Regulation of secretory granule pH in insulin-secreting cells.
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.
Tompkins
Linda S
LS
Department of Physiology, University of Arizona, Health Sciences Center, Tucson, Arizona 85718, USA.
Nullmeyer
Kevin D
KD
Murphy
Sean M
SM
Weber
Craig S
CS
Lynch
Ronald M
RM
D000818:Animals; D002138:Calibration; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D005947:Glucose / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D019382:Human Growth Hormone / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma / Q000378:metabolism* / Q000473:pathology; D008164:Luminescent Proteins; D010190:Pancreatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014407:Tumor Cells, Cultured
D007202:Indicators and Reagents; D007328:Insulin; D008164:Luminescent Proteins; D019382:Human Growth Hormone; D049452:Green Fluorescent Proteins; D006859:Hydrogen; D017868:Cyclic AMP-Dependent Protein Kinases; D005947:Glucose