Inhibition Of cAMP Phosphodiesterase Activity Research Articles

YC-1 potentiates cAMP-induced CREB activation and nitric oxide production in alveolar macrophages

Alveolar macrophages play significant roles in the pathogenesis of several inflammatory lung diseases. Increases in exhaled nitric oxide (NO) are well documented to reflect disease severity in the airway. In this study, we investigated the effect of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on prostaglandin (PG)E1 (a stable PGE2 analogue) and forskolin (a adenylate cyclase activator) induced NO production and inducible NO synthase (iNOS) expression in rat alveolar macrophages (NR8383). YC-1 did not directly cause NO production or iNOS expression, but drastically potentiated PGE1- or forskolin-induced NO production and iNOS expression in NR8383 alveolar macrophages. Combination treatment with YC-1 and PGE1 significantly increased phosphorylation of the cAMP response element-binding protein (CREB), but not nuclear factor (NF)-κB activation. The combined effect on NO production, iNOS expression, and CREB phosphorylation was reversed by a protein kinase (PK)A inhibitor (H89), suggesting that the potentiating functions were mediated through a cAMP/PKA signaling pathway. Consistent with this, cAMP analogues, but not the cGMP analogue, caused NO release, iNOS expression, and CREB activation. YC-1 treatment induced an increase in PGE1-induced cAMP formation, which occurred through the inhibition of cAMP-specific phosphodiesterase (PDE) activity. Furthermore, the combination of rolipram (an inhibitor of PDE4), but not milronone (an inhibitor of PDE3), and PGE1 also triggered NO production and iNOS expression. In summary, YC-1 potentiates PGE1-induced NO production and iNOS expression in alveolar macrophages through inhibition of cAMP PDE activity and activation of the cAMP/PKA/CREB signaling pathway.

Role of β-adrenoceptors, cAMP phosphodiesterase and external Ca2+ on polyamine-induced relaxation in isolated bovine tracheal strips

Polyamines relax several smooth muscles and elicit cardiotonic effects in the rat heart via interactions with β-adrenoceptors. The aim of this work was to establish whether β2-adrenoceptors were involved in polyamine-relaxation of bovine tracheal strips. Endogenous polyamines displaced the specific radioligand, [ H]dihydroalprenolol, but spermine was the most potent. The polyamines elicited an acute transient relaxation, which was independent of β-adrenoceptor activation, followed by a maintained component, which was shown to be dependent on β-adrenoceptor activation because it was antagonized and reversed by propranolol. Polyamines did not alter salbutamol-induced acute relaxation. Polyamines modified the salbutamol-induced long-term effect on airway tone, which was shown by a partial reversal of β-adrenoceptor desensitization. This process was delayed by α-difluoromethylornithine, but spermine increased the latency and time of reversal and decreased receptor desensitization. Putrescine prolonged the time-constant without changes in the desensitization. Spermine, but not putrescine, might block Ca2+ channels, because it relaxed KCl- or electrical stimulated-contractions, which are related to Ca2+ influx, and the inhibition of cAMP phosphodiesterase activity. These differences might explain the functional differences observed between putrescine and spermine. Therefore, polyamines may modulate airway smooth muscle tone and interfere with the mechanism of receptor desensitization via several mechanisms involving β2-adrenoceptors, Ca2+ influx and cAMP phosphodiesterase.

A new insight of anti-platelet effects of sirtinol in platelets aggregation via cyclic AMP phosphodiesterase

Sirtinol, a cell permeable six-membered lactone ring, is derived from naphthol and potent inhibitor of SIR2 and its naphtholic may have the inhibitory effects on platelets aggregation. In this study, platelet function was examined by collagen/epinephrine (CEPI) and collagen/ADP-induced closure times using the PFA-100 system reveal that CEPI-CT and CADP-CT were prolonged by sirtinol. The platelets aggregation regulated by physiological agonists such as: thrombin, collagen and AA and U46619 were significantly inhibited by sirtinol. Increases cAMP level was observed when sirtinol treated with Prostaglandin E1 in washed platelets. Moreover, sirtinol attenuated intracellular Ca 2+ release and thromboxane B2 formation stimulated by thrombin, collagen, AA and U46619 in human washed platelets. This study indicated that sirtinol could inhibit the platelet aggregation induced by physiological agonists, AA and U46619. The mechanism of action may include an increase of cAMP level with enhanced VASP-Ser157 phosphorylation via inhibition of cAMP phosphodiesterase activity and subsequent inhibition of intracellular Ca 2+ mobilization, thromboxane A2 formation, and ATP release during the platelet aggregation.

Negative Metabolic Effects of cGMP Are Enhanced in Obese Rat Hearts

Leptin resistance leads to obesity and may affect responses to the second messenger cGMP. We tested the hypothesis that the myocardial negative metabolic response to cGMP would be enhanced in leptin-resistant animals. This hypothesis was tested in anesthetized open-chest Zucker obese (n = 16) and age-matched control rats (n = 13). Coronary blood flow (microspheres) and O2 extraction (microspectrophotometry) measurements were used to determine myocardial O2 consumption (VO2). Protein phosphorylation by cGMP protein kinase and cAMP phosphodiesterase activity were also determined. Either vehicle (saline) or 8-Br-cGMP (10(-3) M) was topically applied to the left ventricular surface. Body weight was significantly greater in the obese rats (523 +/- 17 versus 322 +/- 12 g). There were no hemodynamic differences between groups. There was no difference in VO2 between lean (52 +/- 13 mL O2/min/100 g) and obese (54 +/- 9) vehicle-treated rats. 8-Br-cGMP significantly lowered VO2 in obese (35 +/- 6) but not lean (45 +/- 7) rats. This was not related to altered protein phosphorylation by the cGMP protein kinase. Cyclic GMP inhibited cAMP phosphodiesterase activity in lean but not obese hearts. Thus, the high myocardial oxygen consumption of lean rats was not significantly affected by cGMP but was reduced in obese hearts. This appeared to be related to a reduced inhibition of cAMP phosphodiesterase activity by cGMP in the Zucker obese rat.

A Biochemical and Functional Assessment of Monocyte Phosphodiesterase Activity in Healthy and Asthmatic Subjects

The aim of this study was to investigate whether cyclic adenosine 3′5-monophosphate (cAMP) phosphodiesterase (PDE) activity is altered in monocytes from mild asthmatic subjects. Total cAMP PDE activity (pmol/min per mg protein) was significantly greater in homogenates prepared from monocytes from asthmatic subjects (68.3 ± 7.0, n=9) compared to healthy individuals (46.3 ± 3.3, n=14, P<0.05). The PDE inhibitors siguazodan (PDE3-selective), rolipram (PDE4-selective) and theophylline (non-selective) produced a concentration-dependent inhibition of cAMP PDE activity in homogenates from monocytes from normal and asthmatic subjects. However, siguazodan produced significantly greater (P<0.05), and rolipram significantly less (P<0.05), inhibition of total cAMP PDE activity in monocytes from asthmatics (n=4) than from healthy individuals (n=5). cAMP PDE activity was inhibited with equal potency by theophylline in monocytes from healthy and asthmatic subjects. We also investigated the functional consequences of the changes in PDE activity in mononuclear cells obtained from asthmatic subjects. There was no significant difference in the ability of PDE4 inhibitors to attenuate TNF α release from monocytes obtained from asthmatic compared with healthy subjects (P>0.05). Despite a significant increase in the biochemical activity of PDE3 in monocytes from asthmatic subjects, the PDE3 inhibitor siguazodan, failed to significantly reduce TNF α release from human monocytes. Thus, total cAMP PDE activity is increased in monocytes taken from mild asymptomatic asthmatics compared to healthy subjects and is reflected by an increase in the proportion of PDE3 and a decrease in the proportion of PDE4. This augmented enzyme activity was not associated with an alteration in the ability of PDE4 inhibitors to attenuate mononuclear cell function from asthmatics compared to healthy individuals.

New steroidal saponins from the bulbs of Allium giganteum exhibiting potent inhibition of cAMP phosphodiesterase activity.

A new spirostanol saponin and two new furostanol saponins were isolated from the fresh bulbs of Allium giganteum together with several known saponins. The structures of the new compounds were determined on the basis of spectroscopic data, the products formed on hydrolysis and by comparing their properties with those of known compounds. The isolated saponins and their derivatives were evaluated for inhibitory activity by examining their action on cAMP phosphodiesterase as a primary screening test to identify new medicinal agents.

Effect of castration on lipid metabolism in porcine isolated adipocytes from two anatomical locations

1. 1. In vitro activities of de novo lipogenesis and lipolysis were compared in isolated adipocytes from male and castrated large white pigs. 2. 2. Castration increased fat cell size and de novo lipogenesis in both subcutaneous and perirenal adipocytes. Lipogenesis was higher in perirenal than in subcutaneous sites. 3. 3. Basal lipolysis was not modified by castration. Addition of theophylline and adenosine deaminase increased the basal rate of lipolysis only in male castrates. 4. 4. Castration reduced the isoproterenol-stimulated lipolysis in both adipose tissues. Theophylline and adenosine deaminase partly restored the isoproterenol-stimulated lipolysis presumably via inhibition of cAMP phosphodiesterase activity. 5. 5. Contrary to male pigs, insulin had no significant antilipolytic action in both adipose tissues in castrated pigs.

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.

Activators of Protein Kinase C Act at a Postreceptor Site to Amplify Cyclic AMP Production in Rat Pinealocytes

Activation of alpha 1-adrenoceptors appears to amplify beta-adrenergic stimulation of cyclic AMP (cAMP) accumulation in rat pinealocytes severalfold by a mechanism involving activation of a Ca2+-, phospholipid-dependent protein kinase (protein kinase C). The mechanism of action of protein kinase C was investigated in this report using intact cells. Activation of protein kinase C with 4 beta-phorbol 12-myristate 13-acetate (PMA; 10(-7) M) or the alpha 1-adrenergic agonist phenylephrine (PE; 10(-6) M) did not inhibit cAMP efflux in beta-adrenergically stimulated cells. The amplification of the beta-adrenergic cAMP response by these agents also occurred in the presence of isobutylmethylxanthine (10(-3) M) and Ro 20-1724 (10(-4) M), an observation suggesting that inhibition of cAMP phosphodiesterase activity is not the mechanism of action. Furthermore, although PMA (10(-7) M) caused a sixfold increase in the magnitude of the cAMP response to isoproterenol, it did not alter the EC50 of the response (1.7 X 10(-8) M), a result indicating that protein kinase C activation does not alter beta-adrenoceptor sensitivity. The cAMP response following cholera toxin pretreatment (60-120 min) was rapidly and markedly enhanced by alpha 1-adrenergic agonists (cirazoline greater than PE greater than methoxamine), by phorbol esters (PMA greater than 4 beta-phorbol 12,13,-dibutyrate much greater than 4 alpha-phorbol 12,13-didecanoate), and by synthetic diacylglycerols (1,2-dioctanoylglycerol greater than 1-oleoyl 2-acetylglycerol much greater than diolein). The cAMP response to forskolin (10(-5)-10(-3) M) was also increased by PE (3 X 10(-6) M) and PMA (10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Follicle-stimulating hormone enhances somatomedin C binding to cultured rat granulosa cells. Evidence for cAMP dependence.

The ovarian granulosa cell has recently been shown to be the site of Somatomedin C (Sm-C) production, reception, and action. To further elucidate the relevance of Sm-C to granulosa cell physiology, we have undertaken to study the regulation of its receptor under in vitro conditions using a primary culture of rat granulosa cells. Granulosa cells cultured without treatment for 72 h displayed limited, albeit measurable, specific Sm-C binding. However, continuous treatment with increasing concentrations of follicle-stimulating hormone (FSH) for the duration of the 72-h incubation period resulted in dose-dependent increments in Sm-C binding (1.7-, 2.9-, 3.9-, and 3.6-fold increases over untreated controls for 50, 100, 180, and 330 ng/ml of FSH, respectively). This apparent up regulatory action of FSH proved time-dependent, with a minimal time requirement of 24-48 h. Granulosa cell Sm-C binding was similarly enhanced following elevation of the intracellular cAMP content by a series of cAMP-generating agonists, inhibition of cAMP-phosphodiesterase activity, or the provision of nondegradable cAMP analogs. Our findings further indicate that high dose forskolin, like FSH, is capable of augmenting Sm-C binding by itself, that a relatively inactive low dose of forskolin synergizes with FSH in this regard, but that combined treatment with maximal stimulatory doses of both agonists does not prove additive. Taken together, these observations indicate that FSH is capable of exerting a stimulatory effect on granulosa cell Sm-C binding and that cAMP, its purported intracellular second messenger, may play an intermediary role in this regard.

Inhibition of cAMP-phosphodiesterase activity from coronary arteries by a new trapidil derivative

Inhibition of cAMP-phosphodiesterase activity from coronary arteries by a new trapidil derivative

Cell type specific inhibition of cAMP phosphodiesterase activity during terminal differentiation in Dictyostelium discoideum

Cyclic AMP phosphodiesterase (PDE) activity reaches a peak during the aggregation stage of development where it functions to regulate extracellular levels of cAMP. During the subsequent differentiation of the two cell types at the culmination stage, the activity reappears but only in stalk cells. We found that extracts from the culmination stage contained PDE which could be activated by preincubation with Mg 2+ and dithiothreitol (DTT), a treatment which is known to release an endogenous inhibitor from the aggregation stage enzyme. When the culmination stage extracts were subjected to chromatography on Biogel P300, two peaks of activity were eluted, PDE-I ( M r > 260,000) and PDE-II ( M r 100,000). Treatment of the fractions with Mg-DTT did not affect the low-molecular-weight enzyme but caused activation of the high-molecular-weight enzyme and the appearance of a third, intermediate form. Kinetic analysis of the two peaks revealed K m values for cAMP of 2 m M and 10 μ M for PDE-I and PDE-II, respectively. We tested the possibility that these forms of the enzyme might be distributed differently in the two cell types by measuring the K m for cAMP and the effect of Mg-DTT treatment on isolated sections of stalk and spore cells. The spore sections contained a high K m form of the enzyme (0.3 m M) which was activated by preincubation with Mg · DTT whereas stalk sections contained a low K m form (3 μ M) which was not affected by the activation treatment. We conclude that both cell types contain enzyme protein and that the apparent localization of PDE activity in stalk cells is due to the inhibition of activity in spore cells.

The antispasmogenic action on guinea-pig ileum of a fraction obtained from the toxic skin secretion of the stonefish, Synanceia trachynis

—The toxic skin tubercle gland secretion of the stonefish, Synanceia trachynis, contains a smooth muscle antispasmogen that is readily distinguished from papaverine, verapamil and both α and β adrenoreceptor agonists. At 15 μg/ml, the toxin containing fraction markedly inhibited the phasic response and had a lesser inhibitory effect on the tonic response of acetylcholine- and KCl-induced contractures of guinea-pig ileum, while at 50 μg/ml, the toxin markedly inhibited both responses. Over the same dose range the toxin fraction markedly inhibited BaCl 2-induced contractile responses but did not distinguish between the phasic and tonic components. Whereas the toxin fraction markedly reduced the tone of previously induced supramaximal KCl and acetylcholine responses of guinea-pig ileum, it had little effect on the tone of previously induced BaCl 2 responses. The toxin non-competitively inhibited acetylcholine-induced contractions of guinea-pig ileum and similarly non-competitively inhibited the response induced by Ca 2+ in high-[K +] Ringer. The toxin is not a chelating agent for Ca 2+ or Mg 2+ and it does not affect ATP-induced contracture of glycerinated guinea-pig ileum. The antispasmogenic effect of the toxin contained in the fraction does not result from inhibition of cAMP phosphodiesterase activity.

Stereoselective interaction of tetrahydroisoquinolines in β-adrenoceptor systems

In selected β 1- (heart, lipolysis) and β 2-adrenoceptor (trachea) systems, the interaction of racemic-trimetoquinol (TMQ) and the erythro- and threo-diastereomers of 1-(3′,4′,5′-trimethoxy-α-hydroxybenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (α-hydroxy TMQ) was investigated. Each tetrahydroisoquinoline possessed agonist activity in these β-adrenoceptor systems. The rank order of potency observed for these compounds was racemic-TMQ > erythro-α-hydroxy TMQ > threo-α-hydroxy TMQ. Using isolated fat adipocytes, a favorable correlation was observed between the elevation in c-AMP and pharmacological response for the TMQ stereoisomers and diastereomers of α-hydroxy TMQ. The rise in intracellular c-AMP produced by (−)- and (+)-TMQ in fat cells was blocked by the presence of propranolol, and not in the presence of phentolamine. Since considerably higher concentrations (>10 −4 M) of these compounds were required to produce a significant inhibition of c-AMP phosphodiesterase activity in adipose tissue, it is proposed that the lipolytic response is a result of stereoselective interaction of these tetrahydroisoquinolines at the level of membrane-bound adenylate cyclase.