Characterization of altered absorptive and secretory functions in the rat colon after abdominal irradiation: comparison with the effects of total-body irradiation.

The aim of this work was to determine the alterations in the absorptive and secretory functions of the rat colon after abdominal irradiation and to compare the effects of abdominal and whole-body irradiation. Rats received an abdominal irradiation with 8 to 12 Gy and were studied at 1, 4 and 7 days after exposure. Water and electrolyte absorption was measured in vivo by insertion of an agarose cylinder into the colons of anesthetized rats. In vitro measurements of potential difference, short-circuit current and tissue conductance were performed in Ussing chambers under basal and agonist-stimulated conditions. Most of the changes appeared at 4 days after abdominal irradiation. At this time, a decrease in water and electrolyte absorption in the colon was observed for radiation doses > or = 9 Gy. The response to secretagogues (VIP, 5-HT and forskolin) was attenuated after 10 and 12 Gy. Epithelial integrity, estimated by potential difference and tissue conductance, was altered from 1 to 7 days after 12 Gy abdominal irradiation. These results show that the function of the colon was affected by abdominal irradiation. Comparison with earlier results for total-body irradiation demonstrated a difference of 2 Gy in the radiation dose needed to induce changes in the function of the colon.     

Functional changes in the rat distal colon after whole-body irradiation: dose-response and temporal relationships

The aim of this study was to determine the acute radiation response of the rat distal colon by in vivo and in vitro measurements of the functions of the colon over a range of radiation doses. Rats received a whole-body irradiation of 2 to 12 Gy and were studied from 1 to 7 days after exposure. In vivo water and electrolyte absorption was measured by insertion of an agarose cylinder in the colon of anesthetized rats. In vitro transepithelial electrical parameters (potential difference, short-circuit current, transepithelial conductance) were measured in Ussing chambers in basal and agonist-stimulated conditions. In vivo and in vitro functional studies were completed by standard histological analyses. The majority of functional modifications appeared at 4 days after exposure. At this time, a dose-dependent decrease in absorption of water and sodium/chloride ions in the colon was noted. In contrast, a twofold increase in potassium secretion was observed for every radiation dose studied. The response to secretagogues was attenuated at doses >8 Gy. Modifications of basal transepithelial electrical parameters together with marked histological alterations were observed at 4 days with the higher doses (>/=10 Gy). In conclusion, these results show that functions of the colon are affected by irradiation and may contribute to diarrhea induced by ionizing radiation.     

Alterations of the VIP-stimulated cAMP pathway in rat distal colon after abdominal irradiation

Ionizing radiation induces hyporesponsiveness of rat colonic mucosa to vasoactive intestinal peptide (VIP). Possible mechanisms responsible for this hyporesponsiveness of the cAMP communication pathway in rat colon were investigated. VIP- and forskolin-stimulated short-circuit current (I(sc)) responses were studied after a 10-Gy abdominal irradiation in Ussing chambers as well as in single, isolated crypts. Adenylyl cyclase (AC) activity and VIP receptor characteristics were determined in mucosal membrane preparations. In addition, alterations in crypt morphology were studied. Impaired secretory responses to VIP and forskolin were observed 4 days after irradiation (decrease of 80%). cAMP analog-stimulated I(sc) responses were unchanged. In isolated crypts, VIP- and forskolin-stimulated cAMP accumulation was markedly reduced by 80 and 50%, respectively. VIP-stimulated AC activity and VIP receptor number were decreased in membrane preparations. No major change of cellularity was associated with these functional alterations. In conclusion, the decreased secretory responses to VIP of rat colon are associated with reduced cAMP accumulation, decreased AC activity, and diminution of VIP receptor numbers without a marked decrease of crypt cell number.     

Alterations in spontaneous contractions in vitro after repeated inflammation of rat distal colon

In inflammatory bowel disease, smooth muscle function reportedly varies with disease duration. The aim of these studies was to determine changes in the control of spontaneous contractions in a model of experimental colitis that included reinflammation of the healed area. The amplitude and frequency of spontaneous contractions in circular smooth muscle were determined after intrarectal administration of trinitrobenzenesulfonic acid in rat distal colon. With the use of a novel paradigm, rats were studied 4 h (acute) or 28 days (healed) after the initial inflammation. At 28 days, rats were studied 4 h after a second inflammation (reinflamed) of the colon. Colitis induced transient increases in the amplitude of spontaneous contractions coincident with a loss of nitric oxide synthase activity. The frequency of contractions was controlled by constitutive nitric oxide in controls. Frequency was increased in healed and reinflamed colon and was associated with a shift in the dominance of neural constitutive nitric oxide synthase control to that of inducible nitric oxide synthase (iNOS). The initial colitis induced a remodeling of the neural control of spontaneous contractions reflecting changes in their regulation by constitutive nitric oxide synthase and iNOS.     

Segmental differences in K-transport across rabbit proximal and distal colon in vivo and in vitro

Net K-transport was investigated in three different segments of rabbit proximal and distal colon. In vivo studies used a dialysis method, and in vitro studies an everted sac preparation. Results from both methods are in good agreement and show net K-absorption in the two segments of the proximal colon, and net K-secretion in the distal colon. K-absorption in the proximal colon seems to be an active mechanism, because K is transported against an electrical potential gradient. The present study is a comparative approach to demonstrate the heterogeneity of an organ function, and represents the first direct comparison of K-transport in various segments of rabbit colon under in vitro and in vivo conditions.     

Intestinal perfusion in vivo for the study of absorptive processes.

1. Intestinal absorption can be studied by in vitro and in vivo techniques. Among the in vivo ones, intestinal perfusion is the one more employed. 2. Intestinal perfusion could be performed by a simple perfusion of an intestinal segment or by a double perfusion of the intestine and the vascular bed simultaneously. 3. The double perfusion has the advantage of measuring the substrate appearance in the vascular circuit. 4. In this review we compare the different techniques described in the literature, paying attention to their advantages. 5. The best method is the one that maintains the animal alive throughout the experiment, because it provides information about intestinal absorption under conditions similar to the natural ones.     

Opioid receptor agonists in the rabbit colon: comparison of in vivo and in vitro studies

Myoelectrical activity was recorded in the proximal and distal colon of rabbits using chronically implanted electrodes. The motility in both the proximal and distal colon was inhibited by the intravenous (IV) administration of the following opioid agonists for mu receptors: morphine and fentanyl, kappa receptors: ethylketazocine (EKC) and U 50 488 H, and delta receptors: D-Ala2 D-Leu5-enkephalin (DADLE) and D-Ser2 Leu-enkephalin-Thr6 (DSLET). In contrast, the myoelectric activity in the distal colon was increased during the infusion of an endogenous kappa opioid agonist, dynorphin (DYN). All of these effects were prevented by naloxone pretreatment. During in vitro studies using extraluminal force transducers, fentanyl, U 50 488 H and DSLET inhibited spontaneous contractions of the proximal colon, but U 50 488 H and DSLET caused a substantial increase in the motility of the distal colon. The observed motor responses in the proximal and distal colon following opioid agonist administration indicate that the control of these two intestinal segments may be different. It is suggested that the stimulatory effect of dynorphin on the distal colon is peripherally-mediated while inhibition of the whole colon by opioid agonists regardless of subtypes seems to be centrally-mediated.     

In vivo effects of tunicamycin on the secretory processes of rat parotid glands

Summary The morphological and functional effects of tunicamycin were studied in rat parotid glands at the stage of the reformation of secretory granules following secretory stimulation by isoproterenol. Tunicamycin inhibited the incorporation of (³H)-mannose into the acid-insoluble fraction but had no effect on total protein synthesis as determined by the incorporation of (¹⁴C)-leucine. Thus the administration of tunicamycin in vivo inhibits the synthesis of mannose-rich glycoproteins in a manner similar to that in an in vitro system. The ultrastructure of the acinar cell showed little change following treatment with this drug, except that the number of reaccumulated secretory granules was greater than in the control. Amylase secretion stimulated by isoproterenol was inhibited in tunicamycin-treated cells, but did not decrease following treatment with N⁶,2-O-dibutyryladenosine 3-5-cyclic monophosphate, a secretory stimulator bypassing the -receptor. A radio-receptor assay using (³H)-dihydroalprenolol and direct localization using the fluorescent -adrenergic blocker 9-amino-acridin propranolol showed a marked reduction in the binding activity of -receptor following treatment with tunicamycin. Thus the inhibition of N-linked glycosylation appears to produce profound effects on the -adrenergic receptor-adenylate cyclase complex of acinar cells, although the steps of the transport and the exocytotic discharge of secretory materials are not affected.     

Cationophore properties of the new polyether antibiotic Salinomycin investigated in distal rabbit colon in vivo and in vitro

The distal rabbit colon was used as a model to investigate the influence of the cationophore Salinomycin in vivo with a single-pass perfusion, and in vitro with a modified Ussing chamber technique. For in vivo experiments with labelled 14C-PEG as a volume marker in the perfusate, a dose of 10E-4 mol/l Salinomycin was used. Net water (53 microliters/h/cm), net chloride (3 mumol/h/cm) and net sodium (3.6 mumol/h/cm) absorption was not significantly influenced, but net potassium secretion (-3 mumol/h/cm) was decreased to zero and transepithelial potential (PD) reduced from -45 mV to -33 mV. 10E-4 mol/l Salinomycin, applied in vitro on the muscosal side, decreased PD in 80 min and 10E-3 mol/l in 30 min from 18 mV to zero. Both concentrations decreased the short-circuit current (Isc = 77 microA/cm2) in 60 min, respectively 30 min to 40 microA/cm2. After 60 min mucosal 10E-4 mol/l Salinomycin the Isc increased, resulting from a transepithelial conductance (Gt) increase from 3 to 40 mS/cm2. A dose-related effect was present on PD, Isc and Gt at concentrations between 10E-7 and 10E-6 mol/l. The unidirectional 22Na fluxes were increased to 20 times the control values and net Na transport disappeared. We conclude that Salinomycin when applied in usual doses (10E-4 mol/l) as a coccidiostatic feed additive, profoundly affects colonic electrolyte transport.     

Electron microscopy study of microcirculation in the rat cerebral cortex after total neutron irradiation

It was established that the ultrastructure of blood capillaries of the brain changes during the first six hours following whole-body neutron irradiation of rats (10 Gy) which was indicative of the capillary wall swelling, the increase in the microcirculatory bed permeability, and pericapillary edema development. Those processes seemed to be reversible since during this period no severe destructive changes were detected in the microvessel wall.     

Kinetics of phenylalanine absorption by the rat intestine in vivo after distal resection

The kinetics of L-phenylalanine absorption across rat small intestine in sham and 50% distal resected animals, in vivo, have been studied by perfusing jejunal loops and monitoring the disappearance of the substrate from the perfusate. After 5 months postresection the total phenylalanine absorption was increased. The relationship between total absorption of substrate and its concentration in the bulk phase shows a non-saturable component and a saturable one that can be inhibited by methionine, both in control and remnant jejunum. The slope of the line that represents the non-saturable component is greater in remnant jejunum, indicating that the apparent mass-transfer coefficient, K'D, was increased by distal resection. The kinetic analysis of the saturable component shows that Jmax was unaltered and the apparent semisaturation constant, K'M, was slightly decreased by distal small intestine resection. Correction of the kinetic constant for the unstirred water layer effects shows that the differences between 'real' KD values of the two experimental groups increase whereas 'real' KM values do not change significantly. This indicates that the observed increase in total intestinal absorption in resected animals appears to result from an increase in the intestinal passive permeability.     

Acute orexin effects on insulin secretion in the rat: in vivo and in vitro studies

Orexin-A and orexin-B are members of a family of newly described orexigenic hypothalamic neuropeptides. Scanty data are available suggesting the involvement of orexins in regulation of the secretion of pituitary hormones and in control of energy homeostasis. Present studies aimed to explain whether orexins affect blood insulin concentration and insulin secretion in the rat. To check this possibility, adult female rats were subcutaneously injected with different doses (1 or 2 nmol) of orexin-A or orexin-B. A bolus administration of orexin-A resulted in an increase in blood insulin (up to min 120) and glucose (60 min after injection) concentration. The higher dose of orexin-B, on the other hand, exerted effect on insulin secretion only at min 60 of experiment and neither doses changed blood glucose level. Only orexin-A stimulated insulin secretion in an in vitro perfusion system of the rat pancreas preparation, while orexin-B was less effective. The results demonstrate that orexins belong to a group of neuropeptides influencing insulin secretion and acting directly on the pancreas. Direct, at least partial, effect of orexin on insulin secretion may be connected with the regulation of metabolism by this peptide.     

Effect of arginine on angiogenesis induced by human colon cancer: in vitro and in vivo studies

This study investigated the effect of arginine (Arg) supplementation on angiogenesis in human colon cancer. The in vitro study investigated the effects of different Arg levels and inducible nitric oxide (iNO) synthase inhibitor on angiogenic protein expressions stimulated by SW480 cells. The results showed that the production of vascular endothelial growth factor (VEGF), basic fibroblast growth factor with 100 and 1000 μmol/L Arg and matrix metalloproteinase (MMP)-2 with 1000 μmol/L Arg was lower than that with 0 and 50 μmol/L Arg. Inhibition of iNO resulted in higher angiogenic protein expressions comparable with groups with low Arg administration, indicating that Arg administration at levels similar to or higher than physiological concentrations reduced the progression of colon cancer, and iNO may partly play a role in reducing angiogenesis. The in vivo study used a human colon cancer xenograft model in nude mice. Mice were inoculated with 1×10⁷ SW480 cells and assigned to two groups. The control group was fed a semipurified diet, while the experimental group was supplied an Arg-supplemented diet. After 5 weeks, tumors were harvested and spleens were excised for further analysis. Results showed that the MMP-2, MMP-9 and VEGF receptor levels in tumors were significantly lower, whereas tumor NO levels and spleen natural killer (NK) cell activities were higher in the Arg group than in the control group. These results were consistent with the in vitro study that dietary Arg supplementation inhibits the progression of colon cancer possibly by increasing NO secretion and consequently enhancing NK cell activity.     

Estrogens and cell multiplication in the adenohypophysis of the male rat: in vivo and in vitro studies

A single dose (1 microgram) of oestradiol sub-cutaneously injected to an immature male rat promotes a transitory increase of the pituitary mitotic activity, the maximum of which is reached between 32 and 48 hours ; the observed fluctuations are similar to those previously described for the thymidine kinase activity. In these conditions, the concentration of blood prolactin remains unaltered, as were those of LH and FSH. It follows that hyperplasy of the pituitary can be quickly induced by doses of oestrogen that do not affect significantly the hormone release. Using Moxestrol, a synthetic oestrogen not bound by the oestradiol plasma binding protein, we show that in the very young rat, the in vivo responsiveness of the pituitary increases and reaches its maximum by day 17. This results can be tentatively related to the ontogeny of the oestradiol receptors in the pituitary described by others ; all our attempts to induce the thymidine kinase in cultured glands remained unsuccessful.     

In vitro and in vivo studies of boron neutron capture therapy: boron uptake/washout and cell death

Boron neutron capture therapy (BNCT) is a binary radiotherapy based on thermal-neutron irradiation of cells enriched with (10)B, which produces α particles and (7)Li ions of short range and high biological effectiveness. The selective uptake of boron by tumor cells is a crucial issue for BNCT, and studies of boron uptake and washout associated with cell survival studies can be of great help in developing clinical applications. In this work, boron uptake and washout were characterized both in vitro for the DHDK12TRb (DHD) rat colon carcinoma cell line and in vivo using rats bearing liver metastases from DHD cells. Despite a remarkable uptake, a large boron release was observed after removal of the boron-enriched medium from in vitro cell cultures. However, analysis of boron washout after rat liver perfusion in vivo did not show a significant boron release, suggesting that organ perfusion does not limit the therapeutic effectiveness of the treatment. The survival of boron-loaded cells exposed to thermal neutrons was also assessed; the results indicated that the removal of extracellular boron does not limit treatment effectiveness if adequate amounts of boron are delivered and if the cells are kept at low temperature. Cell survival was also investigated theoretically using a mechanistic model/Monte Carlo code originally developed for radiation-induced chromosome aberrations and extended here to cell death; good agreement between simulation outcomes and experimental data was obtained.