No abstract

We analyzed the contribution of calcium (Ca2+)-induced Ca2+ release to somatic secretion in serotonergic Retzius neurons of the leech. Somatic secretion was studied by the incorporation of fluorescent dye FM1-43 upon electrical stimulation with trains of 10 impulses and by electron microscopy. Quantification of secretion with FM1-43 was made in cultured neurons to improve optical resolution. Stimulation in the presence of FM1-43 produced a frequency-dependent number of fluorescent spots. While a 1-Hz train produced 19.5+/-5.0 spots/soma, a 10-Hz train produced 146.7+/-20.2 spots/soma. Incubation with caffeine (10 mM) to induce Ca2+ release from intracellular stores without electrical stimulation and external Ca2+, produced 168+/-21.7 spots/soma. This staining was reduced by 49% if neurons were preincubated with the Ca2+- ATPase inhibitor thapsigargin (200 nM). Moreover, in neurons stimulated at 10 Hz in the presence of ryanodine (100 microM) to block Ca2+-induced Ca2+ release, FM1-43 staining was reduced by 42%. In electron micrographs of neurons at rest or stimulated at 1 Hz in the ganglion, endoplasmic reticulum lay between clusters of dense core vesicles and the plasma membrane. In contrast, in neurons stimulated at 20 Hz, the vesicle clusters were apposed to the plasma membrane and flanked by the endoplasmic reticulum. These results suggest that Ca2+-induced Ca2+ release produces vesicle mobilization and fusion in the soma of Retzius neurons, and supports the idea that neuronal somatic secretion shares common mechanisms with secretion by excitable endocrine cells.

Ca efflux from rat brain presynaptic nerve terminals (synaptosomes) was examined after loading the terminals with 45Ca during a brief depolarization, usually in media containing 20 microM Ca labeled with 45Ca, to assure a small (physiological) load. Efflux of 45Ca was very slow in the absence of external Na and Ca (approximately 0.5% of the load/s) and was greatly accelerated by Na and/or Ca (presumably Na+-Ca2+ and Ca2+-Ca2+ exchange, respectively). The dependence of 45Ca efflux on external Na was sigmoid, with a Hill coefficient of approximately 2.5; this implies that more than two external Na ions are required to activate the efflux of one Ca ion. The external Na (Nao)-dependent Ca efflux was inhibited by 1 mM external La, by low temperature (Q10 congruent to 2.3), and by raising external K (to depolarize the synaptosomes). With small Ca loads, the mitochondrial uncoupler, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), had negligible effect on either Ca uptake or efflux; with large loads (greater than or equal to 5 nmol/mg protein), however, FCCP reduced the depolarization-stimulated Ca uptake and increased the Nao-dependent Ca efflux. These effects may be attributed to reduction of mitochondrial Ca sequestration. Mitochondria do not appear to sequester much Ca when the loads are smaller (and more physiological). Estimations of Ca efflux indicate that approximately 20% of a small 45Ca load (approximately 0.75 nmol Ca/mg protein) may be extruded via Na+-Ca2+ exchange within 1 s; this corresponds to a net Ca efflux of approximately 110 pmol Ca X mg protein-1 X s-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotoninergic Retzius neurons reform an inhibitory synapse onto pressure-sensitive mechanosensory (P) neurons when the cells are removed from the nervous system of the leech and are juxtaposed in tissue culture. The somas of P cells in situ and single (uninnervated) P cells in culture have both a depolarizing and Cl-dependent hyperpolarizing response to application of the transmitter serotonin (5-HT). Synaptic release of 5-HT by a Retzius cell in situ and in culture evokes a Cl-dependent postsynaptic response but does not appear to activate the depolarizing receptors. We have characterized the ionic currents induced by synaptically released and applied 5-HT in voltage-clamped P cells in culture in order to determine the responses to transmitter and their modifications following innervation. When 5-HT was applied to single P cells, 2 types of channels were activated by 5-HT and could be distinguished by changing the ionic composition of the superfusion solution. In an impermeant cation (TrisCl) solution, a 5-HT-dependent Cl current was activated. When single P cells were superfused with a Cl-free solution (Cl replaced by impermeant SO4), 5-HT activated a monovalent cation current. Following innervation of a P cell by a cocultured Retzius cell, the reversal potential of the peak postsynaptic current depended on the Cl gradient and the synaptic response was blocked by the Cl channel blocker 9-anthracenecarboxylic acid. Thus, synaptic release of 5-HT activated solely the Cl channels and not the cationic channels. Pipette application of 5-HT onto innervated P cells activated a Cl conductance comparable in magnitude to the synaptic response. In contrast, the cationic conductance was reduced roughly 5-fold. It is concluded that innervation of a P cell by a Retzius cell resulted in clustering of the synaptic 5-HT receptors, which activate Cl channels and reduction of the nonsynaptic, cationic response. The result is a selection of receptors in the cultured P cell that mimics the pattern observed in vivo.

1. We investigated the regulation of intracellular pH (pHi) in rat brain isolated nerve terminals (synaptosomes), using fluorescence pH indicators and time-resolved fluorescence spectroscopy. 2. The resting pHi was not significantly affected by the presence or absence of HCO3-. Removal of external Na+, in the absence or presence of HCO3- caused a rapid acidification of pHi. The recovery from acid loads was primarily due to the activity of the Na+/H+ exchanger, confirming the relevance of this transport system in synaptosomes. 3. Our data revealed that in synaptosomes the activity of the Na+/H+ exchanger was not regulated by either protein kinase C or kinase A. In contrast, Ca2+ played an important role in the regulation of Na+/H+ exchanger. This was supported by the observation that 4Br-A23187 induced a Na(+)-dependent alkalinization of the resting pHi and greatly enhanced the initial rate and the degree of the recovery from acid loads. 4. In most eukaryotic cells, HCO3(-)-based transport mechanisms play an important role in pHi regulation. In synaptosomes, however, HCO3- transport is not significantly involved in pHi regulation, because the presence or absence of HCO3- does not affect resting pHi nor the rate of pHi recovery to acid loads. Further studies to address the role of Cl- and HCO3- in pHi regulation in synaptosomes are discussed in the companion paper. 5. Increasing the concentration of Ko+ also resulted in a rise of steady-state pHi by a processes that is Ca2+ and HCO3- independent. This alkalinization could be due to either K+/H+ exchanger activity, K(+)-induced depolarization, reduction of delta microH+, or a direct reduction of delta microK+. Calculated H+ driving forces suggest that the reduction in the inwardly directed H+ leak is sufficient to explain this K(+)-induced alkalinization because it changes the delta microH+ by virtue of setting the membrane potential difference (Em) to the K+ equilibrium potential (EK+).