The release and depletion of neurotensin in sympathetic preganglionic axon terminals and internalization in principal ganglion cells were investigated in the cat stellate ganglion by means of combined immunohistochemical staining, image analysis and confocal microscopy. Neurotensin stored in preganglionic boutons was released by 40 or 5 Hz electrical stimulation of preganglionic nerves, being depleted to 7.4 and 19.2% of control levels by continuous stimulation lasting 20 or 160 min (both stimuli delivered 48,000 pulses). Once released, neurotensin was internalized by the principal ganglion cells as evidenced by a ring of bright spot-like granules in the perinuclear region indicating the sites of intracellular neurotensin accumulation. Neurotensin internalization was time-dependent, thus, different content was found when the time between the end of stimulation and start of perfusion was varied. The onset of neurotensin internalization appeared in the first minutes, intracellular accumulation was evident at 20 min, maximal internalization occurred at 120 min and, 24 h later internalized neurotensin content had faded. Internalization was partially blocked by the nonpeptide neurotensin antagonist SR48692. These data provide evidence of presynaptic neurotensin release and depletion by electrical stimulation with varied frequencies. They also provide evidence for in situ receptor-mediated internalization of endogenously released neurotensin, raising the possibility that internalization may represent, in addition to some kind of turnover dynamics, an important part of the mechanisms of neuropeptide signaling.
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