Inhibition of forskolin-activated adenylate cyclase by ethanol and other solvents

Ethanol and a variety of solvents are known to activate basal and Gpp(NH)p- and hormone-stimulated adenylate cyclase. We report here that ethanol and other solvents inhibit the activation of adenylate cyclase by forskolin. In the presence of 10 microM forskolin, 2% ethanol gives about 20% inhibition and 5% ethanol gives 40% inhibition of enzyme activity. Analysis of ethanol inhibition at several forskolin concentrations suggests that inhibition is competitive versus forskolin. Thus the effect of ethanol is greater at low forskolin concentrations and minimal at high concentrations. In addition to ethanol, inhibition of forskolin activation was observed with acetone, n-butanol, t-butanol, dimethyl formamide, dioxane, methanol and n-propanol. Dimethyl sulfoxide was inhibitory only at high concentrations (10%). Since some solvent is needed to prepare forskolin solutions and to maintain solubility at higher concentrations, the inhibitory effects reported here are an important consideration in studies employing forskolin activation. To minimize solvent inhibition we recommend that dimethyl sulfoxide be used to prepare forskolin solutions. At concentrations of 5% and less, dimethyl sulfoxide gives little if any inhibition of forskolin activation and causes only small increases in basal activity.     

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.     

Inhibition of hepatic adenylate cyclase by NADH

Adenylate cyclase activity in isolated rat liver plasma membranes was inhibited by NADH in a concentration-dependent manner. Half-maximal inhibition of adenylate cyclase was observed at 120 microM concentration of NADH. The effect of NADH was specific since adenylate cyclase activity was not altered by NAD+, NADP+, NADPH, and nicotinic acid. The ability of NADH to inhibit adenylate cyclase was not altered when the enzyme was stimulated by activating the cyclase was not altered when the enzyme was stimulated by activating the Gs regulatory element with either glucagon or cholera toxin. Similarly, inhibition of Gi function by pertussis toxin treatment of membranes did not attenuate the ability of NADH to inhibit adenylate cyclase activity. Inhibition of adenylate cyclase activity to the same extent in the presence and absence of the Gpp (NH) p suggested that NADH directly affects the catalytic subunit. This notion was confirmed by the finding that NADH also inhibited solubilized adenylate cyclase in the absence of Gpp (NH)p. Kinetic analysis of the NADH-mediated inhibition suggested that NADH competes with ATP to inhibit adenylate cyclase; in the presence of NADH (1 mM) the Km for ATP was increased from 0.24 +/- 0.02 mM to 0.44 +/- 0.08 mM with no change in Vmax. This observation and the inability of high NADH concentrations to completely inhibit the enzyme suggest that NADH interacts at a site(s) on the enzyme to increase the Km for ATP by 2-fold and this inhibitory effect is overcome at high ATP concentrations.     

Mn2+-sensitive, soluble adenylate cyclase in rat testis. Differentiation from other testicular nucleotide cyclases.

In subcellular fractions prepared from homogenate of adult rat testis adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity was found in the particulate, primarily 600 X g for 10 min, fractions, as well as in the cytosol. The properties of the adenylate cyclase in the cytosol differs substantially from the adenylate cyclase system associated with the 600 X g for 10 min particulate fraction. The cytosol enzyme, in contrast to the particulate adenylate cyclase, was found to be fluoride- and gonadotropin hormone-insensitive. The cytosol adenylate cyclase appears to be located in the germ cell while the particulate enzyme system in the non-germ cell component of the seminiferous tubules, The cytosol adenylate cyclase was found to be distinct also from the guanylate cyclase present in the rat testis cytosol. The adenylate cyclase appears to be located in the germ cell component while the guanylate cyclase, in the non-germ cell tubular component. Furthermore, it was found that the cytosol guanylate cyclase develops at an earlier stage of spermatogenesis, and precedes the development of the cytosol adenylate cyclase.     

Activation of adenylate cyclase by forskolin in rat brain and testis

Detergent-dispersed adenylate cyclase from rat cerebrum was detected in two components, one sensitive to Ca2+ and calmodulin and another sensitive to fluoride or guanyl-5'-yl imidodiphosphate (Gpp(NH)p). The enzyme activity of both components was markedly augmented by forskolin assayed in the presence or absence of other enzyme activators (e.g., NaF, Gpp(NH)p, calmodulin). The catalytic subunit fraction in which G/F protein was totally lacking was also activated by forskolin. During 1-35 days of postnatal development, the basal adenylate cyclase activities in either cerebrum and cerebellum particulate preparations progressively increased. While the fluoride sensitivity of the cerebrum and cerebellum enzyme increased during postnatal development, the responsiveness to forskolin remained unaltered. There was no enhancement of soluble adenylate cyclase (from rat testis) by forskolin under the assay conditions in which there was a marked stimulatory action on the particulate enzyme. The results seen with the solubilized enzyme, with either Lubrol PX or cholate, indicate that the effects of forskolin on the cyclase do not require either G/F protein or calmodulin and the results of our study of brain enzymes support this view. Data on soluble testis cyclase (a poor or absent response to forskolin by this enzyme) imply that it lacks a protein (other than the catalytic unit) which could confer greater stimulation. The present results do not rule out an alternative explanation that forskolin stimulates adenylate cyclase by a direct interaction with the catalytic subunit, if the catalytic proteins do differ widely in various species of cells and their response to this diterpene.     

Stimulation of erythroid cells adenylate cyclase by soluble factors

Soluble factors obtained from human, rat and rabbit erythroid cell lysates are capable to stimulate basal and hormone activated adenylate cyclase of erythroid cell membranes from homologous sources. Extensive dialysis and removal of hemoglobin from the soluble factors do not modify their activity. Human erythrocyte soluble factors stimulate the human reticulocyte enzyme. Nevertheless human erythrocyte adenylate cyclase is not stimulated by either of the soluble factors. The presence of active soluble factors in human erythrocytes where the adenylate cyclase is no longer sensitive to these factors, as well as to guanylnucleotides or protaglandins, indicates that the enzyme has been altered during the maturation processes.     

Inhibition of soluble guanylate cyclase by ODQ

The heme in soluble guanylate cyclases (sGC) as isolated is ferrous, high-spin, and 5-coordinate. [1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one] (ODQ) has been used extensively as a specific inhibitor for sGC and as a diagnostic tool for identifying a role for sGC in signal transduction events. Addition of ODQ to ferrous sGC leads to a Soret shift from 431 to 392 nm and a decrease in nitric oxide (NO)-stimulated sGC activity. This Soret shift is consistent with oxidation of the ferrous heme to ferric heme. The results reported here further define the molecular mechanism of inhibition of sGC by ODQ. Addition of ODQ to the isolated sGC heme domain [beta1(1-385)] gave the same spectral changes as when sGC was treated with ODQ. EPR and resonance Raman spectroscopy was used to show that the heme in ODQ-treated beta1(1-385) is indeed ferric. Inhibition of the NO-stimulated sGC activity by ODQ is due to oxidation of the sGC heme and not to perturbation of the catalytic site, since the ODQ-treated sGC has the same basal activity as untreated sGC (68 +/- 12 nmol min(-)(1) mg(-)(1)). In addition, ODQ-oxidized sGC can be re-reduced by dithionite, and this re-reduced sGC has identical NO-stimulated activity as the original ferrous sGC. Oxidation of the sGC heme by ODQ is fast with a second-order rate constant of 8.5 x 10(3) M(-)(1) s(-)(1). ODQ can also oxidize hemoglobin, indicating that the reaction is not specific for the heme in sGC versus that in other hemoproteins.     

Inhibition of neuroblastoma adenylate cyclase by cannabinoid and nantradol compounds

This study was undertaken to ascertain the effects of cannabinoid drugs on prostanoid-stimulated adenylate cyclase in neuroblastoma cells. This report demonstrates that Δ⁹-tetrahydrocannabinol (THC) and levonantradol could decrease initial rate cyclic AMP accumulation in response to prostacyclin in intact cells. Basal accumulation was also diminished. Prostanoid-stimulated adenylate cyclase in a membrane preparation from these cells was inhibited by cannabinoid and nantradol compounds. However, this inhibition was not competitive with prostaglandin E₁ or prostacyclin. Further, inhibition was also observed when the enzyme was stimulated by peptide hormones at the secretin receptor. In contrast, enzyme activated by NaF was not inhibited by cannabinoid compounds. Cyclic AMP phosphodiesterase activity in subcellular fractions was unaltered by these agents. These data demonstrate that cannabinoid and nantradol compounds decrease cyclic AMP accumulation in neuronally derived cells, and that this results from an inhibition of basal and hormone-stimulated adenylate cyclase activity.     

Inhibition of pituitary adenylate cyclase by atrial natriuretic factor

The effect of synthetic rat atrial natriuretic factor (ANF) on adenylate cyclase activity was studied in rat anterior and posterior pituitary homogenates. ANF (Arg 101-Tyr 126) inhibited adenylate cyclase activity in anterior and posterior pituitary homogenates in a concentration dependent manner. The maximum inhibitions observed were 42% in anterior pituitary with an apparent Ki of 10(-10) M, and 25% with an apparent Ki of 10(-11) M in posterior pituitary. Corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP) and prostaglandins (PGE1) stimulated adenylate cyclase to various degrees in anterior pituitary homogenates and ANF inhibited the stimulatory effect of all these hormones. In addition ANF was also able to inhibit the stimulation exerted by NaF and forskolin which activate adenylate cyclase by receptor independent mechanism. Similarly, the stimulatory effects of N-Ethylcarboxamide adenosine (NECA), NaF and forskolin on adenylate cyclase in posterior pituitary homogenates were also inhibited by ANF. This is the first study demonstrating the inhibitory effect of ANF on pituitary adenylate cyclase.     

Activation of Neurospora crassa soluble adenylate cyclase by calmodulin.

The soluble form of adenylate cyclase was extracted and purified from wild-type Neurospora crassa mycelia. Brain or N. crassa calmodulin significantly enhanced this enzyme activity in assay mixtures containing Mg2+-ATP as substrate. EGTA reverses this calmodulin activation.     

Inhibition of soluble guanylate cyclase activity by citrate

Soluble guanylate cyclase activity from guinea pig heart is inhibited by increasing concentrations of sodium citrate. The Ki value was found to be 2.83 +/- 0.05 mM in the presence of 3 mM Mn2+ and 0.6 mM GTP. Citrate acts by lowering Vmax and increasing the apparent values of Km for GTP and K0.5 for Mn2+ and Mg2+. The soluble guanylate cyclase, activated by sodium nitroprusside, was also inhibited by citrate. This inhibitory action of citrate was not restricted to soluble guanylate cyclase activity of the heart and has been demonstrated also in the supernatant of lung, liver, diencephalon and in the homogenate of blood platelets. Since citrate is known to be an important intermediate of metabolism, its intracellular concentration may be also of relevance for guanylate cyclase activity.     

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.     

Inhibition of forskolin-stimulated cardiac adenylate cyclase activity by short-chain alcohols

The diterpene forskolin stimulated rat cardiac adenylate cyclase activity at least 20-fold and potentiated the effect of NaF. The stimulatory effect of forskolin was reduced in the presence of Gpp(NH)p. Ethanol markedly reduced the stimulation of adenylate cyclase by forskolin while potentiating NaF and Gpp(NH)p stimulation. The inhibitory effect of ethanol on forskolin stimulation appeared to be of a mixed type with both a competitive and a non-competitive component. Three other short-chain linear alcohols (methanol, propanol, butanol) also inhibited forskolin-stimulation, this effect being proportional to the number of carbon atoms.     

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

Heparin inhibits (I50 = 2 microgram/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'-(beta,gamma-imido)triphosphate were inhibited to a lesser extent. Human chorionic gonadotropin binding to this membrane preparation was inhibited by heparin (I50 = 6 microgram/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. Heparin (3 microgram/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 heparin molecule interferes with enzyme receptor coupling, by restricting the mobility of these components or by effecting their conformation.     

Inhibition of the catalytic subunit of ram sperm adenylate cyclase by adenosine

Adenosine inhibits ram sperm adenylate cyclase activity which is membrane-bound and comprises only the catalytic subunit. The inhibition parameters of adenylate cyclase by adenosine were not modified when the enzyme was purified 3 to 5,000 fold. Optimal inhibition by adenosine was found to require a high concentration of manganese, and exhibited a noncompetitive pattern up to a concentration of 1 mM adenosine. Adenosine was the most potent inhibitor among various analogs tested with the following rank order of potencies: adenosine greater than 2'O-methyladenosine greater than 2'deoxyadenosine much greater than 2 chloroadenosine. Studies with agonists and antagonists of the "R"-type adenosine receptor led us to conclude that adenosine inhibits ram sperm adenylate cyclase via a "P"-site carried by the catalytic subunit itself.     

Inhibition of soluble guanylate cyclase by bovine serum albumin

Bovine serum albumin (BSA) and to a lesser extent beta-lactoglobulin produced concentration-dependent inhibition of the guanylate cyclase activity in supernatant fraction and partially purified enzyme (PPE) prepared from rat lung homogenates. Ovalbumin had little effect. Some activity was lost when PPE was applied to a BSA-agarose column, however the loss disappeared when the enzyme reaction mixture contained Lubrol PX. Also, BSA no longer inhibited PPE after BSA-agarose treatment. BSA inhibition of PPE was not apparent when activity was maximally stimulated by arachidonate. These data were interpreted as indicating that the enzyme had bound to it an amphiphilic activator, possibly a fatty acid, the removal of which by BSA decreased activity.     

Forskolin-activated adenylate cyclase. Inhibition by guanyl-5'-yl imidodiphosphate

Forskolin activated adenylate cyclase of purified rat adipocyte membranes in the absence of exogenous guanine nucleotides. Guanyl-5'-yl imidodiphosphate (Gpp(NH)p) inhibited the forskolin-activated cyclase immediately upon addition of the nucleotide at concentrations too low to activate adenylate cyclase (10(-9) to 10(-7) M). Inhibition seen with a very high concentration of Gpp(NH)p (10(-4) M) lasted for 3-4 min and was followed by an increase in the synthetic rate which remained constant for at least 15 min. The length of the transient inhibition did not vary with forskolin concentrations above 0.05 microM but low Gpp(NH)p (10(-8) M) exhibited a lengthened (6-7 min) inhibitory phase. The transient inhibitory effects of Gpp(NH)p were eliminated by 10(-7) M isoproterenol, high (40 mM) Mg2+, or preincubation with Gpp(NH)p in the absence of forskolin. While forskolin stimulated fat cell cyclase in the presence of Mn2+, this ion blocked the inhibitory effects of Gpp(NH)p. The well documented inhibitory effects of GTP on the fat cell adenylate cyclase system were also observed in the presence of forskolin. However, the inhibition by GTP is not transitory. These findings indicate that Gpp(NH)p regulation of forskolin-stimulated cyclase has at least two components: 1) an inhibitory component which acts through an undetermined mechanism and which acts immediately to decrease cyclase activity; and 2) an activating component which modulates the inhibited cyclase activity through the guanine nucleotide regulatory protein.     

Inhibition of adenylate cyclase activity of human thyroid membranes by gangliosides

Gangliosides inhibit basal, thyrotropin-induced and fluoride-induced adenylate cyclase activity of human thyroid membranes in physiological conditions. In contrast neutral glycolipids, phospholipids and neuraminic acid containing oligosaccharides show no effect. The efficacy of inhibition is more dependent upon the position of the sialic acid residues than upon their absolute number. In general gangliosides with disialyl groups are more inhibitory than those with single sialyl moieties. The inhibitory effects of the individual gangliosides on the two modes of stimulation are parallel. This parallelism suggests that the inhibitory effect is located at the postreceptor level and that the gangliosides interact directly with the adenylate cyclase system. A possible role of thyroid membrane gangliosides as suppressive cofactors of adenylate cyclase is discussed in relation to recent findings of stimulating anti-ganglioside antibodies in Graves' disease.