Complex regulation of macrophage colony-stimulating factor in the HL-60 promyelocytic cell line.

The regulation of macrophage colony-stimulating factor (M-CSF) gene expression by phorbol esters and calcium ionophore (A23187) was studied in HL-60 cells. In untreated HL-60 cells, M-CSF transcripts were undetectable, but transcripts were present within 2 h of A23187 treatment or within 8 h of phorbol 12,13-dibutyrate (PdBu) treatment. Concurrent treatment of HL-60 cells with A23187 and cycloheximide (CHX) for 6 h led to a superinduction of message over A23187 treatment alone. But concurrent treatment with PdBu and CHX for 24 h abolished expression of the message normally present after 24 h of PdBu treatment. The role of new protein synthesis in transcriptional regulation of M-CSF was studied by run-on transcription assay. Untreated HL-60 cells or cells treated with CHX alone do not transcribe M-CSF mRNA. However, cells treated with A23187 for 6 h and CHX for 1, 3, or 6 h transcribed more M-CSF than cells treated with A23187 alone for 6 h. CHX also regulates M-CSF expression by message stabilization. The t1/2 of M-CSF mRNA was 45 min in cells treated with A23187 for 6 h or in cells treated with PdBu for 24 h, was over 2 h in HL-60 cells treated with A23187 for 6 h and CHX for 1 h, and was 3 h in cells treated with PdBu for 24 h and CHX for 1 h. We conclude that M-CSF gene expression can be differentially regulated in HL-60 cells and that new protein synthesis plays an important role in both the transcriptional and posttranscriptional regulation.

Multiple calcium-mediated mechanisms regulate c-myc expression in HL-60 cells.

Treatment of human promyelocytic leukemia cells (HL-60) with the calcium ionophore A23187 induced a biphasic change in the expression of the c-myc protooncogene. Within 1 h of exposure to a single 2.5-microM dose of A23187, steady state levels of c-myc mRNA increased to 170% of control. After this early increase, c-myc mRNA expression underwent an abrupt decline over the next 2 to 4 h. Between 6 to 18 h of treatment with A23107, steady state levels of c-myc mRNA fell gradually toward zero. The early increase in expression of c-myc mRNA was mediated by both enhanced initiation of c-myc transcription and posttranscriptional stabilization of c-myc mRNA. Stimulation of HL-60 cells with A23187 for 30 min increased transcription of both exons 1 and 3 of c-myc by 125%. After 1 h of treatment with A23187, the half-life of c-myc mRNA increased to 150% of control. The late decrease in c-myc expression induced by A23187 was primarily mediated by interruption of elongation of c-myc nascent mRNA beyond exon 1. The combination of calcium ionophore A23187 and phorbol 12,13-dibutyrate acted synergistically to inhibit expression of c-myc mRNA.

Localization of collagenase mRNA in rheumatoid arthritis synovium by in situ hybridization histochemistry.

Collagenase has been implicated as playing an important role in the connective tissue destruction that is a major feature of rheumatoid arthritis. Numerous cell types in the hyperplastic rheumatoid synovium are capable of synthesizing collagenase. Past studies have used predominantly synovial fibroblasts in culture as a model system for the regulation of collagenase production, but the major cellular source of the enzyme in vivo has not been determined. Using the techniques of in situ hybridization histochemistry and indirect immunofluorescence, we determined the cellular source of collagenase in frozen sections of human synovium. Collagenase mRNA production was localized to cells along the synovial lining layer in rheumatoid arthritis. These were identified as the macrophage-like Type A synovial lining cells by immunofluorescence with antibody LeuM3. Endothelial cells, fibroblasts, and T and B lymphocytes were devoid of detectable collagenase mRNA. Synovial tissue sections from patients with osteoarthritis and trauma did not contain detectable collagenase mRNA. These data identify the Type A macrophage-like synovial lining cell as the primary source of collagenase mRNA in vivo in the rheumatoid arthritis synovium and, potentially, as a major effector cell in the tissue destruction of the disease.

Regulation of human synovial fibroblast collagenase messenger RNA by interleukin-1.

Interleukin-1 (IL-1) may contribute to tissue destruction in rheumatoid arthritis, in part, by inducing messenger RNA (mRNA) that encodes interstitial collagenase. In human synovial fibroblasts in vitro, IL-1 induced collagenase mRNA accumulation 6 hours after being added to the cells. High levels of mRNA remained present for at least 48 hours after treatment. The rate of transcription of collagenase in isolated nuclei peaked after approximately 6 hours of treatment with IL-1 and declined thereafter, becoming nearly undetectable by 24 hours. The persistence of mRNA, in view of the transient peak of transcription, suggested that collagenase mRNA was stable in synovial fibroblasts. The half-life of collagenase mRNA after the synoviocytes were treated with actinomycin D was approximately 27 hours, both in the presence and in the absence of IL-1. It has been noted that induction of the expression of collagenase by phorbol esters requires fos protein synthesis and is mediated through a tetradecanoyl phorbol acetate response element in the 5'-flanking region of the gene. However, we found that cycloheximide, when added to synovial fibroblast cultures up to 6 hours after treatment with IL-1, inhibited the expression of collagenase mRNA. These results suggest that fos alone is unlikely to be sufficient for collagenase expression, and that additional factors, or alternative pathways, are involved in the induction of collagenase by IL-1.

Dynamics of the diradylglycerol responses of stimulated phagocytes.

The generation of diradylglycerols (sn-1,2 diacylglycerols (DAG) and 1-O-alkyl-2-acylglycerols (AAG] was investigated in human polymorphonuclear leukocytes stimulated with fMet-Leu-Phe, phorbol myristate acetate (PMA), or A23187. With each stimulus, the elevations in the mass of DAG clearly preceded that of AAG. The levels of both lipids increased over time, peaked by 15-20 min (fMet-Leu-Phe) or 45-60 min (PMA or A23187) and returned slowly toward base line thereafter. The base-line levels of DAG were some 4-fold higher than levels of AAG. On stimulation, the relative increases in AAG (approximately 4-fold, fMet-Leu-Phe; approximately 20-fold, PMA and A23187) were much greater than the corresponding relative increases in the levels of DAG (approximately 2-fold fMet-Leu-Phe; approximately 5-fold, PMA and A23187). The diradylglycerol responses were dependent upon agonist concentration. Prior treatment with cytochalasin B augmented the fMet-Leu-Phe diradylglycerol responses but did not alter unstimulated or PMA- or A23187-stimulated diradylglycerol responses. Depletion of extracellular Ca2+ blocked responses to fMet-Leu-Phe, but not to PMA. Treatment with pertussis toxin: (a) completely blocked the responses to fMet-Leu-Phe, (b) slightly suppressed the AAG but not the DAG response to PMA, and (c) did not affect the responses to A23187. Gas chromatographic/mass spectral analyses indicated that the AAG generated during cell activation consists of a mixture of species differentiated by 1-O-alkyl chains of 16:0, 18:0, 18:1 and an additional species that remains uncharacterized. Since DAG and AAG are reportedly activators and inhibitors, respectively, of protein kinase C activities, the sequential generation of these lipid messengers may provide for a system to critically control the activation of protein kinase C.

Constitutive production of macrophage colony-stimulating factor by human ovarian and breast cancer cell lines.

Many nonhematologic tumors produce growth factors that may influence cellular proliferation either by autocrine or by paracrine mechanisms. In the current study, human tumor cell lines were investigated for the constitutive production of macrophage colony-stimulating factor (M-CSF). Culture supernatants obtained from cell lines were analyzed using a radioimmunoassay and a radioreceptor assay specific for M-CSF. Among the various cell types analyzed, all the ovarian cell lines and a majority of the breast cancer cell lines secreted significant amount of an M-CSF-like factor. Treatment of mouse bone marrow cultures with culture supernatants from ovarian cancer cells stimulated the production of macrophage colonies. Analysis of total cellular RNA obtained from the ovarian cell lines by Northern blot showed multiple sizes of M-CSF transcripts with an abundance of a 4.2-kb message. The relative amount of M-CSF transcripts correlated with the level of immunoreactive material seen in the culture supernatants.

Human thymic epithelial cells produce granulocyte and macrophage colony-stimulating factors.

The development of culture conditions for growing normal human thymic epithelial (TE) cells free from contamination with other stromal cells has allowed us to identify and characterize TE cell-derived cytokines. In this study, we report that cultured human TE cells produced CSF that supported the growth of clonal hematopoietic progenitor cells in the light density fraction of human bone marrow cells. Thymic epithelial supernatants (TES) induced growth of granulocyte/macrophage colonies (CFU-GM), mixed granulocyte/erythrocyte/monocyte/megakaryocyte colonies (CFU-GEMM), and early burst-forming unit erythroid colonies (BFU-E). In addition, TES induced differentiation of the promyelocyte leukemic cell line HL-60 and stimulated growth of both granulocyte (CFU-G) and monocyte (CFU-M) colonies from murine bone marrow cells. Using anion exchange column chromatography, pluripotent CSF activities in TES were separated and shown to be distinct from an IL-1-like cytokine that has been shown as a TE cell-derived cytokine (TE-IL-1). Colony-stimulating activity supporting the growth of bone marrow CFU-GEMM, BFU-E, and CFU-GM co-eluted at 150 to 180 mM NaCl. A separate peak of CFU-GM-stimulating activity eluted early in the gradient at 20 mM NaCl. In Northern blot analysis of enriched RNA, synthetic oligonucleotide probes complementary to human G-CSF and M-CSF coding sequence each hybridized with a single RNA species of 1.7 and 4.4 kb, respectively. These data suggest that normal human TE cells synthesize G-CSF and M-CSF that promote differentiation of non-lymphoid hematopoietic cell precursors.

Dioctanoylglycerol and phorbol esters regulate transcription of c-myc in human promyelocytic leukemia cells.

Treatment of human promyelocytic leukemia cells (HL-60) with phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate (PDBu) caused a rapid decrease in transcription of the c-myc protooncogene. In the continuous presence of PMA or PDBu, the rate of transcription of c-myc decreased to 20% of control within 2 h and was maintained at 20-30% of the control level for the ensuing 24 h. Cell-permeable sn-1,2-dioctanoylglycerol (diC8), a diacylglycerol analogue, also caused a rapid decrease in c-myc transcription. The decrease in the transcription of c-myc induced by diC8 or PDBu was reversible; prolonged exposure of the cells to either agent for periods greater than 2 h was necessary to maintain the transcription of c-myc below the control rate. With both PDBu and diC8, there was a close correlation between the concentration dependence for binding to the phorbol ester receptor and the concentration dependence for inhibition of c-myc transcription. The decrease in transcription of c-myc induced by PDBu or diC8 appeared to be due to a block of elongation of the nascent mRNA beyond exon 1. The results of this study suggest that prolonged stimulation of protein kinase C (Ca2+/phospholipid-dependent enzyme) is required for persistent inhibition of transcription of c-myc in HL-60 cells.

Transcription interruption may be a common mechanism of c-myc regulation during HL-60 differentiation.

Human promyelocytic leukemia cells (HL-60) differentiate along a monocytoid pathway in response to recombinant human tumor necrosis factor or recombinant human interferon gamma. Together, these agents act synergistically to induce phenotypic differentiation. Since reduced expression of mRNA for the proto-oncogene c-myc correlates with differentiation of HL-60 cells induced by other agents, we tested the abilities of tumor necrosis factor and interferon gamma to regulate expression of c-myc mRNA. Tumor necrosis factor rapidly and effectively reduced c-myc mRNA levels. In contrast, interferon gamma did not affect c-myc mRNA levels, nor did it show synergy with tumor necrosis factor in reducing c-myc. Transcription run-on studies confirmed that tumor necrosis factor caused interruption of c-myc transcription after exon 1. Phorbol diesters also caused interruption of transcription of c-myc. Thus, interruption of transcription may be a common mode of regulation of c-myc during induced differentiation of HL-60 cells.

Phorbol diesters and dioctanoylglycerol stimulate accumulation of both diacylglycerols and alkylacylglycerols in human neutrophils.

Human neutrophils treated with phorbol 12-myristate 13-acetate (PMA) or dioctanoylglycerol exhibited a large (10-fold), sustained accumulation of the mass of diradylglycerol, beginning 1 min after stimulation and continuing for 30 to 60 min. Phorbol dibutyrate was less potent than PMA in stimulating diradylglycerol accumulation, whereas the 4-alpha analogs of PMA and phorbol dibutyrate were inactive. Submaximal concentrations of PMA (0.5 to 2.5 nM) plus the calcium ionophore, ionomycin (15 to 60 nM), led to synergistic accumulation of diradylglycerols. Chlorpromazine and sphingosine, inhibitors of protein kinase C, blocked PMA-stimulated accumulation of diradylglycerol with IC50 concentrations of 32 and 9 microM, respectively, paralleling their inhibition of PMA-stimulated O2- production. These compounds also inhibited the ionomycin-stimulated accumulation of diradylglycerols. A third protein kinase C inhibitor, H-7, was less effective, inhibiting PMA-stimulated accumulation of diradylglycerol by 25% at 100 microM. Differential sensitivity to alkaline hydrolysis suggests that diradylglycerols that accumulate in response to PMA or ionomycin stimulation are composed of a mixture of two distinct diglyceride species, diacylglycerols and alkylacylglycerols. Whereas diacylglycerol may activate cellular protein kinase C, the importance of the production of alkylacylglycerols is uncertain.

Distinct patterns of expression of different protein kinase C mRNAs in rat tissues.

The identification of multiple protein kinase C (PKC) cDNA sequences from rat, bovine, and human tissues has led to the discovery of a family of PKC genes. Using probes complementary to three cDNA sequences from rat brain, designated PKC-I, -II, and -III, the distribution of PKC transcripts in rat tissues was studied by in situ hybridization histochemistry. In brain, PKC-II and PKC-III transcripts colocalized except in the brain stem and spinal cord, where some cells appeared to contain only PKC-III message. PKC-I mRNA had a different, but partially overlapping, pattern of expression. In spleen, PKC-II and PKC-III, but not PKC-I, transcripts were concentrated in the marginal zone of white pulp, the major B-lymphocyte compartment of rat spleen. The distinct distributions of rat PKC mRNAs suggest that different members of the PKC family have specialized functions.

Diacylglycerol accumulation and superoxide anion production in stimulated human neutrophils.

Exogenous diacylglycerols stimulate neutrophil superoxide anion production, suggesting that endogenous diacylglycerols may function as second messengers for this biological response. We have measured the diacylglycerol mass in human neutrophils stimulated by fMet-Leu-Phe, ionomycin, and concanavalin A and have correlated the kinetics and magnitude of the diacylglycerol response with those for superoxide anion production. For each stimulus, no increase in diacylglycerol mass was detected prior to the onset of superoxide anion generation. However, large sustained increases in diacylglycerol concentration (260-2000% of basal levels) occurred in parallel with the rise in superoxide anion. The cessation or continuation of diacylglycerol accumulation and superoxide anion production also correlated. The diacylglycerol response was proportional to the stimulus concentration and correlated with the concentration dependence for superoxide anion. Pretreatment of neutrophils with cytochalasin B enhanced both superoxide anion and diacylglycerol responses with all three stimuli. These data support the hypothesis that diacylglycerol functions as a modulator of superoxide anion generation causing a sustained or augmented respiratory burst.

Diacylglycerol mass measurements in stimulated HL-60 phagocytes.

The mass of sn-1,2-diacylglycerol in crude lipid extracts from differentiated HL-60 phagocytes was measured by quantitative conversion of the diacylglycerol to [32P]-labeled phosphatidic acid catalyzed by E. coli diacylglycerol kinase. The chemotactic peptide N-formyl-Met-Leu-Phe caused a time- and concentration-dependent increase in diacylglycerol that was maximal at 4 min. Diacylglycerol returned toward basal levels by 15 min. The basal level of diacylglycerol was 290 +/- 25 pmol/10(7) cells (n = 36). Maximally effective concentrations of N-formyl-Met-Leu-Phe and N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys increased diacylglycerol to 176% +/- 16 of basal (n = 8) and 198% +/- 15 of basal (n = 4), respectively. t-Boc-Phe-Leu-Phe-Leu-Phe, a competitive antagonist of formyl peptide receptor function, competitively inhibited the N-formyl-Met-Leu-Phe-induced diacylglycerol increase. Pretreatment of the cells with pertussis toxin abolished the stimulated rise in diacylglycerol, whereas depletion of extracellular Ca2+ markedly inhibited the increase. The Ca2+ ionophore A23187 stimulated a large (450% of basal) and persistent (greater than 30 min) increase in diacylglycerol. These data suggest that agents which raise intracellular Ca2+ levels in differentiated HL-60 cells produce a prolonged increase in cellular diacylglycerol which may activate protein kinase C.

Cytochalasin B enhancement of the diacylglycerol response in formyl peptide-stimulated neutrophils.

Diacylglycerol has gained wide acceptance as an important second messenger in the signal transduction mechanism by which occupancy of certain membrane receptors such as the formyl peptide receptor of neutrophils leads to biological responses, but supporting evidence for this proposed role is limited. We have utilized a recently developed diacylglycerol kinase assay (Preiss, J. E., Loomis, C. R., Bishop, W. R., Stein, R., Niedel, J. E., and Bell, R. M. (1986) J. Biol. Chem. 261, 8597-8600) to characterize the diacylglycerol response of normal human neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) and other formyl peptides. fMLP alone stimulated a slow, prolonged 36% rise in diacylglycerol levels above basal levels. Cytochalasin B enhances several fMLP-stimulated neutrophil responses, including aggregation, superoxide production, and degranulation. Pretreatment of neutrophils with cytochalasin B markedly increased the rate and extent of the diacylglycerol response to fMLP stimulation. Diacylglycerol peaked at 5 min at 206 +/- 21% above basal levels with a t1/2 of 45 s. The diacylglycerol response was time- and fMLP and cytochalasin B concentration-dependent, appropriate for the known biological activities of several peptide analogues, and completely inhibited by pretreatment with pertussis toxin. These data demonstrate that diacylglycerol may function as a second messenger for neutrophil activation and suggest that cytochalasin B enhancement of neutrophil biology may be the result of an enhanced diacylglycerol response.

Dibutyryl cyclic adenosine monophosphate reduces expression of c-myc during HL-60 differentiation.

The human promyelocytic leukemia cell line HL-60 is induced to differentiate along a myelocytic pathway by dibutyryl cyclic adenosine monophosphate (dbcAMP). Other cAMP analogs are ineffective as inducing agents. The effect of these compounds on expression of c-myc was investigated using a DNA probe for c-myc to detect RNA transcripts. The dose response and time to commitment for reduction in c-myc expression with dbcAMP was similar to the findings for phenotypic changes. Bromo-cyclic AMP and butyrate alone caused no changes in c-myc expression in 24 hours, but demonstrated dramatic synergism together, suggesting that butyrate contributes in part to the effects of dbcAMP. Evidence for mechanisms of action of cAMP other than activation of the cAMP-dependent protein kinase is reviewed.

Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells.

A simple enzymatic method for the quantitation of the mass of sn-1,2-diacylglycerol (DAG) present in crude lipid extracts was developed to assess the function of DAGs as intracellular "second messengers" of extracellular agents and of oncogene products. The assay employed Escherichia coli DAG kinase which constituted approximately 15% of the membrane protein of a plasmid-bearing strain and defined mixed micellar conditions to solubilize the DAG present and allow its quantitative conversion to [32P]phosphatidic acid. The assay was proportional with the amount of DAG added over the range of 25 pmol to 25 nmol. The rapid rise of DAG in platelets stimulated with thrombin (210% over basal) and in hepatocytes stimulated with vasopressin (230% over basal) was quantitated and the values agreed with previous measurements. The amounts of DAG in normal rat kidney (NRK) cells grown at 34 and 38 degrees C, respectively, were 0.47 and 0.61 nmol/100 nmol of phospholipid. In K-ras-transformed NRK cells grown at 34 or 38 degrees C, DAG levels were elevated 168 or 138%, respectively. When a temperature-sensitive K-ras NRK cell line was investigated, the amount of DAG present was elevated at the permissive but not at the restrictive temperature. These data are consistent with the K-ras protein functioning in transmembrane signalling by activating phospholipase C. Protein kinase C (Ca2+/phospholipid-dependent enzyme) activation by DAG may play an important role in cellular transformation.

Differential stimulation of the respiratory burst and lysosomal enzyme secretion in human polymorphonuclear leukocytes by synthetic diacylglycerols.

Binding of chemoattractants to receptors on human polymorphonuclear leukocytes (PMN) stimulates the phosphodiesteric cleavage of phosphatidylinositol 4,5-bisphosphate to produce inositol 1,4,5-trisphosphate and 1,2-diacylglycerols. To investigate the possible second messenger function of diacylglycerols in PMN activation, we tested the ability of a series of synthetic sn 1,2-diacylglycerols, known to stimulate protein kinase C in other systems, to promote superoxide anion release, oxygen consumption, lysosomal enzyme secretion, and chemotaxis. None of the diacylglycerols initiated the chemotactic migration of PMN. Several of the diacylglycerols however, were, active in stimulating superoxide anion release and lysozyme secretion, with dioctanoylglycerol (diC8) being the most potent. Unexpectedly, didecanoylglycerol (diC10) induced lysosomal enzyme secretion, but failed to stimulate superoxide production or oxygen consumption. All other biologically active diacylglycerols tested displayed similar EC50 for stimulating lysozyme secretion and superoxide production. The ability of the diacylglycerols to compete for phorbol dibutyrate (PDBu) binding in intact PMN suggested a mechanism for the divergent biological activity of diC10. Although the compounds that stimulated both superoxide production and lysosomal enzyme secretion competed for essentially all [3H]PDBu binding from its receptor, diC10, which only stimulated secretion, competed for 45% of the bound [3H]PDBu. Thus diacylglycerols can selectively activate certain functions of leukocyte chemoattractant receptor. The data suggest that a discrete pool of protein kinase C may mediate activation of the respiratory burst in PMN.

Cyclic AMP levels and cellular kinetics during maturation of human promyelocytic leukemia cells.

Differentiation of human promyelocytic leukemia cells (HL-60) in response to several classes of inducing agents is characterized by the sequential appearance of granulocytic or monocytic markers. Compounds that increase intracellular cAMP in HL-60 cells induce a program of maturation in which cells demonstrate early functional phagocytic properties. Cyclic nucleotide metabolism was studied during monocytic and granulocytic differentiation of the HL-60 cell line. In synchronous and nonsynchronous cell cultures, cyclic AMP levels were raised 300-fold or 25-fold in response to N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphate or the combination of prostaglandin E2 and theophylline, respectively. No reproducible changes in intracellular adenosine 3':5'-cyclic monophosphate levels occurred in response to retinoic acid or dimethyl sulfoxide, suggesting that changes in adenosine 3':5'-cyclic monophosphate levels alone do not mediate cell maturation induced by these compounds. Agents that increased intracellular cyclic AMP in HL-60 cells slowed the progress through the S and G2-M phase of the cell cycle, whereas other agents such as dimethyl sulfoxide stimulated cells through this same period.

Cytosolic calcium regulates phorbol diester binding affinity in intact phagocytes.

The mobilization of internally sequestered stores of Ca2+ and activation of protein kinase C appear to be involved in neutrophil activation. We have examined the inter-relationship of these two pathways by investigating the effects of modulating Ca2+ activity on the binding of [3H]phorbol 12,13-dibutyrate (PDBU) to protein kinase C in intact phagocytes. Differentiated HL-60 cells were equilibrated with [3H]PDBU prior to stimulation with various agents known to alter Ca2+ homeostasis in cells. Agents that elevated cytosolic Ca2+, such as f-Met-Leu-Phe and A23187, up-regulated radioligand binding by increasing the affinity of the PDBU/protein kinase C interaction. These effects were time- and agonist concentration-dependent and temperature-sensitive. The kinetics of the up-regulation of binding by f-Met-Leu-Phe coincided with the kinetics of Ca2+ mobilization (by quin2 fluorescence measurements). The putative intracellular Ca2+ antagonist 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate alone down-regulated [3H]PDBU binding and inhibited the up-regulation of ligand binding by f-Met-Leu-Phe and A23187. Low concentrations of La3+ (0.1-10 microM) also inhibited up-regulation of radioligand binding to f-Met-Leu-Phe and A23187, whereas higher concentrations (0.1-1 mM) alone increased [3H] PDBU binding and supported further up-regulation of ligand binding by the Ca2+-mobilizing agents. These data suggest a role for Ca2+ in the regulation of phorbol diester binding to protein kinase C in intact cells.