Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation, DeltaF508, results in the production of a misfolded CFTR protein that is retained in the endoplasmic reticulum and targeted for degradation. Curcumin is a nontoxic Ca-adenosine triphosphatase pump inhibitor that can be administered to humans safely. Oral administration of curcumin to homozygous DeltaF508 CFTR mice in doses comparable, on a weight-per-weight basis, to those well tolerated by humans corrected these animals' characteristic nasal potential difference defect. These effects were not observed in mice homozygous for a complete knockout of the CFTR gene. Curcumin also induced the functional appearance of DeltaF508 CFTR protein in the plasma membranes of transfected baby hamster kidney cells. Thus, curcumin treatment may be able to correct defects associated with the homozygous expression of DeltaF508 CFTR.

Biodegradable polymer nanoparticles (NPs) are a promising approach for intracellular delivery of drugs, proteins, and nucleic acids, but little is known about their intracellular fate, particularly in epithelial cells, which represent a major target. Rhodamine-loaded PLGA (polylactic co-glycolic acid) NPs were used to explore particle uptake and intracellular fate in three different epithelial cell lines modeling the respiratory airway (HBE), gut (Caco-2), and renal proximal tubule (OK). To track intracellular fate, immunofluorescence techniques and confocal microscopy were used to demonstrate colocalization of NPs with specific organelles: early endosomes, late endosomes, lysosomes, endoplasmic reticulum (ER), and Golgi apparatus. Confocal analysis demonstrated that NPs are capable of entering cells of all three types of epithelium. NPs appear to colocalize with the early endosomes at short times after exposure (~2 hr), but are also found in other compartments within the cytoplasm, notably Golgi and, possibly, ER, as time progressed over the period of 4 to 24 hrs. The rate and extent of uptake differed among these cell lines: at a fixed particle/cell ratio, cellular uptake was most abundant in OK cells and least abundant in Caco-2 cells. We present a model for the intracellular fate of particles that is consistent with our experimental data.

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Active K absorption in the rat distal colon is energized by an apical membrane H-K-ATPase, whereas K absorption in the distal collecting duct is generally believed to be modulated by a related renal H-K-ATPase. Experiments were performed to establish the mechanism(s) by which dietary Na depletion (with resulting elevated aldosterone levels) and K depletion stimulate K absorption. A colonic H-K-ATPase-specific cDNA probe and a polyclonal antibody were utilized to measure mRNA (Northern blot analyses) and protein (Western blot and immunofluorescence studies) abundance in the distal and proximal colon and renal collecting ducts and cortex of dietary Na- and K-depleted rats. Dietary Na depletion, but not K depletion, upregulated H-K-ATPase-specific mRNA and protein expression in the distal and proximal colon; Na depletion also stimulated H-K-ATPase activity in the distal colon. In contrast to the distal colon, H-K-ATPase-specific protein level in the outer medulla was enhanced by dietary K depletion, but not by Na depletion. This study establishes that 1) dietary Na depletion stimulates colonic H-K-ATPase activity most likely by a transcriptional process and 2) the regulation of colonic H-K-ATPase expression by dietary Na depletion and dietary K depletion is not identical in the large intestine and differs in the kidney from the colon, suggesting the presence of two (or more) H-K-ATPase isoforms in the rat colon.

Mutations in PKD1 and PKD2, the genes that encode polycystin-1 and polycystin-2 respectively, account for almost all cases of autosomal dominant polycystic kidney disease. Although the polycystins are believed to interact in vivo, the two proteins often display dissimilar patterns and gradients of expression during development. In an effort to understand this apparent discrepancy, we investigated how changes in polycystin-2 expression can affect the subcellular localization of polycystin-1. We show that, when polycystin-1 is expressed alone in a PKD2 null cell line, it localizes to the cell surface, as well as to the endoplasmic reticulum. When co-expressed with polycystin-2, however, polycystin-1 is not seen at the cell surface and co-localizes completely with polycystin-2 in the endoplasmic reticulum. The localization of a polycystin-1 fusion protein was similarly affected by changes in its level of expression relative to that of polycystin-2. This phenomenon was observed in populations as well as in individual COS-7 cells. Our data suggest that the localization of polycystin-1 can be regulated via the relative expression level of polycystin-2 in mammalian cells. This mechanism may help to explain the divergent patterns and levels of expression observed for the polycystins, and may provide clues as to how the function of these two proteins are regulated during development.

A putative cDNA for the colonic K-ATPase has recently been cloned (Crowson, M. S., and G. E. Shull. 1992. J. Biol. Chem. 267:13740-13748). Considerable evidence exists that there are two K-ATPases and active K absorptive processes in the rat distal colon: one that is ouabain sensitive and the other ouabain insensitive. The present study used the baculovirus expression system to express K-ATPase activity in insect Spodoptera frugiperda (Sf 9) cells and a polyclonal antibody (M-1), developed against a fusion protein produced from the 327 nucleotide fragment from 5' coding region of the putative K-ATPase cDNA, to identify the specific localization of the K-ATPase protein. K-ATPase activity (28.7+1.2 nmol inorganic phosphate/mg protein min) was expressed in plasma membranes isolated from Sf 9 cells infected with baculovirus containing recombinant DNA with the putative K-ATPase cDNA. Km for K for the K-ATPase was 1.2 mM. The expressed K-ATPase activity was not inhibited by ouabain (1 mM); while the K; for vanadate inhibition was 8.3 ,uM. Western blot analysis with the M-1 antibody identified a 100-kD protein in apical membranes prepared from distal, but not proximal, rat colon. Immunohistochemical studies with M-1 antibody localized K-ATPase only in the apical membrane of surface cells, while an mAb (c464.6) against Na,K-ATPase localized basolateral membranes of both surface and crypt cells of rat distal colon. In conclusion, the putative K-ATPase cDNA encodes an ouabain-insensitive K-ATPase that is present only in the apical membrane of surface cells of rat distal colon. (J. Clin.

The localization of polycystin (PC)1) to the plasma membrane requires coexpression with PC2 and cleavage at the PC1 G protein-coupled receptor proteolytic site. Neither the PC1 binding capacity of PC2 nor its channel function is required for this effect.