ABBREVIATIONS.

The following abbreviations were used throughout:

SUMMARY OF METHODS.

The sequential isolation of crypt epithelial units from the guinea-pig jejunum.

2.2. METHODS.

Adult male guinea pigs (Duncan Hartley strain) were purchased from Tuck and Son, Battlesbridge, Essex, U.K. and were maintained at 18-21° C and fed ad libitum with a dietary supplementation of vitamin C (Roche). Guinea pigs with a body weight of between 250-350g were sacrificed by cervical dislocation starvation for 24 hours.

Jejunal enterocytes were isolated by a method similar to that previously described by Bjerkness and Cheng for the isolation of murine enterocytes (1981, Anat. Rec. 199, pp 565-574). A 25-30 cm length of jejunum was excised from a cervically located guinea-pig and washed through twice with 20 ml of ice-cold Hanks solution (of composition 140 mM NaCl; 5 mM KCl; 10 mM HEPES; 1.3 mM CaCl₂; 0.5 mM MgCl₂; 0.36 mM K₂HPO₄; 0.44 mM KH₂PO₄; 5.5 mM D-glucose; 4.2 mM NaHCO₃ pH 7.2 with 2.5M Tris), before being everted on and ligated to a 5 mm diameter perspex rod. The preparation was then attached to a Vibromixer (Chemap AG, Switzerland) and operated at 53 Hz in a Ca²⁺-free isolation buffer pre-equilibrated at 4^oC which contained DL-dithiothreitol (DTT) to disaggregate mucus (of composition 30 mM Na₂ EDTA; 10 mM HEPES; 5 mM KCl; 60 mM HCl; 52 mM NaCl; 2 mM DTT; pH 7.1 with 2.5M Tris) for timed periods of 4, 3, 3, 6 and 6 minutes respectively.

Successive fractions were centrifuged at 100 x g for 1 minute in a Super Minor MSE centrifuge before resuspension in Hanks medium at 4^oC to remove the chelating buffer. The fractions were then centrifuged at 100 x g, aspirated and resuspended in 1 ml of Dulbecco's Modified Eagles Medium (DMEM, low glucose, pH 7.4 + 0.1% BSA). The cells were maintained on ice and used within 6 hours. Viability was assessed by the exclusion of 0.03% trypan blue and 5 m g/ml propidium iodide when incubated at room temperature, and by their birefringence when viewed by phase contrast microscopy.

Cellular material from successive fractions isolated as described in 2.2.2. was resuspended in Tris-sucrose buffer to a final volume of 6 ml [0.25 M sucrose; 4 mM MgCl₂; 0.02 M Trisma base pH 7.4 with 1M HCl] and homogenised at 4° C with an Ultraturrax homogeniser (Jankel & Kunkel, Germany) at 2,500 rpm. The surface layer of lipid was removed by Pasteur pipette from the resultant crude enzyme homogenate. A 1ml aliquot from each homogenate was spun at 10,000 x g at 4^oC for 2 min and 0.7 ml of the resulting supernatant was subsequently taken for enzyme assay (Klemperer, Biochem.J. 108: 541-546). The reaction mixture contained 10 m M thymidine (1.96 m Ci/ml ³H thymidine); 5 mM ATP; 2.5 mM MgCl₂ and 0.05 M Tris-HCl, pH 8.0. Assays were started by the addition of an aliquot of enzyme extract to the above medium (0.1 ml/ml reaction mixture) at 37^oC. The reaction was quenched after 30 min by placing assay tubes in a 100^oC water bath for 2 mins. The tubes were then put on ice to cool. Denatured protein was removed by centrifugation (2,000 x g for 10 min). Phosphorylated thymidine was isolated from the "de-proteinised" reaction mixture by binding to Dowex 1x1-100 resin (Gonzalez et al. Anal.Biochem. 114: pp 285-287). Unbound labelled thymidine was removed by repeated washing with 1mM HCl. Bound phosphorylated thymidine was released from the resin with 1M HCl and then counted in a liquid scintillation counter together with appropriate standards. Specific activities were calculated as pmole of [³H] thymidine-P per mg protein per minute.

Incubations were performed as originally described by Weiser et al (J.Biol.Chem. 248: pp 2536-2541, 1973). Incubations were performed using an assay buffer containing 5 mM MgCl₂ and 50 mM Tris pH 10.1 with 0.1 mM para-nitro-phenyl phosphate (PNPP) as substrate. The reaction was started by the addition of crude homogenate (10-100m l/ml reaction mixture) to pre-equilibrated assay buffer and incubated for six minutes at 37° C. Sample assays were performed in duplicate. The reaction was quenched by the addition of 40% w/v trichloroacetic acid, followed by centrifugation at 10,000 x g for 2 min to precipitate any flocculated protein. One ml of supernatant was then mixed with 2.0 ml of 0.4 M NaOH and absorbance readings were taken at 410 nm and related to a 4-nitrophenol standard curve, with PNPP as a blank. Specific activities were calculated as m mole of PNPP hydrolysed per mg of homogenate protein per minute.

Protein concentrations were measured by the bicinchoninic acid method (Pierce commercial kit) and related to a BSA protein standard curve.

Characterisation of a basolateral K⁺ conductance present in isolated jejunal crypts.

The initial stage of preparation of intact crypts was performed as described in 2.2.2., paragraph 1. The fractions shed by mechanical vibration were from timed intervals of 10, 6, 2, 2 and 2 minutes respectively. The latter fractions from minutes 17-22 were centrifuged at 50 x g for 1 minute in a Super Minor MSE centrifuge and resuspended in Hanks medium at 4^oC to wash out the remaining chelating buffer. The fractions were then centrifuged at 50 x g, aspirated and pooled together by resuspension in 25ml of DMEM (pH 7.4 with 0.1M NaOH) containing 2mM DTT at 4° C. The crypt suspension was then placed on a rotary inversion mixer for 20 minutes. The suspension was then spun at 50 x g for 2 minutes and aspirated, before final resuspension in 2ml of ice-cold DMEM. The cells were maintained on ice and used within 6 hours. The crypts were easily identified under phase contrast microscopy by their birefringence and cylindrical morphology and appeared viable as judged by their capacity to exclude 0.03% trypan blue.

The composition of the intra- and extracellular solutions is given in table 3.1. Patch pipettes were filled with an artificial K⁺-rich "intracellular" solution containing 100 m g/ml nystatin. Nystatin-containing pipette solutions were prepared freshly every 1-2 hours (from a frozen stock (10 mg/ml) of nystatin in dimethylsulphoxide (DMSO)) by adding 20m l of nystatin stock to 1ml of pipette solution in an eppendorf, taking precautions to avoid light sensitive reactions. The eppendorf was sonicated for 2 minutes and the pipette solution was then filtered through a Dynaguard (0.2 m m) ME syringe filter (Microgon inc., U.S.A.) into a 5ml beaker kept on ice, shielded from the light with foil. In some experiments pipette Ca²⁺ was buffered to a quasiphysiological intracellular level [93 nM] with EGTA. Neither the spontaneous "zero-current" reversal potential (E_m) nor the action of carbachol upon E_m was affected by this manoeuvre, which can be explained by the impermeability of nystatin to divalent cations (Horn and Marty, 1988). The tip of the pipette was filled by capillary action with nystatin-free pipette solution before the pipette was back-filled with the nystatin-containing solution. The concentrations of Ca²⁺ [2 mM] and EGTA [5 mM] required were calculated according to the stability constants of Martell & Smith (Vol.1 Amino acids. New York: Plenum Press, 1974). The composition of the pipette solution is as given in table 3.1.

Solutions for ion substitution experiments were prepared as described in table 3.1.. The low Na⁺ solution was prepared by isomolar replacement of NaCl by N-methyl-D-glucamine Cl and the low Cl^- solution by an equivalent replacement with Na gluconate. Changes in [K⁺]_o were achieved by isomolar changes with NaCl.

A stock solution of carbachol (100mM) was prepared in distilled water and stored frozen. Atropine was prepared freshly by dissolving in Hanks medium to a final concentration of 1mM, taking precautions to avoid light-sensitive reactions. Final concentrations of antagonist and agonist were achieved by appropriate dilutions with normal Hanks medium. All bath and pipette solutions were filtered through Dynaguard 0.2m m syringe filters prior to use.

A suspension of crypt enterocytes was aliquoted onto type 0 glass 10mm diameter cover slips (Chance Propper) precoated with 0.01% polyethylene imine supported on 2.5cm glass micro-fibre filters (Whatman Ltd., England), placed inside a 135mm petri dish. The crypts were left to equilibrate for 10 minutes at room temperature before being transferred into a purpose-built chamber containing Hanks medium, the bottom comprising of a type 0 glass cover slip. The chamber was mounted on the stage of an inverted microscope. Rapid solution changes were effected by directing a small jet of the desired solution directly to the crypt under study, without substantially changing the composition of the bulk solution, by means of a device similar to that described by Suzuki et al (1990). The bulk solution was constantly removed with a peristaltic pump and replenished by Hanks medium flowing under gravity. All experiments were conducted at room temperature » 25° C.

Patch-pipettes were fabricated from Boralex glass capillaries of outside diameter 1.7mm (Rochester Scientific Co., New York, U.S.A.) using a two-stage vertical pipette puller (PP-83, Narishige, Japan). The tips of pipettes were polished using a microforge before back-filling with "intracellular" solution. Patch pipettes used for current-clamp recordings had resistances of 4-5 MW when filled with KCl-rich solutions.

"Zero-current" reversal potentials and whole-cell currents were recorded according to the method of Hamill et al. (1981) with a List EPC-7 amplifier (List Electromedical, Germany) using the perforated-patch method as previously outlined by Horn & Marty (1988). Isolated crypts attached to 10 mm diameter type O glass cover-slips (Chance-Propper) were mounted in a specifically designed perfusion-chamber, as described above, and the crypts were viewed on the stage of an inverted microscope (Zeiss IM 35, Germany) at a total magnification of x320. A smooth basolateral membrane surface was approached with the patch-pipette without applying positive pressure in the pipette using a hydraulic micromanipulator (Narishige MO 203, Japan). Giga-ohm seals were obtained by applying light-suction to polished patch-pipettes gently pressed against the crypt membrane. A train of small voltage-pulses (0.1-10 mV) was applied to observe the progression of seal formation by monitoring the size of the current-response. Access resistances were estimated in the range of 20-50 MW within 2-5 minutes of seal formation. For whole-cell recordings the series capacitance and series conductance were then adjusted to compensate for the whole-cell capacitative current. Access resistances were monitored and progressively compensated throughout the recordings. Cells were voltage clamped at a holding potential (typically -40 mV) and membrane currents were recorded in response to depolarising and hyperpolarising voltage steps.

In current-clamp recordings the zero-reversal potential (E_m) of the crypt was continuously monitored after clamping the current at 0 mV and recorded after E_m had stabilised at a potential, usually greater than -40 mV. Membrane potential changes evoked in response to ion substitutions and the addition of agonists and blockers to the bathing medium were recorded, with or without the application of fixed amplitude current pulse trains to monitor changes in membrane conductance.

The established sign convention was used throughout and potentials reported with respect to the patch-pipette. The bath electrode consisted of an Ag-AgCl pellet. Junction potentials evoked following bath solution changes were determined and measurements obtained from recordings were corrected for the measured offsets.

An IBM-AT microcomputer equipped with a Lab-PC interface and software programmes (VGEN and VCAN, J. Dempster, Dept. of Physiology and Pharmacology, University of Strathclyde, Glasgow) were used for data acquisition and the analysis of current-clamp and voltage-clamp recordings respectively. Voltage pulse protocols and current pulse trains were applied to the stimulus input of the List EPC-7 following digital-to-analogue conversion using a Lab-PC laboratory interface (National Instruments, U.S.A.). The signal from the patch-clamp amplifier was simultaneously viewed on a storage oscilloscope and recorded on videotape for subsequent analysis together with triggering pulses using an adapted pulse-code modulation encoder (Sony PCM-701ES, Lamb 1985, J.Neurosci.Methods).

Replayed records of whole-cell currents were filtered at 2.5kHz (-3db) using a variable 8-pole Bessel filter (Barr & Stroud) and then digitised using a PC-Lab laboratory interface. Current-clamp recordings were acquired at a frequency of 4 Hz and whole-cell recordings at 2-5kHz. Results are expressed as means ± standard errors of n observations.

Effects of VIP and carbachol upon crypt membrane potential and cell volume.

4.2. METHODS.

Electrophysiological recordings and data acquisition and analysis were performed as described in sections 3.2.3. and 3.2.4. respectively.

4.2.1. Crypt preparation.

For electrophysiological experiments the crypt enterocytes were prepared by the method described in section 3.2.1.. The preparation of crypt enterocytes for crypt volume measurements differed in that the isolated crypts were not resuspended in DMEM containing 2mM DTT, but in normal Hanks medium at 4° C.

4.2.2. Solutions.

The composition of the pipette and extracellular solutions used is given in table 4.1.

Patch pipettes were filled with KCl-rich "intracellular" solution containing 100 m g/ml nystatin prepared as described in 3.2.2.. In experiments conducted with "low" Cl^- (60mM) in the pipette solution, KCl was replaced with K gluconate. Hypotonic medium was prepared as the isotonic medium, omitting mannitol. Osmolarities were measured as 298 ± 5 and 225 ± 8 mosmoles for the isotonic and hypotonic solutions respectively using a freezing point depression osmometer (3MO Advanced Instruments, Massachussetts, U.S.A.).

Stock solutions of carbachol (100mM) and porcine vasoactive polypeptide (VIP, 100m M) were prepared in distilled water and stored frozen. Stock solutions of the Cl^- channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 9-anthracene carboxylic acid (9-AC) were prepared in DMSO at 100mM concentration and stored frozen. Forskolin was prepared as a 20mM stock frozen in DMSO. Final concentrations were achieved by appropriate dilutions with normal Hanks medium. DMSO (final concentration 0.1% v/v) was without effect on cell volume or membrane potential. Precautions against light-sensitive reactions were adopted when using Cl^- channel blockers.

Solution changes and perfusion arrangements during electrophysiological recordings were made as described in section 4.2.2.. Changes in the bathing solution during experiments to measure cell volume were made by the successive addition of two 5ml aliquots of the replacement solution to a chamber containing approximately 1ml of bathing medium, removing excess solution by means of a vacuum pump. All errors quoted are standard errors of the mean of n observations.

An individual isolated crypt was selected by its cylindrical morphology and birefringence when observed under phase contrast optics with an inverted microscope (Wild, Germany, total magnification 320x). The crypts were aliquoted into a perspex chamber (total volume of 1ml), the bottom of which was made from a glass coverslip. Adherence of the crypts was achieved by precoating the glass surface with 0.1% PEI for 4 hours. Crypts were allowed to attach by pre-equilibration for 15 min in Hanks medium and the chamber was subsequently washed with 10 ml of Hanks medium to remove loosely attached cellular material. Photomicrographs of the selected crypt were taken at timed intervals before and after solution changes were effected. Zero time was defined as the time of solution change. Photomicrographs of the crypts were analysed for total area, mean height (h) and mean width (2r) with the aid of a Quantimet 520 image analysis system (Cambridge Instruments, U.K.). Volume was estimated, assuming a cylindrical morphology, as p .h.r², and normalised to the point previous to solution changes. All crypt volume measurement experiments were carried out at 25° C.

High K⁺ crypt isolation method.

Electrophysiological recordings and data acquisition and analysis were performed as described in sections 3.2.3. and 3.2.4. respectively.

Crypt enterocytes were isolated as outlined in 3.2.1. with the following modifications. The composition of the isolation buffer was changed by reducing the Na⁺ and Cl^- concentrations and increasing the K⁺ concentration (see table 5.1. for composition). The enterocytes were sequentially shed by vibration over successive intervals of 10, 10, 3, 3, 3 and 3 minutes. The fractions collected from minutes 24 to 32 were each centrifuged at 100 x g for 1 minute, before being resuspended and pooled in 20mM K⁺ Hanks solution (see table 5.1). The crypts were then centrifuged for 2 minutes at 50 xg and then resuspended in 20mM K⁺ Hanks medium (see table 5.1.) containing 4mM DTT. The crypt suspension was then placed in a rotary inversion mixer for 15 minutes at 4^oC. The crypts were finally centrifuged as before and resuspended in 2ml of 20mM K⁺ Hanks medium.

Bath and pipette solutions were prepared as described previously (see 3.2.2. and 4.2.2.). Stock solutions of quinine and forskolin were prepared in DMSO at 10mM and 20mM respectively and were stored frozen. Solutions containing carbachol were prepared as described in section 3.2.2..

Mechanism of RVI.

Crypts were isolated as described previously in 5.2.1. for electrophysiological experiments and 4.2.1. for cell volume measurements. Electrophysiological recordings and data acquisition and analysis were performed as described in sections 3.2.3. and 3.2.4. respectively.

6.2.1. Solutions.

The composition of all solutions used is given in Table 6.1.. The tonicity of the standard isotonic medium was 298 ± 5 mosmole/l and was increased to 400 ± 5 mosmole/l when required by addition of 100 mM D-mannitol, or to 499 ± 8 mosmole/l by adding either 100 mM NaCl or 200 mM D-mannitol (means ± SD). Osmolality of all solutions was measured with a freezing point depression osmometer (3MO Advanced Instruments, Massachussetts, USA). The low Na⁺ solution was prepared by isomolar replacement of NaCl by N-methyl-D-glucamine Cl and the low Cl^- solution by an equivalent replacement with Na gluconate. The inhibitors ethylisopropyl amiloride, amiloride and bumetanide were prepared as stock solutions in DMSO and frozen. Final concentrations were prepared by appropriate dilutions with the appropriate bathing medium so that the final concentration of DMSO was £ 0.1% (v/v). Experiments using bumetanide were performed so as to minimise light sensitive reactions. All errors quoted are standard errors of the mean of n observations.

Experiments to measure cell volume were performed as described in 4.2.3. except that all experiments were conducted in a room maintained at 30° C. All solutions and the chambers containing crypts were pre-equilibrated at 30° C for 15 minutes before the start of the experiment.

Measurements of cAMP and cGMP levels in the isolated crypt.

Crypts were isolated by the protocol described in 5.2.1. with the following modifications. Four three minute fractions from minutes 20 to 32 from the commencement of the vibration were centrifuged for 1 minute at 50 x g and were then resuspended in 20ml of Hanks medium. The resulting suspensions were then filtered through a 140m m nylon mesh (Henry Simon Ltd., U.K.) into a beaker to remove contaminating villus caps. The filtrates from each fraction were then pelleted at 50 x g and pooled together in the required volume of Hanks medium. The isolated crypt fraction was stored on ice for up to 30 minutes before the start of the experiment.

Agonists were prepared as stock solutions in distilled water and kept frozen. ANP, VIP, STa, histamine and acetylcholine stocks were prepared in distilled water. The forskolin stock was prepared as described in 5.2.2.. The assay solutions containing agonists were prepared at twice the final assay concentration by the appropriate dilution with Hanks medium.

The crypts were resuspended homogeneously in Hanks medium by gentle swirling and 150m l aliquots were pipetted into 15ml polystyrene tubes (Falcon, Becton Dickinson, U.S.A.). Two 150m l aliquots were frozen for the subsequent estimation of protein concentrations according to the Lowry method (J.Biol.Chem., 193, pp. 265-275 (1951)). The crypt aliquots and Hanks medium (with and without the appropriate agonist) were then pre-equilibrated for 15 minutes at 30° C. From zero time 150m l aliquots of Hanks medium containing the appropriate agonist (at twice the final concentration) were added to successive crypt fractions at 5 second intervals, each incubation with agonist being completed in triplicate. After 5 minutes the reactions were terminated at 5s intervals by the addition of 550m l aliquots of ice-cold ethanol (final concentration 65% v/v) and the resulting suspension was kept on ice and left for 5 minutes to settle. The assay tubes were centrifuged for 15 minutes at 4° C in a Mistral 3000 MSE centrifuge and the supernatant decanted into 15 ml polypropylene tubes (Falcon, Becton Dickinson, U.S.A.). The supernatants were then evaporated for 3 hours at 90° C in a vacuum oven (760mm Hg). The tubes were then stored in a desiccator containing dehydrated silica gel for up to 64 hours prior to measurement of cGMP/ cAMP levels by radioimmunoassay.

Measurement of cellular cAMP and cGMP levels were performed using commercial kits (RPA 509 and RPA 525, Amersham International plc, Amersham, U.K.). The assay buffer was reconstituted with distilled water [final composition 0.05 M acetate buffer with 0.01% w/v sodium azide (pH 5.8)]. The radiolabelled tracer guanosine 3',5'-cyclic phosphoric acid 2'-O-succinyl-3-[¹²⁵I] iodotyrosine methyl ester was reconstituted in assay buffer with a final activity of 3.7kBq/ml. The cAMP tracer (adenosine 3',5'-cyclic phosphoric acid 2'-0-succinyl-3-[¹²⁵I] iodotyrosine methyl ester) was reconstituted in assay buffer to a final activity of 5.4 kBq/ml. The cGMP and cAMP acetylation standards were reconstituted to a final concentration of 2.56 pmol/ml.

This assay was performed as directed by the manufacturers instructions. Working standards were prepared by pipetting 500m l aliquots of assay buffer into 7 labelled polypropylene tubes. A 500m l aliquot of acetylated cGMP standard (2.56 pmol/ml) was added to a tube labelled as 128 fmol/tube before vortexing. Subsequent dilutions were performed by pipetting 500m l aliquots to produce 7 working standards of 2, 4, 8, 16, 32, 64 and 128 fmol/tube respectively. The assay unknowns were resuspended in 500m l of assay buffer by vortexing. Two zero standards (B_o) containing 500m l of assay buffer (with labelled cGMP only), and two tubes to measure total counts (Tc) without assay buffer or cGMP were prepared. All reagents were pre-equilibrated at room temperature before the assay. To all tubes 25m l of acetylation reagent (2 parts triethylamine: 1 part acetic anhydride) was added by pipetting gently above the aqueous layer and vortexing immediately. Duplicate 100m l samples from all working standards, B_o and unknowns were pipetted into LP3 tubes (Luckham Ltd., Sussex, U.K.). Antiserum in 100m l aliquots were then added to all tubes except the Tc tubes and immediately vortexed. All tubes were then covered with parafilm and incubated for 1 hour at 25° C. Subsequently 100m l aliquots of [¹²⁵I] labelled cGMP were pipetted into all assay tubes and vortexed. All tubes were incubated overnight at 4° C for 12-18 hours.

The second antibody reagent (Amerlex-M) was resuspended and 500m l aliquots were then added to all tubes except the Tc and vortexed. All tubes were then incubated for 10 minutes at 25° C, before centrifugation for 15 minutes at 2000 x g in an Mistral 3000 MSE centrifuge (Fisons Scientific, Sussex, U.K.). The tubes were then placed in decantation racks and then inverted onto paper towels for 5 minutes to remove fluid. The tubes were finally blotted around the rims to remove any remaining droplets. All tubes were then counted for 1 minute in a gamma counter (Hewlett Packard, U.S.A.).

The protocol followed was the same as that for cGMP determination except that the addition of [¹²⁵I] cAMP preceded the addition of the antiserum containing the first antibody reagent.

Paraformaldehyde was dissolved by heating in phosphate buffered saline (PBS, composition [120mM NaCl; 2.7mM KCl; pH 7.3 with 10mM phosphate buffer]) to a concentration of 4% (w/v). The solution was subsequently cooled and the pH adjusted to 7.2 with 2.0M Tris and 0.8M H₂SO₄. Samples were preserved at 4° C in phosphate buffered saline containing 7% sucrose (w/w) and 0.01% azide (pH 7.2 with 0.8M H₂SO₄/ 2M Tris).

Crypts fractions were isolated as described previously in 5.2.1., but were resuspended after the initial centrifugation step with 4% paraformaldehyde in phosphate buffered saline (pH 7.2) and left on ice for 90 minutes. The crypt suspension was then centrifuged at 100 x g for 3 minutes and resuspended in phosphate buffered saline (PBS) containing 7% sucrose and 0.01% azide (pH 7.2). The suspension was left for 1 hour on ice prior to centrifugation (100 x g for 2 minutes) and then resuspended in 1ml of PBS. Samples were then stored in eppendorfs at 4° C. A 2cm length of guinea pig jejunum was excised and similarly fixed in 4% paraformaldehyde for 90 minutes. The jejunal segment was then washed three times for 20 minutes in PBS solution for 1 hour and then stored at 4° C.

Single channel recordings from the crypt basolateral membrane.

Crypts were prepared as described previously in 4.2.1..

9.2.2. Solutions.

Electrophysiological recordings of single channel currents were obtained as described before (3.2.3) with the following modifications. Small mouth patch-pipettes used for single channel recording were fabricated from Boralex glass capillaries (Rochester Scientific Co., New York, U.S.A., section 3.2.3.) with typical resistances of 8-10 MW when filled with K⁺ or Cl^- rich pipette solutions. Single channel currents were recorded as outlined by Hamill et al. (1981) using a List EPC-7 amplifier (List Electromedical, Germany) or a Biologic RK-300 amplifier (Biologic, Claix, France). Junction potentials arising from entry into the bathing solution were compensated for in the current-clamp mode of the amplifier. Cell-attached recordings were obtained from the basolateral membrane of intact crypt enterocytes once an adequate seal resistance (³ 10 GW ) had been achieved. The series capacitance was then adjusted to compensate for the fast transient capacitative current. To form cell-free patches the electrode was quickly retracted and the excised (inside-out) membrane patches were passed through the air-water interface to avoid vesicle formation. The normal sign convention was observed (3.2.3). Cells were voltage clamped at a pre-determined holding potential and single-channel current events were recorded in response to depolarising and hyperpolarising voltage steps. In the cell-attached configuration the trans-membrane potential of the patch (V_patch) is determined by that of the cell interior (V_m) as well as that of the inside of the patch pipette (V_ref) and is given by the relation:

Usually V_m is unknown. Positive (outward by convention) currents correspond to upward deflections of the illustrated current records.

Single channel currents were acquired using a voltage pulse generator programme (VGEN, J.Dempster 1987) as described in section 3.2.4. Stored single channel records were analysed using a semi-automatic patch-clamp analysis programme (PAT, J.Dempster, 1987). Relay records were low pass filtered at 0.3-0.5kHz (-3db) and digitised (2-4 kHz) as described in 3.2.4. Current flow through single channels was measured from histograms of unitary current amplitude distribution. Channel open- and closed-state events were identified using a detection threshold crossing method with the discriminator positioned midway between the fully open and closed levels.

Channel open-state probability (P_o), defined as the fraction of the total time the channel was open, was computed from the duration of channel open times. When patches contained more than one channel, the detection threshold was set at progressively greater current levels to determine the time interval when 1, 2, 3,...,n channels were open simultaneously. Hence channel open probability for a patch containing more than one channel (nP_o) was calculated as P_o (level 1) + P_o (level 2) ... + P_o (level n).

Only membrane patches containing a single channel were used for stochastic analyses. Histograms of channel residence times were constructed from current records exceeding 40 s duration. Mean open and closed times represent arithmetic means of all observed open or shut times.

Illustrated traces represent records digitised at 2-4 kHz (filtered at 0.3-0.5 kHz (-3db), Bessel 8-pole low-pass filter) using an IBM-AT computer with appropriate software (PAT, J.Dempster 1987) and then imported as ASCII files into a graphics programme (Sigmaplot 5.0, Jandel Corporation 1992).

Table 3.1. Composition of the solutions used in electrophysiological experiments.

Pipette free Ca²⁺ was buffered to 93nM in some experiments. All solutions were adjusted to pH 7.2 with Tris and the N-methyl D-glucamine salts (NMG) were prepared by titrating the appropriate acid. No correction for Ca²⁺ chelation by gluconate was made in preparing the low (8 mM) Cl^- Hanks medium. The 0 Ca²⁺ Hanks solution was prepared by omitting CaCl₂ from normal Hanks solution.

Table 4.1. Composition of solutions.

All concentrations given in mM and solutions titrated to pH 7.2 with Tris. Hypotonic solution was prepared as isotonic solution omitting mannitol. Solutions containing 20mM K⁺ were made by replacing 18mM NaCl with 18mM KCl.

Table 5.1. Composition of solutions.

All concentrations given in mM and solutions titrated to pH 7.2 with Tris (except isolation buffer pH 7.1).

5mM Ba²⁺ and TEA Hanks were prepared by substituting for 5mM NaCl.

Table 6.1. Composition of solutions.

Concentrations given in mM. pH was adjusted to 7.2 with Tris.

Analar grade chemicals were used throughout and unless otherwise indicated were purchased from BDH chemicals Ltd.. The following chemicals are listed in alphabetical order. Compounds not commercially available were generous gifts and are marked with an asterisk. Full addresses of suppliers are given below.

Aldrich Chemical Co. Ltd., New Road, Gillingham, Dorset, England, SP8 4JL.

Amersham International plc, Amersham, U.K..

BDH Chemicals Ltd., Poole, England, BH12 4NN.

Fisons, FSA Laboratory Supplies, Bishop Meadow Road, Loughborough, England, LE11 0RG.

Gibco BRL, Paisley, Scotland, PA3 4EP.

Prof.R. Greger, Alberts-Ludwigs-Universitat, D-7800 Freiburg, Germany.

Hayman Ltd., 70 Eastways Industrial Park, Witham, Essex, England, CM8 3YE.

Merck, Sharp & Dohme Research Laboratories,

Pierce Europe, PO Box 1512, NL-3260 BA, Oud-Beijerland, Netherlands.

Sigma Chemical Co. Ltd., Poole, England, BH17 7TG.

Unipath Ltd., Basingstoke, Hampshire, England.

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