Nuclear calcium gradients in cultured rat hepatocytes.
Ca2+ concentrations ([Ca2+]) in cytosol and nucleus in fura-2-loaded cultured rat hepatocytes were determined by three-dimensional (3-D) optical-sectioning microscopy. After determining the empirical 3-D point spread function of the fluorescence microscope-coupled digital video imaging system, contaminating light arising from optical planes above and below the plane of interest was removed by deconvolution using the nearest-neighboring approach (NNA) algorithm. Although deconvolution resulted in substantial improvement in accuracy of fluorescence intensity determinations in single-wavelength excitation images as well as sharper delineation of boundaries between cellular compartments, the complicated mathematical process did not significantly enhance the precision of [Ca2+] values derived from ratiometric (ratio of dual-wavelength excitation) images. In resting hepatocytes, cytosolic Ca2+ (210 +/- 15 nM) was 1.6- to 2-fold higher than nuclear Ca2+ (128 +/- 12 nM). This difference in Ca2+ between the two compartments was detected both in raw ratiometric images and in those processed with NNA algorithm. Addition of arginine vasopressin or epidermal growth factor resulted in significant increases (2- to 3-fold) in both cytosolic and nuclear Ca2+; however, the nuclear-to-cytosolic Ca2+ gradient was preserved in hepatocytes stimulated with mitogens. We conclude that the hepatocyte nuclear membrane contains Ca2+ permeability barriers and Ca2+ transport mechanisms that may be hormonally sensitive. We postulate that the increase in nuclear Ca2+ may be important in regulation of cell proliferation induced by mitogens, possibly by activating Ca(2+)-dependent endonucleases, nuclear calmodulin, or nuclear protein kinase C.
Waybill
M M
MM
Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
Yelamarty
R V
RV
Zhang
Y L
YL
Scaduto
R C
RC
LaNoue
K F
KF
Hsu
C J
CJ
Smith
B C
BC
Tillotson
D L
DL
Yu
F T
FT
Cheung
J Y
JY
D000465:Algorithms; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016257:Fura-2; D007091:Image Processing, Computer-Assisted; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008856:Microscopy, Fluorescence; D051381:Rats; D011919:Rats, Inbred Strains; D014743:Videotape Recording
D001127:Arginine Vasopressin; D004815:Epidermal Growth Factor; D002118:Calcium; D016257:Fura-2