The Journal of Neurophysiology Vol. 77 No. 1 January 1997, pp. 386-392
Copyright ©1997 by the American Physiological Society

Mediation by Intracellular Calcium-Dependent Signals of Hypoxic Hyperpolarization in Rat Hippocampal CA1 Neurons In Vitro

S. Yamamoto, E. Tanaka, and H. Higashi

Department of Physiology, Kurume University School of Medicine, Kurume 830, Japan

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Yamamoto, S., E. Tanaka and H. Higashi. Mediation by intracellular calcium-dependent signals of hypoxic hyperpolarization in rat hippocampal CA1 neurons in vitro. J. Neurophysiol. 77: 386-392, 1997. In response to oxygen deprivation, CA1 pyramidal neurons show a hyperpolarization (hypoxic hyperpolarization), which is associated with a reduction in neuronal input resistance. The role of extra- and intracellular Ca²⁺ ions in hypoxic hyperpolarization was investigated. The hypoxic hyperpolarization was significantly depressed by tolbutamide (100 µM); moreover, the response was reversed in its polarity in medium containing tolbutamide (100 µM), low Ca²⁺ (0.25 mM), and Co²⁺ (2 mM), suggesting that the hypoxic hyperpolarization is mediated by activation of both ATP-sensitive K⁺ (K_ATP) channels and Ca²⁺-dependent K⁺ channels. The hypoxic depolarization in medium containing tolbutamide, low Ca²⁺, and Co²⁺ is probably due to inhibition of the electrogenic Na⁺-K⁺ pump and concomitant accumulation of interstitial K⁺. Hypoxic hyperpolarizations were depressed in either low Ca²⁺ (0.25 or 1.25 mM) or high Ca²⁺ (5 or 7.5 mM) medium (control: 2.5 mM), indicating that there is an optimal extracellular Ca²⁺ concentration required to producethe hypoxic hyperpolarization. Bis-(o-aminophenoxy)-N,N,N,Ntetraacetic acid (BAPTA)-AM (50-100 µM), procaine (300 µM), or ryanodine (10 µM) significantly depressed the hypoxic hyperpolarization, suggesting that Ca²⁺ released from intracellular Ca²⁺ stores may have an important role in the generation of hypoxic hyperpolarization. The high-affinity calmodulin inhibitor N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonomide hydrochloride (W-7) (5 µM) completely blocked, whereas the low-affinity calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonomide hydrochloride (W-5) (50 µM) did not affect, the hypoxic hyperpolarization. The calmodulin inhibitor trifluoperazine (50 µM) also suppressed the hypoxic hyperpolarization. In addition, calcium/calmodulin kinase II inhibitor 1-[N,O-bis(1,5-isoquinol-inesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN-62) (10 µM) markedly depressed the amplitude and net outward current of the hypoxic hyperpolarization without affecting the reversal potential. In contrast, neither the myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexa-hydro-1,4-diazepin hydrochloride (ML-7) (10 µM) nor the protein kinase A inhibitorN-[2-(p-bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide(H-89) (1 µM) significantly altered the hypoxic hyperpolarization. These results suggest that calmodulin kinase II, which is activated by calmodulin, may contribute to the generation of the hypoxic hyperpolarization. In conclusion, the present study indicates that, in the majority of hippocampal CA1 neurons, the hypoxic hyperpolarization is due to activation of both K_ATP channels and Ca²⁺-dependent K⁺ channels.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Short-term (2-4 min) oxygen deprivation induces a hyperpolarization (hypoxic hyperpolarization) in rat hippocampal CA1 neurons in vitro (Fujiwara et al. 1987; Leblond and Krnjevi 1989). There is general agreement that the hypoxic hyperpolarization is most likely mediated by an enhanced potassium conductance (G_K) (Fujiwara et al. 1987; Hansen et al. 1982; Leblond and Krnjevi 1989). The precise nature of the G_K activation has not yet been elucidated. Two major contending hypotheses ascribe the hyperpolarization to either activation of an ATP-dependent potassium conductance (G_KATP) (Fujimura et al. 1997) or to elevation of the calcium-dependent potassium conductance [G_K(Ca)] (Leblond and Krnjevi 1989). Because anoxia or hypoxia sooner or later leads to both a rise in intracellular calcium concentration ([Ca²⁺]_i) (Dubinsky and Rothman 1991; Duchen 1990; Kass and Lipton 1982; Silver and Erecinska 1990) and a reduction in cytosolic ATP ([ATP]_i) (Lipton and Whittingham 1982; Siesjö 1981), there is, as pointed out by Zhang and Krnjevi (1993), no a priori reason for considering one rather the other as the primary signal.

The calcium hypothesis is favored by the depression of the hyperpolarization (or corresponding outward current during voltage clamp) by blockers of Ca²⁺ release from internal stores, such as dantrolene, heparin, thapsigargin, ruthenium red, and procaine (Belousov et al. 1995; Krnjevi and Xu 1989). Nevertheless, the effects of intracellular Ca²⁺ chelators are variable and inconclusive. (cf. Fujiwara et al. 1987; Leblond and Krnjevi 1989).

Thus the main aim of this study was to investigate the involvement of activation of Ca²⁺-dependent K⁺ channels in hypoxic hyperpolarization. Attempts were made to examine whether or not hypoxic hyperpolarization is dependent on external Ca²⁺ concentrations and able to be depressed by either intracellular Ca²⁺ chelators or inhibitors of release from intracellular Ca²⁺ stores. In addition, the intracellular Ca²⁺-dependent signal transduction systems contributing to the generation of hypoxic hyperpolarization were also examined.

	METHODS

Abstract Introduction Methods Results Discussion References

The methods have been previously described (Fujimura et al. 1997). Briefly, rats were killed under deep ether anesthesia by severing the great vessels of the chest. The brain was removed and a block of tissue that contained the hippocampus was sectioned with a Vibratome (400 µm). The tissue slice was submerged in a flowing (6-8 ml/min) physiological saline that contained (in mM) 117 NaCl, 3.6 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 1.2 NaH₂PO₄, 25 NaHCO₃, and 11 glucose, saturated with 95% O₂-5% CO₂, preheated to 36 ± 0.5°C. Intracellular recordings were made from the pyramidal layer in the hippocampal CA1 region with the use of electrodes that contained potassium chloride (2 M), with electrode resistances of 60-90 M. The membrane potential was determined by the baseline level recorded from an X-Y recorder with low-pass filter.

Slice preparations were made "hypoxic" by superfusing medium equilibrated with 95% N₂-5% CO₂ (hypoxic medium). When switching the superfusing mediums, there was a delay of 15-20 s before the new medium reached the chamber, because of the volume of the connecting tubing.

Drugs used were tolbutamide (Research Biochemicals International), procaine (Sigma), N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonomide hydrochloride (W-7), N-(6-aminohexyl)-1-naphthalenesulfonomide hydrochloride (W-5), 1-[N,O-bis(1,5isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine(KN-62), 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexa-hydro-1,4-diazepine hydrochloride (ML-7), N-[2-(p-bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide (H-89, Seikagaku Kogyo), bis-(o-aminophenoxy)-N,N,N,N-tetraacetic acid (BAPTA)-AM (Dojin), and ryanodine (Calbiochem).

All quantitative results are expressed as means ± SD. The number of neurons examined is given in parentheses. The unpaired Student's t-test was used to compare data, with P< 0.05 considered significant unless specified otherwise.

	RESULTS

Abstract Introduction Methods Results Discussion References

This study was based on recordings from 100 CA1 pyramidal neurons of adult rats with stable membrane potentials more negative than 60 mV. The resting membrane potential and the apparent input resistance were 69.3 ± 2.7 mV and 51.4 ± 8.2 M (n = 100), respectively. In this study, unless specified otherwise, the membrane potential was depolarized to 60 mV before hypoxic exposure by passing depolarizing DC current through the recording electrode.

Ca²⁺-dependent K⁺ conductance is involved in the hypoxic hyperpolarization

Brief applications of hypoxic media (2-4 min) induced hyperpolarizations with a mean amplitude of 10 ± 4 mV(n = 70), which were accompanied by a reduction in the apparent input resistance to 56 ± 12% (n = 70) of the prehypoxic control. Readmittance of oxygen produced a transient hyperpolarization and then restored the membrane potential to the prehypoxic level. To analyze the conductance change of the hypoxic hyperpolarization, hyperpolarizing and subsequent depolarizing ramp command currents were applied before and during the hyperpolarization (Fig. 1A). The resultant steady-state current-voltage curves showed that the membrane conductance during the hypoxic hyperpolarization was increased compared with that of the control (Fig. 1B), and that the net outward current induced by hypoxia had an outward rectification when the membrane potential was made more positive (Fig. 1B, inset). The net outward current was markedly depressed by tolbutamide (100 µM) (Fig. 1C, inset). In the presence of tolbutamide (100 µM), Ca²⁺ (0.25 mM), Mg²⁺ (8 mM), and Co²⁺ (2 mM), exposure to hypoxic medium shifted the membrane potential from 60 mV to approximately 40 mV and markedly decreased the slope conductance (n = 4, Fig. 1D). The net inward current was reduced at hyperpolarizing membrane potentials, but its polarity was not reversed at membrane potentials between 50 and 95 mV (Fig. 1D, inset). These results suggest that hypoxic hyperpolarization is mediated by activation of both ATP-sensitive K⁺ (K_ATP) channels and Ca²⁺-dependent K⁺ channels.

View larger version (36K): [in this window] [in a new window]   FIG. 1. ATP-sensitive K+ (KATP) channel blockers and a reduction in external Ca2+ depress the net outward current produced by hypoxia in hippocampal CA1 neurons. In this and subsequent figures, hypoxic medium was applied between the downward and upward arrows and, in each trace, the dotted line indicates the preexposure level of the membrane potential, unless specified otherwise. A: pairs of traces show current (top) and potential (bottom) recordings under current-clamp condition. Slow hyperpolarizing and depolarizing DC ramp currents (1-2 mV/s) were passed through the recording electrode to obtain steady-state current-voltage relationships before and during hypoxic exposure in the control condition (i); after pretreatment with tolbutamide (100 µM) for 10 min (ii); and with tolbutamide (100 µM), low Ca2+ (0.25 mM), high Mg2+ (8 mM), and Co2+ (2 mM) for 10 min (iii). B-D: steady-state current-voltage curves were obtained before (Control) and during hypoxic exposure (Hypoxia) in the control condition (B); in the presence of tolbutamide (100 µM) (C); and in the presence of tolbutamide (100 µM), low Ca2+ (0.25 mM), high Mg2+ (8 mM), and Co2+ (2 mM) (D). Inset: net outward current produced by hypoxia, which were obtained by subtraction of the steady-state current-voltage relation at preexposure level from that during hypoxic exposure. For subtraction, steady-state current-voltage relations, which were continuously recorded from the most hyperpolarizing level (95 mV) to 60 mV, were used. The net outward current induced by hypoxia in the control condition (B, inset) was markedly depressed by tolbutamide (C, inset), and the response to hypoxia was reversed in polarity, and a net inward current was obtained, in tolbutamide (100 µM), low Ca2+ (0.25 mM), high Mg2+ (8 mM), and Co2+ (2 mM) medium (D, inset). A-D: same neuron.

Effects of extracellular Ca²⁺ concentration on the hypoxic hyperpolarization

To investigate the contribution of Ca²⁺-dependent K⁺ channels to the hypoxic hyperpolarization, the extracellular Ca²⁺ concentration ([Ca²⁺]_o) was varied. Superfusion with low-Ca²⁺ (0.25 or 1.25 mM) medium produced a depolarization ~5-10 mV in amplitude, but the apparent input resistance was not significantly changed (n = 8). In 0.25 and 1.25 mM-Ca²⁺ media, the amplitude of the hypoxic hyperpolarization was significantly reduced to 5.2 ± 2 mV (n = 4, P < 0.01) and 5.8 ± 2 mV (n = 4, P < 0.01), respectively (Fig. 2, A and B). Superfusion with high-Ca²⁺ (5.0 or 7.5 mM) medium produced a hyperpolarization ~2-4 mV in amplitude (n = 10). The apparent input resistance was significantly reduced in Ca²⁺ (7.5 mM) containing medium but not significantly changed in 5.0 mM-Ca²⁺-containing medium. The apparent input resistance was decreased from 48.7 ± 4.6 M in 2.5 mM-Ca²⁺-containing medium to44.7 ± 2.8 M in 7.5 mM-Ca²⁺-containing medium (n =5, P < 0.01). In 5.0 and 7.5 mM-Ca²⁺ media, the amplitude of the hypoxic hyperpolarization was reduced to 4.8 ± 1 mV (n = 5, P < 0.01) and 1.6 ± 1 mV (n = 5, P < 0.01), respectively (Fig. 2B). These results indicate that there is an optimal [Ca²⁺]_o required to produce the hypoxic hyperpolarization.

View larger version (47K): [in this window] [in a new window]   FIG. 2. Effects of various extracellular Ca2+ concentrations ([Ca2+]o) on the hypoxic hyperpolarization. Downward deflections in voltage recordings under current-clamp mode are hyperpolarizing electrotonic potentials elicited by anodal current pulses (0.5 nA applied for 200 ms every 3 s). A: hypoxic hyperpolarization in control medium (2.5 mM Ca2+) (top trace), after pretreatment with low-Ca2+ (1.25 mM) medium for 10 min (middle trace), and after washing out the low-Ca2+ medium for 10 min (bottom trace). B: changes in the amplitude of the hypoxic hyperpolarization at various [Ca2+]o. The amplitude in various [Ca2+]o was normalized with that of the control medium (2.5 mM Ca2+). Error bars: mean ± SD. Note that a rise or fall in [Ca2+]o decreased the amplitude of the hypoxic hyperpolarization.

Effects of intracellular Ca²⁺ on the hypoxic hyperpolarization

To investigate whether internal Ca²⁺ may be importantin the generation of the hypoxic hyperpolarization, theeffects of a Ca²⁺ chelator and an inhibitor of Ca²⁺ releasefrom intracellular stores on the hypoxic hyperpolarizationwere examined. The membrane-permeable Ca²⁺ chelator BAPTA-AM (50-100 µM, Fig. 3A), or the ryanodine receptor agonist ryanodine (10 µM), reduced the peak amplitude of the hypoxic hyperpolarization without affecting the posthypoxic hyperpolarization, whereas the inhibitor of Ca²⁺ release from intracellular stores, procaine (300 µM), reversibly depressed both the hypoxic and posthypoxic hyperpolarizations (Fig. 3B). Table 1 summarizes effects of these drugs on the relative amplitude of the hypoxic hyperpolarization and the reduction of neuronal input resistance during hypoxic hyperpolarization. BAPTA-AM, procaine, and ryanodine significantly reduced the peak amplitude of the hypoxic hyperpolarization and reversed the ratio of the reduction of the neuronal input resistance during the hypoxic hyperpolarization.

View larger version (54K): [in this window] [in a new window]   FIG. 3. Effects of a Ca2+ chelator, intracellular Ca2+ release inhibitors, and calmodulin inhibitors on the hypoxic hyperpolarization. Downward deflections in voltage recordings under current-clamp mode are hyperpolarizing electrotonic potentials elicited by anodal current pulses (in the range of 0.2-0.4 nA, applied for 200 ms every 3 s). A: hypoxic hyperpolarization before treatment (top trace); after pretreatment with 50 µM (2nd trace), 75 µM (3rd trace), and 100 µM (4th trace) bis-(o-aminophenoxy)-N,N,N,N-tetraacetic acid (BAPTA)-AM for 10 min; and after washing out of the drug for 30 min (bottom trace). B: hypoxic hyperpolarization before treatment (top trace), after pretreatment with procaine (300 µM) for 10 min (middle trace), and after washing out of the drug for 10 min (bottom trace). C: hypoxic hyperpolarization before treatment (top trace), after pretreatment with N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonomide hydrochloride (W-7) (50 µM) for 10 min (middle trace), and after washing out of the drug for 30 min (bottom trace).

Contribution of intracellular signal transduction systems in the generation of hypoxic hyperpolarization

To investigate the role of intracellular Ca²⁺-dependent signal transduction systems, the effects of antagonists for the protein kinases and calmodulin (CaM) were examined. The CaM antagonist W-7 (50 µM) completely blocked hypoxic hyperpolarizations and reversed their polarity; the membrane was depolarized during exposure to the hypoxic medium (Fig. 3C). The amplitude of the depolarization at the end of exposure was 5.5 ± 2.7 mV (n = 6). Another CaM inhibitor, trifluoperazine (50 µM), also depressed the hypoxic hyperpolarization and reversed the ratio of the reduction in the neuronal input resistance during the hyperpolarization (Table 2). These CaM inhibitors did not significantly affect the posthypoxic hyperpolarization. On the other hand, W-5 (50 µM), which is a weak, less specific antagonist for CaM (Kanamori et al. 1981), did not show any effects on the hypoxic hyperpolarization (Table 2).

The CaM kinase II antagonist KN-62 (10 µM) also depressed the hypoxic hyperpolarization (Fig. 4A) and reversed the ratio of the reduction in the neuronal input resistance during the hypoxic hyperpolarization (Table 2). In contrast, the myosin light chain kinase inhibitor ML-7 (10 µM) and the protein kinase A inhibitor H-89 (1 µM) did not significantly affect the hypoxic hyperpolarization (Table 2).

View larger version (60K): [in this window] [in a new window]   FIG. 4. Effects of inhibitors of protein kinases on the hypoxic hyperpolarization. A: hypoxic hyperpolarization before treatment (top trace), after pretreatment with 1-[N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN-62) (10 µM) for 10 min (middle trace), and after washing out of the drug for 10 min (bottom trace). B: hypoxic hyperpolarization before treatment (top trace), after pretreatment with ML-7 (10 µM) for 10 min (middle trace), and after washing out of the drug for 10 min (bottom trace). C: hypoxic hyperpolarization before treamtent (top trace), after pretreatment with N-[2-(p-bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide (H-89) (1 µM) for 10 min (middle trace), and after washing out of the drug for 10 min (bottom trace).Note that KN-62 significantly depressed the hypoxic hyperpolarization.

To study further the inhibitory action of KN-62 (10 µM), steady-state current-voltage curves before and during the hypoxic hyperpolarization were obtained by passing ramp currents through the recording electrode (Fig. 5A). The resultant net outward current produced by exposure to hypoxic medium was compared in the absence and presence of KN-62 (Fig. 5, B and C, insets). The chord conductance of the membrane potentials between 80 and 90 mV in the absence of KN-62 (11 ± 4.2 nS, n = 5) was significantly greater than that in the presence of KN-62 (5.0 ± 3.7 nS, n = 5, P < 0.05). In contrast, the reversal potential of the hypoxic hyperpolarization was not different, being 85 ± 2 mV (n = 5) in the absence, and 85 ± 3 mV (n = 5) in the presence, of KN-62.

View larger version (44K): [in this window] [in a new window]   FIG. 5. Effects of KN-62 on the net outward current induced by hypoxia. A: pairs of traces show current (top) and potential (bottom) recordings under current-clamp condition. Slow hyperpolarizing and depolarizing DC ramp currents (1-2 mV/s) were passed through the recording electrode to obtain steady-state current-voltage relationships before and during hypoxic exposure in the control condition (i) and after pretreatment with KN-62 (10 µM) for 10 min (ii). B and C: steady-state current-voltage curves in the control condition were obtained before (Control) and during hypoxic exposure (Hypoxia) in the control condition (B) and in the presence of KN-62 (10 µM) (C). The net outward current induced by hypoxia in the control condition (B, inset) was depressed by KN-62 (C, inset). A-C are from the same neuron.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

The present study shows that the amplitude of the hypoxic hyperpolarization was critically dependent on [Ca²⁺]_o, because it could be reduced by concentrations either greater or less than 2.5 mM. Both intracellular Ca²⁺ chelation and inhibitors of intracellular Ca²⁺ release reduced the hypoxic hyperpolarization, and external Ca²⁺ also has a comparable role in the generation of the hypoxic hyperpolarization. These results suggest that internal Ca²⁺ has an important role in the generation of the hypoxic hyperpolarization. The Ca²⁺ dependency of the hypoxic hyperpolarization and the possible Ca²⁺-dependent signal transduction systems that contribute to the generation are discussed in the following section.

Ca²⁺ dependency of the hypoxic hyperpolarization

In the presence of the K_ATP channel blocker tolbutamide (100 µM), hypoxic hyperpolarizations were markedly depressed, whereas in the presence of Krebs solution containing low Ca²⁺ and Co²⁺ and tolbutamide, the hyperpolarizations were not only completely suppressed, but the polarity of the response was also reversed, causing a membrane depolarization. These results suggest that both K_ATP and Ca²⁺-dependent K⁺ conductances contribute to the generation of the hypoxic hyperpolarization. Fujiwara et al. (1987) have shown that electrogenic Na⁺-K⁺ pump activity is depressed during hypoxia. It is therefore possible that the hypoxic depolarization in tolbutamide and low-Ca²⁺-containing medium may be due to suppression of electrogenic Na⁺-K⁺ pump activity.

If the Ca²⁺-dependent K⁺ conductance were only activated by Ca²⁺ influx from the extracellular environment, the hypoxic hyperpolarization should be increased in high-Ca²⁺ media. As described above, the optimal [Ca²⁺]_o for generation of the hypoxic hyperpolarization was 2.5 mM, a finding that suggests that the hypoxic hyperpolarization may not be simply dependent on Ca²⁺ influx, but may also involve other mechanisms for the regulation of [Ca²⁺]_i, such as intracellular Ca²⁺-dependent signal transduction systems. The BAPTA-AM-reduced amplitude of the hypoxic hyperpolarization suggests that intracellular BAPTA may chelate Ca²⁺ that had flowed into the impaled neuron from the extracellular space and/or been extruded from intracellular stores. This reduction is comparable with the inhibitory effect of the Ca²⁺ chelator ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) on the hypoxic hyperpolarization in the majority of hippocampal CA1 neurons (Leblond and Krnjevi 1989).

Procaine and ryanodine markedly reduced the hypoxic hyperpolarization, suggesting that the hypoxic hyperpolarization may have been due to an increase of [Ca²⁺]_i that is caused by Ca²⁺-induced Ca²⁺ release (Marrion and Adams 1992). In fact, Higashi et al. (1990) have shown that, in hypoxic Fura-2-loaded hippocampal CA1 neurons of the slice preparation, the rise in [Ca²⁺]_i and the initial hyperpolarization occur almost simultaneously. Recently, Belousov et al. (1995), using the whole cell recording method, have reported that procaine, heparin, and thapsigargin markedly reduce the hypoxic hyperpolarization but ryanodine does not. Belousov et al. concluded that the hypoxic hyperpolarization is predominantly mediated by Ca²⁺ release from an inositol 1,4,5-triphosphate-sensitive internal store. It is likely that the lack of effect of ryanodine on the hypoxic hyperpolarization in the whole cell recording mode, which uses relatively large electrodes, is due to inactivation of the intracellular ryanodine-receptor-coupled Ca²⁺ channels following perfusion with internal Ca²⁺-free solutions, because the open probability of the ryanodine receptor channels is dependent on [Ca²⁺]_i (Ashley and Williams 1990).

Intracellular signal transduction systems involved in the generation of the hypoxic hyperpolarization

The present study shows that the CaM inhibitors W-7 and trifluoperazine depressed the hypoxic hyperpolarization. Involvement of CaM in the generation of the hypoxic hyperpolarization therefore seems likely. W-7 reversed the polarity of the response to hypoxia; that is to say, it caused a hypoxic depolarization that was associated with a fall in the apparent input resistance. W-7 is an inhibitor of CaM, phosphodiesterase, and myosin light chain kinase. The value of the half-maximum inhibition for these enzyme activities is ~30-50 µM (Cafouleas et al. 1982; Hidaka et al. 1981). Neither the protein kinase A inhibitor H-89 nor the myosin light chain kinase inhibitor ML-7 affected the hypoxic hyperpolarization, suggesting that the involvement, if any, of phosphodiesterase or myosin light chain kinase in the hypoxic hyperpolarization is minimal. It is therefore likely that the hypoxic depolarization in W-7-containing medium is due to inactivation of the electrogenic Na⁺-K⁺ pump and the concomitant accumulation of interstitial K⁺.

CaM activates various protein kinases, including myosin light chain kinase, phosphorylase kinase, and Ca²⁺/CaM-dependent protein kinases I, II, and III (CaM kinase I, II, III). Immunohistochemical studies show that CaM kinase II is quite plentiful in the hippocampus (Erondu and Kennedy 1985; Ouient et al. 1984). The selective inhibitor for CaM kinase II, KN-62, depressed the hypoxic hyperpolarization. This suggests that the increased internal Ca²⁺ binds to CaM and the Ca²⁺/CaM complex activates CaM kinase II, which may phosphorylate and open K⁺ channels. CaM kinase II can be phosphorylated at low concentrations (3-500 µM) of [ATP]_i (Lai et al. 1986; Miller and Kennedy 1986). In guinea pig hippocampal neurons in vitro, the [ATP]_i could be >1 mM, and the [P]_i is decreased by ~15% 2 min after exposure to hypoxic medium (Lipton and Whittingham 1982). Thus the remaining [ATP]_i during hypoxia would be enough to sustain CaM kinase II phosphorylation of potassium channels.

Contribution of K_ATP channels and Ca²⁺-dependent K⁺ channels to the hypoxic hyperpolarization

Hypoxic hyperpolarization has been found to be depressed by glibenclamide and tolbutamide in 60-80% of hippocampal CA1 neurons tested, whereas the remaining neurons were insensitive to these K_ATP channel blockers (Fujimura et al. 1997). The present study shows that even in the tolbutamide-sensitive hypoxic hyperpolarization, Ca²⁺-dependent K⁺ channels partially contribute to the generation of the hypoxic hyperpolarization. Thus the ratio for the contribution of Ca²⁺-dependent K⁺ channels and K_ATP channels to the hypoxic hyperpolarization is different from neuron to neuron. Two major mechanisms underlying the hypoxic hyperpolarization have been proposed in hippocampal neurons; activation of Ca²⁺-dependent K⁺ channels via a rise in [Ca²⁺]_i (Belousov et al. 1995; Katchman and Hershkowitz 1993; Krnjevi and Xu 1989; Leblond and Krnjevi 1989) and activation of K_ATP channels by depletion of [ATP]_i (Godfraind and Krnjevi 1993; Grigg and Anderson 1989). Because hypoxia leads to a significant depletion of [ATP]_i as well as a rise of [Ca²⁺]_i (Biscoe et al. 1988; Hansen 1985; Higashi et al. 1990; Nishimura 1986; Siesjö 1978), both effects seem equally likely, and both could contribute toward the outward K⁺ currents. An important consideration is changes in the proportions of K_ATP channels and Ca²⁺-dependent K⁺ channels due to aging of experimental animals, because the density of K_ATP channels has been shown to increase with age (Xia et al. 1993).

The present finding that BAPTA-AM depressed the hypoxic hyperpolarization is comparable with the previous report that EGTA depressed the hypoxic hyperpolarization in the majority of hippocampal CA1 neurons (Leblond and Krnjevi 1989). In our previous study, however, we found that intracellular EGTA injection did not affect the hypoxic hyperpolarization, but markedly depressed a slow afterhyperpolarization following spikes in hippocampal CA1 neurons (Fujiwara et al. 1987). It is possible that the amount of injected EGTA was not enough to chelate the increased cytosolic Ca²⁺ that activates the Ca²⁺-dependent K⁺ channel, because the chelating action of BAPTA is more potent and more selective for Ca²⁺ than that of EGTA (Tsien 1981). Alternatively, the activation of the K_ATP channel may be predominant in the EGTA-insensitive neurons.

A protein kinase A inhibitor decreases K_ATP channel activity in pancreatic -cells (Ribalet et al. 1989). It has been reported that activity of cloned fly Ca²⁺-dependent K⁺ channels is augmented by phosphorylation of an A kinaselike protein (Esguerra et al. 1994). In rat cortical neurons, ATP, forskolin, and dibutyl adenosine 3,5-cyclic monophosphate stimulated Ca²⁺-dependent K⁺ channel activity, whereas H-7, a peptide inhibitor of protein kinase A, diminished the activity (Lee et al. 1995). These results suggest that the activation of Ca²⁺-dependent K⁺ channels would be augmented by phosphorylation of A kinase. In contrast, our findings that the A kinase inhibitor H-89 did not affect the hypoxic hyperpolarization suggest that the involvement, if any, of A kinase-mediated phosphorylation in the generation of the hypoxic hyperpolarization is minimal.

In conclusion, the present study indicates that the hypoxic hyperpolarization is due to activation of both K_ATP channels and Ca²⁺-dependent K⁺ channels. The Ca²⁺-dependent K⁺ channels may be activated by the increased [Ca²⁺]_i that results from the Ca²⁺-induced Ca²⁺ release from intracellular stores during exposure to hypoxic medium. Furthermore, activation of CaM and Ca²⁺/CaM kinase II may be involved in the activation of Ca²⁺-dependent K⁺ channels. Study of the isolated individual currents is, however, required to further elucidate the pharmacological characteristics of the channels involved.

	ACKNOWLEDGEMENTS

  We thank Drs. G. M. Lees and S. M. C. Cunningham for valuable comments and suggestions on the manuscript.

  This work was supported in part by a Grant-in-Aid for Scientific Research of Japan and an Ishibashi Foundation Grant.

	FOOTNOTES

  Address for reprint requests: S. Yamamoto, Dept. of Physiology, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830, Japan.

  Received 21 June 1996; accepted in final form 12 September 1996.

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