Cyclic nucleotides and calcium in human lymphocytes induced to divide

Intracellular concentrations of cyclic adenosine 3'-5' monophosphate (cAMP) and cyclic guanosine 3'-5' monophosphate (cGMP) were measured in human lymphocytes induced to divide by the addition of lectins, 12-O-tetra-decanoylphorbol-13-acetate (TPA) and the calcium ionophore A 23187. cGMP levels rose within minutes without concomitant alterations in cAMP concentration. The cAMP and cGMP levels rose during the prereplicative and replicative phases respectively. Under calcium depleting conditions, both the fluctuations in cyclic nucleotide levels and the increase in [3H[ thymidine incorporation into DNA were abolished, suggesting a role for calcium ions in the regulation of lymphocyte proliferation.     

Effect of insulin on metabolism of cyclic adenosine 3',5'-monophosphate by plasma cell tumor (MPC-11).

Addition of insulin to cultured mouse plasma tumor cells (MPC-11) increases the entry of tritiated cyclic adenosine 3',5'-monophosphate (3H-cAMP). No increase of entry of N6-O2-dibutyryl adenosine 3',5' cyclic monophosphate (DBcAMP), 5' adenosine monophosphate (5' AMP) or adenosine was noted in the presence of insulin. The stimulation of cAMP transport by insulin was concentration dependent and inactivated insulin had no effect on nucleotide transport. Intracellular radioactivity after transport of cAMP was largely 5'AMP, while most of the extracellular radioactivity remained as cAMP after incubation.     

Regulation of bacterial motility by cyclic nucleotides and effect of biogenic amines.

When assayed by a newly devised, simple and quantitative method, "motilometry", the motility of E. coli S-26 was found stimulated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) and 8-hydroxy cyclic AMP as well as histamine, catecholamines and inhibited by cyclic guanosine 3':5'-monophosphate (cyclic GMP). The stimulation elicited by cyclic AMP or other biogenic amines was reversed by cyclic GMP. The experimental significance and implicaton of these findings are discussed.     

Intracellular Levels of Cyclic Nucleotides during Cell Enlargement and Cell Division in Excised Tobacco Pith Tissues

Two lines of evidence, one of which is based on the radioimmunoassay and the other on adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-dependent histone phosphorylation, are presented to demonstrate the presence of cyclic AMP in excised tissues of higher plant species. Intracellular levels of this cyclic nucleotide appear to be determined by auxin and a positive correlation has been found to exist between cell enlargement and chromosomal DNA replication, both auxin-dependent processes, and the level of cyclic AMP in tobacco pith cells. Intracellular guanosine 3', 5'-cyclic monophosphate (cyclic GMP) levels, while measurable, did not appear to be affected by either auxin or kinetin, or both, during the cell enlargement or cell division phases of the cell cycle in the tobacco pith system.     

Regulation of intracellular adenosine cyclic 3':5'-monophosphate levels in Escherichia coli and Salmonella typhimurium. Evidence for energy-dependent excretion of the cyclic nucleotide.

Sugars and other energy sources were found to lower intracellular concentrations of adenosine 3':5'-monophosphate (cyclic AMP) in strains of Escherichia coli and Salmonella typhimurium which were deficient for cyclic AMP phosphodiesterase. This effect required the presence of the specific transport system responsible for entry of that sugar into the cell and depended on the intracellular catabolic enzymes. Metabolizable sugars were more effective than nonmetabolizable sugars in reducing cellular cyclic AMP levels, and this reduction was blocked partially by uncouplers of oxidative phosphorylation. Electron donors such as lactate and ascorbate plus phenazine methosulfate reduced internal cyclic AMP levels in bacterial membrane vesicles which had been preloaded with the cyclic nucleotide. Uncouplers of oxidative phosphorylation, but not arsenate, blocked the energy-stimulated loss of intravesicular cyclic AMP. Employing intact cells, sugars were shown to have two primary effects on cyclic AMP metabolism: (a) they inhibited net synthesis of the cyclic nucleotide while promoting its degradation, and (b) they stimulated efflux of cyclic AMP into the extracellular fluid. While the former effect was elicited by metabolizable and nonmetabolizable sugars alike, stimulation of cyclic nucleotide excretion was only observed with metabolizable sugars. The results suggest that the extrusion of cyclic AMP from the bacterial cell is energy-dependent and is driven by an energized membrane state.     

Role of Cyclic Adenosine 3′,5′-Monophosphate in the In Vivo Expression of the Galactose Operon of Escherichia coli

Studies of levels of galactokinase in Escherichia coli with mutations affecting synthesis of, or response to, cyclic adenosine 3',5'-monophosphate show that this nucleotide does not play a major role in expression of the galactose operon, causing at most a twofold stimulation. The discrepancy between our in vivo results and the marked stimulation by cyclic adenosine 3',5'-monophosphate in in vitro systems indicates that current cell-free systems lack a factor which allows efficient expression of the galactose operon even in the absence of cyclic adenosine 3',5'-monophosphate or of the binding protein for this nucleotide.     

Human blood platelet 3': 5'-cyclic nucleotide phosphodiesterase. Isolation of low-Km and high-Km phosphodiesterase.

Human blood platelet contained at least three kinetically distinct forms of 3': 5'-cyclic nucleotide phosphodiesterase (3': 5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) (F I, F II, and F III) which were clearly separated by DEAE-cellulose column chromatography. Although a few properties of the platelet phosphodiesterases such as their substrate affinities and DEAE-cellulose profile resembled somewhat those of the three 3': 5'-cyclic nucleotide phosphodiesterase in rat liver reported by Russell et al. [10], there were pronounced differences in some properties between the platelet and the liver enzymes: (1) the platelet enzymes hydrolyzed both cyclic nucleotides and lacked a highly specific cyclic guanosine 3': 5'-monophosphate (cyclic GMP) phosphodiesterase and (2) kinetic data of the platelet enzymes indicated that cyclic adenosine 3': 5'-monophosphate (cyclic AMP) and cyclic GMP interact with a single catalytic site on the enzyme. F I was a cyclic nucleotide phosphodiesterase with a high Km for cyclic AMP and a negatively cooperative low Km for cyclic GMP. F II hydrolyzed cyclic AMP and cyclic GMP about equally with a high Km for both substrates. F III was low Km phosphodiesterase which hydrolyzed cyclic AMP faster than cyclic GMP. Each cyclic nucleotide acted as a competitive inhibitor of the hydrolysis of the other nucleotide by these three fractions with Ki values similar to the Km values for each nucleotide suggesting that the hydrolysis of both cyclic AMP and cyclic GMP was catalyzed by a single catalytic site on the enzyme. However, cyclic GMP at low concentration (below 10 muM) was an activator of cyclic AMP hydrolysis by F I. Papaverine and EG 626 acted as competitive inhibitors of each fraction with virtually the same Ki value in both assays using either cyclic AMP or cyclic GMP as the substrate. The ratio of cyclic AMP hydrolysis to cyclic GMP hydrolysis by each fraction did not vary significantly after freezing/thawing or heat treatment. These facts also suggest that both nucleotides were hydrolyzed by the same catalytic site on the enzyme. The differences in apparent Ki values for inhibitors such as cyclic nucleotides, papaverine and EG 626 would indicate that three enzymes were different from each other. Centrifugation in a continuous sucrose gradient revealed sedimentation coefficients F I and II had 8.9 S and F III 4.6 S. The molecular weight of these forms, determined by gel filtration on a Sepharose 6B column, were approx. 240 000 (F I and II) and 180 000 (F III). F III was purified extensively (70-fold) from homogenate, with a recovery of approximately 7%.     

A specific cyclic guanosine 3':5'-monophosphate-binding protein in Caulobacter crescentus.

A binding protein specific for cyclic guanosine 3':5'-monophosphate (cyclic GMP) has been partially purified from extracts of the eubacterium Caulobacter crescentus and resolved from cyclic adenosine 3':5'-monophosphate (cyclic AMP)-binding activity. Binding of cyclic GMP is not affected by the addition of cyclic AMP or 5'-GMP, but is inhibited about 50 percent by a 50-fold molar excess of dibutyryl cyclic GMP or cyclic hypoxanthine 3':5'-monophosphate. The apparent dissociation constant for the cyclic GMP-binding protein complex is 1.1 X 10(-6) M.     

Effects of adenosine 3' : 5'-monophosphate and guanosine 3' : 5'-monophosphate on calcium uptake and phosphorylation in membrane fractions of vascular smooth muscle.

The effects of adenosine 3' : 5'-monophosphate (cyclic AMP), guanosine 3' : 5'-monophosphate (cyclic GMP) and exogenous protein kinase on Ca uptake and membrane phosphorylation were studied in subcellular fractions of vascular smooth muscle from rabbit aorta. Two functionally distinct fractions were separated on a continuous sucrose gradient: a light fraction enriched in endoplasmic reticulum (fraction E) and a heavier fraction containing mainly plasma membranes (fraction P). While cyclic AMP and cyclic GMP had no effect on Ca uptake in the absence of oxalate, both cyclic nucleotides inhibited the rate of oxalate-activated Ca uptake when used at concentrations higher than 10(-5) M. The addition of bovine heart protein kinase to either fraction produced an increase in the rate of oxalate-activated Ca uptake which was further augmented by cyclic AMP. Cyclic GMP caused smaller stimulations of protein kinase-catalyzed Ca uptake than cyclic AMP. Mg-dependent phosphorylation, attributable to endogenous protein kinase(s), was inhibited in fraction E by low concentrations (10(-8) M) of both cyclic AMP and cyclic GMP. In fraction P, an inhibition by cyclic AMP occurred also at a concentration of 10(-8) M, while with cyclic AMP a concentration of 10(-5) M was required for a similar inhibition. Bovine heart protein kinase stimulated the phosphorylation of the membrane fractions much more than Ca uptake. In fraction E, in the presence of bovine protein kinase, both cyclic AMP and cyclic GMP stimulated phosphorylation up to 200%. Under these conditions, no stimulation was observed in fraction P. These results are compatible with the hypothesis that in vascular smooth muscle soluble rather than particulate protein kinases are involved in the regulation of intracellular Ca concentration.     

Regulation of adenosine 3' :5'-monophosphate efflux from rat glioma cells in culture*.

Rat glioma cells grown in culture secrete cyclic adenosine 3':5'-monophosphate (cyclic AMP) into the culture medium following stimulation by beta-agonistic catecholamines. Agents which reduced cellular ATP levels such as valinomycin, oligomycin, and uncouplers of oxidative phosphorylation, inhibited cyclic AMP efflux. Secretion of cyclic AMP was also prevented by prostaglandin A-1 and pharmacological agents including probenecid and papaverine. Of the latter agents, only papaverine reduced ATP levels. These results suggest that the transport of cyclic AMP across animal cell membranes is energy-dependent and subject to regulation.     

Ketotifen increases cyclic adenosine 3',5'-monophosphate in intact human lymphocyte and potentiates other adenylate cyclase activating agents

We have investigated the effects of ketotifen on the cyclic adenosine 3',5'-monophosphate (cyclic AMP) response of intact human lymphocyte and its interaction with adenylate cyclase activating agents. In the presence of cyclic AMP phosphodiesterase inhibitor (3-isobutyl-1-methyl-xanthine), ketotifen (10(-8)-10(-4) M) caused an 80% increase in cyclic AMP content of human lymphocyte, a magnitude similar to that observed with hydrocortisone. The cyclic AMP level peaked at about 15 minutes and remained elevated for at least 45 minutes. In addition, ketotifen (10(-6)-10(-4) M) markedly potentiated the effect of several adenylate cyclase stimulating agents, including L-isoproterenol, prostaglandin E1 and forskolin. The biochemical mechanisms underlying these effects are unknown. It may be at least partly related to the ability of ketotifen to reverse and prevent beta 2 adrenoceptor desensitization and to promote the formation of hormone - nucleotide - high affinity receptor complex. These effects may contribute to its prophylactic effect in the treatment of bronchial asthma.     

Metabolism and excretion of exogenous adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate. Studies in the isolated perfused rat kidney and in the intact rat.

Isolated rat kidneys were perfused with a recirculating medium containing exogenous adenosine 3':5'-monophosphate (cyclic AMP) or guanosine 3':5'-monophosphate (cyclic GMP) at an initial concentration of 0.1 mM. Both cyclic nucleotides were rapidly removed from the perfusate. Urinary excretion accounted for about 20% and 40% of the respective cyclic AMP and cyclic GMP lost from the perfusate. The metabolism of the cyclic nucleotides was studied by 14C-labeled cyclic nucleotides in the perfusate. During 60 min, 30% of added cyclic [14C]AMP was metabolized to renal [14C]adenine nucleotides (ATP, ADP, and AMP) and 30% to perfusate [14C]uric acid. Similarly, 20% of cyclic[14C]GMP was metabolized to renal [14C]guanine nucleotides (GTP, GDP, and GMP) and 30% to perfusate [14C]uric acid. Urine contained principally unchanged 14C-labeled cyclic nucleotide. Addition of 0.1 mM cyclic AMP to the perfusate elevated the renal ATP and ADP contents 2-fold. Addition of 0.1 mM of either cyclic AMP or cyclic GMP to the perfusate also elevated the renal production of uric acid 2- to 3-fold. The production and distribution of metabolites of exogenous cyclic nucleotides were also studied in the intact rat. Within 60 min after injection, 3.3 mumol of either 14C-labeled cyclic AMP or cyclic GMP was cleared from the plasma. Kidney cortex and liver were the principal tissues for 14C accumulation. Urinary excretion accounted for about 20 and 45% of the cyclic [14C]AMP and cyclic [14C]GMP lost from the plasma, respectively. The 14C found in the kidney and liver was present almost entirely as the respective purine mono-, di-, and trinucleotides. The other principal metabolite was [14C]allantoin, found in the urine and, to a lesser extent, the liver. The urine contained mostly unchanged 14C-labeled cyclic nucleotide. Unlike the findings with the perfused kidney, [14C]uric acid was not a significant metabolite of the 14C-labeled cyclic nucleotides in these in vivo experiments.     

Cyclic nucleotide hydrolysis in the thyroid gland. General properties and key role in the interrelations between concentrations of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate.

Phosphodiesterase activities of horse (and dog) thyroid soluble fraction were compared with either cyclic AMP (adenosine 3':3'-monophosphate) or cyclic GMP (guanosine 3':5'-monophosphate) as substrate. Optimal activity for cyclic AMP hydrolysis was observed at pH 8, and at pH 7.6 for cyclic GMP. Increasing concentrations of ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid inhibited both phosphodiesterase activities; in the presence of exogenous Ca2+, this effect was shifted to higher concentrations of the chelator. In a dialysed supernatant preparation, Ca2+ had no significant stimulatory effect, but both Mg2+ and Mn2+ increased cyclic nucleotides breakdown. Mn2+ promoted the hydrolysis of cyclic AMP more effectively than that of cyclic GMP. For both substrates, substrate velocity curves exhibited a two-slope pattern in a Hofstee plot. Cyclic GMP stimulated cyclic AMP hydrolysis, both nucleotides being at micromolar concentrations. Conversely, at no concentration had cyclic AMP any stimulatory effect on cyclic GMP hydrolysis. 1-Methyl-3-isobutylxanthine and theophylline blocked the activation by cyclic GMP of cyclic GMP of cyclic AMP hydrolysis, whereas Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), a non-methylxanthine inhibitor of phosphodiesterases, did not alter this effect. In dog thyroid slices, carbamoylcholine, which promotes an accumulation of cyclic GMP, inhibits the thyrotropin-induced increase in cyclic AMP. This inhibitory effect of carbamoylcholine was blocked by theophylline and 1-methyl-3-isobutylxanthine, but not by Ro 20-1724. It is suggested that the cholinergic inhibitory effect on cyclic AMP accumulation is mediated by cyclic GMP, through a direct activation of phosphodiesterase activity.     

Modulation of adenylate cyclase/cyclic AMP response by thyrotropin and prostaglandin E2 in cultured thyroid cells. 2. Positive regulation.

Two different independent processes are operating in cultured thyroid cells to regulate adenylate cyclase/cyclic AMP responsiveness to thyroid stimulators (thyrotropin and prostaglandin E2): firstly, refractoriness or negative regulation [preceding paper], which is specific for each thyroid stimulator, is not mediated by cyclic AMP and is not accompanied by alteration of adenylate cyclase activity; secondly, positive regulation which is characterized by an augmentation of the cyclic AMP response stimulated by thyrotropin and prostaglandin E2. This process is not specific for each thyroid stimulator and is a state of increased susceptibility of cyclic AMP synthesis to stimulation, accompanied by increased activity of the catalytic subunit of adenylate cyclase. Positive regulation is apparently mediated by increased intracellular cyclic AMP levels. It is a time-dependent and dose-dependent process. Very low concentrations (5-50 micronU/ml) of thyrotropin augmented cyclic AMP synthesis stimulated by thyrotropin and prostaglandin E2 whereas higher concentrations (above 0.1 mU/ml) augmented prostaglandin E2 stimulation but induced refractoriness to thyrotropin. Prostaglandin E2 (0.1 to 10 micronM) augmented thyrotropin stimulation and dibutyryl adenosine 3':5'-monophosphate (0.3 to 2 mM) augmented thyrotropin and prostaglandin E2 stimulation. Positive regulation is a slow process which develops within days and increases up to day 5 in culture. Experiments using inhibitors suggested that protein synthesis is required for the full expression of the increase in adenylate cyclase activity induced by the studied thyroid stimulators.     

Induction of phosphodiesterase by cyclic adenosine 3':5'-monophosphate in differentiating Dictyostelium discoideum amoebae.

Cyclic adenosine 3':5'-monophosphate added to the starvation media of Dictyostelium discoideum amoebae induces both intracellular and extracellular phosphodiesterase activities of these cells. The induced enzyme activity appears several hours earlier than that in starved cells which have not been induced with cyclic nucleotide. In both cases, the appearance of enzyme is inhibited by cycloheximide, and actinomycin D, and daunomycin. The KmS for the extracellular enzyme(s) of nucleotide-induced and uninduced control cells are identical. The induction of enzyme activity seems specific for cyclic adenosine 3':5'-monophosphate since cyclic guanosine 3':5'-monophosphate, as well as other nucleotides, have no effect. No differences in the activity or excretion of either N-acetylglucosaminidase or the inhibitory of the extracellular phosphodiesterase are observed between cyclic adenosine 3':5'-monophosphate-induced and control cells. A direct activation of phosphodiesterase by cyclic adenosine 3':5'-monophosphate can be excluded, since the addition of this nucleotide to cell lysates has no effect on the enzyme activity.     

Effects of beta-adrenergic catecholamines on potassium transport in turkey erythrocytes.

Isoproterenol stimulates cellular accumulation of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and produces a 2- to 4-fold increase in bidirectional potassium fluxes in turkey erythrocytes. Ouabain, which does not alter catecholamine-stimulated cellular cyclic AMP, inhibits potassium influx by 50 to 70%, does not alter potassium outflux or isoproterenol-stimulated potassium influx, but increases isoproterenol-stimulated potassium outflux. Stimulation of potassium transport by isoproterenol can be reproduced by adding cyclic AMP to the medium and is inhibited by propranolol or dichloroisoproterenol but not by phentolamine. Theophylline at concentrations which inhibit cyclic nucleotide phosphodiesterase in isolated turkey erythrocyte plasma membranes by greater than 90%, does not augment isoproterenol stimulation of cellular cyclic AMP or of potassium transport but does potentiate stimulation of potassium influx produced by adding cyclic AMP to the medium. Isoproterenol-stimulated cellular cyclic AMP increases steadily for at least 2 hours. Potassium transport, however, increases rapidly, becomes maximal after 20 to 30 min of incubation, and thereafter decreases progressively so that after 2 hours of incubation potassium fluxes are only slightly greater than for the control. Ouabain prolongs the duration of catecholamine-stimulated potassium influx and potassium outflux, reflecting its ability to relieve the refractoriness developed by turkey erythroyctes to endogenous cyclic AMP.     

Hormonal control of cyclic 3':5'-AMP levels and gluconeogenesis in isolated hepatocytes from fed rats.

Glucagon can stimulate gluconeogenesis from 2 mM lactate nearly 4-fold in isolated liver cells from fed rats; exogenous cyclic adenosine 3':5'-monophosphate (cyclic AMP) is equally effective, but epinephrine can stimulate only 1.5-fold. Half-maximal effects are obtained with glucagon at 0.3 nM, cyclic AMP at 30 muM and epinephrine at 0.2 muM. Insulin reduces by 50% the stimulation by suboptimal concentrations of glucagon (0.5 nM). A half-maximal effect is obtained with 0.3 nM insulin (45 microunits/ml). Glucagon in the presence of theophylline (1 mM) causes a rapid rise and subsequent fall in intracellular cyclic AMP with a peak between 3 and 6 min. Some of the fall can be accounted for by loss of nucleotide into the medium. This efflux is suppressed by probenecid, suggesting the presence of a membrane transport mechanism for the cyclic nucleotide. Glucagon can raise intracellular cyclic AMP about 30-fold; a half-maximal effect is obtained with 1.5 nM hormone. Epinephrine (plus theophylline, 1 mM) can raise intracellular cyclic AMP about 2-fold; the peak elevation is reached in less than 1 min and declines during the next 15 min to near the basal level. Insulin (10 nM) does not lower the basal level of cyclic AMP within the hepatocyte, but suppresses by about 50% the rise in intracellular and total cyclic AMP caused by exposure to an intermediate concentration of glucagon. No inhibition of adenylate cyclase by insulin can be shown. Basal gluconeogenesis is not significantly depressed by calcium deficiency but stimulation by glucagon is reduced by 50%. Calcium deficiency does not reduce accumulation of cyclic AMP in response to glucagon but diminishes stimulation of gluconeogenesis by exogenous cyclic AMP. Glucagon has a rapid stimulatory effect on the flux of 45Ca2+ from medium to tissue.     

Adenosine 3':5'-monophosphate in perinatal rat liver. Ontogeny and response to hormones.

Developmental changes in the concentration of adenosine 3':5'-monophosphate (cyclic AMP) and the effects of glucagon and epinephrine were studied in the perinatal rat liver. Hepatic cyclic AMP concentration doubled during the last day of gestation. After birth, the cyclic AMP concentration continued to increase and maximal levels were observed on the fifth postnatal day. Surgical delivery of foetuses on days 20, 21 and 22 of gestation resulted in a rapid increase in cyclic AMP concentration. Maximal concentrations were reached within one hour of delivery in the day-21 and day-22 foetuses. However with surgically delivered day-20 foetuses, the cyclic AMP concentration increased for a least two hours. Glucagon and epinephrine increases the hepatic cyclic AMP concentration in rats delivered surgically on days 20, 21 and 22 of gestation and in postnatal rats. Maximal stimulation by epinephrine was observed in 2-day-old rats. Maximal stimulation by glucagon was observed in 10-day-old rats. The results support the hypothesis that cyclic AMP is the intracellular effector for the synthesis of some enzymes in the perinatal rat. The cyclic AMP concentration in the perinatal rat liver in vivo appears to be controlled by changes in the relative concentrations of plasma glucagon and insulin.     

Bidirectional regulation of lysosomal enzyme secretion and phagocytosis in human neutrophils by guanosine 3',5'-monophosphate and adenosine 3',5'-monophosphate.

The biologic roles of guanosine 3',5'-monophosphate (cyclic GMP) and adenosine 3',5'-monophosphate (cyclic AMP) in the secretion of lysosomal enzymes from, and in phagocytosis by, human neurtrophils were studied. Contact between neurophils and particulate immunologic reactants results in both phagocytosis of the particles and secretion of lysosomal enzymes. These cellular events are accompanied by the accumulation of cyclic GMP and require the presence of extracellular caclium. Acetylcholine, pilocarpine, and cyclic GMP enhance, whereas epinephrine, cyclic AMP, and/or dibutyryl cyclic AMP inhibit, both phagocytosis and lysosomal enzyme secretion. The stimulatory action of cholinergic agents and the inhibitory action of epinephrine are accompanied by the accumulation of cyclic GMP and cyclic AMP, respectively, in human neutrophils. The data suggest that cyclic GMP mediates whereas cyclic AMP inhibits the major functions of human neutrophils. Moreover, by virtue of their effects of cyclic nucleotide accumulation, autonomic neurohormones are capable of modulating human neutrophil function.