Studies on maitotoxin-induced intracellular Ca(2+) elevation in chinese hamster ovary cells stably transfected with cDNAs encoding for L-type Ca(2+) channel subunits.
The aim of the present study was to characterize the role played by different L-type Ca(2+) channel subunits in [Ca(2+)](i) increase induced by maitotoxin (MTX). In the presence of 5 mM extracellular K(+), MTX (0.01-0.5 ng/ml) induced a significant concentration-dependent increase in Fura-2-monitored [Ca(2+)](i) in single Chinese hamster ovary (CHO) cells expressing the alpha(1c) (CHOCalpha9 cells) or the alpha(1c)beta(3)alpha(2)delta (CHOCalpha9beta3alpha2/delta4 cells) subunits of voltage-gated Ca(2+) channels (VGCCs), whereas the effect was much reduced in wild-type CHO cells lacking VGCCs. In addition, MTX effect on CHOCalpha9, CHOCalpha9beta3alpha2/delta4, and GH(3) cells (0.01-0.1 ng/ml) was inhibited by the selective L-type Ca(2+) channel entry-blocker nimodipine (10 microM); a nimodipine-insensitive component was still present, particularly at high (>1 ng/ml) toxin concentrations. In CHOCalpha9beta3alpha2/delta4 cells, depolarizing concentrations of extracellular K(+) (55 mM) reinforced the [Ca(2+)](i) increase induced by MTX (0.1 ng/ml), and this effect was prevented by nimodipine (10 microM). Finally, patch-clamp experiments in CHOCalpha9beta3alpha2/delta4 cells showed that low MTX concentrations (0.03 ng/ml) induced the occurrence of an inward current at -60 mV, which was completely prevented by Cd(2+) (100 microM) and by nimodipine (10 microM), whereas the same dihydropyridine concentration (10 microM) failed to prevent the electrophysiological effects of a higher toxin concentration (3 ng/ml). In conclusion, the results of the present study showed that MTX-induced [Ca(2+)](i) elevation involves two components: 1) an action on L-type VGCCs at the pore-forming alpha(1c) subunit level, which is responsible for the greatest rise of [Ca(2+)](i); and 2) a VGCC-independent mechanism that is present both in excitable and in nonexcitable cells and is responsible for a lower elevation of [Ca(2+)](i).
Cataldi
M
M
Section of Pharmacology, Department of Neuroscience, School of Medicine, University of Naples Federico II, Naples, Italy.
Secondo
A
A
D'Alessio
A
A
Taglialatela
M
M
Hofmann
F
F
Klugbauer
N
N
Di Renzo
G
G
Annunziato
L
L
D000818:Animals; D001464:Barium / Q000378:metabolism; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000096:biosynthesis* / Q000235:genetics; D020746:Calcium Channels, L-Type; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005456:Fluorescent Dyes; D016257:Fura-2; D015640:Ion Channel Gating; D008387:Marine Toxins / Q000633:toxicity*; D009553:Nimodipine / Q000494:pharmacology; D035964:Oxocins; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000494:pharmacology; D014162:Transfection; D000095702:Polyether Toxins
D001464:Barium; D002118:Calcium; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D016257:Fura-2; D008387:Marine Toxins; D009553:Nimodipine; D035964:Oxocins; D011188:Potassium; C020394:maitotoxin; D000095702:Polyether Toxins