Proceedings of The Physiological Society
University of Oxford (2008) Proc Physiol Soc 12, C7 and PC17
Electrophysiological characterisation of murine primary L-cells
G. J. Rogers1, F. Reimann1, G. Tolhurst1, H. Parker1, A. Habib1, F. Gribble1
1. CIMR Clinical Biochemistry, University of Cambridge, Cambridge, United Kingdom.
Glucagon-like peptide-1 (GLP-1) is an enteric hormone released by L-cells in response to nutrient ingestion. Although such cells are present throughout the gastrointestinal tract, they are most abundant in the ileum and colon. GLP-1 is a potent insulinotropic agent that also inhibits beta-cell apoptosis, making it a particularly attractive therapeutic target for the treatment of type 2 diabetes. Pharmacological GLP-1 mimetics and inhibitors of GLP-1 degradation are currently employed to activate the GLP-1 axis therapeutically. An alternative approach would be to enhance the secretion of endogenous GLP-1. However, this has been hampered by a poor understanding of stimulus-secretion coupling in this cell type due largely to difficulties in identifying and culturing primary L-cells. The generation of transgenic mice that express YFP under the control of the proglucagon promoter enables the identification of L-cells, making functional characterisation of such cells possible. The purpose of this study was to characterise the electrical properties of L-cells and their expression of voltage-gated ion channels. This was achieved by performing standard and perforated-patch whole-cell patch-clamp experiments on primary cultured murine colonic L-cells identified by their expression of YFP. Immunocytochemistry confirmed that glucagon and peptide YY (PYY) expression was restricted to YFP-expressing cells and that the cells maintain their differentiated phenotype in culture. L-cells were electrically active, with a resting membrane potential of ~-50 mV and a threshold for firing action potentials of -35 ± 2 mV (n=9). 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 ± 1 mV (n=9), and half-maximal inactivation at -46 ± 1 mV (n=10). FACs-sorted L-cells predominantly expressed scn3a, as assessed by quantitative RT-PCR. In the presence of TTX (0.3µM), the residual inward current was abolished by 5mM Co2+, strongly suggesting that this is a voltage-dependent Ca2+ current (n=3). The use of pharmacological blockers such as isradipine (10µM) and NNC 55-0396 (1µM) partially ablated the TTX-resistant current confirming the presence of L- and T-type calcium channels, respectively. We believe that this is the first demonstration and characterisation of Na+-dependent electrical activity in a primary enteroendocrine cell type. Improving our understanding of L-cell function may have potential implications for the development of new anti-diabetic therapeutics targeting the enteroendocrine axis.
Where applicable, experiments conform with Society ethical requirements