Victor V. Chaban

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.

Estrogen receptor-α mediates estradiol attenuation of ATP-induced Ca2+ signaling in mouse dorsal root ganglion neurons

A mechanism underlying gender‐related differences in pain perception may be estrogen modulation of nociceptive signaling in the peripheral nervous system. In rat, dorsal root ganglion (DRG) neurons express estrogen receptors (ERs) and estrogen rapidly attenuates ATP‐induced Ca2+ signaling. To determine which estrogen receptor mediates rapid actions of estrogen, we showed ERα and ERβ expression in DRG neurons from wild‐type (WT) female mice by RT‐PCR. To study whether ERα or ERβ mediates this response, we compared estradiol action mediating Ca2+ signaling in DRG neurons from WT, ERα knockout (ERαKO), and ERβKO mice in vitro. ATP, an algesic agent, induced [Ca2+]i transients in 48% of small DRG neurons from WT mice. 17β‐Estradiol (E2) inhibited ATP‐induced intracellular Ca2+ concentration ([Ca2+]i) with an IC50 of 27 nM. The effect of E2 was rapid (5‐min exposure) and stereo specific; 17α‐estradiol had no effect. E2 action was blocked by the ER antagonist ICI 182,780 (1 μM) in WT mouse. Estradiol coupled to bovine serum albumin (E‐6‐BSA), which does not penetrate the plasma membrane, had the same effect as E2 did, suggesting that a membrane‐associated ER mediated the response. In DRG neurons from ERβKO mice, E2 attenuated the ATP‐induced [Ca2+]i flux as it did in WT mice, but in DRG neurons from ERαKO mice, E2 failed to inhibit the ATP‐induced [Ca2+]i increase. These results show that mouse DRG neurons express ERs and the rapid attenuation of ATP‐induced [Ca2+]i signaling is mediated by membrane‐associated ERα.

Estrogen and Visceral Nociception at the Level of Primary Sensory Neurons

Clinical studies suggest the comorbidity of functional pain syndromes such as irritable bowel syndrome, painful bladder syndrome, chronic pelvic pain, and somatoform disorders approaches 40% to 60%. The incidence of episodic or persistent visceral pain associated with these “functional” disorders is two to three times higher in women than in men. One of the possible explanations for this phenomenon is estrogen modulation of viscerovisceral cross-sensitization. While a central site of this modulation has been shown previously, our studies suggest a peripheral site, the dorsal root ganglion (DRG). Estrogens have remarkably wide range of functions including modulation of voltage-gated calcium channels (VGCCs) and purinoreceptors (P2Xs). Significantly, inflammation dramatically alters purinoception by causing a several fold increase in ATP-activated current, alters the voltage dependence of P2X receptors, and enhances the expression of P2X receptors increasing neuronal hypersensitivity. Gonadal hormones are thought as indispensable cornerstones of the normal development and function, but it appears that no body region, no neuronal circuit, and virtually no cell is unaffected by them. Thus, increasing awareness toward estrogens appears to be obligatory.

Estradiol attenuates the adenosine triphosphate-induced increase of intracellular calcium through group II metabotropic glutamate receptors in rat dorsal root ganglion neurons.

Estradiol attenuates the ATP‐induced increase of intracellular calcium concentration ([Ca2+]i) in rat dorsal root ganglion (DRG) neurons by blocking the L‐type voltage gated calcium channel (VGCC). Because ATP is a putative nociceptive signal, this action may indicate a site of estradiol regulation of pain. In other neurons, 17β‐estradiol (E2) has been shown to modulate L‐type VGCC through a membrane estrogen receptor‐group II metabotropic glutamate receptor (mGluR2/3). The present study investigated whether the rapid estradiol attenuation of the ATP‐induced increase in [Ca2+]i requires mGluR2/3. Previously we showed that DRG (L1–S3) express ERα, P2X3, and mGluR2/3 receptors. DRG were acutely dissociated by enzyme digestion and grown in short‐term culture for imaging analysis. DRG neurons were stimulated twice, once with ATP (50 μM) for 5 sec and then again in the presence of E2 (100 nM) or E2 (100 nM) + LY341495 (100 nM), an mGluR2/3 inhibitor. ATP induced a transient increase in [Ca2+]i (216.3 ± 41.2 nM). This transient increase could be evoked several times in the same DRG neurons if separated by a 5‐min washout. Treatment with estradiol significantly attenuated the ATP‐induced [Ca2+]i increase in 60% of the DRG neurons, to 163.3 ± 20.9 nM (P < 0.001). Coapplication of E2 and the mGluR2/3 inhibitor LY341495 blocked the 17β‐estradiol attenuation of the ATP‐induced [Ca2+]i transient (209.1 ± 32.2 nM, P > 0.05). These data indicate that the rapid action of E2 in DRG neurons is dependent on mGluR2/3 and demonstrate that membrane estrogen receptor‐α‐initiated signaling involves interaction with mGluRs.

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

Expression of P2X3 and TRPV1 receptors in primary sensory neurons from estrogen receptors-α and estrogen receptor-β knockout mice.

In women, pain symptoms and nociceptive thresholds vary with the reproductive cycle, suggesting the role of estrogen receptors (ERs) in modulating nociception. Our previous data strongly suggest an interaction between ERs and ATP-induced purinergic (P2X3) as well as ERs and capsaicin-induced vanilloid (TRPV1) receptors at the level of dorsal root ganglion (DRG) neurons. In this study, we investigated the expression of P2X3 and TRPV1 receptors by western blotting and immunohistochemistry in lumbosacral DRGs from wild type, ER&agr;, and ER&bgr; knockout mice. We found a significant decrease for both P2X3 and TRPV1 in ER&agr;KO and ER&bgr;KO. This phenomenon was visualized in L1, L2, L4, and L6 levels for P2X3 receptors and in L1, L2, and S2 levels for TRPV1 receptors. This tan interaction between P2X3/TRPV1 and ERs expression in sensory neurons may represent a novel mechanism that can explain the sex differences in nociception observed in clinical practice. The DRG is an important site of visceral afferent convergence and cross-sensitization and a potential target for designing new anti-nociceptive therapies.

Inflammation in the uterus induces phosphorylated extracellular signal-regulated kinase and substance P immunoreactivity in dorsal root ganglia neurons innervating both uterus and colon in rats

In women, clinical studies suggest that pain syndromes such as irritable bowel syndrome and interstitial cystitis, which are associated with visceral hyperalgesia, are often comorbid with endometriosis and chronic pelvic pain. One of the possible explanations for this phenomenon is viscerovisceral cross‐sensitization, in which increased nociceptive input from an inflamed pelvic organ sensitizes neurons that receive convergent input to the same dorsal root ganglion (DRG) from an unaffected visceral organ. Nociception induces up‐regulation of cellular mechanisms such as phosphorylated extracellular signal‐regulated kinase (pERK) and substance P (SP), neurotransmitters associated with induced pain sensation. The purpose of this study was to determine, in a rodent model, whether uterine inflammation increased the number of pERK‐ and SP‐positive neurons that received input from both the uterus and the colon. Cell bodies of colonic and uterine DRG were retrogradely labeled with fluorescent tracer dyes microinjected into the colon/rectum and into the uterus. Ganglia were harvested for fluorescent microscopy to identify positively stained neurons. Approximately 6% of neurons were colon specific and 10% uterus specific. Among these uterus‐ or colon‐specific neurons, up to 3–5% of DRG neurons in the lumbosacral neurons (L1–S3 levels) received input from both visceral organs. Uterine inflammation increased the number of pERK‐ and SP‐immunoreactive DRG neurons innervating specifically colon, or innervating specifically uterus, and those innervating both organs. These results suggest that a localized inflammation activates primary visceral afferents, regardless of whether they innervate the affected organ. This visceral sensory integration in the DRG may underlie the observed comorbidity of female pelvic pain 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.

Nitric oxide synthase inhibitors enhance mechanosensitive Ca2+ 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.

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.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+]iresponses 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.