1 Abbreviations ................................................ 4
1.1 Introduction ............................................ 7
1.1.1 Endothelin ....................................... 7
1.1.2 Endothelin receptors ............................. 7
1.1.3 Endothelin and arterial blood pressure ........... 8
1.1.4 Endothelin action in the kidney and lungs ........ 8
1.1.5 Endothelin role in cardiovascular, pulmonary
and renal diseases ............................... 9
1.1.6 Endothelin action in the brain .................. 11
1.1.7 Endothelin signalling ........................... 11
1.1.8 Neuroprotective effects of Erythropoietin ....... 14
1.1.9 Erythropoietin signalling ....................... 15
1.2 Evidence for the crosstalk between EPO and
Endothelin ............................................. 17
1.3 Purpose of the Study ................................... 18
2 Materials and Methods ....................................... 20
2.1 List of materials ..................................... 20
2.1.1 Cell culture materials .......................... 20
2.1.2 Immunofluorescence materials .................... 20
2.1.3 RNA Isolation and RT-PCR materials .............. 21
2.1.4 LDH Measurement ................................. 21
2.1.5 Measurement of EPO .............................. 21
2.1.6 Measurement of human Endothelin-1 ............... 21
2.2 Methods ................................................ 21
2.2.1 Rat astroglial primary culture .................. 21
2.2.2 Human astrocytes (SV-40 FHAS) culture ........... 22
2.2.3 Hypoxia and stimulation with BQ123, BQ-788 and
EPO ............................................. 22
2.3 Immunocytochemical analysis ............................ 23
2.4 Determination of the cell viability .................... 24
2.5 Cytotoxicity assay ..................................... 24
2.6 RT-PCR ................................................. 25
2.7 Determination of Endothelin-1 .......................... 26
2.8 Determination of Erythropoetin ......................... 27
2.9 Statistical analyses ................................... 27
3 Results ..................................................... 28
3.1 Influence of ETA blockade and EPO on neurons ........... 28
3.2 Influence of ETA blockade and EPO on neural
precursors ............................................. 35
3.3 Influence of BQ123 and EPO on the survival of human
astrocytes ............................................. 36
3.4 Age-related changes in release of EPO from
astrocytes ............................................. 37
3.5 Influence of ETA receptor blockade on the expression
of EPO ................................................. 39
3.6 ETA receptor blockade increases the concentration of
ET-1 released from human astrocytes .................... 41
3.7 Influence of EPO on the release of ET-1 from human
astrocytes ............................................. 42
3.8 Influence of the long term hypoxia on the viability
of human astrocytes .................................... 44
3.9 Effects of ETA receptor blockade on the expression of
EPO receptor in astrocytes and neural precursors ....... 46
4 Table summarizing the results ............................... 50
5 Discussion .................................................. 53
5.1 ETA blockade and EPO increases neuronal survival ....... 53
5.2 ETA blockade and EPO upregulates the population of
neural precursors ...................................... 54
5.3 ETA blockade and EPO increases the survival of human
astrocytes ............................................. 55
5.4 EPO release from astrocytes is affected by aging ....... 56
5.5 ETA receptor blockade upregulates the expression of
EPO .................................................... 57
5.6 ETA receptor blockade increases the concentration of
ET-1 released from human astrocytes .................... 58
5.7 EPO decreases the release of ET-1 from human
astrocytes ............................................. 60
5.8 Long term (48h and 72h) but not 24h hypoxia causes
cell death in human astrocytes culture ................. 61
5.9 ETA receptor blockade affects the expression of EPO
receptor in astrocytes and neural precursors ........... 62
6 Summary ..................................................... 65
7 References .................................................. 66
8 Acknowledgments ............................................. 87
9 Curriculum Vitae ............................................ 88
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