Effect of elite physical exercise by triathletes on seven catabolites of DNA oxidation.

The oxidized nucleoside 8-hydroxy-2'-deoxyguanosine has been widely studied as a marker of DNA oxidation; however, data on the occurrence of other metabolites in plasma that are related to DNA damage are scarce. We have applied an improved, sensitive, robust, and reliable method, involving solid phase extraction and ultrahigh-performance liquid chromatography (UHPLC)-tandem mass spectrometry (MS/MS), to the precise quantitation of seven metabolites in the plasma of 15 elite triathletes after a 2-week training program. All compounds were eluted in the first 1.6 min, with limits of detection and quantification ranging between 0.001 and 0.3 ng.mL(-1) and 0.009 and 0.6 ng.mL(-1), respectively. Four compounds were detected in plasma: guanosine-3'-5'-cyclic monophosphate, 8-hydroxyguanine, 8-hydroxy-2'-deoxyguanosine, and 8-nitroguanosine. After two weeks of training, 8-hydroxyguanine exhibited the highest increase (from 0.031 ± 0.008 nM to 0.036 ± 0.012 nM) (p < 0.05), which could be related to the enhanced activity of DNA-repairing enzymes that excise this oxidized base. Increased levels of guanosine-3'-5'-cyclic monophosphate and 8-hydroxy-2'-deoxyguanosine were also observed. In contrast, levels of 8-nitroguanosine (p < 0.05) were significantly reduced, which might be a protective measure as this compound strongly stimulates the generation of superoxide radicals, and its excess is related to pathologies such as microbial (viral) infections and other inflammatory and degenerative disorders. The results obtained indicate an induced adaptive response to the increased oxidative stress related to elite training, and point to the benefits associated with regular exercise.

Cyclic AMP induces IPC leukemia cell apoptosis via CRE-and CDK-dependent Bim transcription.

The IPC-81 cell line is derived from the transplantable BNML model of acute myelogenic leukemia (AML), known to be a reliable predictor of the clinical efficiency of antileukemic agents, like the first-line AML anthracycline drug daunorubicin (DNR). We show here that cAMP acted synergistically with DNR to induce IPC cell death. The DNR-induced death differed from that induced by cAMP by (1) not involving Bim induction, (2) being abrogated by GSK3ß inhibitors, (3) by being promoted by the HSP90/p23 antagonist geldanamycin and truncated p23 and (4) by being insensitive to the CRE binding protein (CREB) antagonist ICER and to cyclin-dependent protein kinase (CDK) inhibitors. In contrast, the apoptosis induced by cAMP correlated tightly with Bim protein expression. It was abrogated by Bim (BCL2L11) downregulation, whether achieved by the CREB antagonist ICER, by CDK inhibitors, by Bim-directed RNAi, or by protein synthesis inhibitor. The forced expression of BimL killed IPC-81(WT) cells rapidly, Bcl2-overexpressing cells being partially resistant. The pivotal role of CREB and CDK activity for Bim transcription is unprecedented. It is also noteworthy that newly developed cAMP analogs specifically activating PKA isozyme I (PKA-I) were able to induce IPC cell apoptosis. Our findings support the notion that AML cells may possess targetable death pathways not exploited by common anti-cancer agents.

Activation of protein kinase A (PKA) by 8-Cl-cAMP as a novel approach for antileukaemic therapy.

Activation of PKA by cAMP agonists, such as 8-Cl-cAMP activation, selectively causes rapid apoptosis in v-abl transformed fibroblasts by inhibiting the Raf-1 kinase. Here we investigated whether 8-Cl-cAMP is useful for the treatment of chronic myelogenous leukaemia (CML), which is hallmarked by the expression of the p210(bcr/abl) oncogene. Autologous bone marrow transplantation is a feasible alternative for patients with no suitable donor, but hampered by the risk of relapse due to the persistence of leukaemia cells in the transplant. To study the effects of 8-Cl-cAMP on primary leukaemic cells, bone marrow cells (BMCs) from eight CML patients (one at diagnosis, three in chronic and four in accelerated phase) were treated. Ex vivo treatment of BMCs obtained in chronic phase of CML with 100 microM 8-Cl-cAMP for 24-48 h led to the selective purging of Philadelphia Chromosome (Ph1 chromosome) without toxic side effects on BMCs from healthy donors as measured by colony-forming unit (CFU) assays. BMCs from patients in accelerated phase showed selective, but incomplete elimination of Ph1 chromosome positive colony forming cells. The mechanism of 8-Cl-cAMP was investigated in FDCP-mix cells transformed by p210(bcr/abl), a cell culture model for CML. The results showed that 8-Cl-cAMP reduced DNA synthesis and viability independent of Raf inhibition as Raf inhibitors had no effect. MEK inhibitors interfered with DNA synthesis, but not with viability. In summary, our results indicate that 8-Cl-cAMP could be useful to purge malignant cells from the bone marrow of patients with CML and certain other forms of leukaemias.

Action of protein kinase A regulators on secretory activity of porcine granulosa cells in vitro.

To understand the role of protein kinase A (PKA) in the control of ovarian secretory activity, we examined effects of stimulators (db-cAMP, 6-Phe-cAMP, Sp-cDBIMPS) or inhibitors (Rp-cAMPS, KT5720) of PKA on the release of insulin-like growth factor I (IGF-I), progesterone (P) and estradiol (E) by cultured porcine granulosa cells using RIA. All the PKA stimulators db-cAMP (10-10000 ng/ml), 6-Phe-cAMP (10-10000 pmol) or Sp-cDBIMPS (1-10000 pmol) increased IGF-I almost at all doses tested. P release was stimulated by db-cAMP (at doses 100-10000 ng/ml), Sp-cDBIMPS (at 10-1000 pmol) and 6-Phe-cAMP (at 1000 and 10000 pmol). The release of E was stimulated by Sp-cDBIMPS (1-100 pmol), db-cAMP (1000 and 10000 ng/ml) and 6-Phe-cAMP (1000 and 10000 pmol). Since Sp-cDBIMPS, which activates preferentially PKA isozyme type II, showed stimulating effects at doses lower than those of 6-Phe-cAMP, a preferential activator of both, type I and II of PKA, it is assumed that PKA type II is more important for the control of ovarian steroidogenesis than type I. A PKA inhibitor Rp-cAMPS inhibited release of IGF-I (10000 pmol), P (1000 pmol) and E (1000 and 10000 pmol), whereas Rp-cAMPS, at doses higher than 1000 pmol, tended to reverse this inhibitory effect. Other PKA inhibitor KT5720 suppressed P (at 10-1000 ng/ml), but not IGF-I or E release.The stimulation of growth factor and sex steroid release by PKA activators, and suppression of the secretion some of these substances by PKA inhibitors may indicate the implication of PKA (probably site B) in up- and down-regulation of ovarian IGF-I and steroid release.

cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension.

Little is known about the relative role of cAMP-dependent protein kinase (cAPK) and guanine exchange factor directly activated by cAMP (Epac) as mediators of cAMP action. We tested cAMP analogs for ability to selectively activate Epac1 or cAPK and discriminate between the binding sites of Epac and of cAPKI and cAPKII. We found that commonly used cAMP analogs, like 8-Br-cAMP and 8-pCPT-cAMP, activate Epac and cAPK equally as well as cAMP, i.e. were full agonists. In contrast, 6-modified cAMP analogs, like N6-benzoyl-cAMP, were inefficient Epac activators and full cAPK activators. Analogs modified in the 2'-position of the ribose induced stronger Epac1 activation than cAMP but were only partial agonists for cAPK. 2'-O-Alkyl substitution of cAMP improved Epac/cAPK binding selectivity 10-100-fold. Phenylthio substituents in position 8, particularly with MeO- or Cl- in p-position, enhanced the Epac/cAPK selectivity even more. The combination of 8-pCPT- and 2'-O-methyl substitutions improved the Epac/cAPK binding selectivity about three orders of magnitude. The cAPK selectivity of 6-substituted cAMP analogs, the preferential inhibition of cAPK by moderate concentrations of Rp-cAMPS analogs, and the Epac selectivity of 8-pCPT-2'-O-methyl-cAMP was also demonstrated in intact cells. Using these compounds to selectively modulate Epac and cAPK in PC-12 cells, we observed that analogs selectively activating Epac synergized strongly with cAPK specific analogs to induce neurite outgrowth. We therefore conclude that cAMP-induced neurite outgrowth is mediated by both Epac and cAPK.

Direct comparison of the potency of three novel cAMP analogs to induce CREB-phophorylation in rat pinealocytes.

A modified analog of cyclic adenosine 3',5'-monophosphate (cAMP), Sp-adenosine-3',5'-monophosphorothioate, designed to be highly membrane-permeable and resistant towards phosphodiesterases was found to induce the phosphorylation of the cAMP-regulated transcription factor cyclic AMP-responsive element binding protein in cultured rat pinealocytes more efficiently than previously described cAMP analogs.

Effect of four cGMP analogues with different mechanisms of action on hormone release by porcine ovarian granulosa cells in vitro.

The aim of our in-vitro experiments was to examine the role of cGMP-dependent intracellular mechanisms in control of ovarian hormone secretion, as well as to understand, whether cGMP effect on the ovary may be mediated by either protein kinase G (PKG), cGMP-gated ion channels (CGI) or cGMP-specific phosphodiesterases (PDE). We compared the effects of the cGMP analogues 8-pCPT-cGMP, an activator of PKG 1-alpha, 1-beta and type II and of CGI, but not of PDE: Rp-8-pCPT-cGMPS and Rp-8-Br-cGMPS, inhibitors of PKG, stimulators of CGI with no effect of PDE, and Rp-8-Br-PET-cGMPS, an inhibitor of both, PKG and CGI and stimulator of PDE (all at 0.01, 0.1, 1, 10 or 100 nM), on the release of oxytocin (OT) and progesterone (P) by cultured porcine granulosa cells. It was observed, that Rp-8-pCPT-cGMPS significantly (p<0.05) suppressed OT release when given at 1 or 10 nM. Rp-8-Br-cGMPS increased OT output, when given at 1-10 nM too, but decreased it at 100 nM. Rp-8-Br-PET-cGMPS inhibited OT release at 1 nM. No influence of 8-pCPT-cGMP on OT output was found. 8-pCPT-cGMP stimulated P release at 0.1, 10 or 100 nM. All other cGMP analogues studied suppressed P release at all doses used. The present observations suggest the involvement of cGMP-dependent intracellular mechanisms in control of ovarian steroid and nonapeptide hormone release. The lack of association between patterns of influence of cGMP analogues on CGI and PDE, and the coincidence of the majority of effects of cGMP analogues on P, OT and PKG may indirectly indicate that cGMP action on release of ovarian hormones is mediated mainly by PKG, but not by CGI or PDE.

Effect of restricted food intake on production, catabolism, and effects of IGF-I and cyclic nucleotides in cultured ovarian tissue of domestic nutria (Myocastor coypus).

The aims of these in vitro experiments were to examine the effects of short-term food restriction on ovarian secretory activity and the role of IGF-I and cAMP- and cGMP-dependent intracellular mechanisms in the control of ovarian function in domestic nutria. Slices of ovary from sexually mature animals kept under conditions of normal and restricted ((1/2) of standard ration) feeding were cultured with or without IGF-I (50 ng/ml), cAMP analogues (dbcAMP and Rp-cAMPS), and cGMP analogues (8-pCPT-cGMP and Rp-8-Br-PET-cGMPS; all at 100 nM). In nonovarian cells dbcAMP activates and Rp-cAMPS inhibits protein kinase A, while 8-p-CPT-cGMP activates and RP-8-Br-PET-cGMPS inhibits protein kinase G and cGMP-gated ion channels. IGF-I release and catabolism, as well as the release of progesterone (P), estradiol (E), and cAMP by the cultures, were evaluated using RIA. IGF-I did not affect cAMP release, while each of the cAMP and cGMP analogues inhibited IGF-I release in both control and experimental groups. Fasting did not affect cAMP or IGF-I release. It partially prevented the effect of Rp-cAMPS, but not of other cyclic nucleotides, on IGF-I release and inhibited IGF-I catabolism. The Rp-cAMPS and Rp-8-Br-PET-cGMPS also inhibited IGF-I catabolism and the effects were greater with tissue from food-restricted than control animals. Ovaries from the underfed nutria secreted significantly more P and less E than those from normally fed animals. IGF-I and both cAMP analogues, given alone, did not affect P release whereas a combination of IGF-I and Rp-cAMPS increased P output in control, but not in the experimental group. The 8-pCPT-cGMP had no effect P release. Rp-8-Br-PET-cGMPS, given alone or in combination with IGF-I, dramatically increased P secretion by tissue from control but not underfed animals. Estradiol secretion by tissue from underfed animals was stimulated by IGF-I, dbcAMP, Rp-cAMPS, 8-pCPT-cGMP, and Rp-8-Br-PET-cGMPS as well as by combinations of IGF-I and Rp-cAMPS or Rp-8-Br-PET-cGMPS; these effects were not seen with control tissue. The results demonstrate that: (1) ovaries of domestic nutria secrete IGF-I, P, E, and cAMP; (2) cAMP and cGMP can influence IGF-I release and catabolism; (3) the cyclic nucleotides may have an IGF-I-mediated effect on P and E output; (4) IGF-I and cyclic nucleotides can prevent the effect of undernutrition on E, but not on P release; (5) effects of cAMP and cGMP on P and E are probably not mediated by protein kinase A, protein kinase G, or cGMP-gated ion channels; and (6) food restriction can influence ovarian IGF-I catabolism, P, and E release and modulate the effects of cyclic nucleotides and IGF-I on steroidogenesis. It is concluded that ovarian secretory activity may be regulated separately by nutrition and the cyclic nucleotide-IGF-I system, and there may be functional interrelationships between these mechanisms.

CREB phosphorylation and melatonin biosynthesis in the rat pineal gland: involvement of cyclic AMP dependent protein kinase type II.

Phosphorylation of cyclic AMP response element binding protein (CREB) at amino acid serine 133 appears as an important link between the norepinephrine (NE)-induced activation of second messenger systems and the stimulation of melatonin biosynthesis. Here we investigated in the rat pineal gland: 1) the type of protein kinase that mediates CREB phosphorylation: and 2) its impact on melatonin biosynthesis. Immunochemical or immunocytochemical demonstration of serine133-phosphorylated cyclic AMP regulated element binding protein (pCREB) and radioimmunological detection of melatonin revealed that only cyclic AMP-dependent protein kinase (PKA) inhibitors suppressed NE-induced CREB phosphorylation and stimulation of melatonin biosynthesis, whereas inhibitors of cyclic GMP-dependent protein kinase (PKG), mitogen-activated protein kinase kinase, protein kinase C, or calcium-calmodulin-dependent protein kinase (CaMK) were ineffective. Investigations with cyclic AMP-agonist pairs that selectively activate either PKA type I or II link NE-induced CREB phosphorylation and stimulation of melatonin biosynthesis to the activation of PKA type II. Our data suggest that PKA type II plays an important role in the transcriptional control of melatonin biosynthesis in the rat pineal organ.

Substituted cGMP analogs can act as selective agonists of the rod photoreceptor cGMP-gated cation channel.

Cyclic nucleotide-gated (CNG) channels are expressed in many cell types in both the nervous system and nonexcitable tissues. In order to understand the roles of cGMP-gated channels, and to distinguish actions of cGMP mediated through CNG channels from those through cGMP-dependent protein kinase (G-kinase), several new cGMP analogs were tested for potency as CNG channel agonists. Using Xenopus oocytes expressing the rat rod cGMP-gated ion channel alpha-subunit, we showed that an analog containing a pCPT group at the 8-position, 8-pCPT-cGMP, was 80 times more potent than cGMP and 14 times more potent than 8-Br-cGMP. 8-pCPT-cGMP is the most potent CNG channel agonist so far described and also has the advantages of much better membrane permeability as well as much higher resistance to PDE-hydrolysis, as compared with 8-Br-cGMP. Modification of both 8-Br-cGMP and 8-pCPT-cGMP by introduction of a sulphur atom into the cyclic phosphate group gave smaller changes in agonist efficiency. Both Sp-8-Br-cGMPS and Sp-8-pCPT-cGMPS acted as agonists of CNG channels and are also G-kinase activators. In contrast, Rp-8-Br-cGMPS was a channel agonist, with an EC50 of 173.5 microM, but a G-kinase antagonist with a Ki of 4 microM. Finally, Rp-8-pCPT-cGMPS was a channel agonist and showed additional noncompetitive antagonist activity at higher concentrations. The results suggest that 8-pCPT-cGMPS is a highly potent photoreceptor CNG channel agonist with high membrane permeability and PDE-resistance and furthermore Rp-8-Br-cGMPS can be used to test whether the actions of cGMP are selectively mediated by CNG channels.

Fast-atom bombardment tandem mass spectrometry of cyclic nucleotide analogues used as site-selective activators of cyclic nucleotide-dependent protein kinases.

The mass spectrometric behaviour of six cyclic nucleotide analogues which activate cyclic AMP-dependent protein kinase was studied by positive-ion fast-atom bombardment (FAB) and collision-induced dissociation (CID) mass-analysed ion kinetic energy (MIKE) spectrometry. The compounds studied were 1,N6-ethenoadenosine-3',5'-cyclic monophosphate, (epsilon-cyclic AMP) and 2'-aza-1,N6-ethenoadenosine-3',5'-cyclic monophosphate, which each activate both isoforms of cyclic AMP-dependent protein kinase and have similar affinity for both the 'fast' and the 'slow' regulatory site of each isoform, N6-phenyl-cyclic AMP, which is selective for the 'fast' regulatory site of each isoform, and 6-chloropurine riboside-3',5'-cyclic monophosphate, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphate and 8-(4-chlorophenylthio)-adenosine-3',5'-cyclic monophosphate, which are each selective for the 'slow' regulatory site and preferentially activate isoform II. The FAB- and CID/MIKE spectra of the analogues are discussed in relation to their use in studies of the regulation of protein kinase activity by quantitative FAB mass spectrometry.

Endogenous type II cGMP-dependent protein kinase exists as a dimer in membranes and can Be functionally distinguished from the type I isoforms.

In mammalian tissues two types of cGMP-dependent protein kinase (cGK) have been identified. In contrast to the dimeric cGK I, cGK II purified from pig intestine was shown previously to behave as a monomer. However, recombinant rat cGK II was found to have hydrodynamic parameters indicative of a homodimer. Chemical cross-linking studies showed that pig cGK II in intestinal membranes has a dimeric structure as well. However, after purification, cGK II was found to be partly proteolyzed into C-terminal monomeric fragments. Phosphorylation studies in rat intestinal brush borders revealed that the potency of cGMP analogs to stimulate or inhibit native cGK II in vitro (i.e. 8-(4-chlorophenylthio)-cGMP > cGMP > beta-phenyl-1,N2-etheno-8-bromo-cGMP > beta-phenyl-1,N2-etheno-cGMP and Rp-8-(4-chlorophenylthio)-cGMPs > Rp-beta-phenyl-1, N2-etheno-8-bromo-cGMPs, respectively) correlated well with their potency to stimulate or inhibit cGK II-mediated Cl- secretion across intestinal epithelium but differed strikingly from their potency to affect cGK I activity. These data show that the N terminus of cGK II is involved in dimerization and that endogenous cGK II displays a distinct activation/inhibition profile with respect to cGMP analogs, which permits a pharmacological dissection between cGK II- and cGK I-mediated physiological processes.

Determination of lipophilicity by gradient elution high-performance liquid chromatography.

A novel method for the determination of lipophilicity using a simple HPLC protocol based on gradient elution chromatography is presented and compared to the common isocratic log k'(w) procedure. Linear relationships with high correlation coefficients between both methods for biologically active nucleosides and cyclic nucleotides as well as for environmentally relevant aromatic hydrocarbons were found. A mathematical fit to support the empirically determined linear relationship is presented. It is shown that the observed relationship between log k'(w) and the apparent capacity factor (k'(g)) determined by gradient elution is derivable by theoretical considerations as well. Since the gradient method is much less time-consuming compared to other procedures, it represents a convenient alternative for determining lipophilicity data in the future.

Identification of competitive antagonists of the rod photoreceptor cGMP-gated cation channel: beta-phenyl-1,N2-etheno-substituted cGMP analogues as probes of the cGMP-binding site.

cGMP is the natural activator of the cyclic nucleotide-gated channel originally isolated from rod photoreceptors but now known to be expressed in a wide variety of neural and non-neural cells. To identify antagonists of cGMP action and to better understand the interaction between cGMP and the channel protein, experimental studies were undertaken using four synthetic cGMP analogues, PET-cGMP, 8-Br-PET-cGMP, Rp-8-Br-PET-cGMPS, and Sp-8-Br-PET-cGMPS. With excised patches from either Xenopus oocytes expressing a cloned rat rod channel alpha-subunit or from native Xenopus rod photoreceptors, Rp-8-Br-PET-cGMPS competitively suppressed the cGMP-induced current with an IC50 of 25 microM and Sp-8-Br-PET-cGMPS inhibited this current with an IC50 of 105 microM. On the expressed rat rod channel, 8-Br-PET-cGMP behaved as a very weak partial agonist at high concentrations and an antagonist (IC50 = 64 microM) at lower concentrations when coapplied with cGMP. PET-cGMP did not activate channel currents alone but showed a synergism when coapplied with subsaturating concentrations of cGMP. Because Sp-8-Br-PET-cGMPS is a potent activator of type I cGMP-dependent protein kinase, but a competitive antagonist of channel activation, it will be a useful reagent for discriminating between those effects of cGMP that are mediated by a protein kinase and those mediated by channel activation. Because the PET derivatives all contain a phenyl-substituted 5-membered ring system fused to the amino group in position 2 and the nitrogen in position 1 of the guanine ring, the results support the idea that N1 and N2 are important for channel activation. They also suggest a minor role for the cyclic phosphate group in binding or activation.

Regulation of olfactory signalling via cGMP-dependent protein kinase.

Strong odor stimuli elicit a slow and sustained increase of the cGMP concentration in isolated rat olfactory cilia. Elevated cGMP levels appear to attenuate the primary response to odorant stimulation. Incubating cilia with membrane-permeable cGMP derivates caused a significantly reduced cAMP signal in response to odorant stimulation. This inhibitory effect was mimicked by 8-(4-chlorophenlythio)-cGMP, a selective activator of cGMP-activated protein kinases; in contrast, a selective inhibitor, [8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate] of cGMP kinases enhanced the reactivity to odorant stimulation. The data suggest that the responsiveness of olfactory sensory cells is governed by a cGMP-dependent protein kinase. Western-blot analysis using subtype-specific antibodies indicated that cytosolic type-I cGMP kinase, but not the membrane-associated type-II cGMP kinase, is expressed in olfactory sensory neurons.

Inhibition of cyclic GMP-dependent protein kinase-mediated effects by (Rp)-8-bromo-PET-cyclic GMPS.

1. The modulation of the guanosine 3':5'-cyclic monophosphate (cyclic GMP)- and adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase activities by the diastereomers of 8-bromo-beta phenyl-1, N2-ethenoguanosine 3':5'-cyclic monophosphorothioate, ((Rp)- and (Sp)-8-bromo-PET-cyclic GMPS) was investigated by use of purified protein kinases. In addition, the effects of (Rp)-8-bromo-PET-cyclic GMPS on protein phosphorylation in intact human platelets and on [3H]-noradrenaline release and neurogenic vasoconstriction in electrical field stimulated rat tail arteries were also studied. 2. Kinetic analysis with purified cyclic GMP-dependent protein kinase (PKG) type I alpha and I beta, which are expressed in the rat tail artery, revealed that (Rp)-8-bromo-PET-cyclic GMPS is a competitive inhibitor with an apparent Ki of 0.03 microM. The activation of purified cyclic AMP-dependent protein kinase (PKA) type II was antagonized with an apparent Ki of 10 microM. 3. In human platelets, (Rp)-8-bromo-PET-cyclic GMPS (0.1 mM) antagonized the activation of the PKG by the selective activator 8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphate (8-pCPT-cyclic GMP; 0.2 mM) without affecting the activation of PKA by (Sp)-5, 6-dichloro-1-beta-D-ribofurano-sylbenzimidazole- 3':5'-cyclic monophosphorothioate ((Sp)-5,6-DCl-cyclic BiMPS; 0.1 mM). 4. (Rp)-8-bromo-PET-cyclic GMPS was not hydrolysed by the cyclic GMP specific phosphodiesterase (PDE) type V from bovine aorta but potently inhibited this PDE. 5. The corresponding sulphur free cyclic nucleotide of the two studied phosphorothioate derivatives, 8-bromo-beta-phenyl-1, N2-ethenoguanosine-3':5'-cyclic monophosphate (8-bromo-PET-cyclic GMP), had no effect on electrically-induced [3H]-noradrenaline release but concentration-dependently decreased the stimulation-induced vasoconstriction. (Rp)-8-bromo-PET-cyclic GMPS (3 microM) shifted the vasoconstriction response to the right without affecting stimulation evoked tritium overflow. 6. The NO donor, 3-morpholinosydnonimine (SIN-1) relaxed rat tail arteries precontracted with phenylephrine (1 microM). The SIN-1 concentration-relaxation curve was shifted in a parallel manner to the right by (Rp)-8-bromo-PET-cyclic GMPS, suggesting that the relaxation was mediated by a cyclic GMP/PKG-dependent mechanism. 7. The [3H]-noradrenaline release-enhancing effect and stimulation-induced decrease in vasoconstriction of forskolin were unaffected by (Rp)-8-bromo-PET-cyclic GMPS. Moreover, the forskolin concentration-relaxation curve was not changed in the presence of the PKG inhibitor, suggesting a high selectivity in intact cells for PKG- over PKA-mediated effects. 8. The results obtained indicate that (Rp)-8-bromo-PET-cyclic GMPS presently is the most potent and selective inhibitor of PKG and is helpful in distinguishing between cyclic GMP and cyclic AMP messenger pathways activation. Therefore, this phosphorothioate stereomer may be a useful tool for studying the role of cyclic GMP in vitro.

Novel (Rp)-cAMPS analogs as tools for inhibition of cAMP-kinase in cell culture. Basal cAMP-kinase activity modulates interleukin-1 beta action.

Novel (Rp)-cAMPS analogs differed widely in ability to antagonize cAMP activation of pure cAMP-dependent protein kinase I and II and to antagonize actions of cAMP on gene expression, shape change, apoptosis, DNA replication, and protein phosphorylation in intact cells. These differences were related to different abilities of the analogs to stabilize the holoenzyme form relative to the dissociated form of cAMP kinase type I and II. (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS were the most potent cAMP antagonists for isolated type I kinase and for cells expressing mostly type I kinase, like IPC-81 leukemia cells, fibroblasts transfected with type I regulatory subunit (RI), and primary hepatocytes. It is proposed that (Rp)-8-Br-cAMPS or (Rp)-8-Cl-cAMPS should replace (Rp)-cAMPS as the first line cAMP antagonist, particularly for studies in cells expressing predominantly type I kinase. The phosphorylation of endogenous hepatocyte proteins was affected oppositely by (Rp)-8-Br-cAMPS and increased cAMP, indicating that (Rp)-8-Br-cAMPS inhibited basal cAMP-kinase activity. The inhibition of basal kinase activity was accompanied by enhanced DNA replication, an effect which could be reproduced by microinjected mutant cAMP-subresponsive RI. It is concluded that the basal cAMP-kinase activity exerts a tonic inhibition of hepatocyte replication. (Rp)-8-Br-cAMPS and microinjected RI also desensitized hepatocytes toward inhibition of DNA synthesis by interleukin-1 beta. This indicates that basal cAMP-kinase activity can have a permissive role for the action of another (interleukin-1 beta) signaling pathway.

(Rp)-8-pCPT-cGMPS, a novel cGMP-dependent protein kinase inhibitor.

In the present study, the inhibitory effect of the cGMP analog (Rp)-8-(para-chlorophenylthio)guanosine-3',5'-cyclic monophosphorothioate ((Rp)-8-pCPT-cGMPS) on the cGMP-dependent protein kinase-mediated protein phosphorylation in intact human platelets was investigated. In vitro phosphorylation experiments with the substrate kemptide demonstrated an inhibition of the cGMP-dependent protein kinase by (Rp)-8-pCPT-cGMPS with a Ki of 0.5 microM. In intact human platelets, (Rp)-8-pCPT-cGMPS antagonized the activation of the cGMP-dependent protein kinase by 8-pCPT-cGMP without affecting cAMP-dependent protein kinase or cGMP-regulated phosphodiesterases. The data obtained suggest that (Rp)-8-pCPT-cGMPS may be a useful tool for studying the role of cGMP in vitro and in intact cells.

Membrane-permeant derivatives of cyclic AMP optimized for high potency, prolonged activity, or rapid reversibility.

A novel membrane-permeant derivative of cAMP, cAMP acetoxymethyl ester (cAMP/AM), was synthesized via silylated intermediates. Its ability to induce Cl- secretion by T84 cells, a human colon cancer cell line, was compared with that of two other membrane-permeant cAMP derivatives that were recently introduced, N6,O2'-dibutyryl-cAMP acetoxymethyl ester (bt2cAMP/AM) and Sp-5,6-dichlorobenzimidazole-1-beta-D-ribofuranoside 3',5'-cyclic phosphorothioate (Sp-5,6-DCl-cBIMPS). All of these compounds are powerful activators of Cl- secretion when applied extracellularly, with EC50 values of 60 microM, 0.7 microM, and 3 microM, respectively. However, cAMP/AM was expected to be readily degraded inside cells, in contrast to the cyclophosphodiesterase-resistant Sp-5,6-DCI-cBIMPS or the only slowly metabolizable N6-butyryl-cAMP derived from bt2cAMP/AM. Reversibility of cAMP/AM action was demonstrated by wash-out experiments; Cl- secretion induced by high doses of cAMP/AM (100 microM) could be quickly abolished by rinsing of the cells, whereas similar experiments with bt2cAMP/AM and Sp-5,6-DCI-cBIMPS showed much slower decreases. Even more sensitive to residual cAMP derivatives was the synergistic effect of carbachol, which was applied after the incubation with membrane-permeant derivatives and their subsequent wash-out. Although doses of cAMP derivatives that barely activated Cl- secretion were readily capable of inducing a synergistic response with carbachol, cells incubated with high doses of cAMP/AM (100 microM) and subsequently washed showed only a nonsynergistic carbachol response, in contrast to cells incubated with bt2cAMP/AM or Sp-5,6-DCI-cBIMPS. We therefore characterize cAMP/AM as a membrane-permeant derivative of cAMP that is easily metabolizable inside cells and hence is most useful for applications where a transient intracellular cAMP signal is desired. In contrast, completely nonmetabolizable Sp-5,6-DCI-cBIMPS seems to be more useful in longer incubations that require steady levels of cAMP-dependent protein kinase activation. bt2cAMP/AM combines the advantages of intracellular trapping by ester hydrolysis and reduced cyclophosphodiesterase sensitivity of its active intracellular product, which probably lead to its particularly high potency.

Two distinctly regulated events, priming and triggering, during retinoid-induced maturation and resistance of NB4 promyelocytic leukemia cell line.

In t(15;17) acute promyelocytic leukemia, all-trans retinoic acid (RA) induces leukemic cell maturation in vitro and remission in acute promyelocytic leukemia patients, but in vivo treatments invariably lead to relapse with resistance to RA. NB4, a maturation-inducible cell line, and NB4-RAr sublines (R1 and R2) displaying no maturation in the presence of RA have been isolated from a patient in relapse. We show that resistance to maturation is not a mere unresponsiveness to RA: rather, R1 "resistant" cells do respond to RA (1 microM) by sustained growth, become competent to undergo terminal maturation, and up-regulate CD11c/CD18 integrins. Interestingly, maturation of "resistant" cells, rendered competent by RA, can be achieved by cAMP-elevating agents (prostaglandin E, isoproterenol, cholera toxin, or phosphodiesterase inhibitor) or stable agonistic cAMP analogs such as (SP)-8-chloroadenosine cyclic 3',5'-phosphorothioate. This shows that activation of cAMP-dependent protein kinase (cA kinase) can override the RA resistance and suggests interdependent RA and cAMP signaling pathways in acute promyelocytic leukemia maturation. No such cooperation was observed in the R2 resistant cells, though their cA-kinase was functional. (RP)-8-Chloroadenosine cyclic 3',5'-phosphorothioate, which by displacing endogenous cAMP inhibits the basal cA-kinase activity, decreased the response of sensitive cells to RA. This raises the possibility that cA-kinase plays a key role in the maturation also of RA-sensitive cells. Our results define two discrete steps in the maturation process: an RA-dependent priming step that maintains proliferation while cells become competent to undergo maturation in response to retinoids and a cAMP-dependent step that triggers RA-primed cells to undergo terminal maturation. Uncoupling RA and cAMP action might cause the so-called "resistance."