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518. T-Type Voltage-Dependent Calcium Ch<strong>an</strong>nels<br />

Location: South Hall A<br />

Time: Tuesday, October 20, <strong>2009</strong>, 8:00 am - 12:00 noon<br />

Program#/Poster#: 518.6/C60<br />

Topic: B.04.b. Calcium ch<strong>an</strong>nels: Physiology<br />

Support: NIH GM 070726<br />

NIH NINDS 1F31NS059190<br />

NIH NIGMS T32 GM08328<br />

Title: Free radical signaling mediates subunit-specific nitrous oxide inhibition of T-type calcium<br />

ch<strong>an</strong>nels in <strong>the</strong> pain pathway<br />

Authors: *P. ORESTES 1 , D. BOJADZIC 2 , S. M. TODOROVIC 1 ;<br />

1 Anes<strong>the</strong>siol & Neurosci Grad Program, 2 Anes<strong>the</strong>siol, Univ. Virginia, Charlottesville, VA<br />

Abstract: Nitrous oxide (N2O) has been used as <strong>an</strong> <strong>an</strong>es<strong>the</strong>tic <strong>an</strong>d <strong>an</strong>algesic <strong>for</strong> centuries, but its<br />

mech<strong>an</strong>ism of action remains unclear. Previous studies have shown that N2O inhibits native Ttype<br />

Ca 2+ current in sensory neurons <strong>an</strong>d recombin<strong>an</strong>t CaV3.2 ch<strong>an</strong>nels. Fur<strong>the</strong>rmore, N2O<br />

requires a key metal-binding histidine (H191) in <strong>the</strong> extracellular IS3-4 loop <strong>for</strong> its action on<br />

CaV3.2 ch<strong>an</strong>nels.<br />

Here, we show that N2O-mediated inhibition is also dependent on <strong>the</strong> presence of common trace<br />

metal ions. Diethylenetriamine pentaacetic acid (DTPA), a non-specific metal chelator,<br />

effectively reduces N2O block of calcium current through CaV3.2 ch<strong>an</strong>nels by 70%. We also<br />

implicate free radicals as mediators of this effect since free radical scavengers such as catalase<br />

<strong>an</strong>d alpha-tocopherol (vitamin E), are able to reduce <strong>the</strong> inhibitory effects of N2O to 7% <strong>an</strong>d 16%<br />

of control, respectively.<br />

Thus we hypo<strong>the</strong>size that N2O is able to generate free radicals in <strong>the</strong> presence of trace metal<br />

ions, similar to those produced by hydrogen peroxide (H2O2). In accord<strong>an</strong>ce, we show that N2O<br />

is unable to achieve additional block of CaV3.2 ch<strong>an</strong>nels over that of H2O2. Additionally, we<br />

show that N2O is able to initiate <strong>the</strong> free radical-mediated oxidation of epinephrine to<br />

adrenochrome in <strong>the</strong> presence of trace metals. The production of adrenochrome was qu<strong>an</strong>tified<br />

by measurements of absorb<strong>an</strong>ce ch<strong>an</strong>ges at 485nm.<br />

To definitively establish <strong>the</strong> role of CaV3.2 ch<strong>an</strong>nels in N2O <strong>an</strong>algesia, we injected <strong>for</strong>malin into<br />

<strong>the</strong> hindpaws of CaV3.2 knockout mice <strong>an</strong>d <strong>the</strong>ir wild-type littermates. Following injection, <strong>the</strong><br />

wild-type mice showed a signific<strong>an</strong>t decrease in licking/biting time in <strong>the</strong> presence of 20% N2O.<br />

In contrast, knockout <strong>an</strong>imals show little decrease in pain with N2O.<br />

Thus, we suggest that N2O indirectly inhibits CaV3.2 ch<strong>an</strong>nels via <strong>the</strong> free oxygen radicals that<br />

are produced in its presence. These highly unstable molecules <strong>the</strong>n react in <strong>the</strong> immediate<br />

neighborhood of tr<strong>an</strong>sition metals bound to H191, oxidizing <strong>the</strong> histidine to 2-oxo-histidine. This

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