FUNCTIONAL MEASUREMENTS OF [CA2-RETICULUM USING A HERPES-VIRUS TO DELIVER TARGETED AEQUORIN(] IN THE ENDOPLASMIC)

Changes in the free calcium concentration of the endoplasmic reticulum ([Ca2+](er)) play a central role controlling cellular functions like contraction, secretion or neuronal signaling. We recently reported tha t recombinant aequorin targeted to the endoplasmic reticulum (ER) [Mon tero M., Brini M., Marsault R. et al. Monitoring dynamic changes in fr ee Ca2+ concentration in the endoplasmic reticulum of intact cells. EM BO J 1995; 14. 5467-5475, Montero M., Barrero M.J., Alvarez J. [Ca2+] microdomains control agonist-induced Ca2+ release in intact cells. FAS EB J 1997; 11: 881-886] can be used to monitor selectively [Ca2+](er) in intact HeLa cells. Here we have used a herpes simplex virus type 1 (HSV-1) based system to deliver targeted aequorin into a number of dif ferent cell types including both postmitotic primary cells (anterior p ituitary cells, chromaffin cells and cerebellar neurons) and cell line s (HeLa, NIH3T3, GH(3) and PC12 cells). Functional studies showed that the steady state lumenal [Ca2+](er) ranged from around 300 mu M in gr anule cells to 800 mu M in GH(3) cells. InsP(3)-coupled receptor stimu lation with agonists like histamine (in HeLa, NIH3T3 and chromaffin ce lls), UTP and bradykinin (in PC12 cells) or thyrotropin-releasing horm one (TRH, in GH(3) cells) produced a very rapid decrease in lumenal [C a2+](er). Caffeine caused a rapid Ca2+ depletion of the ER in chromaff in cells, but not in the other cell types. Depolarization by high K+ p roduced an immediate and reversible increase of [Ca2+](er) in all the excitable cells (anterior pituitary, GH(3), chromaffin cells and granu le neurons). We conclude that delivery of recombinant aequorin to the ER using HSV amplicon provides the first direct quantitative and dynam ic measurements of [Ca2+](er) in several primary non-dividing cells.