Abstract
Tumor-promoting phorbol esters and histamine induce tissue plasminogen activator (tPA) release from human endothelial cells in a dose- and time-dependent manner. Phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBu) increased tPA concentration in the culture medium by eight to 12 times after 24 h with half-maximal stimulation at 13 and 55 nM, respectively. Maximum release by histamine was only half that of the phorbol esters and required 18 microM for half-maximal response. Kinetics of enhanced release was similar with both types of agonists: a 4-h lag period followed by a period of rapid release (4 h in PMA-treated and 10 h in histamine-treated cultures) followed by a decline toward pretreatment rates. The PMA and histamine effects were additive while histamine and thrombin, which also stimulates tPA release in human endothelial cells, were no more effective together than they were alone. Exposure of the cells to PMA, PDBu, or phorbol 12,13-didecanoate caused a loss of responsiveness to second treatment of the homologous agent that was time- and dose-dependent, sustained, and specific to active tumor promoters (half-maximal desensitization = 52 nM PDBu). A partial desensitized state was also established by histamine which resulted in a 60% lower response to a second challenge dose. Histamine-induced desensitization did not interfere with the PMA response. However, PMA-induced desensitization caused a 75% loss of the histamine and a 67% loss of the thrombin effects. These studies indicate that tumor promoters are potent agonists of tPA release from human endothelial cells and establish a desensitized state to further stimulation. Treatment of these cells with histamine has similar effects which may be mediated at least in part by pathways common to phorbol ester stimulation.
Highlights
Tumor-promoting phorbol esters and histamine in- ety of pathways including the production and release of tissue plasminogen activator (tPA)
TheH1 to Phorbol Esters and Histamine-To compare the effect of receptor antagonist diphenhydramine reduced the final level various tumor promoters on tPA release, dose-titration ex- of tPA in a dose-dependent manner, while cimetidine, a H2 periments were performed with increasing concentrations of receptor antagonist, had only a small effect
Desensitization of tPARelease by Tumor Promoters and Histamine-The transient nature of the increase in tPA release is similar to drug-induced desensitization previously reported to occur following treatment with tumor-promoting agents [13, 15, 16].We examined whether these cells became desensitized to phorbol esters by exposing confluent cultures to several concentrations of PMA or phorbol dibutyrate (PDBu) for 20 h and reexposing them to an optimal dose (100 nM PMA or 1 p~ PDBu) of the homologous agonist for an additional 20 h (Fig. 3)
Summary
Tumor-promoting phorbol esters and histamine in- ety of pathways including the production and release of tPA. Stimulation of tPA release from culturedhuman endothelial cells by thrombin is much slower, requiring 4-6 h to begin [7] This similar with both types of agonists: a 4-h lag period is followed by 12-16 h of rapid release and a decline to followed by a period of rapid release Because protein kinase C appears to be the major cellular receptor for phorbol esters [19,20,21,22] and theloss of protein kinase C activity accompanies the loss of respontamine and a 67%loss of the thrombin effects These siveness to these tumor promoters [23,24,25,26,27], the role of this studies indicatethat tumor promoters are potent ago- enzyme in thecontrol of the synthesis and release of certain nists of t P A release fromhuman endothelial cells and proteins can be assessed. Employing phorbol esters and hisestablish a desensitized state to further stimulation. tamine, we have investigated whether the loss of continued
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