Cyclic nucleotide alterations in mixed leukocyte reaction: a preliminary report

Endogenous cyclic adenosine and guanosine monophosphate (cAMP, cGMP) levels were studied in human peripheral blood lymphocytes during mixed leukocyte reactions (MLR). cAMP level was consistently elevated in one-way MLR, with good correlation to 3H-thymidine uptake in these reactions. In contrast, cGMP level was practically unchanged. Irradiation of reacting cell populations resulted in inhibition of cyclic nucleotide phosphodiesterase (PDE) activity. These results suggest that metabolic alterations in cAMP may be associated with immune reactions of cellular recognition.     

Improvement of pulmonary arterial hypertension,inflammatory response,and epithelium injury by dual activation of cAMP/cGMP pathway in a rat model of monocrotaline-induced pulmonary hypertension

Pulmonary hypertension (PH) is a life-threatening lung disease. PH with concomitant lung diseases, e.g., idiopathic pulmonary fibrosis, is associated with poor prognosis. Development of novel therapeutic vasodilators for treatment of these patients is a key imperative. We evaluated the efficacy of dual activation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) using an active, small-molecule phosphodiesterase (PDE4)/PDE5 dual inhibitor (Compound A). Compound A increased both cAMP and cGMP levels in WI-38 lung fibroblasts and suppressed the expressions of type-1 collagen α1 chain and fibronectin. Additionally, compound A reduced right ventricular weight/left ventricular weight+septal weight ratio, brain natriuretic peptide expression levels in right ventricle, C─C motif chemokine ligand 2 expression levels in lung, and plasma surfactant protein D. Our data indicate that dual activation of cAMP/cGMP pathways may be a novel treatment strategy for PH.     

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.     

Increase of cyclic GMP induced in murine thymocytes by thymosin fraction 5

Using a radioimmunoassay (RIA) for the determination of adenosine 3'5' cyclic monophosphate (cAMP) and an acetylation-RIA procedure to measure guanosine 3'5' cyclic monophosphate (cGMP), we observed that cGMP levels, but not cAMP levels, were significantly elevated in murine thymocytes which had been incubated with preparations containing the thymic hormone, thymosin. Stimulation of intracellular cGMP levels was seen as early as 1 minute after incubation with thymosin fraction 5 and was maximal at approximately 10 minutes. Dose response studies indicated an optimum stimulation of cGMP with a thymosin concentration of 100 microg/ml. A control spleen fraction prepared by an identical procedure as fraction 5 did not affect the levels of either cyclic nucleotide.     

Cyclic nucleotides and haemoglobin concentration in rabbit bone marrow cell suspensions stimulated by purified erythropoietin

This study was conducted to determine the possible correlations between cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP), and haemoglobin (Hb) concentration in nucleated cell suspensions of rabbit bone marrow incubated with erythropoietin (Ep). The levels of cAMP and cGMP were measured following the addition of different Ep concentrations to the suspensions. The Hb concentration was also measured in suspensions treated with Ep, dibutyryl cAMP (db-cAMP) or dibutyryl cGMP (db-cGMP), respectively. The following results were obtained: (1) upon the addition of 1 IU ml-1 Ep, an increase of cAMP levels was related to an increase in Hb concentration; while a decrease of Hb concentration was related to an increase of cGMP levels obtained when 0.1 IU ml-1 Ep was present in the incubation mixture. (2) A mimetic effect on Hb concentration was obtained upon the addition of db-cAMP or db-cGMP to the suspensions. (3) A quantitative correlation was found between the cAMP/cGMP ratio and Hb levels in cellular suspensions. This rapport was reviewed with respect to the controls as a decrease in Hb concentration when the ratio is less than one and an increase in Hb concentration when the ratio is greater than one.     

In Central Obesity,Weight Loss Restores Platelet Sensitivity to Nitric Oxide and Prostacyclin

Central obesity shows impaired platelet responses to the antiaggregating effects of nitric oxide (NO), prostacyclin, and their effectors—guanosine 3′,5′‐cyclic monophosphate (cGMP) and adenosine 3′,5′‐cyclic monophosphate (cAMP). The influence of weight loss on these alterations is not known. To evaluate whether a diet‐induced body‐weight reduction restores platelet sensitivity to the physiological antiaggregating agents and reduces platelet activation in subjects affected by central obesity, we studied 20 centrally obese subjects before and after a 6‐month diet intervention aiming at reducing body weight by 10%, by measuring (i) insulin sensitivity (homeostasis model assessment of insulin resistance (HOMA_IR)); (ii) plasma lipids; (iii) circulating markers of inflammation of adipose tissue and endothelial dysfunction, and of platelet activation (i.e., soluble CD‐40 ligand (sCD‐40L) and soluble P‐selectin (sP‐selectin)); (iv) ability of the NO donor sodium nitroprusside (SNP), the prostacyclin analog Iloprost and the cyclic nucleotide analogs 8‐bromoguanosine 3′,5′‐cyclic monophosphate (8‐Br‐cGMP) and 8‐bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP) to reduce platelet aggregation in response to adenosine‐5‐diphosphate (ADP); and (v) ability of SNP and Iloprost to increase cGMP and cAMP. The 10 subjects who reached the body‐weight target showed significant reductions of insulin resistance, adipose tissue, endothelial dysfunction, and platelet activation, and a significant increase of the ability of SNP, Iloprost, 8‐Br‐cGMP, and 8‐Br‐cAMP to reduce ADP‐induced platelet aggregation and of the ability of SNP and Iloprost to increase cyclic nucleotide concentrations. No change was observed in the 10 subjects who did not reach the body‐weight target. Changes of platelet function correlated with changes of HOMA_IR. Thus, in central obesity, diet‐induced weight loss reduces platelet activation and restores the sensitivity to the physiological antiaggregating agents, with a correlation with improvements in insulin sensitivity.     

Immunocytochemical localization of cAMP and cGMP in cells of the rat carotid body following natural and pharmacological stimulation.

Although the chemoreceptive function of the carotid body has been known for many decades, the cellular mechanisms of sensory transduction in this organ remain obscure. Common elements in the transductive processes of many cells are the cyclic nucleotide second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Studies from our laboratory have revealed stimulus-induced changes in cyclic nucleotide levels in the carotid body as measured by RIA, but such changes in second messenger levels have not been localized to specific cellular elements in the organ. The present immunocytochemical study utilized the avidin-biotin-peroxidase method to investigate the distribution of cAMP and cGMP in the rat carotid body and to assess changes in the intensity of immunostaining following in vitro stimulation by hypoxia, forskolin, sodium nitroprusside, high potassium, and atrial natriuretic peptide. Both cAMP and cGMP immunoreactivity were localized to type I cells of organs maintained in vivo and fixed by perfusion. Organs exposed to 100% O2-equilibrated media in vitro produced low but visible levels of cAMP immunoreactivity in a majority of type I cells; hypoxia (5% O2-equilibrated media) for 10 min moderately increased the level of immunoreactivity; forskolin (10(-5) M), or forskolin combined with hypoxia, dramatically increased cAMP levels in virtually all cells. Moderate levels of cGMP immunoreactivity in control carotid bodies in vitro were strikingly reduced by hypoxia; a significant increase in cGMP levels occurred following incubation in high potassium (100 mM), and under these conditions, the decrease in cGMP immunoreactivity with hypoxia was much more pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of oxytocin and methacholine on cyclic nucleotide levels of rabbit myometrium

The effects of oxytocin and methacholine on cyclic nucleotide levels in estrogen-primed rabbit myometrium were studied in the presence and absence of 1-methyl-3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor. In the absence of MIX, methacholine increased guanosine 3',5'-cyclic monophosphate (cGMP) levels at a time when contraction was decreasing, but had no influence on adenosine 3',5'-cyclic monophosphate (cAMP) levels. In contrast, oxytocin did not elevate cGMP, but rapidly decreased cAMP levels. MIX (1 mM) increased both cAMP and cGMP levels. Oxytocin or methacholine further increased cGMP, indicating activation of guanylate cyclase. Oxytocin- but not methacholine-induced stimulation of guanylate cyclase was abolished in Ca2+-free solution. Oxytocin increased cAMP over the levels produced by MIX alone, whereas methacholine decreased cAMP below the MIX control values; these effects were insensitive to indomethacin. Tissue levels of cGMP and cAMP did not directly correlate with isometric tension. The results also indicate that both oxytocin and methacholine stimulate guanylate cyclase but have opposing effects on adenylate cyclase of rabbit myometrium.     

The effect in vivo of alterations in gonadotropins and cyclic nucleotides on oocyte maturation in the hamster

The temporal relationship of changes in cAMP and cGMP to oocyte maturation was examined in proestrous hamsters (day 4). The first series of experiments showed, in normal cycling hamsters, an increase in cAMP and a decrease in cGMP at 1400 h shortly after the rise in LH with oocyte maturation beginning at 1800 h. When a second group of animals was injected with phenobarbital at 1200 h to block the LH surge, no significant change occurred in either cyclic nucleotide and oocyte maturation was prevented. In the second series of experiments single injections of either saline, hCG (30 IU), LH (10 micrograms) or FSH (10 micrograms) were given each to a group of animals at 0900 h on day 4. Animals were killed at five time intervals between 15 min and 3 h following the injection. LH and hCG stimulated a simultaneous increase in cAMP and decline in cGMP. The injection of FSH, however, did not cause an increase in cAMP but still produced a sharp decline in cGMP. Oocyte maturation occurred at 3 h in those animals injected with gonadotropins. Animals injected with saline showed neither cyclic nucleotide changes nor oocyte maturation. When cAMP and cGMP levels were expressed as a ratio (cAMP/cGMP) a significant increase occurred in the normal cycling animals and in those injected at 0900 h with gonadotropins. Phenobarbital and saline injected control animals showed no significant increase in the cAMP/cGMP ratio and no oocyte maturation. The results of these experiments and previous studies by this investigator indicate that cGMP may play an important role in oocyte maturation in the hamster prior to the LH surge. Since, in the presence of gonadotropins, the cAMP/cGMP ratio increases prior to oocyte maturation, it may be that the cyclic nucleotide ratio is also of importance in this process. Previous work by Hubbard and Terranova (1) has shown that guanosine 3':5' cyclic monophosphate (cGMP), can inhibit spontaneous maturation of hamster oocytes in vitro. This inhibitory action was dose dependent and overcome by LH. The cGMP-mediated inhibition occurred only in cumulus-enclosed oocytes, while adenosine 3':5' cyclic monophosphate (cAMP) inhibited spontaneous maturation in both cumulus-enclosed and denuded oocytes. The results of this study suggested that cGMP may play a role in inhibiting oocyte maturation prior to the LH surge. LH, the initiator of oocyte maturation, has also been shown in the intact proestrous rat and hamster to cause a decrease in cGMP at the same time that cAMP is rising (2,3).(ABSTRACT TRUNCATED AT 400 WORDS)     

Cyclic nucleotides and cyclic nucleotide phosphodiesterase during development of Polysphondylium violaceum

Polysphondylium violaceum is shown to produce and excrete cyclic nucleotides and to produce a cell-associated cyclic nucleotide phosphodiesterase(s). The amount of adenosine 3′,5′-cyclic monophosphate (cAMP) excreted by the amebae reaches a maximum during development when aggregation centers are just forming and then falls off rapidly. Measurements of total cAMP show that the amount synthesized increases more than 15-fold throughout development with the majority of the increase coming during the culmination stages. Guanosine 3′,5′-cyclic monophosphate (cGMP) is either not excreted or is excreted at levels below our limits of detection. An increase in the total cGMP synthesized occurs at mid-aggregation when two or three sharp peaks of synthesis are observed. However, development of P. violaceum is not affected by the addition of high concentrations of either cAMP or cGMP (or their dibutyryl derivatives) to the medium despite the fact that the cells produce these nucleotides. Cell-associated cyclic nucleotide phosphodiesterase activity, which hydrolyses both cAMP and cGMP, is greatest at the onset of starvation with a second increase in activity during aggregation.     

Plasma atrial natriuretic peptide and cyclic nucleotide levels before and after a marathon

Plasma alpha-atrial natriuretic peptide (alpha-ANP) concentration and levels of cyclic nucleotides [guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP)] were studied in 23 runners before and after a marathon race. Blood samples were drawn from an antecubital vein the morning before the race (base line), at 3 P.M. (i.e., 2 h before the start), on arrival, and 12 and 36 h and 7 days later. Compared with the base-line values of plasma alpha-ANP (5 pmol/l), cGMP (3.8 nmol/l), and cAMP (15.8 nmol/l), the plasma levels of alpha-ANP, cGMP, and cAMP were increased immediately after the marathon, respectively, to 12.0 pmol/l, 12.7 nmol/l, and 50.5 nmol/l. The increase in the plasma alpha-ANP concentration was related (r = 0.85; P less than 0.001) to the changes in plasma cGMP, plasma lactate, hematocrit, and body weight. The plasma cGMP and cAMP concentrations had returned to the prerace levels 12 h after the marathon, whereas the plasma alpha-ANP concentration was significantly lower (3.1 pmol/l) than the base-line values and increased above the prerace values 36 h (7.5 pmol/l) and 7 days (6.8 pmol/l) after the marathon. The plasma cGMP level was also higher 36 h (5.4 nmol/l) and 7 days (5.0 nmol/l) after the marathon race.     

Optimization and validation of a reporter gene assay for screening of phosphodiesterase inhibitors in a high throughput system

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and guanosine monophosphate (cGMP) into inactive 5' monophosphates, and exist as 11 families. Inhibitors of PDEs allow the elevation of cAMP and cGMP, which leads to a variety of cellular effects including airway smooth muscle relaxation and inhibition of cellular inflammation or of immune responses. PDE4 inhibitors specifically prevent the hydrolysis of cAMP. We have validated the manually developed reporter gene assay in a high-throughput screening format that allows for fast and cost-effective identification of potential inhibitors of PDE4 isozymes. The assay is sensitive and robust, with a Z' value of >0.5. The assay is also amenable to 384-well format.     

Stimulation of ovarian cyclic guanosine 3',5'-monophosphate levels by the subunits of human chorionic gonadotropin

Injection of human chorionic gonadotropin (HCG) into the tail vein of superovulated rats resulted in a significant (P<0.01) increase in peripheral plasma progesterone without a concomitant increase in ovarian cyclic GMP (cGMP) levels. However, when equimolar quantities of α and β subunits of HCG were injected, a significant increase in plasma progesterone was accompanied by a concomitant and significant (P<0.01) increase in ovarian cGMP levels. The observation that these subunits increase ovarian cGMP levels without increasing cAMP suggests the possibility of cGMP involvement in steroidogenesis induced by subunits.     

A cell-based PDE4 assay in 1536-well plate format for high-throughput screening

The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,'5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease. Although biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. The authors report the development and validation of a new cell-based PDE4 assay using a constitutively active G-protein-coupled receptor as a driving force for cAMP production and a cyclic nucleotide-gated cation channel as a biosensor in 1536-well plates.     

Role of cyclic nucleotides in modulating ovarian hCG action.

The role of cyclic nucleotides in mediating hormonally responsive adenylate cyclase and cAMP-dependent protein kinase was examined in vivo and in vitro when pseudopregnant rats were injected with hCG. Intracellular ovarian levels of cAMP increased, as expected, but no change in cGMP concentrations was observed. However, both cGMP and cAMP activated ovarian CDPK holoenzyme in vitro but cGMP had a lower affinity. The subunits of hCG were without effect. Even though cGMP and cAMP dissociate partially purified ovarian CDPK holoenzyme in vitro, the receptor sites of the regulatory subunit of CDPK would appear to be relatively specific for cAMP. Moreover, cGMP probably does not mediate hCG action in vivo.     

Immunofluorescent localization of cGMP,cGMP-dependent protein kinase,calmodulin and cAMP in the rat uterus

Cyclic guanosine 3',5' monophosphate (cGMP), cGMP-dependent protein kinase, calmodulin and cyclic adenosine 3',5' monophosphate (cAMP) were localized in the uterus of the immature rat by an indirect immunofluorescence technique. cGMP, cGMP-dependent protein kinase and calmodulin were detected predominantly along epithelial and myometrial plasma membranes and in the adjacent cytoplasm. In contrast, cAMP immunoreactive material was found principally in the cytoplasm of connective tissue. After administration of 17 beta-estradiol, similar time-dependent changes were observed in the localization of cGMP, cGMP-dependent protein kinase and calmodulin in all uterine cell types. For the three compounds, nucleolar-like distribution of the immunofluorescence appeared approximately 12 h after treatment. A more dispersed, reticular distribution of the nuclear fluorescent staining was observed 20-24 h after hormonal treatment. Estrogen did not affect the localization of cAMP. The simultaneous mobilization of cGMP, cGMP-dependent protein kinase and calmodulin towards the same nuclear loci suggests concerted roles for these three molecules in nuclear metabolic processes during the development of the uterotrophic action of estrogens.     

Cyclic nucleotide phosphodiesterases (PDEs) in human osteoblastic cells; the effect of PDE inhibition on cAMP accumulation

The regulation of the secondary messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), is crucial in the hormonal regulation of bone metabolism. Both cAMP and cGMP are inactivated by cyclic nucleotide phosphodiesterases (PDEs), a superfamily of enzymes divided into 11 families (PDE1-11). We compared the PDEs of cultured human osteoblasts (NHOst) and SaOS-2 osteosarcoma cells. The PDE activity of NHOst cells consisted of PDE1, PDE3 and PDE7, whereas PDE1, PDE7 and PDE4, but no PDE3 activity was detected in SaOS-2 cells. In line with the difference in the PDE profiles, rolipram, a PDE4 inhibitor, increased the accumulation of cAMP in SaOS-2, but not in NHOst cells. Expression of PDE subtypes PDE1C, PDE3A, PDE4A, PDE4B, PDE7A and PDE7B was detected in both cell types. NHOst cells additionally expressed PDE1A.     

Effects of intracellular cyclic AMP and cyclic GMP levels on DNA synthesis of young-adult rat hepatocytes in primary culture.

Possible roles of dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) and dibutyryl-guanosine 3',5'-cyclic monophosphate (cGMP) in regulation of hepatocyte DNA synthesis were examined using primary cultures of young-adult rat hepatocytes maintained in arginine-free medium. Throughout the experimental period, nonparenchymal cells were hardly observed in the selective medium. When epidermal growth factor (EGF) was added to the cultures, a transient increase in the intracellular cAMP level preceded the elevation of hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was remarkably enhanced by the elevation of the intracellular cAMP level induced by treatment with cAMP alone or a combination of cAMP and theophylline, an inhibitor of cyclic nucleotide phosphodiesterase. Furthermore, the early elevation of intracellular cAMP alone, which was induced by treatment with the combination of cAMP and theophylline, caused a remarkable increase in hepatocyte DNA synthesis. On the other hand, addition of EGF to the cultures caused a rapid decrease in the intracellular cGMP level followed by an increase in hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was severely suppressed or completely inhibited by the elevation of the intracellular cGMP level induced by treatment with cGMP alone or a combination of cGMP and dipyridamole, a specific inhibitor of cGMP phosphodiesterase. These findings indicate that cAMP and cGMP act oppositely on the regulation of DNA synthesis of young-adult rat hepatocytes in primary culture: cAMP plays a positive role, whereas cGMP plays a negative role. Also it is strongly suggested that an early elevation of the intracellular cAMP level is essential for the onset of DNA synthesis in hepatocyte primary cultures.     

Immunocytochemical localization of cAMP and cGMP in cells of the rat carotid body following natural and pharmacological stimulation

Summary Although the chemoreceptive function of the carotid body has been known for many decades, the cellular mechanisms of sensory transduction in this organ remain obscure. Common elements in the transductive processes of many cells are the cyclic nucleotide second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Studies from our laboratory have revealed stimulus-induced changes in cyclic nucleotide levels in the carotid body as measured by RIA, but such changes in second messenger levels have not been localized to specific cellular elements in the organ. The present immunocytochemical study utilized the avidin-biotin-peroxidase method to investigate the distribution of cAMP and cGMP in the rat carotid body and to assess changes in the intensity of immunostaining following in vitro stimulation by hypoxia, forskolin, sodium nitroprusside, high potassium, and atrial natriuretic peptide. Both cAMP and cGMP immunoreactivity were localized to type I cells of organs maintained in vivo and fixed by perfusion. Organs exposed to 100% O₂-equilibrated media in vitro produced low but visible levels of cAMP immunoreactivity in a majority of type I cells; hypoxia (5% O₂-equilibrated media) for 10 min moderately increased the level of immunoreactivity; forskolin (10^–5 M), or forskolin combined with hypoxia, dramatically increased cAMP levels in virtually all cells. Moderate levels of cGMP immunoreactivity in control carotid bodies in vitro were strikingly reduced by hypoxia; a significant increase in cGMP levels occurred following incubation in high potassium (100 mM), and under these conditions, the decrease in cGMP immunoreactivity with hypoxia was much more pronounced. The synthetic analog of atrial natriuretic peptide, atriopeptin III (10^–7 M), greatly elevated cGMP immunoreactivity in the type I cells. On the other hand, sodium nitroprusside (1 mM) elevated cGMP staining mostly in vascular elements of the carotid body in vitro. The data implicate the involvement of cyclic nucleotides in transduction of natural chemosensory stimuli by the type I cells in rat carotid body.