Evidence that increases in lymphocyte tyrosine phosphorylation precede cardiac allograft rejection. Effects of cyclosporine and potential use in clinical management.

Tyrosine phosphorylation is an early, critical event in lymphocyte signal transduction. We measured tyrosine phosphorylation in a porcine experimental transplant model to evaluate its utility in monitoring the allograft immune response. Using flow cytometry, we demonstrate a biphasic increase in phosphotyrosine (ptyr) levels in peripheral blood mononuclear cells (PBMC), and that increases are detectable as early as 1 day posttransplantation in untreated transplanted animals (n = 4). This biphasic response is likely result from the sequestration of ptyr+ cells from the periphery into the graft as graft-infiltrating lymphocytic cells show increased ptyr levels. This suggests possible lymphocyte trafficking between the peripheral compartment and the allograft. A 5-day course of treatment with cyclosporine (CsA) at 20 mg/kg/day (n = 4), but not at 10 mg/kg/day (n = 4), prevents graft rejection in this allograft model. Strikingly, treatment with 20 mg/kg/day CsA, but not with 10 mg/kg/day, suppressed increases in ptyr levels in both PBMC and graft-infiltrating cells. Increases in ptyr levels in PBMC are detectable 2-5 days before histologic and electrocardiographic signs of graft rejection, suggesting a potential diagnostic utility for measuring tyrosine phosphorylation in monitoring and managing transplant rejection.

Identification of a subpopulation of reactive large granular mononuclear cells in allogeneic heart transplantation.

This study is designed to test the hypothesis that specific morphologic attributes of peripheral blood mononuclear cells, measurable by flow cytometry, are correlated with the timing and the intensity of allograft injury during the development of heart rejection. A pig model of major histocompatibility complex-mismatched heterotopic heart transplantation with (n = 5) and without (n = 5) cyclosporine administration was monitored serially be telemetered electrocardiography and endomyocardial biopsies. Flow cytometric analysis of peripheral blood mononuclear cells revealed the emergence of a discrete subpopulation of peripheral blood mononuclear cells (7.8% +/- 1.0% and 8.5% +/- 0.9% before transplantation to 16.5% +/- 1.3% and 19.4% +/- 3.0% after transplantation in the untreated and the cyclosporine-treated groups, respectively, p < 0.05), exhibiting characteristic changes in forward and 90-degree light scatter, indicative of increased cell size and granularity, and possibly representing monocytes or large granular lymphocytes. Lymphocyte cell surface-marker studies indicated that 62% of these cells are DH59B+ (monocyte/granulocyte). Because intracellular free calcium is an important second messenger in lymphocyte activation we measured intracellular free calcium by flow cytometry using fluo-3. This subpopulation of cells was found to have similar intracellular free calcium when compared to normal-sized lymphocytes (104 +/- 7 nmol/L versus 101 +/- 5 nmol/L, respectively). We conclude that this lymphocyte subset detected by flow cytometry represents specifically reactive cells that are associated with incipient allograft rejection.

Pharmacodynamics and pharmacokinetics of cyclosporin A in the newborn pig.

The disposition kinetics of cyclosporin A in the neonates as well as age-related differences in lymphocyte responses to cyclosporin A are unknown. A single intravenous infusion of cyclosporin A was given to neonatal (2.5 or 5 mg/kg) and mature pigs (10 mg/kg) and blood cyclosporin A levels were measured by RIA. The neonates had longer elimination half-life and lower drug clearance than mature animals. Suppression in lymphocyte proliferation was only observed in mixed lymphocyte reaction and phytohemagglutinin-stimulated cultures of the 2-hour samples from neonates receiving 5 mg/kg. We conclude that neonatal pig exhibit different cyclosporin A pharmacokinetics and show higher sensitivity to cyclosporin A than mature animals.

Age-related differences in the effects of cyclosporine on lymphocyte intracellular free calcium.

The effect of cyclosporine on lymphocyte intracellular free calcium [( Ca2+]i) is controversial, and potential age-related differences in lymphocyte CsA sensitivity have not been studied. We measured the mitogen-induced change in [Ca2+]i in peripheral blood lymphocytes (PBLs) following intravenous CsA infusion (5 mg/kg) in neonatal pigs and found a significantly reduced calcium response compared with control (P = 0.02). This was associated with an elevation in resting [Ca2+]i in the neonatal PBLs 24 hr following the CsA infusion (P = 0.02). These changes in lymphocyte [Ca2+]i were associated with suppression of cell proliferation. Neonatal PBLs in mixed lymphocyte cultures showed a greater PHA-induced change in [Ca2+]i (delta[Ca2+]i) compared with mature PBLs (P = 0.0007). The addition of CsA (1 microgram/ml) to mitogenic- and allogeneic-stimulated cultures did not affect resting [Ca2+]i or delta[Ca2+]i in either neonatal or mature PBLs. Our results demonstrate significant differences in calcium responses in neonatal lymphocytes following CsA infusion and allogeneic stimulation. This implies that there are age-related differences in CsA effects at or proximal to the level of calcium release and/or sequestration in the lymphocyte signal transduction pathway, and that elevated resting intracellular calcium levels may be indicative of reduced responsiveness, possibly through feedback inhibition of tyrosine kinase activity.

The transport of digoxin across the perfused human placental lobule.

The cardiac glycoside, digoxin, is clinically used to treat fetal tachyarrhythmias and congestive heart failure. The time course of digoxin transfer across the human placenta was studied by dually perfusing an isolated lobule of the human placenta in vitro. Viability of the placental preparation was validated by measuring the rates of glucose and oxygen consumption, lactate production and synthesis of the protein hormone, chorionic gonadotropin. Following administration of 5 ng/ml digoxin to the maternal circulation, digoxin appeared in the fetal circulation within 5 min. The disappearance of digoxin from the maternal circulation was biexponential and best fit a two-compartment pharmacokinetic model. Mean calculated volume of the central compartment (257 +/- 6.3 ml) was consistent with the actual volume of the in vitro maternal circulation (246 +/- 7.4 ml). The half-life of the distribution phase was 9.7 +/- 3.3 min, and half-life of the terminal elimination phase was 362 +/- 83 min. After 30 min of perfusion, the amount of digoxin leaving the maternal circulation and appearing in the fetal circulation was constant at a fetomaternal mass ratio of 0.36 +/- 0.04. This ratio was maintained through to the end of the 3-hr experiment. All of the digoxin leaving the maternal circulation could be accounted for either in the fetal circulation or bound to placental tissue. The time to achieve equal concentrations on both sides of the placenta was estimated to be 268 +/- 34 min. These data are consistent with in vivo data obtained in humans, and support the relevance of using the in vitro placental perfusion model to obtain information regarding placental drug transfer in humans.

Furosemide increases total calcium in kidney and cytoplasmic free calcium in blood mononuclear cells of guinea pigs.

Nephrocalcinosis has been observed in premature infants treated with furosemide. To see whether furosemide-induced renal calcium accumulation is reflected in easily accessible extrarenal cells, we measured cytosolic free calcium ([Ca2+]i) in blood mononuclear cells and kidney tissue calcium of guinea pigs chronically treated with furosemide. At week 0, the mean [Ca2+]i in blood mononuclear cells using the fluorescent indicator quin2 was 105 +/- 4 nM. After 2 weeks of treatment with furosemide, the high dosage (10 or 20 mg/kg/day) caused a significant increase in [Ca2+]i. There was no change in cell volume after 1 or 2 weeks of treatment with furosemide at this dosage. The kidney total calcium concentration and histological calcium accumulation increased with increasing furosemide dosages. These observations suggest that calcium accumulation in blood mononuclear cells may reflect the calcium accumulation in the kidney after furosemide treatment.

Ionized calcium measurement in vitro: is it necessary?

Experimental data obtained from a study on the inhibition of placental Ca-ATPase by ethacrynic acid were graphically analyzed using either total calcium or ionized calcium as the independent variable. Correct interpretation of the results required actual measurement of ionized calcium in the incubation medium. The assumption that ionized calcium is equal to the total calcium resulted in artifacts and led to erroneous conclusions. These observations emphasize the necessity for accurate measurement of ionized calcium in test systems where calcium effects are being examined.

Transplacental 45Ca and 32P flux in the guinea pig: effect of maternal hypercalcaemia and hypocalcaemia.

Transplacental 45Ca and 32P flux was measured across the in situ perfused guinea-pig placenta under conditions of acute maternal hypocalcaemia and hypercalcaemia. Maternal hypercalcaemia induced acutely by calcium gluconate infusion caused an increase in maternal-to-fetal 45Ca flux which was proportional to the increase in maternal plasma ionized calcium concentration. Acute maternal hypocalcaemia was induced by EGTA infusion and resulted in a decrease in maternal plasma ionized calcium concentration proportional to a corresponding decrease in transplacental 45Ca transfer. A bolus of calcium gluconate caused a transient decrease in 32P flux, whereas EGTA administration was without significant effect on transplacental 32P transfer. Calcium transport across the placenta is not saturated under conditions of maternal normocalcaemia and may be altered according to acute changes in maternal plasma calcium concentration. Thus, control of maternal-to-fetal calcium transfer does not appear to be at the placental level. This suggests that fetal calcium homeostasis may be regulated by the fetus itself.

Hepoxilin A, hydroxyepoxide metabolite of arachidonic acid, stimulates transport of 45Ca across the guinea pig visceral yolk sac.

The effect of hepoxilin A, a newly isolated hydroxyepoxide metabolite of arachidonic acid, on calcium transport across the visceral yolk sac membrane of the guinea pig was investigated in vitro in Ussing chambers. While 1-14C-labelled hepoxilin A itself was not transported across the membrane, it increased the rate of transport of calcium toward the side to which hepoxilin A was added. The degree of increase in calcium transport was similar whether hepoxilin A was added to the maternal side or to the fetal side of the membrane. The observed effect was dependent on the concentration of hepoxilin A over a narrow range (0.5-1.0 X 10(-6) M). It was also dependent on the time of incubation reaching maximal effect by 25 min. We have recently observed that hepoxilin A is formed from platelet-derived 12-hydroperoxyeicosatetraenoic acid (12-HPETE) through hemin and hemoglobin catalysis as well as during perifusion of 12-HPETE through isolated pancreatic islets. The present study suggests that hepoxilin A, if formed in vivo, could play a role in the mobilization of calcium.

Effects of pharmacological and physiological modulators on Ca-ATPase and alkaline phosphatase activities from guinea-pig placenta in vitro.

In a search for modulators of Ca-ATPase and AP activities, we examined three pharmacological agents and the cations Ca2+ and Zn2+. Placental Ca-ATPase specific activity was uncompetitively inhibited in vitro by millimolar concentrations of the diuretics ethacrynic acid and furosemide. Cysteine, a sulphydryl donor, partially reversed the ethacrynic acid inhibition but enhanced the furosemide inhibition, indicating that sulphydryl-binding may be part of the mechanism of the inhibition of Ca-ATPase by ethacrynic acid but not by furosemide. In contrast to Ca-ATPase, AP activity was enhanced by both ethacrynic acid and furosemide. Zinc inhibited Ca-ATPase activity at all concentrations tested, but enhanced and, at higher concentrations, inhibited AP activity. The inhibition of AP activity by D-penicillamine was reversed by Zn, supporting the view that this drug acts by chelating Zn which is essential for AP activity. D-penicillamine had no significant effect on Ca-ATPase activity. Calcium activated both enzyme activities but inhibited only AP activity at higher concentrations. These results indicate that placental Ca-ATPase and AP activities may be distinct and dissociable based on responses to various pharmacological and physiological modulators.

Placental calcium and phosphorus transfer in the guinea pig: lack of effect of modulators of Ca-ATPase and alkaline phosphatase activity.

The effects of modulators of Ca-ATPase and alkaline phosphatase (AP) activity on placental calcium and phosphorus transfer were studied using the in situ perfused guinea pig placenta. The diuretics ethacrynic acid and furosemide had no significant effect on placental calcium and phosphorus transfer when injected into the mother (1.0 or 10.0 mg X kg-1) or added to the solution perfusing the fetal side of the placenta (0.25 or 2.0 mM). These two drugs have previously been shown to inhibit placental Ca-ATPase and enhance AP activity in vitro. D-Penicillamine, which inhibits placental AP but not Ca-ATPase activity in vitro, also had no significant effect on net calcium and phosphorus transfer from mother to fetus either when given to the mother (50 mg X kg-1) or added to the placental perfusion solution (0.25 or 2.0 mM). These results suggest that placental transfer of calcium and phosphorus in the guinea pig may not be directly related to placental Ca-ATPase and AP activities.

Macromineral balances in premature infants fed their own mothers' milk or formula.

The premature infant's own mother's milk (preterm milk) and modified infant formula (SMA, 67 and 80 kcal/dl) were fed to paired groups of seven infants, all of whom were of very low birth weight (VLBW) (less than 1.3 kg) and were studied during the first month of life. Sodium, potassium, magnesium, calcium, and phosphorus status was compared. The apparent retention of sodium from their mother's milk paralleled intrauterine retention rates and was greater than retention from SMA formula (P less than 0.01) during the first two weeks of life. However, when the formula was supplemented with NaHCO3 to intakes of 2.7 mmol Na/kg/24 hr after week 2, the infants retained adequate amounts of sodium. Potassium retention was similar to intrauterine retention rates in both groups throughout the four postnatal weeks. Magnesium intake, but not retention, was consistently higher in the group fed SMA (P less than 0.01), and intrauterine retention rates were achieved only in the group given formula. Calcium and phosphorus intakes from SMA were also higher (P less than 0.01) than from human milk. However, retention of calcium and phosphorus in both groups did not meet intrauterine retention rates, and hypophosphatemia developed in infants who received their mothers' milk. Growth in length and head circumference in both groups approximated intrauterine growth rates. If it is assumed that body composition of the growing VLBW infants should be similar to the composition of the fetus at corresponding gestational ages, then their nutrient requirements should be based on knowledge of intrauterine nutrient accretion rates. Based on this premise, we conclude that, for the growing VLBW infant, early maternal milk provided for sufficient retention of sodium, chloride, and potassium during the first four postnatal weeks. Neither human preterm milk nor SMA supplied adequate calcium and phosphorus for the growing VLBW infant.

Placental transfer of calcium (Ca) and phosphorus (Pi) in the guinea pig: lack of correlation with placental Ca-ATPase and alkaline phosphatase (AP) activities.

In summary, it has been observed that in vitro inhibitors of placental Ca-ATPase and AP activities (EA, F, D-pen) and activators of placental AP (EA,F) are not associated with changes in Ca and Pi transfer across the in situ perfused guinea pig placenta. Assuming that the two enzyme activities were altered in vivo by these drugs, it may be that they are not directly related to active transport of Ca and Pi across the placenta.

Calcium and phosphate fluxes across the fetal membranes of the guinea pig: in vitro measurement.

Bidirectional calcium (45Ca) and phosphate (32P) fluxes across the amnion and visceral yolk sac of the guinea pig were measured in vitro in modified Ussing chambers. The net flux of these ions across both fetal membranes was in the maternal-to-fetal direction. The net flux of 45Ca and 32P across the yolk sac was significantly greater than that across the amnion. This difference was due to a greater maternal-to-fetal flux across the yolk sac. In addition, net 32P flux was greater than net 45Ca flux across the yolk sac, while in the amnion, there was no significant difference in the net flux of the two ions. It is suggested that the fetal membranes, especially the visceral yolk sac, contribute significantly to fetal acquisition of calcium and phosphate in mammals possessing a functional yolk sac placenta.

Effect of salmon calcitonin on plasma calcium and phosphorus in pregnant and non-pregnant guinea pigs.

The effect of salmon calcitonin (SCT) on plasma calcium ion activity (ICa) and inorganic phosphorus (Pi) was investigated in pregnant and non-pregnant guinea pigs. Basal plasma ICa and Pi were lower in pregnant compared to non-pregnant animals. Intravenous administration of SCT in doses ranging from 120 to 520 MRC mU . kg-1 resulted in hypocalcemia in both groups. Pregnant guinea pigs responded with a more pronounced and sustained hypocalcemia compared to non-pregnant animals. The observed effects on plasma Pi were less consistent, with hypophosphatemia evident only in the non-pregnant guinea pigs. It is suggested that the sensitivity of pregnant guinea pigs to the hypocalcemic effect of SCT may be a result of a higher rate of bone turnover.

The effect of porcine calcitonin on intestinal calcium and phosphate fluxes in the young piglet.

The effects of a physiological (3.8 ng.mL-1) and a pharmacological (120 ng.mL-1) concentration of porcine calcitonin (CT) on transmucosal calcium and phosphate flux rates were determined in intestinal mucosa from young piglets (4--40 days) mounted in Ussing flux chambers. The physiological concentration of the hormone inhibited net absorptive calcium flux rates in the proximal jejunum and distal ileum but not in the duodenum. No effect of either concentration of CT on phosphate flux rates was observed. The inhibitory effect of the physiological concentration may indicate a role for CT in the maintenance of calcium homeostasis in the young suckling mammal.

Macro-mineral content of milk obtained during early lactation from mothers of premature infants.

Milk from mothers giving birth prematurely was analyzed for Na, Cl, K, Mg, Ca and P concentrations. The data presented are from analyses of milk samples representative of complete 24-hour expressions and collected serially over the first 29 days of lactation from mothers giving birth at term (FT) and mothers giving birth prematurely (PT). Mineral composition of FT and PT milks was similar during the first month lactation. With the exceptions of Mg and P, the concentrations of the minerals studied were higher initially than at the end of the fourth week of lactation. From these data, intakes of premature infants fed their own mothers' milk can be estimated and compared to predicted mineral requirements for the premature infant. On the basis of this comparison, we suggest that the quantities provided of Na, Cl, K and Mg, but not Ca and P, would be adequate to meet requirements of premature infants during the early weeks of life.