Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1998 Oct;75(4):1635–1647. doi: 10.1016/S0006-3495(98)77606-4

Two-dimensional determination of the cellular Ca2+ binding in bovine chromaffin cells.

M Naraghi 1, T H Müller 1, E Neher 1
PMCID: PMC1299836  PMID: 9746506

Abstract

The spatiotemporal profile of intracellular calcium signals is determined by the flux of calcium ions across different biological membranes as well as by the diffusional mobility of calcium and different calcium buffers in the cell. To arrive at a quantitative understanding of the determinants of these signals, one needs to dissociate the flux contribution from the redistribution and buffering of calcium. Since the cytosol can be heterogeneous with respect to its calcium buffering property, it is essential to assess this property in a spatially resolved manner. In this paper we report on two different methods to estimate the cellular calcium binding of bovine adrenal chromaffin cells. In the first method, we use voltage-dependent calcium channels as a source to generate calcium gradients in the cytosol. Using imaging techniques, we monitor the dissipation of these gradients to estimate local apparent calcium diffusion coefficients and, from these, local calcium binding ratios. This approach requires a very high signal-to-noise ratio of the calcium measurement and can be used when well-defined calcium gradients can be generated throughout the cell. In the second method, we overcome these problems by using calcium-loaded DM-nitrophen as a light-dependent calcium source to homogeneously and quantitatively release calcium in the cytosol. By measuring [Ca2+] directly before and after the photorelease process and knowing the total amount of calcium being released photolytically, we get an estimate of the fraction of calcium ions which does not appear as free calcium and hence must be bound to either the indicator dye or the endogenous calcium buffer. This finally results in a two-dimensional map of the distribution of the immobile endogenous calcium buffer. We did not observe significant variations of the cellular calcium binding at a spatial resolution of approximately 2 micron. Furthermore, the calcium binding is not reduced by increasing the resting [Ca2+] to levels as high as 1.1 microM. This is indicative of a low calcium affinity of the corresponding buffers and is in agreement with a recent report on the affinity of these buffers (Xu, T., M. Naraghi, H. Kang, and E. Neher. 1997. Biophys. J. 73:532-545). In contrast to the homogeneous distribution of the calcium buffers, the apparant calcium diffusion coefficient did show inhomogeneities, which can be attributed to restricted diffusion at the nuclear envelope and to rim effects at the cell membrane.

Full Text

The Full Text of this article is available as a PDF (511.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Allbritton N. L., Meyer T., Stryer L. Range of messenger action of calcium ion and inositol 1,4,5-trisphosphate. Science. 1992 Dec 11;258(5089):1812–1815. doi: 10.1126/science.1465619. [DOI] [PubMed] [Google Scholar]
  2. Chad J. E., Eckert R. Calcium domains associated with individual channels can account for anomalous voltage relations of CA-dependent responses. Biophys J. 1984 May;45(5):993–999. doi: 10.1016/S0006-3495(84)84244-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clapham D. E. Calcium signaling. Cell. 1995 Jan 27;80(2):259–268. doi: 10.1016/0092-8674(95)90408-5. [DOI] [PubMed] [Google Scholar]
  4. Ellis-Davies G. C., Kaplan J. H., Barsotti R. J. Laser photolysis of caged calcium: rates of calcium release by nitrophenyl-EGTA and DM-nitrophen. Biophys J. 1996 Feb;70(2):1006–1016. doi: 10.1016/S0006-3495(96)79644-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Heinemann C., Chow R. H., Neher E., Zucker R. S. Kinetics of the secretory response in bovine chromaffin cells following flash photolysis of caged Ca2+. Biophys J. 1994 Dec;67(6):2546–2557. doi: 10.1016/S0006-3495(94)80744-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hüser J., Lipsius S. L., Blatter L. A. Calcium gradients during excitation-contraction coupling in cat atrial myocytes. J Physiol. 1996 Aug 1;494(Pt 3):641–651. doi: 10.1113/jphysiol.1996.sp021521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Imredy J. P., Yue D. T. Submicroscopic Ca2+ diffusion mediates inhibitory coupling between individual Ca2+ channels. Neuron. 1992 Aug;9(2):197–207. doi: 10.1016/0896-6273(92)90159-b. [DOI] [PubMed] [Google Scholar]
  9. Kaplan J. H., Ellis-Davies G. C. Photolabile chelators for the rapid photorelease of divalent cations. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6571–6575. doi: 10.1073/pnas.85.17.6571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kasai H., Augustine G. J. Cytosolic Ca2+ gradients triggering unidirectional fluid secretion from exocrine pancreas. Nature. 1990 Dec 20;348(6303):735–738. doi: 10.1038/348735a0. [DOI] [PubMed] [Google Scholar]
  11. Llinás R., Sugimori M., Silver R. B. The concept of calcium concentration microdomains in synaptic transmission. Neuropharmacology. 1995 Nov;34(11):1443–1451. doi: 10.1016/0028-3908(95)00150-5. [DOI] [PubMed] [Google Scholar]
  12. Markram H., Sakmann B. Calcium transients in dendrites of neocortical neurons evoked by single subthreshold excitatory postsynaptic potentials via low-voltage-activated calcium channels. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5207–5211. doi: 10.1073/pnas.91.11.5207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mathias R. T., Cohen I. S., Oliva C. Limitations of the whole cell patch clamp technique in the control of intracellular concentrations. Biophys J. 1990 Sep;58(3):759–770. doi: 10.1016/S0006-3495(90)82418-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Naraghi M. T-jump study of calcium binding kinetics of calcium chelators. Cell Calcium. 1997 Oct;22(4):255–268. doi: 10.1016/s0143-4160(97)90064-6. [DOI] [PubMed] [Google Scholar]
  15. Neher E., Augustine G. J. Calcium gradients and buffers in bovine chromaffin cells. J Physiol. 1992 May;450:273–301. doi: 10.1113/jphysiol.1992.sp019127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Neher E. The use of fura-2 for estimating Ca buffers and Ca fluxes. Neuropharmacology. 1995 Nov;34(11):1423–1442. doi: 10.1016/0028-3908(95)00144-u. [DOI] [PubMed] [Google Scholar]
  17. O'Sullivan A. J., Cheek T. R., Moreton R. B., Berridge M. J., Burgoyne R. D. Localization and heterogeneity of agonist-induced changes in cytosolic calcium concentration in single bovine adrenal chromaffin cells from video imaging of fura-2. EMBO J. 1989 Feb;8(2):401–411. doi: 10.1002/j.1460-2075.1989.tb03391.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Smith C., Neher E. Multiple forms of endocytosis in bovine adrenal chromaffin cells. J Cell Biol. 1997 Nov 17;139(4):885–894. doi: 10.1083/jcb.139.4.885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wagner J., Keizer J. Effects of rapid buffers on Ca2+ diffusion and Ca2+ oscillations. Biophys J. 1994 Jul;67(1):447–456. doi: 10.1016/S0006-3495(94)80500-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Williams D. A., Fogarty K. E., Tsien R. Y., Fay F. S. Calcium gradients in single smooth muscle cells revealed by the digital imaging microscope using Fura-2. Nature. 1985 Dec 12;318(6046):558–561. doi: 10.1038/318558a0. [DOI] [PubMed] [Google Scholar]
  21. Xu T., Naraghi M., Kang H., Neher E. Kinetic studies of Ca2+ binding and Ca2+ clearance in the cytosol of adrenal chromaffin cells. Biophys J. 1997 Jul;73(1):532–545. doi: 10.1016/S0006-3495(97)78091-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Zucker R. S. The calcium concentration clamp: spikes and reversible pulses using the photolabile chelator DM-nitrophen. Cell Calcium. 1993 Feb;14(2):87–100. doi: 10.1016/0143-4160(93)90079-l. [DOI] [PubMed] [Google Scholar]
  24. al-Baldawi N. F., Abercrombie R. F. Cytoplasmic calcium buffer capacity determined with Nitr-5 and DM-nitrophen. Cell Calcium. 1995 Jun;17(6):409–421. doi: 10.1016/0143-4160(95)90087-x. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES