Abstract
Luminal acidification is important for the maturation of secretory granules, yet little is known regarding the regulation of pH within them. A pH-sensitive green fluorescent protein (EGFP) was targeted to secretory granules in RIN1046-38 insulinoma cells by using a construct in which the EGFP gene was preceded by the nucleotide sequence for human growth hormone. Stimulatory levels of glucose doubled EGFP secretion from cell cultures, and potentiators of glucose-induced insulin secretion enhanced EGFP release. Thus this targeted EGFP is useful for population measurements of secretion. However, less than ∼4% of total cell EGFP was released after 1.5 h of stimulation. Consequently, when analyzed in single cells, fluorescence of the targeted EGFP acts as an indicator of pH within secretory granules. Glucose elicited a decrease in granule pH, whereas inhibitors of the V-type H+-ATPase increased pH and blocked the glucose effect. Granule pH also was modified by effectors of the protein kinase A pathway, with activation eliciting granule alkalinization, suggesting that potentiation of peptide release by cAMP may involve regulated changes in secretory granule pH.
Original language | English (US) |
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Pages (from-to) | C429-C437 |
Journal | American Journal of Physiology - Cell Physiology |
Volume | 283 |
Issue number | 2 52-2 |
DOIs | |
State | Published - 2002 |
Keywords
- Green fluorescent protein
- Insulin secretion
- Protein kinase A
- V-type H ATPase
- cAMP
ASJC Scopus subject areas
- Physiology
- Cell Biology