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Pharmacological modulation of the acute release of tissue-type plasminogen activator from endothelial cells
l S.th.33.7 ]
Pharmacological modulation of the acute release of tissue-type p|asminogen activator from endothelial cells Tranquille, N. and Emeis, J.,I. Gaubius Institute TNO, P.O. Box 612, 2300 A P Leiden, The Netherlands
Tissue-type plasminogen activator (t-PA) is the key enzyme in the fibrinolytic system and can be released from vascular endothelial cells into the blood by stimulation. The cellular reaction mechanisms involved in the acute release of t-PA are poorly understood. To help elucidate these mechanisms, the modulation of the acute release of rat t-PA by compounds affecting cyclic nucleotide levels was studied. The system used involved isolated rat hindlegs perfused with Tyrode's (Tranquille and Emeis, 1988). Tyrode alone did not induce any release, but subsequently, acute release was induced by adding either platelet-activating factor (PAF, 5 nM; Emeis and Kluft, 1985) or bradykinin (BRA; 0.8/xM) to the Tyrode. The perfusate was collected at 1 rain intervals and assayed for t-PA activity. PAF and BRA induced a rapid (within 1-2 rain), transient release of t-PA. The release of t-PA by BRA and PAl= was altered by pretreatment with both the adenylate cyclase activator, forskolin, and the guanyl cyclase activators sodium nitroprusside and atrial natriuretic factor (ANF). Forskolin had no effect on BRA release but reduced PAl: release by a third. Sodium nitroprusside and A N F reduced both BRA and PAF induced release. Nitro arginine (an inhibitor of nitric oxide formation) had no effect on the release induced by either BRA or PAF. The effects of specific phosphodiesterase inhibitors were also investigated. The ;nhibitors used included isobutylmethylxanthine (IBMX, non-selective), SKF 94120 and Rolipram (both c-AMP selective) and M + B 22948 (c-GMP selective). These were perfused through our system at different doses and in combination with the cyclic nucleotide activators mentioned above. None of these compounds induced any release on their own. Some of the data is shown in the table below. Table 1 t-PA (IU/ml) release induced by PAF (5 nM). Controls (n = 30) plus IBMX plus SKF 94120 plus Rdipram plus Rofipram+ forskolin plus M + B 22948 plus M + B 22948+ sodium ni'~-oprusside plus nitroprusside (1 mM)
10/tM 2.39 + 0.39 10 ltM 2.80 + 0.52 * 2.83 ± 0.22 2.36 + 0.41 2.35 + 0.83 -
100 pM
200 pM
100 pM 2.37 + 0.34 1.43 + 0.23 1.08 + 0.18 0.35 + 0.19 2.27 + 0.32 0.70 + 0.42
200 pM 2.54 + 0.27 2.09 + 0.39 0.20 + 0.13 2.12 + 0.71 -
0.64+0.29
• mean+s.d. (n =4). Our results show that the cyclic nucleotides do not induce but are involved in the release of t-PA from endothelial cells and that altering their levels modulates this release. References Tranq,a~le, N., J.J. Enlcis, 1988, Br. J. Pb.~maool. 93, 156. Emeis, J.J., C. Kluft, 1985, Blood 66, 86.
Pharmacological modulation of the acute release of tissue-type p|asminogen activator from endothelial cells Tranquille, N. and Emeis, J.,I. Gaubius Institute TNO, P.O. Box 612, 2300 A P Leiden, The Netherlands
Tissue-type plasminogen activator (t-PA) is the key enzyme in the fibrinolytic system and can be released from vascular endothelial cells into the blood by stimulation. The cellular reaction mechanisms involved in the acute release of t-PA are poorly understood. To help elucidate these mechanisms, the modulation of the acute release of rat t-PA by compounds affecting cyclic nucleotide levels was studied. The system used involved isolated rat hindlegs perfused with Tyrode's (Tranquille and Emeis, 1988). Tyrode alone did not induce any release, but subsequently, acute release was induced by adding either platelet-activating factor (PAF, 5 nM; Emeis and Kluft, 1985) or bradykinin (BRA; 0.8/xM) to the Tyrode. The perfusate was collected at 1 rain intervals and assayed for t-PA activity. PAF and BRA induced a rapid (within 1-2 rain), transient release of t-PA. The release of t-PA by BRA and PAl= was altered by pretreatment with both the adenylate cyclase activator, forskolin, and the guanyl cyclase activators sodium nitroprusside and atrial natriuretic factor (ANF). Forskolin had no effect on BRA release but reduced PAl: release by a third. Sodium nitroprusside and A N F reduced both BRA and PAF induced release. Nitro arginine (an inhibitor of nitric oxide formation) had no effect on the release induced by either BRA or PAF. The effects of specific phosphodiesterase inhibitors were also investigated. The ;nhibitors used included isobutylmethylxanthine (IBMX, non-selective), SKF 94120 and Rolipram (both c-AMP selective) and M + B 22948 (c-GMP selective). These were perfused through our system at different doses and in combination with the cyclic nucleotide activators mentioned above. None of these compounds induced any release on their own. Some of the data is shown in the table below. Table 1 t-PA (IU/ml) release induced by PAF (5 nM). Controls (n = 30) plus IBMX plus SKF 94120 plus Rdipram plus Rofipram+ forskolin plus M + B 22948 plus M + B 22948+ sodium ni'~-oprusside plus nitroprusside (1 mM)
10/tM 2.39 + 0.39 10 ltM 2.80 + 0.52 * 2.83 ± 0.22 2.36 + 0.41 2.35 + 0.83 -
100 pM
200 pM
100 pM 2.37 + 0.34 1.43 + 0.23 1.08 + 0.18 0.35 + 0.19 2.27 + 0.32 0.70 + 0.42
200 pM 2.54 + 0.27 2.09 + 0.39 0.20 + 0.13 2.12 + 0.71 -
0.64+0.29
• mean+s.d. (n =4). Our results show that the cyclic nucleotides do not induce but are involved in the release of t-PA from endothelial cells and that altering their levels modulates this release. References Tranq,a~le, N., J.J. Enlcis, 1988, Br. J. Pb.~maool. 93, 156. Emeis, J.J., C. Kluft, 1985, Blood 66, 86.