Time-resolved release of calcium from an epithelial cell monolayer during mucin secretion.
A significant amount of Ca²+ is contained in secretory mucin granules. Exchange of Ca²+ for monovalent cations drives the process of mucin decondensation and hydration after fusion of granules with the plasma membrane. Here we report direct observation of calcium secretion with a Ca²+ ion-selective electrode (ISE) in response to apical stimulation with ATP from HT29-Cl.16E cells, a subclone of the human colonic cancer cell line HT29. No increase in Ca²+ level was seen for the sister cell line Cl.19A, which lacks mucin granules, or for Cl.16E cells after inhibition of granule fusion with wortmannin. Further, the measured concentration was used to estimate the time-resolved rate of release of Ca²+ from the cell monolayer, by use of a deconvolution-based method developed previously (Nair and Gratzl in Anal Chem 77:2875-2881, 2005). The results argue that Ca²+ release by Cl.16E cells is associated specifically with mucin secretion, i.e., that the measured Ca²+ increase in the apical solution is derived from granules after fusion and mucin exocytosis. The Ca²+ ISE in conjunction with deconvolution provides a minimally disturbing method for assessment of Ca²+ secretion rates. The release rates provide estimates of exocytosis rates and, when combined with earlier capacitance measurements, estimates of post-stimulation endocytosis rates also.
Department of Biomedical Engineering, Case School of Engineering, Case Western Reserve University, Cleveland, OH 44106, USA.
Sumitha
S
Nair
Rohit
R
Kashyap
Christian
C
Laboisse
Ulrich
U
Hopfer
Miklós
M
Gratzl
D000255:Adenosine Triphosphate / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D004705:Endocytosis; D004847:Epithelial Cells / Q000378:metabolism*; D005089:Exocytosis; D019073:HT29 Cells; D006801:Humans; D017736:Ion-Selective Electrodes; D009077:Mucins / Q000378:metabolism*; D018408:Patch-Clamp Techniques / Q000379:methods*
D002413:Cations, Divalent; D009077:Mucins; D000255:Adenosine Triphosphate; D002118:Calcium