Mammalian pinealocytes are neuroendocrine cells that synthesize and secrete melatonin, these processes being positively controlled by norepinephrine derived from innervating sympathetic neurons. Previously, we showed that pinealocytes contain a large number of microvesicles (MVs) that specifically accumulate L-glutamate through a vesicular glutamate transporter and contain proteins for exocytosis such as synaptobrevin 2 (VAMP2). These findings suggested that the MVs are counterparts of synaptic vesicles and are involved in paracrine-like chemical transduction in the pineal gland. Here, we show that pinealocytes actually secrete glutamate upon stimulation by KCl in the presence of Ca2+ at 37 degrees C. The ability of glutamate secretion disappeared when the cells were incubated at below 20 degrees C. Loss of the activity was also observed on successive stimulation, but it was recovered after 12 hr incubation. A low concentration of cadmium chloride or omega-conotoxin GVIA inhibited the secretion. Botulinum neurotoxin E cleaved synaptic vesicle-associated protein 25 (SNAP-25) and thus inhibited the secretion. The released L-glutamate stimulated pinealocytes themselves via glutamate receptor(s) and inhibited norepinephrine-stimulated melatonin secretion. These results strongly suggest that pinealocytes are glutaminergic paraneurons, and that the glutaminergic system regulates negatively the synthesis and secretion of melatonin. The MV-mediated paracrine-like chemical transduction seems to be a novel mechanism that regulates hormonal secretion by neuroendocrine cells.

1. We have investigated interactions between intracellular pH (pHJ) and the intracellular free calcium concentration ([Ca2+]i) in collagenase-isolated rat lacrimal acinar cells. The fluorescent dyes fura-2 and 2',7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF) were used to measure [Ca2+] Ca2+ from the intracellular agonist-sensitive Ca2+ pool. However, releasing stored Ca2+ via alkalinization does not appear to trigger significant Ca2+ entry, perhaps because intracellular alkalinization inhibits either the Ca2P entry pathway or the mechanism which couples the entry pathway to store depletion.The primary signal controlling fluid and electrolyte Nae and with changes in intracellular pH (see Nauntofte, secretion in exocrine acinar cells is an increase in the intra-1992, for review). The interactions between these changes in cellular free calcium concentration ([Ca2P]

Spatial and temporal changes of cytoplasmic free calcium concentration ([Ca2+]i) in single parietal cells of guinea pig were investigated with a digital imaging microscope equipped with a microspectrofluorometer, using a Ca2+-sensitive dye, fura-2. Intracellular distribution of [Ca2+]i was not homogeneous, but there were two kinds of [Ca2+]i gradient in the resting parietal cells, one a continuous gradient increasing towards the plasma membrane and a second discontinuous gradient (Ca2+ plateau) in some restricted regions of the cytoplasm. When treated with gastrin, only about 40% of parietal cells in the gastric gland responded with an almost twofold increase in the average resting [Ca2+]i of 52.4 +/- 7.1 nM. In the responding cells, the discontinuous plateaus transiently enlarged to the entire cytoplasm. In marked contrast, all of these cells responded to Ca2+ ionophore ionomycin. We also found that when provoked by gastrin Ca2+ transient in the parietal cells in the gastric gland often propagated to some adjacent cells, and occasionally spontaneous Ca2+ transient and oscillation were observed even in the resting state.

The myo-inositol 1,4,5trisphosphate (IPs)-induced Caz + mobilization in single saponin-permeabilixed and fura-2-loaded

We investigated spatiotemporal changes in cytoplasmic free Ca2 concentration ([Ca2 Jj) in norepinephrine (NE)-stimulated and fura-2-loaded individual H-35 rat hepatoma cells, using digital imaging microscopy and high time-resolution microspectrofluorometry. Application of NE (5 x 10 M) resulted in an initial transient increase in [Ca2 jj, followed by a small sustained [Ca2 Ji plateau above the pre-stimulaiion level. The initial peak and the small sustained plateau originated from intracellular stores and the extracellular space, respectively. The initial transient evoked by NE was totally blocked by phentolamine, an a-adrenergic antagonist, but was not blocked by either pre-incubation with nominally Ca2-free medium or by pre-treatment ofcells with La3. On the other hand, the sustained plateau was eliminated by act synergistically (2,7,29).