After onset of depolarization, the level of cortical. ATP and phosphocreatine (PCr) decrease rapidly,. Received March 21, 1989; revised June 12, 1989; accepted.
Journal of Cerebral Blood Flow and Metabolism 10:136-139 © 1990 Raven Press, Ltd., New York
Short Communication
Influence of MK-801 on Brain Extracellular Calcium and Potassium Activities in Severe Hypoglycemia
Entan Zhang, Anker Jon Hansen, *Tadeusz Wieloch, and Martin Lauritzen Department of General Physiology and Biophysics, University of Copenhagen, Copenhagen, Denmark and *Laboratory for Experimental Brain Research, University of Lund, Sweden
Summary: The purpose of the present study was to ex amine the effect of blockade of N-methyl-o-aspartate (NMDA) receptors on the depolarization associated with severe hypoglycemia, which is commonly preceded by one or a few transient depolarizations reminiscent of cor tical spreading depression (CSD). In the cerebral cortices of rats [K+le and [Ca2+le were measured with ion selective microelectrodes. NMDA blockade was achieved by injection of MKSOI in doses that block CSD. In control rats, the latency from the time point when blood glucose reached minimal levels to onset of ionic shifts was 33.2 ± 3.5 min, and [K+le rose from 3.2 ± 0.2 to 55 ± 5 mM. All variables remained unchanged in rats
treated with MKS01. In another four rats treated with MKSOI, [Ca2+1e declined from 1.06 ± 0 22 to 0.12 ± 0.02 mM. Plasma glucose measurements indicated that the cortex depolarized at a plasma glucose concentration be tween 0.7 and O.S mM, i.e., within a narrow range, sug gesting a threshold phenomenon. In conclusion, activa tion of NMDA receptors seems of minor importance for hypoglycemic depolarization. The ionic transients that precede the persistent hypoglycemic depolarization are probably mediated by mechanisms distinct from those of electrically induced CSD. Key Words: Cortical spread ing depression-Extracellular ions-Glutamate-Hypo glycemia.
The aim of the present study was to examine the mechanism of change of extracellular ionic activi ties associated with severe hypoglycemia (Astrup and Norberg 1976; Harris et aI., 1984). Previous studies have shown that the tissue concentrations of labile phosphates and energy charge remain con stant immediately preceding the large cortical changes of [K+]e and [Ca2+]e (Harris et aI., 1984). Therefore, the triggering of hypoglycemic depolar ization is not explained simply by a decline of en ergy metabolism. After onset of depolarization, the level of cortical ATP and phosphocreatine (PCr) decrease rapidly,
accompanied by release of neurotransmitters, in cluding -y-aminobutyric acid (GABA), glutamate, and aspartate (Tossman et aI., 1985; Sandberg et aI., 1986). The exact time relation of transmitter release to the onset of ionic changes is unknown, due to the limited temporal resolution of the mi crodialysis technique. Excitatory amino acids such as aspartate and glu tamate trigger cortical spreading depression (CSD) (Leao, 1944; Van Harreveld, 1959; Curtis and Wat kins, 1963; Bures et aI., 1974; Lauritzen et aI., 1988). This fact is of interest since it has been sug gested that the initial depolarization associated with severe hypoglycemia is a CSD (Harris et aI., 1984). The question now being asked was whether hypo glycemic depolarization could be inhibited by the N-methyl-D-aspartate (NMDA) antagonist MK801, since this compound reliably blocks CSD (Hansen et aI., 1988). To elucidate this problem [Ca2+]e, [K +]e, and the extracellular DC potential were monitored during severe hypoglycemia in rats treated with MK801.
.
Received March 2 1, 1989; revised June 12, 1989; accepted June 14, 1989. Address correspondence and reprint requests to Dr. M. Lau ritzen at Department of General Physiology and Biophysics, Pa num Institute, Blegdamsvej 3c, 2200-DK, Copenhagen, Den mark. Abbreviations used: CSD, cortical s preading depres s ion; GABA, "f-aminobutyric acid; NMDA, N-methyl-D-aspartate; PCr, phosphocreatine.
136
GLUTAMATE RECEPTORS AND HYPOGLYCEMIA
MATERIALS AND METHODS Male Wistar rats of about 300 g were starved overnight before the experiment but allowed access to water. Insu lin (Insulin N ovo Actrapid, N OVO Industries A/S, Copenhagen, Denmark) was given intraperitoneally in a dose of 40 IU/kg just prior to surgery in order to induce hypoglycemia. Anesthesia was induced by intraperitoneal injection of mebumal at 50 mg/kg with supplementary doses of 10--25 mg/kg every hour as required. Polyethyl ene catheters were placed in a femoral artery and in a femoral vein. Rats were placed in a headholder and the cranial bones removed over the parietal cortex on one side. Double-barrelled ion-selective microelectrodes were used to measure [K+lc' and in four experiments [Ca2+le. The microelectrodes were lowered by a motor driven micromanipulator into the parietal cortex to a depth of approximately 500 !-Lm. The construction and calibration of these electrodes have been described in de tail (Hansen and Zeuthen, 1981). Rats were relaxed with 5-15 mg/kg of suxamethonium intraperitoneally and ven tilated by a volume respirator with 02-enriched atmo spheric air. Rectal temperature was adjusted to 37°C by a servo-controlled heating lamp. Blood sugar was monitored by frequent sampling di rectly on Glucostix reagent strips and a reflectometer sys tem (Ames Co., Elkhart, IN, U.S.A.). Latency from in jection of insulin to glucose declined below the detection limit of the Glucostix test was 151 ± 7 min. At this time point MK801 (a generous gift of Merck, Sharpe and Dohme, Harlow, UK) was given intraperitoneally at 3-lO mg/kg in 10 rats. At this dose CSD is completely blocked in this preparation for hours (Hansen et aI., 1988). Eight rats received no drug treatment and served as controls. In five of the control rats 1 ml of 10% glucose was injected intravenously after the hypoglycemic depolariza tion had lasted for a few minutes causing the ionic changes to return to normal. A few minutes later, when blood glucose once more became undetectable by the Glucostix test, MK801 was injected as noted above, and the variables of hypoglycemic depolarization were re corded. In this way the animals served as their own con trols. The effect of MK801 was evaluated by the latency to onset of ionic shifts from the time at which blood glucose was below the detection limit of the Glucostix test; by the amplitude of the ionic shifts; and by the waveform of ionic changes as compared with control animals not re ceiving any drug treatment. In nine of the rats a blood sample was taken for deter mination of plasma glucose by the hexokinase method before and/or after depolarization in order to elucidate the correlation between ionic activities and plasma glucose concentration. Student's t test was used for statistics. Results were accepted as significant at p < 0.05. Values in text and tables are mean ± SEM.
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TABLE 1. Physiological variables of animals exposed to insulin-induced severe hypoglycemia (mean ± SEM)
-------
MABP (mm Hg) Pac02 (mm Hg) Pao2 (mm Hg) pHa
78 41 207 7. 33
=
------
± ± ± ±
3 1 32 0. 01
±
5
±
2 30 0.02
± ±
=
TABLE 2. Characteristics of hypoglycemic depolarizations in control rats and in rats treated with MK801 Untreated
MK801-treated
rats
3.2 55
Baseline [K + Ie
Maximal [K +
(mM) Ie (mM)
during depolarization
MABP and arterial Pao , Paco , and pH were 2 2 measured in all experiments. Values are given in Table 1. MABP usually dropped from 100-120 mm Hg to 70-100 mm Hg during severe hypoglycemia.
82 37 282 7.34
No additional MABP reduction was observed after injection of MK801. Table 2 gives experimental results for [K+]e in untreated rats (n 8). The latency from blood glu cose declined below the detection limit of the Glu costix test to onset of ionic changes (transient or persistent) was 33.2 ± 3.5 min. Baseline [K + ] e was 3.2 ± 0.2 mM, while maximal [K + ] e was 55 ± 5 mM. The median number of transient changes of the extracellular ionic activities was 0 (range 0 to 3). The effect of MK801 was studied in 10 rats that had not experienced any prior episode of hypogly cemia. [K + ] e was measured in six rats. MK801 did not significantly influence baseline [K+]e, latency to onset of depolarization, maximal increase of [K+]e' or number of ionic transients preceding the persistent hypoglycemic depolarization (Table 2). In four rats [Ca2+]e was measured instead of [K+]e. [Ca2+]e declined from 1.06 ± 0.22 mM to 0.12 ± 0.02 mM during cortical depolarization. This finding is similar to previous measurements with ion selective microelectrodes in the rat parietal cortex for untreated animals during severe insulin-induced hypoglycemia (Harris et al., 1984). Five rats were exposed to hypoglycemic depolar ization twice: before drug treatment and again after transient normalization of blood glucose to normal and injection of MK801 when blood glucose was below the Glucostix detection limit. Figure 1 illus trates a typical experiment in which, under un treated conditions, the cortex underwent a monophasic, persistent depolarization accompa nied by a substantial increase of [K + ] e' Mter intra-
(n
RESULTS
MK801-treated rats (n = 10)
Untreated rats (n 8)
33.2
Latency from Glucostix below detection limit to depolarization (min)
o
Median no. of transient a
rats 8)
(n
± 0. 2
3.3 57
=
±
5
±
3.5
(range 0 to
33.9 3)
=
6)
± 0.4 ±
3
±
2.4Q
1 (range 0 to
5)Q
Includes animals in which [Ca2+le was measured.
J
Cereb Blood Flow Metab, Vol. 10, No.1, 1990
138
E. ZHANG ET AL. HYPOGL YCEMIA
K
+
in Cerebral Cortex
[K'] mM
60 30 -
glc.
l MKBOI ..
5 2 -
5min [K+le measured with ion-selective microelectrodes in rat parietal cortex during severe insulin-induced hypoglycemia. Approximately 30 min after blood glucose (glc.) was below the Glucostix detection limit «1.0 mM), the cortex underwent a monophasic depolarization associated with pronounced increases of [K+le. Subsequently, the rat was given 1 ml 10% glucose intravenously immediately followed by return to normal [K+le. When blood glucose concentration decreased below the detection limit again, MKB01 (3 mg/kg) was given intraperitoneally. Twenty minutes later the cortex underwent a persistent FIG. 1. Changes in
depolarization preceded by a short-lasting potassium transient resembling a cortical spreading depression.
venous injection of 1 ml of 10% glucose, [K +]e re turned to normal levels. Shortly thereafter blood glucose once more declined below the Glucostix de tection limit, and MK801 was injected. Twenty min utes later the cortex again depolarized, but this time the persistent cortical depolarization was preceded by a potassium transient reminiscent of CSD. The latency to depolarization for each of the five ani mals was (without/with MK801, in minutes) 19/25, 26/55, 55/22, 15/17, and 23/20. The maximal increase of [K +]e was (in mM) 55/55, 50/60, 65/90, 32/65, and 65/65. Thus, the paired data also suggested absence of effect of MK801 on hypoglycemic depolariza tion. In three rats MK801 at 10 mg/kg was injected after complete depolarization, but the cortex re mained depolarized. The plasma glucose concentration was measured by the hexokinase method before and/or after onset of ionic shifts in nine rats of which four were treated with MK801. The data from treated and untreated animals were lumped since MK801 had no effect on hypoglycemic depolarization. Figure 2 shows the correlation between [K +]e and plasma glucose. The major ionic shifts occurred within a rather narrow range, at plasma glucose concentrations between 0.7 and 0.8 mM.
the observation that the ionic transients preceding the persistent hypoglycemic depolarization were of similar amplitude and duration to those seen during CSD. Furthermore, the ionic changes occurred at different latencies at two sites within the same hemisphere from the time point at which blood glu cose reached minimal levels. This finding was taken to suggest that an episode of CSD triggered the per sistent hypoglycemic depolarization. It has been demonstrated that CSD is completely blocked by competitive and noncompetitive NMDA antagonists (Van Harreveld, 1984; Mody et aI., 1987; Goroleva et aI., 1987; Hansen et aI., 1988; Lauritzen et al., 1988; Marrannes et al., 1988). Therefore, if hypoglycemic depolarization and CSD were triggered by the same mechanism, we would expect some degree of blockage of the persistent 80
0 0
60
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+ �
o
0 20 0 0
J Cereb Blood Flow Metab, Vol. 10, No.1, 1990
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DISCUSSION It has been suggested that the initial depolariza tion during hypoglycemia was a CSD-like episode leading to widespread depolarization of the cortex, triggering the fast depletion of energy stores and degradation of phospholipids (Harris et aI., 1984; Wieloch et aI., 1984). This hypothesis was based on
CO
40
3
PLASMA GLUCOSE (mM) FIG. 2. Changes in
[K+le in rat parietal cortex during severe
insulin-induced hypoglycemia as function of plasma glucose concentration. Plasma glucose was measured on 13 occa sions in nine rats during cortical depolarization and/or dur ing control conditions. Cortical depolarization occurred within a narrow range of plasma glucose values, ranging from about 0.7 to O.B mM, suggesting a threshold phenome non.
GLUTAMATE RECEPTORS AND HYPOGLYCEMIA depolarization in severe hypoglycemia in response to NMDA antagonism. MK801 was entirely ineffec tive as a blocker of hypoglycemic depolarization. Thus, it is unlikely that NMDA receptor activation is of principal importance for the onset of ionic shifts in severe hypoglycemia. The triggering of hypoglycemic depolarization obviously depends on limitation of the energy sup ply to the brain, but the exact mechanism remains elusive. Up to the first ionic transient, energy me tabolites and phospholipids remain essentially nor mal (Harris et aI., 1984; Wieloch et aI., 1984), but thereafter ATP decreases rapidly. This finding is in contrast to CSD, which is associated with only mar ginally affected energy stores (Lauritzen et aI., in press). The release of glutamate (and possibly other) neurotransmitters at a circumscribed site is believed to activate NMDA receptors and initiate the wave of depolarization. The events initiating hy poglycemic depolarization could also be associated with transmitter release (Sandberg et aI., 1986), but the rapid energy depletion and activation of the phosphoinositol cycle (Harris et aI., 1984; Wieloch et aI., 1984) may lead to activation of ion channels or translocators unaffected by NMDA antagonists. In conclusion, the lack of effect of NMDA recep tor blockade on hypoglycemic depolarization sug gests that the ionic shifts associated with severe hypoglycemia are mediated by mechanisms distinct from CSD. More likely, hypoglycemic depolariza tion is triggered by mechanisms dependent on the available substrate supply such as second messen ger-operated ion channels or translocator-coupled ATPases Acknowledgment: This work was supported by the Uni versity of Copenhagen, the Medical Research Council (Denmark), the Carlsberg Foundation, the Foundation for Experimental N eurological Research, Fonden af 1870, and the Danish Migraine Society. T.W. was supported by grants from the Medical Research Council (Sweden) (grant no. 4x-08644) and the U.S. Public Health Service (l ROI N S-25302).
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Bures J, Buresova 0, Krivanek J (1974) The Mechanism and Applications of Leao's Spreading Depression of Electroen cephalographic Activity. New York, Academic Press, pp 1-410.
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Hansen AJ, Lauritzen M, Wieloch T (1988) NMDA antagonists block cortical spreading depression but not anoxic depolar ization. In: Frontiers in Excitatory Amino Acid Research (Cavalheiro EA, Lehmann J, Turski L. eds ) , New York, Alan R. Liss, pp 661-666 Harris RJ, Wieloch T, Symon L, Siesj � BK (1984) Cerebral ex tracellular calcium activity in severe hypoglycemia: relation to extracellular potassium activity and energy state. J Cereb Blood Flow Metab 4:187-193
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