A phenomenologic investigation of pediatric residents' experiences being parented and giving parenting advice.

Factors surrounding pediatricians' parenting advice and training on parenting during residency have not been well studied. The primary purpose of this study was to examine pediatric residents' self-reported experiences giving parenting advice and explore the relationship between parenting advice given and types of parenting residents received as children. Thirteen OUHSC pediatric residents were individually interviewed to examine experiences being parented and giving parenting advice. Phenomenological methods were used to explicate themes and secondary analyses explored relationships of findings based upon Baumrind's parenting styles (authoritative, authoritarian, permissive). While childhood experiences were not specifically correlated to the parenting advice style of pediatric residents interviewed, virtually all reported relying upon childhood experiences to generate their advice. Those describing authoritative parents reported giving more authoritative advice while others reported more variable advice. Core interview themes related to residents' parenting advice included anxiety about not being a parent, varying advice based on families' needs, and emphasis of positive interactions and consistency. Themes related to how residents were parented included discipline being a learning process for their parents and recalling that their parents always had expectations, yet always loved them. Pediatric residents interviewed reported giving family centered parenting advice with elements of positive interactions and consistency, but interviews highlighted many areas of apprehension residents have around giving parenting advice. Our study suggests that pediatric residents may benefit from more general educational opportunities to develop the content of their parenting advice, including reflecting on any impact from their own upbringing.

Gene targeting of a CFTR allele in HT29 human epithelial cells.

HT29 cells endogenously express the cystic fibrosis transmembrane conductance regulator (CFTR) and have been used previously as a model to examine cellular regulation of CFTR expression and chloride secretory function. Homologous recombination has been used to specifically disrupt CFTR transcription in the HT29-18-C1 subclone. Experiments demonstrate successful disruption of a CFTR allele by DNA constructs, which target insertion of the neomycin phosphotransferase gene into CFTR exon 1 via homologous recombination. The mutation of one allele is a partial knockout because this cell line has multiple CFTR alleles. The mutation is confirmed by polymerase chain reaction (PCR) and genomic Southern blot analysis. A 52-68% reduction in CFTR mRNA levels is observed in the mutant cell line by both Northern and PCR analysis. However, Western blots show no decrease in total CFTR protein levels. Consistent with the lack of reduction in CFTR protein, the partial knockout mutant does not demonstrate alterations in cyclic AMP or calcium stimulation of chloride efflux or net osmolyte loss. Results suggest that posttranscriptional regulation of CFTR levels may contribute to maintenance of cellular chloride transport function.

Cellular chloride depletion inhibits cAMP-activated electrogenic chloride fluxes in HT29-18-C1 cells.

Cyclic AMP-activated chloride fluxes have been analyzed in HT29-18-C1 cells (a clonal cell line derived from a human colon carcinoma) using measurements of cell volume (electronic cell sizing), cell chloride content (chloride titrator) and intracellular chloride activity (6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ). HT29-18-C1 was shown to mediate polarized chloride transport. In unstimulated cells, the apical membrane was impermeable to chloride and net chloride flux was mediated by basolateral furosemide-sensitive transport. Forskolin (10 microM) increased furosemide-insensitive chloride permeability of the apical membrane, and decreased steady-state intracellular chloride concentration approximately 9%. Cellular chloride depletion (substitution of medium chloride by nitrate or gluconate), caused greater than fourfold reduction in cellular chloride concentration. When chloride-depleted cells were returned to normal medium, cells regained chloride and osmolytes via bumetanide-sensitive transport, but forskolin did not stimulate bumetanide-insensitive chloride uptake. The inhibition of cAMP-activated chloride reuptake was not explained by limiting cation conductance, cell shrinkage, choice of substitute anion, or decreased generation of cAMP in chloride-depleted cells. When cells with normal chloride content were depolarized (135 mM medium potassium + 10 microM valinomycin), cAMP activated electrogenic chloride uptake permselective for Cl- approximately Br- > NO3- > I-. The electrogenic transport pathway was inhibited in chloride-depleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.

Propionate activates multiple ion transport mechanisms in the HT29-18-C1 human colon cell line.

Short-chain fatty acids (SCFAs) are the major solutes and the major anions in the colonic lumen. We studied the response of suspended HT29-18-C1 cells (an epithelial cell line derived from a human colon carcinoma) to SCFA exposure. Cellular response was evaluated by measurement of cell volume (Coulter counter), intracellular pH [pHi; measured fluorometrically with 2',7'-bis(2-carboxyethyl)-5-(6)-carboxyfluorescein (BCECF)], and intracellular Na+, K+, and Cl- content (flame photometry and chloride titrator). Exposure to 130 mM propionate in isosmotic medium causes a rapid decrease in pHi and activates pHi recovery via amiloride-sensitive Na-H exchange. In the presence of propionate, Na-H exchange also causes cell swelling to a peak volume 11% above control cells and causes a 2.8-fold increase in intracellular Na+ content. After peak swelling, a regulatory-volume decrease (RVD) significantly reduced volume and intracellular Na+ returned to baseline. Other SCFAs (acetate, butyrate, and valerate) also elicit swelling and RVD. Activation of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) is required to return Na+ to normal levels and to indirectly provide ion gradients required for propionate-induced RVD, but Na(+)-K(+)-ATPase activity does not directly mediate RVD. When 1 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) is added in the presence of propionate, RVD was inhibited and cell Na+ content increased. Cl- depletion inhibited propionate-induced RVD and diminished the effect of SITS.

Regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gene transcription and alternative RNA splicing in a model of developing intestinal epithelium.

Transcriptional and post-transcriptional regulation of CFTR (cystic fibrosis transmembrane conductance regulator) gene expression was studied in HT29 cells. It is known that the abundance of CFTR mRNA increases during differentiation of pluripotent HT29-18 cells and is maintained at high levels in the stably differentiated HT29-18-C1 subclone. Nuclear run-on assays suggest that increased transcription of the CFTR gene explains the increased abundance of total CFTR mRNA in differentiated HT29 cells. The increased transcription cannot be ascribed to cell cycle-dependent expression of the CFTR gene or to changes in CFTR gene copy number between subcloned cells. Similar to native tissue cells, differentiated HT29 cells contain low copy numbers of CFTR transcripts (1-5/cell), and a portion of the CFTR transcripts are alternatively spliced to remove exon 9 (and make 9-mRNA). During differentiation of HT29-18 cells, the absolute amount of full-length CFTR mRNA increases 8-fold, whereas the amount of 9- mRNA increases 18-fold. The fraction of 9- mRNA in the CFTR mRNA pool is increased in differentiated HT29 cells. The results show that gene transcription regulates the abundance of CFTR transcripts and that regulatory control of alternative RNA splicing may also be a cellular mechanism to modulate CFTR function.

Assay of apical membrane enzymes based on fluorogenic substrates.

A series of enzymatic rate assays are described. The assays are based on coumarin derivatives that are fluorogenic substrates for the enzymes dipeptidase IV, aminopeptidase N, alkaline phosphatase, and gamma-glutamyltransferase. These simple assays are rapid and offer improved sensitivity over established colorimetric methods. The substrates have apparent affinities for the enzymes of 5-250 microM. L-Glutamic acid gamma-(7-amido-4-methylcoumarin) is characterized as a substrate of gamma-glutamyltransferase on the basis of inhibition of enzymatic cleavage when the glycylglycine acceptor molecule is omitted and inhibition of the enzymatic reaction by addition of glycine. Assay conditions for the four enzymes are established such that less than 0.6% of the substrate is consumed, fluorescence is proportional to enzymatic product, and results may be directly compared to established colorimetric assays. Intestinal epithelial cells are used both to establish appropriate assay conditions and to demonstrate the utility of the assays.