Korean J Physiol Pharmacol.  2022 May;26(3):219-225. 10.4196/kjpp.2022.26.3.219.

Ca²⁺ entry through reverse Na⁺ /Ca²⁺ exchanger in NCI-H716, glucagon-like peptide-1 secreting cells

• Choi KJ
• Hwang JW
• Kim SH
• Park HS

• KMID: 2529404
• DOI: http://doi.org/10.4196/kjpp.2022.26.3.219

Abstract

Glucagon like peptide-1 (GLP-1) released from enteroendocine L-cells in the intestine has incretin effects due to its ability to amplify glucose-dependent insulin secretion. Promotion of an endogenous release of GLP-1 is one of therapeutic targets for type 2 diabetes mellitus. Although the secretion of GLP-1 in response to nutrient or neural stimuli can be triggered by cytosolic Ca²⁺ elevation, the stimulussecretion pathway is not completely understood yet. Therefore, the aim of this study was to investigate the role of reverse Na⁺ /Ca²⁺ exchanger (rNCX) in Ca²⁺ entry induced by muscarinic stimulation in NCI-H716 cells, a human enteroendocrine GLP-1 secreting cell line. Intracellular Ca²⁺ was repetitively oscillated by the perfusion of carbamylcholine (CCh), a muscarinic agonist. The oscillation of cytosolic Ca²⁺ was ceased by substituting extracellular Na⁺ with Li + or NMG + . KB-R7943, a specific rNCX blocker, completely diminished CCh-induced cytosolic Ca²⁺ oscillation. Type 1 Na⁺ /Ca²⁺ exchanger (NCX 1 ) proteins were expressed in NCI-H716 cells. These results suggest that rNCX might play a crucial role in Ca²⁺ entry induced by cholinergic stimulation in NCIH716 cells, a GLP-1 secreting cell line.

Keyword

Calcium entry; Carbamylcholine; Glucagon like peptide-1; NCI-H716; Na⁺ /Ca²⁺ exchanger

Figure

• 

  Fig. 1 Dose-dependent Ca2+ oscillation induced by carbamylcholine (CCh) in NCI-H716 cells. (A) Representative cytosolic Ca2+ oscillation obtained from stepwise increase of various concentration of CCh (0.3 μM–1 mM) every 100 sec. (B) CCh-induced initial Ca2+ peak (% of maximum) and frequency of Ca2+ oscillation (peaks/min). Cytosolic Ca2+ measurement was obtained from seven separate experiments in fura-2 loaded NCI-H716 cells. CCh significantly stimulated initial Ca2+ peak and frequency of Ca2+ oscillation, dose-dependently.

• 

  Fig. 2 Voltage-operated Ca2+ channel (VOC) does not contribute to carbamylcholine (CCh)-induced Ca2+ entry process in NCI-H716 cells. (A) Nifedipine, a VOC antagonist, failed to change CCh-induced Ca2+ oscillation in NCI-H716 cells. (B) CCh-induced frequency of Ca2+ oscillation was not changed by nifedipine treatment. Similar results were obtained from six separate experiments in NCI-H716 cells. VOC might not be involved in the calcium influx in CCh-stimulated NCI-H716 cells.

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  Fig. 3 Effects of extracellular free Na+ on carbamylcholine (CCh)-induced Ca2+ oscillation in NCI-H716 cells. (A) Substitution of Na+ with N-methyl-d-glucamine (NMG+) resulted in cessation of Ca2+ oscillation, which was restored by Na+ reperfusion. (B) Replacing Na+ with Li+ resulted in termination of cytosolic Ca2+ oscillation and restoration of oscillatory Ca2+ signals by Na+ reperfusion. Na+-dependent Ca2+ influx might be important for the generation of oscillatory Ca2+ signaling induced by muscarinic stimulation in stimulus-secretion mechanism of NCI-H716 cells.

• 

  Fig. 4 Effects of KB-R7943 on carbamylcholine (CCh)-induced Ca2+ oscillation in NCI-H716 cells. (A) KB-R7943 significantly blocked CCh-induced Ca2+ oscillation in NCI-H716 cells. Ca2+ oscillation was restored after cessation of KB-R7943 perfusion. (B) Elimination of extracellular Ca2+ resulted in complete inhibition of CCh-induced Ca2+ oscillation. (C) Pretreatment of KB-R7943 failed to inhibit initial Ca2+ peak induced by CCh. (D) Effect KB-R7943 on CCh-induced initial Ca2+ peak obtained from seven separate experiments. rNCX might contribute to the generation of CCh-induced cytosolic Ca2+ oscillation by modulating Ca2+ influx pathway from extracellular fluid in NCI-H716 cells.

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  Fig. 5 Type 1 Na+/Ca2+ exchanger (NCX1) protein expression and distribution in NCI-H716 cells. (A) Western blot analysis of NCX1 expression. NCX1 protein was detected in NCI-H716 cells at band of about 120 kDa, and small sized proteolytic product band of 67 kDa was additionally detected. (B) Immunocytochemistry for NCX1 protein expression. Corresponding bright-field (top) and immunofluorescence (bottom) microscopy images showing the labelling of NCX1 protein. NCX1 protein was remarkably expressed on NCI-H716 cells, scale bars = 10 μm.

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