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Since the 1960's physiologists have upheld the central dogma that structural segregation into surface and crypt cells directly supports a functional segregation of ion transport processes (Powell 1995). The dogma states that absorptive processes are located exclusively in surface epithelial cells and secretory exclusively in crypt cells. This classification resulted from several experimental observations on small intestine, jejunum and colon (Serebro et al 1969, Roggin et al 1972 and Field 1980). Amongst the clearest direct evidence in colonic tissue is that from microelectrode studies recording individual membrane conductances of rabbit colonic crypts. Inhibition of Na"" absorption (via blocking amiloride-sensitive apical Na'’ channels) decreased membrane conductance but in surface cells only. In contrast serosal application of prostaglandin E2 stimulated electrogenic cAMP-mediated Cl' and fluid secretion in crypt cells only. In addition fluid droplets, seen on oil-covered mucosal surface, formed directly over crypt openings only and were not observed in the presence of frusemide, an inhibitor of Cl" secretion (Welsh et al 1982).

However much of the evidence supporting this segregation of ion transport processes arose out of indirect observations of fluid measurement. A novel method, involving the concentration polarisation of impermeant fluorescent dyes visualised by confocal microscopy, demonstrated that colonic crypts in rats exhibit the capacity to absorb fluid (Naftalin & Pedley 1990 and Pedley & Naftalin 1993). Subsequent to this finding the development of a technique permitting direct determination of fluid movement in intestinal crypts, openly challenged the central dogma (Singh et al 1995a). Hand-dissected, perfused isolated crypt preparations, devoid of myofibroblast-containing

pericryptal sheath, exhibited net basal Na'^-dependent fluid absorption. Addition of secretagogues dibutyryl-cyclic AMP, vasoactive intestinal peptide or acetylcholine to the serosal bathing fluid induced reversible net fluid secretion. From this evidence Singh et al (1995a) speculated that absorptive processes are constitutively expressed in crypt epithelial cells with crypt secretion regulated by one or more neurohumoral agonists. These agents, being released insitu from cells in the lamina propria, such as myofibroblasts, directly into the crypts' local environment, can effectively alter crypt basal function. The major advantage of this technique is the ability to isolate crypt transport processes from surface cells and the surrounding cellular environment. The influence and control of the latter increasingly appears to be responsible for the secretory characteristics previously assumed to be constitutive crypt properties. Both these novel techniques provide conclusive evidence of the dual functional nature of colonic crypts.

The supposition that secretory mechanisms are exclusively located in crypt cells has also been called into question. Kockerling & Fromm (1993) looked at the spatial distribution of cAMP-dependent Cl' secretion by measuring local ion conductances in rat colonic crypts and surface epithelium, using voltage-scanning techniques. In control conditions surface and crypt cells contributed 39% and 61 % respectively to total ion conductance. Exposure to theophylline, elevating cAMP, increased both crypt and surface epithelial cell conductance. The effects were Cl' dependent, as theophylline had no effect in Cl'-free buffer and decreased ion conductances were recorded in both cell types when stimulated in the presence of a Cl' channel blocker. It was concluded that cAMP-dependent Cl' secretion was not confined to crypt cells but also occurred in surface cells in rat distal colon. This has also been shown using the isolated crypt preparation (Greger et al 1997).

1.4.2 An old dogma - laid to rest.

Functional segregation within the colonic mucosa no longer holds true. The revised view is that all transporting epithelial cells throughout epithelial layers have the capacity to both absorb and secrete depending on the

nature of the prevailing luminal and submucosal environments (Pedley & Naftalin 1993, Greger et al 1997 and Singh et al 1995a). Further studies, using the isolated crypt preparation in conjunction with morphological studies, have indeed revealed functional heterogeneity's but from within the crypt enterocyte profile itself. Enterocytes at the crypt base constitute poorly differentiated stem cells that frequently divide giving rise to the three major colonic epithelial cell types (section 1.3). Daughter cells migrate over several days to the surface differentiating and, from electrophysiological studies, systematically acquiring various functional transport properties as they travel (Ecke et al 1996a). Consequently several ionic gradients have been observed along the crypt axis. A summary of crypt transport to date arising predominantly from isolated crypts studies on rat distal colon, and the functional significances are outlined below.

1.4.3 Colonic crypt transport 1.4.3.1 Crypt absorption

Colonic crypts in their basal state demonstrate spontaneous Na'^-dependent fluid absorption (Pedley & Naftalin 1993 and Singh et al 1995a). An apical chloride-dependent Na'^-H'' exchanger with partial amiloride-sensitivity has been demonstrated. It appears functionally distinct from the chloride- independent Na^’-H"’ exchanger in surface epithelial cells, which shows comparably greater amiloride-sensitivity (Rajendran et al 1995). In rabbit distal colon crypt luminal cross-sectional surface area was found to increase on exposure to theophylline (a secretagogue) but decrease in control conditions below that seen with octanol (inhibits Na,K-ATPase and therefore water absorption). These findings support the view that in control conditions there is fluid outflow across the crypt wall demonstrated to be generated by a transepithelial osmotic pressure gradient (Bleakman & Naftalin 1990).

1.4.3.2 Crypt secretion

cAMP-mediated Cl' secretion has been proposed to occur in crypt wall but not crypt base cells (Bohme et al 1991 and Siemer & Gogelein 1993). However a recent study recorded cAMP-stimulated Cl' secretion with the most marked response in base cells (Greger et al 1997). From their findings

Greger et al (1997) state that the ability to secrete NaCI appears to be characteristic of colonocytes early in their development being gradually lost and replaced by the ability to absorb NaCI via amiloride-sensitive epithelial Na"" channels as they migrate to the surface. Cells in the mid crypt region demonstrated a capacity for both absorption and Cl' secretion suggesting a transitional zone. Conditions and cellular mechanisms involved that suppress or shift crypt cells between these two transport states remain unknown. Greger et al (1997) proposed that the activation of Cl' channels by cAMP itself inactivates Na"” conductance in the same cell and maybe one key component in determining the direction of NaCI transport.

1.4.4 Functional significance of crypt absorption

The fundamental driving force for water absorption is a high intercellular Na"" concentration generating a transepithelial osmotic gradient (section 1.2.3). Having established the basal absorptive function of colonic crypts, it has been proposed that the specific structure and properties of these crypts confer an ideal microenvironment in which a hypertonic absorbate (high N a \ section 1.2.3.1) can be effectively produced (Bleakman & Naftalin 1990 and Naftalin 1994). To understand the potential functional significance of these crypts in providing isolated microenvironments it is necessary to briefly highlight some of the problems of colonic fluid salvage imposed by the specific nature of colonic luminal contents.

1.4.4.1 Faecal dehydration

It is normally assumed that any resistance to fluid movement in the bulk solution is negligible compared with the hydraulic resistance across the epithelium. The properties and nature of luminal contents in the colon however single colonic salvage out as an exception to this assumption. Faeces are composed of very fine particulate matter (forming the skeleton) and fluid (forming pore-water, occupying the spaces between particles), in ratios of fluid to solid 20:1 in ileal fluid, consolidated to 2:1 in dehydrated formed stools (table 1,1). The following steps occur when pressure is applied,

(i) Pore-pressure increases producing hydraulic pressure gradients between gut contents and surroundings.

(ii) Fluid outflow and compression of particulate matter results

(iii) Pore-pressure subsides and the load is taken up by the ‘skeleton’. Initial reduction in stool water content from 95% to 80% (water to solids 4:1) is relatively easy, requiring a force of only - 5 kPa (50cm H2O). This is achieved through mechanical force from colonic smooth muscle contraction. The fine particulate matter of faeces however, infers a composition similar to clay in which there exists an exponential relationship between the force required to compress water out of faeces and its steady state water content (McKie et al 1990). In terms of faecal dehydration this means that as faeces become more solid the hydraulic resistance offered to further dehydration increases exponentially. Dehydration from fluid to solid 4:1, to the final 2:1 of dehydrated formed stools (68% water content) requires the enormous pressure of -lOOOkPa (~10 atmospheres). As this amount of pressure is beyond that achievable by compressive muscular forces the only other force available is osmotic pressure. It is these properties of the colonic luminal faecal contents that underlie the essential requirement for the colon to be able to generate a hypertonic absorbate and osmotic pressure to produce dehydrated solid stools. Animals that can not generate this hypertonic absorbate such as cattle only dehydrate faeces to -83% water (McKie et al 1991).

1.4.4.2 A specific role for crypt absorption in faecal dehydration?

Overall the colonic mucosa is nearly as permeable as the small intestine to both salt and water (Bleakman & Naftalin 1990). However from its capacity to generate a hypertonic absorbate it can be deduced that discrete regions exist within the mucosa that are very impermeant to both salt and water. Indeed hydraulic conductance across the colon has been shown to be heterogeneous (Pedley & Naftalin 1993). It has been proposed and substantiated that luminal surface epithelium is relatively ‘leaky’ with bi­ directional passage of water and electrolytes (lumen to blood and blood to lumen). Although this route provides a high capacity absorptive pathway it is unable to produce the high suction pressures necessary for the formation of

solid faeces (section 1.4.4.1). This is because the high water flow reduces the ratio of Na'^iwater absorbed so preventing the generation of a hypertonic absorbate (section 1.2.3.1). By contrast crypt epithelium is very ‘tight’ and accordingly of low hydraulic conductance. Local Na,K-ATPase activity necessary for active Na'" absorption is high with cell density approximately eight times that at the mucosal surface. These factors enable colonic crypts to generate and maintain high Na* concentrations as found in the pericryptal space. Colonic crypts are therefore able to generate high osmotic pressure gradients exerting the suction pressures necessary to dehydrate faeces and form solid stools (Pedley & Naftalin 1993 and Naftalin 1994).