- Email: [email protected]
Adenosine triphosphate released from human mononuclear cells
THROMBOSIS RESEARCH 59; 887-890,199O 00493848/90 $3.00 + .OO Printed in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.
ADENOSINE TBIPHOSPHATE BELEXSED FROM HUMAN MONONUCLEAB CELLS
N. Maugeri, E. Bermejo and
M.A. Lazzari.
Department of Hemostasis and Thrombosis. Institute de Investigaoiones Hematologicas. Academia Naoional de Medicina de Buenos Aires. Argentina. (Received 2.7.1990; accepted in original form 2.7.1990 by Editor H. Vinazzer)
We have described that arachidonic acid causes aggregation of mononuclear cells (MNC) (l).It is well known that ATP release from different cells (Z-4) may modulate platelet function and act in concert on vascular smooth muscles to induce vasoconstriction as part of the hemostatic process. In addition, dephosphorylating surfaces may rapidly enzymes present in blood and on vascular initiate platelet generate ADP from ATP (5-8) which may activation. We have now tested whether CMN are able to release ATP during the aggregation response.
MNC were obtained from healthy volunteers according to the Boyum method (7). After washing and resuspending in saline solution they were 95% CMN and 95% viable. Aggregation and ATP release were monitored_ simultaneously using a Crono-Log Lumi aggregometer. The bioluminescence intensity was standardized using known amounts of ATP (Sigma). For each experiment 25-50~1 of MNC suspension (containing 8x10'" MNC/ml) were added to 400~1 of saline solution with Luciferin Luciferase (Zmg/ml) reagent (Sigma) to obtain samples with a final cellular concentration of 4 or 8 xlof' MNC/'ml respectively. The samples were warmed to 37" C _____-----__--_.__ -:
mononuclear
leukocytes,
887
ATP-
release,
ADP.
888
ATP RELEASE
Vol. 59, No. 5
for 1 minute prior to the addition of different agonists to final concentrations of AA (Sigma) (40-160nM), epinephrine (Stago) (5-50nM) and calcium ionophore A23187 (Sigma) (10~M) in volumes of 1 to 51.11.The amount of intracellular ATP was measured after lysis of MNC (8xlF/ml) suspensions with Triton X-100 (Sigma). BIirSULTS Among the agents studied, only AA (40-160~M) was able to induce aggregation and ATP release from the MNC. This response was concentration dependent (Table 1). No measurable response was seen with epinephrine or calcium ionophore A23187. Table 1 shows that the lag time required to reach the maximum response depends on the number of cells in the solution and on the AA concentration. The concentration of ATP in the medium reached a maximum and later decreased. Furthermore when ATP was added to the same medium but without cells the bioluminescence obtained also decreased, at the same rate as in the experiment described above (data not shown). This suggests that the decrease in bioluminescence measured after AA action was due to ATP hydrolysis. The Triton test demonstrated that the ATP released using AA 160bM in samples with 8xlW MNC/ml is approximately 20% of the total cellular ATP. TABLE 1
w
\
ATP RELEASE Amount Lag Time (seconds) (MM)
N" of MNC (l~/ml)
AGGREGATION Maximum (percentage)
n
, 4 4 4 8 8 8
40 80 160 40 80 160
0.28:O.l 0.44kO.l 0.16+0.07 0.45tO.l 0.9OkO.2.
_-602 9 512 3 138k30 72+ 9 48r 9
__-_ 2823 3225 29+3 38+3 4124
8
R" 8 8 * t response to ATP release from human mononuclear cells in + SEM of the arachidcmic acid. Results are expressed as mean amount of ATP released, the lag time to reach the maximum release and the percentage of total aggregation.
smooth muscle, (endothelial, It is known that different cells adenino release selectively can platelets) erythrocytes, nucleotides (2-4). Extraoelfular ATP was reported to exhibit different biological responses such as increase of prostaglandins cells from endothelial (2,813 production and prostacyclin response regulation of vascular tone and modulation of platelet primary functional role in a possible (4,5,9), suggesting
Vol. 59, No. 5
ATP RELEASE
889
hemostasis. In the present study, we analyzed the ATP release from MNC during the aggregation process. Our results, showed that MNC release ATP are stimulated with AA. Whether this only when the cells selective release of ATP is an unespecific response, or whether it represents a secretory mechanism remains to be resolved. Whatever the responsible mechanism may be the release of ATP from MNC in vivo would have great biological significance, because adenine nucleotides and adenosine not only influence platelet aggregation but are also potent vasoactive agents. ACNOHLEDGMENTS We thank Dr. Mirta A. Schattner for fruitful
discussions.
M. l- LAZZARI, M.A., SCHATTNER, M.A., FINIASZ, M. and GIMENO, Prostaglandins, Leukotrienes Human lymphocyte aggregation?. and Med. 15, 303-316, 1984. 2- PEARSON, J.D. and GORDON, J.L. Vascular smoooth muscle cells in culture selectively nucleotides. Nature 281, 384-386, 1979.
endothelial and release adenine
D'ANDREA, G. and RIDDLE, 3- RAJIV, J., WELCH, K.M.A., J.M. Platelet- activating factor and red blood cells. Thromb. Ees. 53, 629-633, 1989. 4- REIMENS, H.J. Adenine nucleotides in blood platelets. In: The Platelets, Physiology and Pharmacology. G. L. Longenecker (Ed.) London: Academic: Press, 1985, pp. 85-112. 5- SOSLAU, G. and Parker J. Modulation of platelet function by extracellular adenosine triphosphate. Blood 74, 984-993, 1989. 6- HASLAM, R.J. and MILLS, D.B.C. The adenilate kinase of human plasma, erythrocytes and platelets in relation to degradation pf adenosine diphosphate in plasma. Bioehem. J. 103, 773, 1967. cells and granulocytes 7- BOYUM, A .Isolation of mononuclear from human blood. Scand. J. Clin. Med. 21, 77-89, 1968. 8- HAUSSINGER,
D.,
BUSSHART,
E.,
STHLE,
T.,
STOLL,
B.,
890
ATP RELEASE
Vol. 59, No. 5
of thromboxane and GEROK, W. Stimulation WETTSTEIN, M. release by extracellular UTP and ATP from perfused rat liver. Role of icosanoids in mediating the nucleotide response. Eur. J. Biochem. 178, 249-256, 1988. I. 9- SPRAGG, R.G., HINSHAW, D.B., HISLOP, D.B., SCHAUSLATTER, Alterations in adenosine U. and COCHRCRANE, C, G. and triphosphate and energy charge in cultured endothelial Invest. 76, P388D1 cells after oxidant injury. J. Clin. 1471-1476, 1985.
ADENOSINE TBIPHOSPHATE BELEXSED FROM HUMAN MONONUCLEAB CELLS
N. Maugeri, E. Bermejo and
M.A. Lazzari.
Department of Hemostasis and Thrombosis. Institute de Investigaoiones Hematologicas. Academia Naoional de Medicina de Buenos Aires. Argentina. (Received 2.7.1990; accepted in original form 2.7.1990 by Editor H. Vinazzer)
We have described that arachidonic acid causes aggregation of mononuclear cells (MNC) (l).It is well known that ATP release from different cells (Z-4) may modulate platelet function and act in concert on vascular smooth muscles to induce vasoconstriction as part of the hemostatic process. In addition, dephosphorylating surfaces may rapidly enzymes present in blood and on vascular initiate platelet generate ADP from ATP (5-8) which may activation. We have now tested whether CMN are able to release ATP during the aggregation response.
MNC were obtained from healthy volunteers according to the Boyum method (7). After washing and resuspending in saline solution they were 95% CMN and 95% viable. Aggregation and ATP release were monitored_ simultaneously using a Crono-Log Lumi aggregometer. The bioluminescence intensity was standardized using known amounts of ATP (Sigma). For each experiment 25-50~1 of MNC suspension (containing 8x10'" MNC/ml) were added to 400~1 of saline solution with Luciferin Luciferase (Zmg/ml) reagent (Sigma) to obtain samples with a final cellular concentration of 4 or 8 xlof' MNC/'ml respectively. The samples were warmed to 37" C _____-----__--_.__ -:
mononuclear
leukocytes,
887
ATP-
release,
ADP.
888
ATP RELEASE
Vol. 59, No. 5
for 1 minute prior to the addition of different agonists to final concentrations of AA (Sigma) (40-160nM), epinephrine (Stago) (5-50nM) and calcium ionophore A23187 (Sigma) (10~M) in volumes of 1 to 51.11.The amount of intracellular ATP was measured after lysis of MNC (8xlF/ml) suspensions with Triton X-100 (Sigma). BIirSULTS Among the agents studied, only AA (40-160~M) was able to induce aggregation and ATP release from the MNC. This response was concentration dependent (Table 1). No measurable response was seen with epinephrine or calcium ionophore A23187. Table 1 shows that the lag time required to reach the maximum response depends on the number of cells in the solution and on the AA concentration. The concentration of ATP in the medium reached a maximum and later decreased. Furthermore when ATP was added to the same medium but without cells the bioluminescence obtained also decreased, at the same rate as in the experiment described above (data not shown). This suggests that the decrease in bioluminescence measured after AA action was due to ATP hydrolysis. The Triton test demonstrated that the ATP released using AA 160bM in samples with 8xlW MNC/ml is approximately 20% of the total cellular ATP. TABLE 1
w
\
ATP RELEASE Amount Lag Time (seconds) (MM)
N" of MNC (l~/ml)
AGGREGATION Maximum (percentage)
n
, 4 4 4 8 8 8
40 80 160 40 80 160
0.28:O.l 0.44kO.l 0.16+0.07 0.45tO.l 0.9OkO.2.
_-602 9 512 3 138k30 72+ 9 48r 9
__-_ 2823 3225 29+3 38+3 4124
8
R" 8 8 * t response to ATP release from human mononuclear cells in + SEM of the arachidcmic acid. Results are expressed as mean amount of ATP released, the lag time to reach the maximum release and the percentage of total aggregation.
smooth muscle, (endothelial, It is known that different cells adenino release selectively can platelets) erythrocytes, nucleotides (2-4). Extraoelfular ATP was reported to exhibit different biological responses such as increase of prostaglandins cells from endothelial (2,813 production and prostacyclin response regulation of vascular tone and modulation of platelet primary functional role in a possible (4,5,9), suggesting
Vol. 59, No. 5
ATP RELEASE
889
hemostasis. In the present study, we analyzed the ATP release from MNC during the aggregation process. Our results, showed that MNC release ATP are stimulated with AA. Whether this only when the cells selective release of ATP is an unespecific response, or whether it represents a secretory mechanism remains to be resolved. Whatever the responsible mechanism may be the release of ATP from MNC in vivo would have great biological significance, because adenine nucleotides and adenosine not only influence platelet aggregation but are also potent vasoactive agents. ACNOHLEDGMENTS We thank Dr. Mirta A. Schattner for fruitful
discussions.
M. l- LAZZARI, M.A., SCHATTNER, M.A., FINIASZ, M. and GIMENO, Prostaglandins, Leukotrienes Human lymphocyte aggregation?. and Med. 15, 303-316, 1984. 2- PEARSON, J.D. and GORDON, J.L. Vascular smoooth muscle cells in culture selectively nucleotides. Nature 281, 384-386, 1979.
endothelial and release adenine
D'ANDREA, G. and RIDDLE, 3- RAJIV, J., WELCH, K.M.A., J.M. Platelet- activating factor and red blood cells. Thromb. Ees. 53, 629-633, 1989. 4- REIMENS, H.J. Adenine nucleotides in blood platelets. In: The Platelets, Physiology and Pharmacology. G. L. Longenecker (Ed.) London: Academic: Press, 1985, pp. 85-112. 5- SOSLAU, G. and Parker J. Modulation of platelet function by extracellular adenosine triphosphate. Blood 74, 984-993, 1989. 6- HASLAM, R.J. and MILLS, D.B.C. The adenilate kinase of human plasma, erythrocytes and platelets in relation to degradation pf adenosine diphosphate in plasma. Bioehem. J. 103, 773, 1967. cells and granulocytes 7- BOYUM, A .Isolation of mononuclear from human blood. Scand. J. Clin. Med. 21, 77-89, 1968. 8- HAUSSINGER,
D.,
BUSSHART,
E.,
STHLE,
T.,
STOLL,
B.,
890
ATP RELEASE
Vol. 59, No. 5
of thromboxane and GEROK, W. Stimulation WETTSTEIN, M. release by extracellular UTP and ATP from perfused rat liver. Role of icosanoids in mediating the nucleotide response. Eur. J. Biochem. 178, 249-256, 1988. I. 9- SPRAGG, R.G., HINSHAW, D.B., HISLOP, D.B., SCHAUSLATTER, Alterations in adenosine U. and COCHRCRANE, C, G. and triphosphate and energy charge in cultured endothelial Invest. 76, P388D1 cells after oxidant injury. J. Clin. 1471-1476, 1985.