CHANGES BY SHORT-TERM HYPOXIA IN THE MEMBRANE-PROPERTIES OF PYRAMIDALCELLS AND THE LEVELS OF PURINE AND PYRIMIDINE NUCLEOTIDES IN SLICES OF RAT NEOCORTEX - EFFECTS OF AGONISTS AND ANTAGONISTS OF ATP-DEPENDENTPOTASSIUM CHANNELS

In a first series of experiments, intracellular recordings were made f rom pyramidal cells in layers II-III of the rat primary somatosensory cortex. Superfusion of the brain slice preparations with hypoxic mediu m (replacement of 95% O-2-5% CO2 with 95% N-2-5% CO2) for up to 30 min led to; time-dependent depolarization (HD) without a major change in input resistance. Short periods of hypoxia (5 min) induced reproducibl e depolarizations which were concentration-dependently depressed by an agonist of ATP-dependent potassium (K-ATP) channels, diazoxide (3-300 mu M). The effect of 30 but not 300 mu M diazoxide was reversed by wa shout. Tolbutamide (300 mu M), an antagonist of K-ATP channels, did no t alter the HD when given alone. IL did, however, abolish the inhibito ry effect of diazoxide (30 mu M) on the HD. Neither diazoxide (3-300 m u M) nor tolbutamide (300 mu M) influenced the membrane potential or t he apparent input resistance of the neocortical pyramidal cells. Curre nt-voltage (I-V) curves constructed at a membrane potential of -90 mV by injecting both de- and hyperpolarizing current pulses were not alte red by diazoxide (30 mu M) or tolbutamide (300 mu M). Moreover, normox ic and hypoxic I-V curves did not cross each other, excluding a revers al of the HD at any membrane potential between -130 and -50 mV. The hy poxia-induced change of the I-V relation was the same both in the abse nce and presence of tolbutamide (300 I IM). In a second series of expe riments, nucleoside di- and triphosphates separated with anion exchang e 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 diphos phates 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 co ntrol values. The tri- to diphosphate ratios progressively declined fo r ATP/ADP and GTP/GDP, but not for UTP/UDP when the duration of hypoxi a was increased from 5 to 30 min, Hence, the rapid fall in the ratios of nucleoside tri- to diphosphates without the induction of a potassiu m current failed to indicate an allosteric regulation of a plasmalemma l K-ATP channel by purine and pyrimidine nucleotides. Diazoxide had no effect on neocortical pyramidal neurons and was effective only in com bination with a hypoxic stimulus; it is suggested that both plasmalemm al and mitochondrial K-ATP channels are involved under these condition s. The hypoxic depolarization may be due to blockade of K+,Na+-ATPase by limitation of energy supplying substrate.