Regulation by cations of adenylate cyclase activity in chicken tissues

The effects of magnesium and sodium ions on adenylate cyclase activity in plasma membranes from chicken heart and eggshell gland mucosa were studied. It was found that the increase in magnesium chloride concentration from 5 to 40 mM results in the stimulation (4.1-fold) of the adenylate cyclase activity. The increase in sodium chloride concentration up to 150 mM stimulated the enzyme activity 2-fold. The stimulation of adenylate cyclase by magnesium and sodium ions was less pronounced in the eggshell gland. GTP did not activate adenylate cyclase. The activating effect of magnesium and sodium ions was accompanied by the attenuation of the enzyme sensitivity to NaF, guanylyl imidodiphosphate and isoproterenol. Activation by guanylyl imidodiphosphate was completely abolished in the presence of 40 mM magnesium chloride. It is assumed that high concentrations of the salt promote subunit dissociation of the adenylate cyclase regulatory protein and its interaction with the catalytic subunit in the presence of endogenous nucleotides. The differences in the adenylate cyclase sensitivity to cations in chicken heart and eggshell gland mucosa correlate with the amount of pertussis toxin substrate.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues

Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I₅₀ = 2 μg/ml) when compared to other naturally occurring glycosaminoglycans. This inhinibition was also appparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E ₂. Heparin was also found to inhibit glucagon-sensitive rat hepatice adenylate cyclase, and the prostaglandin E₁-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfade polysugar dextran sulfate exerts similar effects on adenylate cyclase activity of the rat ovary was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.     

Calmodulin regulation of adenylate cyclase activity

Calmodulin-dependent stimulation of adenylate cyclase was initially thought to be a unique feature of neural tissues. In recent years evidence to the contrary has accumulated, calmodulin-dependent stimulation of adenylate cyclase now being demonstrated in a wide range of structurally unrelated tissues and species. Demonstration of the existence of calmodulin-dependent adenylate cyclase has in nearly all instances required the removal of endogenous calmodulin. It is not yet clear whether calmodulin-dependent and calmodulin-independent forms of the enzyme exist and whether some tissues (such as heart) lack a calmodulin-dependent adenylate cyclase. The presence of calmodulin appears largely responsible for the ability of the adenylate cyclase enzyme to be stimulated by submicromolar concentrations of calcium; it may not be relevant to the inhibition of the enzyme which occurs at higher concentrations of calcium. The physical relationship of calmodulin to the plasma membrane bound enzyme (or to the soluble forms of the enzyme) is not known nor is the mechanism of adenylate cyclase activation by calmodulin clear; current data suggest some involvement with both the N and C units of the enzyme. Finally, it is possible that in vivo calcium contributes to the duration of the hormone stimulated cyclic AMP signal. Thus current in vitro data suggest that optimal hormonal activation of calmodulin-dependent adenylate cyclase occurs at very low intracellular calcium concentrations, comparable to those found in the resting cell; conversely the enzyme is inhibited as intracellular calcium increases, following for example agonist stimulation of the cell. These higher calcium concentrations would then activate calmodulin-dependent phosphodiesterase. Such differential effects of calcium on adenylate cyclase and phosphodiesterase would ultimately restrict the duration of the hormone-induced cyclic AMP signal.     

Impaired adenylate cyclase activity of phenylhydrazine-induced reticulocytes

A method which involves Percoll gradient centrifugation, is described for separating rabbit reticulocytes from other blood cells, including erythrocytes. This permits a quantitative comparison of the adenylate cyclase activity of reticulocyte membranes which had been induced either by bleeding (30% reticulocytosis) or by repeated injections of phenylhydrazine (90% reticulocytosis). Adenylate cyclase activity was greatly impaired on exposure to the hemolytic agent; total activity was reduced about 20-fold. However, a more selective loss was observed in terms of hormonal stimulation. Prostaglandin E1 was 2-fold more effective than either sodium fluoride or Forskolin in stimulating the enzyme from bled reticulocytes, whereas it was 3-fold less effective than either fluoride or Forskolin in the case of membranes from animals which had been exposed to phenylhydrazine. A stimulatory adenosine receptor was detectable only in reticulocytes which had not been treated with the hemolytic agent. These studies suggest that purified reticulocytes from bled animals represent the most suitable model system in which to study the maturation of red blood cells.     

Modulation of adenylate cyclase activity by sulfated glycosaminoglycans. II. Effects of mucopolysaccharides and dextran sulfate on the activity of adenylate cyclase derived from various tissues.

Heparin was found to be the most potent inhibitor of rat ovarian luteinizing hormone-sensitive adenylate cyclase (I50 = 2 microgram/ml) when compared to other naturally occurring glycosamin oglycans. This inhibition was also apparent when this enzyme was stimulated by follicle-stimulating hormone or prostaglandin E2. Heparin was also found to inhibit glucagon-sensitive rat hepatic adenylate cyclase, and the prostaglandin E1-sensitive enzyme from rat ileum and human platelets. In contrast, heparin stimulated the dopamine sensitive adenylate cyclase from rat caudate nucleus. The sulfated polysugar dextran sulfate exerts similar effects on adenylate cyclase activity of the rat ovary and was shown to inhibit hormone binding to rat ovarian plasma membrane in a manner similar to that exerted by heparin. In contrast to heparin, dextran sulfate inhibited dopamine-sensitive adenylate cyclase from rat caudate nucleus.     

Soluble adenylate cyclase activity in Neurospora crassa.

A soluble form of adenylate cyclase was extracted from mycelia of Neurospora crassa wild-type strains. This enzyme activity was purified by chromatography on hexyl-amino-Sepharose, agarose and Blue Sepharose and preparative polyacrylamide-gel electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, peak fractions from the later purification steps showed a main polypeptide band with an apparent molecular weight of about 66 000. The following hydrodynamic and molecular parameters were established for the Neurospora soluble adenylate cyclase activity: sedimentation coefficient, 6.25 S; Stokes radius, 7.3 nm; partial specific volume, 0.74 ml/g; molecular weight, 202 000; frictional ratio, 1.65. The isoelectric point of this enzyme activity was 4.65. The enzyme was not activated by GTP, [beta gamma-imido]GTP, fluoride or cholera toxin.     

Guanylate cyclase activity in Escherichia coli mutants defective in adenylate cyclase.

Guanylate cyclase, which catalyzes the synthesis of guanosine 3',5'-monophosphate, has been assayed in several strains of Escherichia coli. They include wild-type cells and mutants defective in adenylate cyclase, which is responsible for the synthesis of adenosine 3',5'-phosphate. Our results demonstrate that adenylate cyclase and guanylate cyclase are two different enzymes in E. coli and suggest that the gene that encodes adenylate cyclase also plays a regulatory role in the synthesis of guanylate cyclase.     

Atrial natriuretic factor inhibits adenylate cyclase activity

The synthetic atrial natriuretic factor (ANF) (8- 33AA ) inhibited adenylate cyclase activity in aorta washed particles, mesenteric artery, and renal artery homogenates in a concentration dependent manner with an apparent Ki between 0.1 to 1nM . The extent of inhibition of adenylate cyclase by ANF varied from tissue to tissue. The adenylate cyclase from mesenteric artery and renal artery was inhibited to a greater extent as compared to that from aorta. ANF was also able to inhibit the stimulatory effects of hormones on adenylate cyclase activity and of agents such as F- and forskolin which activate adenylate cyclase by receptor- independent mechanism. In addition, ANF showed an additive effect with the inhibitory response of angiotensin II on adenylate cyclase from rat aorta. These studies for the first time demonstrate that ANF is an inhibitor of adenylate cyclase of several systems.     

cAMP and adenylate cyclase activity in Streptomyces granaticolor

Abstract Intracellular and extracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels were determined during the growth of Streptomyces granaticolor . The intracellular level of cAMP represents not more than 10% of the total amount. cAMP synthesis varies in cultures growing on different carbon sources. The activity of adenylate cyclase in intact cells is strictly dependent on the presence of a metabolizable carbon source.     

Ultracytochemical localization of adenylate cyclase activity in rat thymocytes

Summary Ultrastructural localization of adenylate cyclase (AC) activity was investigated in suspensions of unfixed isolated rat thymocytes using a medium containing 0.6 mM 5-adenylylimidodiphosphate (AMP-PNP) as a substrate, 10 mM MgSO₄ as an activator, 5 mM theophylline as an inhibitor of 3,5-AMP-phosphodiesterase and 2 mM lead nitrate as a capturing agent. AC activity was demonstrated in plasma membrane, perinuclear space, endoplasmic reticulum, Golgi complex, centriole microtubules and mitochondria. AC was activated with 10^–4 M adrenalin in the presence of 5-guanylylimido-diphosphate (GMP-PNP) as well as with 10^–2 M NaF. In the cells incubated in a medium devoid of theophylline and containing 5-AMP instead of AMP-PNP, 5-nucleotidase activity was observed in the same cell structures as AC activity. Hydrolysis of 5-AMP in the nucleus was much stronger than that of AMP-PNP. 10 mM NaF markedly inhibited hydrolysis of 5-AMP in all cell structures. No staining was observed with 2 mM -glycerophosphate as a substrate. Incubation of unfixed thymocytes in media containing AMP-PNP, 5-AMP or p-nitrophenyl phosphate, but not -glycerophosphate, induced both in the nucleus and in the cytoplasm in some cells an appearance of a transitory reticular formation consisting of about 30 nm thick strands which could penetrate the nuclear envelope and plasma membrane and form connections with adjacent cells. The transitory reticular formation seems to belong to the cytoskeleton and to be involved in cell aggregation.     

Ultrastructural localization of adenylate cyclase activity in chicken osteoclasts

Using lead citrate as a capture reagent and adenylate-(beta, gamma-methylene) diphosphate (AMP-PCP) as a substrate, we localized adenylate cyclase activity on the non-ruffled border plasma membrane of approximately half of the osteoclasts on trabecular bone surfaces in the tibial metaphyses of chickens fed a low (0.3%)-calcium diet. The enzyme was not detectable in osteoclasts when chickens were fed a normal calcium diet. Activity was observed on the entire plasma membrane of detached osteoclasts that were situated between osteoblasts on the bone surface and blood vessels in the marrow cavity. Detection of activity on detached osteoclasts required the presence of an activator, implying lower levels in these cells than in those with ruffled borders. Staining was greater on the lateral sides of osteoblasts and osteoclasts when they were in contact with each other. Reaction specificity was indicated by the demonstration of stimulation by forskolin, guanylate-(beta, gamma-methylene) diphosphate (GMP-PCP), dimethylsulfoxide, and NaF, inhibition by alloxan and 2',5'-dideoxyadenosine, and absence of activity when sections were incubated in substrate-free medium or when GMP-PCP replaced AMP-PCP as a substrate. The finding of adenylate cyclase in osteoclast plasma membrane provides structural evidence that the adenylate cyclase-cyclic AMP system has a role in regulation of osteoclast cell function. The low-calcium diet appears to have resulted in increased amounts of adenylate cyclase in osteoclasts.     

Cytochemical demonstration of adenylate cyclase activity with cerium

Cerium was applied for the ultrastructural, cytochemical localization of adenylate cyclase (EC 4.6.1.1.). The enzyme activity was stimulated with norepinephrine, prenalterol and cholera toxin in the brown fat cells of newborn rats. The final reaction product was observed in the plasmalemmas of the stimulated adipocytes. The precipitate was finely crystalline, easily visible in the electron microscope and in the X-ray microprobe analysis it yielded cerium and phosphate peaks, respectively. The use of cerium offers a new tool valid for the cytochemical localization of adenylate cyclase enzyme related to the membrane receptors.     

Modulation byn-alkanols of rat cardiac adenylate cyclase activity

Summary n-Alkanols (from methanol to decanol) have a biphasic effect on rat cardiac adenylate cyclase either basal or stimulated by GTP, GppNHp, NaF or hormones (isoproterenol, glucagon, secretin) in the presence of GTP. At high concentration, all the enzyme activities are inhibited. At low concentration, adenylate cyclase activity is either unchanged or potentiated depending on both the stimulus and the alkanols involved. Potentiation is due to an increase of maximum velocity with no change in the activation constant of the enzyme. Basal activity is unchanged as well as the isoproterenol-and glucagon-stimulated enzyme. The secretin-stimulated enzyme is potentiated. It is the guanyl nucleotide regulatory protein-mediated stimulation of adenylate cyclase which is mainly affected. An attempt was made to relate these effects on adenylate cyclase with physical parameters of the alkanols (partition coefficient). From the data obtained as a function of the alkanol chain-length and of temperature on the adenylate cyclase stimulated by GTP, GppNHp, NaF and permanently activated, it is concluded that the increase in efficacy observed in the presence of alkanol is due to an interaction with the protein moeity particularly with the guanyl nucleotide regulatory protein.     

Cytochemical demonstration of adenylate cyclase activity with cerium

Summary Cerium was applied for the ultrastructural, cytochemical localization of adenylate cyclase (EC 4.6.1.1.). The enzyme activity was stimulated with norepinephrine, prenalterol and choleratoxin in the brown fat cells of newborn rats. The final reaction product was observed in the plasmalemmas of the stimulated adipocytes. The precipitate was finely crystalline, easily visible in the electron microscope and in the X-ray microprobe analysis it yielded cerium and phosphate peaks, respectively. The use of cerium offers a new tool valid for the cytochemical localization of adenylate cyclase enzyme related to the membrane receptors.This study was supported by the grant from Reino Lahtikari Foundation     

The control of adenylate cyclase activity inEscherichia coli

Cell-free extract of E. coli possessed an inhibited adenylate cyclase activity after a previous anaerobic incubation of cells with glucose which is transported and metabolized. The degree of the inhibition depends on incubation conditions. Glucose analogues that are only transported but not metabolized, are not inhibitory. To restore the adenylate cyclase activity, the cells have to be cultivated aerobically prior to disintegration for a defined period of time without glucose.     

Mouse neuroblastoma adenylate cyclase. Adenosine and adenosine analogues as potent effectors of adenylate cyclase activity.

1. Intact mouse neuroblastoma NS20 cells, in the presence of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase inhibitor, responded to adenosine (200 muM) and 2-chloroadenosine (200 muM) with a 20-fold increase in intracellular cAMP levels. AMP (200 muM) additions caused only a 3.5-fold cAMP level elevation. ATP, ADP, guanosine, cytidine, uridine, and guanine, all at 200 muM, had no effect on the cAMP level of these cells. 2. Homogenate NS20 adenylate cyclase activity was increased 2.5- to 4-fold by addition of 200 muM adenosine, 2-chloroadenosine, 2-hydroxyadenosine, or 8-methylaminoadenosine. Prostaglandin E1 additions (1.4 muM) produced about an 8-fold stimulation of homogenate cyclase activity. The Km of homogenate cyclase activation by adenosine and 2-chloroadenosine was 67.6 and 6.7 muM, respectively. Addition of 7-deazaadenosine, tolazoline, yohimbine, guanosine, cytosine, guanine, 2-deoxy-AMP, and adenine 9-beta-D-xylopyranoside, all at 200 muM were found to be without effect on homogenate NS20 adenylate cyclase. Two classes of inhibitors of homogenate NS20 adenylate cyclase activity were observed. One class, which included AMP, adenine, and theophylline, blocked 2-chloroadenosine but not prostaglandin E1 stimulation of cyclase. Theophylline was shown to be a competitive inhibitor of 2-chloroadenosine, with a Ki of 35 muM. The second class of inhibitors, which included 2'- and 5'-deoxyadenosine, inhibited unstimulated, 2-chloroadenosine and prostaglandin E1-stimulated homogenate cyclase activity to about the same degree. 3. Activation of NS20 homogenate adenylate cyclase by adenosine appears to be noncooperative. 4. The inhibitory action of putative "purinergic" neurotransmitters is postulated to be due to their effects on adenylate cyclase activity.     

Streptozotocin-induced diabetes produces alterations in adenylate cyclase in rat cerebrum, cerebral microvessels and retina

Eight weeks following streptozotocin-induced diabetes mellitus in rats, the sensitivity of adenylate cyclase to dopamine (DA) and norepinephrine (NE) was reduced in homogenates of retina. Furthermore, the activation of adenylate cyclase in cerebral microvessels (capillaries) by NE, 5'-guanylyl imidodiphosphate (alone or with NE) and forskolin was reduced in diabetic rats versus appropriate controls. In diabetic rats enzyme sensitivity to only NE was attenuated in homogenates of cerebral cortex and cortical piaarachnoid. No differences between controls and diabetics were noted with respect to guanylate cyclase or cyclic AMP phosphodiesterases. The damage observed in retina and microvessels may play an important pathogenic role in diabetes-induced blindness and stroke.