Failure of dideoxynucleosides to inhibit human immunodeficiency virus replication in cultured human macrophages.

Primary human monocyte-derived macrophages (MDM) were shown to have diminished deoxynucleoside kinase activities compared to T lymphoblasts, and a reduced ability to phosphorylate dideoxynucleosides with anti-human immunodeficiency virus (HIV) activity. These drugs, azidothymidine (AZT), dideoxycytidine (ddC), and dideoxyadenosine (ddA), which are potent anti-HIV agents in CD4 lymphocytes, did not inhibit HIV replication in MDM, even at concentrations of 100 microM. This drug concentration of AZT is approximately 100-fold higher than the levels attained in the serum of treated patients and the levels required to inhibit HIV replication in lymphocytes. These observations may explain the failure of AZT therapy to clear viremia, consistent with the presence of a drug-resistant reservoir of infected cells in vivo. New therapeutic approaches to inhibit the replication of HIV in MDM may be needed.

Benzo(a)pyrene-7,8-dihydrodiol 9,10-oxide adenosine and deoxyadenosine adducts: structure and stereochemistry.

The structure and absolute stereoconfigurations of four adenosine adducts with (+/-)-7 alpha,8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) and their deoxyadenosine analogs have been determined. They result from both cis and trans addition of the N6 amino group of ademine to the 10 position of both enantiomers of BDPE. This was determined from studies of the nuclear magnetic resonance spectra, mass spectra, and circular dichroism spectra, as well as from their pKa values and chemical reactivities.

Prostacyclin stimulation of the activation of blood coagulation factor X by platelets.

When platelets were incubated with prostacyclin, prostaglandin E1, or prostaglandin D2 at concentrations insufficient to increase the level of adenosine 3',5'-monophosphate (cyclic AMP), coagulation factor X was activated by a platelet cysteine protease. Prostacyclin or prostaglandin E1 at higher concentrations increased the cyclic AMP level and inhibited the activation of factor X by platelets. Inhibition of platelet adenylate cyclase by 2',5'-dideoxyadenosine allowed the activation of the protease at higher concentrations of the autocoids. Prostaglandins A1, A2, B1, B2, E2, F2 alpha, 6-keto-prostaglandin F1 alpha, and thromboxane B2, which do not affect platelet cyclic AMP level, did not stimulate the protease.

Mechanism of deoxyadenosine and 2-chlorodeoxyadenosine toxicity to nondividing human lymphocytes.

Deoxyadenosine has been implicated as the toxic metabolite causing profound lymphopenia in immunodeficient children with a genetic deficiency of adenosine deaminase (ADA), and in adults treated with the potent ADA inhibitor deoxycoformycin. However, the biochemical basis for deoxyadenosine toxicity toward lymphocytes remains controversial. The present experiments have examined in detail the sequential metabolic changes induced in nondividing human peripheral blood lymphocytes by incubation with deoxyadenosine plus deoxycoformycin, or with 2-chlorodeoxyadenosine (CdA), an ADA resistant deoxyadenosine congener with anti-leukemic and immunosuppressive properties. The lymphotoxic effect of deoxyadenosine and CdA required their phosphorylation, and was inhibited by deoxycytidine. As early as 4 h after exposure to the deoxynucleosides, strand breaks in lymphocyte DNA began to accumulate, and RNA synthesis decreased. These changes were followed by a significant fall in intracellular NAD levels at 8 h, a drop in ATP pools at 24 h, and cell death by 48 h. Incubation of the lymphocytes with 5 mM nicotinamide, a NAD precursor and an inhibitor of poly(ADP-ribose) synthetase, prevented NAD depletion. The nicotinamide treatment also rendered the lymphocytes highly resistant to deoxyadenosine and CdA toxicity, without altering dATP formation or the accumulation of DNA strand breaks. The poly(ADP-ribose) synthetase inhibitor 3-aminobenzamide exerted a similar although less potent effect. These results suggest that NAD depletion, probably triggered by poly(ADP-ribose) formation, is the principle cause of death in normal resting human lymphocytes exposed to deoxyadenosine plus deoxycoformycin, or to CdA.

8-vinyl-deoxyadenosine, an alternative fluorescent nucleoside analog to 2'-deoxyribosyl-2-aminopurine with improved properties.

We report here the synthesis and the spectroscopic characterization of 8-vinyl-deoxyadenosine (8vdA), a new fluorescent analog of deoxyadenosine. 8vdA was found to absorb and emit in the same wavelength range as 2'-deoxyribosyl-2-aminopurine (2AP), the most frequently used fluorescent nucleoside analog. Though the quantum yield of 8vdA is similar to that of 2AP, its molar absorption coefficient is about twice, enabling a more sensitive detection. Moreover, the fluorescence of 8vdA was found to be sensitive to temperature and solvent but not to pH (around neutrality) or coupling to phosphate groups. Though 8vdA is base sensitive and susceptible to depurination, the corresponding phosphoramidite was successfully prepared and incorporated in oligonucleotides of the type d(CGT TTT XNX TTT TGC) where N = 8vdA and X = A, T or C. The 8vdA-labeled oligonucleotides gave more stable duplexes than the corresponding 2AP-labeled sequences when X = A or T, indicating that 8vdA is less perturbing than 2AP and probably adopts an anti conformation to preserve the Watson-Crick H-bonding. In addition, the quantum yield of 8vdA is significantly higher than 2AP in all tested oligonucleotides in both their single strand and duplex states. The steady-state and time-resolved fluorescence parameters of 8vdA and 2AP were found to depend similarly on the nature of their flanking residues and on base pairing, suggesting that their photophysics are governed by similar mechanisms. Taken together, our data suggest that 8vdA is a non perturbing nucleoside analog that may be used with improved sensitivity for the same applications as 2AP.

Imbalance of deoxyribonucleoside triphosphates, DNA double-strand breaks, and cell death caused by 2-chlorodeoxyadenosine in mouse FM3A cells.

The mechanism of the cytotoxic action of 2-chlorodeoxyadenosine in mouse FM3A cells was investigated. Imbalance of the dNTP pools occurred within 3 h of treatment with 20 microM 2-chlorodeoxyadenosine; the dATP and dGTP pools were depleted and the dTTP pool increased. 2-Chlorodeoxyadenosine added to the culture medium broke mature DNA strands, giving fragments of 100-200 kilobase pairs as found by orthogonal-field-alternation gel electrophoresis. DNA strand breaks, measured by this technique, were observed in the treated cells about 12 h after the addition. The cells also lost viability at about 12 h. Breaks in the single and double strands of DNA, as measured by alkaline and neutral filter elution, became evident 18 h after treatment with 20 microM 2-chlorodeoxyadenosine; there were as many single-strand breaks as would be caused by 130 rads of gamma-ray irradiation. Double-strand breaks were equivalent to those caused by 2180 rads of gamma-ray irradiation. Comparison of the ratio of single- and double-strand breaks caused by 2-chlorodeoxyadenosine to that following radiation suggested that 2-chlorodeoxyadenosine broke only double strands. Cycloheximide inhibited the breakage of DNA double strands and the cell death caused by this compound. Flow cytometric studies of cytostasis brought about by 2-chlorodeoxyadenosine in FM3A cells showed that cells accumulated in the earlier part of the S phase. 2-Chlorodeoxyadenosine decreased DNA synthesis more than RNA or protein synthesis. The breaks in double strands of DNA were probably important in the cell death caused by 2-chlorodeoxyadenosine. The intracellular dNTP imbalance may trigger these events.

2,6-Diaminopurinedeoxyriboside as a prodrug of deoxyguanosine in L1210 cells.

The mode of action of the antiproliferative nucleoside analogue 2,6-diaminopurinedeoxyriboside (DAPdR) has been characterized in cultured L1210 cells. A marked concentration-dependent decrease in DNA synthesis and ribonucleotide reductase activity occurred in L1210 cells exposed to 0.05 to 1.0 mM DAPdR. Concomitantly, dGTP levels increased as much as 1100-fold as compared to untreated controls. Adenosine deaminase efficiently catalyzed DAPdR conversion to deoxyguanosine in vitro. In a comparative study, DAPdR and deoxyguanosine gave similar results. A 50% inhibition of cell growth during a 72-h incubation was achieved with 0.14 mM DAPdR or 0.26 mM deoxyguanosine. Deoxycytidine rescued the L1210 cells from DAPdR and deoxyguanosine toxicity to the same extent. DAPdR and deoxyguanosine counteracted the toxic effects of mycophenolic acid with the same efficiency. While DAPdR was not metabolized to its 5'-triphosphate, 2,6-diaminopurine was converted to 2,6-diaminopurineriboside 5'-triphosphate in L1210 cells; accordingly 50% inhibition of cell growth occurred at 0.015 mM 2,6-diaminopurine. Combinations of DAPdR with erythro-9-(2-hydroxy-3-nonyl)adenine or deoxycoformycin resulted in antagonism instead of an expected synergism. These data suggest that DAPdR exerts its toxicity on L1210 cells as a prodrug of deoxyguanosine.

Synergistic inhibition of leukemia L1210 cell growth in vitro by combinations of 2-fluoroadenine nucleosides and hydroxyurea or 2,3-dihydro-1H-pyrazole[2,3-a]imidazole.

9-beta-D-Arabinofuranosyl-2-fluoroadenine (2-F-ara-A) and 2-fluoro-2'-deoxyadenosine (2-FdAdo) were potent inhibitors of L1210 cell growth in culture. Even though these 2-fluoroadenine nucleosides are very poor substrates for adenosine deaminase, erythro-9-(2-hydroxyl-3-nonyl)adenine potentiated the growth-inhibitory properties of 2-FdAdo but not 2-F-ara-A in a synergistic manner. 2-FdAdo and 2-F-ara-A inhibited the conversion of [3H]cytidine to deoxycytidine nucleotides and incorporation into DNA, suggesting that ribonucleotide reductase was an intracellular site of action. 2-F-ara-A (6 microM) in combination with 2,3-dihydro-1H-pyrazole[2,3-a]imidazole gave synergistic inhibition of L1210 cell growth. At lower concentrations of 2-F-ara-A, the inhibition by this combination was only additive. The addition of Desferal to the combination of 2-F-ara-A plus 2,3-dihydro-1H-pyrazole[2,3-a]imidazole provided a strong synergistic combination. Similar results were obtained with combinations which included F-ara-A, hydroxyurea, and Desferal. The combinations of 2-FdAdo plus 2,3-dihydro-1H-pyrazole[2,3-a]imidazole or hydroxyurea gave strong synergistic inhibition of L1210 cell growth, even at the lowest concentration of 2-FdAdo (0.6 microM) studied. The presence of Desferal in the combination served to further potentiate the synergism.

Inhibition of dimethyl sulfoxide-induced differentiation of Friend erythroleukemic cells by 5'-methylthioadenosine.

5'-Methylthioadenosine is a sulfur-containing nucleoside derived from the metabolism of polyamines which is known to exert an antiproliferative effect on several cell systems in vitro, including the Friend leukemia cell system. We have investigated the role of 5'-methylthioadenosine on the dimethyl sulfoxide-induced differentiation of this system. At a concentration of 400 microM, the drug strongly inhibited (80%) the induced differentiation of Friend cells, and this effect was already observable at a concentration as low as 10 microM (36% inhibition), as evidenced by the benzidine staining procedure and by the dot-blot hybridization of globin mRNA with a human beta-globin probe. Similar results have been obtained by using 5'-S-isobutylthioadenosine, which is a synthetic structural analogue of 5'-methylthioadenosine. The block of differentiation produced by these nucleosides was not mediated by adenine (a catabolite of both molecules) and was not reverted by spermine or spermidine, the two polyamines whose synthesis is inhibited by 5'-methylthioadenosine. We report a decrease of the aminopropyltransferases activities (the enzymes responsible for 5'-methylthioadenosine biosynthesis) in dimethyl sulfoxide-treated Friend cells, which could lead to a decrease of the intracellular content of 5'-methylthioadenosine during the erythroid maturation of Friend cells. The results obtained are consistent with the hypothesis that 5'-methylthioadenosine may act as an endogenous regulator of Friend cell differentiation.

Effects of cytotoxicity of 2-chloro-2'-deoxyadenosine and 2-bromo-2'-deoxyadenosine on cell growth, clonogenicity, DNA synthesis, and cell cycle kinetics.

The cytotoxic effects of the adenosine deaminase resistant analogues 2-bromo-2'-deoxyadenosine (2-BrdAdo) and 2-chloro-2'-deoxyadenosine (2-CldAdo) have been compared with those of deoxyadenosine (dAdo). Like 2-CldAdo, 2-BrdAdo is highly effective in inhibiting the growth of many T-lymphoblastoid, B-lymphoblastoid, and myeloid cell lines in culture. Concentrations required to inhibit growth of CCRF-CEM human T-lymphoblastoid cells by 50% (IC50) are: 2-CldAdo, 0.045 microM; 2-BrdAdo, 0.068 microM; dAdo, 0.9 microM in the presence of 5 microM erythro-9-(2-hydroxy-3-nonyl)adenine. Like dAdo, 2-BrdAdo causes a much greater decrease in DNA synthesis than in RNA and protein synthesis. For each of the nucleosides the concentration required to cause 50% inhibition of DNA synthesis (as measured by thymidine incorporation) in an 18-h exposure is very similar to the IC50 for growth and to the concentration required to decrease viability (clonogenicity) over 18 h by 50% (EC50). A fraction of CCRF-CEM cells (approximately equal to 30%) is resistant to killing by exposure to 2-BrdAdo or 2-CldAdo for 4 h at concentrations 100 times the EC50, but 3% of cells are resistant to exposure for 4 h to a concentration of dAdo 3 times the EC50. Each of the three nucleosides causes accumulation of cells in S phase, the accumulation becoming more marked with longer periods of exposure and with higher concentrations of nucleoside. During exposures for 18-24 h at a concentration of nucleoside near the EC50 most cells accumulate in S, with most in early S, whereas exposure to concentrations greater than EC95 accumulates cells at the G1/S border. This suggests that loss of viability is associated with a blockade of some process specifically occurring at the initiation of S phase. At an optimum dosage schedule, 2-BrdAdo and 2-CldAdo have similar therapeutic effects against L1210 in vivo, both producing over 99% cell kill, but the optimum dosage of 2-CldAdo is lower.

Biochemical pharmacology of 2-chlorodeoxyadenosine in malignant human hematopoietic cell lines and therapeutic effects of 2-bromodeoxyadenosine in drug combinations in mice.

Growth of human hematopoietic cell lines showed a 100-fold range of sensitivity to inhibition by 2-chloro-2'-deoxyadenosine (CldAdo), with highly sensitive lines in all three groups: T-lymphoblastic, B-lymphoblastic, and non-T, non-B. Formation of nucleotides from [8-3H]CldAdo was investigated in ten lines. In cells exposed to 0.15 microM CldAdo, CldAdo 5'-phosphate (CldAMP) reached 0.7-14 microM and CldAdo 5'-triphosphate (CldATP) reached 0.05-6 microM in 1 h. In most cases these nucleotide concentrations at 1 h were close to the steady-state concentrations, and the latter concentrations were approximately proportional to extracellular CldAdo concentration. On removal of extracellular CldAdo, intracellular CldAMP and CldATP declined rapidly with half times of 0.56-0.9 and 0.64-1.46 h, respectively. There was no correlation between these rates of catabolism and steady-state levels. The different sensitivities of the lines to CldAdo is explained only in part by the different steady-state concentrations of CldATP, and must be more directly related to differential effects on target enzymes. Mice inoculated with L1210 leukemia were treated with 2-bromo-2'-deoxyadenosine (BrdAdo) paired with one of 18 other therapeutic agents. Eight of the drugs paired with BrdAdo gave therapeutic responses from the combination greater than the sum of the responses of members of the pair. They included alkylating agents, antimetabolites blocking deoxyribonucleotide synthesis, and DNA polymerase inhibitors. Toxic dosages of CldAdo caused damage chiefly to the hemic-lymphatic systems and the kidneys.

Two approaches that increase the activity of analogs of adenine nucleosides in animal cells.

Deamination of many analogs of adenine nucleosides results in the loss of their chemotherapeutic efficacy. Two approaches have been used in this study to overcome this problem. First, some adenine nucleotides, which are resistant to mammalian adenosine deaminase, are more toxic to animal cells than are the respective nucleosides. For toxic to animal cells than are the respective nucleosides. For example, 9-beta-D-arabinofuranosyladenine 5'-phosphate, a molecule that penetrates the cell without degradation, has a more sustained toxicity against mouse fibroblasts (L-cells) than does 9-beta-D-arabinofuranosyladenine (ara-A). Furthermore, L-cells treated with 2',3'-dideoxyadenosine 5'-phosphate are extensively killed after 48 hr, whereas 2',3'-dideoxyadenosine is almost nontoxic to L-cells. Specific inhibition of adenosine deaminase by nontoxic concentrations of erythro-9-(2-hydroxy-3-nonyl)adenine greatly potentiates the biological activity of both ara-A and 3'-deoxyadenosine (cordycepin). Simultaneous administration of cytostatic concentrations of ara-A and the inhibitor of adenosine deaminase to L-cells killed greater than 99.9 percent of cells in 36 hr. A similar concentration of ara-A plus the deaminase inhibitor also markedly extended the mean survival of mice bearing Ehrlich ascites carcinoma as compared to ara-A alone. A cytostatic concentration of cordycepin 1 x 10-4 M), administered in the presence of deaminase inhibitor, killed greater than 99.9 percent of cultured L-cells in only 8 hr. During the latter incubation, accumulation of uridine in acid-insoluble material reached a maximum after 30 min, and incorporation of thymidine into acid-insoluble material was almost totally arrested after 2 hr.

Comparison of theoretical and experimental approaches to determination of conformation of nucleosides about the glycosidic bond.

A study has been made by means of 1H-NMR spectroscopy of the syn in equilibrium anti dynamic equilibrium about the glycosidic bond for 5'-deoxyadenosine and some 8-substituted analogues, in different solvents. The results are compared with those previously obtained for the parent adenosine and its 8-substituted analogues. Quantum chemical calculations, with the aid of the Classical Potential and PCILO procedures, were applied to obtain the energies for different conformations of the base in adenosine and 5'-deoxyadenosine, and their 8-methyl and 8-halogeno derivatives. Good agreement was found between experimentally determined conformations in solution and those corresponding theoretically to the energy minima, particularly those calculated by the PCILO method. Comparison of the quantitative experimental data with the theoretical results was used to evaluate the validity of the latter and their applicability to studies of nucleoside conformation. The experimental and theoretical findings pointed to the existence of a marked flexibility about the glycosidic bond of the parent nucleosides and their 8-substituted analogues, when the 8-substituents were not too bulky, such as methyl or bromine. Considerations is given to possible correlations between conformational parameters in nucleosides and their 5'-deoxy analogues. It is shown that the proposed stabilization of the conformation syn by intramolecular hydrogen bonding, 5'-OH...N(3), is not in accord with the results of the present study.

Poly(8-bromo-2'-deoxyadenylic acid): the syn/anti relationship.

The synthesis of a high-molecular-weight, putatively all-syn DNA analogue, poly(8-bromo-2'-deoxyadenylic acid), is described. The syn----anti transition was shown to be both salt and temperature dependent. Conditions were found which favored 'normal' Watson-Crick pairing and duplex formation with poly(dT).

The binding of 1,N6-ethenoNAD to bovine liver glutamate dehydrogenase: studies using the time-correlated single photon counting fluorescence technique.

The time-correlated single photon counting (TCPC) fluorescence technique has been used as a novel approach to investigate ligand-protein interaction, for the case of the binding of the fluorescent coenzyme analogue 1,N6-ethenoNAD (epsilon NAD) to bovine liver glutamate dehydrogenase in the presence of glutarate, a substrate analogue which stabilizes the complex. System calibration was performed using solutions of epsilon ADP and carefully purified epsilon NAD mixed at variable molar ratios (pH 7.0, 0.05 M sodium phosphate buffer, 20 degrees C). The fluorescence lifetimes obtained after deconvolution were 2.4 ns (for epsilon NAD) and 23 ns (for epsilon ADP), in good agreement with literature values obtained under similar conditions. epsilon NAD binds to glutamate dehydrogenase in the presence of 50 mM glutarate, with a fluorescence quantum yield enhancement factor, Q, of about 17-fold, as previously reported (Favilla, R. and Mazzini, A. (1984) Biochim. Biophys. Acta 48-57). For this system, fluorescence lifetime values were obtained after deconvolution as 2.4 ns for free epsilon NAD and 21 ns for bound epsilon NAD. These values did not vary appreciably with enzyme concentration nor with degree of saturation, thus reflecting the existence of only one spectroscopically relevant type of complex. Addition of either GTP or ADP did not affect the lifetime of epsilon NAD bound to the enzyme, but only its affinity, thus allowing calculations of binding strengths. In the case of a simple binding (i.e., in the absence of GTP) the dissociation constant of the complex could be derived from a simple relationship, in which only the ratio between the pre-exponential factors and the parameter gamma, which represents the molar fraction of epsilon NAD molecules free in solution in the open conformation, are to be taken into account. The results are in good agreement with those reported by some of us (reference above) using a steady-state fluorescence technique, which by itself is, however, unable to resolve the number of relevant species present in the system.

Inhibition of lipolysis and cyclic AMP accumulation by adenosine analogues in hamster epididymal adipocytes exposed to cholera toxin.

The effects of adenosine, N6-phenylisopropyl adenosine and 2',5'-dideoxyadenosine on lipolysis and cyclic AMP accumulation, in hamster adipocytes treated with cholera toxin, were studied. Cholera toxin caused an increase in lipolysis and cyclic AMP accumulation that was dependent upon the concentration of toxin and the length of time cells were exposed to the toxin. When N6-phenylisopropyl adenosine or 2',5'-dideoxyadenosine were present, the lipolytic and cyclic AMP responses to cholera toxin were inhibited. The adenosine analogues were equally effective inhibitors of lipolysis and cyclic AMP accumulation, when they were added 1 or 2 h after exposure to the toxin. Enzymatic removal of endogenously produced adenosine with adenosine deaminase potentiated both the lipolytic and cyclic AMP responses to cholera toxin. In addition, the inhibitory effects of N6-phenylisopropyl adenosine, 2'5'-dideoxyadenosine and clonidine on lipolysis and cyclic AMP were enhanced consequent to enzymatic removal of adenosine. These data show responses of intact fat cells to N6-phenylisopropyl adenosine, 2',5'-dideoxyadenosine or removal of endogenous adenosine and provide evidence for an adenosine sensitivity of fat cells exposed to cholera toxin.

Choline turnover in phosphatidylcholine of pancreatic islets. Implications for CDP-choline pathway.

The CDP-choline pathway is the major route of phosphatidylcholine (PC) biosynthesis in mammalian cells. The incorporation of [14C]choline into PC of isolated pancreatic islets of the rat was time dependent, glucose stimulable, and inhibited by mannoheptulose. Removal of extracellular Ca2+ enhanced glucose-stimulated choline incorporation without affecting basal levels. Glucose stimulated PC synthesis in islets labeled to equilibrium with 32PO4 in the presence or absence of extracellular Ca2+. The water-soluble intermediates of the CDP-choline pathway, phosphorylcholine and CDP-choline, accumulated to a lesser extent under Ca2+-free conditions; however, glucose enhanced the levels of these intermediates in the presence and absence of Ca2+. Thus, glucose stimulates CDP-choline-pathway activity. Ca2+-free conditions may promote flux of choline intermediates through the pathway and retard the hydrolysis of PC. The phospholipase A2-activating agents delta-9-tetrahydrocannabinol and melittin enhanced [3H]choline incorporation into PC and potentiated incorporation in response to a submaximal secretagogic concentration of glucose (8.5 mM); insulin release paralleled the changes in PC. p-Bromophenacyl bromide and mepacrine reduced islet glucose utilization and glucose-stimulated [3H]choline levels in PC. An inhibitor of CTP: phosphorylcholine cytidylyltransferase, 5'-deoxy-5'-isobutylthioadenosine, reduced glucose-stimulated [14C]choline incorporation into PC; insulin release was inhibited in a parallel fashion. Thus, islet PC turnover and CDP-choline pathway activity appear to be modulated by glucose metabolism and membrane phospholipid hydrolysis. PC turnover and insulin release appear to be related.

Regulation of sodium-dependent phosphate transport by parathyroid hormone in opossum kidney cells: adenosine 3',5'-monophosphate-dependent and -independent mechanisms.

The hormonal regulation of Na+-dependent phosphate transport was studied in opossum kidney (OK) cells. PTH caused time- and concentration-dependent decreases in Na+-dependent phosphate transport, with 10 pM PTH-(1-34) producing a 19% decline in phosphate transport. The EC50 for PTH inhibition of phosphate transport was 50 pM. Kinetic analyses of phosphate transport indicated that PTH decreased the maximum velocity without affecting the Km for phosphate. PTH increased cAMP formation with an EC50 of 10 nM. 8-Bromo-cAMP and (Bu)2cAMP also inhibited phosphate transport. Forskolin increased cAMP formation and decreased phosphate transport, whereas the cyclase-inactive forskolin analog 1,9-dideoxyforskolin also inhibited phosphate transport. The PTH analog [8,18-norleucine,34-tyrosinamide]PTH-(3-34) reduced phosphate transport at concentrations from 10 nM to 30 microM, but did not increase cAMP formation at concentrations up to 10 microM. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine produced concentration-dependent decreases in PTH-stimulated cAMP formation, but did not influence PTH inhibition of Na+-dependent phosphate transport. Vasoactive intestinal polypeptide and prostaglandin E1 increased cAMP formation in OK cells, but were weak inhibitors of phosphate transport. This study suggests that cAMP may not be the only transmembrane signaling mechanism involved in the regulation of Na+-dependent phosphate transport by PTH-(1-34) in OK cells.

Adenosine inhibits prolactin and growth hormone secretion in a clonal pituitary cell line.

Although purine nucleosides have been shown to regulate the secretion of several peptide and steroid hormones, effects on pituitary hormone release have not been reported. We show here that in the clonal GH4C1 pituitary cell line maximal concentrations of adenosine (greater than or equal to 50 microM) inhibited PRL and GH secretion by 40%. Adenosine deaminase abolished the inhibitory effect of adenosine but not that of SRIF or (-)N6(R-2-phenylisopropyl)adenosine (PIA), a nonhydrolyzable adenosine analog. Furthermore, this enzyme increased basal secretion by 50%, and analysis of the incubation medium by HPLC demonstrated that the cells secreted biologically effective concentrations of adenosine. These results indicate that adenosine produced in culture tonically inhibits hormone release. In other target cells, adenosine inhibition is mediated by two types of binding sites: an extracellular Ri-site requiring an intact ribose moiety or an intracellular P-site requiring an intact purine ring. Four lines of evidence indicate that in GH4C1 cells, adenosine acts at an Ri-site. PIA, an Ri-site-specific agonist, was a potent inhibitor of hormone release (ED50 = 30 nM). Theophylline, an Ri-site antagonist, competitively inhibited the action of PIA (Ki = 2.4 microM). 3) 2'5'-Dideoxyadenosine, a P-site-specific agonist, did not inhibit PRL release even at a concentration of 1 mM. 4) Dipyridamole, an adenosine uptake inhibitor, did not reduce adenosine inhibition. In addition to its effect on basal secretion, PIA inhibited stimulation of hormone release by vasoactive intestinal peptide and TRH. PIA also reduced vasoactive intestinal peptide-stimulated cAMP accumulation by 75%, consistent with its action to inhibit adenylate cyclase via Ri receptors in other targets. Since PIA inhibition of PRL release and cAMP accumulation was not additive with the effects of SRIF and carbamyl choline, these inhibitors may act via a common rate-limiting step. Our results demonstrate that adenosine activates an Ri-type of adenosine receptor in GH4C1 cells and that the production of adenosine under normal culture conditions causes autocrine inhibition of secretion.

The effects of 1-aminooxy-3-aminopropane and S-(5'-deoxy-5'-adenosyl)methylthioethylhydroxylamine on ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase from Escherichia coli.

1-Aminooxy-3-aminopropane (APA) was shown to be a potent competitive inhibitor (Ki = 1.0 nM) of partially purified Escherichia coli ornithine decarboxylase. APA did not inhibit S-adenosyl-L-methionine decarboxylase and spermidine from E. coli. S-(5'-Deoxy-5'-adenosyl)methylthioethylhydroxylamine (AMA), which is a structural analogue of decarboxylated S-adenosyl-L-methionine, was for the first time shown to be an irreversible inhibitor of bacterial S-adenosyl-L-methionine decarboxylase and a competitive inhibitor (Ki = 47 microM) of bacterial ornithine decarboxylase. AMA had no effect on spermidine synthase.