Ischemia But Not Anoxia Evokes Vesicular and Ca2+-Independent Glutamate Release In the Dorsal Vagal Complex In Vitro

Anna Kulik, Stefan Trapp, and Klaus Ballanyi

II. Physiologisches Institut, Universität Göttingen, D-37073 Göttingen, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Kulik, Anna, Stefan Trapp, and Klaus Ballanyi. Ischemia But Not Anoxia Evokes Vesicular and Ca2+-Independent Glutamate Release In the Dorsal Vagal Complex In Vitro. J. Neurophysiol. 83: 2905-2915, 2000. Whole cell recordings of fura-2 dialyzed vagal neurons of brain stem slices were used to monitor interstitial glutamate accumulation within the dorsal vagal complex. Anoxia produced a sustained outward current (60 pA) and a moderate [Ca2+]i rise (40 nM). These responses were neither mimicked by [1S,3R]-1-aminocyclo-pentane-1,3-dicarboxylic acid nor affected by Ca2+-free solution, 6-cyano-7-nitroquino-xaline-2,3-dione (CNQX), 2-amino-5-phosphonovalerate (APV), or tetrodotoxin. Anoxia or cyanide in glucose-free saline (in vitro ischemia) as well as ouabain or iodoacetate elicited an initial anoxia-like [Ca2+]i increase that turned after several minutes into a prominent Ca2+ transient (0.9 µM) and inward current (-1.8 nA). APV plus CNQX (plus methoxyverapamil) inhibited this inward current as well as accompanying spontaneous synaptic activity, and reduced the secondary [Ca2+]i rise to values similar to those during anoxia. Each of the latter drugs delayed onset of both ischemic current and prominent [Ca2+]i rise by several minutes and attenuated their magnitudes by up to 40%. Ca2+-free solution induced a twofold delay of the ischemic inward current and suppressed the prominent Ca2+ increase but not the initial moderate [Ca2+]i rise. Cyclopiazonic acid or arachidonic acid in Ca2+-free saline delayed further the ischemic current, whereas neither inhibitors of glutamate uptake (dihydrokainate, D,L-threo-beta -hydroxyaspartate, L-transpyrrolidone-2,4-dicarboxylate) nor the Cl- channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid had any effect. In summary, the response to metabolic arrest is due to activation of ionotropic glutamate receptors causing Ca2+ entry via N-methyl-D-aspartate receptors and voltage-activated Ca2+ channels. An early Ca2+-dependent exocytotic phase of ischemic glutamate release is followed by nonvesicular release, not mediated by reversed glutamate uptake or Cl- channels. The results also show that glycolysis prevents glutamate release during anoxia.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Excitotoxicity related to extracellular accumulation of glutamate is believed to be a major cause for the low tolerance of most mammalian neurons to anoxia (Benveniste et al. 1984; Choi 1988). The anoxia-induced vulnerability, in particular of cortical neurons, appears to be related to the profound rise of the free intracellular concentration of Ca2+ ([Ca2+]i) that is associated with increased interstitial levels of glutamate. Such neuronal [Ca2+]i elevation results primarily from influx via Ca2+-permeable glutamate receptors and voltage-activated Ca2+ channels (Kristian and Siesjö 1996; Silver and Erecinska 1990). Also impairment of Ca2+ extrusion and sequestration due to suppression of ion pump activity and mitochondrial function contributes to the excessive anoxic rise of [Ca2+]i (Kristian and Siesjö 1996; Schinder et al. 1996). The neuronal Ca2+ overload in combination with cellular changes such as ATP consumption by depolarized mitochondria activates a cascade of events that leads to cell death (Budd and Nicholls 1996; Schanne et al. 1979). Besides the complexity of cellular events that mediate glutamate-induced neuronal death, it is not clear which processes are responsible for interstitial accumulation of glutamate during anoxia. There is disagreement on whether Ca2+-dependent exocytosis or reduced uptake is the major constituent of the initial phase of the ischemia-related glutamate rise (Drejer et al. 1985; Ikeda et al. 1989; Katayama et al. 1991; Szatkowski and Attwell 1994). It is also under discussion whether the late phase of the anoxic glutamate release is associated with reversed action of the transporters, unspecific membrane damage, or other processes (Bickler and Hansen 1994; Madl and Burgesser 1993; Roettger and Lipton 1996; Sanchez-Prieto and Gonzales 1988).

Here we have investigated the origin and functional consequence of glutamate release during anoxia/ischemia in a brain stem slice preparation that contains the dorsal vagal complex (Loewy and Spyer 1990). This preparation was chosen as the response of dorsal vagal neurons (DVN) to different types of metabolic disturbances is well described. In contrast to the glutamate-related vulnerability of most neuronal structures of the mature brain, DVN are extremely tolerant to anoxia (Cowan and Martin 1992; Trapp and Ballanyi 1995). Block of aerobic metabolism by either anoxia or cyanide (CN-) hyperpolarizes DVN due to a sustained outward current through ATP-sensitive K+ channels (Karschin et al. 1998; Trapp and Ballanyi 1995). Sulfonylurea-induced block of the anoxic outward current unmasks a moderate inward current that remains stable during anoxia periods of at least 20 min (Ballanyi and Kulik 1998; Trapp and Ballanyi 1995). Thus, in the DVN, oxygen depletion alone does not evoke a progressive inward current that underlies the "terminal" depolarization of anoxia-vulnerable neurons (Bures and Buresova 1957; Haddad and Jiang 1993a,b; Hansen et al. 1982). However, block of glycolysis with iodoacetate (Ballanyi and Kulik 1998) and anoxic exposure after preincubation in glucose-free solution that mimics ischemia (Ballanyi et al. 1996) lead to a progressive depolarization of DVN that is accompanied by profound perturbation of ion homeostasis. In the present study, we have used whole cell recordings to indirectly monitor changes of extracellular glutamate (Warr et al. 1999) during anoxia or ischemia by activation of postsynaptic ionotropic glutamate receptors of DVN (Travagli et al. 1991; Willis et al. 1996). In addition, the cells were dialyzed via the patch electrode with fura-2 to assess pathways of putative glutamate receptor-associated [Ca2+]i increases during anoxia and ischemia.


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INTRODUCTION
METHODS
RESULTS
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Preparation and solutions

The experiments were performed on brain stem slices obtained from 14- to 20-day-old Wistar rats of either sex. Rats were anesthetized with ether and decapitated. The brain stem was isolated and kept for 5 min in ice-cold artificial cerebrospinal fluid (standard solution; composition see below; Ca2+ concentration reduced to 0.5 mM). Afterward, six to eight transverse slices (200 µm) were cut from the brain stem around the obex level in the 0.5 mM Ca2+ solution. Before transfer to the recording chamber, the slices were stored at 30°C in standard solution. The recording chamber (volume 3 ml) was superfused at 30°C with oxygenated standard solution (flow rate 5 ml/min) of the following composition (in mM): 118 NaCl, 3 KCl, 1 MgCl2, 1.5 CaCl2, 25 NaHCO3, 1.2 NaH2PO4, and 10 D-glucose. pH was adjusted to 7.4 by gassing with 95% O2 and 5% CO2. Tissue anoxia was evoked by hypoxic superfusate gassed with 95% N2 instead of O2 (Ballanyi et al. 1996; Trapp and Ballanyi 1995). Chemical anoxia was induced by addition of CN- (1 mM) to the standard solution (Ballanyi and Kulik 1998; Karschin et al. 1998). In vitro ischemia was elicited by either anoxic exposure of the brain stem slices after preincubation in glucose-free solution for at least 30 min or by application of CN- subsequent to 30 min preincubation in glucose-free superfusate (Ballanyi et al. 1996). Ca2+-free saline contained 1 mM EGTA and 5 mM Mg2+.

Drugs were purchased from Sigma (Munich, Germany), Biomol (Cologne, Germany) or Tocris Cookson (Bristol, UK). The following drugs were kept as stock solution in standard saline: glutamate (100 mM), the blocker of N-methyl-D-aspartate (NMDA) glutamate receptors 2-amino-5-phosphonovalerate (APV, 10 mM), N-(4-hydroxyphenyl-propanolyl)-spermine (NHPPS, 1 mM), L-transpyrrolidone-2,4-dicarboxylate (PDC, 10 mM), dihydrokainate (DHK, 10 mM), and D,L-threo-beta -hydroxyaspartate (THA, 10 mM). The blocker of alpha -amino-3-hydroxy-5-methyl-4-isoxalone/kainate (AMPA/KA) glutamate receptors 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 mM), methoxyverapamil (D-600, 100 mM), tolbutamide (100 mM), gliquidone (10 mM), cyclopiazonic acid (CPA, 10 mM), and 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB, 100 mM) were dissolved in DMSO. [1S,3R]-1-aminocyclopentane-1,3-dicarboxylic acid ([1S,3R]-ACPD, 1 mM) was dissolved in 0.1 M NaOH and arachidonic acid (100 mM) in ethanol. The other drugs were added to the superfusate.

Patch clamp

Patch pipettes were produced from borosilicate glass capillaries (GC 150TF, Clark Electromedical Instruments, Pangbourne, UK) using a horizontal electrode puller (Zeitz, Munich, Germany). Standard patch pipette solution contained (in mM) 140 potassium gluconate, 1 MgCl2, 10 HEPES, 1 Na2ATP, pH 7.3-7.4. The resistance of the patch electrodes ranged from 4 to 6 MOmega 100 µM fura-2 (Molecular Probes, Eugene, OR) was added to the pipette solution before the experiment. Whole cell recordings were performed on superficial DVN under visual control using an EPC-9 patch-clamp amplifier (HEKA, Lambrecht, Germany). Membrane conductance (gm) was measured by application of hyperpolarizing current (10-100 pA) or voltage (-20 mV) pulses with a duration of 500 ms. The holding potential (Vh) in voltage clamp was -50 mV [corresponding to mean resting membrane potential (Vm)] (Ballanyi et al. 1996; Trapp and Ballanyi 1995) unless stated otherwise.

Ca2+ measurements

[Ca2+]i was measured fluorometrically using a fura-2-based single-detector system. Cells were loaded with fura-2 by dialysis via the patch electrode. An upright microscope (Standard 16, Zeiss, Oberkochen, Germany) was equipped with epifluorescence optics, a monochromator (Polychrome II, T.I.L.L. photonics, Planegg, Germany) to allow for alternating fluorescent excitation at 360 and 390 nm, and a photomultiplier system (Luigs and Neumann, Ratingen, Germany). Emission was measured at 510 nm through a pinhole diaphragm, which limited the region from which light was collected to a circular spot of 20 µm diam. Fluorescence ratios were converted into [Ca2+]i by using Eq. 1, [Ca2+]i = K(R - Rmin)/(Rmax - R) (Grynkiewicz et al. 1985), in which R is the fluorescence ratio (360 nm/390 nm) and K is the effective dissociation constant of fura-2. In vivo calibrations to determine Rmin, Rmax, and K were done according to the method described by Neher (1989). Measurements were performed with three pipette solutions (in mM): 1) 130 KCl, 1 MgCl2, 10 BAPTA, 10 HEPES, and 1 Na2ATP (low calcium; Rmin); 2) 130 KCl, 1 MgCl2, 3 CaCl2, 4 BAPTA, 10 HEPES, and 1 Na2ATP [intermediate calcium; 300 nM, according to a KD of 107 nM for BAPTA (Tsien 1980)]; and 3) 130 KCl, 1 MgCl2, 10 CaCl2, 10 HEPES, and 1 Na2ATP (high calcium; Rmax); to each solution 100 µM fura-2 was added. The intracellular fluorescence ratios were calculated according to Eq. 1. K was calculated as K = 300 nM (Rmax - R)/(R - Rmin).

Data analysis

Electrophysiological and microfluorometrical signals were sampled into a Macintosh PowerPC (Apple Computers, Cupertino, CA) using Pulse/Pulsefit and X-chart/Fura extension from HEKA. Current and voltage signals were also digitized (Instrutech VR-100A, Elmont, NY) and recorded on a VCR. Values are means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Response to anoxia

In an initial series of experiments, the effects of tissue anoxia were investigated on Vm and [Ca2+]i. Spontaneous spike discharge at a frequency of 0.5-5 Hz was observed in 11 of 14 DVN in which [Ca2+]i baseline ranged between 38 and 156 nM. In 10 of these cells, anoxia produced a stable hyperpolarization by up to 20 mV. The anoxia-evoked hyperpolarization to a maximal of -75 mV was accompanied by a modest rise of [Ca2+]i by <100 nM (Fig. 1A). In four cells with spike frequencies >2 Hz and therefore a higher [Ca2+]i baseline, the anoxia-evoked [Ca2+]i rise was preceded by a 15- to 35-nM reduction of [Ca2+]i. In these DVN, reoxygenation first led to a rapid fall of [Ca2+]i that was followed by a reincrease associated with recovery of spike activity (Fig. 1A). In neurons with a spike frequency <2 Hz, no biphasic Ca2+ response was observed (not shown).



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Fig. 1. Anoxia-induced membrane response and modest anoxic Ca2+ rise in dorsal vagal neurons. A: anoxia evoked by nitrogen (N2) gassing of the superfusate led to hyperpolarization-induced block of tonic discharge and a concomitant initial fall and secondary modest rise of intracellular free Ca2+ ([Ca2+]i). B: tolbutamide (100 µM) blocked the anoxic outward current and the increase in membrane conductance (measured by hyperpolarizing voltage pulses), but did not affect the accompanying elevation of [Ca2+]i. C: bars represent [Ca2+]i (±SE) under resting conditions and at steady-state during anoxia as obtained from 35 dorsal vagal neuron (DVN), voltage clamped at -50 mV (*** P < 0.001).

To exclude effects of voltage-activated Ca2+ channels, the anoxia response was analyzed under voltage clamp. A 5-min period of anoxia led to a stable increase of [Ca2+]i by 39 ± 3 nM (mean ± SE, n = 43; Fig. 1, B and C). In 27 of these cells, the [Ca2+]i rise was accompanied by a sustained outward current of 59 ± 7 pA and a gm increase by up to 500% (Figs. 1B and 2). The anoxic [Ca2+]i rise was not affected by block of the outward current by the sulfonylureas tolbutamide (50-200 µM; n = 9; Fig. 1B) or gliquidone (1 µM; n = 3; not shown). In eight of the cells, the sulfonylurea-induced block of the anoxic outward current unmasked a persistent inward current of between -10 and -40 pA and a decrease of gm down to 20% of control (Fig. 1B). This inward current was not accompanied by a significant increase in spontaneous subthreshold synaptic activity (not shown). Both the anoxic [Ca2+]i increase and the outward current were not changed by a solution that contained tetrodotoxin (TTX, 0.5 µM), CNQX (50 µM), and APV (100 µM) to block synaptic transmission as well as ionotropic glutamate receptors (n = 5; Fig. 2A). The anoxic outward current and [Ca2+]i elevation were also unaffected by Ca2+-free solution, which abolished depolarization-induced [Ca2+]i increases (n = 3; Fig. 2B). The unspecific metabotropic glutamate receptor agonist [1S,3R]-ACPD (100 µM) did not change Im, gm, or [Ca2+]i in cells that responded to anoxia with both a moderate [Ca2+]i rise and an outward current (n = 6; not shown). The results suggest that glutamate receptors are not involved in the anoxia response.



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Fig. 2. The anoxic [Ca2+]i rise is not due to ionotropic glutamate receptor activation and caused by release from intracellular stores. A: neither the outward current nor the [Ca2+]i rise during anoxia were affected by combined application of 6-cyano-7-nitroquino-xaline-2,3-dione (CNQX, 50 µM), 2-amino-5-phosphonovalerate (APV, 100 µM), and tetrodotoxin (TTX, 0.5 µM). B: the outward current and the modest rise of [Ca2+]i in response to anoxia were not changed by Ca2+-free superfusate that abolished [Ca2+]i transients caused by 15 s depolarization from -50 to 0 mV (open circle ). Upward deflections of the current (Im) trace are due to an A-type K+ current after the hyperpolarizing voltage steps used for monitoring membrane conductance.

Response to ischemia

The effects of in vitro ischemia on [Ca2+]i and Vm of DVN were tested by anoxic exposure of the slices after preincubation in glucose-free solution for at least 30 min. The cell illustrated in Fig. 3A hyperpolarized with a concomitant gm increase after 20 min of glucose depletion. The hyperpolarization-related suppression of spontaneous spiking caused a fall of [Ca2+]i by ~80 nM. Anoxic exposure during glucose depletion evoked a progressive depolarization by ~30 mV, which was accompanied by a fourfold increase of gm, and a rise of [Ca2+]i by >400 nM. The cell recovered from both the depolarization and the prominent [Ca2+]i rise on reoxygenation of the glucose-free solution. After readdition of glucose, [Ca2+]i and Vm returned to control levels, and spiking reappeared. A similar response was revealed in three further DVN during ischemia.



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Fig. 3. Effects of in vitro ischemia on membrane properties and [Ca2+]i. A: removal of glucose from the standard solution led to a delayed hyperpolarization-induced block of tonic discharge accompanied by a moderate fall of [Ca2+]i. Subsequent anoxic exposure, mimicking ischemia, led to a major depolarization, conductance increase, and concomitant progressive rise of [Ca2+]i. These effects were rapidly reversed on reoxygenation of the glucose-free saline. B: in this voltage-clamp recording glucose was removed from the superfusate after a control anoxic exposure. Subsequent anoxia produced a slight potentiation of the initial [Ca2+]i increase and evoked a delayed progressive inward current and profound [Ca2+]i rise. C: a delayed outward current and conductance increase developed after glucose removal. Subsequent ischemia led to a major inward current, conductance increase and [Ca2+]i rise without recovery.

The mechanism of the ischemia-induced depolarization and [Ca2+]i rise was analyzed under voltage clamp. Eighteen of 29 DVN responded to glucose removal with an outward current of 47 ± 5 pA and a gm increase by 100-500% after a delay of 8-20 min. Im and gm of the remaining cells did not change. Thirty minutes of glucose removal induced a minor (23 ± 3 nM, n = 29) rise in [Ca2+]i baseline. The cell of Fig. 3B responded to anoxia with a moderate increase of [Ca2+]i in the presence of glucose. An ischemia period that was induced after 40 min of glucose depletion led initially to a slow, moderate increase of [Ca2+]i. However, after ~5 min, a strong inward current developed that was accompanied by a rapid increase of [Ca2+]i to >600 nM. Reoxygenation of the glucose-free solution led to complete recovery of both [Ca2+]i and Im. In 9 of 27 DVN, the inward current, which was accompanied by an up to sixfold increase of gm, was larger than 1 nA and [Ca2+]i increased to >1 µM (Fig. 3C). Complete recovery from the ischemic [Ca2+]i rise was critically dependent on immediate reoxygenation after onset of the ischemic current.

Pharmacology of the ischemia response

We next explored the basis for the ischemic depolarization and fast [Ca2+]i rise. A mixture of TTX (0.5 µM) and both CNQX (50 µM) and APV (100 µM) reversibly abolished the ischemia-induced inward current as well as the secondary large [Ca2+]i rise (n = 9; Fig. 4A), whereas TTX plus APV, or TTX plus CNQX only attenuated the ischemic current and Ca2+ rise (not shown). The protection by TTX plus CNQX and APV was also observed when ischemic exposure was prolonged for several minutes compared with control ischemia (Fig. 4A). In >50% of cells, membrane noise increased within 1-2 min before onset and during the initial phase of the ischemic inward current due to increased spontaneous synaptic activity. Such ischemia-induced synaptic activity did not develop on ischemia in the presence of CNQX/APV (not shown, but see Fig. 4, B and C).



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Fig. 4. Suppression of the ischemia current and [Ca2+]i rise by ionotropic glutamate receptor antagonists. A: in contrast to the minor effect on membrane current (Im) and [Ca2+]i in standard saline, anoxia led to a progressive inward current and secondary fast [Ca2+]i elevation ~30 min after glucose removal. The latter responses were blocked on combined application of TTX (0.5 µM), CNQX (50 µM), and APV (100 µM). B: recordings of 4 DVN that were exposed to CN- (1 mM) after 30 min preincubation in glucose-free solution (also mimicking ischemia). In the recordings of cells 3 and 4, CNQX and APV were added to the superfusate 5 min before CN- application. Note that the blockers of ionotropic glutamate receptors abolished the CN--induced inward current as well as the progressive rise of [Ca2+]i. C: current traces of cell 2 in A filtered at 3 kHz instead of 2 Hz as sampled 1 min before (*) and 2 min after (**) start of ischemia show an increase of spontaneous subthreshold synaptic activity.

Quantitative analysis of the effects of glutamate receptor blockers on the ischemia response was hampered by the observation that the onset of the progressive inward current and [Ca2+]i rise varied between 3 and >10 min in different preparations. It turned out that the delay of onset as well as the magnitude of the effects were dependent on both the age of the animal and the time of in vitro storage of the slices. Thus for quantitative pharmacological analysis of the ischemia response, animals of an age between 18 and 20 days were chosen, slices were investigated within 1 to 4 h after isolation, and chemical anoxia was induced by CN-. Under these experimental conditions, 5 min application of 1 mM CN- 30 min after preincubation in glucose-free saline evoked a peak inward current of -1.75 ± 0.27 nA and an average [Ca2+]i rise of 0.912 ± 76 µM after a delay of 2.88 ± 0.12 min (n = 12; Figs. 4B and 5, C and D). In >60% of these recordings, subthreshold synaptic activity increased considerably before development of the fast phase of the ischemic inward current (Fig. 4, B and C). This, as well as the inward current and fast [Ca2+]i rise, were blocked by CNQX and APV (n = 5; Figs. 4, B and C, and 5D). As tested during 5-20 min exposure to CN- in glucose-free solution, either of the drugs alone only attenuated both the inward current and [Ca2+]i rise by <40% (Fig. 5, A and D). CNQX and APV also increased further the time lag (tonset) from the beginning of ischemia until an inward current of -150 pA was reached (Fig. 5, A and C).



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Fig. 5. Pharmacology of the ischemia response. A: after preincubation in glucose-free saline containing 50 µM CNQX, CN- (1 mM) induced an initial modest rise of [Ca2+]i without effect on membrane current. After a delay of ~4.5 min, a progressive inward current was accompanied by a major irreversible [Ca2+]i rise. B: CNQX plus 100 µM APV not only delayed the onset, but also decreased the amplitude of the ischemic inward current and [Ca2+]i transient. tonset: time lag after which the ischemic inward current reached -150 pA. C: CNQX (50 µM), APV (100 µM), methoxyverapamil (D-600, 200 µM) alone as well as combined administration of the drugs increased tonset. Bars represent mean ± SE (n = 5 each). Left bar shows tonset of the CN- response (outward current) in glucose-containing solution (n = 39). D: drug-related reduction of the peak ischemic inward current and [Ca2+]i rise (n = 5 each). Asterisks indicate significance values (* P < 0.05; ** P < 0.01; *** P < 0.001) of drug effects compared with control ischemia.

In addition, the effects of the Ca2+ channel blocker D-600 (200 µM) were tested. D-600 attenuated the ischemic [Ca2+]i increase by ~35% and increased tonset by further 4.8 min, but induced only a minor reduction of the ischemic inward current (Fig. 5D). In contrast, the drug depressed depolarization-induced [Ca2+]i rises by ~90% (n = 6; not shown). In the presence of CNQX plus APV, or CNQX, APV, plus D-600, tonset was increased to 13.3 and 18.0 min, respectively (Fig. 5, B and C). In the presence of these drugs, 20 min of ischemia led to a [Ca2+]i increase that was not significantly larger than during anoxia in glucose-containing solution (Fig. 5D).

Effects of glutamate application

Because the latter results indicated involvement of glutamate in the ischemia response, the effects of exogenous glutamate and glutamate receptor antagonists were tested. Bath application of 0.5 mM glutamate (20 s) led to a rise of [Ca2+]i by 0.30 ± 0.05 µM (n = 16) and an inward current of -0.70 ± 0.06 nA accompanied by a severalfold increase in gm. Glutamate (0.1 mM) evoked an inward current of less than -0.1 nA, a rise of gm by up to 300%, and a [Ca2+]i elevation by ~50 nM. A mean inward current of -1.24 ± 0.2 nA or -1.95 ± 0.1 nA, and a concomitant [Ca2+]i rise of 0.64 ± 0.05 µM or 0.71 ± 0.03 µM were evoked in four DVN in response to 1 and 2 mM glutamate, respectively (not shown). Coapplication of CNQX and APV abolished both the current and the [Ca2+]i transients (Fig. 6, A and B). CNQX alone blocked the glutamate-activated inward current and rise of [Ca2+]i by 80 and 67%, respectively, whereas APV reduced the glutamate-induced current by 40% and the elevation of [Ca2+]i by 50% (Fig. 6B). The blocker of Ca2+-permeable AMPA/KA receptors NHPPS (1 µM) did neither attenuate the inward current nor the [Ca2+]i rise in response to 0.5 mM glutamate in four DVN (Fig. 6B).



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Fig. 6. Glutamate-induced inward currents and Ca2+ increases. A: combined administration of CNQX (50 µM) and APV (100 µM) abolished the response to bath-applied (20 s) glutamate (GLU, 0.5 mM). B: (n = 7) inhibitory effects of CNQX and/or APV as well as of N-(4-hydroxyphenyl-propanolyl)-spermine (NHPPS, 1 µM) on Im and [Ca2+]i (n = 4 for NHPPS). (* P < 0.05; ** P < 0.01; *** P < 0.001). C: changing to voltage clamp considerably reduced the [Ca2+]i rise associated with the depolarization of this cell due to 0.5 mM GLU. D: mean data (n = 6) of the reduction of the GLU-induced [Ca2+]i transient (top panels) in voltage clamp and average depolarization (bottom left panel) and inward current (bottom right panel). E: the [Ca2+]i rise in response to 0.5 mM GLU was diminished on changing holding potential from -50 to -80 mV. F: mean data from recordings as shown in E (n = 3). ** P < 0.01.

The lack of effects of NHPPS suggests that a considerable portion of the [Ca2+]i rise in response to glutamate is caused by imperfect voltage control of (remote) dendritic regions resulting in activation of voltage-activated Ca2+ channels. To test for this possibility, cells were exposed to glutamate under current and voltage clamp. Glutamate (0.5 mM; 20 s) evoked a mean membrane depolarization of 30 ± 0.7 mV (Fig. 6, C and D) that was associated with an average [Ca2+]i increase of 0.5 ± 0.03 µM (n = 6). In these cells, the glutamate-induced [Ca2+]i rise was reduced by ~50% after switching to voltage clamp (Fig. 6, C and D). In individual voltage-clamped cells, the magnitude of the [Ca2+]i transients in response to 0.5 mM glutamate varied considerably (not shown). If insufficient space clamp was responsible for the [Ca2+]i rises in some cells, an attenuated response would be expected at more negative Vh. In contrast, if the Ca2+ transient was due to influx through glutamate receptors, an increased response would be expected because the driving force is increased. Changing Vh from -50 to -80 mV reduced the mean glutamate-induced [Ca2+]i rise by 64%, whereas the accompanying inward current slightly increased (n = 3; Fig. 6, E and F).

Origin of ischemic glutamate release

The facts that 1) the ischemic response of DVN can be blocked by glutamate receptor antagonists and 2) exogenous glutamate mimics the effects of ischemia suggest that ischemia but not anoxia evokes a rise of extracellular glutamate. To investigate whether the glutamate accumulation is caused by Ca2+-dependent vesicular release, slices were exposed to Ca2+-free solution 25 min after removal of glucose. This led to a fall of [Ca2+]i baseline by between 20 and 50 nM (Fig. 7A). Subsequent ischemia evoked the initial moderate rise of [Ca2+]i, whereas the profound [Ca2+]i transient was blocked (Fig. 7A). The Ca2+-free solution doubled tonset from 2.9 min under control (Fig. 5C) to ~6 min (Fig. 7B). Addition of the endoplasmatic reticulum Ca2+ pump blocker CPA (30 µM) to the Ca2+- and glucose-free superfusate further delayed tonset by ~2 min without an effect on the magnitude of the ischemic current (n = 6; Fig. 7, B and D). In contrast to this effect, addition of CPA neither induced spontaneous synaptic activity, nor did it potentiate spontaneous synaptic activity that developed in the initial phase of ischemia (compare Fig. 4, B and C).



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Fig. 7. Origin of the ischemic inward current and [Ca2+]i rise. A: after preincubation in glucose- and Ca2+-free solution CN- (1 mM) evoked an initial moderate rise of [Ca2+]i and an outward current. After ~6 min, a progressive ischemic inward current developed without a major [Ca2+]i increase. B: mean data (n > 3) for tonset (compare with Fig. 5) in control solution and in the absence of extracellular Ca2+. DHK, dihydrokainate; PDC, L-transpyrrolidone-2,4-dicarboxylate; THA, D,L-threo-beta -hydroxyaspartate; AA, arachidonic acid (1 mM each); CPA, cyclopiazonic acid (30 µM); NPPB, 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (0.2 mM). C: both PDC and DHA induced a prominent inward current and [Ca2+]i transient, whereas DHK had only minor effects. D: mean data of the effects of the glutamate transport and Cl- channel blockers (symbols and concentrations as in B) on ischemic inward current and [Ca2+]i rise (n > 3). Asterisks indicate significance values (* P < 0.05; ** P < 0.01; *** P < 0.001) of drug effects.

In another set of experiments, it was studied whether the ischemic glutamate rise is caused by reversed glutamate uptake. Arachidonic acid (200 µM), which inhibits glutamate transporters (Barbour et al. 1989), increased tonset in both Ca2+-containing (4.3 vs. 2.9 min in control) and in Ca2+-free (9 vs. 6 min) solution, but had no significant effect on the peak ischemic current (Fig. 7D). In contrast to arachidonic acid, addition of the glutamate transport blockers THA or PDC (1 mM each) to the standard solution for 1 min evoked an inward current of -1.4 ± 0.3 nA and -1.5 ± 0.2 nA and a [Ca2+]i increase by 1.3 ± 0.4 µM and 1.3 ± 0.1 µM, respectively (n = 5; Fig. 7C). Both the THA-induced inward current and [Ca2+]i rise were reduced by >80% after preincubation with 50 µM CNQX (n = 3; not shown). An inward current of -0.2 ± 0.17 nA and a [Ca2+]i increase of 0.04 ± 0.02 µM were evoked by 1 min exposure to DHK (1 mM), another blocker of glutamate transporters (Fig. 7C). It has been described previously that the transport blockers need to be loaded intracellularly before ischemia (Madl and Burgesser 1993; Roettger and Lipton 1996). Thus slices were incubated for 1 h with THA, PDC, and DHK (1 mM each). CNQX (50 µM) and APV (100 µM) were also added to avoid excessive intracellular Ca2+ accumulation in the presence of THA and PDC. After loading, slices were kept in standard solution for 1 h before use for intracellular recording. There was no apparent difference in the ischemia effects between control and treated slices (compare Fig. 5, C and D, and Fig. 7, B and D). The "cocktail" of glutamate transport blockers was also ineffective in Ca2+-free solution (Fig. 7, B and D). NPPB (200 µM), a blocker of glial volume-sensitive Cl- channels that permit efflux of glutamate (Basarsky et al. 1999), did not change tonset or the magnitude of the ischemic current in Ca2+-free solution (n = 4; Fig. 7, B and D).

Effects of ouabain and iodoacetate

In a final experimental approach, it was tested whether direct block of the Na+/K+ pump or block of glycolysis with iodoacetate (Haddad and Jiang 1993a; Hansen 1985) mimic the effects of ischemia. Ouabain (50 µM), the blocker of the Na+/K+ pump, induced an immediate and stable inward current of between -20 and -100 pA (Fig. 8). After a delay of 6.9 ± 0.5 min (n = 4), a strong inward current (-1.1 ± 0.2 nA) and [Ca2+]i rise (1.6 ± 0.1 µM) developed that were very similar to those seen under ischemia (not shown). Also similar to ischemia, the inward current was preceded by increased synaptic activity (Fig. 8). This as well as the progressive inward current and [Ca2+]i increase were suppressed by preincubation with CNQX plus APV (n = 4). However, within several minutes after washout of CNQX/APV in the presence of ouabain, the inward current and [Ca2+]i rise developed (Fig. 8).



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Fig. 8. Suppression of ischemia-like effects of ouabain by ionotropic glutamate receptor blockers. Block of the Na+/K+ pump with ouabain (50 µM) led to a moderate inward current but had no effect on [Ca2+]i in the presence of CNQX (50 µM) and APV (100 µM). However, in the absence of the blockers the drug induced a progressive inward current and [Ca2+]i increase within several minutes. Current traces of the cell low-pass filtered at 3 kHz instead of 2 Hz and taken at the time indicated by the arrows show that ouabain elicited a prominent CNQX/APV-sensitive increase in spontaneous synaptic activity.

In 12 of 19 DVN, iodoacetate evoked a sustained outward current (0.1 ± 0.04 nA) and a minor rise of [Ca2+]i (45 ± 15 nM) after 5 min. As exemplified for the cell of Fig. 9A that did not show an initial iodoacetate-evoked outward current, a progressive inward current of -1.6 ± 0.2 nA and a concomitant [Ca2+]i rise by 1.3 ± 0.1 µM developed after further 17.5 ± 1.2 min. Addition of CNQX (50 µM) and APV (100 µM) 10 min after start of exposure to iodoacetate prevented both the inward current and rise of [Ca2+]i (n = 5; Fig. 9B).



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Fig. 9. Block of glycolysis has effects similar to ischemia. A: after ~15 min subsequent to bath application of the glycolytic blocker iodoacetate an inward current and a concomitant [Ca2+]i rise developed. B: application of CNQX (50 µM) and APV (100 µM) suppressed the ischemia-like effects of iodoacetate, whereas an inward current and [Ca2+]i rise developed slowly after wash out of the glutamate receptor antagonists.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Anoxia response

As established previously, the anoxic hyperpolarization of DVN is mediated by ATP-sensitive K+ channels (Ballanyi et al. 1996; Karschin et al. 1998; Trapp and Ballanyi 1995). Sulfonylurea-induced block of the underlying anoxic outward current revealed a moderate and sustained inward current (Ballanyi and Kulik 1998; Trapp and Ballanyi 1995). This anoxic inward current was accompanied by a conductance decrease and not an increase as seen on bath application of glutamate. Also spontaneous synaptic activity was not increased under anoxia. Thus it is unlikely that the inward current is caused by elevated levels of interstitial glutamate (see also Haddad and Jiang 1993b). Probably, the inward current is due to the anoxia-induced stable rise of extracellular K+ by 1 mM (Ballanyi et al. 1996). Also the minor stable increase in [Ca2+]i in response to block of aerobic metabolism is not caused by activation of ionotropic glutamate receptors because it was not attenuated by CNQX and APV. Similar observations were made for striatal interneurons (Pisani et al. 1999) and dopaminergic neurons (Guatteo et al. 1998) of brain slices. The finding that CNQX plus APV abolishes the glutamate-induced [Ca2+]i transient indicates that glutamate does not promote Ca2+ release from intracellular stores via metabotropic receptors (Hollmann and Heinemann 1994; Metzger et al. 2000; Schoepp et al. 1990) in DVN. This is substantiated by our observation that [1S,3R]-ACPD does not mimic effects of anoxia (Bickler and Hansen 1994). The anoxia-related [Ca2+]i rise persists in Ca2+-free solution (present study) and is not changed by depletion of CPA-sensitive Ca2+ stores (Kulik and Ballanyi 1998). This suggests that inhibition of Ca2+ uptake into or release from mitochondria (Budd and Nicholls 1996; Schinder et al. 1996) causes the anoxic [Ca2+]i rise in DVN. However, because CPA might not deplete all types of nonmitochondrial Ca2+ stores (Golovina and Blaustein 1997; Meldolesi and Pozzan 1998), drugs such as dantrolene, ryanodine, or caffeine should be used in future studies to address this issue in more detail.

Role of glutamate in the ischemia response

In contrast to anoxia, ischemia leads to a prominent depolarization of DVN, which is very similar to that observed during anoxia alone in cortical neurons (Ben-Ari 1990; Bures and Buresova 1957; Silver and Erecinska 1990). We found that the underlying progressive inward current and conductance increase are abolished by blockers of ionotropic glutamate receptors. This indicates that accumulation of extracellular glutamate (Benveniste et al. 1984; Choi 1988; Roettger and Lipton 1996) is responsible for the ischemia-induced depolarization. Glutamate can accumulate to millimolar concentrations in the synaptic cleft during physiological activity (Clements 1996). Thus the postsynaptic glutamate receptors might be exposed to even higher concentrations during impairment of metabolism, which causes massive release of this neurotransmitter, often associated with impaired glutamate uptake (Hansen 1985; Szatkowski and Attwell 1994). Accordingly, we have shown here that millimolar concentrations of bath-applied glutamate mimic the ischemia-induced inward current, conductance increase, and [Ca2+]i elevation. The ischemic (and the glutamate-induced) inward current and the prominent Ca2+ rise are only blocked by combined application of CNQX and APV. Thus AMPA/KA as well as NMDA receptors appear to contribute to the membrane depolarization of DVN on ischemia as shown for the response to anoxia of cortical neurons (Müller and Somjen 1998; Silver and Erecinska 1990; Tanaka et al. 1997).

The established Ca2+ permeability of the NMDA receptor (MacDermott et al. 1986) makes it plausible that a considerable component of the ischemic [Ca2+]i rise in the DVN is caused by these receptors. However, a major portion of the ischemic elevation of [Ca2+]i persisted during blockade of NMDA receptors. Ca2+-permeable AMPA/KA receptors (Hollmann and Heinemann 1994; Metzger et al. 2000; Pellegrini-Giampietro et al. 1997) do not appear to be responsible for the [Ca2+]i rise remaining after block of NMDA receptors because NHPPS, an inhibitor of such channels (Weigand and Keller 1998), does not affect the glutamate-evoked [Ca2+]i rise. This suggests that insufficient space clamp during AMPA/KA receptor-mediated currents results in activation of voltage-activated Ca2+ channels that promote considerable Ca2+ influx into the DVN (Ballanyi and Kulik 1998). A significant contribution of Ca2+ channels is also indicated by our finding that the glutamate-induced [Ca2+]i transient is greatly reduced under voltage clamp and diminished further at more negative holding potentials (Metzger et al. 2000). Despite lack of direct Ca2+ influx via AMPA/KA receptors, their activation has a major contribution to the ischemia-induced [Ca2+]i rise because the resulting rapid and prominent depolarization opens voltage-activated Ca2+ channels and furthermore removes the Mg2+ block of NMDA receptors (Hollmann and Heinemann 1994). In addition to such channel-mediated Ca2+ influx, inhibition or reversed action of plasmalemmal Ca2+ transporters such as Na+/Ca2+-exchanger or Ca2+/H+-ATPase might constitute a further source of cellular Ca2+ accumulation during ischemia (Blaustein and Lederer 1999; Trapp et al. 1996).

Source of ischemic glutamate release

Removal of extracellular Ca2+ delays the onset of the ischemia-related inward current approximately twofold. This, and also the occurrence of spontaneous CNQX/APV-sensitive EPSCs in the DVN, suggests that the early phase of ischemic glutamate release is caused by Ca2+-dependent exocytosis as also proposed for other neuronal structures (Drejer et al. 1988; Katayama et al. 1991). Ca2+ release from presynaptic stores seems to contribute noticeably to ischemia-induced Ca2+-dependent exocytosis as depletion of Ca2+ stores with CPA before ischemia further delayed the inward current by several minutes in the absence of extracellular Ca2+. A reversed action of glutamate transporters does not appear to play a major role during the early phase of ischemic glutamate release because preincubation in a solution containing DHK, THA, and PDC as uptake blockers (Barbour et al. 1989; Madl and Burgesser 1993; Obrenovitch et al. 1998; Roettger and Lipton 1996) does not delay the inward current. Because the drugs are also ineffective in Ca2+-free saline, glutamate transport apparently has only a minor overall contribution to ischemic glutamate release in the dorsal vagal complex as was also hypothesized for other brain regions (Bickler and Hansen 1994; Drejer et al. 1985; Obrenovitch et al. 1998; O'Reagan et al. 1995). In contrast to DHK, THA, and PDC, arachidonic acid significantly delayed the ischemic inward current in both Ca2+-containing and Ca2+-free solution. Further analysis is required to elucidate whether the protective action of arachidonic acid is due to its inhibitory effect on glutamate transporters (Barbour et al. 1989; Szatkowski and Attwell 1994). As an alternative explanation, arachidonic acid might block vesicular uptake (Roseth et al. 1998), thereby decreasing the compartmentalized presynaptic pool of releasable glutamate. Finally, arachidonic acid might be converted in the slices, for example, into prostaglandins that mediate its protective effect (Barbour et al. 1989). Recently, it was described that glutamate release in response to ischemia (Phillis et al. 1997) or spreading depression (Basarsky et al. 1999) can occur via efflux through anion channels that are activated by cellular swelling secondary to disturbance of ion homeostasis. In the latter reports, in particular NPPB was effective to suppress this source of pathological glutamate release, whereas the drug had no effect in the present study.

Several groups have reported that ischemia-induced glutamate release is associated with a breakdown of membrane function (Bickler and Hansen 1994; Katayama et al. 1991; O'Reagan et al. 1995). One of these studies presented evidence that anoxia- and ischemia-induced Ca2+ influx and ATP loss in neurons of cortical slices coincide with membrane damage (Bickler and Hansen 1994). In glial cells as well as in neuronal somata or presynaptic structures, ischemia-induced membrane damage is likely to result in an increase of extracellular K+ to several tens of millimolar (Ballanyi et al. 1999; Hansen 1985; Völker et al. 1995). However, no major inward current resulting from such putative K+ accumulation (compare Fig. 6 in Trapp and Ballanyi 1995) is revealed during ischemia or block of glycolysis with iodoacetate after preincubation with CNQX and APV. Therefore at least on a time scale of ~30 min, membrane damage does not seem to occur within the dorsal vagal complex. For interpretation of the minor effects of ischemia on the postsynaptic membrane properties and [Ca2+]i of DVN in the presence of CNQX and APV, it needs to be considered that the patch electrodes contained 1 mM ATP. For anoxia, an almost identical membrane response is observed in DVN on whole cell or sharp microelectrode recording (Ballanyi et al. 1996; Cowan and Martin 1992). Furthermore, anoxia produces an almost identical moderate and stable [Ca2+]i rise in intact fura-2 esterloaded and fura-2 dialyzed whole cell recorded DVN (Ballanyi and Kulik 1998). Nevertheless, the effects of long-term ischemia (after block of ionotropic glutamate receptors) on postsynaptic membrane properties and [Ca2+]i in DVN should be studied in detail in future studies in cells that are not dialyzed with ATP.

Metabolism of the dorsal vagal nucleus

No major ischemic inward current as indication of interstitial accumulation of either neuromodulators or K+ develops within >30 min in the DVN during block of ionotropic glutamate receptors. This shows that the cellular elements of the dorsal vagal complex surrounding the recorded postsynaptic neuron are not only extremely tolerant to anoxia, but also to complete metabolic arrest. This can only be explained by a particularly low metabolic rate that is a major constituent of the anoxia tolerance of the brain of cold-blooded vertebrates and perinatal mammals (Ballanyi et al. 1999; Lutz 1992; Völker et al. 1995). A low rate of energy consumption could maintain cellular ATP levels for extended time periods sufficiently high to maintain basic cellular function. Resting Na+/K+ pump activity within the dorsal vagal complex appears to be low because no change in resting current as indication of interstitial K+ accumulation due to perturbance of ion homeostasis was revealed in the presence of CNQX/APV after treating of the slices with ouabain. However, ouabain induced within several minutes interstitial accumulation of glutamate almost identical to that on ischemia. This is consistent with the assumption that depolarization of presynaptic structures due to block of the Na+/K+ pump secondary to a fall of ATP is a major factor of ischemic glutamate release (Haddad and Jiang 1993a; Madl and Burgesser 1993).

The delay of onset (18 min) of the iodoacetate-induced progressive inward current and [Ca2+]i rise of DVN was very similar to that observed for the inward current and perturbation of ion homeostasis of the in vitro respiratory network of newborn rats (Ballanyi et al. 1999; Völker et al. 1995). This supports the above view that the metabolic rate of DVN is similarly low as that of anoxia-tolerant immature brain structures (see also Ballanyi et al. 1996; Ballanyi and Kulik 1998; Trapp and Ballanyi 1995). A low metabolic rate is also suggested by the finding that, in contrast to mature neurons of most brain regions (Calabresi et al. 1997; Haddad and Jiang 1993a,b; Hansen 1985; Takata et al. 1995), glucose-free superfusate neither causes a progressive inward current nor a major rise of [Ca2+]i in the DVN within at least 30 min (see also Knöpfel et al. 1990). However, this tolerance to glucose depletion might also indicate that (glial) glycogen stores allow for sustained glycolytic energy production during anoxia (Tsacopoulos and Magistretti 1996). Stimulation of anaerobic metabolism during anoxia ("Pasteur effect") in combination with a high glycolytic capability of structures involved in homeostasis of glutamate within the dorsal vagal complex is a possible explanation for the lack of a "terminal" glutamate-induced depolarization during oxygen depletion alone (Ballanyi et al. 1999; Lutz 1992; Völker et al. 1995).

Conclusions

The present study investigated the contribution of glutamate release to the response of the medullary dorsal vagal complex to anoxia and ischemia. For this purpose, whole cell recorded DVN in brain stem slices were dialyzed with fura-2, and their glutamate receptors and [Ca2+]i transients were used as potential sensors of extracellular glutamate. We found that glutamate is not involved in the moderate response to anoxia. In contrast, ischemia (as well as iodoacetate and ouabain) promote a profound rise of interstitial glutamate concentration. The ischemic glutamate increase evokes a prominent depolarization and a [Ca2+]i increase caused by Ca2+ influx through both the NMDA receptor and voltage-activated Ca2+ channels. The occurrence of EPSCs and the delay of the ischemic inward current by Ca2+-free superfusate and block of Ca2+ release show that the initial phase of the ischemic glutamate rise is exocytotic. As glutamate transport blockers and an inhibitor of volume-sensitive Cl- channels are not effective, the origin of the late phase of ischemic glutamate release in the dorsal vagal complex remains unclear. The glycolytic blocker iodoacetate produced a similar CNQX/APV-sensitive inward current and [Ca2+]i rise as ischemia. Thus it appears that effective utilization of anaerobic metabolism by structures involved in glutamate homeostasis constitutes an important factor of the anoxia tolerance of dorsal vagal neurons.


    ACKNOWLEDGMENTS

We thank A.-A. Grützner for expert technical assistance, Dr. J. Brockhaus for critical reading of the manuscript, and Dr. B. U. Keller for the generous gift of NHPPS.

This study was supported by the Deutsche Forschungsgemeinschaft and the Hermann und Lilly Schilling-Stiftung.

Present address of S. Trapp: University Laboratory of Physiology, Parks Rd., Oxford OX1 3PT, UK.


    FOOTNOTES

Address for reprint requests: K. Ballanyi, II. Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D-37073 Gottingen, Germany.


E-mail: kb{at}neuro-physiol.med.uni.goettingen.de

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 September 1999; accepted in final form 26 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/00 $5.00 Copyright © 2000 The American Physiological Society