Inhibition of transmitter release from rat sympathetic neurons via presynaptic M(1) muscarinic acetylcholine receptors.

BACKGROUND AND PURPOSE: M(2), M(3) and/or M(4) muscarinic acetylcholine receptors have been reported to mediate presynaptic inhibition in sympathetic neurons. M(1) receptors mediate an inhibition of K(v)7, Ca(V)1 and Ca(V)2.2 channels. These effects cause increases and decreases in transmitter release, respectively, but presynaptic M(1) receptors are generally considered facilitatory. Here, we searched for inhibitory presynaptic M(1) receptors. EXPERIMENTAL APPROACH: In primary cultures of rat superior cervical ganglion neurons, Ca(2+) currents were recorded via the perforated patch-clamp technique, and the release of [(3)H]-noradrenaline was determined. KEY RESULTS: The muscarinic agonist oxotremorine M (OxoM) transiently enhanced (3)H outflow and reduced electrically evoked release, once the stimulant effect had faded. The stimulant effect was enhanced by pertussis toxin (PTX) and was abolished by blocking M(1) receptors, by opening K(v)7 channels and by preventing action potential propagation. The inhibitory effect was not altered by preventing action potentials or by opening K(v)7 channels, but was reduced by PTX and omega-conotoxin GVIA. The inhibition remaining after PTX treatment was abolished by blockage of M(1) receptors or inhibition of phospholipase C. When [(3)H]-noradrenaline release was triggered independently of voltage-activated Ca(2+) channels (VACCs), OxoM failed to cause any inhibition. The inhibition of Ca(2+) currents by OxoM was also reduced by omega-conotoxin and PTX and was abolished by M(1) antagonism in PTX-treated neurons. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that M(1), in addition to M(2), M(3) and M(4), receptors mediate presynaptic inhibition in sympathetic neurons using phospholipase C to close VACCs.

Annexin 5 mediates a peroxide-induced Ca(2+) influx in B cells.

Annexin 5 is a Ca(2+)-binding protein, the function of which is poorly understood. Structural and electrophysiological studies have shown that annexin 5 can mediate Ca(2+) fluxes across phospholipid membranes in vitro [1]. There is, however, no direct evidence for the existence of annexin 5 Ca(2+) channels in living cells. Here, we show that annexin 5 inserts into phospholipid vesicle membranes at neutral pH in the presence of peroxide. We then used targeted gene disruption to explore the role of annexin 5 in peroxide-induced Ca(2+) signaling in DT40 pre-B cells. DT40 clones lacking annexin 5 exhibited normal Ca(2+) responses to both thapsigargin and B-cell receptor stimulation, but lacked the sustained phase of the response to peroxide. This late phase was due to Ca(2+) influx from the extracellular space, demonstrating that annexin 5 mediates a peroxide-induced Ca(2+) influx. Thus, peroxide induces annexin 5 membrane insertion in vitro, and peroxide-induced Ca(2+) entry in vivo in DT40 cells requires annexin 5. Our results are consistent with a role for annexin 5 either as a Ca(2+) channel, or as a signaling intermediate in the peroxide-induced Ca(2+)-influx pathway.

Inhibition of EGF-dependent calcium influx by annexin VI is splice form-specific.

Annexin VI is a widely expressed calcium- and phospholipid-binding protein that lacks a clear physiological role. We now report that A431 cells expressing annexin VI are defective in their ability to sustain elevated levels of cytosolic Ca(2+) following stimulation with EGF. Other aspects of EGF receptor signaling, such as protein tyrosine phosphorylation and induction of c-fos are normal in these cells. However, EGF-mediated membrane hyperpolarization is attenuated and Ca(2+) entry abolished in cells expressing annexin VI. This effect of annexin VI was only observed for the larger of the two annexin VI splice forms, the smaller splice variant had no discernable effect on either cellular phenotype or growth rate. Inhibition of Ca(2+) influx was specific for the EGF-induced pathway; capacitative Ca(2+) influx initiated by emptying of intracellular stores was unaffected. These results provide the first evidence that the two splice forms of annexin VI have different functions.

S100 calcium binding protein affects neuronal electrical discharge activity by modulation of potassium currents.

S100 calcium binding protein has been associated with a variety of intra- and extracellular calcium-mediated functions, including learning and memory. We have previously localized S100-immunoreactive neurons correlated with spontaneous discharge activity in the central nervous system of the mollusc, Helix pomatia. In this study, we further investigated the effects of S100 (S100B and S100A1) on electrical discharge activity and membrane currents of Helix neurons, using current- and voltage-clamp techniques. Extracellular application of disulphide-linked S100B (S100B-s-s) in pico- to nanogram/ml concentrations was found to hyperpolarize the membrane resting potential, to inhibit spontaneous discharge activity of action potentials, to alter the stimulus response behaviour from tonic to phasic, to decrease the duration and increase the afterhyperpolarization of action potentials, and to reduce the cell input resistance. Measurement of membrane currents revealed that the total outward current was increased by S100B-s-s. Separation of outward currents showed that three types of potassium currents were altered: (i) an inward rectifying current, (ii) a calcium-activated potassium outward current, both increased by S100B-s-s, and (iii) a delayed, voltage-dependent potassium outward current which was decreased by the protein. The transient potassium outward and the calcium inward currents were not affected by S100B-s-s. Immunocytochemistry showed intracellular labelling of the cytoplasm after extracellular application of the protein, indicating internalization and suggesting an internal site of action. Injection of S100A1 mimicked the effects of S100B-s-s on discharge activity and action potentials. We conclude from our experiments that S100 calcium binding protein, by modulation of potassium currents, may play a role as a neuromodulator in nervous functions.

Characterisation of calcium signalling in DT40 chicken B-cells.

The chicken DT40 pre-B-cell line is becoming a potent experimental tool in the elucidation of higher organism cellular functions due to its unique genetic tractability. While several publications have described the effects of disruption of a range of genes in DT40 cells on calcium signalling, there has been no general overview of Ca2+ responses in wild-type cells. Here, we present experimental data comparing and contrasting the calcium responses to a range of agonists, such as alphaIgM, H2O2 and thapsigargin, applied singly or consecutively in the presence or absence of extracellular calcium. Briefly, we show that calcium release is from thapsigargin-sensitive and also -insensitive stores. This release results in, or is concomitant with, calcium entry across the plasma membrane through store-operated, receptor-operated and possibly L-type like Ca2+ channels. The agonists activate these pathways differentially producing a wide range of different sized and shaped Ca2+ signals. Furthermore, we report that Ca2+ responses in DT40 cells are dependent on the growth conditions. The presence of 1% chicken serum in the growth medium increased amplitudes of calcium responses and enhanced the sustained phase of the alphaIgM response, while 10 microM beta-mercaptoethanol in the medium (not, however, present during calcium measurements) resulted in more transient H2O2 responses and larger amplitude alphaIgM responses while failing to affect thapsigargin responses. The possible causes of these effects and their importance in comparing data from different studies on DT40 cells is discussed.

S-100-immunoreactivity in spontaneously active snail neurons.

The distribution of S-100-immunoreactive material was examined in the central nervous system of the gastropod snail. Helix pomatia, and electrophysiological properties of S-100-positive neurons were characterized. Immunocytochemical studies revealed S-100-like protein to be present in neurons localized in the cerebral, left parietal, and visceral ganglia, but not in glial cells. Among the immunoreactive neurons we identified the giant cells LPa3 and LPa4. Western blots showed a single S-100-immunoreactive band at 12-14 kDa. S-100-positive neurons are distinguished by spontaneous discharge activity in a beating or bursting mode and a prominent Ca(2+)-activated potassium outward current. Our result show that a S-100-like protein exclusively present in neurons of the Helix central nervous system is correlated with spontaneous discharge activity of these cells.

S-100-immunoreactive protein in Helix neurons.

S-100 proteins are a versatile group of calcium-binding proteins which exert intracellular as well as extracellular functions in a variety of cells. Our immunocytochemical studies, using the central nervous system of the terrestrial snail, Helix pomatia, showed that S-100-like material is present exclusively in a restricted number of neurons distributed in the cerebral ganglia, the left parietal ganglion, and the visceral ganglion. Electrophysiological investigations showed that immunostained neurons spontaneously discharge action potentials in a beating or bursting mode. Voltage clamp experiments further revealed a pronounced N-shaped current-voltage relationship of outward currents, indicating a prominent calcium-activated potassium current in these cells. Extracellular application of the disulfidic form of S-100(s-s) results in suppression of spontaneous discharge activity accompanied by an increase of membrane conductance. The total outward current as well as the inward rectifier current were increased. After removal of the calcium-dependent outward current component, S-100 effects were abolished indicating that S-100 acts on the calcium-activated current component. Our results suggest that S-100-like proteins modulate electrical activity of neurons in the central nervous system of Helix pomatia.