Studies on adenyl cyclase in Necturus gastric mucosa

Adenyl cyclase activity of Necturus gastric mucosa was determined by measuring the amount of radioactive adenosine 3′,5′-cyclic monophosphate formed from ³H-labeled adenosine triphosphate. Histamine, pentagastrin, and fluoride added in vitro significantly increased fundic adenyl cyclase activity. The dose response curves show that the affinity of pentagastrin for adenyl cyclase is greater than that of histamine, whereas the peak response to pentagastrin is less than that to histamine. Additive stimulation was not obtained when maximal doses of pentagastrin and histamine were combined. These findings suggest that gastric mucosa contains a single adenyl cyclase unit which is coupled to distinctive selectivity sites for gastrin and histamine. N-Benzyl-3-pyrrolidyl acetate methobromide (AHR-602), the muscarinic compound, caused a significant reduction in adenyl cyclase activity, indicating a different mechanism of stimulation of gastric acid secretion for cholinergic muscarinic compounds.     

Stimulation of adenyl cyclase in pupal wing epidermis by -ecdysone

The injection of β-ecdysone into chilled Hyalophora gloveri pupae resulted in the stimulation of adenyl cyclase activity in the wing epidermis as measured by the incorporation of label into cyclic AMP from a prelabeled endogenous pool. Stimulation was also obtained in pupal wings in vitro and in wing epidermal homogenates. Although the sequence of responses to β-ecdysone in vitro depended on the composition of the incubation medium, the stimulation of cyclic AMP synthesis always preceded increases in the rates of RNA and protein synthesis. The increase in adenyl cyclase activity is the earliest metabolic event thus far discerned as a result of β-ecdysone action. It is suggested that β-ecdysone stimulates adenyl cyclase (and guanyl cyclase) but that the hormone also exerts effects on target cells independent of the cyclic AMP system.     

Studies on the assay and activities of guanyl and adenyl cyclase of rat liver

In applying recently developed methods for measuring adenyl and guanyl cyclase activities, we found that some modifications produced much better cyclic nucleotide recovery, lower assay backgrounds, and greater reliability than previously reported. The reliability and specificity of the assay methods were confirmed by substrate and product analysis. Kinetic analysis of rat liver guanyl and adenyl cyclase was subsequently performed to investigate regulatory properties of both enzymes. The Michaelis-Menton constant of guanyl cyclase activity of a 30,000g supernatant fraction of rat liver for guanosine 5′-triphosphate (GTP) was 0.04 mm. This enzyme was competitively inhibited by adenosine 5′-triphosphate (ATP) (K_i = 0.011 mM). Guanyl cyclase was activated in vitro by secretin but unaffected by carbamylcholine, hist-amine, methoxamirie, serotonin, glucagon, and pancreozymin. Liver homogenate adenyl cyclase had a Michaelis-Menten constant for ATP of 0.2 mm. This enzyme was activated by secretin, pancreozymin, glucagon, sodium fluoride, and isoproterenol. GTP (0.005 mm) enhanced the activation by both isoproterenol and glucagon. Methoxamine had no effect on adenyl cyclase activity in the presence or absence of GTP. These results suggest that both guanyl cyclase and adenyl cyclase may be mediators of hormone action in the liver.     

Lymphocyte and macrophage adenyl cyclase activity in animals with enhanced cell-mediated resistance to infection and tumors.

As cyclic AMP has been associated with the inhibition of lymphocyte cytotoxicity, studies were performed to investigate adenyl cyclase activity in lymphocytes and macrophages of Toxoplasma-infected mice in which the efferent limb of the cell-mediated immune response had previously been found to be activated. In peritoneal or splenic lymphocytes from $\text{Balb}\text{c}$ mice chronically infected with Toxoplasma in which growth of an isogeneic bladder tumor was found to be inhibited, adenyl cyclase activity was significantly less than in lymphocytes from uninfected control mice. Stimulation by prostaglandin E₁ or NaF in vitro led to higher levels of adenyl cyclase activity in lymphocytes from unifected animals than in cells from Toxoplasma-infected animals. Similar observations were made with peritoneal macrophages from Toxoplasma-infected and uninfected mice. Lower levels of adenyl cyclase activity were also found in lymphocytes from tumor-bearing mice than in lymphocytes from nontumor-bearing controls. These data suggest that production of cyclic AMP by lymphocytes is inhibited with activation of certain cell-mediated immune functions.     

Neurohormones on Brain Adenyl Cyclase Activity <Emphasis Type="Italic">in vivo</Emphasis>

CYCLIC adenosine 3′,5′-monophosphate (cyclic AMP) has been suggested to be the receptor site for neurotransmitters as well as a second messenger which mediates the action of a variety of hormones and neurohormones in animals and human tissues. Cyclic AMP is derived from adenosine triphosphate (ATP) by the activity of adenyl cyclase and it is metabolized into adenosine monophosphate (AMP) by phosphodiesterase. Beta receptor and adenyl cyclase may be the same in the peripheral tissues¹. In vitro activation of adenyl cyclase activity by various putative neurotransmitters such as noradrenaline (NA), 5-hydroxytryptamine (5-HT), acetylcholine (ACh) and histamine in preparations from brain homogenates and slices is well established². The in vivo effect, however, of these neurohormones on adenyl cyclase has not been studied.     

Hymenolepis microstoma: tegumentary disks.

The distribution and concentration of disk-like tegumentary bodies were studied in adult Hymenolepis microstoma using electron microscopy. Two sets of tissue were examined. In the first set, the tissue was post-fixed with osmic acid and stained with uranyl acetate and bismuth subnitrate or with potassium permanganate. In the second set, osmium exposure was avoided and the tissue was stained with either phosphotungstic acid or potassium permanganate. The staining revealed that the electron-opaque disk is composed of a dense central core that is surrounded by an electron-lucid region. Electron-opaque and electron-transparent bodies were found to be present at constant concentrations in the tegument of the immature, mature, and gravid regions. Also, it was found that postfixation with osmic acid concealed the presence of numerous stacks of electron-opaque bodies in the tegument. The stacks of disks have a tendency to be aligned perpendicular to the tegument surface.     

Formation of adenosine 5'-phosphorofluoridate and the assay of adenyl cyclase in barley seeds

Barley seed (Hordeum vulgare L.) homogenates contain an apparent enzymatic activity which catalyzes the synthesis of adenosine 5′-phosphorofluoridate from magnesium-adenosine 5′-triphosphate and sodium fluoride. Formation of this compound may interfere with some adenyl cyclase assays which use fluoride as a component of the incubation medium. Neither adenyl cyclase activity nor endogenous adenosine 3′: 5′-monophosphate was detected in barley seed homogenates or extracts.     

Localization of Adenyl Cyclase in Meristems of Young Pea Hypocotyls

Electron micrographs of Pisum sativum L. hypocotyl tips treated to localize adenyl cyclase revealed discrete deposits on the internal membranes of cytoplasmic vacuoles which correspond to previously localized enzymes described as acid phosphatases. It remains to be determined whether the specificity of the substrate, adenylyl-imidodiphosphate, used in the present study is such as to exclude all phosphatase activity other than adenyl cyclase. The acid phosphatase localized in earlier studies by other investigators may be an adenyl cyclase. In the differentiated cells of the root cap, lead precipitate was localized in distinct areas bound to the smooth endoplasmic reticulum.     

Changes in adenyl cyclase specific activity during differentiation on an established myogenic cell line

The variation of specific activity of adenyl cyclase has been studied during differentiation of an established line of myoblast, strain L₆D and of a temperature sensitive developmental variant strain, H₆, derived from it. The specific activities of both basal and NaF stimulated adenyl cyclase were found to decrease 2 to 3 folds after fusion of myoblasts into myotubes in cultures of L₆D. Cultures of strain H₆ displayed the same decrease in specific activity of adenyl cyclase when grown at temperature which allows differentiation, while no decrease was observed at the temperature which does not allow cell fusion. These results indicare that the decrease in specific activity of adenyl cyclase is associated with cell fusion and reflects membrane changes ocurring during differentiation of myogenic cells.     

A comparison of the subcellular distribution of 5′-nucleotidase, (Na+K+)-ATPase and adenyl cyclase in beef thyroid gland

The validity of 5′-nucleotidase as a plasma membrane marker enzyme in beef thyroid has been tested by comparing the subcellular distribution of its activity to that of (Na⁺K⁺)-activated ATPase and adenyl cyclase. The specific activity and total activity of (Na⁺K⁺)-ATPase and adenyl cyclase were greatest in the 1000 × g (“nuclear”) and 33 000 × g (“mitochondrial and lysosomal”) fractions. In contrast, 5′-nucleotidase activity was concentrated in the 165 000 × g (“microsomal”) pellet and supernatant. Partially purified plasma membranes were separated from the 1000 (N₂), 30 000 (M₂) and 165 000 × g (P₂) pellets by discontinuous sucrose gradient centrifugation. Again a discordant distribution of these enzyme activities was observed. (Na⁺K⁺)-ATPase specific activity was increased approximately 30-fold over the homogenate in Fractions N₂ and M₂. Basal, thyroid-stimulating hormone-and fluoride-stimulated adenyl cyclase activities were concentrated in the same fractions. 5′-Nucleotidase activity was preferentially located in M₂ and P₂. These differences in distribution pattern suggest that 5′-nucleotidase activity is not uniquely located in the plasma membrane in the thyroid.     

Histochemical studies on Raillietina (Raillietina) johri (Cestoda: Davaineidae). I. Nonspecific and specific phosphatases.

Certain phosphatases have been localized by histochemical techniques in various tissues of a pigeon cestode, Raillietina (Raillietina) johri. Acid phosphatase (AcPase), alkaline phosphatase (AlPase) and adenosine triphosphatase (ATPase) were present in almost all structures: tegument; subtegumental muscles; subtegumental cells; excretory canal; testes; sperm ductules; vas deferens; cirrus sac; cirrus; ovary; receptaculum seminis; vagina; vitelline gland cells; oocytes; uterus; embryonated eggs. AlPase was absent in parenchyma, spermatocytes, spermatids and spermatozoa. AlPase activity was more intense in the tegument of mature gravid proglottides. AcPase and ATPase were visualized in various stages of spermatogenesis of the parasite. ATPase activity was also observed in chromosomes. 5'-nucleotidase (AMPase) activity was restricted to embryonated eggs only. Functional significance of these phosphatases is discussed.     

Adenyl cyclase activities in rat erythrocytes during stress erythropoiesis: localization of the enzyme in the reticulocytes

NaF- and isoproterenol-stimulated adenyl cyclase activity found in preparations from rat erythrocytes is obviously localized mainly in the reticulocytes. After treatment with acetylphenyl hydrazine, both reticulocyte counts and adenyl cyclase activity increase dose-dependently. Evidence is given that adenyl cyclase activity in the red blood cell is lost during the maturation process.     

Effect of guanyl nucleotides on the stimulation of adenyl cyclase activity in human thyroid plasma membranes by thyroid-stimulating hormone and prostaglandin E2

Thyroid homogenates and thyroid plasma membranes were prepared from human thyroid and the effects of thyroid-stimulating hormone (thyrotropin), NaF, and prostaglandins E₁ and E₂ on adenyl cyclase activity in these preparations were studied. The basal level of adenyl cyclase activity in plasma membranes was 5–8 times greater than that of the original homogenates. Adenyl cyclase activity in plasma membranes was stimulated 4.7-fold by 100 munits/ml of thyrotropin and 5-fold by 10 mM of NaF, but the activity in the homogenates was only stimulated 2-fold by either thyrotropin or NaF. Prostaglandin E₁ (10^?6?10^?3 M) and prostaglandin E₂ (10^?7?10^?4 M) failed to stimulate adenyl cyclase activity in plasma membranes, but they did stimulate adenyl cyclase activity in the homogenates. A marked stimulatory effect of prostaglandin E₂ (10^?5 M) on adenyl cyclase activity in plasma membranes resumed in the presence of GTP (10^?7?10^?4 M), although GTP itself only slightly stimulated enzyme activity. GDP and GMP were also effective in this respect, although their potencies varied from compound to compound. GTP potentiated slightly the action of thyrotropin on adenyl cyclase in plasma membranes, but it significantly depressed an increase of enzyme activity produced by NaF. Since GTP did not affect the ATP-regenerating system, it seems that GTP, GDP or GMP was required for the manifestation of prostaglandin E₂ action on adenyl cyclases of human thyroid plasma membranes.     

Adenyl cyclase in human platelets: activity and responsiveness

A clinical study was conducted whereby the activity of adenyl cyclase in the human platelet was demonstrated. The study showed that this activity can be stimulated and inhibited in vitro. Platelets were isolated from normal donors. The laboratory procedures involved in the study are described in detail. It seems that many of the biologic processes which occur in the human platelet are dependent on the breakdown of ATP (adenosine-tri-phosphate) to, among other substances, AMP (adenosine-3',5' monophosphate). Activity of the adenyl cyclase was stimulated by fluoride, prostaglandin E1, and glucagon; it was inhibited by thrombin, epinephrine, norepinephrine, and serotonin. PG (prostaglandin) E1 at concentrations of 20 ng/ml and above increased adenyl cyclase in 7 experiments by 3-5 times. Even at concentrations as low as 2 ng/ml., PGE2 caused perceptable stimulation. The PGE, while stimulating adenyl cyclase activity, also inhibited aggregation of platelets by a variety of substances. Results of the study suggest that adenosine-3',5' monophosphate may be important in the regulation of platelet adhesiveness.     

Role of PKCα−p38MAPK−Giα axis in peroxynitrite-mediated inhibition of β-adrenergic response in pulmonary artery smooth muscle cells

In the context of cross-talk between transmembrane signaling pathways, we studied the loci within the β-adrenergic receptor/G protein/adenyl cyclase system at which PKC exerts regulatory effects of peroxynitrite (ONOO^?) on isoproterenol stimulated adenyl cyclase activity in pulmonary artery smooth muscle cells. Treatment of the cells with ONOO^? stimulated PKC-α activity and that subsequently increased p³⁸MAPK phosphorylation. Pretreatment with Go6976 (PKC-α inhibitor) and SB203580 (p³⁸MAPK inhibitor) eliminated ONOO^? caused inhibition on isoproterenol stimulated adenyl cyclase activity. Pretreatment with Go6976, but not SB203580, prevented ONOO^? induced increase in PKC-α activity. Studies using genetic inhibitors of PKC-α (PKC-α siRNA) and p³⁸MAPK (p³⁸MAPK siRNA) also corroborated the findings obtained with their pharmacological inhibitors in eliminating the attenuation of ONOO^? effect on isoproterenol stimulated adenyl cyclase activity. This inhibitory effect of ONOO^? was found to be eliminated upon pretreatment of the cells with pertussis toxin thereby pointing to a G_i dependent mechanism. This hypothesis was reinforced by G_iα phosphorylation as well as by the observation of the loss of the ability of Gpp(NH)p (a measure of G_i mediated response) to stimulate adenyl cyclase activity upon ONOO^? treatment to the cells. We suggest the existence of a pertussis toxin sensitive G protein (G_i)-mediated mechanism in isoproterenol stimulated adenyl cyclase activity, which is regulated by PKCα-p³⁸MAPK axis dependent phosphorylation of its α-subunit (G_iα) in the pulmonary artery smooth muscle cells.     

Effects of adrenergic blocking agents on lipolysis and adenyl cyclase activity induced by vasoactive intestinal polypeptide (VIP).

VIP stimulates lipolysis and adenyl cyclase activity in the rat adipose tissue. VIP-induced lipolysis and adenyl cyclase activity are not affected by phenoxybenzamine. VIP-induced lipolysis is inhibited by propranolol but VIP-induced adenyl cyclase activity is not.     

ONTOGENETIC DEVELOPMENT OF ADENYL CYCLASE AND PHOSPHODIESTERASE IN RAT BRAIN

Abstract— The activities of adenyl cyclase and phosphodiesterase were determined in homogenates of cerebrum, cerebellum and brain stem of rats of 1 day to 9 weeks of postnatal age. The activity of cerebral and brain stem adenyl cyclase measured either in the absence or presence of sodium fluoride increased rapidly for 2 weeks, reached at 20 days a maximum about three times (brain stem) or six times (cerebrum) that seen on day 1 and then declined slightly during the next several weeks. In contrast, activity of cerebrellar adenyl cyclase increased more slowly and reached a maximum at about 32 days. Activity of phosphodiesterase in cerebrum and brain stem increased several-fold during brain maturation; however, enzymic activity in the cerebellum decreased during the entire 9 week period.
In the pineal gland, adenyl cyclase activity measured in the absence of norepinephrine or sodium fluoride did not change significantly with age. However, enzymic activity measured in the presence of these agents increased with the age of the rat. Bilateral removal of the superior cervical ganglia at 1 day of age is known to arrest the sympathetic innervation of the pineal gland but did not prevent the development of this adenyl cyclase system activated by catecholamines or fluoride.     

Effects of Long-acting Thyroid Stimulator (LATS) and LATS Protector on Human Thyroid Adenyl Cyclase Activity

The long-acting thyroid stimulator (LATS) has been thought to be responsible for the hyperthyroidism of Graves''s disease. It is detected by its effect on the mouse thyroid gland but cannot be found in all patients with hyperthyroidism. In an attempt to clarify the problem of LATS-negative hyperthyroidism, serum was obtained from untreated patients and its effect in vitro on human thyroid tissue examined, using the activation of adenyl cyclase as a measure of stimulation. Human thyroid adenyl cyclase was activated by both thyroid-stimulating hormone (TSH) and LATS. Thyroid tissue obtained from patients with Graves''s disease was relatively less responsive to LATS than was non-toxic thyroid tissue. Of the 24 samples studied five contained LATS and all of these activated adenyl cyclase. The presence of LATS protector in LATS-negative hyperthyroid patients was confirmed but LATS-negative sera had no effect on human thyroid adenyl cyclase activity.     

Adenyl Cyclase and the Differentiation of β-Adrenoreceptors

WE have studied the enzyme adenyl cyclase to extend the evidence that β-adrenoreceptors consist of at least two types, β-1 and β-2 (refs. 1-4.) Cyclic 3′,5′-AMP has been shown to be the intracellular mediator of the action of various hormones, including the catecholamines and it has been suggested that adenyl cyclase is an integral part of the β-adrenoreceptor^5,6. Thus adenyl cyclase preparations from certain tissues should differ in their responsiveness to β-adrenoreceptor agonists and antagonists in a manner analogous to the intact tissues. We have therefore examined the differential effects of some sympathomimetic amines and their antagonists on the activity of adenyl cyclase preparations from rat heart and lung.     

Adenyl cyclase of oxyntic cells its association with different cellular membranes

The adenyl cyclase of the oxyntic, or acid-secreting, cells of bullfrog gastric mucosa has been found to be a membrane-bound enzyme. A method has been developed to isolate the adenyl cyclase rich membrane fractions in a hypotonic medium containing dithiothreitol, which has been found to protect the hormonal resposivenes of the adenyl cyclase.Highest specific activity of adenyl cyclase was localized in a light membrane fraction which also had abundant K⁺-stimulated ATPase and K⁺-stimulated $\text{p-}\text{nitrophenyl}$ phophatase and very low cytochrome $\text{c}$ oxidase activty. The three gastric secretagogues tested, namely histamine, pentagastrin and methylcholine, significantly stimulated the adenyl cyclase activity of the light membrane fraction.After treatment with 10 mM Mg⁺ further subfractionation of the light membrane fraction on a sucrose density gradient yielded light membrane subfraction 1, light membrane subfraction 2 and light membrane subfraction 3 in order of increasing densities. The three subfractions had different enzymatic and chemical properties. Adenyl cyclase activity has been found to be distributed in all three subfractions. However, the hormonal responsiveness of the three fractions was quite different. Light membrane subfraction 2 could be stimulated by all three secretagogues, light membrane subfraction 1 by histamine and methylcholine, while light membrane subfraction 3 was refractory to all three secretagogues. On the basis of the cholesterol to phospholipid molar ratio, RNA content, glycoprotein content and the enzymatic data it is suggested that light membrane subfraction 1 and light membrane subfraction 2 are of general plasma-membrane type, while light membrane subfraction 3 is largely of cytoplasmic origin.