Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Summary

Intercellular Ca²⁺-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca²⁺-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Abstract

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca²⁺-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP₃ and Ca²⁺ that initiate the propagation of the Ca²⁺-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca²⁺-wave propagation are provided by gap junction channels through the direct transfer of IP₃ and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca²⁺-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca²⁺-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 μm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca²⁺-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Introduction

Intercellular communication and signaling are essential for the coordination of physiological processes in response to extracellular agonists at the tissue and whole-organ level ^1,2 . The most direct way of intercellular communication is created by the occurrence of gap junctions. Gap junctions are plaques of gap junction channels, which are proteinaceous channels formed by the head-to-head docking of two connexin (Cx) hemichannels of adjacent cells ^3,4 (Figure 1). Gap junctions allow the passage of small signaling molecules with a molecular weight of less than 1.5 kDa, including Ca²⁺ or IP₃ ⁵, causing and modulating Ca²⁺-release from the intracellular stores of the neighboring cells ⁶ (Figure 2). Gap junction channels are tightly regulated by intra- and intermolecular protein interactions and by cellular signaling processes, like redox modification and phosphorylation⁷. GJs facilitate the coordinated response of connected cells, thereby acting as a chemical and electrical syncytium. For example, the spreading of cardiac action potential across the atrial and ventricular myocytes is mediated by Cx-based GJ channels ⁸⁵. Cxs not only have a role as gap junction channels, but also form unpaired hemichannels, thereby functioning as channels in membranes similarly to regular ion channels ^8-10 (Figure 1). Hemichannels participate in paracrine signaling between neighboring cells by controlling the exchange of ions and signaling molecules between the intra- and extracellular environment.

In many cell types (like epithelial cells, osteoblastic cells, astrocytes, endothelial cells, etc.) and organs (like brain, liver, retina, cochlea and the vasculature), intercellular Ca²⁺-waves are fundamental for the coordination of multicellular responses ¹¹. Increases in intracellular Ca²⁺ levels in a certain cell are not limited to this cell, but propagate to the surrounding neighboring cells, thereby establishing an intercellular Ca²⁺-wave ^12,13 . These intercellular Ca²⁺-waves are important for normal physiological regulation of cell layers as a syncytium and their dysregulation has been associated with pathophysiological processes ¹¹. In the corneal endothelium and epithelium, different groups ^14-24, including our own ^25-33, studied the mechanisms and roles of intercellular communication. In non-excitable cells, like corneal endothelial cells, two distinct modes of intercellular communication occur ^28,29 , namely gap junctional intercellular communication and paracrine intercellular communication. Gap junctional intercellular communication involves a direct exchange of signaling molecules via gap junctions ⁷. Gap junctional intercellular communication is critical for maintaining tissue homeostasis, controlling cell proliferation, and establishing a synchronized response to extracellular stress ^10,34,35 . In a number of pathologies, gap junction coupling is reduced due to defective Cxs, and hereby affecting gap junctional intercellular communication ³⁶. This emphasizes the importance and influence of gap junctional intercellular communication in multicellular organisms. In contrast to gap junctional intercellular communication, paracrine intercellular communication is not dependent on cell-cell apposition, since it involves the release of diffusible extracellular messengers (Figure 2). Different types of signaling molecules are released in the extracellular space by signaling cells. The molecule is then transported to the target cell where it is detected by a specific receptor protein. Subsequently the receptor-signal complex induces a cellular response, which is terminated by removal of the signal, inactivation or desensitization. Released lipophilic extracellular signaling messengers penetrate the membrane and act on intracellular receptors. In contrast, hydrophilic messengers do not cross the plasma membrane of the responding cell, but act as a ligand that binds to surface-expressed receptor proteins, which then relay the signal to the intracellular environment. Three major families of cell surface receptor proteins participate in this process: ion-channel-linked, enzyme-linked, and G protein-linked. The released messenger molecule can act on receptors of the same cell (autocrine), on target cells in close proximity (paracrine), or on distant target cells that require the circulatory system (endocrine).

In many cell types, including corneal endothelium ^28,29, ATP is one of the major hydrophilic, paracrine factors that drive the propagation of intercellular Ca²⁺-waves ^37-40. During mechanical deformation, hypoxia, inflammation or stimulation by various agents, ATP can be released from healthy cells ^41-44 in response to shear stress, stretch, or osmotic swelling ^44,45. Different ATP-release mechanisms have been postulated, including vesicular exocytosis ⁴⁴ and a plethora of transport mechanisms, such as ATP-binding cassette (ABC) transporters, plasmalemmal voltage-dependent anion channels ⁴⁶, P2X7 receptor channels ^47,48, as well as connexin hemichannels ^49-52 and pannexin hemichannels ^43,49,53. Extracellular ATP can be rapidly hydrolyzed to ADP, AMP and adenosine ^54,55 by ectonucleotidases that are present in the extracellular environment. The extracellularly released ATP and its metabolite ADP ⁵⁶ will spread through diffusion. The subsequent interaction of these nucleotides with purinergic receptors in the neighboring cells has been implicated in the propagation of intercellular Ca²⁺-waves ^28,37,51. Two different classes of purinergic receptors are present: adenosine is the principal natural ligand for P1-purinoceptors, while both purine (ATP, ADP) and pyrimidine (UTP, UDP) nucleotides act on most P2-purinoceptors ⁵⁷.

Intercellular communication can be investigated by different methods such as scrape loading, dye transfer, local uncaging of agonists like IP₃ and Ca²⁺, mechanical stimulation, etc.. Here we describe the study of Ca²⁺-wave propagation elicited by mechanical stimulation of a single cell. The advantage of studying Ca²⁺-wave propagation by mechanical stimulation is that it provides an easy tool to quantify the spread of the Ca²⁺-wave over time and it allows quantitatively comparing different pretreatments of the cells. In the corneal endothelium, these intercellular Ca²⁺-waves allow a coordinated response from the monolayer, hereby acting as a possible defense mechanism of the non-regenerative corneal endothelium helping the endothelium to withstand extracellular stresses during intraocular surgery, or upon exposure to inflammatory mediators during immune rejection or uveitis ^58,59 .

Protocol

1. Isolation of Corneal Endothelial Cells

Before getting started: Isolate the cells from the fresh eyes, obtained from a local slaughterhouse, as soon as possible after enucleating the eye. Make sure that the eye was enucleated from a cow of maximal 18 months old, five minutes post mortem and preserved in Earle's Balanced Salt Solution - 1% iodine solution at 4 °C for transportation to the laboratory.

1. Take the eye out of the Earle's Balanced Salt Solution - 1% iodine solution and place it in a Petri dish (100 x 20 mm).
2. Sterilize the eye with a solution containing 70% ethanol and rinse with Earle's Balanced Salt Solution containing 1% iodine.
3. From this step on, work in a sterile hood. Carefully dissect the cornea from the eye and place it in a Petri dish (35 x 10 mm) containing Earle's Balanced Salt Solution, with the epithelial cell layer facing upward. Carefully remove any remaining iris tissue still attached to the cornea if needed.
4. Transfer the cornea to another Earle's Balanced Salt Solution containing Petri dish with the endothelial cell layer upward and rinse twice with Earle's Balanced Salt Solution.
5. Transfer the cornea with the endothelial layer facing upward to an hourglass, which is a cup-shaped dish, and cover it with growth medium. The growth medium consists of Dulbecco's Modified Eagle's Medium containing 25 mM glucose, 10% fetal bovine serum, 6.6% L-glutamine, 2.5 μg/ml amphotericin-B and 1% antibiotic-antimycotic mixture containing 10,000 units/ml of penicillin, 10,000 μg/ml of streptomycin, and 25 μg/ml amphotericin B.
6. Remove the medium with a suction pipette.
7. Apply 300 μl of a trypsin solution (0.5 g/L) to the endothelial layer of the cornea (all the steps that include trypsin are done using the same concentration).
8. Place the hourglass containing the cornea in a covered Petri dish and put it in the incubator for 30 min at 37 °C and 5% CO₂.
9. Gently scrape the endothelial cells away from the cornea with a fire-polished hook-shaped glass Pasteur pipette in a sterile hood.
10. Suck off the solution containing the endothelial cells and add it to culture flasks (25 cm²) containing 4 ml of culture medium.
11. Apply 300 μl of growth medium to the cornea and repeat the scraping and add the solution containing the endothelial cells to the culture flask.
12. Repeat this final step (1.11) once more.
13. Place the culture flasks containing the corneal endothelial cells in the growth medium in the incubator at 37 °C and 5% CO₂.
14. After two days, add 6 ml of culture medium.
15. Refresh the growth medium every second day.

2. Cell Culture

1. Remove the culture medium and wash the cells twice with Earle's Balanced Salt Solution, when confluency is reached (within about 10 days after isolation).
2. Add 1.5 ml trypsin solution to the cells to detach them and place the flask in the incubator (37 °C, 5% CO₂) for 3 to 4 min.
3. Add 12 ml of growth medium. Pipette the medium three times in and out to disperse the cells and count the cells.
4. Seed the cells with a variable fraction depending on the cell density and put them in the incubator. Prepare two well-chambered slides (with an area of 4.2 cm²) with a cell count of 165,000 cells (cell density 39,286 per cm²). Prepare 80 cm² culture flasks for a new passage at a density of 6,250 per cm², and add fresh culture medium up to a total volume of 25 ml.
5. Refresh the medium every two days.
6. Confluency of the cell layer is reached after 3 to 4 days. Use cells for experiments.
7. When confluency of the cell layer in the flasks is reached, repeat steps 2.1 to 2.6. Cell cultures up to passage 2 can be used for experiments.

3. Mechanical Stimulation for Inducing Calcium Wave

1. Load the cells in the chambered slide with 10 μM Fluo-4 AM in phosphate-buffered saline for 30 min at 37 °C while gently shaking.
2. Remove the Fluo-4 AM solution, wash the cells five times with phosphate-buffered saline, incubate the cells with phosphate-buffered saline and leave the cells for at least 5 min at room temperature before measurement.
3. Excite at 488 nm with Argon laser and use beam splitter HFT 488, collect the fluorescence emission at 530 nm using a longpass emission filter LP 505, set the pinhole at minimum. Use an oil immersion 40X objective (Air, 1.2 N.A.). In experiments with ARL-67156, use a 10X objective (Air, 0.3 N.A.).
4. Search for a field in which the cells are confluent on the confocal microscope.
5. Position the pipette so that it is at 45° in respect to the chambered slide and touching the cell membrane. Provoke a short (≈ 1 sec) mechanical stimulation to a single cell. The mechanical stimulation consists of an acute, short-lasting deformation of the cell by briefly touching less than 1% of the cell membrane with a glass micropipette (tip diameter <1 μm) coupled to a piezoelectric crystal nanopositioner, operated through an amplifier which is mounted on a micro-manipulator. The glass micropipettes are made with a microelectrode puller. Make sure that the nanopositioner is operated by a voltage between 0.2 and 1.5 V during the mechanical stimulation. A voltage higher than 1.5 V can result in cell damage. For each cell type and condition, the optimal voltage for mechanical stimulation without cell damage must be carefully determined by applying a series of voltages starting from low (0.2 V) to high (1.5 V) voltage. The voltage is a measure for the force of the stimulation since this voltage determines the mechanical stress and strain that are applied to the cell membrane. The force of the mechanical stimulation can be calculated by multiplying the mechanical stress with the area. Since both the area (<3.14 μm²) and the mechanical stress are very low, the force of the mechanical stimulation is low. (Note that when a cell is damaged, the fluorescence leaks out of the cell and the cell turns dark.)
6. Measure spatial changes in [Ca²⁺]_i following mechanical stimulation with the confocal microscope.
7. Collect and store images.
8. Draw a polygonal region of interest to define the total surface area of responsive cells (active area, AA) using the software of the confocal microscope.

Results

All experiments are executed in compliance with all relevant guidelines, regulations and regulatory agencies and the protocol being demonstrated is performed under the guidance and approval of the animal care and use committee of the KU Leuven.

In bovine corneal endothelial cells (BCEC), functional gap junctions are expressed and both gap junctional intercellular communication and paracrine intercellular communication contribute significantly to intercellular communication in an interactive wa...

Discussion

In this manuscript, we describe a simple method to measure intercellular Ca²⁺-wave propagation in monolayers of primary bovine corneal endothelial cells by providing a localized and controlled mechanical stimulation using a micropipette. Mechanically stimulated cells respond with a local increase in intracellular IP₃ and Ca²⁺, both of which are essential intracellular signaling molecules that drive intercellular Ca²⁺-wave propagation ^11,67 . IP₃ is directl...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Earle's Balanced Salt Solution (EBSS)	Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)	14155-048
Iodine	Sigma-Aldrich (Deisenhofen, Germany)	38060-1EA
Dulbecco's Modified Eagle's Medium (DMEM)	Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)	11960-044
L-glutamine (Glutamax)	Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)	35050-038
Amphotericin-B	Sigma-Aldrich (Deisenhofen, Germany)	A2942
Antibiotic-antimycotic mixture	Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)	15240-096
Trypsin	Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)	25300-054
Dulbecco's PBS	Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)	14190-091
Fluo-4 AM	Invitrogen-Gibco-Molecular Probes (Karlsruhe, Germany)	F14217
ARL-67156 (6-N,N-Diethyl-b,g-dibromomethylene-D-ATP)	Sigma-Aldrich (Deisenhofen, Germany)	A265
Apyrase VI	Sigma-Aldrich (Deisenhofen, Germany)	A6410
Apyrase VII	Sigma-Aldrich (Deisenhofen, Germany)	A6535
Gap26 (VCYDKSFPISHVR)	Custom peptide synthesis
Gap27 (SRPTEKTIFII)	Custom peptide synthesis
Control Peptide (SRGGEKNVFIV)	Custom peptide synthesis
siRNA1 Cx43 (sense: 5'GAAGGAGGAGGAACU-CAAAdTdT)	Annealed siRNA was purchased at Eurogentec (Luik, Belgium)
siRNA2 Cx43 (sense: 5'CAAUUCUUCCUGCCGCAAUdTdT)	Annealed siRNA was purchased at Eurogentec (Luik, Belgium)
siRNA scramble: scrambled sequence of siCx43-1 (sense: 5'GGUAAACG-GAACGAGAAGAdTdT)	Annealed siRNA was purchased at Eurogentec (Luik, Belgium)
TAT-L2 (TAT- DGANVDMHLKQIEIKKFKYGIEEHGK)	Thermo Electron (Ulm, Germany)
TAT-L2-H126K/I130N (TAT-DGANVDMKLKQNEIKKFKYGIEEHGK)	Thermo Electron (Ulm, Germany)
Two chambered glass slides	Laboratory-Tek Nunc (Roskilde, Denmark)	155380
Confocal microscope	Carl Zeiss Meditec (Jena, Germany)	LSM510
Piez–lectric crystal nanopositioner (Piezo Flexure NanoPositioner)	PI Polytech (Karlsruhe, Germany)	P-280
HVPZT-amplifier	PI Polytech (Karlsruhe, Germany)	E463 HVPZT-amplifier
Glass tubes (glass replacement 3.5 nanoliter)	World Precision Instruments, Inc. Sarasota, Florida, USA	4878
Micr–lectrode puller	Zeitz Instrumente (Munchen, Germany)	WZ DMZ-Universal Puller