Involvement of Calmodulin in Glucagon‐Like Peptide 1(7‐36) Amide‐Induced Inhibition of the ATP‐Sensitive K⁺ Channel in Mouse Pancreatic β‐Cells

Abstract

The present investigation was designed to examine whether calmodulin is involved in the inhibition of the ATP‐sensitive K+ (KATP) channel by glucagon‐like peptide 1(7‐36) amide (GLP‐1) in mouse pancreatic β‐cells. Membrane potential, single channel and whole‐cell currents through the KATP channels, and intracellular free Ca2+ concentration ([Ca2+]i) were measured in single mouse pancreatic β‐cells. Whole‐cell patch‐clamp experiments with amphotericin‐perforated patches revealed that membrane conductance at around the resting potential is predominantly supplied by the KATP channels in mouse pancreatic β‐cells. The addition of 20 nM GLP‐1 in the presence of 5 mM glucose significantly reduced the membrane KATP conductance, accompanied by membrane depolarization and the generation of electrical activity. A calmodulin inhibitor N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide (W‐7, 20 μM) completely reversed the inhibitory actions of GLP‐1 on the membrane KATP conductance and resultant membrane depolarization. Cell‐attached patch recordings confirmed the inhibition of the KATP channel activity by 20 nM GLP‐1 and its restoration by 20 μM W‐7 or 10 μM calmidazolium at the single channel level. Bath application of 20 μM W‐7 also consistently abolished the GLP‐1‐evoked increase in [Ca2+]i in the presence of 5 mM glucose. These results strongly suggest that the mechanisms by which GLP‐1 inhibits the KATP channel activity accompanied by the initiation of electrical activity in mouse pancreatic β‐cells include a calmodulin‐dependent mechanism in addition to the well‐documented activation of the cyclic AMP‐protein kinase A system.

PKA-dependent potentiation of glucose-stimulated insulin secretion by Epac activator 8-pCPT-2′-O-Me-cAMP-AM in human islets of Langerhans

“…Furthermore, 8-pCPT-2=-O-Me-cAMP-AM exerted a glucose-dependent action to depolarize ␤-cells and to increase [Ca 2ϩ ] i . Such actions of 8-pCPT-2=-O-Me-cAMP-AM in human islets resemble the previously described membrane-depolarizing (11,25,64), [Ca 2ϩ ] i -elevating (3), and insulin secretagogue actions (17, 27) of cAMP-elevating hormone GLP-1 in rodent islets. However, despite the fact that Epac2 activation most likely subserves stimulatory effects of GLP-1 on islet insulin secretion (26,39,54), it is clear that GLP-1 also acts through PKA (13,17,24), and in fact the full insulinotropic action of GLP-1 may arise only under conditions in which there is dual activation of both Epac2 and PKA (47)(48)(49).…”

“…Thus, free cytosolic Ca 2ϩ may play a significant role in support of CICR and also in support of Epac-regulated processes that control the ␤-cell membrane potential. In fact, it was reported previously that the action of cAMP-elevating agent GLP-1 to inhibit K ATP channels and depolarize mouse ␤-cells was disrupted after treatment of these cells with agents that interfere with Ca 2ϩ signaling (11). RT-QPCR for Epac1 and Epac2 mRNA.…”

The Multiple Actions of GLP-1 on the Process of Glucose-Stimulated Insulin Secretion

“…Moreover, others have shown similar results using Rp-8-Br-cAMPS, a more membrane-permeable analog of Rp-cAMPS (38,41). The actions of GLP-1 on K ATP channels may also involve other signaling pathways because K ATP channel inhibition by GLP-1 in mouse ␤-cells was demonstrated to be calmodulin dependent, using the calmodulin inhibitors W-7 and calmidazolium (46).…”

Glucagon-Like Peptide-1 Synthetic Analogs: New Therapeutic Agents for Use in the Treatment of Diabetes Mellitus

“…The cAMPGEFs couple cAMP production to the activation of Rap1, a small molecular weight G protein. Novel signaling properties of the GLP-1 receptor include its ability to activate immediate early genes (IEGs) [80], to increase the number of insulin receptors on insulin-secreting cells [81,82], to stimulate mitogen-activated protein kinases (p38 MAPK, ERK), phosphatidylinositol 3-kinase [83], atypical protein kinase C-ζ [84], Ca 2+ /calmodulin-regulated protein kinase [85], protein kinase B (Akt), and hormonesensitive lipase [86]. It seems likely that at least some of these effects result from activation of Epac by GLP-1.…”