Rat Intestinal Segments Research Articles

Influences of regional differences in activities of brush-border membrane peptidases within the rat intestine on site-dependent stability of peptide drugs

Proteolytic hydrolysis rates of neurotensin and acetyl-neurotensin-(8–13) by brush-border membranes from various rat intestinal segments were as follows: jejunum > duodenum ∼ jejunoileal junction > ileum > caecum. The rank order of endopeptidase-24.11 activity along the intestine was jejunum > duodenum ∼ jejunoileal junction > ileum > caecum. Angiotensin converting enzyme (ACE) had a similar distribution profile as endopeptidase-24.11. Activities of these two enzymes were lower in the distal intestine. Distribution of endopeptidase-2 activity along the intestine was different: ileum > duodenum ∼ jejunum ∼ jejunoileal junction > caecum. The profiles of differential hydrolysis of neurotensin and acetylneurotensin-(8–13) within the gut corresponded to the distribution of endopeptidase-24.11 and ACE. Moreover, effects of enzyme inhibitors confirm that these two enzymes initiated proteolysis of neurotensin and acetylneurotensin-(8–13). These results suggest that the regional differences in the activities of key brush-border membrane peptidases will affect site-dependent stability of peptide drugs.

Mechanism and site dependency of intestinal mucosal transport and metabolism of thymidine analogues.

This study has been undertaken to investigate the mechanisms of intestinal mucosal transport and metabolism of thymidine analogues and to identify any optimal site(s) of the rat intestine particularly involved in the absorption of thymidine analogues. The intestinal absorption of 3'-azido-3'-deoxythymidine (AZT) was studied at three initial concentrations in four segments of the rat intestine using an in situ recirculating perfusion technique. Disappearance of AZT followed first-order kinetics throughout the gastrointestinal (GI) tract at all tested concentrations. The apparent first-order rate constants were found to be relatively invariant over a broad range of concentrations from 0.01 to 1.0 mM. Corrected for the length of each segment, the apparent permeability (Papp) of AZT was 3.01 +/- 0.32 x 10(-5) cm/sec (mean +/- SE) in the duodenum, 2.06 +/- 0.24 x 10(-5) cm/sec in the upper jejunum, 0.76 +/- 0.13 x 10(-5) cm/sec in the combined lower jejunum and ileum, and 0.32 +/- 0.10 x 10(-5) cm/sec in the colon, which indicated that intrinsic absorptivity was greater in the upper GI tract than in the lower portions possibly due to the differences in surface area for absorption. No AZT metabolite appeared in any part of the GI tract. On the other hand, thymidine and other analogues, i.e., 5-iodo-2'-deoxyuridine and 2'-deoxyuridine, were rapidly metabolized into nucleobase and sugar in the upper GI tract, whereas in the colon no metabolite appeared. A free 3'-OH group appears to be necessary for the metabolism (catabolism) of thymidine analogues in the rat intestine mainly by pyrimidine nucleoside phosphorylase.(ABSTRACT TRUNCATED AT 250 WORDS)

Pituitary adenylate cyclase activating peptide: A novel vasoactive intestinal peptide-like neuropeptide in the gut

Pituitary adenylate cyclase activating peptide (PACAP) is a vasoactive intestinal peptide (VIP)-like hypothalamic peptide occurring in two forms, PACAP-27 and the C-terminally extended PACAP-38. The predicted rat and human PACAP sequence is identical to the isolated ovine one. In the present study, the occurrence and distribution of PACAP-like peptides were examined in the gut of several species by immunocytochemistry and immunochemistry using an antibody raised against PACAP-27. PACAP-like immunoreactivity was observed in nerve fibers in the gut wall of all species examined (chicken, mouse, rat, hamster, guinea-pig, ferret, cat, pig, sheep and man). In the chicken and human gut, immunoreactive fibers were numerous in all layers. In the other species examined the fibers were predominantly found in the myenteric ganglia and smooth muscle. Delicate PACAP-immunoreactive fibers were seen in the gastric mucosa of mouse, rat, hamster and man but not in the other species examined. The chicken proventriculus harbored numerous PACAP-immunoreactive endocrine cells which were identical with the serotonin-containing cells storing gastrin-releasing peptide. PACAP-immunoreactive nerve cell bodies were numerous in the submucous ganglia and moderate in number in the myenteric ganglia of the human gut. They were few in the intramural ganglia of the other species examined. Extrinsic denervation (performed on segments of rat and guinea-pig small intestine) did not visibly affect the PACAP innervation, indicating an intramural origin of most PACAP-immunoreactive fibers. Double immunostaining for VIP and PACAP revealed co-existence of the two peptides in nerve cell bodies and nerve fibers of the human and chicken gut and in fibers in the gastric mucosa of mouse and rat. In all other species examined and in all other locations in the gut PACAP-immunoreactive nerve cell bodies and nerve fibers were distinct from those storing VIP; many of them contained gastrin-releasing peptide instead. Immunochemistry revealed PACAP-like peptides in gut extracts of all species studied; upon high performance liquid chromatography the immunoreactive material co-eluted with synthetic PACAP-27. The distribution of PACAP-immunoreactive nerve cell bodies and nerve fibers in the gut wall suggests their involvement in the regulation of both motor and secretory activities.

Hydrogen peroxide reduces β-adrenoceptor fonction in the rat small intestine

Incubation of isolated rat intestinal segments with hydrogen peroxide (H 2O 2) led to a decreased β-adrenoceptor response. The maximal relaxation induced by isoprenaline was lowered while the EC 50 remained unaffected. The effect of H 2O 2 in the small intestine increased slightly from duodenum to ileum. In the ileum, 10 −4 M H 2O 2 led to a 10% decrease of the maximal relaxation due to isoprenaline and 1 mM decreased the maximal response to about 50%. We further investigated the level at which the isoprenaline response was impaired. The relaxation caused by the stable cAMP analog, dibutyryl-cAMP, or by the adenylate cyclase activator, forskolin, was not affected or affected less than by isoprenaline. When the response to isoprenaline was expressed relative to the maximal response to dibutyryl-cAMP or forskolin, pretreatment with H 2O 2 led to a decreased isoprenaline response relative to the response to dibutyryl-cAMP or forskolin. This might indicate that exposure to H 2O 2 leads to a disturbance in receptor-mediated cAMP production. The adenylate cyclase unit is probably not affected since the response to forskolin is relatively resistant to H 2O 2. Our conclusion is that pretreatment of isolated intestinal segments with H 2O 2 leads to disturbed β-adrenoceptor coupling, probably due to altered membrane integrity.

Structural requirements for the intestinal mucosal-cell peptide transporter: the need for N-terminal alpha-amino group.

The requirement for a free alpha-amino group for the intestinal peptide carrier-mediated transport was investigated. A series of dipeptide analogues without the N-terminal alpha-amino group [including phenylpropionylproline, phenylacetylproline, N-benzoylproline, phenylacetyl-alpha-methyldopa, and hippuric acid (N-benzoylglycine)] were studied in the perfused rat intestinal segment. The absorption of phenylpropionylproline, phenylacetyl-alpha-methyldopa, and N-benzoylproline was concentration dependent. The transport parameters (mean +/- SD) of phenylpropionylproline and N-benzoylproline were as follows: Jmax*, 0.037 (+/- 0.019) mM; Km, 0.045 (+/- 0.027) mM; Pc*, 0.830 (+/- 0.130); and Pm*, 0.673 +/- 0.049; and Jmax*, 1.34 (+/- 0.24) mM; Km, 1.31 (+/- 0.30) mM; Pc*, 1.02 (+/- 0.11); and Pm*, 0; respectively. The intestinal permeabilities of phenylpropionylproline, phenylacetylproline, N-benzoylproline, and hippuric acid (N-benzoylglycine) were significantly reduced by dipeptides and cephradine. These results strongly suggest that these dipeptide analogues, without an alpha-amino group, are transported by the peptide carrier and provide more direct evidence that a free alpha-amino group is not absolutely essential for the mucosal-cell peptide carrier-mediated transport.

Non-everted oxygenated rat intestinal segments as a measure of neutral detergent fiber effects on iron absorption

Iron absorption in the presence of varying amounts and sizes of dietary fiber was measured. A method using non-everted rat intestinal segments perfused in oxygen was refined. Neutral detergent fiber (NDF), a component of dietary fiber, was extracted from cooked pinto bean ( Phaseolus vulgaris). The NDF did not affect iron absorption in intestinal segments from iron replete rats. However, 4 and 6 mg of NDF/ml significantly decreased iron absorption in the intestinal segments from anemic rats. NDF with a smaller particle size of 0.125 mm increased iron absorption relative to that absorbed with 0.180 mm particles. Histological examination validated using non-everted intestinal segments perfused with oxygen as a method for studying dietary effects on iron absorption. Segments which are not everted are less prone to damage. Perfusion with oxygen maintained metabolic activity in the tissue during the experiment.

Mucoadhesion of hydroxypropylmethacrylate nanoparticles to rat intestinal ileal segments in vitro.

The purpose of this study was to evaluate the adhesion of HPMA nanoparticles to mucus using a perfused rat ileum test system. Radiolabeled nanoparticles were prepared and deposited onto rat ileal segments in vitro. The segments were perfused and the perfusate was collected in fractions and assayed for radioactivity. Between 10 and 50% of the radioactivity was eliminated over the first 120-sec perfusion, whereas the remaining activity was firmly attached to the ileum. Among the variables tested, the time interval between nanoparticle deposition and perfusion played the major role, indicating that the mucus-nanoparticle interaction is likely to result from the diffusion of polymers into the mucus and of mucin into the polymeric matrix.

Metabolism of [formula omitted] Some structural requirements for the deacetylation and consequences for the oral bioavailability

Rat liver, lung and intestine homogenates deacetylated N- acetyl- l-cysteine . Nearly stoichiometric amounts of l-cysteine were recovered. In rat liver, the enzyme activity was associated with the cytosolic fraction. Liver cytosolic fractions from rat, man and mouse hydrolysed N- acetyl- l-cysteine at similar rates, while dog liver cytosol was much less active. N- acetyl- d-cysteine or the disulphide of N- acetyl- l-cysteine were not deacetylated or in other ways consumed in vitro. Isolated, perfused rat liver did not retain or metabolize N- acetyl- l-cysteine to any measurable extent during single-pass experiments. N- Acetyl- l-cysteine or N- acetyl- d-cysteine were injected into a ligated segment of rat intestine in situ. After 1 hr 2% of the l-isomer and 35% of the d-isomer remained in the intestinal lumen. Systemic plasma levels were <3 μM of the l-form and ⋍40 μ M of the d-form. We conclude that deacetylation in the intestinal mucosa and possibly in the intestinal lumen is the major factor determining the low oral bioavailability of N- acetyl- l-cysteine . The deacetylation is discussed on the basis of the subcellular localization and the structural requirement of the reaction.

Multiple frame processing in automated quantitaive coronary angiography: Cheryl Hagerty, David H. Blankernhorn, Robert H. Selzer, and Stanley P. Azen University of Southern Californa, Los Angeles, Californai(58)

Coreopsis tinctoria Nutt mainly distributed in Hetian region of Xinjiang at an altitude of 3000 m, which is used as Uyghur traditional medicine because of its clearing heat, promoting circulation and removing toxicity and antihypertension, ect. effect.This research was to study the four ingredients in the extracts of Coreopsis tinctoria Nutt. that are absorbed in different intestinal segments in rats to lay the foundation for further study on the effective constituents, tissue distribution, metabolism, and spectrum-effect relationships of these extracts.High, medium, and low concentrations were prepared according to their pharmacological effects. Quantitative analysis multi-components by single marker was used to test the cumulative absorption volume Q, absorption rate constant Ka, and apparent permeability coefficient Papp of the four main ingredients in C. tinctoria Nutt. extract in different intestinal segments in rats using a Ussing chamber model and high-performance liquid chromatography.The Papp of chlorogenic acid and flavanomarein in the duodenum, jejunum, ileum, and colon were 1.0×10−6 to 10×10−6 cm s−1. Papp of marein in the duodenum and jejunum was <1.0×10−6, and was 1.0×10−6 to 10×10−6 cm s−1 in the ileum and colon. Papp of 3,5-O-dicaffeoylquinic acid in the duodenum was <1.0×10−6 cm s−1, while it was 1.0×10−6 to 10×10−6 cm s−1 in the jejunum, ileum, and colon.All four chemical components of the plant extract can be absorbed by the intestinal canal of rats, which conforms to zero-order absorption; the ileum presented the best absorption.

Time-dependent deterioration of active transport in duodenal segments of rat intestine

In this study, a time-dependent deterioration of the active transport in duodenal segments of rat intestine was observed. The everted gut-sac technique was used to quantitate both calcium and glucose transport in intestinal segments. The results indicate that both calcium and glucose transport decreased significantly in intestinal segments of animals killed by cervical dislocation 10–20 min prior to tissue removal. It was further determined that animal anesthesia permitted excision of intestinal segments prior to death and thus avoided transport deterioration.

Treatment of Intestinal Ischemia with Oxygenated Intraluminal Perfluorocarbons

Liquid perfluorocarbons are biologically inert compounds capable of dissolving up to 40 percent oxygen by volume. This remarkable and reversible oxygen solubility has encouraged investigations into therapeutic application in situations where tissue oxygen delivery is impaired. One such setting is intestinal ischemia. Identically prepared devascularized segments of rat intestine were treated with either intraluminal oxygenated perfluorocarbon (perfluorotributylamine) or physiologic saline solution. After timed sacrifice, blinded quantitative histologic evaluation for ischemic injury was performed. The perfluorotributylamine treatment groups had histologic scores indicative of less severe injury between 1 and 4 hours. These scores achieved statistical significance (p less than 0.05). We conclude that intraluminal oxygenated perfluorocarbons have a significant protective effect in this model of intestinal ischemia. This quantitative analysis is unique and is an important aspect of the preclinical evaluation of the perfluorocarbon preparations.

Estimating human oral fraction dose absorbed: a correlation using rat intestinal membrane permeability for passive and carrier-mediated compounds.

Based on a simple tube model for drug absorption, the key parameters controlling drug absorption are shown to be the dimensionless effective permeability, P*eff, and the Graetz number, Gz, when metabolism or solubility/dissolution is not rate controlling. Estimating the Graetz number in humans and assuming that P*aq is not rate controlling give the following equation for fraction dose absorbed: F = 1 - e-2P*w. The correlation between fraction dose absorbed in humans and P*w determined from steadystate perfused rat intestinal segments gives an excellent correlation. It is of particular significance that the correlation includes drugs that are absorbed by passive and carrier-mediated processes. This indicates that P*w is one of the key variables controlling oral drug absorption and that the correlation may be useful for estimating oral drug absorption in humans regardless of the mechanism of absorption.

Survival of transferred intestinal segments after vascular pedicle interruption.

Rat intestinal segments with intact vascular pedicles were transferred from the abdominal cavity into the subcutaneous space. After 30 days, the vascular pedicle was severed. The intestinal segments, now completely dependent on wound neovascularization, survived completely. We conclude that early survival of intestinal transfers requires perfusion through the vascular pedicle. With time in a favorable bed, new vessel ingrowth from the recipient wound into the intestinal segment provides adequate circulation so that the vascular pedicle can be safely divided with no ill effect or at most a minor mucosal slough which quickly heals. Further clinical opportunities are necessary to determine the time course for these phenomena in humans.

Treatment of intestinal ischemia with oxygenated intraluminal perfluorocarbons

Liquid perfluorocarbons are biologically inert compounds capable of dissolving up to 40 percent oxygen by volume. This remarkable and reversible oxygen solubility has encouraged investigations into therapeutic application in situations where tissue oxygen delivery is impaired. One such setting is intestinal ischemia. Identically prepared devascularized segments of rat intestine were treated with either intraluminal oxygenated perfluorocarbon (perfluorotributylamine) or physiologic saline solution. After timed sacrifice, blinded quantitative histologic evaluation for ischemic injury was performed. The perfluorotributylamine treatment groups had histologic scores indicative of less severe injury between 1 and 4 hours. These scores achieved statistical significance (p < 0.05). We conclude that intraluminal oxygenated perfluorocarbons have a significant protective effect in this model of intestinal ischemia. This quantitative analysis is unique and is an important aspect of the preclinical evaluation of the perfluorocarbon preparations.

Intraluminal bile salt increases rate of firing in afferent fibers from the small intestine of the rat.

Perfusion of a rat intestinal segment with a solution containing sodium deoxycholate (8 mM) increases the rate of firing in periarterial afferent nerves from the gut. This observation indirectly supports our earlier proposal that bile salt evokes a net fluid secretion in the small intestine via an activation of the enteric nervous system.

Effect of Melatonin on the Force of Spontaneous Contractions of In Vitro Rat Small and Large Intestine

Segments from various locations of the small and large intestine of the rat were removed, bathed in Tyrode's solution and attached to a force displacement transducer. Melatonin, while not influencing the frequency of contraction, did reduce the force of spontaneous contractions of duodenal and colon segments of rat intestine by 92 and 52%, respectively compared to only 25 and 22% for the ileum and jejunum, respectively. Areas with greatest responsiveness to melatonin were those that previous studies have shown to contain the largest concentrations of endogenous melatonin. Cyclic guanosine monophosphate, when tested in similar preparations, did not produce an inhibitory response characteristic of melatonin. It is hypothesized, therefore, that this hormone has physiological action within the gut, including motility; however, its action may not be directly on smooth muscle contraction but may be through an indirect action inhibiting the contractile response of serotonin, as suggested by other investigators.

Retention of Iron by Rat Intestine in Vivo as Affected by Dietary Fiber, Ascorbate and Citrate

The effects of pH, ascorbate, citrate and dietary fiber on retention of ferrous and ferric iron by jejuno-ileal segments of rat intestine were examined in vivo. Iron was introduced in an isosmotic solution of sodium chloride and dextrose buffered by 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid (BES) and acetate. Stabilization of the iron solutions was aided by use of iron concentrations ≤ 1 µg/ml injected into the intestine for 10-min periods. Iron retention was optimal over a broad pH range from 5 to 7.8. Inclusion of ascorbic acid in the solution injected (5, 25 or 75 µg/ml) did not increase retention of iron in either valence state. A low concentration of sodium citrate (2 mM) had no effect on iron retention, but increasing the concentration to 5 mM released iron from the mucosa. Maize and wheat fibers decreased the retention of ferrous iron by binding and by promoting autoxidation and formation of poorly soluble iron polymers. Bound ferrous iron was released completely at pH below 5. Retention of ferric iron was also lowered in the presence of fiber, presumably as a result of polymerization. Retention of iron by the rat in the absence of ligands was independent of valence state.

Simultaneous vascular and luminal perfusion of rat small intestine.

A method is described which allows simultaneous vascular and luminal perfusion of the jejunal segment of rat intestine. Perfluorochemical artificial blood (FC-43 Emulsion) was utilized as a vascular perfusion medium. The viability of this preparation can be maintained for 1h as indicated by morphological findings, competitive inhibition between L-phenylalanine and L-methionine, and the transport of L-phenylalanine against a concentration gradient. This preparation was used for investigation of the transport behavior of several amino β-lactam antibiotics.

Mucin depletion in the intestine of malnourished rats.

The effects of malnutrition on mucosal goblet cell mucin levels were studied in rats deprived of 50% of their daily intake, as judged by pair-fed, age-matched control animals, for 5 wk. Average daily weight gain was 0.7 g/day compared with 5.8 g in age-matched (AM) rats; final weight was 246 +/- 9 g compared with 406 +/- 4 g. Immunoassayable mucin, sucrase, protein, and DNA were assayed in mucosal scrapings from the proximal, middle, and distal segments of the small intestine in malnourished rats, AM rats, and a third group of low-weight, less mature (LM) rats. Total protein, total DNA, and protein-to-DNA ratios in malnourished rats were unchanged compared with AM control rats and often higher than levels in LM control rats. In malnourished animals, mucin concentration per milligram protein was significantly decreased below AM control animals in the upper two segments and below LM control animals in all segments. Mucin concentration per milligram DNA was significantly lower in malnourished rats than in all segments of both control groups. In contrast, sucrase activity per milligram protein or DNA was either unchanged or increased in the malnourished rats, indicating that the reduction in mucin concentration was selective and did not reflect all surface glycoproteins. Isolated mucins from malnourished and AM control rats were chemically similar, and the affinity and number of antigenic determinants were the same. Malnutrition therefore leads to an absolute decrease in intestinal mucin rather than reduced molecular antigenicity. Impaired capacity to maintain mucosal mucin content may be a factor in reducing intestinal resistance to enteric infection in malnutrition.

Effect of pancreatic secretions on transit in bypassed loops of intestine in rats.

The influence of intraluminal infusions of saline and a pancreatic juice-bile salt mixture on transit was determined in bypassed segments of intestine in rats. Animals underwent bypass of 70% of their jejunoileum and insertion of a catheter into the proximal end of the bypassed segment. After recovery from operation, each animal was placed into a harness that allowed free movement while fluids could be infused into the bypassed segment. Beginning on the first postoperative day, the bypassed loops were infused with either saline or a pancreatic juice-bile salt mixture. Transit in these animals was determined on the third day after operation. Other animals received no infusions and were tested either on the third or the 35th day after operation. Animals that received no infusions had low rates of transit when tested either in the fasted or fed condition three days after operation. Saline infusions resulted in a slight increase in transit in fasted animals only. Transit rates were increased to a greater degree following infusion of the pancreatic juice-bile salt mixture than with saline, especially in fasted animals in whom the rates approached those seen in the uninfused, fasted animals that were tested 35 days after operation. These findings are consistent with the hypothesis that the reduced transit seen in the bypassed loops of rats three days after operation may be due to intestinal contents characterized by deficiencies in pancreatic juice and bile salts.