Temperature-induced variations of blood acid-base status in the lugworm, Arenicola marina (L.): bdI. In vitro study

Respiration Physiology ( 1977) 31, 0

Elsevier/North-Holland

I 39- I49

Biomedical

Press

TEMPERATURE-INDUCED VARIATIONS OF BLOOD ACID-BASE IN THE LUGWORM, ARENZCOLA MARINA (L.): I. IN VITRO

ANDRfi

STATUS STUDY’

TOULMOND2

Laboratoire de Zoologie, UnirersitP Pierre-et-Marie-Curie, Paris; Station Biologique, Roscoff; Loboratoire de Physiologic Respiratoire, C. N. R.S., Strasbourg. France

Abstract.

temperature

Blood of the lugworm Arenicola marina studied in vitro behaved like a Rosenthal system: when whereas [HCOS] remained practically constant. rose, pH decreased and P co1 increased,

pH values were low whatever cantly

different

0 and

30 C. Consequently,

I .53-5.06)

and increased

of protein

imidazole

groups,

The very variable

buffer

molecule,

alkalinity

coefficient

of the blood,

with temperature.

dpH/dt

of the neutral [OH-]/[H+],

Calculated

changes

was always

signiti-

pH of pure water between was very low (range,

in the fractional

dissociation

x,,,,, were smaller. power

of Arenicola hemoglobin was maximum (/Imax) for a strictly defined, ionizable group on the ), suggesting that as yet unidentified

RH, could be responsible pK&

= pH#,,,,

for the pH-dependent

the calculated

fractional

changes

dissociation

of blood buffer of RH,

power in

rRH, was constant

0 and 30 C.

The nature protein

variations

value of pH (pH,,,,

Arenicola. Assuming between

the relative

appreciably

temperature-dependent hemoglobin

the C co2 and So,. The temperature

from the mean temperature-induced

of RH is discussed

imidazole

groups

in relation

in the regulation Acid-base

with Reeves’s

of extra-

Rosenthal relative

concerning

the preeminence

of

pH.

Hemoglobin

balance

Alphastat Constant

hypothesis

and intracellular

system

Temperature

alkalinity

The blood pH of poikilotherms decreases when their body temperature increases. This phenomenon was first observed by Austin et al. (1927) in alligators and was rediscovered by Robin (1962) in the turtle Pseudemys scripta elegans. From these observations, it became obvious that the axiom of a strictly regulated value of blood pH was only true in animals capable of maintaining their body temperature at a Acceptedfor

publication 22 March 1977.

’ This research

was partly

supported

by the Centre

National

de la Recherche

Scientitique.

Paris,

ATP

No. 469906. ’ Reprint

requests

to: Laboratoire

de Zoologie,

Universiti:

Paris Cedex 05, France. 139

Pierre-et-Marie-Curie,

4 place Jussieu,

75230

140

A. TOULMOND

constant level, i.e. in homeotherms. Rahn (1967) suggested that in vertebrate poikilotherms what was held constant was the ‘relative alkalinity of the blood’, conveniently m a given species, whatever the temperature, expressed by the ratio [OH-]/[H+]: the difference between blood pH and pN, the temperature-dependent value of neutral pH in water, remains constant. Later, Reeves (1972) proposed a complementary hypothesis in which the constancy ofthe [OH-]/[H ‘1 ratio was the consequence of a primary regulation of the fractional dissociation of protein imidazole groups. Rahn’s hypothesis has been experimentally tested in several species of fishes, amphibians and reptiles (see Dejours, 1975; Heisler rt al., 1976). Among invertebrate poikilotherms, detailed studies have been made in aquatic and terrestrial arthropods, by Truchot (1973) in Carcinus maenas, and by Howell et al. (1973) in Callinectes sapidus, Car&us maenas, Uca pugilator, Limulus polyphemus, Gecarcinus lateralis and Paralithodes camtschaticus.

The present article reports on the effect of temperature on blood acid-base status in vitro in a polychaete annelid, the lugworm Arenicola marina (L.). Of special interest

is the fact that Arenicoja marina blood composition is simple, since one may approximately describe it as a solution of hemoglobin in sea water (Roche et al., 1960; Florkin, 1969). Moreover, the buffer power of this hemoglobin presents some peculiarities since it varies considerably in the physiological range of pH, showing a maximum value for a strictly defined value of pH. Some of the results of in vitro experiments have been published in abstract form (Toulmond, 1976) and the in vivo variations of blood acid-base status are treated in a separate article (Toulmond, 1977).

Materials and methods

Lugworms were collected in August and September 1975 at the exit of the ‘Vieux Port’ of Roscoff (Nord-Finis&e, France), brought back to the laboratory and kept unfed in running, aerated sea water (15-l 7 ’ C) for 48 h before experiments. Prebranchial blood was withdrawn from the ventral vessel (Ashworth, 1904) into a glass pipette. Pooled blood from several animals was centrifuged at low speed for 10 min at 4’C in order to eliminate the white cells and then stored at - 35 ‘C. Measurements

pH values in blood were measured with a thermostatted Radiometer capillary pHmicroelectrode (G297/G2 or G299A) coupled to a Radiometer PHM 72 pH-meter. The electrode was calibrated for each experimental temperature with Radiometer precision phosphate buffers. The concentrations of combined oxygen in blood when the hemoglobin is fully saturated, Cg&, and ofblood protein [Pr], were determined using methods described elsewhere (Toulmond, 1973). Gas phases were prepared from pure gases with Wiisthoff gas-mixing pumps.

TEMPERATURE Experimental

AND

BLOOD ACID-BASESTATUS~N

141

Arenicola

protocols

variations of the lugworm blood pH with temperature were measured at constant values of carbon dioxide concentration, Ccoz. and percentage saturation of blood pigment, So*. Each of six l-ml aliquots of the pooled lugworm blood was separately equilibrated at 30°C in a 5 ml glass syringe against one of six different gas phases: PO, = 0 or 150 torr, giving corresponding values of So, = 0 and 100%; PCoL= 0,2.2 or 7.3 torr, using a procedure described by Truchot (1973). After equilibration, the gas phase was carefully expelled and the syringe kept tightly closed in melting ice. pH was measured by anaerobically transferring the blood from the syringe into the capillary microelectrode thermostatted at 30, 26, 22, 18, 14, 10 and 6 C. Corresponding concentrations of bicarbonates and total carbon dioxide were calculated by substitution of the pH values into the Henderson-Hasselbalch equation in the form In vitro

pH = pK; + log [HCO:]

-log uco2 . Pco2

Values of pK; and ~~~~appear in table 1. For the low observed values of pH, carbonate concentration may be neglected. We consider the carbamate concentration to have been negligible. Blood titration curves were obtained in the following way: 50 ~1 blood aliquots were placed in an Astrup microtonometer thermostatted at 5, 10, 15,20,25 or 30 “C. Five ~1 of a solution of hydrochloric acid or sodium hydroxide of known concentration were added to the aliquots with vigourous shaking. The pH of each aliquot was measured after 30 min of equilibration against a gas phase composed of pure nitrogen.

TABLE1 Values of constants substituted in the HendersonHasselbalch Temperature ( C)

Ro, ’ (mmol.L~‘.torr-‘)

PK;' Pcol = 2.2 torr

6 IO 14 18 22 26 30

0.067 0.059 0.052 0.046 0.041 0.037 0.033

equation

Pco, = 7.3 torr

sol = o:/,

so, = 100%

so, = oo/,

so, = 100%

6.02 5.99 5.97 5.95 5.93 5.91 5.91 --_~________

6.04 6.01 5.98 5.96 5.94 5.93 5.93

6.02 6.00 5.97 5.95 5.93 5.92 5.91

6.05 6.02 5.99 5.97 5.95 5.94 5.93

’ acO, = 98% of aco2 in sea water of chlorinity = 19.5’/00 (Harvey, 1963; Toulmond, unpublished). ’ at 15°C. pK’r values for Arenicola blood are pH dependent (Toulmond, unpublished). pK’, values of this table were obtained assuming that pK’, variations with temperature, at a given pH, parallel those of ~K’I in sea water of chlorinity = 19.5°/00 (Harvey, 1963).

142

A. TOULMOND

Results In vitro temperature-induced variations of pH, Pco2 and [HCO;] Figure 1A shows six regression lines describing the variations of pH with temperature in six aliquots of a sample of pooled whole blood of Arenicola. In each aliquot, total carbon dioxide content, GoI. and hemoglobin saturation percentage, So2, were held constant for all temperatures (see table 2). Calculated parameters of each regression line are given in table 3. In each case, pH decreased when temperature rose. The slopes dpH/dt are slightly different, ranging from - 0.0137 to - 0.0152, but these differences cannot be correlated with the set initial values of P co2 and So,. Values of pH for a given temperature were strictly dependent on these initial Pco2 and So, values: the higher the PCo2, the lower the pH; and at the same time, for a given value of Pco,, Ccoz was higher in deoxygenated blood (Haldane effect, see table 3). Calculated values of the ratio [OH-]/[H+], q uantifying the relative alkalinity of the blood, vary from 1.53 to 5.06 (table 4). Due to the fact that the slope of the curve describing the temperature-induced variations of neutral pH in water (curve pN, fig. 1A) is slightly less than the slope of any of the regression lines, the value of the [OH-]/[H+] ra t’IO increased significantly with increasing temperature. Calculated variations of Pco2 are shown in table 2 and fig. IB. From the initial values of 2.2 and 7.3 torr at 30 “C, P co2 decreased with temperature, respectively to 0.6 and 2 torr at 6°C. Pco, variations were not significantly different in oxygenated and deoxygenated blood. Pco2 . torr

PH

t

8-

B 6-

4-

2-

/. 2

oL

0

I

I

10

Fig. 1. A. In vitro variations dioxide concentration

I

I

1

20

30

of pH with temperature

and constant

hemoglobin

1

.o

0

t,“C

10

in Arenicolu

oxygen saturation.

30

20 blood

maintained

Parameters

at constant

ofthe regression

carbon lines were

calculated by the method of least squares (table 3) from pH values of table 2. -0-1 So, = 0%; -0-: So, = 100%; (1): Pco, =O; (2): Pco, = 2.2 torr; (3): PFo, = 7 3 torr. pN: pH (or pOH) value of neutral water ([OH-]/[H+] = 1). CT& = 5.1 mmol, L-l, [Pr] = 129 g. L-‘. B. Mean calculated

variations

of P co2 with temperature

in Arenicolu

blood (see table 2). Legend as in A.

TEMPERATURE

AND BLOOD ACIWBASE

TABLE In vitro variations at constant

of pH, Pco, and [HCOF]

carbon

as a function

dioxide concentration

(Cc,,)

2 of temperature

and constant

(%) *Pro2 ca 0 *cco,

PH

100

*Pco, = 2.2 torr

PH

*Ccol = constant

Pco, (torr, talc.) 1.64mmol.

L-r [HCOS]

1.34mmol.

100%

[HCOS]

* initial conditions

7.361 7.419

6

7.548

7.618

7.490 7.550

7.467 7.526 7.565

7.277 7.339 7.385

7.441 7.513

0.97

0.79

0.68

100 2.20

1.77

1.44

1.17

0.98

0.81

0.65

0

1.56

1.57

1.58

1.58

1.59

1.59

1.59

100

1.27

I.28

1.28

1.29

1.29

1.29

1.30

7.321 7.385 7.438 7.504

7.176 7.233 7.291 7.350

7.544

7.405 7.465

0 7.30

5.86

4.69

3.79

3.11

2.53

2.13

100 7.30

5.54

4.60

3.76

3.07

2.55

2.11

0 4.68

4.70

4.73

4.15

4.76

4.76

4.78

3.61

3.62

3.64

3.65

3.66

3.67

(mEq. L-r, talc.)

mmol.L-’

7.297 7.359 7.413

IO

1.17

Pco, (torr, talc.)

Cco,=3.81

14

(So,)

1.43

0 7.197 1.257

Cco,=4.92mmol~L~’

18

7.312 1.367 7.423 7.480

7.185 7.242

100 7.111

forSo,=O% forSo,=

22

saturation

1.74

PH (talc.)

oxygen

0 2.20

(mEq. L-l, talc.)

L-t

*Pco, = 7.3 torr *Ccol = constant

26

100 7.168 7.219

forSo,=lOO% Cco,=

t, in Arenicola blood maintained

hemoglobin

0 7.243 7.297

(talc.)

forSo,=O% Ceo,=

*30

0 7.263

ca 0

143

Arenicola

STATUS IN

100

3.57

(t = 30°C).

Each value of pH = mean of 2 or 3 measurements

TABLE Parameters

3

lines pH = f(t) of fig. IA, calculated

by the method

of least squares

pco,’

cco,1

(torr)

(mm01 . L-

0.

0

0.9979

7.695

-0.0147~0.0011

2.2

1.64

0.9992

7.657

- 0.0137 + 0.0006

3 4

0 0 0

7.3

4.92

0.9982

1.644

-0.0148~0.0010

100

0

0

0.9991

7.637

-0.0152+0.0002

5

100

2.2

1.34

0.9990

7.590

-0.0142+0.0008

6

100

1.3

3.81

0.9999

7.553

-0.0146+0.0002

aliquot No.

of the regression

1 2

SO2 (%)

’ initial conditions * k SEM x

Correlation

’)

coefficient -_

PH fort

Slop& = 0°C

(t = 30°C).

t (Student) fonN = 7 and P = 0.05.

Calculated values of [HCO;] are shown in table 2. [HCO:] constant, whatever the temperature.

remained practically

In vitro temperature-induced variations of Arenicola hemoglobin buffer power Titration curves of whole Arenicola blood are shown in fig. 2. For each value of

144

ATOULMOND Base

added mEq. L-’ of blood

6.5

7.5

ZO

8.0

pH

8.5

blood titration curves. From left to right: curve at 30,25,20, 15,IOand 5 ‘C. Each point is the mean oftwo measurements. Cm”” Hb0~=4.85mmol~L~‘;[~]=126g~L~‘;Pco,=O;So,=O(Po,=0). Dotted curve: titration curve of a solution of human deoxygenated hemoglobin (Cfg, = 5.8 mmol.L-‘), obtained at 37 C in the same conditions (redrawn from Toulmond, 1971). Fig. 2. Arpnic&

7.8 -

r = -0.9966

7.6 -

l

\

.

. . 7.4 -

\

l\

72 -

1 0 Fig. 3. Temperature-induced

I

,

10

20

I t,“C

30

variations of pHg,,,, value of pH for which the buffer power of Arenicola

blood is maximum at a given temperature ~maximum slope of the titration curves of fig. 2). Parameters

of the regression line were calculated by the method of least squares : pH,,,,_ = - 0.0150 f 7.755 ; r : correlation coefficient; SEM (slope) x t (Student) = 0.0017 for N = 6 and P = 0.05.

TEMPERATUREANDBLOOD

ACILFBASESTATUS

IN

Arenicola

145

temperature, the slope of the curves, fl = d [base added]/d pH, i.e. the buffer power of the blood as defined by Van Slyke (1922) abruptly increases in a very narrow range of pH. Values of pH corresponding to maximum buffer power, /Imax,are plotted in fig. 3 against temperature. The calculated slope of the regression line (- 0.0150) is quite comparable to the slopes of the regression lines in fig. 1A.

Discussion

In uiuo, at 15 “C, blood hemoglobin of Arenicola can be either practically fully saturated or desaturated with oxygen, and values of P co2 can vary between 0.5 and 3 torr (Toulmond, 1973,1975). Thus, values of So, and P co2 imposed in the present in vitro experiments cover the whole physiological range of these two variables. In uitro, our results show that Arenicola blood, kept at constant values of So, and Cco2 and exposed to temperature changes, behaves like what Reeves (1972) calls a Rosenthal system: when temperature decreases, pH increases and Pcol decreases, whereas [HCO;] remains almost constant. Such general variations of blood acidbase status in vitro were first observed by Rosenthal (1948) on mammalian blood anaerobically cooled from 38 “C to 18 “C, and have been since demonstrated in the blood of the turtle (Robin, 1962), the bullfrog (Reeves, 1972) and the shore crab (Truchot, 1973). Our similar results on Arenicola present nevertheless some peculiarities which must be examined in detail. Values of Arenicola’s coefficient of pH variation with temperature, dpH/dt, vary between -0.0137 and-0.0152, values lower than that of -0.0130 given by Weber (1972) for Arenicola marina and obtained in unspecified conditions. Our values are of the order of those found in whole blood of mammals (- 0.0147, Rosenthal, 1948) the turtle Pseudemys scripta elegans (-0.0144, Robin, 1962) and the bullfrog Rana catesbeiana (- 0.0157, Reeves, 1972). However, they differ notably from the mean values recalculated from data of Howell et al. (1970) for the bullfrog and the turtle Chelydra serpentina (-0.0171) and of Howell et al. (1973) for Limulus polyphemus (-0.0170). The value of -0.0195 found by Truchot (1973) in the crab Carcinus maenas appears to be exceptionally low. Thus, our values of dpH/dt in Arenicolti are among the highest values recorded in the literature, and they are significantly different (P < 0.001) from the mean value of the temperature-induced variations of pN (dpN/dt = -0.0186 for 0 < t < 30 “C, calculated from data in Handbook of Chemistry and Physics, 1961-62). This, added to the fact that pH values were very low at all temperatures, entails two consequences concerning the value of the blood relative alkalinity in Arenicola. Absolute values of the ratio [OH-]/[H+] calculated for the six curves of fig. 1A (table 4) vary between 1.53 and 5.06, very low compared to those of 20 to 40 found in vertebrate poikilotherms (Howell et al., 1970). Nevertheless, they seem to be the rule in invertebrates, since Truchot (1973) reported a value of 12.5 in Carcinus and Howell et al. (1973) values (calculated from Mangum, 1973) of 6.5 in the sipunculid

146

A. TOULMOND

TABLE Calculated

temperature-inducedvariations,

of fractional

dissociation

tified group

of imidazole

groups

(Q,,,) and of fractional

(xi&. in Arenicola blood

Values of [OH -]/[H

Blood’

4

between Oand 30 ‘C, ofbloodrelativealkalinity([OH-]/[H+]),

‘1’

dissociation

kept in vitro at constant

ofa hypothetical

uniden-

values of Cco, and Sol Values of IaH 4

Values of %,m3

aliquot oc

30 ‘C

:‘, change o-+30

oc

30 c

c

:a change O-30

oc

30 c

“(1change 0+3O’C

c

no. I

2.95

5.06

+72

0.360

0.415

+I5

0.465

0.467

<+I

2 3

2.48 2.33

4.88 3.94

+97 f69

0.339 0.332

0.412 0.386

f22 +I6

0.442 0.437

0.463 0.437

<+5

4

2.26

3.61

+60

0.329

0.375

+ 14

0.433

0.426

<-2

0

5

I.82

3.34

+84

0.306

0.366

+20

0.407

0.417

<+3

6

1.53

2.67

+75

0.286

0.340

+19

0.386

0.389

<+I

’ see table 3. 2 calculated 1961-62)

using pN = 7.460-0.0186

t (0 < t < 30’ C,data

from Handbook qf’Chemistr,v and Physics,

and mean pH values from table 3.

3 I,,,, = [Im]/([HIm+]

+ [Im]) = fractional

dissociation

ofimidazole

groups,

Calculated

using pK;,

from

Reeves (1972) and mean pH values from table 3. 4 xan = [R-]/([RH] regression

+ [R-l)

= fractional

dissociation

of RH. Calculated

using pKkn = pH,,,,,.

from the

line in fig. 3 and mean pH values from table 3

Phascolopsis gouldi and 4 in the polychaete annelid Glycera dibranchiata. In fact, the value of the relative alkalinity of Arenicola blood appears to be very close to the intracellular value of this parameter estimated by Rahn et al. (1974) for vertebrates. Second, Arenicola’s blood relative alkalinity does not remain constant with changing temperature. Table 4 shows that [OH-]/[H+] increased by 60 to almost 100 % when temperature rose from 0 to 30°C. This apparent discordance with Rahn’s (1967) hypothesis is not unique: a rather lesser inconstancy has been observed in bullfrog blood (Reeves, 1972). We have said that Arenicola blood, in vitro, behaves like what Reeves (1972) calls a Rosenthal system, i.e. a mixture of several weak acid-conjugated base buffers, one of which must necessarily account for the observed temperature-induced changes of blood pH. Albery and Lloyd (1967) have demonstrated on the basis of Rahn’s (1967) data, that such a buffer system must have a pK’around 7 and a heat of dissociation of approximately 7 kcal . mol- ‘. Phosphate and carbonic acid-bicarbonate buffers, present in all biological fluids, are thus excluded. Reeves (1972) found that imidazole groups of histidine in proteins were the only possible candidates and he effectively showed that in bullfrog b&d the fractional dissociation of these groups (aim = [Im]/[(HIm+] + [Im]) = l/Cl +antilog (pKi,- pH)]) remains constant whatever the temperature. In Arenicola blood also, it appears that txi,,,changes with temperature are much less important than [OH-]/[H+] changes (table 4).

TEMPERATURE AND

BLOOD

ACID-BASESTATUSIN

Arenicolu

147

Let us now consider the titration curves of Arenicola blood in fig. 2. E&h curve shows that the buffer power /? of the blood strongly increases in a very narrow range of pH. The main characteristics of this phenomenon (Toulmond, 1971) are that the variations of /? must be related to the presence of dissolved hemoglobin in Arenicoh, and that they always occur in the same range of pH at a given temperature. The buffer power is maximum (fimax)for a strictly defined value of pH (pHp,,,) being independent of the hemoglobin percentage saturation. The sharp inflection in the titration curves of Arenicofu hemoglobin may be interpreted as the result of the titration of one particular ionizable group of the protein, which we shall call RH. Since the buffer power of a group is maximum when pH = pK’, perhaps values of pH,,,, = pKkH for each given temperature, so that fig. 3 would describe the variations of pK KHwith temperature. These variations (d pKk”/dt = - 0.0150) so closely follow the temperature-induced variations of Arenicofu blood pH that calculated values of aRHfor given values of Cco2 and So, remain practically constant, whatever the temperature (table 4). Thus, in the particular case of Arenicola, (R-)-(RH) groups on hemoglobin appear to be the weak acid-conjugated base buffer system postulated by Albery and Lloyd (1967). Is RH imidazole? Values of pHB,,, = ~K&Hare significantly different from pK;, values given by Reeves (1972). However, as is well known, numerous factors may affect the intrinsic dissociation constant of a given group at a given temperature, particularly the position of the carrier amino acid residue in the protein molecule, its relations with other residues and, also, the variations of these relations due to conformational changes in the protein structure. Most troublesome is the fact that Arenicolu hemoglobin appears to be relatively poor in histidine, the concentration being approximately half that in human hemoglobin (Roche and Combette, 1937). However, this difference need not argue decisively against imidazole as RH, since the work of Steinhardt et al. (1962) suggests that, in native human hemoglobin, about half of the imidazole groups are masked and electrochemically inactive. Another fact which must be pointed out is that, whereas the position of prnaxon the pH scale at a given temperature is independent of the hemoglobin percentage saturation, its absolute value is strictly dependent on So2, i.e. of the oxy- or deoxy-conformation of the protein molecule (Toulmond, 1971). This observation may perhaps be related with Weber’s (1970) finding that the value of the heme-heme interaction constant in Arenicolu hemoglobin is pH dependent, and shows a maximum for a welldefined value of pH. RH may therefore be related to the oxygen-binding sites of the hemoglobin molecule, and it is known that in human hemoglobin, half ofthe oxygenlinked Bohr protons come from imidazole groups of histidine 1468 (Perutz et al., 1969; Kilmartin and Wootton, 1970). These considerations suggest that RH may well be an imidazole group. However, this remains to be experimentally proved. The determination of the exact nature of RH in Arenicola hemoglobin could provide a good test of the validity of Reeves’ hypothesis on the preeminence of imidazole in extra- and intracellular pH regulation.

148

A. TOULMOND

Acknowledgements

We wish to thank the staff of the Station Biologique de Roscoff for the facilities put at our disposal.

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