Cx43-hemichannel function and regulation in physiology and pathophysiology: insights from the bovine corneal endothelial cell system and beyond

Intercellular communication in primary bovine corneal endothelial cells (BCECs) is mainly driven by the release of extracellular ATP through Cx43 hemichannels. Studying the characteristics of Ca2+-wave propagation in BCECs, an important form of intercellular communication, in response to physiological signaling events has led to the discovery of important insights in the functional properties and regulation of native Cx43 hemichannels. Together with ectopic expression models for Cx43 hemichannels and truncated/mutated Cx43 versions, it became very clear that loop/tail interactions play a key role in controlling the activity of Cx43 hemichannels. Interestingly, the negative regulation of Cx43 hemichannels by enhanced actin/myosin contractility seems to impinge upon loss of these loop/tail interactions essential for opening Cx43 hemichannels. Finally, these molecular insights have spurred the development of novel peptide tools that can selectively inhibit Cx43 hemichannels, but neither Cx43 gap junctions nor hemichannels formed by other Cx isoforms. These tools now set the stage to hunt for novel physiological functions for Cx43 hemichannels in primary cells and tissues and to tackle disease conditions associated with excessive, pathological Cx43-hemichannel openings

Connexins : substrates and regulators of autophagy

Connexins mediate intercellular communication by assembling into hexameric channel complexes that act as hemichannels and gap junction channels. Most connexins are characterized by a very rapid turn-over in a variety of cell systems. The regulation of connexin turn-over by phosphorylation and ubiquitination events has been well documented. Moreover, different pathways have been implicated in connexin degradation, including proteasomal and lysosomal-based pathways. Only recently, autophagy emerged as an important connexin-degradation pathway for different connexin isoforms. As such, conditions well known to induce autophagy have an immediate impact on the connexin-expression levels. This is not only limited to experimental conditions but also several pathophysiological conditions associated with autophagy (dys) function affect connexin levels and their presence at the cell surface as gap junctions. Finally, connexins are not only substrates of autophagy but also emerge as regulators of the autophagy process. In particular, several connexin isoforms appear to recruit pre-autophagosomal autophagy-related proteins, including Atg16 and PI3K-complex components, to the plasma membrane, thereby limiting their availability and capacity for regulating autophagy

RhoA GTPase switch controls Cx43-hemichannel activity through the contractile system

ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho) physiological conditions of stress

Modulation of gap junctional and paracrine intercellular communication by phosphorylation in bovine corneal endothelial cells

The corneal endothelium is a non-regenerative monolayer of polygonal cells at the posterior surface of the cornea. This monolayer plays an important role in regulating the hydration state of the corneal stroma. Loss of an intact endothelial monolayer (e.g., by apoptosis through age, inflammation, corneal endotheliopathies or trauma) induces corneal edema, which causes the cornea to become cloudy, resulting in loss of transparency and visual acuity. In situations of inflammation, thrombin can be generated in the cornea since all components for its production are present in the avascular cornea. Thrombin is known to break down interendothelial junctions, resulting in loss of barrier integrity of the corneal endothelium and formation of interendothelial gaps. Formation of intercellular gaps can diminish the number of gap junctions connecting the endothelial cells, therefore, gap formation is likely to affect intercellular communication (IC). Since IC is important in coordinating the response of cells to external stresses, we investigated the effect of thrombin on IC in corneal endothelial cells. We studied the effect of thrombin on intercellular calcium wave propagation evoked by mechanical stimulation of a single cell in a monolayer of bovine corneal endothelial cells (BCEC), by using calcium fluorescence imaging via laser scanning confocal microscopy and by measuring the area reached by the calcium wave, called active area (AA), the percentage responsive neighboring cells and the normalized fluorescence of the cells. We also investigated the effect on the gap junctional (GJIC) and paracrine (PIC) components of IC. We studied GJIC by measuring fluorescence recovery after photobleaching (FRAP) and by using the gap junction blocking connexin mimetic peptide Gap27. PIC (which was previously shown to be due to ATP release via hemichannels) was studied by investigating the effect of apyrase (which hydrolyzes ATP and ADP), by application of the ectonucleotidase inhibitor ARL-67156, by measuring ATP release, by studying the effects of the hemichannel blocking connexin mimetic peptide Gap26, and by measuring Lucifer Yellow (LY) dye uptake in calcium-free condition, which is an indicator of open hemichannels. Since time in culture can affect cell size and thereby influence the interpretation of measurements of IC, we first investigated the effect of time in culture on cell size and IC in BCEC. We demonstrated that cultured BCEC show a 3.5-fold increase in cell size with time in culture, we also showed that cell size of BCEC in passage 3 is significantly different from cell size of BCEC cultured for the same period in passage 1 or 2. In the intact bovine cornea we also showed an age-related increase in cell size. For the IC studies we therefore only used cultured BCEC established from animals with a maximum age of 18 months, and only used cells cultured for 8 to 14 days of passage 1 or 2. Measurements of active area, percentage responsive neighboring cells as well as normalized fluorescence demonstrated that thrombin application caused a marked reduction of intercellular propagation of calcium waves in BCEC. FRAP measurements and measurements of the effect of Gap27 showed a reduction of GJIC by thrombin. Hydrolysis of extracellular ATP by application of apyrase markedly reduced calcium wave propagation and precluded the effect of thrombin. Application of the ectonucleotidase inhibitor ARL-67156 caused a pronounced enhancement of calcium wave propagation, and pretreatment with the drug markedly enhanced the inhibitory effect of thrombin. Thrombin markedly decreased ATP release and Lucifer Yellow uptake. These experiments demonstrate that thrombin has a strong inhibitory effect on PIC. We showed that thrombin exerts its effect on IC through PAR-1 activation. RT-PCR and immunocytochemistry demonstrated expression of thrombin receptor PAR-1 and the trypsin- activated PAR-2. Experiments with PAR-1 and -4 antagonists and with the selective PAR-1 agonist TRAP-6 demonstrated that the effect of thrombin is mediated via PAR-1 activation. The effects of thrombin on GJIC and PIC could be mimicked by TRAP-6, demonstrating that the effects of thrombin on GJIC as well as on PIC were mediated via PAR-1. Our experiments also provided evidence that the effect of thrombin or TRAP-6 on IC is mediated by enhanced phosphorylation of MLC. Thrombin affects myosin light chain kinase (MLCK) as well as myosin light chain phosphatase (MLCP). Inhibition of MLCK with the MLCK inhibitor ML-7 overcame the effect of thrombin or TRAP-6 on calcium wave propagation, ATP-release, FRAP and LY dye uptake. The activity of the MLCP is regulated by Rho-associated kinase (ROCK) and PKC: inhibition of ROCK with Y-27632 and/or PKC-inhibition with chelerythrine also limited the effect of thrombin or TRAP-6 on calcium wave propagation, ATP-release, FRAP and LY dye uptake. Since application of adenosine is known to preclude the breakdown of barrier integrity by thrombin, we investigated the effect of activation and inhibition of purinergic receptors on the effect of thrombin on IC. We demonstrated that adenosine is able to overcome the inhibitory effect of thrombin or TRAP-6 on calcium wave propagation, ATP-release, FRAP and LY dye uptake in BCEC. While the potent A2B agonist NECA could prevent the inhibitory effect of thrombin on calcium wave propagation and ATP release, the selective A1 agonist CPA did not oppose the thrombin-induced inhibition of IC. Pretreatment of BCEC with forskolin (to directly activate adenylate cyclase) or the combination of forskolin and rolipram (a cAMP-dependent phosphodiesterase inhibitor) significantly inhibited the effect of thrombin. Thus, adenosine overcomes the inhibitory effect of thrombin on IC through A2B activation resulting in cAMP formation, which is known to inhibit activation of RhoA, an activator of ROCK. Previously it was shown that P2Y1 and P2Y2 contributed to PIC in BCEC. By RT-PCR and immunocytochemistry, we showed expression of the ADP-sensitive P2Y12 receptor. Pretreatment with the P2Y12 antagonists AR-C69931MX and MRS2395 showed that P2Y12 activation contributes to IC upon mechanical stimulation. However, in contrast to the potentiation of the effect of PAR-1 activation on platelet aggregation, P2Y12 activation does not potentiate the effect of thrombin on IC in BCEC. In conclusion: Thrombin inhibits intercellular calcium wave propagation in BCEC. This effect is due to activation of PAR-1 receptors and involves MLC phosphorylation by MLCK-, PKC- and Rho kinase-sensitive pathways. Thrombin mainly inhibits the ATP-mediated PIC pathway, and also reduces GJIC to a lesser extent. Adenosine prevents the thrombin-induced inhibition of hemichannel-mediated PIC and of GJIC. The mechanism involves an increase in cAMP concentration, which results in inhibition of RhoA and a subsequent decrease in MLC phosphorylation via enhanced MLCP. Since decrease in MLC phosphorylation causes a decrease in contractility of the actin cytoskeleton, our results suggest possible effects of the actin cytoskeleton on gap junctions and also on hemichannels. These findings are important for understanding the processes involved in corneal inflammation.TABLE OF CONTENTS I LIST OF ABBREVIATIONS V CHAPTER I. INTRODUCTION 1 I. CORNEA 2 A. CORNEA: STRUCTURE AND FUNCTION 2 B. CORNEAL ENDOTHELIUM 5 II. ENDOTHELIAL CELLS AND THROMBIN 24 A. VASCULAR ENDOTHELIUM 24 B. CORNEAL ENDOTHELIUM 30 CHAPTER II. AIMS 33 CHAPTER III. MATERIALS AND METHODS 37 I. PRODUCTS 38 II. ISOLATION AND CELL CULTURES 40 A. ISOLATION AND CULTURE OF BCEC 40 B. ISOLATION AND CULTURE OF BCEPC 42 III. MORPHOLOGIC STUDY 43 A. SCANNING ELECTRON MICROSCOPY 43 B. MEASUREMENT OF CELL AREA 43 C. FLOW CYTOMETRY 43 D. FLUORESCENCE STAINING OF ACTIN AND α-TUBULIN 44 E. IMMUNOCYTOCHEMISTRY FOR VON WILLEBRAND FACTOR 44 IV. IDENTIFICATION OF RECEPTORS 45 A. RT-PCR ASSAY FOR EXPRESSION OF PAR RECEPTORS, P2 RECEPTORS AND VON WILLEBRAND FACTOR (VWF) 45 B. STUDY OF EXPRESSION OF P2X4, P2X7, P2Y12 & PAR RECEPTORS BY IMMUNOCYTOCHEMISTRY 45 V. INTERCELLULAR COMMUNICATION EXPERIMENTS 47 A. FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING 47 B. MECHANICAL STIMULATION FOR INDUCING CALCIUM WAVE 47 C. MEASUREMENT OF INTRACELLULAR CALCIUM 48 D. MEASUREMENT OF ATP RELEASE 48 E. LUCIFER YELLOW UPTAKE ASSAY 49 VI. DATA ANALYSIS 50 CHAPTER IV. RESULTS 51 I. CHANGES IN MORPHOLOGY AND INTERCELLULAR COMMUNICATION WITH AGE AND TIME IN CULTURE 52 A. CHARACTERIZATION OF BOVINE CORNEAL ENDOTHELIAL CELLS 52 B. INTERCELLULAR COMMUNICATION DECREASES WITH TIME IN CULTURE 63 C. CONCLUSIONS 72 II. EFFECT OF THROMBIN ON INTERCELLULAR COMMUNICATION 73 A. EFFECTS OF THROMBIN ON CALCIUM WAVE PROPAGATION 73 B. IDENTITY OF RECEPTORS INVOLVED IN THE EFFECTS OF THROMBIN 77 C. SIGNAL TRANSDUCTION UNDERLYING THE EFFECTS OF THROMBIN 80 D. EFFECTS OF THROMBIN ON GJIC AND PIC 82 E. CONCLUSIONS 89 III. PURINERGIC MODULATION OF THE THROMBIN EFFECT ON INTERCELLULAR COMMUNICATION 90 A. P1 RECEPTORS 90 B. P2 RECEPTORS 100 CHAPTER V. DISCUSSION 103 I. CHANGES IN MORPHOLOGY AND INTERCELLULAR COMMUNICATION WITH AGE AND TIME IN CULTURE 105 A. AGE-RELATED CHANGES IN MORPHOLOGY 105 B. CHANGES IN CELL SIZE WITH TIME IN CULTURE 106 C. EXPRESSION OF VON WILLEBRAND FACTOR 108 D. INTERCELLULAR COMMUNICATION DECREASES WITH TIME IN CULTURE 109 II. EFFECT OF THROMBIN ON INTERCELLULAR COMMUNICATION 111 A. EFFECT OF THROMBIN ON CALCIUM WAVE PROPAGATION 113 B. EFFECTS OF THROMBIN ON GAP JUNCTIONAL AND PARACRINE INTERCELLULAR COMMUNICATION: DEPENDENCE ON MLC PHOSPHORYLATION 113 C. ROLE OF CORTICAL ACTIN IN THE EFFECT OF THROMBIN ON INTERCELLULAR COMMUNICATION 115 D. POTENTIAL EFFECTS OF MLC PHOSPHORYLATION ON CX43 AND ZO-1 INTERACTIONS 116 III. MODULATION OF THE THROMBIN EFFECT ON INTERCELLULAR COMMUNICATION VIA PURINERGIC SIGNALING 117 A. P1 RECEPTORS: ADENOSINE OPPOSES THE EFFECT OF THROMBIN ON INTERCELLULAR COMMUNICATION 117 B. P2 RECEPTORS AND THE EFFECT OF THROMBIN 120 IV. PHYSIOLOGICAL SIGNIFICANCE OF INTERCELLULAR COMMUNICATION AND THE EFFECTS OF THROMBIN AND ADENOSINE IN THE CORNEAL ENDOTHELIUM 122 CHAPTER VI. FUTURE PERSPECTIVES 127 CHAPTER VII. SUMMARY 131 CHAPTER VIII. SAMENVATTING 135 IX. REFERENCES 139 X. CURRICULUM VITAE 163 I. PUBLICATIONS 165 II. ABSTRACTS AND POSTER PRESENTATIONS 166 III. SEMINARS AND TALKS 168status: publishe

Mechanical stimulation-induced calcium wave propagation in cell monolayers: the example of bovine corneal endothelial cells

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 Ca(2+)-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 Ca(2+) that initiate the propagation of the Ca(2+)-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca(2+)-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 Ca(2+)-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca(2+)-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 Ca(2+)-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.status: publishe

Calcium Wave Propagation Triggered by Local Mechanical Stimulation as a Method for Studying Gap Junctions and Hemichannels

Intercellular communication is essential for the coordination and synchronization of cellular processes. Gap junction channels play an important role to communicate between cells and organs, including the brain, lung, liver, lens, retina, and heart. Gap junctions enable a direct route for ions like calcium and potassium, and low molecular weight compounds, such as inositol 1,4,5-trisphosphate, cyclic adenosine monophosphate, and various kinds of metabolites to pass between cells. Intercellular calcium wave propagation evoked by a local mechanical stimulus is one of the gap junction assays to study intercellular communication. In experimental settings, an intercellular calcium wave can be elicited by applying a mechanical stimulus to a single cell. Here, we describe the use of monolayers of primary bovine corneal endothelial cells as a model to study intercellular communication. Calcium wave propagation was assayed by imaging fluorescent calcium in bovine corneal endothelial cells loaded with a fluorescent calcium dye using a confocal microscope. Spatial changes in intercellular calcium concentration following mechanical stimulation were measured in the mechanical stimulated cell and in the neighboring cells. The active area (i.e., total surface area of responsive cells) of a calcium wave can be measured and used for studying the function and regulation of gap junction channels as well as hemichannels in a variety of cell systems.status: publishe

Systematic relevance of pollen and orbicule characters in the tribe Hillieae (Rubiaceae)

Pollen and orbicule morphology of 26 species in the tribe Hillieae is described based on observations by light, scanning and transmission electron microscopy. Pollen and orbicule characters are critically evaluated and discussed in the context of existing hypotheses of systematic relationships within the tribe. Pollen is 3-zonocolporate with a perforate, microreticulate, reticulate or eutectate sexine. In the two species of Blepharidium, however, the pollen has one, four or five apertures. These pollen morphological data were incorporated into an existing macromorphological matrix of the group and cladistically analysed. The resulting phylogenies indicate that it may be appropriate to reduce Cosmibuena to a subgenus of the genus Hillia, while Blepharidium should be removed from Hillieae because of the deviating pollen type that is unique to the Rubiaceae. All species investigated produce orbicules, which are mostly spherical and possess an electron-lucent core that is sometimes characteristically flattened. (C) 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 303-321.status: publishe

Adenosine opposes thrombin-induced inhibition of intercellular calcium wave in corneal endothelial cells

PURPOSE. In corneal endothelial cells, intercellular Ca2+ waves elicited by a mechanical stimulus involve paracrine intercellular communication, mediated by ATP release via connexin hemichannels, as well as gap junctional intercetlular communication. Both mechanisms are inhibited by thrombin, which activates RhoA and hence results in myosin light chain phosphorylation. This study was conducted to examine the effects of adenosine, which is known to oppose thrombin-induced RhoA activation, thereby leading to myosin light chain dephosphorylation, on gap junctional intercellular communication and paracrine intercellular communication in cultured bovine corneal endothelial cells.status: publishe

Reduced intercellular communication and altered morphology of bovine corneal endothelial cells with prolonged time in culture

PURPOSE: Mechanical stimulation induces intercellular Ca(2 +) waves in the corneal endothelium. The extent of the wave propagation is dependent on the activity of gap junctions, hemichannels, and ectonucleotidases. To further establish the use of a cell culture model to investigate intercellular communication, in this study, we have characterized the changes in the Ca(2 +) wave propagation in bovine corneal endothelial cells with prolonged time in culture. MATERIALS AND METHODS: Freshly isolated BCEC were cultured for a short term (8 to 14 days; referred to as "short term") and a long term (21 to 30 days; referred to as "long term"). Cell surface area and size were measured by confocal microscopy and flow cytometry, respectively. Calcium wave propagation was assayed by imaging spread of the Ca(2 +) waves elicited by mechanical stimulation. ATP release was assayed using Luciferin-Luciferase bioluminescence technique. RESULTS: Cells cultured for a long term showed larger surface area and size compared to those cultured for a short term, but a reduced spread of the Ca(2 +) wave. Exposure to exogenous apyrases, which can rapidly hydrolyze extracellular ATP, reduced the spread of the Ca(2 +) wave in both groups. The fractional decrease, however, was smaller in cells cultured for a long term. Exposure to ARL-67156 to inhibit the ectonucleotidases led to a larger enhancement of the active area in cells cultured for a long term. However, the active areas of the two groups were not significantly different in the presence of the drug. Furthermore, ATP release in response to mechanical stimulation was lower in cells cultured for a long term in the absence of ARL-67156 but not in its presence. CONCLUSIONS: BCEC cultured for a long term show an increase in cell surface area and cell size similar to the effect of aging in human corneas. Moreover, the cells cultured for a long term showed a reduced ATP-dependent paracrine intercellular communication, largely due to an increase in the activity of the ectonucleotidases.status: publishe