CHAPTER 6

VARIATION IN THE SPONTANEOUS MEMBRANE POTENTIAL AND RESPONSE TO AGONISTS ALONG THE CRYPT AXIS.

Stem cells located towards the base of the crypt compartment give rise to Paneth, goblet, enteroendocrine and columnar epithelial cells by a process of primary differentiation. As the cells programmed to become columnar enterocytes migrate towards the tips of the villi they differentiate structurally, acquiring an elaborate microvillus membrane containing digestive enzymes, before developing their absorptive function (for review see Smith, 1985). These changes are not confined to absorptive and digestive function however, as microelectrode impalement studies of villus enterocytes reveal that both membrane potential and intracellular K⁺ concentration increase during the structural phase of enterocyte development (Cremaschi et al., 1984).

As the small intestinal crypt is the site of enterocyte production, proliferation and differentiation, it is possible that there is a variation in the functional expression and activity of ionic channels and transport systems along the length of the crypt axis. Measurements of membrane potential and conductance changes in response to secretagogues have thus far been confined to the `mid-region' of the isolated crypt for consistency. In this study the distribution of spontaneous membrane potentials of enterocytes located in the ground, surface and mid-regions has been determined in intact crypts isolated with intracellular-like solutions. The variation in membrane potential and conductance changes evoked by carbachol and forskolin in these three regions has also been established. The data presented here supports the contention that there is a heterogeneity in the functional expression of transport proteins and receptors along the crypt axis.

Electrophysiological recordings and data acquisition and analysis were performed as described in sections 3.2.3. and 3.2.4. respectively. Histological staining of sections of guinea-pig small intestine was performed as described in section 2.2.6..

6.2.1. Crypt preparation.

Crypt enterocytes were isolated as outlined in section 3.2.1. with the following modifications to improve sealing frequency and viability (as judged by their birefringence and capacity to exclude 0.03% trypan blue). The composition of the isolation buffer was changed by reducing the concentration of Na⁺ and Cl^- and increasing the concentration of K⁺ (for composition of isolation buffer see table 6.1). The enterocytes were sequentially shed by vibration over successive intervals of 10, 10, 3, 3, 3 and 3 minutes. The three fractions collected from minutes 24 to 32 were each centrifuged at 100g for 1 minute, before being resuspended and pooled in 20mM K⁺ Hanks solution (see table 6.1). The crypts were then centrifuged for 2 minutes at 50g and resuspended in 20mM K⁺ Hanks medium containing 4mM DTT. The crypt suspension was then placed in a rotary inversion mixer for 15 minutes at 4° C. The crypts were finally centrifuged and resuspended in 2ml of Hanks medium containing 7mM K₂SO₄.

Bath and pipette solutions were prepared as described previously (see sections 4.2.2. and 5.2.2.). 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 4.2.2..

Table 6.1. Composition of solutions.

All concentrations given in mM and solutions titrated to the desired pH with Tris base.

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

The current-clamp mode of the nystatin-perforated patch technique was used to measure E_m at different points along the crypt axis. Crypts could be clearly identified by their cylindrical morphology, with a virtual lumen enclosed at the ground region by a confluent cell monolayer and opening into the bathing medium at the surface region (Fig.2.3). Gigaohm seals were obtained only from intact birefringent crypts. The distribution of zero-current reversal potentials in the surface, middle and ground regions is illustrated in the frequency distribution histogram shown in Fig.6.1. Ground zero-current reversal potentials (Fig.6.1) gave a mean E_m of -77 ± 2 mV (n=12) and were consistently more hyperpolarised (P<0.0001, unpaired t-test) than the E_m values obtained from the mid-region of the crypt (-51 ± 2 mV, n=14), around 100m m from the crypt base. The mean value for E_m obtained from the mid-region of the crypt was not significantly different from the mean E_m obtained from the high Na⁺ isolation method (-49 ± 2 mV, n=35). E_m values obtained from the surface region (approximately 150-200m m from the crypt base) showed a greater variation than those recorded from the crypt mid-region, with a mean E_m of -39 ± 5 mV (P=0.06, n=8, unpaired t-test). The values reported for the ground region are close to the predicted value for E_k (-81mV) calculated for a pipette K⁺ concentration of 145mM assuming complete equilibration between the cell interior and the patch pipette.

Frequency histogram illustrating the distribution of zero-current potentials in the different regions of intact small intestinal crypts. The numbers on the voltage axis indicate the ranges of the individual classes extending from -22 mV to -102 mV in 16 mV steps (n=34 for all three regions).

6.3.2. Variation in membrane potential and conductance changes evoked by carbachol and forskolin along the crypt axis.

Experiments were performed to determine the effect of carbachol addition upon the E_m and membrane conductance of enterocytes located in the various regions along the crypt axis. In the ground region of the crypt 100m M carbachol induced a mean hyperpolarisation of 4.8 ± 0.6 mV (n=4, P<0.005, paired t-test) associated with a slight increase in membrane conductance (Fig.6.2A). In 5 experiments 100m M carbachol induced a mean hyperpolarisation in the mid-region of the crypt of 15.2 ± 2.1 mV (P<0.005, paired t-test), the extent of the carbachol-induced hyperpolarisation being significantly greater than those obtained from the ground region (P<0.01, unpaired t-test). This is presumably because the spontaneous E_m in the ground region approaches the equilibrium potential for K⁺ (E_K). The effect of 100m M carbachol upon membrane potential and conductance in the surface region of the crypt was determined and in 6 separate experiments carbachol evoked no significant change in membrane potential (2.7 ± 3.5 mV) or conductance (P >0.05, paired t-test, Fig.6.2B). The mean changes in E_m evoked by carbachol in all three regions are summarised in the histogram shown in Fig.6.2C.

Effects of carbachol and forskolin upon crypt membrane potential and conductance along the crypt axis. A. Continuous recording of E_m taken from the ground region of the crypt. During the time indicated by the horizontal bars the crypt was successively perfused with 100 m M carbachol (CCH) and then with 20m M forskolin (FSK). B. Continuous recording of E_m taken from the surface region of the crypt showing the responses to the same agonists. The scale bars are representative of both recordings shown. Current pulses of constant amplitude were applied throughout the recordings. Initial E_m values are as indicated by arrows. C. Histogram showing the variations in the magnitude of membrane potential changes evoked by carbachol and forskolin along the crypt axis. Data are expressed as means ± SE from 4-6 separate experiments for both agonists. Sample numbers are given in bars.

The variation in the response of cells in the surface, middle and ground regions to 20m M forskolin was also determined. In 4 experiments 20m M forskolin induced a mean depolarisation of 23.8 ± 4.5 mV in the ground region of the crypt. This depolarisation was reversible upon washout and was not associated with a sustained increase in membrane conductance in two of the four cells tested (see Fig.6.2A). In the mid- and surface regions of the crypt 20m M forskolin evoked mean depolarisations of 25 ± 1.2 mV (n=4, Fig.4.4B) and 28 ± 7.6 mV (n=4, Fig.6.2B) respectively, associated with an increase in membrane conductance. The repolarisation of E_m upon forskolin washout was slow, if present, in all regions of the crypt. The magnitude of the depolarisations induced by 20m M forskolin in the surface and ground regions of the crypt were not significantly different from those observed for the mid-region (P>0.05, unpaired t-test). However the nature of the cellular events underlying the forskolin-induced depolarisation in the ground region may be different from those in the surface and mid-regions.

VIP evoked depolarisations in 4 crypts with values of initial E_m ranging from -20 to -40mV; in 8 crypts with initial E_m values ranging from -40 to -60mV and in 2 crypts from the ground region with initial E_m values of -74 and -75mV. The regional variation in the response to VIP was not quantified, although the responsiveness to VIP appeared to be independent of the initial E_m. The initial and steady-state E_m values (means ± SE) obtained in response to carbachol and forskolin addition in each region are summarised in Fig.6.3..

Changes in membrane potential evoked by carbachol and forskolin along the crypt-villus axis. Membrane potentials recorded before and after the addition of 100m M carbachol (upper graph) or 20m M forskolin (lower graph) are presented as paired values for the ground, middle and surface regions respectively (means ± SE from 4-6 separate experiments).

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

The variation in initial E_m along the crypt axis provides an indication of the balance of all electrogenic transport processes in the membrane of the unstimulated crypt enterocyte. The distribution of E_m values obtained from the ground region, i.e. the least mature crypt enterocytes, is consistently between -70 and -80mV. This is close to the equilibrium potential for K⁺ (E_K) that can be calculated assuming that efficient dialysis of ions occurs between the cell interior and the pipette solution. The efficiency of dialysis of the intracellular milieu in high access resistance nystatin-perforated patches has been reported to vary in intact epithelial monolayers as intracellular Na⁺ and K⁺ activities are not substantially altered by the ionic composition of the pipette solution where there is extensive intercellular gap-junctional communication (Rae & Fernandez, 1991). However extensive dialysis of the intracellular ionic milieu must occur in perforated patches obtained from intact small intestinal crypts, as the magnitude of the depolarisations induced by VIP are dependent upon the concentration of Cl^- in the patch pipette (chapter 4). The hyperpolarised membrane potentials recorded in the ground region of the unstimulated crypt are consistent with a membrane conductance almost completely selective to K⁺ ions. Staining of transverse sections of guinea-pig jejunum with phloxine-tartrazine reveals an abundance of large acidophilic vesicles in the ground region of the small intestinal crypt which indicates the presence of a Paneth cell population (histology not presented here). The large variation in the range of spontaneous membrane potentials recorded in the ground and mid-regions of the crypt may therefore reflect the presence of different cell types in the two regions.

Spontaneous membrane potentials recorded from the mid-region of the unstimulated crypt are consistently depolarised relative to those recorded from the ground region. This may be accounted for by the presence of either a Na⁺ or a Cl^--selective conductance in the mid-region of the unstimulated crypt. Ion substitution experiments (Fig.3.1) are inconsistent with a basolateral location for such a conductance, although it may be present in the apical membrane domain. This would be consistent with the slow membrane hyperpolarisations evoked by NPPB in the unstimulated crypt (Fig.5.7.). The variation in the distribution of spontaneous membrane potentials was greater in the surface region of the crypt than in the mid-region, although the mean E_m (-39 ± 5 mV) was not significantly depolarised relative to the mid-region. The region of the crypt-villus interface has been shown to be associated with large changes in the pattern of gene expression (Trezise et al, 1992), and this may account for the greater variation in the distribution of spontaneous membrane potentials found in the surface region.

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

The magnitude of the hyperpolarisation evoked by carbachol is significantly greater in the mid-region of the crypt than the ground region, presumably because E_m is further from E_K which will limit the extent of the carbachol-induced hyperpolarisation. A similar dependence of the magnitude of the carbachol-induced hyperpolarisation upon the initial resting membrane potential was observed in studies with conventional microelectrodes in intact crypts isolated from rabbit colon (Greenwald et al., 1992). The apparent absence of any effect of carbachol upon E_m or membrane conductance in the surface region of the crypt, even in crypt enterocytes with resting E_m values similar to those observed in the crypt mid-region, suggests either the disappearance of functional muscarinic receptors, the K⁺ conductance responsive to its intracellular mediators or elements of the signalling cascade itself. Muscarinic receptors of similar characteristics have been demonstrated to be present on both villus and crypt enterocytes isolated from the rat small intestine (Isaacs et al., 1982), and therefore the most probable explanation is the loss of functional expression of a carbachol-modulated K⁺ conductance. This argument is further supported by whole-cell and single channel studies in isolated guinea-pig villus enterocytes, which so far have not yielded evidence for the presence of a Ca²⁺-dependent K⁺ conductance (Sepulveda et al., 1991; Mintenig et al., 1992). It is possible that the functional expression of a carbachol-responsive K⁺ channel is confined to the lower and mid-crypt regions and is `switched-off' as the enterocytes begin to differentiate near the crypt-villus interface. This observation also supports the contention that separate K⁺ conductances underlie the spontaneous resting membrane potential and the carbachol-induced membrane hyperpolarisation respectively, as the absence of a carbachol-responsive K⁺ conductance is observed in cells that exhibit spontaneous E_m values similar to those observed in the crypt mid-region which is consistently responsive to carbachol.

6.4.3. Regional variations in the response to forskolin

Forskolin evokes depolarisations of similar magnitude in the surface, middle and ground regions of the small intestinal crypt. However the depolarisation evoked in the ground region of the crypt was not associated with a consistent increase in membrane conductance, whilst the depolarisations evoked in the middle and surface regions of the crypt were associated with large increases in membrane conductance. This suggests that the nature of the depolarisation evoked by forskolin in the ground region may differ from that in the cells of the middle and surface regions of the crypt. Studies in intact crypts isolated from rat colon have demonstrated that forskolin depolarises both the ground and middle regions of the crypt. However, forskolin evoked a decrease in the magnitude of the outward current in the ground region of the colonic crypt, which was attributed to the inhibition of a K⁺ conductance, whilst in the mid-region of the colonic crypt forskolin evoked an increase in Cl^- current that could be inhibited by the Cl^- channel blocker NPPB at a concentration of 100m M (Bohme et al., 1991). The inactivation of a K⁺ conductance may in part account for the absence of consistent increases in membrane conductance in response to forskolin addition in the ground region of the small intestinal crypt. Recent studies in the rat small intestine have revealed high levels of expression of CFTR mRNA along the length of the crypt axis (Trezise & Buchwald, 1992). As the CFTR gene product is believed to be a cAMP-activated Cl^- channel the depolarisations induced by forskolin along the crypt axis may correlate with CFTR expression (Bear et al., 1992). The responsiveness to VIP is apparently independent of the initial crypt E_m, which would be consistent with the observation that VIP receptor expression varies little along the crypt-villus axis (Couvineau et al., 1992). Furthermore crypt enterocytes isolated from rat jejunum express abundant levels of the catalytic subunit of the G-protein (Ó_s) mediating the hormonal (e.g.VIP) stimulation of adenylate cyclase activity (Couvineau et al, 1992).

In summary crypt enterocytes exhibit a remarkable gradient in the distribution of their spontaneous resting membrane potentials along the crypt axis. A comparable variation has been described in isolated rat colonic crypts (Bohme et al., 1991) and in the rabbit ileal mucosa (Cremaschi et al., 1982). The gradient in the distribution of resting membrane potentials probably reflects the relative contributions of Cl^-, Na⁺ and K⁺ channels to the maintenance of the resting membrane potential. Similarly the nature of the response of enterocytes to carbachol is dependent upon the age of the enterocyte. The more fully differentiated enterocytes present in the middle and upper crypt regions show a greater increase in Cl^- conductance in response to forskolin, whilst differentiating enterocytes present in the surface region appear to lose their responsiveness to carbachol.

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