Adult Rat Small Intestine Research Articles

Determination of the panel of reference genes for quantitative real-time PCR in fetal and adult rat intestines

In quantitative real-time PCR (qRT-PCR) detection, the stability of reference genes varies with different organs, tissue locations, sex and developmental stages. This study aimed to screen out and determine the optimal panel of reference genes of the intestine in pre- and post-natal rats of different sex. We used qRT-PCR to detect the mRNA expression of six commonly used reference genes (ACTB, GAPDH, HPRT1, B2M, RPLPO and SDHA) in rat intestines at gestational day 21 (GD21) and postnatal week 12 (PW12). Using GeNorm, BestKeeper and NormFinder software comprehensively analyzed the stability of candidate reference genes and screened out stable reference genes. Further, we used the pathological model of prenatal dexamethasone exposure (PDE) to verify the stability of the selected panel of reference genes. Based on the results of the software analysis, the optimal panel of reference genes in the fetal rat intestine was SDHA + ACTB, and the adult rat small intestine and colon were ACTB + HPRT1 and RPLP0 + GAPDH, respectively. There was no significant sex difference in the above results. Besides, in the PDE model, the results were consistent with those under physiological conditions. Therefore, the stability of intestinal reference genes in fetal rats and adult rats was different, and the intestinal reference genes of adult rats were intestinal segments-specific. The selected panel of reference genes was still stable under pathological conditions. This study determined the optimal panel of reference genes of pre- and post-natal rat intestines and provided reliable reference genes for the qRT-PCR analysis of rat intestines.

Method for Detecting Hyaluronan in Isolated Myenteric Plexus Ganglia of Adult Rat Small Intestine.

The cellular components of the enteric nervous system (ENS), namely enteric neurons and glia, display plasticity and respond to environmental cues deriving from growth factors, extracellular matrix (ECM) molecules, and cell-surface molecules, both in physiological and pathological conditions. ECM, in particular, provides an important framework for the enteric microenvironment and influences the homeostasis of myenteric neuronal circuitries. Isolation of pure myenteric plexus preparations from adult tissue permits to investigate changes in the ENS involving specific ECM, such as hyaluronan. This approach is based upon the possibility to isolate myenteric ganglia from the intestinal wall of either adult animals or humans, after microdissection and subsequent enzymatic digestion of the tissue. Enteric ganglia are free of connective tissue, extracellular collagen, and blood vessels, and thus treatment of intact intestinal segments with highly purified collagenases permits ganglia isolation from the surrounding smooth muscle cells. In this chapter, we describe methods for visualizing HA in isolated primary cultures of adult rat small intestine myenteric ganglia.

Absence of intestinal gluconeogenesis in type 1 diabetic rats: carbon 13 NMR and arteriovenous concentration difference measurements

Recent reports have indicated that 48–72h of fasting, type 1 diabetes and high‐protein feeding induce gluconeogenesis in the small intestine of adult rats in vivo. Since this would (i) represent a dramatic revision of the prevailing view that only the liver and the kidneys are gluconeogenic, and (ii) have major consequences in the metabolism, nutrition and diabetes fields, we have thoroughly re‐examined this question in the situation reported to induce the highest rate of gluconeogenesis. For this, metabolically viable small intestinal segments from 72h‐fasted adult rats were incubated with [3‐13C]glutamine as substrate. After incubation, substrate utilization and product accumulation were measured by enzymatic and NMR methods. Although the segments utilized 13C‐glutamine at high rates and accumulated 13C‐labeled products linearly for 30 min in vitro, no substantial glucose synthesis could be detected. This was not due to the re‐utilization of 13C‐glucose initially synthesized. Arteriovenous metabolite concentration difference measurements across the portal vein‐drained viscera of 72h‐fasted Wistar and Sprague‐Dawley rats clearly indicated that glutamine, the main if not the only gluconeogenic precursor taken up, could not give rise to detectable glucose production in vivo. Therefore, we challenge the view that the small intestine of the adult rat is a gluconeogenic organ.

Absence of gluconeogenesis in the rat small intestine: In vitro carbon 13 NMR and in vivo evidence

Recent reports have indicated that 48–72h of fasting, type 1 diabetes and high-protein feeding induce gluconeogenesis in the small intestine of adult rats in vivo. Since this would represent a dramatic revision of the prevailing view that only the liver and the kidneys are gluconeogenic, and have major consequences in the metabolism, nutrition and diabetes fields, we have thoroughly re-examined this question in the situation reported to induce the highest rate of gluconeogenesis. For this, metabolically viable small intestinal segments from 72h-fasted adult rats were incubated with [3–13C]glutamine as substrate. After incubation, substrate utilization and product accumulation were measured by enzymatic and NMR methods. Although the segments utilized 13C-glutamine at high rates and accumulated 13C-labeled products linearly for 30 min in vitro, no substantial glucose synthesis could be detected. This was not due to the re-utilization of 13C-glucose initially synthesized. Arteriovenous metabolite concentration difference measurements across the portal vein-drained viscera of 72h-fasted Wistar and Sprague-Dawley rats clearly indicated that glutamine, the main if not the only gluconeogenic precursor taken up, could not give rise to detectable glucose production in vivo. Therefore, we challenge the view that the small intestine of the adult rat is a gluconeogenic organ. Supported by the INSERM.

Glutamine gluconeogenesis in the small intestine of 72 h-fasted adult rats is undetectable

Recent reports have indicated that 48-72 h of fasting, Type 1 diabetes and high-protein feeding induce gluconeogenesis in the small intestine of adult rats in vivo. Since this would (i) represent a dramatic revision of the prevailing view that only the liver and the kidneys are gluconeogenic and (ii) have major consequences in the metabolism, nutrition and diabetes fields, we have thoroughly re-examined this question in the situation reported to induce the highest rate of gluconeogenesis. For this, metabolically viable small intestinal segments from 72 h-fasted adult rats were incubated with [3-13C]glutamine as substrate. After incubation, substrate utilization and product accumulation were measured by enzymatic and NMR spectroscopic methods. Although the segments utilized [13C]glutamine at high rates and accumulated 13C-labelled products linearly for 30 min in vitro, no substantial glucose synthesis could be detected. This was not due to the re-utilization of [13C]glucose initially synthesized from [13C]glutamine. Arteriovenous metabolite concentration difference measurements across the portal vein-drained viscera of 72 h-fasted Wistar and Sprague-Dawley rats clearly indicated that glutamine, the main if not the only gluconeogenic precursor taken up, could not give rise to detectable glucose production in vivo. Therefore we challenge the view that the small intestine of the adult rat is a gluconeogenic organ.

Increased expression of vasoactive intestinal polypeptide in cultured myenteric neurons from adult rat small intestine

Adult neurons possess the ability to adapt to a changing environment. Loss of target-derived neurotrophic factors due to axotomy or isolation by culturing is known to induce changes in neuropeptide expression in several types of peripheral neurons. The aim of the present study was to investigate changes in the expression of vasoactive intestinal polypeptide (VIP) and nitric oxide synthase (NOS) in cultured myenteric ganglia and dissociated neurons. Myenteric ganglia and neurons from rat small intestine were dissociated and cultured for up to 21 days. Immunocytochemistry was used to determine the total number of neurons and the proportions of subpopulations containing VIP or NOS or both in preparations of whole mounts (controls used to determine the conditions in vivo), myenteric ganglion culture and dissociated myenteric neuronal culture. In situ hybridization was used to determine changes in the expressions of NOS and VIP mRNA. The relative number of VIP-expressing neurons increased significantly during culturing. The percentage of all neurons expressing VIP was 3.6±0.3% in whole mounts, 22–24% in cultured myenteric ganglia, and up to 35% in cultured dissociated neurons. NOS-expressing neurons constituted approximately 30–40% of all neurons in whole mounts as well as in cultured ganglia or dissociated neurons. A dramatic increase in NOS/VIP-containing neurons were detected in cultured neurons irrespective of whether they were arranged in ganglia or dissociated, as compared to whole mount preparations. This suggests that the NOS-containing neurons are the ones that increase their VIP expression. The induced expression of VIP in cultured adult myenteric neurons indicates that VIP is important for neuronal adaptation, maintenance and survival.

Immunolocalization of Protein 4.1B in the Rat Digestive System

Protein 4.1 family proteins are thought to interact with membrane proteins and also membrane skeletons. In this study, immunohistochemical studies by light and electron microscopy were performed with a specific antibody against protein 4.1B. Specific protein 4.1B immunolabeling was observed in simple columnar epithelium in the adult rat large intestine, small intestine and stomach. Protein 4.1B immunolabeling was localized along the membranes facing the adjacent cells (lateral portion) and also facing the extracellular matrix (basal portion). Moreover, a spatial protein 4.1B expression gradient was observed along the crypt-villus axis of the rat small and large intestinal epithelium: strong protein 4.1B expression was present within the villus, with the crypt showing barely any detectable protein 4.1B. The expression of protein 4.1B was not detected in the stratified squamous epithelium in the forestomach or the esophagus. By immunoelectron microscopy, the immunolabeling of the cells was observed to be restricted to the cytoplasmic side just beneath the plasma membrane, including the membranes adjacent to the next cells, except for the tight junctions. We conclude that the protein 4.1B expression pattern is related to the maturation of simple columnar epithelium in the rat digestive system, probably by the effect of adhesion.

Differential effects of VIP and PACAP on survival of cultured adult rat myenteric neurons

Our knowledge of neuroprotective factors important for the adult enteric nervous system is poor. Changes in expression of vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) in enteric neurons in response to neuronal injury or colchicine treatment, as well as in intestinal adaptation, have been described. Cultured myenteric neurons increase their expression of VIP; furthermore, culturing myenteric neurons in the presence of VIP enhances neuronal survival. The aims of this study were to evaluate possible changes in PACAP expression in dissociated and cultured myenteric neurons from adult rat small intestine, and to determine the ability of PACAP-38 and PACAP-27 to promote survival of cultured myenteric neurons, as compared with that of VIP. A marked decrease in the number of surviving neurons was noted during culturing. No difference in neuronal survival was found after culturing in the presence of PACAP-38 or PACAP-27, whereas VIP significantly increased neuronal survival. In contrast to the marked increase noted in the number of VIP-expressing neurons, culturing caused no change in the number of PACAP-expressing myenteric neurons. We were thus able to demonstrate that VIP, but not PACAP, promoted survival of myenteric neurons in culture. This suggests the presence of a VIP-specific receptor mediating neuroprotection in adult myenteric neurons.

Development of a vitamin D-responsive organ culture system for adult and old rat intestine.

The purpose of this study was to develop an organ culture system for adult and old rat small intestine that is responsive to vitamin D. Explants from F344 rats were cultured on Millipore tissue culture inserts placed in 6-well dishes at a temperature of 28 degrees C and in the presence of 95% oxygen. Explants from young (2 months old), adult (12 months old), and old (22 months old) rats were viable for up to 12 hr as determined by constant rates of DNA and protein synthesis. Hormonal responsiveness was characterized by measuring the capacity of 1,25-dihydroxyvitamin D [1,25(OH)2D], the hormonal form of vitamin D, to increase mRNA levels of the intestinal 24-hydroxylase cytochrome P-450 (CYP24). Jejunal explants from young rats increased CYP24 mRNA levels in a linear fashion with an EC50 of 3 nM in response to 1,25(OH)2D. There was no change with age in the magnitude of the jejunal response with regard to time (0-12 hr) or dose (0.1-100 nM). However, in the duodenum, 1,25(OH)2D increased CYP24 mRNA to significantly higher levels in the adult compared to the young. Since the 24-hydroxylase is the first step in the degradative pathway for 1,25(OH)2D in the intestine, increased duodenal expression of the 24-hydroxylase may contribute to the decreased action of 1,25(OH)2D on the adult duodenum.

Functional Fragments of Ingested Lactoferrin Are Resistant to Proteolytic Degradation in the Gastrointestinal Tract of Adult Rats

Pharmaceutical and food-related applications of lactoferrin, an 80-kDa iron-binding glycoprotein found predominantly in milk, have attracted interest lately, but the process of digestion of lactoferrin has been poorly characterized. The digestive fate of bovine lactoferrin in adult rats after oral administration of a single dose and after dietary supplementation was studied by (125)I-labeling and by surface-enhanced laser desorption/ionization (SELDI) affinity mass spectrometry. The latter method was designed to detect multiple forms of degraded lactoferrin as simple molecular ion peaks corresponding to one of the core regions of lactoferrin, namely, the lactoferricin region (Phe17-Ala42). Radioactive fragments with molecular masses of 42, 36, 33 and 29 kDa were observed at 20, 60 and 180 min postingestion in the contents of the lower small intestine. Rats were given free access to milk enriched with lactoferrin at 482 micromol/L (40 mg/mL). The concentrations of lactoferrin fragments in the contents of the stomach, small intestine and lower small intestine as determined by SELDI affinity mass spectrometry were approximately 200, 20 and 1 micromol/L, respectively. These data indicate that functional fragments of LF such as fragments containing glycosaminoglycan-binding site(s), as well as large fragments with a mass >20 kDa, indeed survive proteolytic degradation in the small intestine of adult rats.

Changes in cell proliferation and differentiation of adult rat small intestine epithelium after adrenalectomy: kinetic, biochemical, and morphological studies.

The effects of 10-day bilateral adrenalectomy on morphometry, proliferation, and apoptosis were determined in the small intestine of 3-month-old Sprague-Dawley rats. The activities of sucrase, lactase, and its respective mRNA, aminopeptidase N, and intestinal alkaline phosphatase were also evaluated. Adrenalectomy lead to partial atrophy and disorganization of the epithelium, with an increased number of goblet and Paneth cells and a reduction of crypt cell proliferation paralleled by a marked increase in villus apoptosis. Biochemical assays revealed that aminopeptidase N and intestinal alkaline phosphatase activities were significantly decreased, whereas disaccharidases were increased by adrenalectomy. The corresponding induction of lactase mRNA suggests an active response of the epithelium. In conclusion, adrenalectomy modified maturation and the differentiation processes of the small intestinal mucosa, especially in the proximal part of the small intestine. This result points to an important role of adrenals and glucocorticoids in the trophic status of the adult small intestinal mucosa.

A Factor Derived from Adult Rat and Cow Small Intestine Reduces Cryptosporidium parvum Infection in Infant Rats

A Factor Derived from Adult Rat and Cow Small Intestine Reduces Cryptosporidium parvum Infection in Infant Rats

Expression of an Intestine-Specific Transcription Factor (CDX1) in Intestinal Metaplasia and in Subsequently Developed Intestinal Type of Cholangiocarcinoma in Rat Liver

CDX1 is a caudal-type homeobox intestine-specific transcription factor that has been shown to be selectively expressed in epithelial cells in intestinal metaplasia of the human stomach and esophagus and variably expressed in human gastric and esophageal adenocarcinomas (Silberg DG, Furth EE, Taylor JK, Schuck T, Chiou T, Traber PG: Gastroenterology 1997, 113: 478-486). Through the use of immunohistochemistry and Western blotting, we investigated whether CDX1 is also uniquely associated with the intestinal metaplasia associated with putative precancerous cholangiofibrosis induced in rat liver during furan cholangiocarcinogenesis, as well as expressed in neoplastic glands in a subsequently developed intestinal type of cholangiocarcinoma. In normal, control adult rat small intestine, specific nuclear immunoreactivity for CDX1 was most prominent in enterocytes lining the crypts. In comparison, epithelium from intestinal metaplastic glands within furan-induced hepatic cholangiofibrosis and neoplastic epithelium from later developed primary intestinal-type cholangiocarcinoma each demonstrated strong nuclear immunoreactivity for CDX1. CDX1-positive cells were detected in hepatic cholangiofibrotic tissue as early as 3 weeks after the start of chronic furan treatment. We further determined that the percentages of CDX1-positive neoplastic glands and glandular nuclei are significantly higher in primary tumors than in a derived, transplantable cholangiocarcinoma serially-propagated in vivo. Western blotting confirmed our immunohistochemical results, and no CDX1 immunoreactivity was detected in normal adult rat liver or in hyperplastic biliary epithelial cells. These findings indicate that CDX1 is specifically associated with early intestinal metaplasia and a later developed intestinal-type of cholangiocarcinoma induced in the liver of furan-treated rats.

Transcriptional and translational control over sodium-glucose-linked transporter (SGLT1) gene expression in adult rat small intestine

We have measured SGLT1 mRNA content and SGLT1-mediated glucose transport at different positions along the small intestine of control and streptozotocin diabetic rats and shown both parameters to be similar but higher in jejunal compared with ileal tissue. No such correlation was seen when comparing measurements of SGLT1 mRNA along jejunal villi with previous estimates of SGLT1 protein and SGLT1-mediated glucose transport [Debnam et al., Eur. J. Physiol. 430 (1995) 151–159]. This is the first time it has been possible to directly relate these three aspects of SGLT1 gene expression in a single species. Results are discussed in terms of a possible time rather than positional control over translation of SGLT1 mRNA. © 1997 Federation of European Biochemical Societies.

170 GENE EXPRESSION OF INSULIN-LIKE GROWTH FACTOR-l IN THE SMALL INTESTINE OF SUCKLING AND ADULT RATS.

Insulin-like growth factor-I (GF-I) is a peptide that induces cell division and differentiation in a wide range of tissues and cell types. IGF receptors are present in almost all organs including the small intestine. A major site for the synthesis of circulating lGF-l is the liver, but lGF-l is also produced in a number of other tissues. Gene expression of IGF-I in fetal and adult rat small intestine was first detected by Lund (1986). However, intestinal levels of lGF-l mRNA during the suckling period, when IGF-I peptide is delivered via milk, are not known. In order to measure IGF-I mRNA levels in intestinal samples of suckling and adult rat, we established a very sensitive quantitative reverse transcription (RT) competitive-PCR assay. A known amount of an IGF-I competitor cDNA was added prior to PCR reaction and served as a quantitative standard. Interestingly, the expression of IGF-I mRNA in jejunum of sucklings was 3-fold lower (p 0.01) than in adult rats. We speculate that this difference may relate to the enteral intake of milk-borne IGF-I. Furthermore, exact cellular localization of IGF-I mRNA transcripts in the small intestine is not defined. To clarify this we used a non-radioactive in situ hybridization technique (digoxigenin labeling). In adult male rats an intense positive signal was evident in the upper crypt enterocytes and goblet cells, and sometimes in the lower villus cells. Probably because of the lesser sensitivity and in agreement with the previous data indicating a low amount of IGF-I mRNA in suckling rats in comparison with adults, no IGF-I transcripts were detected in suckling rats. We have thus demonstrated IGF-I mRNA presence in the intestinal mucosa of suckling rats and characterized its localization in the small intestinal mucosa of adult rats.

GLUTAMINE SYNTHETASE AND GLUTAMINASE IN THE HUMAN PLACENTA: CLUES TO A GLUTAMINE-GLUTAMATE SHUTTLE? • 1830

Based on studies of glutamine flux in the ovine placenta, we hypothesize that the human placenta is capable of substantial glutamine synthesis via glutamine synthetase (GS) and has little capacity to deaminate glutamine via glutaminase (GA). GS and GA activities and GS transcript were examined on freshly obtained homogenates from term human placenta and compared to values obtained from other organs known to be primarily producers or consumers of glutamine. The activity of GS and transcript size (1.8 and 2.8 kB species) in human placenta is comparable to that observed in adult rat lung, an organ known to be an important glutamine producer involved in glutamine homeostasis. GS activity in the placenta is higher than that in adult rat small intestine which is a major glutamine consumer rather than a glutamine producer. Placental GA shows low activity (0.21 ± 0.29 μmoles • mg protein-1 indicating that the human placenta converts very little glutamine to glutamate. Low activity of GA suggests that the placenta has little need to convert glutamine to glutamate. The human placenta may, like the ovine placenta, derive sufficient glutamate from the fetus for incorporation into the citric acid cycle. These results: 1) suggest that the human placenta, like the rat lung, is a glutamine producing organ; and 2) are consistent with the presence of an ovine-like glutamine-glutamate shuttle between the human placenta and fetal liver. Supported by NIH Grant#RO1-HD29279. Table

The potential use of tomato lectin for oral drug delivery: 2. Mechanism of uptake in vitro

Tomato lectin (TL) is a non-toxic dietary glycoprotein of molecular mass 71 kDa. Its interaction with, and uptake by, the adult rat small intestine was investigated using an improved everted gut sac system, to evaluate its potential in oral drug delivery systems. Uptake of [ 125I]TL was compared with two control molecules; 125I-labelled polyvinylpyrrolidone (PVP), an inert polymer often used as a marker for fluid-phase endocytosis, and 125I-labelled bovine serum albumin (BSA), a degradable protein of similar molecular mass to TL. Uptake of substrate associated with gut tissue or in the serosal space was calculated as ng per mg of gut sac protein. The rate of uptake of tomato lectin into gut sac tissue at 37°C was 27 ng/h per mg protein, but passage into the serosal space was much slower (1.7 ng/h per mg protein). The rate of uptake by the tissue was 11-times higher than BSA and 20-times higher than PVP, but transfer into the serosal space was only 4.3- and 5-times greater than BSA and PVP, respectively. This showed that the proportion of lectin transferred across the mucosa to the serosa was less than either control, and indicated accumulation of TL within the enterocytes. Incubation at 4°C and with metabolic inhibitors demonstrated that the mechanism of TL uptake by small intestinal gut sacs in culture was adsorptive endocytosis. Trichloroacetic acid solubility studies showed that TL was more resistant to enzyme degradation during incubation with gut sacs than BSA. The fact that TL was shown to adhere to the gut surface, showed increased uptake when compared with control macromolecules, PVP and BSA, and accumulated in the mucosal cells, may give it potential as a drug carrier for delivery to the gastrointestinal tract.

The distribution of novel intermediate filament proteins defines subpopulations of myenteric neurons in rat intestine

Background/Aims : Recent studies with neurofilament antibodies as neuronal markers have shown subpopulations of myenteric neurons that do not contain neurofilament proteins. Novel neuronal intermediate filament proteins α-internexin, peripherin, and nestin have been identified. The aim of this study was to examine the distribution of these novel intermediate filaments in comparison with neurofilaments in myenteric plexus neurons. Methods : Using indirect immunofluorescence techniques in whole-mount cryostat sections from neonate and adult rat small intestine and in primary cultures of myenteric neurons, the distribution of neurofilaments, α-internexin, peripherin, and nestin was studied in comparison with the neuronal marker protein gene product (PGP) 9.5 in myenteric neurons. Results : Sixty-five percent of neurons contained neurofilament triplet proteins, α-lnternexin and/or peripherin were found in the neurofilament-negative neurons. PGP 9.5 was present in 80% of the myenteric neurons. Of the neurons that were PGP negative,95% contained peripherin or α-internexin. Nestin was not found in either neonate or adult myenteric neurons but was seen in glial cells in culture. Conclusions : The results suggest that a subpopulation of myenteric neurons lacks neurofilament triplet proteins but contains either peripherin, α-internexin, or both. This selective distribution of intermediate filaments in subpopulations of enteric neurons may support differential roles in these structurally unique neurons.

Thyroid hormone responsiveness is developmentally regulated in the rat small intestine: a possible role for the alpha-2 receptor variant.

Thyroid hormone (T3) alters gene expression through binding to a receptor protein located within the nucleus of target cells. Multiple forms of the T3 receptor (TR) have been identified and are encoded by the alpha and beta c-erbA genes. We have previously found that TR beta-1 is the major receptor form expressed in the adult rat small intestine, although there are also moderate levels of c-erbA alpha-2, a nonhormone-binding variant that is thought to inhibit T3 action. In developing rats, we studied the regulation of two small intestinal enterocyte genes previously shown to be T3 responsive, lactase and 3.0-kilobase intestinal alkaline phosphatase (IAP). Animals were treated with six daily ip injections of either saline (control) or 30 micrograms/100 g BW T3 (T3 group) and killed at 10 and 25 days of age. Northern analyses of RNA derived from intestinal tissues showed that the magnitude of the T3-induced changes in lactase and IAP gene expression increased with development. Jejunal 3.0-kilobase IAP messenger RNA (mRNA) levels were unaffected by T3 at 10 days, but increased by 15-fold at 25 days. Similarly, jejunal lactase mRNA levels were unchanged by T3 at 10 days, but decreased by 75% at 25 days. Qualitatively similar results were seen in the duodenum and ileum. Studies of TR expression revealed that TR beta-1 mRNA levels were unchanged during the developmental period, whereas the levels of c-erbA alpha-2 decreased by 90% between 5-25 days after birth. These results indicate that the rat small intestine becomes increasingly T3 responsive during postnatal development. These changes occur in parallel with a decline in c-erbA alpha-2 levels, suggesting that this T3 receptor variant may play a role in this hormonal responsiveness.

Uptake and transport of poly( N-vinylpyrrolidone-co-maleic acid) by the adult rat small intestine cultured in vitro: Effect of chemical structure

To study the structure-activity relationships of polymer uptake and transfer across the gastrointestinal mucosa, poly( N-vinylpyrrolidone-co-maleic acid) (NVP MA) polymers of standardised molecular mass (20000 Da) were synthesised to contain side-chains of different charge or hydrophobicity. All polymers additionally contained tyrosinamide residues to permit radioiodination. Using an improved everted gut sac prepared from adult rat small intestine, the tissue accumulation and serosal transport of the polymers was measured in vitro over a 2 h incubation period. All polymers were captured by the tissue linearly with time (endocytic indices between 1.6 and 16 μl/mg protein per h), and then transferred slowly to the serosal fluid (endocytic indices between 0.18 and 2 μl/mg protein per h). The neutral NVP MA polymer showed the lowest rate of tissue association, but this was increased 5-fold by the presence of either negatively or positively charged groups. The maximum rate of transport across the mucosa was seen for the most negatively charged polymer derivative, this being equivalent to approx. 26% its rate of tissue accumulation. Increasing hydrophobicity of the polymer derivatives had a more pronounced effect on the rate of tissue capture, increasing it by up to 10-fold for the most hydrophobic derivative. However, in this case, the serosal transfer was only 10–15% the rate of tissue uptake. The data presented indicate that NVP MA polymers can be tailor-made for use in oral delivery systems. Substituent groups can be incorporated to promote tissue uptake or translocation across the gastrointestinal mucosa, or a combination of the two.