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.     

Developmental physiology of cestodes: characterization of putative crowding factors in Hymenolepis diminuta

It was shown previously that worm-conditioned saline (WCS) prepared from crowded 10-day-old H. diminuta inhibited the incorporation of 3H-thymidine into DNA in the anterior regions of uncrowded worms and that the inhibition was partially accounted for by succinate and acetate excreted by the worms. The present study describes further characterization of the active components of WCS. An ultrafiltrate was fully as potent as untreated WCS, indicating that all detectable inhibitory components were less than about 500 daltons in molecular mass. Inhibitory factors in WCS were stable to heat (80 C for 30 min), cold (4 C for 48 hr), drying and reconstitution, alkaline pH (11 to 12 for 3 hr), and ethanolic extraction. Active compounds were probably not lipoidal in nature. Although the acidic ethanol extract of WCS was inhibitory, no activity was observed in fractions of WCS that contained basic, acidic and neutral amino acids. Amino compounds in the WCS were further investigated. Twenty-four amino acids were identified, 3 of which (phosphoserine, 1-methylhistidine, and 3-methylhistidine) have not been reported previously for H. diminuta. On a molar basis, alanine accounted for 40-50% of the amino acids released. The amino sugar, D-glucosaminic acid, was found in the WCS and also has not been heretofore reported from H. diminuta or any other cestode. In concentrations comparable to those in the WCS, D-glucosaminic acid inhibited incorporation of 3H-thymidine into the DNA of the tapeworms by 25-35%, suggesting that D-glucosaminic acid may be one of the crowding factors.     

Cyclic GMP and lectin-induced lymphocyte activation.

Purified human peripheral lymphocytes incubated with the mitogenic plant lectins phytohemagglutinin and concanavalin A were examined for alterations in intracellular cGMP and cAMP under a variety of experimental conditions and using multiple techniques for the isolation and purification of cGMP and cAMP before assay of the cyclic nucleotides by radioimmunoassay. In contrast to work reported by others, we have been unable to demonstrate consistent increases in cGMP under any of the experimental conditions used and with any of the various purification schemes. In these same experiments exogenous cGMP added to the lymphocytes could be measured, and the immunoreactive material was destroyed by cyclic nucleotide phosphodiesterase, indicating that our inability to measure increases in cGMP was not caused by our inability to measure cGMP. Under identical experimental conditions, small but consistent and statistically significant increases in cAMP were noted. In addition, other parameters of lymphocyte activation, 45Ca uptake (an early parameter), and incorporation of 3H thymidine into DNA were unimpaired. These data call to question the concept of cGMP as the second messenger in lectin-stimulated human peripheral lymphocytes.     

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.     

In vivo effects of putative crowding factors on development of Hymenolepis diminuta.

During in vitro incubation, Hymenolepsis diminuta secretes substances into the medium that inhibit DNA synthesis in the germinative region of freshly isolated, uncrowded worms. Of the many substances that are released by H. diminuta into the medium, earlier studies indicate that only succinate, acetate, glucosaminic acid, and cGMP are responsible for the inhibition. In the present report, effects of these putative crowding factors on worm development in vivo were examined. At 7 days postinfection the proximal end of the host's intestine was catheterized and perfused with test solution. The test solution contained 28 nM cGMP, 250 microM glucosaminic acid, 120 mM succinate, and 40 mM acetate. The solution was perfused by a peristaltic pump at a rate of 50 ml/day. At 2 wk postinfection, worms were recovered for subsequent analysis. Worms developing in the presence of crowding factors were 53% less in wet weight than control worms. Carbohydrate concentrations in worms from experimental groups were not different from those in control groups; therefore, the inhibition in growth was probably not due to carbohydrate deprivation. Worms from experimental groups had fewer immature, mature, and gravid proglottids than did worms from control groups. The results are consistent with the hypothesis that the tested substances, which inhibit DNA synthesis in H. diminuta in vitro, are a part of the cause of the crowding effect in vivo.     

Regulation of cyclic nucleotide phosphodiesterases in cultured hepatoma cells by dexamethasone and N6,O2'-dibutyryl adenosine 3':k'-monophosphate.

DEAE-Bio-Gel chromatography of 100,000 X g supernatant from cultured HTC hepatoma cells separated cyclic nucleotide phosphodiesterase into three forms, numbered E I, E II, and E III in order of elution from the column, E I had a low Km for cyclic guanosine 3':5'-monophosphate (cGMP) and a high Km for cyclic adenosine 3':5'-monophosphate (cAMP), E II exhibited anomalous kinetics. At low substrate concentrations (0.5 muM) cGMP was hydrolyzed more rapidly than cAMP and hydrolysis of 0.5 muM cAMP was stimulated by 1 muM cGMP. E III had a low Km for cAMP. Incubation of cells with 1 muM dexamethasone for 72 h decreased the activity of E I and E II. In cells incubated with N6,O2'-dibutyryl cAMP plus 3-isobutyl-1-methylxanthine for 14 h the activity of E III was increased approximately 100%. Similar activities of calcium-dependent, heat stable phosphodiesterase activator were recovered from supernatants from all cells. These studies have established the presence, in a homogeneous population of hepatoma cells, of at least three forms of cyclic nucleotide phosphodiesterase, the activities of which can be independently regulated.     

Biochemical aspects of cardiac muscle differentiation. Possible control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3':5'-monophosphate.

A single injection of either isoproternol or N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) results in an inhibition in the rate of [3H]thymidine incorporation into DNA of differentiating cardiac muscle of the neonatal rat. This inhibition is not due to substantially altered cellular uptake or catabolism of [3H]thymidine. Inhibition of [3H]thymidine incorporation by isoproterenol or dibutyryl cyclic AMP is potentiated by theophylline. Maximal inhibition (95%) is observed 24 h after administration of isoproterenol, and the rate of incorporation returns to a value 80% of control by 72 h. Norepinephrine also inhibits [3H]thymidine incorporation whereas cyclic GMP, N2, 02-Dibutyryl guanosine 3':5'-monophosphate (dibutyryl cyclic GMP), and phenylephrine have little effect. Equilibrium sedimentation analysis of cardiac muscle DNA in neutral and alkaline cesium chloride gradients using bromodeoxyuridine as a density label indicate that isoproterenol and dibutyryl cyclic AMP inhibit [3H]thymidine incorporation into DNA that is replicating semiconservatively. Administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats inhibits by approximately 60% the incorporation of [3H]thymidine into DNA of tissue slices of cardiac muscle prepared 16 h later. [3H]Thymidine incorporation into DNA of tissue slices is into chains that were growing in vivo. This incorporation is linear for at least 4 h of incubation and is inhibited by isoproterenol and dibutyryl cyclic AMP. Inhibition is not due to altered cellular uptake of [3H]thymidine nor is it due to a cytotoxic action. Several other compounds which elevate intracellular levels of cyclic AMP (epinephrine, norepinephrine, glucagon, and prostaglandin E1) also inhibit [3H]thymidine incorporation into DNA or cardiac muscle tissue slices. Cyclic GMP, dibutyryl cyclic GMP, sodium butyrate, and phenylephrine have little effect. Isoproterenol administered together with theophylline to neonatal rats signficantly stimulates the in corporation of [3H]phenylalanine into total cardiac muscle protein and into myosin. This enhanced incorporation may be due in part to an increase in the cellular uptake of [3H]phenylalanine. DNA synthesis decreases progressively in differentiating cardiac muscle of the rat during postnatal development and essentially ceases by the middle of the third week (Claycomb, W. C. (1975) J. Biol. Chem. 250, 3229-3235). In reviewing the literature it was found that this decline in synthetic activity correlates temporally with a progressive increase in tissue concentrations of norepinephrine and cyclic AMP and with the anatomical and physiological development of the adrenergic nerves in this tissue. Because of these facts and data presented in this report it is proposed that cell proliferation and cell differentiation in cardiac muscle may be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators.     

Induction of ornithine decarboxylase in guinea pig lymphocytes by the divalent cation ionophore A 23187. Effect of dibutyryladenosine 3',5'-monophosphate

The divalent cation ionophore, A23187, at a concentration of 0.25 microgram/ml, enhanced influx of Ca2+, activity of ornithine decarboxylase and incorporation of [3H]thymidine into DNA of guinea pig lymphocytes. Combined treatment of cells with A23187 and dibutyryladenosine 3',5'-monophosphate (Bt2cAMP) augmented these three events. A23187 at a concentration of 0.06 microgram/ml was insufficient for induction of ornithine decarboxylase stimulated neither Ca2+ influx nor [3H]thymidine incorporation, but stimulated Ca2+ efflux. A23187 (0.06 microgram/ml) in combination with Bt2cAMP caused a marked induction of ornithine decarboxylase and stimulation of [3H]thymidine incorporation into DNA. When the time of Bt2cAMP addition was delayed after A23187, the stimulation of ornithine decarboxylase activity decreased. Washout of Bt2cAMP from cell culture earlier than 4 h of incubation caused a reduction in the stimulatory effect of Bt2cAMP. These results suggest that raising concentrations of cytoplasmic Ca2+ and cellular cAMP are important to some initial events leading to induction of ornithine decarboxylase and these biochemical changes are obligatory sequential steps for stimulation of DNA synthesis.     

Synthesis and enzymic activity of 8-acyl and 8-alkyl derivatives of guanosine 3, 5-cyclic phosphate.

Several new 8-alkyl and 8-acyl derivatives of quanosie 3',5'-cyclic phosphate (cGMP) and inosine 3',5'-cyclic phosphate (cGMP) were prepared by direct alkylation or acylation of the parent cyclic nucleotide via free radicals generated in situ. These compounds have been examined for their ability to stimulate a cGMP-dependent protein kinase, and several of the cGMP derivatives were as active in this regard as cGMP. These compounds proved to be quite ineffective when tested for their ability to activate an adenosine 3',5'-cyclic phosphate (cAMP) dependent protein kinase. In addition, these 8-substituted cGMP derivatives are not substrates for a phosphodiesterase preparation from rabbit kidney, but do show inhibition of the hydrolysis of cAMP by crude phosphodiesterase preparations from rabbit lung and beef heart.     

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.     

Phosphorylation in sealed rod outer segments: effects of cyclic nucleotides

Rod outer segments (ROS) from rat were purified on Percoll gradients. These ROS had intact plasma membranes since they were impermeable to small molecules. Protein phosphorylation in the purified ROS was studied after the plasma membrane was disrupted by freeze/thawing. [gamma-32P]ATP was used as phosphate donor. ATP concentration, time, temperature, and light or dark adaptation were varied in the assays. The 32P-labeled proteins were separated by polyacrylamide gel electrophoresis and autoradiographed. Rhodopsin was the dominant phosphorylated protein, and the addition of adenosine cyclic 3',5'-phosphate (cAMP) or guanosine cyclic 3',5'-phosphate (cGMP) (10(-4) M) did not qualitatively alter the ROS phosphorylation pattern. The only cyclic nucleotide effect we could establish in these experiments was the inhibition of rhodopsin phosphorylation by cGMP. This inhibition did not appear to be competitive with ATP since cAMP was much less inhibitory than cGMP and the phosphorylation in the presence of cGMP reached a plateau at a much lower level than in control conditions. Hypotheses implying an involvement of protein phosphorylation/dephosphorylation in dark adaptation have been formulated [Miller, J. A., & Paulsen, R. (1975) J. Biol. Chem. 250, 4427-4432; Kuhn, H., McDowell, J. H., Leser, K. H., & Bader, S. (1977) Biophys. Struct. Mech. 3, 175-180]; we suggest that cGMP may control this process through the modulation of the extent of inhibition of phosphorylation of the visual pigment.     

Mapping ligand interactions with the hyperpolarization activated cyclic nucleotide modulated (HCN) ion channel binding domain using a soluble construct

Hyperpolarization activated cyclic nucleotide modulated (HCN) ion channel currents are activated by hyperpolarization and modulated in response to changes in cytosolic adenosine 3',5'-cyclic monophosphate (cAMP) concentrations. A cDNA chimera combining the rat HCN2 cyclic nucleotide binding domain and the DNA binding domain of the cAMP receptor protein (CRP) from E. coli and the histidine tag (HCN2/CRP) was expressed and purified. The construct is capable of forming only non-ligand dependent dimers because the C-linker region of the channel is not present in this construct. The construct binds 8-[[2-[(fluoresceinylthioureido) amino] ethyl] thio] adenosine-3',5'-cyclic monophosphate (8-fluo cAMP) with a Kd of 0.299 microM as determined with a monomer binding model. The Ki values of 20 ligands related to cAMP were measured in order to determine the properties necessary for a ligand to bind to the HCN2 binding domain. This is the first report of cAMP and gunaosine 3',5'-cyclic monophosphate (cGMP) affinities to the HCN2 binding domain being equivalent, even though they modulate the channel with a 10-fold difference in K0.5. Furthermore, the array of ligands measured allows the preference rank order for each purine ring position to be determined: position 1, H > NH2 > O; position 2, NH2 > Cl > H > O; position 6, NH2 > Cl > H > O; and position 8, NH2 > Cl > H > O. Finally, the ability of HCN2/CRP to bind cyclic nucleotide pyrimidine rings at concentrations approximately 1.33 times greater than cAMP suggests that ribofuranose is key for binding.     

Studies of the molecular mechanism of discrimination between cGMP and cAMP in the allosteric sites of the cGMP-binding cGMP-specific phosphodiesterase (PDE5).

The regulatory domain of the cGMP-binding cGMP-specific 3':5'-cyclic nucleotide phosphodiesterase (PDE5) contains two homologous segments of amino acid sequence that encode allosteric cyclic nucleotide-binding sites, referred to as site a and site b, which are highly selective for cGMP over cAMP. The possibility that the state of protonation in these sites contributes to cyclic nucleotide selectivity was investigated. The binding of cGMP or cAMP was determined using saturation and competition kinetics at pH values between 5.2 and 9.5. The total cGMP binding by PDE5 was unchanged by variation in pH, but the relative affinity for cGMP versus cAMP progressively decreased as the pH was lowered. Using site-directed mutagenesis, a conserved residue, Asp-289, in site a of PDE5 has been identified as being important for cyclic nucleotide discrimination in this site. It is proposed that deprotonation of Asp-289 enhances the number and strength of bonds formed with cGMP, while concomitantly decreasing the interactions with cAMP.     

Characterization of soluble uterine cyclic nucleotide phosphodiesterase.

Soluble cyclic nucleotide phosphodiesterase of rat uterus displays distinct structural and regulatory properties. Like phosphodiesterases from many mammalian sources the soluble uterine enzyme system exhibits nonlinear Lineweaver--Burk kinetics with cyclic adenosine 3':5'-monophosphate (cAMP) as substrate (apparent Kms congruent to 3 and 20 micron) and linear kinetics with cyclic guanosine 3':5'-monophosphate (cGMP) as substrate (apparent Km congruent to 3 micron). Unlike most other mammalian phosphodiesterases, however, numerous separation procedures reveal only a single form of uterine phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP. A single form of the enzyme is observed upon sucrose gradient centrifugation (7.9 S), agarose gel filtration, and DEAE-cellulose chromatography at either pH 8.0 OR 6.0. Heat denaturation (50 degrees C) of soluble uterine phosphodiesterase causes the loss of both cAMP and cGMP hydrolytic activities at the same rate. Isoelectric focusing reveals major (pI = 5.2) and minor forms (pI = 5.8) of phosphodiesterase which both catalyze the hydrolysis of the two cyclic nucleotide substrates. In vivo administration of estradiol produces identical decreases in the activities of cAMP and cGMP phosphodiesterase. These results raise the possibility that the uterus contains a single form of soluble phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP.     

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)     

cGMP secreted from the tapeworm Hymenolepis diminuta is a signal molecule to the host intestine

3',5'-Cyclic guanosine monophosphate (cGMP), a well-known intracellular second messenger, is released to the intestinal lumen by the tapeworm, Hymenolepis diminuta. Enzyme-linked immunosorbent assay analysis of tapeworm conditioned media shows that cGMP is released at a constant rate. Multidrug resistant (MDR) proteins are efflux transporters for cyclic nucleotides. Two MDR inhibitors, niflumic acid and zaprinast, inhibit cGMP secretion by tapeworms and change the cGMP localization within the tapeworm tegument, as assessed by immunochemistry. cGMP, normally present throughout the tapeworm tegumental cytoplasm, is absent from the outer cytoplasmic band upon treatment with inhibitors. Inhibition of cGMP secretion by colchicine indicates that cGMP secretion is cytoskeleton dependent. Binding studies of [3H]cGMP to ileal segments of intestine demonstrate 2 saturable, reversible, and high-affinity binding sites. These studies demonstrate that cGMP is secreted from the cestode via a cytoskeleton-dependent mechanism and MDR efflux transporters. In addition, cGMP reaching the intestinal lumen can bind to the mucosa via receptors for cGMP. These data, combined with earlier observations of cGMP altering intestinal motility and slowing lumenal transit, indicate that tapeworms alter the physiology of the host digestive process via the secretion and binding of extracellular cGMP to lumenal receptors in the host intestine.     

Substrate and effector specificity of a guanosine 3':5'-monophosphate phosphodiesterase from rat liver.

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.     

Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells

Cyclic nucleotides are known to be effluxed from cultured cells or isolated tissues. Two recently described members of the multidrug resistance protein family, MRP4 and MRP5, might be involved in this process, because they transport the 3',5'-cyclic nucleotides, cAMP and cGMP, into inside-out membrane vesicles. We have investigated cGMP and cAMP efflux from intact HEK293 cells overexpressing MRP4 or MRP5. The intracellular production of cGMP and cAMP was stimulated with the nitric oxide releasing compound sodium nitroprusside and the adenylate cyclase stimulator forskolin, respectively. MRP4- and MRP5-overexpressing cells effluxed more cGMP and cAMP than parental cells in an ATP-dependent manner. In contrast to a previous report we found no glutathione requirement for cyclic nucleotide transport. Transport increased proportionally with intracellular cyclic nucleotide concentrations over a calculated range of 20-600 microm, indicating low affinity transport. In addition to several classic inhibitors of organic anion transport, prostaglandins A(1) and E(1), the steroid progesterone and the anti-cancer drug estramustine all inhibited cyclic nucleotide efflux. The efflux mediated by MRP4 and MRP5 did not lead to a proportional decrease in the intracellular cGMP or cAMP levels but reduced cGMP by maximally 2-fold over the first hour. This was also the case when phosphodiesterase-mediated cyclic nucleotide hydrolysis was inhibited by 3-isobutyl-1-methylxanthine, conditions in which efflux was maximal. These data indicate that MRP4 and MRP5 are low affinity cyclic nucleotide transporters that may at best function as overflow pumps, decreasing steep increases in cGMP levels under conditions where cGMP synthesis is strongly induced and phosphodiesterase activity is limiting.     

Studies on the terminal stages of antibody-complement-mediated killing of a tumor cell. II. Inhibition of transformation of T to dead cells by 3'5' cAMP.

Transformation of T to dead cells was prevented by 3'5' cAMP. The effect of 3'5' cAMP was dose, time, and temperature dependent. T washed free of 3'5' cAMP after short-term incubation proceeded to die to the same extent as control cells. After 3 hr of incubation of T with 3'5' cAMP the level of killing was significantly reduced. The 3'5' cyclic nucleotides of uridine, guanine, cytosine, and thymidine and the 2'3' cyclic adenosine nucleotide were not effective. It was concluded that prolonged treatment of T with 3'5' cAMP either irreversibly blocked the damage-producing process or facilitated the reapir of damaged sites.     

Regulation of DNA synthesis by guanosine-5′-diphosphate, cyclic guanosine-3′,5′-monophosphate, and cyclic adenosine-3′,5′-monophosphate in mouse lymphoid cells

The effect of various adenine and guanine nucleotides and nucleosides on DNA synthesis was studied in various types of mouse lymphoid cells. Two out of the ten compounds tested, namely guanosine-5′-diphosphate (GDP) and cyclic guanosine-3′,5′-monophosphate (cGMP) increased the thymidine incorporation into the DNA of the spleen cells and counteracted completely or partially the inhibitory action of cyclic adenosine-3′,5′-monophosphate (cAMP) on spleen cells stimulated by various B or T cell mitogens. GDP seems to act preferentially on thymus cells while cGMP acts better on bone marrow cells. The possible significance of the results for the mechanism of the mitogenic signal is discussed.