Hirokazu Fujikawa

The linoleic acid derivative DCP-LA selectively activates PKC-ϵ, possibly binding to the phosphatidylserine binding site

This study examined the effect of 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA), a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on protein kinase C (PKC) activity. In the in situ PKC assay with reverse-phase high-performance liquid chromatography, DCP-LA significantly activated PKC in PC-12 cells in a concentration-dependent (10 nM–100 μM) manner, with the maximal effect at 100 nM, and the DCP-LA effect was blocked by GF109203X, a PKC inhibitor, or a selective inhibitor peptide of the novel PKC isozyme PKC-ϵ. Furthermore, DCP-LA activated PKC in HEK-293 cells that was inhibited by the small, interfering RNA against PKC-ϵ. In the cell-free PKC assay, of the nine isozymes examined here, DCP-LA most strongly activated PKC-ϵ, with >7-fold potency over other PKC isozymes, in the absence of dioleoyl-phosphatidylserine and 1,2-dioleoyl-sn-glycerol; instead, the DCP-LA action was inhibited by dioleoyl-phosphatidylserine. DCP-LA also activated PKC-γ, a conventional PKC, but to a much lesser extent compared with that for PKC-ϵ, by a mechanism distinct from PKC-ϵ activation. Thus, DCP-LA serves as a selective activator of PKC-ϵ, possibly by binding to the phosphatidylserine binding site on PKC-ϵ. These results may provide fresh insight into lipid signaling in PKC activation.

Activation of Stress-activated Protein Kinase/c-Jun NH2-terminal Kinase and p38 Kinase in Calphostin C-induced Apoptosis Requires Caspase-3-like Proteases but Is Dispensable for Cell Death

Apoptosis was induced in human glioma cell lines by exposure to 100 nm calphostin C, a specific inhibitor of protein kinase C. Calphostin C-induced apoptosis was associated with synchronous down-regulation of Bcl-2 and Bcl-xL as well as activation of caspase-3 but not caspase-1. The exposure to calphostin C led to activation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) and p38 kinase and concurrent inhibition of extracellular signal-regulated kinase (ERK). Upstream of ERK, Shc was shown to be activated, but its downstream Raf1 and ERK were inhibited. The pretreatment with acetyl-Tyr-Val-Ala-Asp-aldehyde, a relatively selective inhibitor of caspase-3, or benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk), a broad spectrum caspase inhibitor, similarly inhibited calphostin C-induced activation of SAPK/JNK and p38 kinase as well as apoptotic nuclear damages (chromatin condensation and DNA fragmentation) and cell shrinkage, suggesting that caspase-3 functions upstream of SAPK/JNK and p38 kinase, but did not block calphostin C-induced surface blebbing and cell death. On the other hand, the inhibition of SAPK/JNK by transfection of dominant negative SAPK/JNK and that of p38 kinase by SB203580 induced similar effects on the calphostin C-induced apoptotic phenotypes and cell death as did z-VAD.fmk and acetyl-Tyr-Val-Ala-Asp-aldehyde, but the calphostin C-induced PARP cleavage was not changed, suggesting that SAPK/JNK and p38 kinase are involved in the DNA fragmentation pathway downstream of caspase-3. The present findings suggest, therefore, that the activation of SAPK/JNK and p38 kinase is dispensable for calphostin C-mediated and z-VAD.fmk-resistant cell death.

Modulation of P2X receptors via adrenergic pathways in rat dorsal root ganglion neurons after sciatic nerve injury

Abstract The present study examined noradrenaline‐induced modulation of ATP‐evoked currents in dorsal root ganglion (DRG) neurons after sciatic nerve injury (transection). ATP (10 μM) generated fast/mixed type of whole‐cell membrane currents, possibly as mediated via P2X3/P2X3‐like receptors, and slow type of the currents, possibly as mediated via P2X2/3 receptors, in acutely dissociated L4/5 DRG neurons, without significant difference between sham and injury group. For sham group, noradrenaline (10 μM) enhanced fast/mixed type of ATP‐evoked currents in ipsilateral DRG neurons, that is not inhibited by H‐7, a broad inhibitor of protein kinases, but otherwise it had no effect on slow type of the currents. For injury group, noradrenaline (10 μM) significantly potentiated slow type of ATP‐evoked currents in ipsilateral DRG neurons, that is abolished by H‐7 or GF109203X, a selective inhibitor of protein kinase C (PKC), while it depressed fast/mixed type of the currents. In the analysis of real‐time reverse transcription‐polymerase chain reaction, an increase in the mRNAs for α1b, α2a, α2d, and β2 adrenergic receptors was found with the ipsilateral DRGs after sciatic nerve injury. Collectively, the results of the present study suggest that noradrenaline potentiates P2X2/3 receptor currents by activating PKC via α1 adrenergic receptors linked to Gq protein, perhaps dominantly α1b adrenergic receptors, in DRG neurons after sciatic nerve injury. This may account for a nociceptive pathway in response to noradrenergic sprouting after peripheral nerve injury.

Activation of Protein Kinases in Canine Basilar Artery in Vasospasm

Subarachnoid hemorrhage (SAH) often leads to a long-term narrowing of cerebral artery called vasospasm. To understand the molecular mechanisms in vasospasm, signal transduction of tyrosine kinase pathway and phosphorylation of myosin light chain (MLC) and calponin (CaP) in the basilar artery were studied. Vasospasm was produced in the canine basilar artery by a two-hemorrhage method, and vasocontraction was induced by a local application of KCl or serotonin to the basilar artery after a transclival exposure. Intracellular substrates of tyrosine kinase pathway, including Shc, Raf1, and extracellular-regulated kinases in the basilar artery, were activated after SAH, and the activation of Shc suggests stimulation of signal transductions from tyrosine kinase receptors, G-coupled receptors, or both. The activation of tyrosine kinase pathway in vasospasm also was supported by dose-dependent dilation of the spastic basilar artery on days 0 and 7 by topical application of genistein, a tyrosine kinase inhibitor, and associated marked inhibition of tyrosine phosphorylation of intracellular substrates, including Shc. In addition, the generation of protein kinase M, catalytic fragment of protein kinase Cα (PKCα), in vasospasm on days 0 and 7 was inhibited in response to genistein, indicating an inactivation of μ-calpain. It is suggested, therefore, that the reversal of vasospasm by genistein is closely associated with the restoration of intracellular Ca2+ levels. However, the increased activities of Raf1 and extracellular-regulated kinases in vasospasm were declined on day 7 compared with those on day 0 or 2, suggesting that the activation of tyrosine kinase pathway is more closely associated with the early stage of vasospasm than with the late stage of vasospasm. The analysis by pyrophosphate polyacrylamide gel electrophoresis (PPi-PAGE) demonstrated three MLC bands in vasospasm on days 2 and 7, as well as in KCl- and serotonin-induced vasocontraction. Since PPi-PAGE resolves smooth muscle MLC into three bands in the MLC kinase (MLCK)-mediated phosphorylation and into a single band in the PKC-mediated phosphorylation based on the phosphorylation state, the current results suggest that MLC in vasospasm is phosphorylated by MLCK but not by PKC. In basilar artery, CaP was significantly down-regulated, and in addition, significantly phosphorylated on serine and threonine residues only in vasospasm on days 2 and 7. Although the significance of CaP phosphorylations in vivo still is controversial, CaP down-regulation and phosphorylation may attenuate the inhibition of Mg2+-ATPase activity by CaP and induce a potential enhancement of smooth muscle contractility in delayed vasospasm. Since CaP is phosphorylated vivo by PKC, activated PKC in vasospasm may phosphorylate CaP. Thus, SAH stimulates tyrosine kinase pathway to increase intracellular Ca2+ and activate PKC, and the former activates MLCK to phosphorylate MLC, whereas the latter phosphorylates CaP but not MLC.

Brain-specific angiogenesis inhibitor 1 (BAI1) is expressed in human cerebral neuronal cells

Brain-specific angiogenesis inhibitor 1 (BAI1) is a p53-target gene specifically expressed in the brain. We examined the distribution of the endogenous BAI1 protein in normal human brain tissue using a polyclonal antibody against the extracellular region of BAI1. Immunohistochemical study demonstrated that BAI1 was expressed in neuronal cells of the cerebral cortex but not in astrocytes. BAI1 protein was localized in the cellular cytoplasm and membrane. It was predominantly localized in the cellular membrane when expressed in cultured cells by means of gene transfection. BAI1 protein may play an important role in neuronal functions such as synapse formation and signal transduction.

8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid stimulates GABA release from interneurons projecting to CA1 pyramidal neurons in the rat hippocampus via pre-synaptic alpha7 acetylcholine receptors.

Nicotinic acetylcholine (ACh) receptors, such as α7, α3β4 and α4β2 receptors in the hippocampus, are suggested to modulate neurotransmitter release. 8‐[2‐(2‐Pentyl‐cyclopropylmethyl)‐cyclopropyl]‐octanoic acid (DCP‐LA) (100 nm), a linoleic acid derivative, potentiated responses of α7, α3β4 and α4β2 ACh receptors expressed in Xenopus oocytes that are blocked by 3‐(1‐[dimethylaminopropyl] indol‐3‐yl)‐4‐[indol‐3‐yl] maleimide (GF109203X), a selective inhibitor of protein kinase C (PKC), except for α3β4 ACh receptors. DCP‐LA enhanced the nicotine‐triggered release of GABA from rat hippocampal slices in the presence of tetrodotoxin in a bell‐shaped dose‐dependent manner at concentrations ranging from 10 nm to 10 µm, although DCP‐LA by itself had no effect on GABA release. The DCP‐LA action was inhibited by GF109203X or α‐bungarotoxin, an inhibitor of α7 ACh receptors, but not by mecamylamine or dihydro‐β‐erithroidine, an inhibitor of α3β4 and α4β2 ACh receptors. A similar effect on GABA release was obtained with 12‐O‐tetradecanoylphorbol 13‐acetate, a PKC activator. DCP‐LA (100 nm) also enhanced GABA release triggered by choline, an agonist of α7 ACh receptors, but not 3‐[2(s)‐azetidinylmethoxy] pyridine, an agonist of α4β2 ACh receptors. In addition, DCP‐LA (100 nm) increased the rate of nicotine‐triggered GABAA receptor‐mediated miniature inhibitory post‐synaptic currents, monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was also inhibited by GF109203X or α‐bungarotoxin but not by mecamylamine. Thus, the results of the present study indicate that DCP‐LA stimulates GABA release by enhancing activity of pre‐synaptic α7 ACh receptors present on the GABAergic terminals of interneurons that transmit to CA1 pyramidal neurons via a PKC pathway.

Spatial resolution of calpain-catalyzed proteolysis in focal cerebral ischemia.

Transient forebrain ischemia induces calpain-mediated degradation of the neuronal cytoskeleton, alpha-fodrin, and this results in ischemic neuronal death. In this study, we investigated the spatial distribution and temporal changes of calpain-catalyzed alpha-fodrin proteolysis in focal cerebral ischemia and examined the effects of a calpain inhibitor. Ischemia was induced in gerbils by 3-h middle cerebral artery occlusion followed by reperfusion. Animals were divided into four groups: a sham-operated group, an ischemic group, a vehicle-treated group, and a calpain inhibitor-treated group. Intravenous injections of vehicle or calpain inhibitor I were administered 30 min before ischemia. Infarct volumes were measured 1 day after reperfusion and the spatial distribution of calpain-catalyzed alpha-fodrin proteolysis was investigated by immunohistochemistry 15 min, 1 h, 4 h, and 1 day after reperfusion. Infarct volume (mean +/- SD) in the ischemic group and the vehicle-treated group was 204.6 +/- 19.1 mm3 and 212.4 +/- 16.3 mm3, respectively, and the calpain inhibitor I reduced the infarct volume [149.4 +/- 25.2 mm3 (P < 0.05)]. Immunoblot analysis demonstrated that calpain inhibitor reduced proteolysis. Ischemia induced fodrin proteolysis in the ischemic core and the peri-infarct zone within 15 min after reperfusion, with proteolysis developing quickly in the ischemic core and more slowly in the peri-infarct zone. Proteolysis preceded neuronal death in the peri-infarct zone. Calpain inhibitor I ameliorated neuronal death in the peri-infarct zone but not in the ischemic core. Thus, calpain plays a pivotal role on focal ischemia as well as in global ischemia.

The phosphodiesterase III inhibitor olprinone inhibits hippocampal glutamate release via a cGMP/PKG pathway

Olprinone, an inhibitor of cyclic nucleotide phosphodiesterase III, inhibited an increase in intracellular Ca(2+) concentrations for acutely dissociated rat hippocampal pyramidal neurons induced by extracellular high K(+) (35 mM) depolarization. Olprinone (100 microM) significantly reduced spontaneous glutamate release from rat hippocampal slices. Furthermore, olprinone significantly decreased the rate of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated miniature excitatory postsynaptic currents (AMPA-mEPSCs) monitored from CA1 pyramidal neurons of rat hippocampal slices, and the effect was blocked by KT5823, an inhibitor of protein kinase G (PKG), but not by H-89, an inhibitor of protein kinase A (PKA). In the PKA assay using PC-12 cells, olprinone did not activate PKA. Taken together, the results of the present study show that olprinone attenuates intracellular Ca(2+) rise through voltage-sensitive Ca(2+) channels and inhibits presynaptic glutamate release via a cGMP/PKG pathway.

Importance of Rho-Kinase-Mediated Phosphorylation of Myosin Light Chain in Vasospasm

Subarachnoid hemorrhage often induces a longterm narrowing of cerebral artery called vasospasm. Rho-kinase is activated by the small GTPase Rho which phosphorylates not only MLC but also myosin phosphatase at its subunit (myosin binding subunit; MBS). The present study examined 1) an activation of Rho-A and Rho-kinase in vasospasm, 2) phosphorylation of myosin binding subunit (MBS; a subunit of myosin phosphatase) in the basilar artery involved in development of vasospasm. 3) Whether these activities and phosphorylations are inhibited by kinase inhibitors (Y-27632, wortmannin, genistein)?

Generation of the catalytic fragment of protein kinase C alpha in vasospastic canine basilar artery

In previous studies of topical application of calphostin C, a specific inhibitor of the regulatory domain of protein kinase C (PKC), and calpeptin, a selective inhibitor of calpain, to spastic canine basilar artery (BA) researchers have suggested that the catalytic fragment of PKC (known as PKM) is probably formed by a limited proteolysis of continuously activated mu-calpain, but there has been no direct evidence for PKM formation in vasospasm. The present immunoblot study with anti-PKCalpha antibody shows a significant decrease in cytosolic 80-kD PKCalpha and a concomitantly significant increase in membrane PKCalpha in the spastic canine BA. In addition, an immunoblot study in which cleavage site-directed antibodies were used demonstrated a significant increase in immunoreactive 45-kD PKM. The changes in membrane PKCalpha and PKM were enhanced with the lapse of time after subarachnoid hemorrhage. The cleavage site-directed antibodies distinguish the proteolyzed from the unproteolyzed forms of PKC for in situ analyses of enzyme regulation mediated by proteolysis. The data indicate that PKCalpha in spastic canine BA is translocated to the cell membrane, where PKCalpha is rapidly cleaved into PKM as a result of proteolysis of the isozyme by mu-calpain but not by m-calpain. The authors hypothesize that mu-calpain is continuously activated in spastic canine BA and produces PKM by limited proteolysis of PKCalpha.