Stable and diffusible pools of nucleotides in pancreatic islet cells.

Abstract

Adenine nucleotides are thought to serve as second messengers in the control of beta-cell function by glucose, e.g. by regulating the activity of ATP-dependent K+ channels. However, their localization in different intracellular pools may mask the biologically relevant changes and complicate the interpretation of measurements in whole cells. The plasma membrane of mouse islet cells was selectively permeabilized by the alpha-toxin from Staphylococcus aureus to allow diffusion of cytoplasmic nucleotides. After permeabilization of cells from freshly isolated islets, approximately 68% of ATP, 45% of ADP, and 52% of AMP rapidly diffused out of the cells, whereas the insulin content hardly varied. The nondiffusible pool of nucleotides was stable for at least 90 min at 4 C, which suggests that it is contained in cellular organelles. The size of this nondiffusible pool decreased proportionally to insulin stores when these were lowered by stimulating secretion to different degrees during culture before permeabilization. From these results, it can be calculated that nondiffusible nucleotides are mainly contained in insulin secretory granules, with a small proportion in another, probably mitochondrial, compartment. Approximately 80% GTP and 30% GDP were present in the diffusible pool, and their relative proportions in the granular pool were only about 20% that of adenine nucleotides. Incubation of the cells in 20 instead of 2 mM glucose before permeabilization did not affect the nondiffusible pool, which indicates that the increase in the ATP/ADP ratio measured in intact cells occurred in the diffusible pool. Cytoplasmic nucleotide levels could be evaluated by subtracting the nondiffusible pool from the measurements in intact cells. It emerges that glucose induces large changes in the ATP/ADP ratio in the cytoplasmic pool, and that these changes are largely due to a fall in ADP.

Lack of “Hemichannel” Activity in Insulin-Producing Cells

“…Under conditions of resting membrane potential, that easily showed the presence of such channels in N2A cells, we obtained no functional evidence for "hemichannels" in either MIN6 cells or primary pancreatic β-cells. This negative observation was made in spite of a sizable release of ATP during stimulation, which our data demonstrate to essentially occur via an exocytotic route, as anticipated by the presence of the nucleotide within the insulin-containing secretory granules (Detimary et al 1996).…”

“…To evaluate whether MIN6 cells express "hemichannels" that could have escaped detection by the tracer uptake protocol, we tested their release of ATP under conditions favoring or preventing insulin release, since insulin secretory granules contain abundant ATP (Detimary et al 1996). Stimulation of WT MIN6 cells by 20 mM glucose elicited a sizable release of ATP (Fig.…”

Purinergic receptors in the endocrine and exocrine pancreas

“…Another source of ATP and nucleotides are insulincontaining granules of β cells [51][52][53][54]. Obermuller et al [55] showed that adenine nucleotides are released with a very short delay after stimulation, while peptides (insulin) are released later.…”

Increased Glucose Sensitivity of Stimulus-Secretion Coupling in Islets FromPsammomys obesusAfter Diet Induction of Diabetes

“…The effluent was collected (2-min fractions) for measurement of insulin by radioimmunoassay using human insulin as standard (19). At the end, the islets were collected for determination of insulin content (18).…”

“…The supernatant (450 l) was submitted to a triple diethylether extraction, diluted with 450 l of a buffer containing 20 mmol/l HEPES, 3 mmol/l MgCl 2 , and KOH to adjust pH at 7.75, and stored at Ϫ80°C. ATP was measured by a luminometric method, as previously described (18). Blanks and ATP standard curves in Krebs buffer were run through the entire procedure.…”