Indomethacin reduces short-circuit current and oxygen consumption in normal and chronically hypoxic rat colon / La indometacina reduce la corriente de cortocircuito y el consumo de oxígeno en elcolon distal de ratas con hipoxia crónica

Chronic hypobaric hypoxia is a physiological environmental stressor. While its effects on most major organsystems have been extensively studied, few works have addressed hypoxia-induced changes in intestinal transport.The effects of cyclooxygenase blockade with indomethacin on short-circuit current (Isc) and oxygen consumption(QO2) of the distal colonic epithelium ofcontrol rats and rats submitted to hypoxia for 10 days at 0.52 atm were studied. Isolated mucosae weremounted in an Ussing chamber modified for measuring QO2 while preserving transepithelial vectorial transport. Amiloride was added to the mucosal hemichamber to block a sodium component of Isc present in hypoxic rats. In this condition, basal Isc did not differ between the hypoxic and the control group, but QO2 was higher in the former. Indomethacin (30 ìmol/L)reduced Isc to the same extent in both groups, but QO2 reduction was larger in the hypoxic group. Pharmacologicalblockade of chloride secretion and a low-chloride solution abolished the indomethacin-induced reductionsof Isc in both groups, and the reduction of QO2 in controls, and attenuated but did not suppress the QO2 reduction in the hypoxic group. Linear regressionanalysis of QO2 changes versus Isc changes yielded a significant correlation for both groups, with regression lines with the same slope, but a higher position in hypoxic hypoxic animals. Results suggest that spontaneously released prostaglandins are equally important for maintaining colonic chloride secretion in hypoxic as in normoxic rats, but that, in the former, indomethacin has an additional effect on QO2 which is unrelated to ion transport.

La hipoxia hipobárica crónica es un estresante ambiental fisiológico. Aunque sus efectos se han estudiado en lamayoría de los sistemas orgánicos, hay pocos trabajos sobre su influencia en el transporte intestinal. Se estudió el efecto del bloqueo de la ciclooxigenasa con indometacina sobre la corriente de cortocircuito (Isc), el consumo de oxígeno (QO2) del epitelio del colon distal de ratas controles y fueron sometidas a hipoxia durante 10 díasa 0,52 atm. Se montaron preparados de mucosa aislada en una cámara de Ussing modificada para medir QO2 preservando el transporte vectorial transepitelial. Se añadió amilorida a la hemicámara mucosa para bloquear un componente de la Isc debido al sodio presente en ratas hipóxicas. En esta condición, la Isc basal fue similar en ambos grupos, pero el QO2 fue mayor enlos controles. La indometacina (30 mmol/L) redujo igualmente la Isc en ambos grupos; siendo la disminuciónde QO2 mayor en el hipóxico. El bloqueo de la secreció de cloruro (farmacológico y por omisión del ión) suprimió la disminución de Isc en ambos grupos y deQO2 en el control, y redujo, sin abolir, la disminución de QO2 en el hipóxico. El análisis de regresión lineal de cambios en QO2 versus cambios en Isc mostró en ambos grupos correlación significativa con líneas de regresiónde igual pendiente, pero más alta en el hipóxico. Los resultados sugieren que las prostaglandinas liberadas espontáneamente son igualmente importantes en mantener la secreción de cloruro en ratas hipóxicas y normóxicas,pero en las primeras la indometacina tiene además un efecto depresor del QO2 no relacionado con el transporte iónico.

Increased oxygen consumption caused by cAMP- and Ca2+-mediated chloride secretion in rat distal colon

El transporte iónico epitelial exige aporte de ATP provisto por el metabolismo aeróbico. En el colon distal de rata, la secreción de cloruro explica la mayor parte del transporte electrogénico medido como corriente de cortocircuito (ISC). La inhibición de la secreción basal de cloruro reduce el consumo epitelial de oxígeno (QO2), mientras que la serotonina aumenta proporcionalmente ISC y QO2. El efecto de la serotonina es mediado por receptores 5HT4 acoplados a adenilato ciclasa medianteproteína G estimulante (GS). En este trabajo seestudió si el aumento del QO2 asociado con la secreción de cloruro es un efecto común a otros agentes que actúan sobre cAMP o Ca2+. Los efectos del inhibidor de la fosfodiesterasa, 3-isobutil-1-metilxantina (IBMX) y del agonista muscarínico carbacol (ambos a 0.1 mmol/L) se evaluaron en la mucosa aislada del colon distal de rata montado en una cámara de Ussing modificada para determinación continua de la concentración de oxígeno, permitiendo medir QO2. Se compararon la ISC y el QO2 basales con las resultantes del añadido de serotonina (control activo), IBMX, carbacol, o IBMX y carbacol. Todos aumentaron proporcionalmente ISC y QO2. Aunque el efecto de IBMX solo fue modesto y el del carbacol fue breve, se observó una sinergia cuando fueron agregados simultáneamente. El análisis de regresión lineal mostró una correlación significativa entre los incrementos de ISC y de QO2 (r2 = 0.746; P menor que 0.0001). Por tanto, la estimulación de la secreción de cloruro aumenta el QO2 independientemente de la vía efectora intracelular involucrada. Estos resultados corroboran el estrecho acoplamiento entre secreción de cloruro y QO2 en este epitelio.

Epithelial ion transport is dependent on ATP supply provided by aerobic metabolism. In the rat distal colon chloride secretion accounts for the largest portion of electrogenic transport measured as the short-circuit current (ISC). Inhibition of basal chloride secretion decreases epithelial oxygen consumption (QO2) in this tissue, while serotonin (5-hydroxytryptamine) proportionally increases both Isc and QO2. The effect of serotonin in this tissue is mainly mediated by 5HT4 receptors linked to adenylate cyclase through a stimulant G protein (GS). This work assessed whether the chloride secretion-induced increase in QO2 is a common characteristic of secretagogues, which act through either cAMP-dependent or Ca2+-dependent mechanisms. The effects of phosphodiesterase inhibitor 3-isobutyl-1- methylxantine (IBMX) and muscarinic agonist carbachol (both 0.1 mmol/L) were studied in rat distal colon isolated mucosa mounted in an Ussing chamber adapted for continuous measurement of oxygen concentration, allowing determination of QO2. Baseline ISC and QO2 were compared with ISC and QO2 after addition of either serotonin as an active control, IBMX, carbachol or IBMX plus carbachol. Each drug increased proportionally Isc and QO2. Although the effect of IBMX alone was modest and that of carbachol was short-lived, a synergic effect on Isc and QO2 was seen when both drugs were simultaneously added. Linear regression analysis showed a significant correlation between increases in ISC and QO2 (r2 = 0.746; P < 0.0001). Thus, stimulation of chloride secretion increases QO2 regardless of the intracellular pathway involved. These results extend previous findings, corroborating the close coupling between chloride secretion and QO2 in this epithelium

Pharmacological Suppression of rat distal colon chloride secretion requires two blockers

Rat distal colon epithelium is frequently employed to assess the effect of natural and synthetic chemicals on chloride secretion. Inhibition of chloride secretion is often reported as the loop diuretic-sensitive portion of short-circuit current (Isc). The present work challenges the hypothesis that a loop diuretic alone is able to fully abolish chloride secretion. Isolated mucosa preparations were mounted in an Ussing chamber. The effects on short-circuit current of replacement of normal Ringer by a low (2.5 mmol/L) Cl solution and of blockers of basolateral Na, K, 2 Cl symport (bumetanide), apical Cl channels (diphenylamine-2-carboxylate, DPC), and anion exchange (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, SITS) alone and combined were assessed. Low Cl reversibly decreased Isc by 76%. In normal Ringer, bumetanide decreased Isc by 65%. SITS also had a significant effect at the serosal side, but not at the apical side, where DPC caused a 40% decrease. Chloride replacement, bumetanide and DPC, but not SITS, increased epithelial resistivity. Combined blockade of Na, K, 2 Cl symport and apical Cl channels, of Na, K, 2 Cl symport and anion antiport, or of anion antiport and apical Cl channels was needed to achieve reduction of short circuit current to the same extent seen with chloride replacement. Present results indicate that Isc of the unstimulated epithelium is mostly due to chloride secretion, and at least two blockers are required to abolish it. This fact should be taken into account in studies of chloride secretion-stimulating agents.

Sodium-deprived rat distal colon epithelial response to acute hypoxia and reoxygenation

In normal rat distal colon isolated mucosa, basal short-circuit current (Isc) is mostly due to chloride secretion. Isc is depressed by a brief (5 min) acute hypoxia and overshoots above baseline during reoxygenation. Sodium deprivation raises serum aldosterone levels and leads to expression of functional epithelial sodium channels which are amiloride-sensitive. Thus, in sodium-deprived rats (SDRs) Isc is dependent on electrogenic sodium absorption. Since the ion primarily responsible for the Isc is different in each functional condition, it is not known whether hypoxia and reoxygenation affect SDRs epithelial response in the same way as in normal rats. Therefore the electrical behavior of isolated mucosa preparations from normal and SDRs was studied in an Ussing chamber, and the effect of the epithelial sodium channel blocker, amiloride sensitive, basal Isc than controls. Their response to hypoxia (expressed as a fraction of basal Isc) was similar to controls but upon reoxygenation their recovery was incomplete. SDRs response to hypoxia was not affected by amiloride at any concentration tested. However, post-hypoxic recovery was modified by amiloride in a concentration-dependent way: it was incomplete at 10(-8) M, complete at 10(-6) M, and at 10(-4) M it overshooted above baseline values. Therefore, in sodium-deprived rats, sodium channel blockade reverts the pattern of blunted recovery to the overshooting pattern seen normal rats. These results may be explained by two non-mutually exclusive hypotheses: Epithelial sodium channel blockade in sodium-deprived rats might (1) unmask a basal chloride conductance, and (2) interfere with a negative interaction between sodium chloride conductances.

Rat colon epithelium response to hypoxia is modified by intrinsic innervation blockade

Background/Aim: Short-circuit current (Isc) and transepithelial potential difference (PD) of rat distal colon decrease during acute hypoxia and overshoot on reoxygenation. It is not known whether tonic intrinsic nervous activity may influence these responses. Methods: Preparations lacking the submucosal plexus (isolet mucosa) and preparations retaining it (mucosa-submucosa) were mounted in Ussing chambers at 37 degrees Celsius and gassed with 95 percent O2 -5 percent CO2; Isc and PD were monitored. A 5-min hypoxia with 95 percent N2-5 percent CO2 was followed by reoxygenation. The procedure was repeated in the presence of the nervous blocking agent, tetrodotoxin (10(-6)M) in the serosal side of the chamber. Results: In the isolated mucosa (n=10) hypoxia reduced Isc by -55 + 5 percent and PD by -54 + 6 percent below baseline; reoxygenatory overschoots were, respectively, + 60 + 17 percent and + 16 percent. Tetrodotoxin slightly and transiently reduced baseline Isc (-16 + 2 percent) and PD (-14 + 3 percent), with a small resistivity increase. It did not significatively modify the responses to responses to either hypoxia or reoxygenation. In mucosa-submucosa preparations (n=9) hypoxia reduced Isc (-54 + 8 percent) and PD (-61 + 4 percent). On reoxygenation Isc and PD were increased, respectively, +30 + 5 percent and +19 + 6 percent over baseline. Tetrodotoxin reduced baseline Isc (-59,6 + 5 percent) and PD (61,3 + 6 percent). It enhanced hypoxic Isc and PD decreases (-80 + 5 percent), but not the reoxygenatory overschoots. Conclusions: 1) Tetrodotoxin affects baseline Isc and PD more intensely in submucosal plexus innervated preparations than in the isolated mucosa. 2) The epithelial electrical response to acute hypoxia appears to be modulated by tonic neural activity.

Colon epithelial electrical responses to acute hyposia and reoxygenation in vitro

Electrogenic epithelial transport depends on oxidative metabolism. Acute hypoxia and subsequent reoxygenation effects on short-circuit current (Isc), transepithelial potential difference (PD) and tissue resistivity (TR) of rat distal colon were assessed. The tissue was mounted in an Ussing chamber filled with Ringer-HCO3 -solution at 37 degrees Celsius and bubbled with 95 per cent O2- 5 per cent CO2 which was switched to 95 per cent N2- 5 per cent CO2 for inducing hypoxia; afterwards normal oxygenation was resumed. The effect of 5, 10, 15 and 20 min-hypoxic periods was assessed in isolataed mucosa preparations. Recovery was complete after 10- and 15-min hypoxia, but not after 20-min hypoxia. After 5-min hypoxia, an overshoot of Isc and PD was seen on reoxygenation. This effect was further characterized comparatively in mucosa-submucosa and isolated mucosa preparations. In the former (n=10), control values were Isc= 71,7 + 8,6 muA,. cm-2, PD = 9,7 + 1,6 mV and TR = 134,9 + 13,6 (. cm2. A 5-min hypoxia reduced Isc by 47.2 + 7,3 per cen+t and PD by 61,5 + 4,9 per cent. Peak values on reoxygenation were 28,1 + 4,1 per cent for Isc and 16,8 + 5,4 per cent for PD, over controls values. In the osilated mucosa (n=9), control values were Isc= 52,04 + 5,5 muA. cm-2, PD = 5,0 + 0,8 mV and TR = 101,04 + 10,5 (. cm2. In hypoxia, Isc decreased by 64,5 + 7,6 per cent and PD by 57,2 + 7,8 per cent. On reoxygenation peak values of 78,0 + 19,0 per cent and 87,5 + 17,1 per cent, respectively, were seen. The response to a 5 min-hypoxia was comparable, but that to reoxygenation was weaker and slower, in mucosa-submucosa than in isolated mucosa preparations. This may be explained by a hindrance to oxygen diffusion caused by the submucosal tissue. TR did not change with any period of hypoxia tested, but decreased slightly (8,9 + 1,3 per cent) upon reoxygenation in the mucosa-submucosa preparations. Ouabain (10(-3)M) markedly blunted the response to reoxygenation. We conclude that hypoxic periods of 20 min lead to irreversible functional deterioration. Hypoxia decreases electrogenic transepithelial pumping, which may allow sodium to accumulate intracellularly and, if the hypoxia is short enough to prevent damage to the epithelium, increase sodium pump activity when oxygenation is resumed.