E. coli STa enterotoxin is believed to cause secretion through a neural reflex involving nitric oxide. L-NAME, the inhibitor of NO synthase, attenuates (Rolfe & Levin, 1994) the rise in short-circuit current (assumed to reflect secretion) in vitro and restores fluid absorption in short duration in in vivo experiments, assessed by indicator dilution methods. For these reasons, the ability of L-NAME to restore fluid uptake after STa exposure was investigated in vivo in experiments of longer duration in anaesthetised (70 mg kg-1 I.P. Sagatal) Wistar rats using recirculated perfused loops. In addition, luminal acidification was measured since it is a critical variable in an alternative model (Lucas, 2001) for net water absorption. At the end of the experiment, the animals were humanely killed. The experiments accorded with UK legislation.
Indicator dilution estimation using ferrocyanide proved unsatisfactory and a volumetric method was adopted. In a 2 h perfusion protocol, the proximal jejunum was perfused with Krebs-phosphate buffer and STa was added (40 ng ml-1) after 30 min in animals dosed I.P. with either 40 mg kg-1 L-NAME or with isotonic saline. Values given are means ± S.E.M., n = animals. In the STa treated loops, luminal acidification of 0.80 ± 0.07µg H+ cm-1 h-1 (n = 10) reversed to alkalinisation of 0.44 ± 0.10 (10) after STa exposure. In STa plus L-NAME loops, initial acidification of 1.03 ± 0.14 (7) µg H+ cm-1 h-1 was similarly reduced to 0.02 ± 0.21µg H+ cm-1 h-1 by STa. There was no significant difference between rates of luminal acidification after STa exposure with and without L-NAME. Fluid absorption was 40 ± 5 (10) µl cm-1 h-1 with STa and unchanged at 28.4 ± 6 (7) µl cm-1 h-1 with STa and L-NAME.
In a modified 3 h perfusion protocol with STa (80 ng ml-1) present or absent at the start of perfusion, control luminal acidification was 0.85 ± 0.18 (7) vs. 0.42 ± 0.07 (11) for STa and 0.31 ± 0.03 (6) µg H+ cm-1 h-1 for STa + L-NAME in the first hour. During the last hour of perfusion, control luminal acidification was 0.60 ± 0.10 (7) vs. 0.12 ± 0.07 (11) µg H+ cm-1 h-1 for STa alone and -0.05 ± 0.07 (6) µg H+ cm-1 h-1 for STa + L-NAME. L-NAME was therefore unable to reverse the effect of STa on acidification. Net fluid absorption was 92 ± 12 (14) µl cm-1 h-1 in control loops and was reduced as expected to 62 ± 4 (19) µl cm-1 h-1 in STa treated loops. In STa exposed loops of L-NAME-treated animals, fluid absorption was 24.0 ± 4.0 (6) µl cm-1 h-1, i.e. L-NAME (P < 0.02, x2 test) did not restore the fluid absorption to normal values after STa exposure in these in vivo experiments and may even have caused a further reduction in fluid absorption.
A recent model proposes that acidification of the jejunum creates hypotonicity at the brush border (Lucas, 2001). The inability of L-NAME to restore luminal acidification or fluid absorption is compatible with this model. STa seems to be acting through NO. The changes in short-circuit current after STa exposure and their attenuation by L-NAME are more likely to be part of a compensatory response by the jejunum to interruption of normal fluid absorption, possibly mediated by a neural pathway involving nitric oxide.
The authors gratefully acknowledge support from the Royal Society.