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PubMed1996

Effects of nucleotide analogues on human immunodeficiency virus type 1 integrase.

We extended our previous study with 3'-azido-3'-deoxythymidine nucleotides [Proc. Natl. Acad. Sci. USA 91:5771-5775 (1994)] and examined the effects on human immunodeficiency virus type 1 (HIV-1) integrase of the nucleotides of three nucleoside analogues currently under evaluation in clinical trials: beta-D-2',3'-didehydro-3'-deoxythymidine, beta-D-2'-ara-fluoro-2', 3'-dideoxyadenosine, and beta-L-2',3'-dideoxy-3'-thiacytidine. Beta-D-2',3'-Didehydro-3'-deoxythymidine and beta-D-2'-ara-fluoro-2',3'-dideoxyadenosine nucleotides had IC50 values for strand transfer of 100 and 200 microM, respectively, whereas the corresponding 2',3'-dideoxynucleoside triphosphates, ddT triphosphate and ddA triphosphate, did not inhibit the integrase at 800 and 200 microM, respectively. Beta-L-2',3'-Dideoxy-3'-thiacytidine triphosphate had no effect up to 500 microM. The L-enantiomers of 5-fluoro-2',3'-dideoxycytidine monophosphate and triphosphate had IC50 values of approximately 40 microM, whereas their D-enantiomer isomers showed no inhibition at 200 microM. NAD, pyridoxal phosphate, and coumermycin A1, which exhibit no antiviral activity but are typically used to probe nucleotide binding sites, were also tested. NAD was inactive, and its etheno derivative exhibited activity at 1 mM. In contrast, pyridoxal phosphate (IC50 = 18 microM and coumermycin A1 (IC50 = 5 microM were potent inhibitors. None of the coumermycin monomeric derivatives were active integrase inhibitors. The physiological ribonucleotides ATP and GTP inhibited HIV-1 integrase at or near cellular concentrations, suggesting that they may regulate HIV-1 integrase activity in cells. In general, the active nucleotides tested inhibited binding of HIV-1 integrase to its substrate DNA an inhibited an integrase deletion mutant containing only amino acids 50-212, indicating that nucleotides bind to the enzyme catalytic core. Consisently, the choice of nucleophile in the 3'-processing reaction was blocked to the same extent regardless of the nucleotide used (water, glycerol, or the viral DNA hydroxyl) by the enzyme. These observations suggest new strategies for antiviral drug development that could be based on nucleotide analogues as inhibitors of HIV-1 integrase.

Antiviral Agents↗
PubMed2000

Androgen-independent growth is induced by neuropeptides in human prostate cancer cell lines.

BACKGROUND: Androgen-independent growth leads to progressive prostate cancer after androgen-ablation therapy. This may be caused by altered specificity of the androgen receptor (AR), by ligand-independent stimulation of the AR, or by paracrine growth modulation by neuropeptides secreted by neuroendocrine (NE) cells. METHODS: We established and characterized the androgen-independent FGC-DCC from the androgen-dependent LNCaP fast growing colony (FGC) cell line. The androgen-independent DU-145, FGC-DCC, and PC-3, and the androgen-dependent LNCaP and PC-346C cell lines were used to study growth modulation of gastrin-releasing peptide (GRP), calcitonin (CT), serotonin (5-HT), and vasoactive intestinal peptide (VIP) by (3)H-thymidine incorporation. Specificity of the growth-modulating effects was tested with the anti-GRP monoclonal antibody 2A11 and induction of cAMP by neuropeptides. RESULTS: Androgen-independent growth stimulation by neuropeptides was shown in DU-145 and PC-346C. 2A11 inhibited GRP-induced (3)H-thymidine incorporation in DU-145 and PC-346C and inhibited proliferation of the FGC-DCC and PC-3 cell lines. With some exceptions, cAMP induction paralleled growth stimulation. Dideoxyadenosine (DDA) inhibited the GRP-induced growth effect in DU-145 and PC-346C, whereas oxadiazoloquinoxaline-1-one (ODQ) had no effect on (3)H-thymidine incorporation. None of the neuropeptides stimulated growth of LNCaP, FGC-DCC, or PC-3. CONCLUSIONS: GRP-induced growth of DU-145 and PC-346C was specific and cAMP-mediated. Androgen-independent growth of FGC-DCC cells was mainly due to an induction of Bcl-2 expression and possibly through the activation of an autocrine and NE-like pathway, as has been shown also for the PC-3 cell line. Growth induction of non-NE cells by neuropeptides could be a possible role for NE cells in clinical prostate cancer.

Androgens↗
PubMed1999

Induction of autocrine factor inhibiting cell motility from murine B16-BL6 melanoma cells by alpha-melanocyte stimulating hormone.

We have previously reported that neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) successfully inhibited Matrigel invasion and haptotactic migration of B16-BL6 melanoma cells towards both fibronectin and laminin without affecting their growth. In the present study, we investigated the inhibitory mechanism of tumor cell motility by alpha-MSH. Alpha-MSH significantly blocked the autocrine motility factor (AMF)-enhanced cell motility. However, alpha-MSH did neither prevent the secretion of AMF from B16-BL6 cells nor alter the expression level of AMF receptor (gp78). On the other hand, alpha-MSH induced the secretion of the motility inhibitory factor(s) from B16-BL6 cells in a concentration- and time-dependent manner. The induction of the motility inhibitor(s) was proportional to increasing levels of intracellular cAMP induced by alpha-MSH as well as forskolin, and the activity was abolished by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA). The motility-inhibiting activity in conditioned medium (CM) from alpha-MSH-treated B16-BL6 cells was found to have a m.w. below 3 kDa after fractionation. This activity was abolished by boiling but insensitive to trypsin. The treatment of tumor cells with cycloheximide reduced the activity in alpha-MSH-stimulated CM. Our results suggest that alpha-MSH inhibited the motility of B16-BL6 cells through induction of autocrine factor(s).

Adenylyl Cyclase Inhibitors↗
PubMed2000

Determination of lodenosine and its major metabolite in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry.

A sensitive and selective method for the determination of 2'-beta-fluoro-2',3'-dideoxyadenosine (lodenosine, F-ddA), an experimental anti-AIDS drug, and its major metabolite, 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), in human plasma was developed and validated. The procedure employs two internal standards and a simple ultrafiltration step followed by chromatography on a Betasil C(18) minibore column. An in-line valve is used to remove salts before reaching the ion source. Detection is by electrospray ionization tandem mass spectrometry with selected reaction monitoring. The method has a limit of quantitation of 4 ng ml(-1) (16 nM) for F-ddA and 8 ng ml(-1) (32 nM) for F-ddI with a linear range up to 2000 ng ml(-1) (7.9 microM) for each. Predicted concentrations from a three-day validation study were within 5% of the nominal values for F-ddA and 16% for F-ddI. Intra- and inter-assay precision, as measured by relative standard deviation, was 13% or better for both compounds. To achieve good reproducibility, many variables related to the electrospray ionization were optimized for both precision and sensitivity. The method was successfully employed to analyze samples and evaluate plasma pharmacokinetics from a Phase I clinical trial.

Anti-HIV Agents↗
PubMed1986

Prostaglandin E2 stimulates DNA synthesis by a cyclic AMP-independent pathway in osteoblastic clone MC3T3-E1 cells.

The effect of prostaglandin E2 (PGE2) on osteoblastic cell proliferation was investigated using osteoblastic clone MC3T3-E1 cells cultured in serum-free medium. PGE2 at 2 micrograms/ml increased the number of the cells by 2 days after its addition. PGE2 raised the level of DNA synthesis in a dose-related fashion after a constant lag time, the maximal effect being at 2-10 micrograms/ml and the level about fourfold over that of the control at 36 hr after its addition. However, at low doses (below 0.2 microgram/ml), PGE2 rather depressed DNA synthesis. Isobutyl methylxanthine counteracted the stimulation of DNA synthesis by PGE2, and forskolin depressed the synthesis, which was inversely correlated with increasing intracellular cAMP content. These results indicate that an increase in cAMP content inhibits DNA synthesis. In addition, 2',5'-dideoxyadenosine did not negate the stimulatory effect of PGE2 on DNA synthesis, suggesting that PGE2 increases DNA synthesis, probably via a pathway different from the adenylate cyclase/cAMP system. Moreover, at a high dose, PGE2 stimulated both the production and degradation of cAMP; the elevation of cAMP content was rapidly depressed by the stimulated degradation system. Consequently, the stimulatory effect of PGE2 on DNA synthesis would be released from the inhibition by cAMP, resulting in an increase in DNA synthesis. Taken together with data from our previous reports, these results indicate that PGE2 enhances both the proliferation and differentiation of osteoblastic cells in vitro, which are probably mediated by two different second messengers dependent on the concentration of PGE2.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Cyclic AMP stimulation of Cl- secretion by the opercular epithelium: the apical membrane chloride conductance.

Chloride secretion (Isc) by the opercular epithelium of the teleost, Fundulus heteroclitus, is stimulated by elevations in intracellular cyclic AMP (cAMP) elicited by beta-adrenergic agonists, such as isoproterenol, and is accompanied by a small but significant increase in the transepithelial conductance (Gt). Cupric ions (Cu2+) have been shown to block the apical membrane Cl- channels in this epithelium, leading to a reduction in both the Isc and Gt (Degnan, '85). In the present studies, the effects of Cu2+ on cAMP-elevated and cAMP-depleted epithelia were observed to define the actions of cAMP in this stimulatory process. At a concentration of 5 X 10(-4) M in the mucosal solution, Cu2+ inhibited the Isc 79.8% and reduced the Gt 39.2%. Isoproterenol produced an attenuated stimulation of the Isc in these tissues compared to untreated controls, but had no effect on the Gt. In tissues bathed bilaterally with Cl- -free Ringer, the Isc was virtually abolished and the Gt was reduced 37.0%; neither Cu2+ nor isoproterenol had any effects on the Isc or Gt under this condition. Simultaneous 2 2Na and 3 6 Cl unidirectional flux determinations indicated that the only effects of both isoproterenol and Cu2+ were on the active Cl- secretory flux. An inhibitor of adenylate cyclase, 2',5' dideoxyadenosine (DDA), reduced the Isc and Gt 39.8% and 20.8% respectively. This inhibitor had no additional effects in Cu2+ -treated tissues and the action of Cu2+ on the Gt was reduced in DDA-treated tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

A strategy for liquid chromatography/tandem mass spectrometric assays of intracellular drugs: application to the validation of the triphosphorylated anabolite of antiretrovirals in peripheral blood mononuclear cells.

The pharmacokinetics of intracellular drugs have recently aroused new interest because monitoring a drug's behaviour near the site of action can enhance knowledge of its efficacy and toxicity. Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) is particularly attractive for intracellular analytes. Very few papers deal precisely with special features encountered in intracellular drug assay or with how closely the assay matches the actual recommendations. Particular problems are encountered mainly because the analytes are located intracellularly. This mainly concerns the handling of biological media, including provision of blank samples using Ficoll gradient separation, cell counts, optimisation of cell lysis, sample extraction, plotting standard curves using either fmol/10(6) cells or fmol/ml of extract or fmol/sample, the matrix effect as a function of the number of cells, stability before and during cell separation, as well as in storage conditions using clinical samples, biological matrix replacement and interference by endogenous compounds. This paper describes a strategy for the full validation and routine use of an LC/MS/MS assay applied to the simultaneous intracellular determination of the triphosphorylated anabolites of didanosine (2',3'-dideoxyadenosine triphosphate or ddA-TP) and stavudine (2',3'-didehydro-3'-deoxythymidine triphosphate or d4T-TP), two nucleoside reverse transcriptase inhibitors of HIV, in human peripheral blood mononuclear cells (PBMCs), as a guide for further LC/MS/MS assay of intracellular drugs.

Anti-HIV Agents↗
PubMed2003

Evidence for multiple distinctly localized adenylyl cyclase isoforms in mammalian spermatozoa.

In addition to a bicarbonate-regulated soluble adenylyl cyclase (sAC), mammalian spermatozoa, like somatic cells, appear to contain receptor/G protein-regulated AC activity that contributes to the modulation of specialized cell processes. This study provides evidence that agents, known to influence somatic membrane-associated AC (mAC) but apparently not germ cell sAC, can modulate cAMP production and functional state in mouse spermatozoa. Specifically, forskolin significantly enhanced cAMP production and capacitation, while inclusion of 2',5'-dideoxyadenosine significantly blocked these responses. Furthermore, GTPgammaS and NaF stimulated cAMP, but GDPbetaS and mastoparan had no apparent effect, consistent with recent evidence that G(s), but not G(i), contributes to AC/cAMP regulation in uncapacitated cells. In addition, intact mouse spermatozoa were screened for all known mAC isoforms by immunolocalization, using commercially available specific antibodies. The most abundant isoforms appeared to be AC2, AC3, and AC8, each with distinct distributions in the acrosomal and flagellar regions; AC1 and AC4 also appeared to be present, although less abundantly, in the midpiece and acrosomal cap regions, respectively. Intriguingly, however, Western blotting revealed that the major immunoreactive proteins in mouse sperm lysates were considerably smaller (approximately 50-60 kDa) than their somatic cell counterparts, suggesting that mature spermatozoa contain multiple mACs which may function in a shortened form. Of particular interest were AC3 and AC8, located in the same regions as, and hence possibly directly associated with, specific cell surface receptors and G proteins that are able to regulate the spermatozoon's acquisition and maintenance of fertilizing ability via changes in AC/cAMP.

Adenylyl Cyclases↗
PubMed1998

Cyclic AMP mediates endothelial protection by nitric oxide.

Incubation with TNF-alpha (50 ng/ml) for 72 hours markedly reduced viability of endothelial cells. A 6-hour preincubation with S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 3-100 microM) protected cells in a concentration-dependent manner and decreased TNF-alpha-mediated toxicity by up to 70%. Cytoprotection by SNAP was completely abolished by the adenylyl cyclase inhibitor 2', 5'-dideoxyadenosine and mimicked by 8-bromo cyclic AMP or forskolin. SNAP produced significant increases in cyclic GMP and cyclic AMP, both being abrogated in the presence of the NO scavenger 2-phenyl-4, 4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). Moreover, no endothelial protection by SNAP was detected in the presence of the protein kinase A inhibitor KT5720, whereas the protein kinase G inhibitor KT5823 left cytoprotection virtually unaltered. Our results demonstrate a crucial role for cyclic AMP in mediating NO-induced endothelial protection against TNF-alpha, possibly through cyclic GMP-dependent inhibition of cyclic AMP breakdown. NO-dependent endothelial protection may ultimately result from cyclic AMP-induced up-regulation of antioxidant proteins or down-regulation of cytotoxic processes.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Acute cardiotoxicity of nucleoside analogs FddA and FddI in rats.

The acute cardiotoxic potential of single dosages of FddA (2'-fluoro-2',3'-dideoxyadenosine) and FddI (2'-fluoro-2',3'-dideoxyinosine) was investigated in 6- to 9-week-old rats. Both nucleoside analogs were administered orally at 1000 and 2000 mg/kg and intravenously at 500 or 1000 mg/kg. For comparative purposes, additional groups of rats received 2'-deoxyadenosine or the 2-fluororibose moiety common to both the FddA and FddI molecules. The effects of two adenosine receptor antagonists, caffeine and theophylline, on the cardiotoxicity induced by FddA were also investigated. Deaths occurred within a few hours to a few days in FddA-treated rats given 2000 mg/kg orally or 500 mg/kg intravenously and in FddI-treated rats given 1000 mg/kg intravenously. Microscopic examination of the hearts revealed myocardial degeneration and necrosis for all rats that died and myocardial fibrosis for many survivors. No deaths or cardiac lesions were observed after administration of 2'-deoxyadenosine or the 2-fluororibose moiety. FddA was more cardiotoxic than FddI in rats at equivalent dosages administered either orally or intravenously. Based on the anatomic findings, all deaths were attributed to cardiac lesions. The administration of high, oral dosages of caffeine and theophylline accentuated the acute cardiotoxicity of FddA in rats.

Administration, Oral↗
PubMed1995

Acute cardiotoxicity of the Anti-HIV dideoxynucleoside, F-ddA, in the rat.

2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA), an acid-stable, purine dideoxynucleoside with in vitro anti-HIV activity, has been selected by the NCI as a clinical trial candidate. A recent report that high, single doses of F-ddA produce cardiotoxicity in rats prompted the present investigation whose objective was to quantitate this effect and establish a relationship between this toxicity and F-ddA plasma concentrations. Microscopic examination of cardiac tissues for degenerative lesions established the effects of F-ddA and ddA on three iv schedules [daily x 1(2.5-250 mg/kg); daily x 5(125, 250 mg/kg), and BID x 1 (250 mg/kg)] as well as one oral schedule [BID x 1 (500 mg/kg) using 8- to 12-week old female Sprague-Dawley rats. For both F-ddA and ddA, the group mean severity of the cardiac lesions was dose-dependent and proportional to the measured plasma concentrations of the undeaminated parent drugs. F-ddI and ddI, were essentially nontoxic in this study (iv, 250 mg/kg, daily x 1 and daily x 5), since plasma concentrations exceeding 2 mM produced only minimal cardiac lesions. The cardiomyopathy of F-ddA was minimal to mild for all iv doses except 250 mg/kg (daily x 1) and usually was greater than that of ddA at any given dose. This is a consequence of the fact that F-ddA is deaminated 20 times more slowly than ddA, resulting in higher plasma concentrations of F-ddA relative to ddA at any given time for any given dose. Neither F-ddA nor ddA was more cardiotoxic on a repeated iv schedule (daily x 5) than when administered only once, suggesting that rat cardiotoxicity is related Cmax rather than total exposure. In this most sensitive species, the formation of cardiac lesions above the background level is associated with i.v. F-ddA administration when the F-ddA plasma concentration approaches 300 microM, 30-50 times the anticipated therapeutic level in humans.

Administration, Oral↗
PubMed2000

Identification of residues involved in the specificity and regulation of the highly efficient multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster.

In contrast to all known deoxyribonucleoside kinases, a single highly efficient deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) is able to phosphorylate all precursor nucleosides for DNA synthesis. Dm-dNK was mutated in vitro by high-frequency random mutagenesis, expressed in the thymidine kinase-deficient Escherichia coli strain KY895 and clones were selected for sensitivity to the nucleoside analogs 1-beta-d-arabinofuranosylcytosine (AraC, Cytarabine), 3'-azido-2', 3'-dideoxythymidine (AZT, Zidovudine, Retrovir, 2', 3'-dideoxyadenosine (ddA) and 2',3'-dideoxycytidine (ddC, Zalcitabine, Hivid. Thirteen mutants with increased sensitivity compared to the wild-type Dm-dNK were isolated from a relatively small pool of less than 10,000 clones. Eight mutant Dm-dNKs increased the sensitivity of KY895 to more than one analog, and two of these mutants even to all four nucleoside analogs. Surprisingly, the mutations did not map to the five regions which are highly conserved among deoxyribonucleoside kinases. The molecular background of improved sensitivity was characterized for the double-mutant MuD (N45D, N64D), where the LD(100) value of transformed KY895 decreased 316-fold for AZT and more than 11-fold for ddC when compared to wild-type Dm-dNK. Purified recombinant MuD displayed higher K(m) values for the native substrates than wild-type Dm-dNK and the V(max) values were substantially lower. On the other hand, the K(m) and V(max) values for AZT and the K(m) value for ddC were nearly unchanged between MuD and wild-type Dm-dNK. Additionally, a decrease in feedback inhibition of MuD by thymidine triphosphate (TTP) was found. This study demonstrates how high-frequency mutagenesis combined with a parallel selection for desired properties provides an insight into the structure-function relationships of the multisubstrate kinase from D. melanogaster. At the same time these mutant enzymes exhibit properties useful in biotechnological and medical applications.

Animals↗
PubMed1994

Effects of lead on osteoclast-like cell formation in mouse bone marrow cell cultures.

To examine an effect of lead (Pb) on the process of osteoclast-like cell formation from its progenitors, we used a mouse bone marrow culture system in which osteoclast-like multinucleated cells (MNCs) were formed in response to bone-resorbing agents. In a 9-day culture period, Pb dose-dependently stimulated MNC formation over the concentration range 2-10 microM, whereas at 40 microM Pb, MNC formation declined. In an 11-day culture period, MNC formation reached a maximum at 5 microM Pb and decreased with increasing concentration of Pb at 10-40 microM. Pb-stimulated MNC formation was inhibited by both indomethacin and SC19220, an antagonist of prostaglandin E2 (PGE2) receptor. Pb stimulated the production of PGE2 in marrow cell cultures, suggesting that Pb-stimulated MNC formation is dependent on the production of PGE2. 3-Isobutyl-1-methylxanthine potentiated Pb-stimulated MNC formation and 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, inhibited it. A calcium ionophore A23187 increased Pb-induced MNC formation and verapamil, a calcium channel blocker, depressed it. It is possible that a PGE2-induced increase in the levels of cyclic adenosine 3',5'-monophosphate (cAMP) and calcium ions in marrow cells is involved in Pb-induced MNC formation. Pb and parathyroid hormone showed a synergistic stimulation on MNC formation. From these results, Pb is thought to induce osteoclast-like cell formation by a mechanism involving PGE2 which increases the intracellular levels of cAMP and calcium ions.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Effect of adenosine receptor agonists and other compounds on cyclic AMP accumulation in forskolin-treated hippocampal slices.

The effect of adenosine analogues and some putative neurotransmitters have been studied on cyclic AMP accumulation in rat hippocampal slices treated with the adenylate cyclase activator forskolin. The effects of PGE2 and histamine were potentiated by forskolin (0.1 microM). Isoprenaline and NECA had essentially additive effects with 0.1 microM forskolin and serotonin (above 10(-4) M) inhibited forskolin-stimulated cyclic AMP accumulation. The A1-adenosine receptor selective adenosine analogue R-PIA inhibited forskolin stimulated cyclic AMP accumulation in low doses and stimulated in high. NECA, adenosine and 2-chloroadenosine uniformly stimulated cyclic AMP accumulation. 2',5'-dideoxyadenosine inhibited, but only at high concentrations. Both the stimulatory and the inhibitory effects of R-PIA were antagonized by 8-phenyltheophylline (10 microM). Enprofylline (100 microM) selectively inhibited the stimulatory effect. In the presence of enprofylline both 2-chloroadenosine showed an inhibitory effect on cyclic AMP accumulation. It is concluded that the forskolin-treated rat hippocampal slice is a useful preparation to study both stimulatory and inhibitory effects of transmitters and modulators on adenylate cyclase. The results also show that the rat hippocampus has both A1-receptors that are linked to inhibition of cyclic AMP accumulation and A2-receptors that are linked to stimulation. Furthermore, enprofylline is shown to selectively antagonize the stimulatory response, revealing inhibitory effects of compounds such as 2-chloroadenosine and adenosine.

2-Chloroadenosine↗
PubMed1988

Endothelium-mediated coronary dilatation by adenosine does not depend on endothelial adenylate cyclase activation: studies in isolated guinea pig hearts.

Adenosine, applied to the coronary system of guinea pigs at up to 10(-6) M, elicits dilatation solely via an endothelium-mediated process. We investigated the role of coronary A2 receptors in this dilation, since the coronary endothelium possesses adenosine A2-receptors with a stimulatory action on the adenylate cyclase. In situ, A2 receptor stimulation can be assessed by prelabeling the coronary endothelial adenine nucleotide pool with 3H-adenosine and subsequently determining the rate of release of radiolabeled cAMP induced by A2 agonists. Thus, perfusion of isolated hearts with 5'-N-ethylcarboxamidoadenosine (NECA) dose-dependently increased coronary flow and the release of 3H-cAMP from the endothelium. In the presence of 50 microM 2',5'-dideoxyadenosine (ddA), a P-site agonist which inhibits the catalytic activity of adenylate cyclase, coronary flow increases induced by both adenosine and NECA were unaffected. In contrast, ddA reduced the release of labeled cAMP in response to NECA by about 60%. In cultured endothelial cells, ddA likewise inhibited cAMP accumulation due to NECA by about 70%. Moreover, ddA antagonized the adenylate cyclase mediated flow response due to the PGI2 analogue, iloprost, as well as the positive chronotropic and inotropic actions of isoproterenol. The dissociation elicited by ddA between the coronary flow response and the release of cAMP strongly indicates that the endothelial A2 receptors which are linked to adenylate cyclase are not causally involved in endothelium-dependent coronary dilatation induced by adenosine.

Adenosine↗
PubMed1985

Purinergic regulation of basal and arginine vasopressin-stimulated hydraulic conductivity in rabbit cortical collecting tubule.

An extracellular adenosine responsive site that stimulates adenylate cyclase activity has been identified in several tissues. There is limited information on the presence and physiologic significance of adenosine receptors in well-defined segments of the mammalian nephron. We therefore examined the effect of adenosine and selected analogues on basal hydraulic conductivity in rabbit cortical collecting tubules (CCT) perfused in vitro. Adenosine and analogues with an intact ribose moiety produced a significant, sustained increase in hydraulic conductivity. No increase in hydraulic conductivity was seen in either time control CCT's or CCT's exposed to an adenosine analogue with an altered ribose moiety. These experiments are compatible with the presence of a functional adenosine receptor which requires an intact ribose moiety and acts to increase hydraulic conductivity in the mammalian CCT. An intracellular adenosine responsive site, termed the "P site," which inhibits adenylate cyclase activity, has also been described in several tissues. We therefore examined the effect of a P site agonist on hydraulic conductivity responses to arginine vasopressin, forskolin and cAMP. P site stimulation with 2'5' dideoxyadenosine inhibited the effect of AVP and of forskolin but not of cAMP to increase hydraulic conductivity. These results are compatible with a functional P site in the rabbit CCT which acts at the catalytic subunit of adenylate cyclase to inhibit hydraulic conductivity. Together, these results demonstrate purinergic modulation of basal and arginine vasopressin-stimulated water flux in the mammalian collecting tubule.

Adenosine↗
PubMed1995

Human platelets have cholesteryl ester hydrolytic activity resulting in esterification of [1-14C]oleate to individual phospholipids of platelets.

Human platelet cholesteryl ester hydrolytic (CEH) activity was determined toward cholesteryl [1-14C]oleate resulting in esterification of [1-14C]oleate to individual platelet phospholipids: choline-containing phospholipids (PC); ethanolamine-containing phospholipids (PE); phosphatidylserine (PS); phosphatidylinositol (PI); and sphingomyelin (SPH). Liberation of [1-14C]oleate and esterification of [1-14C]oleate to platelet phospholipids was enhanced by 100 nM iloprost (a stable analogue of prostacyclin that increases platelet cyclic adenosine monophosphate (c-AMP)), inhibited by 30 microM H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide)) (a specific c-AMP dependent protein kinase (CADPK) inhibitor) and 500 microM 2',5' dideoxyadenosine (DDA) (an inhibitor of iloprost-induced rise in platelet c-AMP), but unaffected by 150 mM choloroquine diphosphate. These observations suggest that the CEH activity is mediated by a CADPK phosphorylation of an enzyme with the phosphorylated state representing the active form of the enzyme and that the CEH activity is extralysosomal.

Blood Platelets↗
PubMed1987

Pertussis toxin inhibits stimulated motility independently of the adenylate cyclase pathway in human melanoma cells.

The human melanoma cell line, A2058, has previously been shown to respond to an autocrine motility factor (AMF). We have studied biochemical pathways that may be involved in the generation of such a motile response. Pertussis toxin (PT) caused a profound, rapid decrease in stimulated motility that was both dose and time-dependent. Preincubation of cells for 2 hr with as little as 1 ng/ml of PT significantly inhibited motility. A concentration of PT (0.5 microgram/ml) that completely eliminated migration after a 30 min. preincubation had a markedly reduced effect when added 1 hr after the start of the assay. In contrast, agents which selectively modulate or have a role in the adenylate cyclase pathway, e.g., cholera toxin, forskolin, the cAMP analogue 8-bromoadenosine 3':5'-cyclic monophosphate and the cyclase inhibitor 2',5'-dideoxyadenosine, all had negligible effect upon motility. These data are consistent with the presence of a receptor coupled to a PT sensitive G protein initiating motility independently of the adenylate cyclase system.

8-Bromo Cyclic Adenosine Monophosphate↗