Regulation of secretory granule pH in insulin-secreting cells

Linda S. Tompkins, Kevin D. Nullmeyer, Sean M. Murphy, Craig S. Weber, Ronald M. Lynch

Research output: Contribution to journalArticle

19 Scopus citations

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 languageEnglish (US)
Pages (from-to)C429-C437
JournalAmerican Journal of Physiology - Cell Physiology
Volume283
Issue number2 52-2
StatePublished - Jul 30 2002

Keywords

  • Green fluorescent protein
  • Insulin secretion
  • Protein kinase A
  • V-type H ATPase
  • cAMP

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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    Tompkins, L. S., Nullmeyer, K. D., Murphy, S. M., Weber, C. S., & Lynch, R. M. (2002). Regulation of secretory granule pH in insulin-secreting cells. American Journal of Physiology - Cell Physiology, 283(2 52-2), C429-C437.