The epidermal Ca(2+) gradient: Measurement using the phasor representation of fluorescent lifetime imaging.

Ionic gradients are found across a variety of tissues and organs. In this report, we apply the phasor representation of fluorescence lifetime imaging data to the quantitative study of ionic concentrations in tissues, overcoming technical problems of tissue thickness, concentration artifacts of ion-sensitive dyes, and calibration across inhomogeneous tissue. We used epidermis as a model system, as Ca(2+) gradients in this organ have been shown previously to control essential biologic processes of differentiation and formation of the epidermal permeability barrier. The approach described here allowed much better localization of Ca(2+) stores than those used in previous studies, and revealed that the bulk of free Ca(2+) measured in the epidermis comes from intracellular Ca(2+) stores such as the Golgi and the endoplasmic reticulum, with extracellular Ca(2+) making a relatively small contribution to the epidermal Ca(2+) gradient. Due to the high spatial resolution of two-photon microscopy, we were able to measure a marked heterogeneity in average calcium concentrations from cell to cell in the basal keratinocytes. This finding, not reported in previous studies, calls into question the long-held hypothesis that keratinocytes increase intracellular Ca(2+), cease proliferation, and differentiate passively in response to changes in extracellular Ca(2+). The experimental results obtained using this approach illustrate the power of the experimental and analytical techniques outlined in this report. Our approach can be used in mechanistic studies to address the formation, maintenance, and function of the epidermal Ca(2+) gradient, and it should be broadly applicable to the study of other tissues with ionic gradients.

[1]  P. Elias,et al.  Calcium and potassium are important regulators of barrier homeostasis in murine epidermis. , 1992, The Journal of clinical investigation.

[2]  P. Elias,et al.  Ultrastructural localization of calcium in psoriatic and normal human epidermis. , 1991, Archives of dermatology.

[3]  K. Turksen,et al.  Permeability barrier dysfunction in transgenic mice overexpressing claudin 6. , 2002, Development.

[4]  J. Pallon,et al.  Pixe analysis of pathological skin with special reference to psoriasis and atopic dry skin. , 1996, Cellular and Molecular Biology.

[5]  E. Gratton,et al.  Oxygen distribution and migration within Mbdes Fe and Hbdes Fe. Multifrequency phase and modulation fluorometry study. , 1984, Biophysical Journal.

[6]  P. Elias,et al.  Localization of calcium in murine epidermis following disruption and repair of the permeability barrier , 1992, Cell and Tissue Research.

[7]  D. Ausiello,et al.  Effects of the renal medullary pH and ionic environment on vasopressin binding and signaling. , 2008, Kidney international.

[8]  Kaori Inoue,et al.  Calcium ion gradients and dynamics in cultured skin slices of rat hindpaw in response to stimulation with ATP. , 2009, The Journal of investigative dermatology.

[9]  C. Oomens,et al.  Mechanisms that play a role in the maintenance of the calcium gradient in the epidermis , 2007, Skin research and technology : official journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging.

[10]  T. Bunse,et al.  PIXE analysis in uninvolved skin of atopic patients and aged skin. , 1991, Acta dermato-venereologica.

[11]  Karen Holbrook,et al.  Calcium regulation of growth and differentiation of mouse epidermal cells in culture , 1980, Cell.

[12]  J. Lakowicz Emerging applications of fluorescence spectroscopy to cellular imaging: lifetime imaging, metal-ligand probes, multi-photon excitation and light quenching. , 1996, Scanning microscopy. Supplement.

[13]  J. Lakowicz,et al.  Possibility of simultaneously measuring low and high calcium concentrations using Fura-2 and lifetime-based sensing. , 1995, Cell calcium.

[14]  C. V. van Donkelaar,et al.  Distinct developmental changes in the distribution of calcium, phosphorus and sulphur during fetal growth‐plate development , 2007, Journal of anatomy.

[15]  Kenneth R Feingold,et al.  Origin of the epidermal calcium gradient: regulation by barrier status and role of active vs passive mechanisms. , 2002, The Journal of investigative dermatology.

[16]  Enrico Gratton,et al.  NHE1 Regulates the Stratum Corneum Permeability Barrier Homeostasis , 2002, The Journal of Biological Chemistry.

[17]  D. Bikle,et al.  Inactivation of the Calcium Sensing Receptor Inhibits E-cadherin-mediated Cell-Cell Adhesion and Calcium-induced Differentiation in Human Epidermal Keratinocytes* , 2008, Journal of Biological Chemistry.

[18]  P. Elias,et al.  Acute barrier perturbation abolishes the Ca2+ and K+ gradients in murine epidermis: quantitative measurement using PIXE. , 1998, The Journal of investigative dermatology.

[19]  Enrico Gratton,et al.  Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient. , 2002, Biophysical journal.

[20]  A. Cowley,et al.  Reactive oxygen species and molecular regulation of renal oxygenation. , 2003, Acta physiologica Scandinavica.

[21]  E. Gratton,et al.  The phasor approach to fluorescence lifetime imaging analysis. , 2008, Biophysical journal.

[22]  H Honjo,et al.  The sinoatrial node, a heterogeneous pacemaker structure. , 2000, Cardiovascular research.

[23]  T. Pozzan,et al.  Human keratinocyte ATP2C1 localizes to the Golgi and controls Golgi Ca2+ stores. , 2003, The Journal of investigative dermatology.

[24]  W. Idler,et al.  Profilaggrin is a major epidermal calcium-binding protein , 1993, Molecular and cellular biology.

[25]  D. Bikle,et al.  The Extracellular Calcium-sensing Receptor Is Required for Calcium-induced Differentiation in Human Keratinocytes* , 2001, The Journal of Biological Chemistry.

[26]  R. Guy,et al.  Determination of the pH gradient across the stratum corneum. , 1998, The journal of investigative dermatology. Symposium proceedings.

[27]  P. Elias,et al.  Stress alters cutaneous permeability barrier homeostasis. , 2000, American journal of physiology. Regulatory, integrative and comparative physiology.

[28]  H. Green,et al.  Presence in human epidermal cells of a soluble protein precursor of the cross-linked envelope: Activation of the cross-linking by calcium ions , 1979, Cell.

[29]  E. Sideras-Haddad,et al.  Formation of the epidermal calcium gradient coincides with key milestones of barrier ontogenesis in the rodent. , 1998, The Journal of investigative dermatology.

[30]  H. Gerritsen,et al.  Fast fluorescence lifetime imaging of calcium in living cells. , 2004, Journal of biomedical optics.

[31]  B. Hyman,et al.  Synchronous Hyperactivity and Intercellular Calcium Waves in Astrocytes in Alzheimer Mice , 2009, Science.

[32]  Tsutomu Araki,et al.  Finding of Optimal Calcium Ion Probes for Fluorescence Lifetime Measurement , 2005 .