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. 1995 Apr;68(4):1453–1460. doi: 10.1016/S0006-3495(95)80318-8

Calibration of indo-1 and resting intracellular [Ca]i in intact rabbit cardiac myocytes.

J W Bassani 1, R A Bassani 1, D M Bers 1
PMCID: PMC1282040  PMID: 7787031

Abstract

Fluorescent Ca indicators have been extremely valuable in understanding intracellular [Ca] ([Ca]i) regulation in many cell types. The calibration of these indicators in the intracellular environment, however, has been a continuous challenge. We performed in vivo calibrations of indo-1 in isolated rabbit ventricular myocytes loaded with the acetoxymethylester form of indo-1 and used the perforated patch variation of whole cell voltage clamp. Voltage, [Na], and [K] gradients were eliminated to approach equilibrium. We also took advantage of the powerful Na/Ca exchange in cardiac myocytes so that [Ca]i would be equilibrated with [Ca]o (because there was no [Na] or voltage gradient). The equilibration of [Na] and [Ca] across the membrane was tested by measuring the reversal potential of Na current and poking the cell to test for changes in [Ca]i-dependent fluorescence ratio. The apparent dissociation constant, Kd for indo-1 in the cellular environment was 844 nM, which is approximately 2-3 times higher than that in aqueous solutions. In a separate series of experiments, a null point approach was used to determine the [Ca]i in intact cells at rest for very long periods (82 +/- 6 nM). This is lower than that measured 15 s after a train of steady-state twitches ([Ca]i = 294 +/- 53 nM). These experiments also allowed the direct assessment of the shortening versus [Ca]i relationship in intact cells.

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Selected References

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  1. Balke C. W., Egan T. M., Wier W. G. Processes that remove calcium from the cytoplasm during excitation-contraction coupling in intact rat heart cells. J Physiol. 1994 Feb 1;474(3):447–462. doi: 10.1113/jphysiol.1994.sp020036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bassani J. W., Bassani R. A., Bers D. M. Ca2+ cycling between sarcoplasmic reticulum and mitochondria in rabbit cardiac myocytes. J Physiol. 1993 Jan;460:603–621. doi: 10.1113/jphysiol.1993.sp019489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bassani J. W., Bassani R. A., Bers D. M. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J Physiol. 1994 Apr 15;476(2):279–293. doi: 10.1113/jphysiol.1994.sp020130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bassani J. W., Bassani R. A., Bers D. M. Twitch-dependent SR Ca accumulation and release in rabbit ventricular myocytes. Am J Physiol. 1993 Aug;265(2 Pt 1):C533–C540. doi: 10.1152/ajpcell.1993.265.2.C533. [DOI] [PubMed] [Google Scholar]
  5. Bassani R. A., Bassani J. W., Bers D. M. Mitochondrial and sarcolemmal Ca2+ transport reduce [Ca2+]i during caffeine contractures in rabbit cardiac myocytes. J Physiol. 1992;453:591–608. doi: 10.1113/jphysiol.1992.sp019246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bers D. M., Bassani R. A., Bassani J. W., Baudet S., Hryshko L. V. Paradoxical twitch potentiation after rest in cardiac muscle: increased fractional release of SR calcium. J Mol Cell Cardiol. 1993 Sep;25(9):1047–1057. doi: 10.1006/jmcc.1993.1117. [DOI] [PubMed] [Google Scholar]
  7. Bers D. M., Bridge J. H., Spitzer K. W. Intracellular Ca2+ transients during rapid cooling contractures in guinea-pig ventricular myocytes. J Physiol. 1989 Oct;417:537–553. doi: 10.1113/jphysiol.1989.sp017817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bers D. M., Lederer W. J., Berlin J. R. Intracellular Ca transients in rat cardiac myocytes: role of Na-Ca exchange in excitation-contraction coupling. Am J Physiol. 1990 May;258(5 Pt 1):C944–C954. doi: 10.1152/ajpcell.1990.258.5.C944. [DOI] [PubMed] [Google Scholar]
  9. Bers D. M., Patton C. W., Nuccitelli R. A practical guide to the preparation of Ca2+ buffers. Methods Cell Biol. 1994;40:3–29. doi: 10.1016/s0091-679x(08)61108-5. [DOI] [PubMed] [Google Scholar]
  10. Blatter L. A., Blinks J. R. Simultaneous measurement of Ca2+ in muscle with Ca electrodes and aequorin. Diffusible cytoplasmic constituent reduces Ca(2+)-independent luminescence of aequorin. J Gen Physiol. 1991 Dec;98(6):1141–1160. doi: 10.1085/jgp.98.6.1141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Blatter L. A., Wier W. G. Intracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators indo-1 and fura-2. Biophys J. 1990 Dec;58(6):1491–1499. doi: 10.1016/S0006-3495(90)82494-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Brandes R., Figueredo V. M., Camacho S. A., Baker A. J., Weiner M. W. Investigation of factors affecting fluorometric quantitation of cytosolic [Ca2+] in perfused hearts. Biophys J. 1993 Nov;65(5):1983–1993. doi: 10.1016/S0006-3495(93)81275-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Brandes R., Figueredo V. M., Camacho S. A., Baker A. J., Weiner M. W. Quantitation of cytosolic [Ca2+] in whole perfused rat hearts using Indo-1 fluorometry. Biophys J. 1993 Nov;65(5):1973–1982. doi: 10.1016/S0006-3495(93)81274-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gao W. D., Backx P. H., Azan-Backx M., Marban E. Myofilament Ca2+ sensitivity in intact versus skinned rat ventricular muscle. Circ Res. 1994 Mar;74(3):408–415. doi: 10.1161/01.res.74.3.408. [DOI] [PubMed] [Google Scholar]
  15. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  16. Harkins A. B., Kurebayashi N., Baylor S. M. Resting myoplasmic free calcium in frog skeletal muscle fibers estimated with fluo-3. Biophys J. 1993 Aug;65(2):865–881. doi: 10.1016/S0006-3495(93)81112-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Horn R., Marty A. Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol. 1988 Aug;92(2):145–159. doi: 10.1085/jgp.92.2.145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hove-Madsen L., Bers D. M. Indo-1 binding to protein in permeabilized ventricular myocytes alters its spectral and Ca binding properties. Biophys J. 1992 Jul;63(1):89–97. doi: 10.1016/S0006-3495(92)81597-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ikenouchi H., Peeters G. A., Barry W. H. Evidence that binding of Indo-1 to cardiac myocyte protein does not markedly change Kd for Ca2+. Cell Calcium. 1991 Jun;12(6):415–422. doi: 10.1016/0143-4160(91)90067-o. [DOI] [PubMed] [Google Scholar]
  20. Ince C., Coremans J. M., Bruining H. A. In vivo NADH fluorescence. Adv Exp Med Biol. 1992;317:277–296. doi: 10.1007/978-1-4615-3428-0_30. [DOI] [PubMed] [Google Scholar]
  21. Kentish J. C., ter Keurs H. E., Ricciardi L., Bucx J. J., Noble M. I. Comparison between the sarcomere length-force relations of intact and skinned trabeculae from rat right ventricle. Influence of calcium concentrations on these relations. Circ Res. 1986 Jun;58(6):755–768. doi: 10.1161/01.res.58.6.755. [DOI] [PubMed] [Google Scholar]
  22. Konishi M., Olson A., Hollingworth S., Baylor S. M. Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements. Biophys J. 1988 Dec;54(6):1089–1104. doi: 10.1016/S0006-3495(88)83045-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li Q., Hohl C. M., Altschuld R. A., Stokes B. T. Energy depletion-repletion and calcium transients in single cardiomyocytes. Am J Physiol. 1989 Sep;257(3 Pt 1):C427–C434. doi: 10.1152/ajpcell.1989.257.3.C427. [DOI] [PubMed] [Google Scholar]
  24. Mattiazzi A., Hove-Madsen L., Bers D. M. Protein kinase inhibitors reduce SR Ca transport in permeabilized cardiac myocytes. Am J Physiol. 1994 Aug;267(2 Pt 2):H812–H820. doi: 10.1152/ajpheart.1994.267.2.H812. [DOI] [PubMed] [Google Scholar]
  25. Miyata H., Silverman H. S., Sollott S. J., Lakatta E. G., Stern M. D., Hansford R. G. Measurement of mitochondrial free Ca2+ concentration in living single rat cardiac myocytes. Am J Physiol. 1991 Oct;261(4 Pt 2):H1123–H1134. doi: 10.1152/ajpheart.1991.261.4.H1123. [DOI] [PubMed] [Google Scholar]
  26. Morgan K. G. Ca2+i versus [Ca2+]i. Biophys J. 1993 Aug;65(2):561–562. doi: 10.1016/S0006-3495(93)81087-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rousseau E., Meissner G. Single cardiac sarcoplasmic reticulum Ca2+-release channel: activation by caffeine. Am J Physiol. 1989 Feb;256(2 Pt 2):H328–H333. doi: 10.1152/ajpheart.1989.256.2.H328. [DOI] [PubMed] [Google Scholar]
  28. Spurgeon H. A., Stern M. D., Baartz G., Raffaeli S., Hansford R. G., Talo A., Lakatta E. G., Capogrossi M. C. Simultaneous measurement of Ca2+, contraction, and potential in cardiac myocytes. Am J Physiol. 1990 Feb;258(2 Pt 2):H574–H586. doi: 10.1152/ajpheart.1990.258.2.H574. [DOI] [PubMed] [Google Scholar]
  29. Uto A., Arai H., Ogawa Y. Reassessment of Fura-2 and the ratio method for determination of intracellular Ca2+ concentrations. Cell Calcium. 1991 Jan;12(1):29–37. doi: 10.1016/0143-4160(91)90082-p. [DOI] [PubMed] [Google Scholar]
  30. Yue D. T., Marban E., Wier W. G. Relationship between force and intracellular [Ca2+] in tetanized mammalian heart muscle. J Gen Physiol. 1986 Feb;87(2):223–242. doi: 10.1085/jgp.87.2.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zhou Z., Neher E. Mobile and immobile calcium buffers in bovine adrenal chromaffin cells. J Physiol. 1993 Sep;469:245–273. doi: 10.1113/jphysiol.1993.sp019813. [DOI] [PMC free article] [PubMed] [Google Scholar]

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