High Concentrations Of Staurosporine Research Articles

Abstract 186: Advances in 3D cell culture analysis: Utilizing novel flow cytometry technology for rapid analysis of intact spheroids

Abstract Cancer cells grown in two dimensional (2D) monolayers are a convenient model to use for basic research and in generating lead candidates in the drug discovery process. However, cells grown in 2D cultures lack many of the structural, biological, and functional attributes of solid tumors, which contributes to the high clinical failure rates for biologic and small molecule drugs. Multicellular three-dimensional (3D) tumor spheroids are advanced models which more faithfully mimic the physiological tumor milieu and is more predictive of therapeutic efficacy than traditional monolayer cultures. However, the inability to generate and to analyze large numbers of intact spheroids with a simplified workflow remains a barrier in the adoption of 3D models for a wide range of applications. To overcome these barriers, we used the Velocyt® large particle flow cytometer (Bennubio) to evaluate spheroid health in a well-established small molecule spheroid killing assay. The Velocyt technology rapidly and gently analyzes particles from 3 to 300 microns and comes with a suite of novel informatics tools purpose built for large particle analysis. To exemplify the use of large particle flow cytometry for the analysis of intact spheroids we generated uniformed-sized spheroids (~180 microns) from the H1650 human lung and HCT116 human colon cancer cell lines using microwell technology. Two to three days after spheroid generation, the spheroids were treated with increasing concentrations of staurosporine and other compounds. Spheroids were harvested at one to three days post treatment (depending on drug treatment) and stained with fluorescent cell viability and apoptotic dyes. Intact spheroids were analyzed for fluorescence and size using the Velocyt flow cytometer and between one and two thousand spheroids were assessed for each treatment in approximately three minutes. In untreated control spheroids, fluorescence was detected in a subpopulation of spheroids consistent with expected low levels of background apoptosis. In treated spheroids, an increase in the mean fluorescence intensity (MFI) of each particle indicated an increase in the number of non-viable and apoptotic cells in each spheroid. MFI increased nearly 6-fold with high concentrations of staurosporine compared to control spheroids, and dead cells were detected in nearly 100% of spheroids that were administered the highest staurosporine concentration. Using the size-based measurement parameters of the Velocyt, we also showed a decrease in overall spheroid size concurrent with increased numbers of spheroid fragments and single cells with increasing drug concentration. These data highlight that the use of 3D cell models combined with the Velocyt large particle flow cytometer is a powerful tool to help advance basic and translational cancer research. Citation Format: John P. O'Rourke, Mathew Saunders, Valerie Sanchez. Advances in 3D cell culture analysis: Utilizing novel flow cytometry technology for rapid analysis of intact spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 186.

Inhibition of Plasminogen Activators Attenuates the Death of Differentiated Retinal Ganglion Cells and Stabilizes Their Neurite Network In Vitro

Although previous studies have indicated that elevated levels of the tissue plasminogen activator (tPA) and the urokinase plasminogen activator (uPA) associate with the death of retinal ganglion cells (RGCs), it was unclear whether these proteases directly cause cell death. With the use of a transformed and undifferentiated retinal ganglion cell line, RGC-5, which does not express tPA, and by treating this cell line with staurosporine, which induces not only the differentiation of RGC-5 cells but also the expression of uPA and tPA in other neuronal cells, the authors sought to determine whether these proteases regulate the differentiation of RGC-5 cells and whether elevated levels of these proteases directly cause the death of RGC-5 cells. Transformed RGC-5 cells were cultured in serum-free medium and were treated with 0.5 muM to 2.0 muM staurosporine to induce their differentiation. Neurite outgrowth was assessed by phase-contrast microscopy and calcein AM staining and quantified with imaging software. Proteolytic activities of tPA and uPA were determined by zymography assays. Cell viability was determined by LIVE/DEAD viability assay kit. Compared with untreated RGC-5 cells, cells treated with staurosporine differentiated as early as 1 to 6 hours. However, proteolytic activities of neither tPA nor uPA were observed within this time frame. Differentiated RGC-5 cells expressed detectable levels of uPA proteolytic activity starting at 24 hours and tPA proteolytic activity only at 48 hours. RGC-5 cells synthesized and secreted uPA and tPA into the conditioned medium, depending on staurosporine concentration and treatment time. At lower concentrations of staurosporine, differentiated RGC-5 cells had longer neurites and expressed lower levels of tPA and uPA. At higher concentrations of staurosporine, differentiated RGC-5 cells expressed higher levels of tPA and uPA, had smaller neurites, and most of them died. In contrast, when RGC-5 cells were treated with staurosporine along with inhibitors specific to tPA and uPA, proteolytic activities of both PAs were significantly reduced. Under these conditions, a significant number of RGC-5 cells survived, showed increased neurite outgrowth, and established their neurite network in vitro. Results presented in this study indicate that RGC-5 cells do not require tPA and tPA for their differentiation. In fact, differentiated RGC-5 cells synthesize elevated levels of tPA and uPA, and elevated levels of these proteases acting in an autocrine-fashion in turn lead to the death of RGC-5 cells.

Staurosporine-induced apoptosis is independent of p16 and p21 and achieved via arrest at G2/M and at G1 in U251MG human glioma cell line.

The mechanisms involved in the cell cycle and cell death remain unresolved despite much investigation. Staurosporine induces cell death and G1 or G2/M arrest in a dose-dependent manner, but the mechanisms remain unknown. In the present study an adenovirus vector expressing p16 or p21 genes in human glioma cell lines was used to examine cell cycle regulation and cell death induced by staurosporine. A low concentration (</=10 n M) of staurosporine induced G1 arrest of U251MG cells, whereas a high concentration (>/=30 n M) induced G2/M arrest and finally induced apoptosis via a caspase-3-activated pathway from both the G2/M and G1 phases. However, pRb was dephosphorylated and cdc2 was inhibited at both the low and the high concentrations of staurosporine, indicating that the mechanisms of cell cycle regulation are not simply p53-Rb- or cdc2-dependent pathways. Forced G1 arrest by transfection with p16 or p21 genes did not alter the rate of staurosporine-induced cell death. This implies that an unknown pathway of apoptosis occurs from the G1 phase.

Regulation of Bcl2 phosphorylation and potential significance for leukemic cell chemoresistance.

Although considered tightly linked, the linkage effectors for proliferation and antiapoptotic signaling pathways are not clear. Phosphorylation of Bcl2 at serine 70 is required for suppression of apoptosis in interleukin 3 (IL-3)-dependent myeloid cells deprived of IL-3 or treated with antileukemic drugs and can result from agonist activation of mitochondrial protein kinase C alpha (PKCalpha). However, we have recently found that high concentrations of staurosporine up to 1 microM: can only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKCalpha-mediated Bcl2 phosphorylation in vitro, indicating the existence of a non-PKC, staurosporine-resistant Bcl2 kinase (SRK). Although the RAF-1MEK-1-mitogen-activated protein kinase (MAPK) cascade is required for factor-dependent mitogenic signaling, a direct role in antiapoptosis signaling is not clear. In particular, the role of phosphorylation in the regulation of death substrates is not yet clear. Our findings indicate a potential role for the MEK/MAPK pathway in addition to PKC in antiapoptosis signaling, involving Bcl2 phosphorylation that features a role for extracellular signal-regulated kinase (ERK)1 and 2 as SRKs. These findings indicate a novel role for ERK1 and 2 as molecular links between proliferative and survival signaling and may, at least in part, explain the apparent paradox by which Bcl2 may suppress staurosporine-induced apoptosis. Although the effect of phosphorylation on Bcl2 function is not clear, effector molecules that regulate Bcl2 phosphorylation may have clinical significance in patients with acute myelogenous leukemia (AML) who express detectable levels of Bcl2. Preliminary findings suggest that expression of PKCalpha, ERK2, and Bax in leukemic blast cells from patients with AML, although individually not prognostic, appears to have potential clinical value in predicting chemoresistance and survival outcomes.

Effects of protein kinase C and phospholipase C inhibitors on IgE-dependent and IgE-independent basophil histamine release.

This study was aimed to evaluate the effects of two protein kinase C (PKC) inhibitors (staurosporine and chelerythrine) and one phospholipase C (PLC) inhibitor (U73122) on basophil histamine release induced by anti-IgE, N-formyl-methionyl-leucyl-phenylalanine (FMLP), IL-3 and GM-CSF. Leukocytes were suspended in solutions containing physiological or low Na+ concentrations, since IL-3 and GM-CSF were shown to induce histamine release only when the inhibitory effect of extracellular Na+ has been removed. After incubation with PKC and PLC inhibitors, the stimuli were added and histamine release was measured by an automated fluorometric method. Staurosporine and chelerythrine exerted a significant inhibitory effect on histamine release induced by anti-IgE, IL-3 and GM-CSF at concentrations much higher than those required to inhibit PKC. FMLP-induced histamine release in a physiological Na+-containing medium was not significantly modified by staurosporine, although it was reduced by high concentrations of chelerythrine. A slight inhibition by high concentrations of staurosporine was found when basophils were suspended in a low Na+ medium. U73122 exerted a significant and dose-dependent inhibitory effect on basophil histamine release induced by anti-IgE, FMLP, IL-3 and GM-CSF. These results suggest that a prodegranulatory role of PKC in basophil histamine release induced by anti-IgE, FMLP, IL-3 and GM-CSF is unlikely; conversely, it is conceivable that PLC has a role in signal transduction and histamine release induced by the above stimuli.

Human oligodendroglial cell line, MO3.13, can be protected from apoptosis using the general caspase inhibitor zVAD-FMK.

Recent evidence suggests that the oligodendrocyte cell loss observed in multiple sclerosis sufferers is in part mediated by apoptosis. Here we use a human cell line, MO3.13, as a model system to investigate the biochemical processes involved in oligodendroglial cell death. Treatment with staurosporine kills both naive and differentiated cells in a dose-dependent manner; however, much higher concentrations of staurosporine are required to kill differentiated cells compared to their naive progenitors. Dying cells displayed the typical morphological characteristics of apoptosis, including cell shrinkage and chromatin condensation. Biochemical analysis showed that caspases, a group of enzymes intimately involved in the execution of apoptosis, are activated in both naive and differentiated cells. Western blotting analysis revealed that similar subsets of caspase enzymes were operating and that the substrate cleavage patterns were identical in both naive and differentiated cells. Treatment of MO3.13 cells with the general caspase inhibitor zVAD-FMK protected them from toxin-induced cell death. These results indicate that when an oligodendroglial human cell line is exposed to toxin it dies in an apoptotic manner. In addition, we show that cells can be protected from toxin-induced death using an appropriate inhibitor.

Expression and regulation of interferon-gamma-induced tryptophan catabolism in cultured skin fibroblasts.

Interferon-gamma (IFN-gamma)-induced, indoleamine dioxygenase-catalyzed tryptophan catabolism was studied in cultured human foreskin fibroblasts using the increase in cellular kynurenine synthesis as an index of gene expression. The time courses of the inhibition of IFN-gamma-induced kynurenine synthesis by actinomycin D and cycloheximide showed that the indoleamine dioxygenase gene was transcribed as early as 2 h and translated as early as 5 h after initiation of IFN treatment. Expression was completely inhibited by the Ser/Thr kinase inhibitor, H-7 (66 microM), during the first 2 h after IFN-gamma treatment. Prolonged pretreatment of cells with high concentrations of staurosporine (380 nM) or genestein (610 microM) inhibited expression by 38% and 53%, respectively. Genestein also inhibited expression when it was added to cultures between 8 and 24 h after IFN-gamma treatment. The expression of kynurenine synthesis was inhibited by A23817 during the first 4 h after IFN treatment by mechanisms that were independent of cyclooxygenase, calmodulin, and calcineurin. Exogenous gangliosides (bovine brain gangliosides and purified GM1) inhibited IDO expression throughout the first 24 h after IFN-gamma treatment by mechanisms that did not involve effects on Ca2+ channels. Other biologic response modifiers, including phorbol myristic acetate, arachidonic acid, lipopolysaccharide, analogs of cAMP and cGMP, W-7, and sphingosine, did not induce IDO in the absence of IFN-gamma, nor did they modulate IFN-gamma-induced expression. These results indicate that the expression of kynurenine synthesis is modulated at the transcriptional and posttranscriptional levels by protein tyrosine kinase and by a Ser/Thr kinase with properties distinctly different from those of conventional protein kinase C. The capacity for attenuation of this IFN-gamma-induced response over its entire time course by many effectors and through multiple cellular signaling pathways may represent a mechanism for fine-tuning the level of oxidative tryptophan metabolism to meet the needs of a particular cytostatic or antiproliferative response.

The Photoreceptor Guanylate Cyclase Is an Autophosphorylating Protein Kinase

The photoreceptor membrane guanylate cyclase is a member of a family of proteins with a set of four structural motifs: an extracellular ligand binding domain, a transmembrane domain, an intracellular protein kinase-like domain, and an intracellular catalytic domain. Purified preparations of the photoreceptor guanylate cyclase have allowed us to explore the function of the protein kinase-like domain. ATP enhances the guanylate cyclase activity 2-fold in membranes stripped of peripheral proteins. The stimulation can be mimicked by ATPgammaS (adenosine 5'-O-(3-thiotriphosphate)), AMPPNP (5'-adenylyl beta,gamma-imidodiphosphate), and ADP, but not AMP. While this effect is lost by solubilizing guanylate cyclase, ATP binds the purified, solubilized enzyme in a site distinct from the catalytic GTP site as shown by specific labeling with 8-N3[alpha-32P]ATP. The enzyme has a protein kinase activity that is Mg2+-dependent and autophosphorylates serine residues. Myelin basic protein serves as a substrate for the kinase and enables further characterization of the kinase properties. The Km for ATP is 81 microM. The kinase activity is unaffected by calcium, cyclic nucleotides, and phorbol 12-myristate 13-acetate/L-alpha-phosphatidylserine/Ca2+ and is inhibited by high concentrations of staurosporine. These properties are distinct from other Ser/Thr kinases identified in rod outer segment preparations including protein kinase A, protein kinase C, and rhodopsin kinase. The observations offer the first biochemical evidence that a member of the receptor guanylate cyclase family has intrinsic protein kinase activity.

Effects of staurosporine on ACTH-mediated stimulation of aldosterone production

Incubation of rat adrenal glomerulosa cells with low concentrations (up to 50 nM) of the protein kinase (PKC) inhibitor staurosporine (ST) inhibited aldosterone (ALDO) and cyclic AMP (cAMP) production stimulated by adrenocorticotropic hormone (ACTH) and cholera toxin. Only higher concentrations (1.6 μM) of staurosporine inhibited dibutyryl-cAMP- and forskolin-induced stimulation of aldosterone production. cAMP levels were increased only with low concentrations of the PKC inhibitor. This latter increase was avoided by treatment with a maximal concentration of isobutylmethylxanthine (MIX). Our results suggest that: (1) second messengers other than cAMP are involved in ACTH action; (2) staurosporine inhibits different kinases involved in ACTH action in a dose-dependent manner; (3) the protein kinase inhibited by high concentrations of staurosporine appears to be the cAMP-dependent kinase, PKA; and (4) the protein kinase inhibited by low concentrations of staurosporine remains to be identified. This latter species is suggested as being involved in mediating ACTH-induced activation of Gs.

Staurosporine does not prevent adrenergic-induced situs inversus, but causes a unique syndrome of defects in rat embryos grown in culture.

Staurosporine, an alkaloid isolated from Streptomyces species, is commonly used as a protein kinase C (PKC) inhibitor in animal investigations. In the present study, we used this compound to determine whether alpha 1-adrenergic stimulation-induced situs inversus in rats is mediated by PKC. Embryos were explanted at 8 A.M. on day 9 of gestation. Those with a neural groove but with no visible neural folds (Stage 11a) were selected and were cultured in medium containing various concentrations of staurosporine with or without 50 microM of phenylephrine, an alpha 1-adrenergic agonist. At 10 A.M. on day 11 of gestation, embryos were examined for situs inversus and other abnormalities. Staurosporine, tested at 0.0001, 0.001, 0.01, 0.1, 0.375, and 0.5 microM (lethal concentration), did not block phenylephrine-induced situs inversus at any concentration. However, staurosporine alone produced situs inversus at concentrations above 0.1 microM. At 0.5 and 1.0 microM, staurosporine also caused cyst-like lesions projecting dorsally from the mesencephalon that we named "mesencephalic vesicles" and the formation of secondary somites. To confirm and further examine these unique effects of staurosporine both grossly and histologically, we conducted additional experiments using staurosporine from another source. Embryos were explanted between 6 A.M. and 9 P.M. on day 9 of gestation and were placed in one of the following groups according to their stage of development: 10b, 11a, 11b, 11c, 12/s1-2, 12/s3-4, and 12/s5-6. Embryos were then cultured with various concentrations of staurosporine. Those cultured from Stage 11a exhibited similar lesions to those seen in the initial experiment but at somewhat higher concentrations of staurosporine. Embryos cultured from Stage 10b showed a similar pattern of lesions as seen at Stage 11a, except that higher concentrations of staurosporine were required to cause mesencephalic vesicles and secondary somites formation. Embryos cultured from Stage 11b showed similar effects to those cultured from younger stages except that maximum incidences of situs inversus were much lower. Those cultured from Stage 11c showed similar dose-response to those cultured from Stage 11b except that the incidence of secondary somites formation was much higher. In addition, in approximately 40% (n = 25) of embryos treated with greater than 1.0 microM of staurosporine, the growing end of the allantois did not reach the chorion and remained unattached in the exocoelomic cavity.(ABSTRACT TRUNCATED AT 400 WORDS)

CD53, a protein with four membrane-spanning domains, mediates signal transduction in human monocytes and B cells.

CD53 is a member of a novel family of molecules with four presumably membrane-spanning domains. The structure and functional characteristics of these molecules indicate that they may play an important role in transmembrane communication. We therefore investigated whether CD53 is involved in activation of human leukocytes. Cross-linking of cell-bound F(ab')2 fragments of two different anti-CD53 mAb with F(ab')2 anti-mouse Ig led to cytoplasmic calcium fluxes in B cells, monocytes, and granulocytes and activation of the monocyte oxidative burst. These responses were specific for CD53, as cross-linking of CD11a, CD18, CD35, CD43, CD44, CD45, or CDw50 did not induce leukocyte activation. Low concentrations of staurosporine (10 to 20 nM) completely inhibited PMA-mediated activation, but had no effect on CD53-mediated calcium fluxes and inhibited only partially CD53-mediated oxidative burst. This suggests that CD53-mediated signaling is largely independent of protein kinase C. CD53-mediated calcium fluxes were inhibited by high concentrations of staurosporine (300 to 500 nM) but not by ADP-ribosylating toxins, suggesting dependence on tyrosine kinases rather than GTP-binding proteins. The results indicate that CD53, like several other leukocyte Ag with four membrane-spanning regions, has the ability to mediate cell activation, and support the view that these molecules are involved in transmembrane communication.

Multiple effects of staurosporine, a kinase inhibitor, on thymocyte functions: Comparison with the effect of tyrosine kinase inhibitors

The effects of staurosporine, a protein kinase inhibitor, on the signal transduction and proliferation of thymocytes were studied. Signal transduction in response to Concanavalin A (Con A) as well as Concanavalin A (Con A)-induced augmentation of [ 3H]inositol incorporation into phospholipids were inhibited by staurosporine (≥10 −8M). Staurosporine inhibited thymocyte proliferation in response to Con A in the presence or absence of the phorbol ester, phorbol myristate acetate (TPA) (10 nM). This inhibition was observed regardless of whether staurosporine was added together with Con A or 3 hr later. High concentrations of staurosporine (>10 −8 M) inhibited thymocyte proliferation induced by the calcium ionophore A23187 and the phorbol ester TPA, whereas lower concentrations of the inhibitor (≤10 −8M) enhanced thymidine incorporation in response to these activators. This dual effect of staurosporine was also observed in the presence of the staurosporine-related kinase inhibitor, K252a. In contrast, the tyrosine kinase inhibitor, tyrphostin AG490, inhibited the response to A23187 and TPA at all concentrations of the inhibitor and no augmentation was seen. Interleukin 2 (IL-2)-driven mitogenesis in IL-2-dependent cells was also inhibited by staurosporine. We suggest that the inhibition of thymocyte proliferation by staurosporine results from inhibition of both protein kinase C and tyrosine kinase: the augmentation of the response to A23187 and TPA results from inhibition of protein kinase C. Inhibition of signal transduction as well as inhibition of IL-2-driven mitogenesis result from inhibition of tyrosine kinase.

Interleukin 1 and phorbol 12-myristate 13-acetate induce collagenase and PGE 2 production through a PKC-independent mechanism in chondrocytes

In the present study we demonstrate that interleukin 1 (IL 1) and phorbol 12-myristate 13-acetate (PMA) stimulate collagenase production by bovine chondrocytes in monolayer culture. Since it has been well established that PMA stimulates protein kinase C (PKC), we examined whether IL 1 and PMA also stimulate PKC in chondrocytes. In agreement with other studies, PMA induced the translocation of PKC, reflecting PKC activation by PMA. In contrast, IL 1 did not induce the translocation of PKC. Both IL 1 and PMA stimulated the release of [ 14C]arachidonic acid from chondrocyte phospholipids, suggesting that both agents stimulate phospholipase A 2 (PLA 2). Concomitantly, IL 1 and PMA also induced a pronounced increase in the production of PGE 2. Pre-incubation of chondrocytes with staurosporine, a PKC inhibitor, did not affect the stimulation of collagenase production by IL 1 and only minimally that induced by PMA. Similarly, high concentrations of staurosporine did not inhibit prostaglandin E 2 (PGE 2) production induced by IL 1 or PMA. These data show that IL 1 and PMA stimulate the PLA 2 pathway and collagenase production, however, these processes can occur in the absence of PKC activation.

Transformed mammalian cells are deficient in kinase-mediated control of progression through the G1 phase of the cell cycle.

To investigate the role of kinase-mediated mechanisms in regulating mammalian cell proliferation, we determined the effects of the general protein kinase inhibitor staurosporine on the proliferation of a series of nontransformed and transformed cultured rodent and human cells. Levels of staurosporine as low as 1 ng/ml prevented nontransformed cells from entering S phase (i.e., induced G1 arrest), indicating that kinase-mediated processes are essential for commitment to DNA replication in normal cells. At higher concentrations of staurosporine (50-75 ng/ml), nontransformed mammalian cells were arrested in both G1 and G2. The period of sensitivity of nontransformed human diploid fibroblasts to low levels of the drug commenced 3 hr later than the G0/G1 boundary and extended through the G1/S boundary. Interference with activity of the G1-essential kinase(s) caused nontransformed human cells traversing mid-to-late G1 at the time of staurosporine addition to be "set back" to the initial staurosporine block point, suggesting the existence of a kinase-dependent "G1 clock" mechanism that must function continuously throughout the early cycle in normal cells. The initial staurosporine block point at 3 hr into G1 corresponds to neither the serum nor the amino acid restriction point. In marked contrast to the behavior of nontransformed cells, neither low nor high concentrations of staurosporine affected G1 progression in transformed cultures; high drug concentrations caused transformed cells to be arrested solely in G2. These results indicate that kinase-mediated regulation of DNA replication is lost as the result of neoplastic transformation, but the G2-arrest mechanism remains intact.

Evidence that Ca2(+)-dependent activation of airway epithelia Cl- channels is not dependent on phosphorylation

Previous studies have shown that an increase in the cytosolic Ca2+ concentration [( Ca2+]c) activates Cl- channels in airway epithelia but that the effect is indirect. Because adenosine 3',5'-cyclic monophosphate (cAMP) and phorbol myristate acetate (PMA) activate Cl- channels via phosphorylation by cAMP-dependent protein kinase and protein kinase C, respectively, we asked whether Ca2(+)-dependent Cl- channel activation is phosphorylation dependent. We measured 125I- efflux as an assay of Cl- channel activation in the intact cell. We found that depletion of cellular ATP prevented cAMP- and PMA-induced activation but did not alter activation produced by the Ca2+ ionophore A23187. Moreover, addition of high concentrations of staurosporine (5 microM), to nonspecifically inhibit kinase activity, blocked cAMP- and PMA-stimulated 125I- efflux but had no effect on A23187-induced efflux. These results suggest that elevation of [Ca2+]c does not activate Cl- channels via phosphorylation.

Calcium transients in human platelets monitored by aequorin, fura-2 and quin-2: Effects of protein kinase C activation and inhibition

Tumour-promoting phorbol esters and 1,2-dioctanoyl-sn-glycerol both induce calcium transients in platelets. However, these can only be detected in platelets loaded with aequorin, but not in those loaded with the fluorescent probes quin-2 and fura-2 presumably because of intracellular calcium buffering. Several effects induced by phorbol esters and diacylglycerols, including the rise in (Ca 2+) i, the stimulation of Na +/H + transporter and the inhibition of the effects of thrombin alone on (Ca 2+) i are potentently antagonised by staurosporine, a compound known to inhibit protein kinase C. Higher concentrations of staurosporine themselves inhibit the thrombin-induced calcium transient. Staurosporine inhibits the effects of phorbol esters and dioctanoyl glycerol with equal potency although the latter does not cause enzyme translocation of cytosolic protein kinase C to membranes. These results therefore suggest that some, if not all, the effects of protein kinase C activation can occur without translocation of the enzyme.