Inge Michel

Potent magnesium-dependent inhibition of adenylate cyclase activity from guinea pig lung by adenosine and other 9-substituted adenines

Abstract The inhibition of adenylate cyclase activity (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) from guinea pig lung by adenosine and a number of 9-substituted adenines was Mg 2+ -dependent, the compounds being up to 13 times more potent at saturating or near saturating Mg 2+ concentrations (11.8 mM) than at limiting (1.8 mM) concentrations. Inhibition by adenine and 6-mercaptopurine did not show a Mg 2+ dependence. The most potent inhibitors of cyclase activity at 11.8 mM Mg 2+ of the 9-substituted adenines tested were: 9-(tetrahydro-5-methyl-2-furyl)adenine ( I 50 = 8 μ M), 9-(tetrahydro-2-furyl)adenine ( I 50 = 10 μ M), 9-cyclopentyladine I 50 = 20 μ M), and 9-furfuryadenine I 50 = 26 μ M). The inhibition of lung cyclase activity by 9-(tetrahydro-2-furyl)adenine was deduced to be hyperbolic non-competitive at 1.8 mM ( K 1 = 1.2·10 −4 M) and 11.8 mM ( K 1 = 2.5·10 −6 M) Mg + from analysis of double-reciprocal plots; these plots showed a concave downward non-linearity in the presence of inhibitor at both Mg 2+ concentrations. This non-linearity was eliminated under conditions where Mg 2+ levels were never in excess of those of ATP. Hill plots of the inhibition by 9-(tetrahydro-2-furyl)adenine (and by adenosine) suggested that negative co-operativity is involved in binding to the enzyme. The possibility that the Mg 2+ -dependence of inhibitory potency of adenosine and its analogs functions as a cellular control mechanism is discussed.

Adenylate cyclase from guinea pig lung: Further characterization and inhibitory effects of substrate analogs and cyclic nucleotides

Abstract A potent ( K i = 0.01 mM ), competitive inhibition of adenylate cyclase activity in particulate fractions of guinea pig lung by 2′ O -palmitoyl cyclic AMP has been observed, in striking contrast to the inactivity of cyclic AMP and N 6 ,2′ O -dibutyryl cyclic AMP at concentrations of up to 1 m m or more. The possibility that 2′ O -palmitoyl cyclic AMP or similar compounds might function as endogenous regulators of the hormonal stimulation of adenylate cyclase activity is discussed. Several 6- and 8- substituted purine 3′,5′-cyclic ribotides also inhibit, probably by direct interaction with enzymatic sulfhydryl groups. A study of the inhibition by purine bases, nucleosides, and 5′ nucleotides suggests that most of the substrate (ATP) binding determinants reside in the nucleoside. The particulate enzyme fractions were found to have lower ATPase activity relative to cyclase activity than cyclase preparations from either guinea pig heart or bronchial smooth muscle. Lung cyclase fractions were maximally stimulated by 5–15 m m Mg 2+ in the presence of 1.2 m m ATP as substrate. The percentage of stimulation of cyclase activity by 0.01 m m isoproterenol is dependent on the Mg 2+ concentration. Cyclase activity was significantly stimulated not only by the catecholamines (isoproterenol, epinephrine, and norepinephrine) and fluoride ion, but also by prostaglandins E 1 , E 2 , and F 2α , histamine, and glucagon.

Effects of barbiturate derivatives, sulfhydryl reagents, and oxidizing agents on the activity of adenylate cyclase from guinea pig heart and lung

Abstract A series of barbiturates (including known sedatives and hypnotics) has been examined as possible inhibitors of basal adenylate cyclase activity in particulate fractions from guinea pig heart and lung. Two brominated barbiturates, 5,5-dibromobarbiturate (DBB) and 5-bromo-5-phenylbarbiturate (BPB) were potent inhibitors of adenylate cyclase activity. The enzymes from heart and lung were sensitive to inhibition by sulfhydryl group reagents, such as p -chloromercuriphenyl sulfonate, and by oxidizing agents, such as N -bromosuccinimide and N -bromoacetamide. The 1,3-diphenyl derivative of DBB did not inhibit the adenylate cyclases, although, in common with DBB and BPB, it oxidized iodide to iodine. These and other data suggest that inhibition by DBB and BPB is oxidative in nature, irreversible, and the result of a direct interaction between inhibitor and an essential sulfhydryl group of the enzyme.

9α-homo-9,11-epoxy-5,13-prostadienoic acid analogues: Specific stable agonist (SQ 26,538) and antagonist (SQ 26,536) of the human platelet thromboxane receptor

A newly synthesized 9 alpha-homo-9,11-epoxy-5,13-prostadienoic acid analogue, SQ 26, 536, (8(R)9(S)11(R)12(S)-9 alpha-homo-9,11-epoxy-5(Z), 13(E)-15S-hydroxyprostadienoic acid) inhibited arachidonic acid (AA)-induced platelet aggregation with an I50 value of 1.7 microM. SQ 26,536 did not inhibit prostaglandin (PG) synthetase activity of bovine seminal vesicle microsomes or thromboxane (Tx) synthetase activity of lysed human blood platelets. SQ 26,536 also inhibited platelet aggregation induced by epinephrine (secondary phase), 9,11-azoPGH2 and collagen but did not inhibit the primary phase of epinephrine-induced aggregation or ADP-induced platelet aggregation. SQ 26,538 (8(R)9(S)11(R)12(S)-9 alpha-homo-9,11-epoxy-5(Z),13(E)-15R-hydroxyprostadienoic acid), a 15-epimer of SQ 26,536, induced platelet aggregation with an A50 value of 2.5 microM. SQ 26,536 competitively inhibited SQ 26,538-induced platelet aggregation with a Ki value of 3 microM. Neither indomethacin, a PG synthetase inhibitor, nor SQ 80,338 (1-(3-phenyl-2-propenyl)-1H-imidazole), a Tx synthetase inhibitor, inhibited SQ 26,538- or 9,11-azoPGH2-induced platelet aggregation. These data indicate that SQ 26,536 and SQ 26,538 are stable antagonist and agonist, respectively, of the human blood platelet thromboxane receptor.

Divergent effects of theophylline on adenylate cyclase preparations from guinea-pig heart and lung

Theophyllin verringert die Aktivität der Adenyl-Cyclase (AC) in der plasmamembranreichen Fraktion der Meerschweinchenlunge in Konzentrationen von 6–10 mM; es hat dagegen keinen Einfluss auf die Aktivität der AC in Meerschweinchenherzen. Diese Resultate weisen darauf hin, dass in verschiedenen Geweben geringe Unterschiede in der Enzym-Katalyse bestehen können.

Effects of SQ 27,427, a thromboxane A2 receptor antagonist, in the human platelet and isolated smooth muscle

The TxA2 receptor antagonist properties of SQ 27,427 [a cyclohexylcarbinol-7-oxabicyclo(2.2.1)heptenoic acid analog] were studied in vitro both in the human platelet and various isolated smooth muscle preparations. SQ 27,427 was found to be a potent inhibitor of human platelet aggregation induced by arachidonic acid, ADP, epinephrine, collagen and the stable TxA2 agonists 9,11-azoPGH2 and SQ 26,655. Inhibition of platelet aggregation was achieved at concentrations of SQ 27,427 which did not alter TxB2 levels. SQ 27,427 was found to weakly inhibit the formation of TxB2 from arachidonic acid and had no effect on the synthesis of PGE2 or PGI2 from arachidonic acid. SQ 27,427 was also found to be a weak stimulator of platelet adenylate cyclase, being 1000 times less potent than PGI2. In isolated smooth muscle experiments, SQ 27,427 was shown to be a potent and specific TxA2 receptor antagonist. It caused competitive antagonism of 9,11-azoPGH2-induced contractions of vascular, respiratory and gastrointestinal smooth muscles. This antagonism was specific, as responses to norepinephrine, serotonin, PGE2, PGI2, PGF2 alpha, histamine, carbachol and KCl were not altered by SQ 27,427.

Some biochemical activities of 10,10-Difluoro-13-dehydroprostacyclin, a chemically stable analog of prostacyclin, in human blood platelets

Abstract 10,10-Difluoro-13-dehydroprostacyclin (DF 2 -PGI 2 ) is a chemically stable analog of prostacyclin (PGI 2 ). DF 2 -PGI 2 was 6 times more potent than prostaglandin E 1 (PGE 1 ), and 1/10 as potent as PGI 2 as an inhibitor of arachidonic acid (AA)-induced platelet aggregation. DF 2 -PGI 2 also inhibited platelet aggregation induced by collagen, ADP, epinephrine and two thromboxane agonists, 9,11-azoPGH 2 and SQ 24,810 (9,11-epoxy-9α-homo-5(Z), 13(E)-15β-hydroxyprostadienoic acid, racemic mixture). The antiaggregatory effects by DF 2 -PGI 2 and PGI 2 on AA-induced platelet aggregation were potentiated by etazolate (SQ 20,009), a potent inhibitor of cyclic AMP phosphodiesterase (PDE), and were blocked by SQ 22,536 (9-(tetrahydro-2-furyl) adenine), an inhibitor of adenylate cyclase (AC). DF 2 -PGI 2 was also more potent than PGE 1 , and less potent than PGI 2 as a stimulator of AC activity in platelet homogenates. DF 2 -PGI 2 - and PGI 2 -stimulated AC activity was inhibited by a series of AC inhibitors, SQ 22,536, SQ 4,647 (9-furfuryl adenine) and 2′,5′-dideoxyadenosine. Furthermore, SQ 22,536 inhibited DF 2 -PGI 2 - and PGI 2 -stimulated AC activity in a concentration-related manner, with 150 values of 150 and 300 μM, respectively. Finally, PGE 1 , DF 2 -PGI 2 and PGI 2 , at 0.35 μM induced 2-, 10- and 17-fold increases, respectively, in cyclic AMP levels of intact platelets in platelet rich plasma (PRP). The increase in cyclic AMP levels by PGI 2 and DF 2 -PGI 2 in PRP was also blocked by SQ 22,536. We conclude that DF 2 -PGI 2 , like PGI 2 and PGE 1 , inhibits platelet aggregation by elevating platelet cyclic AMP levels.

Interactions of α-methylfluorene-2-acetic acid with adenylate cyclase☆

Abstract α-Methylfluorene-2-acetic acid (MFA), a new anti-inflammatory agent, enhanced the stimulation of adenylate cyclase activity from guinea pig heart by isoproterenol, epinephrine and norepinephrine, but did not cause increases in either basal or histamine-stimulated activity. Propranolol blocked the effects of isoproterenol plus MFA on heart cyclase activity. MFA did not enhance PGE 1 - or PGE 2 -stimulated cyclase activity from guinea pig lung; effects of MFA on isoproterenol-stimulated activity were obscured by a marked inhibition of basal activity. d -MFA and l -MFA were equipotent enhancers of isoproterenol-responsive heart cyclase activity. Increases in isoproterenol-stimulated cyclase activity from heart in the presence of MFA appeared reversible; decreases in basal activity were irreversible. The action of MFA was not due to inhibition of cyclic nueleotide phosphodiesterase activity, nor did MFA appear either to affect catecholamine degradation or to antagonize an endogenous cytoplasmic inhibitor of catecholamine action. It is possible that MFA perturbed the membrane milieu of the cyclase complex so as to have enhanced catecholamine binding to hormone receptors or coupling of the binding event to cyclic AMP synthesis.

Some Biochemical Characteristics of Rodent and Human Mammary Carcinomas

SummaryIn order to establish valid experimental systems for the study of breast cancer, an examination of the biochemical and morphologic characteristics of transplantable and carcinogen-induced mamma

Cyclic nucleotides vs. adenosine analogs as inhibitors of adenylate cyclase activity: Nonidentity of sites of action

Substitution einerN 6-Aminogruppe eines zyklischen Nukleotids führt zu einer erhöhten Hemmwirkung des Nukleotids gegenüber Adenyl-Cyclase von Meerschweinchenlungen, während dieN 6-Amino-Substition von Adenosin-Analogen eine herabgesetzte Inhibitionswirksamkeit gegenüber demselben Enzym zur Folge hat. Die experimentellen Daten führen zu dem Schluss, dass der Inhibitionsmechanismus gegenüber Cyclase für beide Verbindungstypen verschieden ist.