title: Novel technique to load indo-1 free acid into single adult cardiac myocytes to assess cytosolic Ca2+.
abstract: This report introduces a novel method to load indo-1 "free acid" selectively into the cytosol of cardiac myocytes, presumably by diffusion through momentarily permeable gap junction sites during mechanical dissociation after low-Ca2+ collagenase treatment. Calibration of indo-1 fluorescence in these cells has been accomplished after subtracting average autofluorescence (AF) from time-matched non-indo-loaded cells, taking into account apparent changes in cell AF due to indo-1. There is wide variation in the degree of uncertainty of individual intracellular Ca2+ concentration ([Ca2+]i) determinations among cells, related principally to differences in cellular indo-1 content, to nonlinear aspects of the [Ca2+]-to-fluorescence ratio relationship, and to the uncertainty in the AF subtraction. Consequently, a quantitative estimate of uncertainty also may be employed in formulating weighted estimates of cytosolic [Ca2+]i. The following [Ca2+]i values in rat ventricular cells (nM; in 1 mM bathing extracellular Ca2+ concentration, 25 degrees C) are given as weighted means +/- 95% confidence intervals (unweighted values in parentheses): 138 +/- 5 (136 +/- 6, n = 44) in quiescent cells, 435 +/- 74 (482 +/- 76, n = 43) at the [Ca2+]i-transient peak during 0.5 Hz steady-state stimulation, and 760 +/- 124 (1,027 +/- 250, n = 42) at the [Ca2+]i-transient peak, postrest. Moreover, these peak [Ca2+]i values fall near the steepest portion of the force-Ca2+ curve (from intact cardiac muscle), consistent with sensitive inotropic regulation and maximal contractile reserve.
authors.affiliation: Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224.
authors.forename: S J
authors.initials: SJ
authors.lastname: Sollott
authors.forename: B D
authors.initials: BD
authors.lastname: Ziman
authors.forename: E G
authors.initials: EG
authors.lastname: Lakatta
mesh_terms: D000143:Acids; D000818:Animals; D002118:Calcium / Q000032:analysis* / Q000502:physiology; D002138:Calibration; D002309:Cardiology / Q000379:methods; D002469:Cell Separation; D003600:Cytosol / Q000737:chemistry*; D004058:Diffusion; D005453:Fluorescence; D005456:Fluorescent Dyes; D007211:Indoles / Q000008:administration & dosage*; D008433:Mathematics; D008856:Microscopy, Fluorescence; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D000143:Acids; D005456:Fluorescent Dyes; D007211:Indoles; C048960:indo-1; D002118:Calcium