Expression of PTHrP and PTH/PTHrP receptor 1 in the superior cervical ganglia of rats
Introduction
PTHrP is a peptide identical to parathyroid hormone (PTH) in its first 13 amino-acid sequence (Thiede, 1994). Its presence was found in a broad spectrum of normal tissues, including the central nervous system (Weir et al, 1990, Wysolmerski, 2012). PTHrP has different functions, acting normally as an autocrine/paracrine factor, with exception of pregnancy and lactation, when it acts also as a humoral factor (Wysolmerski, 2012). Moreover, in cells and body fluids, PTHrP is normally subjected to posttranslational changes and proteolytic cleavage resulting in occurrence of different peptides with distinctive roles (Orloff et al., 1994). Its amino-terminal peptide (1–37) acts through PTHrP/PTHR1, a G-protein coupled receptor, which is also a common receptor to PTH (Abou-Samra et al, 1992, Wysolmerski, 2012). PTHR1 acts through different signaling pathways and induces the entry of extracellular calcium by regulation of calcium channels (Swarthout et al., 2002).
The presence of PTHrP and PTHR1 in glia of sensory – dorsal root ganglia (DRG) – was observed previously in proliferating Schwann cells (Macica et al., 2006) and their role in nerve regeneration was postulated. Also, a presence of PTHrP and PTHR1 was detected in various parts of the CNS (Swarthout et al, 2002, Weaver et al, 1995, Weir et al, 1990, Wysolmerski, 2012) including cerebral cortex, hippocampus, cerebellar cortex and hypothalamus (Holt et al, 1996, Weir et al, 1990, Yamamoto et al, 2002). Until now, the expression of PTHrP was not investigated in sympathetic neurons, although the presence of PTHrP mRNA in the superior cervical ganglion (SCG) of rats was proved previously by using DNA microarray technology and RT-PCR, along with up-regulation of PTHrP mRNA following axotomy (Boeshore et al., 2004). Nevertheless, the above study did not specify whether PTHrP was expressed in neurons or glia of the SCG, and if expressed in neurons, in which neuronal subpopulations.
It is known that the activity of the sympathetic nervous system varies with the ovarian cycle and pregnancy (Anglin, Brooks, 2003, Greenwood et al, 2001, Kaur et al, 2007, Minson et al, 2000). Ovarian hormones influence the expression of neuropeptides in central, as well as in tissues of the peripheral nervous system (Puri et al, 2005, Puri et al, 2006, Roman et al, 2006, Williams et al, 2011). Sympathetic neurons widely express estrogen receptors and are sensitive to the influence of estrogens (Zoubina and Smith, 2002). Influence of ovarian hormones on the level of sympathetic activity is thought to be related to the lower incidence of cardiovascular disease in young women, in comparison to men and postmenopausal women (Birkhaeuser, 2005, Burt et al, 1995). The SCG is a paravertebral chain ganglion that supplies sympathetic innervations to structures of the head and neck (Arbab et al, 1986, Grkovic, Anderson, 1995, Grkovic, Anderson, 1997). We have recently demonstrated that gender and gonadectomy have a significant influence on density, size of neurons and distribution of populations of neurons in SCG of sexually mature rats (Filipovic et al., 2014). Furthermore, it is known that expression of PTHrP and PTHR1 can be influenced by ovariectomy and estrogens in different tissues (Cros et al, 1998, Paspaliaris et al, 1995) but there are no data linking their expression in neurons and glia with gonadal activity. Also there is some evidence that the PTHrP/PTHR1 system could regulate release of catecholamine from PC12 cells (a cell line derived from a pheochromocytoma of the rat adrenal medulla) (Brines and Broadus, 1999), leaving the possibility for a similar role in sympathetic neurons and potential regulation of overall sympathetic activity.
Since there are no data on the cellular distribution of PTHrP and PTHR1 in the SCG, we investigated their presence and patterns of distribution in neurons and glia of the SCG of rats. In addition, we wanted to find out whether the expression of PTHrP and PTHR1 in neurons of the SCG is linked to a distinct neuronal sub-population, related to the presence/absence of NPY. There is no study on the influence of ovariectomy and/or ovarian hormones on expression of PTHrP and its receptor in neurons of the SCG. Hence, we seek to find out if ovariectomy (and consequent lack of ovarian hormones) influences their expression in neurons of the SCG of rats.
Section snippets
Material and methods
Experimental procedures were approved by the Ministry of Agriculture (UP/I-322-01/11-01/117, 526-06-1-0255/11-1) and were performed according to the European Communities Council Directive of 24 November 1986 (86/609/EEC). A total of 4 male and 10 female Sprague–Dawley rats were used. The animals were raised under controlled conditions (temperature 22 ± 1 °C, under 12/12 light/dark cycle). They were housed individually in plastic cages with sawdust bedding and fed ad libitum with standard
Results
The results of the present study are shown in Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5. PTHrP immunoreactivity was observed in cytoplasm as well as in nuclei of almost all neurons in the SCG. In male rats, intensity of PTHrP fluorescence was significantly higher in cytoplasm of NPY negative somata, in comparison to NPY+ neurons (p < 0.05; Fig. 1). Similarly, immunoreactivity for PTHR1 was also observed in all neurons of the SCG, in cytoplasm, as well as in nuclei, but there was no difference in
Discussion
PTHrP is a peptide that is widely distributed in the central and peripheral nervous system (Weir et al., 1990). Although the presence of PTHrP mRNA in the SCG of rats and its up-regulation following axotomy was proved previously by using DNA microarray and RT-PCR (Boeshore et al., 2004), there are no data on its cellular and sub-cellular distribution. Data from the present study show that almost all neurons of the rat SCG express immunoreactivity for PTHrP, as well as for its receptor,
Acknowledgments
The authors thank Mrs. Asija Miletić for her skillful technical assistance and to Dr. Irena Zakarija-Grković for critical reading of the manuscript. This study was funded by the Ministry of Science, Education and Sports, Republic of Croatia (Project no. 216-2160528-0067) whose support is gratefully acknowledged.
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