- Email: [email protected]
The influence of calcium transients on intracellular pH in cortical neurons in primary culture
tamatc transients,
prcventcd
i, it ~18s proMAy not dub: to wuckrulcd that it was secondary tr) Ca”‘/21~ ’ cxchangc across plasma m~~n~rancs.
the rise, suggesting
KcyworJs: Intracellular
p
; Prrtracellular calcium; BCECF; Fura-,,7. Glutamate; --
There is an intimate relationship between cellular concentrations of Ca2 + and H+ [5,6,17]. For example, Ca2+ is extruded from cells or sequestered in endoplasmic reticulum in exchange for H+. The cycling of Ca2+ across mitochondrial membranes also involves H+ since primary Ca2+/2Na+ exchange is followed by Na+/H+ exchange. Furthermore, H+ and Ca2’ can bind to the same negative groups in macromolecules, meaning that Ca2+ can be displaced by H+, and vice versa ([1,4], see also [13]). Finally, a lowering of extra-
* Corresponding
author. Fax: (46) (46) 15-1480.
0006-8993/95/$09.50 0 1995 Elsevier Science B.V. Ali rights reserved SSDI 0006-8993(95)00056-9
Cortical IMIFOII -w
.) reduces calcium influx through chanDA) receptors nels gated by N-methyl-D-asparate ( n response to [l&20], as well as those which op depolarization [14]. The net result of these interactions is that although acidosis leads to an intracellular release of Ca”+ and to an increase in the free cytosolic calcium concentration (Ca:‘), the rise in Caf + caused ‘-induced depolarization is by glutamate exposure or blunted by acidosis [14,15]. Our results on the effect of changes in extra- and intracellular pH on Cai2+ led to the question of how calcium influx alters intracellular pH. The objective of the present study was to allow calcium entry by agonist-operated and voltage-sensitive calcium channels. and to record changes in pHi. In the midst of this
work, two articles appeared which showed that glutamate- and K’-induced calcium influx into hippoeampal cells in primary culture was associated with a marked decrease in pHi which was assumed to be caused by intracellular Ca’+/H’ exchange 19,101. Our results, obtained on cultures of neocortical neurons, proved to be radically different, suggesting that Ca’+/H+ interaction are different in hippocampal and neocortical neurons. 2, Materials and methods The methods used have previously been described [14,I5]. Primary neuronal cell cultures were prepared from I$- to 15-day-old embryos of Sprague-Dawley rats and grown in monolayers on coverslips precoated with collagen gel and poly+lysine. Cultures were regularly stained for neuron-specific enolase. As judged by phase-contrast microscopy, neurons (phase-bright and bearing extensive processes) were easy to identify morphologically [16]. Experiments were performed on single cells that had been in culture for 5-12 days. Culture-bearing coverslips were mounted in a closed 100 ~1 perfusion chamber, continuously perfused with temperature-controlled buffer at a rate of 125 ~1. min-‘. The chamber was placed on a Nikon inverted microscope (Nikon Diaphot TMD, Nikon Inc., Garden City, NY, USA) equipped with a CF Fluor 100 >( objective (N.A. 1.30). Medium changes were accelerated by increasing the speed of the driving peristaltic pump, allowing a total than e of the chamber conteut in about 5 s. Following recording of background signals at 30”C or 3s”C, cells were loaded by continuous perfusion of 2 FM BCBCF/AM for 25-30 min or 2 PM fur&Z/AM for 1 h. It was not necessary to use neutral density filters at this level of loading since we did not observe any loss of dye during experiments lasting up to two hours, indicating photic cell membrane injury. After washing, 15-30 min passed before any experiment was started. The fluorescence from single cells WBS monitored with a SPEX CM system microspectrofluorometer (model CMlTl lE, SPEX Industries Inc., Edison, NJ USA), connected to an IBM PC/AT computer (PS/2, IBM, USA). A 450 W Xenon lamp was used as the light source. The cytosolic fluorophore was excited by alternating the monochromator excitation at 490 nm and 440 nm for pH measurement or 340 nm and 380 nM for calcium measurement, the emission signals for respective wavelength being collected at 500 nm. Measurements were made every 0.3 s, and were integrated over 2 s. Calibrations for pH or ealeium measurements have been described elsewhere [I4-161. Briefly, at the end of the experiments pH calibration was made by exposing the cells to IO PM nigericin in the presence of 130 mM K+, at pH 6.40,
ducing calibration buffer (in MgCl,, 10 HEPES, PI-I 7.05) and either 2 mM CaCl, or 10 ccnce intensity was convert with the formula: [Ca’+ R)](F,/F,) where K, fura-2, R is the 340 nm/380 nm fluorescence ratio, and Rmin and R,, are the ratios in the absence and presence of saturating concentrations of c spectively. Values for Rmin and R,, were (n = 11) (mean + SD.) and 31.5 & ‘1.6, r F,/F, is the ratio of the 380 nm at zero and saturatin We also preformed the calibr of cells which had been tre ionophore-ionomycin at 2 EGTA) calcium concentrati bration procedure the Rmi, values were very variable, giving a large variation in baseline Ca”+. The values for R min were 0.96 + 0.24, n = 12. We, therefore, decided to use the calibration procedure without cel%s,a procedure which gave more consistant results. In experiments with measurements both of p Caf+ in the same cell, we first loaded the neurons with fura-2, induced the calcium transient by exposure, and then carried out the loading wi for pH, measurement in the same cell during glutamate administration. This procedure avoided an influence of BCECF on the fura- measurements. 2.1. Reagents of analytical grade and were used. BCECF/AM, fura- and fura-2/AM (i.e. the acetoxylmethylester of fura-2) were obtained from Molecular Probes, Eugene, OR, USA. All other chemicals were purchased from Sigma Chemical Co., St. Louis, MO, USA. BCECF/AM, fura-2/AM, and amiloride were dissolved in DMSO (dimethyl sulfonic oxide!, fura- in water, and nigericin, 4-bromo-A23187 and ionomycin in 95% alcohol. A nominally bicarbonate-free, HEPES-buffered solution was used, with the following composition (in mM): 140 Na+, 123 Cl; 5 K+, 1 Ca2+, 1 Mg2+, 5 glucose and 20 HEPES. In some experiments, a bicarbonate buffer was used, similar in composition to the HEPES buffer described above except that it contained 20 mM HCOj instead of HEPES. pH was measured on an ABL 30 Acid-Base Analyzer (Radiometer, Copenhagen, Denmark). The pH of the solution was normally 7.35 f 0.02. When needed, pH was varied by titration with HCl or NaOH. In all calciumfree solutions calcium was removed, and 100 PM EGTA was added. In the solution used to depolarise
f cells (7128) gluta asureable change i
oderatcly dccrcased
all these cells did not respond to glutamate exposure with a rise in Cai2+. Thus when 15 cells were exposed to glutamate after loading with fura-2, all of them responded with a rise in Ca 2+ from a baseline level of about 100 nM to 250-300 nM. It seems likely, therefore, that although all neocortical cells showed a rise in Ca?+ I they either showed no measurable response or
Table 1 The effect of glutamate
exposure (100 PM) on PHi in cortical
neurons
Small response No response Large response
Basline PH i
APHi
I1
7.04kO.13 7.06kO.11 7.1650.20
- 0.06 & 0.01 0 - 0.24 + 0.06
16 7 5
sponse encompassed a small reduction cells were tested for their response to N These results demonstrated that ah responded normally to acid transients, a small acidification in response to glutamate, or none at all. As a fura- and tamate with a no pH, response responded to rise in Ca?+. The cells were first loaded with and expos;d to glutamate to induce Caf ’ and then were loaded with BCECF for p ments. By performing both calcium and p ments on the same cell (n = 3) we could demonstrate that cells which showed a normal rise in Caf ’ (dCa:+
71 nM) responded to utarnatc with the small decrease in pH i (A PHI =ZO,O6 0.03) illustrated in Fig. la. The results obtained following glutamate exposure were thus different from those reported by Hartley and Dubinsky [O] and by Irwin et al. [IO], suggesting that hippocampal and neocortical cells differ in their responses to glutamate exposure and to the associated influx of Ca’+. The only similarity was that when cells responded with a (small) reduction in pH i, pM, increased above control after discontinuation of glutamate exposure. 3.2, Response of pHi to applicationof KC1
“I’iIhk 2 JpHi (+K)
H
HEPES (30” C) HEPES (35” C)
0. I? 4 0.04 0.18 * 0.05
6 8
-0.22_tO.O3 - cr.15It 0.04
6 6
Ca-free
0.21f0.05
3
-0.15~0.04
4
HCO; (35’ C)
0.18 * 0.08 _.
4
-0.21 _tO.W
1
similar in HEPES gesting that the
depolarization,
As illustrated in Fig. 2, exposure of cells to 50 mhl KC1invariably led to a peak rise in pHi. In some cells, there was complete or partial regulation towards normal during the K’-induced depolarization but, in others, the rise in pH, persisted during the exposure to KCl. Discontinuation of exposure to increased K+ concentration invariably gave rise to a ‘rebound’ alkalinization. However, unless pre-exposure pHi was high (about 7.2), pH, then stabilized at a higher value than before exposure. There was no age-related difference between cells showing different pattern in pHi response during KC1 application (cells age in all tree groups was from 7 to 9 days in vitro), Table 2 gives means & S.D. for the rise in pH, during exposure to KCl, and for the decrease in pH i upon discontinuation of exposure to high K+. The peak responses were rapid at 35°C. The responses were
-
demon
neurons. Since application of larisation and to influx of cal
cium ionophores. 3.3. Response Of
PHi
t0
ioflomycin and 4
equally rapid rise in pH i. As shown in Table 3 the rise units. The calcium
lhxiclina Before
a
7.08 f 0.06
b
6.96 ?:0.07
c
6.82 ?:0.18
After
n
7.10 izO.09
4
7.13 f 0.06
5
9.2
7.0 I 9.2
7.08 f 0.12
7
7.0
50 mM KCI
5 mln -
Fig. 2. The changes in pHi induced by exposure of cells to high potassium (SO mM, left panel) :*nd the pre- and post-exposure pH, baseline values. Potassium exposure indued a peak rise in pHi. In some cells, particularly those with more alkaline baseline level (see the right panel), this was followed by complete or partial recovery (a,bI. The removal of 50 mM potassium led to a transient acidification. In most of the cells the rise in pHi persisted during potassium exposure fc). In these cells, when exposure to high potassium was discontinued, pHi stabilized at higher level than pre-exposure baseline (c),
ionomycin exposur Since the rise in pH was not curtailed by amiloride (data not shown) or by removal of Na+ (Fig. 4a), it probably not due to active extrusion of H+ by Na+, or HCOi/Cl exchange. Furthermore, since the rise was equally large (and equally rapid) in the presence or nominal absence of HCO; ( ApH = 0.56 f 0.1 I in HCO;; ApH = 0.57 + 0.10 in HEPES), cotransport of HCO; and Na+ can also be excluded. However, since no rise in pH, occurred in calcium-free solution (Fig. 4b) influx of calcium seemed a prerequisite for the rise in pH i, probably by promoting CaZ+/2H+ exchange. We recognize that, in this respect. the response to ionomycin differs from that induced by exposure to KC1 (see section 4).
In order to discuss the present results, we find it justified to recall results pertaining to metabolism of
CaT’ with respect to p i transients in neocortical ct of exposure of these cells to neurons, and to the glutamate or raised ter that, we will address the question whether calcium influx during glutamate exposure, depolarisation by KCI, or treatment of cells with ionophores is what determines the response of pHi, or if other factors come into play. Finally, we will discuss differences in response bctwccn hippocampal and neocortical neurons.
b
+cB
.coz’
lonomycin
Table 3 The effect of ionomycin on intracellular pH in cortical neurons
(nM)
APHi
Id
339 + 97
0.57 + 0.10
6
ND
0.54+0.13
4
ND ND
0.50 + 0.04 0
4 4
ACaf+ lonomycin + Amiloride Na-free Ca-free
2+
5 min
Fir). 4. The effect of Na ‘-free and Ca’ *-free solutic* on ino induced pH, changes. In Na+-free solution (empty horizontal bar) the pH, decreased and stabilized at a lower pH, level. but the ionomycin-induced increase in pH, was not influenced (a). The alkalinization was dependent on extracellular calcium. since C’a’.free solution completely blocked the ionomycin-induced pH , change.
in
pHi was small, and not entirely CollSiStent. It iS tempting to conclude that the variability in response
reflects differential expression of glutamate receptors in the relatively young cells studied. However, all cells responded with a rise in Caf + upon glutamate exposure, Since changes in membrane potential are not known, we abstain from further analyses. It is of interest, though, that cells responding with a decrease in pII, showed rebound alkalinization (see below). (b) K +-induced depolarisation. Exposure to 50 mM KCl, which should depolarise cells appreciably, consistently increased PHi by about 0.20 units. Since the response was unaltered by removal of calcium from the external medium, and since such a procedure prevents Cat+ from increasing, the rise in pH i during KC1 exposure was clearly unrelated to calcium exchanges. In theory, depolarisation can increase PHi by triggering influx of HCO; via a Na+-2HCO; symporter 171. However, since the rise in PHi was uneffected by the nominal absence of HCO; another explanation must be sought. The most likely one is that the balance between influx and efflux of H+ is altered by the depolarisation. Thus, depolarisation must reduce the driving force for H’ influx and HCO; efflux, probably allowing the extrusion mechanisms to raise pH i, Clearly, this gives no role to Ca2+, which enters when cells are depolarised by KC1 in Ca%ontaining solutions (see above). One can only speculate that the slightly larger ApHi in a CaZf-free solution (albeit not significant) reflects a small acidifying effect of Ca2+ entry (cf. results on glutamate exposure).
4.4. Dijyerences between neocortical an neurons As stated in the introduction demonstrate that exposure of hip 500 PM (NMDA concentration 250 0.40-0.50 units [9, continued, with reb
only a small decrease in p
0.24 units), but all these had high initial p and a falling baseline; besides, PHi did not 6.90. We likewise failed to observe cell acidification upon exposure to KC1 The mechanism pro [9] and by Irwin et al. in response to glutamate or KC1 exposure was calciu influx and internal Ca2”/H+ exchange. One can talc late the corresponding amount of calcium which have to be taken up by cells to by suggcstcd mechanism. ity of cells is m 20 mM i by 0.5 units means th must have been displaced by Ca2+, requiring influx of millimolar amounts of Ca2+. It remains to be explained why hippocampal neurons react differently than neocortical neurons to calcium transients. It doesn’t seem established that the decrease in pH, during glutamate exposure is only due to internal Ca2+/2H+ exchange, since H+ influx through agonist-operated and voltage-sensitive cation channels is also likely, at least as long as the electrochemical potential favours such influx (or efflux of HCO;). The argument was raised by Irwin et al. [lo], though, that since a rise in pH, to 8.0 did not blunt the decrease in pH, during glutamate exposure, this decrease was not likely to reflect passive H+ influx. Perhaps the easiest way to explain the different behaviour of hippocampal and neocortical neurons is to assume that the former have a high density of cation-permeable channels which are activated by glutamate, or KCI, leading to influx of Ca2+ and H+ with subsequent cell acidification. Alternatively, or in addition, receptor activation could increase the intra??
exposure to Ca’+”ionophores tained in hepatocytes [23or in ranulosa cells [3] since calcium influx and the dasociated rise in Ca:+ triggered a large rise in pH,. This was not due stimulation of H” extrusion since neither amiloride, which inhibits the Na.‘/H+ exchanger in neocortical neurons [16], nor removal of Na+ affected the rise. However, since removal of Cal+ prevented the rise, it probably reflected Ca2+/2H+ exchange across the plasma membrane (see [2]). We recognize that calcium influx in exchange for H+ should alkalinize the cell while, once in the cell, Ca*+ can exchange for HS, thereby acidifying the Woplasmic phase. Obviously, the calcium ionophores gave rise to a massive influx of calcium, explaining how PHi could change by as much as OS units. Furthermore, any Ca%nduced release of H+ from internal stores must have been concealed by the massive efflux of H+. The results thus demonstrate that if the mechanism of Ca”+ influx involves exchange between Ca2+ and H” across plasma membranes, calcium influx leads to alkalosis, not acidosis. Furthermore, experiments The results followi
= 0.06 units) follow-
265 t 1977) 867-879. OuYang. Y.B., Kristian.
creases of intracellular calcium cone
Faculty,
University
ration
in cortical
neu-
of Lund. ,, MiStWicitl
overview. Calcium,
ud.
ation of the orad, M., Proc. NilI/. extracellu Acad. Sci. USA, 87 (1990) 6445-6449. Traynelis, S. and Cull-Candy, S., Proton inhibition of N-methylo-aspartate receptors in cerebellar neurms, Numw, 345 (1990) 347-350. Vyklicky, L., Vlachova, V. and Krusek, J., The effect of external pH changes on responses to excitatory amino acids in mouse hippocam\,al neurons, J. Physiol., 430 (1990) 497-517.
and
nel
[l] Abercrombie, RF. and art. C.E., Calcium and proton buffering and diffusion in isolated cytoplasm from myxicola axons, Am. J. Physiol., 250 (1986) C391-C405. [2] Anwer, M.S., M ec h anism of ionomycin-induced intraceiiuiar alkalinization of rat hepatocytes, Hepatology, 18 (1993) 433-439. [3] Asem E.M., Li M. and Tsang B.K., Calcium ionophores increase
isc~~erni~, arKi 661.
, 522 (198
prcventcd
i, it ~18s proMAy not dub: to wuckrulcd that it was secondary tr) Ca”‘/21~ ’ cxchangc across plasma m~~n~rancs.
the rise, suggesting
KcyworJs: Intracellular
p
; Prrtracellular calcium; BCECF; Fura-,,7. Glutamate; --
There is an intimate relationship between cellular concentrations of Ca2 + and H+ [5,6,17]. For example, Ca2+ is extruded from cells or sequestered in endoplasmic reticulum in exchange for H+. The cycling of Ca2+ across mitochondrial membranes also involves H+ since primary Ca2+/2Na+ exchange is followed by Na+/H+ exchange. Furthermore, H+ and Ca2’ can bind to the same negative groups in macromolecules, meaning that Ca2+ can be displaced by H+, and vice versa ([1,4], see also [13]). Finally, a lowering of extra-
* Corresponding
author. Fax: (46) (46) 15-1480.
0006-8993/95/$09.50 0 1995 Elsevier Science B.V. Ali rights reserved SSDI 0006-8993(95)00056-9
Cortical IMIFOII -w
.) reduces calcium influx through chanDA) receptors nels gated by N-methyl-D-asparate ( n response to [l&20], as well as those which op depolarization [14]. The net result of these interactions is that although acidosis leads to an intracellular release of Ca”+ and to an increase in the free cytosolic calcium concentration (Ca:‘), the rise in Caf + caused ‘-induced depolarization is by glutamate exposure or blunted by acidosis [14,15]. Our results on the effect of changes in extra- and intracellular pH on Cai2+ led to the question of how calcium influx alters intracellular pH. The objective of the present study was to allow calcium entry by agonist-operated and voltage-sensitive calcium channels. and to record changes in pHi. In the midst of this
work, two articles appeared which showed that glutamate- and K’-induced calcium influx into hippoeampal cells in primary culture was associated with a marked decrease in pHi which was assumed to be caused by intracellular Ca’+/H’ exchange 19,101. Our results, obtained on cultures of neocortical neurons, proved to be radically different, suggesting that Ca’+/H+ interaction are different in hippocampal and neocortical neurons. 2, Materials and methods The methods used have previously been described [14,I5]. Primary neuronal cell cultures were prepared from I$- to 15-day-old embryos of Sprague-Dawley rats and grown in monolayers on coverslips precoated with collagen gel and poly+lysine. Cultures were regularly stained for neuron-specific enolase. As judged by phase-contrast microscopy, neurons (phase-bright and bearing extensive processes) were easy to identify morphologically [16]. Experiments were performed on single cells that had been in culture for 5-12 days. Culture-bearing coverslips were mounted in a closed 100 ~1 perfusion chamber, continuously perfused with temperature-controlled buffer at a rate of 125 ~1. min-‘. The chamber was placed on a Nikon inverted microscope (Nikon Diaphot TMD, Nikon Inc., Garden City, NY, USA) equipped with a CF Fluor 100 >( objective (N.A. 1.30). Medium changes were accelerated by increasing the speed of the driving peristaltic pump, allowing a total than e of the chamber conteut in about 5 s. Following recording of background signals at 30”C or 3s”C, cells were loaded by continuous perfusion of 2 FM BCBCF/AM for 25-30 min or 2 PM fur&Z/AM for 1 h. It was not necessary to use neutral density filters at this level of loading since we did not observe any loss of dye during experiments lasting up to two hours, indicating photic cell membrane injury. After washing, 15-30 min passed before any experiment was started. The fluorescence from single cells WBS monitored with a SPEX CM system microspectrofluorometer (model CMlTl lE, SPEX Industries Inc., Edison, NJ USA), connected to an IBM PC/AT computer (PS/2, IBM, USA). A 450 W Xenon lamp was used as the light source. The cytosolic fluorophore was excited by alternating the monochromator excitation at 490 nm and 440 nm for pH measurement or 340 nm and 380 nM for calcium measurement, the emission signals for respective wavelength being collected at 500 nm. Measurements were made every 0.3 s, and were integrated over 2 s. Calibrations for pH or ealeium measurements have been described elsewhere [I4-161. Briefly, at the end of the experiments pH calibration was made by exposing the cells to IO PM nigericin in the presence of 130 mM K+, at pH 6.40,
ducing calibration buffer (in MgCl,, 10 HEPES, PI-I 7.05) and either 2 mM CaCl, or 10 ccnce intensity was convert with the formula: [Ca’+ R)](F,/F,) where K, fura-2, R is the 340 nm/380 nm fluorescence ratio, and Rmin and R,, are the ratios in the absence and presence of saturating concentrations of c spectively. Values for Rmin and R,, were (n = 11) (mean + SD.) and 31.5 & ‘1.6, r F,/F, is the ratio of the 380 nm at zero and saturatin We also preformed the calibr of cells which had been tre ionophore-ionomycin at 2 EGTA) calcium concentrati bration procedure the Rmi, values were very variable, giving a large variation in baseline Ca”+. The values for R min were 0.96 + 0.24, n = 12. We, therefore, decided to use the calibration procedure without cel%s,a procedure which gave more consistant results. In experiments with measurements both of p Caf+ in the same cell, we first loaded the neurons with fura-2, induced the calcium transient by exposure, and then carried out the loading wi for pH, measurement in the same cell during glutamate administration. This procedure avoided an influence of BCECF on the fura- measurements. 2.1. Reagents of analytical grade and were used. BCECF/AM, fura- and fura-2/AM (i.e. the acetoxylmethylester of fura-2) were obtained from Molecular Probes, Eugene, OR, USA. All other chemicals were purchased from Sigma Chemical Co., St. Louis, MO, USA. BCECF/AM, fura-2/AM, and amiloride were dissolved in DMSO (dimethyl sulfonic oxide!, fura- in water, and nigericin, 4-bromo-A23187 and ionomycin in 95% alcohol. A nominally bicarbonate-free, HEPES-buffered solution was used, with the following composition (in mM): 140 Na+, 123 Cl; 5 K+, 1 Ca2+, 1 Mg2+, 5 glucose and 20 HEPES. In some experiments, a bicarbonate buffer was used, similar in composition to the HEPES buffer described above except that it contained 20 mM HCOj instead of HEPES. pH was measured on an ABL 30 Acid-Base Analyzer (Radiometer, Copenhagen, Denmark). The pH of the solution was normally 7.35 f 0.02. When needed, pH was varied by titration with HCl or NaOH. In all calciumfree solutions calcium was removed, and 100 PM EGTA was added. In the solution used to depolarise
f cells (7128) gluta asureable change i
oderatcly dccrcased
all these cells did not respond to glutamate exposure with a rise in Cai2+. Thus when 15 cells were exposed to glutamate after loading with fura-2, all of them responded with a rise in Ca 2+ from a baseline level of about 100 nM to 250-300 nM. It seems likely, therefore, that although all neocortical cells showed a rise in Ca?+ I they either showed no measurable response or
Table 1 The effect of glutamate
exposure (100 PM) on PHi in cortical
neurons
Small response No response Large response
Basline PH i
APHi
I1
7.04kO.13 7.06kO.11 7.1650.20
- 0.06 & 0.01 0 - 0.24 + 0.06
16 7 5
sponse encompassed a small reduction cells were tested for their response to N These results demonstrated that ah responded normally to acid transients, a small acidification in response to glutamate, or none at all. As a fura- and tamate with a no pH, response responded to rise in Ca?+. The cells were first loaded with and expos;d to glutamate to induce Caf ’ and then were loaded with BCECF for p ments. By performing both calcium and p ments on the same cell (n = 3) we could demonstrate that cells which showed a normal rise in Caf ’ (dCa:+
71 nM) responded to utarnatc with the small decrease in pH i (A PHI =ZO,O6 0.03) illustrated in Fig. la. The results obtained following glutamate exposure were thus different from those reported by Hartley and Dubinsky [O] and by Irwin et al. [IO], suggesting that hippocampal and neocortical cells differ in their responses to glutamate exposure and to the associated influx of Ca’+. The only similarity was that when cells responded with a (small) reduction in pH i, pM, increased above control after discontinuation of glutamate exposure. 3.2, Response of pHi to applicationof KC1
“I’iIhk 2 JpHi (+K)
H
HEPES (30” C) HEPES (35” C)
0. I? 4 0.04 0.18 * 0.05
6 8
-0.22_tO.O3 - cr.15It 0.04
6 6
Ca-free
0.21f0.05
3
-0.15~0.04
4
HCO; (35’ C)
0.18 * 0.08 _.
4
-0.21 _tO.W
1
similar in HEPES gesting that the
depolarization,
As illustrated in Fig. 2, exposure of cells to 50 mhl KC1invariably led to a peak rise in pHi. In some cells, there was complete or partial regulation towards normal during the K’-induced depolarization but, in others, the rise in pH, persisted during the exposure to KCl. Discontinuation of exposure to increased K+ concentration invariably gave rise to a ‘rebound’ alkalinization. However, unless pre-exposure pHi was high (about 7.2), pH, then stabilized at a higher value than before exposure. There was no age-related difference between cells showing different pattern in pHi response during KC1 application (cells age in all tree groups was from 7 to 9 days in vitro), Table 2 gives means & S.D. for the rise in pH, during exposure to KCl, and for the decrease in pH i upon discontinuation of exposure to high K+. The peak responses were rapid at 35°C. The responses were
-
demon
neurons. Since application of larisation and to influx of cal
cium ionophores. 3.3. Response Of
PHi
t0
ioflomycin and 4
equally rapid rise in pH i. As shown in Table 3 the rise units. The calcium
lhxiclina Before
a
7.08 f 0.06
b
6.96 ?:0.07
c
6.82 ?:0.18
After
n
7.10 izO.09
4
7.13 f 0.06
5
9.2
7.0 I 9.2
7.08 f 0.12
7
7.0
50 mM KCI
5 mln -
Fig. 2. The changes in pHi induced by exposure of cells to high potassium (SO mM, left panel) :*nd the pre- and post-exposure pH, baseline values. Potassium exposure indued a peak rise in pHi. In some cells, particularly those with more alkaline baseline level (see the right panel), this was followed by complete or partial recovery (a,bI. The removal of 50 mM potassium led to a transient acidification. In most of the cells the rise in pHi persisted during potassium exposure fc). In these cells, when exposure to high potassium was discontinued, pHi stabilized at higher level than pre-exposure baseline (c),
ionomycin exposur Since the rise in pH was not curtailed by amiloride (data not shown) or by removal of Na+ (Fig. 4a), it probably not due to active extrusion of H+ by Na+, or HCOi/Cl exchange. Furthermore, since the rise was equally large (and equally rapid) in the presence or nominal absence of HCO; ( ApH = 0.56 f 0.1 I in HCO;; ApH = 0.57 + 0.10 in HEPES), cotransport of HCO; and Na+ can also be excluded. However, since no rise in pH, occurred in calcium-free solution (Fig. 4b) influx of calcium seemed a prerequisite for the rise in pH i, probably by promoting CaZ+/2H+ exchange. We recognize that, in this respect. the response to ionomycin differs from that induced by exposure to KC1 (see section 4).
In order to discuss the present results, we find it justified to recall results pertaining to metabolism of
CaT’ with respect to p i transients in neocortical ct of exposure of these cells to neurons, and to the glutamate or raised ter that, we will address the question whether calcium influx during glutamate exposure, depolarisation by KCI, or treatment of cells with ionophores is what determines the response of pHi, or if other factors come into play. Finally, we will discuss differences in response bctwccn hippocampal and neocortical neurons.
b
+cB
.coz’
lonomycin
Table 3 The effect of ionomycin on intracellular pH in cortical neurons
(nM)
APHi
Id
339 + 97
0.57 + 0.10
6
ND
0.54+0.13
4
ND ND
0.50 + 0.04 0
4 4
ACaf+ lonomycin + Amiloride Na-free Ca-free
2+
5 min
Fir). 4. The effect of Na ‘-free and Ca’ *-free solutic* on ino induced pH, changes. In Na+-free solution (empty horizontal bar) the pH, decreased and stabilized at a lower pH, level. but the ionomycin-induced increase in pH, was not influenced (a). The alkalinization was dependent on extracellular calcium. since C’a’.free solution completely blocked the ionomycin-induced pH , change.
in
pHi was small, and not entirely CollSiStent. It iS tempting to conclude that the variability in response
reflects differential expression of glutamate receptors in the relatively young cells studied. However, all cells responded with a rise in Caf + upon glutamate exposure, Since changes in membrane potential are not known, we abstain from further analyses. It is of interest, though, that cells responding with a decrease in pII, showed rebound alkalinization (see below). (b) K +-induced depolarisation. Exposure to 50 mM KCl, which should depolarise cells appreciably, consistently increased PHi by about 0.20 units. Since the response was unaltered by removal of calcium from the external medium, and since such a procedure prevents Cat+ from increasing, the rise in pH i during KC1 exposure was clearly unrelated to calcium exchanges. In theory, depolarisation can increase PHi by triggering influx of HCO; via a Na+-2HCO; symporter 171. However, since the rise in PHi was uneffected by the nominal absence of HCO; another explanation must be sought. The most likely one is that the balance between influx and efflux of H+ is altered by the depolarisation. Thus, depolarisation must reduce the driving force for H’ influx and HCO; efflux, probably allowing the extrusion mechanisms to raise pH i, Clearly, this gives no role to Ca2+, which enters when cells are depolarised by KC1 in Ca%ontaining solutions (see above). One can only speculate that the slightly larger ApHi in a CaZf-free solution (albeit not significant) reflects a small acidifying effect of Ca2+ entry (cf. results on glutamate exposure).
4.4. Dijyerences between neocortical an neurons As stated in the introduction demonstrate that exposure of hip 500 PM (NMDA concentration 250 0.40-0.50 units [9, continued, with reb
only a small decrease in p
0.24 units), but all these had high initial p and a falling baseline; besides, PHi did not 6.90. We likewise failed to observe cell acidification upon exposure to KC1 The mechanism pro [9] and by Irwin et al. in response to glutamate or KC1 exposure was calciu influx and internal Ca2”/H+ exchange. One can talc late the corresponding amount of calcium which have to be taken up by cells to by suggcstcd mechanism. ity of cells is m 20 mM i by 0.5 units means th must have been displaced by Ca2+, requiring influx of millimolar amounts of Ca2+. It remains to be explained why hippocampal neurons react differently than neocortical neurons to calcium transients. It doesn’t seem established that the decrease in pH, during glutamate exposure is only due to internal Ca2+/2H+ exchange, since H+ influx through agonist-operated and voltage-sensitive cation channels is also likely, at least as long as the electrochemical potential favours such influx (or efflux of HCO;). The argument was raised by Irwin et al. [lo], though, that since a rise in pH, to 8.0 did not blunt the decrease in pH, during glutamate exposure, this decrease was not likely to reflect passive H+ influx. Perhaps the easiest way to explain the different behaviour of hippocampal and neocortical neurons is to assume that the former have a high density of cation-permeable channels which are activated by glutamate, or KCI, leading to influx of Ca2+ and H+ with subsequent cell acidification. Alternatively, or in addition, receptor activation could increase the intra??
exposure to Ca’+”ionophores tained in hepatocytes [23or in ranulosa cells [3] since calcium influx and the dasociated rise in Ca:+ triggered a large rise in pH,. This was not due stimulation of H” extrusion since neither amiloride, which inhibits the Na.‘/H+ exchanger in neocortical neurons [16], nor removal of Na+ affected the rise. However, since removal of Cal+ prevented the rise, it probably reflected Ca2+/2H+ exchange across the plasma membrane (see [2]). We recognize that calcium influx in exchange for H+ should alkalinize the cell while, once in the cell, Ca*+ can exchange for HS, thereby acidifying the Woplasmic phase. Obviously, the calcium ionophores gave rise to a massive influx of calcium, explaining how PHi could change by as much as OS units. Furthermore, any Ca%nduced release of H+ from internal stores must have been concealed by the massive efflux of H+. The results thus demonstrate that if the mechanism of Ca”+ influx involves exchange between Ca2+ and H” across plasma membranes, calcium influx leads to alkalosis, not acidosis. Furthermore, experiments The results followi
= 0.06 units) follow-
265 t 1977) 867-879. OuYang. Y.B., Kristian.
creases of intracellular calcium cone
Faculty,
University
ration
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neu-
of Lund. ,, MiStWicitl
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ud.
ation of the orad, M., Proc. NilI/. extracellu Acad. Sci. USA, 87 (1990) 6445-6449. Traynelis, S. and Cull-Candy, S., Proton inhibition of N-methylo-aspartate receptors in cerebellar neurms, Numw, 345 (1990) 347-350. Vyklicky, L., Vlachova, V. and Krusek, J., The effect of external pH changes on responses to excitatory amino acids in mouse hippocam\,al neurons, J. Physiol., 430 (1990) 497-517.
and
nel
[l] Abercrombie, RF. and art. C.E., Calcium and proton buffering and diffusion in isolated cytoplasm from myxicola axons, Am. J. Physiol., 250 (1986) C391-C405. [2] Anwer, M.S., M ec h anism of ionomycin-induced intraceiiuiar alkalinization of rat hepatocytes, Hepatology, 18 (1993) 433-439. [3] Asem E.M., Li M. and Tsang B.K., Calcium ionophores increase
isc~~erni~, arKi 661.
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