Changes by short-term hypoxia in the membrane properties of pyramidal cells and the levels of purine and pyrimidine nucleotides in slices of rat neocortex; effects of agonists and antagonists of ATP-dependent potassium channels

Abstract

In a first series of experiments, intracellular recordings were made from pyramidal cells in layers II–III of the rat primary somatosensory cortex. Superfusion of the brain slice preparations with hypoxic medium (replacement of 95% O₂–5% CO₂ with 95% N₂–5% CO₂) for up to 30 min led to a time-dependent depolarization (HD) without a major change in input resistance. Short periods of hypoxia (5 min) induced reproducible depolarizations which were concentration-dependently depressed by an agonist of ATP-dependent potassium (K_ATP) channels, diazoxide (3–300 µM). The effect of 30 but not 300 µM diazoxide was reversed by washout. Tolbutamide (300 µM), an antagonist of K_ATP channels, did not alter the HD when given alone. It did, however, abolish the inhibitory effect of diazoxide (30 µM) on the HD. Neither diazoxide (3–300 µM) nor tolbutamide (300 µM) influenced the membrane potential or the apparent input resistance of the neocortical pyramidal cells. Current-voltage (I-V) curves constructed at a membrane potential of –90 mV by injecting both de- and hyperpolarizing current pulses were not altered by diazoxide (30 µM) or tolbutamide (300 µM). Moreover, normoxic and hypoxic I-V curves did not cross each other, excluding a reversal of the HD at any membrane potential between –130 and –50 mV. The hypoxia-induced change of the I-V relation was the same both in the absence and presence of tolbutamide (300 µM). In a second series of experiments, nucleoside di- and triphosphates separated with anion exchange HPLC were measured in the neocortical slices. After 5 min of hypoxia, levels of nucleoside triphosphates declined by 29% (GTP), 34% (ATP), 44% (UTP) and 58% (CTP). By contrast, the levels of nucleoside diphosphates either did not change (UDP) or increased by 13% (GDP) and 40% (ADP). In slices subjected to 30 min of hypoxia the triphosphate levels continued to decrease, while the levels of GDP and ADP returned to control values. The tri- to diphosphate ratios progressively declined for ATP/ADP and GTP/GDP, but not for UTP/UDP when the duration of hypoxia was increased from 5 to 30 min. Hence, the rapid fall in the ratios of nucleoside tri- to diphosphates without the induction of a potassium current failed to indicate an allosteric regulation of a plasmalemmal K_ATP channel by purine and pyrimidine nucleotides. Diazoxide had no effect on neocortical pyramidal neurons and was effective only in combination with a hypoxic stimulus; it is suggested that both plasmalemmal and mitochondrial K_ATP channels are involved under these conditions. The hypoxic depolarization may be due to blockade of K⁺,Na⁺-ATPase by limitation of energy supplying substrate.