Hypoxia-induced Depolarization Research Articles

Perinatal hyperoxia exposure impairs hypoxia-induced depolarization in rat carotid body glomus cells

Chronic post-natal hyperoxia reduces the hypoxic ventilatory response by reducing the carotid body sensitivity to acute hypoxia as demonstrated by a reduced afferent nerve response, reduced calcium response of carotid body glomus cells and reduced catecholamine secretion in response to acute hypoxia. The present study examined whether hyperoxia alters the electrophysiological characteristics of glomus cells. Rats were treated with hyperoxia for 1 week starting at P1 or P7 and for 2 weeks starting at P1 followed by harvesting and dissociation of their carotid bodies for whole cell, perforated-patch recording. As compared to glomus cells from normoxia animals, hyperoxia treated cells showed a significant reduction in the magnitude of depolarization in response to hypoxia and anoxia, despite little change in the depolarizing response to 20mMK+. Resting cell membrane potential in glomus cells from rats exposed to hyperoxia from P1 to P15 and studied at P15 was slightly depolarized compared to other treatment groups and normoxia-treated cells, but conductance normalized to cell size was not different among groups. We conclude that postnatal hyperoxia impairs carotid chemoreceptor hypoxia transduction at a step between hypoxia sensing and membrane depolarization. This occurs without a major change in baseline electrophysiological characteristics, suggesting altered signaling or alterations in the relative abundance of different leak channel isoforms.

Mitochondria in Hypoxic Pulmonary Vasoconstriction

Mitochondria in Hypoxic Pulmonary Vasoconstriction

Functional expression of background K+ channels in rat carotid body cells.

Oxygen‐sensing glomus cells of the carotid body (CB) express a number of K+ channels that responds to hypoxia. TASK‐like K+ channels are expressed in CB cells and have been suggested to be particularly important for hypoxia‐induced depolarization. However, it is still unclear whether the TASK‐like K+ channels in CB cells are functional correlates of TASK‐1, TASK‐3, TASK‐1/TASK‐3 heteromer, or another K+ channel. To address this issue, we studied the properties of active K+ channels in resting CB cells isolated from 6‐7 day‐old rats, and compared them with those of cloned K+ channels. Single channels were recorded from cell‐attached and inside‐out patches of CB cells. In symmetrical 150 mM KCl, ~15‐pS (TASK‐1‐like) and ~32‐pS (TASK‐3‐like) channels were present together in 90% of patches, and were inhibited by acid (pH 6.0) and methanandamide (5 μM), similar to those of cloned TASK‐1 and TASK‐3. In addition to TASK‐like channels, another acid‐sensitive K+ channel with a unitary conductance of ~30‐pS was present in ~15% of patches. Mechano‐ and fatty acid‐sensitive channels with properties of TREK‐1 and TREK‐2 were also recorded in a few patches. These results show that TASK‐like channels in CB cells are functional correlates of both TASK‐1 and TASK‐3. TREK‐1, TREK‐2 and an unidentified acid‐sensing K+ channel also present in CB cells should contribute to the background K+ current. (Funded by NIH HL054621)

Gene expression of TREK K+ channels in rat carotid body (CB)

Oxygen sensing by CB glomus cells shows postnatal development in mammals. Both hypoxia-induced depolarization and [Ca2+]i response are small at birth and increase with age, but mechanisms are poorly understood. We hypothesized that expression of TREK-1, a two-pore domain K+ channel, may regulate glomus cell excitability during development. Therefore, we tested whether a) TREK-1 is expressed in rat CB; b) TREK-1 expression changes with age; and c) chronic hypoxia (CH) from birth alters TREK-1 expression. rTREK-1 and rTREK-2 primers were tested on rat CB of normoxic 0–1day old (N1), 14–16 days old (N14), and 2 weeks of CH after birth with quantitative real time RT-PCR (qRT-PCR) by using SYBR green. 18s rRNA gene was used as the reference gene and relative gene expression ratio was calculated (Pfaffl MW 2001). Results Immunoreactivity for hTREK-1 was found only in glomus cells at both ages. Between P0 and P14, rTREK-1 expression declined, while rTREK-2 expression did not. TREK-1 and TREK-2 relative gene expression ratio for N1 vs. N14 CB were 0.16 ± 0.03 (n=6) (p<0.001, downregulation) and 0.93 ± 0.22 (n=4) (NS), respectively. In sharp contrast, in rats reared in 0.12 FiO2 from P0 to P14, TREK-1 expression showed almost 2 fold upregulation. The sequence of qRT-PCR amplicon of rat CB TREK-1 showed 100% homology with that of rat cerebellum TREK-1. We conclude that a) TREK-1 and TREK-2 are expressed in CB glomus cells; b) TREK-1 expression decreases >80% between P0 and P14; and c) expression of TREK-1 after birth appears to be regulated by oxygen tension. Developmental changes in TREK-1 potassium channel expression may play a role in CB functional maturation. (funded by NIH R01 HL54621 and UAMS Dean's Research Development Fund).

Voltage-gated Calcium Channels Mediate Hypoxic Vasoconstriction in the Human Placenta

Unlike all vascular beds with the exception of the pulmonary circulation, fetoplacental vessels respond to acute hypoxia with vasoconstriction. While this hypoxic fetoplacental vasoconstriction (HFPV) is considered essential in the pathogenesis of intrauterine growth retardation, its mechanism is largely unknown. Hypoxia inhibits potassium channels and thus causes depolarization in fetoplacental vascular smooth muscle. We propose that this hypoxia-induced depolarization leads to vasoconstriction by activating voltage-dependent calcium (Ca) channels and Ca influx. We compared HFPV between isolated perfused human cotyledons treated with an inhibitor of L-type channels, nifedipine, and preparations receiving only vehicle. While the solvent (diluted DMSO) had no inhibitory effect on HFPV, the hypoxic responses were completely abolished even by a relatively low dose of nifedipine (1 nM). We conclude that activation of L-type Ca channels is an essential part of HFPV.

Down regulation of Kv3.4 channels by chronic hypoxia increases acute oxygen sensitivity in rabbit carotid body

The carotid body (CB) chemoreceptors participate in the ventilatory responses to acute and chronic hypoxia (CH). Arterial hypoxaemia increases breathing within seconds, and CB chemoreceptors are the principal contributors to this reflex hyperventilatory response. Acute hypoxia induces depolarization of CB chemoreceptors by inhibiting certain K+ channels, but the role of these channels in CH, as in high-altitude acclimatization, is less known. Here we explored the effects of prolonged (24-48 h) hypoxic exposure of rabbit CB chemoreceptor cells in primary cultures on the voltage-dependent K+ currents and on their response to acute hypoxia. We found that CH induces a decrease in the amplitude of outward K+ currents due to a reduction in a fast-inactivating BDS- and highly TEA-sensitive component of the current. In spite of this effect, acute hypoxic inhibition of K+ currents is increased in CH cultures, as well as hypoxia-induced depolarization. These data suggest that downregulation of this component (that does not contribute to the oxygen-sensitive K+ current (IKO2) participates in the hypoxic sensitization. Pharmacological, immunocytochemical and quantitative PCR (qPCR) experiments demonstrate that CH-induced decrease in outward K+ currents is due to a downregulation of the expression of Kv3.4 channels. Taken together, our results suggest that CH sensitization in rabbit CB could be achieved by an increase in the relative contribution of IKO2 to the outward K+ current as a consequence of the decreased expression of the oxygen-insensitive component of the current. We conclude that acute and chronic hypoxia can exert their effects acting on different molecular targets.

Postnatal development of E-4031-sensitive potassium current in rat carotid chemoreceptor cells

The O2 sensitivity of dissociated type I cells from rat carotid body increases with age until approximately 14-16 days. Hypoxia-induced depolarization appears to be mediated by an O2-sensitive K+ current, but other K+ currents may modulate depolarization. We hypothesized that membrane potential may be stabilized in newborn type I cells by human ether-a-go-go-related gene (HERG)-like K+ currents that inhibit hypoxia-induced depolarization and that a decrease in this current with age could underlie, in part, the developmental increase in type I cell depolarization response to hypoxia. In dissociated type I cells from 0- to 1- and 11- to 16-day-old rats, using perforated patch-clamp and 70 mM K+ extracellular solution, we measured repolarization-induced inward K+ tail currents in the absence and presence of E-4031, a specific HERG channel blocker. This allowed isolation of the E-4031-sensitive HERG-like current. E-4031-sensitive peak currents in type I cells from 0- to- 1-day-old rats were 2.5-fold larger than in cells from 11- to 16-day-old rats. E-4031-sensitive current density in newborn type I cells was twofold greater than in cells from 11- to 16-day-old rats. Under current clamp conditions, E-4031 enhanced hypoxia-induced depolarization in type I cells from 0- to- 1-day-old but not 11- to 16-day-old rats. With use of fura 2 to measure intracellular Ca2+, E-4031 increased the cytosolic Ca2+ concentration response to anoxia in cells from 0- to- 1-day-old but not cells from 11- to 16-day-old rats. E-4031-sensitive K+ currents are present in newborn carotid body type I cells and decline with age. These findings are consistent with a role for E-4031-sensitive K+ current, and possibly HERG-like K+ currents, in the type I cell hypoxia response maturation.

Muscarinic modulation of hypoxia-induced release of catecholamines from the cat carotid body

Chemotransduction of arterial hypoxemia by the cat carotid body is generally thought to begin with a hypoxia-induced depolarization of the glomus cells (GCs) of the carotid body (CB). This depolarization activates voltage-gated calcium channels with the subsequent entry of calcium, movement of transmitter-containing vesicles to the synaptic-like juncture between the GC and apposed sensory afferent neuron. The vesicles exocytotically release their transmitters which then proceed to the receptors on both the postsynaptic neuron and on the GCs themselves (autoreceptors). Action potentials and their modulation in the sensory fibers are the result, along with the modulation of further neurotransmitter release from the GCs. The purpose of the present study was to: (1) determine the parameters of an incubated cat CB preparation capable of releasing measurable amounts of catecholamines (CAs) in response to hypoxia; (2) determine the impact of muscarinic activities on CA release during the hypoxic challenge; (3) determine if the muscarinic activity preferentially modified the release of one CA more than another; (4) determine if there were any differences in the pattern of hypoxia-induced release of dopamine (DA) vs. norepinephrine (NE). CBs were harvested from deeply anesthetized cats. Cleaned of fat and connective tissue, they were incubated in Krebs Ringer bicarbonate solution at 37 °C, and bubbled with a hyperoxic mixture of gases (95% O 2–5% CO 2) for 30 min. The first series of experiments to address the CB’s hypoxia-induced release of CAs explored the effects of incubating CBs for 2 h with hyperoxia vs. normoxia (21% O 2–6% CO 2) followed by a 30 min hypoxic challenge, with or without l-dihydroxyphenylalanine ( l-DOPA). In the second series of experiments the CBs, after the first 30 min of hyperoxia, were next challenged with hypoxia (4% O 2–5% CO 2) for intervals of 3–20 min with intervening recovery periods of hyperoxia to determine the effect of the duration of the hypoxic exposure on CA release. In the third series of experiments the CBs, after the first 30 min of hyperoxia, were challenged with hypoxia for intervals of 10–40 min in the presence or absence of an M1 or M2 muscarinic receptor antagonist. CAs released into the incubation medium were analyzed by means of high performance liquid chromatography–electrochemical detection using standard procedures. Incubated cat CBs challenged for 2 h with hyperoxia followed by 30 min of hypoxia, released much more measurable amounts of CAs in the presence of 40 μM l-DOPA than without it. Moving from hyperoxia to hypoxia produced a better yield than moving from normoxia to hypoxia, and at least 10–20 min exposures were needed for measurable amounts of CAs. The M1 muscarinic receptor antagonist, pirenzepine, reduced the hypoxia-induced release of CAs during each exposure. Further, the reduction appeared to be dose-related. The M2 muscarinic receptor antagonist, methoctramine, enhanced the hypoxia-induced release of CAs during each exposure. These data support a role for acetylcholine (ACh) in the hypoxia-induced release of CAs, and suggest a significant, if modest, muscarinic dimension to it. And although hypoxia induced a greater release of DA than of NE, the muscarinic modulation of the release (both decreasing it and increasing it) may have had a greater impact on NE release than on DA release. Finally, the patterns of hypoxia-induced release of DA and NE from incubated cat carotid bodies are significantly different.

Partial HIF-1alpha deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia.

Chronic hypoxia depolarizes and reduces K+ current in pulmonary arterial smooth muscle cells (PASMCs). Our laboratory previously demonstrated that hypoxia-inducible factor-1 (HIF-1) contributed to the development of hypoxic pulmonary hypertension. In this study, electrophysiological parameters were measured in PASMCs isolated from intrapulmonary arteries of mice with one null allele at the Hif1a locus encoding HIF-1alpha [Hif1a(+/-)] and from their wild-type [Hif1a(+/+)] littermates after 3 wk in air or 10% O2. Hematocrit and right ventricular wall and left ventricle plus septum weights were measured. Capacitance, K+ current, and membrane potential were measured with whole cell patch clamp. Similar to our laboratory's previous results, hypoxia-induced right ventricular hypertrophy and polycythemia were blunted in Hif1a(+/-) mice. Hypoxia increased PASMC capacitance in Hif1a(+/+) mice but not in Hif1a(+/-) mice. Chronic hypoxia depolarized and reduced K+ current density in PASMCs from Hif1a(+/+) mice. In PASMCs from hypoxic Hif1a(+/-) mice, no reduction in K+ current density was observed, and depolarization was significantly blunted. Thus partial deficiency of HIF-1alpha is sufficient to impair hypoxia-induced depolarization, reduction of K+ current density, and PASMC hypertrophy.

Hypoxic chemosensitivity of the cardiorespiratory regions of the rostral ventrolateral medulla (RVLM)

Recently, unique cardiorespiratory regions in the RVLM have been found to be oxygen sensitive [1,2]. However, the mechanism of sensing O2 in these RVLM regions is unknown. Since heme oxygenase (HO) has been shown to be involved in the hypoxic responses of the carotid body and pulmonary artery, the aim of our work has been to determine if HO is present in the RVLM, whether expression of HO is altered by chronic hypoxia, and whether HO is necessary for the excitatory response of RVLM neurons. RT-PCR of the RVLM of rats exposed to hypoxia (10% O2) or normoxia for 10 days, revealed that HO-2 is expressed in the RVLM constitutively during both normoxia and hypoxia and that HO-1 is induced during chronic hypoxia [3]. Immunocytochemistry localized these HO isoforms to the C1 region and pre-Botzinger complex (pre-Bot) of the RVLM. Thus, HO isoforms are present in the RVLM cardiorespiratory regions under control and hypoxic conditions, consistent with a potential role for HO in the O2 sensing function of these cardiorespiratory regions. To determine whether HO is important for the oxygen sensitivity of the RVLM, we examined the response of cultured RVLM neurons to chemical hypoxia (NaCN), before and after blocking HO with SnPP-IX, and correlated the hypoxia-excited response with the expression of HO-2 using immunocytochemistry. Primary cultures were prepared from neonatal rats and studied using the whole-cell perforated patch clamp technique. The hypoxia-induced depolarization of these RVLM neurons in response to NaCN was abolished after blocking HO. Examination of these hypoxia-excited neurons showed that they were immunoreactive for HO-2. Further phenotyping found that RVLM neurons that express HO are located in the tyrosine hydroxylase rich C1 sympathoexcitatory region as well as within the neurokinin-1 receptor (NK-1R) rich pre-Bot. In fact, 70% of the neurons expressing HO-2 in the pre-Bot co-express NK-1R suggesting that oxygen sensitivity may reside in some respiratory rhythm generating neurons in the pre-Botzinger complex. Thus, HO is present in the cardiorespiratory regions of the RVLM, HO-1 is induced by chronic hypoxia, and HO is necessary for the excitatory response to chemical hypoxia in RVLM neurons consistent with the presence of HO-2 in these excited neurons. These findings support an important role for HO in the oxygen sensitivity of cardiorespiratory neurons in the RVLM.

Inhibition of apamin-sensitive K+ current by hypoxia in adult rat adrenal chromaffin cells.

The effect of hypoxia on small-conductance Ca(2+)-activated K+ current was investigated in a study of adult rat adrenomedullary chromaffin cells (AMCs), which were maintained in short-term culture. The nystatin-perforated, whole-cell patchclamp technique was used to study the effect of hypoxia with minimum perturbation of the intracellular milieu. Under voltage-clamp conditions, acute hypoxia (P(O2) approximately equal to 25 mmHg) suppressed the whole-cell outward currents of more than half the AMCs (24/46). This suppression was eliminated after application of apamin (400 nM), a selective inhibitor of small-conductance Ca(2+)-activated K+ current (I(SK)(Ca)) (n=5), suggesting that an apamin-sensitive component of whole-cell currents is suppressed during hypoxia. In contrast to I(SK)(Ca), Ca2+ current (I(Ca)) (n=10) was not affected by hypoxia. Finally, under current-clamp conditions, hypoxia reversibly depolarized the resting membrane potential of adult AMCs (34/40). Apamin, however, eliminated the hypoxia-induced depolarization (400 nM) (7/8), suggesting that hypoxic depolarization is related to the suppression of I(SK(Ca). From the above results, we conclude that adult AMCs are sensitive to hypoxia, and that I(SK)(Ca) contributes to the hypoxia-induced suppression of whole-cell outward current and depolarization of the resting membrane potential in adult AMCs.

Ionic mechanisms underlying depolarizing responses of an identified insect motor neuron to short periods of hypoxia.

Hypoxia can dramatically disrupt neural processing because energy-dependent homeostatic mechanisms are necessary to support normal neuronal function. In a human context, the long-term effects of such disruption may become all too apparent after a "stroke," in which blood-flow to part of the brain is compromised. We used an insect preparation to investigate the effects of hypoxia on neuron membrane properties. The preparation is particularly suitable for such studies because insects respond rapidly to hypoxia, but can recover when they are restored to normoxic conditions, whereas many of their neurons are large, identifiable, and robust. Experiments were performed on the "fast" coxal depressor motoneuron (Df) of cockroach (Periplaneta americana). Five-minute periods of hypoxia caused reversible multiphasic depolarizations (10-25 mV; n = 88), consisting of an initial transient depolarization followed by a partial repolarization and then a slower phase of further depolarization. During the initial depolarizing phase, spontaneous plateau potentials normally occurred, and inhibitory postsynaptic potential frequency increased considerably; 2-3 min after the onset of hypoxia all electrical activity ceased and membrane resistance was depressed. On reoxygenation, the membrane potential began to repolarize almost immediately, becoming briefly more negative than the normal resting potential. All phases of the hypoxia response declined with repeated periods of hypoxia. Blockade of ATP-dependent Na/K pump by 30 microM ouabain suppressed only the initial transient depolarization and the reoxygenation-induced hyperpolarization. Reduction of aerobic metabolism between hypoxic periods (produced by bubbling air through the chamber instead of oxygen) had a similar effect to that of ouabain. Although the depolarization seen during hypoxia was not reduced by tetrodotoxin (TTX; 2 microM), lowering extracellular Na+ concentration or addition of 500 microM Cd2+ greatly reduced all phases of the hypoxia-induced response, suggesting that Na influx occurs through a TTX-insensitive Cd2+-sensitive channel. Exposure to 20 mM tetraethylammonium and 1 mM 3,4-diaminopyridine increased the amplitude of the hypoxia-induced depolarization, suggesting that activation of K channels may normally limit the amplitude of the hypoxia response. In conclusion we suggest that the slow hypoxia-induced depolarization on motoneuron Df is mainly carried by a TTX-resistant, Cd2+-sensitive sodium influx. Ca2+ entry may also make a direct or indirect contribution to the hypoxia response. The fast transient depolarization appears to result from block of the Na/K pump, whereas the reoxygenation-induced hyperpolarization is largely caused by its subsequent reactivation.

Expression of dopamine D2 receptor in PC-12 cells and regulation of membrane conductances by dopamine.

PC-12 cells depolarize during hypoxia and release dopamine. The hypoxia-induced depolarization is due to inhibition of an O2-sensitive K+ current. The role of dopamine released during hypoxia is uncertain, but it could act as an autocrine to modulate membrane conductance during hypoxia. The current study was undertaken to investigate this possibility. Reverse transcription-polymerase chain reaction and sequence analysis revealed that the D2 isoform of the dopamine receptor is expressed in rat PC-12 cells. Exogenously applied dopamine and the D2 agonist quinpirole elicited inhibition of a voltage-dependent K+ current (I(K)) that was prevented by sulpiride, a D2 receptor antagonist. Dopamine and quinpirole applied during hypoxia potentiated the inhibitory effect of hypoxia on I(K). We also found that quinpirole caused reversible inhibition of a voltage-dependent Ca2+ current (I(Ca)) and attenuation of the increase in intracellular free Ca2+ during hypoxia. Our results indicate that dopamine released from PC-12 cells during hypoxia acts via a D2 receptor to "autoregulate" I(K) and I(Ca).

Membrane depolarization in PC-12 cells during hypoxia is regulated by an O2-sensitive K+ current.

The effects of hypoxia on K+ current (IK), resting membrane potential, and cytosolic free Ca2+ in rat pheochromocytoma (PC-12) cells were studied. Whole cell voltage- and current-clamp experiments were performed to measure IK and membrane potential, respectively. Cytosolic free Ca2+ level was measured using the Ca(2+)-sensitive fluorescent dye fura 2. Depolarizing voltage steps to +50 mV from a holding potential of -90 mV elicited a slowly inactivating, tetraethylammonium chloride-sensitive, and Ca(2+)-insensitive IK that was reversibly inhibited by reduced O2 tension. Graded reduction in PO2 (from 150 to 0 mmHg) induced a graded inhibition of O2-sensitive IK [IK(O2)] up to 46% at 0 mmHg. Moreover, hypoxia induced a 19-mV membrane depolarization and a twofold increase in cytosolic free Ca2+. In Ca(2+)-free condition, inhibition of IK(O2) induced an 8-mV depolarization, suggesting that inhibition of IK(O2) was responsible for initiating depolarization. The effect of reduced PO2 on the current-voltage relationship showed a reduction of outward current and a 14-mV shift in the reversal potential comparable with the amount of depolarization measured in current clamp experiments. Neither Ca(2+)-activated IK nor inwardly rectifying IK are responsible for the hypoxia-induced depolarization. In conclusion, PC-12 cells express an IK(O2), inhibition of which leads to membrane depolarization and increased intracellular Ca2+, making the PC-12 clonal cell line a useful model for studying the molecular and biophysical mechanisms that mediate O2 chemosensitivity.

On the mechanisms underlying hypoxia-induced membrane depolarization in striatal neurons.

Clinical and experimental evidence has shown that the striatal neurons are particularly vulnerable to hypoxia and ischaemia. An excessive excitatory action of glutamate, released by the corticostriatal terminals, has been implicated in this peculiar vulnerability of striatal neurons. We have studied the effects of hypoxia on the membrane properties of striatal neurons intracellularly recorded from a corticostriatal slice preparation. Brief (2-10 min) periods of hypoxia produced reversible membrane depolarizations. During the initial phase of the hypoxia-induced depolarization the frequency of action potential discharge was transiently increased; 2-3 min after the onset of hypoxia the firing activity was fully abolished. Brief periods of hypoxia also caused a reversible reduction of the amplitude of the excitatory postsynaptic potentials (EPSPs) evoked by cortical stimulation. Longer period of hypoxia (12-20 min) produced irreversible membrane depolarizations. In voltage-clamp experiments hypoxia caused an inward current coupled with an increased membrane conductance. Tetrodotoxin (TTX) or low calcium (Ca2+)-high magnesium containing solutions blocked synaptic transmission, but they did not reduce the hypoxia-induced electrical changes. Antagonists of excitatory amino acid receptors failed to affect the electrical effects caused by oxygen deprivation. Hypoxia-induced inward currents were reduced either by the potassium (K+) channel blockers, barium and tetraethyl ammonium (TEA) cations, or by lowering external sodium (Na+) concentration. Blockade of ATP-dependent Na(+)-K+ pump by both ouabain and strophanthidin enhanced hypoxia-induced membrane depolarization/inward current. Our findings indicate that the release of excitatory amino acids does not seem to be required for the acute hypoxia-induced electrical changes in striatal neurons. Moreover, TTX-resistant Na+ influx and K+ currents seem to play an important role in the generation of hypoxia-induced electrical changes. These data also suggest that the selective vulnerability of striatal neurons to oxygen deprivation may be caused by their peculiar sensitivity to energy metabolism failure.

Dextromethorphan attenuates hypoxia-induced neuronal dysfunction in rat neocortical slices

We investigated the effects of the antitussive dextromethorphan (DM; 100 μM) on the extracellular DC potential, extracellular calcium concentration ([Ca 2+] o) and on stimulus-evoked field potential (FP) responses in rat neocortical slices during hypoxia. DM significantly reduced the amplitude of the anoxic depolarization (AD) and the associated [Ca 2+] o decrease by 47.6% and 48.5%, respectively, but did not change the onset latency and the duration of the AD. DM did not affect the preservation or recovery of excitatory synaptic transmission and significantly suppressed paired-pulse inhibition in the postanoxic recovery phase. These results indicate that DM exerts its potential neuroprotective action by reducing the hypoxia-induced depolarization and Ca 2+ influx.

Glucose and sulfonylureas modify different phases of the membrane potential change during hypoxia in rat hippocampal slices

Intracellular recording during hypoxia in submerged hippocampal slices revealed an initial hyperpolarization with decreased membrane input resistance followed by complete depolarization. Glibenclamide (1 μM) reduced and tolbutamide (400 μM) completely blocked the hypoxic hyperpolarization and the accompanying increase in conductance. Neither glibenclamide nor tolbutamide altered the time to 25 or 50% depolarization. Sulfonylurea-treated slices completely depolarized during 10 min of hypoxia and did not recover upon reoxygenation. In contrast, 11 mM glucose had no effect on hypoxic hyperpolarization or conductance, but it significantly slowed hypoxic depolarization. In half of the intracellular recordings made during high glucose hypoxia, the membrane potential and input resistance returned during reoxygenation. Extracellular recordings were used to evaluate the effect of sulfonylureas and high glucose on acute neuronal survival from hypoxia. Glibenclamide (0.1–5 μM) did not change the survival rate of slices exposed to hypoxia, whereas, high glucose (11 and 40 mM) dramatically improved the recovery of population spikes during reoxygenation. These findings support the hypothesis that an ATP-sensitive K + channel mediates hypoxic hyperpolarization 14,15,19, but these channels are not regulated by glucose in the brain as in pancreatic islet-cells. These results also suggest that high glucose protects hippocampal slices from hypoxia by slowing the hypoxia-induced depolarization.