Rotavirus infection stimulates the Cl- reabsorption process across the intestinal brush-border membrane of young rabbits

Abstract

Rotavirus is a major cause of infantile gastroenteritis worldwide. However, the mechanisms underlying fluid and electrolyte secretion associated with diarrhea remain largely unknown. We investigated the hypothesis that loss of Cl(-) into the luminal contents during rotavirus infection may be caused by a dysfunction in the chloride absorptive capacity across the intestinal brush-border membrane (BBM). The luminal Cl(-) concentrations in the entire small intestine of young rabbits infected with lapine rotavirus decreased at 1 and 2 days postinfection (dpi), indicating net Cl(-) absorption. At 7 dpi, luminal Cl(-) concentrations were slightly increased, indicating a moderate net Cl(-) secretion. By using a rapid filtration technique, (36)Cl uptake across BBM was quantified by modulating the alkali-metal ion, electrical, chloride, and/or proton gradients. Rotavirus infection caused an identical, 127% +/- 24% increase in all Cl(-) uptake activities (Cl(-)/H(+) symport, Cl(-) conductance, and Cl(-)/anion exchange) observed across the intestinal BBM. The rotavirus activating effects on the symporter started at 1 dpi and persisted up to 7 dpi. Kinetic analyses revealed that rotavirus selectively affected the capacity parameter characterizing the symporter. We report the novel observation that rotavirus infection stimulated the Cl(-) reabsorption process across the intestinal BBM. We propose that the massive Cl(-) reabsorption in villi could partly overwhelm chloride secretion in crypt cells, which possibly increases during rotavirus diarrhea, the resulting imbalance leading to a moderate net chloride secretion.

FIG. 1.

Ionic concentrations in the intestinal fluid of noninfected and experimentally infected rabbits. Potassium and chloride concentrations in the luminal contents from the entire small intestine of each animal were determined as a function of time after infection. Results are expressed in millimolars ± SD, with n = 8 to 43 determinations per point obtained from 4 to 12 rabbits. Because the results for four to nine noninfected rabbits at 0, 8, and 57 dpi were statistically indistinguishable, data have been pooled and are shown as a horizontal line. Identical letters indicate results found to be statistically indistinguishable according to a global F test (P < 0.01).

FIG. 2.

Time course of chloride uptake by intestinal BBM purified from noninfected rabbits: effect of H⁺, electrical, and/or Cl⁻ gradients. Cl⁻ uptake was determined with 15 mM cis ³⁶Cl as substrate. The extra- and intravesicular spaces contained a 20 mM HEPES-40 mM citric acid buffer adjusted with Tris base to give at zero time a pHo/pHi gradient of either 7.5/7.5 (□, ⋄, Δ) or 5.0/7.5 (▪) and was supplemented with appropriate mixtures of either Tris gluconate, K⁺ gluconate, or KCl to obtain the following ionic concentration gradients: [K⁺]o/[K⁺]i = 0/0 mM and [Cl⁻]o/[Cl⁻]i = 15/0 mM (▪, □); [K⁺]o/[K⁺]i = 100/0 mM and [Cl⁻]o/[Cl⁻]i = 15/0 mM (⋄); and [K⁺]o/[K⁺]i = 100/100 mM and [Cl⁻]o/[Cl⁻]i = 15/100 mM (Δ). When K⁺ was present, valinomycin was added at 10 μg · mg protein⁻¹. Results are expressed as absolute uptake values in nanomoles · milligrams of protein⁻¹ ± SD, with n = 16 to 26 determinations per point derived from four different membrane preparations. Because the uptake values at equilibrium (2 h) were identical, data have been pooled (n = 18). So as not to overload the figure, the uptake values at 4 s, 60 s, and equilibrium were used.

FIG. 3.

Rotavirus activating effects on BBM Cl⁻/H⁺ symporter as a function of time after infection. The initial Cl⁻ entry rates were determined with 15 mM cis ³⁶Cl. The extra- and intravesicular spaces contained a 20 mM HEPES-40 mM citric acid-100 mM Tris gluconate buffer adjusted with Tris base to give an initial pH_o/pH_i gradient of 5.0/7.5. Results are expressed in nanomoles · milligrams of protein⁻¹ · second⁻¹ ± SD, with n = 18 to 72 determinations per point obtained from three to seven different membrane preparations. Identical letters indicate results found to be statistically indistinguishable according to a global F test (P < 0.01).