Related Experiment Videos
Phorbol ester differentially regulates oxytocin receptor binding activity in hypothalamic cultured neurons and
M T Strosser1, M E Evrard, C Breton
1Laboratoire de Neurophysiologie Cellulaire et Intégrée, UMR 7519, CNRS ULP, 21 rue René Descartes, 67084, Strasbourg Cedex, France.
Abstract:
Hypothalamic cultured neurons and astrocytes were used to investigate the cellular mechanisms underlying the oxytocin receptor-mediated downregulation through a possible involvement of protein kinase C (PKC). For this purpose, the effects of PKC activators, inhibitor and of OT on OT receptor binding activity were compared in both cultures. In neurons, phorbol-myristate-acetate (PMA), a potent PKC activator, increased the binding of an OT receptor antagonist whereas in astrocytes, a decrease was observed. Pre-treatment of the cells with bisindolylmaleimide (10(-4) M), a PKC inhibitor, prevented the PMA-induced up- and downregulation. In contrast, receptor downregulation resulting from treatment of both cells with OT (10(-9) M) was not affected by the PKC inhibitor. On the other hand, when PMA (10(-7) M) was tested along with OT (10(-9) M), a subsequent decrease in ligand binding was observed in astrocytes. In neurons, PMA attenuated the OT-induced downregulation. Structural analysis of neuron and astrocyte OT receptor mRNA by RT-PCR, subcloning and sequencing, demonstrated identical sequence to rat uterine receptor. In conclusion, these data suggest that activation of PKC has opposite effect on OT receptor binding activity in neurons and astrocytes but they do not support the involvement of PKC in the OT-induced downregulation.
Insights
Protein kinase C (PKC) activation has opposing effects on oxytocin receptors in hypothalamic neurons and astrocytes. However, PKC is not involved in oxytocin-induced receptor downregulation in these cells.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Oxytocin receptors mediate crucial physiological functions.
- Understanding oxytocin receptor regulation is vital for neuroscience.
- Protein kinase C (PKC) is implicated in cellular signaling pathways.
Purpose of the Study:
- To investigate the role of PKC in oxytocin receptor (OTR) downregulation in cultured hypothalamic neurons and astrocytes.
- To determine if PKC activation influences OTR binding activity.
- To elucidate the cellular mechanisms of OTR regulation.
Main Methods:
- Primary cultures of hypothalamic neurons and astrocytes were utilized.
- Effects of PKC activator (phorbol-myristate-acetate, PMA) and inhibitor (bisindolylmaleimide) on OTR binding were assessed.
- Oxytocin (OT) treatment was used to induce receptor downregulation.
- RT-PCR, subcloning, and sequencing were performed for OTR mRNA analysis.
Main Results:
- PKC activation by PMA increased OTR antagonist binding in neurons but decreased it in astrocytes.
- The PKC inhibitor prevented PMA-induced changes in OTR binding.
- OT-induced OTR downregulation was not affected by the PKC inhibitor in either cell type.
- PMA modulated OT-induced downregulation differently in neurons and astrocytes.
- Neuronal and astrocyte OTR mRNA sequences were identical to the rat uterine receptor.
Conclusions:
- PKC activation exerts opposing effects on OTR binding activity in hypothalamic neurons and astrocytes.
- PKC is not involved in the mechanism of OT-induced OTR downregulation.
- Hypothalamic OTRs in neurons and astrocytes share sequence homology with peripheral receptors.