Interactive reportVasopressin-induced cytoplasmic and nuclear calcium signaling in cultured cortical astrocytes
Introduction
Vasopressin, the endogenous ligand for V1a vasopressin receptor (V1aR), is a nonapeptide hormone that is synthesized in a number of sites in the brain, including paraventricular, supraoptic and suprachiasmatic nuclei of the hypothalamus, the bed nucleus of the stria terminalis and the medial amygdala [10], [11], [46]. Vasopressin is known to act as a neurotransmitter [5], mediating a variety of behavioral and cognitive functions in the central nervous system, including scent marking [22], aggression [19] and learning and memory [15], [16]. The central functions of vasopressin are mainly mediated by the V1 vasopressin receptor [1], [2], [5], [23].
Our earlier autoradiographic work using radiolabeled vasopressin, the endogenous ligand, and a selective antagonist binding to the V1aR demonstrated that vasopressin recognition sites were present throughout the cerebral cortex of the mammalian brain [5], [8], [13]. These findings were confirmed by a number of laboratories which detected mRNA for the V1a receptor in the cerebral cortex [36], [37], [38], [50], [56]. In addition, we sought to determine the cell types in the cerebral cortex that express V1a recognition sites. Using differential culturing techniques, we found that neurons, astrocytes and oligodendrocytes from rat cerebral cortex all express V1a vasopressin receptor mRNA [56].
To determine whether V1a receptors in cortical astrocytes are linked to a biochemical signaling cascade, we investigated calcium signaling in cortical astrocytes in response to a specific V1 vasopressin receptor agonist, [Phe2,Orn8]-oxytocin. Fluorescent intracellular calcium imaging with the calcium-sensitive dye Fura-2 AM to analyze intracellular calcium dynamics was conducted and confirmed by Fluo-3 AM intracellular calcium localization by laser scanning confocal microscopy. Tritium-labeled InsP1 ([3H]IP1) accumulation was utilized as an index for activation of phospholipase C. 45Ca2+ uptake was assayed to evaluate calcium influx from the extracellular compartment.
The results of these studies indicated that V1a receptor rapidly increases intracellular calcium levels both within the cytoplasm and the nucleus. The rise in nuclear calcium occurs simultaneously with the calcium rise in the cytoplasm followed by an increase in nuclear calcium concentration which appears to result from translocation of cytoplasmic calcium into the nuclear compartment. The rise in nuclear calcium remains high for several seconds followed by a translocation back to the cytoplasm and an eventual decline in the cytoplasmic calcium concentration. The source of the V1 agonist-induced rise in intracellular calcium is the result of the influx of extracellular calcium and activation of the phosphatidylinositol signaling cascade which releases calcium from endoplasmic reticulum stores. These data indicate that, in cortical astrocytes, vasopressin V1 receptor is functional and that activation of the V1 receptor leads to dynamic calcium signaling in both the cytoplasm and nucleus of cortical astrocytes. The spatial dynamics of the calcium signal may implicate differential gene expression induced by vasopressin in cortical astrocytes.
Section snippets
Cell culture preparation
Cultures of cortical astrocytes were prepared following the method described by Brinton et al. [7]. Cortices were dissected from the brains of embryonic day 18 (E18) Sprague–Dawley rat fetuses. The tissue was treated with 0.05% trypsin in Hank’s balanced salt solution (100 mM NaCl, 2.0 mM KCl, 4.2 mM NaHCO3, 1.0 mM MgCl2·6H2O, 1.0 mM NaH2PO4·H2O, 2.5 mM CaCl2·2H2O, 12.5 mM HEPES, 10.0 mM dextrose) for 5 min at 37 °C. Following incubation, trypsin was inactivated with cold phenol red free
Intracellular calcium rise in response to V1 agonist
Cultured cortical astrocytes were exposed to various concentrations of V1 agonist (1, 10, 100, 250 and 500 nM) and the intracellular calcium concentration was analyzed using fura-2 ratiometric fluorescent imaging. Five parameters were assessed: (1) the number of responsive cells, (2) average lag time of response, (3) average magnitude of intracellular calcium rise, (4) average slope of intracellular calcium rise and (5) average intracellular calcium decay. Dose response analyses demonstrated
Discussion
The functionality of V1a vasopressin receptors in cortical astrocytes was investigated. Results of this study demonstrated that V1 receptor agonist induced a marked intracellular calcium rise in cultured cortical astrocytes with a dynamic calcium nuclear localization. This intracellular calcium rise was composed of both the release of intracellular calcium stores and influx of extracellular calcium.
Acknowledgements
This work was supported by grants from the Norris Foundation and the National Institutes of Aging (PO1 AG1475: Project 2) to RDB. Laser Scanning Confocal Microscopy was supported by the Confocal Microscopy Subcore of the USC Center for Liver Diseases supported by NIH 1 P30 DK48522. Technical consultation on calcium imaging by Dr. Michael Son is gratefully acknowledged.
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2010, Neurochemistry InternationalCitation Excerpt :It is possible that Ca2+ release activated by vasopressin in astrocytes leads to activation of a protein kinase other than PKC, which results in phosphorylation of the PKA/PKG-sensitive Ser111 and consequent upregulation of AQP4 water permeability. Activation of CaMKII, which has been shown to participate in PKG-dependent activation of AQP4 (Gunnarson et al., 2008), was demonstrated in response to V1a activation in astrocytes (Zhao and Brinton, 2002). There is a compelling evidence that vasopressin aggravates brain edema formation, since water accumulation in the brain after focal brain ischemia or traumatic brain injury (TBI) is diminished by vasopressin deficiency or vasopressin receptor antagonists (Dickinson and Betz, 1992; Ikeda et al., 1997; Kleindienst et al., 2006b; Okuno et al., 2008; Taya et al., 2008; Liu et al., 2010).
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2008, European Journal of PharmacologyCa<sup>++</sup> influx is essential for the hypotensive response to arginine vasopressin-induced neuron activation of the area postrema in the rat
2007, Brain ResearchCitation Excerpt :However, there has been no such study so far associating signal processing in AP neurons with changes in BP. Activation of V1A receptors by AVP administration has been documented to initiate an increase in intracellular Ca++ in studies of both cell culture (Hay et al., 1996; Brueggemann et al., 2005; Fan and Byron, 2000; Li et al., 2001; Zhoa and Brinton, 2002) and isolated neurons (Mihara et al., 1999; Sabatier et al., 1998). A large body of evidence has revealed that the binding of AVP to V1A receptors may interact with G protein to activate phospholipase C (PLC), which catalyzes the hydrolysis of phosphatidylinositol phosphate to produce inosital triphosphate (IP3) and diacylglycerol (DAG) in cell culture studies (Byron and Lucchesi, 2002) and isolated neurons (Sabatier et al., 1998; Dayanithi et al., 2000).
Vasopressin-induced translocation and proteolysis of protein kinase Cα in an amphibian brain: Modulation by corticosterone
2007, Brain ResearchCitation Excerpt :The data presented in Fig. 3 suggest that, consistent with current molecular models for the activation of conventional PKC isoforms (Bolsover et al., 2003), vasopressin-induced translocation of PKCα in the amphibian brain is a Ca2+-dependent process. Since changes in extracellular Ca2+ concentration altered PKCα and PKMα localization, it is likely that the effects of vasopressin in our studies are mediated, at least in part, by VTr-induced influx of Ca2+ via plasma membrane Ca2+ channels, as in mammalian cells (Sabatier et al., 1997; Son and Brinton, 2001; Zhao and Brinton, 2002). A novel finding in our study is that vasopressin and vasotocin treatment led to the appearance of the truncated PKCα-immunoreactive band known as PKMα.