Isolated Rat Colon Research Articles

Complement Activation of Electrogenic Ion Transport in Isolated Rat Colon

The complement cascade is an important component in many immune and inflammatory reactions and may contribute to both the diarrhoea and inflammation associated with inflammatory bowel disease. Isolated rat colonic mucosae were voltage clamped in Ussing chambers. Basolateral addition of zymosan-activated whole human serum (ZAS) induced a rapid onset, transient inward short circuit current (SCC). This response was concentration dependent and was significantly attenuated by pre-heating ZAS at 60°C for 30 min. Depletion of complement from normal human serum with cobra venom factor (CVF) significantly lowered SCC responses. Chloride was the primary charge carrying ion as responses to ZAS were abolished in the presence of the loop diuretic bumetanide. The complement component C3a stimulated ion transport but not to the same extent as whole serum. Exogenous C5 was without effect. The cyclooxygenase inhibitor piroxicam significantly attenuated the response to ZAS. These findings support the possibility that complement activation may contribute to the pathophysiology of secretory diarrhoea since activation of electrogenic chloride secretion converts intestinal epithelia to a state of net fluid secretion.

Thromboxane-like actions of prostaglandin D2 on the contractility of the rat colon in vitro.

Prostaglandin D2 (PGD2) concentration-dependently induced a contraction of the longitudinal muscle of the intact isolated rat colon in vitro. The effect of PGD2 increased continuously from the anal to the oral end of the colon. The action of PGD2 was not inhibited but rather enhanced by the neurotoxin, tetrodotoxin, and by the PGD2 antagonist, AH 6809. In contrast, the thromboxane A2 antagonist, SK&F 88046, concentration-dependently inhibited the PGD2 effect. The action of PGD2 was mimicked by the stable thromboxane A2 derivative, carbocyclic thromboxane A2, indicating that PGD2 exerts its action on the smooth muscle by stimulation of thromboxane receptors. Between 18 (distal colon) and 38% (proximal colon) of the preparations exhibited spontaneous phasic myogenic contractions. The thromboxane antagonist, SK&F 88046, completely suppressed these spontaneous contractions. The combined lipoxygenase/cyclo-oxygenase inhibitor nordihydroguaiaretic acid and sulfasalazine mimicked the action of SK&F 88046, whereas the cyclo-oxygenase inhibitor, indomethacin, was ineffective. These results suggest that endogenously produced metabolites of arachidonic acid, e.g. thromboxane A2, contribute to the generation of spontaneous muscle contractions in vitro. The failure of indomethacin to suppress muscular activity, however, requires further studies.

Effect of endothelin 1 on ion transport in isolated rat colon

Background: Endothelin may play a significant role in the regulation of gastrointestinal function because it has a variety of biological activities and because endothelin-like immunoreactivity as well as its specific binding sites have been found in the gastrointestinal tract. This study investigated the secretory effect and mechanism of action of endothelin 1 in mammalian large intestine. Methods: Distal colonic segments from Sprague-Dawley rats were stripped of their muscle layers and mounted in Ussing chambers. The effects of endothelin 1 on short-circuit current in rat colonic mucosa were studied in the absence or presence of specific inhibitors. Transmural unidirectional 22Na+ and 36Cl− fluxes and endothelin 1-induced prostacyclin release were also measured. Results: Serosal addition of endothelin 1 evoked a sustained increase in short-circuit current that was significantly reduced by tetrodotoxin or atropine, and virtually abolished by a selective endothelin A receptor antagonist (BQ-123), furosemide, piroxicam, d,l-verapamil, or removal of serosal calcium. Hexamethonium, amiloride, diphenhydramine, or a specific platelet-activating factor antagonist (CV-6209) did not influence the response to endothelin 1. Endothelin 1 significantly decreased net sodium and net chloride absorption and induced a marked increase in prostacyclin release from the serosal surface of stripped colonic mucosa. Conclusions: Endothelin 1 has a secretory effect in rat colon. Its action seems to be mediated by cyclo-oxygenase products and enteric nerves via the activation of an endothelin A receptor.

Antagonism of platelet‐activating factor in isolated rat colon: Possible mechanism

The contractions of three different regions of rat colon in response to platelet-activating factor (PAF) were compared. The ascending colon was found to be the most responsive. The slow contraction of the ascending colon induced by PAF was dependent on external Ca2+. CV-3988, a structural analog of PAF, slowly induced irreversible inhibition of PAF-induced contraction, whereas FR-900452, which is structurally unrelated to PAF, caused rapid reversible inhibition of PAF-induced contraction. No inhibitory effects of CV-3988 were observed when the strip was washed with Tyrode's solution containing 1% bovine serum albumin (BSA). The results suggest that PAF and CV-3988 penetrate slowly into the outer half of the lipid bilayer of plasma membranes of cells in isolated rat colon, and then rapidly diffuse laterally to associate firmly with specific binding sites.

In vitro anticolon antibody production by mucosal or peripheral blood lymphocytes from patients with ulcerative colitis.

Serum anticolon antibody and in vitro anti-colon antibody production by peripheral blood and mucosal lymphocytes was investigated in patients with ulcerative colitis. The frequency of serum anticolon antibody was 71% in 41 patients with ulcerative colitis, estimated by enzyme linked immunosorbent assay (ELISA) using isolated rat colon epithelial cells. This finding confirms our previous report on the frequency of serum anticolon antibody detected by flow cytometry analysis. The estimated frequencies of IgG anticolon antibody secreting cells were 1.5-12.5/10(6) cells in the colonic mucosa and 0.1-0.5/10(6) cells in peripheral blood, from patients with ulcerative colitis when Epstein-Barr virus (EBV) was used as a B cell polyclonal activator. Poisson analysis of limiting dilution culture showed that about one per 140 IgG cells in the colonic mucosa synthesised anticolon antibody. Two monoclonal IgG antibodies were obtained from EBV transformed anticolon antibody secreting cells by limiting dilution method. One reacted with goblet cells in the intestine, and the other reacted mainly with colonic epithelial cells. These results suggest that heterogeneous anticolon antibodies are present in patients with ulcerative colitis and that colonic mucosa may be the main source of anticolon antibody. Local autoimmune reaction might have an important role in causing the inflammation of colonic mucosa in this disease.

Study of platelet activating factor and its antagonists on rat colon strip with a new method avoiding tachyphylaxis

Platelet activating factor induced slow, but sustained contraction of isolated rat colon in a dose-dependent manner. The contraction persisted even when the strips were washed several times with Tyrode solution. However, the addition of high concentrations of bovine serum albumin to the washing solution led to the rapid relaxation of the strips to the basal level, possibly by removing platelet activating factor tightly bound to rat colon. These strips then responded normally to a second addition of platelet activating factor. Neither the cholinergic nervous system nor release of histamine, serotonin, prostaglandins or leukotriene D 4 were significantly involved in the contraction induced by platelet activating factor. FR-900452 and CV-3988, recently found to be antagonists of platelet activating factor, selectively blocked the contraction of rat colon induced by the active phospholipid, indicating that it induced ontraction by a direct stimulatory effect on the smooth muscle of rat in a receptor-mediated manner.

β-Adrenergic control of motility in the rat colon. I. Evidence for functional separation of the β1- and β2-adrenoceptor-mediated inhibition of colon activity

The β-adrenoceptor-mediated inhibitory response in isolated rat colon strips of β-adrenoceptor agonists (isoproterenol, terbutaline, prenalterol) in the absence and presence of the selective β-adrenoceptor antagonists metoprolol (β1) and IPS 339 (β2) demonstrates that both β1- and β2-adrenoceptors are involved in the inhibition of colonic motility. Neuronal blockade induced by tetrodotoxin suppressed the relatively high (68%) maximal response of prenalterol (partial β-adrenoceptor agonist) to 23%. The concentration-response curves for terbutaline (β2-selective agonist) and isoproterenol (nonselective agonist) were not influenced by tetrodotoxin. The results thus indicate that the β-adrenergic inhibition of spontaneous activity in the rat colon strip may be mediated at two functional levels within the colon wall: either by β2-adrenoceptors in the smooth muscle layer or by β1-adrenoceptors in the intramural ganglionic plexuses, which by neuronal elements are coupled to the effector smooth muscle.

ACTH-(1–24) antagonizes the contractile effect of morphine on the isolated rat colon

ACTH-(1–24) antagonizes the contractile effect of morphine on the isolated rat colon

Effect of prostaglandin (PG)-synthetase-inhibitors on met-enkephalin-,acetylcholine- and glucagon-stimulated motility of the isolated rat colon

Effect of prostaglandin (PG)-synthetase-inhibitors on met-enkephalin-,acetylcholine- and glucagon-stimulated motility of the isolated rat colon

Antiabsorptive effects of 16, 16 dimethyl prostaglandin E 2 and isolated rat colon

Ulcerative colitis is distinguished by abundant prostaglandin E 2 (PGE 2) in the stools and by severe diarrhea. To determine whether luminal PGE 2 alters normal colonic absorption, Na + and Cl −transport across isolated rat proximal colon were studied before and after 16, 16 dimethyl PGE 2 (dmPGE 2) addition to flux chambers. Luminal administration of dmPGE 2 significantly reduced the net mucosal to serosal fluxes of Na + and Cl −. These antiabsorptive tive effects of dmPGE 2 on Na + and Cl − active transport were reflected by a reduced metabolic rate of colonic tissue slices incubated with dmPGE 2. Addition of dmPGE 2 significantly reduced oxidation of glucose by the colon. Structurally, dmPGE 2 reduced the length of colonic mucosal microvilli, thereby decreasing absorptive surface area. These results suggest that PGE 2 released into the colonic lumen of patients with ulcerative colitis exerts antiabsorptive effects on the colon and in this way contributes to the associated diarrhea.

Oxalate transport across the isolated rat colon. A re-examination

A net absorption of oxalate and chloride was observed when isolated, shortcircuited segments of rat colon were bathed by a calcium-containing buffer. Removal of calcium promoted a two-fold decrease in transmural resistance, while the net chloride flux was reduced and the net oxalate transport abolished. It was concluded that net oxalate absorption was not observed in previous studies (employing calcium-free buffers) because calcium is required to maintain the integrity of the conductive pathways across colonic epithelia.

Action of enkephalin analogue FK 33-824 on the motility of the isolated rat colon

Action of enkephalin analogue FK 33-824 on the motility of the isolated rat colon

Inhibition of VIP-stimulated intestinal secretion and cyclic AMP production by somatostatin in the rat

The effect of somatostatin on colonic secretion induced by 10−8m vasoactive intestinal peptide (VIP), 10−2m theophylline, and 2 × 10−3m dibutyryl cyclic AMP was studied in muscle-stripped everted open rat colon sacs. The secretory response to VIP, measured as the decrease in net absorptive flow rate (microliters 30 min−1 mg−1 of dry weight), was maximal and equalled the responses to theophylline or dibutyryl cyclic AMP. Somatostatin (10−5m) blocked completely the secretory response to VTP but only partially the secretory response to theophylline or dibutyryl cyclic AMP. This difference in the extent of inhibition suggested that somatostatin exerted an inhibitory effect both before and after the point of generation of intracellular cyclic AMP. In order to test the hypothesis that one component of the action of somatostatin involved inhibition of the production of cyclic AMP, measurements of this nucleotide were made in isolated rat colon cells. Control levels of cyclic AMP measured by radioimmunoassay (12.6 ± 1.6 pmoles per 106 cells) were not affected by 10−5m somatostatin. VIP (5 × 10−8m) increased cyclic AMP levels 2-fold (P < 0.01) and this increase was blocked by somatostatin. The results indicated that somatostatin inhibits colonic secretion by exerting effects at two sites: one site lies at, and another beyond, the point of generation of intracellular cyclic AMP.

Contraction of the rat colon by sympathomimetic amines: Effect of methysergide and 5-HT desensitization

Metaraminol and phenylephrine induced relaxations of the isolated rat colon which were reversed into contractions following β-adrenergic blockade with oxprenolol. Phentolamine prevented the stimulatory effects of both amines. Pretreatment with methysergide abolished completely the responses to serotonin without altering significantly those induced by acetylcholine, 10 ng/ml, and adrenaline, 100 ng/ml. Tissue desensitization to serotinin abolished completely the responses to test doses of serotonin whereas it only reduced those to adrenaline. It is concluded that stimulatory effects of adrenaline on the rat colon can be dissociated from an action on serotonergic receptors and that these effects would rather involve the participation of α-adrenergic receptors.

Specific antagonists for the myotropic action of angiotensin II and angiotensin I on the isolated rat colon.

Substitutions of 8 phenylalanine with L-alanine and D-phenylalanine abolish the myotropic action of the angiotensin II (AT(II)) analogues and confer inhibitory properties on the molecule. [8-L-Ala]-AT(II) and [8-D-Phe]-AT(II) antagonize specifically the myotropic action of AT(II) and angiotensin I (AT(I)) on the rat colon, while the action of other myotropic agents (acetylcholine, 5 hydroxytryptamine) is not modified.

Action of angiotensins and synthetic renin substrate on isolated rat colon.

AIKEN and Vane have reported1 the independent actions of angiotensin I (AI) and angiotensin II (AII) on various isolated smooth muscle preparations, including rat colon. Tney showed that the apparent contractile action of AI on these preparations depended primarily on its in situ conversion to All by “converting enzyme” and that when the activity of the enzyme was inhibited by a pentapeptide extracted from Bothrops jararaca venom, the residual contractile action, probably due to unchanged AI, was very small. I have investigated the action of synthetic tetradecapeptide renin substrates (TPRS) on rat colon, in the light of the known actions of AI and All.

Stimulatory effects of catecholamines on the isolated rat colon after beta-adrenergic blockade with oxprenolol and propranolol

The relaxation of the isolated rat colon induced by either adrenaline or noradrenaline was generally reversed into a contraction by beta-adrenergic blockade with oxprenolol and propranolol, the former being more effective. Phentolamine, an alpha-receptor blocking agent, greatly diminished the contractions of the tissue to adrenaline and noradrenaline following beta-blockade. It is suggested that stimulation of alpha-receptors can result in contraction of the smooth muscle of the isolated rat colon.

Antagonism by prostaglandins of the responses of various smooth muscle preparations to sympathomimetics.

PROSTAGLANDINS are known to produce two types of effects on smooth muscle. First, they may produce direct and relatively short-lived effects, such as stimulation of the isolated uterus or relaxation of the isolated tracheal chain preparation. Secondly, in doses too low to produce a direct effect, they may produce a long-term potentiation of the effects of other stimulants. These two effects have been investigated in the isolated guinea-pig uterus1. The long-term potentiation of the effects of other stimulants which is induced by brief exposure of the guinea-pig uterus to low concentrations of PGE1 or PGE2 appears to be non-specific with respect to the potentiated stimulant, and can be demonstrated with a variety of stimulants including vasopressin, oxytocin and histamine, and also with electrical field stimulation2. The potentiating effect, however, is fairly specific to prostaglandins of the PGE series. This type of effect is not restricted to the guinea-pig uterus and can be demonstrated on the isolated rat colon or vas deferens (Fig. 1).

The binding of atropine to bovine serum albumin

The binding of atropine to bovine serum albumin (BSA) as a model system was studied by ultrafiltration, spectrophotometry, and biological assay. Binding of atropine increased with an increase in pH from 5 to 8; the number of binding sites was approximately 20 at pH 6 and 100 at pH 8. Exhaustively acetylated BSA still bound some atropine when studied by ultrafiltration, although spectrophotometry did not show the interaction with atropine. Cysteine, which fully inhibited the spectrophotometric interaction of atropine with BSA, reduced the binding only partially, when determined by ultrafiltration. The requirement of the free amino groups of the protein for the interaction could be demonstrated by spectrophotometry. The participation of other functional groups of the protein was discussed. Atropine-protein binding and its inhibition by cysteine was demonstrated biologically on the isolated rat colon.

Antagonism between heparin and 5-hydroxytryptamine (serotonin) in vitro

In the isolated rat colon and uterus, heparin caused inhibition or elimination of contractions occurring after the administration of 5-hydroxytryptamine. In contrast to this, neither Tyrode's solution, nor glycogen, nor dextran produced any change in the response of these organs to 5-hydroxytryptamine.