Characterization of the metabotropic glutamate receptors mediating phospholipase C activation and calcium release in cerebellar granule cells: calcium-dependence of the phospholipase C response.
In this study we have determined the metabotropic glutamate receptors (mGluRs) involved in the glutamate activation of phospholipase C (PLC) and Ca(2+) mobilization in cerebellar granule cells at 9 days in vitro; and studied the Ca(2+) modulation of the PLC response. Both PLC activation and Ca(2+) signalling were found to be mediated exclusively by the mGluR1 subtype, although both group I mGluRs, mGluR1 alpha and mGluR5, could be detected in cell extracts. Exposure of cells to medium devoid of Ca(2+) for various times before agonist stimulation reduced the PLC response, which was quickly recovered following the re-exposure of cells to Ca(2+)-containing medium. The extent of the glutamate response correlated well with changes in the cytosolic Ca(2+) concentration. On the other hand, loading of the intracellular Ca(2+) stores by a transient depolarization followed by washing in nondepolarizing buffer, allowed glutamate to release stored Ca(2+) in the majority of cells and enhanced glutamate activation of PLC. Under such conditions, the absence of extracellular Ca(2+) during stimulation and the chelation of cytosolic Ca(2+) with BAPTA/AM inhibited both glutamate-elicited Ca(2+) response and PLC activation. Overall, these results indicate that the mGluR-mediated activation of PLC depends on the presence of extracellular Ca(2+) and can be modulated by moderate changes of cytosolic Ca(2+). Furthermore, ryanodine reduced PLC stimulation by glutamate in predepolarized cells but not in control cells, suggesting that ryanodine receptors could play a role in the potentiation of the mGluR-mediated activation of PLC by Ca(2+) release in predepolarized cells.
Masgrau
R
R
Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Catalonia, Spain. roser.masgrau@uab.es
Servitja
J M
JM
Young
K W
KW
Pardo
R
R
Sarri
E
E
Nahorski
S R
SR
Picatoste
F
F
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D002614:Chelating Agents / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D018698:Glutamic Acid / Q000494:pharmacology; D009474:Neurons / Q000201:enzymology*; D010716:Phosphatidylinositols / Q000378:metabolism; D051381:Rats; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism*; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*
D002614:Chelating Agents; C578937:Grm5 protein, rat; D010716:Phosphatidylinositols; D064529:Receptor, Metabotropic Glutamate 5; D018094:Receptors, Metabotropic Glutamate; D019837:Ryanodine Receptor Calcium Release Channel; C104077:metabotropic glutamate receptor type 1; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D012433:Ryanodine; D018698:Glutamic Acid; D004533:Egtazic Acid; D010738:Type C Phospholipases; D002118:Calcium