Functional measurements of [Ca2+] in the endoplasmic reticulum using a herpes virus to deliver targeted aequorin.
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 that recombinant aequorin targeted to the endoplasmic reticulum (ER) [Montero M., Brini M., Marsault R. et al. Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells. EMBO J 1995; 14: 5467-5475, Montero M., Barrero M.J., Alvarez J. [Ca2+] microdomains control agonist-induced Ca2+ release in intact cells. FASEB 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 different cell types including both postmitotic primary cells (anterior pituitary cells, chromaffin cells and cerebellar neurons) and cell lines (HeLa, NIH3T3, GH3 and PC12 cells). Functional studies showed that the steady state lumenal [Ca2+]er ranged from around 300 microM in granule cells to 800 microM in GH3 cells. InsP3-coupled receptor stimulation with agonists like histamine (in HeLa, NIH3T3 and chromaffin cells), UTP and bradykinin (in PC12 cells) or thyrotropin-releasing hormone (TRH, in GH3 cells) produced a very rapid decrease in lumenal [Ca2+]er. Caffeine caused a rapid Ca2+ depletion of the ER in chromaffin cells, but not in the other cell types. Depolarization by high K+ produced an immediate and reversible increase of [Ca2+]er in all the excitable cells (anterior pituitary, GH3, chromaffin cells and granule neurons). We conclude that delivery of recombinant aequorin to the ER using HSV amplicon provides the first direct quantitative and dynamic measurements of [Ca2+]er in several primary non-dividing cells.
Alonso
M T
MT
Departamento de Bioquímica y Biología Molecular y Fisiología, Universidad de Valladolid y CSIC, Spain. talonso@cpd.uva.es
Barrero
M J
MJ
Carnicero
E
E
Montero
M
M
Garcia-Sancho
J
J
Alvarez
J
J
D000331:Aequorin / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D019439:Chromaffin Cells / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry* / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008163:Luminescent Measurements; D051379:Mice; D009474:Neurons / Q000378:metabolism / Q000821:virology; D016716:PC12 Cells / Q000187:drug effects / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism / Q000821:virology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D018139:Simplexvirus / Q000235:genetics*; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000494:pharmacology
D011994:Recombinant Proteins; D002110:Caffeine; D000331:Aequorin; D013973:Thyrotropin-Releasing Hormone; D006632:Histamine; D001920:Bradykinin; D002118:Calcium; D014544:Uridine Triphosphate