CONFOCAL NEAR-MEMBRANE DETECTION OF CALCIUM IN CARDIAC MYOCYTES

Near-membrane [Ca2+] may differ significantly from bulk cytosolic [Ca2 +], particularly during rapid Ca2+ signalling events related to cardia c muscle excitation-contraction coupling. We used the lipophilic membr ane-associated Ca2+ indicator Ca2+-Green C-18 (C-18) and laser-scannin g confocal microscopy to detect extracellular [Ca2+] and changes of t- tubular [Ca2+] in cultured neonatal rat myocytes and in freshly isolat ed adult guinea pig ventricular myocytes. Changes of extracellular [Ca 2+] were readily detected by the C-18 located in the cell membrane. Co ntrol experiments were carried out with 100 mM extracellular nickel to rapidly quench the fluorescent indicator accessible form the extracel lular space. After exposure to Ni-2+,Ni- C-18 fluorescence was lower t han measured in Ca2+-free conditions indicating that C-18 was located in the outer leaflet of the cell membrane. In contrast, the lipophilic derivative of Indo-1 (FIP-18) was significantly internalized, as visu alized using two-photon excitation of FIP-18. Surprisingly, in low ext racellular [Ca2+], C-18 located in the outer leaflet of the cell membr ane also reported transient elevations of intracellular [Ca2+] during application of 10 mM caffeine. In the absence of extracellular Na+ to inhibit Ca2+ removal via Na/Ca exchange, the intracellular Ca2+ signal s evoked by caffeine were prolonged, as recorded with Fura-Red, Howeve r, the near-membrane Ca2+ signal simultaneously detected by C-18 did n ot increase during caffeine stimulation in the absence of extracellula r Na+. These results suggest that the C-18 signal reports extrusion of cytosolic Ca2+ from the subsarcolemmal space mediated by Na/Ca exchan ge. C-18 was also used to analyze the extracellular accessibility of t he t-tubular lumen in isolated guinea pig ventricular myocytes. After stepwise increases of [Ca2+](0) with a rapid superfusion device, a wav e-like Ca2+ gradient travelled along the t-tubules at a velocity of 3. 4-16.3 mu m/s. The solution change within the t-tubules was delayed by 0.63-2.3 s and wash-out of Ca2+ from the t-tubules slowed from t(1/2) = 0.9 s at the surface to 1.7 s in deeper regions of the t-tubular sy stem. This slow exchange of the solution within the t-tubules, lasting several seconds, may give rise to spatially inhomogeneous accumulatio n and/or depletion resulting from ion fluxes across the t-tubular memb rane during physiological activity.