Intracellular effects of catecholamines

The catecholamines dopamine and noradrenaline are important neurotransmitters in the brain. They are involved in control of mood, cognition and motor behaviour; antidepressive drugs interfere with the function of noradrenaline, neuroleptics block the action of dopamine and Parkinson`s disease is the result of a degeneration of a specific dopaminergic pathway. Signalling of catecholamines for these functions is based on the interaction of the neurotransmitters with plasmalemmal receptors. We have established a cellular model where catecholamines exert effects independent of classical signalling. SK- N-MC neuroblastoma cells were stably transfected with the human dopamine or noradrenaline transporter cDNA. In these cells, low concentrations of dopamine or noradrenaline (1 to 10 M) have potent antiproliferative effects with signs of apoptosis. The effect depends on the expression level of the transporters and can only be blocked by uptake inhibitors but not by blockers of dopaminergic or adrenergic receptors. Preliminary findings are at variance with another established mechanism independent of G-protein coupled receptors, that is oxidative stress. Thus, the mechanism of this apparently intracellular effect of catecholamines is completely unknown. The main focus of the proposed project is to elucidate the underlying mechanism. For that purpose, the influence of catecholamines on the redox status, indices of apoptosis and the cell cycle of transporter expressing neuroblastoma cells will be investigated. Furthermore, a detailed analysis of the cell cycle checkpoints will be performed and it will be searched for genes selectively upregulated by intracellular catecholamines. Interest in the action of catecholamines on cell growth and death is based on their potential role in the development of dividing neuronal progenitors and on hypotheses that agents which are antiproliferative on dividing cells might be neuroprotective on postmitotic neuronal cells. This hypothesis will be tested in the proposed project by examining the effect of low molar catecholamines on differentiated neuroblastoma cells and rat superior cervical ganglion neurones. Neurotoxic versus neuroprotective actions of catecholamines are of particular importance in questions about dopamine substitution therapy of Parkinson`s disease by levodopa. Our findings could have an impact on the evaluation of this mainstay of Parkinson`s disease therapy in terms of neuronal survival.

The project`s most important result is the discovery of a novel cellular effect of the neurotransmitter dopamine. Dopamine is a neurotransmitter involved in various brain functions such as motor behavior, cognition and reward. Dysfunction can lead to Parkinson`s disease, schizophrenia and addiction. All these functions of dopamine are mediated by special proteins on the surface of neuronal cells called dopamine receptors. We have found that intracellular dopamine has a strong inhibitory effect on cell growth which is unrelated to dopamine receptors. We observed this effect of dopamine if it was taken up into the cell by the dopamine transporter, a membrane protein to be found in all neurons using dopamine as a neurotransmitter. We could explain the molecular mechanism of the inhibitory effect of intracellular dopamine on cell growth. It is due to inhibition of a key enzyme of cellular reproduction. This enzyme is called ribonucleotide reductase and provides deoxyribonucleotides, components required for DNA synthesis. In our experimental set up the effect of dopamine was stronger than the effect of hydroxyurea, the prototypical inhibitor of ribonucleotide reductase and an established cytostatic agent in cancer therapy. This newly discovered effect of dopamine might play a role in the development of dopamine neurons at a stage where they still divide. On the other hand, cellular actions which interfere with cell division have been found to be protective in non-dividing, so-called postmitotic cells. By the same token, the intracellular effect of dopamine might have a protective action on dopamine neurons which are non-dividing in the adult brain. Studies on this aspect are still in progress. They might contribute to our understanding of survival or cell death of dopamine neurons in neurodegenerative disorders, such as Parkinson`s disease, and to a better insight into the longterm effects of the drug levodopa. Levodopa is the precursor of dopamine and on the one hand side the most effect therapy of Parkinson`s disease, which is characterized by a loss of dopamine neurons, on the other hand levodopa is controversial because of toxicity in cell culture. However, levodopa increases intracellular dopamine, and a protective action of dopamine through our newly found mechanism might explain why levodopa does not accelerate but actually slows down the progression of Parkinson`s disease.