Carole M. Liedtke

PKCδ Acts Upstream of SPAK in the Activation of NKCC1 by Hyperosmotic Stress in Human Airway Epithelial Cells

Airway epithelial Na-K-2Cl (NKCC1) cotransport is activated through hormonal stimulation and hyperosmotic stress via a protein kinase C (PKC) δ-mediated intracellular signaling pathway. Down-regulation of PKCδ prevents activation of NKCC1 expressed in Calu-3 cells. Previous studies of this signaling pathway identified coimmunoprecipitation of PKCδ with SPAK (Ste20-related proline alanine-rich kinase). We hypothesize that endogenous PKCδ activates SPAK, which subsequently activates NKCC1 through phosphorylation. Double-stranded silencing RNA directed against SPAK reduced SPAK protein expression by 65.8% and prevented increased phosphorylation of NKCC1 and functional activation of NKCC1 during hyperosmotic stress, measured as bumetanide-sensitive basolateral to apical 86Rb flux. Using recombinant proteins, we demonstrate direct binding of PKCδ to SPAK, PKCδ-mediated activation of SPAK, binding of SPAK to the amino terminus of NKCC1 (NT-NKCC1, amino acids 1–286), and competitive inhibition of SPAK-NKCC1 binding by a peptide encoding a SPAK binding site on NT-NKCC1. The carboxyl terminus of SPAK (amino acids 316–548) pulls down endogenous NKCC1 from Calu-3 total cell lysates and glutathione S-transferase-tagged NT-NKCC1 pulls down endogenous SPAK. In intact cells, hyperosmotic stress increased phosphorylated PKCδ, indicating activation of PKCδ, and activity of endogenous SPAK kinase. Inhibition of PKCδ activity with rottlerin blocked the increase in SPAK kinase activity. The results indicate that PKCδ acts upstream of SPAK to increase activity of NKCC1 during hyperosmotic stress.

Electrolyte transport in the epithelium of pulmonary segments of normal and cystic fibrosis lung.

The epithelium of pulmonary segments from trachea to aveoli actively transports electrolytes and allows osmotic movement of water to maintain the ionic environment in the airway lumen. Models of airway absorption and secretion depict the operation of transporters localized to apical or basolateral membrane. In many epithelia, a variety of electrolyte transporters operate in different combinations to produce absorption or secretion. This also applies to pulmonary epithelium of the large airways (trachea, main‐stem bronchi), bronchioles, and alveoli. Na+ absorption occurs in all three pulmonary segments but by different transporters: apical Na+ channels in large airways and bronchioles; Na+/H+ exchange and Na+ channels in adult alveoli. The Na+ channels in each pulmonary segment share a sensitivity to amiloride, a potent inhibitory of epithelial Na+ channels. Fetal alveoli display spontaneous Cl– secretion, as do the large airways of some mammals, such as dog and bovine trachea. Cl– channels differ in conductance properties and in regulation by intracellular second messengers, osmolarity, and voltage mediate stimulated Cl– secretion, Electroneutral carriers, such as NaCl(K) cotransport, Cl–/HCO3– exchange, and Na+/HCO3– exchange, operate in large airways and alveoli during absorption and secretion. Abnormal ion transport in airways of cystic fibrosis (CF) patients is manifest as a reduced Cl– conductance and increased Na+ conductance. Isolation of the CF gene and identification of its product CFTR now allow investigations into the basic defect. Intrinsic to these investigations is the development of systems to study the function of CFTR and its relation to electrolyte transporters and their regulation.— Liedtke, C. M. Electrolyte transport in the epithelium of pulmonary segments of normal and cystic fibrosis lung. FASEB J. 6: 3076‐3084; 1992.

Chloride Channels in Cystic Fibrosis

Cystic fibrosis (CF), a genetic disease that affects 1 in 2000 Caucasian newborn babies, is often described as a generalized exocrinopathy with altered electrolyte and macromolecular secretion. The syndrome, characterized by chronic obstruction and infection of the respiratory tract, insufficiency of the exocrine pancreas, and elevated levels of sweat electrolytes, was first described in the 1930s,1 although references to signs and symptoms possibly related to CF have appeared as early as the mid-1600s. Thus some reports refer to cases of steatorrhea and pancreatic insufficiency, while folktales2 and European folk literature1,3–6 refer to the ominous significance and early death associated with salty sweat. An 1838 report of meconium ileus and its complications by Rokintansky3 was followed by more complete descriptions of newborns with symptoms typical of CF.7 Pancreatic insufficiency received attention before the turn of the century for its characteristic effects on growth8–10 and was attributed, in some reports, to an inborn error of metabolism.11