Ion channel regulation in small intestinal crypts

1.1. Background and objectives.

1.2. Ion transport across polarised epithelial cells.

1.3. Do distinct intestinal cell populations meditate electrolyte secretion and absorbtion.

1.4. Cl^- movements and their role in intestinal secretion.

1.5. The neurohormonal and endocrine control of small intestinal secretion.

1.6. Intracellular signals regulate small intestinal transport.

1.9. Experimental approaches.

2.1. Introduction.

2.2. Methods.

2.2.1 Animals.

2.2.2. Isolation of intact crypt epithelium.

2.2.3. Measurement of thymidine kinase specific activity as a marker for crypt enterocytes.

2.2.4. Measurement of alkaline phosphatase specific activity as a marker for villus enterocytes.

2.2.5. Estimation of protein concentrations.

2.2.6. Preparation of sections of guinea-pig small intestine.

2.2.7. Staining methods.

2.3. Results.

2.3.1. Biochemical characterisation of isolated epithelial cell populations.

2.3.2. Effects of isolation protocol upon structure of small intestinal mucosa.

2.3.3. Morphology and viability of the isolated crypts.

2.4. Discussion

3.1. Introduction.

3.2. Methods.

3.2.1. Isolation of viable crypts suitable for patch-clamp electrophysiology.

3.2.2. Solutions.

3.2.3. Perfusion arrangements for crypt electrophysiological recordings.

3.2.4. Electrophysiological recordings.

3.2.5. Data Acquisition and Analysis.

3.3. Results.

3.3.1. Effect of ion substitution experiments upon crypt membrane potential.

3.3.2. Whole-cell currents recorded from unstimulated small intestinal crypts.

3.3.3. Effect of K⁺ channel blockers upon the resting membrane potential of small intestinal crypts.

3.4. Discussion

4.1. Introduction.

4.2. Methods.

4.2.1. Crypt preparation.

4.2.2. Solutions.

4.3. Results.

4.3.1. Effects of carbachol upon crypt membrane potential and conductance.

4.3.2. Ca²⁺-dependence of the action of carbachol upon crypt membrane potential.

4.3.3. Effects of phorbol esters upon crypt membrane potential and conductance.

4.3.4. Action of vasoactive intestinal polypeptide upon crypt membrane potential and conductance.

4.3.5. Membrane conductance changes evoked by forskolin in small intestinal crypts.

4.3.6. Sensitivity of the actions of VIP and carbachol to ion channel inhibitors.

4.4. Discussion.

4.4.1. Overview.

4.4.2. Second messenger pathways mediating the response to carbachol.

4.4.3. Second messenger pathways mediating the response to VIP.

4.4.4. Evidence for the activation of K⁺ channels by VIP and CCH.

4.4.5. Evidence for the activation of a Cl^- conductance by VIP in the small intestinal crypt.

4.4.6. Evidence for the activation of a Cl^- conductance by PKC and Ca²⁺.

4.4.7. Evidence for the presence of a cAMP-activated Cl^- conductance in the small intestinal epithelium.

5.1. Introduction.

5.2. Methods.

5.2.1. Crypt preparation.

5.2.2. Solutions.

5.3. Results.

5.4. Discussion.

6.1. Introduction.

6.2. Methods.

6.2.1. Crypt preparation.

5.2.2. Solutions.

6.3. Results.

6.3.1. Variation in zero-current reversal potentials along the crypt axis.

6.3.2. Variation of membrane potential changes in response to carbachol and forskolin along the crypt axis.

6.4. Discussion.

6.4.1. Variation in the distribution of zero-current reversal potentials along the crypt axis.

6.4.2. Variation in the response to carbachol along the crypt axis.

6.4.3. Regional variations in the response to forskolin

7.1. Introduction.

7.2. Methods.

7.2.1. Solutions.

7.3. Results.

7.3.1. Determination of the accuracy of the volume assay.

7.3.2. Effect of hypotonicity on crypt volume.

7.3.3. Effect of increasing extracellular K⁺ concentration upon crypt RVD.

7.3.4. Effect of K⁺ and Cl^- channel blockers upon RVD.

7.3.6. Effects of cell swelling upon crypt membrane potential and conductance.

7.4. Discussion.

8.1. Introduction.

8.2. Methods.

8.2.1. Solutions.

8.2.2. Electrophysiological recordings.

8.2.3. Data Acquisition and Analysis.

8.3. Results.

8.3.1. Inwardly-rectifying single channel activity of the crypt basolateral membrane.

8.3.2. Cation non-selective single channel activity in the crypt basolateral membrane.

8.3.3. Effects of carbachol upon single channel activity in the crypt basolateral membrane.

8.3.4. Single channel recordings obtained with Na⁺ Gluconate rich pipette solutions.

8.4. Discussion.

8.4.1. Inwardly-rectifying single channel activity.

8.4.2. Cationic non-selective single channel activity.

8.4.3. Identification of the carbachol-activated K⁺ conductance.

9.1. Introduction.

9.2. Methods.

9.2.1. Solutions.

9.2.2. Electrophysiological recordings.

9.2.3. Data acquisition and analysis.

9.3. Results.

9.4. Discussion.

10.1. Discussion.

10.1.1. The evolution of small intestinal transport studies.

10.1.2. Regulation of cation-selective channels in the crypt basolateral membrane.

10.1.3. Regulation of Ca²⁺ mobilisation in the crypt enterocyte.

10.1.4. Membrane Cl^- conductance pathways of the intestinal epithelium and their central role in the pathophysiology of diarrhoea and cystic fibrosis.

10.1.5. Proliferation, differentiation and secretion in the small intestinal crypt.

10.1.6. A possible model for small intestinal secretion.

10.1.7. Conclusions.

10.2. Summary.