Stimulated Guanylate Cyclase Activity Research Articles

Adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate: levels and cyclase activities in liver and adipose tissue from diabetic mice (db/db).

The adenylate and guanylate cyclase activities and the adenosine and guanosine 3′:5′‐monophosphate (cyclic AMP and cyclic GMP) contents have been investigated in the liver and adipose tissue of control homozygote (+m/+m), heterozygote (db+/+m) and diabetic homozygote (db+/db+) mice (C57BL/Ks‐J‐db m strain).The response to (−)‐isoproterenol of the adenylate cyclase system in isolated fat cells, as well as in adipocyte plasma membranes, from diabetic mice (db+/db+), is impaired as compared to their thin littermates (+m/+m) and also to heterozygote (db+/+m) mice. The increase in cyclic AMP formation elicited by (−)‐isoproterenol was 10‐fold and 4‐fold higher in isolated adipocytes and in adipocyte plasma membranes respectively from lean control as compared to diabetic mice. The cyclic AMP content of adipose tissue from diabetic mice (44.0 ± 7.6 fmol per mg of wet weight) was markedly lower than that from control and heterozygote mice (128.3 ± 41.5 and 128.4 ± 34.5 fmol per mg of wet weight, respectively). In contrast the basal adenylate cyclase activities of liver homogenates and the activties stimulated by glucagon, epinephrine and fluoride were similar in the three groups of animals. Similarly, the hepatic cyclic AMP content did not differ in the three genotypes of mice. These findings indicate that disturbances in cyclic AMP metabolism in adipose tissue from diabetic mice might result from a phenomenon secondary to the diabetic syndrome.The specific activity of guanylate cyclase in liver and adipocytes progressively decreased from lean control (+m/+m) to heterozygote (db+/+m) to homozygote diabetic (db+/db+) mice; e.g. the guanylate cyclase activity in adipocyte plasma membranes was markedly diminished in diabetic mice (18.9 ± 1.9, 12.7 ± 0.9 and 4.7 ± 1.4 pmol cyclic GMP · min−1· (mg protein)−1 for homozygote control, heterozygote and diabetic mice, respectively). A similar situation was noted in respect of the cyclic GMP levels in the liver and adipose tissue of these animals. Thus, the cyclic GMP content in tissues from diabetic mice represented only 13% and 43% of that in the liver and adipose tissue respectively of control mice. These data are consistent with the existence of a dosage effect of the db gene on cyclic GMP metabolism. On the other hand identical maximal guanylate cyclase activities were obtained after preincubation with Triton X‐100 of adipocyte plasma membranes from the three kinds of mice. At least, there was a 19‐fold and 2.5‐fold stimulation of guanylate cyclase activity by sodium azide in liver homogenates from homozygote diabetic mice and homozygote control, respectively; the hypersensitivity to NaN3 of guanylate cyclase activity in liver and adipose tissue could be related to a disturbance in peroxide metabolism in the tissues of the mutant.

Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by contaminating soluble enzyme, suggesting that physiological aggregating agents may increase cyclic GMP in intact platelets through the effects of intermediary factors. The activated and inhibited states of the enzyme described in the present paper may be relevant to the actions of these factors.

Stimulation of guanylate cyclase activity in cultured osteogenic murine calvarial mesenchymal cells by PTH, calcitonin and insulin

These studies provide the first evidence that parathyroid hormone (PTH), calcitonin (CT), and insulin, all known effectors of bone cell metabolism, stimulate the activity of guanylate cyclase in osteogenic cells derived from fetal mouse calvarial mesenchyme. Adenylate cyclase activity was stimulated by PTH and epinephrine, but not by CT, the latter effect being consistent with an absence of osteoclastpprogenitor cells in this osteogenic mesenchyme. Adenylate cyclase activity was associated entirely with the particulate fraction of the cells while guanylate cyclase, as well as acid and alkaline phosphatase, were present in both soluble and particulate material. The activation of guanylate cyclase by hormones may provide a better basis for understanding the differentiation and regulation of osteogenic cells.

Activation of guanylate cyclase by arachidonic acid: In Mammary gland homogenates from mice

Arachidonic acid stimulated guanylate cyclase activity about two fold in homogenates of mammary glands obtained from midpregnant mice; effects of arachidonic acid were observed during incubation periods between 5 and 20 minuted. Stimulatory effects of arachidonic acid on guanylate cyclase activity were observed when 10 to 100 μg arachidonic acid was added to the reaction mixtures (150 μl). When 250 μg or more arachidonic acid was added to the reaction mixtures, the activity of guanylate cyclase was inhibited. Other fatty acids including linoleic acid, linolenic acid and oleic acid also stimulated guanylate cyclase activity but neither arachidic acid nor stearic acid had an effect. The arachidonic acid stimulation of guanylate cyclase activity was abolished by incubation with indomethacin and aspirin, thus suggesting the arachidonic acid effect may be carried out via the prostaglandins. A variety of prostaglandins, however, at several concentrations did not stimulate guanylate cyclase activity when added to the reaction mixtures. The failure of the prostaglandins to have an effect may be due to several reasons which are discussed.

Methylprednisolone stimulation of guanylate cyclase activity in rat ileal mucosa

Methylprednisolone stimulation of guanylate cyclase activity in rat ileal mucosa

Stimulation of soluble guanylate cyclase activity and cellular accumulation of cyclic guanosine 3',5'-monophosphate by the carcinogen 4-nitroquinoline 1-oxide: brief communication.

4-Nitroquinoline 1-oxide (4NQO), a compound that induces tumors in various rat organs, rapidly increased the cellular accumulation of cyclic guanosine 3',5'-monophosphate (cGMP) to peak values fourfold to 13-fold over basal levels in the liver, lung, renal cortex, and gastric and colon mucosa of rats. This action of 4NQO was expressed in the presence or absence of extracellular calcium. When added directly to the broken cell preparations, 4NQO also stimulated guanylate cyclase activity threefold to sixfold over basal levels in the 100,000 X g soluble fractions of each of these tissues. Dicumarol, which blocks the reduction of 4NQO, inhibited 4NQO stimulation of guanylate cyclase and cGMP. Conversely, phenythydrazine, which enhances the reduction of 4NQO, potentiated the actions of 4NQO on guanylate cyclase and cGMP. These results suggested that the activation of the guanylate cyclase-cGMP system may be mediated by reduction products of 4NQO. The activation of the guanylate cyclase system by 4NQO or its derivatives could function in the expression of carcinogenicity.

Stimulation of human platelet guanylate cyclase by unsaturated fatty acid peroxides.

Guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] activity of human platelet homogenates was stimulated by the addition of phospholipase A2 or unsaturated fatty acids such as oleic, vaccenic, linoleic, linolenic, eicosenoic, eicosadienoic, and arachidonic acids. The addition of lipoxidase potentiated the fatty acid-induced stimulation of guanylate cyclase purified by DEAE-cellulose column chromatography. The extent of the stimulation was dependent on the concentration of the oxidized form of these fatty acids (peroxides). Saturated fatty acids such as stearic and arachidic acids had no effect on the guanylate cyclase activity in the presence or absence of lipoxidase, indicating that human plateletguanylate cyclase is stimulated by unsaturated fatty acid peroxides rather than by fatty acids. Hemoglobin prevented the enzyme stimulation produced by low concentrations of fatty acid peroxides, but enhanced stimulation of the enzyme activity with high concentrations of fatty acid peroxides. 2-Mercaptoethanol, dithiothreitol, and N-ethylmaleimide inhibited the guanylate cyclase activities both in the presence and absence of unsaturated fatty acidperoxide. The stimulation of guanylate cyclase activity by unsaturated fatty acid peroxidesis attributed to oxidation of sulfhydryl residues of the enzyme protein.

Activation of guanylate cyclase in cerebral cortex of rat by hydroxylamine.

Hydroxylamine actived guanylate cyclase in particulate fraction of cerebral cortex of rat. Activation was most remarkable in crude mitochondrial fraction. When the crude mitochondrial fraction was subjected to osmotic shock and fractionated, guanylate cyclase activity recovered in the subfractions as assayed with hydroxylamine was only one-third of the starting material. Recombination of the soluble and the particulate fractions, however, restored guanylate cyclase activity to the same level as that of the starting material. When varying quantities of the particulate and soluble fractions were combined, enzyme activity was proportional to the quantity of the soluble fraction. Heating of the soluble or particulate fraction at 55 degrees for 5 min inactivated guanylate cyclase. The heated particulate fraction markedly activated guanylate cyclase activity in the native soluble fraction, while the heated soluble fraction did not stimulate enzyme activity in the particulate. The particulate fraction preincubated with hydroxylamine at 37 degrees for 5 min followed by washing activated guanylate cyclase activity in the soluble fraction in the absence of hydroxylamine. Further fractionation of the crude mitochondrial fraction revealed that the factor(s) needed for the activation by hydroxylamine is associated with the mitochondria. The mitochondrial fraction of cerebral cortex activated guanylate cyclase in supernatant of brain, liver, or kidney in the presence of hydroxylamine. The mitochondrial fraction prepared from liver or kidney, in turn, activated soluble guanylate cyclase in brain. Activation of guanylate cyclase by hydroxylamine was compared with that of sodium azide. Azide activated guanylate cyclase in the synaptosomal soluble fraction, while hydroxylamine inhibited it. The particulate fraction preincubated with azide followed by washing did not stimulate guanylate cyclase activity in the absence of azide. The activation of guanylate cyclase by hydroxylamine is not due to a change in the concentration of the substrate GTP, Addition of hydroxylamine did not alter the apparent Km value of guanylate cyclase for GTP. Guanylate cyclase became less dependent on manganese in the presence of hydroxylamine. Thus the activation of guanylate cyclase by hydroxylamine is due to the change in the Vmax of the reaction.

Stimulation of guanylate cyclase activity by several fatty acids

Guanylate cyclase of plasma membrane of isolated rat fat cells was activated 7 to 11 fold by oleic acid, linoleic acid, linolenic acid or arachidonic acid. The activation of the enzyme by linoleic acid or oleic acid was influenced by the concentration of enzyme protein and that of the fatty acid. At 158 μg/ml of enzyme protein, 0.6 mM linoleic acid produced maximal activation of 12 fold which was partially reversed by washing. Particulate guanylate cyclase of cerebral cortex and liver was also activated by linoleic acid.

Stimulation of membranous guanylate cyclase by concentrations of calcium that are in the physiological range

Summary Ca 2+ at a concentration as low as 3 μM stimulated guanylate cyclase activity in membranes of cultured Balb 3T3 fibroblasts when Mg 2+ was the major bivalent cation. If the enzyme was assayed with Mn 2+ as the major cation, Ca 2+ caused either inhibition or stimulation depending on conditions but these effects of Ca 2+ occurred at much higher concentrations. The addition of NaCl decreased the activity with Mg 2+ but increased the degree of stimulation of Ca 2+ . We suggest that the activity of guanylate cyclase in vitro with Mg 2+ represents its behavior in vivo and that Ca 2+ regulates cyclic GMP in cells by directly stimulating guanylate cyclase activity.

Stimulation of guanylate cyclase of fibroblasts by free fatty acids.

The membranous guanylate cyclase of Balb 3T3 fibroblasts was stimulated by a fraction of calf serum extracted by ether. Stimulation was observed with Mg2+ as the only bivalent cation in the presence of Lubrol PX. The activator co-chromatographed with free fatty acids, and several of these were found to stimulate guanylate cyclase. Among the saturated fatty acids, myristic acid had the highest activity. Stimulating activity diminished as the hydrocarbon chain of the fatty acid was lengthened or shortened. Introduction of an unsaturated bond enhanced the activation by the longer fatty acids. This pattern of specificity is similar to that observed for the effect of fatty acids on many other membranous functions. Under appropriate conditions fatty acids were found to stimulate guanylate cyclase activity in the absence of Lubrol PX. The relationship among the effects of Mg2+, Mn2+, Lubrol PX, and fatty acids on enzyme activity was examined. On the basis of these studies, it appears that fatty acids stimulate the enzyme by a mechanism different from nonionic detergents or Mn2+.

Increases in cyclic GMP levels in brain and liver with sodium azide an activator of guanylate cyclase

SODIUM azide produces extrapyramidal symptoms with necrosis of the cerebral cortex, cerebellum, and basal ganglia. Toxicity with this metabolic inhibitor may also include hypotension, blindness and hepatic necrosis1–5. Many hormones and drugs mediate their effects by altering the intracellular levels of cyclic AMP and/or cyclic GMP6–10. We found that NaN3 increases cyclic GMP levels in incubations of slices from cerebral cortex, cerebellum or liver, and also stimulates guanylate cyclase activity. In contrast to other agents that can increase cyclic GMP accumulation in tissues the effect of NaN3 is observed in the absence of added calcium.

Myocardial guanylate cyclase: Properties of the enzyme and effects of cholinergic agonists in vitro

The characteristics of myocardial guanylate cyclase (GTP pyrophosphatelyase, EC 4.6.1.2) were studied. Specific activity of the myocardial enzyme in five vertebrate species was guinea pig > man ⪢ cat > dog > rat. In the guinea pig, guanylate cyclase activity was uniformly distributed throughout the anatomical regions of the heart. The major portion of the enzyme activity was retrieved in the supernatant fraction after centrifugation at 12 000 × g. The K m for GTP was similar in supernatant (0.12 mM) and particulate (0.21 mM) preparations, although the K a for Mn 2+ in particulate preparations (0.3–0.6 mM) was less than that observed for guanylate cyclase in the supernatant fraction (0.8–2.0 mM). ATP competitively inhibited supernatant and particulate activity. Addition of 0.005–10.0 mM Ca 2+ to assay incubations did not enhance guanylate cyclase activity. Suspension of 105 000 × g supernatant guanylate cyclase preparations with membrane lipids or phosphatidylserine stimulated activity 1.4–4.3 fold, whereas similar treatment of particulate preparations caused little alteration of enzyme activity. Addition of the cholinergic agonists acetylcholine, carbachol or methacholine (10 −4–10 −8 M) to homogenate, supernatant, particulate and disrupted tissue slice preparations in the presence of 0.0012–1.2 mM GTP, 0.3–10.0 mM Mn 2+ and 0.005–10.0 mM Ca 2+ or 0.0012–1.2 mM ATP did not stimulate guanylate cyclase activity. Similarly, further stimulation of guanylate cyclase activity was not elicited when enzym-lipid suspensions were assayed in the presence of cholinergic agents.

Ion transport in rabbit ileal mucosa. IV. Bicarbonate secretion.

Ion transport in rabbit ileal mucosa. IV. Bicarbonate secretion.

Ion transport in rabbit ileal mucosa. 3. Effects of catecholamines.

Ion transport in rabbit ileal mucosa. 3. Effects of catecholamines.