Selective knockdown of NMDA receptors in primary afferent neurons decreases pain during phase 2 of the formalin test.

The role of NMDA receptors (NMDARs) expressed by primary afferent neurons in nociception remains controversial. The aim of this study was to develop mice with a tissue selective knockdown of NMDARs in these neurons and to evaluate their behavioral responses to different types of painful stimuli. Mice with floxed NMDAR NR1 subunit gene (fNR1) were crossed with mice expressing Cre recombinase under the control of the peripherin promotor (Prph-Cre). Male Prph-Cre+ floxed NR1 mice were compared to Cre- littermates. Both quantitative RT/PCR and Western blotting indicated a ∼75% reduction in NR1 expression in dorsal root ganglia (DRG) extracts with no effect on NR1 expression in spinal cord, brain or the enteric nervous system. Immunocytochemistry with antibodies to NR1 revealed decreased staining in all size classes of DRG neurons. NMDA produced a detectable increase in [Ca2+]i in 60% of DRG neurons cultured from Cre- mice, but only 15% of those from Cre+ mice. Furthermore, the peak [Ca2+]i responses were 64% lower in neurons from Cre+ mice. There was no significant difference between Cre+ and Cre- mice in response latencies to the hotplate or tail withdrawal tests of thermal nociception, nor was there a difference in withdrawal thresholds to mechanical stimuli of the tail or paw. However, compared to Cre- littermates, Cre+ knockdown mice had a 50% decrease in the phase 2 response to formalin injection (P<0.001). There was no effect on phase 1 responses. These results suggest that NMDA receptors expressed by primary afferent nerves play an important role in the development of sensitized pain states.

Sex-dependent differences in the activity and modulation of N-methyl-d-aspartic acid receptors in rat dorsal root ganglia neurons.

Women have greater temporal summation of experimental pain stimuli and also have a higher propensity for developing chronic visceral pain conditions. Sex hormone-mediated regulation of N-methyl-d-aspartic acid receptors (NMDARs) in nociceptive pathways is a plausible mechanism that may underlie these phenomena. The aim of this study was to compare the effect of 17-beta-estradiol (E2) in modulation of NMDAR activity in adult male and female rat dorsal root ganglia (DRG) neurons. DRG neurons were collected from adult male or female rats and grown in short-term culture in steroid-free media. NMDAR currents were recorded on small to medium size neurons by whole cell patch clamp using rapid perfusion with saturating concentrations of N-methyl-d-aspartic acid and glycine in the absence of extracellular Mg(2+). We found that the average density of NMDAR currents was 2.8-fold larger in DRG neurons from female rats compared with male rats (P<0.0001). Addition of 100 nM E2 increased NMDAR currents 55+/-15% in female neurons, but only 19+/-7% in male neurons. Potentiation was maximal after 20-40 min and dose dependent with an apparent 50% excitatory concentration of 17-23 nM. This effect was mimicked by E2 conjugated to BSA and attenuated by pretreatment with the protein tyrosine kinase inhibitor lavendustin A (1 microM) or the estrogen receptor (ER) antagonist, ICI 182,780 (1 microM), strongly suggesting activation of a cell surface ER acting through a non-genomic mechanism involving protein tyrosine kinases to increase NMDAR currents. These results identify sex-based differences in both the basal expression and the regulation of the NMDARs in DRG neurons.

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.

Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism.

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 and, by unknown mechanisms, induce widespread inflammation. We found that a large proportion of primary spinal afferent neurons, which express proteinase-activated receptor 2, also contain the proinflammatory neuropeptides calcitonin gene-related peptide and substance P. Trypsin and tryptase directly signal to neurons to stimulate release of these neuropeptides, which mediate inflammatory edema induced by agonists of proteinase-activated receptor 2. This new mechanism of protease-induced neurogenic inflammation may contribute to the proinflammatory effects of mast cells in human disease. Thus, tryptase inhibitors and antagonists of proteinase-activated receptor 2 may be useful anti-inflammatory agents.

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.

Chemical signaling from colonic smooth muscle cells to DRG neurons in culture.

Transduction mechanisms between target cells within the intestinal wall and peripheral terminals of extrinsic primary afferent neurons are poorly understood. The purpose of this study was to characterize the interactions between smooth muscle cells from the rat distal colon and lumbar dorsal root ganglion (DRG) neurons in coculture. DRG neurons visually appeared to make contact with several myocytes. We show that brief mechanical stimulation of these myocytes resulted in intracellular Ca2+ concentration ([Ca2+]i) transients that propagated into 57% of the contacting neurites. Direct mechanical stimulation of DRG neurites cultured without smooth muscle had no effect. We also show that colonic smooth muscle cells express multiple connexin mRNAs and that these connexins formed functional gap junctions, as evidenced by the intercellular transfer of Lucifer yellow. Furthermore, thapsigargin pretreatment and neuronal heparin injection abolished the increase in neurite [Ca2+]i, indicating that the neuronal Ca2+ signal was triggered by inositol 1,4, 5-trisphosphate-mediated Ca2+ release from intracellular stores. Our results provide evidence for intercellular chemical communication between DRG neurites and intestinal smooth muscle cells that mediates the exchange of second messenger molecules between different cell types.

Substance P induces brief, localized increase in [Ca2+]i in dorsal horn neurons.

We determined the spatial and temporal dynamics of the increase in intracellular Ca2+ levels [Ca2+]i produced by substance P (SP) in dorsal horn neurons. A microinjection technique was used to apply minute amounts of SP to small areas of cultured neurons loaded with the Ca2+ indicator fura-2. Five successive applications of SP to the soma produced short-lasting (< 50 s) increases in [Ca2+]i that became gradually smaller, indicating receptor desensitization. Focal application of SP to a distal locus in a neurite produced a brief (12 s) increase in [Ca2+]i that travelled down the dendrite but did not spread into cell soma. Prolonged application of SP to these neurons caused the appearance of varicosities in their dendrites.

Mechanotransduction in colonic smooth muscle cells.

We evaluated mechanisms which mediate alterations in intracellular biochemical events in response to transient mechanical stimulation of colonic smooth muscle cells. Cultured myocytes from the circular muscle layer of the rabbit distal colon responded to brief focal mechanical deformation of the plasma membrane with a transient increase in intracellular calcium concentration ([Ca2+]i) with peak of 422.7 +/- 43.8 nm above an average resting [Ca2+]i of 104.8 +/- 10.9 nM (n = 57) followed by both rapid and prolonged recovery phases. The peak [Ca2+]i increase was reduced by 50% in the absence of extracellular Ca2+, while the prolonged [Ca2+]i recovery was either abolished or reduced to less than or = 15% of control values. In contrast, no significant effect of gadolinium chloride (100 microM) or lanthanum chloride (25 microM) on either peak transient or prolonged [Ca2+]i recovery was observed. Pretreatment of cells with thapsigargin (1 microM) resulted in a 25% reduction of the mechanically induced peak [Ca2+]i response, while the phospholipase C inhibitor U-73122 had no effect on the [Ca2+]i transient peak. [Ca2+]i transients were abolished when cells previously treated with thapsigargin were mechanically stimulated in Ca2+-free solution, or when Ca2+ stores were depleted by thapsigargin in Ca2+-free solution. Pretreatment with the microfilament disrupting drug cytochalasin D (10 microM) or microinjection of myocytes with an intracellular saline resulted in complete inhibition of the transient. The effect of cytochalasin D was reversible and did not prevent the [Ca2+]i increases in response to thapsigargin. These results suggest a communication, which may be mediated by direct mechanical link via actin filaments, between the plasma membrane and an internal Ca2+ store.

Expression of insulin-like growth factor I receptors and binding proteins by colonic smooth muscle cells.

We recently demonstrated upregulation of insulin-like growth factor I (IGF-I) binding sites in the smooth muscle layer of inflamed rat colon. The increase in binding sites was due to increased expression of IGF binding proteins (IGFBPs), which modulate the effects of IGF. To further study the role of IGF in the colon, we investigated whether cultured colonic smooth muscle cells (SMC) express IGF-I receptors and IGFBPs. SMC were isolated by collagenase digestion from rat colonic smooth muscle and grown in primary culture. Equilibrium binding experiments using (125)I-labeled IGF-I showed the presence of an IGF-I receptor with a dissociation constant of 1.96 nM and a maximal binding constant of 53,000 receptors/cell. Competition binding studies with IGF-II and insulin, together with chemical cross-linking experiments, corroborated this conclusion. Western ligand blotting of conditioned medium and Northern analysis of total RNA demonstrated that the cells expressed and secreted IGFBP-4, -5, and -3 with molecular masses of 25, 31, and 45 kDa, respectively. These results, together with our in vivo studies in the rat, support a role for IGF in tissue fibrosis and stricture formation during chronic intestinal inflammation.

Intercellular communication between dorsal root ganglion cells and colonic smooth muscle cells in vitro.

The mechanism(s) by which intestinal smooth muscle tension is signaled to extrinsic primary afferent neurons is poorly understood. In order to characterize myocyte-neuron communication, we developed a coculture system using rat dorsal root ganglion (DRG) neurons and myocytes obtained from the circular muscle layer of the rat distal colon. Both cell types maintained their phenotype in culture, as demonstrated by positive immunocytochemical staining for neuron-specific enolase and smooth muscle actin. Myocytes showed mechanosensitivity in the form of increases in [Ca2+]i in response to light mechanical touch of the plasma membrane. This increase in [Ca2+]i was independent of extracellular Ca2+ and passed as a propagated wave from muscle cells into adjacent DRG neurites. The inhibitory effect of octanol on this intercellular propagation suggests propagation of [Ca2+]i gradients via heterologous gap junctions. This preparation may serve a useful model system for the study of the interaction of visceral afferents and their target cells.

Propagation of calcium waves between colonic smooth muscle cells in culture.

Intercellular propagation of a diffusible substance through direct cytoplasmic communication between multiple cells could represent an important mechanism for mutual multiple cell signaling between cells in a tissue. The current study was aimed at characterizing the mechanism(s) underlying the intercellular propagation of calcium concentration ([Ca2+]i) transients between colonic smooth muscle cells. Changes in [Ca2+]i in smooth muscle cells from the rabbit distal colon in primary cultures were monitored using videomicroscopy with the fluorescent dye Fura-2. Myocytes responded to light mechanical deformation of the plasma membrane with a localized increase in [Ca2+]i which spread in a wave-like fashion through up to 5 adjacent cells, with little change in wave amplitude. Dye coupling between cells was demonstrated by Lucifer Yellow, and intercellular wave propagation was abolished by octanol, suggesting propagation of Ca2+ waves via gap junctions. Wave propagation was not dependent on extracellular [Ca2+]i suggesting regenerative release of Ca2+ from intracellular stores. Propagation of Ca2+ waves through silent cells suggested a diffusible messenger other than Ca2+. Wave propagation and kinetics were unaffected by ryanodine (50 microM) or caffeine (10 mM), but abolished by depletion of thapsigargin-sensitive Ca2+ stores and by the phospholipase C inhibitor U-73122 (10 microM), implicating inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-sensitive stores as the major Ca2+ source for propagated Ca2+ transients. These results indicate that, in a connected complex of colonic smooth muscle cells in culture, multiple cells can monitor the mechanical status of a single cell through diffusion of Ins(1,4,5)P3, Ca2+, or another intercellular messenger.

Characterization of colonic circular smooth muscle cells in culture.

The receptor-binding properties of isolated rabbit colonic circular smooth muscle cells in primary culture have been investigated. In intact smooth muscle, acetylcholine, acting through M2 muscarinic receptors, and vasoactive intestinal polypeptide (VIP), acting through VIP receptors, are two of the principal neurotransmitters mediating contraction and relaxation, respectively. The muscarinic receptor was present in very high levels (600,000 receptors/cell) on freshly isolated colonic smooth muscle cells as shown by binding of the muscarinic receptor antagonist N-methylscopolamine (NMS). However, NMS binding sites decreased rapidly when the cells were placed in primary culture. After 21 h in culture, specific binding of [3H]NMS decreased to 20%, and after 48 h to less than 10% that of preculture values. This loss was not associated with a change in receptor affinity, since Kd was unchanged for the receptors still present. In contrast, high-affinity VIP receptors were expressed on cultured smooth muscle cells but could not be detected on freshly isolated cells. Cultured cells responded to VIP with an increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP), indicating that the VIP receptors were functionally coupled to adenylate cyclase. Cultured cells also responded to calcitonin gene-related peptide (CGRP) and forskolin with increased production of intracellular cAMP. In contrast, neither VIP nor CGRP elicited an increase in intracellular cAMP when added to freshly isolated cells. Furthermore, freshly isolated cells had a greatly diminished response to forskolin, suggesting that the isolation procedure not only destroyed cell surface receptors for VIP and CGRP, but also damaged the cells sufficiently to decrease cellular adenylate cyclase activity.(ABSTRACT TRUNCATED AT 250 WORDS)