Astrocyte stellation in saline media lacking bicarbonate: possible relation to intracellular pH and tyrosine phosphorylation.
Primary cultures of astrocytes exhibit a polygonal morphology, but on treatment with agents that increase cAMP they change to stellate cells. We found that astrocyte stellation also occurred on replacing the culture medium with saline buffered with HEPES. However, stellation did not occur when the medium was replaced with saline buffered with bicarbonate/CO(2) provided Ca(2+) was present. Since exposure of astrocytes to media lacking bicarbonate results in a decrease in intracellular pH (pH(i)) we sought evidence for an association between pH(i) and morphology. Astrocytic pH(i) was monitored for 60 min after transferring the cells to HEPES or bicarbonate-buffered saline. HEPES-induced stellation was associated with transient acidification which coincided with the morphological changes. Acidification was not observed in cells transferred to bicarbonate-saline. However when cytoplasmic acidification of cells in bicarbonate-saline was induced pharmacologically, rapid stellation occurred. Stellation induced by cAMP is reversed by activation of the RhoA pathway with lysophosphatidic acid (LPA). Here we found that LPA inhibited HEPES-induced stellation, but only with Ca(2+) present. Inhibition of stellation by LPA+Ca(2+) was associated with transient acidification followed by modest alkanization. A close association of tyrosine phosphorylation with stellation and pH(i) was observed. Thus incubation of astrocytes in HEPES-saline with orthovanadate to inhibit dephosphorylation abolished stellation and acidification; conversely incubation of cells in bicarbonate-saline with genistein to inhibit tyrosine kinases caused stellation and major acidification. Acidification may be one of several factors resulting in stellation, but it is not a necessary factor since stellation without acidification was observed in bicarbonate-saline lacking Ca(2+).
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, UFRGS, Porto Alegre, 90.035.003, RS, Brazil.
Sirlene R
SR
Cechin
Carmem
C
Gottfried
Cristina C
CC
Prestes
Leticia
L
Andrighetti
Susana T
ST
Wofchuk
Richard
R
Rodnight
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000166:cytology* / Q000187:drug effects*; D001639:Bicarbonates / Q000494:pharmacology; D002412:Cations / Q000032:analysis; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D004338:Drug Combinations; D006531:HEPES / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D008094:Lithium / Q000494:pharmacology; D008246:Lysophospholipids / Q000494:pharmacology; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012965:Sodium Chloride / Q000494:pharmacology*; D017923:Sodium-Hydrogen Exchangers / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism
D001639:Bicarbonates; D002412:Cations; D003470:Culture Media; D004338:Drug Combinations; D008246:Lysophospholipids; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1; D000643:Ammonium Chloride; D014443:Tyrosine; D012965:Sodium Chloride; D008094:Lithium; D011188:Potassium; D006531:HEPES