nystatin-a1 and Dehydration

In cystic fibrosis (CF), the absence of functional CFTR leads to dysregulated Na(+) absorption across airway epithelia. We established an in vitro model of dysregulated Na(+) absorption by treating polarized normal human bronchial epithelial cells (HBEs) with nystatin (Nys), a polyene antibiotic that enables monovalent cations to permeate biological membranes. Acute mucosal Nys produced a rapid increase in short circuit current (I(sc)) that reflected increased transepithelial Na(+) absorption and required Na(+)/K(+)ATPase activity. The acute increase in I(sc) was associated with increased mucosal liquid absorption. Prolonged mucosal Nys treatment resulted in sustained Na(+) hyperabsorption, associated with increased mucosal liquid absorption in comparison with naïve (nontreated, kept under air-liquid interface conditions) or vehicle-treated cultures. Nys treatment was not toxic. Increased lactate accumulation in Nys-treated culture media suggested a higher metabolic rate associated with the higher energy demand for Na(+) transport. After chronic Nys treatment, the increased I(sc) was rapidly lost when the cultures were mounted in Ussing chambers, indicating that Nys could be rapidly removed from the apical membrane. Importantly, chronic Nys treatment promoted sustained mucosal liquid depletion and caused mucus dehydration, compaction, and adhesion to the apical surface of Nys-treated cultures. We conclude that mucosal Nys treatment of HBEs provides a simple in vitro model to recapitulate the Na(+) and volume hyperabsorptive features of CF airway epithelia.

Topics: Absorption; Biological Transport; Cell Membrane Permeability; Cell Polarity; Cells, Cultured; Dehydration; Electric Conductivity; Epithelial Cells; Gene Expression Regulation; Humans; Inflammation; Lactic Acid; Models, Biological; Mucous Membrane; Nystatin; Organ Size; Respiratory System; RNA, Messenger; Serous Membrane; Sodium; Sodium-Potassium-Exchanging ATPase

We tested the hypothesis that dehydration-induced alterations in red blood cell (RBC) membrane organisation or composition contribute to sickle cell adhesion in sickle cell disease (SCD). To examine the role of RBC hydration in adhesion to the subendothelial matrix protein thrombospondin-1 (TSP), normal and sickle RBCs were incubated in buffers of varying tonicity and tested for adhesion to immobilised TSP under flow conditions. Sickle RBCs exhibited a decrease in TSP binding with increasing cell hydration (P<0.005), suggesting that cellular dehydration may contribute to TSP adhesion. Consistent with this hypothesis, normal RBCs showed an increase in TSP adhesion with increasing dehydration (P<0.01). Furthermore, increased TSP adhesion of normal RBCs could also be induced by isotonic dehydration using nystatin-sucrose buffers. Finally, TSP adhesion of both sickle RBCs and dehydrated normal RBCs was inhibited by the anionic polysaccharides, chondroitin sulphate A and high molecular weight dextran sulphate, but not by competitors of CD47-, band 3-, or RBC phosphatidylserine-mediated adhesion. More importantly, we found increased adhesion of nystatin-sucrose dehydrated normal mouse RBCs to kidney capillaries following re-infusion in vivo. In summary, these findings demonstrate that changes in hydration can significantly impact adhesion, causing normal erythrocytes to display adhesive properties similar to those of sickle cells and vice versa.

Topics: Adolescent; Adult; Anemia, Sickle Cell; Animals; Capillaries; Cell Adhesion; Cells, Cultured; Child; Dehydration; Erythrocyte Membrane; Erythrocytes; Erythrocytes, Abnormal; Flow Cytometry; Humans; Kidney; Mice; Nystatin; Osmosis; Phosphatidylserines; Sucrose; Thrombospondin 1

Activation of K-Cl cotransport is associated with activation of membrane-bound serine/threonine protein phosphatases (S/T-PPases). We characterize red blood cell S/T-PPases and K-Cl cotransport activity regarding protein phosphatase inhibitors and response to changes in ionic strength and cell size. Protein phosphatase type 1 (PP1) activity is highly sensitive to calyculin A (CalA) but not to okadaic acid (OA). PP2A activity is highly sensitive to CalA and OA. CalA completely inhibits K-Cl cotransport activity, whereas OA partially inhibits K-Cl cotransport. Membrane PP1 and membrane PP2A activities are elevated in cells suspended in hypotonic solutions, where K-Cl cotransport is elevated. Increases in membrane PP1 activity (62 +/- 10% per 100 meq/l) result from decreases in intracellular ionic strength and correlate with increases in K-Cl cotransport activity (54 +/- 10% per 100 meq/l). Increases in membrane PP2A activity (270 +/- 77% per 100 mosM) result from volume increases and also correlate with increases in K-Cl cotransport activity (420 +/- 47% per 100 mosM). The characteristics of membrane-associated PP1 and PP2A are consistent with a role for both phosphatases in K-Cl cotransport activation in human erythrocytes.

Topics: Anemia, Sickle Cell; Biological Transport; Carrier Proteins; Cell Size; Cytosol; Dehydration; Enzyme Inhibitors; Erythrocytes; Humans; Ionophores; K Cl- Cotransporters; Marine Toxins; Membrane Proteins; Nystatin; Okadaic Acid; Osmolar Concentration; Oxazoles; Phosphoprotein Phosphatases; Serine; Symporters; Threonine; Water-Electrolyte Balance

Topics: Administration, Oral; Ampicillin; Animals; Bone Marrow Transplantation; Cloxacillin; Cyclophosphamide; Dehydration; Diarrhea; Dogs; Feces; Graft Rejection; Hematuria; Injections, Intravenous; Melena; Neomycin; Nystatin; Pharynx; Polymyxins; Transplantation, Homologous; Urine; Vomiting