title: Acute action of rotenone on nigral dopaminergic neurons--involvement of reactive oxygen species and disruption of Ca2+ homeostasis.
abstract: Rotenone is a toxin used to generate animal models of Parkinson's disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05-1 microm) effects on SNc neurons in acute rat midbrain slices, using whole-cell patch-clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide-sensitive outward current (94 +/- 15 pA) associated with increases in intracellular [Ca(2+)] ([Ca(2+)](i)) (73.8 +/- 7.7 nm) and intracellular [Na(+)] (3.1 +/- 0.6 mm) (all with 1 microm). The outward current was not affected by a high ATP level (10 mm) in the patch pipette but was decreased by Trolox. The [Ca(2+)](i) rise was abolished by removing extracellular Ca(2+), and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N-(p-amylcinnamoyl) anthranilic acid. Other effects included mitochondrial depolarization (rhodamine-123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm) that, by itself, did not evoke a [Ca(2+)](i) rise resulted in a large (46.6 +/- 25.3 nm) Ca(2+) response when baseline [Ca(2+)](i) was increased by a 'priming' protocol that activated voltage-gated Ca(2+) channels. There was also a positive correlation between 'naturally' occurring variations in baseline [Ca(2+)](i) and the rotenone-induced [Ca(2+)](i) rise. This correlation was not seen in non-dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP-gated K(+) channels and TRPM2-like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone-induced [Ca(2+)](i) rise by a small increase in baseline [Ca(2+)](i).
authors.affiliation: Department of Physiology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand.
authors.forename: Peter S
authors.initials: PS
authors.lastname: Freestone
authors.forename: Kenny K H
authors.initials: KK
authors.lastname: Chung
authors.forename: Ezia
authors.initials: E
authors.lastname: Guatteo
authors.forename: Nicola B
authors.initials: NB
authors.lastname: Mercuri
authors.forename: Louise F B
authors.initials: LF
authors.lastname: Nicholson
authors.forename: Janusz
authors.initials: J
authors.lastname: Lipski
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D000975:Antioxidants / Q000494:pharmacology; D055592:Biophysical Phenomena / Q000187:drug effects; D001703:Biophysics; D002118:Calcium / Q000378:metabolism*; D002839:Chromans / Q000494:pharmacology; D002934:Cinnamates / Q000494:pharmacology; D051152:Clusterin / Q000037:antagonists & inhibitors; D004298:Dopamine / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004558:Electric Stimulation; D005260:Female; D005439:Flufenamic Acid / Q000494:pharmacology; D006706:Homeostasis / Q000187:drug effects; D066298:In Vitro Techniques; D007306:Insecticides / Q000494:pharmacology*; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D018408:Patch-Clamp Techniques / Q000379:methods; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012402:Rotenone / Q000494:pharmacology*; D012964:Sodium / Q000378:metabolism; D013378:Substantia Nigra / Q000166:cytology* / Q000187:drug effects; D014044:Tolbutamide / Q000494:pharmacology; D062367:ortho-Aminobenzoates / Q000494:pharmacology
chemical_list: D000975:Antioxidants; D002839:Chromans; D002934:Cinnamates; D051152:Clusterin; D007306:Insecticides; D017382:Reactive Oxygen Species; D062367:ortho-Aminobenzoates; D012402:Rotenone; C085901:4-amylcinnamoylanthranilic acid; D005439:Flufenamic Acid; D000255:Adenosine Triphosphate; D014044:Tolbutamide; D012964:Sodium; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; D002118:Calcium; D004298:Dopamine