Effects of protein kinase C (PK-C) activators and inhibitors on human large granular lymphocytes (LGL): role of PK-C on natural killer (NK) activity

The role of protein kinase C (PK-C) in the early metabolic events involved in human natural killer (NK) cell activation has been studied through the action of PK-C-specific activators and inhibitors. Highly purified human large granular lymphocytes (LGL) were treated for 1 hr with the diacylglycerol analog 1-oleoyl-2-acetyl glycerol (OAG) (10(-4)-10(-5) g/ml) or with 12-O-tetradecanoylphorbol-13-acetate (TPA) (10(-8)-10(-10) g/ml), both specific activators of PK-C. Both these agents consistently increased NK activity against K562 target cells. Suboptimal doses of either OAG or TPA also synergized with Ca2+ ionophores to augment spontaneous cytotoxic activity. Pretreatment of LGL with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrocloride (H7) (5-40 microM), a potent PK-C inhibitor, greatly reduced NK activity in a time- and dose-dependent fashion. By contrast, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA 1004), a potent cAMP- and cGMP-dependent PK inhibitor with almost no effect on PK-C, marginally reduced NK activity. Moreover, almost complete NK activity inhibition was observed when H7 (10 microM), but not HA 1004 (50 microM), was present in the NK assay. Finally, 48 hr stimulation of LGL with TPA (10(-6) g/ml), a treatment able to inactivate most of the PK-C cellular pool, almost completely abrogated NK activity. This functional evidence was supported by phosphorylation of several endogenous substrates which occurs within 5 min in TPA-treated LGL. Two proteins of 70 and 56 kDa have been identified as major PK-C substrates, together with other phosphorylated proteins with MW ranging from 177 to 43 kDa. H7, but not HA 1004, almost completely inhibited the TPA-induced phosphorylation of all of these proteins in the NK cells. These data strongly suggest that selective activation of PK-C plays an essential role in the mechanisms of NK cell activation.     

Examination of the role of the proteolytically-activated form of protein kinase C in the differentiation of human haemopoietic cells

Abstract. In neutrophils, the phorbol ester 12- O -tetrade-canoylphorbol-l3-acetate (TPA) induced the translocation of the Ca⁺⁺- and phospholipid-dependent protein kinase, protein kinase C (PK-C) from the soluble to the particulate fraction. At the same time there was a corresponding increase in the amount of Ca⁺⁺- and phospholipid-independent protein kinase activity recovered in the soluble fraction. This soluble Ca⁺⁺- and phospholipid-independent protein kinase presumably reflects proteolytic activation of the particulate associated PK-C. Bone marrow and undifferentiated HL-60 cells also translocated PK-C to the particulate fraction in response to TPA but did not accumulate the soluble Ca⁺⁺- and phospholipid-independent form of the enzyme. Similar results were obtained using HL-60 cells induced to differentiate with dimethyl sulphoxide (DMSO), recombinant human granulocyte-macrophage colony-stimulating factor (rh GM-CSF) or la,25-dihydroxyvitamin D₃. There was also no significant change in either the number or time of expression of differentiation-specific cell surface antigens observed on HL-60 cells induced to differentiate with either DMSO, 1α,25-dihydroxyvitamin D3 or TPA in the presence of cyclosporin A, an agent reported to inhibit the proteolytic breakdown of PK-C to the Ca⁺⁺- and phospholipid-independent form. Likewise, cyclosporin A did not affect the rate or extent of differentiation of primary bone marrow cell cultures. These results suggest that the proteolytically activated and phospholipid-independent form of PK-C is probably not involved in haemopoietic cell differentiation.     

Phorbol ester prevents the thyroid-stimulating-hormone-induced but not the forskolin-induced decrease of cAMP-dependent protein kinase activity in thyroid cell cultures

The potent tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA) affects several thyroid cell functions and interacts with thyroid-stimulating hormone (TSH) either by inhibiting or potentiating its action on different cellular parameters. Since phorbol ester acts mainly through the activation of protein kinase C, which is its receptor, we studied this activation and its interaction with TSH and forskolin in suspension cultures of porcine thyroid cells. In thyroid cell cultures, TPA has a dual effect on protein kinase C activity: immediately (2-5 min) after exposure of cells to TPA, it began to be translocated from the cytosol to the particulate fraction. The transfer of the cytosolic enzyme was total and could occur with or without a loss of activity. The translocated enzyme still needed Ca2+ and phospholipids for its activation. The basal activity increased transiently (2-4 h) in both the cytosol and particulate fractions during translocation. The peak activity in the particulate fraction was reached 10-30 min after exposure of cells to TPA, and was followed by down-regulation of protein kinase C and almost complete disappearance of its activity. The residual activity was about 13% of control after a 2-day exposure to TPA. It was unequally distributed between cytosol (4%) and particulate fraction (9%). Prolonged exposure of cells to TPA did not affect either the activity or the subcellular distribution of the cAMP-dependent protein kinase activity. TPA interacted with TSH and prevented the decrease of this activity induced by prolonged exposure of cells to the hormone not only when it was introduced simultaneously with TSH, but also when it was added 24 h after TSH. However, the forskolin-induced decrease in cAMP-dependent protein kinase activity was not prevented by the presence of TPA. TPA also affected the increases in cAMP accumulation mediated by TSH and forskolin. The TSH-induced increase was significantly stimulated by TPA after short contacts (5-15 min), while longer preincubations of cells with TPA provoked a very strong inhibition of the TSH action. However, the forskolin-induced stimulation of the cAMP accumulation was maintained and even further increased in the presence of TPA. Consequently, the actions of TSH and TPA are apparently interdependent, while those of forskolin and TPA seem to be parallel and independent. Neither TSH nor forskolin prevented the TPA-induced down regulation of protein kinase C. The biologically inactive phorbol ester analogue 4 alpha-phorbol 12,13-didecanoate had no effect on protein kinase C activity, and did not interact with either TSH or forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation by phorbol esters of protein kinase C in MCF-7 human breast cancer cells

Exposure of MCF-7 human breast cancer cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) leads to the inhibition of cell proliferation. We investigate here the short-term effects of TPA on subcellular distribution of protein kinase C, and on protein phosphorylation in cultured MCF-7 cells. We report a rapid and dramatic decrease in cytosolic protein kinase C activity after TPA treatment. Only 30% of the enzymatic activity lost in the cytosol was recovered in the particulate fraction. These data suggest that subcellular translocation of protein kinase C is accompanied by a rapid down-regulation of the enzyme (70%). Furthermore, TPA and other protein kinase C activators rapidly induce the phosphorylation of a 28 kDa protein in intact MCF-7 cells. Phorbol esters devoid of tumor-promoting activity are ineffective both for inducing these early biochemical events and for inhibiting cell proliferation.     

Early effects of TPA on protein kinase activity in murine thymocytes : Reduction of protein kinase C activity in the cytosol and increase of Ca and phospholipid-independent kinase activity in the particulate fractions

Brief treatment of intact thymocytes with TPA and other tumor promoters causes a reduction in protein kinase C activity from the cytosol and an increase in kinase activity in the pariculate fraction. In contrast to the activity in the cytosol, which is absolutely dependent on the addition of Ca²⁺, phosphatidylserine and diolein, the activity in the particulate fraction is independent of these agents. Analysis of target specificity of the particulate kinase activity using exogenous and endogenous substrates suggests that the increased phosphorylation in the particulate fraction is catalysed by protein kinase C with altered catalytic properties. Although interleukin-1 and TPA are both co-mitogens for murine thymocytes, interleukin-1 does not share with TPA its property to alter protein kinase activity in the cytosolic and particulate fractions.     

Agonist-induced desensitization of cholinergically stimulated phosphoinositide breakdown is independent of endogenously activated protein kinase C in HT-29 human colon carcinoma cells.

Activation of M3 muscarinic receptors in HT-29 cells by carbachol rapidly increases polyphosphoinositide breakdown. Pretreatment of these cells with carbachol (0.1 mM) for 5 h completely inhibits the subsequent ability of carbachol to increase [3H]inositol monophosphate ([3H]InsP) accumulation, paralleled by a total loss of muscarinic binding sites. In contrast, protein kinase C (PK-C)-mediated desensitization by incubation with phorbol esters [PMA (phorbol 12-myristate 13-acetate)], leading to a time- and dose-dependent inhibition of cholinergically stimulated InsP release (95% inhibition after 4 h with 0.1 microM-PMA), is accompanied by only a 40% decrease in muscarinic receptor binding, which suggests an additional mechanism of negative-feedback control. Neither carbachol nor PMA pretreatment had any effect on receptor affinity. Incubation with carbachol for 15 min caused a small increase of membrane-associated PK-C activity (15% increase, P less than 0.05) as compared with the potency of phorbol esters (PMA) (3-4-fold increase, P less than 0.01). Long-term incubation (4-24 h) with PMA resulted in a complete down-regulation of cytosolic and particulate PK-C activity. Stimulation of InsP release by NaF (20 mM) was not affected after a pretreatment with phorbol esters or carbachol, demonstrating an intact function of G-protein and phospholipase-C (PL-C) at the effector side. Determination of PL-C activity in a liposomal system with [3H]PtdInsP2 as substrate, showed no change in PL-C activity after carbachol (13 h) and short-term PMA (2.5 h) pretreatment, whereas long-term preincubation with phorbol esters (13 h) caused a small but significant decrease in PL-C activity (19%, P less than 0.05). Our results indicate that agonist-induced desensitization of phosphoinositide turnover occurs predominantly at the receptor level, with a rapid loss of muscarinic receptors. Exogenous activation of PK-C by phorbol esters seems to dissociate the interaction between receptor and G-protein/PL-C, without major effects on total cellular PL-C activity.     

Protein kinase C activation and down-regulation in relation to phorbol ester-induced differentiation of SH-SY5Y human neuroblastoma cells

The role of protein kinase C activation in changes in muscarinic receptor functions and in the appearance of biochemical properties characteristic of neuronal cells was studied in SH-SY5Y human neuroblastoma cells induced to differentiate with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). A decrease in muscarinic receptor sensitivity with respect to agonist induced Ca2+ mobilization and receptor number parallelled the increase in membrane-associated protein kinase C (PK-C) activity. These changes occurred during the first 6 h of culture, and they were associated with rounding-up of cells. A subsequent decrease in particulate PK-C activity was followed by an increase in noradrenaline content, the appearance of an electrically excitable membrane, and an increase in the level of neuron-specific enolase. These changes were accompanied by a pronounced neurite outgrowth. 1-(5-Isoquinolinesulphonyl)-2-methylpiperazine (H-7), an inhibitor of PK-C and cyclic nucleotide-dependent protein kinases, enhanced the morphological differentiation induced by TPA, whereas N-(2-guanidinoethyl)-5-isoquinolinesulphonamide (HA-1004), which primarily inhibits cyclic nucleotide-dependent protein kinases, had no effect on the TPA-induced phenotypic differentiation. H-7 inhibited the decrease in muscarinic receptor sensitivity and receptor number, but had no effect on the appearance of the electrically excitable membrane or on the increase in the neuron-specific enolase level. Both H-7 and HA-1004 inhibited the TPA-induced increase in noradrenaline content.     

Phorbol esters induce both intracellular translocation and down-regulation of protein kinase C in MCF-7 cells

Exposure of MCF-7 human breast cancer cells to phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) results in a complete inhibition of cell proliferation. We investigated the effects of TPA on protein kinase C activity when cells were exposed to phorbol ester for various lengths of time. TPA induces within 5 min a drastic dose-dependent decrease of the cytosolic protein kinase C activity. The enzyme apparently lost at the cytosolic level was only partially recovered in the particulate fraction. The apparent down-regulation of the translocated enzyme which was only 34% after 1 min reached 72% and 84% after respectively 10 min and 15 min. Moreover, when cells are treated with TPA for longer periods of time, the particulate protein kinase C activity continues to decrease, dropping below control after 1 hour. This progressive decline leads to an almost complete disappearance of protein kinase C activity in MCF-7 cells after 45 hours of TPA treatment. The apparent loss of protein kinase C activity upon short- as well as long-exposure of cells to TPA was not accompanied by a concomitant increase of Ca, PL-independent protein kinase activity. We discuss the implication of these biochemical events in the inhibition of cell proliferation with regard to the respective short- and long-term effects of TPA on protein kinase C activity.     

Reduced Protein Kinase C Activity in Ischemic Spinal Cord

Protein phosphorylation was evaluated in a rabbit spinal cord ischemia model under conditions where cyclic AMP-dependent protein kinase (PK-A) and calcium/phospholipid-dependent protein kinase (PK-C) were activated. One hour of ischemia did not affect PK-A activity significantly; however, PK-C activity was reduced by more than 60%. In vitro phosphorylation of endogenous proteins by endogenous PK-C revealed that eight particulate and five cytosolic proteins showed stimulated phosphorylation by PK-C activators in control tissue, although this stimulation was virtually absent in ischemic samples. When control and ischemic particulate fractions were combined, the endogenous protein phosphorylation pattern under PK-C-activating conditions was similar to the ischemic sample, which suggests that inhibitory molecules may be present in the ischemic particulate fraction. In vitro phosphorylation of endogenous proteins under PK-A-activating conditions in ischemic tissue was similar to that in control tissue. The results suggest that the PK-C phosphorylation system is selectively impaired in ischemic spinal cord. In addition to reduced PK-C-dependent phosphorylation, an Mr 64,000 protein was phosphorylated in ischemic cytosolic samples, but not in control samples. The phosphorylation of the Mr 64,000 protein was neither PK-C-dependent nor PK-A-dependent. These altered phosphorylation reactions may play critical roles in neuronal death during the course of ischemia.     

Involvement of protein kinase C in pulmonary surfactant secretion from alveolar type II cells

The purpose of this study is to clarify the involvement of protein kinase C in pulmonary surfactant secretion from adult rat alveolar type II cells in primary culture. Surfactant secretion in vitro is stimulated by at least two classes of compounds. One class, (e.g. terbutaline) increases intracellular cyclic AMP, whereas the other class (e.g. 12-O-tetradecanoylphorbol 13-acetate (TPA] does not. TPA has been shown to activate protein kinase C in other cell systems. In our studies, 1-oleoyl-2-acetyl-sn-glycerol (OAG), which is a direct activator of protein kinase C, stimulated [3H] phosphatidylcholine secretion by alveolar type II cells in a dose- and time-dependent manner. Tetracaine, which is an inhibitor of protein kinase C, inhibited the TPA-induced secretion of [3H]phosphatidylcholine from alveolar type II cells in a dose-dependent manner. However, tetracaine had no effect on terbutaline-induced secretion. The effects of terbutaline and OAG upon surfactant secretion were significantly more than additive, but those of TPA and OAG were less than additive. The specific activity of protein kinase C was 6-fold higher than cyclic AMP-dependent protein kinase found in type II cells when both kinases were assayed using lysine-rich histone as a common phosphate acceptor. Ninety-four per cent of protein kinase C activity was recovered in the cytosolic fraction of unstimulated type II cells, and 40% of activity in cytosolic fraction was translocated to particulate fraction upon treatment with TPA. As observed in other tissues, protein kinase C of alveolar type II cells was highly activated by 1,2-dioleoyl-sn-glycerol or TPA in the presence of Ca2+ and phosphatidylserine. These results suggest that pulmonary surfactant secretion in vitro is stimulated by both protein kinase C and cyclic AMP-dependent protein kinase.     

Early effects of TPA on protein kinase activity in murine thymocytes. Reduction of protein kinase C activity in the cytosol and increase of Ca2+ and phospholipid-independent kinase activity in the particulate fractions

Brief treatment of intact thymocytes with TPA and other tumor promoters causes a reduction in protein kinase C activity from the cytosol and an increase in kinase activity in the particulate fraction. In contrast to the activity in the cytosol, which is absolutely dependent on the addition of Ca2+, phosphatidylserine and diolein, the activity in the particulate fraction is independent of these agents. Analysis of target specificity of the particulate kinase activity using exogenous and endogenous substrates suggests that the increased phosphorylation in the particulate fraction is catalysed by protein kinase C with altered catalytic properties. Although interleukin-1 and TPA are both co-mitogens for murine thymocytes, interleukin-1 does not share with TPA its property to alter protein kinase activity in the cytosolic and particulate fractions.     

佛波醇酯诱导HL-60细胞巨噬细胞样分化时蛋白激酶C活力及分布的改变

本文对佛波醇酶诱导人早幼粒白血病细胞系HL-60细胞分化为巨噬细胞样细胞对蛋白激酶c活力及其在亚细胞分布的变化进行了研究。蛋白激酶c活力在TPA处理1小时即明显降低,此低水平的酶活力持续整个实验时期。酶的亚细胞分布研究提示TPA处理细胞胞质组分酶活力剧烈降低,而颗粒组分存在一高盐浓度洗脱的酶活力峰。蛋白激酶c抑制剂三氟过(口了)嗪单独处理HL-60细胞导致胞质和颗粒组分酶活力升高,但并不诱导细胞分化;若与TPA合并处理细胞,酶活力又降低,此时细胞又分化为巨噬细胞样细胞。对上述结果的可能机理进行了讨论。     

Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester-Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity

The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two- to fourfold in the presence of PDB or TPA. PDB at 1-100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the particulate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the particulate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the particulate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.     

视黄酸对人肝癌细胞蛋白激酶的影响

 视黄酸(RA)处理SMMC-7721人肝癌细胞株72小时后,胞浆、膜性组分的蛋白激酶C(PK-C)的活力及比活力均下降,活力分别下降44.9％和48.8％,比活力下降42.7％和35.0％。然而,胞浆与膜性组分的活力,比活力比值在RA处理前后并无十分明显的变化,这提示在RA作用过程中,未发生PK-C的膜-浆转位。蛋白激酶A(PK-A)的变化则相反,RA处理72小时,活力、比活力上升了295％,258％。PK-A/PK-C的活力比值则从0.342增加到1.849,比活力比值从0.210增加到0.897。因PKC和PKA分别和细胞的去分化性增殖和分化有关,故上述结果和我们已报道的RA可抑制SMMC-7721细胞株的增殖和促进其分化相一致。     

Stimulation of hexose uptake in rat thymic lymphocytes by phorbol ester. A role for Ca2+ and Na+H+ exchange?

The tumor promoter 12-0-tetradecanoyl phorbol-13-acetate (TPA) stimulates hexose uptake into rat thymocytes. This study explores two possible messengers of this stimulation: changes in cytosolic [Ca2+], and activation of the Na+/H+ antiport. The cytosolic level of Ca2+, determined by the fluorescence of quin-2, was elevated by TPA, and this rise required extracellular Ca2+. In contrast, stimulation of hexose uptake was still observed in Ca2+ -free media even when cytoplasmic [Ca2+] was buffered with quin-2. TPA also raised the cytoplasmic pH, presumably through activation of the Na+/H+ exchange. However, replacement of extracellular Na+ by N-methylglucamine+ or choline+ which prevents the cytoplasmic alkanization did not prevent stimulation of hexose uptake by TPA. Moreover, amiloride, at concentrations that inhibit Na+/H+ exchange in these cells, did not interfere with stimulation of hexose uptake by TPA. In conclusion, stimulation of hexose uptake by phorbol ester in rat thymocytes does not appear to be mediated by changes in cytosolic free Ca2+ or in the activity of the Na+/H+ antiport.     

A calmodulin antagonist inhibits histamine-stimulated acid production by isolated rat parietal cells

The role of calmodulin in the regulation of histamine-stimulated parietal cell function was studied in isolated rat parietal cells using [14C]aminopyrine uptake as a quantitative index of acid production. In enriched (77-87%) intact parietal cells the calmodulin antagonist naphthalene sulfonamide W 7 dose-dependently inhibited the response to 10(-4) M histamine (IC50: 2 X 10(-6) M). The mechanism of this inhibition was examined further with two other stimuli of H+-production: forskolin which directly activates the parietal cell adenylate cyclase without interacting at the histamine H2-receptor and dbcAMP which mimics the biological action of cAMP without preceding activation of adenylate cyclase. W 7 effectively inhibited the responses to 10(-4) M forskolin (IC50: 6 X 10(-7) M), 10(-3) M dbcAMP (IC50: 10(-6) M) and to 10(-2) M K+ (IC50: 3 X 10(-6) M). The action of W 7 followed non-competitive kinetics since the antagonist reduced the entire range of the concentration-response curves without shifting them rightwards towards higher concentrations of the respective stimulants. The effect of W 7 was reversed by washing the cells. ATP-induced [14C]aminopyrine uptake into digitonin-permeabilized oligomycin-inhibited parietal cells reflects H+-production independent of oxidative phosphorylation and was also inhibited by W 7 (IC50: 10(-5) M). Inhibition of K+-stimulated H+/K+-ATPase activity required even higher W 7-concentrations (IC50: 1.4 X 10(-4) M). Our data suggest that calmodulin might be involved in the intracellular mediation of the response to histamine. Between histamine-induced cAMP-generation and the H+-secreting tubulovesicular system W 7 seems to inhibit an intracellular step that finally activates the H+/K+-ATPase. Yet, direct inhibition of the ATPase requires W 7 concentrations of questionable specificity and is unlikely to be the mechanism behind the action of W 7 on the parietal cell response to histamine.     

Parietal cell protein kinases. Selective activation of type I cAMP-dependent protein kinase by histamine

cAMP-dependent protein kinases have been characterized in parietal cells isolated from rabbit gastric mucosa. Both Type I and Type II cAMP-dependent protein kinase isozymes are present in these cells. Type II isozymes were detected in 900, 14,000, and 100,000 X g particulate fractions as well as 100,000 X g cytosolic fractions; Type I isozymes were found predominately in the cytosolic fraction. When parietal cells were stimulated with histamine, an agent that elevates intracellular cAMP content and initiates parietal cell HCl secretion, cAMP-dependent protein kinase activity was increased in homogenates of these cells as measured by an increase in the cAMP-dependent protein kinase activity ratio. Histamine activation of cAMP-dependent protein kinase was correlated with parietal cell acid secretory responses which were measured indirectly as increased cellular uptake of the weak base, [14C]aminopyrine. These results suggest that cAMP-dependent protein kinase(s) is involved in the control of parietal cell HCl secretion. The parietal cell response to histamine may be compartmentalized because histamine appears to activate only a cytosolic Type I cAMP-dependent protein kinase isozyme, as determined by three different techniques including 1) ion exchange chromatography; 2) Sephadex G-25 to remove cAMP and allow rapid reassociation of the Type II but not the Type I isozyme; and 3) 8-azido-[32P]cAMP photoaffinity labeling. Forskolin, an agent that directly stimulates adenylate cyclases, was found to activate both the Type I and Type II isozymes. Several cAMP-dependent protein kinases were also detected in parietal cell homogenates, including a Ca2+-phospholipid-sensitive or C kinase and two casein kinases which were tentatively identified as casein kinase I and II. At least two additional protein kinases with a preference for serine or lysine-rich histones, respectively, were also detected. The function of these enzymes in parietal cells remains to be shown.     

Cyclosporin A inhibits proteolytic cleavage and degradation of membrane-bound protein kinase C in hepatocytes after stimulation by phorbol ester

Stimulation of hepatocytes by the tumor promoter phorbol 12-myristate 13-acetate (PMA) caused translocation of cytosolic Ca2+/phospholipid-dependent protein kinase C (PK-C). The major part of PK-C activity (greater than 80%) was associated with the membrane fraction after 30 min. During the following 6 h protein kinase C activity decreased to less than 10%. Minor amounts of Ca2+/phospholipid-independent PK-C activity were found in the cytosol fraction at all times; they temporarily increased 2.5-fold with PMA and decreased after 1 h. Cyclosporin A did not affect the translocation of PK-C from the cytoplasm to the membrane fraction, but the decrease of PK-C activity following translocation was blocked. No marked increase of Ca2+/phospholipid-independent PK-C activity was observed in the cytosol in the presence of cyclosporin A. Leupeptin, which is known to inhibit Ca2+-requiring non-lysosomal proteinases (e.g. calpain), showed an effect similar to cyclosporin A. Both agents reduced proteolytic degradation of cellular proteins observed in isolated hepatocytes after PMA treatment. Ca2+-ionophore A23187 in high doses (greater than 10(5) M) partly reversed cyclosporin A and leupeptin action.     

Evidence for a role of protein kinase C in luteinizing hormone synthesis and secretion. Impaired responses to gonadotropin-releasing hormone in protein kinase C-depleted pituitary cells

The role of protein kinase C in luteinizing hormone (LH) release was analyzed in studies on the actions of phorbol esters and gonadotropin-releasing hormone (GnRH) in normal and protein kinase C (Ca2+/phospholipid-dependent enzyme)-depleted pituitary cell cultures. LH secretory responses of normal pituitary cells to GnRH were reduced but not abolished in Ca2+-deficient medium, consistent with the existence of extracellular Ca2+-dependent and -independent components of GnRH action. Both of these components could be elicited by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). The LH secretory responses to TPA and GnRH were additive only at low doses and converged to a common maximum at high concentrations of the agonists in the presence or absence of extracellular Ca2+. The release of stored LH by GnRH and TPA was accompanied by secretion of newly synthesized LH from 2 to 5 h during stimulation by either of the agonists. LH synthesis was increased in a progressive and dose-dependent manner by GnRH and TPA, and the ratio between newly synthesized and released hormone was near 1:2. TPA caused rapid and complete translocation of cytosolic protein kinase C to the particulate fraction of pituitary cells, followed by a progressive decrease in total enzyme content to approximately 10% after 6 h. Partial recovery of the cytosolic enzyme (to 20%) occurred after washing and reincubation for 15 h. Such kinase C-depleted cells showed prominent, dose-dependent reductions in the actions of GnRH and TPA on LH release and synthesis in both normal and Ca2+-deficient media. These observations support the hypothesis that protein kinase C participates in LH biosynthesis and secretion in pituitary gonadotrophs and is involved in the actions of GnRH upon these processes.     

Characterization of Na+/H+ exchange in FRTL-5 thyroid cells. Evidence for dependence on activation of protein kinase C.

Na+/H+ exchange activity was investigated in cultured rat thyroid follicular FRTL-5 cells using the pH sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Basal intracellular pH (pHi) was 7.13 +/- 0.10 in cells incubated in Hepes-buffered saline solution. The intracellular buffering capacity beta i was determined using the NH4Cl-pulse method, yielding a beta i value of 85 +/- 12 mM/pH unit. The relationship between extracellular Na+ and the initial rate of alkalinization of acid-loaded cells showed simple saturation kinetics, with an apparent Km value of 44 +/- 26 mM, and an Vmax value of 0.3 +/- 0.01 pH unit/min. The agonist-induced activation of Na+/H+ exchange was investigated in cells acidified with nigericin. Addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) or ATP induced rapid cytosolic alkalinization in acid-loaded cells. The action of both TPA and ATP was abolished by preincubating the cells with 100 microM amiloride, by substituting extracellular Na+ with equimolar concentrations of choline+, and by pretreating the cells with TPA for 24 h. Chelating extracellular Ca2+, or depleating intracellular Ca2+ pools did not affect the ATP-induced alkalinization. The results indicate, that FRTL-5 cells have a functional Na+/H+ exchange mechanism. Furthermore, stimulation of protein kinase C activity is of importance in activating the antiport.