Fluoride-stimulated Activity Research Articles

Tungstate stimulates adenylate cyclase.

Tungstate stimulates adenylate cyclase (E.C. 4.6.1.1) in ovarian homogenates of the rat; maximal stimulation (approximately 3-fold) is achieved at a concentration of 1 mM. This stimulation of cAMP production cannot be explained by the inhibition of phosphodiesterase activity or of ATP hydrolysis. Activation of adenylate cyclase by tungstate is rapid and reversible. The effects of tungstate and hCG on cyclase activity are additive, but tungstate does not augment fluoride-stimulated activity. At higher concentrations (5 and 10 mM), tungstate inhibits basal as well as hCG- and fluoride-stimulated cyclase activity in an irreversible manner. After solubilization by Lubrol-PX, the cyclase enzyme is inhibited by tungstate at concentrations ranging from 0.1-10 mM. Tungstate also activates adenylate cyclase in the brain, heart, lungs, kidneys, and liver of the rat. This suggests that tungstate activation of adenylate cyclase is a general phenomenon and may be mediated by a similar mechanism in different tissues. Tungstate provides an additional tool by which the molecular basis of adenylate cyclase activation can be probed. (Endocrinology 108: 435, 1981)

Restoration of guanine nucleotide- and fluoride-stimulated activity to an adenylate cyclase-deficient cell line with affinity-purified guanine nucleotide regulatory protein.

A guanine nucleotide-binding protein purified from turkey erythrocytes by affinity chromatography confers both F-- and guanine nucleotide-stimulation of adenylate cyclase to membranes from CYC- cells, a mutant cell line deficient in these responses. Interaction of turkey erythrocyte membranes with beta-adrenergic agonists before affinity chromatography, which is essential for binding of the guanine nucleotide regulatory protein to the affinity matrix, was also required for recovery of F--stimulation restoring activity in the affinity eluate.

Glucagon-stimulated adenylate cyclase detects a selective perturbation of the inner half of the liver plasma-membrane bilayer achieved by the local anaesthetic prilocaine.

Prilocaine can increase the fluidity of rat liver plasma membranes, as indicated by a fatty acid spin-probe. This led to the activation of the membrane-bound fluoride-stimulated adenylate cyclase activity, but not the Lubrol-solubilized activity, suggesting that increased lipid fluidity can activate the enzyme. With increasing prilocaine concentrations above 10 mM, the membrane-bound fluoride-stimulated activity was progressively inhibited, even though bilayer fluidity continued to increase and the activity of the solubilized enzyme remained unaffected. Glucagon-stimulated adenylate cyclase was progressively inhibited by increasing prilocaine concentrations. Prilocaine (10 mM) had no effect on the lipid phase separation occurring at 28 degrees C and attributed to those lipids in the external half of the bilayer, as indicated by Arrhenius plots of both glucagon-stimulated adenylate cyclase activity and the order parameter of a fatty acid spin-probe. However, 10 mM-prilocaine induced a lipid phase separation at around 11 degrees C that was attributed to the lipids of the internal (cytosol-facing) half of the bilayer. It is suggested that prilocaine (10 mM) can selectively perturb the inner half of the bilayer of rat liver plasma membranes owing to its preferential interaction with the acidic phospholipids residing there.

Interactions of gonadotropins with corpus luteum membranes: IX. Changes in the specific activities of some plasma-membrane marker enzymes in rat ovarian homogenates and purified membrane fractions at various times after priming with PMSG and hCG

Immature 23–25 day old rats were treated with pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) and a number of luteal cell surface-membrane marker activities were measured in ovarian homogenates and in isolated ovarian light membrane fractions, at different times after priming. Injection of PMSG alone reduced the specific activities of hCG-binding and hCG-stimulated adenylate cyclase in ovarian homogenates, but was without significant effect on other activities tested. Injection of PMSG-primed rats with hCG lowered levels of hCG-binding and hormone-sensitive adenylate cyclase in ovarian homogenates still further. Once again, other enzyme activities were unchanged. These very low levels of hCG-binding and hCG-stimulated cyclase recovered after about 48 h and subsequently increased sharply, reaching maximum levels at about 7–9 days after priming. Both activities then declined slowly thereafter, returning to levels characteristic of unstimulated ovaries by days 16–20, coincident with luteal regression. . Levels of 5'-nucleotidase and Mg 2+-dependent ATPase in whole ovarian homogenates increased 3–4-fold by 8 days after PMSG-hCG treatment and these levels were maintained up to day 20. In contrast to the marked fluctuation in hormone-sensitive adenylate cyclase, basal and fluoride-stimulated activities did not change appreciably throughout the formation, development and regression of rat corpora lutea. Changes in the specific activity of hCG-binding in partially-purified light membrane fractions prepared from PMSG-hCG primed ovaries at various times after priming paralleled changes observed in whole homogenates. Nucleotidase and (Na + + K +)ATPase activities also showed similar changes, but of lower magnitude. In contrast, the levels of alkaline phosphatase, a thecal-interstitial cell membrane marker, did not change. The distribution of 5'-nucleotidase activity between the light and heavy membrane fractions changed markedly at different stages of luteinization. The results indicate that both the relative amounts and the marker-enzyme compositions, of rat luteal cell microvillous and basolateral surface-membranes change throughout luteinization.

Adenylate cyclase in the hindgut of the cockroach, Leucophaea maderae (F.)

An adenylate cyclase from the hindgut of the cockroach, Leucophaea maderae (F.), has been investigated. Several properties of the enzyme, including subcellular distribution, pH optimum, stimulation by NaF, effect of ATP and Mg 2+, effect of divalent cations and effect of hindgut-stimulating neurohormone (HSN) were determined. Studies with isolated hindguts revealed that cyclic AMP potentiated the effect of HSN fourfold. However, HSN had no effect on basal- or fluoride-stimulated activities of adenylate cyclase in vitro. These observations are discussed with reference to the mode of action of HSN.

The selective effects of charged local anaesthetics on the glucagon‐ and fluoride‐stimulated adenylate cyclase activity of rat‐liver plasma membranes

The cationic local anaesthetics carbocaine and nupercaine were found to increase the fluoride-stimulated adenylate cyclase up to a maximum level; above this maximum level further increases in drug concentration inhibited the enzyme. At concentrations where this activity was stimulated, a fatty acid spin label detected an increase in bilayer fluidity, which, it is suggested, is responsible for the activation of the enzyme. A solubilized enzyme was unaffected by the drugs, a finding consistent with this proposal. These cationic drugs began to inhibit the glucagon-stimulated activity at concentrations where they activated the fluoride-stimulated activity. It is suggested that this is due to their effect on the coupling interaction between the receptor and catalytic unit. The anionic drugs, phenobarbital, pentobarbital, and salicylic acid, all inhibited the fluoride-stimulated enzyme. This may be due in part to a direct effect on the protein and in part to the interaction of the drugs with the bilayer. The drugs had small inhibitory effects on the lubrol-solubilized enzyme. The glucagon-stimulated enzyme was initially inhibited by the anionic drugs at low concentrations, then activated, and finally inhibited with increasing drug concentration. The reasons for such changes are complex, but there was no evidence from electron spin resonance studies to suggest that the elevations in activity were due to increases in bilayer fluidity.

Decreased cardiac beta-adrenergic receptors in deoxycorticosterone-salt and renal hypertensive rats.

The development of experimental deoxycorticosterone-salt (DOCA-salt) and renal artery clip hypertension in rats is associated with alterations in the sensitivity of the myocardium to adrenergic stimulation. We studied beta-adrenergic receptors and isoproterenol-stimulated adenylate cyclase in myocardial membranes from hypertensive rats to determine whether this altered sensitivity is associated with any change in beta-adrenergic receptors. The specific binding of the beta-adrenergic antagonist, 125I-iodohydroxybenzylpindolol, was used to measure numbers and affinities of receptors in myocardial membrane preparations. Cardiac membranes from both DOCA-salt and renal hypertensive rats showed significantly fewer beta-receptors than did membranes from control, normotensive rats. Receptor affinity remained unchanged. This decrease was from 110 +/- 19 to 49 +/- 5 fmol/mg protein for DOCA-salt hypertension and from 110 +/- 18 to 75 +/- 16 fmol/mg protein for renal artery clip hypertension. Isoproterenol-stimulated adenylate cyclase activity also was lower in membranes from hypertensive rats, whereas basal and fluoride-stimulated activities were unchanged.

Augmentation of cyclic AMP content induced by lysophosphatidyl choline in rabbit hearts

The accumulation of both lysophosphoglycerides and cyclic AMP in ischaemic myocardium has been associated with electrophysiological deterioration of the heart. Although the increase in concentration of cyclic AMP is mediated in part by adrenergic stimulation, some of the increase occurs despite β-adrenergic blockade. Thus, we considered the possibility that lysophosphoglycerides may contribute to the increases in myocardial cyclic AMP in ischaemic zones by stimulating adenylate cyclase. Accordingly, we examined the effects of albumin-bound lysophosphatidyl choline in concentrations simulating those in extracts of ischaemic myocardium on adenylate cyclase and phosphodiesterase activities in rabbit and cat myocardial broken cell preparations and on cyclic AMP content of isolated perfused rabbit hearts. Lysophosphatidyl choline (0.4 to 1.2 mmol·litre−1) augmented basal activity of adenylate cyclase by approximately 30% in broken cell preparations without affecting phosphodiesterase activity. Lysophosphatidyl choline (1 and 2 mmol·litre−1) did not alter isoprenaline-stimulated activity but reduced fluoride-stimulated activity by 31 and 39% of values obtained in the absence of lysophosphatidyl choline. In isolated perfused hearts from normal rabbits and those pretreated with reserpine to deplete the heart of catecholamines, lysophosphatidyl choline (2 mmol·litre−1) augmented the concentration of cyclic AMP from 4.6 ± 0.3 to 11.4 ± 1.3 pmol·mg protein−1 (P <0.005) and from 4.9 ± .4 to 8.0 ± .4 pmol·mg protein−1 (P <0.005) respectively. Thus, accumulation of lysophosphatidyl choline during ischaemia could contribute to the transiently increased concentration of cyclic AMP seen typically in ischaemic myocardium.

Effect of Multiplication Stimulating Activity (MSA) on intracellular cAMP levels and adenylate cyclase activity in chick embryo fibroblasts

The addition of the somatomedin-like growth factor Multiplication Stimulatory Activity (MSA) to intact chick embryo fibroblasts (CEF) in culture does not decrease the basal intracellular pool of cAMP. As a more sensitive means of determining a possible effect of MSA on cAMP metabolism we have assessed the ability of MSA to alter the accumulation of intracellular cAMP in response to added prostaglandin E 1 (PGE 1) to intact CEF. MSA exhibits a concentration-dependent inhibition of PGE 1-stimulated accumulation of cAMP in CEF, with maximal inhibition observed at 100–400 ng/ml MSA. The MSA inhibitory effect is rapid, occurring within minutes of MSA addition, and the extent of inhibition is not altered with increasing PGE 1 concentration. MSA is also effective in inhibiting both PGE 1- and fluoride-stimulated activities of CEF adenylate cyclase of crude membrane preparations. In the concentration range of 100–400 ng/ml, MSA causes a 20–40% inhibition of cyclase activity. It should be emphasized that it has not been shown that these MSA effects on cAMP metabolism are relevant to the mechanism for growth promotion by this factor.

Effects of phospholipase A2 and filipin on the activation of adenylate cyclase

Rat liver plasma membranes were incubated with phospholipase A2 (purified from snake venom) or with filipin, a polyene antibiotic, followed by analysis of the binding of glucagon to receptors, effects of GTP on the glucagon-receptor complex, and the activity and responses of adenylate cyclase to glucagon + GTP, GTP, Gpp(NH)p, and F-. Phospholipase A2 treatment resulted in concomitant lossess of glucagon binding and of activation of cyclase by glucagon + GTP. Greater than 85% of maximal hydrolysis of membrane phospholipids was required before significant effects of phospholipase A2 on receptor binding and activity response to glucagon were observed. The stimulatory effects of Gpp(NH)p or F- remained essentially unaffected even at maximal hydrolysis of phospholipids, whereas the stimulatory effect of GTP was reduced. Detailed analysis of receptor binding indicates that phospholipase A2 treatment affected the affinity but not the number of glucagon receptors. The receptors remain sensitive to the effects of GTP on hormone binding. Filipin also caused marked reduction in activation by glucagon + GTP. However, in contrast to phospholipase A2 treatment, the binding of glucagon to receptors was unaffected. The effect of GTP on the binding process was also not affected. The most sensitive parameter of activity altered by filipin was stimulation by GTP or Gpp(NH)p; basal and fluoride-stimulated activities were least affected. It is concluded from these findings that phospholipase A2 and filipin, as was previously shown with phospholipase C, are valuable tools for differentially affecting the components involved in hormone, guanyl nucleotide, and fluoride action on hepatic adenylate cyclase.

Lysophosphatidylcholines can modulate the activity of the glucagon-stimulated adenylate cyclase from rat liver plasma membranes

1. Synthetic lysophosphatidylcholines inhibit the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes at concentrations two to five times lower than those needed to inhibit the fluoride-stimulated activity. 2. Specific 125I-labelled glucagon binding to hormone receptors is inhibited at concentrations similar to those inhibiting the fluoride-stimulated activity. 3. At concentrations of lysophosphatidylcholines immediately below those causing inhibition, an activation of adenylate cyclase activity or hormone binding was observed. 4 These effects are essentially reversible. 5. We conclude that the increased sensitivity of glucagon-stimulated adenylate cyclase to inhibition may be due to the lysophosphatidylcholines interfering with the physical coupling between the hormone receptor and catalytic unit of adenylate cyclase. 6. We suggest that, in vivo, it is possible that lysophosphatidylcholines may modulate the activity of adenylate cyclase only when it is in the hormone-stimulated state.

Mode of Action of Ticlopidine in Inhibition of Platelet Aggregation in the Rat

Ticlopidine, when orally administered to rats, resulted in activation of basal and prostaglandin E1 (PGE1)-stimulated adenylate cylase activity through increase in affinity of the cyclase in platelet membrane to PGE1, although it failed to affect adenosine- or sodium fluoride-stimulated activity of the enzyme. In washed platelets, Ticlopidine also activated basal and PGE1-stimulated activity of the cyclase and prevented reduction in the cyclase activity caused by low concentrations of PGE2. Furthermore, Ticlopidine inhibited malondialdehyde formation in platelets induced by thrombin but failed to inhibit that caused by exogenous arachidonic acid. Adenosine 3',5'-cyclic monophosphate (c-AMP): phosphodiesterase activity of platelet lysate was not significantly affected by Ticlopidine treatment. These findings indicate that Ticlopidine inhibits platelet aggregation and prostaglandin synthesis from endogenous substrate through activating basal and PGE1-stimulated activity of the cyclase, preventing PGE2-induced depression of the cyclase activity and thus increasing platelet c-AMP level.

Maturation dependent decline of adenylate cyclase in rabbit red blood cell membranes

Adenylate cyclase (EC 4.6.1.1) was studied in membrane preparations of reticulocyte-rich blood obtained from phenylhydrazine-treated rabbits and compared to that of untreated animals. Basal and fluoride-stimulated activities were decreased 2- and 4-fold, respectively, during the process of maturation. Catalytic parameters such as time course, protein, ATP, Mg 2+ concentration curves and K m have been determined and were found to be similar in the reticulocyte and the erythrocyte. Adenylate cyclase was sensitive to GTP, 5′-guanylyl imidodiphosphate, prostaglandin E 1 and prostaglandin E 2. Activation by prostaglandin E 1 was higher than that produced by prostaglandin E 2. Only additive effect was found when 5′-guanylyl imidodiphosphate or GTP was added to hormone-stimulated activity. The sensitivity of the enzyme to these effectors was decreased over the transition reticulocyte-erythrocyte. In either cell the enzyme was not activated by catecholamines (epinephrine, norepinephrine, isoproterenol).

Adenylate cyclase activity in the epididymal adipose tissue from obese-hyperglycaemic mice

The adenylate cyclase activities in 1,500 × g to 25,000 × g sediments of adipose tissue from 3 month old C57BL 6J/ob and lean mice were compared. Three anomalies were observed in obese mice: 1) a defect in basal (−80%) and fluoridestimulated (−50%) activities suggesting a decreased number of catalytic units per mg protein; 2) an impairment in stimulation by isoproterenol (−85%) with no change in apparent affinity for the hormone; and, 3) no stimulatory effect of ethanol upon guanyl nucleotide-stimulated activity. The two latter effects may be due to a reduced number ofβ-adrenergic receptors per mg protein and/or to an alteration in the transducing process. On the other hand, guanyl nucleotides as well as prostaglandin PGE1 exerted similar effects in the preparations from both lean and obese adipose tissue.

Adenylate cyclase in cardiac microsomal fractions

Adenylate cyclase was examined in microsomal fractions of rabbit heart and compared with activity sedimenting at lower gravitational forces (washed particle fractions). Enzyme activity in the two fractions differed markedly in several respects. The microsomal enzyme (which comprised 5% of the total activity) was dramatically stimulated by low concentrations of the nonionic detergents Lubrol PX and Triton X-100, while basal activity in the washed particle fraction was only modestly stimulated and the fluoride-activated enzyme was inhibited by detergent. Both basal- and fluoride-stimulated activity in the microsomal fraction was enhanced by incubation of the membranes with phospholipase A, whereas basal activity in the washed particle fraction was decreased by this treatment and fluoride activation was unaffected. The rate of activation of adenylate cyclase by the GTP analog, guanylylimidodiphosphate, was much slower at 20°C in the washed particle fraction than in the microsomal preparation. Epinephrine dramatically increased the rate of activation by guanine nucleotide in washed particle membranes, but had a much more modest effect on the microsomal enzyme. It is concluded that adenylate cyclase is present in microsomal fractions of the ventricular myocardium and can be distinguished from the plasma membrane enzyme by virtue of these differences. Since microsomal fractions of the heart constitute membranes of the sarcoplasmic reticulum, it is suggested that a fraction of the heart's adenylate cyclase resides within these intracellular membranes.

Protease inhibitors block hormonal activation of adenylate cyclase

To investigate the mechanism of serine protease stimulation of rat ovarian adenylate cyclase, a variety of synthetic protease inhibitors were used. These inhibitors blocked trypsin, chymotrypsin and hCG stimulation of adenylate cyclase in nearly the same manner. The inhibition of hormone stimulated adnylate cyclase could not be explained by a loss of [ 125I]hCG binding. Cholera toxin and epinephrine stimulation of adenylate cyclase were similarly inhibited, whereas basal and fluoride-stimulated activities were only affected by higher doses of the inhibitors. The results suggest that adenylate cyclase in the ovary may be regulated by membrane protease activity.

Age-related parallel decline in beta-adrenergic receptors, adenylate cyclase and phosphodiesterase activity in rat erythrocyte membranes

The activities of three components of the cyclic AMP system were compared in erythrocyte ghost membranes prepared from the blood of rats at various ages from 1.5 to 15 months. The apparent number of β-adrenergic receptor sites, adenylate cyclase activity and cyclic AMP phosphodiesterase activity all declined about 50% in the membranes from the older animals (>5 months) as compared to the 1.5 month ones. The soluble erythrocyte phosphodiesterase also declined with age, but the decline did not parallel that of the membrane-associated activity. In contrast, there was no age-related change in the number of β-adrenergic receptors in membranes from the brains of the same animals. In erythrocyte ghosts, both the ratio of isoproterenol-stimulated adenylate cyclase activity to basal activity and the ratio of sodium fluoride-stimulated activity to basal were constant with age. Neither the dissociation constant for the β-adrenergic receptor nor the Michaelis constant for the phosphodiesterase changed as a function of age. Together with other data in the literature, these results suggest a close functional association of the components of the cyclic AMP system in the mature erythrocyte membrane, and support a physiological role for the cyclic AMP mediated β-adrenergic effects in the red blood cell.

Exchange of partners in glucagon receptor-adenylate cyclase complexes. Physical evidence for the independent, mobile receptor model

The apparent target sizes of the glucagon receptor and the catalytic unit of adenylate cyclase in rat liver plasma membranes have been measured by the technique of radiation inactivation in an electron beam. When irradiated in the uncoupled state, the apparent target size for the catalytic unit assayed by fluoride-stimulated activity was 160 000, and for the receptor assayed by specific 125I-labelled glucagon binding was 217 000. The corresponding target size estimated from glucagon-stimulated activity after irradiation in the uncoupled state was 389 000. When the complexes were irradiated in the coupled state in the presence of glucagon, the apparent target sizes from 125I-labelled glucagon binding, and fluoride- or glucagon-stimulated activities had similar values of 310 000, 380 000 and 421 000, respectively. However, if the complexes were allowed to uncouple by removing glucagon after irradiation and activity was then assayed after readdition of glucagon, the apparent target size from the glucagon-stimulated activity increases from 421 000 to 811 000. The pattern of apparent target sizes obtained under these different conditions has been tested against the pattern predicted for simple models of the coupling mechanism. The only simple model that is consistent with the pattern of target sizes requires the receptors and catalytic units to be present in approximately equal numbers. On binding glucagon, the receptor forms a locking interaction with the catalytic units, so that the complex and its components are inactivated as a single target with an apparent size of about 380 000 ( ± 15%). After the removal and readdition of glucagon to complexes that were irradiated in the coupled state, the new population of complexes must contain hybrids of active and inactive partners obtained by exchange between active and inactivated complexes, to account for the doubling in apparent target size to 811 000 for glucagon-stimulated activity. This hybridization of catalytic units and receptors is the essential feature of the model that distinguishes it from others in which permanently associated complexes of the two components are activated by lateral dimersation on binding glucagon. Simple models of this type are shown to be physically improbable. It is emphasized that the models described are based only on the relationships between the apparent target sizes of components that are defined by their functions, and the apparent target sizes do not necessarily relate solely to the components that can be defined structurally as the receptor or catalytic unit.

Adenosine and the regulation of insulin secretion by isolated rat islets of Langerhans

The effect of adenosine in insulin secretion and adenylate cyclase activity of rat islets of Langerhans was investigated. Adenosine inhibited insulin secretion stimulated by glucose, glucagon, prostaglandin E2, tolbutamine and theophylline. Adenosine decreased basal adenylate cyclase activity of the islets as well as that stimulated by glucagon prostaglandin E2 and GTP, although fluoride-stimulated activity was not affected. Neither insulin secretion nor adenylate cyclase activity of the islets was affected by adenine, AMP or ADP. The inhibitory effect of adenosine on adenylate cyclase activity was not altered by either phenoxybenzamine (alpha-adrenergic blocker) or propranolol (beta-adrenergic blocker), suggesting that the effect is not mediated through the adrenergic receptors of the islet cells. These results suggest that the intracellular concentration of adenosine in the beta-cell may play a role in regulating insulin secretion and that this effect may be mediated via alterations in the activity of adenylate cyclase in the beta-cell.

Effects of glucose, glucose metabolites and calcium ions on adenylate cyclase activity in homogenates of mouse pancreatic islets

The effects of glucose, a series of glucose metabolites, nicotinamide nucleotides, Ca2+ and p-chloromercuribenzenesulphonate on adenylate cyclase activity in homogenates of mouse pancreatic islets were studied. The basal activity of the adenylate cyclase was approx. 6 pmol of cyclic AMP formed/30 min per microng of DNA at 30 degrees C. The enzyme activity was stimulated by some 150% by fluoride. Starvation of the animals for 48h had no effect on either the basal or the fluoride-stimulated activity. The adenylate cyclase activity was increased by 40-50% when 17 mM-glucose, 10 micronM-phosphoenolpyruvate or 10 micronM-pyruvate was added to the assay medium. The effect of glucose was unchanged in the presence of 17 mM-mannoheptulose, and mannoheptulose alone had no effect. The other glycolytic intermediates, and the coenzymes NAD+, NADH and NADPH, at concentrations up to 1 mM were without any detectable effect on the rate of formation of cyclic AMP. The insulin secretagogue p-chloromercuribenzenesulphonate inhibited the adenylate cyclase markedly even at a concentration of 10 micronM. Calculated concentrations of free Ca2+ of 10 micronM and 0.1 mM inhibited adenylate cyclase by 29 and 71% respectively. It is concluded that both glucose itself and phosphoenolpyruvate and/or pyruvate are true activating ligands for islet and adenylate cyclase and that inhibition of the cyclase by Ca2+ may be of physiological significance.