Ca2+ uptake through voltage-gated L-type Ca2+ channels by polarized enterocytes from Atlantic cod Gadus morhua.
The presence and localization of voltage-gated Ca2+ channels of L-type were investigated in intestinal cells of the Atlantic cod. Enterocytes were loaded with the fluorescent Ca2+ probe, fure-2/AM and changes in intracellular Ca2+ concentrations ([Ca2+]i) were measured, in cell suspensions, in the presence of high potassium levels (100 mm), BAY K-8644 (5 microM), nifedipine (5 microM) or omega-conotoxin (1 microM). L-type Ca2+ channels were visualized on intestinal sections using the fluorescent dihydropyridine (-)-STBodipy. Depolarization of the plasma membrane produced a rapid (within 5 sec) and transient (at basal levels after 21 sec) increase in [Ca2+]i. BAY K-8644 increased the [Ca2+]i by 7.2%. Cells in a Ca2+-free buffer increased [Ca2+]i after addition of 10 mm Ca2+, and this increase was abolished by nifedipine in both depolarizing and normal medium but not by omega-conotoxin. Single cell experiments using video microscopy revealed that enterocytes remained polarized several hours after preparation and that the Ca2+ entry and extrusion occurred at specific and different regions of the enterocyte outer membrane. Fluorescent staining of L-type Ca2+ channels in the intestinal mucosa showed the most intense staining at the brushborder membrane. These results demonstrate the presence of voltage gated L-type Ca2+ channels in enterocytes from the Atlantic cod. The channels are mainly located at the apical side of the cells, and there is a polarized uptake of Ca2+ into the enterocytes. This suggests that the L-type Ca2+ channels are involved in the transcellular Ca2+ entry into the enterocytes.
Larsson
D
D
Fish Endocrinology Laboratory, Department of Zoophysiology, Göteborg University, Box 463, SE-405 30 Göteborg, Sweden.
Lundgren
T
T
Sundell
K
K
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism*; D016764:Cell Polarity; D002478:Cells, Cultured; D005260:Female; D005399:Fishes; D005456:Fluorescent Dyes; D007413:Intestinal Mucosa / Q000378:metabolism* / Q000648:ultrastructure; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D008297:Male; D008564:Membrane Potentials; D018715:Microscopy, Video; D008871:Microvilli / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D005456:Fluorescent Dyes; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; D002118:Calcium