A Highly Ca2+-sensitive Pool of Granules Is Regulated by Glucose and Protein Kinases in Insulin-secreting INS-1 Cells

Abstract

We have used membrane capacitance measurements and carbon-fiber amperometry to assay exocytosis triggered by photorelease of caged Ca2+ to directly measure the Ca2+ sensitivity of exocytosis from the INS-1 insulin-secreting cell line. We find heterogeneity of the Ca2+ sensitivity of release in that a small proportion of granules makes up a highly Ca2+-sensitive pool (HCSP), whereas the bulk of granules have a lower sensitivity to Ca2+. A substantial HCSP remains after brief membrane depolarization, suggesting that the majority of granules with high sensitivity to Ca2+ are not located close to Ca2+ channels. The HCSP is enhanced in size by glucose, cAMP, and a phorbol ester, whereas the Ca2+-sensitive rate constant of exocytosis from the HCSP is unaffected by cAMP and phorbol ester. The effects of cAMP and phorbol ester on the HCSP are mediated by PKA and PKC, respectively, because they can be blocked with specific protein kinase inhibitors. The size of the HCSP can be enhanced by glucose even in the presence of high concentrations of phorbol ester or cAMP, suggesting that glucose can increase granule pool sizes independently of activation of PKA or PKC. The effects of PKA and PKC on the size of the HCSP are not additive, suggesting they converge on a common mechanism. Carbon-fiber amperometry was used to assay quantal exocytosis of serotonin (5-HT) from insulin-containing granules following preincubation of INS-1 cells with 5-HT and a precursor. The amount or kinetics of release of 5-HT from each granule is not significantly different between granules with higher or lower sensitivity to Ca2+, suggesting that granules in these two pools do not differ in morphology or fusion kinetics. We conclude that glucose and second messengers can modulate insulin release triggered by a high-affinity Ca2+ sensor that is poised to respond to modest, global elevations of [Ca2+]i.

A subcellular model of glucose-stimulated pancreatic insulin secretion

“…Such an extended model could be based on the approach by Chen et al (in press), and would be useful for studying pulsatile insulin secretion, which is believed to be the result of calcium and, possibly, metabolic oscillations (Barbosa et al 1998;Nunemaker et al 2006;Bertram et al 2007). In addition, a detailed description of intracellular calcium handling would allow the notion of pools with different calcium sensitivities (Wan et al 2004;Yang & Gillis 2004) to be added to the model.…”

Newcomer insulin secretory granules as a highly calcium-sensitive pool

“…Because both HCSP granules and newcomers fuse away from L-type Ca 2ϩ channels, we hypothesized that they might be overlapping sets of vesicles. Because newcomers are independent of docking proteins such as Syntaxin-1A and granuphilin, we propose further that granules that are still not completely docked to the cell membrane have a higher calcium sensitivity and therefore respond to bulk cytosolic calcium rather than the microdomain calcium that triggers exocytosis of the IRP (21,20). Docking would lower the affinity for calcium, and attachment to L-type Ca 2ϩ channels would become a prerequisite for fusion of docked granules.…”

“…In addition to the fast microdomain controlled exocytosis, another highly calcium-sensitive pool (HCSP) of granules has been described with an EC 50 value of a few micromolar (20,21). This pool is not a subset of the granules residing within microdomains because it is not exhausted by short depolarizations.…”

Mechanisms of action of glucagon-like peptide 1 in the pancreas

“…However, recently it has been demonstrated that intercellular Ca 2+ increases distal to the Ca 2+ channel are instrumental in the exocytosis of a subset of highly Ca 2+ sensitive pool of insulin secretory vesicles. This subset of insulin secretory vesicles have been clearly defined by two laboratories using both membrane capacitance (Wan et al, 2004;Yang and Gillis, 2004) and carbon-fibre amperometry measurements (Wan et al, 2004;Yang and Gillis, 2004). They are responsive to global rather than localized increases in Ca 2+ and are mobilized concurrently with low Ca 2+ sensitivity vesicles that are closely associated with voltage-dependent Ca 2+ channels.…”