ATP-Evoked Intracellular Ca²âº Signaling of Different Supporting Cells in the Hearing Mouse Hemicochlea.

Hearing and its protection is regulated by ATP-evoked Ca(2+) signaling in the supporting cells of the organ of Corti, however, the unique anatomy of the cochlea hampers observing these mechanisms. For the first time, we have performed functional ratiometric Ca(2+) imaging (fura-2) in three different supporting cell types in the hemicochlea preparation of hearing mice to measure purinergic receptor-mediated Ca(2+) signaling in pillar, Deiters' and Hensen's cells. Their resting [Ca(2+)]i was determined and compared in the same type of preparation. ATP evoked reversible, repeatable and dose-dependent Ca(2+) transients in all three cell types, showing desensitization. Inhibiting the Ca(2+) signaling of the ionotropic P2X (omission of extracellular Ca(2+)) and metabotropic P2Y purinergic receptors (depletion of intracellular Ca(2+) stores) revealed the involvement of both receptor types. Detection of P2X2,3,4,6,7 and P2Y1,2,6,12,14 receptor mRNAs by RT-PCR supported this finding and antagonism by PPADS suggested different functional purinergic receptor population in pillar versus Deiters' and Hensen's cells. The sum of the extra- and intracellular Ca(2+)-dependent components of the response was about equal with the control ATP response (linear additivity) in pillar cells, and showed supralinearity in Deiters' and Hensen's cells. Calcium-induced calcium release might explain this synergistic interaction. The more pronounced Ca(2+) leak from the endoplasmic reticulum in Deiters' and Hensen's cells, unmasked by cyclopiazonic acid, may also suggests the higher activity of the internal stores in Ca(2+) signaling in these cells. Differences in Ca(2+) homeostasis and ATP-induced Ca(2+) signaling might reflect the distinct roles these cells play in cochlear function and pathophysiology.

Platelet-activating factor evokes Ca2+ transients after the blockade of ryanodine receptor by dantrolene in RAW 264.7 macrophages.

In the present study we studied platelet-activating factor (PAF)-, and ATP-induced increases in intracellular Ca2+ concentration ([Ca2+]i) using RAW 264.7 macrophages filled with fura-2/AM and imaged with fluorescence video microscopy. We found that the prevalence of detectable [Ca2+]i responses to PAF application was significantly higher in the presence of dantrolene. Dantrolene itself significantly decreased basal [Ca2+]i of macrophages compared to control cases after a 20-min incubation period. In the dantrolene-treated cells even the peak [Ca2+]i in response to PAF (as an average of all cells) was below the baseline of control suggesting that decreased [Ca2+]i plays a permissive role in the Ca2+ rise induced by PAF in macrophages. In contrast to the effect of PAF, neither the amplitude of response to ATP nor the frequency of responding cells changed significantly during dantrolene treatment in our experiments. These cells were able to respond to a standard immune stimulus as well: lipopolysaccharide (LPS) was able to increase [Ca2+]i. Our data indicate that the effectiveness of PAF to increase [Ca2+]i in RAW 264.7 macrophages depends on the resting [Ca2+]i. It has also been shown in this study that PAF and ATP differently regulate Ca2+ homeostasis in macrophages during inflammatory response and therefore they possibly differently modulate cytokine production by macrophages.

Modulatory role of presynaptic nicotinic receptors in synaptic and non-synaptic chemical communication in the central nervous system.

Neuronal nicotinic acetylcholine receptors (nAChRs) belong to a family of ligand-gated channels closely related to but distinct from the muscle nAChRs. Recent progress in neurochemical and pharmacological methods supports the hypothesis of presynaptically located nAChRs on axon terminals and indicates that the major effect of nAChR is the modulation rather than processing of fast synaptic transmission. Strong neurochemical evidence indicate that the most important function of presynaptic nAChRs in either synaptic or non-synaptic localization is to increase transmitter release initiated by axonal firing, or directly induce Na(+) and Ca(2+) influx followed by a depolarization sufficient to activate local voltage-sensitive Ca(2+) channels, as a result transmitter of vesicular origin will be released. Therefore, it is somewhat expected that nicotine-induced transmitter release of different monoamines including norepinephrine (NE), dopamine (DA), serotonin (5-HT) can be tetrodotoxin (TTX)- and [Ca(2+)](o)-sensitive. However, some of the nAChR agonists at higher concentrations (1, 1-dimethyl-4-phenylpiperazinium (DMPP) and lobeline), besides their effects on presynaptic nAChRs, are able to inhibit the uptake of NE and 5-HT into nerve terminals, thereby their transmitter releasing effects are extended in time and space. The effect on the uptake process is different from classical nicotinic actions, not being sensitive to nAChR antagonism, but can be prevented by selective uptake blockers or reduced temperature. Considering neurochemical, pharmacological and electrophysiological evidence it seems likely that presynaptic nAChRs on monoaminergic fibers are composed of alpha3 or alpha4 subunits in combination with the beta2 subunit. This is supported by the observation that nicotinic agonists have no presynaptic effect on transmitter release in knockout mice lacking the beta2 nAChR subunit gene. The essential brain function lies not only in impulse transmission within a hard-wired neuronal circuitry but also within synaptic and non-synaptic communication subjected to presynaptic modulation. Since the varicose noradrenergic, dopaminergic, serotonergic, glutamatergic and cholinergic axon terminals mainly do not make synaptic contact, but their varicosities are equipped with nAChRs and these non-synaptically localized receptors are of high affinity, it is suggested that nicotine inhaled during smoking might exert its behavioral, psychological, neurological and neuroendocrinological effects via these receptors.

Modulation by dantrolene of endotoxin-induced interleukin-10, tumour necrosis factor-alpha and nitric oxide production in vivo and in vitro.

1. Intracellular calcium has been suggested to be an important mediator of the cellular response in endotoxaemia and shock. Dantrolene is an agent that interferes with intracellular calcium fluxes resulting in a decreased availability of calcium in the cytoplasm. Here we have investigated the effect of dantrolene on lipopolysaccharide (LPS)-induced production of interleukin-10 (IL-10), tumour necrosis factor-alpha (TNF-alpha), and nitric oxide (NO) in mice and in cultured RAW 264.7 macrophages in vitro. 2. In BALB/c mice, LPS-induced plasma IL-10 levels were significantly enhanced by pretreatment with dantrolene (20 mg kg(-1), i.p.) (P < 0.005 at the 90 min time-point). On the other hand, dantrolene pretreatment suppressed circulating TNF-alpha and nitrite/nitrate (breakdown products of NO) concentrations. However, dantrolene had no effect on LPS-induced plasma interleukin-6 (IL-6) levels (67.22+/-5.51 ng ml(-1) in vehicle-pretreated mice and 62.22+/-3.66 ng ml(-1) in dantrolene-pretreated mice, n = 9). 3. Dantrolene inhibited TNF-alpha and NO production in C57BL/6 IL-10+/+ mice, as well as in their IL-10 deficient counterparts (C57BL/6 IL-10(0/0)). 4. In RAW 264.7 macrophages, dantrolene (10-300 microM) reduced IL-10, TNF-alpha, and nitrite (breakdown product of NO) production elicited by LPS (10 microg ml(-1)). Dantrolene (300 microM) did not affect the LPS-induced nuclear translocation of transcription factor nuclear factor kappaB in these cells. 5. Although LPS failed to alter the intracellullar concentration of calcium in single macrophages loaded with Fura-2, dantrolene caused a significant decrease of the basal calcium level as determined 30 min after dantrolene treatment (P < 0.005). ATP (1 mM) caused a rapid rise in intracellular calcium levels in both dantrolene-pretreated and vehicle-pretreated cells. 6. These results indicate that unlike the secretion of TNF-alpha and NO, IL-10 production is differentially regulated in vitro and in vivo. The decrease of plasma levels of the pro-inflammatory mediators TNF-alpha and NO, and increase in circulating IL-10 concentrations by dantrolene suggest that this drug might offer a new therapeutic approach in inflammatory diseases and septic shock.

Differential mechanisms involved in the effect of nicotinic agonists DMPP and lobeline to release [3H]5-HT from rat hippocampal slices.

In the present study we investigated the effect of different nicotinic agonists (dimethylphenyl-piperazinium-iodide (DMPP), (-)nicotine, cytisine, (-)-lobeline, and (-)epibatidine) and antagonists (mecamylamine and dihydro-beta-erythroidine) on the release of [3H]5-HT from hippocampal slices. The nicotinic agonists DMPP and lobeline and electrical field stimulation, released [3H]5-HT from the hippocampus; other nicotinic agonists, such as (-)-nicotine, cytisine, and (-)-epibatidine had no effect. Unlike lobeline-induced release of [3H]5-HT, the effect of DMPP (10 and 40 microM) was antagonized by mecamylamine (20 and 10 microM). The effect of DMPP was [Ca2+]o-independent. In experiments carried out at 7 degrees C, i.e. the membrane carrier proteins are inhibited and the release by lobeline was abolished while the DMPP-induced release of 5-HT was rather potentiated. It is proposed that the effect of DMPP and lobeline, to enhance the release of [3H]5-HT from the hippocampus, was mediated by two different mechanisms. While DMPP-induced 5-HT release can be linked to a non-classical nAChR activation ([Ca2+]o-independence), the effect of lobeline was likely mediated by uptake carriers.