Abstract

We studied calcium signaling in a newly described pancreatic cell line, GK-P3, that expresses functional amino acid neurotransmitter receptors. GK-P3 cells express the first strychnine-sensitive glycine receptors reported in a permanent cell line. In addition, GK-P3 cells express α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. Both types of amino acid receptors showed electrophysiological and pharmacological behavior similar to their neuronal counterparts. The glycine receptors were permeable to Cl⁻ and blocked by the selective antagonist strychnine. AMPA receptors showed limited permeability to Ca²⁺, were blocked by 6-cyano-2,3-dihydroxy-7-nitroquinoxaline, and were potentiated by cyclothiazide. Interestingly, activation of either receptor type increased intracellular Ca²⁺ measured by digital imaging of Fura-2 fluorescence. These Ca²⁺ signals were completely blocked by 30 μm La³⁺, suggesting that the Ca²⁺ entered the cells largely through voltage-dependent Ca²⁺ channels. Alterations in the extracellular concentrations of Cl⁻ and/or HCO₃⁻ had only marginal effects on glycine-evoked Ca²⁺ signals. However, increases in intracellular Ca²⁺ mediated by AMPA receptors were absent when the extracellular Na⁺ was replaced with an impermeant cation, N-methyl-d-glucamine. We conclude that activation of ligand-gated cation or anion channels depolarize GK-P3 cells sufficiently to activate their voltage-gated Ca²⁺ channels leading to increases in intracellular Ca²⁺ concentration. Thus, glycine and glutamate receptors may regulate Ca²⁺-dependent secretory mechanisms in islet cells by altering the membrane potential of these cells. Our data in GK-P3 cells support the growing weight of evidence for a role of amino acid neurotransmitters in pancreatic islets and introduce strychnine-sensitive glycine receptors as a novel target of amino acid neurotransmitter regulation in islets.