Interaction between P2X3 and oestrogen receptor (ER)α/ERß in ATP-mediated calcium signalling in mice sensory neurones.

Emerging evidence supports a role of purinergic P2X3 receptors in modulating nociceptive signalling in sensory neurones. Previously, we showed that dorsal root ganglion (DRG) neurones (L1-S1) express both oestrogen receptor (ER)α and ERß receptors. In the present study, we investigated the expression of P2X3 receptors and the effect of 17ß-oestradiol (E(2)) on the ATP-induced [Ca(2+)](i) increase in DRG neurones collected from C57Bl/6J, ERα knockout (KO) and ERßKO mice. Our data showed a significant decrease for P2X3 in ERαKO (all levels) and ERßKO (mostly observed in L1, L2, L4 and L6). Furthermore, E(2) (100 nm) significantly attenuated the ATP (10 µm)-induced [Ca(2+)](i) in C57Bl/6J mice. ER antagonist ICI 182,780 (1 µm) blocked this attenuation. Homomeric P2X3 receptors are plentifully expressed in DRG neurones and contribute to nociceptive signals. α,ß-Methylene (α,ß-me) ATP, which is a specific agonist of P2X2/3 receptors, showed similar responses to the ATP-induced calcium increase in KO mice. A membrane-impermeable E-6-bovine serum albumin (1 µm) had the same effect as E(2) , suggesting action on the membrane. In DRG neurones from ERßKO and wild-type mice, E(2) attenuated the ATP/α,ß-me ATP-induced [Ca(2+)](i) fluxes but, in DRG neurones from ERαKO mice, this hormone had no effect, suggesting that this attenuation depends on membrane-associated ERα receptors. Together, our data indicate an interaction between P2X3 and membrane-associated ERα in primary sensory neurones that may represent a novel mechanism to explain sex differences observed in the clinical presentation of visceral nociceptive syndromes.

N-methyl-D-aspartate receptors enhance mechanical responses and voltage-dependent Ca2+ channels in rat dorsal root ganglia neurons through protein kinase C.

N-methyl-D-aspartate (NMDA)receptors (NMDARs) located on peripheral terminals of primary afferents are involved in the transduction of noxious mechanical stimuli. Exploiting the fact that both NMDARs and stretch-activated channels are retained in short-term culture and expressed on the soma of dorsal root ganglia (DRG) neurons, we examined the effect of NMDA on mechanically mediated changes in intracellular calcium concentration ([Ca2+]i). Our aims were to determine whether NMDARs modulate the mechanosensitivity of DRG neurons. Primary cultures of adult rat lumbosacral DRG cells were cultured for 1-3 days. [Ca2+]i responses were determined by Fura-2 ratio fluorescence. Somas were mechanically stimulated with fire-polished glass pipettes that depressed the cell membrane for 0.5 s. Voltage-activated inward Ca2+ currents were measured by the whole cell patch clamp. Stimulation of neurons with 100 microM NMDA in the presence, but not the absence, of co-agonist (10 microM D-serine) caused transient [Ca2+]i responses (101+/-9 nM) and potentiated [Ca2+]i peak responses to subsequent mechanical stimulation more than two-fold (P < 0.001). NMDA-mediated potentiation of mechanically induced [Ca2+]i responses was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (GFX; 10 microM), which had no independent effects on NMDA- or mechanically induced responses. Short-term treatment with the PKC activator phorbol dibutyrate (1 microM PDBu for 1-2 min) also potentiated mechanically induced [Ca2+]i responses nearly two-fold (P < 0.001), while longer exposure (>10 min) inhibited the [Ca2+]i transients by 44% (P < 0.001). Both effects of PDBu were prevented by prior treatment with GFX. Inhibition of voltage-dependent Ca2+ channels with 25 microM La3+ had no effect on mechanically induced [Ca2+]i transients prior to NMDA, but prevented enhancement of the transients by NMDA and PDBu. NMDA pretreatment transiently enhanced nifedipine-sensitive, voltage-activated Ca2+ currents by a process that was sensitive to GFX. In conclusion, activation of NMDARs on cultured DRG neurons sensitize voltage-dependent L-type Ca2+ channels which contribute to mechanically induced [Ca2+]i transients through a PKC-mediated process.

Estradiol inhibits atp-induced intracellular calcium concentration increase in dorsal root ganglia neurons.

Estrogen has been implicated in modulation of pain processing. Although this modulation occurs within the CNS, estrogen may also act on primary afferent neurons whose cell bodies are located within the dorsal root ganglia (DRG). Primary cultures of rat DRG neurons were loaded with Fura-2 and tested for ATP-induced changes in intracellular calcium concentration ([Ca(2+)](i)) by fluorescent ratio imaging. ATP, an algesic agent, induces [Ca(2+)](i) changes via activation of purinergic 2X (P2X) type receptors and voltage-gated Ca(2+) channels (VGCC). ATP (10 microM) caused increased [Ca(2+)](i) transients (226.6+/-16.7 nM, n = 42) in 53% of small to medium DRG neurons. A 5-min incubation with 17 beta-estradiol (100 nM) inhibited ATP-induced [Ca(2+)](i) (164+/-14.6 nM, P<0.05) in 85% of the ATP-responsive DRG neurons, whereas the inactive isomer 17 alpha-estradiol had no effect. Both the mixed agonist/antagonist tamoxifen (1 microM) and specific estrogen receptor antagonist ICI 182780 (1 microM) blocked the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. Estradiol coupled to bovine serum albumin, which does not diffuse through the plasma membrane, blocked ATP-induced [Ca(2+)](i), suggesting that estradiol acts at a membrane-associated estrogen receptor. Attenuation of [Ca(2+)](i) transients was mediated by estrogen action on VGCC. Nifedipine (10 microM), an L-type VGCC antagonist mimicked the effect of estrogen and when co-administered did not increase the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. N- and P-type VGCC antagonists omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (100 nM), attenuated the ATP-induced [Ca(2+)](i) transients. Co-administration of these blockers with estrogen induced a further decrease of the ATP-induced [Ca(2+)](i) flux. Together, these results suggest that although ATP stimulation of P2X receptors activates L-, N-, and P-type VGCC, estradiol primarily blocks L-type VGCC. The estradiol regulation of this ATP-induced [Ca(2+)](i) transients suggests a mechanism through which estradiol may modulate nociceptive signaling in the peripheral nervous system.

Nitric oxide synthase inhibitors enhance mechanosensitive Ca(2+) influx in cultured dorsal root ganglion neurons.

Nitric oxide (NO) can have opposite effects on peripheral sensory neuron sensitivity depending on the concentration and source of NO, and the experimental setting. The aim of this study was to determine the role of endogenous NO production in the regulation of mechanosensitive Ca(2+) influx of dorsal root ganglion (DRG) neurons. Adult mouse DRG neurons were grown in primary culture for 2-5 days, loaded with Fura-2, and tested for mechanically mediated changes in [Ca(2+)](i) by fluorescent ratio imaging. In the presence of the NOS inhibitors L-NAME, TRIM, or 7-NI, but not the inactive analogue D-NAME, peak [Ca(2+)](i) transients to mechanical stimulation were increased more than 2-fold. Neither La(3+) (25 microM), an inhibitor of voltage activated Ca(2+) channels, or tetrodotoxin (TTX, 1 microM), a selective inhibitor of voltage-gated Na(+) channels, had an effect on mechanically activated [Ca(2+)](i) transients under control conditions. However, in the presence of L-NAME, both La(3+) and TTX partially blocked the [Ca(2+)](i) response. Addition of Gd(3+), a blocker of mechanosensitive cation channels and L-type Ca(2+) channels, at a concentration (100 microM) that markedly inhibited the mechanical response under control conditions, only partially inhibited the response in the presence of L-NAME. The combination of either La(3+) or TTX with Gd(3+) caused near complete inhibition of mechanically stimulated [Ca(2+)](i) transients in the presence of L-NAME. We conclude that focal mechanical stimulation of DRG neurons causes Ca(2+) influx occurs primarily through mechanosensitive cation channels under control conditions. In the presence of NOS inhibitors, additional Ca(2+) influx occurs through voltage-sensitive Ca(2+) channels. These results suggest that endogenously produced NO in cultured DRG neurons decreases mechanosensitivity by inhibiting voltage-gated Na(+) and Ca(2+) channels.

Role of peripheral N-methyl-D-aspartate (NMDA) receptors in visceral nociception in rats.

BACKGROUND & AIMS: N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that have an important role in long-term potentiation and memory processing in the central nervous system. The aims in this study were to determine whether NMDA receptors are expressed in the peripheral nervous system and identify their role in mediating behavioral pain responses to colonic distention in the normal gut. METHODS AND RESULTS: Immunohistochemical localization of the NR1 subunit showed that NMDA receptors are expressed on the cell bodies and peripheral terminals of primary afferent nerves innervating the colon. Dorsal root ganglia neurons retrogradely labeled from the colon in short-term culture responded to addition of NMDA with increased intracellular [Ca2+]. Activation of peripheral NMDA receptors in colonic tissue sections caused Ca2+-dependent release of the proinflammatory neuropeptides, calcitonin gene-related peptide and substance P. Behavioral pain responses to noxious mechanical stimulation were inhibited in a reversible, dose-dependent manner by intravenous administration of memantine, a noncompetitive antagonist of the NMDA receptor. Single fiber recordings of decentralized pelvic nerves showed that colorectal distention responsive afferent nerve activity was inhibited by memantine. CONCLUSIONS: Peripheral NMDA receptors are important in normal visceral pain transmission, and may provide a novel mechanism for development of peripheral sensitization and visceral hyperalgesia.

Mechanical activation of dorsal root ganglion cells in vitro: comparison with capsaicin and modulation by kappa-opioids.

The aim of this study was to characterize plasma membrane pathways involved in the intracellular calcium ([Ca(2+)](i)) response of small DRG neurons to mechanical stimulation and the modulation of these pathways by kappa-opioids. [Ca(2+)](i) responses were measured by fluorescence video microscopy of Fura-2 labeled lumbosacral DRG neurons obtained from adult rats in short-term primary culture. Transient focal mechanical stimulation of the soma, or brief superfusion with 300 nM capsaicin, resulted to [Ca(2+)](i) increases which were abolished in Ca(2+)-free solution, but unaffected by lanthanum (25 microM) or tetrodotoxin (10(-6) M). 156 out of 465 neurons tested (34%) showed mechanosensitivity while 55 out of 118 neurons (47%) were capsaicin-sensitive. Ninty percent of capsaicin-sensitive neurons were mechanosensitive. Gadolinium (Gd(3+); 250 microM) and amiloride (100 microM) abolished the [Ca(2+)](i) transient in response to mechanical stimulation, but had no effect on capsaicin-induced [Ca(2+)](i) transients. The kappa-opioid agonists U50,488 and fedotozine showed a dose-dependent inhibition of mechanically stimulated [Ca(2+)](i) transients but had little effect on capsaicin-induced [Ca(2+)](i) transients. The inhibitory effect of U50,488 was abolished by the kappa-opioid antagonist nor-Binaltorphimine dihydrochloride (nor-BNI; 100 nM), and by high concentrations of naloxone (30-100 nM), but not by low concentrations of naloxone (3 nM). We conclude that mechanically induced [Ca(2+)](i) transients in small diameter DRG somas are mediated by influx of Ca(2+) through a Gd(3+)- and amiloride-sensitive plasma membrane pathway that is co-expressed with capsaicin-sensitive channels. Mechanical-, but not capsaicin-mediated, Ca(2+) transients are sensitive to kappa-opioid agonists.

Calcium waves in colonic myocytes produced by mechanical and receptor-mediated stimulation.

The mechanisms underlying intracellular Ca2+ waves induced by either mechanical or receptor-mediated stimulation of myocytes isolated from the longitudinal muscle layer of the rabbit distal colon were compared using fura 2 and fluorescence videomicroscopy. Light focal mechanical deformation of the plasma membrane or focal application of substance P resulted in localized intracellular Ca2+ concentration ([Ca2+]i) transients that propagated throughout the cell. In both cases, the Ca2+ response consisted of a transient peak response followed by a delayed-phase response. Substance P-mediated [Ca2+]i responses involved generation of inositol 1,4, 5-trisphosphate and release of Ca2+ from thapsigargin-sensitive stores, whereas mechanically induced responses were partially (29%) dependent on La3+-sensitive influx of extracellular Ca2+ and partially on release of intracellular Ca2+ from thapsigargin-insensitive stores gated by ryanodine receptors. The delayed-phase response in both cases was dependent on extracellular Ca2+. However, although the response to substance P was sensitive to La3+, that after mechanical stimulation was not. In the later case, the underlying mechanism may involve capacitative Ca2+ entry channels that are activated after mechanical stimulation but not by substance P.

PKC role in mechanically induced Ca2+ waves and ATP-induced Ca2+ oscillations in airway epithelial cells.

Mechanical stimulation of airway epithelial cells generates the Ca2+ mobilization messenger inositol 1,4,5-trisphosphate and the protein kinase (PK) C activator diacylglycerol. Inositol 1,4,5-trisphosphate diffuses through gap junctions to mediate intercellular communication of the mechanical stimulus (a "Ca2+ wave"); the role that diacylglycerol-activated PKC might play in the response is unknown. Using primary cultures of rabbit tracheal cells, we show that 12-O-tetradecanoylphorbol 13-acetate- or 1, 2-dioctanyl-sn-glycerol-induced activation of PKC slows the Ca2+ wave, decreases the amplitude of induced intracellular free Ca2+ concentration ([Ca2+]i) increases, and decreases the number of affected cells. The PKC inhibitors bisindolylmaleimide and Gö 6976 slowed the spread of the wave but did not change the number of affected cells. We show that ATP-induced [Ca2+]i increases and oscillations, responses independent of intercellular communication, were inhibited by PKC activators. Bisindolylmaleimide decreased the amplitude of ATP-induced [Ca2+]i increases and blocked oscillations, suggesting that PKC has an initial positive effect on Ca2+ mobilization and then mediates feedback inhibition. PKC activators also reduced the [Ca2+]i increase that followed thapsigargin treatment, indicating a PKC effect associated with the Ca2+ release mechanism.

Mechanical stimulation initiates intercellular Ca2+ signaling in intact tracheal epithelium maintained under normal gravity and simulated microgravity.

We investigated mechanically induced cell-to-cell Ca2+ signaling in a preparation of rabbit tracheal epithelium close to its in vivo condition. We used confocal microscopy to analyze changes in intracellular free calcium concentration ([Ca2+]i) in intact ciliated tracheal mucosal explants loaded with the Ca2+-indicator dye, fluo-3. When a single cell in the epithelium was transiently stimulated with a microprobe, [Ca2+]i increased in the stimulated cell and then increased in surrounding cells. In the absence of extracellular Ca2+, the [Ca2+]i increases had a smaller amplitude and spread to fewer cells. Treatment of the cells with thapsigargin, in the presence of extracellular Ca2+, more markedly reduced the spread of elevated [Ca2+]i. These results suggest that the propagated [Ca2+]i increases are due to mobilization of Ca2+ from intracellular stores and, possibly, the influx of extracellular Ca2+. The mechanically stimulated [Ca2+]i increases were accompanied by propagated increases in ciliary beat frequency. Since microgravity has been shown to alter signal transduction, we investigated whether simulated microgravity affects the mechanically stimulated cell-to-cell Ca2+ signaling observed in tracheal epithelium. Tissues were maintained for 3-8 d in a rotating wall vessel which simulates microgravity conditions. Cells maintained in simulated microgravity exhibited mechanically induced [Ca2+]i increases not significantly different in magnitude, in speed of propagation, or in the number of cells involved, from tissue maintained at unit gravity. Our results suggest that intercellular Ca2+ signaling coordinates cellular activity, including ciliary beating, within the tracheal epithelium in vivo and that this function is not compromised in microgravity.

[Electrophysiologic parameters of ion transport systems in early developmental stages of fish and amphibia]. / Elektrofiziologichni parametry iontransportnykh system u ryb i amfibii za period ikh rannógo rozvytku.

It is shown that the membrane potential level, ionic current and membrane conductance depend on the cell cycle stage both in Misgurnus fossilis L. embryos and in Xenopus laevis Daudin embryos by the microelectrode technics. Allowing for the role of the adenylate cyclase system in the membrane potential oscillations and ion conductance of membranes, some series of experiments for analysis of the inhibitor have been carried out.