- Abstract
- Introduction
- Selective enhancement of the permeability of the possum hindgut
- Transport characteristics of the hindgut of the possum
- Conclusion
The Possum Intestine a Barrier & a Target
Grant Butt1, J Wen2, IG Tucker2, N Davies2, R Ledger2, Bj McLeod3
1Department of Physiology, 2School of Pharmacy University of Otago
and 3AgResearch, Invermay.
Abstract
The intestinal epithelium is a significant barrier to the oral delivery of bioactives, such as peptides and proteins. Normally this barrier is overcome by the inclusion of enhancers in the formulation given to the animal, which increase the permeability of the epithelium. However, these enhancers are often non-specific, operating by stripping off the epithelial layer. While this increases the oral delivery of bioactives, it may also be detrimental as it compromises the epithelium, which has a number of essential functions for the survival of the animal. We have determined the ability of enhancers to selectively increase the permeability of the hindgut of the possum without damaging the epithelial cells. We have also investigated in detail the role of the hindgut in fluid and electrolyte balance of the possum and shown that the functional properties of the possum hindgut differ markedly to the hindgut of eutherian mammals. Selective disruption of hindgut function of the possum may allow the development of possum specific control mechanism.
Introduction
The epithelial lining of the intestine represents a significant permeability barrier to the delivery of peptide drugs. The normal approach to overcome this is to include in the formulation an "enhancer", which increases the permeability of the intestinal epithelium. Examples of enhancers include bile salts, Ca chelators and detergents.
The intestinal epithelium consists of a single layer of cells, bound together at their apical poles by a belt like tight junction. Consequently, there are two routes for the movement of solutes across the epithelium, the cellular route and the paracellular route. Movement via the cellular route involves diffusion across the cell membrane and hence favours lipid soluble substances. In contrast, movement via the paracellular route is a hydrophilic pathway and favours water-soluble compounds such as peptides and protein.
Ideally any enhancer used to increase the permeability of the intestinal epithelium to peptide and protein drugs will selectively disrupt the paracellular pathway and cause minimal damage to the epithelial cells. Unfortunately, many enhancers are non-selective, or are used at concentrations that are non-selective. The result is that rather than disrupting the paracellular pathway they strip off the epithelial cells, increasing the permeability of the epithelium but at the same time causing significant damage to the epithelium.
This has consequences. The barrier function of the intestinal epithelium provides protection from pathogens in the external environment, including the bacterial flora and fauna in the gut, especially the hindgut. Furthermore, the epithelial lining of the intestine has a number of essential functions, including the absorption of nutrients and fluid and electrolytes. Consequently, disruption of the epithelial lining of the intestine can expose animals to both infections by pathogens and stress due to compromised intestinal function.
This is of concern when developing formulations for delivery of drugs to wild populations of animals such as the possum. It is of little value to develop a highly specific bioactive that modifies the reproductive potential of a possum and then deliver it with a formulation containing an enhancer that causes extensive damage to the intestinal epithelium. This in itself may kill the animal and, while the bioactive may be specific, the enhancer is likely to cause equivalent damage to the intestine of non-target species.
In this paper we report on studies investigating the ability of a range of enhancers to increase the permeability of the possum hindgut to the small hydrophilic marker, fluroscein. We also report unique aspects of the transport physiology of the possum hindgut, which may be targets for possum specific control strategies.
Selective enhancement of the permeability of the possum hindgut
To date we have tested the effectiveness of a range of compounds to increase the permeability of the hindgut of the possum by measuring the flux of the small hydrophilic compound fluroscein across the isolated epithelium mounted in an Ussing chamber. We have further investigated the extent to which three of these compounds, the bile salt sodium deoxycholate (SDA), the divalent cation chelator (EDTA) and the mucolytic agent dithiothreitol (DTT), damage the intestinal epithelium by measuring the amount of the cytoplasmic enzyme lactate dehydrogenase released from the epithelium when it is exposed to these compounds.
All three compounds significantly increased the permeability of both the proximal colon and the caecum of the possum hindgut as measured by the flux of fluroscein. However, SDA, even at low concentrations (1 mmol L-1) resulted in significant damage to the epithelial cells, as is evident from the release of LDH into the solution bathing the mucosal surface of the epithelium (Fig. 1). In contrast, EDTA increased the permeability of the epithelium to a comparable degree as SDA, but resulted in minimal damage to the epithelium.
Figure 1. Effect of SDA and EDTA on the transepithelial resistance (an index of epithelial permeability) of the possum proximal colon, and mucosal lactate dehydrogenase concentration.
This would suggest that EDTA increases the permeability of the hindgut of the possum by selectively disrupting the paracellular pathway. In contrast SDA appears to increase the permeability of the hindgut through a general disruption of the epithelial layer. This is supported by histological measurements that demonstrate significant damage to the epithelial cells when they are exposed to SDA.
Interestingly, the mucolytic agent, DTT, which significantly reduces the adherent mucus layer in both the proximal colon and the caecum of the possum, also induced the release of significant amounts of the cytoplasmic enzyme LDH. This would suggest that DTT increases the permeability of the intestine not only by reducing the amount of mucus but also by disrupting the epithelial layer.
Transport characteristics of the hindgut of the possum
The hindgut of eutherian mammals plays an important role in overall fluid and electrolyte balance. Furthermore, in animals that derive nutrients through fermentation of indigestible proteins and carbohydrates in the caecum and proximal colon, the hindgut is an important source of nutrients. If there are significant differences in the mechanisms involved in these process in the possum when compared to eutherian mammals, these phsyiological process could be targeted as possible possum specific control mechanisms.
Significantly, to date we have demonstrated marked differences between the properties of the hindgut of the possum and comparable eutherian mammals such as rabbit, rats, guinea pigs and mice. Unlike eutherian mammals, in the possum amiloride sensitive Na absorption is distributed throughout the proximal and distal colon rather than being restricted to the distal colon. This is of interest as, in eutherian mammals, Na absorption in the proximal colon occurs by Na/H exchange and is associated with the absorption of short chain fatty acids, the products of fermentation. This raises questions as to the mechanism of absorption of short chain fatty acids in the hindgut of the possum.
Figure 2. Plasma aldosterone concentrations and amiloride sensitive Na transport in the distal colon of possums maintained on a low and high Na diet for 2 weeks.
Figure 3. Comparison of electrogenic Cl secretion stimulated by the secretagogues forskolin and carbachol in the hindgut of the possum, mouse, guinea pig and rabbit
Furthermore, unlike in eutherian mammals, amiloride sensitive Na absorption in the colon of the possum does not appear to be involved in salt balance. Variations in dietary Na intake, which resulted in four fold differences in plasma concentrations of aldosterone, the hormone responsible for regulation of Na balance in eutherian mammals, had no effect on amiloride sensitive Na transport in the possum colon (Fig. 2). In contrast there were significant differences in the urinary excretion of Na by the two groups of possums.
Finally in eutherian mammals fluid secretion in the gut is driven by the secretion of Cl ions. This is essential for hydration of the intestinal mucus and pathological conditions (e.g., cystic fibrosis) in which fluid secretion is reduced result in compromised intestinal function. It is therefore surprising that agents that stimulate secretion in the colon of eutherian mammals do not stimulate a similar response in the possum (Fig. 3). Furthermore, while the small intestine of the possum responds to secretagogues the mechanism of fluid secretion appears to differ to that in eutherian mammals in that it does not appear to involve the secretion of Cl.
Conclusion
Details of the permeability properties of the possum intestine and the way in which enhancers modify the permeability of the intestine will allow the development of formulations for the oral delivery of bioactive compounds to the possum while minimising damage to the intestinal epithelium. This will ensure effective delivery of the bioactive with minimum stress to the possum and non-target species. Furthermore, there are significant differences in the properties of the intestinal epithelium of the possum and eutherian mammals that may allow the development of possum specific toxins that will exploit these differences to selectively control the possum.
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