David G. Oelberg

Bile salt-induced intracellular Ca++ accumulation in type II pneumocytes

The pathogenesis of pneumonitis associated with meconium aspiration is poorly understood. To explore the possibility of pulmonary cytotoxicity in association with bile salt exposure and calcium accumulation, we compared cell viability, radiolabeled calcium accumulation, and intracellular [calcium] in the presence and absence of bile salts, chenodeoxycholate, and 3β-OH-5-cholenoate. We assessed viability of type II pneumocytes in culture by cell permeability to trypan blue dye, incorporation of leucine into cellular proteins, and cellular morphology. Intracellular calcium concentrations were monitored with fluorescent dye methodology. At micromolar concentrations, the above bile salts increased cell permeability by as much as 9-fold and decreased leucine incorporation by as much as 5-fold. Radiolabeled calcium accumulation increased by as much as 2.5-fold and intracellular [calcium] transiently increased by as much as 6-fold. Studies using bile salts extracted from meconium yielded similar results. Correlation of calcium accumulation to viability studies yielded a direct relationship with cell permeability and an inverse relationship with leucine incorporation. We speculate that bile salt-induced accumulation of intracellular calcium in lung cells may contribute to the pathogenesis of meconium aspiration pneumonitis.

Vitamin concentrations in very low birth weight infants given vitamins intravenously in a lipid emulsion: measurement of vitamins A,D, and E and riboflavin

Because total parenteral nutrition with vitamins added to the glucose-amino acid mixture is often associated with a reduction in blood levels of vitamin A (retinol) during the routine treatment of many very low birth weight (VLBW) infants (less than 1500 gm), and because retinol losses in the plastic delivery system can be prevented by adding the vitamins to an intravenous lipid emulsion, seven VLBW infants with a mean birth weight of 900 gm (range 450 to 1360 gm) were given 40% of a unit dose vial, per kilogram of body weight, of a multivitamin preparation (M.V.I. Pediatric) (280 micrograms retinol; 160 IU vitamin D; 2.8 mg tocopherol; 0.68 mg riboflavin) in a lipid emulsion, Intralipid. After treatment with the intralipid-vitamin mixture for 19 to 28 days, plasma vitamin A (retinol) concentrations increased significantly from 11.0 +/- 0.76 (mean +/- SEM) before intralipid to 19.2 +/- 0.97 micrograms/dl after the intralipid-vitamin mixture (p less than 0.01); 25-hydroxyvitamin D concentrations increased from an initial value of 12.6 +/- 2.6 to 20.2 +/- 1.9 mg/dl (p less than 0.01); alpha-tocopherol concentrations increased from an initial value of 0.31 +/- 0.06 to 2.44 +/- 0.13 mg/dl (p less than 0.01); and riboflavin levels increased from 64.1 +/- 7.8 ng/ml to concentrations between 20 and 100 times the initial level. Erythrocyte riboflavin levels increased from 71.8 +/- 14 initially to 166 +/- 41 ng/gm hemoglobin, and erythrocyte flavin-adenine dinucleotide levels increased similarly from 972 +/- 112 initially to 2005 +/- 294 ng/gm hemoglobin. These results show that the addition of M.V.I. Pediatric to Intralipid decreases the extensive in vivo loss of retinol and is associated with an increase in plasma retinol concentrations in VLBW infants. The daily doses of vitamins D (160 IU/kg) and E (2.8 mg/kg) appear sufficient, but the dose of vitamin A (280 micrograms/kg) is insufficient to raise blood levels of all infants into the normal range. The current dose of riboflavin is excessive and may be harmful.

Surfactant modulates calcium response of neutrophils to physiologic stimulation via cell membrane depolarization.

Pulmonary surfactant (PS) reduces inflammation in the lung by poorly understood mechanisms. We have observed that surfactant-associated proteins (SAP) insert monovalent cation channels in artificial membranes. Neutrophils are primary mediators of acute pulmonary inflammation, and their functions are activated by increases in cytosolic ionized calcium concentration ([Ca2+]) and by changes in membrane potential. We hypothesize that PS inserts SAP-dependent cation channels in neutrophils, causing membrane depolarization, altered [Ca2+] response, and depressed activation. Human neutrophils were isolated, exposed to PS+SAP (1% Survanta), PS−SAP (1% Exosurf), or buffer, and washed before activating with selected stimulants. PS+SAP reduced phorbol ester- and formyl peptide–stimulated adherence and aggregation by 38% (p < 0.05) and 54% (p < 0.02), respectively. PS+SAP also inhibited the formyl peptide–induced [Ca2+] response of neutrophils (p < 0.01), but only in the presence of external Ca2+. Further characterization of this inhibition demonstrated that PS+SAP blocked formyl peptide–induced influx of both Ca2+ and Mn2+, and that this inhibition was present during activation by other neutrophil stimulants (IL-8, immune complexes). Prior depolarization of neutrophils with gramicidin-D similarly inhibited the [Ca2+] response of neutrophils to formyl peptide, and analysis of neutrophil membrane potential by 3,3′-dipentyloxaearbocyanine iodide (diOC5(3)) fluorescence revealed that PS+SAP induced rapid neutrophil depolarization. In contrast, PS−SAP exhibited little effect on neutrophil function, [Ca2+], or membrane potential. We conclude that PS+SAP decreases neutrophil adherence and aggregation responses, blocks Ca2+ influx after physiologic stimulation, and decreases membrane potential. We speculate that these effects are caused by membrane depolarization via SAP-dependent cation channel insertion, and that all of these effects contribute to the antiinflammatory properties of PS+SAP.

Bile salt-induced calcium fluxes in artificial phospholipid vesicles

The ionic permeability of selected biological membranes is increased by bile salts. To examine changes in calcium permeability during the exposure of artificial membranes to bile salts, we investigated calcium uptake by unilamellar and multilamellar phospholipid vesicles. In the presence of 750 microM taurodeoxycholate, uptake of radiolabelled calcium by unilamellar vesicles increased 2.5-fold over control values. Calcium uptake by multilamellar vesicles as measured with a free calcium indicator, arsenazo III, increased 2.2- or 21-fold in the presence of 60 microM lithocholate or 3 beta-hydroxy-5-cholenoate, respectively. Results were directly influenced by experimental variables such as bile salt hydrophobicity, external calcium concentration, and the bile salt/lipid molar ratio. Observed membrane solubilization was minimal despite increased calcium permeability. Comparison of radiolabelled calcium uptake with radiolabelled sodium or radiolabelled rubidium uptake indicated that bile salt-dependent calcium uptake was 60-140-times greater than bile salt-dependent uptake of either monovalent cation. In an effort to delineate forces affecting calcium translocation, vesicles were exposed either to valinomycin, which induced an electrochemical gradient across the membrane, or to nigericin, which induced a proton gradient. Exposure to valinomycin minimally influenced bile salt-induced calcium uptake while exposure to nigericin significantly promoted uptake by 40-70%. The results suggest that bile salts promote calcium uptake by a mechanism which may be similar to those of other carboxylic ionophores.

Surfactant releases internal calcium stores in neutrophils by G protein-activated pathway.

Pulmonary surfactant with surfactant-associated proteins (PS+SAP) decreases pulmonary inflammation by suppressing neutrophil activation. We have observed that PS+SAP inserts channels into artificial membranes, depolarizes neutrophils, and depresses calcium influx and function in stimulated neutrophils. We hypothesize that PS+SAP suppresses neutrophil activation by depletion of internal Ca++ stores and that PS+SAP induces depletion through release of Ca++ stores and through inhibition of Ca++ influx. Our model predicts that PS+SAP releases Ca++ stores through insertion of channels, depolarization of neutrophils, and activation of a G protein–dependent pathway. If the model of channel insertion and membrane depolarization is accurate, then gramicidin—a channel protein with properties similar to those of PS+SAP—is expected to mimic these effects. Human neutrophils were monitored for [Ca++] responses after exposure to one of two different PS+SAP preparations, a PS-SAP preparation, gramicidin alone, and gramicidin reconstituted with phospholipid (PLG). [Ca++] responses were reexamined following preexposure to inhibitors of internal Ca++ release or the G protein pathway. We observed that (i) 1% PS+SAP—but not PS-SAP—causes transient increase of neutrophil [Ca++] within seconds of exposure; (ii) 1% PLG—but not gramicidin alone—closely mimics the effect of PS+SAP on Ca++ response; (iii) PS+SAP and PLG equally depolarize neutrophils; (iv) direct inhibition of internal Ca++ stores releases or of G protein activation suppresses Ca++ responses to PS+SAP and PLG; and (v) preexposure to either PS+SAP or PLG inhibits Ca++ influx following fMLP stimulation. We conclude that PS+SAP independently depolarizes neutrophils, releases Ca++ from internal stores by a G protein-mediated pathway, and alters subsequent neutrophil response to physiologic stimulants by depleting internal Ca++ stores and by inhibiting Ca++ influx during subsequent fMLP activation. The mimicking of these results by PLG supports the hypothesis that PS+SAP initiates depolarization via channel insertion into neutrophil plasma membrane.

Human Milk Effects on Neutrophil Calcium Metabolism: Blockade of Calcium Influx after Agonist Stimulation

Neutrophils are the predominant cellular mediators of acute inflammation, and human milk suppresses multiple neutrophil functions. We sought to determine whether these effects were mediated through disruption of normal intracellular Ca2+ homeostasis. Exposure of human neutrophils to human milk, followed by washing, resulted in altered Ca2+ transient responses to formyl-peptide stimulation in which the peak cytosolic free Ca2+ concentration ([free Ca]) was the same as in unexposed cells, but the postpeak decline in [free Ca] was more rapid. This effect was observed after human milk exposures as brief as 10 s, persisted for up to 4 h after human milk removal, and was concentration dependent. On the basis of experiments examining Ca2+-free conditions followed by Ca2+ supplementation, and experiments examining spontaneous and stimulated manganese and barium influx into neutrophils, the human milk effect was due to blockade of Ca2+ influx. Decreased Ca2+ transient responses to other physiologic stimuli (IL-8, opsonized Staphylococcus aureus, and immune complexes) were observed after human milk exposures. Rat intestinal epithelial cells and HL-60 cells failed to show these effects, suggesting a selective effect on mature inflammatory cells. Characterization of the Ca2+-blocking activity showed it was heat and acid stable in human milk with a molecular mass between 30-100 kD. Commercial human milk lactoferrin exhibited Ca2+ influx blockade activity, but recombinant human lactoferrin showed none. Separation of the activity by heparin affinity chromatography showed that it was distinct from lactoferrin. Human milk-induced blockade of Ca2+ influx provides a potential mechanism for broad suppression of neutrophil functions that may contribute to the antiinflammatory properties of human milk.

Effect of bile acids on calcium efflux from isolated rat hepatocytes and perfused rat livers.

Abstract The changes in intracellular Ca2+ concentration ([Ca2+]i) of hepatocytes induced by certain bile acids are biphasic: an initial increase is followed by a more gradual decrease. This latter decline in [Ca2+]i may be due to an efflux of Ca2+ across the plasma membrane. This hypothesis was tested by studying the effect of different bile acids on the efflux of 45Ca from preloaded rat hepatocytes and isolated perfused rat livers. The following bile acids were studied: cholic (C), ursodeoxycholic (UDC), chenodeoxycholic (CDC), and deoxycholic (DC) acids; their taurine (T) conjugates (TC, TUDC, TCDC, and TDC); and the taurine, sulfate (S), and glucuronide (Glu) derivatives of lithocholic acid (TLC, LS, TLS, and LGIu, respectively). At 0.3 mM, all bile acids except C, TC, TCDC, UDC, and TUDC significantly increased 45Ca efflux from preloaded hepatocytes without affecting cell viability. Dose-response studies revealed that the minimum effective concentration needed to induce 45Ca efflux was 0.06 mM for LS, 0.8 mM for TCDC, and 10 mM for TC. Efflux of 86Rb from preloaded hepatocytes was not significantly altered by 0.1 mM LS, indicating relative specificity for calcium. TDC and DC, but not TC, increased 45Ca efflux from preloaded perfused rat livers. These results showed that bile acids known to increase [Ca2+]i, (CDC, DC, TDC, and TLC) also increased 45Ca efflux from hepatocytes and perfused livers and that efflux was also stimulated by LS, TLS, and LGlu. The extent of this efflux was related to the hydrophobicity of the steroid nucleus of the bile acid. It is speculated that bile acid-induced increases in [Ca2+]i, activate the plasma membrane Ca2+ pump resulting in increased Ca2+ efflux.

Intracranial Hemorrhage in Term or Near-term Newborns With Persistent Pulmonary Hypertension

To substantiate the clinical impression of an increased incidence of intracranial hemorrhage (ICH) in term and near-term infants with persistent pulmonary hypertension (PPH), a retrospective chart review of 35 affected patients was performed. ICH was diagnosed in 40 percent of the patients. Multiple regions of the brain were affected; in many patients, at more than one location. In addition, 43 percent of ICH victims had hemorrhages of the type associated with periventricular bleeding in preterm newborns. Of multiple obstetric and neonatal factors analyzed, many of which are identified risk factors for periventricular hemorrhage, only thrombocytopenia (p = 0.02) was significantly associated with ICH. We conclude that the risk of ICH in newborns with PPH is significant and warrants consideration by clinicians caring for this population. Risk factors (except thrombocytopenia) previously implicated in other types of neonatal ICH, particularly periventricular hemorrhage, do not significantly correlate with ICH in infants with PPH.

EXOGENOUS SURFACTANT PREPARATION INITIATES SINGLE CHANNEL CURRENT FLOW IN ARTIFICAL LIPID BILAYERS. † 2047

EXOGENOUS SURFACTANT PREPARATION INITIATES SINGLE CHANNEL CURRENT FLOW IN ARTIFICAL LIPID BILAYERS. † 2047

Intestinal absorption of bile acid glucuronides in rats

While the intestinal absorption of taurine, glycine, and sulfate conjugates of bile acids has been studied extensively, nothing is known about the absorption of bile acid glucuronides. In the present study, the intestinal phase of the enterohepatic circulation of two bile acid glucuronides was examined. [3β-3H]cholic acid 3-O-β-d-glucuronide or [3β−3H]lithocholic acid 3-O-β-d-glucuronide was perfused through isolated segments of ileum or jejunum with intact blood supply in rats prepared with a biliary fistula. [14C]Taurocholic acid was perfused simultaneously with each glucuronide to compare glucuronide absorption with that of an actively transported bile acid. Intestinal absorption was determined by measuring the rate of secretion of labeled bile acid in bile. The absorption of [3H]cholic acid glucuronide by the ileum and jejunum was one fortieth and one eighth, respectively, that of [14C]taurocholic acid. Comparison of the two glucuronides show that [3H]lithocholic acid glucuronide absorption was 18 and 10 times greater than [3H]cholic acid glucuronide absorption from the jejunum and ileum, respectively. Collectively, the above observations suggest that glucuronidation of bile acids markedly reduces absorption from the small intestine.