivity of P-site-mediated inhibition is associated with increased enzyme activity. In contrast, activation of Mn2+-stimulated adenylate cyclase by 30 mM (NH4)2SO4 also reduced sensitivity to inhibition by 2',5'-ddAdo(IC50 1.1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Acrosin↗

PubMed1989

Cation and structural requirements for P site-mediated inhibition of adenylate cyclase.

The cation and structural requirements of the intracellular inhibitory "P" site of adenylate cyclase were investigated in human platelet membranes, bovine sperm particles, and detergent-solubilized and purified preparations from rat and bovine brain. Sensitivity of adenylate cyclase to P site-mediated inhibition was enhanced by reversible and irreversible activators of the enzyme. The most effective sensitization of the platelet and brain adenylate cyclases was observed with Mn2+ and upon proteolysis with inhibin in the presence of guanosine 5'-O-(3-thiotriphosphate). These resulted in IC50 values for (2',5'dideoxy-adenosine (2',5'-dd-Ado) and 2'-deoxy-3'-AMP of approximately 1-2 microM. The data were consistent with the ideas that P site-mediated inhibition of adenylate cyclase is dependent on divalent cation and is a function of enzyme activity. A number of nucleosides and nucleotides were synthesized and used to define structural requirements for P site-mediated inhibition of a detergent-solubilized adenylate cyclase from rat brain. The data suggest a strict requirement for an intact adenine moiety and a beta-glycosidic linkage for the ribosyl moiety. 2'-Deoxy-and especially 2',5'-dideoxy-ribosyl moieties enhanced sensitivity and a strong preference for phosphate at the 3'-position was exhibited. Substitutions at the 5'-ribose position impaired sensitivity. The order of potency and IC50 values of the more potent adenosine analogs were 2',5'-dideoxy-3'-AMP (congruent to 0.1 microM) greater than 2'-deoxy-3'-AMP (congruent to 1 microM) greater than 2',5'-dd-Ado (congruent to 3 microM) greater than 3'-AMP (congruent to 9 microM) greater than 2'-deoxy-adenosine (congruent to 15 microM) greater than adenosine (congruent to 80 microM). Large substitutions at the 3'-ribose position were tolerated, e.g., dApdN di- and dAp(dN)4 penta-nucleotides and succinyl- and p-fluoro-sulfonyl-benzoyl- moieties. The purified adenylate cyclase from bovine brain was inhibited by P site agonists with IC50 values of 34 and 45 microM for 2'-deoxy-3'-AMP and 2',5'-dd-Ado, respectively. The data imply, first, that the locus of the P site is the catalytic subunit of adenylate cyclase and, second, that the increased sensitivity observed with Mn2+ is due to an effect of the cation on the catalytic subunit. In contrast with adenylate cyclases from other mammalian tissues, the enzyme from bovine sperm exhibited only weak sensitivity to P site agonists; 2'-deoxy-3'-AMP congruent to 2',5'-dd-Ado greater than adenosine, each with IC50 values greater than 1000 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine↗

PubMed1987

Hypertrophy and hyperplasia during goitre growth and involution in rats--separate bioeffects of TSH and iodine.

Goitre growth was investigated in rats receiving a low iodine diet (less than 0.1 microgram iodine/g) and either 1 g/l KClO4 or 1 g/l propylthiouracil (PTU), or a combination of KClO4 or PTU with 50.82 nmol/1 T3 in tap water for 3 weeks. To investigate goitre involution, rats with iodine-deficient goitres were treated for 3 weeks either with T3 (0.5 microgram T3/day = 0.768 nmol/day), iodide (0.5 or 2.7 micrograms KI/day) or a combination of T3 with both iodide doses. Histology together with total DNA distinguished between hypertrophy and hyperplasia of the gland. During goitre growth there was highly significant correlation between goitre weight and TSH serum level (r = 0.93, P less than 0.001). Thyroid total DNA, however, was only weakly correlated to TSH but was inversely related to the degree of iodine deficiency. During goitre regression, TSH levels were normalized, histological signs of hypertrophy had disappeared, and thyroid weight was nearly normalized in all therapy groups. Total DNA, however, was normalized only with 2.7 micrograms KI/day (95 +/- 18 micrograms DNA/gland), and still elevated in all other groups. The highest DNA levels were found under T3 therapy (143 +/- 21 micrograms DNA/gland) and under 0.5 microgram KI/day (161 +/- 19 micrograms DNA/gland). Reduction of total DNA was independent of TSH, but followed replenishment of the iodine content of the glands. We conclude that TSH mainly induces hypertrophy, whereas thyroid hyperplasia is mainly regulated by the intracellular iodine content.

Animals↗

PubMed1987

Inhibition of cAMP formation by EGF in thyroid follicles is mediated by intracellular Ca++.

The mechanism of cAMP-inhibition by EGF was studied in isolated porcine thyroid follicles. EGF inhibited TSH-induced cAMP-formation maximally by 40%, this effect remained up to 1 h of pre-incubation. The calcium-ionophore A 23 187 also inhibited cAMP-formation, but its effect was relieved after 1 h. The phorbolester TPA had a biphasic influence on cAMP-formation, with a transient increase (5 min) before a sustained inhibition (60 min); the inhibitory effect was mimicked by the diacylglycerol 1-oleoyl-2-acetyl-glycerol. Exogenous arachidonic acid had only a small and transient inhibitory effect on cAMP-formation. We conclude, that EGF inhibits cAMP-formation by a raise of intracellular Ca++, as well as by the direct activation of proteinkinase C, indicating, that a phosphorylated product could be a mediator for the inhibition of adenylate cyclase.

Animals↗

PubMed1987

Paracrine interaction between thyrocytes and fibroblasts.

Cell free supernatants (conditioned medium) of isolated porcine thyroid follicles, stimulated with EGF (5 ng/ml) or TSH (1-1000 microU/ml), were tested for a mitogenic activity for fibroblasts. Whereas TSH-conditioned medium dose-dependently stimulated [3H]thymidine incorporation into DNA of fibroblasts, only a weak stimulation was found with EGF. However, when the changes in cell number were determined, a significant increase was only found with EGF-conditioned medium from thyroid follicles. The cause of this discrepancy is a dose-dependent stimulation of [3H]thymidine incorporation into fibroblasts by cAMP and thyroid hormones. Cyclic AMP, however, does not stimulate growth of fibroblasts. IGF I production is stimulated in fibroblasts by basal as well as EGF stimulated conditioned medium of thyroid follicles. In contrast, TSH-conditioned medium inhibited IGF I production in fibroblasts. Conditioned medium itself is free of detectable IGF I. As IGF I stimulates not only growth of fibroblasts, but also of thyrocytes, we conclude, that conditioned medium from thyrocytes stimulates IGF I production in fibroblasts, which itself stimulates fibroblast and thyrocyte growth.

Animals↗

PubMed1985

Preparation of porcine thyroid follicles with preserved polarity: functional and morphological properties in comparison to inside-out follicles.

One of the main problems in establishing isolated thyroid follicles in vitro is their tendency to form inside-out follicles. The reason for this change in polarity is unknown. We describe here a method for the preparation of stable thyroid follicles with preserved polarity for at least 6 days. Isolated follicles were obtained by infusion of collagenase (1.5 mg/ml) dissolved in minimal essential medium into the artery of intact thyroid glands. The morphological and functional properties of these follicles were compared to inside-out follicles. These inside-out follicles were obtained by digestion of minced thyroid tissue in a collagenase (1 mg/ml) solution. The polarity of follicles was proved by morphological criteria. Follicles with preserved polarity did not change polarity for at least 6 days in the presence of 1% or 5% fetal calf serum. As the culture conditions for both preparation methods were identical, we conclude that the preparation method rather than the culture condition is responsible for the preservation of cell polarity of isolated thyroid follicles in our system. Increases in cyclic AMP levels induced either by bovine thyrotropin (10(-3) U/ml) or by isoproterenol (10(-6) M) as well as iodide uptake and organification were rapid and significant in right-side-right follicles but not in inside-out follicles. Therefore the TSH receptor and the beta-adrenergic receptor appear to be exclusively located at the basal membrane of follicular cells. In addition, iodide uptake apparently is unidirectional.

Animals↗