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Tuesday, February 26, 2013

Molecular Tolerance of Voltage-Gated Calcium Channels is Evident After Short Exposures to Alcohol in Vasopressin-Releasing Nerve Terminals





Voltage-gated calcium channels (VGCCs) in rat neurohypophysial terminals exhibit molecular tolerance to alcohol, including desensitization to the drug and increased current density, after 3 weeks of alcohol drinking. Moreover, after this time, terminals from drinking rats exhibit diminished alcohol inhibition of vasopressin (AVP) release.

We took advantage of organotypic cultures (explants) of the hypothalamo-neurohypophysial system (HNS) to extend our analysis of molecular tolerance to 2 classes of the VGCC. The isolated HNS explant allows much finer temporal resolution of molecular tolerance than do voluntary drinking paradigms. After exposure of the HNS explant to alcohol, terminals are isolated by mechanical treatment and plated in a dish. Patch clamp recording techniques are used to obtain VGCC currents, and immunohistochemistry is used to determine VGCC distribution. A release assay is used to provide functional readout of AVP release.

We show that even a brief, 1-hour exposure to a clinically relevant concentration of alcohol is sufficient to evoke similar changes to those observed after several weeks of exposure. Acute ethanol (EtOH) exposure inhibits high K+-induced AVP release from naïve terminals. However, terminals pre-exposed to 20 mM EtOH for 1 hour become tolerant to EtOH, and subsequent exposure has significantly less effect on high K+-induced AVP release. Electrophysiological recordings indicate that among different types of VGCCs present in the neuronal terminal, the L-type is the most affected by alcohol. The current density of L-type current is significantly increased (approximately 50%), while its responsiveness to alcohol is significantly diminished (approximately 50%), after brief alcohol exposure. Fluorescent imaging results were consistent with the electrophysiology and suggest that the increased current density of VGCCs after brief exposure is attributable to combined synthesis of 1.2 and 1.3 subtypes of the L-type VGCC and redistribution of channel protein into terminal plasma membrane.

These data indicate that a brief alcohol exposure affects subsequent alcohol sensitivity of VGCCs and neuropeptide release from presynaptic terminals.
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