Demonstration of Dopamine-sensitive Adenylate Cyclase in Malignant Neuroblastoma Cells and Change in Sensitivity of Adenylate Cyclase to Catecholamines in “Differentiated” Cells

Adenylate cyclase (EC 4.6.1.1) activity was stimulated by low concentrations of dopamine and apomorphine, but not by low concentrations of norepinephrine in homogenates of malignant mouse neuroblastoma cells. In cyclic AMP-induced "differentiated" cells, dopamine concentration required for a maximal increase in adenylate cyclase activity was about 10-fold less than that required for a similar increase in control cells, and norepinephrine-sensitive adenylate cyclase activity became apparent at low norepinephrine concentrations. The pharmacological properties of dopamine-sensitive adenylate cyclase were different from those of norepinephrine-sensitive enzyme. For example, dopamine-stimulated adenylate cyclase activity was markedly reduced by low concentrations of haloperidol and phentolamine, whereas norepinephrine-stimulated enzyme activity required higher concentrations of these blocking agents for a similar amount of inhibition. Norepinephrine-stimulated enzyme activity was markedly blocked by low concentrations of propranolol, whereas dopamine-stimulated enzyme activity required a much higher concentration of this blocking agent for a similar amount of inhibition. Low concentrations of isoproterenol increased adenylate cyclase activity in malignant cells, but in "differentiated" cells even a high concentration failed to do so. The fact that dopamine and norepinephrine produced an additive stimulatory effect on adenylate cyclase activity suggests that they interact at different receptor sites. This suggestion is further supported by the observation that the combination of prostaglandin E(1) and norepinephrine produced an additive stimulatory effect of enzyme activity. The observation that the effects of dopamine and prostaglandin E(1) are not additive, coupled with the observation that a low concentration of phentolamine blocked the effect of prostaglandin E(1), suggests that these two agents may interact at a common site.     

Glucose Inhibition of Adenylate Cyclase in Intact Cells of Escherichia coli B

Previous studies in E. coli B have demonstrated an inverse correlation between the presence of glucose in the medium and the accumulation of cyclic AMP in the medium. This observation could not be explained by the action of glucose as a repressor of adenylate cyclase (EC 4.6.1.1) synthesis, as a stabilizer of cyclic AMP phosphodiesterase (EC 3.1.4.17) activity, or as a direct inhibitor of adenylate cyclase activity in cell-free preparations. The recent development of an in vivo assay for adenylate cyclase has provided a basis for further exploring the inhibitory action of glucose in intact cells. With this assay it has been possible to show that, while glucose does not affect adenylate cyclase in vitro, it rapidly inhibits the enzyme activity in intact cells. Extensive metabolism of glucose is not required, since alpha-methylglucoside also inhibits adenylate cyclase in vivo. When cells are grown on glucose as carbon source, some sugars (mannose, glucosamine) substitute for glucose as adenylate cyclase inhibitors while others (e.g., fructose) do not. Dose-response studies indicate that low concentrations of glucose lead to essentially complete inhibition of adenylate cyclase activity while only moderately decreasing intracellular cyclic AMP concentrations. The evidence presented suggests that the decreased cellular cyclic AMP levels resulting from glucose addition can be accounted for by inhibition of adenylate cyclase without any significant effect on cyclic AMP phosphodiesterase or the transport of cyclic AMP from the cells to the medium.     

Adenylate Cyclase from Brevibacterium liquefaciens. III. In Situ Regulation of Adenylate Cyclase by Pyruvate

In the presence of DL-alanine intracellular cyclic AMP in nonproliferating cells of Brevibacterium liquefaciens increased rapidly to the maximum level of approximately 180 muM, and extracellular cyclic AMP increased to 100 muM within 4 hr at 25 degrees . Adenylate cyclase (EC 4.6.1.1) induction was not observed during this incubation. The concentration of pyruvate in the total culture increased concomitantly with that of cyclic AMP and reached approximately 20 mM after 4 hr of incubation. Since the activity of cyclic nucleotide phosphodiesterase is extremely low in this bacterium, the accumulation of cyclic AMP with DL-alanine appeared to be due to the activation of adenylate cyclase by pyruvate. D-alanine was more effective than L-alanine in producing pyruvate, and a high activity of D-alanine oxidation was detected in the cell lysate of B. liquefaciens.Thus, adenylate cyclase in this bacterium appeared to be regulated in vivo by pyruvate which was formed, in this case, predominantly from D-alanine through the action of D-aminoacid oxidase (EC 1.4.3.3). Pyruvate, added extracellularly, also caused a rapid accumulation of intracellular cyclic AMP. Glucose did not change the level of cyclic AMP significantly. It also did not affect the intracellular accumulation of cyclic AMP with DL-alanine.     

5′-Guanylylimidodiphosphate, A Potent Activator of Adenylate Cyclase Systems in Eukaryotic Cells

5'-Guanylylimidodiphosphate (Gpp(NH)-p) stimulates adenylate cyclase [ATP-pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in plasma membranes isolated from frog and salmon erythrocytes, from rat adrenal, hepatic, and fat cells, and from bovine thyroid cells. The nucleotide acts cooperatively with the various hormones (glucagon, secretin, ACTH, thyrotropin, and catecholamines) that stimulate these adenylate cyclase systems with resultant activities that equal or exceed those obtained with hormone plus GTP or with fluoride ion. In the absence of hormones, Gpp(NH)p is a considerably more effective activator than GTP, and, under certain conditions of incubation, stimulates rat fat cell adenylate cyclase to levels of activity (about 20 nmoles of 3',5'-adenosine monophosphate mg protein per min) far higher than reported hitherto for any adenylate cyclase system examined. The nucleotide activates frog erythrocyte adenylate cyclase when the catecholamine receptor is blocked by the competitive antagonist, propranolol, and activates the enzyme from an adrenal tumor cell line which lacks functional ACTH receptors. In contrast, Gpp(NH)p does not stimulate adenylate cyclase in extracts from Escherichia coli B. Gpp(NH)p appears to be a useful probe for investigating the mechanism of hormone and nucleotide action on adenylate cyclase systems in eukaryotic cells.     

Neurohypophyseal Hormone-Responsive Adenylate Cyclase from Mammalian Kidney

The investigation was undertaken to evaluate the direct stimulatory effects of neurohypophyseal hormones upon adenylate cyclase activity in a cell-free, particulate fraction derived from the kidney medulla of various mammalian species. The relative affinity of neurohypophyseal hormones for the receptor component of the adenylate cyclase system (as defined by the concentration of hormone required for half-maximal stimulation) had the order [8-arginine]-vasopressin > [8-lysine]-vasopressin oxytocin (AVP > LVP OT) for rat, mouse, rabbit, and ox; in the pig, the order was LVP > AVP OT. The relative affinities of the three hormones in rat and pig cyclase systems were found to correspond with the relative antidiuretic potencies of these hormones in the intact rat and pig. These findings show that the renal receptor for neurohypophyseal hormones in a particular species exhibits the highest affinity for the specific antidiuretic hormone that occurs naturally in that species.     

Activation of Membrane-Bound Adenylate Cyclase by Glucagon in Neurospora crassa

Membrane-bound adenylate cyclase in Neurospora crassa is activated by glucagon. Half-maximal effect is observed at hormone concentrations of about 10 nM. After solubilization of the enzyme with Lubrol-PX, the glucagon effect is lost. Incubation of neurospora cells with glucagon leads to a decrease in the activity of glycogen synthetase (EC 2.4.1.11) and to an increase in the activity of glycogen phosphorylase (EC 2.4.1.1) and in the rate of glycogenolysis.     

垂体腺苷环化酶激活肽对胃癌细胞增殖的抑制作用

垂体腺苷环化酶激活肽(PACAP)是一种新的胃肠激素,其参与许多胃肠功能的调节。为了解其是否对胃癌细胞有作用,我们应用细胞培养等方法研究了PACAP对MKN-45细胞的影响。结果发现:10nM～1μM PA-CAP作用3天后即表现出对细胞的生长有抑制效应,且可持续5天;并与PACAP的剂量呈正相关,其机制可能与延缓DNA合成、使细胞同期阻止在S期有关。     

Up-Regulation of H2 Receptor and Adenylate Cyclase in Rabbit Parietal Cells During Prolonged Treatment with H2-Receptor Antagonists

Intragastric hyperacidity occurs after abruptwithdrawal of histamine H₂-receptorantagonists, and the prolonged administration of theseagents induces tachyphylaxis of the inhibitory effectson gastric acid secretion. We examined the effect of theprolonged administration of H₂-receptorantagonists on the H₂-receptor signalingsystem in parietal cells isolated from rabbits that hadreceived H₂-receptor antagonists for 14 days.[¹²⁵I]aminopotentidine (APT) binding sites toH₂ receptors in parietal cell membranes wereincreased without any significant change in the affinityfor [¹²⁵I]APT. The expression of Gs, guanosine triphosphate(GTP)-binding protein coupled to H₂ receptor,was slightly increased. Basal as well as GTP orhistamine-stimulated cAMP production was increased, butno significant change was observed in the presence of anH₂-receptor antagonist. The up-regulation ofthe H₂ receptor and adenylate cyclaseappeared to cause hypersecretion of acid afterwithdrawal of H₂ receptor blockade.     

Expression of Adenylate Cyclase, Catecholamine Receptor, and Cyclic Adenosine Monophosphate-Dependent Protein Kinase in Synchronized Culture of Chang''s Liver Cells

In Chang's liver cells synchronized by exposure to thymidine, both cyclic adenosine monophosphate-dependent and cyclic adenosine monophosphate-dependent and cyclic adenosine monophosphate-independent protein kinase activities increased throughout the cell cycle in a manner that corresponded approximately to the overall increase in cellular protein. Thus, the catalytic and the cyclic adenosine monophosphate-binding regulatory subunits of protein kinase appear to be temporally coexpressed. Adenylate cyclase activity stimulated by catecholamine, as well as basal activity, decreased markedly after release of cells from thymidine blockade during the S phase of the cell cycle; this was followed by recovery and overall net increase to fully expressed activity by the peak of mitosis. After initial decrease early in the S phase, cyclase activity assayed in the presence of fluoride ion began to rise before the rise in basal and catecholamine-stimulated activities, indicating that regulation of membrane receptors for catecholamines and for basal activity may be at least in part separate from that for catalytic activity of adenylate cyclase. The differential expression of membrane receptors for adenylate cyclase during the Chang's liver cell cycle affords a possible mechanism for control of response to hormones during the cell cycle.     

Conditions Leading to Enhanced Response to Glucagon, Epinephrine, or Prostaglandins by Adenylate Cyclase of Normal and Malignant Cultured Cells

HeLa S3 cells, a clonal strain adapted for growth in suspension culture, contain adenylate cyclase activity that is stimulated by glucagon, prostaglandin E(1), and epinephrine. Total enzymatic activity and response to hormones is increased as a result of these cells being in stationary culture for several days. The parental strain of HeLa cells normally grown in stationary culture shows even greater adenylate cyclase activity. Hepatoma (HTC) cells grown in suspension culture have no detectable adenylate cyclase activity, but when grown in stationary culture they contain low, but detectable, amounts of adenylate cyclase, which is stimulated by glucagon, epinephrine, or prostaglandins. Chang's liver cells, both suspension and stationary culture, contain relatively high levels of adenylate cyclase that is stimulated by epinephrine or prostaglandin E(1), with differences in activity depending upon culture conditions. Adenylate cyclase of 3T3 and L929 mouse fibroblasts respond to catecholamines, as well as to prostaglandin E(1), but not to glucagon. Characteristic increases in basal activity of adenylate cyclase and in activity in the presence of hormone occurred as cell density increased during stationary culture of certain cell lines, but do not occur with fluoride ion present. In addition to the influence of growth conditions and rate or phase of cell growth on cyclase activity and hormonal response, the cultured cell lines frequently showed marked differences in activity from one another; malignant cells generally exhibited less activity. It is postulated that adenylate cyclase activity may be enhanced by cell to surface contact or cell to cell interaction and that this phenomenon may represent a form of self regulation or modulation of hormonal response by a tissue.     

Myocardial Adrenergic Receptors and Adenylate Cyclase in the Developing Spontaneously Hypertensive Rat

Cardiac membrane preparations from developing spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats (0 to 125 days of age) were analyzed for the apparent numbers of alpha-and beta-adrenergic receptors and adenylate cyclase activities in an attempt to correlate biochemical changes with the reported functional changes occurring with the development of hypertension in the SHR. Although the apparent number of alpha- and beta-adrenergic receptors were similar in both strains of rats, isoproterenol-stimulated adenylate cyclase activities were significantly higher (P<0.05) in the prehypertensive SHRs when compared to WKY rats and declined to lower values as hypertension appeared. The percent stimulation produced by isoproterenol remained similar in cardiac membranes from normotensive WKY rats at all ages of development whereas this percent stimulation was 40% higher at birth in the SHRs and declined to approximately one half the original value by 100 days of age (P<0.05). The elevated adenylate cyclase activity observed during the prehypertensive state may contribute to the genesis of hypertension.     

Antipsychotic Drugs and Dopamine-Stimulated Adenylate Cyclase Prepared from Corpus Striatum of Rat Brain

Antipsychotic drugs and their clinically impotent congeners were examined as inhibitors of dopamine-sensitive adenylate cyclase (EC 4.6.1.1) in cell-free membrane preparations of the caudate-putamen of rat brain. Of 12 neuroleptic drugs with reported antipsychotic efficacy, all inhibit stimulation of adenylate cyclase by 40 muM dopamine at micromolar concentrations. Among 14 other structurally related drugs that are not clinically effective as antipsychotic agents, 12 were almost ineffective while two drugs were moderate inhibitors of dopamine-sensitive adenylate cyclase.     

Effect of Insulin on the Growth Pattern and Adenylate Cyclase Activity of BHK Fibroblasts

BHK 21/13 fibroblasts grown in the presence of insulin show some characteristics of a transformed strain. Cells broaden, cross each other intricately, and form colonies on both polystyrene dishes and soft agar. The effect of insulin is prevented by addition of N(6)-butyryl adenosine 3':5'-cyclic monophosphate and theophylline to the culture medium. Insulin causes a decrease in the concentration of intracellular adenosine 3':5'-cyclic monophosphate and inhibition of adenylate cyclase activity in BHK fibroblast membranes. This inhibition is observed at insulin concentrations of 0.1 nM or higher.     

Biochemical Characterization and Cytochemical Localization of a Catecholamine-Sensitive Adenylate Cyclase in Isolated Capillary Endothelium

Capillaries were isolated from epididymal fat, and a catecholamine-sensitive adenylate cyclase found in these capillaries was characterized. The effect of various hormones on the accumulation of adenosine 3′:5′-cyclic monophosphate in capillary endothelial cells was determined and the cyclase was found to exhibit mixed alpha and beta characteristics. Cyclase was cytochemically localized in these endothelial cells with 5′-adenylyl-imidodiphosphate as a specific cyclase substrate and alloxan as a specific cyclase inhibitor. Lead imidodiphosphate was precipitated at or near the site of cyclase activity upon hydrolysis of 5′-adenylyl-imidodiphosphate by cyclase. This reaction product was observed primarily on the luminal surface of intact capillaries, in micropinocytic invaginations, in free vesicles within the cytoplasm, and in the intracellular junctions.     

垂体腺苷酸环化酶激活肽对家兔实验性动脉粥样硬化血管重塑的影响

为探讨垂体腺苷酸环化酶激活肽对家兔动脉粥样硬化血管重塑的影响，将60只新西兰雄性家兔随机等分为正常对照组、动脉粥样硬化组和垂体腺苷酸环化酶激活肽组，分别于实验的第4、8和第12周末随机处死每组家兔各5－8只并取胸主动脉。在上述动脉条的头、中、尾部各截取约0．5cm长的组织作石蜡切片，苏木素－伊红染色，光镜下观察并用图像分析系统测量相关形态学参数。余下的动脉条用苏丹Ⅳ染色，作大体形态观察。结果发现，随时间延长，动脉粥样硬化组斑块面积逐渐增大，斑块检出率逐渐增加（P＜0．05），且斑块多分布于头段，而中段、尾段依次减少。管腔面积在早期斑块形成时并无改变，晚期则明显缩小（P＜0．05）。垂体腺苷酸环化酶激活肽组斑块面积、斑块最大厚度均小于同期动脉粥样硬化组，而管腔面积则大于动脉粥样硬化组（P＜0．05）。提示垂体腺苷酸环化酶激活肽可通过抑制负性血管重塑及促进正性血管重塑的作用而延缓动脉粥样硬化病变的进展。     

Mode of Action of Cholera Toxin: Stabilization of Catecholamine-Sensitive Adenylate Cyclase in Turkey Erythrocytes

Preincubating turkey erythrocytes with cholera toxin alters their adenylate cyclase (EC 4.6.1.1) system: basal activity, maximal epinephrine-stimulatable activity, and affinity of the enzyme reaction for epinephrine are all increased. Pretreatment of erythrocytes with choleragenoid prevents these changes. Cholera toxin does not alter [(3)H]epinephrine uptake by intact erythrocytes. The increase in epinephrine-stimulatable cyclase activity appears to occur at the expense of fluoride-stimulatable activity, which is decreased by the toxin. In lysates from both toxin-treated and control cells, maximally stimulating amounts of epinephrine and fluoride, when added in combination, have a nearly additive effect on cyclase activity. These observations suggest that the adenylate cyclase system of the turkey erythrocyte may exist in two interconvertible forms, one that is catecholamine-responsive but fluoride-insensitive, and another that is fluoride-sensitive but not coupled to catecholamine receptors. Cholera toxin appears to stabilize the enzyme in its hormone receptor-coupled form.     

Dopamine-Sensitive Adenylate Cyclase in Mammalian Brain: A Possible Site of Action of Antipsychotic Drugs

Adenylate cyclase (EC 4.6.1.1), selectively stimulated by low concentrations of dopamine, has been found in the olfactory tubercle, the nucleus accumbens, and the caudate nucleus of several mammalian species. Several different classes of drugs effective in the treatment of schizophrenia (antipsychotic drugs) were potent inhibitors of the stimulation by dopamine of the enzyme from these various regions. The drugs studied included representatives of the phenothiazine, butyrophenone, and dibenzodiazepine classes. The inhibition by these antipsychotic drugs was competitive with respect to dopamine. The most potent of the antipsychotic agents tested was fluphenazine, which had a calculated inhibition constant (K(i)) of about 5 x 10(-9) M. For each of several drugs tested, the K(i) for the enzyme from the olfactory tubercle was similar to that for the enzyme from the caudate nucleus. Several compounds closely related structurally to the psychoactive phenothiazines, but which have little or no antipsychotic or extrapyramidal actions clinically, had low relative potencies as inhibitors of dopamine-stimulated adenylate cyclase activity. The results, considered together with other data, raise the possibility that the therapeutic effects, as well as the extrapyramidal side effects, of these antipsychotic agents may be attributable, at least in part, to their ability to block the activation by dopamine of specific dopamine-sensitive adenylate cyclases in the human brain.     

Transformation of Chick-Embryo Fibroblasts by Wild-Type and Temperature-Sensitive Rous Sarcoma Virus Alters Adenylate Cyclase Activity

The activity of the enzyme adenylate cyclase, a component of the plasma membrane, has been determined in chick-embryo fibroblasts and in cells transformed by either Bryan high-titer strain of Rous sarcoma virus (RSV-BH) or a temperature-sensitive mutant of this virus (RSV-BH-Ta). Adenylate cyclase activity is reduced in cells transformed by the wild-type virus and also by the temperature-sensitive mutant when the cells are grown at the permissive temperature (37 degrees ). Transformation results in an altered affinity (K(m)) for the substrate (Mg ATP). The apparent K(m) ATP is 0.23 mM in normal cells and 1.1 mM in cells transformed with wild-type virus. The K(m) ATP of the cells infected with RSV-BH-Ta is 0.67-1.0 mM at 37 degrees and 0.28 mM at 42 degrees . The enzyme from normal cells appears to have two binding sites for Mg(++), one at the catalytic site and a second at a regulatory site. Transformation by RSV-BH or RSV-BH-Ta (37 degrees ) apparently alters this second Mg(++) site. A decrease in adenylate cyclase activity occurs within 10 min after cells infected with RSV-BH-Ta are shifted from 42 degrees to 37 degrees ; the activity falls to one-half that of normal cells 30 min after the temperature shift. Our observations indicate that a viral function lowers cyclic AMP content by lowering the activity of adenylate cyclase, probably through some modification of the plasma membrane.     

Epinephrine Binding to the Catecholamine Receptor and Activation of the Adenylate Cyclase in Erythrocyte Membranes

Turkey erythrocyte membranes showed specific binding of [(3)H]epinephrine. The concentration of hormone required for half-maximal binding (30 muM) was the same as that required for half-maximal activation of the adenylate cyclase located in the same membrane preparation. The binding reaction at 37 degrees C reached completion during the first minute of incubation, which agrees well with the known rapidity of the biological response to catecholamines. Specific binding was abolished by heating the membranes 1 min at 100 degrees C. Chromatography of the bound (3)H, after its extraction from the membranes, indicated that the hormone had fully retained its chemical structure. Epinephrine binding was inhibited by the beta-adrenergic blocking agent propranolol, which also inhibited the activation of adenylate cyclase by the hormone. The specificity of phenethylamine derivatives in displacing [(3)H]epinephrine from the binding sites showed that a typical catecholamine receptor was responsible for the binding. Displacement of the bound hormone by analogs lacking the catechol group was more extensive at 37 degrees C than at 0 degrees C. Some of the analogs that displaced epinephrine from the binding site caused only a feeble activation of the adenylate cyclase, but were able to inhibit the activation of the enzyme by epinephrine. Thus, binding to a catecholamine receptor on a membrane preparation is an essential, but insufficient, condition to elicit a response.