Mechanism of induction of mouse erythrocyte receptor switch in human B cells

An early event in phorbol ester-induced maturation of chronic lymphocytic leukemic (CLL) B cells is a membrane change characterized by the inactivation of a mouse erythrocyte receptor (MER). This event, the MER-switch, is quantified by inhibition of rosette formation. By using [3H]phorbol dibutyrate ([3H]PDBu), both to stimulate MER-switch and assay binding of PDBu to CLL cells, it was shown that MER-switch was an irreversible, time-dependent event which occurred some time after maximal binding of [3H]PDBu to cells. Two classes of binding sites, one of high affinity (Kd 1 to 2 nM) at low frequency (1.5 to 5 X 10(4) sites per cell), and a lower affinity site (Kd 33 to 50 nM) of higher frequency (2 to 3.5 X 10(5) sites per cell), were detected. Binding of [3H]PDBu was inhibited by phorbol ester analogs that stimulated MER-switch, but not by inactive analogs. This, and the similarity in shapes of the binding and rosette inhibition curves over a range of concentrations, suggests that stimulation of MER-switch by phorbol esters is due to this specific binding. The phorbol ester receptor and MER are distinct because MER-ve T cells and MER-ve atypical B cells from a patient with CLL had both classes of PDBu receptor. Solubilized MER did not bind [3H]PDBu. Time-course studies, and the irreversibility of the switch, despite removal of most of the bound [3H]PDBu, indicate that inhibition of rosetting is not due to competitive or steric hindrance by phorbol esters. Equivalent activities of soluble MER were released from fresh and phorbol ester-treated CLL cells, indicating a rearrangement of MER, rather than a loss. A supernatant of phytohemagglutinin-stimulated human spleen cells also induced MER-switch in CLL lymphocytes, suggesting that a lymphokine may be a natural inducer of this event.     

Keloid fibroblasts are refractory to inhibition of DNA synthesis by phorbol esters. Altered response is accompanied by reduced sensitivity to prostaglandin E2 and altered down-regulation of phorbol ester binding sites.

To investigate abnormal growth regulation in keloid fibroblasts, responses to phorbol esters were examined. Treatment of quiescent cultures with phorbol 12-myristate 13-acetate (PMA) blocked a normally occurring (20-24 h) peak of serum-stimulated thymidine incorporation in normal and keloid cells. In keloid fibroblasts PMA induced a delayed peak of DNA synthesis. When indomethacin was added with PMA the delayed peak appeared in normal fibroblasts. The ED50 for inhibition of the 20-24-h peak was 1 nM, whereas the delayed peak required a 50-fold-higher PMA concentration. In both cell types PMA induced prostaglandin E2 (PGE2) synthesis, and exogenous PGE2 caused 50% inhibition of the 20-24-h peak. When PMA and indomethacin were added with PGE2 the delayed peak was inhibited 90% in normal fibroblasts, whereas inhibition of keloid cells was the same as with PGE2 alone. Normal and keloid fibroblasts had the same number of phorbol ester binding sites. However, in normal cells, phorbol 12,13-dibutyrate bound with greater affinity, and down-regulation of phorbol ester binding occurred to a greater extent. These findings suggest that altered expression of protein kinase C isozymes or another molecule that binds phorbol esters may play a role in abnormal growth regulation of keloid cells.     

Regulation of motility in bovine brain endothelial cells

Scatter factor (SF) is a fibroblast-derived cytokine which stimulates motility of epithelial and vascular endothelial cells. We used a quantitative assay based on migration of cells from microcarrier beads to flat surfaces to study the regulation of motility in bovine brain endothelial cells (BBEC). Peptide growth factors (EGF, ECGF, basic FGF) did not stimulate migration. Tumor promoting phorbol esters (PMA, PDD) markedly stimulated migration, while inactive phorbol esters (4a-PDD, phorbol-13,20-diacetate) did not affect migration. Both SF- and PMA-stimulated migration were inhibited by 1) TGF-beta; 2) protein kinase inhibitors (e.g., staurosporine, K-252a); 3) activators of the adenylate cyclase signaling pathway (e.g., dibutyryl cyclic AMP, theophylline); 4) cycloheximide; and 5) anti-cytoskeleton agents (e.g., cytochalasin B, colcemid). However, PMA and SF pathways were distinguishable: 1) PMA induced additional migration at saturating SF concentrations; 2) the onset of migration-stimulation was immediate for PMA and delayed for SF; and 3) down-modulation of protein kinase C (PKC) ablated PMA but not SF responsiveness. Assessment of PKC by (3H)-phorbol ester (PDBu) binding and by immunoblot showed 1) scatter factor does not cause significant redistribution or down-modulation of PDBu binding or alpha-PKC; and 2) PDBu mediates redistribution and down-modulation of both binding and alpha-PKC. These findings suggest two pathways for BBEC motility: a PKC-dependent pathway and an SF-stimulated/PKC-independent pathway.     

The role of protein kinase C in T lymphocyte proliferation. Existence of protein kinase C-dependent and -independent pathways

The mouse cytotoxic T cell clone (CTLL-2) was able to grow in the presence of culture medium supplemented only with transferrin, 2-mercaptoethanol, and recombinant interleukin 2 (IL-2). This lymphokine stimulated the synthesis of DNA in these cells. Similarly, phorbol esters, which activate protein kinase C, induced DNA synthesis in this clone. Furthermore, this later proliferation was not blocked by anti-IL-2 receptor antibodies, which inhibited IL-2-induced proliferation, suggesting that it was not indirectly due to the secretion of IL-2 by the cells. CTLL-2 cells pretreated with high doses of phorbol esters for 48 h down regulated protein kinase C and were depleted of this enzyme. This was shown by: 1) purification and in vitro assay of protein kinase C; 2) the lack of effect of phorbol esters in the stimulation of the Na+/H+ anti-porter which has been directly linked to the activation of protein kinase C. As expected, those protein kinase C-depleted cells no longer synthesized DNA and proliferated in response to phorbol esters. However, they proliferated identically to control cells in response to IL-2. Therefore, our results suggest two different pathways for T cell proliferation, one which involves protein kinase C and the other which does not.     

Phorbol ester treatment inhibits thrombin but not stable GTP analogue-induced platelet granule secretion despite inhibition of phosphatidate formation with both agonists

Previous work has demonstrated that pre-treatment of platelets with phorbol esters that activate protein kinase C eg phorbol 12-myristate 13-acetate (PMA) results in an inhibition of inositol phospholipid breakdown and granule secretion induced by physiological agonists such as thrombin and collagen. In the present study, the effect of pre-treatment with PMA on granule secretion and [32P]-phosphatidate (PA) formation induced by the stable GTP analogue, guanosine 5'-[gamma thio] triphosphate (GTP gamma S) was examined in saponin-permeabilized platelets. A low concentration of PMA ie 1.6nM, that did not induce significant 5-hydroxytryptamine (5HT) secretion on its own, but inhibited low-dose thrombin-induced 5HT secretion totally and PA formation by 30-40% in intact as well as permeabilised platelets was chosen. Our results demonstrate a lack of inhibition of GTP gamma S (40 microM)-induced 5HT secretion by PMA in permeabilised platelets, despite significant inhibition (70%) of PA formation, suggesting that apart from the diacylglycerol pathway of secretion which may be common to thrombin and GTP analogues, secretion induced by physiological agonists such as thrombin may involve another mechanism that is inhibitable by phorbol esters.     

Sphingosine reverses growth inhibition caused by activation of protein kinase C in vascular smooth muscle cells.

In certain cell systems, including neonatal vascular smooth muscle (VSM) cells, phorbol esters are growth inhibitory. Here we show that 1,2-dioctanoyl-sn-glycerol (DiC8), when added 2 h after alpha-thrombin, reverses by greater than 95% the induction of DNA synthesis in VSM cells by alpha-thrombin. Sphingosine, a naturally occurring lysosphingolipid inhibitor of protein kinase C, and its synthetic analogues N-acetylsphingosine and C11-sphingosine were used to investigate this phenomenon further. Neither phorbol 12-myristate 13-acetate (PMA;200 ng/ml) nor sphingosine (up to 10 microM) alone had any effect upon basal DNA synthesis in VSM cells. Like DiC8, PMA totally blocked the induction of DNA synthesis by alpha-thrombin. This inhibitory effect of PMA was reversed by sphingosine in a dose-dependent manner with complete reversal at 10 microM. Neither N-acetylsphingosine nor C11-sphingosine exhibited any effect on DNA synthesis in VSM cells. The effect of sphingosine and its analogues on the activity of protein kinase C extracted from VSM cells was measured by histone III-S phosphorylation. Protein kinase C activity was inhibited 50% by 300 microM sphingosine, but less than 15% by similar concentrations of N-acetylsphingosine and C11-sphingosine. To assess the effects of sphingosine and analogues on protein kinase C in intact cells, we examined the effect of the lipids on [3H]phorbol dibutyrate binding. Sphingosine (at greater than 5 microM), but not N-acetylsphingosine or C11-sphingosine, blocked [3H]phorbol dibutyrate binding in a dose- and time-dependent fashion. Thus the mechanism of growth inhibition by DiC8 and PMA in neonatal VSM cells appears to be through activation of protein kinase C by these compounds. Sphingosine reverses this growth inhibition through interference with the binding to protein kinase C of phorbol esters or other activators of this enzyme.     

Protein kinase C-dependent and -independent inhibition of Ca(2+) influx by phorbol ester in rat pancreatic beta-cells

Phorbol esters were used to investigate the action of protein kinase C (PKC) on insulin secretion from pancreatic beta-cells. Application of 80 nM phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, had little effect on glucose (15 mM)-induced insulin secretion from intact rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, PMA significantly reduced the glucose-induced insulin secretion. PMA decreased the level of intracellular Ca(2+) concentration ([Ca(2+)](i)) elevated by the glucose stimulation when tested in isolated rat beta-cells. This inhibitory effect of PMA was not prevented by BIM. PMA inhibited glucose-induced action potentials, and this effect was not prevented by BIM. Further, 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), a non-PKC-activating phorbol ester, produced an effect similar to PMA. In the presence of nifedipine, the glucose stimulation produced only depolarization, and PMA applied on top of glucose repolarized the cell. When applied at the resting state, PMA hyperpolarized beta-cells with an increase in the membrane conductance. Recorded under the voltage-clamp condition, PMA reduced the magnitude of Ca(2+) currents through L-type Ca(2+) channels. BIM prevented the PMA inhibition of the Ca(2+) currents. These results suggest that activation of PKC maintains glucose-stimulated insulin secretion in pancreatic beta-cells, defeating its own inhibition of the Ca(2+) influx through L-type Ca(2+) channels. PKC-independent inhibition of electrical excitability by phorbol esters was also demonstrated.     

Inhibition of phorbol ester stimulated superoxide production by 1-oleoyl-2-acetyl-sn-glycerol (OAG); fact or artefact?

OAG-stimulated superoxide (O2) production by HL-60 granulocytes showed enantiomeric specificity but reached a maximum of only 5% of that produced by either phorbol myristate acetate (PMA) or phorbol dibutyrate (PDBu). At 10-100 microM, OAG displaced specifically-bound [3H]PDBu from intact HL-60 cells by only 25%, suggesting limited cell penetration. OAG (10-100 microM) also inhibited PDBu-stimulated O2 production by 25%; this inhibition was enantiomerically specific. However, at a lower concentration (3 microM), both enantiomers of OAG fully blocked O2 production stimulated by PMA (0.5 microM). This inhibition is probably artefactual, due to the hydrophobic PMA physically associating with OAG in the extracellular fluid.     

Rat macrophage treatment with lipopolysaccharide leads to a reduction in respiratory burst product secretion and a decrease in NADPH oxidase affinity

The effect of LPS on the respiratory burst in resident rat peritoneal macrophages has been examined. Rat macrophages secreted high levels of both O2- and H2O2 in response to triggering with phorbol esters, opsonized zymosan, and immune complexes. After culture in vitro with LPS these macrophages exhibited a marked diminution in their capacity to secrete high levels of respiratory burst products. The LPS-mediated loss of secretory activity was apparent after 2 hr of exposure to LPS and was inhibitable by polymyxin B in a dose-dependent fashion. The effect was not selective for any triggering agent type as inhibition of secretory activity occurred after triggering with PMA, zymosan and immune complexes. PGE2 added at levels secreted by the macrophages in response to LPS also inhibited respiratory burst product secretion. In addition, indomethacin prevented the LPS-mediated inhibition of secretion. Because the inhibition of secretion was common to all triggering agents tested, this suggested that the basis for the impaired secretion was at a level other than the receptor for the triggering agent. Both LPS and PGE2 treatment of the macrophages increased the Km of the oxidase for NADPH (1.7- to 2.3-fold) without affecting significantly the Vmax of the enzyme. These data suggest that stimulation of rat peritoneal macrophages by LPS results in an impaired ability to secrete respiratory burst products as a result of a PGE2-mediated decrease in NADPH oxidase affinity and that this alteration is independent of alterations in tumoricidal activity.     

The inhibition of phosphoenolpyruvate carboxykinase (guanosine triphosphate) gene expression by insulin is not mediated by protein kinase C

The role protein kinase C plays in the regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression by insulin and phorbol esters was studied in H4IIE hepatoma cells (ATCC CRL 1548). The combined effects of phorbol 12-myristate 13-acetate (PMA) and insulin on the suppression of mRNA coding for PEPCK (mRNAPEPCK) synthesis were additive. A potent inhibitor of both cyclic nucleotide-dependent protein kinases and protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, inhibited the cAMP and PMA-mediated regulation of mRNAPEPCK synthesis, but did not affect the action of insulin. Desensitization of the protein kinase C pathway by exposure to PMA for 16 h abolished the subsequent action of the phorbol ester, but did not affect insulin- or cAMP-mediated regulation of PEPCK gene expression. We conclude that insulin suppresses PEPCK gene expression independently from the protein kinase C-mediated pathway used by phorbol esters.     

Regulation of Na+/K+/Cl- cotransport and [3H]bumetanide binding site density by phorbol esters in HT29 cells

The involvement of protein kinase C in the regulation of Na+/K+/Cl- cotransport was investigated in cultured HT29 human colonic adenocarcinoma cells. We have demonstrated previously the presence of a Na+/K+/Cl- cotransport pathway in HT29 cells (Kim, H.D., Tsai, Y-S., Franklin, C.C., and Turner, J.T. (1989) Biochim. Biophys. Acta 946, 397-404). Treatment of cells with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) caused an increase in membrane-associated protein kinase C activity that was accompanied by a concomitant decrease in cytosolic protein kinase C activity. PMA also produced a rapid transient increase in cotransport to 137% of control values by 5 min followed by a progressive decrease to 19% of control values by 2 h. To determine the underlying mechanism for the reduction in Na+/K+/Cl- cotransport, changes in cotransporter number and/or affinity were determined in radioligand binding studies using [3H]bumetanide. PMA and PDBu produced essentially identical time- and dose-dependent decreases in specific [3H]bumetanide binding that were similar to the observed decreases in cotransport. Analysis of saturation and competition binding data indicated that the decrease in binding was due to a lowered Bmax with no change in affinity. Both the decrease in binding and the changes in cotransport elicited by PMA were prevented by the protein kinase inhibitor H7. These findings suggest that phorbol esters cause a decrease in the number of cotransporters in HT29 cells, resulting in a reduction in Na+/K+/Cl- cotransport activity.     

Tumor-promoting phorbol esters stimulate the proliferation of interleukin-3 dependent cells

To determine whether activation of protein kinase C is involved in the proliferation of interleukin-3 (IL-3) -dependent cells, we examined the effect of tumor-promoting phorbol esters on the in vitro proliferation of the IL-3-dependent cell lines FD and DA-1. The viability of FD and DA-1 cells cultured for 24 hours in 100 nM phorbol myristate acetate (PMA) and 10% FCS was similar to that of cells cultured in 25% WEHI-3 conditioned medium as a source of IL-3, and 10% FCS. FD cells failed to proliferate in concentrations of FCS of up to 50%, while DA-1 cell proliferation was not markedly influenced by FCS. By contrast, PMA promoted the proliferation of FD and DA-1 cells in the absence of FCS and enhanced their proliferation in the presence of 10% FCS, 60- and 20-fold, respectively. Stimulation of proliferation was achieved with as little as 10 nM PMA and was maximal at 100 nM PMA. Low concentrations (0.05-0.1%) of WEHI-3 CM promoted the proliferative response of FD and DA-1 cells to PMA, but at concentrations of WEHI-3 CM greater than 0.8%, no further increment in proliferation was obtained with PMA. As little as 1/2 hour of exposure to phorbol esters was sufficient to cause translocation of protein kinase C from the cytosol to the membranes of DA-1 cells, and 1 hour of exposure to phorbol esters was sufficient to stimulate DNA synthesis. A protein kinase C inhibitor, H-7, at a concentration of 10 microM inhibited phorbol ester-induced stimulation of DA-1 cell proliferation. When DA-1 cells were exposed to the calcium ionophore A23187 in addition to both a phorbol ester and IL-3, their proliferation was enhanced over that stimulated by only the phorbol ester and IL-3. The data indicate that stimulation of proliferation of IL-3-dependent cells involves the activation of protein kinase C.     

Regulation of chromaffin cell secretion and protein kinase C activity by chronic phorbol ester treatment

Bovine adrenal chromaffin cells were exposed to phorbol esters to determine the effects of reduced levels of protein kinase C on secretion of hormones. Treatment with active phorbol esters such as 4 beta-phorbol 12, 13-didecanoate (PDD) reduced levels of protein kinase C activity with a maximal 80-90% reduction in activity after 16-24 h treatment (greater than or equal to 500 nM PDD). Treatment with PDD also inhibited catecholamine secretion from chromaffin cells evoked by nicotine, barium, and scorpion venom (50-70%, t1/2 approximately 6 h) and by veratridine (80%, t1/2 less than 15 min). Secretion induced by these agents in phorbol ester-treated cells returned to that of untreated cells by 3-4 days despite no recovery of protein kinase C activity. Potassium-evoked secretion was not inhibited by phorbol ester treatment. Catecholamine secretion from digitonin-permeabilized cells was more sensitive to calcium between 1 and 24 h, but not greater than or equal to 48 h, after addition of phorbol ester. The results suggest that phorbol esters inhibit secretion by activation of protein kinase C resulting in inhibition of ion channels or receptors but not of the secretory machinery itself; hence, protein kinase C may usually machinery itself; hence, protein kinase C may usually attenuate secretory responses in the adrenal chromaffin cell.     

Rapid priming of calcium mobilization and superoxide anion production in human neutrophils by substimulatory concentrations of phorbol esters: a novel role for protein kinase C and tyrosine phosphorylation in the up-modulation of signal transduction.

The modulatory influences of phorbol esters on the functional responsiveness of human peripheral blood neutrophils have been investigated. These studies focused on measurements of the levels of cytoplasmic free calcium and of tyrosine phosphorylation as well as on their ability to mount an oxidative response. Short incubation times (< 1 min) with low concentrations of phorbol esters (5-50 nM) were shown to enhance the above indices of neutrophil responsiveness to chemotactic factors such as fMet-Leu-Phe and leukotriene B4. On the other hand, a time- and concentration-dependent inhibition of calcium mobilization and superoxide production was also observed. The effects of the phorbol esters were stereo-specific and were antagonized by a novel protein kinase C inhibitor (RO 318220) but were not affected by the oxidative burst inhibitor diphenyleneiodonium. Pre-incubation of the cells with phorbol 12,13-dibutyrate (PDBu) altered in a concentration-dependent manner the tyrosine phosphorylation pattern stimulated by fMet-Leu-Phe. In addition, the tyrosine kinase inhibitor erbstatin inhibited the priming of the mobilization of calcium induced by PDBu. These data demonstrate the rapidity of the effects of the activation of protein kinase C, their potential to modulate positively the early events of the excitation-response coupling sequence and the complexity of the functional interrelationships among the various cellular activation pathways available to human neutrophils and other non-muscle cells.     

Role of DNA synthesis in secretion of immunoglobulin from murine B cells stimulated by T cell derived lymphokines

We have investigated whether cell division is required for induction of Ig secretion from three types of B cells, which represent distinct activation states: normal splenic B cells, anti-Ig-treated B cells, and a monoclonal murine B cell tumor, BCL1. Polyclonal Ig secretion was stimulated in vitro by LPS or by lymphokines produced by EL-4 cells (EL-4 SN), which includes B cell growth factor II (BCGF II). LPS and EL-4 SN were mitogenic for all three cell populations and stimulated substantial IgM secretion from both B cells and anti-Ig blasts. Aphidicolin, a reversible inhibitor of DNA synthesis, abolished IgM secretion from B cells and anti-Ig blasts induced by either mitogen, indicating that Ig-secreting cells in these cultures are part of a cycling population. BCL1 tumor cells respond to BCGF II (but not to interleukin 2 or B cell stimulatory factor 1) with IgM secretion and cell division, allowing a direct assessment of the influence of BCGF II-stimulated cell division on secretion of IgM. Secretion by these cells during the first 24 hr of culture was not substantially affected by aphidicolin, but secretion at 48 or 72 hr was markedly inhibited. Culture of BCL1 cells for 48 hr with aphidicolin alone had no effect on cell viability or on subsequent responsiveness if the drug was removed, eliminating non-specific toxicity as an explanation of the drug's effect. Addition of aphidicolin during the last 24 hr of culture to either normal B cells or BCL1 cells was much less effective at inhibiting IgM secretion. These results indicate that the cells that secrete IgM in response to BCGF II also synthesize DNA when exposed to this factor. Thus, induction of high-rate Ig secretion from murine B cells by some stimuli, including BCGF II, may require at least one round of cell division.     

Activation to IgG secretion by lipopolysaccharide requires several proliferation cycles.

We have investigated the relationship between IgG secretion and cell proliferation after polyclonal activation of murine spleen cells with lipopolysaccharide (LPS). It was found that IgG secretion was optimal when cells proliferated extensively. Under those conditions, DNA synthesis commenced 8 to 12 hr after exposure to LPS. Increased proliferative activity was observed up to day 3, when the majority of the lymphoblasts were mitotically active. Two inhibitors of DNA synthesis, thymidine (TdR) and hydroxyurea (HU) caused a reduction in both the IgM and the IgG response, but the latter was more severely reduced. The inhibition was strongest when TdR and HU were added to cultures early after exposure to LPS, indicating that the cells developing to Ig secretion were continuously proliferating. 5-bromo-2'-deoxyuridine (BrdU) caused a general inhibition of IgM and IgG secretion at high concentrations, and a selective inhibition at low concentrations. The selective inhibition of IgG secretion, when measured on day 4, was also observed after a pulse of BrdU on days 1 and 2. The data suggest that development to IgG secretion is a complex process, which requires several proliferation cycles.     

Activation of protein kinase C inhibits human keratinocyte migration

The involvement of protein kinase C (PKC) in epidermal growth factor (EGF)-induced human keratinocyte migration was studied with the phagokinetic assay. It was concluded that PKC activation does not mediate, but rather inhibits, EGF-induced keratinocyte migration. The following experimental observations support these conclusions: 1) The PKC inhibitor H-7 did not inhibit EGF-induced migration but instead led to a modest enhancement. 2) PKC activators such as phorbol-12-myristate-13-acetate (PMA), phorbol-12,13-dibutyrate (PDBu), and 1,2-dioctanoly-sn-glycerol inhibited migration, but biologically inactive 4α-PMA had no effect. 3) PMA did not inhibit keratinocyte attachment and spreading but blocked migration almost immediately after addition. 4) Migration of PKC-depleted cells, which were produced by prolonged treatment with PDBu, was enhanced similarly to normal cells by EGF. 5) PKC-depleted cells were not susceptible to the inhibitory effects of phorbol esters on migration. Additional experiments, in which cells were preactivated with EGF, suggested that PKC inhibits the EGF effect at a post-receptor level. The inhibitory effect of PKC on keratinocyte migration was not restricted to EGF-induced migration; PKC activation also inhibited keratinocyte migration induced by bovine pituitary extract, insulin, insulin-like growth factor-1, and keratinocyte growth factor. © 1993 Wiley-Liss, Inc.     

Protein kinase C and the stimulation of tissue plasminogen activator release from human endothelial cells. Dependence on the elevation of messenger RNA

Tumor-promoting phorbol esters stimulate tissue plasminogen activator (tPA) release from human endothelial cells, and simultaneous elevation of cyclic AMP potentiates this response 5-fold (Santell, L., and Levin, E. G. (1988) J. Biol. Chem. 263, 16802-16808). A similar effect on tPA mRNA was observed, with phorbol myristate acetate inducing a 3.5-fold increase in steady state tPA mRNA levels and forskolin enhancing that increase to 25-fold. Peak levels occurred at 8 h after agonist addition and returned to baseline levels by 16 h. As was found with tPA antigen secretion, delayed addition of forskolin reduced the level of potentiation, and, at 6 h after phorbol 12-myristate 13-acetate (PMA), forskolin was no longer effective. The protein synthesis inhibitor cycloheximide did not inhibit the rise in tPA mRNA levels in response to PMA/forskolin nor the decline in mRNA levels between 8 and 12 h. However, peak levels (8 h) were approximately 1.5-fold higher than in cultures not treated with cycloheximide. The effect of two inhibitors of protein kinases, H-7 and staurosporine, on PMA-induced tPA antigen secretion and tPA mRNA levels were examined. H-7 and staurosporine inhibited PMA, and PMA/forskolin induced tPA secretion in a dose-dependent manner. This effect was time-dependent; the inhibitory effect was reduced with delayed H-7 addition, and, by 6 h after PMA treatment, no inhibition was observed. H-7 and staurosporine also inhibited the PMA/forskolin-induced increase in tPA mRNA levels and were less effective the later they were added. The same time-dependent effect on the potentiation of PMA-induced tPA mRNA levels by forskolin was observed. Again, delayed addition reduced the effect, and, by 6 h, potentiation was absent. The results of this study indicate that changes in mRNA levels in response to PMA and PMA/forskolin precede and determine those that occur to tPA antigen secretion. In addition, the maximal response is dependent upon the prolonged activation of an H-7- and cAMP-sensitive pathway.