Search

After incubation with 0.5 mM isobutylmethylxanthine, 1 microM dexamethasone, and 1 microM insulin for 72 h, 3T3-L1 cells acquire the phenotypic characteristics of mature adipocytes, including a hormone-sensitive particulate cAMP phosphodiesterase activity. In addition, adipocytes contain soluble cAMP and calmodulin-sensitive and -insensitive cGMP phosphodiesterase activities. After exposure of the differentiated cells to 1 microM epinephrine, cAMP content increased, reaching a maximum in 2-4 min, and then declined to the control level by 20 min. After incubation of adipocytes with 10 nM dexamethasone for 72 h, the initial increment in cAMP produced by epinephrine was not altered, but the decline in cellular cAMP to basal levels was delayed. Treatment with 10 nM dexamethasone prevented hormonal activation of particulate cAMP phosphodiesterase activity without altering basal activity (11). Soluble cAMP and calmodulin-sensitive and -insensitive cGMP phosphodiesterase activities were also reduced by exposure to 10 nM dexamethasone; higher concentrations were required to decrease basal particulate phosphodiesterase activities. Estradiol did not alter phosphodiesterase activities. Incubation of either undifferentiated (fibroblasts) or differentiated (adipocytes) 3T3-L1 cells with 1 microM dexamethasone for 48 h reduced cAMP and cGMP phosphodiesterase activities. After removal of dexamethasone, phosphodiesterase activities were restored to control levels in 4-6 days. The effects of dexamethasone on phosphodiesterase activities could in part account for the observed alterations in hormone-induced accumulation of cAMP in steroid-treated cells and for the permissive effects of glucocorticoids on certain cAMP-mediated processes.

We studied the effects of dialysates from leukocyte lysates containing dialyzable transfer factor activity and other leukocyte dialysates devoid of transfer factor activity on accumulation of cyclic nucleotides in human leukocytes. Dialysates from normal leukocytes produced 4- to 11-fold increases in leukocyte cGMP, and experiments with purified cell populations revealed that the increases were predominantly, if not entirely, in blood monocytes. Substances that increased monocyte cGMP could be obtained from several cell populations including mononuclear cells from Hypaque-Ficoll gradients, plastic-adherent monocytes, nonadherent lymphocytes, and neutrophils, but were not present in dialysates of leukemic lymphocytes from patients with the Sezary syndrome. Moreover, dialysates that increased leukocyte cGMP had essentially no effect on intracellular cAMP. Dialysates of lysed mononuclear cells contained serotonin, ascorbate, and an unidentified cholinergic activity, agents known to increase leukocyte cGMP. After passage of dialyzable transfer factor from mononuclear cells through a gel-filtration column, four fractions were obtained that increased leukocyte cGMP. Two of these fractions contained ascorbate; two other active fractions, including one that also caused conversion of delayed skin tests, did not contain detectable ascorbate or serotonin. The dialysate of lysed neutrophils also increased cGMP, but this activity was limited to the column fractions which contained ascorbic acid. These observations raise the possibility that alterations in monocyte cGMP content could modulate either the specific antigen-dependent, or, more likely, the antigen-independent activities in preparations of transfer factor.

Publisher Summary Multiple forms of phosphodiesterase (PDE) are found in most types of cells; they are found in association with membranes as well as in the cytoplasm. These enzymes differ in amounts and proportions in different cells; some are absent from or highly concentrated in certain cells. The effects of specific PDE inhibitors on 3T3-L1 adipocytes have suggested that the particulate cAMP PDE is important in the regulation of a cAMP pool involved in lipolysis and that activation of this enzyme is an important component in the antilipolytic action of insulin. The particulate, but not soluble, cAMP PDE is sensitive to inhibition by cilostamide and certain cardiotonic drugs, whereas soluble cAMP PDE activity is sensitive to inhibition by RO 20-1724. Cilostamide, but not RO 20-1724, blocked the antilipolytic action of insulin. These observations are consistent with the recent findings of Corbin and associates, who have demonstrated that in rat fat cells, insulin inhibited lipolysis stimulated by cAMP analogs that both activate protein kinase and serve as substrates for particulate cAMP PDE but did not inhibit lipolysis stimulated by cAMP analogs that are poor substrates for particulate cAMP PDE.

We have used cultured 3T3-L1 adipocytes to assess direct effects of T3 on beta-adrenergic-mediated regulation of lipolysis and adenylate cyclase and phosphodiesterase activities. Differentiated 3T3-L1 adipocytes were maintained under four conditions: in the presence of medium containing serum from a hypothyroid calf (hypothyroid medium), hypothyroid medium supplemented with T3 (T3-supplemented hypothyroid medium), medium with serum from a normal calf (control medium), and control medium supplemented with excess T3 (hyperthyroid medium). Compared to the two control groups, i.e. cells maintained in control medium or T3-supplemented hypothyroid medium, cells maintained in hypothyroid medium exhibited lower basal rates of lipolysis and lower sensitivity to isoproterenol. Hyperthyroid cells exhibited higher basal rates of lipolysis and higher sensitivity to isoproterenol. In the presence of maximally effective concentrations of isoproterenol, rates of lipolysis were similar in the four groups. Similarly, basal cAMP content and cAMP accumulation in the presence of isoproterenol were reduced in hypothyroid and increased in hyperthyroid adipocytes compared to those adipocytes maintained in control or T3-supplemented hypothyroid medium. Basal adenylate cyclase activity was similar in the four groups. Sensitivity to isoproterenol and maximal isoproterenol-stimulated cyclase activity were diminished in membrane preparations from hypothyroid adipocytes and increased in preparations from hyperthyroid adipocytes. Cyclase activity in the presence of NaF, however, was similar in preparations from cells maintained in hypothyroid, T3-supplemental hypothyroid, or control medium. NaF-stimulated activity was increased in preparations from hyperthyroid adipocytes. Thyroid status did not affect beta-receptor number of affinity for iodohydroxybenzylpindolol. Compared to control cells or cells maintained in T3-supplemented hypothyroid medium, both soluble and particulate cAMP phosphodiesterase activities were increased in hypothyroid cells and decreased in hyperthyroid cells. These results indicated that in 3T3-L1 adipocytes, some of the effects of thyroid hormone on cAMP content and lipolysis can be explained by alterations in both production and degradation of cAMP.

The cAMP content of cultured human fibroblasts was increased by incubation of the cells with choleragen, prostaglandin E1 (PGE1), or isoproterenol. Propranolol (80μM) markedly reduced the elevation in intracellular cAMP induced by choleragen or isoproterenol but increased the accumulation of cAMP secondary to PGE1. (“Propranolol”) refers to the racemic mixture; when either isomer alone was used, the appropriate designation is given.) Propranolol prolonged the delay in choleragen responsiveness, normally apparent in human fibroblasts; the effects of propranolol on choleragen and PGE1 occurred in the presence or absence of 3-isobutyl-l-methylxanthine and were observed on both intracellular and extracellular cAMP. d- and l-propranolol were equally effective with choleragen and PGE1, whereas l-propranolol was much more effective than d-propranolol in blocking the action of isoproterenol. Propranolol had no significant effect on the activation of adenylate cyclase by choleragen or PGE1. Butoxamine and dichloroisoproterenol did not decrease cAMP accumulation induced by choleragen but enhanced the response to PGE1. A lipid-soluble anesthetic, tetracaine, also increased the accumulation of intracellular cAMP due to PGE1 but inhibited the response to choleragen. These data indicate that propranolol exerts effects independent of its β-adrenergic blocking activity on agonist-induced changes in cAMP metabolism of human fibroblasts. In view of the concentrations of propranolol at which these “nonspecific” effects were observed, and based on similarities to actions of other drugs, it appears that some of the toxic and/or therapeutic effects of the drug may result from “membrane-stabilizing” properties and/or effects on microtubule assembly and may be unrelated to β-adrenergic blockade.

Several new positive inotropic drugs with vasodilating properties for which a major mechanism of action is believed to be inhibition of phosphodiesterase (PDE) have been introduced in the treatment of congestive heart failure. Hydrolysis of cyclic nucleotides is catalyzed by multiple forms of PDE, which may vary between organs and cell-types. These enzymes can be selectively inhibited by various agents, theoretically making it possible to produce tissue-selective responses. An enzyme, which belongs to a subclass of cGMP inhibited low Km cAMP PDE, was recently purified from rat adipose tissue. The enzyme was specifically and potently inhibited by the cilostamide derivative OPC 3911 (OPC) and milrinone (mil). We studied the relaxant effects of OPC and mil on isolated human mesenteric arteries and veins in vitro. In preparations contracted by noradrenaline (NA), both agents produced about 60% maximum relaxation; OPC was 3-4 times more potent than mil. Both OPC and mil caused rightward displacement of the NA concentration-response curve and depressed the maximum responses. Arteries, as compared to veins, were more sensitive to this inhibition of NA contraction. Both drugs relaxed arteries contracted by 30 mM K+, but not 127 mM K+; maximum relaxation was between 60 and 70%. OPC was 10 times more potent than mil. The interactions between mil/OPC and isoprenaline, forskolin and ouabain were also studied. In preparations pretreated with isoprenaline or forskolin, the relaxant effects of mil and OPC were additive to those of isoprenaline and forskolin. Ouabain pretreatment did not affect the actions of the phosphodiesterase inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Choleragen and the isolated A protomer catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. The protein with NADase activity (NAD nucleosidase; NAD glycohydrolase, EC 3-2-2-5) migrated on polyacrylamide gels with choleragen, and chromatographed on Bio-Gel P-60 columns with the A protomer. The NADase activity of choleragen and of the A protomer was increased markedly in acetate and phosphate buffers, and enhanced over 10-fold by dithiothreitol in high concentration. NAD hydrolysis was proportional to choleragen concentration; the Michaelis constant for NAD was about 4 mM with both choleragen and the A protomer. The demonstration that the A protomer of choleragen catalyzes an enzymatic reaction involving activation of the ribosyl-nicotinamide bond of NAD, a reaction analogols to those catalyzed by diphtheria toxin, supports the hypothesis that activation of adenylate cyclase by choleragen involves the ADP-ribosylation of an appropriate acceptor protein.

The interaction of bradykinin (BK) with its specific receptors on intact cultured human fibroblasts results in production of prostaglandins, including prostacyclin (PGI2), and accumulation of cyclic AMP. Incubation of cells with 1 microM BK for 5 min at 37 degrees C led to a marked reduction (75-90%) in BK-induced PGI2 release and in total number of [3H]BK-binding sites with no change in dissociation constant (6.1 and 7.6 nM for control and BK-treated cells, respectively). The decrease in receptor number did not result from BK transferred from the first incubation into the binding assay. BK-induced receptor loss was temperature dependent; exposure of cells to BK at 4 degrees C had little or no effect on receptor number. After incubation with BK for approximately equal to 15 min, further incubation in the absence of BK for 30 min at 37 degrees C almost completely restored both receptor number and BK-induced PGI2 release. With more prolonged exposure to BK (greater than 1 h), restoration of receptors was inversely related to the length of exposure and the concentration of BK. Recovery was unaffected by cycloheximide. During prolonged incubation without removal of BK, cells began to recover receptors by 5 h; greater than 99% of the bradykinin initially present disappeared by 3 h. Bacitracin greatly retarded BK disappearance and totally prevented recovery. These observations provide direct evidence that the number of BK receptors on cultured human fibroblasts can be regulated by BK itself. In addition, it appears that BK-degrading systems, by influencing local concentrations of the peptide, may play an important role in the autoregulation of BK receptors. The presence of highly active degradation systems might serve to protect target tissues from developing chronic insensitivity to BK and, perhaps, similar peptides.

Guanylate cyclase activity and cyclic nucleotide content were studied in individual slices from guinea pig kidneys. Basal guanylate cyclase activity, assayed in homogenates or in particulate fractions (100,000g × 1 h), and the tissue content of cGMP and cAMP were greater in the inner than in the outer (entirely cortical) slices. The fraction of guanylate cyclase activity recovered in the supernatant was greater in the cortex. Taurodeoxycholate increased activity of the particulate cyclase but decreased that of the supernatant enzyme. Activity of the particulate was increased ca. 200% and that of the supernatant >500% by 1 m m NaN3. Supernatant activity was markedly increased by 0.1 m m Co2+, which had no effect on the particulate enzyme. (Incubation of kidney slices with 2 m m Co2+ did not alter their cGMP content, but caused a small increase in the cAMP content of slices containing medullary tissue.) Basal guanylate cyclase activity in fresh supernatants increased linearly with pH from 5.9 to 9, whereas in the presence of Co2+ there was a clear maximum at pH 7.3 to 7.5. Incubation of fresh supernatant fractions at 37 °C for 3 h increased guanylate cyclase activity and abolished Co2+ activation. The relationship between Co2+ activation and that resulting from incubation remains to be defined. It seems probable, however, that these phenomena reflect regulatory properties of the supernatant guanylate cyclases of kidney and other tissues.

Guanosine 3':5'-monophosphate phosphodiesterases, which appear to be under allosteric control, have been partially purified from rat liver supernatant and particulate fractions. The preferred substrate for both phosphodiesterases was cGMP (Km values: cGMP less than cIMP less than cAMP). At subsaturating concentrations of substrate, the phosphodiesterases were stimulated by purine cyclic nucleotides. The order of effectiveness for activation of cyclic nucleotide hydrolysis was cGMP greater than cIMP greater than cAMP greater than cXMP. Using cAMP derivatives as activators of cIMP hydrolysis, modifications in the ribose, cyclic phosphate, and purine moieties were shown to alter the ability of the cyclic nucleotide to activate the supernatant enzyme. cGMP, at concentrations that stimulated cyclic nucleotide hydrolysis, enhanced chymotryptic inactivation of the supernatant phosphodiesterase. At similar concentrations, cAMP was not effective. It appears that on interaction with appropriate cyclic nucleotides, this phosphodiesterase undergoes conformational changes that are associated with increased catalytic activity and enhanced susceptibility to proteolytic attack. Divalent cation may not be required for the nucleotide-phosphodiesterase interaction and resultant change in conformation.

In isolated fat cells, the same maximal rate of glycerol production can be induced by epinephrine or ACTH, alone or in combination with each other or with glucagon. With fat cells from rats weighing 150–175 g, the maximal rate of lipolysis attained with glucagon was 75–80% of that produced by epinephrine or ACTH, and with increasing size of the donor rat, the magnitude of the effect of glucagon relative to that of the other hormones declined markedly. In particulate preparations from fat cells of rats weighing 100–125 g, the maximal effect of glucagon on adenyl cyclase activity was about 60% of that of epinephrine, and was significantly less (30%) in preparations from 350–400 g rats. These data are consistent with the hypothesis that with growth of the rat there is a selective decline in the number of glucagon receptors relative to those for epinephrine or ACTH in the fat cell membrane.

Isolated fat cells were incubated in Krebs-Ringer phosphate medium. Within 1 min after the addition of 1.1 µm epinephrine, the cyclic AMP content of the cells was elevated. It continued to rise, reaching a peak in 4 to 7 min, and fell thereafter, being only slightly above the control levels after 10 to 15 min. A maximal rate of glycerol production was established within 1 to 2 min, well before the peak in cyclic AMP concentration, and was maintained unchanged while the cyclic AMP level continued to rise and then fell. Both in magnitude and time course, the effect of adrenocorticotropin (ACTH), 0.2 unit per ml, on cyclic AMP levels and on lipolysis was exactly like that of epinephrine (or of both hormones together). Addition of more epinephrine or of ACTH at several times during 1 hour of incubation with 1.1 µm epinephrine did not change cyclic AMP levels. The effects of epinephrine on cyclic AMP levels and on glycerol production were unaltered by the presence in the medium of 4 mm glucose. When propranolol (or insulin) was added several times after epinephrine-simulated lipolysis had begun, the rate of glycerol production fell to zero within 1 to 2 min. In the same period, the cyclic AMP concentration fell to a basal level; i.e. the rate of decrease was much greater than that observed after 4 to 7 min of incubation with epinephrine alone. Furthermore, after incubation of cells for 20 min with epinephrine, the addition of 0.8 mm theophylline produced an 8-fold rise in cyclic AMP content measured 5 min later. This increment was, however, only a fraction of that observed during the first 5 min after addition of epinephrine in the presence of theophylline. Since propranolol inhibits the action of epinephrine but does not alter basal adenyl cyclase activity, and, since 0.8 mm theophylline alone did not alter cyclic AMP levels, it may be concluded that adenyl cyclase was still epinephrine-stimulated during the time when cyclic AMP levels were falling. Whether the rate of formation of cyclic AMP during this period was equal to that during the first minutes after addition of the hormone cannot be established. Attempts to show directly alterations in phosphodiesterase activity during incubation of cells with epinephrine have been unsuccessful. When the effect of epinephrine was terminated after 4 min by the addition of propranolol, introduction of ACTH at several times during the following 20 min caused an immediate resumption of glycerol production at a rate equal to that observed with the initial addition of epinephrine. Cyclic AMP levels, however, rose little if at all in 4 to 5 min after addition of ACTH. The ability of cells to elevate cyclic AMP levels a second time in response to hormonal stimulation was restored when cells that had been incubated for 20 min were washed and suspended in fresh medium. There appears to be something accumulated in the medium during incubation of cells with epinephrine that interferes with the effectiveness of the hormone in causing accumulation of cyclic AMP in fresh cells. Whether this material has its effect on the rate of formation or of degradation of cyclic AMP cannot be decided.

To study the effect of phosphodiesterase (PDE) 3 inhibition on plasma insulin and glucose levels, the selective PDE 3 inhibitor milrinone (0.25, 1.0, and 2.5 mg/kg) was given orally to anesthetized CL57Bl/6J mice 10 min before a gastric glucose gavage (150 mg/mouse). It was found that milrinone augmented the glucose-mediated increase in plasma insulin at 1.0 and 2.5 mg/kg without, however, any improvement in glucose elimination. In contrast, when given 10 min before intravenous glucose (1 g/kg), milrinone (1 mg/kg) did not affect the insulin response to glucose. The increase in glucagon-like peptide-1 (GLP-1) levels after gastric glucose was not altered by milrinone. However, the PDE3 inhibitor augmented the insulin response to intravenous GLP-1 (2.8 nmol/kg). We therefore conclude that PDE3 inhibition by milrinone augments insulin secretion in vivo in mice after oral but not after intravenous glucose, which may be explained by enhanced response to the cAMP-dependent insulinotropic action of endogenously released GLP-1.

Stimulation of rat adipocytes with insulin and isoproterenol results in serine phosphorylation and activation of the adipocyte cGMP-inhibited phosphodiesterase (cGI PDE), events believed to be important in the antilipolytic action of insulin (Degerman, E., Smith, C.J., Tornqvist, H., Vasta, V., Manganiello, V.C., and Belfrage, P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,533-537). Here we demonstrate, by two-dimensional phosphopeptide mapping, that the major phosphopeptide generated by trypsin, or trypsin followed by Asp-N protease digestion of [32P]cGI PDE phosphorylated in adipocytes in response to isoproterenol and/or insulin, in each case co-migrates with the phosphopeptide released by the same treatment of M297FRRPS(P)LPCISREQ310. This peptide was synthesized based on the deduced sequence of the cloned rat adipocyte cGI PDE and phosphorylated by cAMP-dependent protein kinase (protein kinase A). Radiosequencing of authentic and synthetic tryptic 32P-peptides showed that a single site in cGI PDE (Ser302) was phosphorylated in adipocytes incubated with isoproterenol and/or insulin. The more than additive phosphorylation and activation of cGI PDE in response to the two hormones found in this report and previously (Smith, C.J., Vasta, V., Degerman, E., Belfrage, P., and Manganiello, V.C. (1991) J. Biol. Chem. 266, 13385-13390) is proposed to reflect cross-talk between their respective signal transduction pathways at the level of the cGI PDE serine protein kinase or upstream regulatory component(s).

Calmodulin is a ubiquitous calcium-binding protein that regulates a variety of enzymes such as adenylate cyclase, cyclic nucleotide phosphodiesterase, ATPase and protein kinase (for review see ref. 1). So far, no enzymatic modification of calmodulin has been shown to affect its function. Another ubiquitous protein, the enzyme protein-carboxyl methylase (PCM), modifies proteins post-translationally by methylating their free carboxyl groups and thus neutralizing negative charges2–6. This enzyme is one of three elements of the protein-carboxyl methylation system; the two others are the substrates, the methyl acceptor proteins (MAP) and the demethylating enzyme, protein methylesterase (PME)7. We report here that calmodulin is an exceptionally good substrate for PCM and that the enzymatic post-translational methylation of calmodulin inhibits its stimulatory effect on cyclic nucleotide phosphodiesterase. Furthermore, we present evidence that carboxyl methylation of calmodulin occurs in intact cells.