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, ca....

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 Sol....

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

Name	Company	Catalog Number	Comments
Name of Reagent/Material	Company	Catalog Number	Column1
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
Piezoelectric 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
Microelectrode puller	Zeitz Instrumente (Munchen, Germany)	WZ DMZ-Universal Puller