The regulation of pH in the central nervous system' W.-R. SCPILLTE A N D R. D ~ R N E R lnsttrut f i r %olr~gie/Neurobiologie~Hehr-ich-Heine-Untversitdt DdisseUorf, D-4080, DiisseCdorf, Germany
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Received November 7, 1991 S c n ~ u e W.-R., , and D ~ R N E R 8 ., 1992. The regulation of pH in the central nervous system. Can. J. Physiol. Pharinacol. 70: S278 - ,9285. The pH, regulation from intracellular acidosis in the central nervous system appears to be mediated by mechanisms driven by the large inwardly directed Na+ gradient. The involvement of these mechanisms in pH, regulation of neurones and glial cells has been investigated in the leech central nervous system using ion-selective rnicroelectrodes. For recovery from acidification, there appear to be three separate mechanisms: Na+/H+ exchange, Na+-dependent Cl-/HC03- exchange, and Na+-HC0,- cotransport. All three meshanisms have a profound effect on the maintenance of pH, homeostasis in glial cells: whereas in leech neurones, as in other neuronal cells studied previously, the predominant mechanisms are Na+/H+ and Na'-dependent Cl-/HCO,- exchange. In addition to acid extrusion mechanisms we also found evidence for Nat-independent CI-/HCO,- exchange. At alkaline pH, this exchanger may mediate some of the pH, recovery from intracellular alkalinization. Key words: central nervous system, pH regulation, neurotransmitter. S c ~ i ~ uW.-R., e, and D ~ R N E R. R , 1992. The regulation of pH in the central nervous system. Can. J. Physiol. Pharmacol. 78: S278 - ,9285. La rCgulation du pH, aprks une acidose intracellulaire dans le systkme nerveux central semble &re mCdiCe par des mCcanismes contrB1Cs par le fort gradient de Na' dirigt vers l'intkrieur. On a examink l'implicatisn de ces mCcanisrnes dam la rkgulation du pH, des neurones et des cellules gliales, dans le systkme nerveux central de la sangsue, en utilisant des microClectrodes sensibles aux ions. Trois mecanisrnes distincts semblent participer au ritablissement de l'acidification : 19CchangeNa+/H+, 1'Cchange CI-/HC03- dependant du Naf et le cotransport de Na+ -HCO,-. Les trois mCcanismes ont un effet significatif sur le maintien de l'homeostasie du pH, dans Ies cellules gliales, alors que dans les neurones de la sangsue, cornme dans d'autres cellules neuronales examinCes antirieurement, les mCcanismes prkdominants sont 1'Cchange Na+/H6 et 1'Cchange Cl-/HC03- &pendant du Na'. En plus des mCcanismes d9Climination de l'acidite, nous avons aussi mis en Cvidence la participation de 1'Cchange Cl-/MCO,- indkpendant du Na+. Au pH, alcalin. tet kchangeur pourrait mCdier une partie du rCtablissement du pH, de l'alcalinisation intracellulaire. Mots c k s : systkme nerveux central, regulation du pH, neurotransmetteur. [Traduit par la rCdaction]
Introduction The regulation af intracellular pH, the pHi, is important because of the large number of pH-sensitive processes (Ross and Boron 1981; Boron 1983; Busa and Nuccitelli 1984; FreIin et al. 1988; Madshus 1988). In the central nervous system changes in pHi modify various ionic conductances of excitable cells, and the electrical coupling between adjacent cells is blocked by decreased pHi (for references see Moody 1984). Nerve cells accumulate H9 after prolonged activity. In neurones of the snail Archidoris trains of action potentials decrease the pHi (Ahmed and Connor 1980). In contrast, the pHi of glial cells increases with electrical stimulation of nerve cells. The alkalinization of cortical rat astrocytes, far example, is triggered by membrane depolarization (Chesler and Kraig 1987, 1989). The pH of the interstitial space, the pH,, decreases during neuronal activity, often after an alkaline shift (Urbanics et al. 1983; Kraig et al. 1983). A full understanding of the role of pHi and pH, changes in affecting the hnctisn of the central nervous system requires a detailed knowledge of the mechanisms by which pH is regulated. The present article summarizes the research on acidbase physiology of nerve and glial cells and presents new data 'This paper was presented at the satellite symposium of the International Brain Research Organization meeting held August 10- 14, 1991, University of Saskatchewan, Saskatoon, Sask., Canada, entitled Ions, Water, and Energy in Brain Cells, and has undergone the Journal's usual peer review. Printed in Canada
1
(mprimC au Canada
with the goal of characterizing ion transporters involved in the pH shifts. 77te leech prep~ration The leech nervous system is particularly suitable for characterizing the ion transporters involved in the pH shifts. The ganglia and connectives show an identical organization, and they contain neuranes and glial cells that can be identified and distinguished according to their location and electrical properties (Schlue et ale 1980; Riehl and Schlue 1990).
pH measurernenFs The involvement of different pH, regulating mechanisms has been investigated in Retzius neurones and neuropile glial cells using ionselective microelectrodes for intracellular recording of ion activities. The neurones and glial tells can be penetrated successfully by several types of ion-selective microelectrodes for measuring rapid ion activity changes. The diagrams in Fig. I illustrate the arrangement for electrical recording with the tip of a double-barreled ion-selective microelectrode in a Retzius neuronae of a leech ganglion. The preparation and dissection procedures used to isolate single ganglia of the leech, Hirudo medicinalis, and the selection of cells have been described before (Schlue and Deitmer 1980). Solutio12~ The normal leech saline solution (nominally HC0,--free) had the following composition: NaCl, 115 mM; KCI, 4 mM; CaCl,, 1.8 8nR/8; HEPES (N-2-kydroxyethylpiperazine-N'-2-ethanesuiphonic acid) (adjusted to pH 7.4 with NaOH), 10 mM; and glucose, 11 mM. HCO,- solutions were equilibrated with 2% CO, and 98% oxygen
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Double - barreled microelectrode
Retzius neursne
Neutral -carrier
FIG. 1. Schematic drawing of the preparation, bath and the electrical recording system. (A) The neutral carrier barrel and the reference barrel of the microelectrode are each connected to two independent probes (E, and E,) of a differential electrometer by chloridized silver wires. The signal from the differential amplifier (DA) giving the pure intracellular ion measurement, aIon, and the signal from probe E,, giving the cell membrane potential, Em, were connected to two independent channels of a pen recorder. (B) Leech ganglion in cross section with the ventral side up. The electrode tip penetrated a Wetzius neurone.
and contained 11 mM NaHCO, instead of HEPES (pH 7.4). Naf -free solutions were made by replacing Na' with M-methyl+ glucamine neutralized with HC1 and (or) CO, as appropriate. The Cl--free solutions were made by equirrmolar replacements of the Clsalts with gluconate salts. Active pH, regulation has been investigated by inducing an intracellular rebound acidification by addition and subsequent removal of NH4CB or C0,-HC0,-. The recovery from intracellular rebound alkalosis was studied following the removal of 60 n.aM acetate from the superhsing saline solution. Solutions containing ammonium or acetate were made by the equimolar replacement of NaCl with either NH4Cl or sodium acetate, respectively. Amiloride (3,5-diamino-6-chloropyruinoy8 guanidine; Sigma Chemie GmbH, Deisenhofen, Germany), SITS (4-acetami.d04'-iso~i~yanatostilbene-2,2'-disulphonic acid; Sigma), DHDS (4,4'- diisothiocyanatostilkene-2,2'-disulphonic acid; Sigma). and the diuretic hrosemide (Sigma) were used within a few minutes of dissolving in solutions.
Microelecfrodes The pH-selective microclectrodes were made as described by Schlue and Thomas (1985). The silanized barrel was filled at the tip with a proton cocktail (tri-n-dodecylamine as H T ionophore: Ammann et al. 1981) and behind that with a Cl--containing citrate buffer, pH 6, saturated with CO,. Finally the reference barrel was filled with 3 M KC1 or with 0.5 M KZSO4 + 5 mM KC1 (in Cl--free experiments), and the microelectrode was tested.
Results Steady-state pl& The steady-state pHi values, measured in Retzius neurones and neuropile glial cells in different buffers, are summarized in Table 1. In both types of cells, a comparison of the naseasured membrane potential, Em, and the calculated equilibrium potential for H4 ions, EH+,showed that the pHi is maintained at values too high to be explained by passive H' ion movements. Therefore, the neurones and glial cells have
TABLEI. Entracellular pH (pH,), membrane potential (Em), and H + equilibrium potential (E,,) in Retzius neurones and neuropile glial cells of the leech nervous system Buffer
pH,
Em (mV)
Wetzius neursnes 7.28+0.10 -42.8k4.2 (n = 20) (n = 20 2% CO, - 11 mM HCO,- 7.20f 0.15 -49.3k3.4 (n = 10) (P? = 10) Neuropile gliaI cells HEPES 6.85f0.06 -68.4k7.5 (n = 23) (n = 23) 2% CO, - 11 mM HCO,- 7.18k0.13 -73.5f 5.6 (n = 25) (n = 25) HEBES
EH+(mV) -7.0 - 11.7
-32.2 -12.2
mechanisms for acid extrusion that maintain pHi at values ell above equilibrium.
Mechanisms qfpf.fi we demonstrated in several investigations that the p ~ i recovery from acidification of both leech neurones and glial cells in HEPES-buffered, nominally H@03--free saline sohtisn is mediated by Na+/HQexchange sensitive to inhibition by amilsride (Schlue and Thomas 1985; Sshlue and Deitmer 1987; Deitmer and Schlue 1987). Recovery o f p H i h m intracellujiar acidosis. by keech neurones in the prexence of C 0 2-HC03( I ) Depeszdence on external HC03In all invertebrate preparations so far investigated, however, H@Bpmions play a role in pHi regulation. In leech Wetzius neursnes the change from HEPES- to COT-HC03--buffered saline solution stimulates the rate of pHi recovery from intracellular acidosis, but the membrane potential remains unchanged (Fig. 2). The pHi recovery at pHi 6.6 was 0.030 pH units/min
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S286
CAN. J. PHYSIOL. PKIARMACOL. VOL. 70, 1992
FIG. 2. The effect of 2% CO, and I I mM HC8,- on the rate of
pH,recovery from acidification induced by NH,CI.
in HEPES-buffered saline solution and 0.101 pH unitstmin in the presence of 2 % C 0 2 and I I mM HC03-. Since the cell membrane is freely permeable to C 0 2 , intracellular buffering is also much higher in the presence of C 0 2 and HC03(Thomas 1976). Allowing for the increased buffering power in this case, acid extrusion was increased by a factor of 3.9 (see Schlue and Thomas (1985) for details).
(2) fie eflect qf amiloride The experiment illustrated in Fig. 3 shows that C02-HC03buffer overcomes inhibition by arniloride following acidification by removal of 20 mM NH4Cl. In a Retzius neurone the pHi recovery in HEPES-buffered saline solution was blocked by 2 mM amiloride. When C02-HC03--buffered saline solution with amiloride was applied, the pHi began to recover. This result also points to a HC03-dependent, but amilorideinsensitive pHi-regulating mechanism. E~ridencefor AJa -dependepilt C%-/HC03- exchange In the presence of C02-HC03- buffer, pHi recovery from intracellular acidosis could be mediated by Na'/H'/Cls/ HC03- exchange. This transport system is dependent on external H@03-, involves the efflux of Cl- ions, is SITS- or BIDS-sensitive, and has an absolute requirement for Na' (Thomas 1977). The system is thought to be electroneutral because pHi recovery occurs with no change in membrane potential, and membrane potential changes do not affect the rate of pHi recovery (Thsnsas 1978). +
(1) 11zfracel/u1arC P depjetiora or SITS and pH, recovery By analogy with other preparations it should be possible to inhibit pH, recovery in Beech neursnes by superfusing the preparation with Cl--free saline solution to deplete internal Cl-. The rate of pH, recovery (the slope of the alkalinization -WC03--induced acidification) was following the initial @02 slowed by the removal of external C1-, as shown in Fig. 4 for a Retzius neurone. The pHi recovery rate at pH, 7.2 in the control was 0.020 pH unitstrnin. At the same pHi in Cls-free saline solution the recovery rate was much slower, 0.006 pH unitstmin. The pHi recovery was totally blocked in Cl--free, amiloride-containing saline solution (Fig. 4). Table 2 summarizes the results of experiments of the type
FIG.3. The effect of arniloride on pHi recovery from acidification with and without 2% CO, and I I mM HC0,-. Acidificaticm was caused by NH,Cl. TABLE2. The pHi recovery of Retzius neurones from acid loads Cell
CO, -HC03-
Cls-free and CB, -HC03-
NOTE:Effect of CI--free saline solutions and C 0 2-HC03addition on dpH,ldt, the rate of pH, recovery, in pH unitstrnin. The dpH,/cft measuremenks were made at the same pH, 7.2. The control measurements summarized in the 2nd column (external buffer was switched from HEPES to @02 -H6O3-) were made at the beginning of each experiment, hefore applying Cl--free, 602-HC8,--buffered saline solution.
shown in Fig. 4 in which C02-HC03--buffered solution was added in the absence of external Cl-. In the neurones, SITS had only a moderate effect on the steady-state pHi in either HEPES- or C 0 2-H@03--buffere$ saline. However, when applied during an intracellular acidification by application and removal of Cq-HC03--buffered saline, SITS slowed the rate of pH, recovery, as shown in Fig. 5 for a Retzius neurone. The pHi recovery rate at pHi 7.2 in the control was 8.033 pH unitstmin. At the same pHi the SITS-containing saline solution reduced the rate of pHi recovery to 0.01%pH unitstmain. Table 3 summarizes the results of experiments of the type shown in Fig. 5 in which C02-HC03--buffered saline solutions were added in the presence of SITS or BIBS.
(2) pHi recovery in Na +ee, C02- NCOj---buflered saline ~o%utdons We have tested the effect of removing Na+ on pHi recovery of leech neurones in @02-HCQ--buffered solutions. In the +
SCHLUE AND D ~ R N E R
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Re tzius neurone
FIG. 4. The effect of removal of external CI- and application of 2 rnM amiloride on the pH, recovery from acidification induced by CO2-HC03-.
> f -LO - -60
e e e e
as
FIG. 5. The effect s f application of 1 mM SITS on the pHi recovery from acidification induced by C8,-HC0,-.
experiment illustrated in Fig. 6, a Retzius neurone was acidified by the addition and subsequent removal of 20 mM NM4Cl. The recoveries of pH, in normal saline solution are shown after the first and third exposure to 20 mM [email protected] the second exposure to NH4C1, there was no pH, recovery in Na -free, C 0 2- HC03--buffered saline; the pHi continued to decrease. As soon as external Na' was restored, the pHi recovered. +
Recovery of pHi $porn intracekduhr acidosis by Eeech gEid cskls in the presence 6.f C02-HC03The presence of CCI2-HCO3- increases the rate s f pHi recovery from intracellular acidosis not only in neursnes, but also in leech glial cells. The stimulation of pHi recovery from intraceilular acidification in the presence of CO2-HCQ3-
buffer appears to be mediated not only by Na+-dependent Cl-/HC03- exchange, but also by Na+ - HCQ3- cotramsport.
'
Evidence for Na -BBCQ3- cc~trarasporr (1) The increase ofpHi recovery from acidi$cafion in CQ2HCdB3- bufler The Na' - HC03- cotransporter depends on the presence sf Na+ and HC03- ions in the external solution, should be blocked by SITS or DIDS, but does not involve the efflux of Cl- ions frown the cell (Boron and Bgaulpaep 2983). This cgatransporter is electrogenic, and thought to tightly couple the nlovement of one Na' to the transport of two (or three) H@03- ions, thus leading to a transfer of net negative charge. The evidence that this cotransport mechanism exists in leech
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CAN. J. PHYSIOL. PHARMACOL. VOL. 70. 1992
-
Na free FIG.6 . The effect of removing external Na+ on pHi recovery from NW,Cl-induced acidification. During the experiment the solutions were buffered with 2% CO, and 11 mM HC8,- except during NH,Cl application.
TABLE3. The rates of pH, recovery (dpPI,/dt) in pH unitshin of Retzius neurones following acid loads under different experimental conditions (a) Effect of SITS (1 mM) in the presence of CO, -HCO,dpH,ldt -
-
Cell
CO, -H60,-
1 2 3 4
0.033 0.0144 0.024 0.065
Mean
8.042
-
- -
-
-
- -
SITS and C 0 2- HC03-
C8,
- HCB, -
(b) Effect of DIDS (1 mM) in the presence of C0,-HCO,dpPIi/dt Cell
CO, -HCO, -
on
BIDS and CO, -HC0,-
alkalinization and increased membrane potential (Fig. 7); (ik) alkalinization and the associated increase in membrane potential were inhibited in the absence of Na+ (Fig. 8) and in the presence of DIBS; and (iii) pHi recovery from intracellular acidosis still occurred in the absence of C1-, although the rate of pHi recovery was slowed.
on
C 0 2-HCO,
"
(2) nze aikalinizwti
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Received November 7, 1991 S c n ~ u e W.-R., , and D ~ R N E R 8 ., 1992. The regulation of pH in the central nervous system. Can. J. Physiol. Pharinacol. 70: S278 - ,9285. The pH, regulation from intracellular acidosis in the central nervous system appears to be mediated by mechanisms driven by the large inwardly directed Na+ gradient. The involvement of these mechanisms in pH, regulation of neurones and glial cells has been investigated in the leech central nervous system using ion-selective rnicroelectrodes. For recovery from acidification, there appear to be three separate mechanisms: Na+/H+ exchange, Na+-dependent Cl-/HC03- exchange, and Na+-HC0,- cotransport. All three meshanisms have a profound effect on the maintenance of pH, homeostasis in glial cells: whereas in leech neurones, as in other neuronal cells studied previously, the predominant mechanisms are Na+/H+ and Na'-dependent Cl-/HCO,- exchange. In addition to acid extrusion mechanisms we also found evidence for Nat-independent CI-/HCO,- exchange. At alkaline pH, this exchanger may mediate some of the pH, recovery from intracellular alkalinization. Key words: central nervous system, pH regulation, neurotransmitter. S c ~ i ~ uW.-R., e, and D ~ R N E R. R , 1992. The regulation of pH in the central nervous system. Can. J. Physiol. Pharmacol. 78: S278 - ,9285. La rCgulation du pH, aprks une acidose intracellulaire dans le systkme nerveux central semble &re mCdiCe par des mCcanismes contrB1Cs par le fort gradient de Na' dirigt vers l'intkrieur. On a examink l'implicatisn de ces mCcanisrnes dam la rkgulation du pH, des neurones et des cellules gliales, dans le systkme nerveux central de la sangsue, en utilisant des microClectrodes sensibles aux ions. Trois mecanisrnes distincts semblent participer au ritablissement de l'acidification : 19CchangeNa+/H+, 1'Cchange CI-/HC03- dependant du Naf et le cotransport de Na+ -HCO,-. Les trois mCcanismes ont un effet significatif sur le maintien de l'homeostasie du pH, dans Ies cellules gliales, alors que dans les neurones de la sangsue, cornme dans d'autres cellules neuronales examinCes antirieurement, les mCcanismes prkdominants sont 1'Cchange Na+/H6 et 1'Cchange Cl-/HC03- &pendant du Na'. En plus des mCcanismes d9Climination de l'acidite, nous avons aussi mis en Cvidence la participation de 1'Cchange Cl-/MCO,- indkpendant du Na+. Au pH, alcalin. tet kchangeur pourrait mCdier une partie du rCtablissement du pH, de l'alcalinisation intracellulaire. Mots c k s : systkme nerveux central, regulation du pH, neurotransmetteur. [Traduit par la rCdaction]
Introduction The regulation af intracellular pH, the pHi, is important because of the large number of pH-sensitive processes (Ross and Boron 1981; Boron 1983; Busa and Nuccitelli 1984; FreIin et al. 1988; Madshus 1988). In the central nervous system changes in pHi modify various ionic conductances of excitable cells, and the electrical coupling between adjacent cells is blocked by decreased pHi (for references see Moody 1984). Nerve cells accumulate H9 after prolonged activity. In neurones of the snail Archidoris trains of action potentials decrease the pHi (Ahmed and Connor 1980). In contrast, the pHi of glial cells increases with electrical stimulation of nerve cells. The alkalinization of cortical rat astrocytes, far example, is triggered by membrane depolarization (Chesler and Kraig 1987, 1989). The pH of the interstitial space, the pH,, decreases during neuronal activity, often after an alkaline shift (Urbanics et al. 1983; Kraig et al. 1983). A full understanding of the role of pHi and pH, changes in affecting the hnctisn of the central nervous system requires a detailed knowledge of the mechanisms by which pH is regulated. The present article summarizes the research on acidbase physiology of nerve and glial cells and presents new data 'This paper was presented at the satellite symposium of the International Brain Research Organization meeting held August 10- 14, 1991, University of Saskatchewan, Saskatoon, Sask., Canada, entitled Ions, Water, and Energy in Brain Cells, and has undergone the Journal's usual peer review. Printed in Canada
1
(mprimC au Canada
with the goal of characterizing ion transporters involved in the pH shifts. 77te leech prep~ration The leech nervous system is particularly suitable for characterizing the ion transporters involved in the pH shifts. The ganglia and connectives show an identical organization, and they contain neuranes and glial cells that can be identified and distinguished according to their location and electrical properties (Schlue et ale 1980; Riehl and Schlue 1990).
pH measurernenFs The involvement of different pH, regulating mechanisms has been investigated in Retzius neurones and neuropile glial cells using ionselective microelectrodes for intracellular recording of ion activities. The neurones and glial tells can be penetrated successfully by several types of ion-selective microelectrodes for measuring rapid ion activity changes. The diagrams in Fig. I illustrate the arrangement for electrical recording with the tip of a double-barreled ion-selective microelectrode in a Retzius neuronae of a leech ganglion. The preparation and dissection procedures used to isolate single ganglia of the leech, Hirudo medicinalis, and the selection of cells have been described before (Schlue and Deitmer 1980). Solutio12~ The normal leech saline solution (nominally HC0,--free) had the following composition: NaCl, 115 mM; KCI, 4 mM; CaCl,, 1.8 8nR/8; HEPES (N-2-kydroxyethylpiperazine-N'-2-ethanesuiphonic acid) (adjusted to pH 7.4 with NaOH), 10 mM; and glucose, 11 mM. HCO,- solutions were equilibrated with 2% CO, and 98% oxygen
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Double - barreled microelectrode
Retzius neursne
Neutral -carrier
FIG. 1. Schematic drawing of the preparation, bath and the electrical recording system. (A) The neutral carrier barrel and the reference barrel of the microelectrode are each connected to two independent probes (E, and E,) of a differential electrometer by chloridized silver wires. The signal from the differential amplifier (DA) giving the pure intracellular ion measurement, aIon, and the signal from probe E,, giving the cell membrane potential, Em, were connected to two independent channels of a pen recorder. (B) Leech ganglion in cross section with the ventral side up. The electrode tip penetrated a Wetzius neurone.
and contained 11 mM NaHCO, instead of HEPES (pH 7.4). Naf -free solutions were made by replacing Na' with M-methyl+ glucamine neutralized with HC1 and (or) CO, as appropriate. The Cl--free solutions were made by equirrmolar replacements of the Clsalts with gluconate salts. Active pH, regulation has been investigated by inducing an intracellular rebound acidification by addition and subsequent removal of NH4CB or C0,-HC0,-. The recovery from intracellular rebound alkalosis was studied following the removal of 60 n.aM acetate from the superhsing saline solution. Solutions containing ammonium or acetate were made by the equimolar replacement of NaCl with either NH4Cl or sodium acetate, respectively. Amiloride (3,5-diamino-6-chloropyruinoy8 guanidine; Sigma Chemie GmbH, Deisenhofen, Germany), SITS (4-acetami.d04'-iso~i~yanatostilbene-2,2'-disulphonic acid; Sigma), DHDS (4,4'- diisothiocyanatostilkene-2,2'-disulphonic acid; Sigma). and the diuretic hrosemide (Sigma) were used within a few minutes of dissolving in solutions.
Microelecfrodes The pH-selective microclectrodes were made as described by Schlue and Thomas (1985). The silanized barrel was filled at the tip with a proton cocktail (tri-n-dodecylamine as H T ionophore: Ammann et al. 1981) and behind that with a Cl--containing citrate buffer, pH 6, saturated with CO,. Finally the reference barrel was filled with 3 M KC1 or with 0.5 M KZSO4 + 5 mM KC1 (in Cl--free experiments), and the microelectrode was tested.
Results Steady-state pl& The steady-state pHi values, measured in Retzius neurones and neuropile glial cells in different buffers, are summarized in Table 1. In both types of cells, a comparison of the naseasured membrane potential, Em, and the calculated equilibrium potential for H4 ions, EH+,showed that the pHi is maintained at values too high to be explained by passive H' ion movements. Therefore, the neurones and glial cells have
TABLEI. Entracellular pH (pH,), membrane potential (Em), and H + equilibrium potential (E,,) in Retzius neurones and neuropile glial cells of the leech nervous system Buffer
pH,
Em (mV)
Wetzius neursnes 7.28+0.10 -42.8k4.2 (n = 20) (n = 20 2% CO, - 11 mM HCO,- 7.20f 0.15 -49.3k3.4 (n = 10) (P? = 10) Neuropile gliaI cells HEPES 6.85f0.06 -68.4k7.5 (n = 23) (n = 23) 2% CO, - 11 mM HCO,- 7.18k0.13 -73.5f 5.6 (n = 25) (n = 25) HEBES
EH+(mV) -7.0 - 11.7
-32.2 -12.2
mechanisms for acid extrusion that maintain pHi at values ell above equilibrium.
Mechanisms qfpf.fi we demonstrated in several investigations that the p ~ i recovery from acidification of both leech neurones and glial cells in HEPES-buffered, nominally H@03--free saline sohtisn is mediated by Na+/HQexchange sensitive to inhibition by amilsride (Schlue and Thomas 1985; Sshlue and Deitmer 1987; Deitmer and Schlue 1987). Recovery o f p H i h m intracellujiar acidosis. by keech neurones in the prexence of C 0 2-HC03( I ) Depeszdence on external HC03In all invertebrate preparations so far investigated, however, H@Bpmions play a role in pHi regulation. In leech Wetzius neursnes the change from HEPES- to COT-HC03--buffered saline solution stimulates the rate of pHi recovery from intracellular acidosis, but the membrane potential remains unchanged (Fig. 2). The pHi recovery at pHi 6.6 was 0.030 pH units/min
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S286
CAN. J. PHYSIOL. PKIARMACOL. VOL. 70, 1992
FIG. 2. The effect of 2% CO, and I I mM HC8,- on the rate of
pH,recovery from acidification induced by NH,CI.
in HEPES-buffered saline solution and 0.101 pH unitstmin in the presence of 2 % C 0 2 and I I mM HC03-. Since the cell membrane is freely permeable to C 0 2 , intracellular buffering is also much higher in the presence of C 0 2 and HC03(Thomas 1976). Allowing for the increased buffering power in this case, acid extrusion was increased by a factor of 3.9 (see Schlue and Thomas (1985) for details).
(2) fie eflect qf amiloride The experiment illustrated in Fig. 3 shows that C02-HC03buffer overcomes inhibition by arniloride following acidification by removal of 20 mM NH4Cl. In a Retzius neurone the pHi recovery in HEPES-buffered saline solution was blocked by 2 mM amiloride. When C02-HC03--buffered saline solution with amiloride was applied, the pHi began to recover. This result also points to a HC03-dependent, but amilorideinsensitive pHi-regulating mechanism. E~ridencefor AJa -dependepilt C%-/HC03- exchange In the presence of C02-HC03- buffer, pHi recovery from intracellular acidosis could be mediated by Na'/H'/Cls/ HC03- exchange. This transport system is dependent on external H@03-, involves the efflux of Cl- ions, is SITS- or BIDS-sensitive, and has an absolute requirement for Na' (Thomas 1977). The system is thought to be electroneutral because pHi recovery occurs with no change in membrane potential, and membrane potential changes do not affect the rate of pHi recovery (Thsnsas 1978). +
(1) 11zfracel/u1arC P depjetiora or SITS and pH, recovery By analogy with other preparations it should be possible to inhibit pH, recovery in Beech neursnes by superfusing the preparation with Cl--free saline solution to deplete internal Cl-. The rate of pH, recovery (the slope of the alkalinization -WC03--induced acidification) was following the initial @02 slowed by the removal of external C1-, as shown in Fig. 4 for a Retzius neurone. The pHi recovery rate at pH, 7.2 in the control was 0.020 pH unitstrnin. At the same pHi in Cls-free saline solution the recovery rate was much slower, 0.006 pH unitstmin. The pHi recovery was totally blocked in Cl--free, amiloride-containing saline solution (Fig. 4). Table 2 summarizes the results of experiments of the type
FIG.3. The effect of arniloride on pHi recovery from acidification with and without 2% CO, and I I mM HC0,-. Acidificaticm was caused by NH,Cl. TABLE2. The pHi recovery of Retzius neurones from acid loads Cell
CO, -HC03-
Cls-free and CB, -HC03-
NOTE:Effect of CI--free saline solutions and C 0 2-HC03addition on dpH,ldt, the rate of pH, recovery, in pH unitstrnin. The dpH,/cft measuremenks were made at the same pH, 7.2. The control measurements summarized in the 2nd column (external buffer was switched from HEPES to @02 -H6O3-) were made at the beginning of each experiment, hefore applying Cl--free, 602-HC8,--buffered saline solution.
shown in Fig. 4 in which C02-HC03--buffered solution was added in the absence of external Cl-. In the neurones, SITS had only a moderate effect on the steady-state pHi in either HEPES- or C 0 2-H@03--buffere$ saline. However, when applied during an intracellular acidification by application and removal of Cq-HC03--buffered saline, SITS slowed the rate of pH, recovery, as shown in Fig. 5 for a Retzius neurone. The pHi recovery rate at pHi 7.2 in the control was 8.033 pH unitstmin. At the same pHi the SITS-containing saline solution reduced the rate of pHi recovery to 0.01%pH unitstmain. Table 3 summarizes the results of experiments of the type shown in Fig. 5 in which C02-HC03--buffered saline solutions were added in the presence of SITS or BIBS.
(2) pHi recovery in Na +ee, C02- NCOj---buflered saline ~o%utdons We have tested the effect of removing Na+ on pHi recovery of leech neurones in @02-HCQ--buffered solutions. In the +
SCHLUE AND D ~ R N E R
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Re tzius neurone
FIG. 4. The effect of removal of external CI- and application of 2 rnM amiloride on the pH, recovery from acidification induced by CO2-HC03-.
> f -LO - -60
e e e e
as
FIG. 5. The effect s f application of 1 mM SITS on the pHi recovery from acidification induced by C8,-HC0,-.
experiment illustrated in Fig. 6, a Retzius neurone was acidified by the addition and subsequent removal of 20 mM NM4Cl. The recoveries of pH, in normal saline solution are shown after the first and third exposure to 20 mM [email protected] the second exposure to NH4C1, there was no pH, recovery in Na -free, C 0 2- HC03--buffered saline; the pHi continued to decrease. As soon as external Na' was restored, the pHi recovered. +
Recovery of pHi $porn intracekduhr acidosis by Eeech gEid cskls in the presence 6.f C02-HC03The presence of CCI2-HCO3- increases the rate s f pHi recovery from intracellular acidosis not only in neursnes, but also in leech glial cells. The stimulation of pHi recovery from intraceilular acidification in the presence of CO2-HCQ3-
buffer appears to be mediated not only by Na+-dependent Cl-/HC03- exchange, but also by Na+ - HCQ3- cotramsport.
'
Evidence for Na -BBCQ3- cc~trarasporr (1) The increase ofpHi recovery from acidi$cafion in CQ2HCdB3- bufler The Na' - HC03- cotransporter depends on the presence sf Na+ and HC03- ions in the external solution, should be blocked by SITS or DIDS, but does not involve the efflux of Cl- ions frown the cell (Boron and Bgaulpaep 2983). This cgatransporter is electrogenic, and thought to tightly couple the nlovement of one Na' to the transport of two (or three) H@03- ions, thus leading to a transfer of net negative charge. The evidence that this cotransport mechanism exists in leech
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CAN. J. PHYSIOL. PHARMACOL. VOL. 70. 1992
-
Na free FIG.6 . The effect of removing external Na+ on pHi recovery from NW,Cl-induced acidification. During the experiment the solutions were buffered with 2% CO, and 11 mM HC8,- except during NH,Cl application.
TABLE3. The rates of pH, recovery (dpPI,/dt) in pH unitshin of Retzius neurones following acid loads under different experimental conditions (a) Effect of SITS (1 mM) in the presence of CO, -HCO,dpH,ldt -
-
Cell
CO, -H60,-
1 2 3 4
0.033 0.0144 0.024 0.065
Mean
8.042
-
- -
-
-
- -
SITS and C 0 2- HC03-
C8,
- HCB, -
(b) Effect of DIDS (1 mM) in the presence of C0,-HCO,dpPIi/dt Cell
CO, -HCO, -
on
BIDS and CO, -HC0,-
alkalinization and increased membrane potential (Fig. 7); (ik) alkalinization and the associated increase in membrane potential were inhibited in the absence of Na+ (Fig. 8) and in the presence of DIBS; and (iii) pHi recovery from intracellular acidosis still occurred in the absence of C1-, although the rate of pHi recovery was slowed.
on
C 0 2-HCO,
"
(2) nze aikalinizwti
