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 microM). The effect of 30 but not 300 microM
diazoxide was reversed by washout.
Tolbutamide (300 microM), an antagonist of K(
ATP) channels, did not alter the HD when given alone. It did, however, abolish the inhibitory effect of
diazoxide (30 microM) on the HD. Neither
diazoxide (3-300 microM) nor
tolbutamide (300 microM) 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 microM) or
tolbutamide (300 microM). 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 microM). 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.