Brain Research 884 2000 155–162 www.elsevier.com locate bres
Research report
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Inhibition of Na H exchange by 5-N-ethyl-N-isopropyl-amiloride
reduces free fatty acid efflux from the ischemic reperfused rat cerebral cortex
a , a
b
John W. Phillis , J. Ren , M.H. O’Regan
a
Department of Physiology , Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201-1928, USA
b
Biomedical Sciences , School of Dentistry, University of Detroit Mercy, 8200 W. Outer Drive, Detroit, MI 48219, USA
Accepted 29 August 2000
Abstract
Brain tissue acidosis is considered to be a contributor to ischemic brain injury. The deleterious effects of marked acidosis may be
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associated with reperfusion and an excessive entry of Na into cerebral neurons and glia as intracellular pH is restored by Na H
exchange. Normalization of pH, with activation of many calcium-dependent and other phospholipases and proteases with pH optima in the neutral or alkaline range, could account for the pronounced elevation in extracellular levels of free fatty acids which occurs during
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reperfusion following cerebral ischemia. In the present investigation we evaluated the effects of inhibition of Na H exchange with
N-N-ethyl-N-isopropyl-amiloride EIPA; 25 mM applied topically onto the rat cerebral cortex prior to and during ischemia. Free fatty acid levels in cortical superfusates, withdrawn at 10-min intervals from bilateral cortical windows, were analyzed by high pressure liquid
chromatography. EIPA application effectively inhibited the increases in arachidonic and linoleic acid release observed in the control rats during reperfusion, and non-significantly depressed that of palmitic and oleic acids. Superfusate levels of glucose, which decline to near
zero levels during ischemia and then rebound during reperfusion, were not affected by EIPA administration. Lactate levels in cortical superfusates from EIPA-treated animals rose more rapidly during reperfusion than did those in the control rats and then significantly
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declined towards basal levels. The data indicate that inhibition of Na H exchange prevented the activation of phospholipases that
usually occurs during reperfusion following a cerebral ischemic episode. These results are the first demonstration of such an effect and may provide an explanation for the cerebroprotective effects that have been observed in stroked animals following administration of
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Na H exchange inhibitors.
2000 Elsevier Science B.V. All rights reserved.
Theme : Disorders of the nervous system
Topic : Ischemia; Neurotoxicity
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Keywords : Cerebral ischemia; Cerebroprotection; Free fatty acid; Phospholipase; Na H
exchange; Amiloride derivative; N-N-ethyl-N-isopropyl- amiloride
1. Introduction protons released in the hydrolysis of ATP and those
resulting from bioenergetic failure with the production of
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The contributions of acidosis to ischemic brain damage lactate and H
[6,9,13,20]. The acidotic hypothesis of have been controversial. Although severe acidosis has been
ischemic injury initially received strong support from the linked to aggravation of ischemic injury, there is also
demonstration that acidosis, equivalent to that occurring evidence that milder acidosis can have cerebroprotective
during ischemia, could damage brain tissue [19]. Explana- effects. Contributors to ischemic acidosis include the
tions to account for the injury include dysfunction of enzymes operating at a pH removed from their pK values
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[16]; cellular swelling due to Na H counter transport
Corresponding author. Tel.: 11-313-577-6745; fax: 11-313-577-
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[7,18] and Ca accumulation due to the Na Ca
5494. E-mail address
: jphillismed.wayne.edu J.W. Phillis.
exchanger operating in a reversed mode [17,22]. ATP
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 3 8 - 3
156 J
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depletion can also lead to an increase in intracellular Ca Penthrane. Body temperature was controlled at 378C with
with activation of calcium-dependent enzymes, including a rectal probe and an abdominal heating pad. One femoral
phospholipases and proteases. Endogenous inhibitors and artery was cannulated for measurement of arterial blood
activators of proteases, which are phosphoproteins, are pressure and to obtain arterial blood samples for pH and
present in cells and a change in their phosphorylation blood gas measurements. Cerebral ischemia was induced
status during ATP depletion could enhance the activity of by coagulating the vertebral arteries via the alar foramina
nonlysosomal proteases. and traction on loops placed around the carotid arteries.
A challenge to the acidotic hypothesis of ischemic injury The dorsal surfaces of both cerebral hemispheres were
arose from the observation that at the pH typically exposed and, after reflection of the dura mater, oval
generated in ischemic brain, NMDA receptor-mediated cortical windows suspended in flexible mounting brackets
currents were almost entirely abolished [38,39]. In that were gently placed on both cortices. The dorsal surface of
NMDA-activated receptors are considered to be amongst the head around the windows was covered with a stabiliz-
the primary mediators of excitotoxic ischemic injury [8], ing gel of 3 agar in artificial cerebrospinal fluid aCSF.
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these findings suggested that acidosis could actually limit The aCSF contained: Na , 155.8 mEq l; K , 2.95 mEq l;
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the extent of excitotoxic injury. Mild acidosis was sub- Ca
, 2.5 mEq l; Mg , 1.85 mEq l; Cl , 141.13 mEq l;
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sequently shown to protect hippocampal and cortical HCO , 22 mEq l; urea, 40.2 mg dl which had been
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cultures from ischemic-like conditions [12,38] and cortical equilibrated with 95 nitrogen, 5 carbon dioxide pH
and cerebellar cultures against glutamate toxicity [2,15]. 7.3. Monopolar EEG electrodes were placed on the
Furthermore, brain acidosis induced by hypercarbic venti- cortical surface within each window. EEGs and arterial
lation attenuated focal ischemic injury in vivo [36] and blood pressure were recorded on a Grass polygraph. Two
hypoxic-ischemic damage to the immature rat brain [42]. hundred ml of warmed 378C aCSF were pipetted into
Sapolsky et al. [35] subsequently referred to the potential each window at 10-min intervals, after removal of the
for acidosis to be neuroprotective under certain circum- previous superfusate sample with Pasteur pipettes. The
stances as a ‘paradoxical wrinkle’. temperature of fluids within the windows was maintained
The term ‘pH paradox’ was used by Bond et al. [4] to at 378C using a heat lamp, and the windows were then
account for the protective action of intracellular acidosis closed with black plastic covers to protect the superfusate
on cultured cardiac myocytes exposed to simulated is- contents from light degradation.
chemia, proposing instead that the injury actually occurs Cerebral ischemia was elicited by occluding the carotid
during the return to normal pH. These investigators arteries for 20 min. Induction of cerebral ischemia was
suggest that although favorable conditions for the activa- manifested by the rapid development of an isoelectric EEG
tion of degradative enzymes such as phospholipases and trace from both cortices. After 20 min the carotid snares
proteases exist during ischemia, they are inhibited by the were withdrawn and reperfusion verified by both the initial
acidotic conditions [5,10]. During reperfusion, the inhibi- sharp decline in arterial blood pressure and visual inspec-
tion is released as intracellular pH recovers, with damage tion of the pial vasculature within the windows. Most
to mitochondrial and plasma membranes initiating necrotic animals ceased to respire spontaneously at some point
or apoptotic processes [21]. Delays in the recovery of during the period of ischemia and were ventilated me-
intracellular pH can be instigated in myocytes using chanically.
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inhibitors of
Na H exchangers,
including di-
Results from three groups of rats are presented in this methylamiloride and HOE 694 [21] or, in the case of
report. One group n59 comprised the control ischemic neuronal tissue cultures, dimethylamiloride or harmaline
animals in which the superfusates were aCSF10.05 [44]. In both instances, powerful protective effects of
dimethylsulfoxide vehicle for EIPA. The cerebral cortices maintaining intracellular acidosis were observed.
of the second group n56 were exposed to EIPA 25 mM The present experiments were designed to evaluate
in 0.05 dimethylsulfoxide aCSF for 15 min after the
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whether the potent and selective inhibitor of Na H completion of superfusate collection 2, and 35 min prior to
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exchange, 5-N-ethyl-N-isopropyl-amiloride EIPA [43], the onset of ischemia. The Na H
exchange inhibitor would block phospholipase activation, measured by the
was then present in all subsequent superfusate samples. efflux of free fatty acids FFAs, in the ischemic re-
For both groups, a standard protocol of superfusate sample perfused rat cerebral cortex as a consequence of its
collections was followed. After two basal 10 min aCSF stabilizing action on the acidotic intracellular pH.
collections the animals were exposed to either aCSF or aCSF plus EIPA for 15 min during which the window
contents were replaced three times. Two more pre-is-
2. Materials and methods chemic samples were then collected, followed by two
