Influence of alkaline buffers on cytoplasmic pH in myocardial cells exposed to metabolic acidosis

resuscitation

Resuscitation 32 (1996) 33-44

Influence of alkaline buffers on cytophsmic pH in myocardial cells exposedto metabolic acidosis Yu-Cai Li, Lars Wiklund, Pekka Tarkkila, Gunnel Bjerneroth* Department of Anaesthesiology, Uppda University Hospital, S-751 85 Vppsakz.SW&PI Received 7 August 1995;revision received 2 Jkember 1995;accqted 2 Dmembcr 1995

Abe&act The iniluence of di&ent clinically usedalkaline btiers on cytoplasmic pH in normalas well as acidotic rat myoaudial C&B was i~estigated in this study by means of the fluorescent intraceilular probe 2’,7’-bis4~xyethy~)-5,6‘n acetoxymethy1ester (BCECF-AM). It was shown that both sodium bkarbcmate PM1Tris buffer C&OX$hOWCCl mixture (TribonaP) causeda sign&ant and dose-dependentaci&&ation of the cytopkn of swpe&d myoc&ial cob with nonnol initial intracellular PH. This deuease was followed by a slow iricremduringthe-ooperiod. The initiaI cytopknic pH value was more easily reached when Tris bufkr mixture was wad. Ringer’s a&ate also causeda ~~JXW of intracelh&r pH, but this change persisted and was further ampli&d during the exyorbmt. Carbicarb in kger dosagesas well as pure trometamol (Tris) causeda pronounoed dv tondba&lgintndhllar tsWioixation. M.radhdar acidosis was achieved by preincubating the cells in sodium acetate.Addition of sodium bicarbow awed an initial and dose-dependent slightly above the induced acidosis. In contrast, T only when huger dosageswere used, and correction o vohunes. Ringer’s acetate produced a lasting and dose-dependentdecreaseof cytow pH, white Carbia~% caused an immediate, pronounced and persistent akalinization. My& C&I with low i&Gal ue to preinmbation in an acid buffer also showed an early decreax of intraceIh&r pH afkr addition of sodium bicarbonate and Tris buffer mixture. In the caseof sodium bicarbonate correztion of the acid-basedisturbance was not achkved during the observation period, while this was accomplished by use of larger volumes of Tris but&r mixture. Carbicarb in larger volumes causedan increasein intracelluhxr pH. The most sign&ant and persistent increws of cytopksmic pH was achieved by use of pure trometamoJ.In ~~IB&?s&, the pnwrt in vitro study implies that Trig b&Ier mixture (T&on&) is well-suited for correction of ktra&&r a&o& s&e it acta without causing a pronounced initial intraceMar acidosis or a later potentially hazardous huge cytopkmic aihalinization. iKqwor&: J!&tabolic acidosis; Alkaline buffers; Fluorescent intra&hdar cells

l

Corresponding author, Tel.: 46 018 664824;Fax: 46 018 559357.

OMO-957XJ64S15.000 1996Ekvier Science Ireland Ltd. All rights reserved SSDI 0300-9572(95)00939-Q

H+-probe, Cytoplaamic pH, My&

34

Y-C. Li et al. /Resuscitation 32 (1996) 33-44

1. Illtmhdon

IntracelhrlarpH is often consideredcrucial with regard to the metabolicresponseto the treatment of acid-basedisturbances[l]. In many hospitals sodium bicarbonate still remains the only drug availablefor correction of metabolic acidosis.Its use- especiallyduring cardiopulmonary resuscitation - has beenquestioneddue to reports of its deleterious effect on intracellular pH [2,3]. In some areas alternative alkaline buffer solutions have been marketed and clinically used with the expectationto cause less sideeffects than traditional sodium bicarbonate [4-61. , The purpose of the present study was to investigatethe influenceof alkaline buffers on cytoplasmic pH in myocardial cells, since it was consideredthat this could give interesting knowledgeabout important intracellular eventsassociated with, for example,myocardial ischemiaand cardiacarresttreatedwith cardiopulmonaryresuscitation. To accomplish this we used the pHsensitive fluorescent probe 2’,7’-bis-(carboxyethyl)-5&carboxyfluoresceinacetoxymethylester (BCECF-AM) [7]. Our hypothesiswasthat the use of Tris buffer mixture (Tribonat@, Pharmacia, Sweden)causesa more appropriate correction of intracellular acidosis than other clinically used buffers, avoiding both an initially aggravated acidosis as well as a sometimeslong-standing alkahnixationthat can be recordedintracellularly after the use of other buffers.

exposedfor 30-40 min and at 37°Cto collagenase (1 mg/mh type V, Sigma Chemicals,St. Louis, MO, USA) and DNAse (0.05 mg/ml; type 2, Sigma)in the F-10 medium containing 1.25 mM Ca2+ in a shaking incubator, as described previously for parathyroid cells [8,9]. The crude cell digests were briefly exposed to a calciumdeficient20 mM HEPES-buffercontaining 1 mM EGTA (ethyleneglycol-bis-[8-aminoethylether]NJ’-tetraacetic acid) and filtered through nylon meshesof 125 pm pore size, whereafter the cells were washed twice in phosphate buffered saline (PBS). Ten-ml test tubes containing 2.5 ml 75% and 2.5 ml 25% Percoll (Pharmacia Biosystems, Uppsala,Sweden),carefully setat layers,werefilled with the diluted cell suspensionand centrifuged for 15 min at 1500rev./mm.The interphasewith myocardial cells was collected and washedtwice with PBS by centrifugating for 10 min at 1500 rev./mm.Finally the cellswerecounted,suspended in Dulbecco’smodified Eagle’smedium(HyClone Europe Ltd, Cramlington, UK) and incubatedfor 1 h at 37°C.Viability in the resulting suspensions of single and small clusters of myocardial cells routinely exceededabout 90% as determinedby trypan blue exclusion.Only negligiblefibroblasts could be seen. The myocardial cells were then transferredto a buffer containing 25 mM HEPES, 3 mM glucose,0.1%bovineserumalbumin (BSA), 125 mM Na+, 5.9 mM K+, 0.5 mM Mg2+ and 1.25 mM Ca2+ with Cl- as the sole anion (pH 7.40).

2.Materialsmdmetbods

2.2. Intracellular acidification of: myocardial cells

2.1. Preparation of myocardial cells

Some of the myocardial cells were acidified prior to addition of different buffersand measurement of cytoplasmic pH. The acidification was achievedin one of two different ways. Twenty ~1 sodium acetatewas added from a 10 mm01stock solution directly into the cuvette containing cells suspendedin 2 ml buffer at the beginning of the experiment,or the cellswereloadedwith the fluorescentindicator in the HEPESbuffer with pH 6.80 for 30 min (seebelow). Both of thesetreatments resulted in an intracellular acidosis thereby mimickingthe metabolicstatusof myocardialcells during cardiac arrest.

The presentexperimentalserieswere performed after due consent by the Institutional Review Board for animal experiments.Myocardial cells were preparedfrom femaleSprague-Dawley rats (B&K Universal AB, Sollentuna, Sweden).The rats were sacrificedby cervical dislocation. Their hearts were removed instantly and minced into smallpieceswith scissorsin chilled (4°C) Ham’s F10 medium (HyClone Europe Ltd, Cramlington, UK) buffered with HEPES (20 mM) to pH 7.40. The mechanically disrupted tissues were then

Y-C. L.i et al. /Resuscitation

32 (19961 33-44

2.3. Akmmvnent of cytoplasmic pH

The pH-sensitive fluorescent probe 2’,7’-bisacetoxymethyl cam (BC!3CF-AM) (Nhxlar Probes, Jib&me,OR, USA) was used to e&hate intracellularpHinthemym&ialceUsbefmeandafter addition of diffma~ a&he buffers. Myocardial in the HEPES b&er (PH 6.80 or cells 7.40,ruagmtivdy) were luaded with the indicator in a cumtnttion of 10 X lo6 cells/mlfor 30 min at 37T uuder gentleagitation [ 111.The final con-

(cprboxysthyl)-5,a

buffer in order cellsin 2 ml of the b&I&r soWkm T&e cedurewas done in ambient air.

Table 1 Initial cytoplasmic pH (PHi) and extracellular pH @I&) as well as subsequentcbangas(A) after addition of b&r

(maat~f S.E.M.)

n

Initial PHI

ApHi immediately after buffer

ApQ after 10 mill

Illitial pH,

ApI% after 10 mifl

A#& lO/ API% 10

TribonaP 10 20 40 80 200

7 9 10 8 8

7.10 + 7.10 + 7.06 4 7.07 f 7.04 f

0.009 0.010 0.013 0.009 0.016

-0.034 -0.043 -0.056 -0.078 -0.112

f f * * 4

0.006 0.005 0.007+ 0.010. 0.0142

-0.040 * -0.037 f -0.018 f -0.002 f 0.001 t

7.28 zt 0.008 7.29 * 0.008 7.28 f 0.010 7.30 4 0.010 7.30 f 0.008

-0.010 f 0.033 f 0.089 * 0.094 f 0.236 i

0.010 0.011 0.008 0.013 0.014

3.OOO4 -2114 * -0.382 t -0.538 j: -0.219 f

1.727 1.230 0.121 0.111 0.065

NaHC03 10 20 30 40 50 80 1W 200

9 8 8 6 11 8 9 8

7.12 * 7.12 f 7.10 4 7.09 * 7.08 * 7.10 4 7.06 f 7.09 f

0.011 0.024 0.019 0.012 0.015 0.012 0.027 0.020

-0.047 -0.069 -0.080 -0.095 -0.102 -0.111 -0.140 -0.176

* * 4 4 * * f

0.007* 0.011+ 0.007* 0.011+ 0.cX-W 0.025, 0.010* 0.021’

-0.063 f 0.007* -0.055 f 0.010’ -0.047 * 0.a-w -0.035 f 0.008 -0.024 zt 0.008 -0.010 f 0.012 0.008 zt 0.014 0.011 * 0.007

7.28 f 7.28 f 7.29 f 7.29 f 7.29 f 7.30 f 7.30 f 7.31 f

0.026 f 0.065 t 0.101 t 0.143 * 0.159 d 0.233 + 0.303 * 0.479 4

0.005 o.aJ8 0.010 0.006 0.014 0.W 0.010 0.009

-3.089 4 -1.205 * -0.545 4 -0.257 4 -0.364 h -aim 4 0.024 4 0.004 f

1.009 0.278 0.155 0.068 0.061 0.057 0.044 0.015

Buffer 64

Ringer’sacetate 50 10 7.11 f 0.015

l

0.008 0.007 0.007 0.004 0.014

0.006 0.011 0.005 0.011 0.007 0.008 0.010 0.005

8 8

7.14 * 0.014 7.09 f 0.014

-0.031 l 0.005 -0.127 * 0.007’ -0.205 f 0.008*

-0.057 f 0.005* -0.146 l 0.011’ -0.211 l 0.010*

7.30 f 0.004 7.31 * 0.006 7.31 f 0.005

8 8 8 14

7.11 f 0.011 7.12 GIE 0.015 7.08 i 0.018 7.00 f 0.013

-0.042 f -0.038 f 0.043 4 0.177 f

0.003 0.007 0.006 0.030.

-0.051 * 0.018 * 0.149 f 0.497 4

0.011* 0.006 o.oNl* 0.045*

7.31 f 7.31 * 7.31 f 7.30 l

0.005 0.007 0.004 0.003

0.115 f 0.239 4 0.477 4 1.126*

0.007 0.009 0.025 0.015

-0.472 f 0.074 4 0.330 4 0.439 4

0.108 0.026 0.061 0.037

Trometamol 5 9 10 8 20 9 40 10

7.07 rt; 0.024 7.06 f 0.017 7.01 * 0.017 6.97 f 0.011

0.036 f 0.070 f 0.094 4 0.152 *

0.010 0.015. 0.010* 0.027+

0.100 f 0.239 f 0.348 f 0.523 f

0.015+ 0.021+ 0.025’ 0.033.

7.29 f 7.31 4 7.30 f 7.30 4

0.008 0.003 0.006 0.004

0.349 f 0.599 l 0.792 i 1.148*

0.009 0.011 0.088 0.009

0.282 4 0.402 * 0.786 4 0.4% *

0.037 0.041 0.389 0.029

200 400 Carbicarb 10 20 40 80

-0.034 f 0.006 -0.053 4 0.007 -0.075 * 0.012

The changes of intracellular pH were considered significant when P < 0.05 (zl&athd* in the table). ApI$ lWAp& 10, quotient betmen changes in cytoplasmic and extracellular pH 10 rain after buffer addition.

2.263 it 0.389 3.176 f 0.441 5.235 zt 1.010

Y-C. L.i et al. / Resuscitahm 32 (19%) 33-44

36

The fluorescencemeasurementswere performed at 37°C in a temperature-regulateddual-excitation-wavelength spectrophotometer (F-2000, Hitachi, Tokyo, Japan)under continuous stirring. The free acid form of the BCECF exhibited an excitation maximum at 500 run (2.5-15 mn slit width), and an emission maximum at 530 mn (2.5- 10mn slit width). Becauseof an unavoidable smallleakageof the fluorescentindicator from the cells,eachexperimentwasrestrictedto 20-30 min. Calibration was performedabout 15 min after the beginningof the experimentby lysing the cellswith Triton X-100 (loo/o v/v, Sigma Chemicals, St. Louis, MO, USA) and titrating medium pH with addition of HCl. From correspondingpH and fluorescencemeasurementsa graph was constructed and usedfor the translation of fluorescencevalues into valuesof cytoplasmicpH [ 141. In each experiment Tris buffer mixture (Tribonat@)[6], sodium bicarbonate (NaHC03), Ringer’s acetate, Tris buffer (THAM) [5] or Carbicarba’(containing l/3 M Na2C03 and l/3 M NaHCO$ [4] was added directly into the cuvette opento ambientair containing the myocardialcell suspension.The dosagesemployedin this experimental setting were grossly chosen to match volumesof buffer given in the clinical situation. Extracellular pH was measured by use of a combination-electrode(ROSS,Bergman& Beving Laboratories, Stockholm, Sweden) inserted through the injection port after each addition.

ApH1

0.4

0

2

4

6

8

LO

I 12 uins after arl.iium d tuffcr

Fig. 1. Changesin cytoplasmic pH @Hi) after addition of 40 d T&on@, 40 d sodium biinatc, 200 d Ringc~‘s acetate, 40 ~1Carbicarb or 20 d tromctamol, rcspectivcly, to ~u~pendcd rat myocardial cells with normal initial cytoplasmic PH.

2.4. Statistical tests

The results are presented as mean f S.E.M. calculated from the original data. Differences within each group from the control value were evaluated by means of Student’s paired t-test, where the changesin intracellular pH after addition of the different buffer substanceswere compared with changes achieved after addition of similarvolumesof the buffer solution that the cells were suspendedin. The results were considered significantif P c 0.05. 3. Results 3.1. Myocardial cells with normal initial cytoplasmic pH (Table 1, Fig. 1)

The initial mean cytoplasmicpH value in the untreated myocardial cells was 7.08 * 0.015.The initial meanexternal pH value was 7.30 f 0.006. Addition of sodium bicarbonatecauseda dosedependentdecreaseof intracellular pH. Dosages from 10 to 200 d were used,and the reduction in cytoplasmicpH then variedfrom 0.047 f 0.007to 0.176 f 0.021 pH units. Ten min after the addition the cytoplasmicpH value was still below the initial control valuewhen the smallerdosageshad beenused,while larger dosagescauseda complete compensationor a slight overcorrectionof the intracellular pH-change. Ringer’s acetategiven in volumesof 50-400 d causeda rapid and dose-dependentacidificationof the cytoplasm varying from 0.031 * 0.005 to 0.205 f 0.008 pH units. The reduction in cytoplasmicpH not only persistedduring the 10 min observation period but was even further aggravated. Smalldosagesof Carbicarb(10 or 20 ~1)induced an insignificant reduction of intracellular pH, while largerdosages(40 or 80 ~1)resultedin an initial pH-increaseof at most 0.177 f 0.030 units. The elevatedpH-valuepersistedduring the period of observation and reached 0.497 & 0.045 units abovethe initial meanvalue 10ruin after addition of 80 4 Carbicarb. Administration of pure trometamol (Tris) diSped,

13 17 14 15 14

n

0.047 0.011 0.028

0.019 0.010 0.012 0.013 0.018 0.016 0.017 0.025

7.12 f 7.05 f 7.04 f 7.05 f 7.06 i 7.08 i 7.07 f 7.11 f

0.014 0.012 O&O34 0.022

7.05 f 7.13 f 7.04 f 1.06 t

f 0.004 * 0.003 f 0.011 f 0.005 zt 0.007* f 0.007. f 0.010+ f 0.009.

0.005 0.004 0.003 o.c@6 0.012*

O‘OM 0.004 o.w6* 0.014*

0.118 f 0.006* 0.182 zt O.OfP 0.270 * 0.014. 0.349 * 0.017+

0.015 f 0.035 f 0.079 f 0.241 f

-0.010 f 0.003 -0.089 f 0.005+ -0.172 f 0.007*

-0.004 -0.016 -0.027 -0.038 -0.058 -0.074 -0.085 -0.141

0.007 f 0.001 * -0.012 f -0.039 * -0.116 *

ApHi immediately after buffer

0.187 i: 0.292 f 0.465 * 0.668 rt

0.005 0.004

0.005

0.005 0.003 0.006 0.003 0.008

0.006 0.005 0.006 0.005 0.004

7.32 f 7.32 f 7.32 f 7.31 f

7.32 f 7.31 f 7.31 f 7.31 f

0.005 0.004 0.004 0.006

0.005 0.004 o.o@l 0.005

7.32 f 0.003 7.31 f 0.005 7.32 t 0.004

7.31 f 7.31 f 7.30 f 7.32 k 7.31 f 7.31 l 7.31 l 7.31 f

7.31 f 7.32 f 7.31 * 7.29 t 7.31 f

Initial pH,

addition.

O.O%* om9+ 0.014* O.Ml‘

0.035 f 0.006 0.079 f o.ow 0.1% f 0.008* 0.418 SEO.@# l*

0.010 f 0.005 -0.057 f 0.006* -0.128 SIG 0.007’

0.005 f 0.004 0.011 l 0.008 0.018 i 0.0% 0.024 * 0.008 0.026 t 0.006 0.028 SE0.009 0.030 f 0.013 0.035 f 0.011

0.058 f 0.007* 0.112 f 0.0102

0.022 l 0.006 0.036 f 0.006

0.013 f 0.005

ApHi after 10 mill

t when P < 0.0-s@arked* in the t&e). and extracehlar pH 10 tnin after b&k

-0.095 t 0.0075 -0.124 t o.lxw -0.133 f o.om*

f 0.007* f 0.008* f o.oa$* ct O.OW

-0.081 -0.093 -0.117 -0.138

0.020 0.017 0.028 0.018

0.008, 0.012* 0.006* om8+ O.OW 0.011. O.WP 0.008*

0.014* 0.010* 0.016* 0.010* 0.007*

7.07 f 7.11 f 7.11 f 7.12 f

f * + f f f f *

f f f f f

-0.082 f 0.011* -0.088 f 0.011* -0.110 f o.WP

-0.091 -0.095 -0.103 -0.099 -0.093 -0.105 -0.112 -0.090

-0.093 -0.095 -0.095 -0.107 -0.119

ApHi after NaAc

7.12 f 0.014 7.15 f 0.010 7.10 * 0.010

0.020

7.15 I 7.01 l 7.10 l 7.07 f 7.13 f

0.043

Initial pHi

0.027 0.004 0.003 0.007 0.003 0.009 0.010 0.010

0.007 0.007

0.004

0.003 0.007

0.353 f 0.592 f 0.863 f 1.136 t

0.005 0.013 0.008 0.014

0.112 f 0.004 0.256 f 0.006 0.516 zt 0.017 1.222 f 0.009

-0.028 f 0.004 -0.046 f 0.006 -0.081 f 0.0@5

0.051 * 0.056 f 0.076 f 0.115 f 0.143 * 0.185 + 0.228 a 0.381 l

0.027 f 0.048 * 0.093 l 0.161 f 0.281 f

APH, after 10 mill

0.333 0.498 0.074 0.045 0.036

0.085 0.043 OSb47 0.055 0.028

0.244 f 0.065 l 0.132 f 0.100 * 0.091 i

-0.912 f 0.503 1.148 * 0.180 1.649 zt 0.128

0.080

0.008 l

0.147 * 0.234 0.137 f 0.208

0.834 A 0.203 f 0.415 * 0.224 I 0.147 f

ApHi IO/ AP& 10

initially, as well as changes (A) after addition of sodium acetate (NaAc) and subsequent addition of buffer

The changes of intracpllular pH were conaidarad ApHi 10/A-p% 10, quotient between changes in crtoplaetaic

80 200 NllHCO~ 10 16 20 14 30 7 40 13 50 6 80 14 100 8 2lm 14 Ringer’s acetate 50 12 200 12 400 12 Carbhrb 10 14 20 13 40 12 80 12 Trometamol 5 12 10 13 20 15 40 14

40

TlibotlaP 10 20

Buffer W

Table2 Cytophmic pH (pHi) and extracellular pH (pw (mean f S.E.M.)

Y

38

Y-C. L.i et al. /Resuscitation 32 (19%) 33-44

caused a prompt, dosedependent and lasting alkalinization of the cytoplasmwhen 5-40 ~1was added to the cuvette. The initial rise of 0.152 * 0.027 after addition of 40 pl further increasedto 0.523 f 0.033units abovethe control value after 10 min. Finally, Tris buffer mixture (Tribonate) resembledsodiumbicarbonatein causingan initial acidificationof the cell interior when dosagesfrom lo-200 fi were used.The decreasein cytoplasmic pII ranged from 0.034 * 0.006 to 0,112 f 0.014 units, but showeda transient nature with quicker reversalto the initial values than in the case of sodium bicarbonate. 3.2. Myocardial cells with low initial cytoplasmic pH due to addition of acetate (T&le 2, Fig. 2)

Addition of 20 ~1 sodium acetateto the 2 ml heartmusclecell suspensionin the cuvetteproduced a decreasein cytoplasmicpH of 0.103 f 0.010 units from an initial mean of 7.09 f 0.017. The initial mean external pH value was 7.31 f 0.005. Administration of lo-200 pl sodium bicarbonate resulted in a dose-dependentacidification of the cytoplasmof up to 0.141 * 0.009pH units. A completecorrection of the acid-basedisturbance was not accomplishedduring the 10 min observation period evenin the caseof larger volumes. Ringer’sacetatewasgivenin volumesof 50-400 d. The addition causeda prompt decreaseof cytoplasmic pH which very slowly returned towards the control value,but remainedsigniticantlybelow the initial value during the observationperiod. When lo-80 ~1 Carbicarb was added to the cuvette contents an alkalinixation of the cell interior was noticed. This increasein cytoplasmic pH varied from 0.015 f 0.004 to 0.241 f 0.014 units, and increasedfurther to 0.035 + 0.006 and 0.418 f 0.011 units, respectively,above the control value during the experimentaltime. Pure trometamol caused the most prominent increaseof intracellular pH with initial values of 0.118 f 0,006 to 0.349 f 0.017 units above the control value when 5-40 ~1were given. After 10 min these elevations had increased further to 0.187 f 0.006 and 0.668 f 0.031 units, respectively.

Fig. 2. Changesin cyt~plasmi~ pH @Hi) after addition of 40 ~1 TribonaP, 40 ~1sodium bicarbonate, 200 4 Ringer’s acctatc, 40 4 Carbicarb or 20 4 tromctamol, respectively, to suspended rat myocardial cells previously made acidotic by addition of sodium acetate.

Finally, Tris buffer mixture (Tribonat@)was investigated in dosages of lo-200 ~1. Smaller dosagesproducedalmostno effecton intracellular pH while larger dosagescauseda moderateacidification. Not only this transient decrease,but also the acidosis induced by addition of acetate was corrected during the observation period when larger volumeswere added. 3.3. Myocardial cells with low initial cytoplasmic pH due to preincubation in acid buffer (Table 3, Fig. 3)

Incubation for 30 min in the buffer usedfor fluorescencemeasurementswith pH 6.80 and further incubation for 30 min after the addition of the fluorescentindicator resultedin an initial meancytoplasmic pH value of 6.91 f 0.018 in the myocardial cells. The initial mean external pH value was 6.70 f 0.005. Addition of 20-80 ~1sodium bicarbonatecaused a dose-dependentacidification of the cytoplasm. The decreasein pH varied from 0.109 f 0.011 to 0.148 f 0.006 units. Correction of this decreasewas achievedby adding at least 40 4 of sodiumbicarbonate,but a completeadjustmentof the initial acid-basedisturbance was not accomplished by evenlarger dosages. Ringer’s acetatecould not be examinedin this experimentalsetting. The combination of a low

12 12 11

11 9 8

8 8 8

NaHCO, 20 40 80

Carbicarb 20 40 80

Trometamol 10 20 40

Loading pH 6.80 Loading pH 6.80 Loading pH 6.80

Loading pH 6.80 Loading pH 6.80 Loading pH 6.80

Loading pH 6.80 Loading pH 6.80 Loading pH 6.80

Loading pH 6.80 Loading pH 6.80 Loading pH 6.80

Acidification

initially

6.94 + 0.020 6.92 zt 0.030 6.88 f 0.018

6.90 zt 0.022 6.92 zt 0.010 6.88 f 0.013

6.92 zt 0.023 6.92 zt 0.017 6.92 zt 0.014

6.90 h 0.019 6.91 i 0.013 6.91 f 0.017

Initial pHi

pH (pm

0.130 f 0.030’ 0.240 f 0.020* 0.330 f o.ow

-0.046 f 0.009* -0.026 f 0.006 0.041 f 0.008

-0.109 l 0.011* -0.123 f O.Olo* -0.148 f 0.006*

-0.075 f 0.009* -0.101 l 0.010* -0.115 f 0.007*

ApHi immediately after buffer

l

0.011

0.011

0.350 f 0.0208 0.650 f 0.030* 1.080 f o.O@*

0.037 f 0.008 0.113 f 0.006* 0.349 f 0.018.

0.021

l

-0.047 f 0.013’ -0.004

6.70 * 0.013 6.71 f 0.008 6.69 f 0.009

6.69 zt 0.005 6.69 ct 0.006 6.69 f 0.005

6.69 f 0.003 6.72 zt 0.004 6.70 f 0.004

6.69 f 0.007 6.72 f 0.004 6.72 f 0.005

Initial pH,

0.597 * 0.012 0.825 = 0.018 1.142 f 0.020

0.3% zt 0.008 0.676 f 0.011 1.128 f 0.007

0.169 * 0.009 0.297 f 0.008 0.491 * 0.006

0.158 zt 0.007 0.263 f 0.008 0.430 f 0.007

ApH, after 10 ruin

0.584 f 0.032 0.792 + 0.028 0.921 f 0.027

0.093 f 0.021 0.168 f 0.010 0.037 f 0.007

-0.312 t 0.088 -0.012 f 0.037 0.043 f 0.022

-0.303 l 0.068 -0.010 zt 0.038 0.079 f 0.016

ApHi lO/ AP& 10

as well as changes (A) after addition of buffer (mean f S.E.M.)

-0.026 t 0.010 0.083 zt O.OlO* 0.175 f 0.007*

ApHi after 10 min

(cells loaded in a buffer at pH indicted)

The changes of intracellular pH were considered significant when P < 0.05 (marked* in the table). ApHi lWApH, 10, quotient between change+ in cytoplasmic and extracellular pH 10 min after buffer addition.

11 12 10

n

TribonatQ 20 40 80

Bt@er 64

Table 3 Cytoplasmic pH (pHi) and extracellular

40

Y-C. Li et al. / Resmcitation 32 (19%) 33-44

0.6 -

Fig. 3. changes in cytoplahc pH (PHi) after addition of 40 4 T&on&, 40 4 sodium bicarbonate, 40 4 Carbicarb or 20 4 trometamol, respcctivcly to rat myocardial cells suspendedin a buffer with pH 6.80.

initial externalpH and addition of this acidic drug resulted in a pH-value that was too low for the fluorescentpH-indicator to be reliable. Smalldosagesof Carbicarb (20-40 4) causeda slight decreaseof intracelldar pH while 80 ~1produceda smallincrease.Ten min after the addition the cytoplasmicpH value was clearly above the initial value in all cases. Addition of pure trometamol in volumes of 10-40 ~1 produced a prompt and lasting alkalinixation of the cell interior. Forty 4 caused an initial increase of 0.330 f 0.050 pH units, which had increased further to 1.080 f 0.060 units above the initial value 10 min after the addition. Finally, Tris butTermixture (Tribonat@)-givenin dosagesof 20-80 4 causedan initial intracellular acidification of 0.075 f 0.009 to 0.115 f 0.007 units. In the cases of 40 and 80 ~1 the decreasewas only transient and 10 min after the addition the cytoplasmicpH valuealmostequalled the normal control value. 4. DltKlmdon The reducedblood flow during ischemiaor cardiac arrest resultsin a critical reduction of tissue perfusion and the conaaquentfailure to maintain oxygendeliverycausesanaerobicmetabolismand lactic acidosis.The reduced blood flow deprives

the heart musclecells of necessarysubstratessuch as oxygen and glucose, and allows metabolites, especiallylactate, H+ and K+ to accumulate[ 151. This causesa metabolicacidosiswhich affectsthe myoflbrils and, together with other factors, leads to reduced myocardial contractility as well as appearanceof arrhythmias [ 16- 181.The negative inotropic effect of acidosishas been explainedas a direct effect of intracellular H+ on the Ca2+sensitivity of the contractile proteins, especially troponin C [ 19-22). Inhibition of different enzyme reactionsmay alsoplay an important part [23], but alterationsin metabolismor supply of high energy phosphatecompounds necessaryfor contraction seemsto be unrelatedto the effect [17,24-261.Accumulation of carbon dioxide causing a respiratory acidosis is also an important feature of myocardial ischemia [15,27]. This is important often is the major determinant of insince PCO~ tracellular pH [25]. Several investigations have shownthat a respiratory acidosisdue to increased PC02produces an even more powerful negative inotropic responseat the same extracellular pH than a metabolicacidosisdue to decreasedbicarbonateconcentration[25,28,29],or at leasta more rapid effect [30]. ExtracellularpH-changesappear to be lessimportant for the contraction modulation [25], even though some effects have been noticed (311. Intracellular Ca2+ accumulation associatedwith ischemia,and probably secondary to raised Na+concentration, also seemsto play an important role for the impaired function of the heart during ischemia[16,32,33].This Ca2+overload will lead to late after-depolarizationsin the myocardiumand potential ventricular arrhythmia or ventricularfibrillation [34]. On the other hand, metabolicalkalosisalso has deleteriouseffectson cells, even if a slight rise of cytoplasmic pH in someinstancescan give a positive inotropic effect [31]. This could be explained by the fact that a moderateintracellular alkalosissensitizesthe contractile elementsto calciumthereby increasingthe contractility [35]. Singlecellshavebeenshownto be more suitable for cytoplasmic pH measurementsthan multicellular preparations [36]. Several authors have describedvarious methods for isolation of myocardial cells 137,381.Many of the proceduresare

Y-C. LA et al. /Resuscitation 32 (19%) 33-44

41

cellsare~posedtophysi-

czanwa~ons. The choice of dictded by the speciesand/or type of tissue to be used. Another drawback of of exposure so exactly that the cell membrane are not

durethathaato ment could either be done nigericinmethod~,~whicb

of the mm to calcium. Also, the were incubated for 1 h at 37°C functional experimentswith TribP

11.

hasprevio&ytifdta pie pI‘I by O.la-o.15 p&I

ie a Tris buf&r mixture consistingof and phos61in order to eliminate the previously noted side effects of sodium bicarbonate and Tris when these sub+%anwswere used alone [42-44]. The main drawback of sodium bicarbonatein the treatment ofmeWalicacidosisisits&ndencytocauaeasign&ant rise of I%DJwhich is further accentuated in situations where carbon dioxide removal is inadequate[45]. Sincecarbon dioxide permeatescell membranesrapidly the rise in arterial PC02furates the intracelhdar acidosis.This is

in extracelhdarpH 128,461.It has been that failure of cardiac resuscitationand of eiactnrmechonicaldissociationmay rapid reduction of force due to dioxide tension [471. A rise in lW4dSObeenllOtiCedftfkSOdi-

[45], and this hyperosrmblity coukl potentially be detrimental to cerebral fimction (481.When Tribonat@is given intra=o&y a normal P,coZ can be maintained in patients with normal ventilatory function. Therefore, TribonatQ has been considered to

When we tried to mimic the early phaseof car-

42

Y-C. Li et al. /Resuscitation 32 (19%) 33-44

disc arrest treated with cardiopulmonary resuscitation by adding acetate to the myocardial cells somewhat different effects were recorded after addition of the alkaline buffers. In this situation administration of sodium bicarbonate and Ringer’s acetatealso resulted in a decreaseof intracellular pH, but this reduction was somewhat lesspronouncedthan in the caseof normal myocardial cells.In combination with the pre-existing decreaseof cytoplasmicpH a substantialacidosis of the cell interior was recorded. A tendency towards an increasewas however noticed during the observationperiod, but completecorrection of cytoplasmicpH was not achievedfor either of the two buffer solutions.Addition of Tris buffer mixture causedalmost no initial etfect in the caseof smallervolumes,but a smalldecreaseof intracellular pH when larger volumeswere given. A rapid correction of the induced acid-basedisturbance was however noted when larger volumes were used. Both Carbicarb and trometamol causedan alkalinixation of the cytoplasm.This rise of intracellular pH continued during the observation period and resultedin valuesfar abovethe normal range in the caseof larger volumes. The clinical situation of late hypodynamicshock wasimitated in vitro by incubating the myocardial cellsin an acid medium.Addition of sodiumbicarbonate or Tris btier mixture caused an initial decreaseof cytoplasmic pH that was more pronouncedthan in the earlier settingswithout extracellularacidosis.In the caseof sodiumbicarbonate correction of the acid-basedisturbance was not achievedduring the observationperiod, while this wasaccomplishedby addition of larger volumesof Tris buffer mixture. Small volumesof Carbicarb increasedthe pre-existingintracellular acidosisinitially, but could partly compensate for the decreasedcytoplasmicpH during the observation period. Large volumesof Carbicarb aswell aspure trometamolcausedan immediateand progressive increaseof cytoplasmic pH to values far above normal valuesin caseof larger dosages. To summa&e the results, Ringer’s acetate seemsto causea lasting acidification of the cytoplasm of myocardial cells in the present experimental setting. Carbicarb and pure trometamol produce a correspondingpronounced alkaliniza-

tion. Addition of both sodium bicarbonate and Tris buffer mixture (TribonatQ) is associatedwith an initial decreaseof intracellular pH followed by a slow increase.In the caseof sodium bicarbonate the correction is somewhat slower than when Tribonato is used. Only addition of Tribonat@ could fully compensatefor the initially induced metabolicacidosis.All changesin cytoplasmicpH seemto be dose-dependent. In conclusion, this in vitro study implies that Tris buffer mixture (Tribonate) is well-suited for cmection of intracellular acidosis,even though benefit from Tribonate in human CPR could not be concluded from a previous clinical trial [51]. Additional studiesare requestedto ascertainthe exact role of buffer therapy in the setting of low flow statestogether with their impact on restoration of myocardial function and, ultimately, on outcome.Tribonat@might potentially offer more advantagesthan other buffers in restoring the intracellularpH without the side-effectsof paradoxical intracellular acidosis of sodium bicarbonate and the marked overcorrectionof THAM. Acknowledgements

This work was financially supported by the Medical Research Council (project 06579), the Laerdal Foundation for Acute Medicine and the Tore Nilsson Foundation for Medical Research. References

111Cameron IR.

Acid-base disorders: analysis and treatment. Clin Endocrinol Metab 1980;9: 529-541. m Ritter JM, Doktor HS, Benjamin N. Paradoxical effect of bicarbonate on cytoplasmic PH. Lancet 1990; 335: 1243-1246. 131 Arieff AI, Leach W, et al. Systemic effects of NaHCQ in experhental lactic acidosis in dogs. Am J Physiol 1982;242: F586-FS91. 141 Filley GF, Kindig NB. Carbicarb, an alkalinixing iongenerating agent of possible clinical usefulness. Tram Am an climato1 Assoc 1984;96: 141-153. r51 Nahas GG. The pharmacology of Tris (hydroxymethyl) aminomethane (THAI@. Pharmaco1 Rev 1962; 14: 441-412. 161Wikhmd L. t)quist L, et al. Clinical buffering of metabolic acidosis: problems and a solution. Resuscitation 1985; 12: 279-293.

Y-C. Li et al. /Resuscitation 32 (19%) 33-44 [fl Ri&TJ,TtiRY,PozzanT.Cy@&micpHandfree r4g2+ in la J cell Biol1982; 95: 189-l%. [Sj Bjemerd G, Juhlin C, et al. MHC class I and II antigen exprdon on parathyroid cells and prospects for their aBo@c txampkation. Transplantation 1993; 56: 717-721. [9] Rudberg C, Grimelius L, et al. Alteration in density, and parathyroid hormone releaseofdispersmorp ed pamthyroid cells from patients with hyperpa@hyroidism. Acta Path Miiobiol Immunol Stand 1986,Sect A, 94: 253-261. [lOI G&s&in S, Furuya W. Cytoplasm& pH regulation in %5~m~-a$vated human neutrophils. Am J Physiol . [ll) Grin&in S, Dixon SJ. Ion transport, membrane potential, and cytop&& pH in lymphocytes: changesduring activation. Physiol Rev 1989; 69: 417-481. [12] Zavti GB, Cragoe EJ Jr, Feinstein MB. Regulation of intracclluhu pH in human platelets. Effects of thrombin, A23187, and ionomycin and evidence for activation of Na%I+ exchange and its inhibition by amiloride analogs. J Biol Chem 1986;261: 13160-13167. 1131 Musgrove E, Rugs C, Hedeley D. Flow cytometric measumment of cytoplasmic pH: a critical evaluation of available fluorochromes. Cytometry 1986;7: 347-355. [14] Reid IR, Civitelli R, et al. Cytoplasm& pH regulation in canine renal proximal tubule cells. Kidney Int 1987;31: 1113-1120. 1151 Gpie LH. Effects of regional ischemia on metabolism of glucose and fatty acids. Circ Res 1976; 38 (Suppl I): 52-74. 1161 Allen DG, Orchard CH. Myocardial contractile function during ischemia and hypoxia. Circ Res 1987; 60: 153-168. [17] JacobusWE, Pores IH, et al. The role of intracellular pH in the control of normal and ischemic myocardial contractility: a 3’P nuclear magnetic resonance and mass spectrometry study. In: Nuccitelli R, Deamer DW, (eds), Intrace&& pH: Its measurement, regulation and utilization in cellular functions. New York, Alan R. Liss Inc., 1982;pp. 537-565. [18] Kleber AG. Extracellular potassium accumulation in acute myccardial ischemia. J Md Cell Cardiol1984,16: 389-394. (191 Blanchard EM, Solar0 RJ. Inhibition of the activation and troponin calcium binding of dog cardiac myofibrils by acidic pH. Circ Res 19&1,55: 382-391. (201 Donaldson SKB, Hermansen L. Differential, direct effects of H+ on Ca*+-activated force of skinned fibers from the soleus,cardiac and adductor magnus musclesof rabbits. Pfliigers Arch 1978;376: 55-65. [21] Fabiato A, Fabiato F. Effects of pH on the myofilaments and the sarcoplasmicreticulum of skinned cells from cardiac and skeletal muscles.J Physiol 1978;276: 233-255. 122) Orchard CH, Hamilton DL, et al. The effect of acidosis on the relationship between Caz+ and force in isolated ferret cardiac muscle. J Physiol 1991;436: 559-578.

47

[23] Ellis D, Thomas RC. Direct ofthe iatracellular pH of mammalian cmdiac muscle. J PhysioI 1976; 262: 755-771. #diollfilll&oa8adme[24] opieLH.Ee!ctoftabolism of isolated pe&sed rat heart. Am 3 Physiol 1965;209: 1075-1080. (251 SteenbergenC, Daleeuw G, et al. Effects of and ischemia on contractility and intrac&ular pH of rat heart. Circ Res 1977;41: 849-858. 1261 WiUiamsonJR, SchatTerSW, et al. ContriBwion oftissue acidosis to is&&c injury in the pe&sed rat heart. Circulation 1976; 53 (Suppl I): 3-14. [27] Graf H, Arieff AI. The use of sodium bicarboaate in the therapy of organic acidosis. Inteus Care Med 196 12: 285-288. [28] Poole-Wilson PA, Langer GA. EfTect of pH on ionic exchange and funetioo in rat aud rabbit myoeardium. Am J Physiol 1975;229: 570-581. [29] Kette F, Weil MH, et al. Buffer agents do not reverse intramyocardial acidosis during cardiac resuscitation. Circulation 1990;81: 1660-1666. 1301 Fry H, Poole-Wilson PA. Effects of acid-basechangeson excitation-contraction coupling in guinea-pig and rabbit cardiacventticularmuscle. J Physioll981; 313: 141-160. [31] Bountm C, Vaughan-Jones RD. El&t of intracellular and extracellular pH on contraction in isolated, mammalian cardiac muscle. J Physiol 1989;418: 163-187. [32] Kohmoto 0, Spitzer KW, et al. Effects of intracellular acidosis on [Ca2+]; transients, transsareolemmal Ca*+ fluxes, and contraction in ventricular myocytes. Circ Res 1990;66: 622-632. [33] Tani M, Neely JR. Na+ accumulation increases Ca2’ overload and impairs function in anoxic rat heart. J Mo’l Cell Cardiol 1990;22: 57-72. [34] Lynch C III. Calcium metabolism in the normal and diseasedheart. Curr Opin Anaesthesiol 1994;7: S- 1I (351 Barry WH, Bridge JHB. Intracellular calcium homeostasis in cardiac myocytes. Circulation 1993;87: 1806- 1815. [36] Bountra C, Powell T, Vaughan-Jones RD. Comparison of intracellular pH transients in single ventricular myocytes and isolated ventricular muscle of puinea-pig. .I Physiol 1990;424: 343-365. I371 Dow JW, Harding NGL, Powell T. Isolated cardiac myocytes. I. Preparation of adult myocytes and their homology with the intact tissue. Cardiovasc Res 1981; 15. 483-514. [38] Mitra R, Morad M. A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates. Am J Physiol 1985;249: H1056-H1060. 1391 Bkaily G, Sperelakis N, Doane J. A new method for preparation of isolated single adult myocytes. Am J Physiol 1984,247: H1018-H1026. 1401 Johansson H, Rastad J, et al. Microfluorometric measurementsof cytoplasmic calcium in chief and oxyphil parathyroid cells of adenomatous and hyperplastic glands and of normal-sized glands associatedwith adenomas. Surgery 1989: 106: 517-524.

44

Y-C. Li et al. /Reau.vcitation 32 (19%) 33-U

[411 Ridefklt P, Hellman P, et al. Neomycin interacts with Cal+ sensing of normal and adenomatous pamthyroid cells. Mol Cell Bndocrinol 1992;83: 211-218. [42] Stacpoole PW. Lactic acid& the case against b&ubonatc tlumpy. Ann Intern Med 1986; 105: 276-279. 1431 Strauss J. Tris (hydroxymethyl) amino-methane ra+& A pediatric evaluation. Pcdiatria 1968; 41: . [44] Wiklund L, Sahlin R. Induction and treatment of metabolic acidosis: a study of pH changesin porcine skeletal muscle and cerebrospinal fluid. Crit Care Med 1985; 13: w-113. [4s] Bishop RL, Weisfeldt ML. Sodium bicarbonate administration during cardiac arrest. J Am Med Assoc 1976;235: 506409. I461 Ng ML, Levy MN, Zkske HA. Effects of changesof pH and carbon dioxide tension on kft ventricular pcrformance. Am J Physiol 1967;213: 115-120.

[47 Wcil MH, Rackow EC, ct al. Difbmce in acid-base state between venous and arterial blood during cardiopulmonary resuscitation. N Bngl J Med 1986; 315: 153-156. 148) Mattar JA, Weil MH, et al. Cardiac arrest in the critically ilk II. Hyperosmolal states following cardiac amst. Am J Med 1974;26: 162-168. 1491 Bidani A, Brown SBS, et al. Cytoplasmic pH in pulmonary macrophagcs:ramcry from acid load is Na+ indeeg6and NEM sensitive. Am J Physiol 1989; 257: [So] Thomas ;A, Buchsbaum RN, et al. Intracellular pH nmaucmmts in Elulkh ascitea tumour cells utilizing spectroscopic probes generated in situ. Biochemistry 1979; 18: 2210-2218. (511 Dybvik T, Strand T, et al. Buffet therapy during out-ofhospital cardiopulmonary rcswcitation. Resuscitation 1995;29: 89-95.