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Lactate efflux and intracellular pH during severe hypoxia in rat cerebral cortex in vitro studied by nuclear magnetic resonance spectroscopy
ELSEVIER
Neuroscience Letters 178 (1994) 111-114
NEUROSCIHCt IEITHS
Lactate efflux and intracellular pH during severe hypoxia in rat cerebral cortex in vitro studied by nuclear magnetic resonance spectroscopy Tiina-R.M. Pirttil/i, Risto A. Kauppinen* NMR Research Group, Department of Biochemistry and Biotechnology, A.I. Virtanen Institute, University of Kuopio, PO Box 1627, SF-70211, Kuopio, Finland
Received 6 June 1994; Revised version received 11 July 1994; Accepted 11 July 1994
Abstract Intracellular pH (pHi) and lactate were monitored in a superfused brain slice preparation using N M R spectroscopy in order to study the role of lactate washout in maintenance of pH i during hypoxia. Data are consistence with a functioning lactate-H + cotransport in the energetically intact cerebral cortex. This pathway is not, however, linked to regulation of pH~ during energy failure with external pH of 6.8 and thus appears not to have physiological impact in H ÷ homeostasis during cerebral hypoxia. Key words: Brain; Hypoxia; Intracellular pH; Lactate; N M R spectroscopy
Drop in intracellular pH (PHi) in vivo during brain ischaemia [3] or hypoxia [1] is accompanied by acidification of extracellular fluid [8,11,13] and thus acid generated by anaerobic metabolism is either directly or indirectly extruded. It is firmly established that intracellular acidification during brain oxygen deprivation in vivo [21] and in vitro [10,15] is detel'mined largely by anaerobic glucose metabolism and the degree of acidification is reflected as brain lactate concentration. It has been previously shown that during normocapnic ischaemia in vivo the drop in the extracellular pH (pHo) is also linearly correlated with brain lactate concentration [8]. Extracellular acidification has been considered to result from lactic acid efflux [8,20] together with reduced buffering capacity of the extracellular fluid [11]. Previous nuclear magnetic resonance (NMR) spectroscopy studies have shown that during hypoxia at a given lactate load pHi is dependent of extracellular Na ÷ [15], bicarbonate [17] and upon pH of the superfusate [16]. These data suggest that transmembrane H + extrusion supported by Na÷-gradient and/or extracellular
*Corresponding author. Fax: (358) (71) 2811-510. E-mail: [email protected]. 0304-3940194/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0304-3940(94)00548-6
HCO3- may be operative in energy compromised cerebral cortex. The mechanisms are sensitive to external rather than internal H ÷ concentration [16] and are different from the amiloride-sensitive Na+/H+-exchange and HCO3--dependent acid-equivalent transport [17]. In the present work we have assessed the possible contribution of lactate transport (reviewed in [18]) in maintenance of pH i during severe hypoxia. Preparation and initial incubations of transverse rat cerebral cortex slices (0.35 mm thick) have been described in our previous articles [10,15]. Medium phosphate was omitted after 3 0 4 5 min in order to monitor and utilize the intracellular phosphate (P) as an indicator of phi using 3~p N M R [22]. Superfusion media used contained either HCO3- or HEPES as a buffering substance and the medium constituents were as follows: (1) Krebs Henseleit buffer (KHB), composition (in mM) NaC1 124, NaHCO3 26, KC1 5, M g S O 4 1.2 and CaC12 1.2; (2) modified KHB with reduced NaHCO3 concentration of 5 mM substituted with 21 mM Na-gluconate; (3) HEPES-buffered medium (in mM) NaC1 124, HEPES 20, Na-gluconate 26, KC1 5, MgSO4 1.2 and CaC12 1.2. pH of the HEPES-buffered media were adjusted to 7.5 or 6.8 with NaOH. 10 mM D-glucose was present in the superfusion media as required. HCO3--containing media
T.-R.M. Pirttilii, R.A. Kauppinen/Neuroscience Letters 178 (19941 111 114
112
were bubbled with either 02/C02 (95%:5%) ('normoxia') or Ne/CO2 (95%/5%) ('hypoxia') whereas HEPES-containing ones with either Oe (100%) or N2 (100%) for respective oxygenation conditions. Severe hypoxia was induced by gassing with N2-containing gas, and simultaneously reducing the flow by 60%, a procedure shown to reduce oxygen tension to the level that effectively inhibits mitochondrial oxidation [10]. After 42 min of hypoxia, D-glucose was omitted from the superfusion medium. k-lactate (15 raM; Sigma, Poole, UK) was added to KHB as a sodium salt and it replaced equimolar amounts of NaC1. c~-Cyano-4-hydroxycinnamate (CHC; Sigma) was used from a stock solution dissolved in deuterated dimethyl sulphoxide, concentration of which in the superfusate did not exceed 0.1%. Dimethylamiloride (DMA) was purchased from Sigma. " P and ~H N M R spectra were recorded using a Bruker 15-mm ~H/broadband (tuned to LH at 400.1 MHz and 3~p at 162.0 MHz) N M R probe in a 9.4 T vertical magnet interlaced with a Bruker AM-400 spectrometer. Acquisitions of the resonances of the two nuclei were interleaved on a pulse by pulse basis in order to improve temporal resolution and to obtain true simultaneous recordings as described previously [10]. 3~p N M R (80 ° pulse angle, interpulse interval of 3 s, spectral width of 7.5 kHz) were acquired and data processed as described previously [15]. pH~ was calculated from the chemical shift of Pi relative to PCr at 0 ppm using the titration curve [23]. Chemical shift of the peaks were determined by software provided by Bruker. ]H N M R spectra (spectral width of 5 kHz, 1024 data points) were recorded using a binomial spin-echo sequence incorporating solvent suppression pulses with optimum excitation adjusted for lactate resonance at 1.33 ppm [6] and with the water signal set on resonance. The spin-echo delay was set at 60 ms, and an interpulse time of 3 s was used. Chemical shifts are expressed relative to a value of 2.02 ppm for the methyl resonance of N-acetylaspartate (NAA). lntracellular lactate was
quantified in absolute terms from the lactate/NAA peak area ratios from ~H N M R spectra using an NAA concentration of 6 mmol/kg [19] and taking into account the non-uniform excitation of the spin-echo sequence as described previously [17]. Peak areas after manual baseline correction were determined using deconvolution software in the W I N - N M R program (Bruker). Lactate concentration in the superfusate was determined enzymatically by the method of Hohorst [5]. The volume-averaged pH in the oxygenated KHB was 7.29 + 0.03 and lactate was 1.4 + 0.1 mmol/kg wet weight (n = 20). Hypoxia induced in the presence of glucose reduced pH~ to 6.92 + 0.02 (n = 27) and intracellular lactate concentration (lac0 accumulated to 14.7 + 0.6 mmol/kg (n = 22). pHi and lactate efflux was determined in hypoxia following removal of glucose from the superfusion medium. Switching to glucose-free KHB resulted in alkalinisation ofpH~ to 7.39 + 0.06 (n = 5) by 10.5 rain and it stabilised to 7.47 + 0.05, a value that is indistinguishable from the pH of the superfusion medium. The change in pH~ during the first 3 rain (ApHi3mi~) from the calculated, statistically-weighted linear regression lines (time points between 0 and 10.5 min) was 0.20 + 0.05 pH units. The respective decrease in lac~ (AlaCi3min) was 3.4 + 0.3 mmol/kg. Lactate concentration in the superfusate matched to that expected from the decrease of la< (data not shown) and it decreased reflecting reduction in the tissue lactate concentration. At lowered medium pH (6.8) in the presence of 5 mM HCO3-, lac~ decreased with similar kinetics as in KHB with 26 mM HCOC (Table 1). Despite the lactate washout, no alkalinisation of pH~ was observed (Table 1). The simultaneous omission of glucose from the superfusion medium in the absence of external bicarbonate did not affect significantly either ApHi3mi. (0.13 + 0.05 pH units), Alac13mm (3.4 + 0.3 mmol/kg) (Table 1) or lactate efflux to the superfusate. This indicates that bicarbonatedependent anion-exchange system is not responsible for transport of H + and/or lactate across the cell membrane
Table I lntracellular pH and lactate concentration in rat brain slices during severe hypoxia. Effects of D-glucose omission Condition
end phi
End laci
KHB pH 7.5 HEPES pH 7.5 HEPES pH 6.8 HCO) 5 raM, pH 6.8 KHB+DMA KHB+Lac 15 mM KHB+CHC
6.92 _+ 0.04
14.5 _+0.6
7.00 _+0.03
ApHi3mm
Alaci~,ni,,
pH, 16.5
laci 16.5
0.20 _+ 0.05
3.4 +_ 0.3
7.41 ± 0.06
1.5 _+ 0.2
13.8 ± 1.2
0.13 _+ 0.05
3.4 _+ 0.3
7.45 _+ 0.08
1.4 ± 0.3
6.94 ± 0.02
16.2 _+ (1.6
0.00 _+ 0.0"
2.8 _+ 0.3
6.94 _+ 0.01"
1.6 _+ 0.4
6.86 _+ 0.04
17.5 ± 0.5*
- 0.04 _+ 0.02*
3.5 _+ 0.5
6.73 _+0.01 *
2.2 _+0.3
6.86 ± 0.03 6.95 ± 0.05
13.7 ± 0.6 N.D.
0.12 ± 0.03 0.02 ± 0.05*
2.9 + 0.2 N.D.
7.27 ± 0.(13 7.26 ± 0.02*
1.5 ± 0.7 N.D.
6.96 ± 0.04
13.8 ± 0.8
0.10 ± 0.02
2.8 ± 0.3
7.25 ± 0.03
2.4 ± 0.1
m
Values are means _+ S.E.M. lk~r l\mr to six independent experiments. "end pHi' and "end lac," indicate respective values at 42 min of hypoxia in the presence of 10 mM D-glucose and 'pH~ 16.5' and "lac~ 16.5' respective values at 16.5 rain of hypoxic superfusion in the absence of D-glucose. D M A and CHC were added to KHB with pH 7.5. *P < 0.05 relative to KHB, pH 7.5, Student's t test. N.D., not determined.
T.-R.M. Pirttilgi, R.A. KauppinenlNeuroscience Letters 178 (1994) 111-114
A 8,00
7,75 ' 7,50' 7,25 ' 7,00'
X7 I"
6,75
•
35
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•
45
•
I
•
•
l
55
•
65
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•
•
75
I
•
•
85
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•
95
B
7,75 7,50 7,25 7,00 6,75 35
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!
I
I
45
55
65
75
C 8,00
7,75 7,50-
7,25 7,00 -
6,75
I
35
40
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45
I
50
I
55
I
60
65
Time (min) Fig. 1. Effects of exogenous lactate (A,B) and glucose removal (C) on pHi in normoxic (A) and hypoxic (B,C) brain slices. In A brain slices were initially superfused in oxygenated KHB, until switched to HEPES as indicated by an open arrow head. Superfusion was continued in the HEPES-buffered medium as indicated by a horizontal bar. 15 m M L-lactate was present the time in between closed arrow heads. Data are representative for three independent experiments. In B slices were superfused in the HEPES-buffered medium gassed with N2. 15 m M Llactate was present the time in between closed arrow heads. Data are representative for four independent experiments. In C K H B was used as a superfusion medium and glucose was removed at the time point indicated by a closed arrow head. 15 m M L-lactate was present after glucose removal in the experiments marked with ( * ). Data are representative for five to seven independent experiments. Error bars are omitted for the shake of clarity.
in hypoxia at a medium pH of 7.5. In the HEPES-buffered medium with a pH of 6.8, reduction of lac~ was not
113
accompanied with pH i change (Table 1). Taking together the data above, lactate washout was not, but pH i alkalinisation was, inhibited by low medium pH indicating that these two processes were mediated by independent mechanisms in the cerebral cortex. Dimethylamiloride (DMA, 150 pM), an inhibitor of Na+/H + exchanger, at a medium pH of 7.5 did neither affect zJpHi3mi n n o r Alaci3min and pHi reached a level that was not significantly different from that detected under control conditions (Table 1). Exposure of isolated spinal roots to lactate has been observed to cause transient alkalinisation of pHo followed by acidification upon removal of the anion and these pH changes have been shown to be due to lactateH + transport [20]. Our previous study has indicated that superfusion of cerebral cortical brain slices in the presence of 10 mM lactate and 3 mM pyruvate in the HCO3buffered medium acidifies one pHi compartment after a delay [9]. Thus it may be that brain cortex also transports lactate and/or pyruvate through a similar mechanism as in peripheral nerve [20]. In the oxygenated HEPES-buffered medium, replacement of 26 mM bicabonate/CO2 with 20 mM HEPES in normoxic conditions resulted in a rise ofpH i from-7.25 to-7.7 in 3 min followed by return to -7.3 (Fig. 1A). Addition of 15 mM L-lactate caused an acidification by ~0.3 pH units in the first 3~p NMR spectrum (Fig. 1A). This is consistent with a lactate-H + transport in the brain slices during normoxia. During aglycaemic hypoxia induced in the HEPES-buffered medium, pH i was -0.1 pH units more acidic in the first time point (Fig. 1B) suggesting that effect of lactate in pHi was retarded in the absence of ATE In the presence of 15 mM L-lactate during severe hypoxia in the HCO3-buffered superfusate, alkalinisation of pHi started later than in the control experiments but pH~ was at the control level by 20 min (Fig. 1C). This was as expected from the experiment in Fig. 1B, where lactate slightly acidified pHi. We used CHC, a potent inhibitor of lactate-H + cotransport and mono-carboxylate carrier, during transition from glucose-containing to glucose-free medium. CHC (2 mM) did not significantly affect the rate of reduction in laci but it inhibited the change in pHi (Table 1). This inhibition is unexpected and the unaffected rate of lactate removal shows that that it cannot be due to a specific action of the drug on lactate-H+-carrier. Three different mechanisms for cellular uptake and release of lactate have been described in non-neural cells; i.e. (i) an anion exchange system, (ii) non-ionic diffusion and (iii) lactate-H + cotransport [4]. Lactate-H + cotransport has been reported to be the major mechanism responsible for lactate movements across the plasma membrane in rat peripheral nerves [20] and cultured neurons and astrocytes [14]. In the intact peripheral nerve this pathway has been shown to be largely responsible for extracellular acidification during hypoxia [20]. The
114
T.-R. M. Pirttilii, R.A. Kauppinen / Neuroscience Letters 178 (1994) 111 414
mechanisms of proton extrusion are particularly interesting as it has been previously shown that low external pH may reduce Ca 2+ entry through agonist-operated ion channels [22] and improve survival of energy deprived neurons [7]. The present data show, consistent with previous studies on hippocampal slices [2] and cultured neural cells [14], that intact cerebral cortex transports lactate through a lactate-H + carrier. This transporter appears, however, not to be of significance in alkalinisation of brain pH~ during severe hypoxia. Lactate washout and rise in pHi take place in concert in KHB and HEPESbuffered medium with pH of 7.5, but reduction of medium pH to 6.8 results in a dissociation of the relationship between the two variables. We have previously shown that lactate washout from brain slices proceeds in the absence of ATP and external Na + suggesting that lactate is exchanging to the external medium through passive diffusion [15]. Interestingly, microelectrode studies have shown that pH o starts to fall immediately following onset of ischaemia [8,13] whereas extracellular lactate rises after a delay after intracellular lactate has reached a relatively high level [12]. [1] Allen, K., Busza, A.L., Crockard, H.A. and Gadian, D.G., Brain metabolism and blood flow in acute cerebral hypoxia studied by NMR spectroscopy and hydrogen clearance, NMR Biomed., 5 (1992) 48 52. [2] Assaf, H.M., Ricci, A.J., Whittington, T.S., LaManna J.C.. Ratcheson, R.A. and Lust, W.D., Lactate compartmentation in hippocampal slices: evidence for a tranporter, Metab. Brain Dis., 5 (1990) 143 154. [3] Crockard, H.A., Gadian, D.G., Frackowiak, R.S.J., Proctor, E.. Allen, K., Williams, S.R. and Russell, R.W.R., Acute cerebral ischaemia: concurrent changes in cerebral blood flow, energy metabolites, pH, and lactate measured with hydrogen clearance and ~Lp and ~H nuclear magnetic resonance spectroscopy. II. Changes during ischaemia, J. Cereb. Blood Flow Metab., 7 (I 987) 394402. [4] Deuticke, B., Beyer, E., and Forst, B., Discrimination of three parallel pathways of lactate transport in the human erythrocyte membrane by inhibitors and kinetic properties, Biochim. Biophys. Acta, 684 (1982) 96-1 t0. [5] Hohorst, H.J., L,-(+)-lactate. Determination withlactic dehydrogenase and DNR In H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, NY, 1963, pp. 26(~270. [6] Hore, P.J., Solvent suppression in Fourier transform nuclear magnetic resonance, J. Magn. Resort., 55 (1983) 283-300. [7] Kaku, D.A., Giffard, R.G., and Choi, D.W., Neuroprotective effects of glutamate antagonists and extracellular acidity, Science. 260 (1993) 1516 1518. [8] Katsura, K., Asplund B., Ekholm, A. and Siesj6 B.K., Extra- and intracellular pH in the brain during ischaemia, related to tissue
lactate content in normo- and hypercapnic rats, Eur. J. Neurosci., 4 (1992) 166-176. [9] Kauppinen, R.A., Williams, S.R., Brooks, K.J. and Bachelard, H.S., Effects of ammonium on energy metabolism and intracellular pH in guinea pig cerebral cortex studied by 3~p and ~H nuclear magnetic spectroscopy, Neurochem. Int. 19 (1991)495-504. [10] Kauppinen, R.A. and Williams, S.R., Cerebral energy metabolism and intracellular pH during severe bypoxia and recovery. A study using ~H, ~lp and ~H{~3C} nuclear magnetic resonance spectroscopy in the guinea pig cerebral cortex in vitro, J. Neurosci. Res., 26 (1990) 359 369. [11] Kraig, R.R, Pulsinelli, W.A., and Plum, F., Carbonic acid buffer changes during complete brain ischemia, Am. J. Physiol., 250 (1986) R348-R357. [12] Kuhr, W.G. and Korl; J., Extracellular lactic acid as an indicator o1" brain metabolism: continuous on-line measurement in conscious, freely moving rats with intrastriatal dialysis, J. Cereb. Blood Flow Metab., 8 (19881 130 137. [13] Much, W.A. and Hansen, A.J., Extracellular pH changes during spreading depression and cerebral ischemia: mechanisms of brain pH regulation, J. Cereb. Blood Flow Metab., 4 (1984) 17-27. [14] Nedergaard. M. and Goldman, S.A., Carrier-mediated transport of lactic acid in ctdtured neurons and astrocytes, Am. J. Physiol., 265 (1993) R282-R289. [15] Pirttil/i, T.-R. and Kauppinen, R.A., Recovery ofintracellular pH in cortical brain slices following anoxia studied by nuclear magnetic resonance spectroscopy: role of lactate removal, exracellular sodium and sodium/hydrogen exchange, Neuroscience, 47 (1992) 155 164. [ 16] Pirttilfi, T.-R. M. and Kauppinen, R.A., Extracellular pH and buffering power determine intracellular pH in cortical brain slices during and following hypoxia, NeuroReport, 5 (1993) 213-216. [17] Pirttilfi, T.-R. M. and Kauppinen, R.A., Regulation ofintracellular pH in guinea pig cerebral cortex ex vivo studied by 3~p and JH nuclear magnetic resonance spectroscopy: role of extracellular bicarbonate and chloride, J. Neurochem., 62 (1994) 656-664. [18] Poole. R.C. and Halestrap, A.R, Transport of lactate and other monocarboxylates across mammalian plasma membranes, Am. J. Physiol., 264 (1993) C761-C782. [19] Preece, N.E., Jackson, G.D., Houseman, J.A., Dunchan, J.S. and Williams, S.R., Elevated cerebral GABA in the vigabatrin-treated rat: measurement in vivo by ~H nuclear magnetic resonance spectroscopy, Epilepsia, 35 (1994) 431-436. [20] Schneider, U., Poole, R.C., Halestrap, A.P. and Grafe, P., Lactateproton co-transport and its contribution to interstitial acidification during hypoxia in isolated rat spinal roots, Neuroscience, 53 (1993) 1153 1162. [21] Siesj6. B.K., Cell damagc in the brain: a speculative synthesis, J. Ccreb. Blood Flow Metab.. I (1981) 155 185. [22] Tang, C.-M., Dichter M., and Morad, M., Modulation of the Nmethyl-D-aspartate channel by extracellular H +, Proc. Natl. Acad. Sci. USA, 87 (1990) 6445 6449. [23] Taylor, D.J.. Bore, P.J., Styles, P.. Gadian, D.G. and Radda, G.K., Bioenergetics of intact human muscle. A 3~p nuclear magnetic resonance study, Mol. Biol. Med., 1 (1983) 77 94.
Neuroscience Letters 178 (1994) 111-114
NEUROSCIHCt IEITHS
Lactate efflux and intracellular pH during severe hypoxia in rat cerebral cortex in vitro studied by nuclear magnetic resonance spectroscopy Tiina-R.M. Pirttil/i, Risto A. Kauppinen* NMR Research Group, Department of Biochemistry and Biotechnology, A.I. Virtanen Institute, University of Kuopio, PO Box 1627, SF-70211, Kuopio, Finland
Received 6 June 1994; Revised version received 11 July 1994; Accepted 11 July 1994
Abstract Intracellular pH (pHi) and lactate were monitored in a superfused brain slice preparation using N M R spectroscopy in order to study the role of lactate washout in maintenance of pH i during hypoxia. Data are consistence with a functioning lactate-H + cotransport in the energetically intact cerebral cortex. This pathway is not, however, linked to regulation of pH~ during energy failure with external pH of 6.8 and thus appears not to have physiological impact in H ÷ homeostasis during cerebral hypoxia. Key words: Brain; Hypoxia; Intracellular pH; Lactate; N M R spectroscopy
Drop in intracellular pH (PHi) in vivo during brain ischaemia [3] or hypoxia [1] is accompanied by acidification of extracellular fluid [8,11,13] and thus acid generated by anaerobic metabolism is either directly or indirectly extruded. It is firmly established that intracellular acidification during brain oxygen deprivation in vivo [21] and in vitro [10,15] is detel'mined largely by anaerobic glucose metabolism and the degree of acidification is reflected as brain lactate concentration. It has been previously shown that during normocapnic ischaemia in vivo the drop in the extracellular pH (pHo) is also linearly correlated with brain lactate concentration [8]. Extracellular acidification has been considered to result from lactic acid efflux [8,20] together with reduced buffering capacity of the extracellular fluid [11]. Previous nuclear magnetic resonance (NMR) spectroscopy studies have shown that during hypoxia at a given lactate load pHi is dependent of extracellular Na ÷ [15], bicarbonate [17] and upon pH of the superfusate [16]. These data suggest that transmembrane H + extrusion supported by Na÷-gradient and/or extracellular
*Corresponding author. Fax: (358) (71) 2811-510. E-mail: [email protected]. 0304-3940194/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0304-3940(94)00548-6
HCO3- may be operative in energy compromised cerebral cortex. The mechanisms are sensitive to external rather than internal H ÷ concentration [16] and are different from the amiloride-sensitive Na+/H+-exchange and HCO3--dependent acid-equivalent transport [17]. In the present work we have assessed the possible contribution of lactate transport (reviewed in [18]) in maintenance of pH i during severe hypoxia. Preparation and initial incubations of transverse rat cerebral cortex slices (0.35 mm thick) have been described in our previous articles [10,15]. Medium phosphate was omitted after 3 0 4 5 min in order to monitor and utilize the intracellular phosphate (P) as an indicator of phi using 3~p N M R [22]. Superfusion media used contained either HCO3- or HEPES as a buffering substance and the medium constituents were as follows: (1) Krebs Henseleit buffer (KHB), composition (in mM) NaC1 124, NaHCO3 26, KC1 5, M g S O 4 1.2 and CaC12 1.2; (2) modified KHB with reduced NaHCO3 concentration of 5 mM substituted with 21 mM Na-gluconate; (3) HEPES-buffered medium (in mM) NaC1 124, HEPES 20, Na-gluconate 26, KC1 5, MgSO4 1.2 and CaC12 1.2. pH of the HEPES-buffered media were adjusted to 7.5 or 6.8 with NaOH. 10 mM D-glucose was present in the superfusion media as required. HCO3--containing media
T.-R.M. Pirttilii, R.A. Kauppinen/Neuroscience Letters 178 (19941 111 114
112
were bubbled with either 02/C02 (95%:5%) ('normoxia') or Ne/CO2 (95%/5%) ('hypoxia') whereas HEPES-containing ones with either Oe (100%) or N2 (100%) for respective oxygenation conditions. Severe hypoxia was induced by gassing with N2-containing gas, and simultaneously reducing the flow by 60%, a procedure shown to reduce oxygen tension to the level that effectively inhibits mitochondrial oxidation [10]. After 42 min of hypoxia, D-glucose was omitted from the superfusion medium. k-lactate (15 raM; Sigma, Poole, UK) was added to KHB as a sodium salt and it replaced equimolar amounts of NaC1. c~-Cyano-4-hydroxycinnamate (CHC; Sigma) was used from a stock solution dissolved in deuterated dimethyl sulphoxide, concentration of which in the superfusate did not exceed 0.1%. Dimethylamiloride (DMA) was purchased from Sigma. " P and ~H N M R spectra were recorded using a Bruker 15-mm ~H/broadband (tuned to LH at 400.1 MHz and 3~p at 162.0 MHz) N M R probe in a 9.4 T vertical magnet interlaced with a Bruker AM-400 spectrometer. Acquisitions of the resonances of the two nuclei were interleaved on a pulse by pulse basis in order to improve temporal resolution and to obtain true simultaneous recordings as described previously [10]. 3~p N M R (80 ° pulse angle, interpulse interval of 3 s, spectral width of 7.5 kHz) were acquired and data processed as described previously [15]. pH~ was calculated from the chemical shift of Pi relative to PCr at 0 ppm using the titration curve [23]. Chemical shift of the peaks were determined by software provided by Bruker. ]H N M R spectra (spectral width of 5 kHz, 1024 data points) were recorded using a binomial spin-echo sequence incorporating solvent suppression pulses with optimum excitation adjusted for lactate resonance at 1.33 ppm [6] and with the water signal set on resonance. The spin-echo delay was set at 60 ms, and an interpulse time of 3 s was used. Chemical shifts are expressed relative to a value of 2.02 ppm for the methyl resonance of N-acetylaspartate (NAA). lntracellular lactate was
quantified in absolute terms from the lactate/NAA peak area ratios from ~H N M R spectra using an NAA concentration of 6 mmol/kg [19] and taking into account the non-uniform excitation of the spin-echo sequence as described previously [17]. Peak areas after manual baseline correction were determined using deconvolution software in the W I N - N M R program (Bruker). Lactate concentration in the superfusate was determined enzymatically by the method of Hohorst [5]. The volume-averaged pH in the oxygenated KHB was 7.29 + 0.03 and lactate was 1.4 + 0.1 mmol/kg wet weight (n = 20). Hypoxia induced in the presence of glucose reduced pH~ to 6.92 + 0.02 (n = 27) and intracellular lactate concentration (lac0 accumulated to 14.7 + 0.6 mmol/kg (n = 22). pHi and lactate efflux was determined in hypoxia following removal of glucose from the superfusion medium. Switching to glucose-free KHB resulted in alkalinisation ofpH~ to 7.39 + 0.06 (n = 5) by 10.5 rain and it stabilised to 7.47 + 0.05, a value that is indistinguishable from the pH of the superfusion medium. The change in pH~ during the first 3 rain (ApHi3mi~) from the calculated, statistically-weighted linear regression lines (time points between 0 and 10.5 min) was 0.20 + 0.05 pH units. The respective decrease in lac~ (AlaCi3min) was 3.4 + 0.3 mmol/kg. Lactate concentration in the superfusate matched to that expected from the decrease of la< (data not shown) and it decreased reflecting reduction in the tissue lactate concentration. At lowered medium pH (6.8) in the presence of 5 mM HCO3-, lac~ decreased with similar kinetics as in KHB with 26 mM HCOC (Table 1). Despite the lactate washout, no alkalinisation of pH~ was observed (Table 1). The simultaneous omission of glucose from the superfusion medium in the absence of external bicarbonate did not affect significantly either ApHi3mi. (0.13 + 0.05 pH units), Alac13mm (3.4 + 0.3 mmol/kg) (Table 1) or lactate efflux to the superfusate. This indicates that bicarbonatedependent anion-exchange system is not responsible for transport of H + and/or lactate across the cell membrane
Table I lntracellular pH and lactate concentration in rat brain slices during severe hypoxia. Effects of D-glucose omission Condition
end phi
End laci
KHB pH 7.5 HEPES pH 7.5 HEPES pH 6.8 HCO) 5 raM, pH 6.8 KHB+DMA KHB+Lac 15 mM KHB+CHC
6.92 _+ 0.04
14.5 _+0.6
7.00 _+0.03
ApHi3mm
Alaci~,ni,,
pH, 16.5
laci 16.5
0.20 _+ 0.05
3.4 +_ 0.3
7.41 ± 0.06
1.5 _+ 0.2
13.8 ± 1.2
0.13 _+ 0.05
3.4 _+ 0.3
7.45 _+ 0.08
1.4 ± 0.3
6.94 ± 0.02
16.2 _+ (1.6
0.00 _+ 0.0"
2.8 _+ 0.3
6.94 _+ 0.01"
1.6 _+ 0.4
6.86 _+ 0.04
17.5 ± 0.5*
- 0.04 _+ 0.02*
3.5 _+ 0.5
6.73 _+0.01 *
2.2 _+0.3
6.86 ± 0.03 6.95 ± 0.05
13.7 ± 0.6 N.D.
0.12 ± 0.03 0.02 ± 0.05*
2.9 + 0.2 N.D.
7.27 ± 0.(13 7.26 ± 0.02*
1.5 ± 0.7 N.D.
6.96 ± 0.04
13.8 ± 0.8
0.10 ± 0.02
2.8 ± 0.3
7.25 ± 0.03
2.4 ± 0.1
m
Values are means _+ S.E.M. lk~r l\mr to six independent experiments. "end pHi' and "end lac," indicate respective values at 42 min of hypoxia in the presence of 10 mM D-glucose and 'pH~ 16.5' and "lac~ 16.5' respective values at 16.5 rain of hypoxic superfusion in the absence of D-glucose. D M A and CHC were added to KHB with pH 7.5. *P < 0.05 relative to KHB, pH 7.5, Student's t test. N.D., not determined.
T.-R.M. Pirttilgi, R.A. KauppinenlNeuroscience Letters 178 (1994) 111-114
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Time (min) Fig. 1. Effects of exogenous lactate (A,B) and glucose removal (C) on pHi in normoxic (A) and hypoxic (B,C) brain slices. In A brain slices were initially superfused in oxygenated KHB, until switched to HEPES as indicated by an open arrow head. Superfusion was continued in the HEPES-buffered medium as indicated by a horizontal bar. 15 m M L-lactate was present the time in between closed arrow heads. Data are representative for three independent experiments. In B slices were superfused in the HEPES-buffered medium gassed with N2. 15 m M Llactate was present the time in between closed arrow heads. Data are representative for four independent experiments. In C K H B was used as a superfusion medium and glucose was removed at the time point indicated by a closed arrow head. 15 m M L-lactate was present after glucose removal in the experiments marked with ( * ). Data are representative for five to seven independent experiments. Error bars are omitted for the shake of clarity.
in hypoxia at a medium pH of 7.5. In the HEPES-buffered medium with a pH of 6.8, reduction of lac~ was not
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accompanied with pH i change (Table 1). Taking together the data above, lactate washout was not, but pH i alkalinisation was, inhibited by low medium pH indicating that these two processes were mediated by independent mechanisms in the cerebral cortex. Dimethylamiloride (DMA, 150 pM), an inhibitor of Na+/H + exchanger, at a medium pH of 7.5 did neither affect zJpHi3mi n n o r Alaci3min and pHi reached a level that was not significantly different from that detected under control conditions (Table 1). Exposure of isolated spinal roots to lactate has been observed to cause transient alkalinisation of pHo followed by acidification upon removal of the anion and these pH changes have been shown to be due to lactateH + transport [20]. Our previous study has indicated that superfusion of cerebral cortical brain slices in the presence of 10 mM lactate and 3 mM pyruvate in the HCO3buffered medium acidifies one pHi compartment after a delay [9]. Thus it may be that brain cortex also transports lactate and/or pyruvate through a similar mechanism as in peripheral nerve [20]. In the oxygenated HEPES-buffered medium, replacement of 26 mM bicabonate/CO2 with 20 mM HEPES in normoxic conditions resulted in a rise ofpH i from-7.25 to-7.7 in 3 min followed by return to -7.3 (Fig. 1A). Addition of 15 mM L-lactate caused an acidification by ~0.3 pH units in the first 3~p NMR spectrum (Fig. 1A). This is consistent with a lactate-H + transport in the brain slices during normoxia. During aglycaemic hypoxia induced in the HEPES-buffered medium, pH i was -0.1 pH units more acidic in the first time point (Fig. 1B) suggesting that effect of lactate in pHi was retarded in the absence of ATE In the presence of 15 mM L-lactate during severe hypoxia in the HCO3-buffered superfusate, alkalinisation of pHi started later than in the control experiments but pH~ was at the control level by 20 min (Fig. 1C). This was as expected from the experiment in Fig. 1B, where lactate slightly acidified pHi. We used CHC, a potent inhibitor of lactate-H + cotransport and mono-carboxylate carrier, during transition from glucose-containing to glucose-free medium. CHC (2 mM) did not significantly affect the rate of reduction in laci but it inhibited the change in pHi (Table 1). This inhibition is unexpected and the unaffected rate of lactate removal shows that that it cannot be due to a specific action of the drug on lactate-H+-carrier. Three different mechanisms for cellular uptake and release of lactate have been described in non-neural cells; i.e. (i) an anion exchange system, (ii) non-ionic diffusion and (iii) lactate-H + cotransport [4]. Lactate-H + cotransport has been reported to be the major mechanism responsible for lactate movements across the plasma membrane in rat peripheral nerves [20] and cultured neurons and astrocytes [14]. In the intact peripheral nerve this pathway has been shown to be largely responsible for extracellular acidification during hypoxia [20]. The
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T.-R. M. Pirttilii, R.A. Kauppinen / Neuroscience Letters 178 (1994) 111 414
mechanisms of proton extrusion are particularly interesting as it has been previously shown that low external pH may reduce Ca 2+ entry through agonist-operated ion channels [22] and improve survival of energy deprived neurons [7]. The present data show, consistent with previous studies on hippocampal slices [2] and cultured neural cells [14], that intact cerebral cortex transports lactate through a lactate-H + carrier. This transporter appears, however, not to be of significance in alkalinisation of brain pH~ during severe hypoxia. Lactate washout and rise in pHi take place in concert in KHB and HEPESbuffered medium with pH of 7.5, but reduction of medium pH to 6.8 results in a dissociation of the relationship between the two variables. We have previously shown that lactate washout from brain slices proceeds in the absence of ATP and external Na + suggesting that lactate is exchanging to the external medium through passive diffusion [15]. Interestingly, microelectrode studies have shown that pH o starts to fall immediately following onset of ischaemia [8,13] whereas extracellular lactate rises after a delay after intracellular lactate has reached a relatively high level [12]. [1] Allen, K., Busza, A.L., Crockard, H.A. and Gadian, D.G., Brain metabolism and blood flow in acute cerebral hypoxia studied by NMR spectroscopy and hydrogen clearance, NMR Biomed., 5 (1992) 48 52. [2] Assaf, H.M., Ricci, A.J., Whittington, T.S., LaManna J.C.. Ratcheson, R.A. and Lust, W.D., Lactate compartmentation in hippocampal slices: evidence for a tranporter, Metab. Brain Dis., 5 (1990) 143 154. [3] Crockard, H.A., Gadian, D.G., Frackowiak, R.S.J., Proctor, E.. Allen, K., Williams, S.R. and Russell, R.W.R., Acute cerebral ischaemia: concurrent changes in cerebral blood flow, energy metabolites, pH, and lactate measured with hydrogen clearance and ~Lp and ~H nuclear magnetic resonance spectroscopy. II. Changes during ischaemia, J. Cereb. Blood Flow Metab., 7 (I 987) 394402. [4] Deuticke, B., Beyer, E., and Forst, B., Discrimination of three parallel pathways of lactate transport in the human erythrocyte membrane by inhibitors and kinetic properties, Biochim. Biophys. Acta, 684 (1982) 96-1 t0. [5] Hohorst, H.J., L,-(+)-lactate. Determination withlactic dehydrogenase and DNR In H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, Academic Press, New York, NY, 1963, pp. 26(~270. [6] Hore, P.J., Solvent suppression in Fourier transform nuclear magnetic resonance, J. Magn. Resort., 55 (1983) 283-300. [7] Kaku, D.A., Giffard, R.G., and Choi, D.W., Neuroprotective effects of glutamate antagonists and extracellular acidity, Science. 260 (1993) 1516 1518. [8] Katsura, K., Asplund B., Ekholm, A. and Siesj6 B.K., Extra- and intracellular pH in the brain during ischaemia, related to tissue
lactate content in normo- and hypercapnic rats, Eur. J. Neurosci., 4 (1992) 166-176. [9] Kauppinen, R.A., Williams, S.R., Brooks, K.J. and Bachelard, H.S., Effects of ammonium on energy metabolism and intracellular pH in guinea pig cerebral cortex studied by 3~p and ~H nuclear magnetic spectroscopy, Neurochem. Int. 19 (1991)495-504. [10] Kauppinen, R.A. and Williams, S.R., Cerebral energy metabolism and intracellular pH during severe bypoxia and recovery. A study using ~H, ~lp and ~H{~3C} nuclear magnetic resonance spectroscopy in the guinea pig cerebral cortex in vitro, J. Neurosci. Res., 26 (1990) 359 369. [11] Kraig, R.R, Pulsinelli, W.A., and Plum, F., Carbonic acid buffer changes during complete brain ischemia, Am. J. Physiol., 250 (1986) R348-R357. [12] Kuhr, W.G. and Korl; J., Extracellular lactic acid as an indicator o1" brain metabolism: continuous on-line measurement in conscious, freely moving rats with intrastriatal dialysis, J. Cereb. Blood Flow Metab., 8 (19881 130 137. [13] Much, W.A. and Hansen, A.J., Extracellular pH changes during spreading depression and cerebral ischemia: mechanisms of brain pH regulation, J. Cereb. Blood Flow Metab., 4 (1984) 17-27. [14] Nedergaard. M. and Goldman, S.A., Carrier-mediated transport of lactic acid in ctdtured neurons and astrocytes, Am. J. Physiol., 265 (1993) R282-R289. [15] Pirttil/i, T.-R. and Kauppinen, R.A., Recovery ofintracellular pH in cortical brain slices following anoxia studied by nuclear magnetic resonance spectroscopy: role of lactate removal, exracellular sodium and sodium/hydrogen exchange, Neuroscience, 47 (1992) 155 164. [ 16] Pirttilfi, T.-R. M. and Kauppinen, R.A., Extracellular pH and buffering power determine intracellular pH in cortical brain slices during and following hypoxia, NeuroReport, 5 (1993) 213-216. [17] Pirttilfi, T.-R. M. and Kauppinen, R.A., Regulation ofintracellular pH in guinea pig cerebral cortex ex vivo studied by 3~p and JH nuclear magnetic resonance spectroscopy: role of extracellular bicarbonate and chloride, J. Neurochem., 62 (1994) 656-664. [18] Poole. R.C. and Halestrap, A.R, Transport of lactate and other monocarboxylates across mammalian plasma membranes, Am. J. Physiol., 264 (1993) C761-C782. [19] Preece, N.E., Jackson, G.D., Houseman, J.A., Dunchan, J.S. and Williams, S.R., Elevated cerebral GABA in the vigabatrin-treated rat: measurement in vivo by ~H nuclear magnetic resonance spectroscopy, Epilepsia, 35 (1994) 431-436. [20] Schneider, U., Poole, R.C., Halestrap, A.P. and Grafe, P., Lactateproton co-transport and its contribution to interstitial acidification during hypoxia in isolated rat spinal roots, Neuroscience, 53 (1993) 1153 1162. [21] Siesj6. B.K., Cell damagc in the brain: a speculative synthesis, J. Ccreb. Blood Flow Metab.. I (1981) 155 185. [22] Tang, C.-M., Dichter M., and Morad, M., Modulation of the Nmethyl-D-aspartate channel by extracellular H +, Proc. Natl. Acad. Sci. USA, 87 (1990) 6445 6449. [23] Taylor, D.J.. Bore, P.J., Styles, P.. Gadian, D.G. and Radda, G.K., Bioenergetics of intact human muscle. A 3~p nuclear magnetic resonance study, Mol. Biol. Med., 1 (1983) 77 94.