Hemocyanin Function in Active and Dormant Land ...

Hemocyanin Function in Active and Dormant Land Snails, Otala lactea Author(s): M. Christopher Barnhart Source: Physiological Zoology, Vol. 59, No. 6 (Nov. - Dec., 1986), pp. 725-732 Published by: The University of Chicago Press Stable URL: http://www.jstor.org/stable/30158618 . Accessed: 05/09/2013 09:42 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp

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HEMOCYANIN FUNCTION IN ACTIVE AND DORMANT LAND SNAILS, OTALA LACTEA' M. CHRISTOPHER BARNHART2 of California, LosAngeles,California 90024 of Biology,University Department (Accepted 6/9/86) The respiratoryfunction of hemocyaninwas assessedin active and dormantindividualsand in activeindividualsat differentbodytemperatures.Hemocyaninoxygen affinityand oxygen-carryingcapacitywere low (P50= 43 torr at pH 7.9, body tem= 0.11 mM). The Bohr shift was negative below pH 7.9, perature = 25 C, and CHCO2

so that oxygen affinityincreasedas pH declined.The negativeBohr shift appearsto increaseoxygen transportin dormantsnails,whichare acidoticand frequentlyhave very low PO2in the lung and hemolymph.In activesnails, only - 10%of the hemocyanin oxygen-carryingcapacityis utilizedat 5 C, but >70% is utilized at 25 C. INTRODUCTION The pulmonate land snail Otala lactea (Helicidae) survives periods of dry weather by retracting into its shell and entering a state of dormancy, in which water loss and metabolism are greatly reduced. Partial pressures of 02 (P02) and of CO2 (PCO2) differ greatly between active and dormant individuals (Barnhart 1986b). Restriction of lung ventilation during dormancy results in low values of P02 in the lung and hemolymph, while PCo2 is elevated and pH declines. The changes in hemolymph 02 tensions and pH during dormancy are of interest in relation to the function of hemocyanin (Hc), the O2-carrier protein of molluscan hemolymph (reviewed by Mangum 1980). In gastropods, He typically exhibits a negative or "reversed" Bohr effect over some range of pH, so that 02 affinity increases with decrease in pH (Mangum and Lykkeboe 1979; Brix 1982 and references

'This work is part of a dissertationsubmittedin partialfulfillmentof the requirementsfor the degree of Doctorof Philosophyat the Universityof California, Los Angeles,and was supportedby a Universityof CaliforniaChancellor'sIntern Fellowshipand NSF grantDEB 78-03471 to G. A. Bartholomew.I thank G. A. Bartholomew,M. S. Gordon, and B. R. McMahonfor theiradviceand encouragement. 2 Present address: Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California 92717.

Physiol. Zool. 59(6):725-732. 1986. a 1986 by The University of Chicago. All rights reserved. 0031-935X/86/5906-8588$02.00

therein). In some marine gastropods and in the horseshoe crabs (Xiphosura), the negative Bohr effect improves 02 loading and transport during hypoxia, because hypoxia induces acidosis and He 02 affinity increases (Johansen and Petersen 1975; Brix 1982; Mangum 1983). In contrast, the physiological significance of the negative Bohr shift has been discounted in the helicid land snails, because the known range ofpH in Helix lies above the point of reversal (Spoek, Bakker, and Wolvekamp 1964; Mangum 1980). However, the low Po2 and acidosis that occur in Otala during dormancy provide a different context for interpretation of He function. Temperature may also affect hemolymph 02 transport, because decrease of temperature causes increase of He 02 affinity and of 02 solubility (So2). In southern California, individuals of Otala are active in the field at body temperatures ranging between 4 and 24 C (M. C. Barnhart, personal observation). Low temperatures stimulate activity in Otala (Herreid and Rokitka 1976). However, the increase of He 02 affinity at low temperature appears likely to inhibit 02 unloading at physiological values of Po2 in active snails. It is therefore desirable to examine He function over the range of body temperature. In the present study, the in vitro 02binding curves of whole hemolymph were compared with in vivo lung and hemolymph gas tensions and pH (Barnhart 1986b) in order to assess hemolymph 02 transport in relation to dormancy and to changes in body temperature in Otala.

725


726

M. C. BARNHART MATERIAL AND METHODS 02-BINDING CURVES

02-binding curves were measured specusing a modified trophotometrically Aminco Hem-O-Scan apparatus (Arp and Childress 1981). Thin layers of whole hemolymph were held between two teflon membranes (each membrane 6 ctmthick). The hemolymph films were prepared in a gas bag containing 5% CO2 in air, in order to prevent loss of CO2 from the hemolymph and precipitation of CaCO3(Campbell and Boyan 1976). The samples were placed in the beam path of a dual-wavelength spectrophotometer within a gas flow-through chamber, and the ratio of optical absorbance at 360 nm and 420 nm was used to determine oxygenation. Po2 in the chamber was changed in increments, rather than continuously, to allow sufficient time for complete equilibration between chamber and sample Po2'S at each point. Po2 was controlled by regulating the flow rates from two pressurized gas cylinders, one containing 21% 02, and the other 0%02. The two gas mixtures had identical CO2 content. The two gas streams were mixed, humidified, and then directed into the chamber containing the sample. Fractional He oxygenation and the chamber PO2 were recorded simultaneously by an XY recorder. Approximately 10 points were measured for each curve. Values of Po2 at specific levels of saturation, such as P50, were obtained by interpolation between the measured points. Bohr and Root shifts.-The Bohr shift was determined by measuring the O2-binding curve of each sample at four to six different levels of Pco2 between 3 and 97 torr, which spans the physiological range of PCo2 in Otala (Barnhart 1986b). The pH was determined from the relationship between pH and PCo2, which was determined by equilibrating a portion of each hemolymph sample at two levels of Pco2 and measuring pH (details in Barnhart 1986a). No attempt was made to determine pH-independent effects of CO2, which were assumed to be small or absent (see Mangum 1980; Wells and Weber 1982). The Root shift was tested spectrophotometrically by monitoring oxygenation at 25 C as Pco2 was varied between 3 and 97 torr at P02 = 150 torr. Temperature effect.-02-binding curves of three samples of hemolymph were mea-

sured at 5 and 25 C at PC02 = 14 torr. The pH of these samples was not measured, but the mean pH of similar samples at the same PCo2 increased from 7.69 at 5 C to 7.81 at 25 C. The Bohr shift over this range of pH was very small, and the effects ofpH change on 02 affinity were therefore ignored. The effect of temperature on He 02 affinity was expressed as A log Po2/AT at He saturations from 10%to 95%. 02 CAPACITY OF HEMOLYMPH

Hemolymph samples from active snails were equilibrated in a tonometer at 156 torr P02, 13 torr Pc02. 02 content was then measured using a method similar in principle to the methods of Tucker (1967) and Bridges, Bicudo, and Lykkeboe (1979). A 10-g/liter aqueous solution of KCN was deoxygenated by shaking with N2 gas and then stored in a large glass syringe. The large syringe was used to fill a 1-ml syringe (reaction chamber). Hemolymph (50 l1) was injected into the reaction chamber using a microsyringe. A glass bead in the chamber was shaken to mix the KCN and hemolymph, and the change in Po02was determined in a Radiometer Blood Micro System Mk II blood-gas analyzer calibrated to double the normal sensitivity. The method was calibrated by using 02-saturated water in place of hemolymph, and precision was within 0.01 mmol 02/liter. The He 02 cOntent was determined by subtracting the calculated dissolved 02 from the total 02 content. Protein content (g/liter) of hemolymph samples was determined using the Bradford method (Biorad reagents) as described elsewhere (Barnhart 1986a). So2 was determined at 25 C from the slope of hemolymph 02 content versus PO2 between 150 and 500 torr, a range above the point of He saturation. So2'S at 15 and 5 C were estimated from the 25 C result by interpolation using the table provided by Hitchman (1978, table B2), which shows combined solute and temperature effects on So2. RESULTS 02-DISSOCIATION CURVES

shape of the 02Effects of pH.-The binding curve changed from sigmoidal to hyperbolic as pH declined (fig. 1). The Bohr shift differed in direction and magnitude at


HEMOCYANIN

727

FUNCTION IN ACTIVE AND DORMANT SNAILS

100

-

pH

C

_

-iro

50 -

L_

_

,PCO2

o

o

3.9

8.23

13.3

7.84

51.5

7.30

-

4--)

cn

100

50

0

Po02, Torr FIG. 1.-02 equilibrium curves in a representative sample of hemolymph at three levels of PCo2 and pH (ambient temperature = 25 C). The Bohr shift is positive between the higher and the middle pH and negative between the middle and the lower pH.

Effect of temperature.--02 affinity decreased with increasing temperature. The effect of temperature on 02 affinity was lower at high He saturation than at low saturation (fig. 3). Thus, the shape of the binding curve varied with temperature, becoming more sharply inflected at high saturations as temperature increased (fig. 4).

different levels of saturation. At Ps0o,the Bohr shift was positive at high pH and negative below approximately pH 7.9 at 25 C (fig. 2). Over the physiological range ofpH, Ps50declined from -43 torr at pH 7.9 to 21.2 torr at pH 7.0, and the Hill coefficient declined from about 6 to 1.2 (table 1). No Root shift was detected. 2.0 Pso

1.5 O

"P50

0)

1.0

A

A

P20 0.5a 6.5

/

7.5

8.5

pH FIG. 2.-The Bohr shift of a representative sample of hemolymph at three levels of 02 saturation (ambient temperature = 25 C). The Bohr shift is negative below pH 7.9 at 50% and 20% saturation.


728

M. C. BARNHART TABLE 1 Hc 02-BINDING

PROPERTIES IN WHOLE HEMOLYMPH

AT 25 C

lactea

OF Otala

PCo2

(torr) 02-BINDING PROPERTY

pH ................. Ps0 (torr)............ Hill coefficient (ns5o) ... n ...................

3.9

13.3

26.5

51.5

97.3

8.34 a .07 34.7 + 4.0 6.28 a 2.15 4

7.88 + .03 45.4 a+7.0 5.99 + 1.78 6

7.60 a .02 35.2 a 5.5 3.28 a .50 6

7.33 + .02 25.2 + 6.0 1.66 + .29 6

7.03 + .06 20.6 + 3.0 1.19 a .12 6

NOTE.-pH, P50, and Hill coefficient values are means a SD. n = Sample size. Bohr coefficient (pH 7.0-7.9): .40 + .052, (n = 6); CHcO2(mmol/liter)

= .107 a .018 (n = 8).

Y= 0.0083X- 0.0016,

SO2 AND 02 CAPACITY

The So2 in the pooled sample of hemolymph from active snails (0.00162 mmol/1 torr; table l) was slightly lower than So2 in distilled water (0.00166 mmol/1 torr at 25 C [Dejours 1981]). Calculated values of hemolymph So2 at 5 and 15 C are, respectively, 0.00243 and 0.00194 mmol/1 torr. The He 02-binding

capacity

(CHcO2)

(r2 = .98) where Y is CHcO2 (mmol/liter) and Xis hemolymph protein concentration (g/liter). DISCUSSION

The 02-binding curves of whole hemolymph from Otala showed a much lower 02 affinity than those previously reported in other terrestrial pulmonates. In Helix pomatia, Ps50is -~ 11-15 torr at 20 C and pH 7.6-8.2 (Spoek et al. 1964; Konings et

Of

hemolymph from active snails was variable but generally low (table 1). CHcO2was directly related to total protein content of the hemolymph, according to the linear regression equation:

.04 .0 3 H-

*

-

* 0

o .0 2 -

_J

.~i.I

2

Y= 2.42* 10

0

-62

-4

+

3.7810-4

X-

6.47" 0-6

X2

50

100

Saturation, %0 FIG. 3.-Effect of temperature on He 02 affinity in whole hemolymph. A Log Po2/AT was calculated for each of three samples from dissociation curves measured at 5 and 25 C (pH - 7.8). The line and equation were fit by curvilinear regression.


729

HEMOCYANIN FUNCTION IN ACTIVE AND DORMANT SNAILS

10

C o O a

50

i

"

I,

a

15 25

00

"

5 15 25

5 "C

50

'venous

.

arterial 100

Po2, Torr FIG.4.-Effect of temperatureon 02 transportby hemocyaninin active Otala.The curveat rightis the mean dissociationcurveat 25 C, pH 7.9. The horizontalbarindicatesa SE(n = 6). At middleandleftarethe estimated dissociationcurvesat 15 and 5 C, which werecalculatedfromthe 25 C curve using the equationin fig. 3. The meanarterialand venous Po2'Sat each temperature(fromBarnhart1986b)are indicated.The verticalarrowsat left showthe predictedarterial-venousdifferencein saturation,whichincreasesfrom - 10%at 5 C to >70% at 25 C.

al. 1969), and, in the pulmonate slug Arion,

it is -~-16.8 torr at 20 C, pH 7.94 (Wells and Weber 1982). Mean Ps50in Otala hemolymph in similar conditions is ~ 40 torr. Preliminary measurements yielded Psovalues -~ 10 torr lower (Barnhart and Arp 1980), possibly owing to loss of CO2 from the hemolymph during the procedure. CO2 loss can result in precipitation of CaCO3 (Campbell and Boyan 1976), which would reduce [Ca++], and of pH, both of which may affect the O2-binding curve. Loss of CO2 may also have influenced values obtained for other species. Formation of precipitates in Helix hemolymph was noted by Spoek et al. (1964), who deoxygenated the hemolymph by exposing it to a vacuum prior to measuring binding curves. Hemolymph of Otala and Helix is supersaturated with respect to CaCO3 (Burton 1976) so that once precipitated, CaCO3 is unlikely to redissolve when CO2 is restored. EFFECT OF TEMPERATURE

ON He FUNCTION

The effect of temperature on P50 of Hc in Otala appears to be similar to that in

other species (Mangum 1980). If the small change in pH at different temperatures is ignored (see Material and Methods), the apparent heat of oxygenation (AH) is S-10 at Pso0.Temperature effect on affinity at other levels of saturation is seldom reported, and it is not known whether the decrease of temperature effect with increased saturation (fig. 3) is unusual. The binding curves for Helix hemolymph show similar change in shape with changing temperature (Spoek et al. 1964; Zolla et al. 1981). The O2-transportfunction of He in active snails is apparently minor at low temperature but increases with increasing temperature. Comparison of the binding curves with arterial and venous P02 (values from Barnhart 1986b) shows that only ~ 10%of the Hc 02 capacity is utilized at 5 C but that >70% is utilized at 25 C (fig. 4). Dissolved-O2 transport accounts for -~0.1 mmol/liter at each temperature, whereas He 02 transport increases from --0.01 at 5 C to 0.07 mmol/liter at 25 C. Thus, the fraction of 02 carried by He apparently in-


730

M. C. BARNHART

creases from 40% at 25 C. 02 transport by He also increases with temperature in a marine prosobranch gastropod (Polites and Mangum 1980).

sured), and Pvo2 usually