Differential regulation of metabotropic glutamate receptor 5-mediated phosphoinositide hydrolysis and extracellular signal-regulated kinase responses by protein kinase C in cultured astrocytes

The metabotropic glutamate receptor 5 (mGluR5) exhibits a rapid loss of receptor responsiveness to prolonged or repeated agonist exposure. This receptor desensitization has been seen in a variety of native and recombinant systems, and is thought to result from receptor-mediated, protein kinase C (PKC)-dependent phosphorylation of the receptor, uncoupling it from the G protein in a negative feedback regulation. We have investigated the rapid PKC-mediated desensitization of mGluR5 in cortical cultured astrocytes by measuring downstream signals from activation of mGluR5. These include activation of phosphoinositide (PI) hydrolysis, intracellular calcium transients, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation. We present evidence that PKC plays an important role in rapid desensitization of PI hydrolysis and calcium signaling, but not in ERK2 phosphorylation. This differential regulation of mGluR5-mediated responses suggests divergent signaling and regulatory pathways which may be important mechanisms for dynamic integration of signal cascades.     

Comparative studies of phosphoinositide hydrolysis induced by endothelin-related peptides in cultured cerebellar astrocytes, C6-glioma and cerebellar granule cells

Effects of endothelin (ET) homologues (ET-1, 2, 3 and sarafotoxin S6b) and its precursor (big ET-1) on phosphoinositide (PI) turnover were compared in neurally-related cell cultures. All ET-related peptides induced a robust increase of PI turnover in cerebellar astrocytes, C6-glioma and cerebellar granule cells. The rank order of potency in stimulating PI turnover was ET-1 = ET-2 greater than or equal to S6b greater than ET-3 greater than big ET-1 for granule cell neurons, while it was ET-1 greater than or equal to ET-2 greater than or equal to S6b greater than big ET-1 greater than ET-3 for astrocytes and C6-glioma cells. Short-term pretreatment with phorbol dibutyrate (PDBu) attenuated the ET-1-induced PI response in all three types of cultures. However, long-term pretreatment with PDBu attenuated the response in granule cells and C6-gliomas, but enhanced responses to ET and ATP in astrocytes. Long-term exposure of cells to pertussis toxin (PTX) attenuated the PI response to ET in astrocytes and C6-gliomas, but not in granule cells. Thus, phospholipase C-coupled ET receptors are expressed in both neurons and glial cells, but they differ considerably in their pharmacological selectivity and signal transduction mechanisms in stimulating PI hydrolysis.     

Regulation of phosphoinositide hydrolysis in cultured astrocytes by sphingosine and psychosine.

The effects of sphingosine and psychosine on phosphoinositide hydrolysis in primary cultured astrocytes were determined. Exposure to sphingosine produced a dose-dependent stimulation of phosphoinositide hydrolysis requiring the presence of external Ca++ for optimal activity. The addition of 10 microM norepinephrine resulted in a stimulation additional to that with sphingosine. The alpha 1-antagonist prazosin completely inhibited norepinephrine-induced phosphoinositide hydrolysis but had no effect on that produced by sphingosine. Psychosine (108 microM), when co-incubated with sphingosine, produced complete inhibition of sphingosine-induced phosphoinositide hydrolysis at all doses of sphingosine tested (33-668 microM). Likewise, psychosine totally inhibited norepinephrine-induced phosphoinositide hydrolysis. The protein kinase C inhibitor staurosporine (1 microM) had no effect on sphingosine-induced phosphoinositide hydrolysis. These findings suggest that lysosphingolipids such as sphingosine and psychosine may play an important role in the regulation of phosphoinositide turnover in astrocytes by a mechanism dependent on extracellular Ca++ and independent of the alpha 1-adrenergic receptor and protein kinase C.     

Epidermal growth factor activates phosphoinositide turnover and protein kinase C in BALB/MK keratinocytes

BALB/MK is a nontransformed epithelial cell line derived from primary BALB/c mouse keratinocytes that requires epidermal growth factor (EGF) for growth. Using a defined-medium culture system, we investigated the role of physiological concentrations of EGF on phosphoinositide metabolism in these cells. The results show that EGF rapidly activates phospholipase-C mediated phosphoinositide metabolism resulting in the generation of the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. These metabolites control intracellular Ca2+ levels and activate protein kinase C, respectively. Protein kinase C activation in response to EGF was evidenced by the phosphorylation of the acidic 80 kilodalton endogenous protein substrate (p80) specific for this kinase. In contrast, insulin, which acts in concert with EGF to cause BALB/MK cell proliferation, had no effect on phosphoinositide metabolism nor led to any additional stimulation when added in combination with EGF. Taken together, our results show that rapid alterations in phosphoinositide metabolism and protein kinase C activation are associated with the normal mitogenic response of keratinocytes to EGF.     

Nongenomic mechanism of glucocorticoid inhibition of bradykinin-induced calcium influx in PC12 cells: possible involvement of protein kinase C

Many stimulants, including bradykinin (BK), can induce increase in [Ca(2+)](i) in PC12 cells. Bradykinin induces an increase in [Ca(2+)](i) via intracellular Ca(2+) release and extracellular Ca(2+) influx through the transduction of G protein, but not through voltage-sensitive calcium channels. In this experiment, We analyzed how corticosterone (Cort) influences BK-induced intracellular Ca(2+) release and extracellular Ca(2+) influx, and further studied the mechanism of glucocorticoid's action. To dissociate the intracellular Ca(2+) release and extracellular Ca(2+) influx induced by BK, the Ca(2+)-free/Ca(2+)- reintroduction protocol was used. The results were as follows: (1) The Ca(2+) influx induced by BK could be rapidly inhibited by Cort, but intracellular Ca(2+) release could not be affected significantly. (2) The inhibitory effect of Cort-BSA (BSA -conjugated Cort) on Ca(2+) influx induced by BK was the same as the effect of free Cort. (3) Protein kinase C (PKC) activator (phorbol 12-myristate 13-acetate) could mimic and PKC inhibitor G?6976 could reverse the inhibitory effect of Cort. (4) There was no inhibitory effect of Cort on Ca(2+) influx induced by BK when pretreated with pertussis toxin. The results suggested, for the first time, that Cort might act via a putative membrane receptor and inhibit the Ca(2+) influx induced by BK through the pertussis toxin -sensitive G protein-PKC pathway.     

A possible role of protein kinase C in signal-induced lysosomal enzyme release

In platelets, activation of protein kinase C and mobilization of Ca2+ were selectively induced by the addition of 1-oleoyl-2-acetyl-glycerol and a low concentration of A23187, respectively (Kaibuchi, K., Takai, Y., Sawamura, M., Hoshijima, M., Fujikura, T. and Nishizuka, Y. (1983) J. Biol. Chem. 258, 6701-6704). Using this procedure evidence was obtained suggesting that the protein phosphorylation and Ca2+ mobilization were both essential and synergistically effective to cause release of lysosomal acid hydrolases such as N-acetylglucosaminidase. A similar observation was made for the lysosomal enzyme release from rat neutrophils.     

Involvement of protein kinase C in prostaglandin D2 synthesis by cultured astrocytes

The role of protein kinase C (PKC) and calcium in the stimulation of prostaglandin D₂ (PGD₂) synthesis was investigated in primary rat astroglial cultures using the phorbol esters phorbol 12-myristate, 13-acetate (PMA), phorbol 12,13-dibutyrate (PDB) and the calcium ionophore A₂₃₁₈₇. Both phorbol esters and the ionophore were able to stimulate PGD₂ synthesis in a concentration dependent manner. The inactive stereoisomers of PMA and PDB had no significant effect. Combinations of subthreshold concentrations of phorbol esters (10 nM PMA or 10 nM PBD) potentiated PG formation induced by 100 nM A₂₃₁₈₇. An even more pronounced effect was observed when phorbol ester concentrations were increased to 100nM. The contribution of extra- and intracellular calcium in phorbol ester or A₂₃₁₈₇ stimulated PGD₂ synthesis was evaluated by carrying out experiments with calcium-free media plus EGTA or with the intracellular calcium-chelating agent TMB-8. Ionophore stimulated PGD₂ release was shut down to basal values upon removal of extracellular calcium, whereas phorbol ester stimulated PGD₂ formation persisted at a reduced level. It was unabated also upon further addition of EGTA. In the presence of TMB-8, however, phorbol ester stimulated PGD₂ synthesis was completely suppressed. These data strongly suggest that PKC has an additional effect on the activation of phospholipase A₂ and subsequent prostanoid synthesis, which is independent from extracellular calcium and, thus, support the concept of more than one metabolic pathway in astrocytes that synergistically regulate phospholipase A₂ activity.     

Glutamate-induced protein phosphorylation in cerebellar granule cells: Role of protein kinase C

Protein phosphorylation in response to toxic doses of glutamate has been investigated in cerebellar granule cells.³²P-labelled cells have been stimulated with 100 M glutamate for up to 20 min and analysed by one and two dimensional gel electrophoresis. A progressive incorporation of label is observed in two molecular species of about 80 and 43 kDa (PP80 and PP43) and acidic isoelectric point. Glutamate-stimulated phosphorylation is greatly reduced by antagonists of NMDA and non-NMDA glutamate receptors. The effect of glutamate is mimicked by phorbol esters and is markedly reduced by inhibitors of protein kinase C (PKC) such as staurosporine and calphostin C. PP80 has been identified by Western blot analysis as the PKC substrate MARCKS (myristoylated alanine-rich C kinase substrate), while antibody to GAP-43 (growth associated protein-43), the nervous tissue-specific substrate of PKC, failed to recognize PP43. Our results suggest that PKC is responsible for the early phosphorylative events induced by toxic doses of glutamate in cerebellar granule cells.Abbreviations (NMDA) N-methyl-D-aspartate - (PKC) protein kinase C - (EAA) excitatory aminoacids - (GAMSA) -D-glutamylaminomethylsulfonate - (MK801) (+)-10,11-dihydro-5-methyl-5-H-dibenzo-(a,d)-cyclohepten-5,10imine - (TPA) phorbol 12-myristate 13-acetate - (MARCKS) myristoylated alanine-rich C kinase substrate - (GAP-43) growth-associated protein-43 - (SDS) sodium dodecyl sulfate - (PAGE) polyacrylamide gel electrophoresis - (H7) 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - (DIV) days in vitro     

Regulation of protein kinase C by lysophospholipids. Potential role in signal transduction

Certain lysophospholipids, lysophosphatidylcholine (lyso-PC) in particular, stimulated protein kinase C at low concentrations (less than 20 microM) but, conversely, inhibited it at high concentrations (greater than 30 microM). Protein kinase C stimulation by lyso-PC required the presence of phosphatidylserine (PS) and Ca2+ and was associated with a decreased Ka for PS and increased Ka for Ca2+ of the enzyme. Cardiolipin and phosphatidic acid could partially substitute for PS in supporting the stimulatory effect of lyso-PC. Lyso-PC also biphasically regulated protein kinase C activated by diolein. Of several synthetic lyso-PC preparations tested, the oleoyl, myristoyl and palmitoyl derivatives were most active. Data from the Triton X-100 mixed micellar assay indicated that 1.4 and 14.0 mol of lyso-PC/micelle produced a maximal stimulation and a complete abolishment of the stimulation of protein kinase C, respectively. Protein kinase C stimulation by lyso-PC, with a pH optimum of about 7.5, was observed for phosphorylation of histone H1, myelin basic protein, and the 35- and 47-kDa proteins from the rat brain, but not for that of other histone subfractions and protamine. Lyso-PC acted synergistically with diacylglycerol in stimulating protein kinase C, whereas the stimulation by lyso-PC was additive to that by oleic acid. Protein kinase C inhibitors (alkyllysophospholipid, sphingosine, tamoxifen, and polymyxin B) inhibited more potently the protein kinase C activity stimulated by PS/Ca2+/lyso-PC than that stimulated by PS/Ca2+. The stimulatory and inhibitory effects of lyso-PC were not observed for myosin light chain kinase and cAMP-dependent protein kinase, indicating a specificity of its actions. The present findings suggested that lyso-PC, likely derived from membrane PC by the action of phospholipase A2, might play a role in signal transduction via a dual regulation of protein kinase C, and that it could further modulate the enzyme and hence the cellular activity by interplaying with diacylglycerol and unsaturated fatty acid, the two other classes of cellular mediators also shown to be activators of protein kinase C.     

Regulation of neutrophil apoptosis: A role for protein kinase C and phosphatidylinositol-3-kinase

Neutrophils play a central role in host defense and are recruited in vast numbers to sites of infection where they phagocytose and kill invading bacterial pathogens. Neutrophils have a short half-life that is extended at the inflamed site by pro-inflammatory cytokines and contact with bacterial cell walls. Normal resolution of inflammation involves the removal of neutrophils and other inflammatory cells by the induction of apoptosis. Spontaneous neutrophil apoptosis does not require Fas ligation, but is mediated by caspases 3, 8 and possibly caspase 9 and also involves activation of protein kinase C-. With chronic inflammatory disease, neutrophil apoptosis is delayed by pro-inflammatory cytokines, leading to persistence of neutrophils at the inflamed site and non-specific tissue damage. Here we discuss the evidence for inhibition of neutrophil apoptosis via signaling though PI-3-kinase and downstream pathways, including PDK-1 and PKB. Therapeutic strategies to resolve chronic inflammation could therefore usefully target neutrophil apoptosis and the PI-3-kinase or PKC- signaling pathways.     

Oxidized low density lipoprotein inhibits bradykinin-induced phosphoinositide hydrolysis in cultured bovine aortic endothelial cells.

Vascular endothelial cells, in response to various neurohumoral and physical stimuli, produce an endothelium-derived relaxing factor, a substance which regulates vascular tone. We have demonstrated that oxidized low density lipoprotein (LDL) inhibits endothelium-dependent relaxation. We studied the effect of oxidized LDL on inositol phosphates formation stimulated with bradykinin (BK) in cultured bovine aortic endothelial cells. BK elicited a rapid generation of inositol phosphates from inositol phospholipids. Accumulation of inositol 1,4,5-trisphosphate (IP3) stimulated with BK (0.1 microM) was markedly inhibited by oxidized LDL. However, native LDL had little effect on BK-induced accumulation of IP3. From these results, oxidized LDL inhibits receptor-mediated phosphoinositides hydrolysis and modulates the endothelial function.     

A possible role of protein kinase C in regulating prostaglandin synthesis of mouse peritoneal macrophages

The addition of the analogue of diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG), to resident macrophages isolated from the peritoneal cavity of mice led to a dose and time dependent increase in the synthesis of prostaglandin E. This was likely due to an enhanced amount of arachidonic acid available for eicosanoid synthesis as OAG suppressed the incorporation of arachidonic acid into cellular phospholipids by inhibiting acyl-CoA:lysophosphatide acyltransferase. Since OAG has been shown to activate protein kinase C in various cells, these data lead us to suggest that synthesis of eicosanoids in peritoneal macrophages is mediated by the activation of protein kinase C.     

Stimulation of hexose transport by human polymorphonuclear leukocytes: a possible role for protein kinase C

The protein C kinase activators 1-O-oleoyl, 2-O-acetylglycerol, 12-O-tetradecanoyl phorbol-13-acetate, and mezerein, stimulated deoxyglucose uptake in human neutrophils. The responses were stimulus specific since no effect was noted with the diether analogues 1-O-hexadecyl-2-O-ethylglycerol, 1-O-palmitoyl-2-O-acetyl or 1-O-palmitoyl-3-O-acetyl diesters of propanediol, or with 1,2-diolein. Stimulation of deoxyglucose uptake had the characteristics of carrier facilitated hexose transport. Stimulated uptake of deoxy-glucose was inhibited by trifluoperazine (10-30 microM). Activation of protein kinase C therefore appears to trigger events involved in hexose transport.     

Regulation of heat shock protein 70 release in astrocytes: role of signaling kinases

The ability to mount a successful stress response in the face of injury is critical to the long-term viability of individual cells and to the organism in general. The stress response, characterized in part by the upregulation of heat shock proteins, is compromised in several neurodegenerative disorders and in some neuronal populations, including motoneurons (MNs). Because astrocytes have a greater capacity than neurons to survive metabolic stress, and because they are intimately associated with the regulation of neuronal function, it is important to understand their stress response, so that we may to better appreciate the impact of stress on neuronal viability during injury or disease. We show that astrocytes subjected to hyperthermia upregulate Hsp/c70 in addition to intracellular signaling components including activated forms of extracellular-signal-regulated kinase (ERK1/2), Akt, and c-jun N-terminal kinase/stress activated protein kinase (JNK/SAPK). Furthermore, astrocytes release increasing amounts of Hsp/c70 into the extracellular environment following stress, an event that is abrogated when signaling through the ERK1/2 and phosphatidylinositol-3 kinase (PI3K) pathways is compromised and enhanced by inhibition of the JNK pathway. Last, we show that the Hsp/c70 is released from astrocytes in exosomes. Together, these data illustrate the diverse regulation of stress-induced Hsp/c70 release in exosomes, and the way in which the balance of activated signal transduction pathways affects this release. These data highlight how stressful insults can alter the microenvironment of an astrocyte, which may ultimately have implications for the survival of neighboring neurons.     

Regulation of cytochrome P4501A by protein kinase C: the role of heat shock protein70

Carbofuran is a pesticide, which is used throughout the world as a nematicide and an acaricide. This pesticide integrates into living organisms through aquatic ecosystem. In earlier report, we had demonstrated that cytochrome P4501A was induced in cultured catfish hepatocytes in response to carbofuran, which might be responsible for the detoxification of this pesticide. As the underlying signaling mechanism associated with induction and regulation of cytochrome P4501A has not yet been well defined, we therefore in the present study have investigated to identify the regulatory network of cytochrome P4501A in catfish liver or cultured hepatocytes by targeting several key signaling molecules such as phosphatidyl inositol (PI) or protein kinase C (PKC), which are critical molecules for many important pathways. PKC and heat shock protein70 (HSP70) have been shown to be induced in response to carbofuran in catfish hepatocytes. Results also indicate that induction of CYP1A is modulated by HSP70 and PKC in fish hepatocytes. Thus our data shed light on the regulation of EROD activity, which has been used as a bio-monitoring tool for measuring aquatic pollution.