Nonmethylated CG motifs packaged into virus-like particles induce protective cytotoxic T cell responses in the absence of systemic side effects.

DNA rich in nonmethylated CG motifs (CpGs) greatly facilitates induction of immune responses against coadministered Ags. CpGs are therefore among the most promising adjuvants known to date. Nevertheless, CpGs are characterized by two drawbacks. They have unfavorable pharmacokinetics and may exhibit systemic side effects, including splenomegaly. We show in this study that packaging CpGs into virus-like particles (VLPs) derived from the hepatitis B core Ag or the bacteriophage Qbeta is a simple and attractive method to reduce these two problems. CpGs packaged into VLPs are resistant to DNase I digestion, enhancing their stability. In addition, and in contrast to free CpGs, packaging CpGs prevents splenomegaly in mice, without affecting their immunostimulatory capacity. In fact, vaccination with CpG-loaded VLPs was able to induce high frequencies of peptide-specific CD8(+) T cells (4-14%), protected from infection with recombinant vaccinia viruses, and eradicated established solid fibrosarcoma tumors. Thus, packaging CpGs into VLPs improves both their immunogenicity and pharmacodynamics.

An extranuclear locus of cAMP-dependent protein kinase action is necessary and sufficient for promotion of spiral ganglion neuronal survival by cAMP.

We showed previously that cAMP is a survival-promoting stimulus for cultured postnatal rat spiral ganglion neurons (SGNs) and that depolarization promotes SGN survival in part via recruitment of cAMP signaling. We here investigate the subcellular locus of cAMP prosurvival signaling. Transfection of GPKI, a green fluorescent protein (GFP)-tagged cAMP-dependent protein kinase (PKA) inhibitor, inhibits the ability of the permeant cAMP analog cpt-cAMP [8-(4-chlorophenylthio)-cAMP] to promote survival, indicating that PKA activity is necessary. Transfection of GFP-tagged PKA (GPKA) is sufficient to promote SGN survival, but restriction of GPKA to the nucleus by addition of a nuclear localization signal (GPKAnls) almost completely abrogates its prosurvival effect. In contrast, GPKA targeted to the extranuclear cytoplasm by addition of a nuclear export signal (GPKAnes) promotes SGN survival as effectively as does GPKA. Moreover, GPKI targeted to the nucleus lacks inhibitory effect on SGN survival attributable to cpt-cAMP or depolarization. These data indicate an extranuclear target of PKA for promotion of neuronal survival. Consistent with this, we find that dominant-inhibitory CREB mutants inhibit the prosurvival effect of depolarization but not that of cpt-cAMP. SGN survival is compromised by overexpression of the proapoptotic regulator Bad, previously shown to be phosphorylated in the cytoplasm by PKA. This Bad-induced apoptosis is prevented by cpt-cAMP or by cotransfection of GPKA or of GPKAnes but not of GPKAnls. Thus, cAMP prevents SGN death through a cytoplasmic as opposed to nuclear action, and inactivation of Bad proapoptotic function is a mechanism by which PKA can prevent neuronal death.

Inhibition of spontaneous acetylcholine secretion by 2-chloroadenosine as revealed by a protein kinase inhibitor at the mouse neuromuscular junction.

1. Previous studies have reported discrepancies in the potencies of A(1) adenosine receptor agonists at mouse motor nerve terminals. In addition, conflicting results on the role of protein kinase A (PKA) in mediating the inhibitory effects of A(1) receptor agonists have been published. We thus decided to investigate the possibility of endogenous control of adenosine receptor sensitivity by protein kinases, using a variety of protein kinase inhibitors in conjunction with the adenosine receptor agonist 2-chloroadenosine (CADO). 2. CADO, at the concentration employed previously to study spontaneous ACh release in the mouse (1 microM), did not inhibit spontaneous ACh release in our experiments. However, a higher concentration of CADO (10 microM) produced highly statistically-significant reductions in spontaneous ACh release. 3. In the presence of the non-selective protein kinase inhibitor, H7 (50 microM), the potency of CADO was increased such that 1 microM CADO now reduced spontaneous quantal ACh release to approximately 63% of control. 4. Both H7, and the selective PKA inhibitor, KT5720 (500 nM) prevented increases in ACh release produced by CPT cyclic AMP (250 microM), suggesting these kinase inhibitors were blocking PKA. In contrast to H7, however, KT5720, did not reveal an inhibitory effect of 1 microM CADO. A number of other non-selective PKA inhibitors also failed to increase the potency of CADO. 5. The results suggest that an endogenous H7-sensitive process modulates the sensitivity of the mouse A(1) adenosine receptor and that the inhibitory effects of CADO are independent of cyclic AMP accumulation or PKA inhibition.

Evaluation of reactive blue 2 derivatives as selective antagonists for P2Y receptors.

P2Y receptor pharmacology is hampered by a lack of subtype selective antagonists. However, a recent study evaluated series of compounds, structurally related to the dye reactive blue 2, for their antagonist selectivity at P2X vs. P2Y receptors. Acid blue 129, acid blue 80, acid blue 25 and acid violet 34 were found to be the most potent of the antagonists studied, at P2Y receptors [Naunyn Schmiedeberg's Arch. Pharmacol. 357 (1998) 111]. In this study, we have determined the ability of these four agents to selectively antagonize inositol phosphate turnover mediated by P2Y1 and P2Y2 receptors that are natively expressed in bovine aortic endothelial (BAE) cells. Acid blue 129, acid blue 80, and acid violet 34 shifted the dose-response curve of the P2Y1 agonist 2-methylthio adenosine trisphosphate (2MeSATP) to the right. Acid blue 129 and acid blue 80 were also very weak antagonists of the P2Y2 agonist uridine 5'-triphosphate (UTP). At 30 and 100 microM, acid violet 34 failed to have any significant effect on the dose-response to UTP. However, at 10 microM, acid violet 34 enhanced the UTP responses. Acid blue 80, acid blue 129 and acid violet 34 are P2Y vs. P2X selective, but show poor selectivity between P2Y1 and P2Y2 receptors and are therefore of limited use in the field of P2Y receptor pharmacology. Furthermore, contrary to previous reports, acid blue 25 is not a P2Y-selective antagonist.

The role of cell surface receptors in the activation of human B cells by phosphorothioate oligonucleotides.

Phosphorothioate oligodeoxynucleotides (sODN) containing the CpG motif or TCG repeats induce T cell-independent polyclonal activation of human B cells. To elucidate the mechanism of this response, the role of cell surface receptors was investigated. Sepharose beads coated with stimulatory but not nonstimulatory sODNs induced B cell proliferation comparably with soluble sODNs. The B cell stimulatory activity of Sepharose-bound sODN did not result from free sODN released from the beads since media incubated with coated beads were inactive. Using FITC-labeled sODNs as probes, binding to human B cells could be detected by flow cytometry. Binding was rapid, saturable, initially temperature independent, but with a rapid off-rate. Competition studies indicated that both stimulatory sODNs and minimally stimulatory sODNs bound to the same receptor. By contrast, phosphodiester oligonucleotides with the same nucleotide sequence as sODNs and bacterial DNA inhibited the binding of sODNs to B cells minimally. Charge appeared to contribute to the binding of sODNs to B cells since binding of sODNs was competitively inhibited by negatively charged molecules, including fucoidan, poly I, and polyvinyl sulfate. These data indicate that human B cells bind sODNs by a receptor-mediated mechanism that is necessary but not sufficient for polyclonal activation.

Adenosine-derived non-phosphate antagonists for P2Y(1) purinoceptors.

Novel type antagonists for P2Y(1) adenine nucleotide receptors were synthesized by coupling of adenosine 5'-OH group with oligo-aspartate chain via a carbonyl linker. All these conjugates (AdoOC(O)Asp(n), n = 1-4) inhibited the 2MeSADP-stimulated synthesis of inositol phosphates in 1321N1 human astrocytoma cells stably expressing human P2Y(1) receptors. This inhibitory effect followed the rank order AdoOC(O)Asp(2)> AdoOC(O)Asp(3)> AdoOC(O)Asp(1)> AdoOC(O)Asp(4) with antagonistic constant pA(2) = 5.4 for AdoOC(O)Asp(2). Potency of this non-phosphate inhibitor was comparable with the previously known adenosine 3',5'- and 2', 5'-bisphosphates. Chemical and biological stabilities of these novel adenosine derived antagonists of the nucleotide receptor provide perspectives of their pharmacological implication.

Adenosine triphosphate is full antagonist at human P2Y(1) purinoceptors.

Both agonistic and antagonistic effects have been reported for ATP at P2Y(1) purinoceptors at micromolar ligand concentrations. These conflicting data hamper specification of the true pharmacological profile as well as structural requirements for antagonistic ligands of this receptor. In this report the type of ATP activity at human P2Y(1) receptors in hP2Y(1)-1321N1 cells was revisited. In parallel, kinetics of degradation of ATP in the assay mixture was analysed. It was found that transformation of this ligand to ADP was responsible for initiation of synthesis of inositol phosphates, observed in the presence of ATP in hP2Y(1)-1321N1 cells. This agonistic effect was abolished in the presence of the triphosphate regeneration system (CP/CPK). On the other hand, if the agonistic effect caused by degradation product of ATP was taken into consideration, this ligand behaved as a full antagonist at P2Y(1) receptors and was characterized by the apparent inhibitory constant 5 microM.

Benzoyl ATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation.

The effects of 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) on intracellular Ca2+ mobilization and cyclic AMP accumulation were investigated using rat brain capillary endothelial cells which express an endogenous P2Y1 receptor, human platelets which are known to express a P2Y1 receptor, and Jurkat cells stably transfected with the human P2Y1 receptor. In endothelial cells, BzATP was a competitive inhibitor of 2-methylthio ADP (2-MeSADP) and ADP induced [Ca2+]i responses (Ki = 4.7 microM) and reversed the inhibition by ADP of adenylyl cyclase (Ki = 13 microM). In human platelets, BzATP inhibited ADP-induced aggregation (Ki = 5 microM), mobilization of intracellular Ca2+ stores (Ki = 6.3 microM), and inhibition of adenylyl cyclase. In P2Y1-Jurkat cells, BzATP inhibited ADP and 2-MeSADP-induced [Ca2+]i responses (Ki = 2.5 microM). It was concluded that BzATP is an antagonist of rat and human P2Y1 receptors and of platelet aggregation. In contrast to other P2Y1 receptor antagonists (A2P5P and A3P5P) which inhibit only ADP-induced Ca2+ mobilization, BzATP inhibits both the Ca2+- and the cAMP-dependent intracellular signaling pathways of ADP. These results provide further evidence that P2Y1 receptors contribute to platelet ADP responses.

Antagonism of immunostimulatory CpG-oligodeoxynucleotides by quinacrine, chloroquine, and structurally related compounds.

Phosphorothioate oligodeoxynucleotides containing CpG (CpG-ODN) activate immune responses. We report that quinacrine, chloroquine, and structurally related compounds completely inhibit the antiapoptotic effect of CpG-ODN on WEHI 231 murine B lymphoma cells and inhibit CpG-ODN-induced secretion of IL-6 by WEHI 231. They also inhibit IL-6 synthesis and thymidine uptake by human unfractionated PBMC induced by CpG-ODN. The compounds did not inhibit LPS-induced responses. Half-maximal inhibition required 10 nM quinacrine or 100 nM chloroquine. Inhibition was noncompetitive with respect to CpG-ODN. Quinine, quinidine, and primaquine were much less powerful. Quinacrine was effective even when added after the CpG-ODN. Near-toxic concentrations of ammonia plus bafilomycin A1 (used to inhibit vesicular acidification) did not reduce the efficacy of the quinacrine, but the effects of both quinacrine and chloroquine were enhanced by inhibition of the multidrug resistance efflux pump by verapamil. Agents that bind to DNA, including propidium iodide, Hoechst dye 33258, and coralyne chloride did not inhibit CpG-ODN effect, nor did 4-bromophenacyl bromide, an inhibitor of phospholipase A2. Examination of the structure-activity relationship of seventy 4-aminoquinoline and 9-aminoacridine analogues reveals that increased activity was conferred by bulky hydrophobic substituents on positions 2 and 6 of the quinoline nucleus. No correlation was found between published antimalarial activity and ability to block CpG-ODN-induced effects. These results are discussed in the light of the ability of quinacrine and chloroquine to induce remission of rheumatoid arthritis and lupus erythematosus.

Evidence for independent Cl- and HCO3- secretion and involvement of an apical Na(+)-HCO3- cotransporter in cultured rat epididymal epithelia.

Electrogenic chloride and bicarbonate secretion by cultured rat epididymal epithelia was studied using the short-circuit current (ISC) technique. When incubated in normal solution, 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (cpt-cAMP) caused a rise in the ISC, which was attributable to Cl- and HCO3- secretion. Cl- secretion was found to contribute to the initial transient phase, whereas HCO3- secretion contributed to the sustained phase of the response. HCO3- secretion involves a basolaterally placed Na(+)-H+ exchanger and apical anion channel, most probably the cystic fibrosis transmembrane conductance regulator (CFTR). There is also evidence that an apical electrogenic Na(+)-HCO3- cotransporter is involved in HCO3- exit. CFTR accounted for 70% of HCO3- secretion, while the Na(+)-HCO3- cotransporter accounted for 30%. The possibility that the cotransporter may serve as an alternative pathway for HCO3- secretion in cystic fibrosis is discussed.

P2-purinoceptors mediating spasm of the isolated uterus of the non-pregnant guinea-pig.

1. The isolated uterus of the non-pregnant guinea-pig has been suggested to contain P1-, and possibly P2-purinoceptors mediating spasm. The presence of P1-purinoceptors has been confirmed and these receptors have been further characterized. 2. In the presence of the adenosine uptake inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI, 300 nM) and a pA100 concentration of the P1-purinoceptor antagonist 8-sulphophenyltheophylline (140 microM), the potency order of agonists as spasmogens was: 2 methylthio ATP >> alpha,beta methylene ATP = UTP = ATP >> beta,gamma methylene ATP. This order is not consistent with any single recognised P2-purinoceptor subtype. 3. Indomethacin (1 microM) treatment abolished responses to 2 methylthio ATP, alpha,beta methylene ATP and UTP, while spasm to ATP was significantly inhibited. When the endometrial and circular smooth muscle cell layers were removed, spasmogenic responses to ATP, 2 methylthio ATP, alpha,beta methylene ATP and UTP were significantly reduced. 4. 2-methylthio ATP was able to cause desensitization to itself, but not to UTP, indicating that these agonists act at different receptor sites. 5. The P2-purinoceptor antagonist, suramin antagonized 2 methylthio ATP with a PA2 of 5.9 +/- 0.3. Suramin was also an antagonist of ATP and UTP. In the case of ATP, the antagonism was not dependent on suramin concentration, while for UTP the interaction appeared to be non-equilibrium. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 10 microM) had no effect on spasm to ATP, UTP or 2 methythio ATP. 6. In the presence of indomethacin, responses to ATP were unaffected by 8-sulphophenyltheophylline (140 microM) or by suramin (100 microM), but PPADS (10 microM) antagonized ATP. 7. These results suggest that the isolated uterus of the non-pregnant guinea-pig contains a mixture of P2-purinoceptors. P2U- (or UTP-selective pyrimidinoceptors) and P2Y-purinoceptors appear to be present, probably mainly located on the endometrial or circular smooth muscle layer. Activation of these receptors leads to spasm via increases in prostanoid generation. There appears also to be a third class of non-P2X-, non p2Y-purinoceptor present, at which ATP is an agonist and PPADS is an antagonist, located on the longitudinal smooth muscle, activation of which causes spasm independent of changes in prostanoids.

Liddle's disease: abnormal regulation of amiloride-sensitive Na+ channels by beta-subunit mutation.

Liddle's disease is an autosomal dominant genetic disorder characterized by severe low renin hypertension ("pseudoaldosteronism") that has been genetically linked to a locus on chromosome 16 encoding the beta-subunit of an amiloride-sensitive Na+ channel (ASSC) (15). Peripheral blood lymphocytes (PBL) express ASSC that are functionally indistinguishable from those expressed by Na(+)-reabsorbing renal epithelial cells (3, 5). The amiloride-sensitive Na+ conductance in PBL from affected and unaffected individuals from the original Liddle's pedigree was examined using whole cell patch clamp. Typically, the basal Na+ currents in cells from affected individuals were maximally activated. Basal Na+ currents in cells from unaffected individuals were minimal and could be maximally activated by superfusion with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Affected cells could not be further stimulated with CPT-cAMP. Superfusion with a supermaximal concentration of amiloride (2 microM) inhibited both the cAMP-activated Na+ conductance in unaffected cells and the constitutively activated inward conductance in affected cells. Cytosolic addition of a peptide identical to the terminal 10 amino acids of the truncated beta-subunit normalized the cAMP-mediated but not the pertussis toxin-induced regulation of the mutant ASSC. The findings show that lymphocyte ASSC are constitutively activated in affected individuals, that a mutation of the beta-subunit alters ASSC responsiveness to specific regulatory effectors, and that the cellular mechanism responsible for the pathophysiology of Liddle's disease is abnormal regulation of Na+ channel activity. These findings have important diagnostic and therapeutic implications and provide a cellular phenotype for the diagnosis of pseudoaldosteronism.

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

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

Osteoblast-like cells have a variable mixed population of purino/nucleotide receptors.

Osteoblast-like UMR 106.06 cells respond to extracellular application of nucleotides with a fast intracellular calcium pulse (latency of about 20 s, half-width of about 10 s), as measured with fluo-3 on a confocal laser scanning system. Cross-inhibition experiments at 50 microM show that, on a cell population basis, adenosine triphosphate (ATP) strongly inhibits the effect of uridine triphosphate (UTP) or 2-methylthio-ATP (2-MeSATP) applied within 2 min after the end of the ATP-induced pulse, while prior application of UTP or 2-MeSATP only weakly inhibits the ATP effect, and UTP and 2-MeSATP weakly inhibit each other. Furthermore, there are clear differences in cross-inhibition between individual cells. Our measurements provide strong evidence that these cells have at least two types of purino/nucleotide receptors, probably P2y and P2u, with a proportion that varies between individual cells.

1,25-dihydroxyvitamin D3 attenuates TSH and 8-(4-chlorophenylthio)-cAMP-stimulated growth and iodide uptake by rat thyroid cells (FRTL-5).

The effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on TSH and the 3',5'-cyclic adenosine monophosphate (cAMP) analogue 8-(4-chlorophenylthio)-cAMP-stimulated cell growth and iodide uptake were studied in a rat thyroid cell line (FRTL-5). 1,25-(OH)2D3 inhibited both TSH and 8-(4-chlorophenylthio)-cAMP-induced cell proliferation with the maximum effect at 100 nmol/L. The inhibitory effect of 1,25-(OH)2D3 (10 nmol/L) on TSH and 8-(4-chlorophenylthio)-cAMP-stimulated iodide uptake was observed after 2 days of incubation, and the effect was maximal after 4 days. The inhibition was dose-dependent and maximal at 100 nmol/L 1,25-(OH)2D3. 1,25-(OH)2D3 (10 nmol/L, 4 days) increased the median concentrations of TSH required to stimulate both cAMP production and iodide uptake half-maximally by 124 and 187%, respectively, whereas the median 8-(4-chlorophenylthio)-cAMP concentration was not changed. Lineweaver-Burke plots revealed that 1,25-(OH)2D3 reduced the Vmax of the sodium-driven iodide carriers to 30% of the control cells without effect on the Km. Iodide efflux was only slightly increased in the 1,25-(OH)2D3-treated cells. In conclusion, 1,25-(OH)2D3 potently inhibited the TSH-stimulated growth and iodide uptake by FRTL-5 cells both by reducing the TSH-stimulated cAMP production and by attenuating the stimulatory effects of cAMP.

PGE2 inhibits AVP-induced water flow in cortical collecting ducts by protein kinase C activation.

It is well known that prostaglandin E2 (PGE2) both inhibits arginine vasopressin (AVP)-stimulated water permeability (hydraulic conductivity, Lp) in the cortical collecting duct (CCD) or, if administered alone, modestly increases Lp in the CCD. These bifunctional effects on Lp correspond to PGE2's capacity to inhibit AVP-stimulated adenylate cyclase (AC) activity, or to singularly stimulate AC activity in the collecting duct. The present studies suggest that the inhibitory effect of PGE2 on Lp may also be mediated by phosphatidylinositol (PI) hydrolysis. Using in vitro microperfused rabbit CCDs, we show that PGE2 releases Ca from intracellular stores. We also demonstrate that the inhibitory effect of PGE2 on AVP-stimulated Lp in the CCD is significantly reversed by the protein kinase C (PKC) inhibitor, staurosporine (SSP). Although PGE2 does not reduce an established water flow response to 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (8-CPTcAMP), when the sequence of addition is reversed and PGE2 is added first, marked inhibition of 8-CPTcAMP-induced Lp is observed. This provides independent evidence that PGE2 can act through a mechanism separate from modulating AC activity. PGE2 inhibition of 8-CPTcAMP-induced Lp is reversed by SSP pretreatment. Finally, SSP pretreatment also markedly potentiates the capacity of PGE2 itself to increase Lp. We conclude that PGE2 releases Ca from intracellular stores and, by activating PKC, inhibits AVP-induced osmotic water flow. This suggests an important role for PI hydrolysis in mediating PGE2's effects on the CCD.

Aminoglycoside toxicity: pH dependent inhibition of ADH response.

The effect of aminoglycoside antibiotics on the response of the isolated toad urinary bladder to antidiuretic hormone (ADH) was investigated. Gentamicin and neomycin both acidify the serosal bathing solution and cause a dose-dependent inhibition of the hydroosmotic response to ADH, while streptomycin has minimal effect on media pH and causes no inhibition of the response to ADH. Detailed studies employing gentamicin indicate that acidification stimulates production of PGE2, a known inhibitor of the hydroosmotic response of the toad bladder to ADH. When media pH is rigidly controlled or PGE2 production is inhibited by indomethacin, the inhibitory effect of gentamicin on the response to ADH is ameliorated. These studies suggest that the defect in renal concentrating ability seen as part of aminoglycoside nephrotoxicity could be due, in part, to an acidification-induced, prostaglandin-mediated resistance to the action of ADH.

Competitive inhibition by adenosine 5'-triphosphate of the actions on human platelets of 2-chloroadenosine 5'-diphosphate, 2-azidoadenosine 5'-diphosphate and 2-methylthioadenosine 5'-diphosphate.

1 Adenosine 5'-diphosphate (ADP) induces human platelet aggregation and noncompetitively inhibits stimulated human platelet adenylate cyclase; these two effects are mediated by the same ADP receptor, at which adenosine 5'-triphosphate (ATP) is a competitive antagonist. 2 Two ADP analogues, 2-azidoadenosine 5'-diphosphate (2-azido-ADP) and 2-methylthioadenosine 5'-diphosphate (2-methylthio-ADP) have been reported to be more potent as inhibitors of adenylate cyclase than they are as aggregating agents, but no evidence has been presented that these actions are mediated solely by the ADP receptor. 3 We therefore tested the ability of ATP to inhibit the actions of these compounds and of another ADP analogue, 2-chloroadenosine 5'-diphosphate (2-chloro-ADP). 4 2-Chloro-ADP, 2-azido-ADP and 2-methylthio-ADP each induced aggregation and inhibited stimulated adenylate cyclase. Both of these actions were competitively inhibited by ATP (50 microM) with pA2 values similar to those previously found for inhibition by ATP of these effects of ADP. 5 The reported greater potency of 2-azido-ADP and of 2-methylthio-ADP as inhibitors of adenylate cyclase than as aggregating agents is therefore due only to their greater efficacy for this effect, not to some extra actions elsewhere.

Therapy of mouse leukemia L1210 with combinations of nebularine and nitrobenzylthioinosine 5'-monophosphate.

Earlier reports from this laboratory showed that: (a) in the presence of nitrobenzylthioinosine (NBMPR), a potent, tightly bound inhibitor of nucleoside transport, cells proliferating in culture were protected against a number of cytotoxic nucleosides; and (b) mice were protected against potentially lethal dosages of nebularine (and other toxic nucleosides) by coadministration of NBMPR. The present study, which used nitrobenzylthioinosine 5'-phosphate (NBMPR-P), a readily soluble "prodrug" form of NBMPR, extended the in vivo protection studies and showed that the half-life of the protection effect was about 4 hr. In chemotherapy experiments, mice bearing transplanted neoplasms were treated with high dosages of nebularine together with protecting doses of NBMPR-P. When mice bearing leukemia L1010 were treated with a potentially lethal regimen of nebularine administered together with NBMPR-P, a substantial kill of leukemic cells resulted (some mice were long-term survivors). The therapeutic effect was optimal at dosage levels of the protecting agent in excess of those required in nonleukemic mice for protection against the lethal nebularine dosages used, suggesting that the therapeutic effect was due to the joint presence in the leukemic cells of a metabolite of NBMPR-P and nebularine; NBMPR-P protection of the leukemic host against nebularine lethality was necessary for the therapeutic effect to be manifested.