Concentrations Of Phorbol 12-myristate 13-acetate Research Articles

Hyperglycemia in vitro attenuates insulin-stimulated chemokinesis in normal human neutrophils. Role of protein kinase C activation.

This study was performed to test whether the inhibitory effect of elevated D-glucose concentrations on insulin-stimulated chemokinesis in normal human neutrophils is mediated by increase in protein kinase C (PKC) activity. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) at 0-100 nM dose-dependently inhibited neutrophil random locomotion in the absence of insulin. Sub-optimal concentrations of PMA (0.1-0.5 nM) inhibited the chemokinetic effect of 160 microU/mL insulin in a dose-dependent way. The specific PKC inhibitor bisindolylmaleimide (GF 109203x) did not affect the insulin-stimulated chemokinesis at 5 mM glucose but restored the chemokinetic effect of insulin at 15 mM glucose. These results therefore suggest that glucose-induced PKC activation may mediate the inhibitory effects of high glucose levels on insulin-stimulated chemokinesis in normal human neutrophils.

The role of protein kinase C α and ε isozymes in DNA synthesis induced by muscarinic receptors in a glial cell line

Acetylcholine has been shown to induce proliferation of human astrocytoma cells by activating muscarinic receptors, particularly the m3 subtype. In the present study the role of protein kinase C in DNA synthesis induced by carbachol has been investigated. Carbachol-induced [methyl-3H]thymidine incorporation was inhibited by the protein kinase C inhibitors GF 109203X and staurosporine. However, carbachol-induced DNA synthesis was only partially reduced by protein kinase C down-regulation by phorbol 12-myristate 13-acetate (PMA), and maximal concentrations of carbachol and PMA had an additive effect on [methyl-3H]thymidine incorporation. Exposure for 24 h to maximally effective concentrations of carbachol did not induce down-regulation of protein kinase C α, and caused a small but significant down-regulation of protein kinase C ε; cells exposed for 24 h to carbachol were still able to respond with protein kinase C translocation to PMA stimulation. Carbachol caused a significant increase of phorbol ester binding, but did not stimulate protein kinase C α translocation, while it caused a short-lasting translocation of protein kinase C ε; however, protein kinase C ε translocation was not correlated with the time-course of carbachol-induced increase in [methyl-3H]thymidine incorporation. On the other hand, the time-course of translocation/down-regulation of protein kinase C α and protein kinase C ε induced by PMA was in good correlation with the time-course of PMA-induced [methyl-3H]thymidine incorporation. These results suggest that protein kinase C α may not be involved in DNA synthesis induced by muscarinic receptors stimulation in 132-1N1 astrocytoma cells, while protein kinase C ε appears to play a role in the initial exit from G0/G1 phase, though it cannot be considered the major determinant for sustained proliferation.

Expression of Protein Kinase C Isozymes in Human Basophils: Regulation by Physiological and Nonphysiological Stimuli

The expression of protein kinase C (PKC) isozymes in human basophils and the regulation of PKC isozymes during basophil activation by phorbol 12-myristate 13-acetate (PMA) ± ionomycin, f-met-leu-phe (FMLP), and anti-IgE antibody were examined. In human basophils (> 98% purity), PKCβΙ, βΙΙ, δ, and  were expressed, PKC was difficult to detect, and PKCγ and η were undetectable. In unstimulated basophils, PKCβI and βII were found primarily in the cytosol fraction (95% ± 3% of total and 98% ± 1%, respectively). Within 5 minutes of stimulation with PMA (100 ng/mL), both PKCβI and βII were translocated to the membrane fraction (85% ± 4% and 83% ± 6%, respectively). In resting cells, 48% ± 3% and 61% ± 10% of PKCδ and , respectively, existed in the membrane fraction. Within 1 minute of stimulation with PMA, 90% ± 6% of PKC was found in the membrane fraction, however, no translocation of PKCδ was apparent. Stimulation with FMLP caused modest translocation (≈20%) of all PKC isozymes by 1 minute, whereas stimulation with anti-IgE antibody led to no detectable changes in PKC location throughout a 15-minute period of measurement. However, concentrations of PMA and ionomycin that alone caused no PKC translocation and little histamine release, together caused significant histamine release but no apparent PKC translocation. Studies with bis-indolylmaleimide analogs showed inhibition of PMA-induced, but not anti–IgE-induced, histamine release. These pharmacological studies suggest that PKC does not play a prodegranulatory role in human basophil IgE-mediated secretion.© 1998 by The American Society of Hematology.

Expression of Protein Kinase C Isozymes in Human Basophils: Regulation by Physiological and Nonphysiological Stimuli

AbstractThe expression of protein kinase C (PKC) isozymes in human basophils and the regulation of PKC isozymes during basophil activation by phorbol 12-myristate 13-acetate (PMA) ± ionomycin, f-met-leu-phe (FMLP), and anti-IgE antibody were examined. In human basophils (> 98% purity), PKCβI, βII, δ, and ε were expressed, PKCα was difficult to detect, and PKCγ and η were undetectable. In unstimulated basophils, PKCβI and βII were found primarily in the cytosol fraction (95% ± 3% of total and 98% ± 1%, respectively). Within 5 minutes of stimulation with PMA (100 ng/mL), both PKCβI and βII were translocated to the membrane fraction (85% ± 4% and 83% ± 6%, respectively). In resting cells, 48% ± 3% and 61% ± 10% of PKCδ and ε, respectively, existed in the membrane fraction. Within 1 minute of stimulation with PMA, 90% ± 6% of PKCα was found in the membrane fraction, however, no translocation of PKCδ was apparent. Stimulation with FMLP caused modest translocation (≈20%) of all PKC isozymes by 1 minute, whereas stimulation with anti-IgE antibody led to no detectable changes in PKC location throughout a 15-minute period of measurement. However, concentrations of PMA and ionomycin that alone caused no PKC translocation and little histamine release, together caused significant histamine release but no apparent PKC translocation. Studies with bis-indolylmaleimide analogs showed inhibition of PMA-induced, but not anti–IgE-induced, histamine release. These pharmacological studies suggest that PKC does not play a prodegranulatory role in human basophil IgE-mediated secretion.

Opposing regulatory effects of protein kinase C on the cAMP cascade in human HL-60 promyelocytic leukemia cells

The functional role of protein kinase C in the cAMP signaling cascade was investigated in human promyelocytic leukemia (HL-60) cells. Protein kinase C activation after short exposure to 100 nM phorbol 12-myristate 13-acetate (PMA) increased the intracellular cAMP level up to 3- to 5-fold after 30 min. Such enhancement was almost completely blocked by the selective protein kinase C inhibitor bisindolylmaleimide (GF 109203X). In addition, PMA, but not 4-α-PMA, synergistically elevated cAMP levels when adenylyl cyclase was activated directly by forskolin or indirectly by G protein activation after cholera toxin treatment or guanosine 5′- O-(3-thiotriphosphate) (GTPγS) treatment in digitonin-permeabilized cells. The results indicate that protein kinase C directly increases adenylyl cyclase activity and synergistically enhances it, when it is simultaneously activated otherwise. On the other hand, a 10-min treatment with PMA cut the cAMP accumulation induced by histamine, prostaglandin E 2, or isoproterenol by 50–70%. However, the binding affinity and total binding of [ 3 H ]histamine to membrane receptors was not effected by PMA, suggesting that the site of protein kinase C's action is not at the receptor level. Western blot analysis of protein kinase C isozymes revealed that PMA (100 nM) caused translocation of cytosolic protein kinase C such as α, β and ε to the particulate/membrane fraction. Treatment with a lower concentration of PMA (10 nM) translocated the protein kinase C-ε within 2 min, while it had little effect on the translocation of protein kinase C-α and -β up to 20 min. However, simultaneous treatment with 10 nM PMA plus histamine for 5 min significantly inhibited the histamine-mediated cAMP generation, indicating that the protein kinase C-ε could be involved in the inhibition of receptor-mediated cAMP generation. Taken together, we conclude that PMA, through the activation of protein kinase C, has two opposite effects on the cAMP signaling cascade in HL-60 cells: a direct activation of adenylyl cyclase and an inhibition of receptor-mediated signal transduction.

Angiotensin II- and phorbol ester-induced steroidogenesis by ovine adrenocortical cells: effect of sex and the gonadal status of the donor animal.

1. Angiotensin II (AngII) is well recognized as a regulator of adrenal cortisol production in the sheep in vivo, but studies to date have failed to reveal this action on cultured ovine adrenocortical cells. Similarly, phorbol myristate acetate (PMA), an activator of protein kinase C, also has little effect on cortisol production from ovine adrenocortical cells in vitro. Previous studies have, however, only looked at the responses to single concentrations of AngII or PMA, but have led to the suggestion that important differences exist between adrenocortical cells of the sheep and other species. 2. We have extended previous studies by examining cortisol production by ovine adrenocortical cells over 3 h in response to a broad range of concentrations of either AngII (10(-10) to 10(-6) mol/L) or PMA (10(-10) to 10(-5) mol/L). In addition, we have investigated the possible role of gonadal factors in regulating these responses by comparing cultures derived from intact and gonadectomized male and female sheep. 3. Angiotensin II caused concentration-dependent increases (P < 0.05) in cortisol production in all cultures, with cortisol production in response to AngII being greater (P < 0.05) in cultures from intact male sheep than in the cultures from orchidectomized males or intact females. Cortisol production in response to AngII was modest in all groups (two- to 2.6-fold increases). 4. Phorbol myristate acetate elicited a concentration-dependent inhibition (P < 0.01) of basal cortisol production in adrenocortical cultures from all groups. Phorbol myristate acetate produced a greater inhibition (P < 0.05) of cortisol production in cultures from intact male sheep than either the orchidectomized males or intact females. 5. These findings demonstrate that ovine adrenocortical cells are responsive in a concentration-dependent fashion to AngII and PMA. In addition, sex and gonadal factors may play an important role in regulating the responsiveness of adrenocortical cells to these factors.

Intracellular calcium requirements for beta1 integrin activation.

Human polymorphonuclear leukocytes (PMNs) express beta1 integrins that mediate adhesion to extracellular matrix proteins following stimulation with agonists that induce an increase in intracellular calcium. The purpose of these studies was to determine the contribution made by alterations in intracellular calcium ([Ca++]i) to inside-out activation of beta1 integrins using dimethyl sulfoxide (DMSO)-differentiated granulocytic HL60 cells as a model of human PMNs. Activation of beta1 integrins was determined by measuring the expression of an activation-dependent epitope on the beta1 subunit that is recognized by monoclonal antibody (mAb) 15/7. Exposure of granulocytic HL60 cells to calcium ionophore ionomycin (800 nM) alone did not increase the binding of mAb 15/7 to the cell surface, nor did it increase beta1 integrin-mediated adhesion of the cells to fibronectin. Similarly, exposure of the cells to the direct protein kinase C (PKC) activator, dioctanoylglycerol (di-C8) at 100 microM, neither increased binding of mAb 15/7 to these cells nor adhesion to fibronectin. Simultaneous addition of di-C8 and ionomycin, however, caused a significant increase in the expression of the 15/7 epitope and cell adhesion, suggesting synergy between elevating [Ca++]i and stimulating PKC in beta1 integrin activation. Chelation of [Ca++]i with Quin-2 and EGTA reduced both basal (unstimulated) expression of the 15/7 epitope and basal adhesion of granulocytic HL60 cells to fibronectin. In addition, chelation of [Ca++]i caused a significant decrease in 15/7 binding and adhesion stimulated by low (1 ng/ml) concentrations of phorbol myristate acetate (PMA). The inhibitory effect of [Ca++]i chelation on beta1 integrin activation was reversed by repleting [Ca++]i with ionomycin in a Ca++-containing buffer, or by the addition of higher concentrations of PMA (10 ng/ml). These data suggest a role for [Ca++]i in inside-out activation of beta1 integrins, probably through a synergistic effect with PKC activation.

Phorbol esters and synthetic diacylglycerols inhibit gamma-aminobutyric acid uptake in human spermatozoa: an effect mediated by protein kinase C?

Protein kinase C (PKC) and protein kinase A (PKA) are present in human spermatozoa and probably involved in the cascade systems leading to acrosomal exocytosis. We have investigated the possibility that these two protein kinases are involved in the regulation of gamma-aminobutyric acid (GABA) uptake in human spermatozoa. Swim-up preparations of human spermatozoa were exposed to phorbol 12-myristate 13 acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG), which are activators of PKC, and to dibutyryl cyclic AMP (dbcAMP), a PKA activator, and subsequently incubated with radiolabelled GABA. Accumulated intracellular GABA was measured in a scintillation counter. Both PMA and OAG caused inhibition of GABA uptake while dbcAMP was without effect. The inhibitory effect on GABA uptake observed following exposure to the PKC activators could, however, not be counteracted by preincubation of the sperm samples with the PKC inhibitors staurosporine or H-7. The inhibitory effect on GABA uptake following addition of PMA was enhanced by prolonged exposure to the reagent and by increased capacitation prior to addition of the reagent. A small numerical increase in the percentage of acrosome-reacted cells was observed following exposure of the sperm cells to the highest concentrations of PMA, OAG and dbcAMP. No sperm motility parameters were affected by the treatment protocols as measured using computer-assisted semen analysis (CASA).

Protein kinase C activity following exposure to magnetic field and phorbol ester

We examined the separate and combined effects of 60 Hz sinusoidal magnetic fields (MFs) and a phorbol ester on protein kinase C (PKC) activity in HL60 cells. No enhancement in PKC activity was observed when a cell culture was exposed to a 1.1 mT (rms) MF alone or to a combination of MF and 2 microM phorbol 12-myristate 13-acetate (PMA) for 1 h. In a second set of experiments, cells were preexposed to a less than optimal concentration of PMA (50 nM) for 45 min, followed by a 15 min exposure to both PMA and MF. The data showed a greater decrease in cytosolic PKC activity and a larger increase in membrane activity than was induced by either 1 h PMA treatment alone or PMA and sham MF exposure. One logical conclusion from these data is that MFs may be acting in a synergistic manner on a pathway that has already been activated. Therefore, we suggest that MFs, rather than producing biological effects by a new pathway or mechanism of interaction, exert their effect(s) by interacting with already functioning reactions or pathways. If correct, the question of an MF's mechanism of interaction refocuses on how weak fields might enhance or depress a molecular reaction in progress, rather than on finding a new transduction pathway.

Protein kinase C activity following exposure to magnetic field and phorbol ester

We examined the separate and combined effects of 60 Hz sinusoidal magnetic fields (MFs) and a phorbol ester on protein kinase C (PKC) activity in HL60 cells. No enhancement in PKC activity was observed when a cell culture was exposed to a 1.1 mT (rms) MF alone or to a combination of MF and 2 μM phorbol 12-myristate 13-acetate (PMA) for 1 h. In a second set of experiments, cells were preexposed to a less than optimal concentration of PMA (50 nM) for 45 min, followed by a 15 min exposure to both PMA and MF. The data showed a greater decrease in cytosolic PKC activity and a larger increase in membrane activity than was induced by either 1 h PMA treatment alone or PMA and sham MF exposure. One logical conclusion from these data is that MFs may be acting in a synergistic manner on a pathway that has already been activated. Therefore, we suggest that MFs, rather than producing biological effects by a new pathway or mechanism of interaction, exert their effect(s) by interacting with already functioning reactions or pathways. If correct, the question of an MF's mechanism of interaction refocuses on how weak fields might enhance or depress a molecular reaction in progress, rather than on finding a new transduction pathway. Bioelectromagnetics 19:469–476, 1998. © 1998 Wiley-Liss, Inc.

Flow cytometric quantitation of oxidative product formation by heterophils from orange-winged Amazon parrots (Amazona amazonica amazonica)

Oxidative metabolic burst activity by heterophils from orange-winged Amazon parrots (Amazona amazonica amazonica) was indirectly evaluated by measuring the oxidation of non-fluorescent intracellular 2′,7′-dichlorofluorescin (DCFH) to fluorescent 2′,7′-dichlorofluorescein (DCF) with flow cytometry. Heterophils were isolated from whole blood samples by preconcentrating the white blood cells with 3% hetastarch before running the samples through a polysucrose and disodium diatrizoate centrifugation. Phorbol myristate acetate (PMA) was used to activate the heterophils. Results show that heterophils from orange-winged Amazon parrots can oxidise DCFH to a fluorescent product and a wide range of DCF production was observed among individual birds. The production of DCF increased with PMA concentrations and significant differences were seen between the highest and lowest concentrations of PMA. These results support the concept that heterophils from orange-winged Amazon parrots can produce oxidative metabolites and that these heterophils appear to have functional heterogenicity.

Differential superoxide anion generation by equine eosinophils and neutrophils

Equine eosinophils and neutrophils are believed to play an important part in the protection of horses against parasitic and bacterial invasion. Eosinophils may also play a key role in the pathogenesis of equine inflammatory conditions such as the allergic skin disease, insect hypersensitivity. The factors which stimulate the respiratory burst of equine eosinophils and neutrophils are poorly understood. The first aim of the present study was to determine the effects of the phorbol ester, phorbol myristate acetate (PMA), which is believed to activate intracellular protein kinase C, and opsonised particles of serum-treated zymosan (STZ), on the production of superoxide anions by equine eosinophils and neutrophils. Since histamine has been detected after antigen challenge in the skin of horses with insect hypersensitivity, the second aim was to establish the effects of this mediator on superoxide anion production by equine eosinophils and the receptor sub-type(s) that mediate histamine-induced responses. For comparison, responses of neutrophils from the same horses were also examined. PMA and STZ induced significant increases in superoxide anion generation by equine eosinophils and neutrophils. The estimated maximum ( E MAX) superoxide anion production by eosinophils in the presence of PMA was significantly greater than that of neutrophils; the estimated concentration of PMA inducing 50% of the maximum response (EC 50) by eosinophils was significantly less. The E MAX values for superoxide anion production by neutrophils in the presence of STZ were significantly greater than those for eosinophils. Histamine induced superoxide anion generation by equine eosinophils which was inhibited by the histamine-1 receptor antagonists chlorpheniramine and mepyramine, but not the histamine-2 and histamine-3 receptor antagonists, cimetidine and thioperamide, respectively. Histamine did not cause superoxide anion production by equine neutrophils. These studies demonstrate that equine granulocytes vary in their ability to produce a respiratory burst in the presence of different stimuli, with eosinophils being more responsive to protein kinase C activators and neutrophils to opsonised particles. They also show that histamine selectively induced the generation of superoxide anions by equine eosinophils via histamine-1 receptor activation. Thus, in horses with insect hypersensitivity, histamine released from cutaneous mast cells after antigen challenge could activate eosinophils which have migrated into the dermis.

Protein kinase C- and Ca2+ ionophore-stimulated production of reactive oxygen species in mechanically dispersed isolated bovine luteal cells.

We measured the production of reactive oxygen species (ROS) using luminol-horseradish peroxidase-induced chemiluminescence in mechanically dispersed cell suspensions from bovine corpus luteum (CL). Since other cell types besides luteal cells were present in crude cell suspensions from CL, cell preparations were purified by centrifugation on Percoll. Only cell suspensions that gave no significant response when stimulated with formyl-methionyl-leucyl-phenylalanine, a potent stimulator of ROS production by phagocytes, were used routinely. Basal ROS production by purified bovine luteal cell preparations was low but could be stimulated rapidly and in a dose-dependent manner by nanomolar concentrations of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), though only in cells purified by fractionation on Percoll. Luteal ROS responses to PMA were quenched by returning bovine erythrocytes to purified luteal cells, or by exogenous catalase or superoxide dismutase. The magnitude of the response to PMA varied markedly from one luteal cell preparation to another but appeared to be unrelated to the stage of the luteal phase of the CL from which the cells were prepared. The luteal ROS response to PMA was blocked by staurosporine, an inhibitor of PKC. Although the inactive phorbol ester (4alpha-phorbol didecanoate; 4alphaPDD) alone had little or no effect on luteal ROS production, 4alphaPDD significantly potentiated the effects of submaximal concentrations of PMA in a dose-dependent manner. ROS production could also be stimulated by the Ca2+ ionophore A23187. This response was rapidly abolished by treatment with EDTA or EGTA. A23187 also augmented the response to submaximal PMA levels: however, pretreatment with 4alphaPDD did not significantly enhance the ROS response to A23187. In conclusion, we have shown that isolated bovine luteal cell suspensions are capable of generating a marked acute ROS response triggered by activation of PKC and/or elevation of cytosolic calcium.

Effect of phorbol ester on the regulation of [formula omitted] receptors

Granulosa cells have been used to study the regulation of LH hCG receptor protein and mRNA expression. Phorbol 12-myristate 13-acetate (PMA) dose-dependently attenuates the increases in LH hCG receptor mRNA and protein induced by FSH and forskolin (FSK). The presence of PMA caused a decrease in cAMP production stimulated by FSH and FSK. These results suggest that PMA-mediated decreases in cAMP are a major factor in PMA-mediated decreases in LH hCG receptor mRNA. On the other hand, in the presence of 8-Br-cAMP, PMA significantly increased LH hCG receptor mRNA and protein, with maximal stimulation between PMA concentrations of 3 to 30 nM (1.5 fold) with 8-Br-cAMP. These findings suggest that activation of protein kinase C by PMA attenuates the increase in cAMP accumulation induced by FSH but enhances the effect of cAMP on LH hCG receptor expression, and that the inhibitory and stimulatory effects of PMA on LH hCG receptor content are correlated with regulation of LH hCG receptor mRNA levels. Since the half-life study revealed no change in the stability of the LH hCG receptor mRNA following PMA treatment, a change in the rate of LH hCG receptor gene transcription must be responsible for the change in the LH hCG receptor mRNA levels.

U937 CELLS AS A MODEL TO STUDY THE EFFECT OF PHYTOCHEMICALS ON SUPEROXIDE ANION PRODUCTION

The ability of phytochemicals to suppress production of superoxide anion (O 2 −̇) in a cell culture model was assessed. Differentiated U937 cells and human polymorph nuclear leukocytes (PMN) were stimulated with increasing concentrations of phorbol myristate acetate (PMA) and O 2 −̇ release was measured by cytochrome c reduction. The overall trend in O 2 −̇ production in response to PMA stimulation was similar in both U937 cells and PMN. The effect of the phytochemicals, chlorogenic acid, caffeic acid, curcumin, quercetin, (-)-epicatechin and glycyrrhizic acid (50, 100 and 500 nM) on O 2 −̇ formation from PMA stimulated U937 cells was examined. All phytochemicals suppressed O 2 −̇ release from U937 cells with chlorogenic acid, (-)-epicatechin and glycyrrhizic acid showing dose dependent effects. In conclusion, U937 cells are a useful model to screen phytochemicals for their ability to suppress O 2 −̇ formation. © 1997 Elsevier Science Inc.

Effect of calcitonin on calcium transport by the luminal and basolateral membranes of the rabbit nephron

In the rabbit, calcitonin has been shown to enhance calcium (Ca2+) reabsorption in the early distal tubule. The aim of the present study was to investigate the mechanism of this action, using isolated luminal and basolateral membranes of distal tubules. The tubule suspensions were preincubated in the presence or absence of 10(-7) M calcitonin. The luminal or basolateral membranes were subsequently purified and 45Ca transport through the vesicles was measured using the rapid filtration technique. Results were compared with those obtained from proximal tubule membranes. In the proximal tubules, calcitonin had no effect on Ca2+ uptake by luminal membranes. In the distal tubules, the presence of Na+ in the incubation medium strongly decreased the uptake of Ca2+ by luminal membranes. Preincubation of distal tubules with calcitonin partially restored this uptake. We previously reported a dual kinetics of Ca2+ uptake by the distal luminal membranes. Calcitonin enhanced Ca2+ transport by the low affinity component, increasing the Vmax and leaving the K(m) unchanged. Renal calcitonin receptors usually couple to both adenylate cyclase and phospholipase C. To determine through which messenger(s) calcitonin enhances Ca2+ transport by the distal tubules, we first confirmed that the hormone stimulates cAMP and IP3 release. Incubation of the distal tubules with 10(-7) M calcitonin significantly increased both messengers. In contrast, calcitonin did not influence the IP3 nor the cAMP content of proximal tubules. Therefore, we studied the actions of cAMP and phorbol 12-myristate 13 acetate (PMA) on Ca2+ transport by the distal luminal membranes. Incubation of distal tubule suspensions with dibutyryl cAMP significantly increased Ca2+ uptake by the luminal membranes. However, incubation of these tubules with various concentrations of PMA (10 nM, 100 nM and 1 microM) had no effect on this uptake. Calcitonin also influenced Ca2+ transport by the distal basolateral membrane. Incubation of distal tubule suspensions with 10(-7) M calcitonin activated the Na+/Ca2+ exchanger activity, almost doubling the Na+ dependent Ca2+ uptake. Here again this action was mimicked by cAMP. We conclude that calcitonin increases Ca2+ transport by the distal tubule through two mechanisms: the opening of low affinity Ca2+ channels in the luminal membrane and the stimulation of the Na+/Ca2+ exchanger in the basolateral membrane, both actions depending on the activation of adenylate cyclase.

Acetylated LDL endocytosis by the human monocytic Mono Mac 6sr cells is not mediated by the macrophage type I and II scavenger receptors.

We recently reported that the human monocytic Mono Mac 6sr cell line constitutively takes up and degrades acetylated (acLDL) and oxidized LDL through receptor-specific pathways. The present studies were undertaken to further characterize the acLDL binding site on a functional and molecular basis. The degradation of acLDL increased during differentiation of Mono Mac 6sr cells with lipopolysaccharide (10 ng/mL, 72 hours) and low concentrations of phorbol 12-myristate 13-acetate (PMA; 0.1 to 1.0 ng/mL, 72 hours). Higher doses of PMA (5 or 10 ng/mL), however, decreased acLDL degradation. Scatchard plots of acLDL binding in untreated and LPS-differentiated Mono Mac 6sr cells were nonlinear and suggested the presence of more than one binding site. Although the ligand specificity of the acLDL receptor in Mono Mac 6sr cells resembles that of the macrophage type I and type II scavenger receptors, we did not detect mRNA of either receptor type in untreated or differentiated Mono Mac 6sr cells by means of Northern blotting and reverse transcription polymerase chain reaction. Furthermore, ligand blotting with 125I-acLDL failed to detect the 220-kD types I and II scavenger receptor protein. Thus, Mono Mac 6sr cells express an acLDL receptor that is distinct from the type I and type II scavenger receptor found in human monocyte-derived macrophages but that, like the latter, is induced during monocytic differentiation.

Activation of p42erk2MAPK and p90rskby IL-2 Occurs Independently of Protein Kinase C

Stimulation of the human interleukin-2 (IL-2)-dependent cell line, Kit225, with IL-2 resulted in the rapid activation of microtubule-associated protein-2 kinase (MAPK), as demonstrated by the tyrosine phosphorylation of p42erk2, the reduced mobility of MAPK in SDS–PAGE gels, and the increased ability of lysates from these cells to phosphorylate myelin basic protein. Measurements of kinase activity in immunoprecipitates of p90 ribosomal S6 kinase (p90rsk) demonstrated that stimulation of this cell line with IL-2 also resulted in the activation of p90rsk. Further, increased MAPK activity occurred following IL-2 stimulation, but not following phorbol 12-myristate 13-acetate (PMA) stimulation in cells depleted of PKC by prolonged treatment with a high concentration of PMA. These results demonstrate that IL-2 stimulation results in the activation of MAPK and p90rskand that this activation occurs in a PKC-independent manner.

Possible involvement of protein kinase C-ϵ in phorbol ester-induced growth inhibition of human lymphoblastic cells

Sustained activation of members of the protein kinase C (PKC) family is known to influence the growth and differentiation of various cell types, however, the specific roles for individual isoforms mediating these cellular events have yet to be elucidated. Activation of PKC by phorbol esters leads to growth inhibition in certain cell lines. The HT58 human B lymphoblastic cell may serve as a cellular model system to investigate the participation of individual isoforms in the initial events of growth arrest induced by phorbol ester. Determination of cell cycle and investigation of apoptosis were performed by flow cytometric measurements. Phorbol ester-induced translocation and down-regulation of the conventional α, β and the novel ϵ isoforms of PKC were demonstrated by Western blot analysis. At lower concentrations (0.5 ng/ml) phorbol myristate acetate (PMA) stimulated a G1 arrest with retention of viability in the human HT58 B lymphoblastic cell. The protein kinase inhibitor staurosporine at a concentration of 25 nM did not significantly alter HT58 cell viability. However, staurosporine (25 nM) induced apoptosis in cells preincubated for 4 hr with 0.5–1.0 ng/ml PMA. The translocation of PKC-ϵ was observed within 30 min exposure to 0.5 ng/ml PMA. After a 4 hr treatment, evidence for down-regulation and an altered phosphorylation state of PKC-ϵ was seen. In contrast, the conventional α and β isoforms were practically uneffected by this PMA treatment. At higher PMA concentrations (50 ng/ml) the α and β isoforms showed a significant down-regulation. The preferential alterations in PKC-ϵ observed under the conditions required for PMA to influence the growth and survival of HT58 cells suggest a role for the Ca 2+-independent ϵ isoform in mediating the initial events of the phorbol ester stimulated cellular responses.

Lineage commitment of transformed haematopoietic progenitors is determined by the level of PKC activity.

Our previous work showed that haematopoietic precursors transformed by the E26 avian leukemia virus undergo multilineage differentiation in response to the phorbol ester phorbol 12-myristate 13-acetate (PMA). Treatment of the cells with high concentrations of PMA (100 nM) favours myelomonocytic differentiation, while lower concentrations (20 nM) induce predominantly eosinophil differentiation. Here we have investigated the role of protein kinase C (PKC) in this process and found that 100 nM, but not 20 nM, PMA dramatically down-regulates total cellular PKC activity, indicating that high PMA concentrations result in less efficient signalling than lower PMA concentrations. Consistent with these findings is the observation that very low PMA concentrations (1 nM), which presumably only moderately activate PKC, induce myeloid differentiation. This suggests the existence of two PKC thresholds which play a role in lineage commitment. To test the model, alpha- and epsilon-PKC isoforms were expressed in E26-transformed progenitors. These cells exhibited myelomonocytic differentiation even in the absence of PMA, while treatment with concentrations of PMA as high as 100 nM led to the differentiation of predominantly eosinophils and failed to downregulate the exogenous PKC. Our results suggest that different levels of PKC activity result in three different phenotypes: (i) no PKC activity maintains the progenitor phenotype; (ii) low PKC activity favours myelomonocytic differentiation; (iii) high PKC favours eosinophil differentiation.