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Besides affecting intracellular calcium signaling, 2-APB reversibly blocks gap junctional coupling in confluent monolayers, thereby allowing measurement of single-cell membrane currents in undissociated cells¹

ERIK G. A. HARKS^*, JESUS P. CAMIÑA^*,,, PETER H. J. PETERS^*, DIRK L. YPEY^*,, WIM J. J. M. SCHEENEN, EVERARDUS J. J. VAN ZOELEN^* and ALEXANDER P. R. THEUVENET^*,2

^* Department of Cell Biology, University of Nijmegen, The Netherlands;
Department of Physiology, Leiden University Medical Center, The Netherlands;
Department of Medicine, Molecular Endocrinology Laboratory, Complejo Hospitalario Universitario de Santiago (CHUS) and University of Santiago de Compostela, Spain; and
Department of Cellular Animal Physiology, University of Nijmegen, The Netherlands

²Correspondence: Toernooiveld 1, 6525 ED Nijmegen, The Netherlands. E-mail: ATheuv{at}sci.kun.nl

SPECIFIC AIMS

In the present study we aimed to use 2-aminoethoxydiphenyl borate (2-APB; see inset, Fig. 2 ) as a blocker of the IP₃ receptor and store-operated calcium channels to analyze agonist-induced intracellular calcium and membrane potential responses in confluent monolayers of normal rat kidney fibroblasts (NRK/49F). However, an unexpected finding was that 2-APB also effectively blocked gap junctional intercellular coupling of the cells.

View larger version (26K): [in this window] [in a new window]   Figure 2. Membrane currents recorded in NRK cells by applying the voltage-clamp step protocols as illustrated. A) Membrane currents measured in a monolayer of electrically coupled cells and B) under conditions where gap junctions were completely blocked by 50 µM 2-APB and C) by 100 µM meclofenamic acid (MFA). D) Membrane currents measured in a single NRK cell that had been dissociated from a confluent monolayer by trypsinization. E) The inward L-type calcium currents at voltage steps to 0 mV are replotted from panels B (monolayer) and D (single cell) for better resolution. The inset shows the chemical structures of 2-APB and MFA.

PRINCIPAL FINDINGS

1. 2-APB inhibits prostaglandin F2- induced intracellular calcium oscillations in NRK monolayer cells
NRK cells in confluent monolayers exhibit a stable membrane potential as a result of extensive electrical coupling. Fura measurements of intracellular calcium showed that addition of prostaglandin F2 (PGF2) to these cells induces unsynchronized calcium oscillations (Fig. 1 A, B) in addition to a steady depolarization of the monolayer as a whole (Fig. 1C ) that results from opening of calcium-activated chloride channels (cf. Fig. 3 ). 2-APB (75 µM) rapidly and reversibly blocked these oscillations (Fig. 1A, B ) and the corresponding calcium-dependent depolarization (Fig. 1C ). However, within a few minutes after the initial hyperpolarization, the cells depolarized again and developed strong membrane potential fluctuations (Fig. 1C ), indicative of electrical uncoupling of the cells.

View larger version (18K): [in this window] [in a new window]   Figure 1. 2-APB blocks the PGF2 response in confluent monolayers of NRK fibroblasts. A) The effect of PGF2 (100 nM) on the intracellular calcium level of an individual cell from a panel of 39 cells in the monolayer. The PGF2-induced intracellular calcium oscillation was blocked by 2-APB (75 µM). B) The average response of 39 cells from the same monolayer. C) Effect of 2-APB (75 µM) on the PGF2 (50 nM)-induced depolarization of the membrane of an NRK cell monolayer. The dashed line represents the effect on the membrane potential of PGF2 (50 nM) alone in the same monolayer determined after the 2-APB experiment.

View larger version (23K): [in this window] [in a new window]   Figure 3. Schematic representation of the PGF2 response in NRK monolayers and the effect of 2-APB on this response. PGF2 binds to the FP receptor, resulting in an activation of phospholipase C (PLC), which liberates IP3 from membrane phospho-inositides. IP3 then binds to its receptor (IP3-R) on the endoplasmic reticulum, thereby inducing a release of stored calcium. The increase of the intracellular calcium concentration causes the opening of calcium-dependent chloride channels (Gcl(ca)), which results in membrane depolarization from a resting membrane potential as determined by inward rectifier K+ channels (GKIR, cf. Fig. 2B, C ). 2-APB inhibits both the IP3 and SOC-mediated increase in the intracellular calcium concentration, thereby repolarizing the membrane. However, 2-APB also blocks gap junctional channels (GJ), resulting in uncoupling and fluctuating membrane depolarization. The L-type calcium channels in the diagram (GcaL, cf. Fig. 2E ) determine fibroblast excitability, which is not considered here.

2. 2-APB completely and reversibly blocks gap junctional intercellular communication
Single-electrode voltage-clamp experiments revealed that 2-APB indeed fully blocks gap junctional conductance between a patched NRK fibroblast and its surrounding cells in a confluent monolayer (see Fig. 2 A, B; note the difference in scale), because voltage-step evoked capacitive current transients changed from a coupled-cell to a single-cell transient. Half-maximal inhibition (IC₅₀) of electrical coupling in NRK cells was achieved at 5.7 µM, such that 2-APB blocked gap junctional coupling only when applied extracellularly but not via the patch pipette (n=18). Similar results were obtained for human embryonic kidney epithelial cells (HEK293/tsA201) with an IC₅₀ of 10.3 µM. Thus, the electrical uncoupling action described here is a new 2-APB property that complicated measurement of its effects on intracellular calcium signaling and membrane potential (see Fig. 1C ).

3. 2-APB allows measurement of single-cell membrane currents under voltage-clamp conditions in intact monolayers
Based on the above observation, we could use 2-APB as an electrical uncoupler of monolayer cells and measure inwardly rectifying potassium, L-type calcium, and calcium-dependent chloride membrane currents in individual cells of confluent NRK monolayers with properties similar to those observed in dissociated NRK cells in the absence of 2-APB (Fig. 2B, D ). Thus, the uncoupling action of 2-APB allowed us to uncover the single-cell excitability mechanism and its inward and outward currents in the intact fibroblastic monolayer culture. In contrast, in NRK monolayers electrically uncoupled by 100 µM meclofenamic acid (MFA; see inset, Fig. 2 ), no distinct single cell currents could be measured because of nonspecific effects of this type of drugs on ion channel conductances (Fig. 2C ).

CONCLUSIONS AND SIGNIFICANCE

An important conclusion from the present work is that effects of 2-APB on intracellular signaling and membrane potential via IP₃-sensitive stores and store-operated channels may also include effects of 2-APB on electrical coupling between the cells (Fig. 3 ), because 2-APB turned out to be an effective gap junctional blocker in two widely used cell lines (NRK and HEK). Because 2-APB seems to lack nonspecific effects on other ion channels, this observation provides the unique possibility to solve the long-standing problem in cellular electrophysiology of measuring single-cell properties from cells in intact tissues. To our knowledge, the present study shows for the first time reliable voltage-clamped ion currents from individual cells in an intact culture after electrically isolating the measured cell from its surrounding cells with the use of an uncoupler (2-APB). This procedure may open new possibilities to voltage-clamp individual cardiac myocytes in the myocardium and thereby separate excitability properties of the cells from conductive properties of the tissue, as explored here in fibroblastic cultures. It may also allow studies of the electrical properties of polarized cells in intact epithelia. The procedure is reversible and does not cause damage to the cells as in enzymatic cell dissociation. Finally, chemical uncoupling of cells with 2-APB may allow a discrimination between intra- and intercellular signaling processes (other than calcium signaling) in cell biological experiments.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0786fje; to cite this article, use FASEB J. (March 5, 2003) 10.1096/fj.02-0786fje

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