- Email: [email protected]
Intracellular PH of the amphibian brain incubated in vitro
Life Sciences Vol . 7, Part I, pp. 499-504, 1968 Printed in Great Britain .
Pergamon Press
INfRACSLLUIAR PH OF THB AMPHIBLf1N BRAIN I1~UBATSD IN VITRO Glen Grayman, Michael W. B . Bradbury and Charlu R. iClsosn Division of Medicine, Cedars-Sinai Radical Center and Research Instituts, and Departrnts of Physiology and Medicine, University of California at Los Angeles Medical Cantor, Los Angela, California
(Received 22 December 1967; in final form 19 February 1968)
la ,
Thn distribution of a weak acid, S,5-disethylaacasolidine-2,4- thons-C
(DMO), batwun intracellular and a:traeallular fluid (MCF), a rthod introduced by Aaddell and Butler (1), has bun used to calculate a value for the pH of the intracellular fluid of a nuober of tissues, both in vivo and in v rn . The rthod has bean applied to mosvalian brain~~n - y,~~ (2,3) . iwo difficulties are inherent in this approach .
Firstly, it is hard to arrive
at a reliable estimate of the extracnllular space of brain ~a vivo and secondly, the concentration of Di!!0 in the SCF is apt accessible oven to indirect measurement, since the DMO concentrations in blood pissed and in CSF may be different and not ratted to the pH difference betwun the fluids (4) . The frog brain may be well snintainad ~ vitro without swsl-liag or loss of intracel.luLr potassium (5,6) .
Further, its extracnllular space may ba
measured with fair precision from the voler of distribution of S35 -sulphate (6) .
Hence, the frog brain seemed an excellent in vitro preparation with
which to measure intncelluLr pH from the distribution of DMO and is which to study thn affect of various factors on it . Methods Thn'axperimants warn performed on whole brains from frogs, Raaa pipieos, of 8.5 to 9 .5 cm in body length, during the months of July, August and September .
The methods of removing the brain, of incubation in vitro , of attracting
and estimating chloride, sodium and potassium have been described (6) . All incubating fluids contained l~l 2 .0 mti, l~I2P04 3.0 ~1, CaC12 0 .9 d4, MgC1 2
499
AMPHIBIAN BRAIN
500
Vol . 7, No. 9
1 .2 eài, Ha 2S04 1 .8 mèl and glucose 13 .8 m!! and vara kept saturated during use with 95x 02 Sx C02. The total sodium concentration vas 118 .5 mEq/L in all fluide, but the aaounts of chloride and bicarbomte vnre varied to give fluids of different pH, thw HaHC03, mM
pH
(added)
(added)
(measured)
Horen1
90
25
7 .26
Alkaline
45
70
7 .72
105
10
6.87
HaCl, mH
Acid
Tha incubating fluids all caatainad C14-~, O .lpc/ml, and S 35 -sulphate, g350 .25pc/sl . The contributions to the radiaactivlty from C14 -DHO and sulphate in the diluted fluids and wtar sxtracts of brain, ware nstiosted by the technique already described for separating C14 -inulin and S35 -sulphate (6) . The axpariaeats ware run at a rocs ta~paratura of 22-24oC .
The pH of
the imubating fluid ws detersdnad with a Radiasetnr capillary microelectroda.
IntracalluLr pH, pHi, ws calculated froze the equation (~i01) (1 + 10~ - ~)
pHi ~ p& + log
(tt~ioe) whew pHa is the pH of the incubating fluid, D!!Oe is the total concentration of radioactive DZiO, ionised and non-ioni:ed, in the fluid, IIiO i is the total concentration of iatracallular radioactive DMO, calculated from the DMO content, the roter contant and thn sulphate space of the brain, and pK is the pH at which ~!D is half dissociated. aesults In Fig. 1 ara recorded mean values for pHi in incubation fluide of different pH and for times of incubation between 30 minutes and 3 hours . The values of pHi ara imrariably leas at 30 min than at Lter times, probably because of incosplata equilibration of the ßi0 in the cells, but thereafter
Vol. 7, No . 9
AMPHIBIAN BRAIN
501
ze
yH
(~t ._______ ...._.I 1
0
s.s Houes FIG . 1
Mean calculated values of pH i (.~~- .) during incubation of frog brains in media of different pH e ( ---- ), Limita are _+ 2 S,E, of 4-6 brains . pH i was fairly stable .
Thus S 35 -sulphate, C1 36 and Na24 are normally cam-
pletely equilibrated in the ECF of the isolated frog brain at 30 min (6), but entry of solutes into the cells may be slaver, urea reaching 80-85Z of its equilibrium value at this time (unpublished observations) .
At extra-
cellular pH of 7 .26, the intracellular fluid sas slightly acre alkaline, mean 7 .29 (1, 2 and 3 hours values), than the medisa .
In the acid fluid of
pH 6 .87, pH i was markedly more alkaline at 7 .01, and in the alkaline medisas of 7 .72 sas acre acid at 7 .62 .
There was no significant change in the intra-
cellular potassium concentration from the control value of 124 .2 ± 1 .6 mEq/L in the alkaline fluid, 122 .6 ± 4 .8 mEq/L, or in thn acid fluid s 113 .9 ± 7 .4 r~Eq/L, though in the latter case the value was suggestively less . Both ouabain 10 -5M and the combined metabolic inhibitora~ NaCN 5x10 -3M and Na iodoacetate 10 -3M (Table 1) caused a considerable fall in the ratio ~i/~e~ the mean values being .67 and .48 respectively (2 and 3 hour
502
AMPHIBIAN BRAIN
values) . the cells.
Vol. 7, No. 9
Under these conditions there may be penetration of sulphate into Hence the calculated values of pHi, 7 .1 and 7 .0, are likely to
be, if anything, too alkaline .
Under these conditions the intracellular
potassium concentration fall to 26 .5 ± 2 .1 mSq/L (ouabain) and 20 .6 ± 0 .9 mBq/L (NaCN and Na iodoacetate) . TABLB 1 . The Effect of Inhibitors on the Ratio, DI~i/DlSOn for Frog Brain in vitro 1 2 hour
1 hour
2 hours
3 hours
Normal madiuo
.90 + .02
1 .05 + .OL
1 .10 + .02
1 .10 + .04
Ouabaia LO -5!1
.66 + .02
.68 + .02
.68 + .02
.66 + .O1
NaCN, 5x10 -3M -3 Na iodoacetata 10 M
.44 + .03
.53 + .O1
.46 + .09
.50 + .04
Valuaa are means
standard errors of 4-6 brains . Discussion
The value of 7 .29 for the intracellular pH of frog brain in vitro is conaidarably more alkaline than figures given for mammrlian brain in vivo (2,3), despite a presumed mor~ acid pH of the extracnllular fluid in the in vitro axperimant .
Kiblar, O'Neill and Robin (2) and Rooa (3) give figures of
7 .08 and 7 .13 for dog and cat cerebral cortex reapactively .
These values
were calculated on the assumption of a 4-SX extracellular space .
As both
groups point out, s larger apace would give a somewhat lower pH .
Whether
our higher value represents a species difference or is dun to the very differont tachaique is not completely clear.
It should be noted, however, that
the concentration of uadiatociatad D!W in CSF may be late than that in either arterial or va~ua blood in some special (4) .
Thus, if the arterial or
venous blood concentration of D~l10 ie used for DMOn, the calculated value for pHi of brain may be too low because the CSF conentratioa of 0!!0 probably more nearly represents the concentration in the extracelluLr fluid of
Vol. 7, No . 9
brain (7) .
AMPHIBIAN BRAIN
&abler et al . (2) did sot measure CSF D!!O .
503
Row (3) found the
uadissx sated iglO in CSF to be .97 of that in arterial blood and .88 of that is venous blood in the cat . The changes of pHi in Low and high bicarbonate media warn much greater in the isolated frog brainy than occurred in vivo whether the pHa ws altered by varying the plasma HC03 or the C02 taosion.
This may ba due to
the circumventing of the blood-brain barriers which at least in the mammalian brain appears to be an important fu for in stabilising the pH of the astracellular fluid of brain .
Ia our preparation pHi is prasarbly solely
determined by events at the membranes of the brain calls, whereas in vivo , the obse:vad behavior is likely to ba the sum of transport activity at the blood-brain barrier and at the brain call membranes . ~n air case, is coon with pH i for other tissues in vivo and in r~itro measured from the distribution of ixi0, the value for pH i of frog brain in vitro is much more alkaline than would occur motor a passive distribution of
/ H y is .93 at a pHa of 7 .26. CHiJ C a It increases to 1 .26 in the alkaline medium and decreases to :73 in the uid H+ across the call membrane . medium .
The ratio
If the discrepancies in the ratios from that predicted on the basis
of passive distribution, i .a . about 24 for a -80s~V membrane potential, is due to active transport of H+~ OH
or HC03
ions, than the increase is the
ratio towards a passive value at higher pH wind ba more compatible with soma inhibition of an inward OH or HC03 pump at higher concentrations of these ions, than same inhibition of an outward H+ pump at a larar concentration of H+ ions . The mechanism of action of the metabolic inhibitors on pHi is likely to be complex .
However, the results do not favor substantial tis'sua binding of
D?f0 as being the reason why n!q imrariably gives a much higher value for pHi than the passive distribution value found by Carter, Rector y Campion and Seldin with intracellular pH electrodes in rat muscle (8) .
If absorption on
504
AMPHIBIAN BRAIN
Vol. 7, No. 9
some substance in the tissue were the major factor in determining
DM01 ,
then
metabolic inhibitors might be anticipated to bave D~M0 1 unaffected or to increase it b~ause of a rise in the component of
DMO
in true solution which
Would increase with a less negative membrane potential and a lower passively determined
H i ~ . That DlfO i /DèiOe does significantly decrease both in LO -5M L ouabaia and in the presence of IiaCN and Na iodoacetate strongly favors the mechanism being due to an inhibition of outward pumping of H + or inward pumping of OH
or AC03 , the affect of this on the distribution of hydrogen
ion being greater than that due to the lese negative mmbrane potential . Another factor might be the acctissulation of acid products of catabolism in the celle under conditions of metabolic inhibition . Açknovled~~eats This Work woes supported by U .S . Public Health Service Grant t~ 05905 . References W . J . WADDELL and T . C . BUTLER, J . Clin . Invest . 38, 720 (1959) .
R.
F . 1CIBLER,
R. P.
O'NEILL and E . D, ROBIN, J . Clin . Invest . 43, 431
(1964) . 3.
A . ROOS, Aver . J . Physiol . 209, 1233 (1965) .
4.
D . J . REED, C . D . WITHROW and D . M . WOODBURY, Exp . Brain Res . 3, 212 (1967) .
5.
D . J . CAIARANDINI and J . A . 7~DUNAISK7f, Exp . Naurol . 15, 319 (1966) .
6.
M, W . 8 . BRA~BURY, K . VILLAMIL and C . R, KLEEliAN, Aver, J . Physiol . In press .
7.
V . FENCL, T . B . MILLER sad J . R . PAPPElU~L?~R, Amer . J . Physiol . 210, 459 (1966) .
8.
N . W . CARTER, F . C, RECTOR, D, S . CAMION and D . W . SELDIN, J . Clin . Invest . ~¢, 920 (1967) .
Pergamon Press
INfRACSLLUIAR PH OF THB AMPHIBLf1N BRAIN I1~UBATSD IN VITRO Glen Grayman, Michael W. B . Bradbury and Charlu R. iClsosn Division of Medicine, Cedars-Sinai Radical Center and Research Instituts, and Departrnts of Physiology and Medicine, University of California at Los Angeles Medical Cantor, Los Angela, California
(Received 22 December 1967; in final form 19 February 1968)
la ,
Thn distribution of a weak acid, S,5-disethylaacasolidine-2,4- thons-C
(DMO), batwun intracellular and a:traeallular fluid (MCF), a rthod introduced by Aaddell and Butler (1), has bun used to calculate a value for the pH of the intracellular fluid of a nuober of tissues, both in vivo and in v rn . The rthod has bean applied to mosvalian brain~~n - y,~~ (2,3) . iwo difficulties are inherent in this approach .
Firstly, it is hard to arrive
at a reliable estimate of the extracnllular space of brain ~a vivo and secondly, the concentration of Di!!0 in the SCF is apt accessible oven to indirect measurement, since the DMO concentrations in blood pissed and in CSF may be different and not ratted to the pH difference betwun the fluids (4) . The frog brain may be well snintainad ~ vitro without swsl-liag or loss of intracel.luLr potassium (5,6) .
Further, its extracnllular space may ba
measured with fair precision from the voler of distribution of S35 -sulphate (6) .
Hence, the frog brain seemed an excellent in vitro preparation with
which to measure intncelluLr pH from the distribution of DMO and is which to study thn affect of various factors on it . Methods Thn'axperimants warn performed on whole brains from frogs, Raaa pipieos, of 8.5 to 9 .5 cm in body length, during the months of July, August and September .
The methods of removing the brain, of incubation in vitro , of attracting
and estimating chloride, sodium and potassium have been described (6) . All incubating fluids contained l~l 2 .0 mti, l~I2P04 3.0 ~1, CaC12 0 .9 d4, MgC1 2
499
AMPHIBIAN BRAIN
500
Vol . 7, No. 9
1 .2 eài, Ha 2S04 1 .8 mèl and glucose 13 .8 m!! and vara kept saturated during use with 95x 02 Sx C02. The total sodium concentration vas 118 .5 mEq/L in all fluide, but the aaounts of chloride and bicarbomte vnre varied to give fluids of different pH, thw HaHC03, mM
pH
(added)
(added)
(measured)
Horen1
90
25
7 .26
Alkaline
45
70
7 .72
105
10
6.87
HaCl, mH
Acid
Tha incubating fluids all caatainad C14-~, O .lpc/ml, and S 35 -sulphate, g350 .25pc/sl . The contributions to the radiaactivlty from C14 -DHO and sulphate in the diluted fluids and wtar sxtracts of brain, ware nstiosted by the technique already described for separating C14 -inulin and S35 -sulphate (6) . The axpariaeats ware run at a rocs ta~paratura of 22-24oC .
The pH of
the imubating fluid ws detersdnad with a Radiasetnr capillary microelectroda.
IntracalluLr pH, pHi, ws calculated froze the equation (~i01) (1 + 10~ - ~)
pHi ~ p& + log
(tt~ioe) whew pHa is the pH of the incubating fluid, D!!Oe is the total concentration of radioactive DZiO, ionised and non-ioni:ed, in the fluid, IIiO i is the total concentration of iatracallular radioactive DMO, calculated from the DMO content, the roter contant and thn sulphate space of the brain, and pK is the pH at which ~!D is half dissociated. aesults In Fig. 1 ara recorded mean values for pHi in incubation fluide of different pH and for times of incubation between 30 minutes and 3 hours . The values of pHi ara imrariably leas at 30 min than at Lter times, probably because of incosplata equilibration of the ßi0 in the cells, but thereafter
Vol. 7, No . 9
AMPHIBIAN BRAIN
501
ze
yH
(~t ._______ ...._.I 1
0
s.s Houes FIG . 1
Mean calculated values of pH i (.~~- .) during incubation of frog brains in media of different pH e ( ---- ), Limita are _+ 2 S,E, of 4-6 brains . pH i was fairly stable .
Thus S 35 -sulphate, C1 36 and Na24 are normally cam-
pletely equilibrated in the ECF of the isolated frog brain at 30 min (6), but entry of solutes into the cells may be slaver, urea reaching 80-85Z of its equilibrium value at this time (unpublished observations) .
At extra-
cellular pH of 7 .26, the intracellular fluid sas slightly acre alkaline, mean 7 .29 (1, 2 and 3 hours values), than the medisa .
In the acid fluid of
pH 6 .87, pH i was markedly more alkaline at 7 .01, and in the alkaline medisas of 7 .72 sas acre acid at 7 .62 .
There was no significant change in the intra-
cellular potassium concentration from the control value of 124 .2 ± 1 .6 mEq/L in the alkaline fluid, 122 .6 ± 4 .8 mEq/L, or in thn acid fluid s 113 .9 ± 7 .4 r~Eq/L, though in the latter case the value was suggestively less . Both ouabain 10 -5M and the combined metabolic inhibitora~ NaCN 5x10 -3M and Na iodoacetate 10 -3M (Table 1) caused a considerable fall in the ratio ~i/~e~ the mean values being .67 and .48 respectively (2 and 3 hour
502
AMPHIBIAN BRAIN
values) . the cells.
Vol. 7, No. 9
Under these conditions there may be penetration of sulphate into Hence the calculated values of pHi, 7 .1 and 7 .0, are likely to
be, if anything, too alkaline .
Under these conditions the intracellular
potassium concentration fall to 26 .5 ± 2 .1 mSq/L (ouabain) and 20 .6 ± 0 .9 mBq/L (NaCN and Na iodoacetate) . TABLB 1 . The Effect of Inhibitors on the Ratio, DI~i/DlSOn for Frog Brain in vitro 1 2 hour
1 hour
2 hours
3 hours
Normal madiuo
.90 + .02
1 .05 + .OL
1 .10 + .02
1 .10 + .04
Ouabaia LO -5!1
.66 + .02
.68 + .02
.68 + .02
.66 + .O1
NaCN, 5x10 -3M -3 Na iodoacetata 10 M
.44 + .03
.53 + .O1
.46 + .09
.50 + .04
Valuaa are means
standard errors of 4-6 brains . Discussion
The value of 7 .29 for the intracellular pH of frog brain in vitro is conaidarably more alkaline than figures given for mammrlian brain in vivo (2,3), despite a presumed mor~ acid pH of the extracnllular fluid in the in vitro axperimant .
Kiblar, O'Neill and Robin (2) and Rooa (3) give figures of
7 .08 and 7 .13 for dog and cat cerebral cortex reapactively .
These values
were calculated on the assumption of a 4-SX extracellular space .
As both
groups point out, s larger apace would give a somewhat lower pH .
Whether
our higher value represents a species difference or is dun to the very differont tachaique is not completely clear.
It should be noted, however, that
the concentration of uadiatociatad D!W in CSF may be late than that in either arterial or va~ua blood in some special (4) .
Thus, if the arterial or
venous blood concentration of D~l10 ie used for DMOn, the calculated value for pHi of brain may be too low because the CSF conentratioa of 0!!0 probably more nearly represents the concentration in the extracelluLr fluid of
Vol. 7, No . 9
brain (7) .
AMPHIBIAN BRAIN
&abler et al . (2) did sot measure CSF D!!O .
503
Row (3) found the
uadissx sated iglO in CSF to be .97 of that in arterial blood and .88 of that is venous blood in the cat . The changes of pHi in Low and high bicarbonate media warn much greater in the isolated frog brainy than occurred in vivo whether the pHa ws altered by varying the plasma HC03 or the C02 taosion.
This may ba due to
the circumventing of the blood-brain barriers which at least in the mammalian brain appears to be an important fu for in stabilising the pH of the astracellular fluid of brain .
Ia our preparation pHi is prasarbly solely
determined by events at the membranes of the brain calls, whereas in vivo , the obse:vad behavior is likely to ba the sum of transport activity at the blood-brain barrier and at the brain call membranes . ~n air case, is coon with pH i for other tissues in vivo and in r~itro measured from the distribution of ixi0, the value for pH i of frog brain in vitro is much more alkaline than would occur motor a passive distribution of
/ H y is .93 at a pHa of 7 .26. CHiJ C a It increases to 1 .26 in the alkaline medium and decreases to :73 in the uid H+ across the call membrane . medium .
The ratio
If the discrepancies in the ratios from that predicted on the basis
of passive distribution, i .a . about 24 for a -80s~V membrane potential, is due to active transport of H+~ OH
or HC03
ions, than the increase is the
ratio towards a passive value at higher pH wind ba more compatible with soma inhibition of an inward OH or HC03 pump at higher concentrations of these ions, than same inhibition of an outward H+ pump at a larar concentration of H+ ions . The mechanism of action of the metabolic inhibitors on pHi is likely to be complex .
However, the results do not favor substantial tis'sua binding of
D?f0 as being the reason why n!q imrariably gives a much higher value for pHi than the passive distribution value found by Carter, Rector y Campion and Seldin with intracellular pH electrodes in rat muscle (8) .
If absorption on
504
AMPHIBIAN BRAIN
Vol. 7, No. 9
some substance in the tissue were the major factor in determining
DM01 ,
then
metabolic inhibitors might be anticipated to bave D~M0 1 unaffected or to increase it b~ause of a rise in the component of
DMO
in true solution which
Would increase with a less negative membrane potential and a lower passively determined
H i ~ . That DlfO i /DèiOe does significantly decrease both in LO -5M L ouabaia and in the presence of IiaCN and Na iodoacetate strongly favors the mechanism being due to an inhibition of outward pumping of H + or inward pumping of OH
or AC03 , the affect of this on the distribution of hydrogen
ion being greater than that due to the lese negative mmbrane potential . Another factor might be the acctissulation of acid products of catabolism in the celle under conditions of metabolic inhibition . Açknovled~~eats This Work woes supported by U .S . Public Health Service Grant t~ 05905 . References W . J . WADDELL and T . C . BUTLER, J . Clin . Invest . 38, 720 (1959) .
R.
F . 1CIBLER,
R. P.
O'NEILL and E . D, ROBIN, J . Clin . Invest . 43, 431
(1964) . 3.
A . ROOS, Aver . J . Physiol . 209, 1233 (1965) .
4.
D . J . REED, C . D . WITHROW and D . M . WOODBURY, Exp . Brain Res . 3, 212 (1967) .
5.
D . J . CAIARANDINI and J . A . 7~DUNAISK7f, Exp . Naurol . 15, 319 (1966) .
6.
M, W . 8 . BRA~BURY, K . VILLAMIL and C . R, KLEEliAN, Aver, J . Physiol . In press .
7.
V . FENCL, T . B . MILLER sad J . R . PAPPElU~L?~R, Amer . J . Physiol . 210, 459 (1966) .
8.
N . W . CARTER, F . C, RECTOR, D, S . CAMION and D . W . SELDIN, J . Clin . Invest . ~¢, 920 (1967) .