Rectification of oxygen transfer through the rat colonic epithelium.

AIM: To assess whether higher sensitivity of colonic epithelium to hypoxia at the serosal side is associated with oxygen transfer asymmetry. METHODS: Rats were fed either with normal chow or a low-sodium diet. Tissues were mounted as flat sheets in a modified, airtight Ussing chamber with oxygen meters in each hemichamber. Mucosal samples from normal diet animals were studied under control conditions, in low-chloride solution and after adding chloride secretion inhibitors and chloride secretagogues. Samples from sodium-deprived rats were studied before and after ouabain addition. In separate experiments, the correlation between short-circuit current and oxygen consumption was analyzed. Finally, hypoxia was induced in one hemichamber to assess the relationship between its oxygen content and the oxygen pressure difference between both hemichambers. RESULTS: In all studied conditions, oxygen consumption was larger in the serosal hemichamber than in the mucosal one (P = 0.0025 to P < 0.0001). Short-circuit current showed significant correlation with both total oxygen consumption (r = 0.765; P = 0.009) in normoxia and oxygen consumption in the serosal hemichamber (r = 0.754; P = 0.011) during mucosal hypoxia, but not with oxygen consumption in the mucosal hemichamber. When hypoxia was induced in the mucosal hemichamber, an oxygen pressure difference of 13 kPa with the serosal hemichamber was enough to keep its oxygen content constant. However, when hypoxia was induced in the serosal hemichamber, the oxygen pressure difference with the mucosal hemichamber necessary to keep its oxygen content constant was 40 kPa (P < 0.0001). CONCLUSION: Serosal oxygen supply is more readily available to support short-circuit current. This may be partly due to a rectifying behavior of transepithelial oxygen transfer.

The effects of amiloride and age on oxygen consumption coupled to electrogenic sodium transport in the human sigmoid colon.

BACKGROUND/AIM: Aerobic metabolism is necessary for ion transport in many transporting epithelia, including the human colonic epithelium. We assessed the effects of the epithelial sodium channel blocker, amiloride, on oxygen consumption and short-circuit current of the human sigmoid epithelium to determine whether these effects were influenced by the age of the subject. MATERIALS AND METHODS: Segments of the sigmoid colon were obtained from the safety margin of resections performed in patients of 62-77 years of age. Isolated mucosa preparations were obtained and mounted in airtight Ussing chambers, fit for simultaneous measurement of short-circuit current and oxygen concentration, before and after blocking epithelial sodium channels with amiloride (0.1 mmol/L). Regression analyses were performed to assess the associations between short-circuit current, oxygen consumption, and age of the subject as well as to define the relationship between the decreases in short-circuit current and oxygen consumption after blockade. RESULTS: Epithelial sodium channel blockade caused an 80% reduction in short-circuit current and a 26% reduction in oxygen consumption. Regression analysis indicated that both changes were significantly related (r = 0.884;P = 0.0007). Oxygen consumption decreased by 1 m mol/h/cm2 for each 25 m A/cm2 decrease in short-circuit current. Neither short-circuit current nor oxygen consumption had any significant relationship with the age of the subjects. CONCLUSION: The decrease in epithelial oxygen consumption caused by amiloride is proportional to the decrease in short-circuit current and independent of the age of the subject.

Aldosterone increases oxygen consumption of rectal epithelia of normal, sodium-deprived and sodiumloaded rats.

INTRODUCTION: The colonic epithelium is a classical aldosterone target, but the effect of the hormone on the oxygen consumption rate (QO2 ) of this tissue is unknown. Objectives. We aimed at assessing, in the rectal epithelium of rats fed with diets of different sodium content, the effect of epithelial sodium channel (ENaC) blockade on short-circuit current (ISC ) and QO2 , and the acute effect of aldosterone incubation on ISC and QO2 . METHODS: Adult male rats were fed with normal, low or high-sodium diets for 8 days. Plasma sodium and serum aldosterone were measured. Isolated mucosa preparations from the rectal portion of the colon were mounted in Ussing chambers modified to measure ISC and QO2. RESULTS: Baseline ISC and QO2 were highest in sodium-deprived rats. Both were proportionally reduced by amiloride (0.1 mM) in this group and in the normal sodium group, but not in sodium-loaded rats. In separate experiments, incubation with aldosterone (10 nM) for 7 h increased ISC and QO2 in all groups; increases were larger in the normal and sodium-loaded groups. Amiloride decreased both ISC and QO2 , abolishing the differences between groups. Linear regression of the decrease in QO2 and ISC after amiloride showed the steepest slope for the sodium-deprived group and the flattest one for the sodium-loaded group. CONCLUSIONS: Baseline epithelial QO2 of sodium-deprived and control rats is reduced by ENaC blockade. Aldosterone increased QO2 proportionally to ISC augmentation in all groups, but the coupling between aerobic metabolism and electrogenic transport seems more efficient in sodium-deprived animals.

The effect of acute hypoxia on short-circuit current and epithelial resistivity in biopsies from human colon.

BACKGROUND AND AIMS: In isolated colonic mucosa, decreases in short-circuit current (ISC) and transepithelial resistivity (RTE) occur when hypoxia is either induced at both sides or only at the serosal side of the epithelium. We assessed in human colon biopsies the sensitivity to serosal-only hypoxia and mucosal-only hypoxia and whether Na, K-ATPase blockade with ouabain interacts with hypoxia. MATERIALS AND METHODS: Biopsy material from patients undergoing colonoscopy was mounted in an Ussing chamber for small samples (1-mm2 window). In a series of experiments we assessed viability and the electrical response to the mucolytic, dithiothreitol (1 mmol/l). In a second series, we explored the effect of hypoxia without and with ouabain. In a third series, we evaluated the response to a cycle of hypoxia and reoxygenation induced at the serosal or mucosal side while keeping the oxygenation of the opposite side. RESULTS: 1st series: Dithiothreitol significantly decreased the unstirred layer and ISC but increased RTE. 2nd series: Both hypoxia and ouabain decreased ISC, but ouabain increased RTE and this effect on RTE prevailed even during hypoxia. 3rd series: Mucosal hypoxia caused lesser decreases of ISC and RTE than serosal hypoxia; in the former, but not in the latter, recovery was complete upon reoxygenation. CONCLUSIONS: In mucolytic concentration, dithiothreitol modifies ISC and RTE. Oxygen supply from the serosal side is more important to sustain ISC and RTE in biopsy samples. The different effect of hypoxia and Na, K-ATPase blockade on RTE suggests that their depressing effect on ISC involves different mechanisms.

Electrogenic transport, oxygen consumption, and sensitivity to acute hypoxia of human colonic epithelium.

INTRODUCTION: It is recognized that epithelial ion transport depends on oxygen supply, but this dependence has not been characterized in the human colon in vitro despite its surgical and clinical implications. PURPOSES: The aim of this study is to measure the oxygen consumption of colonic epithelium under conditions which preserve vectorial ion transport and to assess the sensitivity of the human colonic epithelium short-circuit current (I (sc)) to acute hypoxia induced in vitro. METHODS: Isolated mucosa preparations from human sigmoid colon were placed in a modified Ussing chamber which allows simultaneous measurement of short-circuit current (I (sc)) and oxygen consumption (QO(2)). In separate experiments, the sensitivity to acute hypoxia induced in a conventional Ussing chamber was assessed. RESULTS: Basal mean ± SEM values (n = 8) were I (sc) = 3.3 ± 0.5 µEq h(-1) cm(-2) and QO(2) = 8.09 ± 0.55 µmol h(-1) cm(-2). The contribution of the serosal side to the oxygen supply was higher than that of the mucosal side (p = 0.0023). Ouabain reduced I (sc) by 70% (P < 0.0001) and QO(2) by 26% (n = 8; P = 0.0009), suggesting that a large fraction of QO(2) is needed to support ouabain-sensitive electrogenic transport. Induction of hypoxia at both sides of the Ussing chamber caused a rapid decrease in I (sc) after 2 min. I (sc) was also significantly depressed when hypoxia was induced by 5 min in the serosal side (n = 6, P < 0.0001), but was unaffected by hypoxia induced in the mucosal side. CONCLUSION: The present results allow a better understanding of the clinical consequences of acute hypoxia on intestinal ion transport.

Effects of chronic hypoxia on electrogenic transport and transport-related oxygen consumption in rat distal colon.

The distal colon epithelium of rats submitted to chronic hypoxia shows higher short-circuit current (Isc) which, unlike non-hypoxic rat epithelium, has an amiloride-sensitive component despite low serum aldosterone levels. Isc and oxygen consumption (QO2) were simultaneously measured in mucosae from rats submitted to 0.5 atm for 10 days and from control rats in a modified Ussing chamber. Hypoxia increased Isc but not QO2. The slope of the regression line between Isc and QO2 reduction after ouabain addition was decreased in epithelia from hypoxic rats (P = 0.03). Chloride secretion blockade reduced Isc and QO2 in both groups, while sodium channel blockade did so only in the hypoxic group. Dual blockade in hypoxic rat epithelia caused correlated (P = 0.0025) additive decreases in Isc and QO2. Presented results suggest that chronic hypoxia induces an improved coupling between QO2 and electrogenic ion transport, and electrogenic sodium absorption despite low aldosterone levels.

Single-channel response of hamster oocytes to fertilization with homologous spermatozoa.

Electrophysiological events occur early after fertilization, along with changes in intracellular Ca2+ concentration. Passive electrical parameters were determined in golden hamster oocytes by whole cell patch-clamp method. In separate experiments the effect of 4-aminopyridine on resting oocytes was tested. The single-channel patch clamp configuration was employed to assess the electrical response to fertilization with homologous sperm. Structure of oocytes submitted to patch clamp was evaluated with scanning electron microscopy and found to be preserved. Oocyte diameter was 70.2 +/- 2.2 microm; their resting parameters were: membrane potential 23.8 +/- 0.8 mV; total membrane specific resistance 519.1 +/- 94.6 ohms.cm2, and specific capacity 0.99 +/- 0.03 microF.cm(-2). Total membrane current was decreased by 42 % by 4-aminopyridine. Control oocytes and oocytes exposed to sperm differed in their membrane currents in response to a voltage ramp clamping membrane potential from - 100 mV to + 100 mV. In both cases, currents were largest at the most negative potentials, but sperm-exposed oocytes had larger currents. Additionally, while in control oocytes the current was inward at negative potentials but outward at positive potentials, in the presence of spermatozoa oocytes was inward within the whole voltage range tested. This latter current may represent Ca2+ entry.

Single-channel response of hamster oocytes to fertilization with homologous spermatozoa

Electrophysiological events occur early after fertilization, along with changes in intracellular Ca2+ concentration. Passive electrical parameters were determined in golden hamster oocytes by whole cell patch-clamp method. In separate experiments the effect of 4-aminopyridine on resting oocytes was tested. The single-channel patch clamp configuration was employed to assess the electrical response to fertilization with homologous sperm. Structure of oocytes submitted to patch clamp was evaluated with scanning electron microscopy and found to be preserved.Oocyte diameter was 70.2 ± 2.2 µm; their resting parameters were: membrane potential 23.8 ± 0.8 mV; total membrane specific resistance 519.1 ± 94.6 Ù.cm2, and specific capacity 0.99 ± 0.03 µF.cm-2. Total membrane current was decreased by 42 % by 4-aminopyridine.Control oocytes and oocytes exposed to sperm differed in their membrane currents in response to a voltage ramp clamping membrane potential from - 100 mV to + 100 mV. In both cases, currents were largest at the most negative potentials, but sperm-exposed oocytes had larger currents. Additionally, while in controloocytes the current was inward at negative potentials but outward at positive potentials, in the presence of spermatozoa oocytes was inward within the whole voltage range tested. This latter current may represent Ca2+ en try

Single-channel response of hamster oocytes to fertilization with homologous spermatozoa

Electrophysiological events occur early after fertilization, along with changes in intracellular Ca2+ concentration. Passive electrical parameters were determined in golden hamster oocytes by whole cell patch-clamp method. In separate experiments the effect of 4-aminopyridine on resting oocytes was tested. The single-channel patch clamp configuration was employed to assess the electrical response to fertilization with homologous sperm. Structure of oocytes submitted to patch clamp was evaluated with scanning electron microscopy and found to be preserved.Oocyte diameter was 70.2 ± 2.2 Am; their resting parameters were: membrane potential 23.8 ± 0.8 mV; total membrane specific resistance 519.1 ± 94.6 Ù.cm2, and specific capacity 0.99 ± 0.03 AF.cm-2. Total membrane current was decreased by 42 % by 4-aminopyridine.Control oocytes and oocytes exposed to sperm differed in their membrane currents in response to a voltage ramp clamping membrane potential from - 100 mV to + 100 mV. In both cases, currents were largest at the most negative potentials, but sperm-exposed oocytes had larger currents. Additionally, while in controloocytes the current was inward at negative potentials but outward at positive potentials, in the presence of spermatozoa oocytes was inward within the whole voltage range tested. This latter current may represent Ca2+ en try(AU)

Single-channel response of hamster oocytes to fertilization with homologous spermatozoa

Electrophysiological events occur early after fertilization, along with changes in intracellular Ca2+ concentration. Passive electrical parameters were determined in golden hamster oocytes by whole cell patch-clamp method. In separate experiments the effect of 4-aminopyridine on resting oocytes was tested. The single-channel patch clamp configuration was employed to assess the electrical response to fertilization with homologous sperm. Structure of oocytes submitted to patch clamp was evaluated with scanning electron microscopy and found to be preserved.Oocyte diameter was 70.2 ± 2.2 Am; their resting parameters were: membrane potential 23.8 ± 0.8 mV; total membrane specific resistance 519.1 ± 94.6 Ù.cm2, and specific capacity 0.99 ± 0.03 AF.cm-2. Total membrane current was decreased by 42 % by 4-aminopyridine.Control oocytes and oocytes exposed to sperm differed in their membrane currents in response to a voltage ramp clamping membrane potential from - 100 mV to + 100 mV. In both cases, currents were largest at the most negative potentials, but sperm-exposed oocytes had larger currents. Additionally, while in controloocytes the current was inward at negative potentials but outward at positive potentials, in the presence of spermatozoa oocytes was inward within the whole voltage range tested. This latter current may represent Ca2+ en try(AU)

Indomethacin reduces short-circuit current and oxygen consumption in normal and chronically hypoxic rat colon.

Chronic hypobaric hypoxia is a physiological environmental stressor. While its effects on most major organ systems 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 of control rats and rats submitted to hypoxia for 10 days at 0.52 atm were studied. Isolated mucosae were mounted 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 micromol/L) reduced Isc to the same extent in both groups, but QO2 reduction was larger in the hypoxic group. Pharmacological blockade of chloride secretion and a low-chloride solution abolished the indomethacin-induced reductions of 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 regression analysis 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 bypoxic animals. Results suggest that spontaneously releasedprostaglandins 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.

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.

Indomethacin reduces short-circuit current and oxygen consumption in normal and chronically hypoxic rat colon

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 ýmol/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.(AU)

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.(AU)

Increased oxygen consumption caused by cAMP- and Ca(2+)-mediated chloride secretion in rat distal colon.

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 (I(SC)). 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 Ca(2+)-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 I(SC) and QO2 were compared with I(SC) 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 I(SC) 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.

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

Increased oxygen consumption caused by cAMP- and Ca(2+)-mediated chloride secretion in rat distal colon.

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 (I(SC)). 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 Ca(2+)-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 I(SC) and QO2 were compared with I(SC) 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 I(SC) 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.

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. (AU)

Oxygen consumption and chloride secretion in rat distal colon isolated mucosa.

The aerobic metabolic cost of chloride secretion was studied in rat distal colon isolated mucosa under several conditions by simultaneous measurement of short-circuit current and oxygen consumption under conditions that preserve vectorial ion transport. A low-chloride solution and the presence of bumetanide plus diphenylamine-2-carboxylate reduced short-circuit current by 75% and oxygen consumption by 25%. Ouabain decreased short-circuit current by 93% and oxygen consumption by 32%. Serotonin increased both variables by 59% and 33%, respectively. Bumetanide and diphenylamine-2-carboxylate reduced but did not abolish the effect of serotonin on short-circuit current and oxygen consumption. Changes in short-circuit current and oxygen consumption were linearly correlated under all conditions tested. It is concluded that, in the unstimulated rat distal colon epithelium, chloride secretion accounts for about 75% of ouabain-sensitive short-circuit current and oxygen consumption. Stimulated chloride secretion may demand over 40% of total oxygen consumption.