Theoretical analysis of the consequences of cyclic nucleotide phosphodiesterase negative co-operativity. Amplification and positive co-operativity of cyclic AMP accumulation.

Most tissues contain multiple forms of cyclic nucleotide phosphodiesterases (3':5'-cyclic-nucleotide 5' nucleotidohydrolase, EC 3.1.4.17). Consequently, in most, if not in all, tissues, substrate-velocity curves deviate from Michaelian kinetics and exhibit an apparent negative co-operativity. We have studied the possible theoretical consequences of this property on the quantitative features of cyclic AMP accumulation in response to activation of adenylate cyclase. Negative co-operativity of phosphodiesterases tends to generate a "positively co-operative" cyclic AMP accumulation curve. It amplifies the stimulation of cyclic AMP accumulation as compared with the stimulation of cyclic AMP synthesis. It enhances the sensitivity of cyclic AMP accumulation to slight variation of phosphodiesterase maximal velocity. It tends to shift the cyclic AMP accumulation curve to higher concentrations of stimulator as compared with the adenylate cyclase activation curve. This accounts for much of the data in the literature of hormonal effects on phosphodiesterase activity. It shows that the characteristics of cyclic nucleotide phosphodiesterases are as important as those of adenylate cyclase in determining the response of the system.     

Activities and some properties of adenylate cyclase and phosphodiesterase in muscle, liver and nervous tissues from vertebrates and invertebrates in relation to the control of the concentration of adenosine 3'':5''-cyclic monophosphate.

1. The basal and fluoride-stimulated activities of adenylate cyclase, and the maximal activities of 3':5'-cyclic AMP phosphodiesterase and 3':5'-cyclic GMP phosphodiesterase, together with the Km values for their respective substrates, were measured in muscle, liver and nervous tissues from a large range of animals to provide information on the mechanism of control of cyclic AMP concentrations in these tissues. High activities of adenylate cyclase and cyclic AMP diesterase are found in nervous tissues and in the more aerobic muscles (e.g. insect flight muscles, cardiac muscle and some vertebrate skeletal muscles). The activities of these enzymes in liver are similar to those in the heart of the same animal. The Km values for the enzymes from different tissues and animals are remarkably similar. 2. The comparison of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase activities suggests that in vertebrate tissues only one enzyme (the high-Km enzyme), which possesses dual specificity, exists, whereas in invertebrate tissues there are at least two phosphodiesterases with separate specificities. 3. A simple quantitative model to explain the control of the steady-state concentrations of cyclic AMP is proposed. The maximum increase in cyclic AMP concentration predicted by comparison of basal with fluoride-stimulated activities of adenylate cyclase is compared with the maximum increases in concentration produced in the intact tissue by hormonal stimulation: reasonable agreement is obtained. The model is also used to predict the actual concentrations and the rates of turnover of cyclic AMP in different tissues and, where possible, these values are compared with reported values. Reasonable agreement is found between predicted and reported values. The possible physiological significances of different rates of turnover of cyclic AMP and the different ratios of high- and low-Km phosphodiesterases in different tissues are discussed.     

Short-term changes in levels of cyclic AMP, adenylate cyclase, and phosphodiesterase during the initiation of sperm motility in rainbow trout

In order to clarify the role of the system that generates and degrades cyclic AMP during the initiation of motility of trout sperm, short-term changes in levels of intraspermatozoal cyclic AMP, adenylate cyclase, and phosphodiesterase were measured. Levels of cyclic AMP and the activity of adenylate cyclase increased and reached a maximum level 1 sec after transfer of sperm to K+-free medium, where they became motile, and then decreased rapidly. However, there were no changes in either parameter in sperm which remained immotile in K+-rich medium. In addition, an increase in the activity of phosphodiesterase was observed 4 sec later than the increase in levels of cyclic AMP and adenylate cyclase. These findings suggest that a very rapid change in the level of intracellular cyclic AMP occurs within 1 sec, at the moment of spawning, by the activation of adenylate cyclase and phosphodiesterase, and regulates the initiation of trout sperm motility.     

Translocation of phospholipid/Ca2+-dependent protein kinase in B-lymphocytes activated by phorbol ester or cross-linking of membrane immunoglobulin.

Treatment of hepatocytes with islet activating protein (pertussis toxin) from Bordetella pertussis blocked the ability of insulin to inhibit adenylate cyclase activity both in broken plasma membranes and in intact hepatocytes. Such treatment of intact hepatocytes with pertussis toxin did not prevent insulin from activating the peripheral plasma membrane cyclic AMP phosphodiesterase although it did inhibit the ability of insulin to activate the 'dense-vesicle' cyclic AMP phosphodiesterase. The ability of glucagon pretreatment of hepatocytes to block insulin's activation of the plasma membrane cyclic AMP phosphodiesterase was abolished in pertussis toxin-treated hepatocytes. It is suggested that the ability of insulin to manipulate cyclic AMP concentrations by inhibiting adenylate cyclase and activating the plasma membrane and 'dense-vesicle' cyclic AMP phosphodiesterases involves interactions with the guanine nucleotide regulatory protein system occurring in liver plasma membranes.     

An insulin mediator preparation serves to stimulate the cyclic GMP activated cyclic AMP phosphodiesterase rather than other purified insulin-activated cyclic AMP phosphodiesterases

An insulin mediator preparation was obtained from rat hepatocytes which had been treated with insulin. This preparation inhibited adenylate cyclase activity. It stimulated the activity of homogeneous preparations of both the cytosolic and membrane-bound forms of rat liver cyclic GMP-activated cyclic AMP phosphodiesterase. It failed to activate homogeneous preparations of both the peripheral plasma membrane and 'dense-vesicle' cyclic AMP phosphodiesterases. The insulin mediator preparation stimulated cyclic GMP-activated cyclic AMP phosphodiesterase activity in a dose-dependent fashion with a hill coefficient of 0.46. Insulin caused the dose-dependent production of mediator activity in intact hepatocytes with a Ka of 9 pM, although concentrations of insulin greater than 10 nM progressively reduced stimulatory activity.     

Receptor-specific threshold effects of cyclic AMP are involved in the regulation of enzyme release and superoxide production from human neutrophils

We have investigated the sequence of events leading from the activation of adenylate cyclase and increases in intracellular cyclic AMP to the modulation of enzyme release and superoxide production in human neutrophils. In the isolated plasma membrane, adenylate cyclase is activated by both prostaglandin E1 and isoproterenol. In the whole cell only a small increase in cyclic AMP is observed, though in the presence of the phosphodiesterase inhibitor, methylisobutylxanthine a substantial amplification in intracellular cyclic AMP is observed with both isoproterenol and prostaglandin E1. These conditions are relevant to the regulation of cell function, since fMet-Leu-Phe-stimulated superoxide production is inhibited by either prostaglandin E1 or isoproterenol in the absence of methylisobutylxanthine, while enzyme release is inhibited only via the prostaglandin E1 receptor and then only in the presence of methylisobutylxanthine. For enzyme release and superoxide production, the order of potency for three prostaglandins tested was prostaglandin E1 greater than prostaglandin D2 much greater than prostaglandin F2 alpha. Our results suggest that (a) superoxide production is more sensitive to regulation by cyclic AMP than enzyme release, (b) the type of receptor occupied as well as the threshold level of cyclic AMP attained are important to the regulation of enzyme release, and (c) although elevation in cyclic AMP is inhibitory to neutrophil function, phosphodiesterase inhibition is required in addition to adenylate cyclase activation to effect maximal inhibition.     

Large potentiation of agonist response in intact cells is produced by increases only in GTP-dependent adenylate cyclase activity

Treatment of cultured SV40-transformed normal rat kidney cells with the drug, 2-pyridine carboxylic acid, results in a pronounced potentiation in the ability of isoproterenol, prostaglandin E1, and cholera toxin to elevate cyclic AMP levels. With isoproterenol, the initial rate of cyclic AMP accumulation and the maximum cyclic AMP attainable are increased, and also the time of maximum cyclic AMP is prolonged. GTP-dependent adenylate cyclase activities are potentiated in crude membranes from the treated cells, but no evidence for alterations in cyclic nucleotide phosphodiesterase or release of cyclic AMP into the medium could be demonstrated. Results show that augmented adenylate cyclase activity alone, without changes in phosphodiesterase, can lead to dramatic alterations in cyclic AMP accumulation in response to cyclase agonists.     

The periodic change in adenosine 3',5'-monophosphate concentration in sea-urchin eggs

The level of adenosine 3',5'-monophosphate (cyclic AMP) in the eggs of the sea urchin, Anthocidaris crassispina, was found to change periodically after fertilization. The minimum and maximum levels of cyclic AMP were 1.0 X 10(-7)M and 1.5 X 10(-6)M, respectively. The activity of adenylate cyclase in a 105 000 X g precipitate reached a plateau at 20 min after fertilization and stayed constant for at least 2 h. It was also found that 1.0 mM CaCl2 increased the activity of adenylate cyclase in the same precipitate from unfertilized eggs. In contrast, phosphodiesterase activity changed periodically and correlated with cyclic AMP levels in the eggs. Up to a concentration of 1.5 X 10(-6)M cyclic AMP, phosphodiesterase activity was low, but it became activated when the level of cyclic AMP rose beyond this level. These results indicate that the change in the intracellular level of cyclic AMP is regulated mainly by the change in phosphodiesterase activity.     

Studies in vivo on the effects of parathyroid hormone upon kidney cyclic adenosine 3',5'-monophosphate content using rapid tissue fixation by microwave irradiation.

Microwave irradiation is shown to be a useful method for simultaneously killing chicks and fixing tissues. Renal adenylate cyclase and phosphodiesterase activities were rapidly abolished by microwaving. The increase in chick kidney cyclic adenosine 3',5'-monophosphate (cyclic AMP) content produced by intravenous bovine parathyroid hormone (PTH) injection was much greater in microwaved birds than in those killed by cervical dislocation with subsequent tissue fixation in liquid nitrogen. After PTH injection there was a prolonged elevation of renal cyclic AMP content. At the time of maximum response (2 minutes), log. dose-response curves were linear in the dose range 0.1-10 U. The responses to three different bovine PTH preparations were indistinguishable. Arginine vasopressin, arginine vasotocin, salmon calcitonin and prostaglandin E1 did not affect kidney cyclic AMP content within 2 minutes. Because of its specificity and precision, the method is of use for the in vivo bioassay of PTH. Injection of CaCl2 (20 mumoles) 1 minute before, or conjointly with, bovine PTH inhibited the subsequent increase in kidney cyclic AMP content. The synthetic bovine PTH peptide fragments BPTH (1-34) and BPTH (2-34) both increased chick kidney cyclic AMP content. The use of such fragments allows investigation of the structural requirements of PTH for interaction with the systems regulating cyclic AMP metabolism in the kidney in vivo.     

Role of Silicon in Diatom Metabolism : Cyclic Nucleotide Levels, Nucleotide Cyclase, and Phosphodiesterase Activities during Synchronized Growth of Cylindrotheca fusiformis

Adenylate cyclase, guanylate cyclase, and the cyclic nucleotide phosphodiesterases of Cylindrotheca fusiformis were characterized in crude and partially purified preparations. Both cyclases were membrane-bound and required Mn²⁺ for activity, though Mg²⁺ gave 50% activity with adenylate cyclase. Properties of adenylate cyclase were similar to those of higher eukaryotic cyclases in some respects, and in other respects were like lower eukaryotic cyclases. Guanylate cyclase was typical of other lower eukaryotic enzymes.

Two phosphodiesterase activities were found, one selective for cyclic AMP, the other for cyclic GMP. The 5′-nucleoside monophosphate was the major product of both activities and each of the enzymes had distinctive divalent cation requirements, pH optima, and kinetic parameters. Both phosphodiesterases were similar to those of other lower eukaryotes with one notable difference: the cyclic AMP enzyme was inhibited by calcium.

Changes in the cyclic nucleotide levels were quantitated in light-dark and silicon-starvation synchronized cultures using a more sensitive radioimmunoassay than used in a previously published study (Borowitzka and Volcani 1977 Arch Microbiol 112: 147-152). Contrary to the previous report, the cyclic GMP level did not change significantly in either synchrony. The cyclic AMP level increased dramatically very early in the period of DNA replication with the peak cyclic AMP accumulation substantially preceding that of DNA synthesis in both synchronies. There was no significant change in the activity of either cyclase or either phosphodiesterase during either synchrony. Thus, the mechanism for the rise in cAMP level remains unclear.

     

The adenylate cyclase-cyclic AMP-protein kinase system in different cell populations of the guinea pig gastric mucosa

In isolated guinea pig gastric mucous and enriched parietal cells it was tested whether or not cyclic AMP in response to histamine stimulation might reach concentrations sufficiently high to activate an intracellular cyclic AMP-dependent protein kinase and thereby mediate the acid response. Although histamine stimulated parietal cell adenylate cyclase to a greater extent than mucous cell adenylate cyclase, cyclic AMP levels in response to maximal histamine stimulation reached higher levels in mucous than in parietal cells. This had to be attributed to a five times higher phosphodiesterase activity in parietal cell than in mucous cell populations. In the absence of the phosphodiesterase inhibitor isobutylmethylxanthine exposure of the cells to histamine only in mucous cells produced an increase in cyclic AMP-dependent protein kinase activity ratio, but not in parietal cells. Dibutyryl-cyclic AMP induced cyclic AMP accumulation in parietal cell populations was compared to dibutyryl-cyclic AMP induced H+ secretion, as measured by 14C-aminopyrine uptake. A maximal acid response was associated with an intracellular cyclic AMP level of approximately 300 pmol/10(6) cells, which was never reached by maximal histamine stimulation even not in the presence of the phosphodiesterase inhibitor. It is concluded that activation of the parietal cell cyclic AMP-dependent protein kinase is one way for stimulating H+ secretion, but that the acid response elicited by histamine requires another intracellular pathway.     

Photolytic studies on the carbon monoxide complex of sulphaemoglobin.

Salivary-gland homogenates contain 5-hydroxytryptamine-stimulated adenylate cyclase. Half-maximal stimulation was obtained with 0.1 microM-5-hydroxytryptamine in the presence of added guanine nucleotides. Gramine antagonized the stimulation of cyclase caused by 5-hydroxytryptamine. In the presence of hormone, guanosine 5'-[gamma-thio]triphosphate produced a marked activation of adenylate cyclase activity. Stimulation of adenylate cyclase by forskolin or fluoride did not require the addition of guanine nucleotides or hormone. In the presence of EGTA, Ca2+ produced a biphasic activation of cyclase activity. Ca2+ at 1-100 microM increased activity, whereas 2000 microM-Ca2+ inhibited cyclase activity. The neuroleptic drugs trifluoperazine and chlorpromazine non-specifically inhibited adenylate cyclase activity even in the absence of Ca2+. The cyclic AMP phosphodiesterase activity in homogenates was not affected by Ca2+ or exogenous calmodulin. This enzyme was also inhibited by trifluoperazine in the absence of Ca2+. These results indicate that Ca2+ elevates adenylate cyclase activity, but had no effect on cyclic AMP phosphodiesterase of salivary-gland homogenates.     

Enzymes of cyclic nucleotide metabolism in invertebrate and vertebrate sperm.

Sperm from several invertebrates contained guanylate cyclase activity several-hundred-fold greater than that in the most active mammalian tissues; the enzyme was totally particulate. Activity in the presence of Mn²⁺ was up to several hundred-fold greater than with Mg²⁺ and was increased 3–10-fold by Triton X-100. Sperm from several vertebrates did not contain detectable guanylate cyclase. Sperm of both invertebrates and vertebrates contained roughly equal amounts of Mn²⁺-dependent adenylate cyclase activity; in invertebrate sperm, this enzyme was generally several hundred-fold less active than guanylate cyclase. Adenylate cyclase was particulate, was unaffected by fluoride, and was generally greater than 10-fold more active with Mn²⁺ than with Mg²⁺. Invertebrate sperm contained phosphodiesterase activities against 1.0 μm cyclic GMP or cyclic AMP in amounts greater than mammalian tissues. Fish sperm, which did not contain guanylate cyclase, had high phosphodiesterase activity with cyclic AMP as substrate but hydrolyzed cyclic GMP at a barely detectable rate. In sea urchin sperm, phosphodiesterase activity against cyclic GMP was largely particulate and was strongly inhibited by 1.0% Triton X-100. In contrast, activity against cyclic AMP was largely soluble and was weakly inhibited by Triton. The cyclic GMP and cyclic AMP contents of sea urchin sperm were in the range of 0.1–1 nmol/g. Sea urchin sperm homogenates possessed protein kinase activity when histone was used as substrate; activities were more sensitive to stimulation by cyclic AMP than by cyclic GMP.⁵     

Hormonal activation of adenylate cyclase in mouse melanoma metastatic variants.

The ability of melanocyte stimulating hormone (MSH), adrenocorticotropic hormone (ACTH), and prostaglandin E1 (PGE1) to stimulate the accumulation of cyclic AMP was examined in intact mouse melanoma cells of varying metastatic potential. F1 cells (low metastatic potential) had significantly greater cyclic AMP levels in response to all three hormones than F5 (intermediate metastatic potential) and F10 (high metastatic potential) cells. The ranking of the response was as follows: MSH, F1 greater than F5 greater than F10, ACTH, F1 greater than F5 greater F10, PGE, F1 greater than F10 greater F5. In contrast to the above, the degree of hormonal stimulation of adenylate cyclase in broken cell preparations was virtually identical in all three melanoma cell lines. Control enzyme activity was depressed in both F5 and F10 relative to F1. The conflicting results between studies of intact vs. broken cell preparations could not be explained by increased cyclic AMP phosphodiesterase activity in F5 and F10 cells. We conclude that as the melanoma cells increase in metastatic potential, there is a significant loss in the ability of their cyclic AMP system to respond appropriately to hormonal stimuli.     

Activation of adenylate cyclase in renal medulla by bovine growth hormone. An artifact attributable to vasopressin.

The effect of bovine growth hormone on adenylate cyclase activity was studied in bovine and rat renal medulla. Highly purified growth hormone (lot B1003A) increased adenylate cyclase activity in plasma membranes from bovine renal medulla from 132+/-6 pmol cyclic AMP formed/mg protein per 10 min to 364+/-10 pmol cyclic AMP formed/mg protein per 10 min. Similar results were seen with homogenates of rat renal medulla. The minimum effective concentration of bovine growth hormone required to activate adenylate cyclase was 0.5 mug/ml and maximum activation was detected at 500 mug/ml. The amount of vasopressin determined by radioimmunoassay to contaminate the growth hormone caused an increase in adenylate cyclase activity comparable to that of the corresponding concentration of growth hormone that was tested. Dialysis of growth hormone and vasopressin resulted in parallel reductions in the effect of each hormone on adenylate cyclase activity. Similarly, both growth hormone and vasopressin produced increases in short circuit current in isolated toad bladders but these effects were not detectable after dialysis of the hormones. In contrast, the effect of growth hormone on the uptake of 35SO2-4 by cartilage from hypophysectomized rats was not decreased after dialysis. These results indicate that available preparations of growth hormone are contaminated by small but physiologically significant amounts of vasopressin and that the activation of adenylate cyclase activity in renal medulla in response to growth hormone can be explained by this contamination rather than by an effect of growth hormone per se.     

Relationship of Adenosine 3′,5′-Monophosphate to Growth and Metabolism of Tetrahymena pyriformis

The concentration of adenosine 3',5'-monophosphate (cyclic AMP) and the activity of adenylate cyclase were determined for the first time in conjuncation with cyclic 3',5'-nucleotide phosphodiesterase (phosphodiesterase) during the growth cycle of Tetrahymena pyriformis. High levels of cyclic AMP observed during early exponential and late stationary phases were associated with elevated adenylate cyclase and decreased phosphodiesterase activities. Adenylate cyclase and cyclic AMP were decreased and phosphodiesterase was increased in cells grown in glucose-supplemented medium. In contrast to findings in mammalian liver, cyclic AMP was decreased during active gluconeogenesis in Tetrahymena. This suggests a different modulation of carbohydrate metabolism in the two species. The results illustrate that both the content of cyclic AMP and its action as a regulatory agent in Tetrahymena are uniquely suited to the metabolism of this organism.     

Evidence that the mitochondrial activator of phosphorylated branched-chain 2-oxoacid dehydrogenase complex is the dissociated E1 component of the complex

The ability of glucagon (10 nM) to increase hepatocyte intracellular cyclic AMP concentrations was reduced markedly by the tumour-promoting phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate). The half-maximal inhibitory effect occurred at 0.14 ng/ml TPA. This action occurred in the presence of the cyclic AMP phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) indicating that TPA inhibited glucagon-stimulated adenylate cyclase activity. TPA did not affect either the binding of glucagon to its receptor or ATP concentrations within the cell. TPA did inhibit the increase in intracellular cyclic AMP initiated by the action of cholera toxin (1 microgram/ml) under conditions where phosphodiesterase activity was blocked. TPA did not inhibit glucagon-stimulated adenylate cyclase activity in a broken plasma membrane preparation unless Ca2+, phosphatidylserine and ATP were also present. It is suggested that TPA exerts its inhibitory effect on adenylate cyclase through the action of protein kinase C. This action is presumed to be exerted at the point of regulation of adenylate cyclase by guanine nucleotides.     

Activation of adenylate cyclase in renal medulla by bovine growth hormone: An artifact attributable to vasopressin

The effect of bovine growth hormone on adenylate cyclase activity was studied in bovine and rat renal medulla. Highly purified growth hormone (lot B1003A) increased adenylate cyclase activity in plasma membranes from bovine renal medulla from 132 ± 6 pmol cyclic AMP formed/mg protein per 10 min to 364 ± 10 pmol cyclic AMP formed/mg protein per 10 min. Similar results were seen with homogenates of rat renal medulla. The minimum effective concentration of bovine growth hormone required to activate adenylate cyclase was 0.5 μg/ml and maximum activation was detected at 500 μg/ml. The amount of vasopressin determined by radioimmunoassay to contaminate the growth hormone caused an increase in adenylate cyclase activity comparable to that of the corresponding concentration of growth hormone that was tested. Dialysis of growth hormone and vasopressin resulted in parallel reductions in the effect of each hormone on adenylate cyclase activity. Similarly, both growth hormone and vasopressin produced increases in short circuit current in isolated toad bladders but these effects were not detectable after dialysis of the hormones. In contrast, the effect of growth hormone on the uptake of ³⁵SO₄²⁻ by cartilage from hypophysectomized rats was not decreased after dialysis. These results indicate that available preparations of growth hormone are contaminated by small but physiologically significant amounts of vasopressin and that the activation of adenylate cyclase activity in renal medulla in response to growth hormone can be explained by this contamination rather than by an effect of growth hormone per se.     

Formation of adenosine 3',5'-monophosphate from adenosine in mouse thymocytes.

The effect of adenosine on the mouse thymocyte adenylate cyclase-adenosine 3':5'-monophosphate (cyclic AMP) system was examined. Adenosine, like prostaglandin E1, can cause 5-fold or greater increases in thymocyte cyclic AMP content in the presence but not in the absence of certain cyclic phosphodiesterase inhibitors. Two non-methylxanthine inhibitors potentiated the prostaglandin E1 and adenosine responses, while methylxanthines selectively inhibited the adenosine response. Adenosine increased cyclic AMP content significantly within 1 min and was maximal by 10 to 20 min with approx. 2 and 10 muM adenosine being minimal and half-maximal effective doses, respectively. Combinations of prostaglandin E1, isoproterenol and adenosine were near additive and not synergistic. Of the adenosine analogues tested, only 2-chloro- and 2-fluoroadenosine significantly increased cyclic AMP. Thymocytes prelabeled with [14C]adenine exhibited dramatic increases in cyclic [14C]AMP 10 min after addition of adenosine or prostaglandin E1 which corresponded to simultaneously determined increases in total cyclic AMP. Using [14C]adenosine, the percent of total cyclic AMP increase due to adenosine was only 16%. Adenosine was also shown to elicit a 40% increase in particulate thymocyte adenylate cyclase activity. Therefore, the increased content of cyclic AMP seen in mouse thymocytes after incubation with adenosine was due primarily to stimulation of adenylate cyclase and only partially to conversion of adenosine to cyclic AMP. The increased cellular content of cyclic AMP may be, in part, responsible for various immunosuppressive effects of adenosine.