Metabolic Brain Disease, [Iol. 2, No. 2, 1987

Brain Cellular and Mitoehondrial Respiration in Media of Altered pH D. Holtzman, ~'3 J. E. Olson, ~ H. Nguyen, ~ J. Hsu, ~ and N. Lewiston 2 Received August 10, 1986; accepted February 20, 1987

This study was designed to investigate the effects of altered pH on cellular aerobic energy metabolism in the immature and adult rat cerebral cortex. Cerebral cortical slice respiration was measured polarographically in acid and alkaline media. In separate experiments, the extracellular pH was changed by altering the HCO;- concentration or the intracellular pH and extracellular pH were changed by altering the CO 2. Respiratory rates and oxidative phosphorylation in adult rat cerebral mitochindria also were measured in media with an altered pH. Increased intracellular pH inhibited respiratory rates in cortical slices from immature rats more than in tissue from adults. Decreasing the pH to 6.7 produced no changes in respiration in mature cortical slices and moderate inhibition of immature tissue respiration. In cerebral mitochondria, altered pH caused inhibition of State 3 respiration, respiratory control ratios, and ADP/O ratios. These changes were greater and occurred with smaller pH changes in the alkaline compared to the acid direction. From the results of these studies, we conclude that brain cellular respiration is not affected by moderate decreases in intracellular pH. With increased pH, there is inhibition of cellular and mitochondrial respiration, which may be the mechanism for the rise in lactic acid previously observed to result from hypocarbia in vivo. KEY WORDS: brain; cell respiration; hypocarbia; hypercarbia; pH.

INTRODUCTION

Ischemic cerebral cell damage is increased if there is a coincident hyperglycemia or if the interruption of blood supply is not complete (Pulsinelli et al., 1982; Plum, 1983; Meyer et aI., 1986; von Hanwehr et al., 1986). Recent studies have suggested that Abbreviations used: BSA, bovine serum albumin; DNP, dinitrophenol; RCR, respiratory control ratio. Department of Psychiatry and Neurology, Tulane University School of Medicine, New Orleans, Louisiana 70112. 2Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305. 3To whom correspondence should be addressed at Francis Bitter National Magnet Laboratory, Massachusetts Institute of Technology, 170 Albany Street, Cambridge, Massachusetts 02139. 127 0885-7490/'87/0600-0127505~00/0 9 1987 Plenum Publishing Corporation

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abnormal mitochondrial oxidative phosphorylation, secondary to tissue acidosis, is important in the pathogenesis of this enhanced cell damage (Hillered et al., 1984a, b). Ischemia plus hypoglycemia results in a reversible inhibition of brain mitochondrial respiration and oxidative phosphorylation (Hillered et al., 1984b). These metabolic changes are irreversible with the more exaggerated lactic acidosis of normoglycemic ischemia or partial ischemia (Rehncrona et al., 1979; Hillered et al., 1984b). Isolated brain mitochondria, like mitochondria from other tissues, show inhibition of State 3 respiration and oxidative phosphorylation in media of decreased pH (Hillered et al., 1984a). Alkaline pH changes, produced by hypocarbia, also may alter cellular aerobic energy metabolism in the brain (Siesjo, 1972, 1978). In mature and immature animals and in humans, hypocarbia results in an increase in cerebral lactic acid production (Alexander et al., 1968; Young and Yagel, 1984; Petroff et al., 1985). While the mechanism of this increase in lactate production is not known, it is proposed to be a physiologically important component of the buffer capacity of the brain in response to the alkaline pH changes (Kjallquist et at., 1969; Siesjo, 1972). A mechanism for increased anaerobic glycolysis relative to aerobic glycosis during alkalosis is suggested in studies of oxidative phosphorylation in heart, liver, and kidney mitochondria (Chance and Conrad, 1959; Tobin et al., 1972; Fry et al., 1980). In mitochondria from each of these tissues, respiratory rates, respiratory control, and oxidative phosphorylation are inhibited to a greater degree and with smaller changes in pH in the alkaline compared to the acid direction. The present study was designed to investigate further the effects of altered pH on brain cellular aerobic energy metabolism in order to understand mechanisms of increased ischemic brain cell damage with acidosis and of increased lactate production with alkalosis. Respiration was measured in acid and alkaline media in cerebral cortical slices from 5-day-old rat pups and from adult animals. The pH changes were effected by changing the HCO3 concentration to alter primarily the extracellular pH or by changing the CO 2 to alter both the extracellular and the intracellular pH. In parallel studies, respiratory rates, respiratory control ratios, and oxidative phosphorylation were measured in cerebral mitochondria from adult animals under conditions of altered pH. From the results of these studies, we conclude that brain cellular respiration is not affected by moderate decreases in intracellular pH. With moderate increases in pH, there is inhibition of cellular and mitochondrial respiration, which may be important in the increased lactic acid production resulting from hypocarbia in vivo.

MATERIALS AND M E T H O D S

Materials. Mannitol, sucrose, EDTA, Trizma (Tris)-HC1, Tris-PO4, ADP, defatted bovine serum albumin (BSA), dinitrophenol (DNP), oligomycin, and metabolic substrates were obtained from Sigma Chemical Co (St. Louis, MO). Bacillus proteinase came from Nagase and Co, Ltd. (Osaka, Japan). All inorganic chemicals and solvents were reagent grade.

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129

Tissue Preparation. Sprague~Dawley albino rats were used in all studies. For experiments with pups, pregnant females were obtained. Litters were reduced to eight pups on the day of birth. Animals were maintained with one litter per cage at an ambient temperature of 22 23~ For preparation of cerebral cortical slices, the animals were sacrificed by decapitation and the brains were removed immediately. Ten slices, each 300/~m thick, were taken from the frontal lobe using a tissue slicer. After teasing away white matter, the slices were cut into smaller pieces by hand. These pieces were suspended in about 2.0ml of a well-aerated medium at 37~ containing 137mM NaC1, 2.7raM KC1, 1.0raM CaC12, and 0.5 m M MgC12 with either 6.5, 13, 26, or 52 m M NaHCO3. The pH of this medium was adjusted by bubbling with 100% CO2. Cerebral cortical slices from one brain were divided between the control medium at pH 7.3 and one of the experimental media at another pH. Tissue suspensions were capped and the pH was measured again after the incubation to be sure CO2 was not lost. Cerebral mitochondria were isolated as previously described (Holtzman et al., 1978). Brains were removed immediately after sacrifice and the cerebrum was isolated by sectioning through the diencephalon. All subsequent isolation steps were carried out at 3-5~ in a medium containing 225 m M mannitol, 75 m M sucrose, 0.2 m M EDTA, and 5 m M Tris-HC1 (pH 7.4). The white matter and basal ganglia were partially removed before the remaining tissue was homogenized with two strokes in a glass homogenizer. Bacillus proteinase (0.1 mg/g brain tissue), BSA (0.05 rag/g), and KHCO3 (0.1 rag/g) were added and homogenization was completed with three additional strokes. After standing for 2-3 min, the mitochondria were collected by differential centrifugation. The white fluffy layer, consisting mostly of myelin overlying the final mitochondrial pellet, was removed by hand and the pellet resuspended in the isolation medium withouth EDTA to a final concentration of 5-10 mg protein/ml. Polarographic Studies. Respiration in cortical slices was studied at 37~ using a Gilson oxygraph with a 2-ml reaction chamber and a Clarke platinum electrode assembly polarized to - 0 . 8 V. The system was calibrated for 02 concentration as previously described (Holtzman and Moore, 1971). The slices were kept in suspension by a rotating magnet on the chamber floor. Glucose was added to a final concentration of 50 mM. In most experiments, the respiratory rate was constant within a few minutes. If the rate was not constant within 10min, the respiration data were discarded. The initial stable respiratory rate is termed the basal rate. After the basal rate was established, DNP, an uncoupler of oxidative phosphorylation, was added in small quantities (50nmol in 1.0/~1 EtOH) until the maximal DNP-stimulated rate was reached. In a second set of experiments, oligomycin, an inhibitor of respiration coupled to oxidative phosphorylation, was added after the basal rate was established. Oligomycin was added in small quantities (25 nmol in 1.0#1 EtOH) until maximal inhibition of respiration was reached. This rate is termed the oligomycin-insensitive respiration. After each experiment, the chamber contents were removed, the pH was measured, and the suspension was frozen for later measurement of protein quantity (Lowry et al., 1951). Mitochondrial 02 consumption also was measured in the Gilson oxygraph at 22-23~ Freshly isolated mitochondria (1.0-2.0mg protein) were added to the well-aerated respiration medium containing 225 m M mannitol, 75 m M sucrose, 5 m M

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KC1, 0 . 2 m M E D T A , 1 0 m M Tris-PO4, 5 m M Tris-HC1, a n d 0 . 1 2 5 m g / m l fresh bovine serum a l b u m i n (BSA). The p H was a d j u s t e d between 5.4 a n d 9.0 with HC1 or N a O H before a d d i n g the m i t o c h o n d r i a . A l i q u o t s o f m i t o c h o n d r i a f r o m the same isolate were studied at the same time in an e x p e r i m e n t a l m e d i u m a n d in the s t a n d a r d m e d i u m o f p H 7.4. R e s p i r a t o r y rates were m e a s u r e d either with the N A D - l i n k e d substrate pair, g l u t a m a t e plus m a l a t e (1.25 m M each), o r with succinate (10 r a M ) in the presence o f r o t e n o n e (5 #g/ml), an i n h i b i t o r o f N A D - l i n k e d s u b s t r a t e o x i d a t i o n (Slater, 1967). The substrates, dissolved in the r e s p i r a t i o n m e d i u m at the a p p r o p r i a t e p H , were a d d e d at a c o n c e n t r a t i o n o f 125 m M . W i t h each substrate, 02 c o n s u m p t i o n was m e a s u r e d in the presence o f a limiting q u a n t i t y o f A D P (State 3 respiration) a n d after c o n s u m p t i o n o f the a d d e d A D P (State 4 respiration). The r e s p i r a t o r y c o n t r o l ratio ( R C R ) is the r a t i o o f State 3 to State 4 rates. A D P / O ratios were c a l c u l a t e d as the ratio o f A D P a d d e d to the q u a n t i t y o f 02 c o n s u m e d d u r i n g State 3. A f t e r the c o m p l e t i o n o f each experiment, the p H o f the m i t o c h o n d r i a l suspension was m e a s u r e d a n d the suspension then was frozen for p r o t e i n m e a s u r e m e n t ( L o w r y et al., 1951). In b o t h the cerebral slice a n d the m i t o c h o n d r i a l studies, the p H values at the c o m p l e t i o n o f the e x p e r i m e n t did n o t v a r y m o r e t h a n 0.05 p H unit f r o m that o f the original r e s p i r a t i o n m e d i u m . Statistical significance for the differences between e x p e r i m e n t a l a n d c o n t r o l m i t o c h o n d r i a l or tissue samples was d e t e r m i n e d by S t u d e n t ' s t test for n o n i n d e p e n dent samples. Table I. Respiratory Rates in Cerebral Cortical Slices from 5-Day-Old and Adult Rats Measured in Media of Varying HCO 3 Concentrations with Fixed pCO2a Respiratory rate (natom O2/mg protein min)

5 days old

pH

Basal

6.7

9.7 +_ 0.7 (12) 9.9 • 0.7 (12) 9.7 + 0.6 (12) 9.8 -b 0.6 (12) 23.4 ! 1.4 (16) 23.8 • 1.4 (15) 23.2 • 1.7 (15) 22.3 • 1.4 (14)

7.0 7.3 7.6 Adult

6.7 7.0 %3 7.6

Oligomycin insensitive

DNP stimulated

6.1 _+ 0.4 (6) 5.3 + 0.5 (6) 5.1 _+ 0.5 (6) 4.2 i 0.6 (6) 19.4 + 1.2 (6) 18.4 -t- 1.2 (6) 16.7 i 1.6 (6) 12.3 • 0.8 (6) (P < 0.02)

10.2 • 1.0 (6) ll.l • 0.8 (6) 11.4 + 1.4 (6) 11.5 _+ 1.0 (6) 34.9 • 3.3 (10) 39.6 + 4.5 (9) 36.1 _+ 2.0 (9) 34.2 • 2.3 (8)

"Respiratory rates are defined in Materials and Methods. Each value is the mean ! SE of the number of experiments shown in parentheses. Significance values are shown comparing rates in media of altered pH to rates measured at pH 7.3 in cortical slices from the same animal.

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RESULTS

Cerebral Cortical Slice Respiration. The effects o f c h a n g i n g the p H b y v a r y i n g the H C O 3 c o n c e n t r a t i o n are shown in T a b l e I. The m a t u r a t i o n a l increase in all r e s p i r a t o r y rates at p H 7.3 was o b s e r v e d as p r e v i o u s l y described ( H o l t z m a n et al., 1982). W i t h a decrease in p H f r o m 7.3 to 6.7, there were no changes in b a s a l or D N P - s t i m u l a t e d r e s p i r a t o r y rates in i m m a t u r e o r a d u l t cortical tissue. W i t h an increase in p H f r o m 7.3 to 7.6, the oligomycin-insensitive r e s p i r a t o r y rate was signific a n t l y inhibited in the a d u l t tissue b u t n o t in the i m m a t u r e tissue. T h e effects o f c h a n g i n g the p H by c h a n g i n g the pCO2 are shown in T a b l e II. D e c r e a s i n g the p H f r o m 7.3 to 6.7 resulted in no changes in r e s p i r a t o r y rates in m a t u r e cerebral cortical tissue. In i m m a t u r e cortex, a decrease in p H to 6.7 inhibited the m a x i m a l r e s p i r a t o r y c a p a c i t y by a b o u t 18%. R a i s i n g the p H to 7.6 f r o m 7.3 resulted in a 25% i n h i b i t i o n o f oligomycin-insensitive r e s p i r a t i o n in m a t u r e cortical tissue. This same change in p H p r o d u c e d a 15% i n h i b i t i o n o f m a x i m a l r e s p i r a t o r y c a p a c i t y a n d a 2 0 % i n h i b i t i o n o f oligomycin-insensitive r e s p i r a t i o n in i m m a t u r e cerebral cortical tissue. Mitochondrial Respiration. The effects o f altered a m b i e n t p H on m i t o c h o n d r i a l r e s p i r a t i o n with the N A D - l i n k e d s u b s t r a t e pair, g l u t a m a t e plus malate, are shown in Table II. Respiratory Rates in Cerebral Cortical Slices from 5-Day-Old and Adult Ratsa Respiratory rate (natom O2/mg protein min)

5 days old

pH

Basal

Oligomycin insensitive

DNP stimulated

6.7

14.9 _+ 0.6 (21)

9.8 _+ 0.7 (8)

7.0

14.8 _+ 0.5 (20) 13.8 _+ 0.5 (33) 12.6 _+ 0.5 (27)

9.4 _+ 0.5 (8) 8.7 _+ 0.5 (15) 6.7 _+ 0.4 (14) (P < 0.01) 13.7 _ 0.5 (13) 13.2 _+ 0.8 (6) 13.0 + 0.8 (14) 9.7 _+ 0.9 (8) (P < 0.005)

12.4 +_ 0.6 (13) (P < 0.01) 15.8 _+ 0.8 (1 I) 15.1 -- 0.6 (17) 13.0 _+ 0.7 (13) (P < 0.03) 34.9 + 1.0 (6) 34.6 _+ 2.3 (4) 31.0 + 2.1 (5) 31.7 _+ 2.2 (6)

7.3 7.6 Adult

6.7 7.0 7.3 7.6

21.7 _+ 0.9 (19) 22.1 _+ 1.8 (10) 21.6 _+ 1.1 (19) 20.2 + 1.3 (14)

~The pH of the medium was changed by varying the pCO 2 with a fixed HCO 3 concentration. Respiratory rates are defined in Materials and Methods. Each value is the mean _+ SE of the number of experiments shown in parentheses. Significance values are shown comparing mean respiratory rates in media of altered pH to rates measured at pH 7.3 in cortical slices from the same animals.

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Respiratory Control Ratios (RCR) in Isolated Cerebral Mitochondria Measured in Media of Varied pH c' Respiratory control ratio

pH

Olutamate/malate

Succinate

5.4

27.4 _+_ 5.8 (5) (P < 0.001)

52.3 _% 3.1 (4) (P < 0.001)

5.8

23.8 +_ 4.7 (4) (P < 0.001)

65.3 7- 3.5 (4) (P < 0.01)

6.2

55.4 _+ 3.9 (5) (P < 0.001)

68.4 q- 3.5 (5) (P < 0.001)

6.6

70.6 _+ 8.6 (5)

86.4 _+ 4.4 (5)

7.0

77.4 + 5.0 (5) (P < 0.02)

95.6 + 2.8 (5)

7.4

100

100

7.8

52.5 + 2.8 (5) (P < 0.001)

70.4 _+ 3.2 (5) (P < 0.00t)

8.2

40.8 4- 5,0 (4) (P < 0.01)

48.5 + 3.1 (4) (P < 0.001)

8.6

19.0 _+ 1.2 (4) (P < 0.001)

37.5 ___ 1.5 (4) (P < 0.001)

aEach value is the mean _+ SE of the number of experiments shown in parentheses for RCRs measured either with glutamate plus malate or with succinate as substrate. The means are expessed as a percentage of the RCRs measured at pH 7.4 in cerebral mitochondria from the same animal. Significance values < 0.05 are shown comparing the mean RCRs in media of altered pH to RCRs in the matched samples at pH 7.4.

Fig. 1. At pH 7.4, the pH value commonly used for studies of oxidative phosphorylation in isolated mitochondria, the RCR was 4.5-6.5. Mean State 3 respiratory rates were 75-95 natom O2/mg protein min. State 4 rates ranged from 15 to 20 natom O2/mg protein rain. At pH 7.0, a value closer to the physiologic intraceltular pH (Petroff et al., 1985), both the mean State 3 rate and the mean RCR were significantly lower than at pH 7.4. State 3 rates were stable with further lowering of pH until marked inhibition appeared at pH values of 5.8 and 5.4. In contrast, at moderate alkaline pH values, 7.8 and 8.2, State 3 respiration was markedly inhibited. State 4 rates generally were moderately, but less consistently, increased at both alkaline and acid pH values. These combined effects on State 3 and State 4 rates resulted in large decreases in RCRs (Table III).

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200 -

//~"

Glutamate-Malate

150 n>,,

co o

100

50

I

I

5.4

5.8

6.2

[

I

I

I

I

I

6.6

7.0

7.4

7.8

8.2

8.6

pH

Fig. 1. Respiratory rates, measured with glutamate plus malate as substrate, in mitochondria isolated from brains of adult rats. State 3 rates (filled circles) and State 4 rates (open circles), as defined in Materials and Methods, were measured in media of various pH values. Each point is the mean _. SE of at least four experiments in separate mitochondrial isolates. Mean values in each medium of altered pH are expressed as a percentage of the respiratory rates measures in matched mitochondrial samples at pH 7.4. Each significant difference (P < 0.05), determined by Student's t test for nonindependent samples comparing the mean experimental value with the value measured at pH 7.4, is indicated with an asterisk.

The effects of altered pH on isolated brain mitochondrial respiration with succinate as the substrate are shown in Fig. 2. At pH 7.4, State 3 respiratory rates ranged from about 180 to 220 natom O2/mg protein min, while the range of State 4 rates was about 70-90 natom O2/mg protein min. RCRs were between 2 and 3. With succinate as substrate, State 3 respiration showed significant inhibition with moderate acid or alkaline pH changes. In contrast, State 4 respiration was not affected by acid pH changes and was significantly increased only at alkaline pH 8.2. RCRs were less affected by changes in pH with succinate as substrate compared to the changes seen with g l u t a m a t e plus m a l a t e (Table III). A s with the N A D - l i n k e d substrates, the decreases in R C R s c o m p a r e d to values at p H 7.4 were greater with alkaline t h a n with acid p H changes. T h e effects o f changes in p H on A D P / O ratios are shown in T a b l e IV. W i t h g l u t a m a t e plus malate, A D P / O ratios were between 2.1 a n d 2.7 at p H 7.4. W i t h succinate as substrate, A D P / O ratios were between 1.2 a n d 1.7 at p H 7.4. These ratios decreased with altered p H to a greater degree a n d with smaller p H changes in the alkaline c o m p a r e d to the acid direction. W h e n the R C R b e c a m e very low (i.e., in the m o r e acid o r alkaline media), the State 3-to-State 4 t r a n s i t i o n was n o t clear a n d the A D P / O r a t i o was c o n s i d e r e d " n o t m e a s u r a b l e " .

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Holtzman, Olson, Nguyen, Hsu, and Lewiston

200

-

Succinate

150

IZ

to

o

100 e~

50

I

I

I

I

I

I

I

I

I

5.4

5.8

6.2

6.6

7.0

7.4

7.8

8.2

8.6

pH Fig. 2. Respiratory rates, measured with succinate as substrate, in cerebral mitochondria from adult rats. State 3 (filled circles) and State 4 rates (open circles), as defined in Materials and Methods, were measured in media of various p H values. Each point is the mean + SE of at least four experiments in separate mitochondria] isolates. TSe mean value in each medium of altered p H is expressed as a percentage of the value measured in matched mitochondrial samples at pH 7.4. Each significant difference (P < 0.05), determined by Student's t test for nonindependent samples comparing the mean experimental value with the value measured at p H 7.4, is indicated with an asterisk.

DISCUSSION The following conclusions can be drawn from the studies ofpH effects on aerobic glycolysis in cerebral cortical slices. First, the oligomycin-insensitive respiration (i.e., respiration not coupled to oxidative phosphorylation) is the respiratory component most consistently inhibited by altered pH. In the immature cortical slices, pH changes also result in inhibition of the DNP-stimulated respiratory rate (i.e., maximal respiratory capacity). This difference between the responses of mature and those of immature cortical tissue to pH changes may be due to the smaller respiratory capacity relative to the oligomycin-insensitive respiratory rate in the immature compared to the mature brain (Holtzman et al., 1982). Second, inhibition of cell respiration, particularly in immature tissue, is greater with a change in intracellular compared to extracellular pH. This conclusion is based on the greater respiratory inhibition seen when the pH is changed by varying the pC02 compared to the effects of changing the HCO3 concentration (Roos and Boron, 1981). Finally, inhibition of cell respiration in

pH and Brain Cell Respiration

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Table IV. ADP/O Ratios in Isolated Cerebral Mitochondria Measured in Media of Various pH Valuesa ADP/O pH

Glutamate/malate

Succinate

5.4

Not measurable (5)

Not measurable (4)

5.8

Not measurable (4)

78.5 _+ 4.7 (4) (P < 0.02)

6.2

93.6 +_ 4.3 (5)

85.4 _+ 2.7 (5) (P < 0.01)

6.6

95.0 ___ 3.7 (5)

94.0 +_ 6.7 (5)

7.0

91.2 _+ 3.8 (5)

93.8 _+ 4.5 (5)

7.4

100

100

7.8

93.0 +_ 2.7 (5)

85.8 _+ 2.0 (5) (P < 0.01)

8.2

86.3 _+ 7.2 (4)

80,7 _+_2.3 (4) (P < 0.01)

8.6

Not measurable (4)

Not measurable (4)

~Each value is the mean _+ SE of the number of experiments shown in parentheses either with glutamate plus malate or with succinate as substrate. Only values in which a clear State 3 State 4 transition occurred are included. Significance values P < 0.05 are shown comparing ADP/O ratios in media of altered pH to ratios in matched mitochondrial samples measured at pH 7,4. cerebral cortical slices is greater with smaller p H changes in the alkaline c o m p a r e d to the acid direction. T h e effects o f altered a m b i e n t p H on m i t o c h o n d r i a l r e s p i r a t i o n are similar, only in part, to the effects seen in cortical slices. U n l i k e the slices, there is no p H - d e p e n d e n t i n h i b i t i o n o f o x i d a t i v e p h o s p h o r y l a t i o n - i n d e p e n d e n t m i t o c h o n d r i a l r e s p i r a t i o n (State 4), suggesting t h a t an e x t r a m i t o c h o n d r i a l e n e r g y - c o u p l e d process m a y be inhibited by altered p H in the b r a i n cells. As in the cortical slices, the m a x i m a l r e s p i r a t o r y rate (State 3) is inhibited with smaller alkaline p H changes c o m p a r e d to acid changes o f similar m a g n i t u d e . W i t h the N A D - l i n k e d substrates, the greater sensitivity o f m i t o c h o n d r i a l r e s p i r a t i o n to alkaline p H changes is a p p a r e n t even when the e x p e r i m e n t a l rates are c o m p a r e d to those m e a s u r e d at p H 7.0, a value close to the p h y s i o l o g i c a l i n t r a c e l l u l a r p H (Siesjo, 1972; P e t r o f f et al., 1985). The effects o f acid p H changes o n b r a i n m i t o c h o n d r i a l r e s p i r a t i o n are very similar to those previously described in isolated b r a i n m i t o c h o n d r i a (Hillered et at., 1984a), in m i t o c h o n d r i a from the ischemic b r a i n ( R e h n c r o n a et al., 1979; Hillered et al., 1984b), a n d in isolated liver,

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Holtzman, Olson, Nguyen, Hsu, and Lewiston

heart, and kidney mitochondria (Chance and Conrad, 1959; Lowenstein and Chance, 1968; Tobin et al., 1972; Fry et al., 1980). In mitochondria from these other tissues, as we have observed in brain mitochondria, the inhibition of respiratory rates and oxidative phosphorylation is greater with alkaline compared to acid pH changes. Inhibition of mitochondrial oxidative phosphorylation has been proposed as a mechanism for the greater cell damage occurring in the brain with partial ischemia or hyperglycemic ischemia compared to that occurring with complete normooglycemic ischemia (Siesjo, 1978; Hillered et al., 1984a, b). It has been proposed that this inhibition is due to the extreme cellular acidosis secondary to lactate accumulation under these conditions. This conclusion is not supported fully by our results. Calculations of intracellular pH and pH measurements with a fluorescent indicator have suggested that intracellular pH drops to about 6.1 with severe partial ischemia (von Hanwehr et al., 1986; Meyer et al., 1986). In our studies, brain mitochondrial respiratory rates and ADP/O ratios were not markedly altered at that pH. Hillered and co-workers (1984a) found moderate inhibition of State 3 respiration at pH 6.4, but as in our studies, they observed even more marked inhibition of respiration and of oxidative phosphorylation at pH values below 6.1. These mitochondrial studies are consistent with the stable ATP levels found in a recent study of hypercarbia-induced brain acidosis (Petroffet al., 1985). Of potential importance, our results using cerebral slices suggest that the immature brain may be more susceptible than the adult brain to inhibition of cell respiration with acidosis. The association of hypocarbia and cerebral alkalosis with increased brain lactic acid concentration is well known (van Vaerenbergh et al., 1965; Plum and Posner, 1967; Alexander et al., 1968; Kjallquist et al., 1968; Miller et al., 1970; Weyne et al., 1970; Siesjo, 1972, 1978). While the mechanism for the increased lactate production is not known, it is not the result of tissue hypoxia and is not associated with a decrease in cell ATP or PCr concentrations (Miller et al., 1970; Siesjo, 1972, 1978; Young and Yagel, 1984; Petroffet al., 1985). Siesjo and co-workers (Kjallquist et al., 1968; Siesjo, 1972, 1978) proposed that lactate production is an important metabolic component in the physiological buffering for alkaline pH changes in the brain. Our studies suggest that intracellular alkalosis causes inhibition of aerobic metabolism, which with the described increased glycolysis (Siesjo, 1978; Young and Yagel, 1984), results in increased lactate production. Such a metabolic sequence may contribute to the behavioral depression seen with hypocarbia (Siesjo, 1978). If the cellular response to alkalosis is the same in brain and smooth muscle, inhibition of cellular aerobic energy metabolism also could contribute to the irreversibility of the intracellular pH change in an alkaline medium reported in uterine muscle (Wray, 1986). Finally, our studies again suggest that the immature brain may be more sensitive than the mature brain to these metabolic and functional effects of hypocarbia and alkalosis.

ACKNOWLEDGMENTS This work was supported by a Public Health Service research grant to DH (NS 16256) and the Laboratory Development Fund of the Department of Psychiatry and Neurology, Tulane University School of Medicine.

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