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Am J Physiol Cell Physiol 283: C429-C437, 2002; doi:10.1152/ajpcell.01066.2000
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Vol. 283, Issue 2, C429-C437, August 2002

Regulation of secretory granule pH in insulin-secreting cells

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

Departments of Physiology and Pharmacology, University of Arizona, Health Sciences Center, Tucson, Arizona 85718

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.

insulin secretion; cAMP; protein kinase A; V-type H+ ATPase; green fluorescent protein


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