In our early studies, we assessed the barrier integrity in terms of flux of FITC Dextran (10 kDa) and/or horse radish peroxidase (40 kDa), applied at the apical side of the corneal endothelial cells (Fig. 9A). To increase the throughput of our experiments and enable rapid testing of other bioactive factors in the aqueous humor, we began to examine TER by measurement of electrical impedance (Fig. 9B) as a measure of barrier integrity of confluent monolayers. We used electrical cell-substrate impedance sensing (ECIS, Applied Biophysics, NY), a commercial device designed to assess TER in up to 16-indepedent monolayers at a time. For such measurements, cells are grown to confluence on small, gelatin-coated gold electrodes. A small AC current is applied across the cells. The impedance for the current flow is measured by a lock-in amplifier at ∼0.1 Hz. The resistive component of the impedance for current flow is taken as TER because the plasma membrane resistance is relatively large.76 Fig. 9C shows evolution of the resistive component of the measured impedance with cell spreading on the gold electrodes. The steady state resistance is indicative of the barrier integrity. Fig. 9D shows the dynamics of TER measurements in response to cytochalasin D, which is known to break down barrier integrity in epithelia.60 As an inhibitor of actin polymerization, cytochalasin D breaks apart the PAMR, and consequently the barrier integrity is lost. These results clearly show that TER, as measured using the ECIS system, reflects the status of the barrier integrity. Similar findings have been reported contemporaneously by Yin and Watsky.77 Moreover, the ECIS protocol offers a sensitive and high-throughput approach to assess the barrier integrity of the leaky corneal endothelium.
I thank all my mentors (Drs. J. A. Bonanno, OD, PhD, D. Maurice, PhD, G. Lowther, OD, PhD, R. Mutharasan, PhD, S. Soni, OD, MS), collaborators (Drs. D. Jans, PhD, B. Himpens, MD, PhD, J. Vereecke, PhD, W. Van Driessche, PhD), postdoctoral fellow (Dr. M. Satpathy, PhD), and students (Dr. C. D'hondt, PhD, Dr. Gomes, PhD, Dr. Y. Guo, PhD, S. Jalimarada, R. Ponsaerts, C. Ramachandran, Dr. M. Shivanna, PhD). I also thank Dr. Tony Adams for organizing this review series and the anonymous reviewer for many useful suggestions and revisions.
Sangly P. Srinivas
1.Edelhauser HF. The balance between corneal transparency and edema: the Proctor Lecture. Invest Ophthalmol Vis Sci 2006;47:1754–67.
2.Maurice DM. The location of the fluid pump in the cornea
. J Physiol 1972;221:43–54.
3.Dikstein S, Maurice DM. The metabolic basis to the fluid pump in the cornea
. J Physiol 1972;221:29–41.
4.Riley M. Pump and leak in regulation of fluid transport in rabbit cornea
. Curr Eye Res 1985;4:371–6.
5.Fischbarg J, Maurice DM. An update on corneal hydration control. Exp Eye Res 2004;78:537–41.
6.Bonanno JA. Identity and regulation of ion transport mechanisms in the corneal endothelium
. Prog Retin Eye Res 2003;22:69–94.
7.Srinivas SP, Guan Y, Bonanno JA. Swelling activated chloride channels in cultured bovine corneal endothelial cells. Exp Eye Res 1999;68:165–77.
8.Srinivas SP, Bonanno JA, Lariviere E, Jans D, Van Driessche W. Measurement of rapid changes in cell volume by forward light scattering. Pflugers Arch 2003;447:97–108.
9.Srinivas SP, Bonanno JA, Hughes BA. Assessment of swelling-activated Cl- channels using the halide-sensitive fluorescent indicator 6-methoxy-N-(3-sulfopropyl)quinolinium. Biophys J 1998;75:115–23.
10.Srinivas SP, Bonanno JA. Measurement of changes in cell volume based on fluorescence quenching. Am J Physiol 1997;272:C1405–14.
11.Riley MV, Winkler BS, Peters MI, Czajkowski CA. Relationship between fluid transport and in situ inhibition of Na(+)-K+ adenosine triphosphatase in corneal endothelium
. Invest Ophthalmol Vis Sci 1994;35:560–7.
12.Bonanno JA, Yi G, Kang XJ, Srinivas SP. Reevaluation of Cl-/HCO3
- exchange in cultured bovine corneal endothelial cells. Invest Ophthalmol Vis Sci 1998;39:2713–22.
13.Bonanno JA, Srinivas SP, Brown M. Effect of acetazolamide on intracellular pH and bicarbonate transport in bovine corneal endothelium
. Exp Eye Res 1995;60:425–34.
14.Bonanno JA, Srinivas SP. Cyclic AMP activates anion channels in cultured bovine corneal endothelial cells. Exp Eye Res 1997;64:953–62.
15.Srinivas SP, Satpathy M, Guo Y, Anandan V. Histamine-induced phosphorylation of the regulatory light chain of myosin II disrupts the barrier integrity of corneal endothelial cells. Invest Ophthalmol Vis Sci 2006;47:4011–8.
16.Srinivas SP, Satpathy M, Gallagher P, Lariviere E, Van Driessche W. Adenosine induces dephosphorylation of myosin II regulatory light chain in cultured bovine corneal endothelial cells. Exp Eye Res 2004;79:543–51.
17.Shivanna M, Srinivas SP. Microtubule stabilization opposes the (TNF-alpha)-induced loss in the barrier integrity of corneal endothelium
. Exp Eye Res 2009;89:950–9.
18.Satpathy M, Gallagher P, Lizotte-Waniewski M, Srinivas SP. Thrombin-induced phosphorylation of the regulatory light chain of myosin II in cultured bovine corneal endothelial cells. Exp Eye Res 2004;79:477–86.
19.Satpathy M, Gallagher P, Jin Y, Srinivas SP. Extracellular ATP opposes thrombin-induced myosin light chain phosphorylation and loss of barrier integrity in corneal endothelial cells. Exp Eye Res 2005;81:183–92.
20.Riley MV, Winkler BS, Starnes CA, Peters MI, Dang L. Regulation of corneal endothelial barrier function by adenosine, cyclic AMP, and protein kinases. Invest Ophthalmol Vis Sci 1998;39:2076–84.
21.Ma L, Kuang K, Smith RW, Rittenband D, Iserovich P, Diecke FP, Fischbarg J. Modulation of tight junction properties relevant to fluid transport across rabbit corneal endothelium
. Exp Eye Res 2007;84:790–8.
22.Jalimarada SS, Shivanna M, Kini V, Mehta D, Srinivas SP. Microtubule disassembly breaks down the barrier integrity of corneal endothelium
. Exp Eye Res 2009;89:333–43.
23.Fischbarg J, Diecke FP, Iserovich P, Rubashkin A. The role of the tight junction in paracellular fluid transport across corneal endothelium
. electro-osmosis as a driving force. J Membr Biol 2006;210:117–30.
24.Sanchez JM, Li Y, Rubashkin A, Iserovich P, Wen Q, Ruberti JW, Smith RW, Rittenband D, Kuang K, Diecke FP, Fischbarg J. Evidence for a central role for electro-osmosis in fluid transport by corneal endothelium
. J Membr Biol 2002;187:37–50.
25.Levin MH, Verkman AS. Aquaporin-dependent water permeation at the mouse ocular surface: in vivo microfluorimetric measurements in cornea
and conjunctiva. Invest Ophthalmol Vis Sci 2004;45:4423–32.
26.Kuang K, Yiming M, Wen Q, Li Y, Ma L, Iserovich P, Verkman AS, Fischbarg J. Fluid transport across cultured layers of corneal endothelium
from aquaporin-1 null mice. Exp Eye Res 2004;78:791–8.
27.Diamond JM, Bossert WH. Standing-gradient osmotic flow. A mechanism for coupling of water and solute transport in epithelia. J Gen Physiol 1967;50:2061–83.
28.Hill AE. Fluid transport: a guide for the perplexed. J Membr Biol 2008;223:1–11.
29.Hill AE, Shachar-Hill B. A new approach to epithelial isotonic fluid transport: an osmosensor feedback model. J Membr Biol 2006;210:77–90.
30.Murakami M, Murdiastuti K, Hosoi K, Hill AE. AQP and the control of fluid transport in a salivary gland. J Membr Biol 2006;210:91–103.
31.Shachar-Hill B, Hill AE. Paracellular fluid transport by epithelia. Int Rev Cytol 2002;215:319–50.
32.Fischbarg J. Mechanism of fluid transport across corneal endothelium
and other epithelial layers: a possible explanation based on cyclic cell volume regulatory changes. Br J Ophthalmol 1997;81:85–9.
33.Srinivas SP, Maertens C, Goon LH, Goon L, Satpathy M, Yue BY, Droogmans G, Nilius B. Cell volume response to hyposmotic shock and elevated cAMP in bovine trabecular meshwork cells. Exp Eye Res 2004;78:15–26.
34.Mehta D, Malik AB. Signaling mechanisms regulating endothelial permeability. Physiol Rev 2006;86:279–367.
35.Maurice DM, Srinivas SP. Fluorometric measurement of light absorption by the rabbit cornea
. Exp Eye Res 1994;58:409–13.
36.Chiba H, Osanai M, Murata M, Kojima T, Sawada N. Transmembrane proteins of tight junctions
. Biochim Biophys Acta 2008;1778:588–600.
37.Takakuwa R, Kokai Y, Kojima T, Akatsuka T, Tobioka H, Sawada N, Mori M. Uncoupling of gate and fence functions of MDCK cells by the actin-depolymerizing reagent mycalolide B. Exp Cell Res 2000;257:238–44.
38.Mandel LJ, Bacallao R, Zampighi G. Uncoupling of the molecular ‘fence’ and paracellular ‘gate’ functions in epithelial tight junctions
. Nature 1993;361:552–5.
39.Pleyer U, Schlickeiser S. The taming of the shrew? The immunology of corneal transplantation. Acta Ophthalmol 2009;87:488–97.
40.Tan DT, Anshu A, Mehta JS. Paradigm shifts in corneal transplantation. Ann Acad Med Singapore 2009;38:332–8.
41.Patel SV, Bachman LA, Hann CR, Bahler CK, Fautsch MP. Human corneal endothelial cell transplantation in a human ex vivo model. Invest Ophthalmol Vis Sci 2009;50:2123–31.
42.Fu H, Larkin DF, George AJ. Immune modulation in corneal transplantation. Transplant Rev (Orlando) 2008;22:105–15.
43.Patel SV, Hodge DO, Bourne WM. Corneal endothelium
and postoperative outcomes 15 years after penetrating keratoplasty. Am J Ophthalmol 2005;139:311–9.
44.Niederkorn JY. Immune mechanisms of corneal allograft rejection. Curr Eye Res 2007;32:1005–16.
45.George AJ, Larkin DF. Corneal transplantation: the forgotten graft. Am J Transplant 2004;4:678–85.
46.Bourne WM, McLaren JW. Clinical responses of the corneal endothelium
. Exp Eye Res 2004;78:561–72.
47.Bourne WM. Biology of the corneal endothelium
in health and disease. Eye (Lond) 2003;17:912–8.
48.Koch S, Nusrat A. Dynamic regulation of epithelial cell fate and barrier function by intercellular junctions. Ann N Y Acad Sci 2009;1165:220–7.
49.Capaldo CT, Nusrat A. Cytokine regulation of tight junctions
. Biochim Biophys Acta 2009;1788:864–71.
50.Bruewer M, Samarin S, Nusrat A. Inflammatory bowel disease and the apical junctional complex. Ann N Y Acad Sci 2006;1072:242–52.
51.Utech M, Bruwer M, Nusrat A. Tight junctions
and cell-cell interactions. Methods Mol Biol 2006;341:185–95.
52.Ivanov AI, Nusrat A, Parkos CA. The epithelium in inflammatory bowel disease: potential role of endocytosis of junctional proteins in barrier disruption. Novartis Found Symp 2004;263:115–24.
53.Bruewer M, Luegering A, Kucharzik T, Parkos CA, Madara JL, Hopkins AM, Nusrat A. Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms. J Immunol 2003;171:6164–72.
54.Nusrat A, Turner JR, Madara JL. Molecular physiology and pathophysiology of tight junctions
. IV. Regulation of tight junctions
by extracellular stimuli: nutrients, cytokines, and immune cells. Am J Physiol Gastrointest Liver Physiol 2000;279:G851–7.
55.Walsh SV, Hopkins AM, Nusrat A. Modulation of tight junction structure and function by cytokines. Adv Drug Deliv Rev 2000;41:303–13.
56.Dudek SM, Garcia JG. Cytoskeletal regulation of pulmonary vascular permeability. J Appl Physiol 2001;91:1487–500.
57.Garcia JG, Davis HW, Patterson CE. Regulation of endothelial cell gap formation and barrier dysfunction: role of myosin light chain phosphorylation. J Cell Physiol 1995;163:510–22.
58.Turner JR. ‘Putting the squeeze’ on the tight junction: understanding cytoskeletal regulation. Semin Cell Dev Biol 2000;11:301–8.
59.Turner JR. Molecular basis of epithelial barrier regulation: from basic mechanisms to clinical application. Am J Pathol 2006;169:1901–9.
60.Madara JL, Barenberg D, Carlson S. Effects of cytochalasin D on occluding junctions of intestinal absorptive cells: further evidence that the cytoskeleton may influence paracellular permeability and junctional charge selectivity. J Cell Biol 1986;102:2125–36.
61.Madara JL, Moore R, Carlson S. Alteration of intestinal tight junction structure and permeability by cytoskeletal contraction. Am J Physiol 1987;253:C854–61.
62.Turner JR, Rill BK, Carlson SL, Carnes D, Kerner R, Mrsny RJ, Madara JL. Physiological regulation of epithelial tight junctions
is associated with myosin light-chain phosphorylation. Am J Physiol 1997;273:C1378–85.
63.Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 2003;83:1325–58.
64.Ding HL, Ryder JW, Stull JT, Kamm KE. Signaling processes for initiating smooth muscle contraction upon neural stimulation. J Biol Chem 2009;284:15541–8.
65.Kamm KE, Stull JT. Dedicated myosin light chain kinases with diverse cellular functions. J Biol Chem 2001;276:4527–30.
66.Ramachandran C, Satpathy M, Mehta D, Srinivas SP. Forskolin induces myosin light chain dephosphorylation in bovine trabecular meshwork cells. Curr Eye Res 2008;33:169–76.
67.D'Hondt C, Ponsaerts R, Srinivas SP, Vereecke J, Himpens B. Thrombin inhibits intercellular calcium wave propagation in corneal endothelial cells by modulation of hemichannels and gap junctions. Invest Ophthalmol Vis Sci 2007;48:120–33.
68.D'Hondt C, Ponsaerts R, Srinivas SP, Vereecke J, Himpens B. Reduced intercellular communication and altered morphology of bovine corneal endothelial cells with prolonged time in cell culture. Curr Eye Res 2009;34:454–65.
69.D'Hondt C, Srinivas SP, Vereecke J, Himpens B. Adenosine opposes thrombin-induced inhibition of intercellular calcium wave in corneal endothelial cells. Invest Ophthalmol Vis Sci 2007;48:1518–27.
70.Guo Y, Ramachandran C, Satpathy M, Srinivas SP. Histamine-induced myosin light chain phosphorylation breaks down the barrier integrity of cultured corneal epithelial cells. Pharm Res 2007;24:1824–33.
71.Guo Y, Satpathy M, Wilson G, Srinivas SP. Benzalkonium chloride induces dephosphorylation of myosin light chain in cultured corneal epithelial cells. Invest Ophthalmol Vis Sci 2007;48:2001–8.
72.Ponsaerts R, D'Hondt C, Bultynck G, Srinivas SP, Vereecke J, Himpens B. The myosin II ATPase inhibitor blebbistatin prevents thrombin-induced inhibition of intercellular calcium wave propagation in corneal endothelial cells. Invest Ophthalmol Vis Sci 2008;49:4816–27.
73.Ramachandran C, Srinivas SP. Actomyosin contraction
regulates formation and disassembly of adherens and tight junctions
in the corneal endothelium
. Invest Ophthalmol Vis Sci 2009 Dec 17, PMID: 20019371. (Epub)
74.Shivanna M, Rajashekhar G, Srinivas S. Barrier dysfunction of the corneal endothelium
in response to TNF-α involves activation of p38 MAP kinase. Invest Ophthalmol Vis Sci 2009 Sep 24. PMID: 19797215. (Epub)
75.Shivanna M, Jalimarada SS, Srinivas SP. Effects of lovastatin on actin cytoskeleton in cultured bovine corneal endothelial cells. J Ocul Pharmacol Ther 2009. (In Press)
76.Tiruppathi C, Malik AB, Del Vecchio PJ, Keese CR, Giaever I. Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function. Proc Natl Acad Sci U S A 1992;89:7919–23.
77.Yin F, Watsky MA. LPA and S1P increase corneal epithelial and endothelial cell transcellular resistance. Invest Ophthalmol Vis Sci 2005;46:1927–33.
78.Rayner SA, Larkin DF, George AJ. TNF receptor secretion after ex vivo adenoviral gene transfer to cornea
and effect on in vivo graft survival. Invest Ophthalmol Vis Sci 2001;42:1568–73.
79.Rayner SA, King WJ, Comer RM, Isaacs JD, Hale G, George AJ, Larkin DF. Local bioactive tumour necrosis factor (TNF) in corneal allotransplantation. Clin Exp Immunol 2000;122:109–16.
80.Watsky MA, Guan Z, Ragsdale DN. Effect of tumor necrosis factor alpha on rabbit corneal endothelial permeability. Invest Ophthalmol Vis Sci 1996;37:1924–9.
81.Petrache I, Birukova A, Ramirez SI, Garcia JG, Verin AD. The role of the microtubules in tumor necrosis factor-alpha-induced endothelial cell permeability. Am J Respir Cell Mol Biol 2003;28:574–81.
82.Birukova AA, Smurova K, Birukov KG, Usatyuk P, Liu F, Kaibuchi K, Ricks-Cord A, Natarajan V, Alieva I, Garcia JG, Verin AD. Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: role of Rho-dependent mechanisms. J Cell Physiol 2004;201:55–70.
83.Gomes P, Srinivas SP, Vereecke J, Himpens B. Gap junctional intercellular communication in bovine corneal endothelial cells. Exp Eye Res 2006;83:1225–37.
84.Gomes P, Srinivas SP, Vereecke J, Himpens B. ATP-dependent paracrine intercellular communication in cultured bovine corneal endothelial cells. Invest Ophthalmol Vis Sci 2005;46:104–13.
85.Gomes P, Srinivas SP, Van Driessche W, Vereecke J, Himpens B. ATP release through connexin hemichannels in corneal endothelial cells. Invest Ophthalmol Vis Sci 2005;46:1208–18.