Proceedings of The Physiological Society
AstraZeneca (2010) Proc Physiol Soc 18, C08 and PC08
Voltage-gated ion channels in primary murine L-cells
G. Rogers1, A. Ramzan1, A. Habib1, G. Tolhurst1, F. Reimann1, F. Gribble1
1. Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom.
Background and aims: Glucagon-like peptide-1 (GLP-1) is an enteric hormone secreted by L-cells and is an attractive therapeutic target for the treatment of Type 2 Diabetes. Recently, primary murine L-cells were shown to be electrically excitable, firing action potentials in the presence of glucose, suggesting that voltage-gated ion channels play an important role in stimulus-secretion coupling in this cell type. The purpose of this study was to identify the voltage-gated ion channels expressed in murine L-cells and investigate their role in GLP-1 release. Materials and methods: Transgenic mice expressing the Venus fluorescent protein under the control of the proglucagon promoter were used as a model system. Quantitative real-time PCR (qPCR) was used to quantify the expression of voltage-gated ion channels in Venus-expressing L-cells, purified by flow cytometry. Standard whole-cell patch-clamp experiments and fluorescence calcium imaging were performed on primary cultured colonic L-cells, identified by their expression of Venus. GLP-1 secretion from primary cultures of adult mouse colon was measured by ELISA. Results: Results are expressed as mean ± SEM. Whole-cell voltage-clamp recordings revealed large rapidly-inactivating, tetrodotoxin (TTX)-sensitive sodium currents (-850±123pA cell-1 at 0mV, n=9), which exhibited half maximal activation at -17±1mV (n=9), and half-maximal inactivation at -46±1mV (n=10). In agreement with these findings, qPCR analysis showed that L-cells predominantly express scn3a (n=3), which is a TTX-sensitive sodium channel isoform. In the presence of TTX (0.3μM), the residual inward current persisted in the absence of Na+ (n=4) but was eliminated by 5mM Co2+, strongly suggesting that this is a voltage-dependent Ca2+ current (p<0.001 by Student's t test, n=10). GLP-1 secretion in the presence of 75mM KCl was markedly inhibited by the L-type Ca2+ channel blocker nifedipine (10μM; p<0.01 by Student's t test, n=5). Furthermore, this selective antagonist attenuated KCl-induced elevation in [Ca2+]i, further confirming that murine L-cells express L-type Ca2+ channels. Ω-Conotoxin MVIIC (1μM) also reduced the secretion of GLP-1 (p<0.05 by Student's t test, n=6). Although this toxin is a recognised blocker of Q-type channels, it also non-selectively blocks N- and P-type channels. However, ω-Conotoxin GVIA (1μM) and ω-Agatoxin IVA (200nM), which block N- and P-type channels respectively, had no effect upon hormone release in the presence of KCl, indicating that murine L-cells express Q-type Ca2+ channels. Conclusion: L-cells are electrically excitable and changes in membrane potential play an important role in the regulation of GLP-1 release by opening voltage-gated Ca2+ channels. Improving our understanding of the stimulus-secretion coupling pathways in L-cells will hopefully facilitate the development of novel therapeutics for the treatment of Type 2 Diabetes.
Where applicable, experiments conform with Society ethical requirements