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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

Effect of mefloquine (MFQ, a Cx36 inhibitor) on the islet content and release of GABA (A) and taurine (Tau) (B) induced by plasma membrane depolarization with 70 mM KCl (70 KCl) and extracellular Ca2+-omission.Ca2+-omission (0 mM CaCl2 + 100 μM EGTA = 0 Ca2+) and 70 mM KCl depolarization (70 KCl + 250 μM diazoxide = 70 KCl) were tested on content and release of GABA and Tau in incubated rat islets. Three groups each of 30 rat islets were pre-incubated with 10 mM Gln and 5 mM glucose for 1 hour. After washing three times with 100 μl PBS to withdraw extracellular Gln, islets were further incubated for 1 hour at 37°C (in 70 μl of KRBH) with 5 mM glucose alone (control) or under Ca2+-omission and KCl-depolarization (0 Ca2+ + 70 KCl) in the absence or presence of 25 and 50 μM mefloquine. At the end of incubation, an aliquot (50 μl) of incubation medium was taken off and the islets washed again three times with cold PBS (100 μl) to wash out remaining extracellular amino acids. Islets were finally extracted with 30 μl 10% (w/v) sulfosalycilic acid and stored frozen. Amino acids in the incubation media and islets extracts were separated by HPLC after derivatization with o-phtalaldialdehyde and quantified by fluorescence detection. (N.S., † p< 0.04, and ‡ p< 0.01 compared with the corresponding control in the absence of MFQ).
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pone.0140096.g004: Effect of mefloquine (MFQ, a Cx36 inhibitor) on the islet content and release of GABA (A) and taurine (Tau) (B) induced by plasma membrane depolarization with 70 mM KCl (70 KCl) and extracellular Ca2+-omission.Ca2+-omission (0 mM CaCl2 + 100 μM EGTA = 0 Ca2+) and 70 mM KCl depolarization (70 KCl + 250 μM diazoxide = 70 KCl) were tested on content and release of GABA and Tau in incubated rat islets. Three groups each of 30 rat islets were pre-incubated with 10 mM Gln and 5 mM glucose for 1 hour. After washing three times with 100 μl PBS to withdraw extracellular Gln, islets were further incubated for 1 hour at 37°C (in 70 μl of KRBH) with 5 mM glucose alone (control) or under Ca2+-omission and KCl-depolarization (0 Ca2+ + 70 KCl) in the absence or presence of 25 and 50 μM mefloquine. At the end of incubation, an aliquot (50 μl) of incubation medium was taken off and the islets washed again three times with cold PBS (100 μl) to wash out remaining extracellular amino acids. Islets were finally extracted with 30 μl 10% (w/v) sulfosalycilic acid and stored frozen. Amino acids in the incubation media and islets extracts were separated by HPLC after derivatization with o-phtalaldialdehyde and quantified by fluorescence detection. (N.S., † p< 0.04, and ‡ p< 0.01 compared with the corresponding control in the absence of MFQ).

Mentions: 70 mM KCl depolarization and Ca2+-omission separately increased the release of GABA and taurine (Tau) and correspondingly depleted their islet content (Fig 3A and 3B). These separate effects were strongly potentiated when the two conditions were applied together. The content and release of Asp and Glu were not so consistently affected by 70 mM KCl depolarization and Ca2+-omission (results not shown). Mefloquine, a specific inhibitor of connexin36, partially blocked the release of GABA stimulated by 70 mM KCl depolarization and Ca2+-omission at 50 but not at 25 μM and correspondingly increased islet amine content (Fig 4A). These data indicate that exchange of some amino acids occur via connexin–36 hemichannels in response to depolarization and Ca2+ removal. In contrast, the drug had no effect on either islet Tau depletion or release induced by 70 mM KCl depolarization and Ca2+-omission.


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)

Effect of mefloquine (MFQ, a Cx36 inhibitor) on the islet content and release of GABA (A) and taurine (Tau) (B) induced by plasma membrane depolarization with 70 mM KCl (70 KCl) and extracellular Ca2+-omission.Ca2+-omission (0 mM CaCl2 + 100 μM EGTA = 0 Ca2+) and 70 mM KCl depolarization (70 KCl + 250 μM diazoxide = 70 KCl) were tested on content and release of GABA and Tau in incubated rat islets. Three groups each of 30 rat islets were pre-incubated with 10 mM Gln and 5 mM glucose for 1 hour. After washing three times with 100 μl PBS to withdraw extracellular Gln, islets were further incubated for 1 hour at 37°C (in 70 μl of KRBH) with 5 mM glucose alone (control) or under Ca2+-omission and KCl-depolarization (0 Ca2+ + 70 KCl) in the absence or presence of 25 and 50 μM mefloquine. At the end of incubation, an aliquot (50 μl) of incubation medium was taken off and the islets washed again three times with cold PBS (100 μl) to wash out remaining extracellular amino acids. Islets were finally extracted with 30 μl 10% (w/v) sulfosalycilic acid and stored frozen. Amino acids in the incubation media and islets extracts were separated by HPLC after derivatization with o-phtalaldialdehyde and quantified by fluorescence detection. (N.S., † p< 0.04, and ‡ p< 0.01 compared with the corresponding control in the absence of MFQ).
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Related In: Results  -  Collection

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pone.0140096.g004: Effect of mefloquine (MFQ, a Cx36 inhibitor) on the islet content and release of GABA (A) and taurine (Tau) (B) induced by plasma membrane depolarization with 70 mM KCl (70 KCl) and extracellular Ca2+-omission.Ca2+-omission (0 mM CaCl2 + 100 μM EGTA = 0 Ca2+) and 70 mM KCl depolarization (70 KCl + 250 μM diazoxide = 70 KCl) were tested on content and release of GABA and Tau in incubated rat islets. Three groups each of 30 rat islets were pre-incubated with 10 mM Gln and 5 mM glucose for 1 hour. After washing three times with 100 μl PBS to withdraw extracellular Gln, islets were further incubated for 1 hour at 37°C (in 70 μl of KRBH) with 5 mM glucose alone (control) or under Ca2+-omission and KCl-depolarization (0 Ca2+ + 70 KCl) in the absence or presence of 25 and 50 μM mefloquine. At the end of incubation, an aliquot (50 μl) of incubation medium was taken off and the islets washed again three times with cold PBS (100 μl) to wash out remaining extracellular amino acids. Islets were finally extracted with 30 μl 10% (w/v) sulfosalycilic acid and stored frozen. Amino acids in the incubation media and islets extracts were separated by HPLC after derivatization with o-phtalaldialdehyde and quantified by fluorescence detection. (N.S., † p< 0.04, and ‡ p< 0.01 compared with the corresponding control in the absence of MFQ).
Mentions: 70 mM KCl depolarization and Ca2+-omission separately increased the release of GABA and taurine (Tau) and correspondingly depleted their islet content (Fig 3A and 3B). These separate effects were strongly potentiated when the two conditions were applied together. The content and release of Asp and Glu were not so consistently affected by 70 mM KCl depolarization and Ca2+-omission (results not shown). Mefloquine, a specific inhibitor of connexin36, partially blocked the release of GABA stimulated by 70 mM KCl depolarization and Ca2+-omission at 50 but not at 25 μM and correspondingly increased islet amine content (Fig 4A). These data indicate that exchange of some amino acids occur via connexin–36 hemichannels in response to depolarization and Ca2+ removal. In contrast, the drug had no effect on either islet Tau depletion or release induced by 70 mM KCl depolarization and Ca2+-omission.

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