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 cardiac muscle excitation-contraction coupling. We used the lipophilic membrane-associated Ca2+ indicator Ca(2+)-Green C-18 (C-18) and laser-scanning confocal microscopy to detect extracellular [Ca2+] and changes of t-tubular [Ca2+] in cultured neonatal rat myocytes and in freshly isolated adult guinea pig ventricular myocytes. Changes of extracellular [Ca2+] were readily detected by the C-18 located in the cell membrane. Control experiments were carried out with 100 mM extracellular nickel to rapidly quench the fluorescent indicator accessible form the extracellular space. After exposure to Ni2+, C-18 fluorescence was lower than measured in Ca(2+)-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 visualized using two-photon excitation of FIP-18. Surprisingly, in low extracellular [Ca2+], C-18 located in the outer leaflet of the cell membrane 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+ signals evoked by caffeine were prolonged, as recorded with Fura-Red. However, the near-membrane Ca2+ signal simultaneously detected by C-18 did not increase during caffeine stimulation in the absence of extracellular Na+. These results suggest that the C-18 signal reports extrusion of cytosolic Ca2+ from the subsarcolemmal space mediated by Na/Ca exchange. C-18 was also used to analyze the extracellular accessibility of the t-tubular lumen in isolated guinea pig ventricular myocytes. After stepwise increases of [Ca2+]o with a rapid superfusion device, a wave-like Ca2+ gradient travelled along the t-tubules at a velocity of 3.4-16.3 microns/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 t1/2 = 0.9 s at the surface to 1.7 s in deeper regions of the t-tubular system. This slow exchange of the solution within the t-tubules, lasting several seconds, may give rise to spatially inhomogeneous accumulation and/or depletion resulting from ion fluxes across the t-tubular membrane during physiological activity.
Department of Physiology, Loyola University, Maywood Illinois, USA. LBLATTE@LUC.EDU
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D000818:Animals; D000831:Animals, Newborn; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002451:Cell Compartmentation; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006168:Guinea Pigs; D007211:Indoles / Q000378:metabolism; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport; D008563:Membrane Lipids / Q000378:metabolism; D018613:Microscopy, Confocal; D009206:Myocardium / Q000737:chemistry* / Q000166:cytology; D009532:Nickel / Q000494:pharmacology; D009930:Organic Chemicals; D051381:Rats; D017208:Rats, Wistar; D012508:Sarcolemma / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism
D000697:Central Nervous System Stimulants; D002614:Chelating Agents; C113827:FIP-18; D005456:Fluorescent Dyes; D007211:Indoles; D008563:Membrane Lipids; D009930:Organic Chemicals; D019831:Sodium-Calcium Exchanger; C071336:calcium green; D002110:Caffeine; D004533:Egtazic Acid; D009532:Nickel; D012964:Sodium; D002118:Calcium