Ca transients in cardiac myocytes measured with a low affinity fluorescent indicator, furaptra.
Intracellular calcium ion ([Ca2+]i) transients were measured in single rat ventricular myocytes with the fluorescent indicator furaptra. Cells were voltage clamped with a single patch electrode containing the K+ salt of furaptra and fluorescence at 500 nm was measured during illumination with 350 and 370 nm light. Depolarizing voltage-clamp pulses elicited [Ca2+]-dependent fluorescent transients in 30 of 33 cells tested. The peak change in [Ca2+]i elicited by 50-ms depolarizations from -70 to +10 mV was 1.52 +/- 0.25 microM (mean +/- SEM, n = 7). The size of the [Ca2+]i transient increased in response to 10 microM isoproterenol, prolongation of the depolarization, and increasing pipette [Na+]. Because furaptra is sensitive to Ca2+ and Mg2+, changes in [Mg2+]i during the [Ca2+]i transient could not be measured. Instead, a single-compartment model was developed to simulate changes in [Mg2+] during [Ca2+] transients. The simulations predicted that a 2 microM [Ca2+] transient was accompanied by a slow increase in [Mg2+] (14-29 microM), which became larger as basal [Mg2+] increased (0.5-2.0 mM). The [Mg2+] transient reached a peak approximately 1 s after the peak of the [Ca2+] transient with the slow changes in [Mg2+] dominated by competition at the Ca2+/Mg2+ sites of Troponin. These changes in [Mg2+], however, were so small and slow that they were unlikely to affect the furaptra fluorescence signal at the peak of the [Ca2+]i transient. The [Ca2+]i transient reported by furaptra appears to be larger than that reported by other Ca2+ indicators; however, we conclude this larger transient is at least as accurate as [Ca2+]i transients reported by the other indicators.
Bockus Research Institute, Graduate Hospital, Philadelphia, Pennsylvania 19146.
M
M
Konishi
J R
JR
Berlin
D000818:Animals; D001572:Benzofurans; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives*; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008564:Membrane Potentials; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010080:Oxazoles; D051381:Rats; D013050:Spectrometry, Fluorescence
D001572:Benzofurans; D005456:Fluorescent Dyes; D010080:Oxazoles; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D008274:Magnesium; D002118:Calcium; D016257:Fura-2