Elsevier

Brain Research

Volume 943, Issue 1, 5 July 2002, Pages 117-131
Brain Research

Interactive report
Vasopressin-induced cytoplasmic and nuclear calcium signaling in cultured cortical astrocytes

https://doi.org/10.1016/S0006-8993(02)02484-8Get rights and content

Abstract

We sought to determine whether vasopressin V1a receptor (V1aR) mRNA detected in cortical astrocytes [Brain Res. Mol. Brain Res. 45 (1997) 138] was translated into functional receptors by investigating the effector calcium signaling cascade associated with the vasopressin V1a receptor subtype. Analysis of intracellular calcium dynamics using the calcium-sensitive dye fura-2 AM indicated that exposure of cortical astrocytes to V1 vasopressin receptor agonist, [Phe2,Orn8]-oxytocin, induced a marked dose-dependent increase in intracellular calcium which was abolished by depletion of extracellular calcium. V1 agonist treatment induced a rapid increase in calcium signal in both the cytoplasm and nucleus, which was followed by an accumulation of the calcium signal in the nucleus, suggesting translocation of cytoplasmic calcium into the nucleus. The nuclear calcium signal was sustained for several seconds followed by translocation back to the cytoplasm. Following the nuclear-to-cytoplasmic calcium translocation, total free intracellular calcium concentration decreased. The dynamic calcium cytoplasmic and nuclear localization was confirmed by laser scanning confocal microscopy coupled with the calcium-sensitive dye fluo-3 AM. To determine the source of calcium, V1 agonist-induced 45Ca2+ uptake and [3H]IP1 accumulation were investigated. V1 agonist induced significant and rapid uptake of 45Ca2+ and a significant dose-dependent increase in [3H]IP1 accumulation in cortical astrocytes. To our knowledge, this is the first documentation of a vasopressin receptor-induced calcium signaling cascade in cortical astrocytes and the first documentation of vasopressin receptor induction of nuclear calcium signaling.

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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