QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Marshall, W. S. Articles by Lynch, E. M. Search for Related Content PubMed PubMed Citation Articles by Marshall, W. S. Articles by Lynch, E. M.

NaCl and fluid secretion by the intestine of the teleost Fundulus heteroclitus: involvement of CFTR

W. S. Marshall^*, J. A. Howard, R. R. F. Cozzi and E. M. Lynch

Department of Biology, St Francis Xavier University, Antigonish, Nova Scotia, Canada B2G 2W5

View larger version (23K): [in a new window]   Fig. 1. Gravimetric measurement of fluid flow (Jv) across Fundulus heteroclitus intestine for time controls (treated with drug vehicle alone) and drug-treated preparations (B) (test; 0.1 mmol l–1 IBMX + 0.5 mmol l–1 db-cAMP + 1 µmol l–1 ionomycin, added at 60 min; arrow in A) from animals before (unfed) or after (fed) daily feeding. Results for a typical experiment appear in A and include control (open symbols) and test (filled symbols) membranes from fed animals. The lines in A are least-squares linear regressions of mass on time. The combined treatment reversed the normal fluid absorption to net fluid secretion in fed animals (P<0.01, Bonferroni post-hoc test following one-way analysis of variance, P=0.005, N=8). In the unfed animals, there was a non-significant trend towards secretion (P>0.05). Values are means + S.E.M., N=8.

View larger version (23K): [in a new window]   Fig. 2. Effects of treatment with 0.1 mmol l–1 IBMX + 0.5 mmol l–1 db-cAMP + 1 µmol l–1 ionomycin on tissue conductance (Gt), unidirectional Cl– fluxes (JCl) and short-circuit current (Isc). (A) Treatment (at time zero) significantly increased tissue Gt and Isc. The effect was reversed by mucosal application of the anion channel blocker diphenylamine-2-carboxylate (DPC; 1 mmol l–1). (B) Drug treatment significantly increased both unidirectional Cl– fluxes and reversed ion uptake to net Cl– secretion (P<0.005, paired t-test compared with control period, N=7). Jsm, serosal-to-mucosal Cl– flux; Jms, mucosal-to-serosal Cl– flux. Apical DPC significantly decreased serosal-to-mucosal unidirectional flux (P<0.02, paired t-test compared with previous period, N=7) and restored Cl– uptake to control levels. (C) Similar post treatment (i.e. after db-cAMP + IBMX + ionomycin) with 100 µmol l–1 DIDS was without effect on the stimulated Isc and Gt. Subscripts ser and muc indicate application of drug to the serosal and mucosal sides, respectively. Values are means ± S.E.M.

View larger version (24K): [in a new window]   Fig. 3. Effects of 0.1 mmol l–1 IBMX + 0.5 mmol l–1 db-cAMP (no ionomycin) on short-circuit current (Isc) and tissue conductance (Gt) (A) and serosal-to-mucosal (Jsm) and mucosal-to-serosal (Jms) Cl– fluxes. Values are means ± S.E.M. Hr1, hour 1; Hr2, hour 2. Other details as in Fig. 2. Jnet, net Cl– flux.

View larger version (27K): [in a new window]   Fig. 4. (A) Effects of 1 µmol l–1 ionomycin alone on short-circuit current (Isc) and tissue conductance (Gt). When post-treated with 0.1 mmol l–1 IBMX + 0.5 mmol l–1 db-cAMP, Gt and Isc increased markedly. (B) Effects of 1 µmol l–1 ionomycin alone and subsequent addition of 0.1 mmol l–1 IBMX + 0.5 mmol l–1 db-cAMP on unidirectional Cl– fluxes (Jsm, serosal-to-mucosal flux; Jms, mucosal-to-serosal flux) and net Cl– secretion (Jnet). Hr1, hour 1; Hr2, hour 2. Values are means ± S.E.M. Other details as in Fig. 2.

View larger version (9K): [in a new window]   Fig. 5. There was no apparent difference between the posterior and anterior portions of intestine in the basal or stimulated (ionomycin+db-cAMP+IBMX) short-circuit current (Isc), suggesting that the two sections of the intestine responded in a similar manner to stimulation. Values are means ± S.E.M., N=7.

View larger version (14K): [in a new window]   Fig. 6. Plot of spontaneous variation in tissue conductance (Gt) versus unidirectional Cl– flux (JCl). There was a linear relationship with r2=0.6692, d.f.=54, a slope of 0.345±0.132 µequiv mS–1 h–1 and a y-intercept of –0.91±1.94 µequiv cm–2 h–1 (mean ± 95 % confidence limit) that was not significantly different from zero; hence, all Cl– flux would appear to be conductive. The solid line is the regression equation and the dotted lines are the 95 % confidence limits.

View larger version (157K): [in a new window]   Fig. 7. Confocal laser scanning images of killifish cystic fibrosis transmembrane conductance regulator (kfCFTR) immunofluorescence of mouse anti-human CFTR and goat anti-mouse IgG Oregon Green 488 in seawater-adapted killifish posterior intestine showing the distribution of kfCFTR (green fluorescence) in the enterocytes of an intestinal villus. (A) Red and green fluorescence overlaid. In approximately 80 % of sections, kfCFTR immunofluorescence is evenly distributed throughout enterocytes but is absent from goblet cells (asterisk) and present at low levels only over enterocyte nuclei (white arrow). Brush-border microvilli stain (non-specifically) with Mitotracker Red. (B) Same image as A but with bright field overlaid with green fluorescence. (C) In a minority (approximately 20 %) of sections, kfCFTR immunofluorescence was also present in the apical membrane (area between arrows). (D) Same image as C but with bright field overlaid with green fluorescence. Scale bars, 20 µm.

View larger version (11K): [in a new window]   Fig. 8. Western blot analysis of cystic fibrosis transmembrane conductance regulator (CFTR) in intestine and gill tissue from seawater-adapted killifish (20 µg total protein per lane). Proteins were separated on a 7 % polyacrylamide gel, transferred onto an Immobilon-P membrane and probed with anti-human CFTR monoclonal antibody (1 µg ml–1). Proteins were visualized following incubation in BCIP/NBT Blue substrate development solution. Lane A, heart tissue (negative control); lane B, gill tissue; lane C, posterior intestine. In gill and intestine, the CFTR antibody cross-reacts with a 175 kDa protein and two lighter bands at 90.3 and 94.2 kDa (N=3). Molecular mass markers (kDa) (on left): 205, 116, 97.4, 84, 66, 55, 45 and 33 (from top to bottom).

View larger version (99K): [in a new window]   Fig. 9. (A,B) Immunofluorescence of mouse anti-human Na+/K+/2Cl– cotransporter (NKCC) and goat anti-mouse IgG Oregon Green 488 in seawater-adapted killifish posterior intestine shows the distribution of Na+/K+/2Cl– cotransporter (green fluorescence) in an intestinal villus. (A) In approximately 20 % of sections, NKCC immunofluorescence is evenly distributed throughout the enterocyte cytoplasm but is absent from the brush border and nuclei (white arrow) and from goblet cells (asterisk). In some cells, NKCC immunofluorescence is seen in the brush border (broad yellow arrow). (B) Same section, bright field overlaid with green fluorescence. (C) NKCC immunofluorescence in most sections (approximately 80 %) is present in the cytoplasm and in the brush border (yellow arrow) and is absent from nuclei (white arrow) and goblet cells (asterisk). (D) Same section but with bright field overlaid with green fluorescence. (E,F) Immunofluorescence of mouse anti-chicken Na+/K+-ATPase and goat anti-mouse IgG Oregon Green 488 in seawater-adapted killifish posterior intestine showing the distribution of Na+/K+-ATPase (green fluorescence) in an intestinal villus. (E) Na+/K+-ATPase immunofluorescence is distributed throughout the basal two-thirds of the enterocytes but not in the nuclei (white arrow), the goblet cells (asterisk) or the brush border, which stains (non-specifically) with Mitotracker Red. (F) Na+/K+-ATPase immunofluorescence overlaid with bright field. Scale bars, 20 µm.

View larger version (112K): [in a new window]   Fig. 10. Control images of seawater-adapted killifish posterior intestine, where the primary antibody (to killifish cystic fibrosis transmembrane conductance regulator, kfCFTR, in this case) was omitted, showing the absence of the secondary antibody goat anti-mouse IgG Oregon Green 488 signal (green fluorescence). Scale bar, 20 µm. (A) Negative control of kfCFTR. Note the sole presence of the Mitotracker Red stain in the enterocytes and its absence from the goblet cells (asterisk). (B) Green channel fluorescence overlaid with bright field has no detectable green signal from the secondary antibody’s fluorescence, indicating a lack of non-specific binding of the second antibody and of ‘bleed-through’ from the red channel.

View larger version (26K): [in a new window]   Fig. 11. Model of transporter distribution in enterocytes of killifish intestinal epithelium, showing brush border (BB) on the luminal side and cells overlying the basement membrane (BM) on the serosal side. In the model, all cells have Na+/K+-ATPase only in the basolateral membrane. The cell on the left is modelled for uptake of ions and fluid, while that on the right is modelled for secretion. The Na+/K+/2Cl– cotransporter (NKCC) is present in the basal portion of all enterocytes and, in most cells (left), also in the apical brush-border membrane. Cystic fibrosis transmembrane conductance regulator (CFTR) immunofluorescence is present in the basal portion of most cells (left) and in the apical membrane of some enterocytes (right).