8-Substituted derivatives of adenosine 3',5'-cyclic phosphate require an unsubstituted 2'-hydroxyl group in the ribo configuration for biological activity.

Derivatives of adenosine 3',5'-cyclic phosphate (cAMP) with modifications in both the 2' and the 8 positions were synthesized and their enzymic activities as activators of cAMP-dependent protein kinase and as substrates for and inhibitors of cAMP phosphodiesterases were determined. Three types of derivatives were investigated: 8-substituted derivatives of O2'-Bt-cAMP, 8-substituted derivatives of 9-beta-D-arabinofuranosyladenine 3',5'-cyclic phosphate (ara-cAMP), and 8-substituted derivatives of 8,2'-anhydro-9-beta-D-arabinofuranosyladenine 3,'5'-cyclic phosphate (8,2'-anhydro-cAMP). The 8-substituted O2'-Bt-cAMP derivatives were synthesized by acylation of the preformed 8-substituted cAMP (8-HS-cAMP, 8-MeS-cAMP, and 8-PhCH2S-cAMP). 8-Br-O2'-tosyl-cAMP was sued as an intermediate for the preparation of 8,2'-anhydro-cAMP derivatives (8-HO-, 8-SH-, 8-H2N-, and 8-H3 CHN derivatives of 8,2'-anhydro-cAMP). 8-Substituted ara-cAMP derivatives were obtained by ring opening of 8-HO-8,2'-anhydro-cAMP with H+/H2O, NH3/MeOH, or MeONa/MeOH (to yield the 8-HO-, 8-H2N-, and 8-MeO-ara-cAMP derivatives). All of these doubly modified derivatives of cAMP are less than one-hundredth as active as cAMP at activating protein kinase and did not serve as substrates for the phosphodiesterase. These data show that the general inactivity of 2' derivatives of cAMP with kinase was not overcome by addition of an 8-substituent, even though many 8-substituted derivatives of cAMP activate the kinase more efficiently than does cAMP itself. In addition they show that while 2'-modification were tolerated by the phosphodiesterase, addition of an 8-substituent countermanded the allowable 2'-modification. The 8-substituted derivates of 02'-Bt-cAMP were found in general to be slightly better inhibitors of phosphodiesterase than the parent compounds containing no o2'-Bt substitution. As a group, the 8-substituted ara-cAMP derivatives were poorer inhibitors of phosphodiesterase than 8-substituted cAMP derivatives while the 8,2'-anhydro-cAMP derivatives were much poorer inhibitors than the 8-substituted ara-cAMP derivatives.     

Three new potential cAMP affinity labels. Inactivation of human platelet low Km cAMP phosphodiesterase by 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate

Three new analogues of cAMP have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (2-BDB-TcAMP), 2-[(3-bromo-2-oxopropyl)thio]-adenosine 3',5'-cyclic monophosphate (2-BOP-tcAMP), and 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP). The bromoketo moiety has the ability to react with the nucleophilic side chains of several amino acids, while the dioxobutyl group can interact with arginine. These cAMP analogues were tested for their ability to inactivate the low Km (high affinity) cAMP phosphodiesterase from human platelets. The 2-BDB-TcAMP and 2-BOP-TcAMP were competitive inhibitors of cAMP hydrolysis by the phosphodiesterase with Ki values of 0.96 +/- 0.12 and 0.70 +/- 0.12 microM, respectively. However, 2-BDB-TcAMP and 2-BOP-TcAMP did not irreversibly inactivate the phosphodiesterase at pH values from 6.0 to 7.5 and at concentrations up to 10 mM. These results indicate that although the 2-substituted TcAMP analogues bind to the enzyme, there are no reactive amino acids in the vicinity of the 2-position of the cAMP binding site. In contrast, incubation of the platelet low Km cAMP phosphodiesterase with 8-BDB-TcAMP resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.031 +/- 0.009 min-1 mM1. Addition of the substrates, cAMP and cGMP, and the product, AMP, to the reaction mixture resulted in marked decreases in the inactivation rate, suggesting that the inactivation was due to reaction at the active site of the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences and similarities between guanosine 3',5'-cyclic monophosphate phosphodiesterase and adenosine 3',5'-cyclic monophosphate phosphodiesterase activities in neuroblastoma cells in culture.

There are phosphodiesterase activities in both particulate and supernatant fractions which hydrolyze guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) with an apparent Km of 2-8 muM and with an apparent Km of 44-222 muM. 4-(3-Butoxy-4-methoxybenzyl-2-imidazolidinone (RO20-1724) did not inhibit cGMP phosphodiesterase activity in homogenates of mouse neuroblastoma cells, but markedly inhibited cAMP phosphodiesterase activity. Papaverine and theophylline inhibited both cGMP and cAMP phosphodiesterase activities to about the same extent. The former was more potent than the latter. The specific activity of cGMP phosphodiesterase as a function of protein concentrations first increased and then decreased. The specific activity of cAMP phosphodiesterase decreased under a similar experimental condition.     

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.     

2-substituted derivatives of adenosine and inosine cyclic 3',5'-phosphate. Synthesis, enzymic activity, and analysis of the structural requirements of the binding locale of the 2-substituent on bovine brain protein kinase.

A number of 2-substituted cyclic nucleotide derivatives were synthesized and investigated as activators of cAMP-dependent protein kinase and as substrates for and inhibitors of cAMP phosphodiesterase. Ring closure of 5-amino-1-beta-D-ribofuranosylimidazol-4-carboxamide cyclic 3',5'-phosphate (1) with various aldehydes according to a new procedure (Meyer, R. B., Jr., Shuman, D.A., and Robins, R. K. (1974), J. Am. Chem. Soc. 96, 4962) gave new derivatives of adenosine cyclic 3',5'-phosphate with the following 2-substituents: n-propyl, n-hexl, n-octyl, n-decyl, styryl, o-methoxyphenyl, and 2-thienyl. Alkylation of 2-mercaptoadenosine cyclic 3',5'-phosphate (20, Meyer et al., 1974) gave new cAMP derivatives with the following 2-substituent: ethylthio, n-propylthio, isopropylthio, allylthio, n-decylthio, and benzylthio. Deamination of 2-methyl-,2-n-butyl-, and 2-ethylthioadenosine cyclic 3',5'-phosphate. Using multiple regression analysis, a striking relationship was found between the relative potency of the compounds as activators of bovine brain cAMP-dependent protein kinase and parameters describing the hydrophobic, steric, and electronic character of the substituents on these compounds. All compounds were substrates for a cyclic nucleotide phosphodiesterase preparation from rabbit kidney. Additionally, the compounds were as a group, good inhibitors of the hydrolysis of cAMP by phosphodiesterase preparations from rabbit lung, beef heart, and dog heart.     

Effects of 8-substituted analogs of cyclic adenosine 3'',5''-monophosphate on in vivo and in vitro syntheses of beta-galactosidase in Escherichia coli.

Several 8-substituted alkylthio and alkylamino cyclic adenosine 3',5'-monophosphate (cAMP) derivatives were tested for their ability to stimulate beta-galactosidase synthesis in Estherichia coli in vivo and in vitro and to inhibit the cAMP phosphodiesterase activity of E. coli. Stimulation of beta-galactosidease synthesis in vivo by cAMP derivatives decreased with increasing length of the unbranched carbon chain of the substituent. On the other hand, the stimulation in vitro was increased as the carbon chain elongated. The 8-decylthio- and 8-dodecylthio-cAMP compounds stimulated beta-galactosidase synthesis almost eight-fold compared with cAMP, whereas 8-undecyl-, 8-dodectyl-, and 8-tridecylamino-cAMP stimulated beta-galactosidase synthesis about threefold. However, in in vitro experiments with a phosphodiesterase-deficient strain of E. coli, the Crooks strain, the stimulatory effects of the derivatives disappeared, except for 8-dodecylthio cAMP which stimulated beta-galactosidase about 1.4- to 1.6-fold. All derivatives were quite resistant to hydrolysis by phosphodiesterase. Most derivatives competitively inhibited the hydrolysis of cAMP by phosphodiesterase.     

cGMP-mediated inhibition of cardiac L-type Ca(2+) current by a monoclonal antibody against the M(2) ACh receptor

The effects of a monoclonal antibody (B8E5) directed against the second extracellular loop of the muscarinic M(2) receptor were studied on the L-type Ca(2+) currents (I(Ca,L)) of guinea pig ventricular myocytes using the whole cell patch-clamp technique. Similar to carbachol, B8E5 reduced the isoproterenol (ISO)-stimulated I(Ca,L) but did not significantly affect basal I(Ca,L). Atropine blocked the inhibitory effect of B8E5. The electrophysiological parameters of ISO-stimulated I(Ca,L) were not modified in presence of B8E5. Inhibition of I(Ca,L) by B8E5 was still observed when intracellular cAMP was either enhanced by forskolin or maintained constant by using a hydrolysis-resistant cAMP analog (8-bromoadenosine 3',5'-cyclic monophosphate) or by applying the phosphodiesterase inhibitor IBMX. The effect of B8E5 was mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate, a potent stimulator of cGMP-dependent protein kinase, and prevented by a selective inhibitor of nitric oxide-sensitive guanylyl cyclase [1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one]. These results indicate that the antibody B8E5 inhibits the beta-adrenergic-stimulated I(Ca,L) through activation of the M(2) muscarinic receptor and further suggest that the antibody acts not via the classical pathway of decreasing intracellular cAMP, but rather by increasing cGMP.     

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.     

Binding of adenosine 3',5'-monophosphate dependent protein kinase regulatory subunit to immobilized cyclic nucleotide derivatives.

Several cyclic nucleotide derivatives with aminoalkyl side chains attached to the purine ring were synthesized and their interactions with adenosine 3',5'-monophosphate (cAMP) dependent protein kinase were studied before and after immobilization to CNBr-activated Sepharose 4B. The soluble N6-substituted derivatives were as effective as cAMP itself in activating protein kinase and were more effective than 8-substituted cAMP derivatives, whereas the 2-substituted cAMP derivatives and the cGMP derivatives were the least effective. All of the synthetic derivatives tested were poor substrates for beef heart phosphodiesterase being hydrolyzed at rates less than 2% for that of cAMP itself. Utilizing methodology developed to evaluate the affinity of protein kinase for immogilized cyclic nucleotides it was found that all of the immobilized cyclic nucleotides interacted with protein kinase in a biospecific manner as judged by the following criteria: (1) the immobilized cyclic nucleotides competed with cAMP for the binding sites on protein kinase; (2) the analogous spacer-arm did not compete; and (3) the effects of enzyme concentration, MgATP, and cleavage of the cyclic phosphate ring on the interactions of protein kinase with the immobilized cyclic nucleotides were the same as previously shown for free cAMP. In addition, the immobilized ligands were bound with the same order of effectiveness as the analogous soluble ligand. The observed Ka for the activation of 0.005 muM protein kinase by N6-H2N(CH2)2-cAMP was increased from 0.23 to 3 muM by the process of immobilization. This increase was unaffected by the coupling density and spacer-arm length. The observed Kb for 0.10 muM protein kinase binding to immobilized N6-H2N(CH2)2-cAMP was increased as the molecular sieving exclusion limit of the matrix used was decreased indicating that at least part of this decrease in apparent affinity upon immobilization is due to exclusion of the enzyme from a portion of the matrix and therefore of the immobilized ligand molecules.     

PKA from Saccharomyces cerevisiae can be activated by cyclic AMP and cyclic GMP.

Analysis of Saccharomyces cerevisiae genome revealed no sequence homologous to cyclic GMP (cGMP) dependent protein kinase from other organisms. Here we demonstrate that cyclic AMP (cAMP) dependent protein kinase purified from S. cerevisiae was almost equally activated by cAMP and cGMP in 3 x 10(-6) M concentrations of either nucleotide in the presence of Mg2+ ions. Interestingly, if Mn2+ ions were used instead of Mg2+, cGMP was only 30% as effective as cAMP in the activation of cAMP-dependent protein kinase. Analogs of cAMP such as 8-chloro-cAMP and 3':5'-cyclic monophosphate of ribofuranosylbenzimidazole were as potent as cAMP in the enzyme activation, while N6,2'-O-dibutyryl-cAMP activated the enzyme to a lower extent. It was also found that yeast cAMP-dependent protein kinase can be activated by limited proteolytic digestion. The results presented were obtained with protamine and ribosomal protein S10 used as phosphorylation substrates.     

Mapping adenosine cyclic 3',5'-phosphate binding sites on type I and type II adenosine cyclic 3',5'-phosphate dependent protein kinases using ribose ring and cyclic phosphate ring analogues of adenosine cyclic 3',5'-phosphate

A series of adenosine cyclic 3',5'-phosphate (cAMP) derivatives containing modifications or substitutions in either the 2',3',4', or 5' position or the phosphate were examined for their abilities to activate type I isozymes of cAMP-dependent protein kinase (PK I) from rabbit or porcine skeletal muscle and type II isozymes of cAMP-dependent protein kinase (PK II) from bovine brain and heart. The studies revealed that the activation of both PK I and PK II isozymes requires a 2'-hydroxyl group in the ribo configuration, a 3' oxygen in the ribo configuration, and a charged cyclic phosphate. The two isozymes appeared to differ in those portions of their respective cAMP-binding sites that are adjacent to the 4' position of the ribose ring and the 3' position, 5' position, and phosphate portion of the cyclic phosphate ring.     

Increased cyclic GMP levels lead to a stimulation of elastin production in ligament fibroblasts that is reversed by cyclic AMP

The effects of cyclic nucleotides on elastin synthesis were studied in ligamentum nuchae fibroblasts by adding exogenous cyclic nucleotide derivatives or beta-adrenergic agents to cell culture medium. Elastin synthesis was enhanced (approximately 80%) by dibutyryl cGMP (Bt2cGMP) in concentrations ranging from 0.01 to 100 nM. Two other cGMP derivatives, 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP) and 2'-deoxy-cGMP, were also potent stimulators of elastin synthesis. In the absence of calcium, basal elastin production was substantially decreased (40% of control) and cGMP analogs no longer stimulated elastin synthesis, suggesting a role for calcium in the cGMP response. Bt2cAMP had no demonstrable effect on elastin production except at high concentrations which produced a nonspecific decrease equivalent to the decrease in total protein synthesis. Similarly, elevation of endogenous cellular cAMP levels by beta-adrenergic stimulation produced no change in elastin production. When 8-Br-cGMP was added to cells together with Bt2cAMP, cGMP-dependent stimulation of elastin production was abolished by cAMP in a dose-dependent fashion. These results suggest a coordinated means by which elastin production is controlled in ligament cells, i.e. increased cGMP levels lead to a stimulation of elastin production that is reversed by cAMP.     

Protein S-guanylation by the biological signal 8-nitroguanosine 3',5'-cyclic monophosphate

The signaling pathway of nitric oxide (NO) depends mainly on guanosine 3',5'-cyclic monophosphate (cGMP). Here we report the formation and chemical biology of a nitrated derivative of cGMP, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), in NO-mediated signal transduction. Immunocytochemistry demonstrated marked 8-nitro-cGMP production in various cultured cells in an NO-dependent manner. This finding was confirmed by HPLC plus electrochemical detection and tandem mass spectrometry. 8-Nitro-cGMP activated cGMP-dependent protein kinase and showed unique redox-active properties independent of cGMP activity. Formation of protein Cys-cGMP adducts by 8-nitro-cGMP was identified as a new post-translational modification, which we call protein S-guanylation. 8-Nitro-cGMP seems to regulate the redox-sensor signaling protein Keap1, via S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1. This study reveals 8-nitro-cGMP to be a second messenger of NO and sheds light on new areas of the physiology and chemical biology of signal transduction by NO.     

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.     

Direct regulation of smooth muscle contractile elements by second messengers

The effects of adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP) and phorbol 12,13 dibutyrate (PDBu) on the Ca2+ sensitivity of the contractile elements in the rat mesenteric artery were investigated, using a method of permeabilizing smooth muscle with Staphylococcal alpha-toxin. Both cAMP and cGMP relaxed the permeabilized rat mesenteric artery at the intracellular Ca2+ concentrations [( Ca2+]i) held constant with Ca2+ EGTA buffer and Ca2+ ionophore, ionomycin. In addition, forskolin and sodium nitroprusside which activate adenylate and guanylate cyclases, respectively, also induced relaxation at a fixed [Ca2+]i. In contrast PDBu which stimulates protein kinase C caused an increase in force at a constant [Ca2+]i which could be partially reversed by cAMP or cGMP. These results indicate that second messengers exert direct control over smooth muscle Ca2+ sensitivity of the contractile elements, which is of physiologic and pharmacologic importance.     

Cerebellar cAMP levels following acute and chronic morphine administration.

Acute administration of morphine sulfate at 20 mg/kg decreased mouse cerebellar adenosine 3',5'-cyclic phosphate (cAMP) levels while not affecting cAMP phosphodiesterase (EC 3.1.4.17). The cAMP levels and cAMP phosphodiesterase activies were not affected by chronic treatment. However, cAMP levels increased during abrupt withdrawal both with and without naloxone precipitation, with cAMP phosphodiesterase activities being correspondingly decreased. Propanolol prevented the cAMP increase during abrupt withdrawal.     

Mechanisms by which endothelin 1 induces pulmonary vasoconstriction in the rabbit.

To investigate the mechanisms by which endothelin 1 (ET-1) causes pulmonary vasoconstriction, we studied the effect of synthetic ET-1 on pulmonary vascular tone in the buffer-perfused isolated rabbit lung. In nanomolar concentrations (1.2-8 nM), ET-1 causes a dose-dependent increase in pulmonary arterial pressure that persists for greater than or equal to 1 h (increase in pressure 19 +/- 2 mmHg with ET-1 vs. 2 +/- 1 with vehicle, P less than 0.0001). Reduction of calcium availability with verapamil, cadmium, or a calcium-free buffer significantly blunts the increase in pressure caused by ET-1. Pretreatment with a calcium-free buffer plus the chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid (EGTA) completely eliminates the vasoconstriction. Three different inhibitors of protein kinase C, phloretin, staurosporine, and dihydrosphingosine, significantly diminish the response to ET-1. Indomethacin and a thromboxane synthase inhibitor partially decrease the response to the highest concentration of ET-1. Isoproterenol and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) are significantly more effective in preventing the vasoconstriction caused by ET-1 than are nitroprusside and guanosine 5'-cyclic monophosphate (cGMP) analogues. ET-1 in doses of 1.2-8 nM is a potent pulmonary vasoconstrictor in the isolated rabbit lung. ET-1 appears to cause pulmonary vasoconstriction by increasing calcium entry and by activating protein kinase C. Vasodilators that increase cAMP are substantially more effective in preventing the increase in pressure than are drugs that increase cGMP.     

A nonhydrolyzable reactive cAMP analogue, (S(p))-8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic S-(methyl)monophosphorothioate, irreversibly inactivates human platelet cGMP-inhibited cAMP phosphodiesterase at micromolar concentrations

We previously showed that 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate inactivates cAMP phosphodiesterase (PDE3A); however, millimolar concentrations were needed to inactivate PDE3A because of ongoing hydrolysis. We have now synthesized a nonhydrolyzable reactive cAMP analogue, (S(p))-8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic S-(methyl)monophosphorothioate (S(p)-8-BDB-TcAMPSMe). S(p)-8-BDB-TcAMPSMe inactivates PDE3A in a time-dependent, irreversible manner, exhibiting saturation kinetics with a k(max) of (19.5 +/- 0.3) x 10(-3) min(-1) and a K(I) of 3.5 +/- 0.3 muM. To ascertain whether S(p)-8-BDB-TcAMPSMe reacts in the active site, nonhydrolyzable analogues of the substrate cAMP, or the competitive inhibitor cGMP, were included to protect against the inactivation of PDE3A. The order of effectiveness of protectants in decreasing the rate of inactivation (with K(d) values in micromolar) is as follows: S(p)-cAMPS (18) > R(p)-cGMPS (560) and S(p)-cGMPS (1260) > 5'-AMP (17 660), R(p)-cAMPS (30 110), and 5'-GMP (42 170). We docked S(p)-8-BDB-TcAMPSMe into PDE3A, based on the structural model of PDE3A-cAMP and the kinetic data from site-directed mutants. The S(p)-8-BDB-TcAMPSMe fits into the active site in the model. These results suggest that inactivation of PDE3A by the affinity reagent is a consequence of reaction at the overlap between cAMP and cGMP binding regions in the active site. S(p)-8-BDB-TcAMPSMe has proven to be an effective active site-directed irreversible cAMP affinity label for platelet PDE3A and can be used to identify amino acids in the active site of PDE3A as well as in other cAMP phosphodiesterases.     

Studies of cGMP analog specificity and function of the two intrasubunit binding sites of cGMP-dependent protein kinase

The specificity of the two intrasubunit cGMP binding sites of cGMP-dependent protein kinase was determined by measuring the ability of 46 cGMP analogs to compete with [3H]cGMP. Both sites of the enzyme exhibited high specificity for the ribose cyclic phosphate moiety, and lower specificity for the guanine moiety. Effects of modifications in the ribose cyclic phosphate moiety suggested that cGMP is bound at both sites by three hydrogen bonds at 2'-OH, 3'-O, and 5'-O. A negative charge in the cyclic phosphate is apparently required. Modifications of the pyrimidine part of guanine, particularly at C-1, generally caused selectivity for the rapidly exchanging site while modifications of the imidazole part of guanine at C-7 and C-8 caused selectivity for the slowly exchanging site. These increases in selectivity for a site were mainly due to losses in affinity of the other site. There was an apparent requirement of the intact amino group at C-2, particularly for the slowly exchanging site. Comparison of the molecular interactions of cAMP and cGMP with their specific protein kinases showed that both nucleotides are bound by similar forces in the 2', 3' and 5' region, both bases may be bound in syn conformation, but that each base moiety is bound by different molecular interaction, thus leading to the selectivity of the two enzymes. cGMP analogs which possessed strong selectivity for the rapidly exchanging site, but not those selective for the slowly exchanging site, stimulated the binding of [3H]cGMP. Only a few cGMP analogs were more potent than cGMP in stimulating protein kinase activity. The potency of cGMP analogs as stimulators of kinase activity correlated better with the mean binding affinity for both binding sites than with the affinity for either site alone. Two analogs added in combination were synergistic in kinase activation, particularly if one analog was selective for the slowly exchanging site and the other for the rapidly exchanging site. These observations are suggestive that cGMP binding at the rapidly exchanging site stimulates cGMP binding at the slowly exchanging site and that both sites are involved in the activation process.     

PKA-dependent activation of PDE3A and PDE4 and inhibition of adenylyl cyclase V/VI in smooth muscle

Regulation of adenylyl cyclase type V/VI and cAMP-specific, cGMP-inhibited phosphodiesterase (PDE) 3 and cAMP-specific PDE4 by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) was examined in gastric smooth muscle cells. Expression of PDE3A but not PDE3B was demonstrated by RT-PCR and Western blot. Basal PDE3 and PDE4 activities were present in a ratio of 2:1. Forskolin, isoproterenol, and the PKA activator 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole 3',5'-cyclic monophosphate, SP-isomer, stimulated PDE3A phosphorylation and both PDE3A and PDE4 activities. Phosphorylation of PDE3A and activation of PDE3A and PDE4 were blocked by the PKA inhibitors [protein kinase inhibitor (PKI) and H-89] but not by the PKG inhibitor (KT-5823). Sodium nitroprusside inhibited PDE3 activity and augmented forskolin- and isoproterenol-stimulated cAMP levels; PDE3 inhibition was reversed by blockade of cGMP synthesis. Forskolin stimulated adenylyl cyclase phosphorylation and activity; PKI blocked phosphorylation and enhanced activity. Stimulation of cAMP and inhibition of inositol 1,4,5-trisphosphate-induced Ca(2+) release and muscle contraction by isoproterenol were augmented additively by PDE3 and PDE4 inhibitors. The results indicate that PKA regulates cAMP levels in smooth muscle via stimulatory phosphorylation of PDE3A and PDE4 and inhibitory phosphorylation of adenylyl cyclase type V/VI. Concurrent generation of cGMP inhibits PDE3 activity and augments cAMP levels.