Boutilier, 1986; Milligan & Wood, 1986). In contrast, much less is known about the adrenergic responses of cyprinid red cells. The adrenergic response is absent ...
J. exp. Biol. 136, 405-416 (1988) Printed in Great Britain © The Company of Biologists Limited 1988
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THE ADRENERGIC RESPONSES OF CARP (CYPRINUS CARPIO) RED CELLS: EFFECTS OF P Ol AND pH BY ANNIKA SALAMA AND MIKKO NIKINMAA Division of Physiology, Department of Zoology, University of Helsinki, Arkadiankatu 7, SF-00100 Helsinki, Finland Accepted 7 December 1987 Summary Carp (Cyprinus carpio) red cells do not show /J-adrenergic responses when incubated with 10~ 5 moH" 1 adrenaline at atmospheric oxygen tension and a pH value close to the in vivo resting pH (approx. 8-1). However, when either the pH or the oxygen tension of the incubation medium is decreased, the adrenergic responses appear, showing that oxygen or an oxygen-linked phenomenon has a direct influence on the response. Once present, the adrenergic red cell response is similar to that of trout: cellular water content, sodium content and intracellular pH all increase. Quantitatively the effect appears to be much smaller in carp than in trout. Adrenaline induces an increase in red cell oxygen content when the oxygen content is plotted as a function of extracellular pH. This effect coincides with the onset of the Root effect and is caused by the adrenaline-induced increase in intracellular pH, since it disappears when the oxygen content is plotted as a function of intracellular pH. The red cell ATP content decreases metabolically during adrenaline incubations. In contrast, cellular GTP content is not metabolically reduced in adrenaline-treated cells, showing that the rapid and selective decrease in red cell GTP concentration, observed in hypoxic cyprinids, is not adrenergically induced. Introduction
The adrenergic response of trout {Salmo gairdneri) red cells is well characterized. In vitro results (e.g. Nikinmaa & Huestis, 1984; Baroin, Garcia-Romeu, Lamarre & Motais, 1984; Cossins & Richardson, 1985) have shown that adrenaline activates N a + / H + exchange across the red cell membrane. Proton extrusion via this pathway leads to a decrease in the transmembrane pH gradient. The influx of Na + , and Cl~ influx through the anion exchange pathway, cause cell swelling. Also, the concentrations of red cell organic phosphates and haemoglobin decrease. All the above responses shift the oxygen dissociation curve to the left (Nikinmaa, 1983). The in vitro response occurs at atmospheric PO2, at normal plasma pH, PCO2 and bicarbonate concentration, but is markedly enhanced by a decrease in pH (Borgese, Garcia-Romeu & Motais, 1987; Heming, Randall & Key words: nucleated red cells, adrenergic response, oxygen transport, Cyprinus carpio.
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Mazeaud, 1987; Nikinmaa, Steffensen, Tufts & Randall, 1987ft). Borgese etal. (1987) have shown that the pH-dependence of the adrenergic activation of sodium/proton exchange follows a bell-shaped curve, reaching a maximum at pH7-3. In vivo, adrenaline-activated N a + / H + exchange occurs both in hypoxia (Fievet, Motais & Thomas, 1987) and in exercise (Primmett, Randall, Mazeaud & Boutilier, 1986; Milligan & Wood, 1986). In contrast, much less is known about the adrenergic responses of cyprinid red cells. The adrenergic response is absent in exercised tench, Tinea tinea (Jensen, 1987), but present in hypoxic carp, Cyprinus carpio (Nikinmaa, Cech, Ryhanen & Salama, 1987a). However, carp red cells swelled and their intracellular Na + concentration increased only when blood PO2 decreased below lOmmHg (1 mmHg = 133-3Pa). These observations suggest, albeit indirectly, that P O2 may have a direct influence on the adrenergic response. Recently, Motais, GarciaRomeu & Borgese (1987) have shown that, although present at atmospheric PQJ, the adrenergic response of trout is enhanced by decreased O 2 tension. We have investigated the role of P O2 and pH on the adrenergic response of carp red cells. Particular emphasis was paid to the effect of adrenaline on the oxygen content of the red cells and the relationship between the Root effect and the adrenergic response.
Materials and methods
Female carp (Cyprinus carpio, 1-2-2-5 kg, N =30) were obtained from Evo Fisheries Experimental Station, transported to the Department of Zoology, University of Helsinki, and acclimated to laboratory conditions (11-12°C, oxygen tension >120mmHg, pH7-2-7-4, 12h:12h L:D rhythm) for at least a month before use. Blood samples were taken into heparinized syringes by caudal puncture from anaesthetized (MS-222, 0-1 gI" 1 , 5min) fish. The blood was centrifuged (2min, 10 000g), and the buffy coat and plasma were discarded. The red cells were washed twice in the physiological saline used and left overnight in the saline at 5°C. This ensured that the cells were not in a catecholamine-stimulated condition (see Bourne & Cossins, 1982). The following day the red cells were again washed and then suspended in the physiological saline at a haematocrit of about 20%. The composition of the saline (in mmoll" 1 ) was: NaCl, 128; KC1, 3; MgCl 2 ,1-5; CaCl2, 1-5; and NaHCO 3 , 10. Each 1-2-ml sample was divided in two parts: l O ^ m o l P 1 adrenaline (final concentration) was added to one, the other served as control. A high concentration of adrenaline was used to ensure a maximal response. lO^tl of 14C-labelled DMO (5,5-dimethyl-2,4-oxazolidine-dione, lO^Ciml" 1 ) was added to each subsample at the onset of the incubations. The subsamples were incubated in a shaking tonometer for 30min at 20 °C in the conditions given in Table 1. The humidified gas mixtures were obtained using Wosthoff gas-mixing pumps.
Adrenaline
and carp red cells
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The extracellular pH and the oxygen content of the suspensions were measured immediately after the end of the incubation. The extracellular pH (pHe) was measured using Radiometer BMS3Mk2, PHM72 apparatus, and the oxygen content using Tucker's (1967) method and subtracting the oxygen dissolved in the incubation medium. The O 2 solubility coefficient of the ferricyanide reagent at 20°C was extrapolated from Tucker's data and the values for 0-119moll" 1 NaCl (Altman & Dittmer, 1971) were used as the solubility coefficient of the incubation medium. 10/xl of the suspension was taken for measurement of the haemoglobin concentration by the cyanmethaemoglobin method. The rest of the suspension was then centrifuged for 2min at 10 000 g in two Eppendorf tubes, and the supernatants transferred into a third Eppendorf tube. The topmost layer of the red cells was then discarded to ensure effective removal of the incubation medium. One of the red cell pellets was used for determination of cell water. It was weighed, dried to a constant weight and reweighed. The water content of the cell pellet is given in kg water kg" 1 dry mass of the cell pellet. The other cell pellet was deproteinized in 0-6moll" 1 perchloric acid and stored in liquid nitrogen until the determinations of red cell ATP, GTP, Na + , K + , Cl~ and DMO contents were made. The supernatant was also deproteinized in 0-6moll" 1 perchloric acid and the Na + , K + , Cl" and DMO concentrations were measured. Na + and K + were determined by flame photometry (Radiometer FLM3), and Cl~ by Radiometer CMT10 chloride titrator. All the intracellular ion contents are given in mmolkg~ 1 drymass of the cell pellet. ATP and GTP contents were measured enzymatically as described by Albers, Goetz & Hughes (1983) and are Table 1. Incubation conditions used in the experiments Constant PQ 2 , varying PCo2 and PH Po, (mmHg) 150
Pco, (mmHg) 0-3 3-8 7-6 15 30
8
0-3 3-8 7-6 15 30
Constant P cc , 2 and pH, varying F pH 81 7-6 7-5 7-3 7-1
8-5 7-7 7-5 7-4 7-1
Pco, (mmHg)
pH
(mmHg)
7-6
7-5
0 3 8 15 30 60 150
7-1
0 3 8 15 30 60 150
30
Po,
The pH values given are those measured at the end of the incubation
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given in mmolkg^'dry mass of the cell pellet. Intracellular pH (pHi) was calculated from the distribution of [ 14 C]DM0 across the cell membrane using the formula: pHi = pK DMO + log{[DMO]1/[DMO]e(10»'He-PK'»«> + 1) - 1}, in which pK DMO at 20°C was taken as 619 (Boutilier, Heming & Iwama, 1984) and [DMO], is taken as disintsmin~ 1 ml~ 1 H 2 O in the red cell pellet. Since our estimations of water and ion contents included the contribution from the extracellular fluid, we estimated, in a separate experiment, the proportion of the extracellular fluid in the cell pellets of adrenaline-treated cells at low pH (7-1) and control cells at high pH (8-1), using [14C]polyethylene glycol as a marker. With the centrifugation used, the proportion of extracellular fluid was independent of the treatment and was 6-5 ±0-2% of the packed cell volume for the whole material (N= 16). Thus, the adrenaline-induced changes observed in the water and ion contents give a realistic picture of the changes taking place in the intracellular compartment. Small effects of adrenaline may be lost in methodological inaccuracies. With regard to the estimation of pHi, the use of the water content of the cell pellet in the calculation of [DMO],, instead of the water content of the red cells (corrected for 6-5% trapped medium), gives values that are 0-001-0-015 units higher than the corrected values. Since the proportion of trapped extracellular fluid was similar for both control and adrenaline-treated cells, the conclusions about the effects of adrenaline on the red cell pH are not affected. Results and discussion Cellular water content At atmospheric PO2 and at pHe7-5 or above, adrenaline did not cause a significant increase in the cellular water content. However, adrenergic swelling appeared either if pHe was decreased below 7-5 at atmospheric PO2 or if at pH 7-5 the PQ 2 of the incubation was decreased to 30mmHg or below (Fig. 1). These results show that the adrenergic response in carp is dependent on both pH and P O2 , and may explain why, in tench, neither exercise nor adrenaline infusion caused adrenergic swelling of red cells (Jensen, 1987). In the exercised tench, the plasma pH values remained higher than 7-5 and blood PQ, was above 30mmHg. It should be noted that different individuals were used at different times for determinations at the different PO2 and pH values. Thus, in addition to the effects of PQ 2 or pH on the water content perse, the water content was affected by differences in, for example, the organic phosphate and haemoglobin concentrations among the different individuals and sampling times. The apparently bellshaped curve in Fig. IB may be caused by such variation. In contrast, at a given pH or PO2, the effects of adrenaline were always tested using a single blood sample, divided into two portions, and thereby give a realistic picture of the effects of adrenaline on the red cell function at any experimental condition.
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Ion contents Similar to the effect of adrenaline on the cellular water content, and as expected from the role of N a + / H + exchange in the adrenergic response of trout (e.g. Borgese etal. 1987), the adrenaline-induced increase in the red cell Na + content was dependent on both pH and PQ 2 (Fig. 2). Also the red cell Cl~ content increased as a result of adrenergic stimulation, although much more marked 2-5 r
A
2-3 M
o
2-1 1-9 1-7 1-5'—
| 7-0
7-4
L 30
8-2 0 pHe
I I 60 90 Po, (mmHg)
I 120
J 150
Fig. 1. The water content (kgH2Okg ' dry mass of the cell pellet) of adrenalinetreated ( • ) and control (O) carp red cells as a function of extracellular pH at atmospheric oxygen tension (A, N = 6-8) and as a function of oxygen tension at extracellular pH7-5 (B, N=8). Asterisks indicate statistically significant (P
