RESUMEN

Dithiothreitol (DTT), at 1 mmol/L or higher, is widely used as a mucolytic in gastrointestinal research. Previous observations suggest that it may be toxic to the mucosa. DTT effects on the mucosal electrical behavior were assessed. Cumulative concentration-response relationships of DTT effects on rat distal colon mucosa were studied. Isolated mucosa preparations were mounted in an Ussing chamber under short-circuit conditions. The effects of concentrations ranging from 1 mumol/L to 1 mmol/L, applied to either the mucosal or serosal side, were studied. As compared with control, untreated preparations, DTT depressed short-circuit current at 10 mumol/L and higher when applied to the serosal side, and at 50 mumol/L and higher when applied to the mucosal side of the epithelium. On the other hand, transepithelial resistivity showed a progressive increase with DTT applied to either side at a concentration of up to 500 mumol/L, while at the highest concentration (1 mmol/L) a marked decrease in resistivity occurred. Neither the short-circuit current decrease, nor the resistivity collapse showed recovery after repeated rinsing with DTT-free solution. It is concluded that DTT affects epithelial electrical properties at low concentrations, and therefore its use as a mucolytic for electrophysiological studies should be discouraged.

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RESUMEN

Dithiothreitol (DTT), at 1 mmol/L or higher, is widely used as a mucolytic in gastrointestinal research. Previous observations suggest that it may be toxic to the mucosa. DTT effects on the mucosal electrical behavior were assessed. Cumulative concentration-response relationships of DTT effects on rat distal colon mucosa were studied. Isolated mucosa preparations were mounted in an Ussing chamber under short-circuit conditions. The effects of concentrations ranging from 1 mumol/L to 1 mmol/L, applied to either the mucosal or serosal side, were studied. As compared with control, untreated preparations, DTT depressed short-circuit current at 10 mumol/L and higher when applied to the serosal side, and at 50 mumol/L and higher when applied to the mucosal side of the epithelium. On the other hand, transepithelial resistivity showed a progressive increase with DTT applied to either side at a concentration of up to 500 mumol/L, while at the highest concentration (1 mmol/L) a marked decrease in resistivity occurred. Neither the short-circuit current decrease, nor the resistivity collapse showed recovery after repeated rinsing with DTT-free solution. It is concluded that DTT affects epithelial electrical properties at low concentrations, and therefore its use as a mucolytic for electrophysiological studies should be discouraged.(AU)

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RESUMEN

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.

Asunto(s)

RESUMEN

Dithiothreitol (DTT), at 1 mmol/L or higher, is widely used as a mucolytic in gastrointestinal research. Previous observations suggest that it may be toxic to the mucosa. DTT effects on the mucosal electrical behavior were assessed. Cumulative concentration-response relationships of DTT effects on rat distal colon mucosa were studied. Isolated mucosa preparations were mounted in an Ussing chamber under short-circuit conditions. The effects of concentrations ranging from 1 mumol/L to 1 mmol/L, applied to either the mucosal or serosal side, were studied. As compared with control, untreated preparations, DTT depressed short-circuit current at 10 mumol/L and higher when applied to the serosal side, and at 50 mumol/L and higher when applied to the mucosal side of the epithelium. On the other hand, transepithelial resistivity showed a progressive increase with DTT applied to either side at a concentration of up to 500 mumol/L, while at the highest concentration (1 mmol/L) a marked decrease in resistivity occurred. Neither the short-circuit current decrease, nor the resistivity collapse showed recovery after repeated rinsing with DTT-free solution. It is concluded that DTT affects epithelial electrical properties at low concentrations, and therefore its use as a mucolytic for electrophysiological studies should be discouraged.

Asunto(s)

RESUMEN

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.

RESUMEN

Dithiothreitol (DTT), at 1 mmol/L or higher, is widely used as a mucolytic in gastrointestinal research. Previous observations suggest that it may be toxic to the mucosa. DTT effects on the mucosal electrical behavior were assessed. Cumulative concentration-response relationships of DTT effects on rat distal colon mucosa were studied. Isolated mucosa preparations were mounted in an Ussing chamber under short-circuit conditions. The effects of concentrations ranging from 1 mumol/L to 1 mmol/L, applied to either the mucosal or serosal side, were studied. As compared with control, untreated preparations, DTT depressed short-circuit current at 10 mumol/L and higher when applied to the serosal side, and at 50 mumol/L and higher when applied to the mucosal side of the epithelium. On the other hand, transepithelial resistivity showed a progressive increase with DTT applied to either side at a concentration of up to 500 mumol/L, while at the highest concentration (1 mmol/L) a marked decrease in resistivity occurred. Neither the short-circuit current decrease, nor the resistivity collapse showed recovery after repeated rinsing with DTT-free solution. It is concluded that DTT affects epithelial electrical properties at low concentrations, and therefore its use as a mucolytic for electrophysiological studies should be discouraged.

RESUMEN

The contributions of subepithelial tissue, mucosa, and mucus gel layer as restraints for oxygen diffusion in rat distal colon in vitro were assessed by comparing oxygen transfer through preparations of isolated submucosa, isolated mucosa with and without the superficial mucus gel layer, and mucosa-submucosa mounted as flat sheets in a diffusion chamber. One side of the chamber was gassed with 95% O2-5% CO2 while the time course of oxygen concentration rise was measured in the continuously stirred opposite side, initially equilibrated with near-zero oxygen solution. The procedure does not affect epithelial viability. Diffusion in isolated mucosa was the same before and after KCN (5 mM) treatment, suggesting that epithelial oxygen consumption does not influence transfer rates. Subepithelial tissue, mucosa, and mucus gel layer are roughly responsible, respectively, for 12%, 56%, and 32% of oxygen diffusive hindrance. Diffusion coefficients range from 13% (mucosa-submucosa) to 54% (isolated submucosa) of that of water. Subepithelial tissue accounts for about 12% of total diffusive restraint.

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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 (islet mucosa) and preparations retaining it (mucosa-submucosa) were mounted in Ussing chambers at 37 degrees C and gassed with 95% O2-5% CO2; Isc and PD were monitored. A 5-min hypoxia with 95% N2-5% 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% and PD by -54 +/- 6% below baseline; reoxygenatory overshoots were, respectively, +60 +/- 17% and +/- 16%. Tetrodotoxin slightly and transiently reduced baseline Isc (-16 +/- 2%) and PD (-14 +/- 3%), 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%) and PD (-61 +/- 4%). On reoxygenation Isc and PD were increased, respectively, +30 +/- 5% and +19 +/- 6% over baseline. Tetrodotoxin reduced baseline Isc (-59.6 +/- 5%) and PD (61.3 +/- 6%). It enhanced hypoxic Isc and PD decreases (-80 +/- 5%), but not the reoxygenatory overshoots. 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.

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Short-circuit current (Isc) and transepithelial potential difference (PD) of the rat distal colon mucosa are sensitive to acute hypoxia in vitro. The relative contribution of luminal and serosal oxygenation in sustaining Isc and PD was assessed. Rat distal colon Isc and PD responses to hypoxia and reoxygenation of preparations of mucosa-submucosa, and of isolated mucosa (with and without the mucus gel layer), mounted in an Ussing chamber, and of sacs of everted and non-everted isolated mucosa, were measured. In Ussing chambers, a 5-min total (bilateral) hypoxia reduces Isc and PD by 50 to 70%, while an overshoot was observed on reoxygenation. Serosal hypoxia caused about the same effect as total hypoxia, with complete recovery on reoxygenation. Luminal hypoxia had no effect in either Isc or PD. After total hypoxia, selective serosal reoxygenation allowed complete recovery of Isc and PD; addition of luminal reoxygenation did not further increase Isc and PD. Luminal reoxygenation after total hypoxia did not modify the decrease in Isc and PD, but addition of serosal reoxygenation led to complete recovery. A similar behaviour was seen in isolated mucosa preparations without the mucus gel layer. Baseline Isc and PD of everted sacs were about 45% of those of non-everted sacs, but their response to a hypoxic challenge was slightly attenuated. On reoxygenation, both everted and non-everted sacs showed complete recovery. Summing up: serosal oxygenation is both necessary and sufficient to sustain rat distal colon Isc and PD, while luminal oxygenation is not; there seems to exist a barrier, different from the mucus gel layer, for oxygen access from the luminal side of the epithelium; and distal colon isolated mucosa everted sac preparations are suboptimally oxygenated.

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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 C and bubbled with 95% O2- 5% CO2 which was switched to 95% N2- 5% CO2 for inducing hypoxia; afterwards normal oxygenation was resumed. The effect of 5, 10, 15 and 20 min-hypoxic periods was assessed in isolated 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 microA. cm-2, PD = 9.7 +/- 1.6 mV and TR = 134.9 +/- 13.6 omega cm2. A 5-min hypoxia reduced Isc by 47.2 +/- 7.3% and PD by 61.5 +/- 4.9%. Peak values on reoxygenation were 28.1 +/- 4.1% for Isc and 16.8 +/- 5.4% for PD, over controls values. In the isolated mucosa (n = 9), control values were Isc = 52.04 +/- 5.5 microA. cm-2, PD = 5.0 +/- 0.8 mV and TR = 101.04 +/- 10.5 omega. cm2. In hypoxia, Isc decreased by 64.5 +/- 7.6% and PD by 57.2 +/- 7.8%. On reoxygenation peak values of 78.0 +/- 19.0% and 87.5 +/- 17.1%, 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%) 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.

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