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Slack (Slo 2.2), a member of the Slo potassium channel family, is activated by both voltage and cytosolic factors, such as Na(+) ([Na(+)](i)) and Cl(-) ([Cl(-)](i)). Since the Slo family is known to play a role in hypoxia, and since hypoxia/ischemia is associated with an increase in H(+) and CO(2) intracellularly, we hypothesized that the Slack channel may be affected by changes in intracellular concentrations of CO(2) and H(+). To examine this, we expressed the Slack channel in Xenopus oocytes and the Slo 2.2 protein was allowed to be inserted into the plasma membrane. Inside-out patch recordings were performed to examine the response of Slack to different CO(2) concentrations (0.038%, 5%, 12%) and to different pH levels (6.3, 6.8, 7.3, 7.8, 8.3). In the presence of low [Na(+)](i) (5 mM), the Slack channel open probability decreased when exposed to decreased pH or increased CO(2) in a dose-dependent fashion (from 0.28+/-0.03, n=3, at pH 7.3 to 0.006+/-0.005, n=3, P=0.0004, at pH 6.8; and from 0.65+/-0.17, n=3, at 0.038% CO(2) to 0.22+/-0.07, n=3, P=0.04 at 12% CO(2)). In the presence of high [Na(+)](i) (45 mM), Slack open probability increased (from 0.03+/-0.01 at 5 mM [Na(+)](i), n=3, to 0.11+/-0.01, n=3, P=0.01) even in the presence of decreased pH (6.3). Since Slack activity increases significantly when exposed to increased [Na(+)](i), even in presence of increased H(+), we propose that Slack may play an important role in pathological conditions during which there is an increase in the intracellular concentrations of both acid and Na(+), such as in ischemia/hypoxia.

Prostaglandins (PGs) are bioactive lipid mediators released following brain hypoxic-ischemic injury. Clearance and re-uptake of these prostaglandins occur via a transmembrane prostaglandin transporter (PGT), which exchanges PG for lactate. We used Western blot analyses to examine the PGT developmental profile and its regional distribution as well as changes in transporter expression during chronic hypoxia in the neonatal mouse brain. Microsomal preparations from four brain regions (cortex, hippocampus, cerebellum and brainstem/diencephalon) showed gradual increases in prostaglandin transporter expression in all brain regions examined from postnatal day 1 till day 30. There was a significant regional heterogeneity in the prostaglandin transporter expression with highest expression in the cortex, followed by cerebellum and hippocampus, and least expressed in the brainstem/diencephalon. To further delineate the pattern of prostaglandin transporter expression, separate astrocytic and neuronal microsomal preparations were also examined. In contrast to neurons, which had a robust expression of prostaglandin transporters, astrocytes had very little PGT expression under basal conditions. In response to chronic hypoxia, there was a significant decline in PGT expression in vivo and in neurons in vitro, whereas cultured astrocytes increased their PGT expression. This is the first report on PGT expression in the CNS and our studies suggest that PGTs have 1) a widespread distribution in the CNS; 2) a gradual increase and a differential expression in various regions during brain development; and 3) striking contrast in expression between glia and neurons, especially in response to hypoxia. Since PGTs play a role as prostaglandin-lactate exchangers, we hypothesize that PGTs are important in the CNS during stress such as hypoxia.

Large-conductance voltage- and calcium-sensitive channels are known to be expressed in the plasmalemma of central neurons; however, recent data suggest that large-conductance voltage- and calcium-sensitive channels may also be present in mitochondrial membranes. To determine the subcellular localization and distribution of large-conductance voltage- and calcium-sensitive channels, rat brain fractions obtained by Ficoll-sucrose density gradient centrifugation were examined by Western blotting, immunocytochemistry and immuno-gold electron microscopy. Immunoblotting studies demonstrated the presence of a consistent signal for the alpha subunit of the large-conductance voltage- and calcium-sensitive channel in the mitochondrial fraction. Double-labeling immunofluorescence also demonstrated that large-conductance voltage- and calcium-sensitive channels are present in mitochondria and co-localize with mitochondrial-specific proteins such as the translocase of the inner membrane 23, adenine nucleotide translocator, cytochrome c oxidase or complex IV-subunit 1 and the inner mitochondrial membrane protein but do not co-localize with calnexin, an endoplasmic reticulum marker. Western blotting of discrete subcellular fractions demonstrated that cytochrome c oxidase or complex IV-subunit 1 was only expressed in the mitochondrial fraction whereas actin, acetylcholinesterase, cadherins, calnexin, 58 kDa Golgi protein, lactate dehydrogenase and microtubule-associated protein 1 were not, demonstrating the purity of the mitochondrial fraction. Electron microscopic examination of the mitochondrial pellet demonstrated gold particle labeling within mitochondria, indicative of the presence of large-conductance voltage- and calcium-sensitive channels in the inner mitochondrial membrane. These studies provide concrete morphological evidence for the existence of large-conductance voltage- and calcium-sensitive channels in mitochondria: our findings corroborate the recent electrophysiological evidence of mitochondrial large-conductance voltage- and calcium-sensitive channels in glioma and cardiac cells.

Acid-base transporters, such as the sodium-hydrogen exchangers (NHEs) and bicarbonate-dependent transporters, play an important role in the regulation of intracellular pH (pH(i)) in the CNS. Previous studies from our laboratory have shown that the absence of the major NHE isoform 1 (NHE1) reduced the steady-state pH(i) and recovery rate from an acid load in the hippocampal neurons not only in HEPES but also in HCO(3)(-) solutions (Yao et al., 1999). The purpose of the current study was to determine whether the NHE1 null mutation affects the expression of pH-regulatory transporters in the mouse CNS. Immunoblotting and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) were performed to examine the protein and mRNA levels of NHE1-4, electrogenic sodium-bicarbonate cotransporter 1 variants (NBCe1), and brain-specific anion exchanger 3 (AE3) in four brain regions (cerebral cortex, hippocampus, cerebellum and brainstem-diencephalon). NHE1 null mutant mice were compared with their wild type controls at the average age of approximately 4 weeks. Our results revealed that the NHE1 null mutation caused a significant increase in NHE3 in the cerebellum (84% for protein, 105% for mRNA), an increase in NBCe1 expression in the brainstem-diencephalon (approximately 40-50% for protein, 9-15% for mRNA), as well as a decrease in AE3 in the hippocampus (approximately 60% for protein, 24% for mRNA). We conclude that the NHE1 null mutation does alter the expression of other membrane transporters at both protein and mRNA levels. The alteration is region-specific. An increase in acid extruders (e.g. NHE3) and a decrease in acid loaders (e.g. AE3) suggest that there are some compensatory mechanisms that occur in NHE1 null mutant mice.

The Na(+)/Ca(2+) exchanger (NCX) participates in the regulation of neuronal Ca(2+) homeostasis and is also believed to be involved in the neuronal responses to hypoxia. However, there are very limited data on how NCX mRNA and protein expression are regulated during brain development. In the present study, we sought to elucidate the developmental expression of NCX1 and NCX2 in the rat cortex from late fetal to adult stages using reverse transcription-polymerase chain reaction and western blot assays. The primers for NCX1 mRNA targeted the alternative splicing domain to allow differentiation between NCX1 splice variants. Our results show that: (1) only two NCX1 mRNA splice variants (NCX1.5 and NCX1.4) are present in the cortex and their expression is age-dependent; (2) total NCX1 mRNA levels are low in fetal tissue, reach maximum density at postnatal day 8 and substantially decline with further maturation; (3) NCX2 mRNA density is significantly greater than total NCX1 mRNA for all ages and increases markedly during maturation from fetus/neonate to adult; and (4) NCX1 protein expression is lowest in late fetal cortex and reaches maximum levels after 2 weeks postnatally, even though expression levels are not significantly different between newborn and adult animals. Also, we found a similar NCX1 protein trend in the subcortical and cerebellar regions during development. From these data we suggest that NCX1 and NCX2 are differentially expressed in the cortex with a predominance of NCX2 levels during postnatal development. We speculate that the developmental increase in NCX2 expression is responsible for the overall increase in Na(+)/Ca(2+) exchange capacity during maturation.

We used western blotting to examine the developmental profiles (at embryonic day 16 and postnatal days 1, 13, 23, 33 and 105) of protein expression for three sodium-hydrogen exchanger isoforms (1, 2 and 4) and for a sodium-bicarbonate co-transporter in three CNS regions (cortex, cerebellum and brainstem-diencephalon). In microsomal preparations, sodium-hydrogen exchanger isoform 1 and sodium-bicarbonate co-transporter protein expression in the CNS increases gradually from embryonic day 16 (25-40% of the adult level) to postnatal day 105. In contrast, sodium-hydrogen exchanger isoform 2 and 4 expression reaches a maximum (three to 20 times the adult level) at around three to four weeks of age. There is significant regional heterogeneity in the expression of sodium-hydrogen exchanger and sodium-bicarbonate co-transporter proteins in the rat CNS. Sodium-hydrogen exchanger isoform 1 was highly expressed in the brainstem-diencephalon, whereas the sodium-bicarbonate co-transporter was robustly expressed in the cerebellum and brainstem-diencephalon. These data indicate that the expression of sodium-hydrogen exchanger and sodium-bicarbonate co-transporter proteins varies as a function of both development and specific brain region.