Translocation Of Protein Kinase C Research Articles

Impaired α1‐adrenergic responses in aged rat hearts

To determine age-related changes in the cardiac effect of alpha1-adrenergic stimulation, both cardiomyocyte Ca2+-transient and cardiac protein kinase C (PKC) activity were measured in 3-month- (3MO) and 24-month- (24MO) old Wistar rats. Ca2+ transients obtained under 1 Hz pacing by microfluorimetry of cardiomyocyte loaded with indo-1 (405/480 nm fluorescence ratio) were compared in control conditions (Kreb's solution alone) and after alpha1-adrenergic stimulation (phenylephrine or cirazoline, an alpha1-specific agonist). PKC activity and PKC translocation index (particulate/total activity) were also assayed before and after alpha1-adrenergic stimulation. In 3MO, cirazoline induced a significant increase in Ca2+ transient for a 10(-9) M concentration which returned to control values for larger concentrations. In contrast, in 24MO, we observed a constant negative effect of cirazoline on the Ca2+ transient with a significant decrease at 10(-6) M compared with both baseline and Kreb's solution. Preliminary experiments showed that, in a dose-response curve to phenylephrine, the response of Ca2+ transient was maximal at 10(-7) M. This concentration induced a significant increase in Ca2+ transient in 3MO and a significant decrease in 24MO. The same concentration was chosen to perform PKC activity measurements under alpha1-adrenergic stimulation. In the basal state, PKC particulate activity was higher in 24MO than that in 3MO but was not different in cytosolic fractions; so that the translocation index was higher in 24MO (P < 0.01). After phenylephrine, a translocation of PKC toward the particulate fraction was observed in 3MO but not in 24MO. In conclusion, cardiac alpha1-adrenoceptor response was found to be impaired in aged hearts. The negative effect of alpha1-adrenergic stimulation on Ca2+ transient in cardiomyocytes obtained from old rats can be related to an absence of alpha1-adrenergic-induced PKC translocation.

Effect of adenosine triphosphate on phosphate uptake in renal proximal tubule cells: Involvement of PKC and p38 MAPK

ATP has been known to act as an extracellular signal and to be involved in various functions of kidney. Renal proximal tubular reabsorption of phosphate (Pi) contributes to the maintenance of phosphate homeostasis, which is regulated by Na+/Pi cotransporter. However, the effects of ATP on Na+/Pi cotransporters were not elucidated in proximal tubule cells (PTCs). Thus, the effects of ATP on Na+/Pi cotransporter and its related signal pathways are examined in the primary cultured renal PTCs. In the present study, ATP inhibited Pi uptake in a time (> 1 h) and dose (>10(-6)M) dependent manner. ATP-induced inhibition of Pi uptake was correlated with the decrease of type II Na+/Pi cotransporter mRNA. ATP-induced inhibition of Pi uptake may be mediated by P2Y receptor activation, since suramin (non-specific P2 receptor antagonist) and RB-2 (P2Y receptor antagonist) blocked it. ATP-induced inhibition of Pi uptake was blocked by neomycin, U73122 (phospholipase C (PLC) inhibitors), bisindolylmaleimide I, H-7, and staurosporine (protein kinase C (PKC) inhibitors), suggesting the role of PLC/PKC pathway. ATP also increased inositol phosphates (IPs) formation and induced PKC translocation from cytosolic fraction to membrane fraction. In addition, ATP-induced inhibition of Pi uptake was blocked by SB 203580 [a p38 mitogen activated protein kinase (MAPK) inhibitor], but not by PD 98059 (a p44/42 MAPK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK, which was not blocked by PKC inhibitor. In conclusion, ATP inhibited Pi uptake via PLC/PKC as well as p38 MAPK in renal PTCs.

An Essential Role for Phospholipase D in the Activation of Protein Kinase C and Degranulation in Mast Cells

Activation of phospholipase D (PLD) and protein kinase C (PKC) as well as calcium mobilization are essential signals for degranulation of mast cells. However, the exact role of PLD in degranulation remains undefined. In this study we have tested the hypothesis that the PLD product, phosphatidic acid, and diacylglycerides generated therefrom might promote activation of PKC. Studies were conducted in two rodent mast cell lines that were stimulated with Ag via FcepsilonRI and a pharmacologic agent, thapsigargin. Diversion of production of phosphatidic acid to phosphatidylbutanol (the transphosphatidylation reaction) by addition of l-butanol suppressed both the translocation of diacylglyceride-dependent isoforms of PKC to the membrane and degranulation. Tertiary-butanol, which is not a substrate for the transphosphatidylation, had a minimal effect on PKC translocation and degranulation, and 1-butanol itself had no effect on PKC translocation when PKC was stimulated directly with phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. Also, in cells transfected with small inhibitory RNAs directed against PLD1 and PLD2, activation of PLD, generation of diacylglycerides, translocation of PKC, and degranulation were all suppressed. Phorbol ester, which did not stimulate degranulation by itself, restored degranulation when used in combination with thapsigargin whether PLD function was disrupted with 1-butanol or the small inhibitory RNAs. However, degranulation was not restored when cells were costimulated with Ag and phorbol ester. These results suggested that the production of phosphatidic acid by PLD facilitates activation of PKC and, in turn, degranulation, although additional PLD-dependent processes appear to be critical for Ag-mediated degranulation.

Protein kinase C δ and ε mediate positive inotropy in adult ventricular myocytes

To examine cardiac contractile regulation and protein kinase C (PKC) translocation in parallel, the δ and ε isoforms of PKC were fused to green fluorescent protein (GFP) and expressed in adult rat ventricular myocytes maintained in short term culture. PKC-δ-GFP and PKC-ε-GFP were predominantly cytosolic until phorbol dibutyrate (PDBu) was introduced. PKC-δ-GFP redistributed preferentially to perinuclear structures that co-localized with a Golgi marker, whereas PKC-ε-GFP redistributed preferentially to the surface sarcolemma. Myocyte contractile function was assessed by monitoring twitch shortening with field stimulation at 0.5 Hz, 22 °C. In myocytes expressing PKC-δ-GFP, PDBu caused a transient negative inotropic response followed by a robust and sustained positive inotropic response that paralleled perinuclear PKC-δ accumulation. In PKC-ε-GFP myocytes, PDBu caused a sustained negative inotropic response that paralleled accumulation at the surface sarcolemma, but this response did not differ from myocytes expressing GFP alone. At higher expression levels, PKC-ε-GFP myocytes responded more like PKC-δ-GFP myocytes including perinuclear accumulation and a sustained positive inotropic response. Positive inotropic responses were markedly attenuated if PKC translocation was biased toward the surface sarcolemma by use of a more hydrophobic PKC activator, and were completely and selectively blocked by the PKC antagonist bis-indoylmaleimide. In contrast, transient and sustained negative inotropic responses were selectively blocked by the Ca 2+-dependent PKC isoform antagonist Go6976. The data indicate that the novel PKC isoforms δ and ε have little effect on contractility when accumulating at the cell surface, but produce a strong positive inotropic response upon accumulation at the Golgi or other intracellular sites.

Differential Effects of Commercial Polybrominated Diphenyl Ether and Polychlorinated Biphenyl Mixtures on Intracellular Signaling in Rat Brain in Vitro

Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants and have been detected in human blood, adipose tissue, and breast milk. Developmental and long-term exposures to these contaminants may pose a human health risk, especially to children. Previously, we demonstrated that polychlorinated biphenyls (PCBs), which are neurotoxic and structurally similar to PBDEs, perturbed intracellular signaling events, including calcium homeostasis and subsequent events such as protein kinase C (PKC), which are critical for the normal function and development of the nervous system. The objective of the present study was to test whether commercial PBDE mixtures (DE-71, a pentabrominated dipheyl ether mixture, and DE-79, a mostly octabromodiphenyl ether mixture) affected intracellular signaling mechanisms in a similar way to that of PCBs and other organohalogens, as an attempt to understand the common mode of action for these persistent chemicals. PKC translocation was studied by determining (3)H-phorbol ester ((3)H-PDBu) binding in rat cerebellar granule cells, and calcium buffering was determined by measuring (45)Ca(2+) uptake by microsomes and mitochondria isolated from adult male rat brain (frontal cortex, cerebellum, and hippocampus). As seen with PCBs, DE-71 increased PKC translocation and inhibited (45)Ca(2+) uptake by both microsomes and mitochondria in a concentration-dependent manner. The effect of DE-71 on (45)Ca(2+) uptake seems to be similar in all three brain regions. Between the two organelles, DE-71 inhibited mitochondrial (45)Ca(2+) uptake to a greater extent than microsomal (45)Ca(2+) uptake. DE-79 had no effects on either neurochemical event even at 30 mug/ml. Aroclor 1254 altered both events to a greater extent compared to DE-71 on a weight basis. When the results were compared on a molar basis, Aroclor 1254 altered PKC translocation and microsomal (45)CaP(2+) uptake to a greater extent than DE-71, however, Aroclor 1254 and DE-71 equally affected mitochondrial (45)Ca(2+) uptake. These results indicate that PBDEs perturbed intracellular signaling mechanisms in rat brain as do other organohalogen compounds and the efficacy between the commercial PCB and PBDE mixtures seem to vary with different endpoints.

Isoform-specific modulation of coronary artery PKC by glucocorticoids

Glucocorticoids (GC) exert diverse cellular effects in response to both acute and chronic stress, the functional consequences of which have been implicated in the development of cardiovascular pathology such as hypertension and atherosclerosis. However, the mechanisms by which GCs activate divergent signaling pathways are poorly understood. The present study examined the direct effects of natural (cortisol) and synthetic (dexamethasone) GCs on protein kinase C (PKC) isoform expression in coronary arteries. Porcine right coronary arteries were treated in vitro for 18 h in the presence and absence of either dexamethasone (10, 100, or 500 nM) or cortisol (50, 125, 250, or 500 nM). PKC isoform levels and subcellular distribution were determined by immmunoblotting of whole cell homogenates and immunocytofluorescence using PKC-α, -β II, -ε, -δ, and -ζ specific antibodies. Dexamethasone caused a ∼4-fold increase in PKC-α, a ∼2.5-fold increase in PKC-β II, and a 2-fold increase in PKC-ε ( p < 0.05). In contrast, dexamethasone had no effect on PKC-δ or PKC- ζ levels. Dexamethasone also caused an increase in the activity of PKC-α (285%), -β II (170%), and -ε (210%). Cortisol produced similar effects on PKC isoform expression. Confocal microscopy revealed that while dexamethasone altered localization patterns for PKC-α, -β II, and -ε, no such effect was observed for PKC-δ or PKC-ζ. The stimulatory effects of dexamethasone and cortisol on coronary PKC levels and translocation were prevented by the GC receptor (GR) blocker, RU486. These results demonstrate, for the first time, that GCs modulate coronary PKC expression and subcellular distribution in an isoform-specific manner through a GR-dependent mechanism.

Phosphorylation of PKC activation loop plays an important role in receptor‐mediated translocation of PKC

Protein kinase C (PKC) is translocated to various cellular regions in a subtype and stimulation-dependent manner. Thereafter, the activated PKC phosphorylates its substrate and causes subsequent cellular responses (PKC targeting). The 3-phosphoinositide-dependent protein kinase-1 (PDK1) has an essential role in the maturation of PKC by phosphorylating a threonine residue in the PKC activation loop. To elucidate the role of PDK1 in PKC targeting, we expressed mutant gamma- or delta-PKC fused with GFP (gamma- or delta-PKC-ALM (activation loop mutant)-GFP), whose threonine residue in the activation loop was replaced with alanine, and compared their P2Y receptor-mediated translocation with wild-type PKC-GFP in CHO cells. ATP (1 mm) induced the transient translocation of wild-type gamma- or delta-PKC-GFP from cytoplasm to plasma membrane and following retranslocation from membrane to the cytoplasm. gamma- or delta-PKC-ALM-GFP was also translocated to plasma membrane, which was, however, retained at the membrane for a longer period than wild type. Similar results were observed in kinase-negative PKC mutants, indicating that the phosphorylation by PDK1 affects the retranslocation step of PKC by regulating the kinase activity. The simultaneous monitoring of [Ca2+]i and diacylglycerol (DG) levels with the translocation of PKC demonstrated that PKC-ALM induced the prolonged accumulation of DG, resulting in the prolonged retention of PKC-ALM at the plasma membrane. It is possible that PKC-ALM with decreased kinase activity could delay the conversion of DG at the plasma membrane. Our present study suggests that the activation loop phosphorylation plays an important role in receptor-mediated PKC targeting.

Subcellular Alterations of Protein Kinase C Isozymes in the Rat Brain after Organophosphate Poisoning

The protein kinase C (PKC) signaling pathway has been associated with modulation of N-metyl-D-aspartate receptor activity, motor behavior, learning, and memory, all of which are severely impaired in organophosphate (OP) intoxication. Nevertheless, the role of PKC in OP intoxication is largely unknown. The present study attempted to characterize alterations in the immunoreactivity levels of PKC isozymes expressed in different brain areas in the rat following exposure to the nerve agent sarin (1x LD(50)). Furthermore, possible neuroprotective effect of selective PKC regulating peptide after such insult was evaluated. The results indicated that a significant reduction in the immunoreactivity level of the conventional betaII-PKC and the atypical zeta-PKC was observed in frontal cortex up to 24 h postsarin and in the striatum up to 5 days postsarin exposure. This reduction was in contrast to the increase in the immuno-reactivity level of both isozymes seen in the hippocampus or thalamus. Treatment with the anticonvulsant midazolam (0.5 mg/kg) 10 min postsarin exposure markedly reduced zeta-PKC immunoreactivity level and betaII-PKC in the membrane fractions in the hippocampus. betaII-PKC peptide (380 ng/kg), known to inhibit PKC translocation and activation, attenuated sarin-induced neuropathology. These observations suggest a role for both conventional and atypical PKC isozymes in OP-induced neuropathy in the rat and further support their involvement in cell death.

Investigation of PKC isoform‐specific translocation and targeting of the current of the late afterhyperpolarizing potential of myenteric AH neurons

AH neurons in the enteric nervous system play an essential role in initiating intestinal reflexes and factors that control AH neuron excitability therefore influence the state of the digestive system. Prominent afterhyperpolarizations that follow action potentials in these neurons strongly affect their excitability. In the present work, we have investigated the regulation of the afterhyperpolarizing current (I(AHP)) by protein kinase C (PKC). Electrophysiological responses and protein translocation were investigated in AH neurons of freshly dissected preparations of myenteric ganglia from the guinea-pig ileum. The activator of conventional and novel PKCs, phorbol dibutyrate, but not the activator of novel PKCs, ingenol, blocked the I(AHP). Phorbol dibutyrate had no effect on the hyperpolarization-activated current (I(h)) or on the A current (I(A)). Stimulation of synaptic inputs to the neurons also reduced the I(AHP), and had no effect on I(h) or I(A). Phorbol dibutyrate also reduced a background outward current that was present after the I(AHP) current had been blocked by clotrimazole. Both phorbol dibutyrate and ingenol caused translocation of the novel PKC, PKCepsilon, in these neurons. Only phorbol dibutyrate caused translocation of PKCgamma, a conventional PKC. The studies thus indicate that the activation of PKC by phorbol esters and by nerve stimulation affects AH neurons in a similar way, and that PKC activation targets both the I(AHP) and another background K(+) current. The I(AHP) is targeted by a conventional PKC, suggested to be PKCgamma, as this is the only conventional PKC that is prominent in AH neurons.

The NK1 Receptor Localizes to the Plasma Membrane Microdomains, and Its Activation Is Dependent on Lipid Raft Integrity

The spatial targeting of receptors to discrete domains within the plasma membrane allows their preferential coupling to specific effectors, which is essential for rapid and accurate discrimination of signals. Efficiency of signaling is further increased by protein and lipid segregation within the plasma membrane. We have previously demonstrated the importance of raft-mediated signaling in the regulation of smooth and skeletal muscle cell contraction. Since G protein-coupled receptors (GPCRs) are key components in the regulation of smooth muscle contraction-relaxation cycles, it is important to determine whether GPCR signaling is mediated by lipid rafts and raft-associated molecules. Neurokinin 1 receptor (NK1R) is expressed in central and peripheral nervous system as well as in endothelial and smooth muscle cells and involved in mediation of pain, inflammation, exocrine secretion, and smooth muscle contraction. The NK1 receptor was transiently expressed in HEK293 and HepG2 cell lines and its localization in membrane microdomains investigated using biochemical methods and immunofluorescent labeling. We show that the NK1 receptor, similar to the earlier described beta(2)-adrenergic receptor and G proteins, localizes to lipid rafts and caveolae. Protein kinase C (PKC) is one of the downstream effectors of the NK1 activation. Its active form translocates from the cytoplasm to the plasma membrane. Upon stimulation of the NK1 receptor with Substance P, the activated PKC relocated to lipid rafts. Using cholesterol extraction and replenishment assays we show that activation of NK1 receptor is dependent on the microarchitecture of the plasma membrane: NK1R-mediated signaling was abolished after cholesterol depletion of the receptor-expressing cells with methyl-beta-cyclodextrin. Our results demonstrate that reorganization of the plasma membrane has an effect on the activation of the raft-associated NK1R and the down-stream events such as recruitment of protein kinases.

Myricetin inhibits matrix metalloproteinase 2 protein expression and enzyme activity in colorectal carcinoma cells

Colorectal carcinoma is a leading cause of human mortality due to its high metastatic ability. Because the activation of matrix metalloproteinases (MMP) is a key factor in the metastatic process, agents with the ability to inhibit MMP activity have potential in the treatment of colorectal carcinoma. In the present study, among 36 flavonoids examined, myricetin was found to be the most potent inhibitor of MMP-2 enzyme activity in COLO 205 cells (IC50 = 7.82 micromol/L). Myricetin inhibition of MMP-2 enzyme activity was also found in the human colorectal carcinoma cell lines COLO 320HSR, COLO 320DM, HT 29, and COLO 205-X (IC50 = 11.18, 11.56, 13.25, and 23.51 micromol/L, respectively). In contrast, no inhibitory effect of MMP-2 protein expression or enzyme activity was observed in myricitrin (myricetin-3-rhamnoside)-treated cells. In 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated COLO 205 cells, an increase in MMP-2 protein expression and enzyme activity, as well as of protein kinase C (PKC) alpha protein translocation, extracellular signal-regulated kinase (ERK) 1/2 protein phosphorylation, and c-Jun protein expression was observed. ERK inhibitor (PD98059) and PKC inhibitors (GF-109203X and H-7), but not p38 inhibitor (SB203580) or c-jun-NH2-kinase inhibitor (SP600125), significantly inhibited TPA-induced MMP-2 protein expression, with reduced ERK phosphorylation and c-Jun protein expression. Addition of myricetin but not myricitrin suppressed TPA-induced MMP-2 protein expression in COLO 205 cells by blocking the TPA-induced events, including translocation of PKCalpha from cytosol to membrane, phosphorylation of ERK1/2 protein, and induction of c-Jun protein expression. Addition of PD98059 or GF-109203X significantly enhanced the inhibitory effect of myricetin on MMP-2 enzyme activity induced by TPA. Furthermore, myricetin, but not myricitrin, suppressed TPA-induced invasion of COLO 205 cells in an in vitro invasion assay using Engelbreth-Holm-Swarm sarcoma tumor extract Matrigel-coated Transwells. Results of the present study indicate that myricetin significantly blocked both endogenous and TPA-induced MMP-2 enzyme activity by inhibiting its protein expression and enzyme activity. The blockade involved suppression of PKC translocation, ERK phosphorylation, and c-Jun protein expression.

Trypsin stimulates the phosphorylation of p42,44 mitogen-activated protein kinases via the proteinase-activated receptor-2 and protein kinase C epsilon in human cultured prostate stromal cells

The pathogenesis of benign prostatic hyperplasia (BPH) is not well understood. It involves the proliferation of prostate stromal cells. The proteinase-activated receptor subtype 2 (PAR-2) receptor is expressed by human prostate tissue and can be stimulated by serine proteases. Prostate epithelial cells secrete serine proteases such as trypsin, prostate specific antigen (PSA), and human glandular kallikrein (hK2). The p42,44 mitogen activated protein kinase (MAP kinase) pathway regulates cell proliferation. Trypsin can stimulate this pathway via the PAR-2 receptor and protein kinase C (PKC) in other tissues. Serine proteases secreted by prostate epithelial cells may interact with PAR-2 receptors expressed by prostate stromal cells causing them to proliferate. The aim of the present study was to establish whether functional PAR-2 receptors are expressed by human prostate stromal cells (HPSCs) and to determine whether PAR-2 stimulation can activate p42,44 MAP kinase via a pathway involving PKC. HPSCs were cultured from patients undergoing trans urethral resection of the prostate (TURP). HPSCs were stimulated with PAR agonists. Immunoblotting of HPSC lysate with anti-p42,44 MAP kinase and -PKC isoforms. Data were analyzed with densitometry. Trypsin and the PAR-2 synthetic peptide SLIGKV caused significant increases in MAP kinase phosphorylation and calcium mobilization in HPSCs. The MAP kinase response was attenuated by pertussis toxin (PTX), phorbol 12,13 dibutyrate, Go6983, and Ro 318220. The PKC isoforms alpha, delta, epsilon, and zeta were detected in HPSCs. Trypsin caused the translocation of PKC(epsilon) from the cytosol to the membrane in HPSCs and was able to stimulate cellular proliferation. The PAR-2 selective serine protease trypsin activates p42,44 MAP kinase phosphorylation via PKC(epsilon). This may be an important mechanism of BPH pathophysiology.

Selective Inhibition of Juxtanuclear Translocation of Protein Kinase C βII by a Negative Feedback Mechanism Involving Ceramide Formed from the Salvage Pathway

In a previous study, we showed that protein kinase C betaII (PKC betaII) translocated to a novel juxtanuclear compartment as observed in several cell types (Becker, K. P., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 52747-52754). In this study, we noted the absence of this translocation in MCF-7 breast cancer cells, and we examined the mechanisms underlying this selectivity of response. We show that sustained stimulation of PKC betaII with 4beta-phorbol 12-myristate 13-acetate (PMA) resulted in accumulation of ceramide in MCF-7 cells but not in those cells that showed juxtanuclear translocation of PKC betaII. Addition of exogenous ceramides or formation of endogenous ceramide by the action of bacterial sphingomyelinase prevented PMA-induced translocation of PKC betaII in HEK 293 cells. On the other hand, inhibition of ceramide accumulation with fumonisin B1 restored the ability of PMA to induce translocation of PKC betaII in MCF-7 cells. Taken together, the results showed that endogenous ceramide is both necessary and sufficient for preventing juxtanuclear translocation of PKC betaII in response to PMA. Investigation of the mechanisms of ceramide generation in response to PMA revealed that PMA activated the salvage pathway of ceramide formation and not the de novo pathway. This conclusion was based on the following: 1) the ability of fumonisin B1 but not myriocin to inhibit ceramide formation, 2) the ability of PMA to induce increases in palmitate-labeled ceramide only under chase labeling but not acute pulse labeling, 3) the induction of the levels of sphingosine but not dihydrosphingosine in response to PMA, and 4) induction of sphingomyelin hydrolysis in response to PMA. Together, these results define a novel pathway of regulated formation of ceramide, the salvage pathway, and they define a role for this pathway in regulating juxtanuclear translocation of PKC betaII.

Docosahexaenoic acid inhibits protein kinase C translocation/activation and cardiac hypertrophy in rat cardiomyocytes

Phenylephrine (PE) induces cardiac hypertrophy through multiple signaling pathways including pathways involving protein kinase C (PKC) activation. Docosahexaenoic acid (DHA), an omega-3 fatty acid, has been shown to reduce the PE-induced hypertrophic responses. However, the effects of DHA on PKC activation and translocation are controversial. The present study investigates the effect of DHA on PE-induced activation of PKC. The results indicate that PE induces PKCalpha translocation (from cytosol to plasma membranes) and activation in cardiomyocytes during the hypertrophic responses. Although DHA itself has no significant effect on basal PKC translocation and activation, it effectively reduced PE-stimulated PKC translocation and activation. The results of the present study suggest a possible mechanism explaining how dietary fish oil may inhibit development of cardiac hypertrophy and therefore may be an attractive dietary agent for preventing cardiac hypertrophy in patients with heart failure.

Modulation of Muscarinic K+ Channel by Protein Kinase C in Ischemic Rat Atrial Myocytes

Background and Objectives：Recent studies have shown that many kinds of K + channels, including the muscarinic K + channel (KACh), are activated in the ischemic heart. It is known that these channels can be modulated by phosphorylation. However, little is known about the function of the KACh in ischemic hearts. In this study, we examined whether the KACh channel is mediated by protein kinase C (PKC) activation in rat atrial myocytes under ischemic conditions. Materials and Methods：Single atrial cells of adult rat heart were prepared by collagenase digestion. Channel activity of KACh was recorded by cell-attached configuration from single atrial cells under ischemic conditions, using a patch clamp technique. To simulate ischemia, adenosine or potassium cyanide (KCN) was applied to atrial myocytes, and Western blot was performed to specify PKC isoforms. Results：Adenosine and KCN markedly increased KACh channel activity. The responses to adenosine and KCN were increased 3-fold at mean open time from that observed with control. Channel activity of KACh was blocked by pretreatment with PKC antagonists such as sphingosine, Go 6976, and rottlerin. PKC α and PKC β I isoform levels were increased in the membrane fraction of ischemic heart, indicating that ischemic stress might trigger translocation of cytosolic PKC to the cell membrane. Conclusion：These results show that KACh channels are modulated by PKC activation under ischemic conditions induced by adenosine or KCN. Therefore, the channels can protect the heart from ischemic stress by increasing channel activity. (Korean Circulation J 2005;35:812-820)

Role of phorbol ester localization in determining protein kinase C or RasGRP3 translocation: Real-time analysis using fluorescent ligands and proteins

The diacylglycerol signaling pathway, involving protein kinase C (PKC) and RasGRP, is a promising therapeutic target for both cancer and other indications. The phorbol esters, ultrapotent diacylglycerol analogues, bind to and activate PKC and RasGRP. Here, using fluorescent phorbol esters and complementary fluorescent PKC and RasGRP constructs, we determined the localization of the phorbol ester as a function of time after addition and how the resultant PKC or RasGRP3 translocation related to ligand localization. For these studies, we prepared fluorescently labeled phorbol esters of varying lipophilicities based on the BODIPY FL (green) or BODIPY 581/591 (red) fluorophores, and by using fusion constructs of green fluorescent protein or DsRed with PKC isoforms or RasGRP3 expressed in Chinese hamster ovary cells, we simultaneously compared the kinetics and pattern of localization of PKC or RasGRP3 with that of the fluorescent red or green phorbol esters. Binding assays showed that the fluorescent derivatives were potent ligands. Uptake followed a one-compartment pharmacokinetic model with a half-time of minutes to hours, depending on the ligand, and all of the fluorescent phorbol esters localized primarily to intracellular membranes, with little plasma membrane localization. The fluorescent phorbol esters induced translocation of and generally colocalized with PKCdelta or RasGRP3. However, PKCalpha and, initially, PKCdelta, translocated to the plasma membrane, in which little phorbol ester accumulated. The findings argue that the rate of uptake of phorbol esters influences the subsequent pattern of PKCdelta translocation, and that the specificity for PKCalpha translocation is dominated by factors other than the localization of the ligand.

Lithium and valproic acid treatments reduce PKC activation and receptor-G protein coupling in platelets of bipolar manic patients

Dysregulated protein kinase C (PKC) distribution and activation, and abnormal receptor-G protein coupling, have been implicated in the pathophysiology of bipolar affective disorder (BD). The therapeutic effectiveness of lithium has also been correlated with its ability to reduce PKC activation and G protein-mediated signaling. We examine the cellular distribution and activation of PKC and receptor-G protein coupling in blood platelets from normal controls, patients with BD mania or schizophrenia during treatment-free state, and after lithium or valproic acid administration. PKC activity was measured under basal and 50 nM phorbol 12-myristate, 13-acetate (PMA), 1 μM serotonin or 0.5 U/ml thrombin-stimulated conditions. The coupling of G proteins to serotonin or thrombin receptors were assessed by serotonin or thrombin-mediated [ 35S]GTPγS binding to membrane Gα proteins. The results demonstrate that membrane-associated PKC activity and stimulus-induced PKC translocation are increased in BD manic, whereas stimulus-elicited PKC translocation is attenuated in schizophrenic patients. Lithium and valproic acid treatments attenuated the stimulus-induced PKC translocations to a similar degree and decreased PKC activity in both cytosolic and membranous fractions after two weeks of drug administration. An increase in 5-HT or thrombin stimulated [ 35S]GTPγS binding to Gα proteins was detected in BD manic but not in schizophrenic patients although basal [ 35S]GTPγS binding was not different across the diagnostic groups. Lithium and valproic acid treatments similarly reduced receptor-G protein coupling with comparable time courses. Thus, increased membrane-associated PKC, cytosol to membrane PKC translocation and receptor-G protein coupling in platelets of BD manic patients were alleviated by lithium or valproic acid treatments.

Selective translocation of protein kinase c isozymes by PAF in rabbit platelets

The action of platelet activating factor (PAF) on subcellular distribution and activity of protein kinase C (PKC) isoforms in rabbit platelets was analyzed. The results showed an increase of PKC α in membrane fraction, concomitantly with a decrease in cytosolic fraction after 5 min PAF treatment, indicating that a translocation of PKC α occurred. In addition, PKC ζ was redistributed in a “reverse” form, from the membrane to cytosolic fraction after PAF treatment. PAF induced an increase of PKC α activity, whereas a decrease rather than increase in PKC ζ was observed by using immunoprecipitation assays. In addition, some results indicated that PI3 kinase activation was not involved in PAF-induced PKC ζ translocation as occur in several cells and with other agonists. These actions were time- and concentration-dependent, and were inhibited by the treatment with a PAF antagonist. No translocation was observed when the platelets were incubated with lysoPAF, a PAF related compound.The redistribution of PKC isoforms take place through the activation of high specificity PAF binding sites. The pretreatment of the rabbit platelets with staurosporine, a putative inhibitor of PKC, completely blocked the PAF-evoked aggregation without affecting to PAF-evoked shape change and serotonin release. All together, these data could suggest that the specific translocation of PKC isoforms play an important role in the activation of rabbit platelets.

Protein kinase C distribution and translocation in rat myocardium: Methodological considerations

Introduction: Protein kinase C (PKC) is an important modifier of several cardiovascular phenomena, including cardioprotection, apoptosis, and hypertrophy. Although pharmacological activation of PKC is often assessed by translocation, the effects of isolation procedures on left ventricular (LV) PKC distribution have not been systematically examined. Accordingly, we sought to determine whether homogenization methods (Polytron, glass–glass tissue grinder), detergent selection and concentration, or centrifugation protocols affect PKC (α, ɛ) distribution or phorbol-12-myristate-13-acetate (PMA)-induced translocation. Methods: Hearts of male F344 or Wistar rats were Langendorff perfused with either 100 nM PMA or vehicle, and LV cytosolic and particulate PKC (α, ɛ) distributions were assessed by differential centrifugation and Western blotting. Results: Following 100 000 × g centrifugation of the homogenate, resuspension of the pellet (P 1) in 0.1% sodium dodecyl sulfate (SDS) increased electrophoretic mobility of PKC (α, ɛ) such that PKCɛ comigrated with a nonspecific band. Resuspension of P 1 in Triton X-100 (TX) did not affect mobility but decreased P 1 PKC (α, ɛ) levels in a TX-concentration-dependent manner; however, this decrease was found to be due to differential protein solubilization. Decreased levels of PKC (α, ɛ) were also noted in soluble and P 2 (supernatant of 100 000 × g centrifugation of P 1) fractions due to increased Polytron burst and total homogenization times. Interestingly, the P 2 fraction also revealed Polytron-dependent decreases (47% vs. glass–glass tissue grinder; p<0.05) in PKCɛ following an initial 1000 × g centrifugation and an increased PMA-dependent translocation of PKC (α, ɛ; 2.4-fold and 1.6-fold, respectively, vs. P 1; p<0.05). Discussion: Taken together, these results suggest that PKC isolation procedures should be carefully considered when designing or comparing LV PKC studies due to the potential effects isolation may have on PKC distribution and translocation.

Effects of hyperglycemia and hypercapnia on lipid metabolism during complete brain ischemia

Ischemic damage is greatly enhanced by preischemic hyperglycemia or hypercapnia, which affects many intracellular responses including protein kinase C (PKC) translocation. We explored whether hyperglycemic or hypercapnic ischemia affects lipid metabolism, especially ischemia-induced release of free fatty acids (FFAs) and diacylglycerols (DAGs). A change in intraischemic level of acidosis was induced either by injecting glucose (hyperglycemic, HG) or by adding CO 2 (hypercapnic, HC). Complete cerebral ischemia was induced, and the brain was frozen in situ after 3, 5, and 10 min at 37 °C. Frontoparietal neocortex was dissected for FFA and DAG lipid analysis by thin-layer chromatography and gas–liquid chromatography. Significant differences were shown between normoglycemic and either hypercapnic or hyperglycemic values for individual and total FFAs. A significant delay in the release of FFA in ischemia with hyperglycemia or hypercapnia was observed. Significant differences were also shown in individual DAG-acyl groups and total DAGs. Hyperglycemic or hypercapnic ischemia resulted in a significant decrease of DAG at 10 min of ischemia. This was unexpected because a previous study showed that PKC translocation was significantly enhanced under similar condition at this time point. Upon cellular depolarization, massive influx of calcium and FFA accumulation may decrease the PKC dependence of DAG for translocation. In addition, PKC activation may lead to a negative feedback inhibition of phospholipase C.