Loading of fura-2/AM with an aid of DFP on single smooth muscle cells prepared from guinea pig taenia coli.
Procedures for loading fura 2 acetoxymethyl ester (fura 2/AM) into smooth muscle cells isolated from guinea pig taenia coli have been investigated. It was difficult to load these cells with fura 2 in the absence of diisopropylfluorophosphate (DFP). The presence of DFP, a potent cholinesterase (ChE) inhibitor during the loading, significantly enhanced the incorporation of the fura 2 into the cells. More than 80% of the single cells treated with DFP and fura 2/AM were viable. DFP did not affect the ability of the cell to shorten in response to either Ca2+ or carbachol (CCh). The single cells contracted transiently with caffeine and the intracellular Ca2+ concentration increased simultaneously. The results indicate that the amount of fura 2/AM incorporated into the single smooth muscle cells depends on the activity of ChE or various serine proteases located outside the cells and suppression of these enzymes induces more efficient incorporation, which permits shorter incorporation periods. Since the presence of DFP may shorten the incubation time significantly, the viability of these cells is improved. The procedure might be applicable for measuring simultaneously the contraction of cells and the behavior of intracellular Ca2+ in the same cells.
Maruyama
I
I
Department of Pharmacology, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Oyamada
H
H
Hasegawa
T
T
Ohtsuka
K
K
Kobayashi
M
M
Momose
K
K
D000818:Animals; D001572:Benzofurans / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002802:Cholinesterases / Q000378:metabolism; D003106:Colon / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D004533:Egtazic Acid / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives*; D006168:Guinea Pigs; D006651:Histocytochemistry; D066298:In Vitro Techniques; D007531:Isoflurophate / Q000494:pharmacology*; D007700:Kinetics; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D013050:Spectrometry, Fluorescence / Q000379:methods
D001572:Benzofurans; D005456:Fluorescent Dyes; C049925:fura-2-am; D007531:Isoflurophate; D002110:Caffeine; D004533:Egtazic Acid; D002217:Carbachol; D002802:Cholinesterases; D002118:Calcium; D016257:Fura-2