Research reportEffect of ethanol on calcium regulation in rat fetal hypothalamic cells in culture
Introduction
The ability of ethanol to activate opiate systems in the central nervous system is an important component in the reinforcing properties of ethanol. This has been shown in both animal 4, 18and human studies 4, 14and has lead to the introduction of the opiate antagonist naltrexone to treat alcohol craving in recovering alcoholics [12]. However, exactly how ethanol activates endogenous opioid systems is not understood. We have recently developed a primary culture system of hypothalamic neurons in which such questions can be addressed at the cellular level [10]. Using this system we have shown that ethanol exposure causes the release of β-endorphin in a calcium-dependent manner [9]. The identification of events activated by ethanol that lead up to the release β-endorphin is a focus of our current research efforts.
One possibility is that ethanol causes the release of β-endorphin via alterations in cellular calcium homeostasis. Ethanol has been shown to increase intracellular calcium in several neuronal cell preparations including PC12 cells 1, 8, NCB-20 cells [2], septohippocampal neurons 20, 21and hippocampal neurons [6]. However, in several of these studies concentrations of ethanol greater than 200 mM where required to observe effects 2, 8raising questions as to the biological significance of this effect in the pharmacological actions of ethanol. On the other hand, in the more recent studies in primary cultures of septohippocampal neurons and hippocampal neurons 21, 6, respectively, an effect of ethanol to increase calcium has been observed at more biologically relevant concentrations (<50 mM). In the case of septohippocampal neurons [21]the effect of ethanol on basal calcium levels was biphasic, occurring only at moderate ethanol concentrations (35–70 mM), and the effect was not observed if the cells were pretreated with nerve growth factor [20]. This biphasic dose–response curve is somewhat in agreement with a study in PC12 cells where ethanol concentrations up to 120 mM increased intracellular calcium whereas greater concentrations decreased intracellular calcium [1]. On the other hand, the effect of ethanol in hippocampal cells was not biphasic [6]. A further disagreement in these studies is that whereas the effect of ethanol to increase intracellular calcium reported in PC12 cells was dependent on extracellular calcium [1], the effect in hippocampal neurons was independent of extracellular calcium [6]. Thus it would appear that the ability of ethanol to alter calcium homeostasis is not only dependent on the cell type under study and the particular state of these cells, but that the very basis for the response may be different in different cells.
What effect ethanol might have on calcium homeostasis in cells from hypothalamic cultures, and whether such an action would be consistent with stimulating the release of β-endorphin, cannot be deduced by these previous studies and must be examined directly. Our first objective in the present study was to establish whether acute ethanol exposure could alter calcium homeostasis in primary cultures of hypothalamic cells, and whether this could occur at pharmacologically relevant concentrations. In pursuing this objective we grew the cells under conditions in which we have previously shown that ethanol can induced the release of β-endorphin [9]. We found that ethanol at concentrations as low as 34 mM could increase calcium in cells contained in our preparation. We further found that in most cells the response was eliminated if extracellular calcium was removed and that blockers of different subtypes of voltage-dependent calcium channels interfered with the ethanol-induced calcium response.
Section snippets
Hypothalamic cell cultures
Primary cultures of hypothalamic cells were obtained as previously described [10]with slight modifications. Briefly, hypothalami were isolated from day 19–21 Sprague-Dawley rat fetuses, and placed in ice cold Hank's Balanced Salt Solution (HBSS) containing 1% Fungi-bact, 0.1% BSA, and 200 mM ascorbic acid. The block of hypothalamic tissue extended about 1 mm rostral to the optic chiasm to just caudal to the mammilary bodies, laterally by the hypothalamic salci, and dorsally by approximately 2
Results
The first two parameters we established in our culture system was the effects of repeated exposure of the cells to a constant concentration of ethanol and a dose–response effect for ethanol. We found that if cells responded to ethanol, most gave a consistent response to repeated challenges with a constant concentration (Fig. 1, top panel). This observation justified us to perform repeated measurements on a given set of cells without the complication that responses would either rapidly sensitize
Discussion
The results presented are the first report that fetal rat hypothalamic cells in culture respond to an acute exposure to ethanol with an increase in cytoplasmic calcium levels. This effect is dose-dependent, it can occur at pharmacologically relevant ethanol concentrations, is repeatable, is rapidly reversible, but does not occur in all cells. The inability of all cells to respond is similar to the observations reported previously in hippocampal neurons in which only 75% of the cells responded
Acknowledgements
This work support by NIH grants RO1 AA08757 and KO2 AA00220 awarded to D.K.S.
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