scholarly journals Cytosolic free Ca2+ oscillations induced by diadenosine 5′,5′′′-P1,P3-triphosphate and diadenosine 5′,5′′′-P1,P4-tetraphosphate in single rat hepatocytes are indistinguishable from those induced by ADP and ATP respectively

1995 ◽  
Vol 310 (2) ◽  
pp. 629-635 ◽  
Author(s):  
A K Green ◽  
P H Cobbold ◽  
C J Dixon
Keyword(s):  
Cyclic Amp ◽  
Short Duration ◽  
Phorbol Ester ◽  
Individual Cell ◽  
Rat Hepatocytes ◽  
Low Concentrations ◽  
High Concentrations ◽  
The Individual ◽  
Single Response

Diadenosine 5′,5‴-P1,P3-triphosphate (Ap3A) and diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) induce distinctive patterns of [Ca2+]i oscillations in single rat hepatocytes. We show here that [Ca2+]i oscillations induced by Ap3A and ADP are indistinguishable and that [Ca2+]i oscillations induced by Ap4A closely resemble those induced by ATP. These similarities embrace the following: (1) ADP and Ap3A invariably induce [Ca2+]i transients of short duration (approx. 9 s). Ap4A, like ATP, can induce, depending upon the individual cell, either transients of short duration (approx. 9 s), transients of much longer duration or a mixture of short and long transients within a single response. We show here that the pattern of oscillations induced by Ap4A is similar to that induced by ATP in the same hepatocyte. (2) Elevated intracellular cyclic AMP concentration modulates Ap3A-induced transients, like ADP-induced transients, through an increase in both the peak [Ca2+]i and the frequency of the transients. In contrast, Ap4A-induced transients, like ATP-induced transients, develop an increased duration or a sustained rise in [Ca2+]i, with no rise in peak [Ca2+]i. (3) Ap3A-induced transients, like ADP-induced transients, are abolished by low concentrations of the phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDB; 5-10 nM), whereas long Ap4A-induced transients, like long ATP-induced transients, are refractory to high concentrations of PDB (100 nM). We propose that the [Ca2+]i oscillations induced in rat hepatocytes by Ap3A are mediated by the same purinoceptor that mediates the effects of ADP, whereas the oscillations induced by Ap4A are mediated by the same purinoceptor(s) that mediate the effects of ATP.

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

1989 ◽  
Vol 61 (02) ◽  
pp. 254-258 ◽  
Author(s):  
Margaret L Rand ◽  
Peter L Gross ◽  
Donna M Jakowec ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Inhibitory Effects ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
In Vitro Effects ◽  
Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

scholarly journals THE TUMOR-PROMOTER PHORBOL ESTER (12-0-TETRADECANOYL-PHORBOL-13-ACETATE), A POTENT AGGREGATING AGENT FOR BLOOD PLATELETS

1974 ◽  
Vol 60 (2) ◽  
pp. 325-336 ◽  
Author(s):  
Marjorie B. Zucker ◽  
Walter Troll ◽  
Sidney Belman
Keyword(s):  
Platelet Aggregation ◽  
Cyclic Amp ◽  
Phorbol Ester ◽  
Platelet Rich Plasma ◽  
Direct Action ◽  
Blood Platelets ◽  
Tumor Promoter ◽  
Metabolic Inhibitors ◽  
Rapid Phase ◽  
Low Concentrations

The phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate, a potent tumor-promoting agent, caused irreversible platelet aggregation when more than 0.02 µM was stirred with human citrated or heparinized platelet-rich plasma (PRP). With washed platelets, 1 nM was effective. The alcohol phorbol, which has little tumor-promoting activity, failed to cause platelet aggregation. With all but low concentrations of phorbol ester, aggregation was succeeded by a rapid phase. The latter was prevented or reduced by enzymes which destroy ADP and by aspirin, was associated with a change in platelet shape, and was presumably due to released ADP. At higher concentrations, only a rapid phase was seen, and these inhibitors were not effective. Low concentrations did not aggregate platelets in PRP containing sufficient EDTA or EGTA to chelate ionized calcium or in PRP from thrombasthenic patients; higher concentrations caused slight aggregation. Both the primary, non-ADP-dependent aggregation and the rapid ADP-dependent aggregation were markedly inhibited by substances which increase cyclic AMP, metabolic inhibitors, and the sulfhydryl inhibitor N-ethylmaleimide. Phorbol ester reduced platelet cyclic AMP only when it had been previously elevated by prostaglandin E1. 1 µM did not release ß-glucuronidase, lactic dehydrogenase, or inflammatory material from platelets in 4–5 min despite marked aggregation, but liberated all three in 30 min. The possibility is discussed that low phorbol ester concentrations cause primary aggregation by a direct action on platelet actomyosin.

scholarly journals Oscillations in cytosolic free Ca2+ induced by ADP and ATP in single rat hepatocytes display differential sensitivity to application of phorbol ester

1995 ◽  
Vol 309 (1) ◽  
pp. 145-149 ◽  
Author(s):  
C J Dixon ◽  
P H Cobbold ◽  
A K Green
Keyword(s):  
Phorbol Ester ◽  
Rat Hepatocytes ◽  
Differential Sensitivity ◽  
Long Duration ◽  
Low Concentrations

We have previously described differences in the oscillatory responses of cytosolic free Ca2+ concentration ([Ca2+]i) in hepatocytes to ADP and ATP, which we have interpreted as evidence that these two nucleotides are acting at distinct receptors. We show here that ADP- and ATP-induced oscillations are differentially sensitive to application of the phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDB). ADP-induced [Ca2+]i oscillations are abolished by low concentrations of PDB (5-10 nM), whereas ATP-induced oscillations of long duration are refractory to PDB, even at greatly elevated concentrations (100 nM). The data illustrate a further difference in the actions of ADP and ATP, strengthening the argument that these agonists are not acting at the same receptor on rat hepatocytes.

scholarly journals Adenosine 3':5'-monophosphate content and actions in the division cycle of synchronized HeLa cells.

1976 ◽  
Vol 71 (2) ◽  
pp. 515-534 ◽  
Author(s):  
C E Zeilig ◽  
R A Johnson ◽  
E W Sutherland ◽  
D L Friedman
Keyword(s):  
Cell Cycle ◽  
Cyclic Amp ◽  
Hela Cells ◽  
S Phase ◽  
Intracellular Camp ◽  
Specific Effects ◽  
Low Concentrations ◽  
High Concentrations ◽  
Camp Levels ◽  
Stimulation Of

The involvement of adenosine 3':5'-monophosphate (cAMP) in the regulation of the cell cycle was studied by determining intracellular fluctuations in cAMP levels in synchronized HeLa cells and by testing the effects of experimentally altered levels on cell cycle traverse. Cyclic AMP levels were lowest during mitosis and were highest during late G-1 or early S phase. These findings were supported by results obtained when cells were accumulated at these points with Colcemid or high levels of thymidine. Additional fluctuations in cAMP levels were observed during S phase. Two specific effects of cAMP on cell cycle traverse were found. Elevation of cAMP levels in S phase or G-2 caused arrest of cells in G-2 for as long as 10 h and lengthened M. However, once cells reached metaphase, elevation of cAMP accelerated the completion of mitosis. Stimulation of mitosis was also observed after addition of CaCl2. The specificity of the effects of cAMP was verified by demonstrating that: (a) intracellular cAMP was increased after exposure to methylisobutylxanthine (MIX) before any observed effects on cycle traverse; (b) submaximal concentrations of MIX potentiated the effects of isoproterenol; and (c) effects of MIX and isoproterenol were mimicked by 8-Br-cAMP. MIX at high concentrations inhibited G-1 traverse, but this effect did not appear to be mediated by cAMP. Isoproterenol slightly stimulated G-1 traverse and partially prevented the MIX-induced delay. Moreover, low concentrations of 8-Br-cAMP (0.10-100 muM) stimulated G-1 traverse, whereas high concentrations (1 mM) inhibited. Both of these effects were also observed with the control, Br-5'-AMP, at 10-fold lower concentrations.

scholarly journals Calcium-mobilizing hormones and phorbol myristate acetate mediate heterologous desensitization of the hormone-sensitive hepatic Na+/K+ pump

1987 ◽  
Vol 248 (3) ◽  
pp. 807-813 ◽  
Author(s):  
C J Lynch ◽  
S B Bocckino ◽  
P F Blackmore ◽  
J H Exton
Keyword(s):  
Cyclic Amp ◽  
Rat Hepatocytes ◽  
Previous Exposure ◽  
High Concentration ◽  
Isolated Rat Hepatocytes ◽  
Pump Activity ◽  
Heterologous Desensitization ◽  
Tumour Promoters ◽  
High Concentrations ◽  
Stimulation Of

The Na+/K+ pump in rat hepatocytes is stimulated in response to Ca2+-mobilizing hormones such as [arginine]vasopressin (AVP), angiotensin II and adrenaline, as well as tumour promoters such as 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). The ability of these agents to increase cellular contents of diacylglycerol and activate protein kinase C may be necessary to observe this response. In the present work, ouabain-sensitive 86Rb+ uptake was studied in isolated rat hepatocytes to help to explain why stimulation of the Na+/K+ pump by Ca2+-mobilizing hormones and tumour promoters is not temporally sustained relative to other hormone responses. A transient stimulation (3-4 min) of the Na+/K+ pump was observed in hepatocytes exposed to high (10 nM), but not low (0.1 nM), concentrations of AVP. Experiments with the Ca2+ chelator EGTA and the Na+ ionophore monensin indicate that the rapid secondary decrease in Na+/K+-pump activity which occurs after AVP stimulation is not due to changes in cytosolic Ca2+ and Na+ concentrations. When added after the stimulation and rapid decrease in Na+/K+-pump activity induced in hepatocytes by a high concentration of AVP, a second challenge with AVP or PMA failed to stimulate the pump. Similarly, previous exposure of hepatocytes to angiotensin, adrenaline or PMA attenuated the subsequent Na+/K+-pump responses to AVP and PMA. In contrast, previous exposure to AVP had no significant effect on subsequent stimulation of the Na+/K+-pump by monensin, glucagon, forskolin or 8-p-chlorophenylthio cyclic AMP. In addition, exposure to monensin had no effect on subsequent responses to AVP and PMA. These data indicate that high concentrations of Ca2+-mobilizing hormones and PMA result in heterologous desensitization of the hepatic Na+/K+ pump to subsequent stimulation by Ca2+-mobilizing hormones and PMA, but not by cyclic-AMP-dependent agonists or monensin.

Hormone-stimulated glycogenolysis in isolated goldfish hepatocytes

1976 ◽  
Vol 231 (1) ◽  
pp. 191-197 ◽  
Author(s):  
MJ Birnbaum ◽  
J Schultz ◽  
JN Fain
Keyword(s):  
Cyclic Amp ◽  
Blocking Agent ◽  
Ionophore A23187 ◽  
Bacterial Collagenase ◽  
Goldfish Carassius Auratus ◽  
Low Concentrations ◽  
Cyclic Amp Accumulation ◽  
The Common ◽  
High Concentrations ◽  
Very High

Hepatocytes isolated from the liver of the common goldfish Carassius auratus L. with crude bacterial collagenase maintained ATP levels for at least 2 h. Glycogenolysis was maximally activated by 1 X 10(-6) M epinephrine and 5.8 X 10(-9) M glucagon. In liver cells incubated in calcium-free buffer containing 1 mM ethylene glycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid, basal glycogenolysis was enhanced by the addition of 1-4 mM calcium but the elevation of cyclic AMP and glycogenolysis due to epinephrine was unaffected by calcium. The divalent cation ionophore A23187 did not alter basal or hormone-stimulated glycogenolysis. Isoproterenol was approximately as potent as epinephrine but phenylephrine was glycogenolytic only at very high concentrations. l-Propranolol competitively inhibited the increased glycogenolysis due to catecholamines but phentolamine was ineffective as a blocking agent. Isoproterenol and epinephrine stimulated glycogenolysis at lower concentrations than those required to elevate cyclic AMP accumulation. Phenylephrine was without effect on cyclic AMP. Propranolol competitively inhibited both epinephrine- and isoproterenol-stimulated cyclic AMP accumulation, but phentolamine did not block either response. Catecholamine-stimulated glycogenolysis in goldfish liver is apparently a beta-adrenergic effect. However, low concentrations of epinephrine enhance glycogenolysis without affecting total cyclic AMP.

scholarly journals 1,2-Diacylglycerols overcome cyclic AMP-mediated inhibition of phosphatidylcholine synthesis in GH3 pituitary cells

1990 ◽  
Vol 267 (1) ◽  
pp. 17-22 ◽  
Author(s):  
R N Kolesnick
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Phorbol Esters ◽  
Pituitary Cells ◽  
Dibutyryl Cyclic Amp ◽  
Kinase C ◽  
Low Concentrations ◽  
High Concentrations ◽  
Phosphatidylcholine Synthesis

Previous studies showed that phorbol esters and thyrotropin-releasing hormone (TRH) stimulated phosphatidylcholine synthesis via protein kinase C in GH3 pituitary cells [Kolesnick (1987) J. Biol. Chem. 262, 14525-14530]. In contrast, 1,2-diacylglycerol-stimulated phosphatidylcholine synthesis appeared independent of protein kinase C. The present studies compare phosphatidylcholine synthesis stimulated by these agents with inhibition via the cyclic AMP system. The potent phorbol ester phorbol 12-myristate 13-acetate (PMA, 10 nM) increased [32P]Pi incorporation into phosphatidylcholine at 30 min to 159 +/- 6% of control. The adenylate cyclase activator cholera toxin (CT; 10 nM) and the cyclic AMP analogue dibutyryl cyclic AMP (1 mM) abolished this effect. CT similarly abolished TRH-induced phosphatidylcholine, but not phosphatidylinositol, synthesis. This is the first report of inhibiton of receptor-mediated phosphatidylcholine synthesis by the cyclic AMP system. The 1,2-diacylglycerol 1,2-dioctanoylglycerol (diC8) also stimulated concentration-dependent phosphatidylcholine synthesis. DiC8 (3 micrograms/ml) induced an effect quantitatively similar to that of maximal concentrations of PMA and TRH, whereas a maximal diC8 concentration (30 micrograms/ml) stimulated an effect 3-4-fold greater than these other agents. CT decreased the effect of diC8 (3 micrograms/ml) by 80%. Higher diC8 concentrations overcame the CT inhibition. Similar results were obtained with dibutyryl cyclic AMP. Additional differences were found between low and high concentrations of diC8. Low concentrations of diC8 failed to induce additive phosphatidylcholine synthesis with maximal concentrations of PMA, whereas high concentrations were additive. Hence, low concentrations of 1,2-diacylglycerols appear to be regulated similarly to phorbol esters, and higher concentrations appear to act via a pathway unavailable to phorbol esters.

scholarly journals The kinetics of effector binding to phosphofructokinase. The influence of effectors on the allosteric conformational transition

1980 ◽  
Vol 189 (3) ◽  
pp. 569-579
Author(s):  
D Roberts ◽  
G L Kellett
Keyword(s):  
Cyclic Amp ◽  
Conformational Transition ◽  
Slow Phase ◽  
Protein Fluorescence ◽  
Allosteric Transition ◽  
Low Concentrations ◽  
Inhibitory Site ◽  
High Concentrations ◽  
Isomerization Pathway ◽  
Fructose 1,6 Bisphosphate

1. The extent of the allosteric transition from the R into the T conformation of rabbit skeletal muscle phosphofructokinase induced by Mg2+-1,N6-etheno-ATP was determined by stopped-flow fluorimetry from the amplitude of the slow phase of the Mg2+-1,N6-etheno-ATP fluorescence enhancement [Roberts & Kellet (1979) Biochem. J. 183, 349–360]. 2. The amplitude of the slow phase was decreased by low concentrations of the activators cyclic AMP and fructose 1,6-bisphosphate, but increased in a complex manner by the inhibitor citrate. 3. Mg2+-1,N6-etheno-ATP and Mg2+-ATP are unable to induce the T conformation to a detectable extent in the presence of saturating cyclic AMP, but can do so readily in the presence of saturating fructose 1,6-bisphosphate. 4. The conformational transitions induced in enzyme alone by different ligands were observed by changes in intrinsic protein fluorescence. In general, an R-type conformation has diminished protein fluorescence compared with a T-type conformation. 5. Mg2+-ATP exerts a complex effect on protein fluorescence; both the enhancement at low concentrations and the quenching at high concentrations of Mg2+-ATP result from the binding of Mg2+-ATP to the inhibitory site and the ensuing allosteric transition. Enhancement reflects the extent of the allosteric transition and involves both tyrosine and tryptophan, probably in the region of the active site; quenching reflects occupation of the inhibitory site and involves tyrosine at the inhibitory site. 6. The mechanism of the allosteric transition from the R into the T conformation induced by Mg2+-1,N6-etheno-ATP at low concentrations occurs predominantly by a ‘prior-isomerization’ pathway; at higher concentrations a limited contribution from a ‘substrate-guided’ pathway occurs. 7. The allosteric behaviour of phosphofructokinase with respect to Mg2+-ATP and Mg2+-1,N6-ethenol-ATP binding may be accounted for in terms of the simple, concerted model.

STUDIES ON CYCLIC NUCLEOTIDES IN THE ADRENAL GLAND

1979 ◽  
Vol 90 (1) ◽  
pp. 139-146 ◽  
Author(s):  
Sumio Shima ◽  
Yoshiko Kawashima ◽  
Masanao Hirai
Keyword(s):  
Cyclic Amp ◽  
Adrenal Cortex ◽  
Calcium Ionophore ◽  
Extracellular Calcium ◽  
Zona Fasciculata ◽  
Acth Stimulation ◽  
Steroid Production ◽  
Low Concentrations ◽  
Cyclic Amp Accumulation ◽  
High Concentrations

ABSTRACT Effects of ACTH and calcium on cyclic AMP and steroid production by the zona fasciculata-reticularis (the decapsulated fraction) from the rat adrenal cortex have been studied. Increasing concentrations of extracellular calcium enhanced the action of ACTH on cyclic AMP and steroid production. These effects of ACTH with calcium were prevented by lanthanum, but not by tetracaine or verapamil, suggesting that ACTH stimulation may be mediated by calcium through a process not involving the tetracaine- or verapamil-vulnerable step(s) of the calcium current. High concentrations of external calcium itself increased cyclic AMP accumulation without any increase in steroidogenesis. A calcium ionophore, X537A was stimulatory for steroidogenesis but inhibitory with respect to cyclic AMP accumulation. Considered together with the findings of steroidogenic stimulation by low concentrations of ACTH without cyclic AMP increase, these results suggest that ACTH primarily increases intracellular calcium mobilization thus stimulating directly the steroidogenesis, which is independent of the cyclic AMP system. Relatively high concentrations of ACTH activate the adenylate cyclase, which depends on extracellular calcium to increase cyclic AMP levels and stimulation of steroidogenesis by the decapsulated fractions of the adrenal cortex.

scholarly journals A cytosolic sperm factor triggers calcium oscillations in rat hepatocytes

1996 ◽  
Vol 313 (2) ◽  
pp. 369-372 ◽  
Author(s):  
Christopher P. BERRIE ◽  
K. S. Roy CUTHBERTSON ◽  
J. PARRINGTON ◽  
F. Anthony LAI ◽  
Karl SWANN
Keyword(s):  
Rat Hepatocytes ◽  
Calcium Oscillations ◽  
Dorsal Root Ganglion Neurons ◽  
Protein Factor ◽  
Mammalian Sperm ◽  
Low Concentrations ◽  
Sperm Factor ◽  
High Concentrations ◽  
Ganglion Neurons ◽  
Free Media

Previously it has been shown that injecting a cytosolic sperm protein factor into mammalian eggs induces sustained repetitive transients of cytosolic free Ca2+ ([Ca2+]i), or [Ca2+]i oscillations [Swann (1990) Development 110, 1295-1302]. These sperm-factor (SF)-induced [Ca2+]i oscillations are similar to those seen at fertilization. Here we demonstrate that injecting the same cytosolic extracts of mammalian sperm into single rat hepatocytes induces a series of [Ca2+]i oscillations, as measured by aequorin luminescence. SF injection into hepatocytes induced [Ca2+]i oscillations that were of longer duration, lower frequency and greater amplitude than those seen with the Ins(1,4,5)P3-generating agonist phenylephrine. The SF-induced [Ca2+]i responses appeared to be due to internal release of Ca2+, since transients could occur in Ca2+-free media. Addition of the phorbol ester phorbol 12,13-dibutyrate (PDBu) at low concentrations did not inhibit the SF-induced [Ca2+]i oscillations; high concentrations of PDBu led to a sustained increase in [Ca2+]i concentrations. These data demonstrate that sperm contain a protein factor capable of inducing a characteristic series of [Ca2+]i oscillations in a somatic cell, the hepatocyte. Along with previous observations in dorsal root ganglion neurons, the data suggest a widespread efficacy of the factor in triggering Ca2+ oscillations.

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