Actin filaments play a permissive role in the inhibition of store-operated Ca2+ entry by extracellular ATP in rat brown adipocytes.
Stimulation of P2 receptors with micromolar concentration of ATP evokes a transient increase in [Ca2+]i (intracellular free Ca2+ concentration), primarily due to release of Ca2+ from intracellular stores; such stimulation also triggers almost complete suppression of thapsigargin-evoked sustained [Ca2+]i increase mediated through a store-operated Ca2+ entry pathway in rat brown adipocytes. We investigated the role of cytoskeletal actin in the inhibitory effect of the extracellular ATP on store-operated Ca2+ entry, using fura 2 fluorescence for continuous measurement of [Ca2+]i, and using Alexa fluor 488-phalloidin staining of actin. Disassembly of actin networks by cytochalasin D (1 microM) or latrunculin A (3 microM) prevented the inhibitory effect of ATP (10 microM) on the thapsigargin (100 nM)-evoked store-operated Ca2+ entry, without changing the effect of ATP in increasing [Ca2+]i. In normal cells, bath application of ATP induced a transient [Ca2+]i increase, consisting of a rapid increase (the rising phase) and the subsequent decrease (the declining phase) to a lower steady level despite the continued presence of the agonist. Disruption of actin assemblies did not significantly affect the rising phase, but prevented the declining phase. Cells incubated with 10 microM ATP for 4 min demonstrated marked accumulations of actin filaments at the cell periphery, showing protrusions at the cell surface; this actin-assembly process is mediated through P2 receptors. In cells treated with cytochalasin D or latrunculin A, extracellular ATP did not induce actin redistribution. These results suggest that the actin reorganization plays a role in ATP-induced inhibition of store-operated Ca2+ entry in rat brown adipocytes.
Omatsu-Kanbe
Mariko
M
Department of Physiology, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan. m_omatsu@belle.shiga-med.ac.jp
Shibata
Masaru
M
Yamamoto
Takefumi
T
Isono
Takahiro
T
Matsuura
Hiroshi
H
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000502:physiology*; D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000502:physiology*; D017667:Adipocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D002001:Adipose Tissue, Brown / Q000166:cytology* / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D020013:Calcium Signaling; D048430:Cell Shape / Q000502:physiology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism; D005110:Extracellular Space / Q000737:chemistry*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D019284:Thapsigargin / Q000494:pharmacology
D000199:Actins; D015220:Calcium Channels; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D002118:Calcium