The thermal lability of adenylate cyclase: Mechanisms of stabilization

The thermal inactivation of adenylate cyclase was investigated in human lymphocytes and in the N-protein deficient cyc-S49 mouse lymphoma cell line. The enzyme is rapidly inactivated at 37C with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 5.5 and 4.5 min respectively in human and cyc^? membranes. Thermal inactivation is prevented by at least two mechanisms. The first mechanism involves ATP which stabilizes adenylate cyclase in a concentration dependent manner similar to the K_m of ATP for cAMP formation. However, the inhibition of inactivation does not require Mg⁺⁺ while the enzyme catalysis of ATP to cAMP does. The second mechanism involves substances which activate the enzyme. The human lymphocyte enzyme is equally stabilized by either NaF, GppNHp, or forskolkin. In contrast, the cyc^? enzyme is fully stabilized by forskolin but only partially stabilized by NaF. When human erythrocyte N-protein extract is added to cyc^? membranes, NaF fully stabilizes the enzyme. These data suggest that an activated N-protein is instrumental in stabilizing adenylate cyclase and that there is some N-protein component in cyc^? membranes through which NaF may be exerting its stabilizing action.     

Activation of rat adipocyte plasma membrane adenylate cyclase by sodium azide

The adenylate cyclase of rat adipocyte plasma membrane is stimulated by sodium azide with a half maximal activation of 100–150% occuring at 50 mM NaN₃. Studies of the effects of azide and fluoride indicate different mechanisms of stimulation of the enzyme by these ions. Comparable stimulation of the activity is obtained by 100 mM NaN₃ or 10 mM NaF but unlike azide, higher concentrations of fluoride cause inhibition of the enzyme. Fluoride activated adenylate cyclase is further stimulated by azide. Epinephrine stimulation of the enzyme is absent in the presence of fluoride but the hormone enhances the activity in the presence of azide. Reversal of the inhibitory action of GTP on adenylate cyclase by epinephrine is demonstrated even in the presence of azide but not in the presence of fluoride.     

Effects of calcium on a parathyroid hormone-sensitive adenylate cyclase inhibitor

Inhibition of parathyroid hormone (PTH)-sensitive adenylate cyclase by {Nle-8, Nle-18, Tyr-34} bPTH-(3–34) amide was studied in thyroparathyroid-ectomized dogs. The inhibitory effect was shown to be markedly enhanced by the addition of calcium ions into the $\text{in}\text{,}\text{vitro}$ assay system. At 0.1 mM Ca²⁺, complete inhibition by the antagonist was obtained. Chelation of exogenous Ca²⁺ by EGTA eliminated the Ca²⁺-induced inhibition. Both the basal and hormone-stimulated activities were decreased in the presence of 0.1 mM Ca²⁺, whereas the addition of EGTA increased both activities. Our results suggest that Ca²⁺ modulates canine renal PTH-sensitive adenylate cyclase and its inhibition by substituted bPTH-(3–34).     

Errata

Optimal conditions for activation of adenylate cyclase in membrane particles were studied. Enzyme activation with serotonin (5-hydroxytryptamine), NaF, and guanosine $\text{5′-(3-O-}\text{thio}\text{)-}\text{triphosphate}$ (GTPγS) was time- and temperature-dependent. Mg²⁺ was required for enzyme activation. Adenylate cyclase that was activated by NaF or GTPγS was gradually inhibited by $\text{N-}\text{methylmaleimide}$ while enzyme activated with serotonin and GTP responded faster to inhibition by the same sulfhydryl reagent. The enzyme responded in a similar fashion to a spin-labeled $\text{N-}\text{methylmaleimide}$ analog 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrolidinyloxyl (i.e., $\text{N-}\text{methylmaleimide}$ nitroxide). Binding of the spin label was enhanced following enzyme activation by serotonin, NaF, or GTPγS in the presence of Mg²⁺. Activation of the enzyme was accompanied by an increase in the strong immobilization peaks in the EPR spectra. Both effects, the increase in binding and in the strong immobilization peaks, can be induced by Mg²⁺ alone. The results indicate that a general conformational change induced by Mg²⁺ may be essential for adenylate cyclase activation.     

Interactions of prostaglandins with subpopulations of ovine luteal cells. I. Stimulatory effects of prostaglandins E1, E2 and I21

Preparations of small and large steroidogenic cells from enzymatically dispersed ovine corpora lutea were utilized to study the $\text{in}$$\text{vitro}$ effects of luteinizing hormone (LH) and prostaglandins (PG) E₁, E₂ and I₂. Cells were allowed to attach to culture dishes overnight and were incubated with either LH (100 ng/ml), PGE₂, PGE₂, or PGI₂ (250 ng/ml each). The secretion of progesterone by large cells was stimulated by all prostaglandins tested (P < 0.05) while the moderate stimulation observed after LH treatment was attributable to contamination of the large cell population with small cells. Prostaglandins E₁ and E₂ had no effect on progesterone secretion by small cells, while LH was stimulatory at all times (0.5 to 4 hr) and PGI₂ was stimulatory by 4 hr. Additional studies were conducted to determine if the effects of PGE₂ upon steroidogenesis in large cells were correlated with stimulated activity of adenylate cyclase. In both plated and suspended cells PGE₂ caused an increase (P < 0.05) in the rate of progesterone secretion but had no effect upon the activity of adenylate cyclase or cAMP concentrations within cells or in the incubation media. Exposure of luteal cells to forskolin, a nonhormonal stimulator of adenylate cyclase, resulted in marked increases in all parameters of cyclase activity but had no effect on progesterone secretion. These data suggest that the actions of prostaglandins E₁, E₂ and I₂ are directed primarily toward the large cells of the ovine corpus luteum and cast doubt upon the role of adenylate cyclase as the sole intermediary in regulation of progesterone secretion in this cell type.     

The dual effects of aluminum as activator and inhibitor of adenylate cyclase in the liver fluke Fasciola hepatica

The liver fluke, $\text{Fasciola hepatica}$, has a very active adenylate cyclase which can be stimulated by NaF or by serotonin and guanine nucleotides. Micromolar amounts of AlCl₃ augment the activation by F-. In contrast, when the enzyme is activated with serotonin and guanine nucleotides, AlCl₃ inhibits the activation. Aluminum also inhibits the activation by forskolin. Gallium mimics the effects of aluminum.     

In vitro stimulation of tadpole tail regression by cyclic AMP.

Treatment of $\text{Rana catesbeiana}$ tail fin tissue $\text{in vitro}$ with 0.1 mM or 0.5 mM cyclic AMP or with triiodothyronine induces an increase in the specific activity of hexosaminidase, a lysosomal marker enzyme, and a decrease in tissue area. Lithium chloride (8 mM), an inhibitor of adenylate cyclase, inhibits these changes when initiated by triiodothyronine but not when initiated by cyclic AMP. The levels of cyclic AMP, determined by radioimmunoassay techniques, increased 110 ± 10% over matched discs in culture after only one day's exposure to triiodothyronine. These results indicate the effect of triiodothyronine on fin resorption may be mediated by cyclic AMP.     

Effects of morphine on dopamine-stimulated adenylate cyclase and on cyclic GMP formation in primate brain amygdaloid nucleus.

In homogenates of $\text{Macaca}$$\text{mulatta}$ (Rhesus) or $\text{Cebus}$$\text{apella}$ amygdaloid nuclear complex, adenylate cyclase activity was approximately doubled by either 10μ$\text{M}$ dopamine or 8m$\text{M}$ NaF. In the presence of morphine, the stimulation by dopamine was reduced. A 90–100% inhibition of the dopamine stimulation was obtained with 20μ$\text{M}$, and a 50% inhibition, with 5μ$\text{M}$ morphine. The effects of 10μ$\text{M}$ morphine on dopamine stimulation were reversed by 10μ$\text{M}$ naloxone. Morphine itself did not significantly affect the basal adenylate cyclase activity, but in the presence of 10μ$\text{M}$ morphine the stimulation by 8m$\text{M}$ NaF was reduced approxiamtely 50%. The data suggest an action of morphine at a receptor site which is distinct from the dopamine receptor, but which inhibits the dopamine-stimulated adenylate cyclase. In addition, the cyclic GMP content of $\text{Cebus}$ amygdala slices was reduced by 50–75% during incubation for 5–20 minutes with morphine. Maximum effects on cyclic GMP were obtained with 10μ$\text{M}$, and half-maximum effects, with 0.1μ$\text{M}$ morphine. The effect of morphine on amygdala cyclic GMP was not reversed by naloxone. Thus, this action of morphine may not be receptor mediated, or may involve the interaction of morphine with receptors other than the opiate receptor.     

Regulation by cations of adenylate cyclase activity in chicken tissues

The effects of magnesium and sodium ions on adenylate cyclase activity in plasma membranes from chicken heart and eggshell gland mucosa were studied. It was found that the increase in magnesium chloride concentration from 5 to 40 mM results in the stimulation (4.1-fold) of the adenylate cyclase activity. The increase in sodium chloride concentration up to 150 mM stimulated the enzyme activity 2-fold. The stimulation of adenylate cyclase by magnesium and sodium ions was less pronounced in the eggshell gland. GTP did not activate adenylate cyclase. The activating effect of magnesium and sodium ions was accompanied by the attenuation of the enzyme sensitivity to NaF, guanylyl imidodiphosphate and isoproterenol. Activation by guanylyl imidodiphosphate was completely abolished in the presence of 40 mM magnesium chloride. It is assumed that high concentrations of the salt promote subunit dissociation of the adenylate cyclase regulatory protein and its interaction with the catalytic subunit in the presence of endogenous nucleotides. The differences in the adenylate cyclase sensitivity to cations in chicken heart and eggshell gland mucosa correlate with the amount of pertussis toxin substrate.     

Reconstitution of dopamine-sensitive adenylate cyclase from dissociated components in canine caudate nucleus

The catalytic component of adenylate cyclase and [${}^3\text{H}$]dopamine binding protein were solubilized with 2% Lubrol PX in the presence of NaF from the synaptic membranes of canine caudate nucleus and were separated into distinct fractions by gel exclusion chromatography on a Sephadex G-200 column. The dissociated adenylate cyclase was no longer responsive to dopamine but was considerably stimulated by 10 mm NaF. Dissociated [${}^3\text{H}$]-dopamine binding protein possessed the apparent dissociation constant of 3.2 μm for dopamine, almost identical to that of the particulate preparations. The affinities of [${}^3\text{H}$]-dopamine binding protein to catecholamines and neuroleptics were also very similar to those of particulate preparations. After the adenylate cyclase and [${}^3\text{H}$]dopamine binding protein were preincubated together at 4 °C for 30 min, the cyclase activity displayed a dose-dependent increase by dopamine with the K_a of 1.6 μm, the concentration of dopamine to stimulate half-maximally. Stimulation of the reconstituted adenylate cyclase by dopamine was maximally 2.7-fold and was strongly inhibited by neuroleptics such as chlorpromazine and haloperidol. These results suggest that [${}^3\text{H}$]dopamine binding protein is identical to the regulatory subunit of dopamine-sensitive adenylate cyclase in the synaptic membranes of canine caudate nucleus.     

Effect of vanadate on phosphoryl transfer enzymes involved in glucose metabolism

Different types of enzymes from yeast and from rabbit muscle which catalyze phosphoryl transfer reactions involved in glucose metabolism differ in their sensitivity to vanadate. Phosph$\text{o}$ glucomutase and phosphoglycerate mutase are inhibited at the μM range. 2,3-Bisphosphoglycerate phosphatase is completely inhibited by 0.5 mM vanadate. 2,3-Bisphosphoglycerate synthase, hexokinase, phosphoglycerate kinase and fructose-1,6-P₂ phosphatase are partially inhibited by mM vanadate. Phosphofructokinase and pyruvate kinase are not affected. The glycolytic enzymes which mechanism does not involves phosphoryl transfer step are not affected by vanadate.     

Sensitivity of rat brain adenylate cyclase to activation by calcium and ethanol after chronic exposure to ethanol

Rats were fed ethanol (Lieber-DeCarli diet) for three weeks. Stimulation of cerebellar adenylate cyclase by calcium was measured in control (pair-fed), chronic-alcohol and alcohol-withdrawn animals. No differences in the sensitivity or maximal stimulation of this enzyme were observed among these groups. Ethanol $\text{in}$,$\text{vitro}$ (1%) stimulated brain adenylate cyclase approximately 50% in the presence or absence of calcium. Chronic alcohol exposure $\text{in}$,$\text{vivo}$ did not alter the sensitivity of adenylate cyclase to stimulation by alcohol $\text{in}$,$\text{vitro}$.     

ADP-ribosylation of brain synaptosomal proteins correlates with adenylate cyclase activation

ADP-ribosylation of the adenylate cyclase $\text{G}\text{F}$ regulatory subunit by cholera toxin is a major tool for the study of this enzyme. Investigation of the brain enzyme has been hampered up to now by the failure to demonstrate cholera toxin-dependent ADP-ribosylation of membrane-bound proteins. Synaptosomes prepared by flotation from fresh brains homogenized in the presence of protease inhibitors yielded membranes of which several proteins could be ADP-ribosylated by the toxin. The same membranes subjected to mild proteolysis could not be ADP-ribosylated. Adenylate cyclase activation and ADP-ribosylation were simultaneous processes. The major labeled species was of 47,000 M_r. It was solubilized by Lubrol-PX, together with other labeled polypeptides. As analyzed on sucrose gradients, the 47,000 M_r protein was found both in the 3S region, and in the adenylate cyclase containing fraction (9.1S).     

Adenosine receptor-mediated stimulation of GTP hydrolysis in adipocyte membranes

The adenosine derivative, N⁶-phenylisopropyladenosine (PIA), which inhibits adenylate cyclase in adipocyte membranes by a GTP-dependent and sodium-amplified process, was studied on GTPase activity in hamster adipocyte ghosts. PIA stimulated a high affinity GTPase without apparent lag phase. Both unstimulated and PIA-stimulated GTPases exhibited very similar K_m values of about 0.2 μM GTP. PIA-induced low K_m GTPase stimulation was amplified by sodium ions and was half-maximal and maximal at about 0.02 and 0.1 μM PIA, respectively. Stimulations of the low K_m GTPase by PIA and PGE₁, both inhibiting adipocyte adenylate cyclase, were not additive. Similar to PIA-induced adenylate cyclase inhibition, stimulation of the GTPase by PIA but not by PGE₁ was prevented by the adenosine receptor antagonist, 3-isobutyl-1-methylxanthine. The data suggest that PIA-induced stimulation of a high affinity GTPase is an essential mechanism of adenosine receptor-mediated adipocyte adenylate cyclase inhibition.     

Adenylate cyclase and phosphodiesterase in transitional epithelium of the urinary bladder.

Adenylate cyclase activities in cell-free preparations of isolated transitional epithelium from rabbit urinary bladders were shown to be stimulated by epinephrine, prostaglandin E₁ (PGE₁), 5-guanylyl imidodiphosphate (GMP-PNP), and NaF. ACTH, aldosterone, insulin, glucagon, oxytocin, parathyroid hormone and vasopressin were without effect at the concentrations tested. The effects of epinephrine, PGE₁, and GMP-PNP appeared to be additive. Essentially all of the adenylate cyclase activity was particulate, while approximately 70% of the cyclic nucleotide 3':5'-phosphodiesterase activity was soluble. Single reciprocal plots of the phosphodiesterase data revealed non-linear kinetics.     

On the mechanism of tolerance to isoproterenol-induced accumulation of cAMP in rat pineal in vivo.

Less cyclic adenosine 3′:5′ monophosphate (cAMP) accumulated in rat pineal gland, $\text{in}$$\text{vivo}$, after two doses of l-isoproterenol (5mg/kg, i.p.) than after one dose. A single injection of l-isoproterenol decreased the ability of l-isoproterenol to activate adenylate cyclase and increased the activity of the low Km phosphodiesterase (PDE). Tolerance to l-isoproterenol-induced accumulation of cAMP in rat pineal $\text{in}$$\text{vivo}$ may be due to decreased responsiveness of adenylate cyclase as well as to increased activity of PDE.     

Modulation of human platelet adenylate cyclase by prostacyclin (PGX)

Prostacyclin (PGX) ($\text{5Z}\text{)-9-}\text{deoxy}\text{-6,9α-}\text{epoxy}\text{-Δ}{}^5\text{-}\text{PGF}{}_{\mathrm{1\alpha }}$ has been found to be a potent stimulator of cAMP accumulation in human platelet rich plasma (PRP), and a direct stimulator of platelet microsome adenylate cyclase. Prostacyclin is, on a molar basis, at least 10 times more potent a stimulator of cAMP accumulation in platelets than PGE₁. The prostacyclin stimulation of platelet cAMP accumulation can be antagonized by the prostaglandin endoperoxide PGH₂, and a PGH₂-induced platelet aggregation is antagonized by prostacyclin. A model of platelet homeostasis is proposed that suggests platelet aggregation is controlled by a balance between the adenylate cyclase stimulating activity of prostacyclin, and the cAMP lowering activity of PGH₂.     

Selective photoinduced uncoupling of the response of adenylate cyclase to gonadotropins by 5-iodonaphthyl 1-azide

5-Iodonaphthyl 1-azide (INA) has been previously shown to selectively label, on photolysis, only those proteins in contact with the membrane lipids. Low concentrations (less than 10 microM) of INA added to rat ovarian plasma membranes induced, on photoactivation, a selective and complete loss of the response of the adenylate cyclase to stimulation by human chorionic gonadotropin (hCG) or luteinizing hormone (LH). In contrast, this treatment affected neither hCG binding to the receptor nor the stimulation of the enzyme by NaF. That the uncoupling of the receptor from the enzyme by INA occurred within the lipid bilayer can be derived from the finding that the prior presence neither of saturating concentrations of hCG nor of the aqueous nitrene-scavenger glutathione (GSH) prevented this effect. Photolysis at higher concentrations of INA (0.1-1 mM) led to the inhibition of the adenylate cyclase stimulated by fluoride. This effect was totally prevented by glutathione. A similar behavior was obtained with a water-soluble analogue of INA, namely, 5-diazonionapthyl 1-azide (DAN). On photoactivation with 30 microM DAN, the NaF-stimulated adenylate cyclase was inhibited, but this effect was completely prevented by added GSH. At low concentrations where its effects are restricted to the lipid core, INA may represent a useful tool to define receptor coupling with the adenylate cyclase. The capacity of INA at low concentrations to uncouple the hormone receptor from the adenylate cyclase is not restricted to the LH/hCG receptor. Other hormone receptors tested behaved similarly. Therefore, the reported findings appear to represent a general phenomenon.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans I. Inhibition by heparin of gonadotropin- stimulated ovarian adenylate cyclase

Heparin inhibits (I₅₀ = 2 μg/ml) the activity of luteinizing hormone and human chorionic gonadotropin-stimulated adenylate cyclase in purified rat ovarian plasma membranes. Unstimulated enzyme activity and activity stimulated by NaF, GTP or guanosine 5′-(β,γ-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by hepatin (I₅₀ = 6 μg/ml). The inhibition with respect to hormone concentration was of a mixed type for hormone binding and adenylate cyclase stimulation. Inhibition by heparin was not eliminated at saturating hormone concentration. The degree of inhibition was unaffected by the order in which enzyme, hormone and heparin were introduced into the assay system. Herapin (3 μg/ml) did not affect the pH activity relationship of basal and hormone-stimulated adenylate cyclase activity and did not change the dependence of enzyme activity on magnesium ion concentration. The inhibitory action of heparin cannot be solely attributed to interference with either catalysis or hormone binding. The possibility is considered that the highly charged herapin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation.     

Changes in pH sensitivity of adenylate cyclase specifically induced by fluoride and vanadate

The interdependent effects of divalent cations, pH, and various activators of adenylate cyclase were examined in partially purified plasma membranes from rat liver. This adenylate cyclase was found to exhibit largely alkaline pH optima, in the range of 8.3 to 9.3, for the expression of basal activity, and activities with GTP, GPP(NH)P, prostaglandin E₁ and GTP, and N⁶-(phenylisopropyl)adenosine and GTP. Glucagon and GTP, while increasing activity 8- to 10-fold, shifted the optimum activity to about pH 7.5. However, stimulation of the enzyme by 10 mm NaF or 3 mm Na₃VO₄ was strikingly dependent on pH. In both cases activation was optimal at pH values between 6.3 and 7.3, though above about pH 8.5 fluoride was barely stimulatory and vanadate was slightly inhibitory. This effect of elevated pH to reduce fluoride- or vanadate-stimulated activity could be prevented by glucagon plus guanine nucleotide, but could not be reversed once activity was lowered during preincubation. The data suggest that this effect was not due to the formation of an inhibitor of adenylate cyclase per se, nor to an artifact of assay methods. The effect of elevated pH was more pronounced with Mn²⁺ as activating cation than with Mg²⁺. With fluoride and lower pH adenylate cyclase was essentially Mn²⁺ requiring, whereas with fluoride and higher pH activity was comparable with either cation. The data suggested that combinations of pH, divalent cation, and activating ligand dictate the interactions of the constitutive subunits of the adenylate cyclase and provide additional criteria with which current models for the regulation of adenylate cyclase may be tested.