UBC Theses and Dissertations
Cellular mechanisms of acid/base transport in an insect excretory epithelium Thomson, Robert Brent
The cellular mechanisms responsible for rectal acidification in the desert locust, Schistocerca gregaria, were investigated in isolated recta mounted as flat sheets in modified Ussing chambers. In the absence of exogenous CO₂, HCO₃⁻, and phosphate, the isolated rectum (under both open- and short-circuit current conditions) was capable of rates of net acid secretion (J[subscript]H+) similar to those observed in vivo, demonstrating the viability of the preparation and suggesting that rectal acidification was due to proton secretion rather than selective movements of HCO₃⁻ or phosphate. The possibility that trace levels of metabolic CO₂ might be generating sufficient HCO₃⁻ to account for the observed rates of rectal acidification (via HCO₃⁻ reabsorption) was assessed by adding exogenous CO₂/HCO₃⁻ to the contraluminal bath. The small increases in J[subscript]H+ observed after addition of 2% or 5% CO₂ were shown to be due to simple hydration of CO₂ which had diffused into the lumen (from the contraluminal bath), rather than changes in rates of HCO₃⁻ reabsorption. Since measurable quantities of luminal HCO₃⁻ did not directly affect the apical acid/base transport mechanism per se, it was concluded that metabolic CO₂ could not generate sufficient HCO₃⁻ in the lumen to account for the rates of rectal acidification observed under nominally CO₂/HCO₃⁻-free conditions and that J[subscript]H+ must be due to a proton secretory rather than bicarbonate reabsorptive mechanism. Microelectrode measurements of intracellular pH (pHi) and apical and basolateral membrane potentials (Va and Vb respectively) indicated that luminal pH was not in equilibrium with either contraluminal pH or pHi and that the mechanism responsible for active luminal acid secretion resided on the apical membrane. Preliminary measurements of bath total ammonia (ie. NH₃ + NH₄+) levels in the previous experiments suggested that the rectum was actively secreting ammonia at significant rates across the apical membrane into the lumen. If the ammonia crossed the apical membrane as NH₃ rather than NH₄+, rates of luminal ammonia secretion (J[subscript]Amm) would have to be added to J[subscript]H+ to obtain corrected values of luminal proton secretion. In the absence of exogenously added ammonia and CO₂, ammonia was preferentially secreted into the lumen under both open- and short-circuit current conditions. J[subscript]Amm was dependent on the presence of luminal amino acids and was relatively unaffected by K[superscript]+ removal or changes in luminal pH from 7.00 to 5.00. Bilateral Na+ substitution or luminal addition of ImM amiloride reduced J[subscript]Amm by 63% and 65% respectively. The data consistently demonstrate that the rectum secretes significant quantities of endogenously produced ammonia preferentially into the lumen as NH₄+ rather than NH₃ via an apical Na[superscript]+/NH₄[superscript]+ exchange mechanism. Clearly, rates of net acid secretion estimated by titratable acidity do not have to include a correction for luminal ammonia secretion. Although J[subscript]H+ was completely unaffected by changes in contraluminal pH, it could be progressively reduced (and eventually abolished) by imposition of either transepithelial pH gradients (lumen acid) or transepithelial electrical gradients (lumen positive). Under short-circuit current conditions, the bulk of J[subscript]H+ was not dependent on Na[superscript]+, K[superscript]+, CI⁻, Mg₂+, or Ca+ and was due to a primary electrogenic proton translocating mechanism located on the apical membrane. A small component (10-16%) of J[subscript]H+ measured under these conditions could be attributed to an apical amiloride-inhibitable Na[superscript]+/H[superscript]+ exchange mechanism. Inhibition of JH+ by anoxia or reduction of luminal pH unmasked a significant proton diffusional pathway on the apical membrane in parallel with the active proton pump. The fact that J[subscript]H+ was significantly inhibited (42%-66%) by contraluminal addition of ImM cAMP and relatively unaffected by changes in contraluminal pCO₂ or pH suggests that net acid secretion in the locust rectum in vivo is modulated by circulating hormonal factors rather than haemolymph pH or pCO₂ per se.
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