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

Abstract

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