Protein phosphorylation in permeabilized pancreatic islet cells

Area of Science:

• Endocrinology
• Cell Biology
• Biochemistry

Background:

• Insulin secretion is a complex process regulated by intracellular calcium levels.
• The role of protein phosphorylation in calcium-mediated insulin release requires further elucidation.

Purpose of the Study:

• To develop and validate a digitonin-permeabilized islet cell system.
• To investigate calcium- and calmodulin-dependent protein phosphorylation in intact islet cells.
• To determine if this membrane-bound process initiates calcium-stimulated insulin secretion.

Main Methods:

• Digitonin permeabilization of pancreatic islet cells.
• Assessment of membrane integrity using Trypan Blue exclusion and lactate dehydrogenase leakage.
• Measurement of [gamma-32P]ATP incorporation into endogenous substrates.
• Quantification of insulin release in response to varying calcium concentrations.

Main Results:

• Digitonin effectively permeabilized islet cells without altering ultrastructure.
• Calcium- and calmodulin-dependent phosphorylation of membrane-bound substrates was observed.
• Half-maximal insulin release occurred at 0.1 microM free calcium, maximal at 0.2 microM.
• Calmodulin enhanced insulin release in the absence of free calcium.

Conclusions:

• The digitonin-permeabilized islet cell model is a valuable tool for studying insulin secretion.
• This model allows for the characterization of calcium- and calmodulin-dependent signaling pathways.
• The findings provide insights into the initiation of calcium-stimulated insulin secretion.