Effects of local anesthetics on guanyl nucleotide modulation of the catecholamine-sensitive adenylate cyclase system and on beta-adrenergic receptors

Tetracaine and other local anesthetics exert multiple actions on the catecholamine-sensitive adenylate cyclase system of frog erythrocyte membranes. Tetracaine (0.2--20 mM) reduces the responsiveness of adenylate cyclase to (a) guanyl-5'-yl-imidodiphosphate and (b) isoproterenol in the presence of GTP or guanyl-5'-yl-imidodiphosphate. Local anesthetics did not affect (a) basal enzyme activity, and (b) enzyme responsiveness to NaF. Tetracaine inhibited stimulation of adenylate cyclase by guanyl-5'-yl-imidodiphosphate over the whole range of nucleotide concentrations. By contrast, inhibition by tetracaine of isoproterenol activity in the presence of GTP was significant only if GTP concentrations exceeded 10(-7) M. Tetracaine also competitively inhibited binding of both the antagonist [3H]dihydroalprenolol and the agonist [3H]hydroxybenzylisoproterenol to beta-adrenergic receptors. However, it was twice as potent in inhibiting [3H]hydroxybenzylisoproterenol as [3H]dihydroalprenolol binding. The greater potency for inhibition of agonist binding was due to the ability of the anesthetics to promote dissociation of the high-affinity nucleotide sensitive state of the beta-adrenergic receptor induced by agonists. Other local anesthetics mimicked the effects of tetracaine on adenylatecyclase and in dissociating high-affinity agonist-receptor complexes. The other of potency for both processes was dibucaine greater than tetracaine greater than bupivacaine greater than lidocaine which agrees with their relative potencies as local anesthetics. By contrast, a different order of potency was observed for competitive inhibition of [3H]dihydroalprenolol binding: dibucaine greater than tetracaine greater than greater than lidocaine greater than bupivacaine.     

Effects of local anesthetics of guanyl nucleotide modulation of the catecholamine-sensitive adenylate cyclase system and on β-adrenergic receptors

Tetracaine and other local anesthetics exert multiple actions on the catecholamine-sensitive adenylate cyclase system of frog erythrocyte membranes. Tetracaine (0.2–2.0 mM) reduces the responsiveness of adenylate cyclose to (a) guanyl-5′-yl-imidodiphosphate and (b) isoproterenol in the presence of GTP or guanyl-5′-yl-imidodiphosphate. Local anesthetics did not affect (a) basal enzyme activity, and (b) enzyme responsiveness to NaF. Tetracaine inhibited stimulation of adenylate cyclase by guanyl-5′-yl-imidodiphosphate over the whole range of nucleotide concentrations. By contrast, inhibition by tetracaine of isoproterenol activity in the presence of GTP was significant only if GTP concentrations exceeded 10^?7 M.Tetracaine also competitively inhibited binding of both the antagonist [³H]-dihydroalprenolol and the agonist [³H]hydroxybenzylisoproterenol to β-adrenergic receptors. However, it was twice as potent in inhibiting [³H]-hydroxybenzylisoproterenol as [³H]dihydroalprenolol binding. The greater potency for inhibition of agonist binding was due to the ability of the anesthetics to promote dissociation of the high-affinity nucleotide sensitive state of the β-adrenergic receptor induced by agonists.Other local anesthetics mimicked the effects of tetracaine on adenylate     

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.     

Characterization of an ATP-Mg2+-dependent guanine nucleotide-stimulated adenylate cyclase from Neurospora crassa

A novel adenylate cyclase activity was found in crude homogenates of Neurospora crassa. The adenylate cyclase had substantial activity with ATP-Mg2+ as substrate differing significantly from the strictly ATP-Mn2+-dependent enzyme characterized previously. Additionally, the ATP-Mg2+-dependent activity was stimulated two- to fourfold by GTP or guanyl-5'-yl-imido-diphosphate (Gpp(NH)p). We propose that the ATP-Mg2+-dependent, guanine nucleotide-stimulated activity is due to a labile regulatory component (G component) of the adenylate cyclase which was present in carefully prepared extracts. The adenylate cyclase had a pH optimum of 5.8 and both the catalytic and G component were particulate. The Km for ATP-Mg2+ was 2.2 mM in the presence of 4.5 mM excess Mg2+. Low Mn2+ concentrations had no effect on adenylate cyclase activity whereas high concentrations of Mn2+ or Mg2+ stimulated the enzyme. Maximal Gpp(NH)p stimulation required preincubation of the enzyme in the presence of the guanine nucleotide and the K1/2 for Gpp(NH)p stimulation was 110 nM. Neither fluoride nor any of a variety of glycolytic intermediates or hormones, including glucagon, epinephrine, and dopamine, had an effect on ATP-Mg2+-dependent adenylate cyclase activity. However, the enzymatic activity was stimulated not only by GTP but also by 5'-AMP and was inhibited by NADH.     

Inhibition of adenylate cyclase from luteinized rat ovary by monovalent cations: roles of the stimulatory guanine nucleotide-binding regulatory component and stimulatory hormone receptor

Sodium and other monovalent cations (added as chloride salts) inhibited adenylate cyclase of luteinized rat ovary. Sodium chloride (150 mM) inhibited basal enzyme activity by 20%. Sodium chloride inhibition was enhanced to 34-54% under conditions of enzyme stimulation by guanine nucleotides (GTP and its nonhydrolyzable analog 5'-guanylyl imidodiphosphate), fluoride anion, and agonists (ovine luteinizing hormone (oLH) and the beta-adrenergic catecholamine isoproterenol) acting at stimulatory receptors linked to adenylate cyclase. Sodium chloride inhibition was dependent on salt concentration over a wide range (25-800 mM) as well as the concentrations of GTP and oLH. Inhibition by NaCl was of rapid onset and appeared to be reversible. The order of inhibitory potency of monovalent cations was Li+ greater than Na+ greater than K+. The role of individual components of adenylate cyclase in the inhibitory action of monovalent cations was examined. Exotoxins of Vibrio cholerae and Bordetella pertussis were used to determine respectively the involvement of the stimulatory and inhibitory guanine nucleotide-binding regulatory components (Ns and Ni) in NaCl inhibition. Sodium chloride inhibited cholera toxin-activated adenylate cyclase activity by 29%. Ni did not appear to mediate cation inhibition of adenylate cyclase because pertussis toxin did not attenuate inhibition by NaCl. Enzyme stimulation by agents (forskolin and Mn2+) thought to activate the catalytic component directly was not inhibited by NaCl but was instead significantly enhanced. Sodium chloride (150 mM) increased both the Kd for high-affinity binding of oLH to 125I-human chorionic gonadotropin binding sites and the Kact for oLH stimulation of adenylate cyclase by sevenfold. In contrast, NaCl had no appreciable effect on either isoproterenol binding to (-)-[125I]iodopindolol binding sites or the Kact for isoproterenol stimulation of adenylate cyclase. The results suggest that in luteinized rat ovary monovalent cations uncouple, or dissociate, Ns from the catalytic component and, in a distinct action, reduce gonadotropin receptor affinity for hormone. Dissociation of the inhibitory influence of Ni from direct catalytic activation could account for NaCl enhancement of forskolin- and Mn2+-associated activities. On the basis of these results, the spectrum of divergent stimulatory and inhibitory effects of monovalent cations on adenylate cyclase activities in a variety of tissues may be interpreted in terms of differential enzyme susceptibilities to cation-induced uncoupling of N and catalytic component functions.     

Mechanism of molybdate activation of adenylate cyclase

Molybdate activation of rat liver plasma membrane adenylate cyclase has been examined and compared with the effect of glucagon, Gpp(NG)p and fluoride. Glucagon does not stimulate the detergent solubilized enzyme, though molybdate, fluoride, and Gpp(NH)p are effective in this regard. The stimulatory effects of either fluoride or molybdate are additive with those of GTP and do not require guanyl nucleotide to evoke their activation. Neither fluoride nor molybdate can substitute for GTP when glucagon is the activator of rat liver adenylate cyclase. The stimulatory effects of either ion on adenylate cyclase are additive with that produced by glucagon. Activation of adenylate cyclase by either molybdate or fluoride occurs by a mechanism distinct from that of glucagon or guanyl nucleotide. The data presented here suggest that fluoride and molybdate may act via a similar mechanism of action. Neither ion displays a lag in activation of adenylate cyclase. The pH profiles of fluoride and molybdate-stimulated adenylate cyclase activity are similar, and distinct from guanyl nucleotide-stimulated activity. Cholera toxin treatment of adenylate cyclase blocks fluoride and molybdate stimulation of the enzyme to the same extent, while enhancing the activation obtained with GTP and hormones.     

Detergent-induced distinctions between fluoride- and vanadate-stimulated adenylate cyclases and their responses to guanine nucleotides

The influence of detergents on fluoride- and vanadate-stimulated adenylate cyclases was investigated with enzyme from liver and adipocyte plasma membranes. Stimulation of the adipocyte cyclase by Na3VO4 was maximal (sixfold) at 3 mM, was not additive with fluoride stimulation, and was readily reversed by washing of the membranes. Vanadate stimulation of the hepatic cyclase was specifically blocked by catechol, which had no effect on basal activity or on fluoride- or glucagon-stimulated activities. The hepatic enzyme, stimulated by fluoride ion, guanyl-5'-yl-(beta,gamma-imino)diphosphate (GPP(NH)P), or GPP(NH)P and glucagon, was inhibited by vanadate with 50% inhibition seen with 2 to 6 mM vanadate. The fluoride-activated adipocyte adenylate cyclase was inhibited by guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S) more potently than by GPP(NH)P, with 50% inhibition being seen with 10 nM GTP gamma S or 100 nM GPP(NH)P. These nucleotides also inhibited the vanadate-stimulated enzyme, but with one-third the potency seen with the fluoride-activated cyclase. Dispersion of the adipocyte cyclase by Lubrol-PX into a 30,000g supernatant fraction caused no change in activation of the enzyme by fluoride, but reduced vanadate-stimulated activity 80%. By comparison, this treatment enhanced stimulation by GPP(NH)P twofold and by GTP gamma S threefold. More importantly, perhaps, the treatment with detergent blocked inhibition of the basal enzyme by GTP, blocked inhibition of fluoride- and vanadate-stimulated cyclases by GTP, GPP(NH)P, or GTP gamma S, and rendered vanadate-stimulated activity sensitive to enhancement by guanine nucleotides. The data indicate differences in the actions of vanadate and fluoride, made evident by the influence of guanine nucleotides and detergent treatment. The observations would be consistent with the idea that the effects of vandate may be due to the formation of GDP X V on the enzyme. The data strongly suggest that treatment of adenylate cyclase with Lubrol-PX causes a functional blockade in the guanine nucleotide-dependent inhibitory regulation (mediated by Ni), thereby allowing activation by the stimulatory guanine nucleotide-dependent regulatory component (Ns).     

GTP-dependent anion-sensitive adenylate cyclase in snail ganglia potentiation of neurotransmitter effects

Snail ganglia possess an anion-sensitive adenylate cyclase. This enzyme was stimulated 100% by chloride in a strictly GTP-dependent manner. The apparent affinity of chloride for adenylate cyclase was 2 X 10(-4) M. Halogens were found to be the most active anions. Some inorganic anions such as SO4(2-) and H2PO4- were inactive, as were all the organic anions tested. Stimulation was not cumulative for any maximal concentration of the active anions except fluoride. Chloride potentiated the effect of fluoride, indicating that the anion effect is not fluoride-like. Another striking result is that chloride enhanced adenylate cyclase sensitivity to the neurotransmitters serotonin and dopamine. The absence of chloride stimulation when Mg2+ was replaced by Mn2+ further indicates a role of the GTP-binding protein (the G/F unit). Chloride could reversibly stimulate the adenylate cyclase activity already maximally stimulated by guanyl 5'-imidodiphosphate. We therefore suggest that, in snail ganglia, chloride raises the activity of the G/F unit-catalytic unit complex at some stage after its formation. The same specific anion-sensitive adenylate cyclase was also found in some of the rat tissues tested.     

Inhibitory effects of pentacaine and some related local anaesthetics on rat hepatic adenylate cyclase

In the present study effects of a new local anaesthetics, pentacaine (trans-2-pyrolidinocyclohexylester of 3-pentyloxyphenylcarbamic acid), and of some chemically related compounds on rat hepatic adenylate cyclase activity were studied under various experimental conditions. As compared with tetracaine, the local anaesthetics tested showed stronger inhibitory effects, regardless of the type of stimulating agents used to activate adenylate cyclase. The most potent effect was observed with pentacaine. Its inhibitory effects on glucagon, guanylylimidodiphosphate (Gpp/NH/p), sodium fluoride or forskolin stimulated activity suggest that it may directly act on the catalytic unit of adenylate cyclase. The same conclusion can be drawn based on its inhibitory effects on adenylate cyclase, regardless ATP concentrations used as the enzyme substrate, and on octylpyranoside solubilized enzyme activated by preincubation of the enzyme preparation with Gpp/NH/p. Structure-activity studies have suggested that the pentacaine molecule as a whole and none of its parts alone or its analogs are responsible for the inhibitory effect. However, the inhibitory effects of these compounds on the rat adenylate cyclase activity do not correlate with their local anaesthetic properties. The possibility of using adenylate cyclase inhibitors to decrease cyclic AMP production under pathological conditions, like in cholera, known to be due to a high adenylate cyclase activity, is discussed.     

Studies on the mechanism of action of local anesthetics on proton translocating ATPase from Mycobacterium phlei

We have measured the inhibitory potencies of local anesthetics (procaine, lidocaine, tetracaine and dibucaine) on ATP-mediated H+-translocation, Ca2+-transport and ATPase activity in membrane vesicles from Mycobacterium phlei. Procaine and lidocaine up to 1 mM concentration did not inhibit ATP-dependent H+-translocation, Ca2+-transport and ATPase activity. However, tetracaine and dibucaine at 0.2 mM concentration caused dissipation of the proton gradient, measured by the reversal of the quenching of fluorescence of quinacrine, and inhibition of active Ca2+-transport. Tetracaine (1 mM) inhibited membrane-bound ATPase activity without affecting solubilized F1-ATPase activity. Studies show that these local anesthetics do not prevent the inactivation of F0-F1 ATPase by dicyclohexylcarbodiimide (DCCD). Binding of [14C]DCCD to F0-proteolipid component remained unchanged in the presence of tetracaine indicating that DCCD and tetracaine do not share common binding sites on the F0-proteolipid sector. The inhibition of H+-translocation and membrane-bound ATPase activity by tetracaine was substantially additive in the presence of vanadate.     

Stimulation of rat liver adenylate cyclase by halothane.

Rat liver membranes prepared by a modification of the procedure of Neville were exposed to clinical and toxic concentrations of the general anesthetic, halothane, for 10 minutes. Basal, glucagon (5 × 10⁻⁵M) and sodium fluoride (20 mM) stimulated adenylate cyclase activity was assayed. Clinical and toxic concentrations of halothane augmented basal adenylate cyclase activity. Glucagon and sodium fluoride stimulated adenylate cyclase activity was enhanced at greater than clinically useful halothane concentrations only. The study provides direct evidence that halothane stimulates adenylate cyclase, the extent of augmentation of enzyme activity is halothane concentration dependent, and modified by other drugs.     

The role of phospholipids in TSH stimulation of adenylate cyclase in thyroid plasma membranes

Treatment of bovine thyroid plasma membranes with phospholipase A or C inhibited the stimulation of adenylate cyclase activity by thyroid-stimulating hormone (TSH). In general, basal and NaF-stimulated adenylate cyclase activity was not influenced by such treatment. When plasma membranes were incubated with 1–2 units/ml phospholipase A, subsequent addition of phosphatidylcholine or phosphatidylserine but not phosphatidylethanolamine partially restored TSH stimulation. Phosphatidylcholine was more effective than phosphatidylserine in that it caused greater restoration of the TSH response and smaller amounts of phosphatidylcholine were active. However, when the TSH effect was obliterated by treatment of plasma membranes with 10 units/ml phospholipase A, phospholipids were unable to restore any response to TSH. Lubrol PX, a nonionic detergent, inhibited basal, TSH- and NaF-stimulated adenylate cyclase activities in thyroid plasma membranes. Although phosphatidylcholine partially restored TSH stimulation of adenylate cyclase activity in the presence of Lubrol PX, it did not have a similar effect on the stimulation induced by NaF. These results indicate that phospholipids are probably essential components in the system by which TSH stimulates adenylate cyclase activity in thyroid plasma membranes. The effects do not seem to involve the catalytic activity of adenylate cyclase but the data do not permit a distinction between decreased binding of TSH to its receptor or impairment of the signal from the bound hormone to the enzyme activity.     

Effects of drugs on pigeon erythrocyte membrane and asymmetric control or adenylate cyclase by the lipid bilayer.

In pigeon erythrocyte membrane, the beta-adrenergic receptor and the enzyme adenylate cyclase can be uncoupled in two different ways depending on the type of drug used. Cationic drugs: chlorpromazine, methochlorpromazine, tetracaine, n-octylamine and a neutral alcohol, octanol, abolished alprenolol receptor binding ability and in the same range of concentration of the drug, sensitized adenylate cyclase to fluoride or Gpp(NH)p stimulation. Anionic drugs: di- and trinitro-phenols, indomethacin and octanoic acid did not affect the total number of beta-adrenergic receptor sites and, with the exception of trinitrophenol, did not change the association constant for alprenolol but they abolished the stimulation of adenylate cyclase by isoproterenol, fluoride or Gpp(NH)p. These modifications of the adenylate cyclase system occurred in a range of drug concentration where cell shape and protection against hemolysis were also affected. As chemical composition varies widely from one drug to another, it is suggested that these effects are largely nonspecific and mediated by the lipid bilayer. They are probably related to a preferential sidedness of action of the drugs in the lipid bilayer, displaying the role of an asymmetric control of the adenylate cyclase system in the membrane by the two halves of this bilayer.     

Activation of adenylate cyclase by molybdate.

The effect of molybdate on adenylate cyclase (EC 4.6.1.1) in rat liver plasma membranes has been examined. The apparent K alpha for molybdate activation of the enzyme is 4.5 mM, and maximal, 7-fold stimulation is achieved at 50 mM. The observed increase in cAMP formation in the adenylate cyclase assay is not due to: (a) an inhibition of ATP hydrolysis; (b) a molybdate-catalyzed conversion of ATP to cAMP; (c) an inhibition of cAMP hydrolysis; or (d) an artifact in the isolation of cAMP formed in the reaction. Molybdate activation of adenylate cyclase is a general phenomenon exhibited by the enzyme in brain, cardiac, and renal tissue homogenates and in erythrocyte ghosts. However, like fluoride and guanyl-5'-yl imidodiphosphate (Gpp(NH)p), molybdate does not activate the soluble rat testicular adenylate cyclase. Molybdate is a reversible activator of adenylate cyclase. Activation is not due to an increase in ionic strength and is independent of the salt used to introduce molybdate. Molybdate does not activate adenylate cyclase previously stimulated with Gpp(NH)p or fluoride. At concentration greater than 20 mM, molybdate inhibits fluoride-stimulated adenylate cyclase, and at concentrations greater than 100 mM, molybdate stimulation of basal adenylate cyclase activity is diminished.     

The guanine nucleotide-binding regulatory component of adenylate cyclase in human erythrocytes

The guanine nucleotide-binding regulatory component of adenylate cyclase (G/F) has been purified from human erythrocyte membranes. It is composed of two major polypeptides with molecular weights of 35,000 and 45,000. When cyc- S49 lymphoma cell plasma membranes are reconstituted with purified human erythrocyte G/F, stimulation of adenylate cyclase by beta-adrenergic agonists, guanine nucleotides, and fluoride is restored. Binding of GTP gamma S to human erythrocyte G/F and GTP gamma S-mediated activation of the protein are closely correlated. The agreement between the apparent dissociation constants for these two reactions suggests that the measured binding site is identical to the site responsible for activation. A 41,000-dalton protein has been identified as a contaminant of preparations of G/F that have been purified by four successive chromatographic steps. This protein serves as a specific substrate for ADP-ribosylation and labeling by islet activating protein (IAP) and [32P]NAD, and it appears to contribute an additional high-affinity guanine nucleotide binding site to such preparations.     

Liver membrane adenylate cyclase. Synergistic effects of anions on fluoride, glucagon, and guanyl nucleotide stimulation.

Some effects of salts on the adenylate cyclase of partially purified plasma membranes from rat liver have been studied. Under conditions where cyclic adenosine 3':5'-monophosphate formation was linear with respect to time and protein concentration, the enzyme was stimulated 3- to 6-fold by 10 mM NaF, 10- to 30-fold by 1 muM glucagon, 4- to 5-fold by 0.1 mM 5'-guanylylimidodiphosphate, and in the presence of 3 muM GTP, 2-fold by 10 mug/ml of prostaglandin E1. Various salts were found to stimulate basal activity slightly, but enhanced the response to NaF 3- to 4-fold, to glucagon 1.5- to 2-fold, to 5'-guanylylimidodiphosphate 2- to 3-fold, and to prostaglandin E1 1.5-fold. This enhancement was observed at maximally effective concentrations of each of the respective activators. Of the salts tested, NaN3 and the Na- or K-halides were most effective. Their action appeared to be due to the respective anions. Stimulation was detectable with 1.5 mM NaN3 or 3 mM NaCl and was maximal with 30 mM NaN3 or 60 mM NaCl. The stimulatory effect of NaN3 was not due to ATP-sparing, nor to an altered cyclic adenosine 3':5'-monophosphate recovery. It was independent of the chromatography and assay methods used, and was therefore not due to procedural artifact. Fluoride-stimulated cyclase activity was enhanced by salts to a greater degree than were 5'-guanylylimidodiphosphate-, glucagon-, or (prostaglandin E1 + GTP)-stimulated activities. The effects of NaN3 were not the result of significant changes in the enzyme's responses to GTP, which increased basal and glucagon-stimulated activities but inhibited F--stimulated activity. The effects of NaN3 were greater when cyclase was assayed with Mn2+ than with Mg2+. The facilitatory effect of NaN3 or NaCl on fluoride-stimulated adenylate cyclase activity was partially reversible as was the stimulatory effect of fluoride in the presence of NaN3. Enhancement of hormonal stimulation by NaN3 was also demonstrable with cardiac and adipose tissue adenylate cyclase. However, NaN3 did not stimulate detergent-dispersed adenylate cyclases from either liver plasma membranes or brain. The data suggest that stimulation of adenylate cyclase by salts may require the added presence of other stimulatory agents and an intact membrane structure.     

Stimulation and inhibition of rat basophilic leukemia cell adenylate cyclase by forskolin

The influence of the diterpene, forskolin, was studied on adenylate cyclase activity in membranes of rat basophilic leukemia cells. Forskolin increased basal adenylate cyclase activity maximally 2-fold at 100 microM. However, adenylate cyclase activity stimulated via the stimulatory guanine nucleotide-binding protein, Ns, by fluoride and the stable GTP analog, guanosine 5'-O-(3-thiotriphosphate), was inhibited by forskolin. Half-maximal and maximal inhibition occurred at about 1 and 10 microM forskolin, respectively. The inhibition occurred without an apparent lag phase, whereas the enzyme stimulation by forskolin was preceded by a considerable lag period. The inhibition was not affected by treating intact cells or membranes with pertussis toxin and proteolytic enzymes, respectively, which have been shown in other cell types to prevent adenylate cyclase inhibition mediated by the guanine nucleotide-binding regulatory component, Ni. The forskolin inhibition of the stable GTP analog-activated adenylate cyclase was impaired by increasing the Mg2+ concentration and was reversed into a stimulation by Mn2+. Under optimal inhibitory conditions, forskolin even decreased basal adenylate cyclase activity. Finally, forskolin largely reduced the apparent affinity of the rat basophilic leukemia cell adenylate cyclase for its substrate, MgATP, which reduction resulted in an apparent inhibition at low MgATP concentrations and a loss of the inhibition at higher MgATP concentrations. The data indicate that forskolin can cause both stimulation and inhibition of adenylate cyclase and, furthermore, they suggest that the inhibition may not be mediated by the Ni protein, but may be caused by a direct action of forskolin at the adenylate cyclase catalytic moiety.     

Adenylate cyclase activity and cyclic nucleotide content in human adrenal tumors under Icenko-Cushing syndrome

The adenylate cyclase activity and cyclic nucleotide content in excised human adrenal tumours (Icenko-Cushing syndrome) were determined. The experimental data were compared to those obtained for hyperplastic adrenals. All adrenal tumours under study revealed a decreased cAMP level, an increased cGMP level and a resulting decrease of the cAMP/cGMP ratio. In malignant adrenal tumours the adenylate cyclase activity was sharply increased in comparison with that in hyperplastic adrenals. In the majority of malignant tumours the adenylate cyclase response to ACTH was either altogether absent or sharply decreased. In benign adrenal tumours the basal activity of the enzyme was unchanged and the enzyme response to ACTH was essentially normal. The decrease of adenylate cyclase response to ACTH in malignant tumours is apparently not due to the impaired catalytic activity of the enzyme, since its response to stimulation by sodium fluoride remains unaffected. In some tumours (one malignant and two benign ones) a non-specific stimulation of adenylate cyclase by hormones, which are not natural activators of the enzyme was observed. It was assumed that these changes are due to the damage of hormonal receptors in adrenal tumours.     

Calcitonin-sensitive adenylate cyclase in rat renal tubular membranes.

1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 x 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F- -and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F- -sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F- -stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors.     

Effects of drugs on pigeon erythrocyte membrane and asymmetric control of adenylate cyclase by the lipid bilayer

In pigeon erythrocyte membrane, the β-adrenergic receptor and the enzyme adenylate cyclase can be uncoupled in two different ways depending on the type of drug used.Cationic drugs: chlorpromazine, methochlorpromazine, tetracaine, $\text{n-}\text{octylamine}$ and a neutral alcohol, octanol, abolished alprenolol receptor binding ability and in the same range of concentration of the drug, sensitized adenylate cyclase to fluoride or Gpp(NH)p stimulation. Anionic drugs: di- and trinitrophenols, indomethacin and octanoic acid did not affect the total number of β-adrenergic receptor sites and, with the exception of trinitrophenol, did not change the association constant for alprenolol but they abolished the stimulation of adenylate cyclase by isoproterenol, fluoride or Gpp(NH)p. These modifications of the adenylate cyclase system occurred in a range of drug concentration where cell shape and protection against hemolysis were also affected.As chemical composition varies widely from one drug to another, it is suggested that these effects are largely nonspecific and mediated by the lipid bilayer. They are probably related to a preferential sidedness of action of the drugs in the lipid bilayer, displaying the role of an asymmetric control of the adenylate cyclase system in the membrane by the two halves of this bilayer.