Potassium Conductance Causing Hyperpolarization of CA1 Hippocampal Neurons During Hypoxia

G. Erdemli, Y. Z. Xu, and K. Krnjevic'

Anaesthesia Research Department, McGill University, Montreal, Quebec H3G 1Y6, Canada

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Erdemli, G., Y. Z. Xu, and K. Krnjevic'. Potassium conductance causing hyperpolarization of CA1 hippocampal neurons during hypoxia. J. Neurophysiol. 80: 2378-2390, 1998. In experiments on slices (from 100- to 150-g Sprague-Dawley rats) kept at 33°C, we studied the effects of brief hypoxia (2-3 min) on CA1 neurons. In whole cell recordings from submerged slices, with electrodes containing only KMeSO4 and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and in the presence of kynurenate and bicuculline (to minimize transmitter actions), hypoxia produced the following changes: under current clamp, 36 cells were hyperpolarized by 2.7 ± 0.5 (SE) mV and their input resistance (Rin) fell by 23 ± 2.7%; in 30 cells under voltage clamp, membrane current increased by 114 ± 22.3 pA and input conductance (Gin) by 4.9 ± 0.9 nS. These effects are much greater than those seen previously with K gluconate whole cell electrodes, but only half those seen with "sharp" electrodes. The hypoxic hyperpolarizations (or outward currents) were not reduced by intracellular ATP (1-5 mM) or bath-applied glyburide (10 µM): therefore they are unlikely to be mediated by conventional ATP-sensitive K channels. On the other hand, their depression by internally applied ethylene glycol-bis-(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (1.1 and 11 mM) and especially 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (11-33 mM) indicated a significant involvement of Ca-dependent K (KCa) channels. The beta -adrenergic agonist isoprenaline (10 µM) reduced hypoxic hyperpolarizations and decreases in Rin (n = 4) (and in another 11 cells corresponding changes in Gin); and comparable but more variable effects were produced by internally applied 3':5'-adenosine cyclic monophosphate (cAMP, 1 mM, n = 6) and bath-applied 8-bromo-cAMP (n = 8). Thus afterhyperpolarization-type KCa channels probably take part in the hypoxic response. A major involvement of G proteins is indicated by the near total suppression of the hypoxic response by guanosine 5'-O-(3-thiotriphosphate) (0.1-0.3 mM, n = 23) and especially guanosine 5'-O-(2-thiodiphosphate) (0.3 mM, n = 26), both applied internally. The adenosine antagonist 8-(p-sulfophenyl)theophylline (10-50 µM) significantly reduced hypoxic hyperpolarizations and outward currents in whole cell recordings (with KMeSO4 electrodes) from submerged slices but not in intracellular recordings (with KCl electrodes) from slices kept at gas/saline interface. In further intracellular recordings, antagonists of gamma -aminobutyric acid-B or serotonin receptors also had no clear effect. In conclusion, these G-protein-dependent hyperpolarizing changes produced in CA1 neurons by hypoxia are probably initiated by Ca2+ release from internal stores stimulated by enhanced glycolysis and a variable synergistic action of adenosine.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Brief periods of hypoxia (2-3 min) result in a total, but reversible, loss of CA1 neuronal function. After a transient initial depolarization, the characteristic changes are cessation of firing, moderate hyperpolarization, and marked decrease in input resistance (Rin), probably caused by enhanced potassium conductance (GK) (Belousov et al. 1995; Croning et al. 1995; Fujimura et al. 1997; Fujiwara et al. 1987; Haddad and Jiang 1993; Hansen et al. 1982; Hyllienmark and Brismar 1996; Krnjevic' 1993; Krnjevic' and Leblond 1989; Yamamoto et al. 1997). Under voltage clamp, the predominant effect is a corresponding outward current and increased input conductance (Gin) (Haddad and Jiang 1993; Krnjevic' 1993; Krnjevic' and Leblond 1989).

The initial depolarization reflects an inward current that probably persists throughout the hypoxic period, as indicated by the sustained inward current seen in many cells during hypoxia when outward current are suppressed by K channel blockers such as Ba and carbachol (Krnjevic' and Xu 1990). According to our unpublished observations, the associated conductance changes (<0.5 nS) are only some 3% of the mean input conductance of our cells and <10% of the observed hypoxic changes in conductance (Table 3). Although not negligible (they explain why the hypoxic outward current often has a more positive reversal potential than would be expected of a pure K current), they are not a major component of the overall response and therefore are not further analyzed in the present paper.

 
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TABLE 3. Resting membrane properties and hypoxic responses of CA1 neurons recorded under voltage clamp with different internal electrode solutions

The nature of the GK responsible for the prevailing hyperpolarization and how it is triggered by hypoxia has not been fully clarified. Obvious possibilities include a Ca-sensitive GK (GK(Ca)) activated by cytosolic Ca2+ ([Ca2+]i) (Krnjevic' 1993; Krnjevic' and Lisiewicz 1972); a ATP-sensitive GK (GK(ATP)) opened by ATP depletion (Ashcroft 1988); and a G-protein-dependent GK activated by adenosine, which is known to be released during hypoxia (Berne et al. 1974), or some other endogenous ligand, such gamma -aminobutyric acid (GABA) or serotonin (Nicoll 1988).

The hypoxic response of CA1 neurons is suppressed by dantrolene, heparin, thapsigargin, procaine and, less consistently, by ryanodine (Belousov et al. 1995; Krnjevic' and Xu 1989; Yamamoto et al. 1997)---agents that prevent Ca2+ release from internal stores (Henzi and MacDermott 1992; Hill et al. 1987; Kobayashi et al. 1988; Mody and MacDonald 1995; Thastrup et al. 1990). In the hippocampus, as in the locus coeruleus (Murai et al. 1997), internal Ca2+ release thus plays an important role in the cellular response to hypoxia. But why should hypoxia elicit an early release of Ca2+? A likely answer is suggested by recent evidence that reduced nicotinamide adenine dinucleotide (NADH; formed by glycolysis during hypoxia, the Pasteur effect) (Clarke et al. 1989) enhances Ca2+ release from InsP3-sensitive stores (Kaplin et al. 1996).

In experiments with "sharp" electrodes, Ca chelators do not suppress hypoxic hyperpolarizations very consistently (Leblond and Krnjevic' 1989; Yamamoto et al. 1997), either because of inadequate injection of chelators or because other types of GK, such as GK(ATP), also are involved. There is convincing evidence from single channel studies that KATP channels are present in some central neurons (Ashford et al. 1990; Jiang and Haddad 1997; Jiang et al. 1994). But for hippocampal neurons, the evidence has been indirect, relying mainly on tests of suphonylurea blockers of GK(ATP) (Fujimura et al. 1997; Grigg and Anderson 1989; Riepe et al. 1992), although sulfonyureas (especially tolbutamide) can block other K channels (Crépel et al. 1993; Erdemli and Krnjevic' 1996; Godfraind and Krnjevic' 1993; Yamada et al. 1997); moreover, tolbutamide does not block some hypothalamic KATP channels (Ashford et al. 1990).

Whole cell recording might be expected to resolve these issues by allowing more efficient internal applications of agents such as ATP and Ca chelators. However, in recordings with gluconate- or Cl-containing electrodes (Zhang and Krnjevic' 1993), hypoxia had much weaker effects than in recordings with sharp electrodes. Subsequently, Zhang et al. (1994) and Velumian et al. (1997) found that hippocampal K and Ca currents soon run down when electrodes contain K gluconate or KCl but not KMeSO4.

In the present experiments with KMeSO4 electrodes, we observed substantial hypoxic changes in membrane properties, under both current and voltage clamp. The underlying mechanisms were investigated by whole cell recording with electrodes containing various combinations of relevant agents such as Ca chelators [ethylene glycol-bis-(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis (2 - a m i n o p h e n o x y)   e t h a n e - N, N, N', N' - t e t r a a c e t i c   a c i d (BAPTA)], ATP, and cyclic AMP, and an activator or blocker of G proteins. In further attempts to identify the main GK involved in the hypoxic responses, we also recorded (with whole cell or sharp electrodes) the effects produced by isoprenaline and antagonists of adenosine, GABAB, and serotonin receptors. Preliminary reports of some of these results have appeared as abstracts (Erdemli and Krnjevic' 1994a, 1997).

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

The brain was removed from young Sprague-Dawley rats (100-150 g) fully anesthetized with halothane. The hippocampus was dissected out in ice-cold oxygenated saline, and 400-µm transverse slices were cut with a Vibroslice (Campden Instruments, Loughborough, UK). They were kept for >= 1 h at room temperature. The standard artificial cerebrospinal fluid (ACSF) contained (in mM) 124 NaCl, 3.0 KCl, 2.0 CaCl2, 2.0 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose. It was aerated continually with carbogen (95% O2-5% CO2), which kept its pH approx  7.3. Slices then were transferred to a Haas-type chamber (Medical Systems, Greenvale, NY) where their upper surface was either exposed to humidified carbogen (for intracellular recordings) or submerged under 0.1-0.2 mm of flowing ACSF (for whole cell recordings). Both the ACSF and the carbogen were warmed to 33 ± 0.5°C.

The sharp microelectrodes were pulled from thin-walled borosilicate glass tubes (1.2 mm OD, WP Instruments, New Haven, CT). After filling with 3 M KCl, they had a resistance of 60-90 MOmega . The 2.5- to 3-µm-tip patch electrodes were made from 1.5 mm OD borosilicate glass. The "simple" internal solution (sIS) contained only 150 mM potassium methylsulfate (KMeSO4) buffered to pH 7.2 with 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). The following were added to some electrodes (alone or in various combinations, Table 1): adenosine 3':5'-cyclic monophosphate (cAMP, 1 mM); ATP di-K salt (ATP, 1-5 mM); MgCl2 (1-5 mM); ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 1.1 and 11 mM); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-K salt (BAPTA, 11 and 33 mM); Na4-BAPTA (11 mM, Fluka Chemical, Ronkonkoma, NY); CaCl2 (0.1, 1.1 or 3.3 mM); GTP tris salt (GTP, 0.2 mM); guanosine 5'-O-(3-thiotriphosphate) tetralithium salt (GTPgamma S, 0.1-0.3 mM); guanosine 5'-diphosphate Na salt (GDP, 1 mM); and guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDPbeta S, 0.1 mM). The pH always was adjusted to 7.2 with KOH. The osmolality (measured with a micro-osmometer, Precision Systems, Natick, MA) was kept within 280-310 mosm/kg by changing [KMeSO4] as needed. The patch electrodes initially had a resistance of 2-3 MOmega . All recordings were done "blind" in the stratum pyramidale. The electrode series resistance was monitored by applying brief current pulses; in useful whole cell recordings, it remained <15 MOmega .

 
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TABLE 1. Composition of filling solutions for patch electrodes

Because all the internal solutions of patch electrodes contained the same concentration of the predominant electrolyte (150 mM KMeSO4), it was assumed that whatever junctional potential was formed at the tip would not vary systematically between different cells. According to our own measurements, 0.15 M KMeSO4 and 0.15 M NaCl differ by 7% in specific conductivity and 1.5% in osmotic coefficients. Using the Henderson equation, one can calculate a junction potential of ~15 mV between the KMeSO4 electrodes and ACSF. On the other hand, unknown, but possibly comparable, Donnan potentials may be generated during whole cell recording, owing to the presence of "immobile" anions in the cell. Therefore no attempt was made to correct the recorded values of membrane potential.

The signals were amplified by an Axoclamp 2 (Axon Instruments, Burlingame, CA) in current ("bridge")- or voltage-clamp mode. The discontinuous voltage-clamp operated at a frequency of 3 kHz, a gain of 25 nA/mV, and an upper bandwidth limit of 300 Hz; the usual precautions were taken to optimize the efficacy of the clamp.

In bridge mode, the cell input resistance (Rin) was assessed by measuring the peak voltage produced by hyperpolarizing current pulses (0.1-0.2 nA, lasting 200 ms). To generate afterhyperpolarizations (AHPs), spike trains were evoked by 200-ms depolarizing pulses. Medium AHPs (Storm 1990) were measured at the early peak, slow AHPs 1 s after the start of the AHP. Under voltage clamp, the baseline current (Ib) was minimized by initially holding the membrane potential (Vm) near its resting level (about equal to -55 mV). The input conductance (Gin) was calculated by fitting a first-order regression to the linear portion of "instantaneous" current-voltage (I-V) plots, in the region negative to the holding potential (Vh). Voltage-dependent currents were elicited with voltage pulses lasting 500 ms.

Slices were made hypoxic with 95% N2-5% CO2, and the superfusate was bubbled vigorously with this gas. Indirect synaptic effects were minimized by adding kynurenic acid (1-2 mM), bicuculline (10 µM) and, in some experiments, tetrodotoxin (1 µM).

The following agents also were tested: by balanced iontophoresis from three-barrelled micropipettes, AMP (from a 0.2 M electrode solution at pH 7) and serotonin [5-hydroxytryptamine (5-HT), from a 40 mM 5-HT creatine sulfate solution at pH 3.2], the third barrel containing 1 mM NaCl; by bath-application, carbachol (20 µM), isoprenaline (5-10 µM, diluted from Isuprel; given by Sterling-Winthrop, Markram, Ontario, Canada), tolbutamide (1 mM, Research Biochemicals, Natick, MA and Sigma, St. Louis, MO; from a 100 mM stock solution in 150 mM NaOH), glyburide [10 µM, given by Hoechst-Roussel Canada (Dr. B. G. Carter, Montréal, Québec) from a stock solution in dimethyl sulfoxide (DMSO; ICN Biomedicals, Aurora, OH)], 8-(p-sulfophenyl)theophylline (8-SPT, 10 µM, Research Biochemicals), 8-bromo-cyclic AMP Na salt (8-Br-cAMP, 1 mM), the GABAB antagonist CGP-35348 [Ciba-Geigy, Basel, Switzerland; given by Dr. R. Ticku and Ciba-Geigy Canada, Calgary (Dr. J. A. Zidichouski)], and the serotonin antagonist spiperone. To eliminate possible artifacts, all control and test media contained the same amounts of DMSO (0.10%) or NaOH. Chemicals were purchased from Sigma, except where indicated. Means ± SE are given throughout. The significance of differences was assessed by Student's t-test (for paired results) or the d-statistic and its Fisher-Behrens distribution (Campbell 1989, p. 420) for unpaired differences with unequal population variances.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

In agreement with previous reports (Belousov et al. 1995; Zhang and Krnjevic' 1993), when compared with conventional intracellular recordings, the whole cell recordings with simple electrodes (sIS, Table 1) were characterized by large action potentials (approx 100 mV), a more positive Vm (-58 ± 0.80 mV) and high Rin (76 ± 3.7 MOmega ; Table 2).

 
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TABLE 2. Resting membrane properties and hypoxic responses of CA1 neurons recorded under current clamp with different internal electrode solutions

Effects of brief hypoxia under current clamp

As mentioned in the INTRODUCTION, after a brief depolarization, which can be seen in several figures (e.g., Figs. 1, A and B, 4A, 6C, and 9A), there is a sustained, predominantly hyperpolarizing change in membrane potential (Vm). In 36 CA1 neurons recorded with sIS electrodes, 2-3 min of hypoxia evoked a mean hyperpolarization of 2.7 ± 0.50 mV, accompanied by a 23 ± 2.7% fall in Rin (Fig. 1A; see also Table 2).


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FIG. 1. Hypoxic changes in Vm and Rin are not abolished by either internal ATP or external glyburide. Current-clamp traces show the responses to 2 min of hypoxia (N2) in 3 cells recorded with patch electrodes containing: simple internal solution [sIS(KMeSO4 and HEPES) A]; sIS(ATP 5 mM) (B), and sIS---for this cell, effect of hypoxia was recorded before (C) and during bath application of 10 µM glyburide (D): note inconspicuous hypoxic hyperpolarization in C was enhanced by glyburide in D. Input resistance was measured with current pulses lasting 200 ms and intensity 0.1 nA for A and B and 0.2 nA for C and D; only those for A and C actually are illustrated. These pulses and the corresponding potential changes are shown on 100-fold accelerated traces before, during, and after hypoxic tests. All recordings were in presence of kynurenic acid (KYN) and bicuculline (BIC), with 0.1% dimethyl sulfoxide (DMSO) added for both C and D. Initial membrane potentials are indicated by numbers.


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FIG. 4. Variable effects of Ca chelators on hypoxic responses of 3 CA1 neurons. Whole cell current-clamp recordings (in presence of KYN and BIC) with ethylene glycol-bis-(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)- or BAPTA-containing electrodes; 0.1-nA pulses (lasting 0.2 s), applied to monitor Rin, are shown (top). Internal solution was sIS(EGTA 11 mM) for A; sIS(BAPTA 11 mM) for B; and sIS(BAPTA 33 mM) for C; [Ca2+]i was always 1/10 of [chelator]. Note absence of hyperpolarization and relatively small Rin changes in B and C.


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FIG. 6. Effects of 5-hydroxytryptamine (5-HT), adenosine 5'-monophosphophate (AMP) and hypoxia recorded with sharp microelectrode from slice in interface-type chamber. Note marked hyperpolarization and conductance increase produced by iontophoretic applications of 5-HT (40 nA for 60 s, A) or AMP (80 nA for 30 s, B); and initial depolarization then small hyperpolarization during 2 min of hypoxia (C). Identical hyperpolarizing current pulses (0.2 nA, 0.2 s; illustrated top) were injected at regular 5-s intervals to monitor input resistance; a constant stimulus, at same frequency, was applied to stratum radiatum to evoke synaptic response.


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FIG. 9. Isoprenaline---but not gamma -aminobutyric acid-B (GABAB)-receptor antagonist---depresses effects of hypoxia on Vm and Rin. A and B: intracellular recording with 3 M KCl electrode, in presence of TTX, shows marked effects of hypoxia (for 2 min, as indicated by arrows): initial depolarization, followed by hyperpolarization and resistance fall, and then sharp postanoxic hyperpolarization, before return to initial condition. A: initial control run; B: 21 min after start of application of GABAB antagonist, CGP 35348 (0.5 mM). C and D: whole cell recording from another experiment with sIS-electrode, in presence of KYN and BIC, shows response evoked by 2 min of hypoxia before (C) and 12 min after (D) start of 10 µM isoprenaline bath application. Current pulses (0.2 s) applied to measure Rin were 0.5 nA for A and B and 0.1 nA for C and D.

Tests of possible activation of KATP channels

HIGH INTERNAL ATP. To counteract any depletion of cellular ATP during hypoxia, 1-5 mM ATP was added to sIS. This reduced but did not abolish the effects of hypoxia (Fig. 1B). The mean hypoxic fall in Rin (Delta Rin) diminished by half---the unpaired difference from the control mean of -23 ± 2.7% was -11 ± 4.8% (for n = 7, P < 0.05); but Delta Vm was slightly increased (Table 2).

In these cells, both Vm and Rin were significantly higher than in cells recorded with sIS electrodes. Thus ATP/Mg may suppress a GK that is at least partly open in the presence of sIS. This would be consistent with block of KATP channels, but also with phosphorylation- and/or G-protein-dependent modulation of some other K channels (Brown 1990; Egan et al. 1993; Sadoshima et al. 1988). A further possibility is significant chelation by ATP (Sellers et al. 1992) of the substantial residual Ca2+ present in such nominally Ca-free solutions as sIS (could be approx 40 µM) (Zhang et al. 1995). The fact that adding EGTA/Ca (which should hold [Ca2+]i well <100 nM) restored the lower Rin suggests that these channels are indeed Ca sensitive.

SULFONYLUREAS. The KATP channel blocker glyburide (10 µM) was applied to 12 cells. As reported by Godfraind and Krnjevic' (1993), glyburide tended to reinforce the hyperpolarizing responses of some cells. Thus the inconspicuous potential change elicited initially by hypoxia in the cell shown in Fig. 1C became a clear hyperpolarization after the application of glyburide (Fig. 1D). But overall, the hypoxic Delta Vm and Delta Rin were not significantly altered by glyburide: the respective paired differences from control values were 0.4 ± 1.8 mV and 0.3 ± 3.5%. Glyburide was equally ineffective as blocker of the outward current induced by hypoxia in cells under voltage clamp (cf. Fig. 2, A and B).


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FIG. 2. Hypoxic outward current recorded under voltage-clamp shows little effect of glyburide but it is blocked by Ba and carbachol. In presence of tetrodotoxin (TTX), KYN, and BIC, with potential held at -40 mV, outward currents were evoked by 20-mV depolarizing pulses or by 2 min of hypoxia (between vertical arrows); before, during, and after hypoxic tests, 20-mV hyperpolarizing pulses monitored input conductance [at bottom the voltage steps, which lasted 500 ms]; 0 current levels are indicated by horizontal arrows (left). A: control traces. B: after 12 min of glyburide (10 µM) application. C: 5 min after start of Ba (1 mM) and carbachol (20 µM) application, depolarizing pulse evokes only inward current and hypoxia minimal outward current and conductance increase.

Tolbutamide, another KATP channel blocker (Ashford et al. 1990; Henquin and Meissner 1982), suppresses both hypoxic outward currents (Godfraind and Krnjevic' 1993) and slow AHPs in hippocampal slices (Erdemli and Krnjevic' 1996), especially the stable AHPs recorded with KMeSO4 electrodes (Zhang et al. 1994). In some hypothalamic neurons, glyburide prevents blockage of GK(ATP) by tolbutamide (Ashford et al. 1990). To exclude a possible hypoxic activation of such KATP channels in our slices, we applied 1 mM tolbutamide to five cells in the presence of glyburide: as in the previous experiments (Erdemli and Krnjevic' 1996; Godfraind and Krnjevic' 1993), the hypoxic responses were suppressed fully, Delta Vm being reduced to 0.2 ± 3.1 mV and Delta Rin to 0.2 ± 2.2%. Thus glyburide did not block the effect of hypoxia nor did it prevent its suppression by tolbutamide.

Internal Ca chelators: tests of possible activation of KCa channels

HYPERPOLARIZING ACTION OF BAPTA. During the first 5-15 min after the break through the membrane, cells recorded with BAPTA-containing electrodes typically showed a gradual hyperpolarization and substantial fall in Rin (Fig. 3, A and B). Similar changes were seen with electrodes containing 11 mM K4-BAPTA (n = 13), 33 mM K4-BAPTA (n = 15), or 11 mM Na4-BAPTA (n = 4) (Table 1). According to the pooled data from 32 cells, Vm (initially -60 ± 0.85 mV) changed by -7.7 ± 1.1 mV (P < 0.001) and Rin (initially 91 ± 5.8 MOmega ) dropped by 40 ± 3.4% (P < 0.001). Most of the Rin change was not secondary to hyperpolarization because it persisted when the initial Vm was restored by current injection (Fig. 3B). Similar changes in Vm and Rin were recorded in 0.2 mM tetraethylammonium (TEA; n = 2). Low concentrations of TEA block a K current that appears to be suppressed by Ca2+ (Constanti et al. 1993) and therefore could mediate the slow hyperpolarizing shift, but they were abolished by 20 µM carbachol (n = 6; Fig. 3C). In its presence, Delta Vm was -0.25 ± 0.57 mV (from -44 ± 3.4 mV) and Delta Rin 12 ± 6.7% (from 124 ± 19.4 MOmega ). No comparable hyperpolarizing trend was seen with EGTA-containing electrodes (Fig. 3D).


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FIG. 3. Internally applied 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) causes hyperpolarization and decrease in input resistance. Whole cell recording from CA1 neurons in presence of kynurenic acid (KYN, 1 mM) and bicuculline (BIC, 10 µM) with electrodes containing sIS(ATP, K4BAPTA 11 mM) (A); sIS(ATP, Na4BAPTA 11 mM) (B); sIS(ATP, K4BAPTA 33 mM) (C); and sIS(ATP, EGTA 11 mM) (D). Bottom of each trace, membrane potential (initial value is indicated). Top of each trace, 0.1 nA current pulses lasting 0.2 s, applied at regular 10-20 s intervals to monitor Rin. Pulses are displayed on greatly accelerated traces just after breakthrough as well as later. Cells in A and B show characteristic hyperpolarization (after transient depolarization) and marked drop in Rin---which was not abolished by restoring initial Vm with 330 pA depolarizing current injection (B). C: in similar recording from another cell, carbachol (20 µM) prevented such BAPTA-induced hyperpolarization and decrease in Rin. D, in recording with EGTA-containing electrode, there was no comparable hyperpolarization and decrease in Rin after the initial membrane breakthrough.

Under voltage clamp, resting values of Vm, Gin and baseline current (Ib) did not differ significantly when recorded with sIS or sIS(EGTA) electrodes (Table 3). With BAPTA-containing electrodes, Ib and Gin rose gradually after the onset of recording, resulting in a higher steady Ib and Gin than was seen with any other type of electrode (Table 3).

EFFECTS OF BRIEF HYPOXIA. Under current clamp. Simply adding EGTA (1.1 mM with 0.1 mM CaCl2) to sIS reduced Delta Rin by nearly half (-11 ± 5.2%; unpaired difference, P = 0.05), although it made no difference to the hypoxic Delta Vm (Table 2). Thus either ATP or EGTA alone depressed Delta Rin. However, when both were added (with or without GTP), the hypoxic changes were virtually identical to those recorded with sIS alone (Table 2).

When electrodes contained BAPTA (11 or 33 mM), the hypoxic hyperpolarization was abolished, but Delta Rin was not significantly depressed (Table 2). Figure 4 illustrates depolarizing responses to hypoxia recorded with 11 and 33 mM BAPTA electrodes (B and C) and, for comparison, a small but clear hyperpolarization seen with an 11 mM EGTA electrode (A).

Under voltage clamp. The substantial increases in Gin and Ib observed with sIS electrodes (Fig. 2A) were two-thirds smaller when EGTA was added to the electrodes (Table 3)---the unpaired reductions (-3.2 ± 1.07 nS for Delta Gin and -80 ± 26.4 pA for Delta Ib) were both significant at P < 0.05. Further addition of ATP to the electrodes restored the larger hypoxic changes obtained with sIS electrodes (Table 3): Delta Gin was increased by 3.2 ± 0.83 nS (P < 0.01) and Delta Ib by 58 ± 23 pA (P < 0.05). Although Delta Gin was not diminished, the hypoxic outward currents seen with BAPTA electrodes were significantly smaller than those recorded with sIS electrodes (by 70 ± 34.9 pA, P = 0.05) (Table 3). The voltage- and current-clamp observations are thus in good agreement.

Tests of G-protein involvement in hypoxic response

EFFECTS OF GDPbeta S AND GTPgamma S. G-protein-mediated activations of GK are well known (Brown 1990; Jan and Jan 1997; Nicoll 1988). A relevant example is the pertussis toxin-sensitive K current elicited by adenosine (Trussell and Jackson 1987). If such a GK contributes significantly to the hypoxic response, it should be sensitive to agents that stimulate or inhibit G proteins, especially GTPgamma S and GDPbeta S, the actions of which are irreversible (Eckstein et al. 1979). Indeed the effects of hypoxia were either undetectable or greatly diminished in both current- and voltage-clamp recordings with electrodes containing GTPgamma S or GDPbeta S.

Under current clamp. Cells recorded with GDPbeta S- or GTPgamma S-containing electrodes showed no hypoxic hyperpolarization and a greatly reduced Delta Rin, especially with GDPbeta S (Fig. 5C and Table 4): mean Delta Rin diminished by 71% (unpaired difference was -18 ± 3.1%, P < 0.01). Although sometimes equally striking (Fig. 5B), the reductions seen with GTPgamma S were less consistent (means down by 31%; unpaired differences -8.0 ± 4.7%, P < 0.1).


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FIG. 5. Internal guanosine 5'-O-(3-thiotriphosphate) tetralithium salt (GTPgamma S) or guanosine 5'-O-(2-thiodiphosphate) trilithium salt (GDPbeta S) suppresses hypoxia-induced changes in Vm and Rin. Whole cell recordings from different CA1 neurons with electrodes containing sIS(ATP, EGTA) alone (A) or with addition of GTPgamma S (B) or GDPbeta S (C) (both at 0.3 mM). In A and C, artificial cerebrospinal fluid contained KYN and BIC throughout. As in all the current-clamp recordings, Rin was measured with current pulses lasting 200 ms; the pulses and corresponding potential changes are shown on 100-fold accelerated traces before, during, and after hypoxic tests.

 
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TABLE 4. Hypoxia-induced changes in membrane properties (under current clamp) recorded with patch-electrodes containing GDPbeta S, GTPgamma S, or cAMP

Under voltage clamp. Both agents abolished the hypoxic outward current; GDPbeta S also fully suppressed the increase in Gin, whereas GTPgamma S reduced it by 69% (the unpaired difference, -3.4 ± 0.78 nS, was very significant, P < 0.01; Table 5). The reversible ligands GTP and GDP were less effective (Table 3), the only notable change being the suppression of the outward current by GDP (down by 110 ± 42 pA, P < 0.05).

 
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TABLE 5. Hypoxia-induced changes in membrane properties (under voltage clamp) recorded with patch-electrodes containing GDPbeta S, GTPgamma S, or cAMP

The impressive actions of GDPbeta S and GTPgamma S, which confirm earlier tests of GTPgamma S (Krnjevic' and Xu 1990), are strong evidence that G-protein activation is an early consequence of hypoxia. Several ligands that open GK via a G protein could be released during hypoxia. They include GABA and serotonin (Andrade et al. 1986) and particularly adenosine (Trussell and Jackson 1987).

Adenosine. In previous intracellular recordings from slices in an interface-type chamber (Leblond and Krnjevic' 1989), adenosine antagonists such as 8-SPT did not suppress the hypoxic changes in Vm and Rin; and 1 mM adenosine, which had a pronounced hyperpolarizing effect, did not occlude the hypoxic conductance increase. Further tests under the same conditions have confirmed these results. A marked hyperpolarization, associated with a sharp conductance increase, could be evoked reproducibly by iontophoretic applications of AMP (AMP; Figs. 6 and 7).


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FIG. 7. In same CA1 neuron (cf. Fig. 6), selective antagonists greatly diminish the responses to 5-HT and adenosine but not the effects of hypoxia. Similar iontophoretic tests of 5-HT and AMP show that spiperone (20 µM, bath-applied) largely blocked effect of 5-HT but not that of AMP or hypoxia (A); whereas 8-(p-sulfophenyl)theophylline (8-SPT; 10 µM, also bath-applied) largely blocked effect of AMP, but small hyperpolarization and marked drop in resistance were still elicited by 2 min of hypoxia (B). As in other current-clamp recordings, input resistance was measured throughout with hyperpolarizing current pulses: these 0.2-nA pulses, which lasted 200 ms, are monitored on top trace (only for A); they are shown on 100-fold accelerated traces shortly before and during hypoxic tests in both A and B.

The traces in Fig. 6 illustrate well the contrast between the purely hyperpolarizing action of adenosine (or serotonin) and the mixed effects of hypoxia: initial depolarization, smaller hyperpolarization, and clear posthypoxic hyperpolarization (not seen after AMP or 5-HT applications). The hypoxic hyperpolarization is relatively small because of an underlying continued depolarizing effect (inward current), which is revealed when, as in Fig. 10B, the hyperpolarizing action (or outward current) is suppressed (Krnjevic' and Xu 1990).


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FIG. 10. In some neurons, 3':5'-adenosine cyclic monophosphate (cAMP) suppresses hypoxic hyperpolarization or outward current as well as AHP or outward after-current. A and B: sharp electrode recordings from a CA1 cell show AHP (after 8-spike burst) and response to 2 min of hypoxia obtained initially (A) and 19 min after start of 8-Br-cAMP (1 mM) bath application (B). All traces were recorded at same resting potential (-52 mV). In B, 8-spike burst, which was evoked by smaller current pulse (top), was followed by much smaller AHP, and hypoxia had a depolarizing effect, not associated with fall in resistance. C and D: in another slice, 2 CA1 cells were recorded with whole cell electrodes containing sIS, either with no cAMP (C) or with cAMP (D, 1 mM) added. For both cells, holding potential was -40 mV and horizontal arrows mark 0 current. Left: are the outward currents evoked by 30-mV depolarizing pulses lasting 500 ms (shown below); note prominent slow outward tail in C and its absence in D. Changes in input conductance during hypoxia were monitored by applying 20-mV hyperpolarizing pulses seen on bottom traces. Note in D, hypoxia induced small inward current and no conductance increase; it was followed by typical postanoxic outward current.

The adenosine antagonist 8-SPT markedly enhanced both ongoing firing and synaptically evoked responses and virtually abolished the effect of AMP but not the small hyperpolarization and large conductance increase induced by hypoxia (Fig. 7B). Comparable tests of 10-50 µM 8-SPT on another 12 cells under current- or voltage-clamp consistently showed clear preservation of hypoxic hyperpolarizations (or outward currents) and especially the conductance changes.

By contrast, in a recent study on submerged slices, including whole cell recording, 8-SPT partly reduced the hypoxic outward current and conductance (Zhu and Krnjevic' 1997); and in the current experiments on submerged slices, 8-SPT eliminated the hypoxic changes in recordings under both current and voltage clamp (n = 5 and 4, respectively). In 10 µM 8-SPT, hypoxia elicited depolarizations and no drop in Rin, as illustrated in Fig. 8. Under voltage clamp, the hypoxic outward current and increase in Gin were similarly abolished. These data are summarized in Table 6. A relevant point is that 8-SPT also depressed (reversibly) both the medium and the slow AHP evoked by bursts of spikes (Fig. 8D).


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FIG. 8. In whole cell recording from submerged slices, 8-SPT depresses both medium AHP and hypoxic hyperpolarization. All traces were obtained from same CA1 neuron recorded with sIS(ATP, EGTA) electrode, in presence of KYN and BIC; 0.2-s, 0.1-nA pulses applied to monitor Rin are shown top. A: hyperpolarization and Rin drop during 2 min of hypoxia in control conditions. B: in 10 µM 8-SPT (10 min), hypoxia elicited depolarization and Rin rise; these were largely reversed by 35-min wash (C). D: examples of afterhyperpolarizations (AHPs) induced by depolarizing pulses that evoked 9 spikes, recorded before, during 8-SPT (10 µM) application, and after wash; all were recorded at initial Vm. Note greater depression of mAHP than sAHP.

 
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TABLE 6. Effects of 8-SPT (10 µM, bath-applied) on resting membrane properties and hypoxia-induced changes

Serotonin (5-HT). In recordings with 3 M KCl-containing sharp electrodes, the 5-HT antagonist spiperone (6-50 µM) selectively suppressed hyperpolarizations evoked by microiontophoretic applications of 5-HT (Figs. 6 and 7A); but the hypoxic responses were little changed (Fig. 7A). Comparable observations were made on four other cells tested under similar conditions.

GABA. The GABAB receptor antagonist CGP-35348 was tested on five cells, two recorded under current clamp, three under voltage clamp, mainly during intracellular (sharp electrode) recordings. When bath-applied at 0.5 mM, this agent did not depress the hypoxic hyperpolarization and Rin fall (Fig. 9, A and B) or the corresponding outward current and Gin rise (not shown). Overall, for the five cells, the hypoxic Delta Gin changed by -0.4 ± 9.3%, Delta Vm by -0.35 ± 0.35 mV and Delta Ib by -13 ± 14.9 pA; none of these was significant. In three other cells, there was no sign of occlusion between marked increases in Gin produced by hypoxia and by the GABAB agonist baclofen (not shown).

Role of AHP-type KCa channels in hypoxic response

Several findings suggested the possibility that a GK(Ca) is activated by hypoxia, more specifically that responsible for the slow component of AHPs: notably the suppression of hypoxic hyperpolarizations by carbachol (Krnjevic' and Xu 1990), by dantrolene (Krnjevic' and Xu 1996) and by 8-SPT (earlier text), all of which depress slow AHPs (sAHPs). The well-known blocker of the AHP conductance, isoprenaline, which acts via beta  receptors and cyclic AMP (Madison and Nicoll 1986; Pedarzani and Storm 1993), might therefore also be expected to depress the hypoxic changes.

ISOPRENALINE. When it was applied to 15 cells under various conditions of recording, mostly in the whole cell mode, isoprenaline (5-10 µM) either abolished (n = 3) or reduced by about half (n = 11) hypoxic hyperpolarizations and outward currents (Fig. 9, C and D, and Table 7). Most cells also showed a corresponding reduction in hypoxic Delta Gin. In agreement with previous reports, isoprenaline markedly depressed sAHPs (by 61 ± 13.9%, P < 0.01). In three cells, slowly decaying tail currents after depolarizing pulses to approximately equal to -10 mV (probably largely consisting of IAHP) (Lancaster and Adams 1986) were reduced by 52 ± 14.7% (P < 0.05).

 
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TABLE 7. Effects of isoprenaline (Isoprel, 10 µM, bath applied) on resting membrane properties and hypoxia-induced changes

CYCLIC AMP. We also tested the effects of cAMP, the internal mediator of isoprenaline's action on AHPs (Madison and Nicoll 1986; Pedarzani and Storm 1993). A series of current-clamp recordings were made with electrodes containing either plain sIS or sIS with 1 mM cAMP added. When compared with 12 control cells (Table 4), six cells recorded in the same group of slices with cAMP-containing electrodes showed no significant change in resting membrane properties and no reduction of hypoxic hyperpolarization, but the associated drop in Rin was diminished by 40%. Under voltage clamp, the hypoxic changes were significantly smaller (by 50-70%) when recorded with cAMP-containing electrodes (n = 6) than in nine control cells (Fig. 10, C and D, and Table 5).

As a further test, eight cells were treated with 1 mM 8-Br-cAMP, the membrane permeable derivative of cAMP, applied in the bath. In its presence, the hypoxic response was either abolished or reversed (as in Fig. 10, A and B): overall the potential changes were no longer significant (0.14 ± 2.2 mV), in contrast to the clear hyperpolarizations seen in the same neurons during preceding control hypoxic tests (-3.7 ± 0.87 mV, P < 0.005). The hypoxic resistance fall also was depressed by 8-Br-cAMP, from a mean of -29 ± 5.3 (P < 0.005) to a less consistent -22 ± 9.9% (from Rin= 76 ± 12 MOmega ).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

By providing better access to the neuronal interior, whole cell recording should clarify the mechanism(s) underlying the GK-mediated hypoxic hyperpolarization of CA1 neurons. However, as reported by previous authors (Kurachi et al. 1992; Lenz et al. 1997; Morita et al. 1980; Velumian et al. 1997; Zhang et al. 1994), K currents, especially those that are activated via a G protein, are very sensitive to the anion content of recording electrodes. The hypoxic K current is no exception. By substituting MeSO4 for gluconate as the main anion (Velumian et al. 1997; Zhang et al. 1994), more substantial hypoxic effects were obtained, confirming the usefulness of KMeSO4, at least for such experiments (cf. Belousov et al. 1995; Zhang and Krnjevic' 1993). Nevertheless, even the largest changes in Rin (-26%; Table 2) were only half the near-50% reductions in Rin typically seen with intracellular electrodes (Croning et al. 1995; Fujiwara et al. 1987; Hansen et al. 1982; Leblond and Krnjevic' 1989; Yamamoto et al. 1997).

Ca-dependent GK and effects of chelators

ON RESTING-MEMBRANE PROPERTIES. The carbachol-sensitive hyperpolarization (or outward current) that developed gradually after the start of whole cell recording with BAPTA-containing electrodes is a curious phenomenon. Similar changes in dorsal vagal and hippocampal neurons were ascribed respectively to activation of GK(ATP) (Trapp et al. 1994) or GK(Ca) (Zhang et al. 1995)---the latter because the outward current was blocked by carbachol and Ba. Zhang et al. (1995) suggested that GK(Ca) somehow might be activated by redistribution of internal Ca2+ by the diffusible BAPTA (see also Schwindt et al. 1992, who also observed lower values of Vm and Rin in neocortical recordings with "high-BAPTA" sharp electrodes).

Another possibility is activation of a GK that is normally suppressed by cytoplasmic Ca2+, such as r-eag-type K channels (Stansfeld et al. 1996), which also are blocked by muscarinic agents. Why EGTA, an equally potent and diffusible buffer, does not have a similar effect is not clear: its slower kinetics should not affect such a slow process.

ON HYPOXIC HYPERPOLARIZATION. Considering the strong circumstantial evidence in favor of Ca2+-release as an important factor (Belousov et al. 1995; Krnjevic' 1993; Yamamoto et al. 1997), it is surprising that Ca chelators do not have more conclusive effects; Duchen (1990) also observed only variable effects of BAPTA on hypoxic responses of sensory neurons. This may be explained if some other messenger also plays an important role in the activation of KCa channels (Lasser-Ross et al. 1997).

Alternatively, like KATP channels, KCa channels may be regulated in a highly localized "microdomain." In both cases, the critical signals are probably generated by glycolysis: ATP for KATP channels (Weiss and Lamp 1989), and NADH for InsP3 receptors (Kaplin et al. 1996). If the ATP-forming enzyme phosphoglycerate kinase is situated close to KATP channels (Schackow and Ten Eick 1994; Weiss and Lamp 1989), its neighbor in the glycolytic pathway, the glyceraldehyde-3-phosphate dehydrogenase that converts NAD to NADH, may release Ca2+ from very superficial InsP3-sensitive stores (Berridge 1993, 1997; Henkart 1980; Lièvremont et al. 1996; Mody and MacDonald 1995; Takei et al. 1992), in a microdomain that is not fully accessible to exogenous chelators (Adachi-Akahane et al. 1996; Matthews 1997; Naraghi and Neher 1997).

Chelators may not obviously suppress the hypoxic GK rise for another reason: by lowering cytosolic [Ca2+], they would diminish any ongoing suppression of a r-eag type of GK (Stansfeld et al. 1996). A combination of Ca2+-induced decrease of a r-eag GK and increase in GAHP could generate the bidirectional changes in Gin and membrane current elicited by hypoxia (Krnjevic' and Xu 1990), although other explanations (Krnjevic' 1993; Nieber et al. 1995; Tanaka et al. 1997) have not been excluded.

Another intriguing possibility is that hypoxia releases an agent like beta -amyloid precursor protein, which activates GK(Ca), although it does not raise [Ca2+]i and is insensitive to chelators (Furukawa et al. 1996).

KATP channels

The weak effects of internally applied 1-5 mM ATP are in agreement with some previous reports (Duchen 1990; Hyllienmark and Brismar 1996). Together with the absence of any depression by glyburide, they strongly argue against a major involvement of conventional KATP channels in our experiments, in keeping with the minimal hyperpolarizing actions that we have observed with KATP channel openers (Erdemli and Krnjevic' 1994b, 1995). The partial disagreement with other authors, who found some effects of glyburide, although much less consistently than with tolbutamide (Fujimura et al. 1997; Grigg and Anderson 1989), may be explained by technical differences; for example, any changes produced by hypoxic glutamate and GABA release largely were eliminated in our experiments. On the other hand, in spite of the use of KMeSO4 electrodes for our whole cell recordings (Lenz et al. 1997; Zhang et al. 1994), there may have been some selective loss of a KATP component of the hypoxic response.

A possible objection might be that unconventional KATP channels are present in CA1 neurons (as in hypothalamic glucoceptive cells) (Ashford et al. 1990) that have a low sensitivity to ATP and glyburide. In the hypothalamic cells, however, glyburide prevents the blocking action of tolbutamide, which was clearly not the case in our CA1 neurons.

Could these K channels be both Ca and ATP sensitive (Hunter and Giebisch 1988; Jiang et al. 1994; Sellers et al. 1992)? But in our recordings with electrodes containing both ATP and a chelator, the hypoxic changes were not diminished. Moreover, according to Jiang et al. (1994), currents of this type in nigral neurons are blocked by glyburide.

Is also unlikely that 2-3 min of hypoxia would cause a major drop of ATP level (cf. Lipton and Whittingham 1984; Takata and Okada 1995) to well <1 mM, where KATP channels open (Jiang et al. 1994). As Hochachka (1996) has emphasized, perhaps the most remarkable feature of energy depletion is the quick suppression of ATP utilization. The tight coupling between ATP production and utilization prevents a rapid fall in cellular ATP, in spite of a large reduction in ATP turnover. Hence changes in ATP level do not accurately reflect the almost instantaneous cellular adaptation to hypoxia.

Only a minor role of KATP channels in the early hypoxic response of CA1 neurons is not surprising in view of the very low incidence of KATP channels in neocortical and hippocampal neurons (Ashford et al. 1988, 1990; Lee et al. 1996): nearly all the KATP channels observed by Lee et al. (1996) were on nerve terminals and not on cell bodies (although cf. Jiang and Haddad 1997). Such a presynaptic localization would be fully consistent with previous evidence that KATP channel blockers reduce hypoxic hyperpolarizations and enhance glutamate release-mediated depolarizations (Ben-Ari 1990; Krnjevic' 1990; Mourre et al. 1989).

Suppressant action of GDPbeta S and GTPgamma S

The powerful effect of GTPgamma S, and especially that of GDPbeta S, are strong evidence that the hypoxic response involves a G protein (Eckstein et al. 1979). This suggests that hypoxia releases a ligand which, acting via a G protein, either directly opens GK or enhances the release of internal Ca2+. Of the numerous ligand-triggered activations of GK that are G-protein-mediated (Brown 1990; Jan and Jan 1997; Nicoll 1988), those that do not involve a second messenger typically are sensitive to pertussis toxin. In Spuler and Grafe's (1989) sharp electrode study of submerged hippocampal slices, pertussis toxin did not prevent the effects of hypoxia, though hyperpolarizations via adenosine or GABAB receptors were abolished. Their conclusion that the anoxic effects could not be mediated by adenosine is in good agreement with the lack of effect of 8-SPT in our intracellular recordings (see also Croning et al. 1995; Leblond and Krnjevic' 1989) but in contradiction with the clear effects of 8-SPT on submerged slices (seen also by Zhu and Krnjevic' 1997). This may be explained if 8-SPT reaches its target more quickly in submerged slices or if adenosine accumulates faster because the slices are underoxygenated (Fredholm et al. 1984).

An alternative possibility is that adenosine, acting on A1 receptors but via a nonpertussis-sensitive G protein, initiates the ligand-triggered production of InsP3 (Burnatowska-Hledin and Spielman 1991; Kohl et al. 1990; Ogata et al. 1994; Rugolo et al. 1993; Yakel et al. 1992), the Ca2+-releasing action of which (Berridge 1993) would be reinforced greatly during hypoxia by increased glycolysis and NADH production (Kaplin et al. 1996). Without the synergistic effect of adenosine-triggered InsP3 formation, NADH may not be able to induce a significant release of Ca2+. This scheme is more plausible, being consistent with the evidence that hypoxic hyperpolarizations are suppressed by blockers of InsP3-mediated Ca2+ release (Belousov et al. 1995; Murai et al. 1997; Yamamoto et al. 1997) and that they are variably sensitive to adenosine antagonists. As already mentioned, it does not exclude the participation of some other G-protein ligand(s) that activate(s) phospholipase C.

Nature of the GK(Ca) postulated to be activated by hypoxia

Several features favor an AHP-type conductance: the greater hypoxic changes recorded with MeSO-4 than with gluconate-containing electrodes, the strong block by carbachol, the depression of both AHPs and hypoxic hyperpolarizations by 8-SPT, and the substantial block by the beta -adrenergic agonist isoprenaline. The suppression of slow AHPs by beta  agents being mediated mainly by cAMP (Blitzer et al. 1994; Madison and Nicoll 1986; Pedarzani and Storm 1993), internally applied cAMP, and its membrane-permeable analogue 8-bromo-cAMP were expected to be consistently effective.

There are several possible explanations for the partial dissociation between the effects of isoprenaline and those of cAMP. Isoprenaline partly may affect AHP-type channels by a more direct action, only G-protein mediated (Welling et al. 1992). At the relatively high concentration of 1 mM, cAMP may have some other action that counteracts the suppression of IAHP. For example, cAMP strongly potentiates maxi-KCa channel activity of olfactory bulb neurons (Egan et al. 1993). Another relevant target for cAMP is the enzyme phosphofructokinase, the principal regulator of glycolysis (Siesjö 1978, p. 204-206). By reinforcing glycolysis, stimulation of phosphofructokinase would enhance the hypoxic production of NADH enhancement and thus potentiate any rise in cytoplasmic [Ca2+]. Alternatively, isoprenaline may be more effective because it produces a high local concentration of cAMP at the critical site (Blitzer et al. 1994).

Near resting potential, hypoxia would activate mainly KAHP channels (SK type), which are sensitive to relatively small increases in [Ca2+]i (Gurney et al. 1987; Lancaster and Zucker 1994; Prakriya et al. 1996); but large, voltage-dependent BK channels (large conductance KCa channels) (Brown and Griffith 1983; Egan et al. 1993; Lee et al. 1995) are also likely to be involved in view of the marked voltage dependence of the hypoxic outward current and its depression by TEA (Krnjevic' and Leblond 1989).

In conclusion, the present findings show that for all its advantages, whole cell recording has some significant drawbacks in studies of cellular mechanisms of hypoxia. Nevertheless, in spite of not wholly conclusive effects of chelators, overall the weight of evidence favors a Ca2+-mediated process---probably initiated by enhanced glycolysis and production of NADH and requiring a G-protein-dependent synergistic action, at least partly mediated by adenosine.

    ACKNOWLEDGEMENTS

  This research was supported by the Medical Research Council of Canada. Two experiments with the GABAB antagonist were done in collaboration with Dr. Peter Miu.

  Present address: Y. Xu, Biology Dept., University of Science and Technology of China, Hefei, Anhui-230026, P.R. China.

    FOOTNOTES

  Address for reprint requests: G. Erdemli, University of Wales, College of Cardiff, Museum Avenue, PO Box 911, Cardiff CF1 3US, Wales, UK.

  Received 20 April 1998; accepted in final form 15 July 1998.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society