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The regulation of intracellular pH studied by 31P- and 1H-NMR spectroscopy in superfused guinea-pig cerebral cortex slices
Neurochem. Int. Vol. 21, No. 3, pp. 375-379, 1992
0197-0186/9255.00+0.00 Copyright © 1992 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
THE REGULATION OF INTRACELLULAR pH STUDIED BY 31p. AND IH-NMR SPECTROSCOPY IN SUPERFUSED GUINEA-PIG CEREBRAL CORTEX SLICES* KEITH J. BROOKS a n d HERMAN S. BACl-mLARO~f Division of Biochemistry, U.M.D.S. (St. Thomas's Campus), Lambeth Palace Road, London SEI 7EH, U.K. (Received 2 December 1991 ; accepted 15 March 1992)
AImtrm:t--(1) The intracellular pH (PHi) of superfused slices of guinea-pig cerebral cortex was measured in ~IP-NMR spectra using the chemical shifts of intracellular inorganic phosphate (Pi) and of 2-deoxyglucose 6-phosphate (D(XTr6P). The pHi was found to be 7.30+0.04 (SD, n = 15) in bicarbonate-buffered medium and 7.20+0.05 (n = 10, P < 0.001) in bicarbonate-free HEPES buffer of the same pH (7.4). (2) Decreases in pHc below 7.05 resulted in pH~ falling to similar values, with a decrease in the energy state. There was no change in intracellular lactate as assessed by ~H-NMR. (3) The tissues showed an. ability to buffer higher pH : increasing pH~ to 8.0 had no effect on pHi, PCr or lactate. (4) In order to characterize possible mechanisms of pH regulation in the tissue, the recovery from acid insult was investigated under various conditions. Initially pHi was decreased to 6.44+0.15 (n = 15) by exposure to media containing 6 mM bicarbonate gassed with Oz/CO2, 80: 20 (pHc 6.4). When this medium was replaced by normal bicarbonate buffer (pH 7.4) there was full recovery ofpH~ to 7.31 +0.05 (n = 15), whereas replacing the buffer with HEPES resulted in incomplete recovery of pHi to 6.884-0.15 (n = 15, P < 0.001). (5) In the presence of the carbonic anhydrase inhibitor, acetazolamide (1 raM), or the sodium/proton exchange inhibitor, amiloride (1 raM), there was an incomplete return of pH~ to the control value (pH~ 6.904-0.20, n = 5, P < 0.001). (6) There was partial recovery of pill in the presence of an inhibitor of the sodium/bicarbonate cotransporter, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), to 7.1 ! 4- 0.15 (n = 5, P < 0.02). (7) These results indicate that both exchangers, bicarbonate/chloride and sodium/proton, are involved in pH~ regulation in the intact metabolizing cerebral cortex. The role of the sodium/bicarbonate cotransporter is less clear.
In previous investigations of pHi regulation, T h o m a s (1976) used pH-sensitive electrodes and reported that the average pHi of snail neurones was 7.4. Increasing the external concentration of CO2 caused a rapid transient fall in p H i - - t h e higher the concentration used the greater was the decrease in phi. In a further study, T h o m a s (1977) found that the intracellular injection of HCI also decreased pHi. Recovery from these *This paper is dedicated to the memory of Professor D. Biesoid who died on the 29 May 1991 at the age of 65. Dr Volker Bigl, long time colleague and friend of Dietmar Biesold acted as Executive Editor in the processing of this paper. tAuthor to whom correspondence should be addressed, at : Department of Physics, University of Nottingham, University Park, Nottingham, NG7 2RD.
insults was totally inhibited by a bicarbonate/chloride exchange blocker, 4-acetoamido-4'-isothioeyanostilbene-2-2'-disulphonic acid (SITS), or a sodiumfree environment, though bis(2-hydroxyethyl) dimethyl a m m o n i u m bicarbonate was present in the latter case. T h o m a s therefore suggested that pHi regulation in the snail neurone involved a bicarbonate/chloride exchange mechanism which was dependent upon the p r e - ~ c e o f external sodium. M o o d y (1981) also used intracellular electrodes to measure pHi in crayfish neurones and found it to be 7.12+0.09. During recovery from an acid load, he observed an increase in intracellular sodium and a decrease in chloride. I f external bicarbonate was omitted, the rate o f recovery Was decreased by a b o u t 45%. Application of SITS slowed the rate o f recovery to 375
376
KHrH J
BROOKS a n d
the same extent as seen in bicarbonate-free conditions (HEPES buffered). Moody concluded that the crayfish neurones possess two separate mechanisms for pH~ regulation : N a + / H ~ exchange and Na +-dependent H C O f / C I - exchange. Deitmer and Schlue (1987), also using microelectrodes, compared the properties of glia and neurones in vitro. They found the pH~ of neuropile glial cells to be 6.87_+0.13 in HEPES-buffer (pH 7.4) and 7.18+_0.19 in 11 mM bicarbonate buffer, gassed with 2% CO2 (pH also 7.4). The pH~ of Retzius neurones was found to be the same (7.28___0.10) whichever of the two media was used. In bicarbonatefree media, the recovery of pH~ from intracellular acidification was completely prevented by removing the external sodium, or by the presence of a m i l o r i d e - this occurred in both cell types and indicated the presence of a N a + / H + exchanger. The effect of SITS was to decrease the rate of recovery in bicarbonate containing media in both cell types, and hence suggested the presence also of a H C O ; / C I - exchanger. As noted above, these investigators found that the two cell types responded differently to media buffered with H E P E S or with bicarbonate. The pH~ of the glia varied with the buffer used, but that of the neurones did not. If CO2 had entered the cells this would have formed carbonic acid which would then dissociate into bicarbonate and a proton, due to activity of carbonic anhydrase ; pH~ would then be expected to fall. As this was not the case, the entry must have been of bicarbonate which would result in the reverse of the above process and an increase in phi. Deitmar and Schlue (1987) found that this bicarbonate-induced alkalinization was not blocked by SITS, which suggested that a mechanism other than H C O ~ / C I exchange was involved. They subsequently showed (Schlue and Deitmer, 1988) that it could be blocked using D I D S , an inhibitor of N a + / H C O ; / C 1 - co-transport in kidney epithelial cells (Jentsch et al., 1986). In a previous study from this laboratory (Brooks et al., 1989a) it was noted that replacement of a bicarbonate-buffered medium with bicarbonate-free HEPES-buffer in superfused guinea-pig cerebral cortex slices caused the pH~ to be decreased by 0.12 4- 0.05 units, significantly different from control ( P < 0.05). These results prompted us to investigate the mechanisms of pH regulation in this intact metabolizing tissue, using the pH-sensitive chemical shifts of the resonances of inorganic phosphate (Pi) and of 2-deoxyglucose 6-phosphate (DOG6P) in 3~p-NMR spectra (Brooks et al., 1989b). ~H-NMR spectroscopy was also employed to monitor any changes in intracellular lactate.
[~IERMAN S. BACHELARD EXPERIMENTAl. PROCEDURES
Tissue preparation. The slices of guinea-pig Cclebral cortex were prepared and superfused as previously described (Bachelard et al., 1985). The superfusion medium contained (raM) : NaCl, 124; KC1, 5 ; KH2PO4, 1.2 ; MgSO4, 1.2 ; CaCI2, 1.2 ; NaHCO~, 26 and glucose, 10, gassed with O2/CO: (95:5) at 37C. For preloading of 2-deoxyglucose (DOG), a mixture of 2 raMDOG and 10 mM glucose was included in the superfusing medium for 1 h. After this, DOG and KH2PO4 were removed from the medium, enabling the NMR signals of intracellular Pi and DOG6P to be observed in the spectra (Brooks et al.. 1989b). To vary the external pH (pHc) the concentrations of bicarbonate and of CO2 were altered as shown in Table 1 ; bicarbonate-free HEPES-buffered media were adjusted by the addition of HCI or NaOH. N M R spectroscopy. The experiments were performed using a Bruker AM 400 spectrometer as previously (Brooks et al., 1989b). 3~P-NMR spectra were obtained using a rapid accumulation technique with an interpulse delay of 0.7 s. and a total of 2048 free induction decays was acquired over 26 min. To maximize the signal from the phosphorous-containing metabolites a pulse length of 35 ps was used, which gave an effective pulse angle of 45 °. The energy state of the tissue was assessed by comparing the areas of the phosphocreatine (PCr) resonances in experimental and control conditions, expressed as % of control. ~H-NMR spectra were collected using a water suppression 1331-2662 pulse sequence with a 60 ms delay, and a total of 128 free induction decays were acquired over 5 min. The water peak was placed at the centre of the field on resonance which effectively suppressed the signal from this component of the ~H-NMR spectrum. To compare the levels of lactate under control and experimental conditions, the area of the lactate methyl resonance (La, 1.33 ppm) was divided by the area of the N-acetyl aspartate methyl resonance (NA, 2.05 ppm) and expressed as the La/NA ratio. NA was used as the internal standard for the ~H-NMR resonances as it has been shown to be relatively metabolically stable (Mcllwain and Bachelard, 1985 ; Birken and Oldendorf, 1989).
RESULTS
The pH~ of our superfused cerebral cortex slices was 7.30+0.04 (n = 15) in 26 m M bicarbonate-buffered medium gassed with O2/CO2 (95:5), as measured Table 1. Bicarbonate buffers used to vary external oH (pHi) Gas
(O2/CO2)
pH~
mM-NaHCO~
7.8
48
95/5
7.4 6.9 6.4
26 6 6
95/5 95/5 80/20
The media contained the other constituents described in Experimental Procedures. Bicarbonate-free buffers were made up in 15 mM HEPES adjusted with N a O H or HCI to give pH values between 6 and 8.
The regulation of intracellular pH Z6 Z4
~
100
._ 7.0 ~ 6,8
~ 50
K6 K4 62 I 63 6,5
25 I 6.7
I I I I I I I 6.9 7.1 Z3 7,5 77 7,9 8.1 PHe
Fi8. I. Effectso f alterations in external pH (pH=) on intracellular pH (pHi) and on levels of phosphocrcatine (Per). I-I, Bicarbonate buffer; A, HEPES buffer; O, PCr as % of control. Error bars indicate + SD.
from the chemical shifts of the resonances of the Pi and DOG6P peaks in the 3'p-NMR spectra (Fig. l). Values for pHi were derived from the chemical shifts of both Pi and DOG6P for greater accuracy, as sometimes either resonance may be poorly resolved. We previously showed them to be equally suitable for monitoring pHi in this superfused slice preparation (Brooks et al., 1989b). In bicarbonate-free HEPES-buffered medium at the same external pH (7.4), the pHi was significantly lower (P < 0.001) at 7.20 + 0.05. Under both conditions, the energy state (as measured by PCr, Fig. 1) and lactate (from La/NA ratio, Fig. 2) were the same. When the pH= was decreased to 7.05, there was a similar decrease in phi, but no change in PCr or lactate.
377
Decreases in pH= below 7.05 resulted in similar decreases in pHi, but here there was a progressive fall in PCr (Fig. 1). This occurred to the same extent whether bicarbonate was present or not in the superfusing medium. These results indicate an inability of the tissue to regulate pHi in decreased pHc. On returning the tissues to normal bicarbonate-buffered medium (pH 7.4) there was full recovery of pHi and PCr. Further experiments were performed in attempts to characterize possible mechanisms of pH regulation in the tissue. An acid insult was produced by exposing the tissues to a pH= of 6.4, using a superfusing medium containing 6 mM bicarbonate gassed with O2/CO2 (80:20; Table l). The pHi decreased to 6.44+0.15 and the PCr decreased to 70 + 5% of control (Fig. 3). There was no change in intracellular lactate which remained at the control level (LA/NA --- 0.12 + 0.08, Fig. 2). Recovery in normal bicarbonate medium resulted in pHi returning to 7.31 +_0.05 (n = 15, Table 2) and restoration of the normal energy state. The roles of potential regulating mechanisms were then assessed by following the ability of the tissue to recover from the acid insult in the presence of selected
a
.
I PCr
I
I I
b
I I
315
31o
2:~
PPH
2:0
tl.
ilo
Fig. 2. IH-NMR spectra of cerebral cortex slices superfused with bicarbonate buffers. (a) pH, 7.4 ; (b) pH, 6.4. La : lactate; NA : N-acetyl aspartate.
'
'
~
:s
PPM
-;o
-~5
Fig. 3.3'P-NMR spectra of cerebral cortex slices supeffu~ with bicarbonate buffers. (a) pile 7.4; (b) phi 6.4. Pi : inorganic phosphate ; PCr : phosphocreatine ; DOG6P: 2-deoxyglucose 6-phosphate.
378
K[ITn J. BR(X)KS and HERMAN S. BACHELARD Table 2. Recoveryofintracellular pH (pH 0 alier acid insull
Conditions
pH,
Normal (with bicarbonate) Bicarbonate-free (HEPES) 1 mM acetazolamide I mMamiloride lmMDIDS
7.31 ± 0.05 (I 5) 6,88 _+0.15 (15)* 6.90~ 0.20 (5)* 6.95_+0.15 (5)* 7.11 ±0.15 (5)**
The extracellular pH was decreased to 6.40. giving a pHi of 6.44_+0. I5. The tissues were then exposed to the different conditions shown and the pHi was measured. Values of pHi are means+ SD (number of experiments). DIDS : 4,4'-diisothiocyanatostilbene-2,2'-disulphonicacid. *P < 0.001 : **P < 0.02. inhibitors b f these regulatory processes. Recovery in the presence of bicarbonate-free H E P E S buffer at pH~ 7.4 was incomplete, as pH~ returned to only 6.88 ___0.15 (n = 15, P < 0.001, Table 2), indicating involvement of the H C O 3 / C I - exchanger. This mechanism is linked to carbonic anhydrase which catalyses the reaction : H 2 0 + C O 2 = H2CO~ = H + + H C O ~ The tissues were allowed to recover in normal bicarbonate buffer in the presence of the carbonic anhydrase inhibitor, acetazolamide, at 1 raM. As can be seen in Table 2, the pH~ did not recover (6.90 + 0.20, n = 15, P < 0.001) which provides further evidence for the role of the H C O 3 / C I - exchanger. The inhibitor of the N a ÷ / H + exchanger, amiloride, has been applied to 3~p-NMR studies on pH regulation in kidney cells (Jans et aL, 1987). In our study it also caused an incomplete recovery of pH~ to 6.95+0.15 (17 = 5, P < 0.001, Table 2). D I D S , which is believed to inhibit the N a ÷ / H C 0 3 co-transporter (Jentsch et al., 1986) had a milder effect on recovery; the pH~ rose to 7.11___0.15 (n = 5) which was just significantly different from control at P < 0.02 (Table
2). DISCUSSION
Previous studies on pH regulation in cerebral preparations have been essentially confined to isolated or cultured cells, where the processes in neurones and glial cells can be compared as noted in the introduction. However in the intact tissue there is likely to be interaction between neurones and g l i a - - t h e r e is increasing awareness of the various roles played by glial cells in contributing to regulation o f neuronal function. The studies described here are based on noninvasive direct N M R measurement ofpH~ in the whole tissue where its metabolic state can be constantly monitored by observing concentrations of lactate and
energy phosphates. Though this preparation does 1)ol allow for distinguishing between neuronal and gtial contributions, it takes into account the mterpla5 between these cell types in pH regulation in the intact metabolizing tissue. Understanding the mechanisms for the regulation of pH~ in the central nervous system is essential for our assessment of the role played by changes in pH in neurotoxic events. Much of the neurodegenerative damage has been thought to be related to lactic acidosis, though many recent N M R studies have reported disparities between pH~ and intracellular levels of lactate (Behar et al., 1985 ; H o p e et al., 1988 ; Corbett and Laptook, 1990). In the present stud,,' it is also clear that intracellular levels of lactate are unchanged when pH~ has been decreased by lowered pHi. Modest decreases in pile to 7.05 caused a similar reduction in pH~ with no change in energy state or lactate. Further decreases in pH~ below 7.05 were followed by similar changes in pHi and decreases in the energy state, but with no change in lactate. The energy-regulating processes were thus able to cope with decreases in pile down to 7.05, but below this were compromised. This decline in the energy state may be linked to changes in trans-membrane cation concentrations, particularly of sodium, which can be remedied by N a + / K + transport processes at the expense of the energy reserves. Inclusion of inhibitors which interfere with the potential pH regulating mechanisms has given some insight into their relative importance. The HCO~-/C1- exchanger, seen in CNS-derived celt preparations (see introduction), has now been shown to be involved in actively-metabolizing whole tissue preparations of the guinea-pig cerebral cortex, since the recovery from acid insult was significantly decreased by the absence of bicarbonate or by the presence of the carbonic anhydrase inhibitor, acetazolamide. The role of the N a + / H + exchange mechanism has also been indicated by the effect of amiloride in preventing recovery from the acid insull. The role of the N a + / H C O f co-transport system is Less cleat', since D I D S exerted a milder effect on recovery. Indeed it is difficult to envisage a separate N a + / H C O 3 co-transport process: if both N a + / H + exchange and H C O 3 / C I - exchange processes are operating (perhaps in unison), these could result in an apparent Na + / H C O 3 co-transport. It seems of interest that increases in pH~ to 8.0 had no detectable effect on phi, or on PCr or lactate ; the tissue appeared to be able to withstand alkaline insult. Also the presence of inhibitors of putative pH regulating mechanisms had no cffect on this buffering
The regulation of intracellular pH ability. So at present it is unclear what mechanisms operate to buffer at higher than normal pH. It would be of interest and importance to identify these, in view of observations of alkaline p H shifts in some cerebral tumours (Oberhaensli et al., 1987). It may be that such understanding may prove useful in combating the growth of cerebral tumours. Acknowledgement--We are grateful to the Research (Endowments) Committee, St. Thomas's Hospital, for financial support. REFERENCES
Bachelard H. S., Cox D. W. G., Feeney J. and Morris P. G. 0985) ~P nuclear magnetic resonance studies on superfused cerebral tissues. Biochem. Soc. Trans. 13, 835-839. Behar K. L., den Hollander J. A., Petroff O. A. C., Hetherington H. P., Prichard J. W. and Shulman R. G. (1985) Effect of hypoglycemic encephalopathy upon amino acids, high-energy phosphates, and pH~ in the rat brain in vivo: detection by sequential ~H and 3~p NMR spectroscopy. J. Neurochem. 44, 1045-1055. Birken D. L. and OidendorfW. H. (1989) N-acetyl-L-aspartic acid : a literature review of a compound prominent in tHNMR spectroscopic studies of brain. Neurosci. biobehav. Rev. 13, 23-3 I. Brooks K. J., Kauppinen R. A., Williams S. R., Bachelard H. S., Bates T. E. and Gadian D. G. (1989a) Ammonia causes a drop in intracellular pH in metabolizing brain slices. A "~P and JH nuclear magnetic resonance study. Neurosci. 33, 185-192. Brooks K. J., Porteous R. and Bachelard H. S. (1989b) Effects of hypoglycaemia and hypoxia on the intracellular pH of cerebral tissue measured by a~p nuclear magnetic resonance spectroscopy. J. Neurochem. 52, 604-610. Corbctt R. J. T. and Laptook A. R. (1990) Acid homeostasis following partial ischaemia in neonatal brain in vivo by
379
~P and ~H nuclear magnetic resonance spectroscopy. J. Neurochem. 54, 1208-1217. Deitmer J. W. and Schlue W.-R. (1987) The regulation of intracellular pH by identified glial cells and neurones in the central nervous system ot" the leech. J. Physiol. 388, 261-283. Hope P. L., Cady E. B., Delpy D. T., Ives N. K., Gardiner R. M. and Reynolds E. O. R. (1988) Brain metabolism and intracellular pH during ischaemia : effects of systemic glucose and bicarbonate administration studied by 3~p and ~H nuclear magnetic resonance spectroscopy in vivo in the lamb. J. Neurochem. 50, 1394-1402. Jans A. W. H., Krijnen E. S., Luig J. and Kinne R. K. H. (1987) A 3~P-NMR study on the recovery of intracellular pH in LU-PK)/CI4 cells from intracellular alkalinization. Biochim. biophys. Acta 931, 326-334. Jentsch T. L., Schill B. S., Schwartz P., Matthes H., Keller S. K. and Wiederholt M. (1986) Kidney epithelial cells of monkey origin (BSC-I) express a sodium bicarbonate cotransport. J. biol. Chem. 260, 15,554-15,560. McIlwain H. and Bachelard H. S. (1985) Biochemistry and the Central Nervous System (5th ed.). London, ChurchillLivingstone. Moody W. J. (1981) The ionic mechanism of intracellular pH regulation in crayfish neurones. J. Physiol. 316, 293308. Oberhaensli R. D., Galloway G. F., Hilton-Jones D., Bore P. J., Styles P., Rajagopalan B., Taylor D. J. and Radda G. K. (1987) The study of human organs by phosphorous31 topical magnetic resonance spectroscopy. Br. J. Radiol. 60, 367-373. Schlue W.-R. and Deitmer J. W. (1988) Ionic mechanisms of intracellular pH regulation in the nervous system. In Proton Passage Across Cell Membranes (Ciba Foundation Symposium 139), pp. 47-69, Chichester, Wiley. Thomas R. C. (1976) The effect of carbon dioxide on the intracellular pH and buffering power of snail neurones. J Physiol. 255, 715-735. Thomas R. C. (1977) The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurones. J. Physiol. 273, 317-338.
0197-0186/9255.00+0.00 Copyright © 1992 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
THE REGULATION OF INTRACELLULAR pH STUDIED BY 31p. AND IH-NMR SPECTROSCOPY IN SUPERFUSED GUINEA-PIG CEREBRAL CORTEX SLICES* KEITH J. BROOKS a n d HERMAN S. BACl-mLARO~f Division of Biochemistry, U.M.D.S. (St. Thomas's Campus), Lambeth Palace Road, London SEI 7EH, U.K. (Received 2 December 1991 ; accepted 15 March 1992)
AImtrm:t--(1) The intracellular pH (PHi) of superfused slices of guinea-pig cerebral cortex was measured in ~IP-NMR spectra using the chemical shifts of intracellular inorganic phosphate (Pi) and of 2-deoxyglucose 6-phosphate (D(XTr6P). The pHi was found to be 7.30+0.04 (SD, n = 15) in bicarbonate-buffered medium and 7.20+0.05 (n = 10, P < 0.001) in bicarbonate-free HEPES buffer of the same pH (7.4). (2) Decreases in pHc below 7.05 resulted in pH~ falling to similar values, with a decrease in the energy state. There was no change in intracellular lactate as assessed by ~H-NMR. (3) The tissues showed an. ability to buffer higher pH : increasing pH~ to 8.0 had no effect on pHi, PCr or lactate. (4) In order to characterize possible mechanisms of pH regulation in the tissue, the recovery from acid insult was investigated under various conditions. Initially pHi was decreased to 6.44+0.15 (n = 15) by exposure to media containing 6 mM bicarbonate gassed with Oz/CO2, 80: 20 (pHc 6.4). When this medium was replaced by normal bicarbonate buffer (pH 7.4) there was full recovery ofpH~ to 7.31 +0.05 (n = 15), whereas replacing the buffer with HEPES resulted in incomplete recovery of pHi to 6.884-0.15 (n = 15, P < 0.001). (5) In the presence of the carbonic anhydrase inhibitor, acetazolamide (1 raM), or the sodium/proton exchange inhibitor, amiloride (1 raM), there was an incomplete return of pH~ to the control value (pH~ 6.904-0.20, n = 5, P < 0.001). (6) There was partial recovery of pill in the presence of an inhibitor of the sodium/bicarbonate cotransporter, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), to 7.1 ! 4- 0.15 (n = 5, P < 0.02). (7) These results indicate that both exchangers, bicarbonate/chloride and sodium/proton, are involved in pH~ regulation in the intact metabolizing cerebral cortex. The role of the sodium/bicarbonate cotransporter is less clear.
In previous investigations of pHi regulation, T h o m a s (1976) used pH-sensitive electrodes and reported that the average pHi of snail neurones was 7.4. Increasing the external concentration of CO2 caused a rapid transient fall in p H i - - t h e higher the concentration used the greater was the decrease in phi. In a further study, T h o m a s (1977) found that the intracellular injection of HCI also decreased pHi. Recovery from these *This paper is dedicated to the memory of Professor D. Biesoid who died on the 29 May 1991 at the age of 65. Dr Volker Bigl, long time colleague and friend of Dietmar Biesold acted as Executive Editor in the processing of this paper. tAuthor to whom correspondence should be addressed, at : Department of Physics, University of Nottingham, University Park, Nottingham, NG7 2RD.
insults was totally inhibited by a bicarbonate/chloride exchange blocker, 4-acetoamido-4'-isothioeyanostilbene-2-2'-disulphonic acid (SITS), or a sodiumfree environment, though bis(2-hydroxyethyl) dimethyl a m m o n i u m bicarbonate was present in the latter case. T h o m a s therefore suggested that pHi regulation in the snail neurone involved a bicarbonate/chloride exchange mechanism which was dependent upon the p r e - ~ c e o f external sodium. M o o d y (1981) also used intracellular electrodes to measure pHi in crayfish neurones and found it to be 7.12+0.09. During recovery from an acid load, he observed an increase in intracellular sodium and a decrease in chloride. I f external bicarbonate was omitted, the rate o f recovery Was decreased by a b o u t 45%. Application of SITS slowed the rate o f recovery to 375
376
KHrH J
BROOKS a n d
the same extent as seen in bicarbonate-free conditions (HEPES buffered). Moody concluded that the crayfish neurones possess two separate mechanisms for pH~ regulation : N a + / H ~ exchange and Na +-dependent H C O f / C I - exchange. Deitmer and Schlue (1987), also using microelectrodes, compared the properties of glia and neurones in vitro. They found the pH~ of neuropile glial cells to be 6.87_+0.13 in HEPES-buffer (pH 7.4) and 7.18+_0.19 in 11 mM bicarbonate buffer, gassed with 2% CO2 (pH also 7.4). The pH~ of Retzius neurones was found to be the same (7.28___0.10) whichever of the two media was used. In bicarbonatefree media, the recovery of pH~ from intracellular acidification was completely prevented by removing the external sodium, or by the presence of a m i l o r i d e - this occurred in both cell types and indicated the presence of a N a + / H + exchanger. The effect of SITS was to decrease the rate of recovery in bicarbonate containing media in both cell types, and hence suggested the presence also of a H C O ; / C I - exchanger. As noted above, these investigators found that the two cell types responded differently to media buffered with H E P E S or with bicarbonate. The pH~ of the glia varied with the buffer used, but that of the neurones did not. If CO2 had entered the cells this would have formed carbonic acid which would then dissociate into bicarbonate and a proton, due to activity of carbonic anhydrase ; pH~ would then be expected to fall. As this was not the case, the entry must have been of bicarbonate which would result in the reverse of the above process and an increase in phi. Deitmar and Schlue (1987) found that this bicarbonate-induced alkalinization was not blocked by SITS, which suggested that a mechanism other than H C O ~ / C I exchange was involved. They subsequently showed (Schlue and Deitmer, 1988) that it could be blocked using D I D S , an inhibitor of N a + / H C O ; / C 1 - co-transport in kidney epithelial cells (Jentsch et al., 1986). In a previous study from this laboratory (Brooks et al., 1989a) it was noted that replacement of a bicarbonate-buffered medium with bicarbonate-free HEPES-buffer in superfused guinea-pig cerebral cortex slices caused the pH~ to be decreased by 0.12 4- 0.05 units, significantly different from control ( P < 0.05). These results prompted us to investigate the mechanisms of pH regulation in this intact metabolizing tissue, using the pH-sensitive chemical shifts of the resonances of inorganic phosphate (Pi) and of 2-deoxyglucose 6-phosphate (DOG6P) in 3~p-NMR spectra (Brooks et al., 1989b). ~H-NMR spectroscopy was also employed to monitor any changes in intracellular lactate.
[~IERMAN S. BACHELARD EXPERIMENTAl. PROCEDURES
Tissue preparation. The slices of guinea-pig Cclebral cortex were prepared and superfused as previously described (Bachelard et al., 1985). The superfusion medium contained (raM) : NaCl, 124; KC1, 5 ; KH2PO4, 1.2 ; MgSO4, 1.2 ; CaCI2, 1.2 ; NaHCO~, 26 and glucose, 10, gassed with O2/CO: (95:5) at 37C. For preloading of 2-deoxyglucose (DOG), a mixture of 2 raMDOG and 10 mM glucose was included in the superfusing medium for 1 h. After this, DOG and KH2PO4 were removed from the medium, enabling the NMR signals of intracellular Pi and DOG6P to be observed in the spectra (Brooks et al.. 1989b). To vary the external pH (pHc) the concentrations of bicarbonate and of CO2 were altered as shown in Table 1 ; bicarbonate-free HEPES-buffered media were adjusted by the addition of HCI or NaOH. N M R spectroscopy. The experiments were performed using a Bruker AM 400 spectrometer as previously (Brooks et al., 1989b). 3~P-NMR spectra were obtained using a rapid accumulation technique with an interpulse delay of 0.7 s. and a total of 2048 free induction decays was acquired over 26 min. To maximize the signal from the phosphorous-containing metabolites a pulse length of 35 ps was used, which gave an effective pulse angle of 45 °. The energy state of the tissue was assessed by comparing the areas of the phosphocreatine (PCr) resonances in experimental and control conditions, expressed as % of control. ~H-NMR spectra were collected using a water suppression 1331-2662 pulse sequence with a 60 ms delay, and a total of 128 free induction decays were acquired over 5 min. The water peak was placed at the centre of the field on resonance which effectively suppressed the signal from this component of the ~H-NMR spectrum. To compare the levels of lactate under control and experimental conditions, the area of the lactate methyl resonance (La, 1.33 ppm) was divided by the area of the N-acetyl aspartate methyl resonance (NA, 2.05 ppm) and expressed as the La/NA ratio. NA was used as the internal standard for the ~H-NMR resonances as it has been shown to be relatively metabolically stable (Mcllwain and Bachelard, 1985 ; Birken and Oldendorf, 1989).
RESULTS
The pH~ of our superfused cerebral cortex slices was 7.30+0.04 (n = 15) in 26 m M bicarbonate-buffered medium gassed with O2/CO2 (95:5), as measured Table 1. Bicarbonate buffers used to vary external oH (pHi) Gas
(O2/CO2)
pH~
mM-NaHCO~
7.8
48
95/5
7.4 6.9 6.4
26 6 6
95/5 95/5 80/20
The media contained the other constituents described in Experimental Procedures. Bicarbonate-free buffers were made up in 15 mM HEPES adjusted with N a O H or HCI to give pH values between 6 and 8.
The regulation of intracellular pH Z6 Z4
~
100
._ 7.0 ~ 6,8
~ 50
K6 K4 62 I 63 6,5
25 I 6.7
I I I I I I I 6.9 7.1 Z3 7,5 77 7,9 8.1 PHe
Fi8. I. Effectso f alterations in external pH (pH=) on intracellular pH (pHi) and on levels of phosphocrcatine (Per). I-I, Bicarbonate buffer; A, HEPES buffer; O, PCr as % of control. Error bars indicate + SD.
from the chemical shifts of the resonances of the Pi and DOG6P peaks in the 3'p-NMR spectra (Fig. l). Values for pHi were derived from the chemical shifts of both Pi and DOG6P for greater accuracy, as sometimes either resonance may be poorly resolved. We previously showed them to be equally suitable for monitoring pHi in this superfused slice preparation (Brooks et al., 1989b). In bicarbonate-free HEPES-buffered medium at the same external pH (7.4), the pHi was significantly lower (P < 0.001) at 7.20 + 0.05. Under both conditions, the energy state (as measured by PCr, Fig. 1) and lactate (from La/NA ratio, Fig. 2) were the same. When the pH= was decreased to 7.05, there was a similar decrease in phi, but no change in PCr or lactate.
377
Decreases in pH= below 7.05 resulted in similar decreases in pHi, but here there was a progressive fall in PCr (Fig. 1). This occurred to the same extent whether bicarbonate was present or not in the superfusing medium. These results indicate an inability of the tissue to regulate pHi in decreased pHc. On returning the tissues to normal bicarbonate-buffered medium (pH 7.4) there was full recovery of pHi and PCr. Further experiments were performed in attempts to characterize possible mechanisms of pH regulation in the tissue. An acid insult was produced by exposing the tissues to a pH= of 6.4, using a superfusing medium containing 6 mM bicarbonate gassed with O2/CO2 (80:20; Table l). The pHi decreased to 6.44+0.15 and the PCr decreased to 70 + 5% of control (Fig. 3). There was no change in intracellular lactate which remained at the control level (LA/NA --- 0.12 + 0.08, Fig. 2). Recovery in normal bicarbonate medium resulted in pHi returning to 7.31 +_0.05 (n = 15, Table 2) and restoration of the normal energy state. The roles of potential regulating mechanisms were then assessed by following the ability of the tissue to recover from the acid insult in the presence of selected
a
.
I PCr
I
I I
b
I I
315
31o
2:~
PPH
2:0
tl.
ilo
Fig. 2. IH-NMR spectra of cerebral cortex slices superfused with bicarbonate buffers. (a) pH, 7.4 ; (b) pH, 6.4. La : lactate; NA : N-acetyl aspartate.
'
'
~
:s
PPM
-;o
-~5
Fig. 3.3'P-NMR spectra of cerebral cortex slices supeffu~ with bicarbonate buffers. (a) pile 7.4; (b) phi 6.4. Pi : inorganic phosphate ; PCr : phosphocreatine ; DOG6P: 2-deoxyglucose 6-phosphate.
378
K[ITn J. BR(X)KS and HERMAN S. BACHELARD Table 2. Recoveryofintracellular pH (pH 0 alier acid insull
Conditions
pH,
Normal (with bicarbonate) Bicarbonate-free (HEPES) 1 mM acetazolamide I mMamiloride lmMDIDS
7.31 ± 0.05 (I 5) 6,88 _+0.15 (15)* 6.90~ 0.20 (5)* 6.95_+0.15 (5)* 7.11 ±0.15 (5)**
The extracellular pH was decreased to 6.40. giving a pHi of 6.44_+0. I5. The tissues were then exposed to the different conditions shown and the pHi was measured. Values of pHi are means+ SD (number of experiments). DIDS : 4,4'-diisothiocyanatostilbene-2,2'-disulphonicacid. *P < 0.001 : **P < 0.02. inhibitors b f these regulatory processes. Recovery in the presence of bicarbonate-free H E P E S buffer at pH~ 7.4 was incomplete, as pH~ returned to only 6.88 ___0.15 (n = 15, P < 0.001, Table 2), indicating involvement of the H C O 3 / C I - exchanger. This mechanism is linked to carbonic anhydrase which catalyses the reaction : H 2 0 + C O 2 = H2CO~ = H + + H C O ~ The tissues were allowed to recover in normal bicarbonate buffer in the presence of the carbonic anhydrase inhibitor, acetazolamide, at 1 raM. As can be seen in Table 2, the pH~ did not recover (6.90 + 0.20, n = 15, P < 0.001) which provides further evidence for the role of the H C O 3 / C I - exchanger. The inhibitor of the N a ÷ / H + exchanger, amiloride, has been applied to 3~p-NMR studies on pH regulation in kidney cells (Jans et aL, 1987). In our study it also caused an incomplete recovery of pH~ to 6.95+0.15 (17 = 5, P < 0.001, Table 2). D I D S , which is believed to inhibit the N a ÷ / H C 0 3 co-transporter (Jentsch et al., 1986) had a milder effect on recovery; the pH~ rose to 7.11___0.15 (n = 5) which was just significantly different from control at P < 0.02 (Table
2). DISCUSSION
Previous studies on pH regulation in cerebral preparations have been essentially confined to isolated or cultured cells, where the processes in neurones and glial cells can be compared as noted in the introduction. However in the intact tissue there is likely to be interaction between neurones and g l i a - - t h e r e is increasing awareness of the various roles played by glial cells in contributing to regulation o f neuronal function. The studies described here are based on noninvasive direct N M R measurement ofpH~ in the whole tissue where its metabolic state can be constantly monitored by observing concentrations of lactate and
energy phosphates. Though this preparation does 1)ol allow for distinguishing between neuronal and gtial contributions, it takes into account the mterpla5 between these cell types in pH regulation in the intact metabolizing tissue. Understanding the mechanisms for the regulation of pH~ in the central nervous system is essential for our assessment of the role played by changes in pH in neurotoxic events. Much of the neurodegenerative damage has been thought to be related to lactic acidosis, though many recent N M R studies have reported disparities between pH~ and intracellular levels of lactate (Behar et al., 1985 ; H o p e et al., 1988 ; Corbett and Laptook, 1990). In the present stud,,' it is also clear that intracellular levels of lactate are unchanged when pH~ has been decreased by lowered pHi. Modest decreases in pile to 7.05 caused a similar reduction in pH~ with no change in energy state or lactate. Further decreases in pH~ below 7.05 were followed by similar changes in pHi and decreases in the energy state, but with no change in lactate. The energy-regulating processes were thus able to cope with decreases in pile down to 7.05, but below this were compromised. This decline in the energy state may be linked to changes in trans-membrane cation concentrations, particularly of sodium, which can be remedied by N a + / K + transport processes at the expense of the energy reserves. Inclusion of inhibitors which interfere with the potential pH regulating mechanisms has given some insight into their relative importance. The HCO~-/C1- exchanger, seen in CNS-derived celt preparations (see introduction), has now been shown to be involved in actively-metabolizing whole tissue preparations of the guinea-pig cerebral cortex, since the recovery from acid insult was significantly decreased by the absence of bicarbonate or by the presence of the carbonic anhydrase inhibitor, acetazolamide. The role of the N a + / H + exchange mechanism has also been indicated by the effect of amiloride in preventing recovery from the acid insull. The role of the N a + / H C O f co-transport system is Less cleat', since D I D S exerted a milder effect on recovery. Indeed it is difficult to envisage a separate N a + / H C O 3 co-transport process: if both N a + / H + exchange and H C O 3 / C I - exchange processes are operating (perhaps in unison), these could result in an apparent Na + / H C O 3 co-transport. It seems of interest that increases in pH~ to 8.0 had no detectable effect on phi, or on PCr or lactate ; the tissue appeared to be able to withstand alkaline insult. Also the presence of inhibitors of putative pH regulating mechanisms had no cffect on this buffering
The regulation of intracellular pH ability. So at present it is unclear what mechanisms operate to buffer at higher than normal pH. It would be of interest and importance to identify these, in view of observations of alkaline p H shifts in some cerebral tumours (Oberhaensli et al., 1987). It may be that such understanding may prove useful in combating the growth of cerebral tumours. Acknowledgement--We are grateful to the Research (Endowments) Committee, St. Thomas's Hospital, for financial support. REFERENCES
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