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.
Footnotes
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Send reprint requests to: Dr. C. David Weaver, Bristol-Myers Squibb, Pharmaceutical Research Institute, 5 Research Parkway, Wallingford, CT 06492. E-mail: dweaver{at}bms.com
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This work was supported by PHS grants NS 30945 (T.A.V) and DK42612 (M.A.M.), American Heart Association EI 95002450 (T.A.V), and The Vanderbilt Diabetes Research and Training Center DK20593. C.D.W. was supported by NS 09788 and the Juvenile Diabetes Foundation.
- Abbreviations:
- CNS
- central nervous system
- AMPA
- α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
- CNQX
- 6-cyano-2,3-dihydroxy-7-nitroquinoxaline
- GABA
- γ-aminobutyric acid
- NMDA
- N-methyl-d-aspartic acid
- EGTA
- ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- SDS
- sodium dodecyl sulfate
- RT
- reverse transcription
- PCR
- polymerase chain reaction
- nt
- nucleotide
- GluR
- glutamate receptor
- PBS
- phosphate-buffered saline
- TBST
- Tris-buffered saline/Tween 20
- I-V
- current-voltage
- [Ca2+]i
- intracellular calcium concentration
- Received March 30, 1998.
- Accepted June 30, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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