title: Whole cell patch-clamp recordings of rat midbrain dopaminergic neurons isolate a sulphonylurea- and ATP-sensitive component of potassium currents activated by hypoxia.
abstract: The effects of brief (2-4 min) hypoxia on presumed dopaminergic "principal" neurons of the rat ventral mesencephalon were investigated by using either intracellular or whole cell patch-clamp recordings in in vitro conditions. Under single-electrode voltage clamp, with sharp microelectrode (Vh -60 mV), a brief hypoxia caused an outward current (hypoOUT) of 110.2 +/- 15.2 (SE) pA (n = 18), which was followed by a posthypoxic outward current (posthypoOUT) of 149.6 +/- 10.6 pA (n = 18). Although the hypoOUT reversed at -83.7 +/- 3.8 mV (n = 18), the posthypoOUT did not reverse. The K+ATP-blocking sulphonylureas tolbutamide (100 microM) and glibenclamide (30 microM), significantly reduced the peak of the hypoOUT by 47.6 +/- 7.7% (n = 16) and 54.18 +/- 7.5% (n = 3), respectively. In contrast, they did not affect the posthypoOUT. Extracellular barium (300 microM to 1 mM) almost abolished the hypoOUT, leaving the posthypoOUT unchanged. The large K+ channel blocker charybdotoxin (10-50 nM), depressed the hypoOUT after tolbutamide treatment. To investigate whether or not cytosolic factors might control the development of the hypoOUT, we dialyzed the principal neurons by patch-clamp recordings (Vh -60 mV). Under whole cell recordings hypoxia evoked an hypoOUT of 70.2 +/- 14.5 pA that reversed polarity at -87.9 +/- 5.1 mV (n = 8). A small posthypoxic response was detected upon reoxygenation in a few neurons (4 out of 14). Three different sulphonylureas, tolbutamide (100 microM), glibenclamide (10-30 microM), and glipizide (100 nM) completely blocked the hypoOUT in patch-clamped neurons. The hypoOUT was also abolished by extracellular BaCl2 (300 microM). When the content of ATP in the dialyzate was raised from 2 to 10 mM no outward current/hyperpolarization was evoked by hypoxia. These data suggest that the hypoOUT, in principal neurons, is a complex response sustained by at least two barium-sensitive components: 1) an ATP-dependent, sulphonylurea-sensitive K+ conductance which could be isolated by the patch-clamp techniques and 2) a K+ conductance remaining after tolbutamide in intracellularly recorded neurons, which is sensitive to charybdotoxin and dependent on dialyzable cytosolic factors.
authors.affiliation: Istituto di Ricovero e Cura a Carattere Scientifico Santa Lucia, Laboratorio di Neurologia Sperimentale, Rome, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Guatteo
authors.forename: M
authors.initials: M
authors.lastname: Federici
authors.forename: A
authors.initials: A
authors.lastname: Siniscalchi
authors.forename: T
authors.initials: T
authors.lastname: Knöpfel
authors.forename: N B
authors.initials: NB
authors.lastname: Mercuri
authors.forename: G
authors.initials: G
authors.lastname: Bernardi
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D017609:Barium Compounds / Q000494:pharmacology; D015687:Cell Hypoxia; D018999:Charybdotoxin / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism*; D005913:Glipizide / Q000494:pharmacology; D005905:Glyburide / Q000494:pharmacology; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008636:Mesencephalon / Q000502:physiology*; D008839:Microelectrodes; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D011930:Reaction Time; D013453:Sulfonylurea Compounds / Q000494:pharmacology*; D014044:Tolbutamide / Q000494:pharmacology
chemical_list: D017609:Barium Compounds; D002712:Chlorides; D015221:Potassium Channels; D013453:Sulfonylurea Compounds; C024986:barium chloride; D018999:Charybdotoxin; D000255:Adenosine Triphosphate; D014044:Tolbutamide; D005905:Glyburide; D004298:Dopamine; D005913:Glipizide