Robert Castro

Shear-induced permeability changes in a polymer grafted silica membrane

Abstract The hydrodynamic response of a graft-polymerized membrane was demonstrated for a microporous silica-poly(vinylpyrrolidone) (silica-PVP) membrane. The membrane pores were modified by graft polymerizing vinyl pyrrolidone onto the membrane pore surface, resulting in a polymer surface layer of covalently tethered polymer chains. The hydraulic permeability of the modified membrane increased with increasing transmembrane pressure owing to flow-induced deformation of the grafted polymer chains. The dynamics of the modified pores was investigated by membrane hydraulic permeability studies along with a two-region hydrodynamic pore flow model. The thickness of the grafted polymer layer decreased with increasing pore-wall shear rate by up to about 47%, relative to the thickness at the zero shear limit, depending on the surface density and length of the grafted chains. Although the effective pore size of the polymer-grafted membrane was reduced by 5–36% (at the zero shear rate limit), about 18–59% of the pore size loss was regained at high pore-wall shear rates. Increasing the degree of shear-induced permeability change is feasible by increasing the ratio of the polymer chain length/pore size ratio as well as the surface density of the grafted polymer phase. The present results suggest that hydrodynamic pore size control could provide an additional useful degree of freedom in operating polymer-modified filtration membranes.

The permeability behavior of polyvinylpyrrolidone-modified porous silica membranes

Abstract The surface of 4100 A-pore-size silica membranes was modified with a covalently bonded polyvinyl-pyrrolidone brush layer by a novel graft polymerization process. Hydraulic permeability measurements performed with six different solvents and both unmodified and modified membranes suggest that the permeability of the modified membrane is determined by the configuration of the terminally anchored polymer chains. In the modified ceramic-supported polymeric (CSP) membrane, the swelling of the polymer brush layer increases as the solvent power increases, resulting in a decrease in the pore radius and subsequently, the permeability.

Silica-suported polyvinylpyrrolidone filtration membranes

A hybrid ceramic-polymeric membrane was developed by the growth of covalently-bonded polyvinylpyrrolidone (PVP) chains from the surface of a porous silica support membrane via a graft polymerization process. These ceramic-supported polymeric (CSP) membranes can be produced with variable surface density and length of the terminally anchored polymer chains. Hydraulic permeability measurements demonstrated the effect of the grafted chain density and length on the water permeability of the CSP membranes. The potential application of these CSP membranes for the treatment of oil-in-water emulsions, was evaluated in cross-flow filtration experiments. This preliminary evaluation indicated improved performance of a CSP membrane, relative to an unmodified support, since the CSP membrane produced a lower permeate concentration at an equivalent permeability. The improved selectivity and reduced fouling tendency is attributed to the increased hydrophilicity of the membrane surface provided by the grafted PVP chains.

Immunosuppressive properties of surfactant in alveolar macrophage NR8383

Abstract.Objective:To evaluate the anti-inflammatory effects of exogenous surfactants and surfactant phospholipid without surfactant proteins (SP-A and SP-D) on the lipopolysaccharide- (LPS) stimulated rat alveolar macrophage (AM) cell line NR8383.Methods:Exogenous surfactants (beractant, calfactant or colfosceril) and surfactant phospholipid (dipalmitoyl phosphatidylcholine, DPPC), standardized to phospholipid content of 25–1,000 μg/ml were incubated with LPS- (1 μg/ml) stimulated NR8383 AMs.Results:TNF-α and IL-1β secretion and nitric oxide (NO) formation following LPS stimulation were inhibited by treatment with surfactants or DPPC. Furthermore, LPS-dependent NO production and iNOS protein levels were significantly suppressed in cells pretreated for one hour with beractant compared to beractant added simultaneously with or following LPS. Additionally, LPS-stimulated oxidative burst, measured by flow cytometry, was significantly decreased by beractant. Finally, beractant inhibited the translocation of NF-κB from cytoplasmic into nuclear extract in LPS-stimulated NR8383 AMs.Conclusions:Exogenous surfactants and surfactant phospholipid inhibit secretion of proinflammatory cytokines and NO in NR8383 AMs. The inhibitory effects of beractant on oxygen radical and LPS-induced NO formation may result from unique mechanisms of decreasing cell signaling. The anti-inflammatory activity of surfactant products used in the treatment of neonatal respiratory distress syndrome (RDS) may depend upon the specific preparation or dose used.

Protein Kinase A and Mitogen-Activated Protein Kinase Pathways Mediate cAMP Induction of α-Epithelial Na+ Channels (α-ENaC)

A major mechanism for Na+ transport across epithelia occurs through epithelial Na+ channels (ENaC). ENaC is a multimeric channel consisting of three subunits (α, β, and γ). The α‐subunit is critical for ENaC function. In specific culture conditions, the rat submandibular gland epithelial cell line (SMG‐C6) demonstrates minimal Na+ transport properties and exposure to dibutyryl cAMP (DbcAMP) for up to 48 h caused an elevation of α‐ENaC mRNA and protein expression and amiloride‐sensitive short‐circuit current (ISC). Here we examined the early signaling pathways evoked by DbcAMP which contribute to the eventual increase in Na+ transport is present. Treatment with either of the protein kinase A (PKA) inhibitors KT5720 or H‐89 followed by exposure to 1 mM DbcAMP for 24 h markedly attenuated DbcAMP‐induced α‐ENaC protein formation and ISC. Exposure of SMG‐C6 cells to 1 mM DbcAMP induced a rapid, transient phosphorylation of the cAMP response element binding protein (CREB). This response was attenuated in the presence of either KT5720 or H‐89. Dominant‐negative CREB decreased DbcAMP‐induced α‐ENaC expression. Suppression of the extracellular signal‐regulated protein kinase (ERK 1,2) with PD98059 or the p38 mitogen‐activated protein kinase (MAPK) pathway with SB203580 reduced DbcAMP‐induced α‐ENaC protein levels in SMG‐C6 cells. DbcAMP‐induced phosphorylation of CREB was markedly attenuated by PD98059 or SB203580. DbcAMP‐induced activation of the either the p38 or the ERK 1,2 MAPK pathways was abolished by either of the PKA inhibitors, H‐89 or KT5720. Cross talk between these signaling pathways induced by DbcAMP via the activation of CREB appears to contribute to increased levels of α‐ENaC observed after 24 h of treatment in SMG‐C6 epithelial cells. J. Cell. Physiol. 215: 101–110, 2008.

Induction of serum- and glucocorticoid-induced kinase-1 (SGK1) by cAMP regulates increases in α-ENaC

α‐ENaC expression and activity is regulated by a variety of hormones including β‐adrenergic agonists via the second messenger cAMP. We evaluated the early intermediate pathways involved in the up‐regulation of SGK1 by DbcAMP and whether SGK1 is a prerequisite for induction of α‐ENaC expression. Submandibular gland epithelial (SMG‐C6) cells treated with DbcAMP (1 mM) induced both SGK1 mRNA and protein expression. DbcAMP‐stimulated SGK1 mRNA expression was decreased by actinomycin D and mRNA and protein expressions were attenuated by PKA inhibitors (H‐89 and KT5720). Inhibition of PI3‐K with either LY294002 or dominant negative PI3‐K reduced DbcAMP‐stimulated SGK1 protein and mRNA levels, attenuated the phosphorylation of CREB (a cAMP‐activated transcription factor) and decreased α‐ENaC protein levels and Na+ transport. In addition, the combination of PKA inhibitors with dominant negative PI3‐K synergistically inhibited DbcAMP‐induced Na+ transport. Inhibition of SGK1 expression by siRNA decreased but did not obliterate DbcAMP‐induced α‐ENaC expression. Thus, in a cell line which endogenously exhibits minimal α‐ENaC expression, induction of SGK1 by DbcAMP occurs via the PI3‐K and PKA pathways. Increased α‐ENaC levels and function are partly dependent upon the early induction of SGK1 expression. J. Cell. Physiol. 217: 632–642, 2008.

Ovine fetal lung fluid response to intravenous saline solution infusion fetal atrial natriuretic factor effect

The fetal lung, a significant source of in utero fluid production, has been postulated to serve a regulatory role in maintenance of fetal body fluid homeostasis. Whereas the fetus responds to intravascular saline solution infusions with increased urine output, the fetal lung fluid response to this stimulus is unclear. Tracheal fluid output was measured in four chronically catheterized ovine fetuses (mean gestation, 129 +/- 1 days) subjected to successive 40-minute intravenous 0.9% saline solution infusions at rates of 0.5 and 1 ml/min/per kilogram of body weight. Tracheal fluid output decreased significantly (1.7 +/- 0.1 to 1.1 +/- 0.1 ml/10 min, p less than 0.01) during the infusion and returned to basal levels during the recovery period. Lung fluid osmolality and electrolyte concentration did not change. Fetal plasma atrial natriuretic factor increased significantly in response to the saline solution infusion (364 +/- 90 to 790 +/- 286 pg/ml, p less than 0.05) and returned to basal levels during the recovery period. There was a significant inverse correlation between plasma atrial natriuretic factor levels and tracheal fluid output. These results suggest that increased fetal plasma atrial natriuretic factor decreases lung fluid production. Lung fluid does not appear to compensate for fetal body water excess. Rather, lung fluid production appears to promote intrauterine pulmonary growth and to facilitate the transition to the extrauterine environment.

Regulation of epithelial Na+ channel (ENaC) in the salivary cell line SMG-C6.

Glucocorticoids and mineralocorticoids modulate Na+ transport via epithelial Na+ channels (ENaC). The rat submandibular epithelial cell line, SMG-C6, expresses α-ENaC mRNA and protein and exhibits amiloride-sensitive Na+ transport when grown in low-serum (2.5%) defined medium, therefore, we examined the effects of altering the composition of the SMG-C6 cell growth medium on ENaC expression and function. No differences in basal or amiloride-sensitive short-circuit current (Isc) were measured across SMG-C6 monolayers grown in the absence of thyroid hormone, insulin, transferrin, or EGF. In the absence of hydrocortisone, basal and amiloride-sensitive Isc significantly decreased. Similarly, monolayers grown in 10% serum-supplemented medium had lower basal Isc and no response to amiloride. Adding hydrocortisone (1.1 μM) to either the low or 10% serum medium increased basal and amiloride-sensitive Isc, which was blocked by RU486, the glucocorticoid and progesterone receptor antagonist. Aldosterone also induced an increase in α-ENaC expression and Na+ transport, which was also blocked by RU486 but not by the mineralocorticoid receptor antagonist spironolactone. Thus, in the SMG-C6 cell line, hydrocortisone and aldosterone increased ENaC expression and basal epithelial Na+ transport. The absence of endogenous ENaC expression in culture conditions devoid of steroids makes the properties of this cell line an excellent model for investigating pathways regulating ENaC expression and Na+ transport.

Ovine maternal and fetal glomerular atrial natriuretic factor receptors : response to dehydration

In pregnancy, dehydration produces marked effects on maternal and fetal body water homeostasis including an increase in fetal urinary sodium concentration and excretion. To examine the role of fetal plasma atrial natriuretic factor (ANF) and glomerular ANF receptors in dehydration-induced natriuresis, we compared plasma ANF levels and glomerular ANF binding characteristics in dehydrated and control maternal and fetal sheep. Mean (+/- SEM) maternal and fetal plasma ANF levels in control animals (n = 9) at 132-136 days gestation were 37 +/- 3 pg/ml and 138 +/- 20 pg/ml, respectively. Although mean ANF receptor maximum binding capacities (Bmax) were significantly higher in maternal than in fetal glomeruli (83 +/- 11 vs 34 +/- 12 fmol/mg protein, respectively), the dissociation constants (Kd) for ANF binding were not different (2.7 +/- 0.6 and 3.7 +/- 1.7 x 10(-10) M, respectively). In an additional 9 animals studied after 63 +/- 4 h of water deprivation, maternal plasma ANF levels were significantly lower than in the control group (14 +/- 4 vs. 37 +/- 3 pg/ml), maternal glomerular ANF receptor Bmax values were significantly higher (732 +/- 203 vs. 83 +/- 11 fmol/mg protein), and Kd values were six-fold higher (17.0 +/- 7.1 vs. 2.7 +/- 0.6 x 10(-10) M), although this difference was only marginally significant (p = 0.06). In contrast to the adult, there was a small, nonsignificant decrease in plasma ANF levels and no difference in Bmax or Kd values between the dehydrated and euhydrated fetal animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Ca2+ signals in a parotid cell line Par-C5

The Ca(2+) signaling system in an established immortalized rat parotid acinar cell line, Par-C5, was examined using the Ca(2+)-sensitive fluorescent indicator fura-2 and by measuring inositol 1,4,5-trisphosphate (IP(3)) formation. Agonist-induced increase in intracellular Ca(2+) ([Ca(2+)](i)) by mobilization of intracellular stores and influx across the cell membrane was stimulated by acetylcholine (ACh) and ATP, whereas noradrenaline-(NA)-induced a small [Ca(2+)](i) increase mediated primarily by release from intracellular Ca(2+) stores. [Ca(2+)](i) increase by ACh and ATP was mediated through the phosphoinositide signal pathway since both agonists significantly increased 1,4,5-IP(3) formation and Ca(2+) mobilization was abolished by the phospholipase C inhibitor U73122. In Ca(2+)-free medium, ACh or ATP discharged the IP(3)-sensitive Ca(2+) store and essentially abolished subsequent [Ca(2+)](i) response to thapsigargin (TG). Exposure to ionomycin and monensin after TG induced a further mobilization of Ca(2+), suggesting IP(3)-insensitive stores are present. Furthermore, depletion of IP(3)-sensitive Ca(2+) stores by TG, ACh and ATP enhanced plasmalemmal Ca(2+)-entry pathways. Exposure to tumor necrosis factor-alpha (TNF-alpha), a cytokine associated with lymphocyte invasion of salivary epithelial cells in autoimmune disorders, significantly reduced ACh-stimulated Ca(2+) mobilization. TNF-alpha inhibitory effect on Ca(2+) mobilization was not directly due to an interaction on muscarinic receptors since ACh-induced 1,4,5-IP(3) formation was not altered. These results in the Par-C5 cell line indicate 1) [Ca(2+)](i) is regulated by muscarinic and P2Y-nucleotide receptors and partly by alpha(1)-adrenergic receptors; 2) IP(3)-sensitive and -insensitive Ca(2+) stores exist; 3) Ca(2+) influx activated by ACh, ATP or TG is mediated by the store-operated Ca(2+) entry pathway; and 4) muscarinic agonist-stimulated Ca(2+) mobilization is altered by the cytokine TNF-alpha.