Generation Of Calcium Spikes Research Articles

The Influence of Exogenous ATP on Functional Responses of Murine Bone Marrow Granulocytes

Extracellular ATP is a potent inflammatory mediator and regulator of neutrophil activity in an inflammatory site. The role of ATP in the regulation of bone marrow granulocyte functions remains unexplored. The aim of this work was to study effects of ATP on the cytoplasmic Ca2+ concentration ([Ca2+]i), generation of reactive oxygen species (ROS) and adhesion of bone marrow granulocytes. Changes of [Ca2+]i were estimated with Fura-2 AM ratiometric probe. To reveal cells with calcium activity in a population we developed an automated procedure based on the calculation of skewness and kurtosis of the [Ca2+]i value distribution. ROS generation was estimated by luminol-dependent chemiluminescence. Cell adhesion was evaluated by a standard absorbance (492 nm) method. The population of bone marrow granulocytes contained cells that generated calcium spikes in response to 10 μM ATP (35.2 ± 4.0%). The automated scanning allowed us to identify four groups of cells: those responding to ATP (23.4 ± 4.9%) or to the addition of Hanks’ solution (16.9 ± 2.5%) or to both ATP and Hanks’ solution (6.7 ± 1.5%), and cells not responding to either stimulus (53.1 ± 8.2%). ATP increased the adhesion of granulocytes, while apyrase, which hydrolyzes ATP, did not significantly change the adhesion. ATP did not influence the respiratory burst initiated by the bacterial peptide N-formyl-MLF (fMLF). Apyrase significantly enhanced the response to fMLF, and cytochalasin D inhibited this effect. In summary, exogenous ATP enhances adhesion and generation of calcium spikes in the population of bone marrow granulocytes. Endogenous ATP likely reduces respiratory burst through a mechanism involving the cytoskeleton. The studied granulocyte population contains cells with different ATP sensitivity.

NaV1.9 Potentiates Oxidized Phospholipid-Induced TRP Responses Only under Inflammatory Conditions.

Oxidized phospholipids (OxPL) like oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) were recently identified as novel proalgesic targets in acute and chronic inflammatory pain. These endogenous chemical irritants are generated in inflamed tissue and mediate their pain-inducing function by activating the transient receptor potential channels TRPA1 and TRPV1 expressed in sensory neurons. Notably, prototypical therapeutics interfering with OxPL were shown to inhibit TRP channel activation and pain behavior. Here, we asked how OxPL excite primary sensory neurons of dorsal root ganglia (DRG neurons from mice of either sex). Acute stimulation of sensory neurons with the prototypical OxPL 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) evoked repetitive calcium spikes in small-diameter neurons. As NaV1.9, a voltage-gated sodium channel involved in nociceptor excitability, was previously shown to be essential for the generation of calcium spikes in motoneurons, we asked if this channel is also important for OxPL mediated calcium spike and action potential generation in nociceptors. In wild-type and NaV1.9-deficient neurons, the action potential firing rate and the calcium spike frequency to an acute PGPC stimulus was similar. When preincubated with inflammatory mediators, both, the action potential firing rate and the calcium spike frequency were markedly increased in response to an acute PGPC stimulus. However, this potentiating effect was completely lost in NaV1.9-deficient small-diameter neurons. After treatment with inflammatory mediators, the resting membrane potential of NaV1.9 KO neurons was slightly more negative than that of wild-type control neurons. This suggests that NaV1.9 channels are active under this condition and therefore increases the ease with which action potentials are elicited after OxPL stimulation. In summary, our data suggest that NaV1.9 has a switch function to potentiate the receptor potentials induced by OxPL under inflammatory conditions. Since human NaV1.9 has been shown to mediate painful and painless channelopathies, this study provides new insights into the mechanism by which NaV1.9 amplifies stimuli of endogenous irritants under inflammatory conditions.

Dedifferentiation of intrinsic response properties of motoneurons in organotypic cultures of the spinal cord of the adult turtle.

Explant cultures from the spinal cord of adult turtles were established and used to study the sensitivity of the intrinsic response properties of motoneurons to the changes in connectivity and milieu imposed by isolation in culture. Transverse sections 700 microm thick were explanted on cover slips and maintained in roller-tube cultures in medium containing serum and the growth factors brain-derived neurotrophin factor (BDNF), neurotrophin-3 (NT3), glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF). The gross morphology of acute sections was maintained after 4 weeks in culture. Cell bodies of motoneurons remained stainable in fixed cultures with an antibody against choline acetyltransferase (ChAT) throughout the culture period. During culture, motoneurons maintained stable resting membrane potentials and were contacted by functional synapses. The ability to generate action potentials was also preserved as was delayed inward rectification and generation of calcium spikes in the presence of tetra-ethyl ammonium (TEA). In response to depolarization, however, motoneurons presented strong outward rectification, and only 41% of the cells recorded from maintained the ability to fire repetitively. By the second week in culture, a fraction of motoneurons displayed fast and slow transient outward rectification and low-threshold calcium spikes, features not seen in turtle motoneurons in acute slices. On the other hand, properties mediated by L-type Ca2+ channels disappeared during the first few days in culture. Our observations show that the phenotypical intrinsic response properties of mature spinal motoneurons are modified in explant cultures. The properties acquired resemble the properties in juvenile motoneurons in several species of terrestrial vertebrates.

Electrophysiological properties of rat pontine nuclei neurons In vitro. I. Membrane potentials and firing patterns.

We used a new slice preparation of rat brain stem to establish the basic membrane properties of neurons in the pontine nuclei (PN). Using standard intracellular recordings, we found that pontine cells displayed a resting membrane potential of -63 +/- 6 mV (mean +/- SD), an input resistance of 53 +/- 21 MOmega, a membrane time constant of 5.3 +/- 2.4 ms and were not spontaneously active. The current-voltage relationship of most of the PN neurons showed the characteristics of inward rectification in both depolarizing and hyperpolarizing directions. A prominent feature of the firing of pontine neurons was a marked firing rate adaptation, which eventually caused the cells to cease firing. Several types of membrane conductances possibly contribute to this feature. For one, a medium and a slow type of afterhyperpolarization (AHP) control the pattern of firing. The medium AHP was partly susceptible to blockade of calcium influx, whereas it was abolished completely by blockade of potassium channels with tetraethylammonium, indicating that it is based on at least two conductances: a calcium-dependent and a calcium-independent one. The slow AHP was carried by potassium ions and could be blocked effectively by preventing calcium influx into the cell. It was present after single spikes but was strongest after a high-frequency spike train. Calcium entry into the cell was mediated by high-threshold calcium channels that were detected by the generation of calcium spikes under blockade of potassium channels. Furthermore, the early phase of the firing rate adaptation was shown to be related to the time course of a slow, tetrodotoxin (TTX)-sensitive, persistent sodium potential, which was activated already in the subthreshold range of membrane potentials. This potential was time dependent and imposed as a depolarizing "hump" with a maximum occurring in most cases between 50 and 100 ms after stimulus onset. In the suprathreshold range, it generated plateau potentials following fast spikes, if potassium channels were blocked. After the complete adaptation of the firing rate, PN neurons were observed to display irregular fluctuations of the membrane potential, which sometimes reached firing threshold thereby eliciting an irregular low-frequency spike train. As these fluctuations could be blocked with TTX, they probably are based on the persistent sodium currents. The opposing drive in hyperpolarizing direction may be provided by strong outward currents that generated a marked outward rectification in the current-voltage relationship under TTX. In conclusion, PN neurons show complex membrane properties that are reminiscent in many ways to cerebrocortical "regular firing" neurons.