scholarly journals A simple assay for adenylate cyclase in intact cells by affinity elution chromatography

1978 ◽  
Vol 174 (3) ◽  
pp. 699-702
Author(s):  
A K Sinha ◽  
R W Colman
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Platelet Rich Plasma ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Intact Cells ◽  
Atp Pool ◽  
Dependent Protein ◽  
Elution Chromatography ◽  
Cellulose Column

A method using the principle of affinity elution chromatography is described for the assay of adenylate cyclase in intact human platelets. By incubating platelet-rich plasma in the presence of radioactively labelled adenine, the ATP pool of the cells was prelabelled. Formation of labelled cyclic AMP from ATP was determined by extracting the platelets with HC1O4. After removal of the latter as KC1O4, the extract containing cyclic AMP and other adenine nucleotides was adsorbed in a NN-diethyl-N-2-hydroxypropylamino (QAE)-cellulose column. The column was washed, and subsequently cyclic AMP was specifically eluted with a cyclic AMP-dependent protein kinase and the radioactivity of the eluate was determined.

scholarly journals Characterization of Sp-5,6-dichloro-1-β-d-ribofuranosylbenzimidazole- 3′,5′-monophosphorothioate (Sp-5,6-DCl-cBiMPS) as a potent and specific activator of cyclic-AMP-dependent protein kinase in cell extracts and intact cells

1991 ◽  
Vol 279 (2) ◽  
pp. 521-527 ◽  
Author(s):  
M Sandberg ◽  
E Butt ◽  
C Nolte ◽  
L Fischer ◽  
M Halbrügge ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Prostaglandin E1 ◽  
Human Platelets ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Cell Extracts ◽  
Platelet Membranes ◽  
Camp Analogue ◽  
Dependent Protein

A newly designed cyclic AMP (cAMP) analogue, Sp-5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole-3′,5′-monophosphorothioate (Sp-5,6-DCl-cBiMPS), and 8-(p-chlorophenylthio)-cAMP (8-pCPT-cAMP) were compared with respect to their chemical and biological properties in order to assess their potential as activators of the cAMP-dependent protein kinases (cAMP-PK) in intact cells. Sp-5,6-DCl-cBiMPS was shown to be both a potent and specific activator of purified cAMP-PK and of cAMP-PK in platelet membranes, whereas 8-pCPT-cAMP proved to be a potent activator of cAMP-PK and cyclic-GMP-dependent protein kinase (cGMP-PK) both as purified enzymes and in platelet membranes. Sp-5,6-DCl-cBiMPS was not significantly hydrolysed by three types of cyclic nucleotide phosphodiesterases, whereas 8-pCPT-cAMP (and 8-bromo-cAMP) was hydrolysed to a significant extent by the Ca2+/calmodulin-dependent phosphodiesterase and by the cGMP-inhibited phosphodiesterase. The apparent lipophilicity, a measure of potential cell-membrane permeability, of Sp-5,6-DCl-cBiMPS was higher than that of 8-pCPT-cAMP. Extracellular application of Sp-5,6-DCl-cBiMPS to intact human platelets reproduced the pattern of protein phosphorylation induced by prostaglandin E1, a cAMP-increasing inhibitor of platelet activation. In intact platelets, Sp-5,6- DCl-cBiMPS was also more effective than 8-pCPT-cAMP in inducing quantitative phosphorylation of the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP), a major substrate of cAMP-PK in platelets. As observed with prostaglandin E1, pretreatment of human platelets with Sp-5,6-DCl-cBiMPS prevented the aggregation induced by thrombin. The results suggest that Sp-5,6-DCl-cBiMPS is a very potent and specific activator of cAMP-PK in cell extracts and intact cells and, in this respect, is superior to any other cAMP analogue used for intact-cell studies. In contrast with 8-pCPT-cAMP, Sp-5,6-DCl-cBiMPS can be used to distinguish the signal-transduction pathways mediated by cAMP-PK and cGMP-PK.

scholarly journals Inhibition of adenylate cyclase by adenosine analogues in preparations of broken and intact human platelets. Evidence for the unidirectional control of platelet function by cyclic AMP

1978 ◽  
Vol 176 (1) ◽  
pp. 83-95 ◽  
Author(s):  
R J Haslam ◽  
M M L Davidson ◽  
J V Desjardins
Keyword(s):  
Platelet Aggregation ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Arginine Vasopressin ◽  
Prostaglandin E1 ◽  
Platelet Rich Plasma ◽  
Kinetic Studies ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
High Concentrations

Whereas adenosine itself exerted independent stimulatory and inhibitory effects on the adenylate cyclase activity of a platelet particulate fraction at low and high concentrations respectively, 2-substituted and N6-monosubstituted adenosines had stimulatory but greatly decreased inhibitory effects. Deoxyadenosines, on the other hand, had enhanced inhibitory but no stimulatory effects. The most potent inhibitors found were, in order of increasing activity, 9-(tetrahydro-2-furyl)adenine (SQ 22536), 2′,5′-dideoxyadenosine and 2′-deoxyadenosine 3′-monophosphate. Kinetic studies on prostaglandin E1-activated adenylate cyclase showed that the inhibition caused by either 2′,5′-dideoxyadenosine or compound SQ 22536 was non-competitive with MgATP and that the former compound, at least, showed negative co-operativity; 50% inhibition was observed with 4 micron-2′,5′-dideoxyadenosine or 13 micron-SQ 22536. These two compounds also inhibited both the basal and prostaglandin E1-activated adenylate cyclase activities of intact platelets, when these were measured as the increases in cyclic [3H]AMP in platelets that had been labelled with [3H]adenine and were then incubated briefly with papaverine or papaverine and prostaglandin E1. Both compounds, but particularly 2′,5′-dideoxyadenosine, markedly decreased the inhibition by prostaglandin E1 of platelet aggregation induced by ADP or [arginine]vasopressin as well as the associated increases in platelet cyclic AMP, so providing further evidence that the effects of prostaglandin E1 on platelet aggregation are mediated by cyclic AMP. 2′-Deoxyadenosine 3′-monophosphate did not affect the inhibition of aggregation by prostaglandin E1, suggesting that the site of action of deoxyadenosine derivatives on adenylate cyclase is intracellular. Neither 2′,5′-dideoxyadenosine nor compound SQ 22536 alone induced platelet aggregation. Moreover, neither compound potentiated platelet aggregation or the platelet release reaction when suboptimal concentrations of ADP, [arginine]vasopressin, collagen or arachidonate were added to heparinized or citrated platelet-rich plasma in the absence of prostaglandin E1. These results show that cyclic AMP plays no significant role in the responses of platelets to aggregating agents in the absence of compounds that increase the platelet cyclic AMP concentration above the resting value.

The Increased Sensitivity of Platelets to Prostacyclin in Marathon Runners

1984 ◽  
Vol 51 (03) ◽  
pp. 385-387 ◽  
Author(s):  
Clive J Dix ◽  
David G Hassall ◽  
K Richard Bruckdorfer
Keyword(s):  
Cardiovascular Disease ◽  
Endothelial Cells ◽  
Platelet Aggregation ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Platelet Rich Plasma ◽  
Marathon Runners ◽  
Inhibition Of Platelet Aggregation ◽  
Increased Sensitivity ◽  
Membrane Bound

SummaryPlatelet-rich plasma was obtained 24 hr after the race ended from athletes who ran in the London marathon. The platelets were only marginally less sensitive to adrenaline than were those of non-runners using conventional aggregation tests. However, the runners’ platelets were much more sensitive to inhibition by prostacyclin, a prostaglandin synthesized by endothelial cells. It appeared that this effect was due to a greater activity in the platelets of the membrane-bound adenylate cyclase enzyme which generates intracellular cyclic AMP. Cyclic AMP production is known to be stimulated by prostacyclin and to cause the inhibition of platelet aggregation. The results indicate another possible protective effect of exercise against cardiovascular disease which is independent of the known changes in lipoprotein concentrations previously observed in athletes.

scholarly journals Phosphorylation of β-Catenin by Cyclic AMP-Dependent Protein Kinase Stabilizes β-Catenin through Inhibition of Its Ubiquitination

2005 ◽  
Vol 25 (20) ◽  
pp. 9063-9072 ◽  
Author(s):  
Shin-ichiro Hino ◽  
Chie Tanji ◽  
Keiichi I. Nakayama ◽  
Akira Kikuchi
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Wnt Signaling ◽  
Protein Level ◽  
Glycogen Synthase ◽  
Wnt Signaling Pathway ◽  
Prostaglandin E ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Dependent Protein

ABSTRACT The mechanism of cross talk between the Wnt signaling and cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) pathways was studied. Prostaglandin E1 (PGE1), isoproterenol, and dibutyryl cAMP (Bt2cAMP), all of which activate PKA, increased the cytoplasmic and nuclear β-catenin protein level, and these actions were suppressed by a PKA inhibitor and RNA interference for PKA. PGE1 and Bt2cAMP also increased T-cell factor (Tcf)-dependent transcription through β-catenin. Bt2cAMP suppressed degradation of β-catenin at the protein level. Although PKA did not affect the formation of a complex between glycogen synthase kinase 3β (GSK-3β), β-catenin, and Axin, phosphorylation of β-catenin by PKA inhibited ubiquitination of β-catenin in intact cells and in vitro. Ser675 was found to be a site for phosphorylation by PKA, and substitution of this serine residue with alanine in β-catenin attenuated inhibition of the ubiquitination of β-catenin by PKA, PKA-induced stabilization of β-catenin, and PKA-dependent activation of Tcf. These results indicate that PKA inhibits the ubiquitination of β-catenin by phosphorylating β-catenin, thereby causing β-catenin to accumulate and the Wnt signaling pathway to be activated.

scholarly journals Forskolin refractoriness. Exposure to the diterpene alters guanine nucleotide-dependent adenylate cyclase and calcium-uptake activity of cells cultured from the rat aorta

1987 ◽  
Vol 241 (2) ◽  
pp. 463-467 ◽  
Author(s):  
J F Krall ◽  
N Jamgotchian
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cultured Cells ◽  
Rat Aorta ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Morphological Properties ◽  
Guanine Nucleotide ◽  
Intact Cells ◽  
Uptake Activity

Cells with the morphological properties of endothelial cells were cultured from the rat aorta. The cultured cells accumulated 45Ca2+ from the medium in a manner which was stimulated by forskolin and by 8-bromo-cyclic AMP. Pretreating the cultures for 20 h with forskolin diminished forskolin-dependent Ca2+-uptake activity. Adenylate cyclase activity of cultured cell homogenates was stimulated by guanosine 5′-[beta, gamma-imido]triphosphate (p[NH]ppG) and forskolin, and by isoprenaline in the presence, but not in the absence, of guanine nucleotide. p[NH]ppG increased forskolin sensitivity and caused a leftward shift in the forskolin dose-response curve. Pretreating the cultured cells with forskolin for 20 h, conditions that decreased forskolin-dependent Ca2+ uptake, increased basal and guanine nucleotide-dependent adenylate cyclase activity, but not forskolin-dependent activity determined in the absence of p[NH]ppG. Forskolin pretreatment diminished p[NH]ppG's capacity to increase forskolin sensitivity, but did not have a significant effect on either the sensitivity of adenylate cyclase to p[NH]ppG or its responsiveness to isoprenaline. These results suggest that the Ca2+-uptake mechanism is cyclic AMP-dependent and that guanine nucleotides mediated forskolin-dependent cyclic AMP production by the intact cells. In addition, there may be different guanine nucleotide requirements for hormone-receptor coupling and forskolin activation.

Activation of human platelets by 2-arachidonoylglycerol is enhanced by serotonin

2003 ◽  
Vol 89 (02) ◽  
pp. 340-347 ◽  
Author(s):  
Monica Bari ◽  
Domenico Del Principe ◽  
Alessandro Finazzi-Agrò ◽  
Mauro Maccarrone
Keyword(s):  
Cyclic Amp ◽  
Cannabinoid Receptors ◽  
Cannabinoid Receptor ◽  
Platelet Rich Plasma ◽  
Maximum Velocity ◽  
Human Platelets ◽  
Platelet Surface ◽  
Therapeutic Implications ◽  
Inositol 1,4,5 Trisphosphate ◽  
Dependent Mechanism

SummaryThe endocannabinoid 2-arachidonoylglycerol (2-AG) has been shown to activate human platelets in platelet-rich plasma, by binding to a “platelet-type” cannabinoid receptor (CBPT). Here, washed human platelets were used to characterize the binding of [3H]2-AG to CBPT, showing a dissociation constant (Kd) of 140 ± 31 nM and a maximum binding (Bmax) of 122 ± 10 pmol.mg protein-1. Selective antagonists of both CB1 and CB2 cannabinoid receptors inhibited this binding, which was enhanced up to ~230% over the controls by 1 µM serotonin (5-hydroxytryptamine, 5-HT). Human platelets were also able to bind [3H]5-HT (Kd = 79 ± 17 nM, Bmax = 14.6 ± 1.3 pmol.mg protein-1), and 1 µM 2-AG enhanced this binding up to ~150%. Moreover, they were able to take up [3H]5-HT through a high affinity transporter (Michaelis-Menten constant = 22 ± 2 nM, maximum velocity = 344 ± 15 pmol.min-1.mg protein-1), which was not affected by 2-AG. Interestingly, 5-HT did not affect the activity of the 2-AG transporter of human platelets. Treatment of washed platelets with 1 µM 2-AG led to increased intracellular inositol-1,4,5-trisphosphate (up to ~300%) and decreased cyclic AMP (down to ~50%). Furthermore, treatment of pre-loaded platelets with 1 µM 2-AG induced a ~300% increase in [3H]2-AG release, according to a CBPT-dependent mechanism. Also, 1 µM 5-HT enhanced the effect of 2-AG on inositol-1,4,5-trisphosphate (~500% of the controls), cyclic AMP (~20%) and [3H]2-AG release (~570%), and the latter process was shown to be partly (~50%) involved in the 5-HT-dependent platelet activation. Taken together, reported findings represent the first demonstration that 2-AG and 5-HT can mutually reinforce their receptor binding on platelet surface, which might have therapeutic implications.

Depressed Responsiveness To Adrenaline In Platelets From Apparently Normal Human Donors – A Familial Trait

1981 ◽  
Author(s):  
M C Scrutton ◽  
K R Bruckdorfer ◽  
R A Hutton
Keyword(s):  
Adenylate Cyclase ◽  
Platelet Function ◽  
Divalent Cation ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
The Other ◽  
Stimulus Response ◽  
A 23187 ◽  
Phospholipid Classes ◽  
Normal Human

Decreased responsiveness to adrenaline has been observed in 5 out of approximately 150 apparently normal unrelated human donors. In 4 donors, familial studies have shown that this trait is inherited. Three of the donors, as well as their affected relatives, exhibit depressed responsiveness to collagen and vasopressin but normal responsiveness to ADP and thrombin in association with the decreased responsiveness to adrenaline. The other two affected donors exhibit normal responsiveness to most other agonists although in one instance depression of responsiveness to vasopressin and absence of a secretory response to ADP may be associated with the decreased adrenaline response.Normal responsiveness can be restored in all instances either by incubating the platelet-rich plasma at 20°C or by addition at 37°C of a low concentration of the divalent cation ionophore, A-23187. No such effect results from addition of an adenylate cyclase inhibitor. All affected platelets have normal ATP and ADP contents, cholesterol to phospholipid ratios, and composition of the phospholipid classes. Mixing experiments demonstrate the absence of a circulating inhibitor of platelet function and suggest that the defect resides in the platelets. We conclude that depressed responsiveness of human platelets to adrenaline may result from a defect in Ca2+ mobilisation to the cytosol. The observed selectivity in the agonists affected may indicate that the stimulus-response coupling pathways converge at the level of an increase in cytosolic Ca2+ concentration.

scholarly journals Labeling of the releasable adenine nucleotides of washed human platelets

Blood ◽  
1977 ◽  
Vol 49 (1) ◽  
pp. 89-99 ◽  
Author(s):  
HJ Reimers ◽  
MA Packham ◽  
JF Mustard
Keyword(s):  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Energy Charge ◽  
Human Platelets ◽  
Transport Processes ◽  
Adenylate Energy Charge ◽  
Prolonged Incubation ◽  
Atp Pool ◽  
Adenine Nucleotide Pool

Abstract In rabbit platelets, the metabolically active ATP pool equilibrates with the releasable ATP pool within 1 day. The studies showing this have now been extended to human platelets. Human platelets labeled with 14C-adenosine or 14C-adenine were incubated for up to 10 hr in vitro at 37 degrees C. After 10 hr, about 12% of the total platelet 14C-ATP and 14C-ADP had become releasable with thrombin (4.2 units/ml). Lysis of platelets did not occur, since less than 1% of the platelet-bound 51Cr from platelets labeled with this radioisotope appeared in the ambient fluid upon thrombin treatment. The 14C-ATP/14C-ADP ratio of the released adenine nucleotides (7.6) was similar to the 14C-ATP/14C-ADP ratio of the nonreleasable adenine nucleotides (7.1) 2 hr after the labeling with 14C-adenosine. However, upon prolonged incubation (10 hr) in vitro, the 14C-ATP/14C-ADP ratio of the releasable adenine nucleotides decreased to 2.7. The adenylate energy charge and the 14C- ATP/14C-ADP ratio of the metabolic adenine nucleotide pool did not change significantly during the time of observation. The 14C-ATP content of the platelets decreased by less than 1% hr of incubation at 37 degrees C. These observations are interpreted to mean that the 14C is transferred from the metabolically active, nonreleasable adenine nucleotide pool of human platelets into the releasable adenine nucleotide pool as ATP and is partially hydrolyzed there to yield ADP. The transfer of ATP across the storage organelle membrane of platelets may be similar to transport processes in the chromaffin cells of the adrenal medulla and may represent a general phenomenon in cells that possess storage organelles containing adenine nucleotides.

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