Altered iPSC-derived neurons' sodium channel properties in subjects with Monge's disease.

Monge's disease, also known as chronic mountain sickness (CMS), is a disease that potentially threatens more than 140 million highlanders during extended time living at high altitudes (over 2500m). The prevalence of CMS in Andeans is about 15-20%, suggesting that the majority of highlanders (non-CMS) are rather healthy at high altitudes; however, CMS subjects experience severe hypoxemia, erythrocytosis and many neurologic manifestations including migraine, headache, mental fatigue, confusion, and memory loss. The underlying mechanisms of CMS neuropathology are not well understood and no ideal treatment is available to prevent or cure CMS, except for phlebotomy. In the current study, we reprogrammed fibroblast cells from both CMS and non-CMS subjects' skin biopsies into the induced pluripotent stem cells (iPSCs), then differentiated into neurons and compared their neuronal properties. We discovered that CMS neurons were much less excitable (higher rheobase) than non-CMS neurons. This decreased excitability was not caused by differences in passive neuronal properties, but instead by a significantly lowered Na(+) channel current density and by a shift of the voltage-conductance curve in the depolarization direction. Our findings provide, for the first time, evidence of a neuronal abnormality in CMS subjects as compared to non-CMS subjects, hoping that such studies can pave the way to a better understanding of the neuropathology in CMS.

Review: Hypoxic and oxidative stress resistance in Drosophila melanogaster.

Oxygen (O(2)) is essential for aerobic life; however, the level of O(2), whether too low (hypoxia) or too high (hyperoxia), can induce oxidative injury and increase morbidity and mortality. Disruption of O(2) homeostasis represents a major aspect of many disease etiologies and pathobiology. In the past, our laboratory has been using Drosophila melanogaster to investigate the cellular and molecular aspects of the response to hypoxia and oxidative stress. There are several advantages for using Drosophila as a model system, the most important one being an evolutionary conservation of genetic and signaling pathways from Drosophila to mammals. As a proof of this concept, we have shown that we can substantially improve the tolerance of human cells in culture by transfecting these cells with particular Drosophila genes. In this review, we summarize the recent findings from our laboratory using Drosophila as a model system to investigate the genetic basis of hypoxia/hyperoxia tolerance. We have done microarray studies and identified several oxidative stress resistance genes that play an important role in individual paradigms such as constant or intermittent hypoxia, short term (days) or long term (generations) hypoxia/hyperoxia. Our studies provide evidence that a pattern of oxidative stress is specific in inducing a gene expression profile which, in turn, plays an important role in modulating the phenotype. To improve our understanding of oxidative and hypoxic stress as well as its associated diseases, multi-disciplinary approaches are necessary and critical in the study of complicated issues in systems biology.

The sodium-activated potassium channel Slack is modulated by hypercapnia and acidosis.

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.

Prostaglandin transporter expression in mouse brain during development and in response to 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.

Beta-subunit-dependent modulation of hSlo BK current by arachidonic acid.

In this study, we examined the effect of arachidonic acid (AA) on the BK alpha-subunit with or without beta-subunits expressed in Xenopus oocytes. In excised patches, AA potentiated the hSlo-alpha current and slowed inactivation only when beta2/3 subunit was co-expressed. The beta2-subunit-dependent modulation by AA persisted in the presence of either superoxide dismutase or inhibitors of AA metabolism such as nordihydroguaiaretic acid and eicosatetraynoic acid, suggesting that AA acts directly rather than through its metabolites. Other cis unsaturated fatty acids (docosahexaenoic and oleic acid) also enhanced hSlo-alpha + beta2 currents and slowed inactivation, whereas saturated fatty acids (palmitic, stearic, and caprylic acid) were without effect. Pretreatment with trypsin to remove the cytosolic inactivation domain largely occluded AA action. Intracellularly applied free synthetic beta2-ball peptide induced inactivation of the hSlo-alpha current, and AA failed to enhance this current and slow the inactivation. These results suggest that AA removes inactivation by interacting, possibly through conformational changes, with beta2 to prevent the inactivation ball from reaching its receptor. Our data reveal a novel mechanism of beta-subunit-dependent modulation of BK channels by AA. In freshly dissociated mouse neocortical neurons, AA eliminated a transient component of whole cell K(+) currents. BK channel inactivation may be a specific mechanism by which AA and other unsaturated fatty acids influence neuronal death/survival in neuropathological conditions.

The calcium-sensitive large-conductance potassium channel (BK/MAXI K) is present in the inner mitochondrial membrane of rat brain.

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.

Hypoxia induces major effects on cell cycle kinetics and protein expression in Drosophila melanogaster embryos.

Hypoxia induces a stereotypic response in Drosophila melanogaster embryos: depending on the time of hypoxia, embryos arrest cell cycle activity either at metaphase or just before S phase. To understand the mechanisms underlying hypoxia-induced arrest, two kinds of experiments were conducted. First, embryos carrying a kinesin-green fluorescent protein construct, which permits in vivo confocal microscopic visualization of the cell cycle, showed a dose-response relation between O2 level and cell cycle length. For example, mild hypoxia (Po2 approximately 55 Torr) had no apparent effect on cell cycle length, whereas severe hypoxia (Po2 approximately 25-35 Torr) or anoxia (Po2 = 0 Torr) arrested the cell cycle. Second, we utilized Drosophila embryos carrying a heat shock promoter driving the string (cdc25) gene (HS-STG3), which permits synchronization of embryos before the start of mitosis. Under conditions of anoxia, we induced a stabilization or an increase in the expression of several G1/S (e.g., dE2F1, RBF2) and G2/M (e.g., cyclin A, cyclin B, dWee1) proteins. This study suggests that, in fruit fly embryos, 1) there is a dose-dependent relationship between cell cycle length and O2 levels in fruit fly embryos, and 2) stabilized cyclin A and E2F1 are likely to be the mediators of hypoxia-induced arrest at metaphase and pre-S phase.

Activation of voltage-sensitive sodium channels during oxygen deprivation leads to apoptotic neuronal death.

Sodium (Na(+)) entry into neurons during hypoxia is known to be associated with cell death. However, it is not clear whether Na(+) entry causes cell death and by what mechanisms this increased Na(+) entry induces death. In this study we used cultures of rat neocortical neurons to show that an increase in intracellular sodium (Na(i)(+)) through voltage-sensitive sodium channels (VSSCs), during hypoxia contributes to apoptosis. Hypoxia increased Na(i)(+) and induced neuronal apoptosis, as assessed by electron microscopy, annexin V staining, and terminal UDP nick end labeling staining. Reducing Na(+) entry with the VSSC blocker, tetrodotoxin (TTX), attenuated apoptotic neuronal death via a reduction in caspase-3 activation. Since the attenuation of apoptosis by TTX during hypoxia suggested that the activation of VSSCs and Na(+) entry are crucial events in hypoxia-induced cell death, we also determined whether the activation of VSSCs per se could lead to apoptosis under resting conditions. Increasing Na(+) entry with the VSSC activator veratridine also induced neuronal apoptosis and caspase-3 activation. These data indicate that a) Na(+) entry via VSSCs during hypoxia leads to apoptotic cell death which is mediated, in part, by caspase-3 and b) activation of VSSCs during oxygen deprivation is a major event by which hypoxia induces cell death.

Expression of Na+/H+ and HCO3- -dependent transporters in Na+/H+ exchanger isoform 1 null mutant mouse brain.

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.

Chronic intermittent hypoxia decreases the expression of Na/H exchangers and HCO3-dependent transporters in mouse CNS.

Chronic intermittent hypoxia (CIH) is a component of several disease states, including obstructive sleep apnea, which results in neurocognitive and cardiovascular morbidity. Because chronic hypoxia can induce changes in metabolism and pH homeostasis, we hypothesized that CIH induces changes in the expression of acid-base transporters. Two- to three-day-old mice, exposed to alternating cycles of 2 min of hypoxia (6.0-7.5% O2) and 3 min of normoxia (21% O2) for 8 h/day for 28 days, demonstrated decreases in specific acid-base transport protein expression in most of the central nervous system (CNS). Sodium/hydrogen exchanger isoform 1 (NHE1) and sodium-bicarbonate cotransporter expression were decreased in all regions of the CNS but especially so in the cerebellum. NHE3, which is only expressed in the cerebellum, was also significantly decreased. Anion exchanger 3 protein was decreased in most brain regions, with the decrease being substantial in the hippocampus. These results indicate that CIH induces downregulation of the major acid-extruding transport proteins, NHE1 and sodium-bicarbonate cotransporter, in particular regions of the CNS. This downregulation in acid-extruding capacity may render neurons more prone to acidity and possibly to injury during CIH, especially in the cerebellum and hippocampus. Alternatively, it is possible that O2 consumption in these regions is decreased after CIH, with consequential downregulation in the expression of certain cellular proteins that may be less needed under such circumstances.

Genetic models in applied physiology: invited review: effect of oxygen deprivation on cell cycle activity: a profile of delay and arrest.

One of the most fascinating fields that have emanated in the past few decades is developmental biology. This is not only the case from a research point of view but also from the angle of clinical care and treatment strategies. It is now well demonstrated that there are many diseases (some believe all diseases) that have their roots in embryogenesis or in early life, where nature and environment often team up to facilitate the genesis of disease. There is probably no better example to illustrate the interactions between nature and environment than in early life, as early as in the first several cell cycles. As will be apparent in this review, the cell cycle is a very regulated activity and this regulation is genetic in nature, with checkpoint proteins playing an important role in controlling the timing, the size, and the growth of daughter cells. However, it is also very clear, as will be discussed in this work, that the microenvironment of the first dividing cells is so important for the outcome of the organism. In this review, we will focus on the effect of one stress, that of hypoxia, on the young embryo and its cell division and growth. We will first review some of the cell cycle definitions and stages and then review briefly our current knowledge and its gaps in this area.

The role of HCO3(-)-dependent mechanisms in pHi regulation during O2 deprivation.

We have reported in our previous work that, in the absence of HCO(3)(-), Na(+)/H(+) exchanger is responsible for an anoxia-induced alkalinization in hippocampal CA1 neurons. HCO(3)(-)-dependent mechanisms have been reported to play a key role in pH(i) regulation in nerve cells, but how their function is affected by O(2) deprivation has not been well studied. In this work, pH(i) measurements (obtained from dissociated neurons loaded with carboxy-seminaphthorhodafluor-1 and using confocal microscopy) and whole-cell patch clamp recording techniques were used to investigate the role of HCO(3)(-)-dependent membrane exchangers on CA1 neurons during O(2) deprivation. Anoxia (5 min) induced a small acidification in neurons in the presence of HCO(3)(-) and this acidification was changed to a significant alkalinization when neurons were bathed with Hepes buffer or when 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was applied in a HCO(3)(-) solution, indicating that HCO(3)(-)-dependent mechanisms were involved. A marked anoxia-induced acidification (0.33+/-0.11 pH unit) was seen when the Na(+)/H(+) exchange was blocked with 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate in the presence of HCO(3)(-), but the same anoxia did not cause a significant pH(i) change in a Na(+) free, HCO(3)(-) solution, suggesting that the anoxia-induced acidification in the presence of 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate is dependent on both Na(+) and HCO(3)(-). Furthermore, anoxia did not cause a significant pH(i) change when both 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate were present. Current clamp recordings showed a significant membrane depolarization following anoxia in HCO(3)(-) solution but not in Hepes buffer. Our data suggest that, in hippocampal neurons: a) pH(i) regulation during O(2) deprivation is affected not only by metabolism but also by membrane exchangers, and b) besides the activation of Na(+)/H(+) exchange, anoxia activates a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive, Na(+)-dependent acid loader (possibly electrogenic).

Na(+)/Ca(2+) exchanger expression in the developing rat cortex.

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.

Role of Na(+)/H(+) exchanger during O(2) deprivation in mouse CA1 neurons.

To determine the role of membrane transporters in intracellular pH (pH(i)) regulation under conditions of low microenvironmental O(2), we monitored pH(i) in isolated single CA1 neurons using the fluorescent indicator carboxyseminaphthorhodafluor-1 and confocal microscopy. After total O(2) deprivation or anoxia (PO(2) approximately equal to 0 Torr), a large increase in pH(i) was seen in CA1 neurons in HEPES buffer, but a drop in pH(i), albeit small, was observed in the presence of HCO(3)(-). Ionic substitution and pharmacological experiments showed that the large anoxia-induced pH(i) increase in HEPES buffer was totally Na(+) dependent and was blocked by HOE-694, strongly suggesting the activation of the Na(+)/H(+) exchanger (NHE). Also, this pH(i) increase in HEPES buffer was significantly smaller in Na(+)/H(+) exchanger isoform 1 (NHE1) null mutant CA1 neurons than in wild-type neurons, demonstrating that NHE1 is responsible for part of the pH(i) increase following anoxia. Both chelerythrine and H-89 partly blocked, and H-7 totally eliminated, this anoxia-induced pH(i) increase in the absence of HCO. We conclude that 1) O(2) deprivation activates Na(+)/H(+) exchange by enhancing protein kinase activity and 2) membrane proteins, such as NHE, actively participate in regulating pH(i) during low-O(2) states in neurons.

Decreased neuronal excitability in hippocampal neurons of mice exposed to cyclic hypoxia.

To study the physiological effects of chronic intermittent hypoxia on neuronal excitability and function in mice, we exposed animals to cyclic hypoxia for 8 h daily (12 cycles/h) for approximately 4 wk, starting at 2-3 days of age, and examined the properties of freshly dissociated hippocampal neurons in vitro. Compared with control (Con) hippocampal CA1 neurons, exposed (Cyc) neurons showed action potentials (AP) with a smaller amplitude and a longer duration and a more depolarized resting membrane potential. They also have a lower rate of spontaneous firing of AP and a higher rheobase. Furthermore, there was downregulation of the Na(+) current density in Cyc compared with Con neurons (356.09 +/- 54.03 pA/pF in Cyc neurons vs. 508.48 +/- 67.30 pA/pF in Con, P < 0.04). Na(+) channel characteristics, including activation, steady-state inactivation, and recovery from inactivation, were similar in both groups. The deactivation rate, however, was much larger in Cyc than in Con (at -100 mV, time constant for deactivation = 0.37 +/- 0.04 ms in Cyc neurons and 0.18 +/- 0.01 ms in Con neurons). We conclude that the decreased neuronal excitability in mice neurons treated with cyclic hypoxia is due, at least in part, to differences in passive properties (e.g., resting membrane potential) and in Na(+) channel expression and/or regulation. We hypothesize that this decreased excitability is an adaptive response that attempts to decrease the energy expenditure that is used for adjusting disturbances in ionic homeostasis in low-O(2) conditions.

Increased neuronal excitability and seizures in the Na(+)/H(+) exchanger null mutant mouse.

Mice lacking the Na(+)/H(+) exchanger isoform 1 (NHE1) manifest neurological diseases that include ataxia, motor deficits, and a seizure disorder. The molecular basis for the phenotype has not been clear, and it has not been determined how the lack of NHE1 leads, in particular, to the seizure disorder. We have shown in this work that hippocampal CA1 neurons in mutant mice have a much higher excitability than in wild-type mice. This higher excitability is partly based on an upregulation of the Na(+) current density (608.2 +/- 123.2 pA/pF in NHE1 mutant vs. 334.7 +/- 63.7 pA/pF in wild type in HCO/CO(2)). Alterations in Na(+) channel characteristics, including steady-state inactivation (shift of 18 mV in the depolarization direction in the mutant), recovery from inactivation (tau(h) = 5.22 +/- 0.49 ms in wild-type neurons and 2.20 +/- 0.20 ms in mutant neurons), and deactivation (at -100 mV, tau(d) = 1.75 +/- 0.53 ms in mutant and 0.21 +/- 0.05 ms in wild-type neurons) further enhance the differences in excitability between mutant and wild-type mice. Our investigation demonstrates the existence of an important functional interaction between the NHE1 protein and the voltage-sensitive Na(+) channel. We hypothesize that the increased neuronal excitability and possibly the seizure disorder in mice lacking the NHE1 is due, at least in part, to changes in Na(+) channel expression and/or regulation.

Major difference in the expression of delta- and mu-opioid receptors between turtle and rat brain.

The reptilian turtle brain has a remarkably higher endurance for anoxia than mammalian brains. Since the response to O(2) deprivation is dependent in a major way on the expression and regulation of membrane proteins, differences in such proteins may play a role in the species-related differences in hypoxic responses. Because opioid system is involved in the regulation of hypoxic responses, we asked whether there are differences between rat and turtle brains in terms of opioid receptor expression. In this work, we compared the expression and distribution of delta-and mu-opioid receptors in the turtle and rat brains. Our results show that (1) the dissociation constant (K(d)) for delta-receptor binding was approximately four times lower and B(max) was more than double in the turtle brain homogenates than in rat ones; (2) the delta-receptor binding density was heterogeneously distributed in the turtle brain, with a higher density in the rostral regions than in the brainstem and spinal cord, and was generally much higher than in rat brains from the cortex to spinal cord; (3) the delta-opioid receptors in the rat brains were mostly located in the cortex, caudate putamen, and amygdala with an extremely low density in most subcortical (e.g., hippocampus and thalamus) and almost all brainstem regions; and (4) in sharp contrast to delta-opioid receptors, mu-opioid receptor density was much lower in all turtle brain regions compared with the rat ones. Our results demonstrate that the turtle brain is actually an organ of delta-opioid receptors, whereas the rat brain has predominantly mu-opioid receptors. Because we have recently found that delta-opioid receptors protect neurons against glutamate and hypoxic stress, we speculate that the unique pattern of delta-receptor receptor expression and distribution plays a critical role in the tolerance of turtle brain to stressful situations characterized by glutamate excitotoxicity.

Cell cycle progression and cell division are sensitive to hypoxia in Drosophila melanogaster embryos.

We and others recently demonstrated that Drosophila melanogaster embryos arrest development and embryonic cells cease dividing when they are deprived of O2. To further characterize the behavior of these embryos in response to O2 deprivation and to define the O2-sensitive checkpoints in the cell cycle, embryos undergoing nuclear cycles 3-13 were subjected to O2 deprivation and examined by confocal microscopy under control, hypoxic, and reoxygenation conditions. In vivo, real-time analysis of embryos carrying green fluorescent protein-kinesin demonstrated that cells arrest at two major points of the cell cycle, either at the interphase (before DNA duplication) or at metaphase, depending on the cell cycle phase at which O2 deprivation was induced. Immunoblot analysis of embryos whose cell divisions are synchronized by inducible String (cdc25 homolog) demonstrated that cyclin B was degraded during low O2 conditions in interphase-arrested embryos but not in those arrested in metaphase. Embryos resumed cell cycle activity within ~20 min of reoxygenation, with very little apparent change in cell cycle kinetics. We conclude that there are specific points during the embryonic cell cycle that are sensitive to the O2 level in D. melanogaster. Given the fact that O2 deprivation also influences the growth and development of other species, we suggest that similar hypoxia-sensitive cell cycle checkpoints may also exist in mammalian cells.

Mutation in pre-mRNA adenosine deaminase markedly attenuates neuronal tolerance to O2 deprivation in Drosophila melanogaster.

O2 deprivation can produce many devastating clinical conditions such as myocardial infarct and stroke. The molecular mechanisms underlying the inherent tissue susceptibility or tolerance to O2 lack are, however, not well defined. Since the fruit fly, Drosophila melanogaster, is extraordinarily tolerant to O2 deprivation, we have performed a genetic screen in the Drosophila to search for loss-of-function mutants that are sensitive to low O2. Here we report on the genetic and molecular characterization of one of the genes identified from this screen, named hypnos-2. This gene encodes a Drosophila pre-mRNA adenosine deaminase (dADAR) and is expressed almost exclusively in the adult central nervous system. Disruption of the dADAR gene results in totally unedited sodium (Para), calcium (Dmca1A), and chloride (DrosGluCl-alpha) channels, a very prolonged recovery from anoxic stupor, a vulnerability to heat shock and increased O2 demands, and neuronal degeneration in aged flies. These data clearly demonstrate that, through the editing of ion channels as targets, dADAR, for which there are mammalian homologues, is essential for adaptation to altered environmental stresses such as O2 deprivation and for the prevention of premature neuronal degeneration.

Sodium-hydrogen exchangers and sodium-bicarbonate co-transporters: ontogeny of protein expression in the rat brain.

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.