Einfluss von Wachstumsfaktoren und Moxonidin auf den intrazellulären pH und die Proliferation bei kardialen Fibroblasten und HeLa Zellen

The aim of this dissertation was to examine the activity of pH-regulating iontransporters of cardiac fibroblasts and tumorcells independent of the Na(+)-H(+)-exchanger (NHE). As the importance of NHE for alkalinisation is already thoroughly described in literature, most experiments have been performed under inhibition of the NHE with HOE694. First of all, we tried to characterise the influence of different substances (growth factors as in fetal calf serum (FCS) and moxonidine) on the activity of iontransporters. Recently, the identity of the imidazoline-1-receptor (I1R), to which moxonidine is binding, has been identified. It is most probably a S1P1-receptor. The imidazoline derivate moxonidine is a clinically established antihypertensive agent and has in comparison to clonidine a very high affinity to the I1R. Furthermore, it inhibits the sympathicotonus and increases the sensitivity for insuline. The main aspect of our studies was to enquire in how far the activity of iontransporters, which is modified by growth factors, could be additionally influenced by moxonidine. As S1P is a component of FCS the influence of moxonidine has been examined in dependence of the stimulation with FCS. Firstly, we discovered that a 30-minutes incubation of cardiac fibroblasts with FCS causes an alkalinisation, which persists even with a 24-hours incubation with FCS. HeLa cells had a higher baseline-pH in comparison with cardiac fibroblasts. But even here we noticed a similar alkalinisation persisting over 24 hours. Furthermore, we examinated those mechanisms leading to alkalinisation. We could show that FCS caused an inhibition of the recovery from the alkaline, an effect caused most likely by inhibition of anionexchangers by FCS. On the other hand, FCS caused an increased recovery from acidity, an effect that is most likely due to the activation of the NBC when inhibiting the NHE with HOE694. Therefore, these mechanisms seem to be mainly responsibly for the alkalinisation when stimulated with growth factors. Furthermore, we could show that there is a difference between pH-regulation of cardiac fibroblasts and HeLa cells. HeLa cells showed an alkalinisation when stimulated by FCS. This alkalinisation seems to be due to the stimulation of the NBC, whereas FCS did not have an influence on the recovery from alkaline. Moxonidine inhibited the anionexchanger (AE) under the precondition that cardiac fibroblasts were incubated with a so-called minimal medium with only 0.4% FCS. When stimulated by 10% FCS we could not show a significant inhibition of the AE by moxonidine. Moxonidine could only inhibit the NBC when stimulated by incubation with FCS, but not when incubated with the minimal medium. Moxonidine did not have an influence on the pH-regulation of HeLa cells. In proliferation-experiments we could show that cultivation of cells in acid environment leeds to inhibition of cell growth. Moxonidine and HOE694 in combination inhibited the proliferation of HeLa cells, whereas these substances alone did not have any significant effect. We concluded that the iontransporters NHE and NBC are able to substitute one another even in HeLa cells. Furthermore, we could confirm the experiments of Wong, Kleemann and Tannock, who regard the pH-regulation of cells by affecting iontransporters an important target for the developement of cytostatic agents.