Effects of Nicotine on PTHrP and PTHrP Receptor Expression in Rat Coronary Endothelial Cells

Abstract

Aims: 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. Methods: 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. Results: 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. Conclusion: 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.

Effect of free running wheel exercise on renal expression of parathyroid hormone receptor type 1 in spontaneously hypertensive rats

“…Furthermore, we have recently shown that nicotine reduces PTHR1 expression in microvascular endothelial cells by activating acetylcholine receptors (Röthig et al. ). Considering that high physical activity is associated with an activation of the parasympathetic nervous system as indicated by lower resting heart rate (Schreckenberg et al.…”

“…Blots were incubated with a monoclonal antibody directed against PTHR1 (antibody SAB4502493, Sigma‐Aldrich, St. Louis, USA) as described before (Röthig et al. ) and a second antibody coupled to horseradish peroxidase (anti‐Actin [antibody A2668, Sigma‐Aldrich, St. Louis, USA]) and anti‐GAPDH (antibody 6C5 from Calbiochem EMD Chemicals, San Diego, USA). Specificity of the PTH‐1R antibody was proven by strong exposure of the samples and the absence of upcoming unspecific bands under these conditions and downregulation of the receptor in microvascular endothelial cells (see Röthig et al.…”

“…Specificity of the PTH‐1R antibody was proven by strong exposure of the samples and the absence of upcoming unspecific bands under these conditions and downregulation of the receptor in microvascular endothelial cells (see Röthig et al. ). Linearity of the signal was also confirmed by diluting the samples (see Fig.…”

Effect of high fat diet on pulmonary expression of parathyroid hormone-related protein and its downstream targets

“…Protein samples were loaded on NuPAGE Bis-Tris Precast gels (10%; Life Technology, Darmstadt, Germany) and subsequently transferred onto nitrocellulose membranes. Primary antibodies directed against PTHrP, PTH-1 receptor and α-actin (loading control) were used as described before [20].…”

Effect of nitric oxide deficiency on the pulmonary PTHrP system

“…Samples (100 μg) were loaded on a 12.5% SDS‐PAGE and blotted onto membranes as described before 22. Blots were incubated with a monoclonal antibody directed against PTHrP (antibody GF08; Calbiochem, Bad Soden, Germany) or PTH‐1 receptor (antibody 3D1.1; Upstate biotechnology, Eschborn, Gerrmany) as described before 28 and a second antibody coupled to horse radish peroxidase. Surfactant production was quantified by immunoblots detecting surfactant protein‐C (SP‐C; antibody FL‐197, sc‐13979 Santa Cruz Biotechnology) and lipofibroblast‐to‐myofibroblast transdifferentiation was analysed by up‐regulation of α‐smooth muscle protein (antibody clone 1A4, C6198 from Sigma‐Aldrich; St. Louis, USA).…”