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INTESTINAL ISCHAEMIA - A PATHOPHYSIOLOGIC STUDY OF BIOCHEMICAL MODIFICATION OCCURING
L Santacroce, S Gagliardi, R Lovero*
Dept. of Dentistry and Surgery - Sect. of General Surgery (CLOPD); *DIMO - Sect. of Clinical Pathology, University of Bari-Italy.
Nowadays intestinal ischaemia is a pathologic event, well known and well described above all in its morphological and pathophysiological aspects. But, while the approach which has been used till now has been based on the observation of the damage suffered by the whole organ, using physiological methods, or on tissues, using optic and electron microscopy, few studies have been done to investigate the alterations induced by ischaemia on cellular components.
In this study we have investigated the possible modifications occurring in the intestinal ephitelium during intestinal ischaemia.
INTRODUCTION
Clinical disorders of the gastro-intestinal tract caused by vascular diseases are rare in clinical practice. In this group of disorders the main syndromes are caused by intestinal ischaemia sufficient to cause cell death. This determines three main pathological modifications: 1) transient tissue damage with mucosal ulceration and inflammatory response which disappears completely, leaving structure and function unimpaired; 2) more severe damage causing structural changes, in the submucosal tissues as well as in the mucosal cells, determining the development of strictures; 3) necrosis of all layers of the intestinal wall leading to perforation of the gut.
Gastroenterologists have become increasingly aware of the clinical relationships among these pathological processes but slowly progressed in developing methods to evaluate the consequences on gastro-intestinal tract.
Nowadays intestinal ischaemia is a pathologic event, well known and well described above all in its morphological and pathophysiological aspects.[9] But, while the approach which has been used till now has been based on the observation of the damage suffered by the whole organ, using physiological methods, or on tissues, using optic and electron microscopy,[13] few studies have been done to investigate the alterations induced by ischaemia on cellular components.
In this study we have investigated the possible modifications occurring in the intestinal epithelium during ischaemia. Overall we have investigated if, in the ischaemic intestine of rats, the damage imply the alteration of: 1) enzymatic activity of either apical (maltase, leucin-aminopeptidase and alkaline phosphatase) or basolateral (Na+ -K+ -ATPase) membrane enzymes, or 2) Na+ -K+ permeability of apical membrane.
In order to measure enzymatic activity and Na+ -K+ permeability, homogenate from scraped intestinal mucosa and purified brush-border membrane vesicles were used.
In fact brush-border membranes can be isolated as vesicles1 and used to study ionic permeabilities and fluxes of several substrates.[1-2]
MATERIAL AND METHODS
All chemicals were of analytical grade and Wistar rats were used. Any material was purchased from the commercial source.
Preparation of brush-border membrane vesicles
A group of 34 Winstar rats (both sex, average poid 350-400 g), previously fasted for 18-24 hours, were divided into two groups of 15 and 19 rats respectively, and were initially anaesthetised with 50 mg/kg of ketamine.
Then, all animals underwent midline laparotomy and the last ileal loop was isolated (about 4 cm in length). The 15 animals of the first group were used as a control; the 19 animals of the other group had strangulation of the vascular pedicle and this situation was maintained for 60 min. All the animals were successively killed, prior taking the isolated last loop.
The intestinal segments were immediately excised, sectioned in their length and rinsed in ice-cold solution 0.9% NaCl. Three grams (fresh weight) of scraped intestinal mucosa (from 3-4 animals) were homogenized for three minutes in a blender, in 30 ml of a medium containing 300 mM mannitol and 12 mM tris-(hydroxymethyl)-aminomethane (Tris), adjusted to pH 7.1 with ethyleneglycol-bis (b-aminoethylether)-N, N’-tetra acetic acid (EGTA).
Just before starting the homogenization, 120 ml of cold distilled water were added to make the homogenate medium hypoosmotic.
Five milliliters of this homogenate were taken for the enzymatic activity measurements: the rest of the homogenate was employed to prepare brush-border membrane vesicles (BBMV) according to a slightly modified Mg2-EGTA precipitation technique.2
The homogenate, after addition of 1.5 ml of MgCl2, 1.2 M, was left on ice for 15 minutes and centrifuged at 4,500 rpm for 15 minutes; the resulting supernatant was centrifuged again at 18,000 rpm for 30 minutes.
The pellet was re-suspended in 35 ml of a solution containing 60 mM of mannitol and 5 mM of EGTA, adjusted to pH 7.4 by Tris.
A second Mg2+ precipitation was performed as described above with two centrifugation steps at 7,000 rpm for 15 min and 18,000 rpm for 30 min.
The resulting pellet was re-suspended in a proper buffer for the permeability experiments and centrifuged at 18,000 rpm for 30 min. The final pellet (BBMV fraction) was re-suspended in the permeability buffer by passing it 30 times through a fine-gauge needle. All procedures were performed at temperature from 0 to 4 Co. Centrifugations were carried out using a J2-21 Beckman centrifuge with a JA-20 rotor.
ENZYME ASSAYS
Enzymatic activities measurements
All enzymatic activities were measured in the starting homogenate.
The enzymatic activity of leucin-aminopeptidase (EC 3.4.11.1) was measured spectrophotometrically at 405 nm and 37oC by using as substrate L-leucin-4-nitronilide (LEUPA) and by measuring the absorbance of p-nitroaniline.[11]
The enzymatic activity of maltase (EC 3.2.1.20) was measured at pH 6.7 in the presence of 100 mM imidazole, 15 mM, MgCl2 and 25 mM maltose; glucose production was assayed using the Sigma kit 16-UV.
The enzymatic activity of Na+ -K+ - stimulated ATPase (EC 3.6.1.3) was measured at 340 nm and 37oC by the redox reaction of NADH +H+/NAD+.1 This enzymatic activity was evaluated as the difference of ATP-asic activity without and in presence of ouabain (g-strofantin) that completely inhibits the Na-K ATPase. Isolation of rat brush-border membrane vesicles
The isolation of rat intestinal BBMV was followed by the measuring of the activity of enzymes known to be characteristic markers of different cellular organelles or components in the starting homogenate and in the final membrane fraction.
Alkaline phosphatase1, leucine aminopeptidase11 and maltase were measured as the marker enzymes for the BBM.
Maltase was measured at pH 6.7 in the presence of 100 mM imidazole, 15 mM MgCl2 and maltase; glucose production was assayed using the Sigma kit 16-UV.
The enrichment factors for these three markers (enzyme activities of final purified membrane pellet compared with those of the initial tissue homogenate) were 13, 14 and 11, respectively.
The methods used for evaluating enrichment factors for these enzymes were the same previously described.
Na+ -K+ stimulated adenosintriphosphatase was taken as the marker enzyme for the basolateral membranes.
Contamination by endoplasmic reticulum was monitored analyzing KCN-insensitive NADH oxidoreductase activity in the presence of 0,9 mM KCN.
Succinate cytochrome-c oxidoreductase activity was measured as the marker enzyme for mitochondria. Negligible enrichment was found for marker enzymes of other cellular components, suggesting minimal contamination by basolateral or organelle membranes.
All the enzyme assays were carried out at 37oC.
Protein concentration was measured by a Bio-Rad kit using lyophilized bovine plasma gamma-globulin as standard.
All the measurements were carried out by a III Trace Beckman spectrophotometer.
Measurements of fluorescence quenching
Ionic permeability studies were carried out by using the Dis-C2-(5) fluorescent cyanine dye, then was measured with a Perkin Elmer LS-5 spectrofluorometer, equipped with an electronic stirring system and a thermostatized (37oC) cuvette holder; fluorescence signals were continuously recorded (by a Hitachi-Perkin Elmer 561).
Excitation and emission wavelengths were 645 and 665 nm and both slit widths were set to 10 nm. 10 ml of a 0.6 mM dye solution in ethanol, 10 ml of a 0.89 mM valinomycin solution in ethanol (or ethanol only for controls), 1960 ml of a cuvette buffer were injected into a glass cuvette; valinomycin/protein ratio was 62 mg/mg and dye/protein ratio was 19.4 mg/mg.
The fluorescence value was arbitrary set to 90 fluorescence units and 20 ml (160 mg of protein) of suspension vesicles were injected into the cuvette to start the experiment.
Intra and extravesicular buffer had the same ionic strength, pH, anion concentration and osmolarity.
RESULTS
Enzymatic activity rate
To establish the possibility, during the ischaemic period, of any damage in intestinal epithelium, and its extent we compared the enzymatic activities (in the starting homogenate from the scraped mucosa) of leucin-aminopeptidase, alkaline phosphatase and maltase, (as enzymes strictly associated with the enterocyte basolateral membrane) either in control rats or in rats in whose ischaemia was induced for one hour.
The measurement of the enzymatic activity of these enzymes was assumed as a parameter for the evaluation of the ischaemic damage due to the strangulation of the vascular peduncle. In the evaluation of the enzymatic activity in the homogenate, both in control and ischaemic rats, we have compared the enzymatic activities of leucin-aminopeptidase, alkaline phosphatase, maltase and Na-K ATPase.
The enzymatic activity values for the same enzyme were different in different experiments : a possible explanation could be the different functional state of the intestinal mucosa in any individual or the different amount of the mucosal proteins from the scraped mucosa in the single experiment.
Thus we compared the enzymatic activities by referring enzymatic activities from ischaemic rats as the per cent value of the enzymatic activity from control rats.
The averaged values (referred to n experiments) of the percentage values referred to the control for:
1)the enzymatic activity of the alkaline phosphatase was reduced to 46% of the control values;
2)the enzymatic activity of the leucin-aminopeptidase was reduced to 53% of the control value;
3)the enzymatic activity of the maltase was reduced to 41% of the control value;
4)the enzymatic activity of the Na+ -K+ -ATPase is reduced to 71% of the control value.K+ permeability
Brush-border membrane vesicles are a suitable system to study metabolite transports. In fact, they are as right-side out vesicles and so the orientation of the membrane and of its enzymes is the same which naturally occurs.
These vesicles can also maintain an inner negative membrane potential when the internal concentration of Na+ and K+ is higher than the external.
DiS-C2 (5) is fluorescent only when it is present outside the vesicles. On the contrary it is not fluorescent when it binds to the membrane (aspecific fluorescent quenching) or when it is distributed inside the membrane in response to an inner negative membrane potential (the dye is lipophylic and positively charged).
So, more negative the inside membrane potential is, more the external fluorescence decays. On the other hand, the width of membrane potential is directly related to:1)The ion concentration gradient existing between the inside and the outside of the vesicles (artificially created).
2)Ion permeabilities which naturally exist in the biologic membranes.
In order to generate an inside-negative membrane potential, vesicles were prepared in a buffer containing 100 nM KCl and diluted into a cuvette buffer to obtain an extravesicular KCl concentration of 1 nM.
Vesicles prepared in the buffer as described before were injected in the same buffer (trace a); the fluorescence quenching depends on the distribution of the dye inside the lipidic bilayer (aspecific quenching) and not by membrane potential that, in these conditions, was zero. Again, the vesicles were injected in a buffer containing 1 mM KCl (trace c); the quenching of fluorescence depends on the aspecific quenching and on the distribution of the dye inside the membrane due to membrane potential created by K+ gradient (inside negative); in fact K+ can pass through the membrane according to K+ permeability. When valinomycin causes K+ equilibrium across the membrane because valinomycin is a specific ionophore for K+. In these conditions we can obtain the maximal quenching of fluorescence; to this signal we can refer the signal obtained in trace c.
Under these experimental conditions by doing:
(trace c - trace a) (trace d-trace b)
We can measure K+ permeability both in control and ischaemic vesicles. Assuming 100% the value of K+ permeability in control vesicles. We can evaluate K+ permeability as per cent value of the control vesicles. Experiments carried out in the ischaemic rats showed that K+ permeability increases 50% with respect to the control value.DISCUSSION
The results of these experiments indicate:
1.Specific activities of brush-border enzymes maltase, leucin-aminopeptidase, alkaline phosphatase decrease in the homogenate of the scraped mucosa from rats in which intestinal ischaemia was induced, with respect to the homogenate from control rats.This means that the composition of the enzymatic pool of the homogenate from ischaemia and control rats is different. As specific activity of these enzymes remains almost unvaried for 24-48 hours the decreasing of the enzymatic proteins content, but not the decrease of their activity.
In particular, our findings about the decrease of alkaline phosphatase activity (during the ischaemia) confirms the hypothesis of Rosato and colleagues.6 who found that the intestinal fraction of this enzyme (which is inhibited by L-phenylalanine) disappears from the seric enzyme pool;
2.We have found that the enzymatic activity of basolateral enzyme Na+ -K+ ATPase is reduced to 71% of the control value; this reduction is smaller than that founded for the brush border enzymes; this means that a smaller amount of basolateral membranes, with respect to brush border membranes, is present in the starting homogenate and this can be due only to a smaller loss of basolateral membranes, with brush border membrane preservation due to the ischaemic conditions. So we may conclude that the greater damage induced by one hour of ischaemia is focalized above all on the apical membranes (brush border) rather than in the basolateral ones.[7-8]
In conclusion, as enzymes are membrane proteins not directly connected to cellular metabolism and as their activity is related to the integrity of membrane itself, our results could indicate a loss of membrane enzymes due to 1) a loss of microvilli or 2) to a loss of the whole epithelial cells.
3)K+ permeability increases in brush border membrane obtained from ischaemic rats with respect to control. This clearly indicates that, during the ischaemic period, an alteration of the organization of apical membranes occurred. The studies with the fluorescent dye, marker of the ionic permeability, lead us to conclude that K+ permeability (that is considered an index of functional integrity) is markedly altered and seems indicate alterations of membrane structure and early damage (in one hour) induced by ischaemia in the structure of the brush border membranes.[10-12] In fact K+ permeability increases 50% with respect to the control. Since the amount of proteins used in each experiment was the same in vesicles from control and ischaemic rats, the only possibility for a different change in fluorescence quenching is that brush border vesicles from ischaemic rats are more leaky than those from control. The reason because these vesicles are more leaky needs to be further investigated, but this study tributes it an important role in the evolution of the ischaemic damage related to the alteration of the membrane structure, induced by superoxide anion and hydroxyl radicals.[10, 12-13] This hypothesis supported by the observation, in the same experimental model, of an increase of the extravascular peroxydasic activity that is closely connected with the removal of superoxide radicals.
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