Submitted on May 8, 2005
Accepted on June 29, 2006
IONOTROPIC GLUTAMATE RECEPTOR ACTIVATION INCREASES INTRACELLULAR CALCIUM IN PROLACTIN-RELEASING CELLS OF THE ADENOHYPOPHYSIS
Frederick P Bellinger1*, Bradely K Fox2, Wing Yan Chan3, Lori K Davis2, Marilou A Andres3, Tetsuya Hirano2, E. Gordon Grau4, and Ian M. Cooke5
1 Cell and Molecular Biology, University of Hawaii School of Medicine, Honolulu, United States
2 Hawaii Institute of Marine Biology, University of Hawaii, Kaneohe, United States
3 Pacific Biosciences Reseach Center, University of Hawaii, Honolulu, United States
4 Dept of Zoology, University of Hawaii, Honolulu, United States; Hawaii Institute of Marine Biology, University of Hawaii, Kaneohe, United States
5 Pacific Biosciences Reseach Center, University of Hawaii, Honolulu, United States; Dept of Zoology, University of Hawaii, Honolulu, United States
* To whom correspondence should be addressed. E-mail: rikkugon{at}pbrc.hawaii.edu.
Endocrine cells of the anterior pituitary are controlled by the central nervous system through hormonal interactions and are not believed to receive direct synaptic connections from the brain. Studies suggest that some pituitary cells may be modulated by the neurotransmitter glutamate (5, 16). We investigated prolactin (PRL)-releasing cells of the anterior pituitary of a euryhaline fish, the tilapia (Oreochromis mossambicus) for the presence of possible glutamate receptors. Fura-2 imaging addressed the ability of glutamate to increase intracellular calcium. We observed a dose-dependent increase in intracellular calcium with transient perfusion (1-2 min) of glutamate (10 nM-1 mM) in 2/3 of imaged cells. This increase was attenuated by the ionotropic GluR antagonist kynurenic acid (0.5-1.0 mM). The increase was also blocked or attenuated by antagonists of L-type voltage-gated calcium channels. The glutamate receptor agonist AMPA (100 µM) produced intracellular calcium increases that were reversibly blocked by the selective AMPA antagonist CNQX. In contrast, the selective agonist NMDA (100 µM to 1 mM in magnesium-free solution with 10 µM glycine) had no effect on intracellular calcium. Radioimmunoassays demonstrated that glutamate stimulated PRL-release. CNQX but not the NMDA-receptor antagonist APV blocked this release. Antibodies for mammalian AMPA- and NMDA-type glutamate receptors produced a similar punctate immunoreactivity in the periphery of PRL cells. However, the NMDA antibody recognized a protein of a different molecular weight in PRL cells compared to brain cells. These results clearly indicate the presence of glutamate receptors on tilapia PRL cells that can stimulate PRL release.
