2',5'-Oligoadenylate trimer core and the cordycepin analog augment the tumoricidal activity of human natural killer cells

Interferon (IFN) augments the lytic activity of natural killer (NK) cells, inhibits the transformation of human peripheral blood lymphocytes (PBL) by Epstein Barr virus (EBV), and induces a 2',5'-oligoadenylate (2',5'-An) synthetase. Exogenous 2',5'-An by itself can inhibit the transformation of human PBL by EBV. The present studies report that 2',5'-An and its cordycepin analog also augmented the tumoricidal activity of human NK cells. Incubation of nylon wool-passed PBL for 1 to 2 hr with the 5'-dephosphorylated core trimer of 2',5'-An boosted natural killing of tumor target cells modestly, but consistently. The cordycepin analog (3'-deoxyadenylate) also augmented NK activity. The optimal concentration both of 2',5'-A3 core and of 2',5'-3'dA3 core was 50 microM, and the optimal time for this effect was 2 hr of treatment. Kinetic analysis revealed that 2',5'-A3 core increased the lytic rate of NK cells by about one-third. This increase was due to an even greater increase (about 50%) in the lytic activity of individual NK cells, coupled with a slight decrease in the number of actual NK effector cells. In contrast, 3',5'-A3 core did not increase NK activity even at 300 microM, at which point it was toxic. In addition, to rule out a pro-drug effect as the basis for the boosting of NK activity by 2',5'-A3 core and by 2',5'-3'dA3 core, the effect of adenosine and cordycepin monomers on NK activity was tested. Neither adenosine nor cordycepin, tested at 150 microM (three times the optimal concentration of the trimer cores), boosted NK activity. The addition of 2'-deoxycoformycin (2 microM) had no effect on the actions of adenosine and cordycepin monomers. The data presented here demonstrate that 2',5'-A3 core and its analog 2',5'-3'dA3 core have another IFN-like action, augmentation of NK activity, in addition to inhibiting EBV-induced transformation.     

Synthesis, lipophilicity and anti-HIV activity of a new brominated analog of zidovudine.

A novel cyclic bromine zidovudine analog, (-)-trans-(5S,6S)-5-bromo-6,5'- epoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (2), and its diastereoisomer (+)-trans-(5R,6R)-(3) were synthesized and characterized by spectroscopic methods, obtaining 3 in very low yields. The major product 2 presents a selectivity index (CCID50/IC50) similar to zidovudine but 55.5 times with higher lipophilicity, which should increase the ability of 2 to cross the blood-brain barrier by a non facilitated diffusion mechanism.     

Synthesis and in vitro antileishmanial activity of 5-substituted-2'-deoxyuridine derivatives

We report herein the synthesis and the in vitro antileishmanial evaluation of 5-substituted-2'-deoxyuridine nucleosides. The most active compound against Leishmania donovani promastigotes was Thia-dU (3a) with an IC50 =3 microM. This compound exhibited the same activity as zidovudine (3'-azido-2'-deoxythymidine) used as nucleoside reference compound. Considering the cytotoxicity of synthetic compounds on peritoneal murine macrophages, the most toxic compound was MeThio-dU (3d) with a MTC at 10 microM. Only Methia-dU (3b) was active against intramacrophagic amastigotes with an IC50 =6.5 microM. This latter can now be evaluated in vivo, for further investigations through structure-based drug design.     

Adriamycin disrupts phosphatidylserine import into the mitochondria of permeabilized CHO-K1 cells

The action of adriamycin (an inhibitor of precursor protein import into mitochondria) upon phosphatidylserine (PtdSer) import into mitochondria was examined in permeabilized CHO-K1 cells. The decarboxylation of nascent PtdSer to phosphatidylethanolamine was used as an indicator reaction for the lipid translocation process. Adriamycin was without effect upon new PtdSer synthesis but blocked the time- and translocation-dependent decarboxylation of this lipid at the mitochondrial inner membrane of permeabilized cells. The effect of adriamycin was concentration-dependent with an IC50 of 150 microM and was not due to direct inhibition of PtdSer decarboxylase. To determine at which level of PtdSer transport adriamycin was working, the adriamycin-treated permeabilized cells were incubated with 1-acyl-2-[N-(6-[(7-nitrobenz-2-oxa-1,3-diazo-4-yl)] aminocaproyl)]phosphatidyl[1'-14C] serine (NBD-Ptd[1'-14C]Ser), and its decarboxylation was determined. Since the NBD-Ptd[1'-14C]Ser freely partitions into all cell membranes, it can partition into the outer mitochondrial membrane in an ATP-independent fashion. The NBD-Ptd[1'-14C]Ser was readily decarboxylated in an ATP-independent manner in permeabilized cells. Adriamycin inhibited the decarboxylation of NBD-Ptd[1'-14C]Ser, thereby indicating that it can act upon lipid transport processes between the outer and inner mitochondrial membrane.     

Intracellular metabolism of beta-L-ddAMP-bis(tbutylSATE), a potent inhibitor of hepatitis B virus replication.

beta-L-ddAMP-bis(tbutylSATE) is a potent inhibitor of HBV replication with an EC50 = 0.1 microM. Following a 0- to 72-hrs exposure of human hepatocytes to a 10 microM [2',3'-3H] beta-L-ddAMP-bis(tbutylSATE), the pharmacologically active beta-L-ddATP was the predominant metabolite attaining a concentration of 268.53 +/- 107.97 pmoles/10(6) cells at 2 hrs. In Hep-G2 cell, beta-L-ddATP accounted for 146.8 +/- 29.8 pmoles/10(6) cells at 2 hrs with an half life of approximately 5.4 hrs. This study reveals that extensive intracellular concentrations of beta-L-ddATP after incubation of cells to the parent drug is accounting for its potent antiviral activity.     

Adenine nucleotides modulate phosphatidylcholine metabolism in aortic endothelial cells

ATP and ADP, in concentrations ranging from 1-100 microM, increased the release of [3H]choline and [3H]phosphorylcholine (P-choline) from bovine aortic endothelial cells (BAEC) prelabelled with [3H]choline. This action was detectable within 5 minutes and was maintained for at least 40 minutes. ATP and ADP were equiactive, and their action was mimicked by their phosphorothioate analogs (ATP gamma S and ADP beta S) and adenosine 5'-(beta, gamma imido) triphosphate (APPNP), but not by AMP, adenosine, and adenosine 5'-(alpha, beta methylene)triphosphate (APCPP): these results are consistent with the involvement of P2Y receptors. ATP also induced an intracellular accumulation of [3H]choline: the intracellular level of [3H]choline was increased 30 seconds after ATP addition and remained elevated for a least 20 minutes. The action of ATP on the release of choline metabolites was reproduced by bradykinin (1 microM), the tumor promoter phorbol 12-myristate 13-acetate (PMA, 50 nM), and the calcium ionophore A23187 (0.5 microM). Down-regulation of protein kinase C, following a 24-hour exposure of endothelial cells to PMA, abolished the effects of PMA and ATP on the release of choline and P-choline, whereas the response to A23187 was maintained. These results suggest that in aortic endothelial cells, ATP produces a sustained activation of a phospholipase D hydrolyzing phosphatidylcholine. The resulting accumulation of phosphatidic acid might have an important role in the modulation of endothelial cell function by adenine nucleotides. Stimulation of phospholipase D appears to involve protein kinase C, activated following the release of diacylglycerol from phosphatidylinositol bisphosphate by a phospholipase C coupled to the P2Y receptors (Pirotton et al., 1987a).     

Augmentation of transient low-threshold Ca2+ current induced by GTP-binding protein signal transduction system in GH3 pituitary cells.

We characterized the effects of thyrotropin-releasing hormone (TRH; 500 nM) and guanosine 5'-0-3-thiotriphosphate (GTP gamma S; 50 microM) on two types of Ca2+ currents in pituitary-hormone-secretory GH3 cells and were surprised to find marked increases in transient, low-threshold Ca2+ currents (T currents) induced by extracellularly applied TRH or intracellularly applied GTP gamma S. The effect of TRH was blocked by intracellularly applied guanosine 5'-0-2-thiodiphosphate (GDP beta S; 100 microM). The increase in the T current was found to be accompanied by a decrease in long-lasting, high-threshold Ca2+ current (L-current), in response to both TRH or GTP gamma S. These indicate that the enhancement of Ca2+ influx by TRH (500 nM) is largely conferred by T currents in GH3 cells. A reduced concentration of TRH (5 nM) still markedly increased the T current, but failed to decrease the L current. These data suggest that the augmentation of the T currents as well as depression of the L currents by TRH (500 nM), through the activation of a GTP-binding protein, may constitute an important regulatory mechanism of sustained pituitary hormone secretion in GH3 cells.     

Influence of inositol 1,4,5-trisphosphate and guanine nucleotides on intracellular calcium release within the N1E-115 neuronal cell line

The Ca2+ accumulating properties of a nonmitochondrial intracellular organelle within cultured N1E-115 neuroblastoma cells containing an (ATP + Mg2+)-dependent Ca2+ pump were recently described in detail (Gill, D. L., and Chueh, S. H. (1985) J. Biol. Chem. 260, 9289-9297). Using both saponin-permeabilized N1E-115 cells and microsomal membranes from cells, this report describes the effectiveness of both inositol 1,4,5-trisphosphate (IP3) and guanine nucleotides in mediating Ca2+ release from this internal organelle, believed to be endoplasmic reticulum. Using permeabilized N1E-115 cells, 2 microM IP3 effects rapid release (t1/2 less than 20 s) of approximately 40% of accumulated Ca2+ releasable with 5 microM A23187. Half-maximal Ca2+ release occurs with 0.5 microM IP3, and maximal release with 3 microM IP3. Using a frozen microsomal membrane fraction isolated from lysed cells, 2 microM IP3 rapidly releases (t1/2 less than 30 s) 10-20% of A23187-releasable Ca2+ accumulated within nonmitochondrial Ca2+-pumping vesicles, although only in the presence of 3% polyethylene glycol (PEG). 10 microM GTP, but not guanosine 5'-(beta, gamma-imido)triphosphate (GMPPNP), increases the extent of release in the presence of IP3. Importantly, however, GTP alone induces a substantial release of Ca2+ (up to 40% of releasable Ca2+) with a t1/2 value (60-90 s) slightly longer than that for IP3. The effects of IP3 and GTP are approximately additive, and both effects require 3% PEG. Half-maximal Ca2+ release occurs with 1 microM GTP, with maximal release at 3-5 microM GTP; 20 microM GMPPNP has no effect on release and only slightly inhibits 5 microM GTP; 20 microM GDP promotes full release, but only after a 90-s lag, and initially inhibits the action of 5 microM GTP. Using permeabilized N1E-115 cells, 5 microM GTP with 3% PEG releases greater than 50% of releasable Ca2+; without PEG, GTP still mediates approximately 30% release of Ca2+ from cells. Neither IP3, GTP, or both together (with or without PEG) effects release of Ca2+ accumulated within synaptic plasma membrane vesicles. The profound effectiveness of GTP on Ca2+ release has important implications for intracellular Ca2+ regulation and is probably related to Ca2+ release mediated by IP3.     

Protein kinase G activation of K(ATP) channels in human-cultured prostatic stromal cells

In this study, we identify and investigate the role of protein kinase G (PKG) in cells cultured from human prostatic stroma. Cells were used for immunocytochemistry, contractility or K(+) fluorescent imaging studies. All cultured prostatic stromal cells showed PKG immunostaining. Phorbol 12,13 diacetate (PDA, 1 microM) elicited contractions from human-cultured prostatic stromal cells that could be blocked by both the L-type Ca(2+) channel blocker, nifedipine (3 microM), and the protein kinase C inhibitor, bisindolylmaleimide (1 microM). The nitric oxide donor, sodium nitroprusside (SNP, molar pIC(50) 5.16+/-0.17) and the cGMP-phosphodiesterase inhibitor, zaprinast (50 microM), inhibited PDA (1 microM)-induced contractions. The PKG activator beta-phenyl-1, N(2)-ethenoguanosine-3',5'-cyclic monophosphate (PET-cGMP, molar pIC(50) 6.96 +/- 0.25) also inhibited PDA (1 microM)-induced contractions. Glibenclamide (10 microM) and Rp-8-Br-cGMPS (5 microM), but not iberiotoxin (100 nM) or Rp-cAMP (5 microM), reversed this inhibition. In human-cultured prostatic stromal cells loaded with the K(+) fluorescent indicator, 1,3-Benzenedicarboxylic acid, 4,4'-[1,4,10,13-tetraoxa-7,16-diazacyclooctadecane-7,16-diylbis(5-methoxy-6,2-benzofurandiyl)]bis-, tetrakis [(acetyloxy) methyl] ester (PBFI), PET-cGMP (300 nM) caused a reduction in intracellular K(+) that was blocked by glibenclamide (10 microM) and Rp-8-Br-cGMPS (5 microM), but not by iberiotoxin (100 nM). These data are consistent with the hypothesis that, in human-cultured prostatic stromal cells, PKG inhibits contractility through the activation of K(ATP) channels.     

Anti-allergic principles from Thai zedoary: structural requirements of curcuminoids for inhibition of degranulation and effect on the release of TNF-alpha and IL-4 in RBL-2H3 cells

The 80% aqueous acetone extract of the rhizomes of Curcuma zedoaria cultivated in Thailand (Thai zedoary) was found to inhibit release of beta-hexosaminidase, as a marker of antigen-IgE-mediated degranulation, in RBL-2H3 cells and passive cutaneous anaphylaxis reaction in mice. From the active fraction, four curcuminoids (curcumin, dihydrocurcumin, tetrahydrodemethoxycurcumin, and tetrahydrobisdemethoxycurcumin) were isolated together with two bisabolane-type sesquiterpenes, and the effects of four curcuminoids from Thai zedoary and several related compounds on the degranulation were examined. Among them, curcumin showed the highest activity against beta-hexosaminidase release with IC(50) of 5.3 microM, followed by bisdemethoxycurcumin (IC(50) = 11 microM). With regard to the structural requirements of curcuminoids for the activity, the conjugated olefins at the 1-7 positions and the 4'- or 4'-hydroxyl groups of curcuminoids were suggested to be essential for the strong activity, whereas the 3'- or 3'-methoxyl group only enhanced the activity. Furthermore, effects of curcumin and bisdemethoxycurcumin on calcium ionophores (A23187 and ionomycin)-induced degranulation and antigen-induced release of TNF-alpha and IL-4 were examined.     

Effects of certain 5'-substituted adenosines on polyamine synthesis: selective inhibitors of spermine synthase

A number of nucleosides related to S-adenosylmethionine were tested for their inhibitory action on three enzymes involved in the biosynthesis of polyamines. The particular objective of the experiments was to determine whether any of the compounds could be used as selective inhibitors of the synthesis of spermine by spermine synthase. None of the nucleosides examined were potent inhibitors of S-adenosylmethionine decarboxylase. 5'-[(3-Aminopropyl)amino]-5'-deoxyadenosine dihydrochloride was quite a strong inhibitor of spermidine synthase (I50 of 7 microM) but was more than an order of magnitude less active than S-adenosyl-1,8-diamino-3-thiooctane, which is a mechanism-based inhibitor of this enzyme. 5'-[(3-Aminopropyl)amino]-5'-deoxyadenosine also inhibited spermine synthase with an I50 of 17 microM, but more selective inhibition of spermine synthase was produced by 9-[6(RS),8-diamino-5,6,7,8-tetradeoxy-beta-D-ribo-octofuranosyl]-9 H-purin-6- amine (I50 of 12 microM) and by dimethyl(5'-adenosyl)sulfonium perchlorate (I50 of 8 microM) since these compounds were much less active against spermidine synthase. Both 9-[6(RS),8-diamino-5,6,7,8-tetradeoxy-beta-D-ribo-octofuranosyl]-9 H-purin-6- amine and dimethyl(5'-adenosyl)sulfonium perchlorate were able to reduce the synthesis of spermine in SV-3T3 cells, but there was a compensatory increase in the concentration of spermidine, and there was no effect on cell growth. These results and those from experiments in which these spermine synthesis inhibitors were combined with inhibitors of spermidine synthase and ornithine decarboxylase indicated that the cells compensated for the inhibition of the aminopropyltransferases by increasing the production of decarboxylated S-adenosylmethionine and putrescine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bisquaternary Pyridinium Oximes as Presynaptic Agonists and Postsynaptic Antagonists of Muscarinic Receptors

A study of the effects of bisquaternary pyridinium oximes on calcium-dependent potassium-evoked [3H]acetylcholine release from rat brain slices revealed that at presynaptic autoreceptors these drugs function like muscarinic agonists, as they mimic the effects of acetylcholine in their inhibition of the evoked [3H]-acetylcholine release in an atropine-sensitive and dose-dependent manner. Since the bisquaternary pyridinium oximes are mild muscarinic antagonists at postsynaptic muscarinic receptors, they constitute a category of muscarinic ligands that are characterized by inverse dual activity at pre- and postsynaptic muscarinic receptors. These drugs may have dual function on cholinergic transmission by acting as presynaptic agonists and as postsynaptic antagonists. The most potent inhibitor of the evoked [3H]acetylcholine release was 1,1'-(4-hydroxyiminopyridinium)trimethylene (TMB-4) (I50 = 8 microM) and the weakest were 1-(2-hydroxyiminoethylpyridinium) 1-(3-cyclohexylcarboxypyridinium) dimethylether (HGG-42) and 1-(2-hydroxyiminoethylpyridinium) 1-(3-phenylcarboxypyridinium) dimethylether (HGG-12) (I50 = 150 microM). As postsynaptic antagonists, the latter drugs are more potent (K1 = 1.3-3.3 microM) than TMB-4 (K1 = 50 microM). Combined therapy with two drugs such as TMB-4 and HGG-12 might be effective in blocking severe hyperactivity of the cholinergic system.     

Antiallergic principles from Alpinia galanga: structural requirements of phenylpropanoids for inhibition of degranulation and release of TNF-alpha and IL-4 in RBL-2H3 cells

The 80% aqueous acetone extract of the rhizomes of Alpinia galanga was found to inhibit release of beta-hexosaminidase, as a marker of antigen-IgE-mediated degranulation in RBL-2H3 cells. Nine known phenylpropanoids and p-hydroxybenzaldehyde were isolated from the extract. Among them, 1'S-1'-acetoxychavicol acetate and 1'S-1'-acetoxyeugenol acetate exhibited potent inhibitory activity with IC(50) values of 15 and 19 microM. From the effects of various related compounds, both the 1'- and 4-acetoxyl groups of 1'S-1'-acetoxychavicol acetate and 1'S-1'-acetoxyeugenol acetate were essential for their strong activity, and the 2'-3' double bond enhanced the activity. In addition, 1'S-1'-acetoxychavicol acetate and 1'S-1'-acetoxyeugenol acetate inhibited ear passive cutaneous anaphylaxis reactions in mice and the antigen-IgE-mediated TNF-alpha and IL-4 production, both of which participate in the late phase of type I allergic reactions, in RBL-2H3 cells.     

Structural requirements of flavonoids for inhibition of antigen-Induced degranulation,TNF-alpha and IL-4 production from RBL-2H3 cells

To clarify the structure-activity relationships of flavonoids for antiallergic activity, the inhibitory effects of various flavonoids on the release of beta-hexosaminidase, as a marker of degranulation of RBL-2H3 cells, were examined. Among them, luteolin (IC(50)=3.0 microM), diosmetin (2.1 microM), and fisetin (3.0 microM) were found to show potent inhibitory activity, and the results suggested the following structural requirements of flavonoids: (1) the 2-3 double bond of flavones and flavonols is essential for the activity; (2) the 3- or 7-glycoside moiety reduced the activity; (3) as the hydroxyl groups at the 3'-, 4'-, 5-, 6-, and 7-positions increased in number, the inhibitory activities become stronger; (4) the flavonols with a pyrogallol type moiety (the 3',4',5'-trihydroxyl groups) at the B ring exhibited less activity than those with a phenol type moiety (the 4'-hydroxyl group) or catechol type moiety (the 3',4'-dihydroxyl groups) at the B ring; (5) the activities of flavones were stronger than those of flavonols; and (6) methylation of flavonols at the 3-position reduced the activity. However, (7) several flavones and flavonols with the 4'- and/or 7-methoxyl groups did not obey rules (3), (4), and (5). In addition, several flavonoids, that is apigenin, luteolin, diosmetin, fisetin, and quercetin, inhibited the antigen-IgE-mediated TNF-alpha and IL-4 production from RBL-2H3 cells, both of which participate in the late phase of type I allergic reactions.     

Transport of physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs by recombinant Escherichia coli nucleoside-H(+) cotransporter (NupC) produced in Xenopus laevis oocytes

The recently identified human and rodent plasma membrane proteins CNT1, CNT2 and CNT3 belong to a gene family (CNT) that also includes the bacterial nucleoside transport protein NupC. Heterologous expression in Xenopus oocytes has established that CNT1-3 correspond functionally to the three major concentrative nucleoside transport processes found in human and other mammalian cells (systems cit, cif and cib, respectively) and mediate Na(+) - linked uptake of both physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs. Here, one describes a complementary Xenopus oocyte transport study of Escherichia coli NupC using the plasmid vector pGEM-HE in which the coding region of NupC was flanked by 5'- and 3'-untranslated sequences from a Xenopus beta-globin gene. Recombinant NupC resembled human (h) and rat (r) CNT1 in nucleoside selectivity, including an ability to transport adenosine and the chemotherapeutic drugs 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxycytidine (ddC) and 2'-deoxy-2',2'-difluorocytidine (gemcitabine), but also interacted with inosine and 2',3'- dideoxyinosine (ddl). Apparent affinities were higher than for hCNT1, with apparent K(m) values of 1.5-6.3 microM for adenosine, uridine and gemcitabine, and 112 and 130 microM, respectively, for AZT and ddC. Unlike the relatively low translocation capacity of hCNT1 and rCNT1 for adenosine, NupC exhibited broadly similar apparent V(max) values for adenosine, uridine and nucleoside drugs. NupC did not require Na(+) for activity and was H(+) - dependent. The kinetics of uridine transport measured as a function of external pH were consistent with an ordered transport model in which H(+) binds to the transporter first followed by the nucleoside. These experiments establish the NupC-pGEM-HE/oocyte system as a useful tool for characterization of NupC-mediated transport of physiological nucleosides and clinically relevant nucleoside therapeutic drugs.     

Interactions of azidothymidine triphosphate with the cellular DNA polymerases alpha, delta, and epsilon and with DNA primase.

The interactions of azidothymidine triphosphate, the metabolically active form of the anti-AIDS drug azidothymidine (zidovudine), with the cellular DNA polymerases alpha, delta, and epsilon, as well as with the RNA primer-forming enzyme DNA primase were studied in vitro. DNA polymerase alpha was shown to incorporate azidothymidine monophosphate into a growing polynucleotide chain. This occurred 2000-fold slower than the incorporation of natural dTTP. Despite the ability of polymerase alpha to use azidothymidine triphosphate as an alternate substrate, this compound was only marginally inhibitory to the enzyme (Ki greater than 1 mM). Furthermore, the DNA primase activity associated with DNA polymerase alpha was barely inhibited by azidothymidine triphosphate (Ki greater than 1 mM). Inhibition was more pronounced for DNA polymerases delta and epsilon. The type of inhibition was competitive with respect to dTTP, with Ki values of 250 and 320 microM, respectively. No incorporation of azidothymidine monophosphate was detectable with these two DNA polymerases because their associated 3'- to 5'-exonuclease activities degraded primer molecules prior to any measurable elongation. Template-primer systems with a preformed 3'-azidothymidine-containing primer terminus inhibited the three replicative polymerases rather potently. DNA polymerase alpha was inhibited with a Ki of 150 nM and polymerases delta and epsilon with Ki values of 25 and 20 nM, respectively. The type of inhibition was competitive with respect to the unmodified substrate poly(dA).oligo(dT) for all DNA polymerases tested. Performed 3'-azidothymidine-containing primers hybridized to poly(dA) were rather resistant to degradation by the 3'- to 5'-exonuclease of DNA polymerases epsilon and more susceptible to the analogous activity that copurified with DNA polymerase delta. It is proposed that the repair of 3'-azidothymidine-containing primers might become rate-limiting for the process of DNA replication in cells that have been treated with azidothymidine triphosphate.     

Involvement of pertussis-toxin-sensitive G protein in muscarinic-receptor-mediated inhibition of K(+)-activated 4-nitrophenylphosphatase activity of cardiac sarcolemma

The effects of the cholinergic agonist carbachol on ouabain-sensitive K(+)-activated 4-nitrophenylphosphatase (K(+)-O2NPhPase) activity of rabbit and pig ventricular sarcolemma were examined. Carbachol (0.01-1000 microM) alone had no effect on K(+)-O2NPase. However, in the presence of GTP (100 microM) or its analog guanosine 5'-[gamma-thio]triphosphate (GTP[S], 1 microM) the agonist reduced this enzymatic activity (IC50 = 0.3 microM) by about 45% in a concentration-dependent manner. The GTP[S]-dependent effect of carbachol was blocked by 10 microM atropine, an antagonist of muscarinic acetylcholine receptor (mAcChoR). In the presence of micromolar concentrations of ATP or the GDP analog guanosine 5'-[beta-thio]diphosphate, carbachol did not change sarcolemmal K(+)-O2NPhPase activity. GTP[S] alone reduced this activity (IC50 = 2 microM) by about 40% in a concentration-dependent manner with a lag period of about 3 min. This lag disappeared in the presence of carbachol. Treatment of sarcolemmal membranes with 20 micrograms/ml pertussis toxin, which catalyzed ADP-ribosylation of the 40-41-kDa alpha-subunits of inhibitory GTP-binding protein (Gi), abolished the GTP[S]-promoted inhibitory effect of carbachol. Immunochemically, these alpha-subunits were identified as alpha 12- and alpha i3-subunits. It is suggested that the carbachol-induced inhibition of ouabain-sensitive K(+)-O2NPhPase activity of mammalian myocardial sarcolemma is a result of a negative coupling between mAcChoR and Na+/K(+)-ATPase via Gi protein.     

Solid phase combinatorial synthesis of benzothiazoles and evaluation of topoisomerase II inhibitory activity

To investigate one possible mechanism of action of the cytotoxic activity of benzothiazoles, we synthesized 2-(substituted-phenyl)benzothiazoles and evaluated their ability to inhibit topoisomerase II activities. Solid phase combinatorial method using trityl resin was employed and benzothiazole derivatives with various substitution on 2'-, 3'-, or 4'-position of phenyl group were obtained in ca. 30 mg scale (7-96% yield). Most of the compounds synthesized exhibited topoisomerase II inhibitory activity at 100 microM. 2-(3-Amino-4-methylphenyl)benzothiazole showed high activity (IC(50) = 71.7 microM), comparable to etoposide (IC(50) = 78.4 microM).     

Regulatory properties of magnesium-dependent guanylate cyclase in Dictyostelium discoideum membranes

We have characterized a magnesium-dependent guanylate cyclase in homogenates of Dictyostelium discoideum cells. 1) The enzyme shows an up to 4-fold higher cGMP synthesis in the presence of GTP analogues with half-maximal activation at about 1 microM guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) or 100 microM guanosine 5'-(beta, gamma-imido)triphosphate; little or no stimulation was observed with GTP, guanosine mono- and diphosphates or with adenine nucleotides, with the exception of the ATP analogue adenosine 5'-(beta, gamma-imido)triphosphate. 2) Both basal and GTP gamma S-stimulated guanylate cyclase activity were rapidly lost from homogenates as was the ability of GTP gamma S to stimulate the enzyme after cell lysis. 3) Inclusion of 25 microM GTP gamma S during cell lysis reduced the KM for GTP from 340 to 85 microM and increased the Vmax from 120 to 255 pmol/min.mg protein, as assayed in homogenates 90 s after cell lysis. 4) Besides acting as an activator, GTP gamma S was also a substrate for the enzyme with a KM = 120 microM and a Vmax = 115 pmol/min.mg protein. 5) GTP gamma S-stimulated, Mg2+-dependent guanylate cyclase was inhibited by submicromolar concentrations of Ca2+ ions, and by inositol 1,4,5-trisphosphate in the absence of Ca2+ chelators. 6) Guanylate cyclase activity was detected in both supernatant and pellet fractions after 1 min centrifugation at 10,000 x g; however, only sedimentable enzyme was stimulated by GTP gamma S. We suggest that the Mg2+-dependent guanylate cyclase identified represents the enzyme that in intact cells is regulated via cell surface receptors, and we propose that guanine nucleotides are allosteric activators of this enzyme and that Ca2+ ions play a role in the maintenance of the enzyme in its basal state.