Anion transport in embryonic and adult chicken red blood cells

Anion transport in embryonic and adult chicken red blood cells

Life Sciences Vol . 20, pp . 1565-1570, Printed in the U.S .A . 1977 . Pergamon Press ANION TRANSPORT IN EMBRYONIC AND ADULT CHICKEN RED BLOOD CELL...

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Life Sciences Vol . 20, pp . 1565-1570, Printed in the U.S .A .

1977 .

Pergamon Press

ANION TRANSPORT IN EMBRYONIC AND ADULT CHICKEN RED BLOOD CELLS Michael J. Weise and Joseph F . Hoffman Department of Physiology, Yale University School of Medicine 333 Cedar Street, New Haven, Connecticut 06510 (Received in final form March 30, 1977) Summary The rates of Cl and S0 4 transport at 0° and 37 °C, respectively, have been measured under exchange cotdl.tions for red blood cells of It was found that the rate of self embryonic and adult chickens . exchange of S04 in embryonic red cells decreases as the embryo matures, and that the S04 transport rate was lower in adult compared to embryonic red cells. In contrast, no difference in the rate of C1 self-exchange was found between adult and embryonic red cells. Anion transport in human red cells has been shown to exhibit kinetics and it has been proposed that anion movements occur by a mechanism (1,2) . Studies with irreversible inhibitors of S04 (3) transport suggest that the predominant molecular species of human membrane polypeptide is intimately involved in anion transport .

saturation facilitated and P04 (4) red cell

It is of interest to determine whether anion transport is altered during red cell maturation and, if it is, to try to identify in turn those factors For this study red cells at different stages of causing the alteration . The first erythroid elements maturation can be obtained from chick embryos. of the embryo are the "primitive" cells which appear at approximately 2 days However, at 5-6 days of incubation the second erythroid of incubation (5) . series, the "definitive" cells, begin to replace the primitive series and, as development continues, definitive cells become the only red blood cells of the organism . Between 9 and 19 days of incubation the majority of circulating red cells are of the definitive series, and the populations of these cells mature from approximately 55-60 percent polychromatophilic erythroThe red cells blasts at 9 days to mainly mature erythrocytes at 19 days (6) . of the adult chicken are also of the definitive series . We have measured rates of both Cl and S04 self-exchange in embryonic Although little or no difference is and adult chicken definitive red cells . found for Cl transport between embryonic and adult cells, there is a lower rate of S0 4 transport for adult compared to embryonic red cells as well as a tendency for the S04 transport rate to decrease during embryonic development . Methods Red Cells : Fertile White Leghorn eggs (SPAFAS, Norwich, Connecticut) were incubated at dry and wet bulb temperatures of 39° and 30 °C, respectively . Embryonic red cells were obtained by opening the eggs to remove extra embryonic fluids and then bleeding the embryos into C1 flux solution, Blood from described below, to which had been added 50ug/ml sodium heparin. adult hens was obtained from a wing vein using heparin as the anticoagulant . 1565

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Before loading with 3601 or 35 S04, red cells were freed of white cell contamination by centrifugation of cell suspensions at 4° for tit minutes at 8,000 x g, removal of the buffy coat from the red cell pellet, and resuspension of the cells with 5-10 volumes of the appropriate flux medium . This washing procedure was repeated at least 3 times . C1 self-exchange: Red cells (ti25% hematocrit) were labelled with 36 C1 (ICN Pharmaceutical #63005) by incubation at 37 ° C for 30 minutes in Cl flux medium (mM: NaCl-150, KC1-5, HEPES-10, glucose-5 (pH 7 .4) containing lu Ci 36C1/ml . In preparation for measuring the Cl self-exchange flux at 0°C the cell suspensions were incubated an additional 30 minutes in an ice/water bath . The efflux of 36 C1 into isotope-free Cl flux medium was measured using a rapid filtration method similar to that described by Dalmark and Wieth (7) . The hematocrit during flux measurements was 0 .5%. The efflux of 36 C1 was corrected for label trapped in the extracellular space of the packed, unwashed cells (about 30% of the total radioactivity present) by subtracting the counts present in the first supernatant time sample from all subsequent samples . S04 self-exchange : Red cells (ti10% hematocrit) were equilibrated in S04 flux medium (mM : NaCl-150, KC1-5, Na2S0y-10, HEPES-5, glucose-5 (pH 7 .4)) containing 20pCi 35 S04/ml (New England Nuclear, NE%-041) by incubation at 37 °C for 3 hours . Longer incubations indicated that S04 was at equilibrium since there was no further change in the distribution of S04 between medium and cells . After incubation, cells were washed twice with cold S04 flux solution in preparation for the measurement of 35 S04 efflux from cells at 0 .25% hematocrit under exchange conditions at 37 ° C . Details of the technique have previously been described (8) . Radioactivity in the first time sample (t-0) was no more than 10% of that present in an equivalent volume of suspension and was mainly due to efflux occurring in the time necessary to add labelled cells to all flasks and take t-0 samples . Calculation of transport rate constants: In some experiments, the amount of 35 S04 in chicken red cells during self-exchange fluxes was measured for 70 minutes at which time the cells had lost more than 70% of their initial radio activity . For these experiments, plots .of ln(1-CO/CT) vs time were found to be linear (correlation coefficient (r) - .99 with 8 time points ; probability of non-linearity (P) < 0 .01) . (CO - counts in time sample minus counts in t-0 sample ; CT - counts in suspension sample minus counts in t-0 sample .) Experiments also showed that plots of ln(1-CO/CT) vs time were linear (r - .99 with 5 time points ; P < 0.01) for Cl exchange fluxes with chicken cells The above measured over a 40 second time span with 40% exchange of tracer . data are consistent with the exchange fluxes in chicken cells being first order processes fitting the two compartment model described by Gardos, Hoffman and Passow (8) . For routine measurements of C1 or 904 flux rates, five cell supernatant samples (one at zero time and four over 30-40 seconds or 30-60 minutes for Cl Times or S0y, respectively) and a sample of the cell suspension were used . for supernatant samples were taken as the beginning of the centrifugation to obtain that sample (S04 fluxes) or as the moment the cell-free medium One ml 10% trichloroacetic appeared in the collection syringe (Cl fluxes) . acid was added to a 1 ml aliquot of each sample and the precipitate in the cell suspension sample was removed by centrifugation . The amount of radioactivity in each sample was determined by adding 1 ml of the acidified samples to 10 ml scintillation fluid (Aquasol, New England Nuclear) and counting in a Acidification of all Searle Instruments Mark III scintillation counter. Rate constants were samples eliminated the need'for quench corrections . taken as the slope of the line fitted to plots of ln(1-CO/CT) vs time by the

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Anion Transport in Chicken Red Cells

method of linear least squares . or greater .

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Correlation coefficients were generally .99 Results

The rates of 504 transport for definitive red cells from adults or from embryos at various stages of development are shown in Figure 1 . There appears to be a tendency for the transport rate to decrease with embryonic maturation and there is a definite difference between embryonic and adult self-exchange rates . Comparing mean rate constants for cells of 13 and 19 day embryos and adults (k aoy t S .E .M . (hr-1 ) a 1 .85 ± 0 .11 (n-3), 1 .13 ± 0 .10 (n-4) and 0 .83 0.10 (n-3) for 13 day, 19 day, and adult, respectively), we find significant differences between 13 day embryos and both 19 day embryos and adults (P < 0.01 for both comparisons ; two sided t-test) . The scatter that is apparent in the embryonic rate constants is most likely due to variation in development rates from one batch of eggs to another . Correcting the transport rates for variation in cell volume, measured with a Coulter Counter (embryonic : 124p 3 ; adult 119u 3 ), would increase the difference in S0,, transport observed between embryonic and adult cells .

2.0

k304

(hr -1)

0

FIG. 1 Rate constants for 35 S04 efflux from embryonic and adult chicken red blood cells . The rates of S04 transport were measured as described in Methods for red cells obtained from chick embryos (9-19 days incubation) or from adult hens . Embryonic cells were obtained from the same batch of eggs at two or three times during the 9-19 day incubation time and results from several batches of eggs are plotted (different symbols) . The rate of transport shown for the adult is the mean of three determinations done in triplicate . Table 1 shows the rates of 36 C1 self-exchange from cells of 12 day embryos and adults . In contrast to results with SOq, the difference in the Cl transport rate for adult compared to embryonic red cells is not statistically significant .

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TABLE 1 Rates of Cl Transport for Embryonic and Adult Chicken Red Blood Cells kCl ± S .E .M . Cells 12 day embryos Adult

(min=i)

(n)

1 .06 ± 0 .15

3

1 .24 ± 0.14

2

Discussion Both Cl (9) and P0 4 (10) transport have been studied in red cells from a variety of animals . In most cases (ox red cells being an exception) the rates of transport for monovalent and divalent anions appear to change coordinately : for a given percentage change in the rate of Cl transport from one animal to another there is a similar percentage change in the P04 transport rate (compare data from 9,10) . This is also true of Cl and S04 transport in dog and cat red cells (11) . In contrast to the differences in anion transport between animals, we find what appear to be non-coordinate changes in monovalent and divalent anion transport between embryonic and adult chicken red cells . This assumes that anion transport in chicken cells is similar to that in mammalian species . If this is true, as there are no genetic differences between embryonic and adult chickens, it may be easier to identify the factors and/or membrane changes that affect anion movements in chicken cells than to find the causes for changes in transport rate in red cells from different animals . A variety of studies have dealt with embryonic and adult chicken cells . Although the same hemoglobins are found in chick definitive red cells before and after hatching, the relative amounts of the hemoglobins are different in embryonic and adult cells (6) . This could produce different membrane potentials in embryonic and adult cells, but it is unlikely that this type of variation could cause differences in the exchange rate of divalent anions only . Other studies show that embryonic cells have lower levels of carbonic anhydrase than adult cells (12) and that there are differences in surface antigens between the two types of cells (13,14), but further work is required to determine the relationship of these differences to anion transport. Of these two variables, the difference regarding carbonic anhydrase is possibly the more important since this enzyme appears to influence anion movements under certain conditions (15) . SDS acrylamide gel studies show that there are similar proteins in the plasma membranes from embryonic and adult chicken red cells and that these In proteins are also similar to the proteins of human red cells (16,17) . particular, chicken cells contain membrane proteins similar in size to the If, species of human red cell membrane protein implicated in anion transport . as has been proposed (1), the movements of both monovalent and divalent anions are mediated by different forms of the same carrier and if the carrier is assumed to be a membrane protein, then the differences in S0 4 transport between embryonic and adult chicken cells could result from alterations in membrane proteins . However, since no changes are detected in SDS gel studies, more detailed analyses of membrane proteins will be required to determine what, Observed differences between if any, protein modifications have occurred .

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embryonic and adult S04 transport regarding pH dependence and SITS inhibition (18) might be explained by some changes in the structure of transport proteins rather than by changes in the amounts of those proteins in the membrane . In addition to further work on anion transport, we are also characterizing cation transport in chicken definitive red cells. Preliminary results indicate that the K influx, measured with 42K, is some 20-fold higher in em bryonic compared to adult definitive cells (34 .9 to 1 .78 mM K/liter cells x hr for 15 day embryos and adult hens, respectively) and that the ouabainsensitivity approximates 60% in both instances . A more complete characterization may point to the factors responsible for the changes in transport reported here . References 1. 2. 3. 4. 5. 6. 7. 8. 9.

10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 .

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