Thapsigargin-sensitive Ca(2+)-ATPases account for Ca2+ uptake to inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive Ca2+ stores in adrenal chromaffin cells.
In chromaffin cells of adrenal medulla, heterogeneity of Ca2+ stores has been suggested with respect to the mechanisms of Ca2+ uptake and release. We have examined Ca(2+)-ATPases responsible for loading of Ca2+ stores in these cells for their sensitivity to thapsigargin, a highly selective inhibitor of the SERCA [sarco(endo)plasmic reticulum calcium ATPase] family of intracellular Ca2+ pumps. Using immunostaining, we studied the distribution of Ca(2+)-ATPases, and of receptors for inositol 1,4,5-trisphosphate (InsP3) and ryanodine, in the density-gradient fractions of microsomes from bovine adrenal medulla. In parallel, we examined distribution profiles of ATP-dependent Ca2+ uptake in the same fractions, along with subcellular markers for plasma membranes and endoplasmic reticulum (ER). Two Ca(2+)-ATPase-like proteins (116 and 100 kDa) were detected, consistent with the presence of SERCA 2b and SERCA 3 isoenzymes of Ca2+ pumps. The distribution of these putative Ca(2+)-ATPase iso-enzymes paralleled that of InsP3 and ryanodine receptors. This distribution of ER Ca(2+)-ATPases, as determined immunologically, was consistent with that of thapsigargin-sensitive, but not of thapsigargin-insensitive, ATP-dependent Ca2+ uptake. In contrast, the distribution profile of the thapsigargin-insensitive Ca2+ uptake was strongly correlated to that of plasma membranes, and co-distributed with plasma membrane Ca(2+)-ATPase detected immunologically. In isolated, permeabilized chromaffin cells, InsP3 and caffeine induced Ca2+ release following an ATP-dependent Ca2+ accumulation to the stores. This accumulation was abolished by thapsigargin. Together, these data strongly indicate that the thapsigargin-sensitive, presumably SERCA-type Ca(2+)-ATPases account for Ca2+ uptake to InsP3-sensitive, as well as to caffeine-sensitive, Ca2+ stores in bovine adrenal chromaffin cells.
Poulsen
J C
JC
Department of Medical Physiology, Panum Institute, University of Copenhagen, Denmark.
Caspersen
C
C
Mathiasen
D
D
East
J M
JM
Tunwell
R E
RE
Lai
F A
FA
Maeda
N
N
Mikoshiba
K
K
Treiman
M
M
D000313:Adrenal Medulla / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism* / Q000493:pharmacokinetics*; D015220:Calcium Channels / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000187:drug effects* / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002837:Chromaffin Granules / Q000187:drug effects* / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015151:Immunoblotting; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism / Q000494:pharmacology*; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008861:Microsomes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel; D012680:Sensitivity and Specificity; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D014638:Vanadates / Q000494:pharmacology
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009124:Muscle Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D019837:Ryanodine Receptor Calcium Release Channel; D013729:Terpenes; D002110:Caffeine; D014638:Vanadates; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium