Hormonal specificity of the melanotropin-sensitive adenylate cyclase of mouse melanoma and effect of cyclic amp on the tyrosinase activity of mouse melanoma cells,in vitro

Transplantable mouse melanomas possess a melantropin-sensitive adenylate cyclase system which is responsive to α-melanotropin, β-melanotropin, adrenocorticotropin (ACTH) and prostglandin E₁. It was found that sensitivity to ACTH was not directed towrds the ACTH activity but to the intrinsic melanotropin activity of the ACTH molecule. Therefore, the melanotropin-sensitive adenylate cyclase system is hormonally specific to the intrinsic melanotropin activity of peptide hormones and is unique in the melanoma tissue. The significance of the sensitivity to prostaglandin E₁ is obscure at present. The melanotropin-sensitive adenylate cyclase requires the presence of Mg²⁺ or Mn²⁺ for its enzymatic activity. Ca²⁺ inhibit the enzyme in the presence of a wide range of concentrations of Mg²⁺. The enzymic activity is ATP concentration-dependent and the saturation concentration appears to be 1 mM. The enzyme is very labile in the unfractionated tumor homogenates. A washed 11 000 × g particulate fraction, representing about 30–60% of the total enzymic activity, was found to be more stable and could be stored at 5°C for 2 h without appreaciable loss of the activity. This fraction retained sensitivity to melanotropin, prostaglandin E₁ and NaF. About 20% of the activity of the tumor homogenate could not be sedimented by centrifugation at 105 000 × g for 60 min. This “soluble” fraction was not responsive to melanotropin, prostglandin E₁ and NaF and might be a degradative product produced by the fractionation. Cyclic AMP and α-melanotropin were able to increase the tyrosinase activity of isolated mouse melanoma-cells in vitro under the same conditions.     

Cytochemical localization of adenylate cyclase and of calcium ion, magnesium ion-activated ATPases in the dense tubular system of human blood platelets.

Cytochemical techniques have been employed to study the localization of adenylate cyclase and (Ca2+ + Mg2+)-stimulated ATPase activities in platelets after fixation. Biochemical analysis of adenylate cyclase demonstrated a 70% reduction in activity in homogenates from fixed cells, but the residual activity could be stimulated 10--20 times by prostaglandin E1 (1 micrometer) under the same incubation conditions as employed in the cytochemical studies (e.g. media containing 2 mM lead nitrate and 10 mM NaF). Adenylate cyclase activity employing 5'-adenylyl-imiodiphosphate (AMP-P(NH)P) as substrate was found to be associated with the dense tubular system (smooth endoplasmic reticulum) in intact fixed platelets, and was apparent only when the cells were incubated with prostaglandin E1. Less activity was found along the membranes of the surface connected open canalicular system and occasionally at the outer cell surface. Enzymatic activity was blocked by the adenylate cyclase inhibitor 9-(tetrahydro-2-furyl) adenine and was not due to AMP-P(NH)P phosphohydrolase activity. The low adenylate cyclase activity in the surface membranes may be due to enzyme inactivation as a result of fixation, since a surface membrane fraction obtained by the glycerol lysis technique from unfixed cells had an adenylate cyclase specific activity equivalent to that in the microsomal membrane fraction. (Ca2+ + Mg2+)-stimulated ATPase activity was found associated with the membranes of the surface connected open canalicular system in unfixed cells. After brief fixation (5--15 min) with glutaradehyde, strong (Ca2+ + Mg2+)ATPase activity became apparent in the dense tubular system. Longer periods of fixation inactivated enzymatic activity. Addition of Ca2+ (1.0 mM) to incubation medium with low Mg2+ (0.2 mM), or increasing Mg2+ to 4.0 mM, in both cases strongly stimulated enzyme activity. The ATPase activity in the platelet membranes was not inhibited by ouabain. It is suggested that the Ca2+-stimulated ATPase and adenylate cyclase activities in the dense tubules may possibly be involved in regulation of intracellular Ca2+ transport.     

A synthetic analog of the calmodulin-binding domain of myosin light chain kinase inhibits melanotropin receptor function and activation of adenylate cyclase

In this study a synthetic analog of the calmodulin-binding domain of myosin light chain kinase, a 17-amino-acid peptide (M5) was used to examine the possible role of calmodulin in melanotropin receptor function. Binding of beta-melanocyte-stimulating hormone to its membrane receptor and subsequent stimulation of adenylate cyclase (AC) were found to be specifically inhibited by M5 in a dose-dependent and noncompetitive manner, both in intact M2R melanoma cells and in a plasma membrane preparation derived thereof. Half-maximal inhibition of both hormone binding and melanotropin-sensitive AC activity was shown to occur at approximately 1 microM M5. In contrast, stimulation of AC by prostaglandin E1, guanosine 5'-O-(3-thio)triphosphate, forskolin, and unstimulated enzyme activity were unaffected by the presence of M5, under the same assay conditions. Furthermore, addition of a molar excess of calmodulin to the AC assay completely abolished the inhibitory effects of the peptide on melanotropin-stimulated AC activity. Other peptides of similar size, which bind to calmodulin with low affinity (e.g. glucagon, somatostatin, and vasoactive intestinal peptide), were shown to be totally ineffective in inhibiting melanotropin-sensitive AC. These findings, along with those shown previously for other antagonists of calmodulin, suggest a role for an M5-binding protein, as of yet unidentified, involved in the regulation of the melanotropin receptor function.     

Properties of cholera toxin- and NaF-stimulated adenylate cyclase from mouse thymocytes.

Kinetic parameters of mouse thymocyte adenylate cyclase activity were determined. NaF and cholera toxin stimulated adenylate cyclase. Stimulation by either agent did not change the pH or Mg2+ optima relative to control (unstimulated cyclase). The Km value for ATP of adenylate cyclase stimulated by NaF was significantly reduced from control. By contrast, cholera toxin treatment did not change the Km relative to control. Adenylate cyclase, when stimulated by NaF, had an optimum for Mn2+ alone, or Mn2+ in combination with Mg2+, at least twice that of control. In contrast, cyclase activity prepared from cells treated with cholera toxin remained unchanged with regard to these divalent cations when compared to control. Addition of NaF to adenylate cyclase prepared from cells treated with cholera toxin resulted in a significant reduction (30%) in activity suggesting that both NaF and cholera toxin were acting on the same cyclase. NaF inhibition of cholera toxin-stimulated activity was shown to be a direct interaction of fluoride on the stimulated cyclase enzyme. This inhibition appeared to be immediate and independent on pH, Mg2+ or ATP concentrations. Although NaF inhibition was lost when Mn2+ was present in the reaction mixture, the activity expressed by addition of NaF to cyclase prepared from cholera toxin-treated cells was much less than by addition of NaF to control. As observed with cholera toxin stimulation alone, activity expressed by the inhibited enzyme (cholera toxin treated + NaF) exhibited a Km for ATP and an optimum for Mn2+ alone or in combination with Mg2+ similar to control.     

Adenylate cyclase and cAMP-dependent protein kinase activity in the adrenal glands of animals after chronic administration of ACTH

ACTH, a prolonged action hormone, in a dose of 2.5 mu. was injected into guinea pigs daily for 5-35 days. The adenylate cyclase activity of the crude adrenal membrane fraction and the activity of cAMP-dependent protein kinases in the cytoplasmic fraction were determined. Cyclic changes in the basal and stimulated adenylate cyclase activities occurring with 15-20-day intervals have been established for the first time. The sensitivity of adenylate cyclase to ACTH, NaF and GTP did not change in the course of two cycles. The activity of cAMP-dependent protein kinases increased during the first few days after ACTH administration and decreased after further injections of the hormone. The role of cyclic changes of the enzyme activity in the mechanism of proliferative effect of ACTH is discussed.     

Regulation of platelet adenylate cyclase by adenosine.

The stimulatory and inhibitory effects of adenosine on the adenylate cyclases of human and pig platelets were studied. Stimulation occurred at lower concentrations than did inhibition, and the stimulatory effect was prevented by methylxanthines. Stimulation by adenosine was immediate in onset and was reversible, under conditions when cyclic AMP formation was linear with respect to time and protein concentration. The stimulatory and inhibitory effects could be distinguished further by the use of various analogues of adenosine and could be prevented by adenosine deaminase. The data suggest that both stimulation and inhibition were due to adenosine itself and not one of its degradation products and that in the platelet preparation, neither formation nor degradation of adenosine during the adenylate cyclase incubation appreciably influenced measured activity. Stimulation by adenosine was additive with the effects of GMP-P(NH)P, and alpha- or beta-adrenergic stimulation, but was abolished by prostaglandin E1 or by NaF. Prostaglandin E1 and NaF increased the sensitivity of adenylate cyclase to inhibition by adenosine. The data suggest that guanyl-5'-yl-(beta-gamma-imino)diphosphate and/or adrenergic stimulation and adenosine exert their effects on adenylate cyclase by distinct mechanisms, but that prostaglandin E1 or F- and adenosine increase enzyme activity by mechanisms which may involve common intermediates in the coupling to adenylate cyclase.     

Forskolin-resistant Y1 mutants harbor defects associated with the guanyl nucleotide-binding regulatory protein, Gs

The properties of the adenylate cyclase from forskolin-resistant mutants of Y1 adrenocortical tumor cells was compared with the properties of the enzyme from parental Y1 cells in order to localize the site of mutation. In parental Y1 cells, forskolin stimulated adenylate cyclase activity with kinetics suggestive of an interaction at two sites; in mutant cells, forskolin resistance was characterized by a decrease in enzymatic activity at both sites. Forskolin potentiated the enzyme's responses to NaF and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) in parent and mutant clones, and the mutant enzyme showed the same requirements for Mg2+ and Mn2+ as did the parent enzyme. The adenylate cyclase associated with forskolin-resistant mutants was insensitive to ACTH and was less responsive to Gpp(NH)p than was the parent enzyme. In parental Y1 cells and in the forskolin-resistant mutants, cholera toxin catalyzed the transfer of [32P]ADP-ribose from [32P]NAD+ into three membrane proteins associated with the alpha subunit of Gs; however, the amount of labeled ADP-ribose incorporated into mutant membranes was reduced by as much as 70%. Both parent and mutant membranes were labeled by pertussis toxin to the same extent. The insensitivity of the mutant adenylate cyclase to ACTH and Gpp(NH)p and the selective resistance of the mutant membranes to cholera toxin-catalyzed ADP-ribosylation suggest that a specific defect associated with Gs is involved in the mutation to forskolin resistance in Y1 cells.     

Cholera toxin and adenylate cyclase: properties of the activated enzyme in liver plasma membranes.

Adenylate cyclase (EC 4.6.1.1) activity in mouse liver plasma membranes is increased fivefold when animals are pretreated with cholera toxin. The increase in activity is detectable within 20 min of an intravenous injection of the toxin. The response of the control and cholera-toxin-activated adenylate cyclase to hormones, GTP, and NaF is complex. GTP causes the same fold stimulation of control and toxin-activated cyclase, but glucagon and NaF remain the most potent activators of liver adenylate cyclase irrespective of whether the enzyme is activated by cholera toxin. Determination of kinetic parameters of adenylate cyclase indicates that cholera toxin, hormones, and NaF do not change the affinity of the enzyme for ATP-Mg nor do they alter the Ka for free Mg2+. High concentrations of Mg2+ inhibit adenylate cyclase that is stimulated by either cholera toxin, glucagon, or NaF. These same Mg2+ concentrations have no effect on the basal activity of the enzyme or its activity in the presence of GTP.     

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.     

Purification of bovine brain inositol 1,4,5-trisphosphate 3-kinase. Identification of the enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.

Treatment of human platelets with concentrations of benzyl alcohol up to 50 mM augmented adenylate cyclase activity when it was assayed in the basal state and when stimulated by prostaglandin E1 (PGE1), isoprenaline or NaF. Benzyl alcohol antagonized the stimulatory effect exerted on the catalytic unit of adenylate cyclase by the diterpene forskolin. Benzyl alcohol did not modify the magnitude of the inhibitory response when the catalytic unit of adenylate cyclase was inhibited by using either low concentrations of guanosine 5'-[beta gamma-imido]triphosphate, which acts selectively on the inhibitory guanine nucleotide-regulatory protein Gi, or during alpha 2-adrenoceptor occupancy, by using adrenaline (+ propranolol). Some 34% of the potent inhibitory action of adrenaline on PGE1-stimulated adenylate cyclase was obliterated in a dose-dependent fashion (concn. giving 50% inhibition = 12.5 mM) by benzyl alcohol, with the residual inhibitory action being apparently resistant to the action of benzyl alcohol at concentrations up to 50 mM. Treatment of membranes with benzyl alcohol did not lead to the release of either the alpha-subunit of Gi or G-protein subunits. The alpha 2-adrenoceptor-mediated inhibition of adenylate cyclase was abolished when assays were performed in the presence of Mn2+ rather than Mg2+ and, under such conditions, dose-effect curves for the action of benzyl alcohol on PGE1-stimulated adenylate cyclase activity were similar whether or not adrenaline (+propranolol) was present. We suggest that (i) alpha 2-adrenoceptor- and Gi-mediated inhibition of PGE1-stimulated adenylate cyclase may have two components, one of which is sensitive to inhibition by benzyl alcohol, and (ii) the Gi-mediated inhibition of forskolin-stimulated adenylate cyclase exhibits predominantly the benzyl alcohol-insensitive component.     

Proof of adenylate cyclase activity in the coronary artery of cattle]

In two fractions obtained from the bovine A. coronaria adenylate cyclase activity was identified and characterized. The adenylate cyclase activity of the 75,000 X g sediment shows a pH optimum at 7.4. The temperature dependence of this adenylate cyclase activity is linear when represented in the Arrhenius plot, and an Arrhenius activation energy of 13.2 kcal Mol-1 can be calculated for the enzyme reaction. The Km-value of the enzyme to ATP is 6 +/- 0.6 - 10(-4) M. The adenylate cyclase activity of the 75,000 X g sediment can be stimulated by NaF. 5'AMP and adenosine inhibit the adenylate cyclase activity of the 75,000 X g sediment. With regard to the enzyme activity, Mn++ and Co++ replace Mg++, but not Ca++. The monovalentcations Na+ and K+ do not influence the adenylate cyclase activity. In a particulate fraction containing plasma membranes, adenylate cyclase activity was also identified. This adenylate cyclase activity can be stimulated by catecholamines, noradrenaline, and isoproterenol. This stimulation can, however, only be proved for the enzyme in the coronaries of 9-week-old and 2-year-old animals. The adenylate cyclase activity from the coronaries of adult animals is not affected by catecholamines. These findings are discussed with regard to hypertension frequently found in adult animals.     

Preparation of hormone-sensitive airway smooth muscle adenylate cyclase from dissociated canine trachealis cells

Conventional homogenizing methods produced membrane preparations of canine trachealis airway smooth muscle which contained adenylate cyclase activity that was stimulated by fluoride but not by isoproterenol. We have devised methods using collagenase digestion of minced trachealis which destroy most of the tough connective tissues but leave dissociated canine trachealis cells in suspension. Gentle homogenization of these cells permitted preparation of a particulate fraction containing adenylate cyclase that was readily stimulated by beta-adrenergic agonist of prostaglandin E2. Isoproterenol stimulation was 2.34 +/- 0.58 (S.E.) times basal and 122 +/- 25% of the stimulation induced by NaF. The beta-adrenergic blocking agent propranolol prevented isoproterenol-induced stimulation of the cyclase but had no effect on prostaglandin E2 stimulation. Catecholamine order of potency was isoproterenol greater than epinephrine greater than norepinephrine. These methods enable demonstration of stimulatory effects of hormones in broken cell preparations of airway smooth muscle that are comparable to those when hormone-stimulated cyclic AMP formation is measured in intact muscle strips.     

Angiotensin stimulates adenylate cyclase activity via prostaglandins in the adrenal glomerulosa membrane

The effects of angiotensin II (A II) on adenylate cyclase activities in membranes of the zona glomerulosa (the capsular fraction) and the zona fasciculata (the decapsulated fraction) from rat adrenocortical glands were investigated. A time- and GTP-dependent stimulation by A II of adenylate cyclase activity was observed in the capsular fraction but not in the decapsulated fraction. The activation of adenylate cyclase by ACTH and A II was additive. Stimulation by A II was inhibited by an angiotensin antagonist, DD-3487 (DD). Moreover, the addition of a prostaglandin antagonist, a mixture of polyesters of polyphloretin phosphate (PPP) and an inhibitor of prostaglandin synthesis, indomethacin, was effective in inhibiting A II-induced stimulation of the capsular adenylate cyclase activity, suggesting that the activation of A II receptors located on the capsular membrane leads to the release of prostaglandins, which in turn stimulates the adenylate cyclase.     

Subcellular localization and partial characterization of bovine corpus luteum adenylate cyclase.

The subcellular localization of adenylate cyclase (ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1) in bovine corpus luteum was studied using isotonic and hypotonic homogenization and fractionation conditions. All fractions prepared were assayed for adenylate cyclase, marker enzymes and DNA. Only plasma membrane marker enzyme, 5'-nucleotidase paralleled the distribution of adenylate cyclase under both isotonic and hypotonic conditions (conditionsoth isotonic and hypotonic conditions (coefficient of correlation = 0.95). Two main fractions prepared under hypotonic conditions were subfractionated by discontinuous sucrose gradient centrifugation. The highest amount of adenylate cyclase was found in a fraction having a density approximately equal to 1.13 g/cm3. The specific activity of this fraction was 4--6 times higher than that of the homogenate. The electron microscopic study of this fraction revealed the presence of a single type of particulate material consisting of small vesicles exhibiting a typical unit membrane structure. It is concluded that this adenylate cyclase is primarily localized in the plasma membranes. Basal adenylate cyclase activity of plasma membranes was stimulated 2--3 times by luteinizing hormone (10 mug/ml), 3--4 times by prostaglandin E2 (10 mug/ml), 4--6 times by NaF (0.01 M) and two times by methanol (0.2%).     

Mechanism of adenylate cyclase activation by the rat lung cytoplasmic factors

Epinephrine, histamine and prostaglandin E₁ stimulated adenylate cyclase activity in lung membranes and their stimulation of the enzyme activity was completely blocked by propranolol, metiamide and indomethacin, respectively. A partially-purified activator from the adult rat lung also enhanced adenylate cyclase activity in membranes. However, stimulation of adenylate cyclase by the rat lung activator was not abolished by the above receptor antagonists. Further, epinephrine, NaF and Gpp(NH)p stimulated adenylate cyclase activity rather readily, whereas stimulation of the enzyme activity by the lung activator was evident after an initial lag phase of 10 min. Also, the lung activator produced additive activation of adenylate cyclase with epinephrine, NaF and Gpp(NH)p. These results indicate that the lung activator potentiates adenylate cyclase activity in membranes by a mechanism independent from those known for epinephrine, NaF and Gpp(NH)p. Incubation of lung membranes for 30 min at 40°C resulted in a loss of adenylate cyclase activation by NaF and Gpp(NH)p. Addition of the released proteins to the heat-treated membranes did not restore the enzyme response to these agonists. However, heat treatment of lung membranes in the presence of 2-mercaptoethanol or dithiothreitol prevented the loss of adenylate cyclase response to NaF and Gpp (NH)p. N-ethylmaleimide abolished adenylate cyclase activation by epinephrine, NaF, Gpp(NH)p and the lung activator. These results indicate that the sulfhydryl groups are important for adenylate cyclase function in rat lung membranes.Abbreviations Gpp(NH)p 5-Guanylimidodiphosphate     

Fluoride activation of rat brain adenylate cyclase: the requirement for a protein co-factor.

Activation of particulate adenylate cyclase and detergent-soluble (lubrol PX) adenylate cyclase occurred when the enzyme was preincubated at 37 degrees in the presence of 5 mM NaF and 5mM MgSO4. Under these conditions the specific activity of the enzyme increased more than twofold in 8 to 12 min. Activation also occurred in the presence of 5 mM NaF alone, but the rate of activation was slower. Under these conditions, activation was inhibited by 1mM EDTA, but this inhibition was prevented byMg++. No activation was observed at 0 degrees or in the absence of F. After repeated extraction with detergent, particulate adenylate cyclase was not stimulated by mM NaF, and activation by preincubation with Mg++ and F- was significantly reduced. Activation was restored by recombination of this particulate fraction with the initial detergent extract. This activating effect appeared to be mediated by one or more proteins present in the detergent extract.     

Multiple effects of guanine nucleotides on human platelet adenylated cyclase.

We report that the adenylate cyclase system in human platelets is subject to multiple regulation by guanine nucleotides. Previously it has been reported that GTP is either required for or has little effect on the response of the enzyme to prostaglandin E1. We have found that when platelet lysates were prepared in the presence of 5 mM EDTA, GTP lowered the basal and prostaglandin E1-stimulated adenylate cyclase activity, but at a higher concentration of Mn2+, it caused an increase in enzyme activity exceeding that occurring in the presence of prostaglandin E1. In the presence of Mn2+, dGTP mimics the effect of GTP and is 50% as effective as GTP. Our data suggest that the inhibitory effect of GTP on prostaglandin E1-stimulated adenylate cyclase is mainly due to its direct effect on the enzyme itself, whereas the stimulatory effect of GTP on prostaglandin E1-stimulated adenylate cyclase is due to enhancement of the coupling between the prostaglandin E1 receptor and adenylate cyclase. These studies also indicate that the method of preparation of platelet lysates can profoundly alter the nature of guanine nucleotide regulation of adenylate cyclase.     

Isolation and characterization of 9-hydroxy-10-trans,12-cis-octadecadienoic acid, a novel regulator of platelet adenylate cyclase from Glechoma hederacea L. Labiatae

We have identified and characterized a fatty acid, (9S,10E,12Z)-9-hydroxy-10,12-octadecadienoic acid (9-HODE) as a regulator of adenylate cyclase activity of human platelet membranes. This fatty acid was isolated from a methanolic extract of the plant Glechoma hederacea L. Labiatae (commonly known as 'lierre terrestre', 'ground ivy' or 'creeping Charlie'; it was identified by nuclear magnetic resonance and mass spectroscopy. This compound increased basal adenylate cyclase activity in platelet membranes about threefold and had an EC50 of 10-20 microM. This increase in adenylate cyclase activity occurred without a temporal lag, was reversible, and represented an increase in Vmax without a substantial change in Km for ATP, Mg2+ or Mn2+. In addition, 9-HODE additively or synergistically increased platelet adenylate cyclase activity in response to guanosine 5'-[beta,gamma-imido]triphosphate and forskolin, but the fatty acid failed to alter inhibition of adenylate cyclase activity mediated by epinephrine (alpha 2-adrenergic receptor). Studies of the interaction of 9-HODE with activation of platelet adenylate cyclase activity mediated by prostaglandin E1 (PGE1) and prostaglandin D2 (PGD2) indicated that this fatty acid produced a parallel shift in the concentration/response curve of PGE1 and PGD2 without altering maximal response, which was substantially greater than that observed with 9-HODE alone. From these results, we conclude that 9-HODE appears to be a partial agonist at PGE1 and PGD2 receptors on human platelets. We believe that this is a novel example of a plant-derived fatty acid which acts on cells to regulate adenylate cyclase via prostaglandin receptors.     

Demonstration of adenylate cyclase activity in human red blood cell ghosts.

The presence of adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity was demonstrated in human erythrocyte ghosts and was found to be around 3 pmol adenosine 3',5'-monophosphate (cyclic AMP) - 2 h-1 - mg-1 protein. This enzymatic activity is strongly stimulated by NaF and 5'-guanylimidodiphosphate, is slightly stimulated by epinephrine, norepinephrine, isoproterenol, and prostaglandin E1 and is inhibited by calcium. The hormone stimulation is not potentiated by 5'-guanylylimidodiphosphate.     

Regulation of adenylate cyclase of neuroblastoma x glioma hybrid cells by alpha-adrenergic receptors. I. Inhibition of adenylate cyclase mediated by alpha receptors.

(-)-Norepinephrine and other catecholamines inhibit basal and prostaglandin E1-stimulated adenylate cyclase activities by 35 to 60% in homogenates of NG108-15 neuroblastoma x gloma hybrid cells and markedly reduce adenosine 3'35:'-monophosphate levels of intact cells, but do not affect guanosine 3':5'-monophosphate levels. The specificity of the NG108-15 receptor for ligands is that of an alpha receptor, possibly a presynaptic alpha 2 receptor. The inhibition of adenylate cyclase by norepinephrine is reversed by alpha receptor antagonists such as dihydroergotamine or phentolamine, but not by the beta receptor antagonist propranolol. The effect of norepinephrine on adenylate cyclase activity initially is dependent on GTP; half-maximal inhibition of enzyme activity by norepinephrine is obtained with 0.2 micron GTP. The inhibition of adenylate cyclase activity by norepinephrine is reduced by 10 mM NaF and is abolished by 0.05 mM guanyl-5'-yl imidodiphosphate. Inhibitions of NG108-15 adenylate cyclase mediated by alpha receptors, opiate receptors, and muscarinic acetylcholine receptors are not additive; this suggests that the three species of receptors can be functionally coupled to the same adenylate cyclase molecules or molecules regulating the enzyme.