Ganglioside inhibition of glutamate-mediated protein kinase C translocation in primary cultures of cerebellar neurons.

Abstract

In primary cultures of cerebellar granule cells, protein kinase C (PKC) translocation and activation can be triggered by the stimulation of excitatory amino acid neurotransmitter receptors. Glutamate evokes a dose-related translocation of 4-fi-[3H~phorbol 12,13-dibutyrate {[3H]-P(BtO)2} binding sites from the cytosol to the neuronal membrane and stimulates the incorporation of 32p into a number of membrane proteins, particularly protein bands in the range of 80, 50, and 40 kDa. The glutamate-evoked PKC translocation is Mg2" sensitive, is prevented by 2-amino-5-phosphonovalerate and phencyclidine, is not inhibited by nitrendipine (a voltage-dependent Ca2+-channel blocker) but is abolished by the removal of Ca2+ from the incubation medium, suggesting that glutamate-mediated Ca21 influx is operative in the redistribution of PKC. Exposure of granule cells to the gangliosides trisialosylgangliotetraglycosylceramide (GT1b) or monosialosylgangliotetraglycosylceramide (GM1) inhibits the translocation and activation of PKC evoked by glutamate. These glycosphingolipids fail to interfere with glutamate binding to its high-affinity recognition site or with the [3H]P(BtO)2 binding, nor do they affect the Ca2+ influx. These gangliosides may prevent PKC translocation by interfering with the PKC binding to the neuronal membrane phosphatidylserine.An increase of cytosolic free Ca2l may promote protein kinase C (PKC) translocation from the cytosol to the membrane and may prime the diacylglycerol activation of PKC by facilitating the binding of the enzyme to phosphatidylserine, a component of the inner lipid bilayer (1-3). Since diacylglycerol can be produced by phospholipase C activation mediated by transmitter receptor stimulation in neurons, the activation and the translocation of PKC can be considered a long-term component of the cascade of events triggered by transmitter activation of metabotropic receptors (4, 5). In addition to diacylglycerol and phospholipids, other classes of lipids located in neuronal membranes, such as lysosphingolipids and gangliosides, appear to participate in the modulation of membrane translocated PKC activity (6,7). This raises the possibility that in neuronal membranes, sialycation of gangliosides by enzymes of the outer or inner membrane surfaces (8) promotes a transmembrane "sphingoglycolipid cycle" (7) that may function as a negative effector system of PKC activity. Studies of synthetic diacylglycerol derivatives and phorbol esters have marshalled indirect evidence supporting a role for PKC in neurotransmitter synthesis and secretion and in the regulation of transmitter receptors and ion channels (9-11). However, the physiological mechanisms by which PKC is translocated are bypassed by the use of these two classes of synthetic PKC effectors. The aim of the present study was to explore whether excitatory amino acid receptor agonists, in particular glutamate, stimulate PKC translocation from the cytosol to the neuronal membrane and allow the subsequent activation of this enzyme. Glutamate...

Staurosporine, a Novel Protein Kinase C Inhibitor, Prevents Postischemic Neuronal Damage in the Gerbil and Rat

“…, using the same rat model as the present study, reported that phorbol ester binding sites in the rat CAl subfield increased 1-12 h after the recirculation period. The excitatory amino acid released during and/or after ischemia may play a role in the translocation of PKC, since glutamate can translocate PKC from the cytosol to the membrane fraction (Vaccarino et at., 1987). Moreover, factors affecting enzyme activity and translocation (Kraft and Anderson, 1983; Go- , 1986) are noticed during and after ischemia.…”

Contribution of central neuroplasticity to pathological pain: review of clinical and experimental evidence

“…1986) and the activation of PLC-related second messenger systems (Sugiyama et al 1987), it is possible that agents which inhibit second messenger cascades may be useful for the treatment of pathological pain. An intriguing possibility is the use of gangliasides, which block glutamate-stimulated translocation of PKC (Vaccarino et al 1987), and have recently been found to reduce hyperalgesia in a rat model of peripheral neuropathy (Hayes et al 1992). It is hoped that a combination of new pharmaceutic developments, careful clinical trials, and an increased understanding of the contribution and mechanisms of noxious stimulusinduced neuroplasticity, will lead to improved clinical treatment and prevention of pathological pain.…”

“…Furthermore, a contribution of PKC to persistent pain is consistent with the findings of Hayes et a1. (1992) who demonstrated that monosialoganglioside, which inhibits the translocation of PKC (Vaccarino et al 1987), reduces behavioral hyperalgesia in rats with peripheral neuropathy.…”

Dynamics of glycolipid domains in the plasma membrane of living cultured neurons, following protein kinase C activation: a study performed by excimer-formation imaging

“…Experimental conditions were adopted under which the bulk of pyrene-G M" was still at the plasma-membrane level, as herein inferred, and under which the effects of PMA and glutamate could be exerted on cerebellar granule cells [13,41]. The treatment with PMA, a drug able to induce PKC activation in many cell types, cerebellar granule cells included [13,41,42], induces an increase of glycolipid segregation in cell bodies, suggesting that activation of the kinase can modulate ganglioside domains. In order to substantiate the involvement of PKC in the observed phenomenon, we carried out experiments with PMA in the presence of BIM and experiments with glutamate.…”