The inositol 1,4,5-trisphosphate-binding site in adrenal cortical cells is distinct from the endoplasmic reticulum.
The distribution of binding sites for the calcium-mobilizing second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) was investigated in subcellular fractions of bovine adrenal cortex. The [3H]Ins(1,4,5)P3-binding capacity was enriched in the microsomal fraction, which contained a single class of high affinity binding sites with a Kd of 21.6 +/- 3.0 nM. The specific [3H]Ins(1,4,5)P3 binding appeared to be sharply pH dependent and was inhibited by millimolar concentrations of ATP. Upon fractionation of microsomes on sucrose density gradient there was a clearcut separation of the Ins(1,4,5)P3 receptor-containing fractions from those enriched in specific endoplasmic reticulum markers such as sulfatase C activity or RNA content. The microsomes enriched in Ins(1,4,5)P3-binding sites were of lower density than the endoplasmic reticulum and co-purified partly with the plasma membrane. In addition, Ins(1,4,5)P3-sensitive 45Ca2+ uptake into the microsomes was maximal in the lighter fractions. This distinction between Ins(1,4,5)P3-binding sites and endoplasmic reticulum-derived microsomes was confirmed upon fractionation according to their electrophoretic mobilities by free flow electrophoresis. These results indicate that in adrenal cortical cells, the source of Ca2+ mobilized by Ins(1,4,5)P3 upon stimulation with an agonist is not located in the endoplasmic reticulum. Our data support the hypothesis that a specialized vesicular organelle, distinct from endoplasmic reticulum and in close apposition with the plasma membrane, is involved in intracellular Ca2+ homeostasis.
Rossier
M F
MF
Department of Medicine, University Hospital, Geneva, Switzerland.
Capponi
A M
AM
Vallotton
M B
MB
D000302:Adrenal Cortex / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D015220:Calcium Channels; D002417:Cattle; D002499:Centrifugation, Density Gradient; D004586:Electrophoresis; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D015544:Inositol 1,4,5-Trisphosphate; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000032:analysis*; D018160:Receptors, Cytoplasmic and Nuclear; D013403:Sugar Phosphates / Q000378:metabolism*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D007295:Inositol Phosphates; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D013403:Sugar Phosphates; D015544:Inositol 1,4,5-Trisphosphate