Search

Inhibition of adenylyl cyclases from Bacillus anthrasis and Bordetella pertussis by polyadenylate and by the most potent "P"-site agonists was investigated. These bacterial adenylyl cyclases differed in their sensitivity to inhibition by nominal "P"-site agents and in the effect of divalent cations on this inhibition. The enzyme from Bordetella pertussis was relatively insensitive to inhibition by "P"-site agonists, exhibiting a rank order of potency of 2'd3'AMP greater than 3'-AMP greater than 2',5'-ddAdo approximately Ado approximately 2'-dAdo, with IC50 values for 2'd3'AMP and 3'-AMP of 1-3 mM. Inhibition by 2'd3'AMP, however, was not affected by divalent cation, making it distinct from "P"-site-mediated inhibition of most mammalian adenylyl cyclases. The sensitivity to these nucleosides was comparable with potency for inhibition of bovine sperm adenylyl cyclase but was 3 orders of magnitude less potent than for activated enzyme from bovine or rat brain. The Bordetella pertussis enzyme was similarly insensitive to inhibition by polyadenylate, with 16 microM inhibiting less than 20%. By comparison, Bacillus anthrasis adenylyl cyclase was more potently inhibited by 2'd3'AMP (IC50 approximately 85 microM) but not by the other nucleosides (less than 15% inhibition at 1 mM), and inhibition by 2'd3'AMP was optimally enhanced by 5-10 mM Mg2+ or Mn2+, as is typical for inhibition by "P"-site agonists. The Bacillus anthrasis enzyme was potently inhibited by polyadenylate (IC50 approximately 0.3 microM), comparable to inhibition of brain adenylyl cyclases. Sensitivity of Bacillus anthrasis adenylyl cyclase to poly(A) was diminished somewhat by Ca2+/calmodulin (to IC50 approximately 1 microM) although Ca2+/calmodulin was without effect on inhibition by 2'd3'AMP. In contrast to inhibition of mammalian adenylyl cyclases via the "P"-site, inhibition of both bacterial adenylyl cyclases by 2'd3'AMP was competitive with respect to substrate MgATP. The data indicate basic differences in susceptibilities of these bacterial adenylyl cyclases to inhibition by poly(A), by adenosine analogs, and the effects of divalent cations. Although the potency of 2'd3'AMP and the metal-dependent nature of inhibition of Bacillus anthrasis adenylyl cyclase shared characteristics of "P"-site-mediated inhibition, the fact that inhibition of both bacterial adenylyl cyclases was competitive with respect to substrate strongly suggests that this inhibition was at the catalytic site and that these bacterial enzymes do not contain a distinct "P"-site.

We have investigated the effects of 2',5'-dideoxyadenosine (DDA), 9'-beta-D-arabinofuranosyladenine (ARA), and 9-beta-D-xylofuranosyladenine (XFA), which have been classified as P-site adenosine agonists, on the cyclic adenosine 3',5'-monophosphate (cAMP) metabolism of human lymphocytes and polymorphonuclear leukocytes (PMNs). DDA (10(-5)-2 x 10(-4) M), ARA and XFA caused a dose-dependent decrease in cAMP content of human lymphocytes. In addition to decreasing lymphocyte cAMP levels, DDA, ARA, and XFA markedly inhibited the effects of many adenylate cyclase-stimulating agents including beta-adrenergic stimuli, prostaglandin E1 (PGE1), histamine, adenosine, forskolin and cholera toxin. Theophylline and 3-isobutyl-1-methylxanthine, which are known antagonists of adenosine A1/Ri and A2/Ra receptors, did not modify the inhibiting effects of DDA. Mn2+ (1 mM) increased the sensitivity to inhibition of adenylate cyclase agonists by DDA. We also search for the presence of adenosine P-sites in human PMNs. DDA caused a significant decrease of PMN cAMP levels only at the highest concentrations used (2 x 10(-4) M). In contrast, even low concentrations of DDA (10(-6)-10(-4) M) concentration-dependently blocked the stimulatory effect of PGE1 and forskolin on PMN cAMP accumulation. The results support the existence of a purine P-site that regulates cAMP metabolism of human lymphocytes and PMNs.

The capacity of PGE2 to inhibit human T cell responses was examined by investigating its effect on mitogen-induced IL-2 production and proliferation of highly purified CD4+ T cells. PGE2 inhibited both PHA and anti-CD3 induced proliferation and IL-2 production by an action directly on the responding T cell. Inhibition of IL-2 production reflected decreased accumulation of mRNA for IL-2. A variety of other cAMP elevating agents exerted similar inhibitory effects. Inhibition of proliferation could be overcome by supplemental IL-2, PMA, or the anti-CD28 mAb 9.3. Although PMA and 9.3 markedly increased the amount of IL-2 produced by mitogen-stimulated T cells, the percentage inhibition of IL-2 secretion caused by PGE2 and other cAMP elevating agents remained comparable in these costimulated cultures. Rescue of T cell DNA synthesis by these agents appeared to reflect the finding that, although PGE2 markedly inhibited IL-2 production, the absolute amount of IL-2 produced was increased sufficiently to sustain mitogen-induced proliferation. As anticipated, PGE2, forskolin, and cholera toxin increased T cell cAMP levels. The quantity of cellular cAMP generated in response to PGE2, cholera toxin, and forskolin could be inhibited by PMA or 2',5'-dideoxyadenosine. Using these reagents, the inhibitory effects of PGE2 were found to reflect intracellular cAMP levels, but only within a very narrow range. The results indicate that by elevating cAMP levels, PGE2 inhibits human T cell IL-2 production at a point that is common to both the CD3 and CD28 signaling pathways.

Apolipoprotein B-100 (apoB) is a constituent of low density lipoproteins that has been implicated in the development of coronary artery disease (Editorial (1988) Lancet 1, 1141-1142). It is produced primarily in the liver, but mechanisms of secretory control are unclear. We examined the possibility that rapid reuptake of newly secreted lipoproteins regulates the net output of apoB by cultured liver cells. Polyclonal blocking antibodies to the low density lipoprotein receptor markedly increased apoB output, and varying the width of the unstirred water layer around the cells also changed apoB output, consistent with local reuptake. Labeled apoB added to the bulk fluid phase of the incubation media was not detectably taken up, implying that re-uptake is predominantly local. We conclude that a major site of apoB secretory control resides in the unstirred water layer, external to the cell. Because many cells secrete products for which they possess receptors, local re-uptake from the unstirred water layer may be a general mechanism for secretory control.

In cultured rat inner medullary collecting tubule (RIMCT) cells, arginine vasopressin (AVP) stimulates adenylyl cyclase (AC) activity in dose-dependent fashion, with no response at concentrations of 10(-10) M or below and with peak activity at 10(-7) M AVP. In contrast, AVP-stimulated phospholipase (PLC) activity is greatest at concentrations at which there is no effect on AC and decreases at higher concentrations of AVP, becoming undetectable at 10(-7) M. Increasing cellular adenosine 3',5'-cyclic monophosphate (cAMP) content with either exogenous ClPheScAMP or forskolin eliminates inositol trisphosphate production in response to 10(-13) M AVP. Conversely, inhibition of AC by 2',5'-dideoxyadenosine (DDA) unmasks PLC activity in response to 10(-7) M AVP that is not observed in the absence of DDA. Similarly, DDA prevents inhibition of epidermal growth factor-stimulated PLC by AVP. These findings demonstrate the reciprocal relationship between AVP-stimulated AC and PLC activities in cultured RIMCT cells, which may explain previous divergent results regarding the ability of AVP to stimulate PLC in this tissue.

AIDS has remained a significant and worsening medical problem since its first description as a new clinical entity in 1981. In the past 6 years, substantial progress has been made in the chemotherapy for this disease; such progress is likely to exert a major effect on the epidemic of human immunodeficiency virus infection in the coming decade. In this article, we overview the preclinical development of an antiretroviral drug, 2',3'-dideoxyinosine (didanosine; ddI), which has recently been shown in early phase I studies to have activity against human immunodeficiency virus in patients with AIDS or AIDS-related complex. Although we will not know the full clinical potential of ddI until we have the results of ongoing controlled clinical trials, this drug appears to possess desirable features for clinical use.

The mechanism for the inhibition of platelet functions by a prostaglandin I2 analog, OP-41483, was studied with guinea pig platelets. OP-41483, and PGI2 as well, inhibited aggregation, ADP release and thromboxane formation of platelets with IC50 values of 4.3-5.8 ng/ml and 0.6-0.9 ng/ml, respectively. The ligand binding study using [3H]-OP-41483 suggested that OP-41483 bound with different affinities to two classes of binding sites on platelets. The dissociation constant of OP-41483 for the higher affinity site corresponded to the IC50 values of its antiplatelet effect. PGI2 as well as OP-41483 displaced [3H]-OP-41483 previously bound to platelets, thus indicating that both agents exerted their antiplatelet effects by binding to the same site on platelets. OP-41483 and PGI2 activated adenylate cyclase and raised cyclic AMP levels in platelets. However, their inhibitory effect on platelet aggregation was not fully antagonized by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA), at a concentration completely inhibiting the increase of cyclic AMP. Moreover, this DDA-resistant effect of OP-41483 disappeared in the presence of calcium chloride (10(-4)-10(-3) M). OP-41483 and PGI2 inhibited thrombin-induced Ca++ influx into platelets. The inhibition of Ca++ influx was not reversed by DDA. Based on these results, we speculate that the inhibitory effects of OP-41483 and PGI2 on platelet functions are produced through dual mechanisms: one mediated by activation of adenylate cyclase and the other by an inhibition of Ca++ influx; and these two mechanisms seem to be independent of each other.

We have previously reported that the isolated rat branch pulmonary artery (PA) contracts when made hypoxic and that the contraction is dependent in large part on the presence of a functioning endothelium. This study tested if the hypoxic contraction was caused by reduced endothelium-derived relaxing factor (EDRF) activity. To do so we tested if chemical inhibitors of EDRF mimicked the effect of hypoxia, if PA guanosine 3',5'-cyclic monophosphate (cGMP) fell during hypoxic contraction, and if stimulation of smooth muscle cGMP attenuated hypoxic contraction. We found that the EDRF inhibitors hemoglobin and methylene blue caused a concentration-dependent increase in PA force that equaled that produced by hypoxia. PA cGMP decreased in endothelium-intact rings from 105 +/- 14 pM/g (wet wt) during normoxia to 41 +/- 9 pM/g during hypoxia. In endothelium-denuded rings normoxic cGMP was reduced to 32 +/- 10 pM/g with no further decrease during hypoxia. The endothelium-independent stimulators of cGMP, nitric oxide, and 8-bromo-cGMP, reduced maximum hypoxic contraction by 80 +/- 11 and 93 +/- 3%, respectively, whereas the endothelium-dependent stimulator acetylcholine did not. PA adenosine 3',5'-cyclic monophosphate (cAMP) fell only slightly during hypoxia and cAMP inhibitors failed to mimic the hypoxic contraction. We conclude that the hypoxic contraction of isolated rat PA is caused largely by decreased EDRF activity.

It is uncertain whether adenosine 3',5'-cyclic monophosphate (cAMP) or the inositol-calcium pathway mediates the stimulation of bone resorption by parathyroid hormone (PTH). Incubation of bone organ cultures with cAMP analogues and forskolin has not resolved this question because of the cellular inhomogeneity of bone and the consequent presence of adenylate cyclase-linked receptors for both PTH and calcitonin, hormones with opposite effects on bone resorption. We have used two new inhibitors of adenylate cyclase, 9-(tetrahydro-2-furyl)adenine (SQ 22536) and 2',5'-dideoxyadenosine (DDA), to directly reassess the role of cAMP in PTH-stimulated osteolysis. SQ 22536 (0.01-1.0 mM) and DDA (0.01-1.0 mM) completely blocked PTH stimulation of cAMP production measured in the absence of a phosphodiesterase blocker. In the presence of 1 mM 3-isobutyl-1-methylxanthine, half-maximal inhibition of PTH-induced cAMP production occurred with 0.2 mM SQ and 0.1 mM DDA, respectively. These concentrations of SQ and DDA had no effect on PTH-stimulated 45Ca release from calvaria, although both agents inhibited bone resorption when present at concentrations of 1-2 mM. At these levels, SQ and DDA caused equivalent inhibition of 45Ca release stimulated by 1,25-dihydroxyvitamin D3 but did not affect basal 45Ca release or [3H]-phenylalanine incorporation. It is concluded that substantial blockade of PTH-induced cAMP production does not affect this hormone's stimulation of bone resorption, which is therefore likely to be mediated by another intracellular messenger system, possibly calcium. In millimolar concentrations, SQ and DDA appear to be nonspecific blockers of osteoclastic bone resorption.

In the search for compounds capable of inducing endogenous production of colony-stimulating factor (CSF) and possessing activity against human immunodeficiency virus (HIV), an immunomodulator, muramyl dipeptide (MDP), was investigated. MDP can enhance monocyte-macrophage CSF in serum and promote nonspecific resistance against a variety of microbial pathogens. MDP exhibited an inhibitory activity against HIV infection of CD4+ H9 lymphocytes and U937 monocytoid cells. An inhibitor of viral reverse transcriptase, 2', 3'-dideoxyadenosine, produced potent inhibition in cultures which were similarly infected with HIV. MDP could partially reduce antigen production in persistently HIV-infected KE37/1 lymphocyte cultures.

(+-)-cis-[4-[(2,5-Diamino-6-chloropyrimidinyl)amino]-2- cyclopentenyl]carbinol (5a) was synthesized from 2-amino-4,6-dichloropyrimidine and cis-4-(hydroxymethyl)cyclopentenylamine (2a) by subsequent preparation of the 5-[(4-chlorophenyl)azo] derivative of the resulting pyrimidine (3a) and reduction of the azo moiety with zinc and acetic acid. The carbocyclic analogue of 2',3'-didehydro-2',3'-dideoxy 2-amino-6-chloropurine (6a) and the corresponding 8-azapurine (9a) were prepared from 5a. The carbocyclic 2',3'-didehydro-2',3'-dideoxy analogues of guanine (7a) and 2,6-diaminopurine (8a), and 8-azaguanine (10a) and 8-aza-2,6-diaminopurine (11a) were prepared from 6a and 9a, respectively. The corresponding 2',3'-saturated series of 2-amino-6-substituted-purine carbocyclic nucleosides was prepared following the same scheme starting with cis-4-(hydroxymethyl)cyclopentylamine (2b). Carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine (carbovir, 7a) emerged as a potent and selective anti-HIV agent. Its hydrolytic stability and its ability to inhibit the infectivity and replication of HIV in T-cells at concentrations of approximately 200-400-fold below toxic concentrations make carbovir an excellent candidate for development as a potential antiretroviral agent.

This study investigated the mechanisms by which adenosine stimulates proliferation of microvascular endothelial cells. The metabolic byproducts of adenosine, inosine and hypoxanthine were unable to stimulate proliferation. When adenosine uptake was prevented, the stimulation of proliferation was unchanged, suggesting that uptake of adenosine with subsequent incorporation into the nucleotide pool is not the mechanism for increasing proliferation. Treatment of endothelial cells with adenosine analogues, presumably selective for either the A1 or A2 receptor, stimulated proliferation equally. This suggested that adenosine 3', 5'-cyclic monophosphate (cAMP) might not mediate the proliferative response to adenosine. However, radioimmunoassay of cell extracts after treatment with either analogue showed an increase in cAMP. In addition, adenylate cyclase blockade with 2', 5'-dideoxyadenosine prevented the proliferative response brought about by these analogues. These data suggest that the proliferative response to adenosine depends on an increase in cAMP. A 2-h pulse of cholera toxin stimulated endothelial cell proliferation, further supporting a role for cAMP. Pretreatment of endothelial cells with pertussis toxin blocked the stimulation of proliferation, indicating that a Gi or similar G protein is also involved in proliferation. We conclude that the proliferative response to adenosine involves a pertussis toxin-sensitive substrate as well as an increase in cAMP.

Common bacterial infections are increasingly being diagnosed in HIV-infected individuals. Cells of the monocyte-macrophage lineage kill invading bacterial pathogens and subsequently release immunoadjuvant components from the degraded cell walls. Since monocytes can be infected with HIV, effects of bacterial immunomodulators on infected promonocytic U937 cells were investigated. Synthetic muramyl peptide, mycobacterial trehalose dimycolate, and detoxified endotoxin exhibited an initial reduction followed by a rapid increase in HIV p24 antigen production. The upregulation of virus expression was correlated with enhanced interleukin-1 beta levels and a decrease in TNF-alpha production.

2',3'-Dideoxyinosine (DDI) and 2',3'-dideoxy-2,6-diaminopurine riboside (ddDAPR) are potent and selective inhibitors of human immunodeficiency virus (HIV) replication in MT-4 cells. They are also inhibitory to the transformation of C3H/3T3 cells by Moloney murine sarcoma virus (MSV). In vivo, they are only marginally effective in delaying MSV-induced tumor formation, and mortality associated therewith in newborn NMRI mice. When combined with ribavirin, DDI and ddDAPR become much more effective in inhibiting MSV and HIV replication in vitro and MSV-induced tumor formation in vivo. These observations point to the potential role of ribavirin in potentiating the anti-HIV activity of DDI in AIDS patients.

Effects of prostaglandin E2 (PGE2) on murine B-cell activation induced by two distinct B-cell differentiation factors, B151-TRF1/IL-5 and B151-TRF2, were examined. A final differentiation of unprimed B cells into IgM-producing cells induced by B151-TRF2 was markedly inhibited by PGE2 at physiological concentrations (around 10(-8) M), whereas B151-TRF1/IL-5- induced antibody responses of unprimed as well as activated B cells were not affected by PGE2, even at 10(-6) M. B-cell responses induced by B151-TRF2-like factors from autoimmune-prone MRL/lpr mice were also inhibited by PGE2. Biphasic increases in intracellular cyclic AMP (cAMP) levels were induced by culturing B cells with 10(-6) or 10(-8) M PGE2: rapid increases within 8 min and delayed increases around 16 hr. The direct addition of dibutyryl cAMP to cultures of B cells resulted in marked inhibition of antibody responses when stimulated with B151-TRF2 but not with B151-TRF1/IL-5. The B151-TRF2-induced antibody responses were also inhibited by cAMP-elevating reagents such as forskolin, cholera toxin and theophylline. Furthermore, 2'.5'-dideoxyadenosine, which is an inhibitor of adenylate cyclase, prevented the PGE2-mediated cAMP accumulation in unprimed B cells as well as the PGE2-mediated inhibition of B151-TRF2-induced B-cell responses when added at the initiation of culture. These results suggest that PGE2 inhibits B151-TRF2-induced antibody responses through the activation of adenylate cyclase and subsequent accumulation of intracellular cAMP, whereas B151-TRF1/IL-5-responsive B cells are resistant to the inhibitory effect of PGE2 and cAMP.

The antiviral activity of 3'-azido-2',3'-dideoxynucleoside 5'-phosphate analogues: 5'-phosphonomethylene-3'-azido-2',3'-dideoxythymidine, 5'-methylphosphonate and 5'-phosphite of 3'-azido-2',3'-dideoxythymidine, 5'-phosphites of 3'-azido-2',3'-dideoxyadenosine and guanosine was investigated in HIV-infected cell cultures (human lymphoblastoid cells). The effectivity of inhibition of HIV-reproduction in cells by these substances was close or even higher than that for the corresponding 3'-azido-2',3'-dideoxynucleosides, whereas their toxicity was lower than that of nucleosides. These substances are supposed to be transported into the cells and to be transformed into the corresponding 5'-triphosphate analogues under the action of cell kinases. It is possible that such agents are terminator substrates of virus reverse transcriptases and thus inhibit the biosynthesis of DNA chains.