Ingeborg C. Radde

Calcium-stimulated ATPase of guinea pig placenta

1. 1. A Ca2+-activated ATPase of isolated membranes of guinea pig placenta is characterized. 2. 2. This enzyme is preferentially activated by calcium ions. In the presence of 5 mM ATP, maximal enzyme activity being obtained at 3–5 mM Ca2+. The maximal rate of ATP hydrolysis varies between 15 and 22 μmoles Pi per mg protein in 30 min. 3. 3. Mg2+ also activates the enzyme, but always to a lesser degree than Ca2+. Mn2+, but not Sr2+, activates the enzyme. At optimal Ca2+ concentration, addition of Mg2+ is always inhibitory. Ca2+ and Mg2+ seem to act on the same site. 4. 4. The enzyme does not require Na+ or K+ for activation by Ca2+. 5. 5. The optimal pH for Ca2+ activation of the enzyme lies between 8.2 and 8.5; at pH 7.1 and 9.5 only 50 % of maximal activation occurs. 6. 6. Addition of increasing amounts of Na2H2EDTA leads to progressive decreases of activity, complete inhibition occurring at 5 mM when the incubation fluid contains 5 mM Ca2+. 7. 7. Ethacrynic acid inhibits the enzyme but ouabain does not. 8. 8. Other triphosphates may serve as substrate; but the νmax for Na2ATP is highest. 9. 9. We suggest that this enzyme aids in maintaining the uphill gradient of Ca2+ between maternal and fetal circulation.

Indomethacin and renal function in premature infants with persistent patent ductus arteriosus

Renal function during indomethacin treatment was studied in 12 premature infants with patent ductus arteriosus. Decreases in urinary flow rate, GFR, and CH2O by 56, 27, and 66%, respectively, occurred during Indo therapy. Urinary excretion rates of ions were also reduced: Na by 70%, Cl by 79%, K by 40%. These changes were accompanied by slight decreases in plasma sodium concentration and osmolality. Except for GFR and urinary Na and osmolality, all these functions returned to pretreatment values one to two weeks after stopping the drug.

Properties of a Ca2+- and Mg2+-activated ATP-hydrolyzing enzyme in rat kidney cortex

Abstract 1. 1. The properties of an ATP-hydrolysing enzyme (ATP phosphohydrolase, EC 3.6.1.3) in rat kidney cortex, activated by bivalent cations, have been defined. 2. 2. This ATPase is activated by both Ca2+ and Mg2+; but Mg2+ is not necessary for Ca2+ activation. 3. 3. Neither Na+ nor K+ are necessary for Ca2+ activation. 4. 4. Inhibition occurs at a bivalent cation concentration above 5 to 10 mM. 5. 5. GTP and ITP are also hydrolysed by the enzyme but not ADP. 6. 6. Mn2+ but not Sr2+ may also stimulate hydrolysis of ATP. 7. 7. The pH optimum for activation lies between 7.5 and 8.2. 8. 8. Ouabain (1 mM) and ethacrynic acid (1 mM) do not inhibit activation by Ca2+ 9. 9. The stimulation of ATP hydrolysis by both Ca2+ and Mg2+ suggests that the enzyme may be involved in the transport of bivalent cations in the renal tubule.

Late hyponatremia in very low birthweight infants. (less than 1.3 kilograms).

Extract: Late hyponatremia (plasma Na+ <130 mEq/liter) occurred frequently (on 54 of 159 occasions) in 46 very low birthweight (VLBW) infants (<1.3 kg at birth) between 2 and 6 weeks of age while receiving a sodium intake of ≤2 mEq/kg/24 hr. To elucidate possible pathogenetic mechanisms five groups of such infants were studied while receiving a commercially available formula reconstituted to give two different volumes and two different Na+ concentrations. Sodium intake in the nonsupplemented (NS) infants (n = 23) was less than 2 mEq/kg/24 hr. Supplemented (S) infants (n = 16) received approximately 3 mEq Na+/kg/24 hr. A further group of seven infants given a high volume (200 ml/kg/24 hr), high caloric (100 cal/dl) formula and Na+ supplementation (to 3 mEq/kg/24 hr) was also included. Infants were studied from age 14 days until they weighed 1.80 ± 0.05 kg at a mean age of 47 days.At the time of start of the study, 6 of 20 NS and 6 of 19 S infants were hyponatremic. After supplementation only two episodes of hyponatremia occurred in S infants, both during the first study week, whereas the high incidence of hyponatremia in NS infants remained unchanged throughout the first 3 weeks of the study period.During baseline urine collections all infants excreted between 80 and 100 ml/kg/24 hr urine, but those receiving 150 ml/kg/24 hr formula decreased their urinary output rapidly to 50 ml/kg/24 hr, whereas infants receiving high volume feeds (200 ml/kg/24 hr) did not decrease their urinary output until the third balance at an average age of 45 days. All infants excreted between 1.0 and 1.2 mEq/kg/24 hr of sodium in their urine during the initial collection. Nonsupplemented infants reduced their urinary Na+ excretion more rapidly than supplemented babies (NS: from 1.03 to 0.55 mEq/kg/24 hr, first vs second balance; S: from 1.00 to 0.80 mEq/kg/24 hr, first vs third balance). Mean potassium excretion remained unchanged in NS and S infants during the study period and was not affected by the volume or caloric content of the formula.Extracellular volume (ECV) and total body water (TBW) were measured serially, and there were no differences between S and NS infants in the distribution of body water. The percentage of TBW and ECV decreased in all groups with increasing postnatal age.Speculation: VLBW infants are prone to hyponatremia in the first 6 weeks of life because of the combined influence of renal immaturity, which permits relatively high urinary sodium loss in the presence of low plasma [Na+], and low intake of sodium (≤2 mEq/kg/24 hr), the amount provided by some current formulas based on breast milk. Dilutional factors are not involved, but the role of aldosterone remains unresolved. Supplementation of the formula to provide a daily total sodium intake of 3 mEq/kg/24 hr until a weight of 1.5 kg is reached is corrective.

Postnatal growth of infants of <1.3 kg birth weight: Effects of metabolic acidosis, of caloric intake, and of calcium, sodium, and phosphate supplementation

Weekly increments of length, weight, head circumference, and skinfold thickness in response to a series of dietary changes were measured in 108 healthy infants who weighed

Calcium Ion Activity in the Sick Neonate: Effect of Bicarbonate Administration and Exchange Transfusion

Extract: Calcium ion activity was measured in plasma obtained by venous or arterial puncture using a calcium-selective flow-through electrode. Mean level of ionized calcium in umbilical venous plasma was 2.48 ± 0.04 mEq/liter. Within 30 hr after birth, the values decreased in sick infants to 1.35 ± 0.11 mEq/liter. Total calcium concentra tions were in the normal adult range at birth (5.20 ± 0.08 mEq/liter), showing a subsequent decline to subnormal values (3.47 ± 0.28 mEq/liter at age 30–40 hr). A calcium ion activity of less than 1.4 mEq/liter was associated with total calcium level at or below 3 mEq/liter in 80% of patients. Symptoms and signs attributable to hypocalcemia (ionized fraction) or hypomagnesemia, or both, were found only in infants in whom plasma levels of both divalent cations were below the lower limit of normal. Administration of NaHGO3 for acidosis caused a slight rise in pH (from 7.20 to 7.28 mEq/liter) and a decrease in plasma calcium ion activity (from 1.68 to 1.51 mEq/liter). During exchange transfusion with acid-citrate-dextrosc (ACD)-Tham buffered blood, calcium ion activity decreased significantly from 1.90 ± 0.08 to 1.20 ± 0.09 mEq/liter, whereas total calcium levels increased consistently (from 4.22 ± 0.13 to 5.33 ± 0.15 mEq/liter).Speculation: The temporary hypocalcemia, observed in sick newborn infants, may also occur in healthy newborns. It is thought to initiate normal calcium homeostasis by stimulating parathormone secretion and by bringing into play the two feedback mechanisms for calcitonin and parathormone secretion.

Vitamin D deficiency in a calcium-supplemented very low-birth-weight infant.

1. Lewin PK, Reid M, Reilly BJ, Swyer PR, and Fraser D: Iatrogenic rickets in low-birth-weight infants, J PEDIATR 78:207, 1971. 2. Leape LL, and Valaes T: Rickets in low birth weight infants receiving total parenteral nutrition, J Pediatr Surg 11:665, 1976. 3. Kooh SW, Fraser D, Reilly B J, Hamilton JR~ Gall DG, and Bell L: Rickets due to calcium deficiency, N Engl J Med 297:1264, 1977. 4. Gutcher GR, and Chesney RW: Iatrogenic rickets as a complication of a total parenteral nutrition program, Clin Pediatr 17:817, 1978. 5. Geg~el RL, Pereira GR, and Spackrnan TJ: Fractured ribs: Unusual presentation of rickets in premature infants, J PED1ATR 93:680, 1978. 6. Rowe JC, Wood DH, Rowe DW, and Reisz LG: Nutritional hypophosphatemic rickets in a premature infant fed breast milk, N Engl J Med 300:293, 1979. 7. Hoff N, Hoddard J, Teitelbaum S, McAlister W, and Hillman LS: Serum concentrations of 25-hydroxyvitamin D in rickets of extremely premature infants, J PEDIATR 94:460, 1979. 8. American Academy of Pediatrics, Committee on Nutrition: Nutritional needs of low birth weight infants, Pediatrics 60:519, 1977. 9. Fomon SJ, and Filer LJ: Milk and formulas, in Fomon S J: Infantnutrition, ed 2, Philadelphia, 1974, WB Saunders Co., p 388. 10. Day GM, Chance GW, Radde IC, Reilly B J, Park E, and Sheepers J: Growth and mineral metabolism in very low birth weight infants II. Effect of calcium supplementation on growth and divalent cations, Pediatr Res 9:568, 1975.

The effect of the Ca2+/Mg2+ concentration ratio on placental (Ca2+-Mg2+)-ATPase activity

Abstract The velocity of the reaction of a (Ca 2+ -Mg 2+ )stimulated ATPase in placental plasma membranes of the guinea pig was found to depend on the [Mg 2+ ]/[Ca 2+ ] concentration ratio. The observed activation curve of the enzyme while changing the concentration ratio agrees with the theoretical equation, derived for the case of two ions activating the same site while the enzyme is saturated. Because of the good agreement, we conclude that the two divalent cations, Ca 2+ and Mg 2+ , activate this placental ATPase at the same site. The equation may be used for other enzymes to determine whether or not two ions activate the same site, and to predict the velocity of the reaction for a given ion concentration ratio.

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.

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.