Mechanisms of Net Chloride Secretion During Rotavirus Diarrhea in Young Rabbits : Do Intestinal Villi Secrete Chloride?

Abstract

Rotaviral diarrheal illness is one of the most common infectious diseases in children worldwide, but our understanding of its pathophysiology is limited. This study examines whether the enhanced net chloride secretion during rotavirus infection in young rabbits may occur as a result of hypersecretion in crypt cells that would exceed the substantial Cl^- reabsorption observed in villi. By using a rapid filtration technique, we evaluated transport of ³⁶Cl and D-¹⁴C glucose across brush border membrane (BBM) vesicles purified from villus tip and crypt cells isolated in parallel from the entire small intestine. Rotavirus infection impaired SGLT1-mediated Na⁺-D-glucose symport activity in both villus and crypt cell BBM, hence contributing to the massive water loss along the cryptvillus axis. In the same BBM preparations, rotavirus failed to stimulate the Cl^- transport activities (Cl^-/H⁺ symport, Cl^-/anion exchange and voltage-activated Cl^- conductance) at the crypt level, but not at the villus level, questioning, therefore, the origin of net chloride secretion. We propose that the chloride carrier might function in both normal (absorption) and reversed (secretion) modes in villi, depending on the direction of the chloride electrochemical gradient resulting from rotavirus infection, agreeing with our results that rotavirus accelerated both Cl^- influx and Cl^- efflux rates across villi BBM.

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“…However, the cellular and molecular bases by which rotavirus and NSP4 induce a moderate net chloride secretion remain unclear. Recently, Lorrot et al reported that rotavirus infection in vivo in young rabbits failed to stimulate the Cl - transport activities at the crypt level, but not at the villus level, questioning, therefore, the origin of net chloride secretion at the onset of diarrhea [8,9]. Because rotavirus stimulated both Cl - influx and Cl - efflux in villi, Lorrot et al proposed that the Cl - /H + symporter might function in both normal (absorption) and reversed (secretion) modes, depending on the direction of the chloride electrochemical gradient resulting from rotavirus infection [9].…”

“…Intestinal villi BBM vesicles were prepared from specific pathogen-free, four-week-old New Zealand albino hybrid rabbit by using the magnesium precipitation method as described [6-10]. They were suspended at about 20 mg of membrane protein/ml in membrane buffer (20 mM Hepes/40 mM citric acid/100 mM Tris gluconate/0.02% LiN3, supplemented to a total osmolarity of 560 mOsM with sorbitol and adjusted to pH 7.5 with Tris base) and stored in liquid nitrogen until the day of transport assay, as described [6-9]. Chloride transport was assayed by using 36 Cl and a rapid filtration technique as described [8,9,11].…”

“…They were suspended at about 20 mg of membrane protein/ml in membrane buffer (20 mM Hepes/40 mM citric acid/100 mM Tris gluconate/0.02% LiN3, supplemented to a total osmolarity of 560 mOsM with sorbitol and adjusted to pH 7.5 with Tris base) and stored in liquid nitrogen until the day of transport assay, as described [6-9]. Chloride transport was assayed by using 36 Cl and a rapid filtration technique as described [8,9,11]. To test the effect of a peptide on proton-coupled Cl - transport, BBM vesicles were mixed in the appropriate volume of membrane buffer in either the absence or presence of a given peptide.…”

“…We earlier showed that an alkaline-inside pH gradient can furnish the energy necessary for the uphill transport of Cl - across intestinal villi BBM purified from four week-old rabbits, indicating the presence of a Cl - /H + symporter in these vesicles [8,9]. To investigate the possible direct effect of each of these peptides on chloride influx, the initial Cl - entry rates were quantified by using the v = f [I] approach in the presence of a pHo/pHi = 5.0/7.5 gradient (o (out) and i (in) indicate the extra- and intravesicular spaces, respectively).…”

“…Whereas in vitro NSP4 114-135 inhibits the Cl - /H + symporter, the situation in vivo is different. Because rotavirus accelerated both Cl - influx and Cl - efflux rates across villi BBM whereas it failed to stimulate Cl - transport in crypt BBM [8,9], we conclude that NSP4 has no direct effect on either intestinal absorption or secretion of chloride.…”