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Effects of nicotine on PTHrP and PTHrP receptor expression in rat coronary endothelial cells.

Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology (2012-04-18)
Anja Röthig, Rolf Schreckenberg, Kerstin Weber, Charlotte Conzelmann, Rui Manuel da Costa Rebelo, Klaus-Dieter Schlüter
ABSTRACT

The study was aimed to investigate whether nicotine affects endothelial expression of PTHrP and PTHrP receptor, a peptide system involved in endothelial protection against apoptosis. Isolated and cultured rat coronary endothelial cells were used. Immunoblot techniques were used to study activation of mitogen-activated protein (MAP) kinases and to quantify PTHrP and PTHrP receptor expression. Real-time RT-PCR was used to quantify PTHrP, PTHrP-receptor, bcl-2, and bax mRNA expression. The rate of apoptosis was determined by HOE33258 staining and confirmed by quantification of the bcl-2-to-bax ratio. In vitro data were compared to hearts from rats exposed to cigarette smoking. Nicotine induced PTHrP protein expression at nanomolar levels and small increases of PTHrP release (≈8%). Antagonists directed against the α7 subunit of cholinergic receptors, the most prominent isoform, attenuated nicotine-dependent increases of PTHrP expression. This effect of nicotine was p38 MAPK dependent. Nicotine at micromolar concentrations reduced PTHrP receptor expression. In vitro and in vivo we found a correlation between PTHrP receptor expression and bcl-2 expression. Nicotine induces PTHrP expression in endothelial cells but excessive concentrations of nicotine reduce PTHrP receptor expression thereby attenuating any protective effects of PTHrP against apoptosis.

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Sigma-Aldrich
Hoechst 33258 solution, 1 mg/mL in H2O, ≥98.0% (HPLC)
Sigma-Aldrich
bisBenzimide H 33258, powder, BioReagent, suitable for cell culture, ≥98% (HPLC and TLC)
Sigma-Aldrich
bisBenzimide H 33258, for fluorescence, ≥98.0% (HPLC)
Sigma-Aldrich
bisBenzimide H 33258, ≥98% (HPLC and TLC)
Sigma-Aldrich
Anti-PTH/PTHrP Receptor Antibody, clone 3D1.1, clone 3D1.1, Upstate®, from mouse