neuropeptide-y and fura-2-am

Neuropeptide Y (NPY) is a potent inhibitory neuropeptide expressed by amacrine cells in the rat retina. NPY modulates the release of multiple neurotransmitters in mammalian retina, yet the mechanisms mediating this regulation are not well defined. To further understand the action of NPY in the retina, Y receptor coupling to voltage-dependent Ca(2+) channels was investigated using Ca(2+) imaging with fura-2 AM to measure [Ca(2+)](i) increases in rod bipolar cell terminals. Y receptor expression was studied in rat retinal tissue with reverse transcription-polymerase chain reaction (RT-PCR). NPY inhibited the depolarization-evoked Ca(2+) influx into rod bipolar cell axon terminals and caused a dose-dependent reduction and an average maximal inhibition of 72% at 1 microM, which was reversed upon washout. K(+)-evoked Ca(2+) increases were also inhibited by the selective Y2 receptor agonists, C2-NPY and NPY(13-36), at concentrations of 1 microM, but not by the selective Y1 receptor agonist, [Leu(31)Pro(34)]NPY, selective Y4 receptor agonist, rPP, or the selective Y5 receptor agonist, [d-Trp32]-NPY. Y receptor expression was determined using RT-PCR for all known Y receptor subtypes. Y2 receptor mRNA, as well as Y1, Y4, and Y5 receptor mRNAs, are present in the rat retina. Like the rod bipolar cell, other studies in central neurons have shown that the Y2 receptor is expressed predominantly as a presynaptic receptor and that it modulates transmitter release. Together, these findings suggest that NPY activates presynaptic Y2 receptors to inhibit voltage-dependent Ca(2+) influx into rod bipolar cell terminals, and establishes one mechanism by which NPY may reduce l-glutamate release from the rod bipolar cell synapse.

Topics: Animals; Calcium; Calcium Channels; Dose-Response Relationship, Drug; Female; Fura-2; Immunohistochemistry; Male; Neuropeptide Y; Protein Isoforms; Rats; Rats, Sprague-Dawley; Receptors, Neuropeptide Y; Retinal Rod Photoreceptor Cells; Reverse Transcriptase Polymerase Chain Reaction; Synapses

Stimulation of neuropeptide Y (NPY) Y2 receptors induced an intracellular free Ca2+ ([Ca2+]i) increase in a human neuroblastoma cell line, CHP-234. When NPY in a Ca(2+)-free solution was applied, this increase was abolished. Depolarization with high KCl evoked no response, suggesting that the responses were not mediated by voltage-gated Ca2+ channels. There was no evidence that the NPY response consisted of a capacitative Ca2+ entry sensitive to internal Ca2+ store levels. The [Ca2+]i elevation was diminished by Ni2+, a blocker of Ca2+ entry. Mn2+ induced a quench of the fura-2 fluorescence, which ceased promptly upon the removal of NPY, indicating that Ca2+ entry was linked tightly to receptor activation. Although thapsigargin- and ryanodine-sensitive Ca2+ stores were present, NPY-induced responses were not impaired by pretreatment with either drug. Furthermore, NPY had no effect on the thapsigargin-sensitive store. Pertussis toxin did not affect the NPY-stimulated [Ca2+]i increase, although it abolished the NPY-dependent inhibition of cAMP production. It is concluded that the Y2 receptors couple directly to receptor-operated Ca2+ channels without the involvement of intracellular Ca2+ stores. The results also indicate that Y2 receptors can activate both pertussis toxin-sensitive and -insensitive mechanisms in the same cell.

Topics: Calcium; Calcium Channels; Calcium-Transporting ATPases; Cell Line; Cyclic AMP; Fluorescent Dyes; Fura-2; Humans; Kinetics; Microscopy, Fluorescence; Neuroblastoma; Neuropeptide Y; Nickel; Pertussis Toxin; Potassium Chloride; Receptors, Neuropeptide Y; Terpenes; Thapsigargin; Time Factors; Tumor Cells, Cultured; Virulence Factors, Bordetella