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PubMed1995

Effects of non-nucleoside inhibitors of human immunodeficiency virus type 1 in cell-free recombinant reverse transcriptase assays.

We have employed a cell-free human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) assay to study the effects of non-nucleoside inhibitors of RT (NNRTI) by directly monitoring specific HIV DNA products using a HIV-1 genome-derived template and an oligodeoxynucleotide primer. As previously shown by ourselves and others, nucleoside analog triphosphates, e.g. 3'-azido-3'-deoxythymidine triphosphate and 2',3'-dideoxyadenosine triphosphate, could directly inhibit HIV RT RNA-dependent DNA polymerase activity by causing chain termination, as visualized in a RT reaction that yields specific DNA products. In contrast, each of two NNRTIs, nevirapine and delavirdine, directly inhibited RT activity without causing chain termination effects. We also analyzed interactions between nucleoside analogs and NNRTIs or among NNRTIs by chain elongation/dNTP incorporation and/or steady-state kinetic assays. Combinations of nevirapine with the triphosphates of either the (-)-strand of 2',3'-dideoxy-3'-thiacytidine or 2',3'-dideoxyadenosine yielded additive/synergistic effects on RT activity. However, only an additive effect was observed when combinations of nevirapine and 3'-azido-3'-deoxythymidine triphosphate were employed. Combinations of nevirapine and delavirdine had an antagonistic effect on the inhibition of HIV-1 RT activity.

Cell-Free System↗
PubMed1996

The bioactive phospholipid, lysophosphatidylcholine, induces cellular effects via G-protein-dependent activation of adenylyl cyclase.

The naturally occurring phospholipid, lysophosphatidylcholine (lyso-PC), regulates a broad range of cell processes, including gene transcription, mitogenesis, monocyte chemotaxis, smooth muscle relaxation, and platelet activation. Despite the growing list of cellular effects attributable to lyso-PC, the mechanism(s) by which it alters cell function have not been elucidated. In this report, we have examined the effects of exogenous lyso-PC on signal transduction processes within a variety of lyso-PC-responsive cells, including human platelets, monocyte-like THP-1 cells, and the megakaryoblastic cell line, MEG-01. Pretreatment of each of these cells with increasing concentrations of lyso-PC (25-150 microg/ml) was associated with a progressive increase in the cytosolic concentration of cAMP. The accumulation of cAMP in platelets correlated closely with the ability of lyso-PC to inhibit multiple platelet processes, including platelet aggregation, agonist-induced protein kinase C activation, thromboxane A2 generation, and the tyrosine phosphorylation of platelet proteins. In each of the cell types examined, the ability of lyso-PC to increase the cellular levels of cAMP was synergistically enhanced by pretreating the cells with the cAMP phosphodiesterase inhibitor, theophylline (5 mM), and was specifically inhibited by the P-site inhibitor of adenylyl cyclase, 2,5-dideoxyadenosine. A role for the stimulatory G-protein, Gs, in the lyso-PC-induced activation of adenylyl cyclase was suggested by the ability of the GTPase inhibitor, guanylyl 5'-thiophosphate (0.2 mM), to inhibit the lyso-PC-stimulated increase in cAMP, and also by the ability of cholera toxin to inhibit increases in membrane GTPase activity in response to lyso-PC. The functional significance of lyso-PC-induced activation of adenylyl cyclase was investigated in MEG-01 cells. Treatment of these cells with either lyso-PC or dibutyryl cAMP for 36-40 h resulted in a 3-5-fold increase in the surface expression of the natural anticoagulant protein, thrombomodulin (TM). The ability of lyso-PC to increase TM expression was abolished by pretreating these cells with the adenylyl cyclase inhibitor, 2,5-dideoxyadenosine, whereas the dibutyryl cAMP-induced increase in TM remained insensitive to adenylyl cyclase inhibition. These studies define an important role for the adenylyl cyclase signaling system in mediating cellular effects induced by lyso-PC.

Adenylyl Cyclases↗
PubMed2004

Modulation of L-type calcium channels in Drosophila via a pituitary adenylyl cyclase-activating polypeptide (PACAP)-mediated pathway.

Modulation of calcium channels plays an important role in many cellular processes. Previous studies have shown that the L-type Ca(2+) channels in Drosophila larval muscles are modulated via a cAMP-protein kinase A (PKA)-mediated pathway. This raises questions on the identity of the steps prior to cAMP, particularly the endogenous signal that may initiate this modulatory cascade. We now present data suggesting the possible role of a neuropeptide, pituitary adenylyl cyclase-activating polypeptide (PACAP), in this modulation. Mutations in the amnesiac (amn) gene, which encodes a polypeptide homologous to human PACAP-38, reduced the L-type current in larval muscles. Conditional expression of a wild-type copy of the amn gene rescued the current from this reduction. Bath application of human PACAP-38 also rescued the current. PACAP-38 did not rescue the mutant current in the presence of PACAP-6-38, an antagonist at type-I PACAP receptor. 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase, prevented PACAP-38 from rescuing the amn current. In addition, 2',5'-dideoxyadenosine reduced the wild-type current to the level seen in amn, whereas it failed to further reduce the current observed in amn muscles. H-89, an inhibitor of PKA, suppressed the effect of PACAP-38 on the current. The above data suggest that PACAP, the type-I PACAP receptors, and adenylyl cyclase play a role in the modulation of L-type Ca(2+) channels via cAMP-PKA pathway. The data also provide support for functional homology between human PACAP-38 and the amn gene product in Drosophila.

Adenylyl Cyclases↗
PubMed2005

Interplay of Ca2+ and cAMP signaling in the insulin-secreting MIN6 beta-cell line.

Ca2+ and cAMP are important second messengers that regulate multiple cellular processes. Although previous studies have suggested direct interactions between Ca2+ and cAMP signaling pathways, the underlying mechanisms remain unresolved. In particular, direct evidence for Ca2+-regulated cAMP production in living cells is incomplete. Genetically encoded fluorescence resonance energy transfer-based biosensors have made possible real-time imaging of spatial and temporal gradients of intracellular cAMP concentration in single living cells. Here, we used confocal microscopy, fluorescence resonance energy transfer, and insulin-secreting MIN6 cells expressing Epac1-camps, a biosynthetic unimolecular cAMP indicator, to better understand the role of intracellular Ca2+ in cAMP production. We report that depolarization with high external K+, tolbutamide, or glucose caused a rapid increase in cAMP that was dependent on extracellular Ca2+ and inhibited by nitrendipine, a Ca2+ channel blocker, or 2',5'-dideoxyadenosine, a P-site antagonist of transmembrane adenylate cyclases. Stimulation of MIN6 cells with glucose in the presence of tetraethylammonium chloride generated concomitant Ca2+ and cAMP oscillations that were abolished in the absence of extracellular Ca2+ and blocked by 2',5'-dideoxyadenosine or 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase. Simultaneous measurements of Ca2+ and cAMP concentrations with Fura-2 and Epac1-camps, respectively, revealed a close temporal and causal interrelationship between the increases in cytoplasmic Ca2+ and cAMP levels following membrane depolarization. These findings indicate highly coordinated interplay between Ca2+ and cAMP signaling in electrically excitable endocrine cells and suggest that Ca2+-dependent cAMP oscillations are derived from an increase in adenylate cyclase activity and periodic activation and inactivation of cAMP-hydrolyzing phosphodiesterase.

Animals↗
PubMed1990

The signal transducer for the dopamine-1 regulated sodium transport in renal cortical brush border membrane vesicles.

We have reported the presence of dopamine-1 (DA-1) and dopamine-2 (DA-2) receptors in renal brush border and basolateral membranes. DA-1 agonists stimulate adenylate cyclase (AC) and phospholipase C (PLC) activity in both membranes. Moreover, the ability of a DA-1 agonist (fenoldopam) to stimulate PLC activity is independent of AC activity. A DA-2 agonist (LY171555) by itself was without effect and did not enhance the ability of the DA-1 agonist to stimulate PLC activity. The DA-1 but not DA-2 agonists inhibit Na+/H+ exchange activity in brush border membrane vesicles (BBMV) and Na+/K(+)-ATPase activity in basolateral membranes. However, cAMP inhibits, while protein kinase C (presumably via PLC activity) stimulates, Na+/H+ exchange activity. We therefore determined the effect of DA-1 agonists on Na+/H+ exchange activity when PLC or AC activity was blocked using neomycin or dideoxyadenosine, respectively. The drugs were incubated with minced renal cortex prior to preparation of BBMV by differential centrifugation and MnCl2 precipitation. Enrichment of BBMV was not affected by drug treatment. The Na+/H+ exchange activity was assessed by measuring amiloride (1 mmol/L) sensitive 22Na+ uptake in BBMV (pHi = 5.5, pHo = 7.5, Nai+ = O, Nao+ = 1 mmol/L). Neomycin inhibited DA and DA-1-stimulated PLC activity in BBMV in a concentration dependent manner (10(-6) to 10(-4) mol/L). Neomycin (10(-4) mol/L) completely blocked the ability of DA and DA-1 agonist to stimulate PLC activity but had no consistent effect on DA-1 inhibited Na+/H+ exchange activity. Dideoxyadenosine inhibited DA and DA-1 simulated AC activity without affecting DA-1 stimulated PLC activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Telomerase inhibition by 3'-azido-2', 3'-dideoxynucleoside 5'-triphosphates and telomere shortening in human cultured cells by the corresponding nucleosides.

Telomerase is believed to be a good target for the development of antitumor agents. In this study, 3'-azido-2',3'-dideoxy-2-aminoadenosine (AZddAA), 3'-azido-2',3'-dideoxyadenosine (AZddA), 9-(3-azido-2,3-dideoxy-beta-D-ribofuranosyl)-2-aminopurine (AZddAP), 3'-azido-2-chloro-2',3'-dideoxyadenosine (AZddClA) and their triphosphate derivatives were synthesized. Telomerase assay studies showed that the 2-amino group plays an important role in the inhibitory activity of these compounds. In addition, AZddAA was found to cause telomere shortening in of HL60 cells in culture.

Antineoplastic Agents↗
PubMed1998

Marked inhibitory activity of masked aryloxy aminoacyl phosphoramidate derivatives of dideoxynucleoside analogues against visna virus infection.

Lipophilic masked aryloxyaminoacylphosphoramidate derivatives of 2',3'-dideoxynucleoside (ddN) analogues with potent anti-HIV activity (i.e., stavudine [d4T], azidothymidine [AZT], dideoxycytidine [ddC], 3'thio-2',3'-dideoxy cytidine [3TC], dideoxyadenosine [ddA], and 2',3'-didehydro-2',3'-dideoxyadenosine [d4A]) activity were evaluated for their activity against visna virus (VV) in sheep choroid plexus (SCP) cells. The activity of several prodrug derivatives against VV proved markedly superior to that of the corresponding free ddN analogues. In particular, the d4A and ddA prodrug derivatives were exquisitely inhibitory in this model system (50% effective concentration [EC50], < or = 0.003 microM), and their anti-VV potency exceeded by at least 200-fold the antiviral potency of the corresponding free nucleosides. Marked differences were noted in the anti-VV potencies of several of the test compounds depending on the nature of the amino acid linked to the 5'-phosphate moiety, the nature of the nucleoside, or both. In view of the stability of the prodrugs in lamb serum, the VV infection model in lambs may be considered highly useful for investigating the in vivo antiretroviral efficacy of these type of drugs, particularly the d4T, ddA, and d4A prodrug derivatives.

Animals↗
PubMed1975

The utilization of nucleotides by animal cells.

We have extended our studies on the toxicity of several arabinosyl nucleotides to L cells. In contrast to a short-term toxic effect of 9-beta-D-arabinofuranosyladenine (araA), both 9-beta-D-arabinofuranosyladenine 5'-phosphate (araAMP) and 9-beta-D-arabinofuranosyladenine 3',5'-cyclic phosphate produced sustained killing of the fibroblasts. 9-beta-D-Arabinofuranosyladenine 2',5'-cyclic phosphate and 9-beta-D-arabinofuranosyladenine-N1-oxide-5'-phosphate were not toxic, whereas 9-beta-D-arabinofuranosylhypoxanthine 5'-phosphate and adenosine 3',5'-cyclic phosphate had relatively slight effects on cell growth. 2',3'-Dideoxyadenosine 5'-phosphate was lethal to L cells, whereas 2',3'-dideoxyadenosine inhibited growth only slightly. The survival time of mice bearing Ehrlich ascites carcinoma was prolonged beyond controls equally well by treatment with either araAMP or araA. To explore the mechanism of nucleotide toxicity, we studied the metabolism of exogenous [3H, 32P]-araAMP. The double-labeled compound entered the cells very slowly and accumulated in both acid-soluble and acid-insoluble materials. 32P and 3H were associated with the adenine nucleotide fractions in ratios similar to that of the exogenous compound. After dephosphorylation of the acid-soluble fraction, greater than 85% of the 3H was associated with araA. Following degradation of the adenine-containing triphosphates with apyrase, all of the 32P and 97% of the 3H were associated with araAMP. Only small quantities of each label were associated with RNA and DNA. Following enzymatic degradation of DNA to 5'-monophosphates, most of both 3H and 32P were associated with the dAMP fraction in a ratio that was similar to that of the exogenous araAMP. After dephosphorylation of the 5'-monophosphates, greater than 90% of the 3H was contained in arabinosyl nucleosides. DNA was also enzymatically degreded to 3'-monophosphates. 3H was found primarily in the 3'-adenine nucleotide fraction with a slight amount in the nucleoside, whereas 32P was distributed among all the 3'-mononucleotides. After dephosphorylation of the 3'-monophosphates, 93% of the 3H was associated with araA. These results indicate that a small amount of araAMP penetrated the cell as the intact nucleotide, was phosphyorlated to the triphosphate and was subsequently incorporated within DNA.

Adenosine↗
PubMed1989

In vitro and in vivo comparison of the abilities of purine and pyrimidine 2',3'-dideoxynucleosides to inhibit duck hepadnavirus.

Four purine and two pyrimidine 2',3'-dideoxynucleosides were studied for their ability to inhibit duck hepadnavirus replication. The purine 2',3'-dideoxynucleosides were more potent antiviral agents than the pyrimidine 2',3'-dideoxynucleosides. The concentration for 50% inhibition of viral replication (IC50) was determined for each of the effective agents. Two drugs with low IC50s, 2,6-diaminopurine 2',3'-dideoxyriboside and 2',3'-dideoxyadenosine, were chosen for in vivo studies. Animals received 10 mg/kg intramuscularly twice daily. Rapid clearance of hepadnavirus DNA from the sera of the animals was seen as a result of treatment with 2,6-diaminopurine 2',3'-dideoxyriboside; however, treatment with 2',3'-dideoxyadenosine did not clear the virus.

Animals↗
PubMed1996

Direct inhibitory effect of thyrotropin-releasing hormone on isolated cecal circular smooth muscle cells of guinea pig.

Smooth muscle cells isolated from cecal circular smooth muscle of the guinea pig were used to determine whether thyrotropin-releasing hormone (TRH) can inhibit the contractile response produced by 10(-6) M carbachol by exerting a direct action on muscle cells. In addition, the inhibitory effect of 2',5'-dideoxyadenosine (an inhibitor of adenylate cyclase), phorbol 12-myristate 13-acetate (an inhibitor of particulate guanylate cyclase), 6-anilinoquinoline-5,8-quinone (an inhibitor of nitric oxide synthase) on the TRH-induced relaxation of cecal circular smooth muscle cells was examined. TRH inhibited the contractile response produced by 10(-6) M carbachol in a concentration-dependent manner, with an IC50 value of 4 nM, 2',5'-Dideoxyadenosine and phorbol 12-myristate 13-acetate did not have any significant effect on the TRH-induced relaxation. On the other hand, 6-anilinoquinoline-5,8-quinone and N omega-nitro-L-arginine methyl ester significantly inhibited the relaxation produced by TRH. Our findings show that TRH has a direct inhibitory effect on the isolated cecal circular smooth muscle cells via activation of nitric oxide synthase and soluble guanylate cyclase.

Aminoquinolines↗
PubMed1980

Derivatives of 3'- and 5'-deoxyadenosine: their inhibitory activity against DNA viruses.

3'-Deoxyadenosine, 3'-amino-3'-deoxyadenosine (3'3'), nine derivatives therefrom, two derivatives of 5'-amino-5'-deoxyadenosine and three derivates of 3', 5'-dideoxyadenosine were tested in cell culture for antiviral activity against three DNA viruses: adenovirus 5, herpesvirus hominis 1, and vaccinia virus. Cytotoxicity was also assessed. (3'3') derivatives affected the multiplication of all three viruses similarly. Those which were effective were also cytotoxic at the same or slightly higher concentration. Substitution or other molecular modification of (3'3') tend to lower the biological activity. The presence of an adenosine deaminase inhibitor enhanced both antiviral activity against adenovirus 5 and cytotoxicity in (3'3') compounds, but not in the others. Both 5'-amino-5'-deoxyadenosine compounds were active against vaccinia virus only. Of the three 3', 5'-dideoxyadenosines, only one had both cytotoxic and antiviral activity. Most, if not all, seemingly antiviral effects appear to be caused by inhibition of the cell metabolism.

Adenosine Deaminase Inhibitors↗
PubMed1994

Parathyroid hormone analogues inhibit calcium mobilization in cultured vascular cells.

Parathyroid hormone and parathyroid hormone-related protein lower blood pressure and relax contracted arteries. Parathyroid hormone also attenuates angiotensin II-induced vasoconstriction. To determine the cellular mechanism or mechanisms by which parathyroid hormone analogues antagonize pressor effects, we examined the effect of these peptides on angiotensin II-induced calcium mobilization in fura 2-AM-loaded cultured rat vascular smooth muscle cells. Either 100 nmol/L parathyroid hormone or parathyroid hormone-related protein significantly reduced the amount of calcium mobilized by 100 nmol/L angiotensin II. The attenuating effect of these peptides was mimicked by 10 mmol/L forskolin and 10 mmol/L isobutylmethylxanthine and was not dependent on the presence of extracellular calcium. This effect of the parathyroid hormone analogues was reduced when cells were pretreated with 100 mmol/L 2',5'-dideoxyadenosine, an adenylate cyclase inhibitor. Combined inhibition of cyclic nucleotide-dependent protein kinases eliminated the inhibitory effect of parathyroid hormone, whereas protein kinase C inhibition had no effect. Parathyroid hormone analogues decreased the amount of calcium released by inositol 1,4,5-trisphosphate in digitonin-permeabilized vascular smooth muscle cells. This effect was inhibited by treatment with 2',5'-dideoxyadenosine. These results suggest that these peptides attenuate inositol 1,4,5-trisphosphate-sensitive calcium mobilized by angiotensin II via an adenylate cyclase-dependent mechanism. This may be a mechanism by which acute administration of parathyroid hormone or parathyroid hormone-related peptide antagonizes vasoconstriction.

Animals↗
PubMed1997

Activation of phospholipase D in FRTL-5 thyroid cells by forskolin and dibutyryl-cyclic adenosine monophosphate.

We demonstrated previously that TSH activates phospholipase D (PLD) via stimulation of protein kinase C (PKC) in Fischer rat thyroid line (FRTL)-5 thyroid cells. To examine the role of the cAMP pathway in the regulation of PLD, we studied the effects of forskolin (0-100 microM; 30 min) and dibutyryl cAMP (dbcAMP; 0-1 mM; 30 min) on PLD activation. FRTL-5 thyroid cells were labeled mainly in phosphatidylcholine with [3H]myristate followed by incubation with 200 mM ethanol before the addition of agonist. PLD was assessed by the measurement of [3H]phosphatidylethanol. Forskolin (100 nM to 100 microM) and dbcAMP (100 pM to 100 microM) increased PLD activity significantly. Maximal responses to forskolin and dbcAMP exceed the PLD responses produced by 100 microU/ml of TSH. To determine whether the effects of forskolin and dbcAMP on PLD occurred as a consequence of PKC activation, FRTL-5 thyroid cells were preincubated for 10 min with the PKC inhibitors, chelerythrine (1 microM) or calphostin C (1 microM), or they were pretreated for 24 h with phorbol myristate acetate (100 nM) to down-regulate PKC. Unlike TSH-mediated PLD activation, these treatments had no effect on PLD activation by cAMP agonists. Forskolin (10 microM; 30 min) had no effect on the subcellular distribution of PKC alpha-, epsilon-, or zeta-isoforms, confirming the lack of involvement of PKC. The protein kinase A (PKA) inhibitors, H-89 (10 microM; 30 min) and dideoxyadenosine (5 nM; 10 min) significantly decreased the forskolin- and dbcAMP-mediated PLD activation without any effect on the phorbol ester-mediated PLD response. Following pretreatment with H-89 or dideoxyadenosine, the TSH-mediated PLD response was also significantly reduced. These studies indicate that forskolin and dbcAMP stimulate PLD in FRTL-5 thyroid cells directly via PKA without involvement of PKC. Studies of cells in the presence and absence of ethanol revealed approximately 60% of the phosphatidate plus diacylglycerol produced via TSH occurs via PLD activation. Although TSH-mediated inositol phosphate generation occurred with similar concentrations of TSH that led to PLD activation, 10-fold higher TSH concentrations were required to increase intracellular Ca2+. These results and the lack of a rapid Ca2+ transient following physiological TSH concentrations suggest that alternatives to conventional hydrolysis of phosphatidylinositol 4,5-bisphosphate may initiate PKC activation. Thus, the two major signal transduction systems in the FRTL-5 thyroid cell (PKA and PKC) appear to converge on PLD activation. Stimulation of both of these pathways by TSH may be required for optimal physiological activation of PLD.

Alkaloids↗
PubMed2004

Thyrotropin (TSH)-induced production of vascular endothelial growth factor in thyroid cancer cells in vitro: evaluation of TSH signal transduction and of angiogenesis-stimulating growth factors.

Thyroid tumor growth requires angiogenesis, and vascular endothelial growth factor (VEGF) has been shown to be the most important endothelial mitogen. TSH is the major thyrotropic hormone, but its impact to modulate VEGF production has not yet been studied. Several other growth factors have also been shown to affect thyroid cancer cell growth and function in vitro. Therefore, the aim of the current study was to 1) establish the effect of TSH on VEGF as well as 2) evaluate the TSH signal transduction of this effect, and 3) screen other growth factors for the ability to modulate VEGF in thyroid cancer cell lines. HTC, a follicular cancer cell line lacking endogenous TSH receptor (TSHr), its receptor positive variant (HTC TSHr), and a cell line of Huerthle cell origin (XTC) were used. After stimulation with growth factors in vitro [TSH; epidermal growth factor (EGF), IGF, placenta growth factor, TGF-alpha, TGF-beta1, fibroblast growth factor, platelet-derived growth factor, and hepatocyte growth factor] cells were analyzed for VEGF gene expression by Northern blotting and for VEGF protein by enzyme immunoassay. TSHr signal transduction was evaluated by analyzing the effect of stimulators (cholera toxin, 8-bromo-cAMP, forskolin, and 12-O-tetradecanoyl-phorbol-13-acetate) and inhibitors (2',5'-dideoxyadenosine and staurosporine) on VEGF protein levels under basal and TSH-stimulated conditions. TSH increased VEGF mRNA and protein in a dose-dependent manner in HTC TSHr and XTC cells by up to 40%. The effects of TSH were mediated by protein kinase C (PKC), rather than protein kinase A (PKA), stimulation, because inhibition of PKC by staurosporine resulted in a decrease in VEGF production of up to 65%, whereas inhibition of the PKA signal transduction pathway (2',5'-dideoxyadenosine) resulted in only a minor decrease. TSH was not the most powerful stimulator of VEGF production. TGF-beta1 and EGF were 1.5- to 2-fold more potent. Placenta growth factor and TGF-alpha did not induce VEGF production in TSHr-positive HTC cells, whereas they did induce VEGF production in TSHr-negative HTC cells. In thyroid cancer cell lines, TSH induces VEGF production involving the PKC, rather than the PKA, pathway. However, EGF and TGF-beta increase the capacity of thyroid cancer cells to provide VEGF more effectively than TSH. In the absence of a functioning TSHr, additional growth factors, such as TGF-alpha, increase capacity for VEGF stimulation.

Cell Line, Tumor↗
PubMed2005

Mechanisms of action of angiotensin II on mammalian sperm function.

Angiotensin II (AII) stimulates capacitation and fertilizing ability in mammalian spermatozoa, with the binding of AII to its receptors resulting in stimulation of cAMP production in both uncapacitated and capacitated cells. This study investigated possible mechanisms whereby AII affects cAMP availability. The first question was whether extracellular Ca2+ is required for responses in mouse spermatozoa and, using chlortetracycline fluorescence analysis, it was clear that cells responded to AII only when the medium contained CaCl2, with both 90 microM and 1.80 mM supporting a significant acceleration of capacitation. Consistent with those results, AII significantly stimulated cAMP production in both CaCl2-containing media tested, the response being greater in that containing 1.80 mM. Several different agents that might affect the signalling pathway stimulated by AII were then evaluated in uncapacitated suspensions. Chlortetracycline analysis revealed that pertussis toxin abolished responses to AII, suggesting the involvement of an inhibitory Galpha subunit; dideoxyadenosine, a specific membrane-associated adenylyl cyclase (mAC) P-site inhibitor, also blocked responses, suggesting involvement of an mAC. cAMP determinations confirmed that both reagents also abolished AII's stimulation of cAMP. In contrast, nifedipine, a Ca2+ channel blocker, did not inhibit AII's effects on spermatozoa. Finally, in capacitated suspensions, both pertussis toxin and dideoxyadenosine were again shown to block AII's stimulation of cAMP. These results suggest that responses to AII involve an inhibitory G protein and an mAC, but it is likely that AII-receptor coupling does not stimulate directly mAC but rather does so in an indirect manner, perhaps by altering the intracellular Ca2+ concentration.

Angiotensin II↗
PubMed1975

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.

Adenine↗
PubMed1984

Progesterone inhibition of Xenopus oocyte adenylate cyclase is not mediated via the Bordetella pertussis toxin substrate.

Preincubation of intact Xenopus laevis oocytes with islet-activating protein (IAP) stimulates plasma membrane adenylate cyclase activity measured in the presence of either GTP or 5'-guanylyl imidodiphosphate. The toxin appears to act by catalyzing the ADP-ribosylation of a 41,000-D protein in the oocyte plasma membrane that is distinct from the 45,000- and 52,000-D proteins labeled by cholera toxin. Preincubation of intact oocytes with IAP for 2.5 hr inhibits 80% of the ADP-ribosylation of oocyte membranes subsequently prepared and treated in vitro with IAP in the presence of [alpha-32P]NAD. The abilities of progesterone and the P site agonist, 2',5'-dideoxyadenosine, to inhibit plasma membrane adenylate cyclase are not inhibited by IAP treatment, but IAP pretreatment abolishes inhibition by acetylcholine. In addition, IAP pretreatment of oocytes slows the time course of germinal vesicle breakdown induced by suboptimal concentrations of progesterone. This effect of toxin upon oocyte maturation is apparently due to its effect upon basal cyclase activity, since IAP has no effect upon the time course of maturation induced by 10 microM progesterone. These results indicate that, even though the oocyte adenylate cyclase does contain a functional Ni subunit, inhibition of Xenopus oocyte adenylate cyclase by progesterone or 2',5'-dideoxyadenosine is not mediated via the Bordetella pertussis toxin substrate.

Adenosine Diphosphate Ribose↗
PubMed1994

Divergent anti-human immunodeficiency virus activity and anabolic phosphorylation of 2',3'-dideoxynucleoside analogs in resting and activated human cells.

The mechanism of divergent anti-human immunodeficiency virus type 1 (HIV-1) activity of various 2',3'-dideoxynucleoside analogs (ddNs) in peripheral blood mononuclear cells (PBM) was studied. We demonstrate that the in vitro anti-HIV-1 activity of various ddNs varies profoundly and that the divergent antiviral activity is related to the extent of anabolic phosphorylation of each ddN and its counterpart 2'-deoxynucleoside (dN). We also show that certain ddNs cause a reduction of their counterpart dNTP formation in PBM in the following order: 2',3'-dideoxycytidine (ddC) >> 2',3'-didehydro-2',3'-dideoxythymidine (d4T), 3'-thia-2',3'-dideoxycytidine (3TC), 2',3'-dideoxyinosine (ddI), 2',3'-dideoxyguanosine (ddG) > 3'-azido-2',3'-dideoxythymidine (AZT) > 2'-beta-fluoro-2',3'-dideoxyadenosine (F-ara-ddA). Based on the phosphorylation profiles, anti-HIV-1 ddNs can be classified into two groups: (i) cell-activation-dependent ddNs such as AZT and d4T that are preferentially phosphorylated, yield higher ratios of ddNTP/dNTP, and exert more potent anti-HIV-1 activity in activated cells than in resting cells; and (ii) cell-activation-independent ddNs including ddI (and 2',3'-dideoxyadenosine), F-ara-ddA, ddG, ddC, and 3TC that produce higher ratios of ddNTP/dNTP and exert more potent anti-HIV-1 activity in resting cells. These data should provide a basis for the elucidation of the mechanism of the divergent antiretroviral activity of ddNs.

Cells, Cultured↗