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1. Glucose and inosine transport by erythrocytes from new-born lambs and adult sheep were compared. Uptake of both permeants was considerably faster in the new-born. Inosine uptake by erythrocytes from nucleoside-permeable and impermeable lambs were not significantly different at birth. The difference between the two phenotypes was first apparent 30 days after birth.2. The post-natal changes in glucose and inosine transport activity closely paralleled the progressive decrease in the percentage of fetal erythrocytes (i.e. cells containing fetal haemoglobin) in the circulation. Cell fractionation studies confirmed that the permeability changes were directly related to changes in the relative proportions of fetal and adult haemoglobin containing erythrocytes.3. The results demonstrate that fetal cells are highly permeable to both glucose and inosine. These cells are replaced by erythrocytes which contain adult haemoglobin and which have a much lower, but still significant, glucose permeability and either low or negligible inosine transport activity depending on the genotype of the animal.4. Inosine transport by fetal erythrocytes from both nucleoside-permeable and impermeable animals was mediated by a nucleoside transport system which had similar properties to that responsible for nucleoside transport in adult nucleoside-permeable cells. Glucose transport in both fetal and adult cells was highly stereo-specific, indicating the presence of a selective transport system.5. It is suggested that the regulatory mechanism responsible for initiating the switch from fetal to adult haemoglobin synthesis may also be responsible for the changes in glucose and nucleoside transport activity.

1. Inosine-fed human red cells, pre-loaded with calcium with the use of the ionophore A23187, and reincubated at 37 degrees C after ionophore and external calcium removal, pumped calcium out initially fast and then substantially slower when the residual calcium was still very high (García-Sancho & Lew, 1988 a, b). Particularly surprising was the finding of such residual calcium pools in a subpopulation of cells (L cells, García-Sancho & Lew, 1988b) with normal ATP, high active calcium pumping rates, and K+ channels which remained inactive during the density separation procedure (SCN- treatment; García-Sancho & Lew, 1988a). The purpose of the present experiments was to investigate the properties and possible origins of the residual calcium pools. 2. At each ionophore concentration, the pool size increased with the duration and magnitude of the calcium load. At comparable calcium loads, the pool size was smaller the higher the ionophore concentration used. 3. In cells from the same sample, residual calcium pools were present in cells that became dense after SCN- treatment (H cells) as well as those that remained in the light-cell fraction (L cells). Residual calcium was always higher in H cells than in L cells. 4. Re-exposure of cells to ionophore in Ca2+-free media could rapidly extract over 99% of their residual calcium. Residual calcium is therefore in a rapidly mobilizable form within a membrane-bound compartment. 5. Iodoacetamide-induced ATP depletion of H cells with high residual calcium-stimulated calcium loss. Such stimulation could only occur if ATP depletion inhibited a calcium-retaining process within the cells. 6. L cells with high residual calcium may have failed to dehydrate during SCN- treatment because of irreversible K+ channel inactivation or because K+ permeabilization would no longer generate a dehydrating net cation loss. These possibilities were tested and ruled out since it was found that virtually all cells became dense in low-K+, SCN- media after valinomycin addition or re-exposure to A23187 + calcium. 7. The results suggest that part if not all of the residual calcium is contained within compartments with the properties of endocytic inside-out vesicles capable of ATP-dependent calcium accumulation, such as those found in normal and abnormal human red cells (Lew, Hockaday, Sepúlveda, Somlyo, Somlyo, Ortiz & Bookchin, 1985).

1. Plasma and adipose tissue purine nucleosides were assayed by reversed phase high-performance liquid chromatography after purification of the samples on phenylboronate affinity gel. 2. The adenosine content of unstimulated subcutaneous adipose tissue was close to 1 n-mole/g. The concentrations of adenosine and inosine in canine arterial plasma were 0.26 +/- 0.03 and 0.16 +/- 0.03 microM, respectively. In venous plasma from the canine subcutaneous adipose tissue the corresponding values were 0.32 +/- 0.04 and 0.28 +/- 0.06 microM under basal conditions. The arterio-venous concentration difference of adenosine was linearly dependent upon the arterial adenosine concentration. At arterial concentrations below 0.3 microM there was a net production of adenosine; above 0.3 microM there was a net extraction of approximately 77% of the adenosine. Adenosine was extensively eliminated in blood. The major part of this elimination could be accounted for by metabolism to inosine, hypoxanthine and uric acid. 3. Following sympathetic nerve stimulation (4 Hz for 20 min) the rate of adenosine outflow from adipose tissue increased from 0.33 +/- 0.22 to a peak value of 1.2 +/- 0.26 n-mole/min. This corresponds to a net release of 8.7 +/- 3.0 n-mole/100 g tissue. Inosine outflow rose from 0.64 +/- 0.37 to 5.3 +/- 1.4 n-mole/min, corresponding to a net release of 24.6 4/- 8.7 n-mole/100 g. Nerve stimulation also increased the release of [3H]purines from [3H]adenine pre-labelled adipose tissue. The fractional release increased 15-fold after stimulation. The radioactivity was mainly in the form of hypoxanthine, inosine and uric acid while adenosine was a minor component. When metabolism in blood was inhibited by dipyridamole and an adenosine deaminase inhibitor nerve-stimulation-induced release of [3H]purines was mainly in the form of adenosine. 4. Noradrenaline injection also induced a release of radioactive purines and of inosine. On the other hand, the outflow of endogenous adenosine was very small. 5. The present results demonstrate that under basal conditions adenosine is present in arterial and venous canine plasma. The free extracellular tissue level may be similar to the basal arterial adenosine concentration. Sympathetic nerve stimulation and noradrenaline induces a marked release of adenosine which is rapidly metabolized in the tissue and blood stream to inosine, hypoxanthine and uric acid. In adipose tissue the levels of adenosine reached after adrenergic stimulation appear high enough to be of physiological relevance.

1. When inosine-fed human red cells are permeabilized to calcium by exposure to the ionophore A23187, progressively larger proportions of the cell population become irreversibly depleted of ATP as calcium influx is increased (Brown & Lew, 1983; García-Sancho & Lew, 1988b). When calcium influx is over 30 mmol/(l cells.h), all cells become ATP depleted and calcium equilibrated (E cells) (García-Sancho & Lew, 1988b). When calcium influx is lower, E cells co-exist with cells able to maintain normal ATP and low calcium contents in vigorous pump-leak balance (B cells). The experiments reported here investigate why calcium-induced ATP depletion of E cells is irreversible. 2. The inosine monophosphate (IMP) content of cells after 30 min of calcium permeabilization increased with the magnitude of the calcium load, roughly in inverse proportion to the fall in ATP. The calcium-induced increase in IMP was confined to the fraction of cells which became osmotically resistant after SCN- treatment (H cells), and which contained the E cells. 3. Cell nucleotides were measured after calcium permeabilization [( A23187]c = 100 mumol/l cells) in substrate-free media with different [Ca2+]o (0-0.5 mM). Calcium entry caused rapid ATP fall, AMP and IMP accumulation, and delayed ADP fall at all [Ca2+]o concentrations. Initial IMP formation increased with [Ca2+]o along a sigmoid saturation-like curve whereas AMP accumulation and ATP fall were maximal at [Ca2+]o = 20 microM and declined at the higher [Ca2+]o. The rate of IMP formation correlated positively with cell ATP and negatively with cell AMP at all [Ca2+]o values. 4. The AMP deaminase activity of red cell lysates was reversibly increased over tenfold by calcium. Half-maximal stimulation was observed at a Ca2+ concentration of about 50 microM. 5. These results suggest that the irreversibility of calcium-induced ATP depletion results from irreversible trapping of the adenine nucleotide as IMP, and help explain the mechanism of E cell formation.

1. Calcium permeabilization of inosine-fed human red cells using the divalent cation ionophore A23187 induces pump-leak steady states in which the mean total calcium content of the cells may be held below electrochemical equilibrium for hours. A new method developed to detect and separate cells with different calcium contents revealed a striking heterogeneity of calcium contents in subpopulations of cells in pump-leak steady state (García-Sancho & Lew, 1988a). Most of the mean total cell calcium was found within a fraction of cells rendered dense by the separation procedure (H cells), with relatively little within the remaining light cells (L cells). The experiments in this paper were designed to study the nature and origin of the observed heterogeneity. 2. The fraction of steady-state H cells increased, and the mean ATP content of the cells fell, both linearly, as calcium influx was increased. The H/L divide is therefore the result of a continuous variation in cell properties. When calcium influx was above about 30 mmol/(l cells.h), all cells became dense, calcium distribution was at or near equilibrium, and cell ATP was 0.1-0.2 mmol/l cells. 3. Inosine-fed cells, subjected to ionophore-mediated net calcium influx of 13-15 mmol/(l cells.h), attained a steady state with mean calcium contents far below equilibrium. After ionophore removal and reincubation in calcium-free media, the initial calcium efflux was only a fraction of that required to sustain the previous steady state (less than 25% for H cells, and less than 2% for L cells). The ATP content of L cells was normal whereas that of H cells was irreversibly reduced. These results revealed a paradoxical discrepancy between leak influx and calcium pump efflux in H and L cells which were supposed to have been in steady-state pump-leak balance. 4. The changes in cell calcium and ATP were followed in time after calcium permeabilization to characterize the development of steady-state heterogeneity. Calcium influx triggered a sharp peak in the H cell fraction within 15 s of permeabilization. The mean calcium content of H cells increased towards steady-state values as their fraction decreased; most other cells transferred from H to L density fractions (HL cells) within the first 5 min of permeabilization. 5. In substrate-starved cells calcium influx triggered an immediate fall in cell ATP, steeper in H cells than in L cells. The initial calcium and density transients were unattected.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Uptake and metabolism of adenosine were investigated from both maternal (M) and fetal (F) circulations of the isolated, dually perfused guinea-pig placenta by using a single-circulation paired-tracer [( 14C]sucrose as extracellular reference, and [3H]adenosine) dilution technique. 2. Maximal [3H]adenosine uptakes (percentage of dose) from adenosine-free perfusates were 75 +/- 1 and 87 +/- 2% (mean +/- S.E. of mean) at maternal and fetal blood-tissue interfaces respectively. Rapid backflux (percentage of influx) of tritium (labelled adenosine and/or adenosine derivatives) from the placental tissue into the ipsilateral circulation was higher at the fetal (24 +/- 2%) than at the maternal side (11 +/- 2%). 3. Tritium uptakes were reduced to 50 +/- 4 (M) and 60 +/- 6% (F) when the perfusion medium contained 100 microM-unlabelled adenosine; backflux was highly stimulated (44% M and 84% F). Neither uptake nor backflux were affected by inosine, uridine, adenine or hypoxanthine present in the perfusion medium (1 mM). 4. Tissue sequestration of tritium (5-6 min) was approximately 60% of the injected dose when perfusates were adenosine-free and 20% or less in the presence of 100 microM-adenosine. 5. Cellular uptake of [3H]adenosine at both sides of the placenta was markedly reduced by the nucleoside transport inhibitors dipyridamole (DIP, 10 microM) and nitrobenzylthioinosine (NBMPR, 5 microM). 6. Thin-layer chromatographic separation of [3H]inosine, [3H]hypoxanthine and [3H]phosphorylated derivatives in venous effluents following a bolus arterial injection of [3H]adenosine showed a greater fraction of metabolites at the fetal side (about 0.75) than at the maternal side (about 0.50). The percentage of [3H]inosine increased when perfusates contained 100 microM-adenosine and the effect was more marked in the fetal circulation. In the presence of DIP and NBMPR the fractional recovery of 3H-labelled metabolites was greatly reduced. 7. During steady-state perfusion of [3H]adenosine (100 microM) a maintained (5-60 min) tritium uptake of about 55% was observed and all the effluent activity was 3H-labelled metabolites [( 3H]adenosine was only 2.8 +/- 0.2%). Under these conditions high-performance liquid chromatography (HPLC) showed that effluents contained xanthine and urate at 16 +/- 1 and 23 +/- 2 microM respectively. 8. Transplacental transfer (6 min) of tritiated compounds (of which only 10-20% was [3H]adenosine) was often less than that of the extracellular marker [14C]sucrose in both maternal-to-fetal and fetal-to-maternal directions.(ABSTRACT TRUNCATED AT 400 WORDS)