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KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion.

Pizarro-Delgado J, Deeney JT, Martín-del-Río R, Corkey BE, Tamarit-Rodriguez J - PLoS ONE (2015)

Bottom Line: These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine.The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1).ATP acts independently of KATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion.

View Article: PubMed Central - PubMed

Affiliation: biochemistry Department, Medical School, Complutense University, Madrid, Spain.

ABSTRACT
Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in β-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS-1 cells. Taking advantage of hemicannels'opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 μM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 μM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1). ATP acts independently of KATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion.

No MeSH data available.


Related in: MedlinePlus

Dose-response relationship of light emission by INS–1 cells expressing the fire-fly luciferase gene with the medium D-luciferin concentration.INS–1 cells transduced with an adenovirus carrying the gene of fire-fly luciferase from Photinus Piralis were incubated at 2 mM glucose and varying concentrations of luciferin under control (4.7 mM KCl) or depolarizing conditions (70 mM KCl).
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pone.0140096.g001: Dose-response relationship of light emission by INS–1 cells expressing the fire-fly luciferase gene with the medium D-luciferin concentration.INS–1 cells transduced with an adenovirus carrying the gene of fire-fly luciferase from Photinus Piralis were incubated at 2 mM glucose and varying concentrations of luciferin under control (4.7 mM KCl) or depolarizing conditions (70 mM KCl).

Mentions: In order to explore changes in plasma membrane permeability induced by 70 mM KCl-depolarization dispersed islet cells or INS–1 cells were transduced with an adenovirus carrying the fire-fly luciferase gene. Luciferin was added to the incubation medium at 5 mM glucose and light production followed under control conditions (4.7 mM KCl) and after depolarization with 70 mM KCl. As shown in Fig 1, the amount of emitted light by transduced INS–1 cells augmented “exponentially” with the luciferin concentration (0.1 to 1.0 mM) in the incubation medium after depolarization with 70 mM KCl at 5 mM glucose. By contrast, non-depolarized control cells showed a flat response (Fig 1). In order to optimize the sensitivity, the highest luciferin concentration (1.0 mM) was systematically used in subsequent experiments. In these cells, light emission was not decreased by 20 mM glucose but it was diminished by 50 μM mefloquine (a specific connexin–36 inhibitor); 250 μM carbenoxolone (more specific pannexin inhibitor) had no effect (Fig 2). Transduced rat islet cells also showed an increased light emission after depolarization with 70 mM KCl in the presence of 1.0 mM luciferin (Fig 2). Light emission was significantly mitigated by 20 mM glucose and 50 μM mefloquine but not carbenoxolone.


KCl -Permeabilized Pancreatic Islets: An Experimental Model to Explore the Messenger Role of ATP in the Mechanism of Insulin Secretion.

Pizarro-Delgado J, Deeney JT, Martín-del-Río R, Corkey BE, Tamarit-Rodriguez J - PLoS ONE (2015)

Dose-response relationship of light emission by INS–1 cells expressing the fire-fly luciferase gene with the medium D-luciferin concentration.INS–1 cells transduced with an adenovirus carrying the gene of fire-fly luciferase from Photinus Piralis were incubated at 2 mM glucose and varying concentrations of luciferin under control (4.7 mM KCl) or depolarizing conditions (70 mM KCl).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4596849&req=5

pone.0140096.g001: Dose-response relationship of light emission by INS–1 cells expressing the fire-fly luciferase gene with the medium D-luciferin concentration.INS–1 cells transduced with an adenovirus carrying the gene of fire-fly luciferase from Photinus Piralis were incubated at 2 mM glucose and varying concentrations of luciferin under control (4.7 mM KCl) or depolarizing conditions (70 mM KCl).
Mentions: In order to explore changes in plasma membrane permeability induced by 70 mM KCl-depolarization dispersed islet cells or INS–1 cells were transduced with an adenovirus carrying the fire-fly luciferase gene. Luciferin was added to the incubation medium at 5 mM glucose and light production followed under control conditions (4.7 mM KCl) and after depolarization with 70 mM KCl. As shown in Fig 1, the amount of emitted light by transduced INS–1 cells augmented “exponentially” with the luciferin concentration (0.1 to 1.0 mM) in the incubation medium after depolarization with 70 mM KCl at 5 mM glucose. By contrast, non-depolarized control cells showed a flat response (Fig 1). In order to optimize the sensitivity, the highest luciferin concentration (1.0 mM) was systematically used in subsequent experiments. In these cells, light emission was not decreased by 20 mM glucose but it was diminished by 50 μM mefloquine (a specific connexin–36 inhibitor); 250 μM carbenoxolone (more specific pannexin inhibitor) had no effect (Fig 2). Transduced rat islet cells also showed an increased light emission after depolarization with 70 mM KCl in the presence of 1.0 mM luciferin (Fig 2). Light emission was significantly mitigated by 20 mM glucose and 50 μM mefloquine but not carbenoxolone.

Bottom Line: These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine.The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1).ATP acts independently of KATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion.

View Article: PubMed Central - PubMed

Affiliation: biochemistry Department, Medical School, Complutense University, Madrid, Spain.

ABSTRACT
Our previous work has demonstrated that islet depolarization with KCl opens connexin36 hemichannels in β-cells of mouse pancreatic islets allowing the exchange of small metabolites with the extracellular medium. In this study, the opening of these hemichannels has been further characterized in rat islets and INS-1 cells. Taking advantage of hemicannels'opening, the uptake of extracellular ATP and its effect on insulin release were investigated. 70 mM KCl stimulated light emission by luciferin in dispersed rat islets cells transduced with the fire-fly luciferase gene: it was suppressed by 20 mM glucose and 50 μM mefloquine, a specific connexin36 inhibitor. Extracellular ATP was taken up or released by islets depolarized with 70 mM KCl at 5 mM glucose, depending on the external ATP concentration. 1 mM ATP restored the loss of ATP induced by the depolarization itself. ATP concentrations above 5 mM increased islet ATP content and the ATP/ADP ratio. No ATP uptake occurred in non-depolarized or KCl-depolarized islets simultaneously incubated with 50 μM mefloquine or 20 mM glucose. Extracellular ATP potentiated the secretory response induced by 70 mM KCl at 5 mM glucose in perifused rat islets: 5 mM ATP triggered a second phase of insulin release after the initial peak triggered by KCl-depolarization itself; at 10 mM, it increased both the initial, KCl-dependent, peak and stimulated a greater second phase of secretion than at 5 mM. These stimulatory effects of extracellular ATP were almost completely suppressed by 50 μM mefloquine. The magnitude of the second phase of insulin release due to 5 mM extracellular ATP was decreased by addition of 5 mM ADP (extracellular ATP/ADP ratio = 1). ATP acts independently of KATP channels closure and its intracellular concentration and its ATP/ADP ratio seems to regulate the magnitude of both the first (triggering) and second (amplifying) phases of glucose-induced insulin secretion.

No MeSH data available.


Related in: MedlinePlus