Dipeptide-induced Cl- secretion in proximal tubule cells

During a survey of dipeptides that might be transported by therenal PEPT2 transporter in proximal tubule cells, we discovered thatacidic dipeptides could stimulate transient secretory anion current andconductance increases in intact cell monolayers. The stimulatory effectof acidic dipeptides was observed in several proximal tubule cell linesthat have been recently developed by immortalization of early proximaltubule primary cultures from the Wistar-Kyoto and spontaneouslyhypertensive rat strains and humans, suggesting that this phenomenon isa characteristic of proximal tubule cells. The electrical currentinduced in intact monolayers by Ala-Asp, a representative of theseacidic dipeptides, must representCl secretion rather thanNa⁺ orH⁺ absorption, because1) it wasNa⁺ independent,2) it showed a pH dependencedifferent from that of the PEPT2 cotransporter, and3) it correlated with anAla-Asp-induced increase inCl conductance of theapical membrane in basolaterally amphotericin B-permeabilizedmonolayers. The secretory current could be inhibited by stilbenedisulfonates, but not diphenylamine-2-carboxylates, suggesting anon-cystic fibrosis transmembrane conductance regulator type ofCl conductance. The effectof Ala-Asp was dose dependent, with an apparent 50% effectiveconcentration of ~1 mM. Ala-Asp also produced intracellularacidification, suggesting that acidic dipeptides are also substratesfor an H⁺-peptide cotransporter.

     

Separation of cell organelles in density gradients based on their permeability characteristics

The buoyant density of intracellular organelles is dependent in part on the nature of the buffer composition of the density gradient and the permeability characteristics of the organelle membrane to the constituents of this buffer. Therefore, knowledge of the transport properties of different organelles allows the design of density gradients useful for their purification. We have used this approach to significantly decrease mitochondrial contamination of pancreatic zymogen granules in a one-step purification procedure on a 40% Percoll density gradient. These gradients, prepared with isoosmotic sucrose, yield a narrow band of zymogen granules and mitochondria. However, by substitution of sucrose with salts to which mitochondria but not zymogen granules are permeable, the densities of mitochondria are altered to give a significant separation. For example, the incorporation of 100 mM sodium succinate in the Percoll gradient can produce a 70% reduction in mitochondrial contamination. The increased ionic strength has an additional beneficial effect on zymogen granule yield by 5-10%. The recognition and utilization of transport pathways in organelle membranes is the principal feature of this technique and should prove to be widely applicable to other isolation procedures.     

Time-resolved release of calcium from an epithelial cell monolayer during mucin secretion

A significant amount of Ca²⁺ is contained in secretory mucin granules. Exchange of Ca²⁺ for monovalent cations drives the process of mucin decondensation and hydration after fusion of granules with the plasma membrane. Here we report direct observation of calcium secretion with a Ca²⁺ ion-selective electrode (ISE) in response to apical stimulation with ATP from HT29-Cl.16E cells, a subclone of the human colonic cancer cell line HT29. No increase in Ca²⁺ level was seen for the sister cell line Cl.19A, which lacks mucin granules, or for Cl.16E cells after inhibition of granule fusion with wortmannin. Further, the measured concentration was used to estimate the time-resolved rate of release of Ca²⁺ from the cell monolayer, by use of a deconvolution-based method developed previously (Nair and Gratzl in Anal Chem 77:2875–2881, 2005). The results argue that Ca²⁺ release by Cl.16E cells is associated specifically with mucin secretion, i.e., that the measured Ca²⁺ increase in the apical solution is derived from granules after fusion and mucin exocytosis. The Ca²⁺ ISE in conjunction with deconvolution provides a minimally disturbing method for assessment of Ca²⁺ secretion rates. The release rates provide estimates of exocytosis rates and, when combined with earlier capacitance measurements, estimates of post-stimulation endocytosis rates also.