Fluorescent markers to study membrane retrieval in antidiuretic hormone-treated toad urinary bladder

Area of Science:

• Cell biology
• Membrane trafficking
• Hormone signaling

Background:

• Antidiuretic hormone (ADH) stimulates water reabsorption in toad urinary bladders by increasing apical membrane water permeability.
• This increase is attributed to the fusion of intracellular aggrephores with the apical plasma membrane.
• ADH removal is hypothesized to involve the retrieval of these aggrephores from the apical membrane into the granular cell cytoplasm.

Purpose of the Study:

• To investigate the intracellular processing of membrane components retrieved during antidiuretic hormone (ADH) washout.
• To utilize fluorescently labeled dextran and horseradish peroxidase as markers for studying membrane retrieval and intracellular trafficking in toad urinary bladder granular cells.

Main Methods:

• Granular cell uptake of fluorescently labeled dextran and horseradish peroxidase was monitored during ADH washout.
• Experiments involved varying dextran concentrations and transepithelial osmotic gradients.
• Ultrastructural analysis using electron microscopy and fluorescent microscopy was employed to correlate cellular uptake with membrane structures.

Main Results:

• Fluorescent dextran uptake was dependent on prior ADH exposure, dextran concentration, and osmotic gradients.
• Following ADH removal, fluorescence initially appeared dispersed near the apical surface, then coalesced into larger intracellular bodies.
• Electron microscopy revealed that retrieved membrane components were initially in tubular vesicles that transferred their contents to multivesicular bodies, resembling aggrephores.

Conclusions:

• Fluorescent compounds serve as effective markers for luminal contents of membrane retrieved during ADH washout.
• These markers enable detailed study of the intracellular processing and trafficking of retrieved membrane components.
• The findings support the model of aggrephore retrieval and processing following ADH stimulation and removal, contributing to understanding water permeability regulation.