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Radioligand binding to A\(_1\) adenosine receptors at brain membranes from seven species was investigated. The antagonist 8-cyclopentyl-1 ,3-[\(^3\)H]dipropylxanthine ([\(^3\)H]DPCPX) bound with affinities between 0.17 nM in sheep brain and 2.1 nM in guinea pig brain. Competition of several antagonists for [\(^3\)H]DPCPX binding showed that the most potent compounds were DPCPX with K\(_i\) values of 0.05 nM in bovine brain and 1.1 nM in guinea pig brain and xanthine amine congener (XAC) with K\(_i\) values of 0.03 nM in bovine brain and 5.5 nM in guinea pig brain. The differences in affinity of the agonist radio Iigand 2-chloro-N\(^6\) -[\(^3\)H]cyclopen tyladenosine ([\(^3\)H]CCP A) were less pronounced, rauging from a K\(_D\) value of 0.12 nM (hamster brain) to 0.42 nM (guinea pig brain). Agonist competition for [\(^3\)H]DPCPX binding of photoaffinity labelling, however, exhibited marked species differences. N-Ethylcarboxamidoadenosine (NECA) and S-N\(^6\)-phenylisopropyladenosine (S-PIA) showed 20 to 25-fold different K\(_D\) values in different species. NECA had a particularly high affinity in guinea pig brain and was only two-fold less potent than R-PIA. Thus, the difference from the "classical" A\(_1\) receptor profile (R-PIA > -NECA > S-PIA) is not sufficient to speculate that A\(_1\) receptor subtypes may exist that are coupled to different effector systems. Our data show that these difference can easily be explained by species differences.
The covalent binding of tritiated benzo(a)pyrene (BP) to DNA has been determined in rat liver in vivo, in rat liver perfused in situ, after incubation of BP with liver single cells, with liver homogenate, with liver microsomes and DNA, with fibroblasts from a rat granulorna pouch, and with · 2 cell lines. Li ver single cells were found to be a valuable compromise between the rnost sensitive system (microsomal incubation of BP with DNA) and the biologically most relevant system (in vivo ).
The rate limiting step in 5-fluorouracil catabolism is catalyzed by the enzyme dihydropyrimidine dehydrogenase. Since degradation of 5-fluorouracil decreases its efficacy in chemotherapy, the inhibition of its catabolism is a promising tool. We investigated the formation of micronuclei in vitro in mouse L5178Y cells. 5-fluorouracil induced an increase in micronucleus frequency, which could significantly be enhanced by the concurrent application of 2,6-dihydroxypyridine, an inhibitor of dihydropyrimidine dehydrogenase. The 5-fluorouracil concentration necessary to reach maximal genotoxic effects could be reduced to half in the presence of inhibitor. 2,6-Dihydroxypyridine alone and the naturally occuring enzyme substrate uracil did not induce micronucleus formation. Combined application of the chemotherapeutic agent 5-fluorouracil and an inhibitor of its could reduce side-effects by lowering the effective dose of the active drug. With this study we provide further support for the usefulness of this concept.
Chemical modification of amino acid residues was used to probe the ligand recognition site of A\(_1\) adenosine receptors from rat brain membranes. The effect of treatment with group·specific reagents on agonist and antagonist radioligand binding was investigated. The histidine-specific reagent diethylpyrocarbonate (DEP) induced a loss of binding of the agonist R-N\(^6\)-[\(^3\)H]phenylisopropyladenosine ([\(^3\)H]PIA), which could be prevented in part by agonists, but not by antagonists. DEP treatment induced also a loss of binding of the antagonist [\(^3\)H]8- cyclopentyl-1 ,3-dipropylxanthine ([\(^3\)H]DPCPX). Antagonists protected A\(_1\) receptors from this inactivation while agonists did not. This result provided evidence for the existence of at least 2 different histidine residues involved in ligand binding. Consistent with a modification of the binding site, DEP did not alter the affinity of [\(^3\)H]DPCPX, but reduced receptor number. From the selective protection of [\(^3\)H] PIA and [\(^3\)H]DPCPX binding from inactivation, it is concluded that agonists and antagonists oocupy different domains at the binding site. Sulfhydryl modifying reagents did not influence antagonist binding, but inhibited agonist binding. This effect is explained by modification of tbe inhibitory guanine nucleotide binding protein. Pyridoxal 5-phosphate inactivated both [\(^3\)H]PIA and [\(^3\)H]DPCPX binding, but the receptors could not be protected from inactivation by ligands. Therefore, no amino group seems to be located at the Iigand binding site. In addition, it was shown that no further amino acids witb polar side chains are present. The absence of bydrophilic amino acids frout the recognition site of the receptor apart from histidine suggests an explanation for the lack of hydrophilic ligands with high affinity for A\(_1\) receptors.
The intake of known dietary carclnogens was compiled and the cancer risk was estlmated on the basis of carcinogenic potencies in animals as derived from the Carcinogenic Potency Database by Gold and co-workers. The total cancer risk was compared with the number of cancer cases attributed by epidemiologists to dietary factors (one-third of all cancer cases, i.e. -80 000 per one million Jives). Except for alcohol, the known dietary carcinogens could not account for more than a few bundred cancer cases. Tbis was seen both with tbe DNA-reactive carcinogens (beterocyclic aromatic amines, polycyclic aromatic hydrocarbons, N-nitroso compounds, estragole, aflatoxin B., ethyl carbamate, to name the most important factors) as wen as with those carclnogens wbich have not been shown to react with DNA (e.g. caffelc acid and the carcinogeruc metals arsenic and cadmium). Residues and contaminants turned out to be negligible. Among the various pmsibilities to explain the discrepancy we investigated the roJe of ovemutritlon. Dietary restriction in animals is weil known for its strong reducing effect on spontaneous tumor formation. These data can be used to derive a carcinogenic potency for excess macronutrients: tbe tumor incidence seen with the restrlcted animals is taken as a control value and the increased tumor incidence in the animals fed ad libitum is attributed to the additional feed iotake. For excess standard diet in rats, a carcinogenic potency TD50 of 16 glkg/day was deduced from a recent study. Ovemutrition in Switzerland, estimated to be 5.5 kcallkg/day, was converted to excess food (1.9 g/kg/day) and tbe cancer incidence was calculated. The result, 60 000 cancer cases per one million Jives, is provocatively close to the number of cases not explained by the known dietary chemical carcinogens. Mechanistic studies will be required to test our hypothesis and investigate the role of different types of macronutrients in ovemutrition.
A\(_1\) adenosine receptors from rat brain membranes were solubilized with the zwitterionic detergent 3-[3-( cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized receptors retained all the characteristics of membrane-bound A\(_1\) adenosine receptors. A high and a low agonist affinity state for the radiolabelled agonist (R)-\(N^6\)-[\(^3\)H]phenylisopropyladenosine([\(^3\)H]PJA) with K\(_D\) values of 0.3 and 12 nM, respectively, were detected. High-affinity agonist binding was regulated by guanine nucleotides. In addition agonist binding was still modulated by divalent cations. The solubilized A\(_1\) adenosine receptors could be labelled not only with the agonist [\(^3\)H]PIA but also with the antagonist I ,3-diethyi-8-[\(^3\)H]phenylxanthine. Guanine nucleotides did not affect antagonist binding as reported for membrane-bound receptors. These results suggest that the solubilized receptors are still coupled to the guanine nucleotide binding protein N; and that all regulatory functions are retained on solubilization. Key Words: A1 adenosine receptors - Solubilization- Rat brain membranes. Klotz K.-N. et al. Characterization of the solubilized A1 adenosine receptor from rat brain membranes. J. Neurochem. 46, 1528-1534 (1986).
In the present work we studied the pharmacological profile of adenosine receptors in guinea pig atria by investigating the effect of different adenosine analogues on 86Rb + -efflux from isolated left atria and on binding of the antagonist radioligand 8-cyclopentyl-1 ,3-[\(^3\)H]dipropylxanthine ([\(^3\)H]DPCPX) to atrial membrane preparations. The rate of \8^{86}\)Rb\(^+\) -effiux was increased twofold by the maximally effective concentrations of adenosine receptor agonists. The EC50-values for 2-chloro-N\(^6\)-cyclopentyladenosine (CCPA), R-N\(^6\)-phenylisopropyladenosine (R-PIA), 5'-Nethylcarboxamidoadenosine (NECA), and S-N\(^6\)-phenylisopropyladenosine (S-PIA) were 0.10, 0.14, 0.24 and 12.9 \(\mu\)M, respectively. DPCPX shifted the R-PIA concentration-response curve to the right in a concentration-dependent manner with a K\(_B\)-value of 8.1 nM, indicating competitive antagonism. [\(^3\)H]DPCPX showed a saturable binding to atrial membranes with a Bmax·value of 227 fmol/mg protein and a K\(_D\)-value of 1.3 nM. Competition experiments showed a similar potency for the three agonists CCPA, R-PIA and NECA. S-PIA is 200 times less potent than R-PIA. Our results suggest that the K\(^+\) channel-coupled adenosine receptor in guinea pig atria is of an A\(_1\) subtype.
Insulin receptors were solubilized from rat liver microsomes by the nonionic detergent Triton X-100. After gel filtration of the extract on Sepharose CL-6B, two insulin-binding species (peak I and peak li) were obtained. The structure and binding properties of both peaks were characterized. Gel filtration yielded Stokes radii of 9.2 nm (peak I) and 8.0 nm (peak Il). Both peaks were glycoproteins. At 4°C peak 1 showed optimal insulin binding at pH 8.0 and high ionic strength. In contrast, peak li bad its binding optimum at pH 7.0 and low ionic strength, where peak I bindingwas minimal. For peak I the change in insulin binding under different conditions of pH and ionic strength was due to a change in receptor affinity only. For peak 11 an additional change in receptor number was found. Both peaks yielded non-linear Scatchard plots under most of the buffer conditions examined. At their binding optima at 4 oc the high affinity dissociation constants were 0.50 nM (peak I) and 0.55 nM (peak II). Sodium dodecyl sulfatejpolyacrylamide gel electrophoresis of peak I revealed five receptor bands with Mr 400000, 365000, 320000, 290000, and 245000 under non-reducing conditions. For peak II two major receptor bands with M\(_r\) 210000 and 115000 were found. The peak II receptor bands were also obtained aftermild reduction of peak I. After complete reduction both peaks showed one major receptor band with M\(_r\) 130000. The reductive generation of the peak II receptor together with molecular mass estimations suggest that the peak I receptor is the disulfide-linked dimer of the peak II receptor. Thus, Triton extracts from rat liver microsomes contain two receptor species, which are related, but differ considerably in their size and insulin-binding properties.
Streptococcus pneumoniae is a common pathogen that causes various infections, such as sepsis and meningitis. A major pathogenic factor of S. pneumoniae is the cholesterol-dependent cytolysin, pneumolysin. It produces cell lysis at high concentrations and apoptosis at lower concentrations. We have shown that sublytic amounts of pneumolysin induce small GTPase-dependent actin cytoskeleton reorganization and microtubule stabilization in human neuroblastoma cells that are manifested by cell retraction and changes in cell shape. In this study, we utilized a live imaging approach to analyze the role of pneumolysin’s pore-forming capacity in the actin-dependent cell shape changes in primary astrocytes. After the initial challenge with the wild-type toxin, a permeabilized cell population was rapidly established within 20–40 minutes. After the initial rapid permeabilization, the size of the permeabilized population remained unchanged and reached a plateau. Thus, we analyzed the non-permeabilized (non-lytic) population, which demonstrated retraction and shape changes that were inhibited by actin depolymerization. Despite the non-lytic nature of pneumolysin treatment, the toxin’s lytic capacity remained critical for the initiation of cell shape changes. The non-lytic pneumolysin mutants W433F-pneumolysin and delta6-pneumolysin, which bind the cell membrane with affinities similar to that of the wild-type toxin, were not able to induce shape changes. The initiation of cell shape changes and cell retraction by the wild-type toxin were independent of calcium and sodium influx and membrane depolarization, which are known to occur following cellular challenge and suggested to result from the ion channel-like properties of the pneumolysin pores. Excluding the major pore-related phenomena as the initiation mechanism of cell shape changes, the existence of a more complex relationship between the pore-forming capacity of pneumolysin and the actin cytoskeleton reorganization is suggested.
5-Azacytidine was originally developed to treat human myelogenous leukemia. However, interest in this compound has expanded because of reports of its ability to affect cell differentiation and to alter eukaryotic gene expression. In an ongoing attempt to understand the biochemical effects of this compound, we examined the effects of 5-azacytidine on mitosis and on micronucleus formation in mammalian cells. In L5178Y mouse cells, 5-azacytidine induced micronuclei at concentrations at which we and others have already reported its mutagenicity at the tk locus. Using CREST staining and C-banding studies, we showed that the induced micronuclei contained mostly chromosomal fragments although some may have contained whole chromosomes. By incorporating BrdU into the DNA of SHE cells, we determined that micronuclei were induced only when the compound was added while the cells were in S phase. Microscopically visible effects due to 5-azacytidine treatment were not observed until anaphase of the mitosis following treatment or thereafter. 5-Azacytidine did not induce micronuclei via interference with formation of the metaphase chromosome arrangement in mitosis, a common mechanism leading to aneuploidy. SupravitalUV microscopy revealed that chromatid bridges were observed in anaphase and, in some cases, were sustained into interphase. In the first mitosis after 5-azacytidine treatment we observed that many cells were unable to perform anaphase separation. All of these observations indicate that 5-azacytidine is predominantly a clastogen through its incorporation into DNA.
In addition to its tumor-promoting activity in honnone-receptive tissue, the carcinogenic estrogen diethylstilbestrol (DES) has been found to induce cell transformation, aneuploidy and micronucleus formation in mammalian cells. The majority of these micronuclei contained whole chromosomes and were fonned during mitosis. Here a possible relationship between a disturbance in cell cycle progression and micronucleus fonnation is investigated by exposing Syrian hamster embryo (SHE) cells to DES. Continuous bromodeoxyuridine labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry was employed for analysis of cell cycle transit and related to the time course of micronucleus formation. Treatment of SHE cells with DES resulted in delayed and impaired cell activation (exit from the GO/G 1 phase), impaired S-phase transit and, mainly, G2-phase traverse. Cells forming micronuclei, on the other hand, were predominantly in G2 phase during DES treatment. These results suggest that impairment of Sand G2 transit may involve a process ultimately leading to micronucleus formation.
In addition to hormonal activity, genetic darnage has been proposed as an important factor in oestrogen-mediated carcinogenesis. However, as short-term tests for oestrogens usually fail to show DNA mutations, lesions other than dassie nuclear DNA mutation have to be considered. Oestrogeninduced mitochondrial darnage was studied in the yeast Saccharomyces cerevisiae. Stilbene-type, but not steroidal, oestrogens were found to induce respiration-dcficient petite mutation. The effect was inversely correlated with cytotoxicity and required aromatic hydroxyl groups at the stilbene molecule. It only occurred under growth conditions and apparently was not due to the A TPase inhibitory qualities of stilbene oestrogens. Other studies have shown that petite mutation clones, which can be induced by a variety of substances, contain altered mitochondrial DNA. The mechanism of petite mutation induction might be important in tumorigenesis by also acting on nuclear DNA or facilitating carcinogenesis by disturbance of mitochondrial function.
The mechanism of the therapeutic and prophylactic effects of carbamazepine (CBZ) in affective psychoses is unknown but may in part be related to the potent competitive interaction of CBZ with adenosine-binding sites in the brain. The antioonvulsant and sedative properties of CBZ are reminiscent of the effects evoked by adenosine-agonists and contrast sharply with the opposite aclions of adenosine-antagonists like caffeine. However. indirect evidence suggests an antagonist- rather than an agonist-like activity of CBZ at adenosi11e-receptors. We have used various model systems, in which adenosine receptor subtypes mediate different second messenger-responses, to investigate this apparent paradox. CBZ was found to antagonize the A\(_1\) receptor-mediated inhibition of cydic AMP accumulation in cultured astroblasts and in GH3-cells. Furthermore, CBZ also inhibits the adenosine-induced increase in the level of cyclic AMP in cultured astroblasts, which is mediated by low-affinity A\(_{2b}\)-receptors. ln contrast, CBZ does not block the inhibition elicited by adenosine-agonists of the agonist-induced increased formation of inositolphosphates in human neutrophils, which is mediated by high-affinity A\(_{2a}\)-receptors. The specific antagonism by CBZ of A\(_1\)- but not of high-affinity A\(_{2a}\)-receptors was further supported by binding experiments using rat brain membranes. These results suggest tbat the paradox of CBZ's antagonistic effects at adenosine-receptors might be at least partially reconciled by a selective antagonistic action of CBZ at A\(_1\)recertors but not at high-affinity A\(_{2a}\)-receptors.
Barbiturates in pharmacologically relevant . concentrations inhibit binding of (R)-\(N^6\)-phenylisopropyl[\(^3\)H]adenosine ([\(^3\)H]PIA) to solubilized A\(_1\) adenosine receptors in a concentration-dependent, stereospecific, and competitive manner. K\(_i\) values are similar to those obtained for membrane-bound receptors and are 31 \(\mu\)M for ( ± )-5-(1 ,3-dimethyl)-5-ethylbarbituric acid [( ± )DMBB] and 89 \(\mu\)M for ( ± )-pentobarbital. Kinetic experiments demoostrate that barbiturates compete directly for the binding site of the receptor. The inhibition of rat striatal adenylate cyclase by unlabelled (R)-\(N^6\)-phenylisopropyladenosine [(R)-PIA] is antagonized by barbiturates in the same concentrations that inhibit radioligand binding. The Stimulation of adenylate cyclase via A\(_2\) adenosine receptors in membranes from NIE 115 neuroblastoma cells is antagonized only by 10-30 times higher concentrations of barbiturates. lt is concluded that barbiturates are selective antagonists at the A1 receptor subtype. In analogy to the excitatory effects of methylxanthines it is suggested that A\(_1\) adenosine receptor antagonism may convey excitatory properties to barbiturates. Key Words: Adenosine receptors-Barbiturates - Adenylate cyclase-Receptor solubilization-[3H]PIA binding-N1E 115 cells. Lohse M. J. et al. Barbiturates are selective antagonists at A1 adenosine receptors.
A literature review has shown that the daily intakes of various N -nitroso-precursor classes in a typical European diet span five orders of magnitude. Amides in the form of protein, and guanidines in the form of creatine and creatinine, are the nitrosatable groups found most abundantly in the diet, approaching Ievels of 100 g/day and 1 gjday, respectively. Approximately 100 mg of primary amines and amino acids are consumed daily, whereas aryl amines, secondary amines and ureas appear to lie in the 1-10 mg range. The ease of nitrosation of each precursor was estimated, the reactivities being found to span seven orders of magnitude, with ureas at the top and amines at the bottom of the scale. From this infonnation and an assessment of the carcinogenicity of the resulting N-nitroso derivatives, the potential health risk due to gastric in vivo nitrosation was calculated. The combined effects of these risk variables were analysed using a simple mathematical model: Risk = [daily intake of precursor] x [gastric concentration of nitrite]\(^n\) x [nitrosatability rate constant} x [carcinogenicity of derivative]. The risk estimates for the various dietary components spanned nine orders of magnitude. Dietary ureas and aromatic amines combined with a high nitrite burden could pose as great a risk as the intake of preformed dimethylnitrosamine in the diet. In contrast, the risk posed by the in vivo nitrosation of primary and secondary amines is probably negligib1y small. The risk contribution by amides (including protein), guanidines and primary amino acids is intermediate between these two extremes. Thus three priorities for future work are a comprehensive study of the sources and Ievels of arylamines and ureas in the diet, determination of the carcinogenic potencies of key nitrosated products to replace the necessarily vague categories used so far, and the development of short-term in situ tests for studying the alkylating power or genotoxicity of N-nitroso compounds too unstable for inclusion in long-term studies.
Tbe benzodiazepines are a class of d.rugs that are widely used in the treatment of various psychiatric disorders. One member of um ~' oxazepam, is also a common metabolite of sevmd other benzod.iazepines. Since the evidence for the genetic toxicity and carcinogenic properties of these compounds is incol:lsb1ent, we investigated the oxazepam-induced fonnation of micronuclei in Syrian Hamster embryo fibroblast (SHE) cells, human amniotic fluid fibroblast-like (AFFL) cells and LS178Y mouse cells. A dose-dependent increase in micronucleus fractions was found in all tbree ceU llnes. The time course of micronucleus induction in L5178Y cells showed a maximum at 5 h after treatment, suggesting that the micronuclei were fonned in the first mitosis after treatment. Kinetochore staining (CREST -antiserum) revealed the presence of kinetochores in -SO% of the micronuclei in aU tbree ceU types. ThJs resu1t was further confinned by in situ bybridization in LS178Y cells and indicates tbe presence of wbole Chromosomes or centric fragments as weU as acentric fragments in the oxazepam-induced micronuclei. The LS178Y cells did not show a mutagenic response to oxazepam at any of the doses or expression times used.
The effects of barbiturates on the GABA·receptor complex and the A\(_1\) adenosine receptor were studied. At the GABA-receptor complex the barbiturates inhibited the binding of [\(^{35}\)S]t-butylbicyclophosphorothionate [\(^{35}\)S]TBPT) and enhanced the binding of [\(^3\)H]diazepam. Kinetic and saturation experiments showed that both effects were allosteric. Whereas all barbiturates caused complete inhibition of [\(^{35}\)S]TBPT binding, they showed varying degrees of maximal enhancement of [\(^3\)H]diazepam binding; (±)methohexital was idenafied as the most efficacious compound for this enhancement. At the A\(_1\) adenosine receptor all barbiturates inhibited the binding of [\(^3\)H]N\(^6\)-phenylisopropyladenosine (\(^3\)H]PIA) in a competitive manner. The comparison of the effects on [\(^3\)H]diazepam and [\(^3\)H]PIA binding showed that excitatory barbiturates interact preferentially with the A\(_1\) adenosine receptor, and sedative/anaesthetic barbiturates with the GABA-receptor complex. It is speculated that the interaction with these two receptors might be the basis of the excitatory versus sedative/ anaesthetic properties of barbiturates.
Adenosine receptor agonists: Synthesis and biological evaluation of 1-deaza analogues of adenosine
(1988)
In a search for more selective A\(_1\) adenosine receptor agonists, N\(^6\)-[(R)-(-)-1-methyl-2-phenethyl]-1-deazaadenosine (1-deaza-R-PIA, 3a), N\(^6\)-cyclopentyl-1-deazaadenosine (1-deazaCPA, 3b), N\(^6\)-cyclohexyl-l-deazaadenosine (1-deazaCHA, Sc), and the corresponding 2-chloro derivatives 2a-c were synthesized from 5,7-dichloro-3-ß-D-ribofuranosyl-3Himidazo[ 4,5-b]pyridine (1). On the other band, N-ethyl-1'-deoxy-1'-(1-deaza-6-amino-9H-purin-9-yl)-ß-D-ribofuranuronamide (1-deazaNECA, 10) was prepared from 7-nitro-3-ß-D-ribofuranosyl-3H-imidazo[4,5-b]pyridine (4), in an attempt to find a more selective A\(_2\) agonist. The activity of all deaza analogues at adenosine receptors has been determined in adenylate cyclase andin radioligand binding studies. 1-DeazaNECA (10) proved tobe a nonselective agonist at both subtypes of the adenosine receptor. It is about 10-fold less active than NECA but clearly more active than the parent compound 1-deazaadenosine as an inhibitor of platelet aggregation and as a stimulator of cyclic AMP accumulation. The N\(^6\)-substituted 1-deazaadenosines largely retain the A\(_1\) agonist activity of their parent compounds, but lose some of their A\(_2\) agonist activity. This results in A\(_1\)-selective compounds, of which N\(^6\)cyclopentyl- 2-chloro-1-deazaadenosine (1-deaza-2-Cl-CPA, 2b) was identified as the most selective agonist at A\(_1\) adenosine receptors so far known. The activity of all 1-deaza analogues confirms that the presence of the nitrogen atom at position 1 of the purine ring is not critical for A\(_1\) receptor mediated adenosine actions.
Two forms of a DNA polymerase have been purified from microplasmodia of Physarum polycephalum by poly(ethyleneimine) precipitation and chromatography on DEAE-Sephacel, phosphocellulose, heparin Sepharose, hydroxyapatite, DNA-agarose, blue-Sepharose. They were separated from DNA polymerase cx on phosphocellulose and from each other on heparin-Sepharose. Form HS1 enzymewas 30-40% pure and form HS2 enzyme 60% with regard toprotein contents of the preparations. Form HS2 enzymewas generated from form HS1 enzyme on prolonged standing of enzyme preparations. The DNA polymerases were obtained as complexes of a 60-kDa protein associated with either a 135-kDa (HS1) or a 110-kDa (HS2) DNA-polymerizing polypeptidein a 1:1 molar stoichiometry. The biochemical function of the 60-kDa protein remained unknown. The complexes tended to dissociate during gradient centrifugation and during partition chromatography as weil as during polyacrylamide gradient gel electrophoresis under nondenaturing conditions at high dilutions of samples. Both forms existed in plasmodia extracts, their proportions depending on several factors including those which promoted proteolysis. The DNA polymerases resembled eucaryotic DNA polymerase ß by several criteria and were functionally indistinguishable from each other. It is suggested that lower eucaryotes contain repair DNA polymerases, which are similar to those of eubacteria on a molecular mass basis.
In the inhalation system described an animal can be kept in the same atmosphere of a 2-liter desiccator for up to 24 h. The expired carbon dioxide is adsorbed with soda lime and the resulting reduced pressure is balanced by a supply of oxygen also used for the inflow of the chemical to be investigated. Urine and faeces can be collected ~eparately and the system allows a periodical control of the concentration of the chemical by sampling the air with needle and syringe.
The properties of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as an antagonist ligand for A\(_1\) adenosirre receptors were examined and conipared with other radioligands for this receptor. DPCPX competitively antagonized both the inhibition of adenylate cyclase activity via A\(_1\) adenosirre receptors and the stimulationvia A\(_2\) adenosirre receptors. The K\(_i\)-values of this antagonism were 0.45 nM at the A\(_1\) receptor of rat fat cells, and 330 nM at the A\(_2\) receptor of human platelets, giving a more than 700-fold A\(_1\)-selectivity. A similar A\(_1\)-selectivity was determined in radioligand binding studies. Even at high concentrations, DPCPX did not significantly inhibit the soluble cAMPphosphodiesterase activity of human platelets. [\(^3\)H]DPCPX (105 Ci/mmol) bound in a saturable manner with high affinity to A\(_1\) receptors in membranes of bovine brain and heart, and rat brain and fat cells (K\(_D\) -values 50-190 pM). Its nonspecific binding was about 1% of total at K\(_D\) , except in bovine myocardial membranes (about 10%). Binding studies with bovine myocardial membranes allowed the analysis of both the high and low agonist affinity states of this receptor in a tissue with low receptor density. The binding properties of [\(^3\)H]DPCPX appear superior to those of other agonist and antagonist radioligands for the A\(_1\) receptor.
lt is known that 5-azacytidine (5-AC) induces tumors in several organs of rats and mice. The mechanisms of these effects are still poorly understood although it is known that 5-AC can be incorporated into DNA. Furthermore, it can inhibit DNA methylation. The known data on its clastogenic andjor gene mutation-inducing potential are still controversial. Therefore, we have investigated the kinds of genotoxic effects caused by 5-AC in Syrian hamster embryo (SHE) fibroblasts. Three different endp6ints (micronucleus formation, unscheduled DNA synthesis (UDS) and cell transforrnation) were assayed under similar conditions of metabolism and dose at target in this cell system. 5-AC induces morphological transformation of SHE cells, but not UDS. Therefore, 5-AC does not seem to cause repairable DNA lesions. Furthermore, our studies revealed that 5-AC is a potent inducer of mkronuclei in the SHE system. Immunocytochemical analysis revealed that a certain percentage of these contain kinetochores indicating that 5-AC may induce both clastogenic events and numerical chromosome changes.
2-Chloro-N\(^6\)-cyclopentyladenosine: a highly selective agonist at A\(_1\) adenosine receptors
(1988)
2-Chloro-N\(^6\)-cyclopentyladenosine (CCPA) was synthesized as a potential high affinity ligand for At adenosine receptors. Binding of [\(^3\)H]PIA to A1 receptors of rat brain membranes was inhibited by CCP A with a Ki-value of 0.4 nM, compared to a Ki-value of 0.8 nM for the parent compound N\(^6\)-cyclopentyladenosine (CPA). Binding of [\(^3\)H]NECA to A\(_2\) receptors of rat striatal membranes was inhibited with a Ki-value of 3900 nM, demonstrating an almost 10,000-fold A\(_1\)-selectivity of CCPA. CCP A inhibited the activity of rat fat cell membrane adenylate cyclase, a model for the A\(_1\) receptor, with an IC\(_{50}\)-value of 33 nM, and it stimulated the adenylate cyclase activity of human platelet membranes with an EC\(_{50}\)-value of 3500 nM. The more than 100-fold A\(_1\)-selectivity compares favourably with a 38-fold selectivity of CPA. Thus, CCPA is an agonist at A\(_1\) adenosine receptors with a 4-fold higher selectivity and 2-fold higher affinity than CPA, and a considerably higher selectivity than the standard At receptor agonist R-N\(^6\) -phenylisopropyladenosine (R-PIA). CCP A represents the agonist with the highest selectivity for A\(_1\) receptors reported so far.
The tritiated analogue of 2-chloro-N6-cyclopentyladenosine (CCPA), an adenosine derivative with subnanomolar affinity and a 10000-fold selectivity for A1 adenosine receptors, has been examined as a new agonist radioligand. [3H]CCP A was prepared with a specifi.c radioactivity of 1.58 TBqjmmol ( 43 Ci/mmol) and bound in a reversible manner to A1 receptors from rat brain membranes with a high affinity K0 -value of 0.2 nmol/1. In the presence of GTP a K0 -value of 13 nmol/1 was determined for the low affinity state for agonist binding. Competition of several adenosine receptor agonists and antagonists for [3H]CCPA binding to rat brain membranes confrrmed binding to an A1 receptor. Solubilized A1 receptors bound [3H]CCPA with similar affinity for the high affinity state. At solubilized receptors a reduced association rate was observed in the presence of MgC12, as has been shown for the agonist [ 3H]N6-phenylisopropyladenosine ([3H]PIA). [3H]CCPA was also used for detection of A1 receptors in rat cardio myocyte membranes, a tissue with a very low receptor density. A K0 -value of 0.4 nmol/1 and a Bmax-value of 16 fmol/ mg protein was determined in these membranes. In human platelet membranes no specific binding of [3H]CCPA was measured at concentrations up to 400 nmoljl, indicating that A2 receptors did not bind [3H]CCPA. Based on the subnanomolar affinity and the high selectivity for A1 receptors [ 3H]CCPA proved to be a useful agonist radioligand for characterization of A 1 adenosine receptors also in tissues with very low receptor density.
In the search for more selective A2-receptor agonists and on the basis that appropriate substitution at C2 is known to impart selectivity for A\(_2\) receptors, 2-alkynyladenosines 2a-d were resynthesized and evaluated in radioligand binding, adenylate cycla.se, and platelet aggregation studies. Binding of [\(^3\)H]NECA to A\(_2\) receptors of rat striatal membranes was inhibited by compounds 2a-d with K\(_i\) values ranging from 2.8 to 16.4 nM. 2-Alkynyladenosines also exhibited high-affmity binding at solubilized A\(_2\) receptors from human platelet membranes. Competition of 2-alkynyladenosines 2a-d for the antagonist radioligand [\(^3\)H]DPCPX and for the agonist [\(^3\)H]CCPA gave K\(_i\) values in the nanomolar range, and the compounds showed moderate A\(_2\) selectivity. In order to improve this selectivity, the correaponding 2-alkynyl derivatives of adenosine-5'-N-ethyluronamide 8a-d were synthesized and tested. A\(_1\) expected, the 5'-N-ethyluronamide derivatives retained the A\(_2\) affinity whereas the A\(_1\) affinity was attenuated, resulting in an up to 10-fold increase in A\(_2\) selectivity. A similar patternwas observed in adenylate cyclase assays andin platelet aggregation studies. A 30- to 45-fold selectivity for platelet A\(_2\) receptors compared to A\(_1\) receptors was found for compounds 8a-c in adenylate cyclase studies.
Tbe 2',3'-dideoxy analogue of the potent A\(_1\) receptor agonist, N\(^6\)-cyclohexyladenosine (CHA), was synthesized as a potential antagonist for the A\(_1\) adenosine receptor. In sturlies on adenylate cyclase 2',3'-dideoxy-N\(^6\)-cyclohexyladenosine (ddCHA) did not show agonist properties at A\(_1\) or at A\(_2\) receptors. However, it antagonized the inhibition by R-PIA of adenylate cyclase activity of fat cell membranes via A\(_1\) receptors with a K\(_i\) value of 13 \(\mu\)M. ddCHA competed for the binding of the selective A1 receptor antagonist, [\(^3\) HJ8-cyclopentyl-1,3-dipropylxantbine ([\(^3\)H]DPCPX), to rat brain membranes with a K\(_i\) value of 4.8 \(\mu\)M; GTP did not affect the competition curve. In contrast to the marked stereoselectivity of the A\(_1\) receptor for the cx- and the natural ß-anomer of adenosine, the cx-anomer of ddCHA showed a comparable affinity for the A\(_1\) receptor (K\(_i\) value 13.9 \8\mu\)M). These data indicate that the 2'- and 3'-hydroxy groups of adenosine and its derivatives are required foragonist activity at and high affinity binding to A\(_1\) adenosine receptors and for the distinction between the cx- and ß-forms.