Research report
Differential regulation of phospholipases C and D by phorbol esters and the physiological activators carbachol and glutamate in astrocytes from chicken embryo cerebrum and cerebellum

https://doi.org/10.1016/0165-3806(95)00047-HGet rights and content

Abstract

Primary astrocytic cultures derived from day-15 chick embryo (E15) cerebral hemispheres (CH) or cerebellum (CB) express a calcium/phospholipid-dependent isoform as the major protein kinase C (PKC-α/β). PKC was activated (translocation of activity from cytosol to membrane) following stimulation with carbachol, so we tested for activation of phospholipase C (PLC) as the source of diacylglycerol released from polyphosphoinositide (PIP2) hydrolysis. Carbachol activated PLC (inositol phosphate release) 4-fold in a time- and dose-dependent manner in cortical (CH) astrocytes, but there was no activation of PLC in astrocytes from cerebellum (CB). Pirenzepine, but not gallamine, attenuated both carbachol-induced PKC translocation and PIP, hydrolysis in E15CH astrocytes, arguing for contribution of M1 subtype. The phorbol ester TPA completely inhibited PIP2 hydrolysis, both basal and carbachol-stimulated, and elicited a stronger, but shorter (10 min) activation of PKC than that observed with carbachol. We investigated phospholipase D (PLD) activation as an alternate source of diacylglycerol in astrocytes, since the ratio of PLC to PKC activation by carbachol was lower in astrocytes than observed in neurons. We observed a dramatic (10-fold) time- and dose-dependent activation of PLD by TPA in CH and a 3-fold increase in CB. The duration of TPA-dependent PLD activation correlated well with increased cell proliferation and changes in astrocytic phenotype markers. Carbachol-stimulated PLD activation was observed in CH but not in CB astrocytes, being mostly dependent on the M3 receptor subtype in the former. In contrast, glutamate elicited a greater PLD activation in CB astrocytes than in CH astrocytes. TPA activation of PLD was totally blocked by staurosporine (PKC inhibitor) and genistein (a tyrosine kinase inhibitor) in cerebellar (CB) astrocytes; however, total inhibition of TPA-dependent PLD activation was only achieved in cortical (CH) astrocytes after addition of EGTA. Thapsigargin activated PLD in both populations, further emphasizing the PLD activation dependency on [Ca2+]i. Taken together with our previous observations that TPA induces proliferation, cytoskeleton changes, and decreases of glutamine synthetase activity, these data suggest that phospholipase D is a differential but important participant in the regulation of the signalling of mitosis and differentiation in astrocytes during their development.

References (47)

  • E.A. Martinson et al.

    Rapid protein kinase C-dependent activation of phospholipase D leads to delayed 1,2-diglyceride accumulation

    J. Biol. Chem.

    (1990)
  • J.K. Pai et al.

    Phispholipase D catalyzes phospholipid metabolism in chemotactic peptide-stimulated HL-60 granulocytes

    J. Biol. Chem.

    (1988)
  • S.H. Ryu et al.

    Feedback regulation of phospholipase C-β by protein kinase C

    J. Biol. Chem.

    (1990)
  • N. Sakellaridis et al.

    Developmental profiles of glial enzymes in the chick embryo; in vivo and in culture

    Neurochem. Intern.

    (1983)
  • N. Sakellaridis et al.

    Effects of neuron conditioned medium and fetal calf serum contnent on glial growth in dissociated cultures

    Dev. Brain Res.

    (1986)
  • J. Sandmann et al.

    Coupling of transfected muscarinic acetylcholine receptor subtypes to phospholipase D

    J. Biol. Chem.

    (1991)
  • K.M. Tietje et al.

    Embryonic chick heart expresses multiple muscarinic acetylcholine receptor subtypes. Isolation and characterization of a gene encoding a novel m2 muscarinic receptor with high affinity for pirenzepine

    J. Biol. Chem.

    (1991)
  • L.C. Wilkes et al.

    Endothelin1 stimulated phospholipase D in A10 vascular smooth muscle derived cells is dependent on tyrosine kinase. Evidence for involve ment in stimulation of mitogenesis

    FEBS Lett.

    (1993)
  • R.H. Amundson et al.

    Uptake of [3H] serotonin and [3H] glutamate by primary astrocyte cultures

    GLIA

    (1992)
  • A. Ashkenazi et al.

    Acetylcholine analogue stimulates DNA synthesis in brain-derived cells via specific muscarinic receptor subtypes

    Nature

    (1989)
  • E. Babcock-Atkinson et al.

    Calcium/calmodulin-dependent protein kinase activity in primary astrocyte cultures

    GLIA

    (1989)
  • M.J. Berridge

    Inositol triphosphate and diacylglycerol as second messengers

    Biochem. J.

    (1984)
  • H.M. Blau

    Differentiation requires continuous active control

    Ann. Rev. Biochem.

    (1992)
  • Cited by (24)

    • Multiple mechanistically distinct modes of endocannabinoid mobilization at central amygdala glutamatergic synapses

      2014, Neuron
      Citation Excerpt :

      Thus, in CeAL neurons, mAChR stimulation can initiate multimodal eCB signaling depending on the duration of Gq-receptor stimulation. The mechanistic basis for this temporal “switch” remains to be determined, but it is likely related to temporal differences in the coupling of mAChRs to distinct signaling pathways important for AEA and 2-AG synthesis (Mangoura et al., 1995; McKenzie et al., 1992; Schmidt et al., 1995). Recent studies have begun to highlight the dissociable roles of AEA and 2-AG signaling on multiple levels.

    • Prolactin concurrently activates Src-PLD and JAK/Stat signaling pathways to induce proliferation while promoting differentiation in embryonic astrocytes

      2000, International Journal of Developmental Neuroscience
      Citation Excerpt :

      Activation was apparent by 1 min, reached a peak at 10 min, and then declined, returning to basal levels by 3 h (Fig. 3). Compared to other physiological agonists [34], PRL is not only the most potent physiological agonist of PLD activation in astrocytes, but also produces the longest duration of activation. To determine if PRL-stimulated PLD activation was dependent on PKC, we examined the effect of PKC inhibitors staurosporine (St) and calphostin C (Cal C) on phosphatidylethanol synthesis corresponding to PLD activation.

    • Activation of astroglial phospholipase D activity by phorbol ester involves ARF and Rho proteins

      2000, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
    View all citing articles on Scopus
    View full text