The muscarinic M1 agonist xanomeline increases soluble amyloid precursor protein release from Chinese hamster ovary-m1 cells

The functionally selective M1 agonist xanomeline, which is currently undergoing clinical trials as a therapy for Alzheimer's disease, was compared to the muscarinic agonist carbachol for effects on secretion of soluble amyloid precursor protein (APPs) from Chinese hamster ovary cells transfected with the human m1 receptor (CHO-m1). Release of APPs from CHO-m1 cells was increased maximally (4-10 fold) by 100 microM carbachol (EC50 = 11 microM) and by 100 nM xanomeline (EC50 = 10 nM). Stimulation of APPs secretion by xanomeline and carbachol was blocked by preincubation with 1 microM atropine. Carbachol did not stimulate APPs secretion from non-transfected CHO cells. Pilocarpine at 1 mM also increased APPs release. The efficacy of carbachol, xanomeline and pilocarpine for stimulating APPs secretion did not differ significantly. Activation of protein kinase C (PKC) in m1 transfected cell lines by 1 microM phorbol dibutyrate (PDBu) increased APPs release, and this was inhibited 97% by the PKC inhibitor bisindolemalemide. The PKC inhibitor decreased xanomeline and carbachol-stimulated APPs secretion by only 25-30%. These results demonstrate that xanomeline increased APPs release by activation of m1 muscarinic receptors and support the possibility that cholinergic replacement therapy for Alzheimer's Disease may reduce amyloid deposition.     

Regulation of H1-receptor coupling and H1-receptor mRNA by histamine in bovine tracheal smooth muscle

1. Pretreatment of bovine tracheal smooth muscle (BTSM) with histamine (1-100 microM, 1 h) induced a concentration-dependent desensitization of the contractile response to subsequently administered histamine, with a reduction of the maximum response of 72 +/- 8% (n = 5) following pre-exposure to 100 microM histamine. In contrast, concentration-response curves to the muscarinic agonist, methacholine were not affected following histamine pretreatment, indicating a homologous desensitization. Furthermore, concentration-response curves to NaF, a G-protein activator, were not altered following histamine pre-incubation. 2. The histamine H1-receptor (H1R) desensitization could be antagonized by mepyramine (an H1-receptor antagonist, 1 microM) but not by cimetidine (an H2-receptor antagonist, 10 microM), indicating that the desensitization occurred via stimulation of histamine H1-receptors, without evidence for the involvement of histamine H2-receptors. 3. Indomethacin (10 microM) did not block the H1R desensitization, suggesting no involvement of prostaglandins. Furthermore, histamine pre-incubation in calcium free medium still induced a functional uncoupling of H1R. 4. GF 109203X, a protein kinase C (PKC) inhibitor, and H-7, a non-selective kinase inhibitor, did not antagonize the homologous H1R desensitization. 5. The steady-state level of H1R mRNA, assessed by Northern blot analysis, was not affected by prolonged histamine exposure (100 microM, 0.5, 1, 2, 4, 16 and 24 h). 6. These results suggest that histamine induces desensitization of the H1R at the level of the receptor protein, which involves a mechanism independent of PKC, PKA, PKG and calcium influx, suggesting the involvement of a receptor-specific kinase.     

Protein phosphorylation at a postreceptor site can block desensitization and induce potentiation of secretion in chromaffin cells

Desensitization or habituation to repeated or prolonged stimulation is a common property of secretory cells. Phosphorylation of receptors mediates some desensitization processes, but the relationship of phosphorylation to desensitization at postreceptor sites is not well understood. We have tested the effect of protein phosphorylation on desensitization in bovine chromaffin cells. To increase protein phosphorylation, we have used the protein phosphatase inhibitor okadaic acid at 12.5 nM, 100 microM 8-bromo-cyclic AMP to activate protein kinase A, and 10 nM phorbol 12,13-dibutyrate to activate protein kinase C. During repeated 6-s stimulation at 5-min intervals, catecholamine secretion from control cells decreases. Cells exposed to 8-bromo-cyclic AMP or okadaic acid alone show slightly decreased rates of desensitization. In cells pretreated with phorbol 12,13-dibutyrate, desensitization is blocked. Okadaic acid-treated cells stimulated in the presence of 8-bromo-cyclic AMP show potentiation of secretion with repeated stimulation. The protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) increases the desensitization rate. Because these phenomena are observed during secretion evoked with elevated K+ as well as by a nicotinic agonist, the effect of phosphorylation is at a postreceptor site. In contrast to desensitization to the repeated stimulations, desensitization to prolonged stimulation with high K+ is not altered by the above protocols in chromaffin cells.     

Norepinephrine stimulates mitogen-activated protein kinase activity in GT1-1 gonadotropin-releasing hormone neuronal cell lines

The GT1-1 GnRH neuronal cell lines exhibit highly differentiated properties of GnRH neurons. We have used GT1-1 cells to study the roles of norepinephrine (NE), membrane depolarization, calcium influx, and phorbol esters in the regulation of mitogen-activated protein (MAP) kinase. NE, which is known to stimulate the release of GnRH, induced MAP kinase activity, the tyrosine phosphorylation of MAP kinase, and MAP kinase kinase activity. Forskolin led to activation of MAP kinase comparable with that induced by NE, and a selective inhibitor of cAMP-dependent protein kinase, H8, attenuated the NE-induced activation of MAP kinase. On the other hand, elimination of extracellular calcium by EGTA completely blocked NE-induced tyrosine phosphorylation of MAP kinase, and a selective inhibitor of calcium/calmodulin-dependent protein kinase, KN-62, attenuated the NE-induced activation of MAP kinase. Furthermore, depolarization of GT1-1 cells with 75 mM KCl, 10 microM BayK 8644, or 1 microM calcium ionophore (A23187) induced rapid tyrosine phosphorylation of MAP kinase. The omission of calcium from the extracellular medium completely abolished these effects of tyrosine phosphorylation of MAP kinase. Phorbol 12-myristate 13-acetate (PMA) also induced MAP kinase activity, but pretreatment of the cultured cells with PMA to down-regulate protein kinase C did not abolish the activation of MAP kinase by NE. In addition, although phosphorylation of Raf-1 kinase was stimulated by PMA, this phosphorylation was not induced by either NE or A23187. These results demonstrate that NE activates MAP kinase directly in GT1-1 cells, and that the effect of NE is mediated by increase in the cAMP level and by calcium influx, but not by PMA-sensitive protein kinase C or Raf-1 kinase.     

Negative regulation of MAP kinase by diacylglycerol-dependent mechanisms via G protein-coupled receptors in rat basophilic RBL-2H3 (ml) cells

Carbachol and 5'-(N-ethylcarboxamido)-adenosine (NECA), stimulants of G protein-coupled receptors, induce MAP kinase activation in the muscarinic ml receptor-transfected mast cell line, RBL-2H3 (ml) cells. The phospholipase C inhibitor neomycin and the phosphatidate phosphohydrolase inhibitor propranolol augmented MAP kinase activation induced by carbachol and NECA without affecting the antigen-induced MAP kinase activation. Furthermore, the duration of MAP kinase activation induced by carbachol or NECA was also prolonged by neomycin and propranolol. The specific protein kinase C inhibitor Ro 31-8425 enhanced the carbachol- or NECA-induced MAP kinase activation. These findings suggest that the MAP kinase activation mediated by the G protein-coupled receptors is negatively regulated by diacylglycerol and activated protein kinase C(s).     

Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells

Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.     

Characterization of [3H]staurosporine binding in protein kinase C-II purified from rat brain

The type II protein kinase C (PKC-II) densely present in mammalian brain plays functional roles in CNS. We examined the characteristics of [3H]staurosporine binding to PKC-II purified from rat brain, compared to [3H]phorbol 12, 13-dibutyrate (PDBu) binding. In brief, [3H]staurosporine binding increased by phosphatidylserine (PtdSer) in a concentration-dependent manner and the binding was enhanced by Ca2+ and phorbol 12-myristate 13-acetate (PMA). In the presence of Ca2+, PMA and PtdSer, Bmax of these bindings markedly increased, but KD did not change. These characteristics of binding were similar to [3H]PDBu binding to PKC-II. Although [3H]PDBu binding was not affected by protein kinase inhibitors such as staurosporine, H-7, K-252a and K-252b, [3H]staurosporine binding was inhibited by these inhibitors. [3H]staurosporine binding was inhibited by several ATP analogues, but was not by guanine nucleotides. PtdSer-induced increase in [3H]PDBu binding was inhibited by Zn2+, but Zn2+ induced increase in [3H]staurosporine binding as well as PtdSer and/or Ca2+. Staurosporine would thus appear to bind to a domain different from phorbol ester-binding one in PKC, interactions between both domains may regulate kinase activity, and 1 mol staurosporine and 4 mol phorbol ester may bind to 1 mol PKC-II.     

Distribution and translocation of isoforms of protein kinase C in rat submandibular acinar cells

The distribution of six isoforms of protein kinase C (PKC) in seromucous acinar cells of rat submandibular gland was examined and their translocation from the cytosolic- to the membrane fraction after different stimuli investigated. Western blotting, immunostaining with isoform-specific antibodies and scanning densitometry showed that PKC-alpha and epsilon were distributed fairly evenly between the cytosol and membranes in resting cells, while isoforms- beta, delta and zeta were all predominantly localized (over 80%) in membranes. PKC-gamma was not detected. PKC-alpha was mobilized to the membrane fraction by the phorbol ester, TPA, but not by the phosphoinositide-coupled agonists carbachol, methoxamine and substance P (SP). PKC-epsilon was translocated by TPA and carbachol but not by SP or methoxamine. Biochemical assay of total PKC confirmed that cytosolic enzyme activity was significantly reduced by TPA and carbachol to 29% and 75% respectively of control levels. These results suggest that muscarinic regulation of the mucosecretory response in the rat submandibular gland may be mediated by the PKC-epsilon isoform.     

HA1077, a protein kinase inhibitor, inhibits calponin phosphorylation on Ser175 in porcine coronary artery

Calponin is a thin filament-associated protein which has been implicated in the modulation of the contractile state of smooth muscle via its interaction with actin and inhibition of the actin-activated myosin Mg-ATPase. This inhibitory effect is alleviated by phosphorylation of calponin at Ser175 in vitro by protein kinase C. The issue of calponin phosphorylation in intact smooth muscle in response to agonists that activate protein kinase C is controversial. We have produced a monoclonal antibody that specifically recognizes calponin phosphorylated at Ser175 and used it to analyze calponin phosphorylation in porcine coronary arterial smooth muscle stimulated with prostaglandin F2alpha or phorbol 12,13-dibutylate (PDB). Calponin phosphorylation increased rapidly in response to prostaglandin F2alpha concomitant with the increase in tension. Calponin was then dephosphorylated while force was maintained. Tension development in response to PDB was significantly slower, but again calponin phosphorylation paralleled force development. In this case, calponin dephosphorylation was very slow, consistent with prolonged activation of protein kinase C. The protein kinase inhibitors, HA1077 (1-5-(isoquinoline sulfonyl)-homopiperazine HCl) and HA1100 (1-hydroxy HA1077; 1-(hydroxy-5-isoquinoline sulfonyl-homopiperazine), inhibited tension development and calponin phosphorylation in a concentration-dependent manner with similar ED50 values in response to prostaglandin F2alpha and PDB. These results support physiological roles for calponin in force development in smooth muscle in response to agonists which trigger protein kinase C activation and in the latch state, i.e., force maintenance at low energy cost. Furthermore, the vasodilator effect of HA1077 and HA1100 is more likely due to inhibition of protein kinase C than of myosin light chain kinase.     

Desensitization of muscarinic receptor-coupled inositol phospholipid hydrolysis in human detrusor cultured smooth cells

PURPOSE: The aim of this study was to investigate the desensitization characteristics of muscarinic M3 receptors in primary cultures of human detrusor smooth muscle cells. MATERIALS AND METHODS: Cell cultures were prepared from cold cup pinch biopsies of the human detrusor muscles by explant culture methods. Accumulation of (3)H-inositol phosphates was measured on confluent monolayers as described previously in detail. Desensitization was achieved by preincubating the cells with carbachol or histamine for 5 to 60 minutes. RESULTS: Carbachol induced a concentration-dependent increase in phosphoinositide turnover in naive cells, the response being rapid and evident after only a 30-second exposure to the agonist. Preincubation of the cells with carbachol produced a concentration-dependent decrease in the inositol phosphate response to a second challenge with carbachol. Preexposure to carbachol for only 5 minutes prior to a rechallenge reduced the mean size of response of the second stimulation to 49% of that observed in naive cells. Preexposure of the cells to histamine did not alter the response of the cells to a subsequent challenge with carbachol and vice versa. CONCLUSIONS: The muscarinic receptors retained by human detrusor smooth muscle cells in culture are susceptible to a desensitization of the carbachol-induced increase phosphoinositide turnover observed in these cells. This desensitization is rapid, and the results indicate that it is homologous and does not occur via a postreceptor mechanism but at the level of the receptor itself.     

Dynamic complexes of beta2-adrenergic receptors with protein kinases and phosphatases and the role of gravin

Signals mediated by G-protein-linked receptors display agonist-induced attenuation and recovery involving both protein kinases and phosphatases. The role of protein kinases and phosphatases in agonist-induced attenuation and recovery of beta-adrenergic receptors was explored by two complementary approaches, antisense RNA suppression and co-immunoprecipitation of target elements. Protein phosphatases 2A and 2B are associated with the unstimulated receptor, the latter displaying a transient decrease followed by a 2-fold increase in the levels of association at 30 min following challenge with agonist. Protein kinase A displays a robust, agonist-induced association with beta-adrenergic receptors over the same period. Suppression of phosphatases 2A and 2B with antisense RNA or inhibition of their activity with calyculin A and FK506, respectively, blocks resensitization following agonist removal. Recycling of receptors to the plasma membrane following agonist-promoted sequestration is severely impaired by loss of either phosphatase 2B or protein kinase C. In addition, loss of protein kinase C diminishes association of phosphatase 2B with beta-adrenergic receptors. Overlay assays performed with the RII subunit of protein kinase A and co-immunoprecipitations reveal proteins of the A kinase-anchoring proteins (AKAP) family, including AKAP250 also known as gravin, associated with the beta-adrenergic receptor. Suppression of gravin expression disrupts recovery from agonist-induced desensitization, confirming the role of gravin in organization of G-protein-linked signaling complexes. The Ht31 peptide, which blocks AKAP protein-protein interactions, blocks association of beta-adrenergic receptors with protein kinase A. These data are the first to reveal dynamic complexes of beta-adrenergic receptors with protein kinases and phosphatases acting via an anchoring protein, gravin.     

Activation of protein kinase C increases neuronal excitability by regulating persistent Na+ current in mouse neocortical slices

Effects of the protein kinase C activating phorbol ester, phorbol 12-myristate 13-acetate (PMA), were studied in whole cell recordings from layer V neurons in slices of mouse somatosensory neocortex. PMA was applied intracellularly (100 nM to 1 microM) to restrict its action to the cell under study. In current-clamp recordings, it enhanced neuronal excitability by inducing a 10- to 20-mV decrease in voltage threshold for action-potential generation. Because spike threshold in neocortical neurons critically depends on the properties of persistent Na+ current (INaP), effects of PMA on this current were studied in voltage clamp. After blocking K+ and Ca2+ currents, INaP was revealed by applying slow depolarizing voltage ramps from -70 to 0 mV. Intracellular PMA induced a decrease in INaP at very depolarized membrane potentials. It also shifted activation of INaP in the hyperpolarizing direction, however, such that there was a significant increase in persistent inward current at potentials more negative than -45 mV. When tetrodotoxin (TTX) was added to the bath, blocking INaP and leaving only an outward nonspecific cationic current (Icat), PMA had no apparent effect on responses to voltage ramps. Thus PMA did not affect Icat, and it did not induce any additional current. Intracellular application of the inactive PMA analogue, 4 alpha-PMA, did not affect INaP. The specific protein kinase C inhibitors, chelerythrine (20 microM) and calphostin C (10 microM), blocked the effect of PMA on INaP. The data suggest that PMA enhances neuronal excitability via a protein kinase C-mediated increase in INaP at functionally critical subthreshold voltages. This novel effect would modulate all neuronal functions that are influenced by INaP, including synaptic integration and active backpropagation of action potential from the soma into the dendrites.     

Modulation of dopamine release from rat striatum by protein kinase C: interaction with presynaptic D2-dopamine-autoreceptors

1. Interactions between dopamine receptors and protein kinase C (PKC) have been proposed from biochemical studies. The aim of the present study was to investigate the hypothesis that there is an interaction between protein kinase C and inhibitory D2-dopamine receptors in the modulation of stimulation-induced (S-I) dopamine release from rat striatal slices incubated with [3H]-dopamine. Dopamine release can be modulated by protein kinase C and inhibitory presynaptic D2 receptors since phorbol dibutyrate (PDB) and (-)-sulpiride, respectively, elevated S-I dopamine release. 2. The protein kinase C inhibitors polymyxin B (21 microM) and chelerythrine (3 microM) had no effect on stimulation-induced (S-I) dopamine release. However, when presynaptic dopamine D2 receptors were blocked by sulpiride (1 microM), an inhibitory effect of both PKC inhibitors on S-I dopamine release was revealed. Thus, sulpiride unmasks an endogenous PKC effect on dopamine release which suggests that presynaptic D2 receptors normally suppress endogenous PKC activity. This is supported by results in striatal slices which were pretreated with PDB to down-regulate PKC. In this case the facilitatory effect of sulpiride was completely abolished. 3. The inhibitory effect of the dopamine D2/D3 agonist quinpirole on S-I dopamine release was partially attenuated by PKC down-regulation. Since the effect of sulpiride was completely abolished under the same conditions, this suggests that exogenous agonists may target a PKC-dependent as well as a PKC-independent pathway. The inhibitory effect of apomorphine was not affected by either polymyxin B or PKC down-regulation, suggesting that it operated exclusively through a PKC-independent mechanism. 4. These results suggest that there are at least two pathways involved in the inhibition of dopamine release through dopamine receptors. One pathway involves dopamine receptor suppression of protein kinase C activity, perhaps through inhibition of phospholipase C activity and this is preferentially utilized by neuronally-released dopamine. The other pathway which seems to be utilized by exogenous agonists does not involve PKC.     

Carbachol stimulates c-fos expression and proliferation in oligodendrocyte progenitors

To determine if muscarinic receptor-activation plays a role in oligodendrocyte development, the effect of carbachol a stable acetylcholine analog, on gene expression and proliferation was investigated. Using Northern blot analysis we showed that carbachol caused a time and concentration-dependent increase in c-fos mRNA. This effect was blocked by atropine, a non-selective muscarinic antagonist. In addition, the muscarinic-stimulated c-fos increase was inhibited by 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C (PKC), but not by N-2-(p-bromocinnamylamino)-ethyl-5-isoquinoline-sulfonamide (H-89), a potent inhibitor of protein kinase A, suggesting the involvement of PKC in mediating the response. Down-regulation of PKC by overnight pre-treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) blocked only the phorbol ester-stimulated c-fos accumulation while no effect was observed in the carbachol-induced response. These results suggested that carbachol stimulated an H-7 sensitive PKC pathway which may be different than that activated by TPA. Further evidence for two separate mechanisms of proto-oncogene induction was provided by the additive effect of carbachol and TPA. Induction of c-fos mRNA by carbachol was dependent on both influx of extracellular Ca2+ and release from intracellular stores, as both EDTA and BAPTA blocked the response. Since activation of muscarinic receptors can affect cell division in other cellular systems, the effect of carbachol on [3H]thymidine and bromodeoxyuridine incorporation into oligodendrocyte DNA was measured. Carbachol stimulated DNA synthesis in oligodendrocyte progenitors. This effect was mediated by muscarinic receptors as [3H]thymidine incorporation was prevented or significantly reduced by the addition of atropine. In conclusion, the present findings suggest that, the neurotransmitter, acetylcholine may act as a trophic factor in developing oligodendrocytes, regulating their growth and development in the central nervous system.     

Role of protein kinase C (PKC) in agonist-induced mu-opioid receptor down-regulation: I. PKC translocation to the membrane of SH-SY5Y neuroblastoma cells is induced by mu-opioid agonists

Phosphorylation of specific amino acid residues is believed to be crucial for the agonist-induced regulation of several G protein-coupled receptors. This is especially true for the three types of opioid receptors (mu, delta, and kappa), which contain consensus sites for phosphorylation by numerous protein kinases. Protein kinase C (PKC) has been shown to catalyze the in vitro phosphorylation of mu- and delta-opioid receptors and to potentiate agonist-induced receptor desensitization. In this series of experiments, we continue our investigation of how opioid-activated PKC contributes to homologous receptor down-regulation and then expand our focus to include the exploration of the mechanism(s) by which mu-opioids produce PKC translocation in SH-SY5Y neuroblastoma cells. [D-Ala2,N-Me-Phe4,Gly-ol]enkephalin (DAMGO)-induced PKC translocation follows a time-dependent and biphasic pattern beginning 2 h after opioid addition, when a pronounced translocation of PKC to the plasma membrane occurs. When opioid exposure is lengthened to >12 h, both cytosolic and particulate PKC levels drop significantly below those of control-treated cells in a process we termed "reverse translocation." The opioid receptor antagonist naloxone, the PKC inhibitor chelerythrine, and the L-type calcium channel antagonist nimodipine attenuated opioid-mediated effects on PKC and mu-receptor down-regulation, suggesting that this is a process partially regulated by Ca2+-dependent PKC isoforms. However, chronic exposure to phorbol ester, which depletes the cells of diacylglycerol (DAG) and Ca2+-sensitive PKC isoforms, before DAMGO exposure, had no effect on opioid receptor down-regulation. In addition to expressing conventional (PKC-alpha) and novel (PKC-epsilon) isoforms, SH-SY5Y cells also contain a DAG- and Ca2+-independent, atypical PKC isozyme (PKC-zeta), which does not decrease in expression after prolonged DAMGO or phorbol ester treatment. This led us to investigate whether PKC-zeta is similarly sensitive to activation by mu-opioids. PKC-zeta translocates from the cytosol to the membrane with kinetics similar to those of PKC-alpha and epsilon in response to DAMGO but does not undergo reverse translocation after longer exposure times. Our evidence suggests that direct PKC activation by mu-opioid agonists is involved in the processes that result in mu-receptor down-regulation in human neuroblastoma cells and that conventional, novel, and atypical PKC isozymes are involved.     

Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages

The modulatory effects of protein kinase C (PKC) on the activation of cytosolic phospholipase A2 (cPLA2) and adenylyl cyclase (AC) have recently been described. Since the signalling cascades associated with these events play critical roles in various functions of macrophages, we set out to investigate the crosstalk between PKC and the cPLA2 and AC pathways in mouse RAW 264.7 macrophages and to determine the involvement of individual PKC isoforms. The cPLA2 and AC pathways were studied by measuring the potentiation by the phorbol ester PMA of ionomycin-induced arachidonic acid (AA) release and prostagladin E1 (PGE1)-stimulated cyclic AMP production, respectively. PMA at 1 microM caused a significant increase in AA release both in the presence (371%) and absence (67%) of ionomycin induction, while exposure of RAW 264.7 cells to PMA increased PGE1 stimulation of cyclic AMP levels by 208%. Treatment of cells with staurosporine and Ro 31-8220 inhibited the PMA-induced potentiation of both AA release and cyclic AMP accumulation, while Go 6976 (an inhibitor of classical PKC isoforms) and LY 379196 (a specific inhibitor of PKCbeta) inhibited the AA response but failed to affect the enhancement of the cyclic AMP response by PMA. Long term pretreatment of cells with PMA abolished the subsequent effect of PMA in potentiating AA release, but only inhibited the cyclic AMP response by 42%. Neither PD 98059, an inhibitor of MEK, nor genistein, an inhibitor of tyrosine kinases, had any effect on the ability of PMA to potentiate AA or cyclic AMP production. The potentiation of AA release, but not of cyclic AMP formation, by PMA was sensitive to inhibition by wortmannin. This effect was unrelated to the inhibition of PKC activation as deduced from the translocation of PKC activity to the cell membrane. Western blot analysis revealed the presence of eight PKC isoforms (alpha, betaI, betaII, delta, epsilon, mu, lambda and xi) in RAW 264.7 cells and PMA was shown to induce the translocation of the alpha, betaI, betaII, delta, epsilon and mu isoforms from the cytosol to the cell membrane within 2 min. Pretreatment of cells with PMA for 2-24 h resulted in a time-dependent down-regulation of PKCalpha, betaI, betaII, and delta expression, while the levels of the other four PKC isozymes were unchanged after PMA treatment for 24 h. A decrease in the potentiation of AA release by PMA was observed, concomitant with the time-dependent down-regulation of PKC. These results indicate that PKCbeta has a crucial role in the mediation of cPLA2 activation by the phorbol ester PMA, whereas PMA utilizes PKC epsilon and/or mu to up-regulate AC activity.     

Tissue equivalent vessel phantoms for intravascular ultrasound

The presence of the non-selective protein kinase C (PKC) inhibitors, staurosporine (100 nM) and polymyxin B (100 microM) in cultured human RPE cells for more than 24 h triggers apoptotic death. Apoptosis is characterized by a diminishing number of cells, a labelling of nuclei by the TUNEL method and by observable morphological changes. An inhibitor of PKC and cyclic nucleotide-dependent protein kinases, 1-(5-isoquinolinesulphonyl)-2-methyl piperazine (H-7; 100 microM), was without effect, as was the specific PKC inhibitor, calphostin C (100 nM). The PKC-activating phorbol esters, phorbol-12-myristate-13-acetate (PMA; 1 microM) and phorbol-12,13-dibutyrate (PDB; 1 microM) and the non-tumour-promoting phorbol ester, 4 alpha-PMA (1 microM) were without effect, as was the diacyl glycerol analogue, 1,2-dioctanoyl-snglycerol (DOG; 10 microM). The PKC activators did not attenuate the apoptosis induced by staurosporine or polymyxin B. Furthermore, deprivation of glucose and oxygen (simulated ischemia) for 72 h induced apoptosis: this could be prevented by inclusion of 10% (v/v) foetal bovine serum (FBS) but not by a variety of PKC activators. Six PKC isoenzymes were shown to be present in RPE cells (alpha, beta 1, beta 2, delta, epsilon, E) and only the calcium-dependent cPKC levels changed after treatment with staurosporine or simulated ischaemia. Since only the less selective inhibitors of PKC induced apoptosis, it is suggested that PKC is not involved directly in the induction process of apoptosis in RPE cells. It is possible that the staurosporine and polymyxin B-induced effects of apoptosis in RPE cells are triggered by an unknown kinase-dependent pathway, but whether the 'ischaemia'-induced death is related to this same process remains to be elucidated.     

Atypical protein kinase C isozyme zeta mediates carbachol-stimulated insulin secretion in RINm5F cells

Carbachol-stimulated insulin release in the RINm5F cell is associated with elevation of the cytosolic Ca2+ concentration ([Ca2+]i) through mobilization of Ca2+ from thapsigargin-sensitive intracellular stores and with the generation of diacylglycerol (DAG). Thus carbachol activates phospholipase C, and this was thought to be the means by which it stimulates insulin secretion. However, when the elevation of [Ca2+]i was blocked by thapsigargin, the effect of carbachol to stimulate insulin release was unchanged. Thus the effect of carbachol to increase [Ca2+]i was dissociated from the stimulation of release. When the role of protein kinase C (PKC) was examined, carbachol-stimulated insulin release was found to be unaffected by phorbol ester-induced downregulation of PKC, using 12-O-tetradecanoylphorbol-13-acetate (TPA), and by the PKC inhibitors staurosporine, bisindolylmaleimide, and 1-O-hexadecyl-2-O-methylglycerol (AMG-C16). These treatments abolished the stimulation of release by TPA. Thus the carbachol activation of PKC appeared also to be dissociated from the stimulation of insulin release. However, when the activation of several different PKC isozymes was studied, an atypical PKC isozyme, zeta, was found to be translocated by carbachol. By Western blotting analysis, carbachol selectively translocated the conventional PKC isozymes alpha and beta (the activation of which is dependent on Ca2+ and DAG) from the cytosol to the membrane. Carbachol also translocated the atypical PKC isozyme zeta, which is insensitive to Ca2+, DAG, and phorbol esters. The PKC inhibitors staurosporine, bisindolylmaleimide, and AMG-C16 blocked the stimulated translocation of PKC-alpha and -beta, but not that of PKC-zeta. Prolonged treatment of the cells with TPA downregulated PKC-alpha and -beta, but not PKC-zeta. Under all these conditions, carbachol-stimulated insulin release was unaffected. However, a pseudosubstrate peptide inhibitor specific for PKC-zeta inhibited the translocation of PKC-zeta and 70% of the carbachol-stimulated insulin secretion. The data indicate that carbachol-stimulated insulin release in RINm5F cells is mediated to a large degree by the activation of the atypical PKC isozyme zeta.