Network-based activity induced by 4-aminopyridine in rat dorsal horn in vitro is mediated by both chemical and electrical synapses.

This study investigated the role of electrical and chemical synapses in sustaining 4-aminopyridine (4-AP)-evoked network activity recorded extracellularly from substantia gelatinosa (SG) of young rat spinal cord in vitro. Superfusion of 4-AP (50 microM) induced two types of activity, the first was observed as large amplitude field population spiking activity and the second manifested within the inter-spike interval as low amplitude rhythmic oscillations in the 4-12 Hz frequency range (mean peak of 8.0 +/- 0.1 Hz). The AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) abolished field population spiking and disrupted 4-12 Hz rhythmic oscillatory activity whereas the NMDA receptor antagonist D-AP5 (50 microM) had no significant effect on either activity component. The glycine receptor antagonist strychnine (4 microM) and the GABA(A) receptor antagonist bicuculline (10 microM) diminished and abolished, respectively, field population spiking and both antagonists reduced the power of 4-12 Hz oscillations. The non-specific gap junction blockers carbenoxolone (100 microM) and octanol (1 mM) attenuated both types of 4-AP-induced activity. By comparison, the neuronal-specific gap junction uncouplers quinine (250 microM) and mefloquine (500 nM) both disrupted 4-12 Hz oscillations but only quinine reduced the frequency of field population spiking. These data demonstrate the existence of 4-AP-sensitive neuronal networks within SG that can generate rhythmic activity, are differentially modulated by excitatory and inhibitory ionotropic neurotransmission and are at least partly reliant on neuronal and/or glial-mediated electrical connectivity. The physiological significance of these putative intrinsic SG networks and the implications in the context of processing of nociceptive inputs are discussed.

Differential expression and regulation of nuclear oligomerization domain proteins NOD1 and NOD2 in human endometrium: a potential role in innate immune protection and menstruation.

Nuclear oligomerization domains (NODs) are cytosolic pattern recognition receptors (PRRs), present in epithelial cells, monocytes and dendritic cells. This study details their expression, regulation and role in human endometrium. Real-time PCR showed that NOD1 mRNA is constitutively expressed in endometrium. NOD2 is up-regulated in the late secretory phase of the menstrual cycle suggesting a role in menstruation. Both proteins are immunolocalized in endometrial epithelium, stroma and endothelium. In first trimester, decidua NODs are present in decidualized stroma. NOD function was examined in endometrial stromal cells (ESCs) and endometrial epithelial cells (EEpCs) in vitro. IkappaBalpha is up-regulated by stimulation of ESC and EEpC with an NOD1 ligand. IkappaBalpha, IL-8 and TNFalpha mRNA expression is increased in EEpC by a NOD2 ligand. NOD2 mRNA expression increases in response to IL-1 treatment while NOD1 transcripts are unaltered. NOD1 mRNA is increased in an in vitro model of decidualization of ESC. In summary, we report expression of NOD1 and NOD2 in human endometrium and show that they are differentially regulated. NOD2 and, to a lesser extent, NOD1 can function to increase expression of innate immune molecules in endometrium. NODs may have a role in innate immune protection in the uterus and NOD2 may regulate inflammation associated with menstruation.

Expression of secretory leukocyte protease inhibitor and elafin in human fallopian tube and in an in-vitro model of Chlamydia trachomatis infection.

BACKGROUND: Secretory leukocyte protease inhibitor (SLPI) and elafin are anti-protease and anti-microbial molecules with a role in innate immune defence. They have been demonstrated at multiple mucosal surfaces including those of the female reproductive tract. METHODS AND RESULTS: This study details their expression in human Fallopian tubes (ampullary region) throughout the menstrual cycle (n = 18) and from women with ectopic pregnancy (n = 6), and examined their regulation by infection with Chlamydia trachomatis in an in-vitro model. Quantitative real-time PCR analysis showed that SLPI and elafin were constitutively expressed in the Fallopian tube during the menstrual cycle but were increased in ectopic pregnancy (P < 0.05 versus early-mid luteal phase, P < 0.01 versus all phases, respectively). SLPI and elafin were immunolocalised to the Fallopian tube epithelium in biopsies from non-pregnant women and those with ectopic pregnancy. An in-vitro culture model of C. trachomatis infection of the OE-E6/E7 oviductal epithelial cell line showed that elafin mRNA expression was upregulated in response to chlamydial infection. CONCLUSION: These data suggest that SLPI and elafin have a role in the innate immune defence of the Fallopian tube in infection and ectopic pregnancy. Their role is likely to include regulation of protease activity, wound healing and tissue remodelling.

Prokineticin-1: a novel mediator of the inflammatory response in third-trimester human placenta.

Prokineticin-1 (PK1) is a recently described protein with a wide range of functions, including tissue-specific angiogenesis, modulation of inflammatory responses, and regulation of hemopoiesis. The aim of this study was to investigate the localization and expression of PK1 and PK receptor-1 (PKR1), their signaling pathways, and the effect of PK1 on expression of the inflammatory mediators cyclooxygenase (COX)-2 and IL-8 in third-trimester placenta. PK1 and PKR1 were highly expressed in term placenta and immunolocalized to syncytiotrophoblasts, cytotrophoblasts, fetal endothelium, and macrophages. PK1 induced a time-dependent increase in expression of IL-8 and COX-2, which was significantly reduced by inhibitors of Gq, cSrc, epidermal growth factor receptor (EGFR), and MAPK kinase. Treatment of third-trimester placenta with 40 nm PK1 induced a rapid phosphorylation of cSrc, EGFR, and ERK1/2. Phosphorylation of ERK1/2 in response to PK1 was dependent on sequential phosphorylation of cSrc and EGFR. Using double-immunofluorescent immunohistochemistry, PKR1 colocalized with IL-8 and COX-2 in placenta. These data suggest that PK1 may have a novel role as a mediator of the inflammatory response in placenta.

Innate immunity and disorders of the female reproductive tract.

Sexually transmitted infections, and their associated sequelae, such as tubal infertility, ectopic pregnancy and preterm labour, are a major worldwide health problem. Chlamydia trachomatis infection is thought to be the leading global cause of tubal infertility and tubal ectopic pregnancy. Preterm birth occurs in around 10% of all deliveries, and nearly 30% of preterm deliveries are associated with intrauterine infection. The mucosal innate immune system of the female reproductive tract has evolved to eliminate such sexually transmitted pathogens whilst maintaining its ability to accommodate specialized physiological functions that include menstruation, fertilization, implantation, pregnancy and parturition. The aim of this review was to describe the role and distribution of key mediators of the innate immune system, the natural antimicrobial peptides (secretory leukocyte protease inhibitor, elafin and the defensins) and the pattern recognition toll-like receptors in the normal female reproductive tract and in the context of these pathological processes.

A Novel Role for Lymphotactin (XCL1) Signaling in the Nervous System: XCL1 Acts via its Receptor XCR1 to Increase Trigeminal Neuronal Excitability.

Chemokines are known to have a role in the nervous system, influencing a range of processes including the development of chronic pain. To date there are very few studies describing the functions of the chemokine lymphotactin (XCL1) or its receptor (XCR1) in the nervous system. We investigated the role of the XCL1-XCR1 axis in nociceptive processing, using a combination of immunohistochemical, pharmacological and electrophysiological techniques. Expression of XCR1 in the rat mental nerve was elevated 3 days following chronic constriction injury (CCI), compared with 11 days post-CCI and sham controls. XCR1 co-existed with neuronal marker PGP9.5, leukocyte common antigen CD45 and Schwann cell marker S-100. In the trigeminal root and white matter of the brainstem, XCR1-positive cells co-expressed the oligodendrocyte marker Olig2. In trigeminal subnucleus caudalis (Vc), XCR1 immunoreactivity was present in the outer laminae and was colocalized with vesicular glutamate transporter 2 (VGlut2), but not calcitonin gene-related peptide (CGRP) or isolectin B4 (IB4). Incubation of brainstem slices with XCL1 induced activation of c-Fos, ERK and p38 in the superficial layers of Vc, and enhanced levels of intrinsic excitability. These effects were blocked by the XCR1 antagonist viral CC chemokine macrophage inhibitory protein-II (vMIP-II). This study has identified for the first time a role for XCL1-XCR1 in nociceptive processing, demonstrating upregulation of XCR1 at nerve injury sites and identifying XCL1 as a modulator of central excitability and signaling via XCR1 in Vc, a key area for modulation of orofacial pain, thus indicating XCR1 as a potential target for novel analgesics.

Nucleoside transporters: from scavengers to novel therapeutic targets.

Hydrophilic purine and pyrimidine nucleosides rely on specialized carrier proteins for their membrane translocation. The recent identification of two gene families encoding equilibrative and concentrative nucleoside transporters in mammals and other organisms has provided the essential breakthrough to a more complete understanding of the biological significance of nucleoside transport. Although nucleoside salvage is a primary function of these proteins, recent data indicate functions beyond metabolic recycling. In brain and spinal cord, for example, nucleoside transporters have the potential to regulate synaptic levels of neuroactive purines such as adenosine and, thereby, indirectly modulate physiological processes through G-protein-coupled purine P1 receptors. As described in this review, recent research indicates novel putative functions for CNS nucleoside transporters in sleep, arousal, drug and alcohol addiction, nociception and analgesia. The therapeutic use of nucleoside analogue drugs and nucleoside transporter inhibitors in viral, neoplastic, cardiovascular and infectious disease is also described.

The role of Cx36 and Cx43 in 4-aminopyridine-induced rhythmic activity in the spinal nociceptive dorsal horn: an electrophysiological study in vitro.

Connexin (Cx) proteins and gap junctions support the formation of neuronal and glial syncytia that are linked to different forms of rhythmic firing and oscillatory activity in the CNS. In this study, quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to profile developmental expression of two specific Cx proteins, namely glial Cx43 and neuronal Cx36, in postnatal lumbar spinal cord aged 4, 7, and 14 days. Extracellular electrophysiology was used to determine the contribution of Cx36 and Cx43 to a previously described form of 4-aminopyridine (4-AP)-induced 4-12 Hz rhythmic activity within substantia gelatinosa (SG) of rat neonatal dorsal horn (DH) in vitro. The involvement of Cx36 and Cx43 was probed pharmacologically using quinine, a specific uncoupler of Cx36 and the mimetic peptide blocker Gap 26 which targets Cx43. After establishment of 4-12 Hz rhythmic activity by 4-AP (25 µmol/L), coapplication of quinine (250 µmol/L) reduced 4-AP-induced 4-12 Hz rhythmic activity (P < 0.05). Preincubation of spinal cord slices with Gap 26 (100 µmol/L), compromised the level of 4-AP-induced 4-12 Hz rhythmic activity in comparison with control slices preincubated in ACSF alone (P < 0.05). Conversely, the nonselective gap junction "opener" trimethylamine (TMA) enhanced 4-12 Hz rhythmic behavior (P < 0.05), further supporting a role for Cx proteins and gap junctions. These data have defined a physiological role for Cx36 and Cx43 in rhythmic firing in SG, a key nociceptive processing area of DH. The significance of these data in the context of pain and Cx proteins as a future analgesic drug target requires further study.

Localization of the NBMPR-sensitive equilibrative nucleoside transporter, ENT1, in the rat dorsal root ganglion and lumbar spinal cord.

ENT1 is an equilibrative nucleoside transporter that enables trans-membrane bi-directional diffusion of biologically active purines such as adenosine. In spinal cord dorsal horn and in sensory afferent neurons, adenosine acts as a neuromodulator with complex pro- and anti-nociceptive actions. Although uptake and release mechanisms for adenosine are believed to exist in both the dorsal horn and sensory afferent neurons, the expression profile of specific nucleoside transporter subtypes such as ENT1 is not established. In this study, immunoblot analysis with specific ENT1 antibodies (anti-rENT1(227-290) or anti-hENT1(227-290)) was used to reveal the expression of ENT1 protein in tissue homogenates of either adult rat dorsal horn or dorsal root ganglia (DRG). Immunoperoxidase labeling with ENT1 antibodies produced specific staining in dorsal horn which was concentrated over superficial laminae, especially the substantia gelatinosa (lamina II). Immunofluorescence double-labeling revealed a punctate pattern for ENT1 closely associated, in some instances, with cell bodies of either neurons (confirmed with NeuN) or glia (confirmed with CNPase). Electron microscopy analysis of ENT1 expression in lamina II indicated its presence within pre- and post-synaptic elements, although a number of other structures, including myelinated and unmyelinated, axons were also labeled. In sensory ganglia, ENT1 was localized to a high proportion of cell bodies of all sizes that co-expressed substance P, IB4 or NF, although ENT1 was most highly expressed in the peptidergic population. These data provide the first detailed account of the expression and cellular distribution of ENT1 in rat dorsal horn and sensory ganglia. The functional significance of ENT1 expression with regard to the homeostatic regulation of adenosine at synapses remains to be established.

Adenosine contributes to mu-opioid synaptic inhibition in rat substantia gelatinosa in vitro.

The purpose of this study was to investigate the cellular basis of the synergistic anti-nociceptive interaction between adenosine and opioids reported for spinal cord in vivo. Patch clamp recordings from rat substantia gelatinosa neurons in vitro were used to assess whether adenosine receptor antagonists impact upon mu-opioid receptor (MOR)-mediated inhibition of glutamatergic synaptic transmission. The MOR agonist DAMGO inhibited evoked EPSCs and this inhibition was partly reversed by DPCPX, an A1 receptor (A1R) antagonist. The A2a receptor antagonist, ZM241385 had mixed effects on DAMGO-mediated inhibition, producing either a further inhibition or a reversal of the inhibition. These data show that activation of A1R as a secondary consequence of MOR-activation and putative adenosine release will potentiate opioid synaptic inhibition of nociceptive circuitry.

Elafin in human endometrium: an antiprotease and antimicrobial molecule expressed during menstruation.

Elafin is an antiproteinase and antimicrobial molecule that is expressed at epithelial sites (for example, cervix). This study details the expression and regulation of elafin in the human endometrium. Elafin mRNA and protein expression were examined in endometrium throughout the menstrual cycle and in first-trimester decidua. Real-time quantitative PCR showed that expression of elafin mRNA peaked during menstruation. Elafin protein was localized to leukocytes scattered in the endometrial stroma during the late secretory and menstrual phases. Faint immunostaining was also present in glandular epithelium at these cycle stages. Immunofluorescent colocalization of elafin with neutrophil elastase confirmed that elafin was expressed by endometrial neutrophils around the time of menstruation. This is consistent with the expression profile observed from immunohistochemical studies. Primary endometrial epithelial cells were treated with proinflammatory molecules, and elafin mRNA was studied. A combination of the proinflammatory mediators, IL-1 beta and TNFalpha, increased elafin mRNA levels by 4.6-fold. These results show that endometrium expresses elafin in a menstruation-dependent manner. This is attributable to the presence of infiltrating leukocytes and increased inflammatory signaling. Elafin will regulate proteolytic enzymes during menstruation and will contribute to the innate defense against uterine infection.

Inflammatory mediators and endometrial function--focus on the perivascular cell.

Human endometrium has a unique vascular architecture that allows menstruation, the shedding of a well-vascularised tissue layer, with limited bleeding. Blood loss is controlled at least in part by constriction of the perivascular cells, myofibroblasts that surround the spiral arterioles and have contractile activity. These perivascular cells, which are coupled to endothelial cells by processes, are responsive to changes in progesterone levels and express chemokines, cytokines and prostaglandins (PG) crucial to the control of leukocyte entry into endometrium. In this location the chemokine interleukin-8 (IL-8) and prostaglandin E (PGE) will have synergistic effects on leukocyte entry. CD40 is also expressed on the perivascular cells. Activation of CD40 by CD40 ligand is known to increase COX-2 and IL-8 expression in endometrial fibroblasts. The likely source of CD40 ligand in the uterus is platelets. Thus ingress of platelets will up-regulate NFkappaB by activating CD40 and increase agents such as PGE which will stimulate further the ingress of platelets. There is thus the possibility of a spiralling inflammatory response. This response however, is normally modulated by progesterone raising the threshold of the NFkappaB pathway and in the presence of high progesterone levels activation of CD40 will be ineffective. When progesterone falls at the end of the ovarian cycle and the restrictions on activation are lost, the perivascular cells will respond, initiating leukocyte entry, vasoconstriction-vasodilatation cycles with associated hypoxia and consequent sloughing off of the endometrium. The perivascular cell in endometrium is pivotal in both menstruation and early pregnancy and we need to understand this cell better to devise more effective medical treatment for menstrual dysfunction.

Differential expression of the natural antimicrobials, beta-defensins 3 and 4, in human endometrium.

beta-Defensins are small cationic molecules that have antimicrobial actions against bacteria, fungi and viruses and contribute to mucosal immune responses at epithelial sites. The female reproductive tract is an important site of defensin production and innate defences are crucial to the preservation of fertility and successful pregnancy. This study details the expression of the recently characterized defensins, HBD3 and 4, in human endometrium. Using real-time quantitative RT-PCR, we have shown that HBD3 mRNA expression is highest during the secretory phase of the menstrual cycle while HBD4 mRNA levels peak in the proliferative phase. Both antimicrobials are expressed by endometrial epithelium. Exogenous steroid hormones in the form of the combined oral contraceptive pill (COCP) alter expression of both defensins in vivo, while treatment of endometrial explants with progesterone in vitro does not alter expression of HBD3 or HBD4. In in vitro cultures of primary endometrial epithelial cells, HBD3 mRNA expression is upregulated by treatment with inflammatory molecules including IL-1 beta+TNF alpha, IFN gamma and phorbol ester. HBD4 mRNA was not expressed in these primary cell cultures. These results show that the human endometrium expresses both HBD3 and HBD4 in a cycle-dependent manner. These natural antimicrobials will contribute to innate defences present in human endometrium protecting against uterine infection. Expression is altered as a result of hormonal contraceptive use and this may contribute to differential infection rates in COCP users relative to non-users. In addition, expression of HBD3 will be upregulated during infection allowing an increased innate immune response at this time.

Innate immune defences in the human endometrium.

The human endometrium is an important site of innate immune defence, giving protection against uterine infection. Such protection is critical to successful implantation and pregnancy. Infection is a major cause of preterm birth and can also cause infertility and ectopic pregnancy. Natural anti-microbial peptides are key mediators of the innate immune system. These peptides, between them, have anti-bacterial, anti-fungal and anti-viral activity and are expressed at epithelial surfaces throughout the female genital tract. Two families of natural anti-microbials, the defensins and the whey acidic protein (WAP) motif proteins, appear to be prominent in endometrium. The human endometrial epithelium expresses beta-defensins 1-4 and the WAP motif protein, secretory leukocyte protease inhibitor. Each beta-defensin has a different expression profile in relation to the stage of the menstrual cycle, providing potential protection throughout the cycle. Secretory leukocyte protease inhibitor is expressed during the secretory phase of the cycle and has a range of possible roles including anti-protease and anti-microbial activity as well as having effects on epithelial cell growth. The leukocyte populations in the endometrium are also a source of anti-microbial production. Neutrophils are a particularly rich source of alpha-defensins, lactoferrin, lysozyme and the WAP motif protein, elafin. The presence of neutrophils during menstruation will enhance anti-microbial protection at a time when the epithelial barrier is disrupted. Several other anti-microbials including the natural killer cell product, granulysin, are likely to have a role in endometrium. The sequential production of natural anti-microbial peptides by the endometrium throughout the menstrual cycle and at other sites in the female genital tract will offer protection from many pathogens, including those that are sexually transmitted.

Hormonal contraception can suppress natural antimicrobial gene transcription in human endometrium.

OBJECTIVE: To determine the effect of hormonal contraception with a combined oral contraceptive pill and levonorgestrel intrauterine system on the expression of the natural antimicrobials secretory leukocyte protease inhibitor, beta-defensins 1 and 2, and granulysin in human endometrium. DESIGN: Observational study. SETTING: Day case ward in a department of obstetrics and gynecology. PATIENT(S): Fifty seven women undergoing gynecologic procedures for benign conditions; 24 received no contraception for more than 3 months, 20 received a combined oral contraceptive for more than 3 months, and 13 wore a levonorgestrel intrauterine system for more than 3 months. MAIN OUTCOME MEASURE(S): Endometrial samples were collected from all women. Messenger RNA was extracted and quantitative polymerase chain reaction was used to investigate expression of secretory leukocyte protease inhibitor, beta-defensin 1, beta-defensin 2, and granulysin. Immunohistochemistry for secretory leukocyte protease inhibitor was performed. RESULT(S): All antimicrobials varied cyclically. The level of secretory leukocyte protease inhibitor was maximal in the late secretory and menstrual phase, beta-defensin 1 in the mid secretory phase, granulysin in the late secretory phase, and beta-defensin 2 in the menstrual phase. Use of a combined oral contraceptive or levonorgestrel intrauterine system use decreased messenger RNA expression of beta-defensin 1 and 2 and granulysin but not secretory leukocyte protease inhibitor. CONCLUSION(S): Endogenous and exogenous sex-steroid hormones, in the form of a combined oral contraceptive or levonorgestrel intrauterine system, influence gene transcription of secretory leukocyte protease inhibitor, beta-defensin 1, beta-defensin 2, and granulysin in the endometrium.

Fos expression induced by activation of NMDA and neurokinin-1 receptors in the trigeminal subnucleus caudalis in vitro: role of protein kinases.

Activity-induced neuronal plasticity is partly facilitated by the expression of the immediate-early gene c-fos and the resulting transcription factor Fos. Expression of Fos is associated with nociceptive afferent activation, but a detailed stimulation-transcription pathway for Fos expression has not yet been determined in the trigeminal system. This study utilized a novel in vitro model to determine whether Fos expression can be induced in trigeminal subnucleus caudalis by NMDA or neurokinin-1 receptor activation, and whether inhibition of intracellular kinases has any effect on Fos expression induced by activation of these receptors. Brainstems of male Wistar rats were excised and maintained in artificial cerebrospinal fluid at 37°C. NMDA or the specific neurokinin-1 receptor agonist [Sar(9),Met(O(2))(11)]-SP was applied. These agonists were subsequently tested in the presence of the protein kinase A inhibitor Rp-cAMP or protein kinase C inhibitor chelerythrine chloride. In all experiments the sodium channel blocker tetrodotoxin was used to prevent indirect neuronal activation. Brainstems were processed immunocytochemically for Fos expression, and positive cells were counted in the trigeminal subnucleus caudalis. NMDA and [Sar(9),Met(O(2))(11)]-SP significantly increased Fos expression, but these increases could be prevented by chelerythrine chloride. Rp-cAMP had no effect on Fos induced by NMDA but caused a significant reduction in Fos induced by [Sar(9),Met(O(2))(11)]-SP. These data demonstrate that in trigeminal subnucleus caudalis activation of either NK1 or NMDA receptors alone induces Fos expression; protein kinases A and C are involved in NK1R-induced Fos while protein kinase A is not required for NMDA receptor-induced Fos.

Oestrogen and progesterone regulation of inflammatory processes in the human endometrium.

The human endometrium is a unique tissue that has to undergo cycles of proliferation, differentiation, destruction and repair. This ensures that the endometrium is optimally prepared for potential embryo implantation but in the absence of an embryo, menstruation occurs to allow endometrial regeneration. These cycles of tissue remodelling occur under the sequential influence of the sex steroid hormones, oestrogen and progesterone. The physiological events of implantation and menstruation display features of inflammation, tightly regulated by oestrogen and progesterone. After menstruation cellular proliferation and blood vessel growth is modulated by oestrogen while after ovulation progesterone is the dominant hormone. In preparation for implantation, progesterone regulates decidualization of the endometrium, uterine natural killer cell numbers within the endometrium and chemokine and cytokine expression. Menstruation, in contrast, is preceded by progesterone withdrawal, which results in an influx of leukocytes into the endometrium and increased production of chemokines and matrix metalloproteinases allowing tissue degradation. The aim of this article is to review the current knowledge on the regulation of inflammatory events within the endometrium by oestrogen and progesterone, in relation to two pivotal events for human reproduction, implantation and menstruation.

Attenuated sex steroid receptor expression in fallopian tube of women with ectopic pregnancy.

CONTEXT: Sex steroid hormone receptor (SHR) dynamics are well-documented in human endometrium but have not been comprehensively studied in Fallopian tube (FT). OBJECTIVE: The aim of the study was to compare expression patterns and hormonal regulation of SHR in FT with that described in endometrium and to determine whether SHR expression is altered in FT of women with ectopic pregnancy (EP). DESIGN: Tissue was analyzed and cultured. PATIENTS OR OTHER PARTICIPANTS: Women undergoing surgery for benign gynecological conditions (n = 14) and EP (n = 6) participated in the study. INTERVENTIONS: Quantitative RT-PCR and immunohistochemistry were used to determine SHR mRNA expression and protein localization, respectively. SHR levels were measured in tubal explant cultures stimulated with estrogen and progestogen. RESULTS: ERalpha and ERbeta mRNAs were constitutively expressed in FT during the menstrual cycle. PR-AB and PR-B mRNAs were decreased in midluteal phase compared to follicular phase. ERalpha, PR-AB, and PR-B mRNAs were down-regulated in human FT in vitro by treatment with progestogen. ERalpha, ERbeta1, ERbeta2, PR, and AR proteins localized to cell nuclei of epithelium, stroma, and smooth muscle of nonpregnant FT. In FT from women with EP, PR-B mRNA was decreased when compared to midluteal FT, and ERalpha protein was not detected. CONCLUSIONS: SHR expression in FT is different from that observed in endometrium recovered at similar stages of the menstrual cycle, and expression in FT from women with EP is also altered compared with normal FT. These data are an important benchmark for furthering the understanding of normal human FT physiology, changes in expression of SHR in FT in response to progesterone, and disorders of FT function, such as EP.

Neurotrophin-induced preprotachykinin-A gene promoter modulation in organotypic rat spinal cord culture.

To study regulation of the preprotachykinin-A gene promoter, we utilised a biolistic gene transfer protocol to deliver a DNA construct that incorporates a portion of the preprotachykinin-A gene promoter and an enhanced green fluorescent protein reporter gene into neonatal rat spinal cord organotypic slices. The ability of the neurokinin-1 receptor agonist [Sar9,Met(O2)11]-substance P, nerve growth factor and brain derived neurotrophic factor to modulate positively preprotachykinin-A gene promoter construct activity, as indicated by de novo enhanced green fluorescent protein expression, was determined. Treatment of organotypic slices with [Sar9, Met(O2)11]-substance P (10 microm, P < 0.05), nerve growth factor (200 ng/mL, P < 0.001) or brain derived neurotrophic factor (200 ng/mL, P < 0.02) significantly increased the proportion of cytomegaloviral promoter-DsRed transfected cells (used to visualise total transfected cells) that co-expressed enhanced green fluorescent protein. The distribution of enhanced green fluorescent protein/DsRed-positive neurones across spinal laminae was broadly in line with the known distribution of spinal Trk and neurokinin-1 receptors. These data suggest a modulated activity of the preprotachykinin-A gene promoter in spinal neurones in vitro by substance P and/or neurotrophins. The functional consequences of such transcriptional changes within central peptidergic circuitry and their relevance to chronic pain are considered.

Serotoninergic-mediated inhibition of substance P sensitive deep dorsal horn neurons: a combined electrophysiological and morphological study in vitro.

Dorsal horn neurons that express the neurokinin 1 receptor (NK-1R) play an important role in nociceptive processing. The targetting of NK-1R neurons by serotoninergic (5-hydroxytryptamine, 5-HT) axons would provide a straightforward means to exert an inhibitory analgesic effect at spinal level. This study used single cell electrophysiology to analyse and correlate the responses of rat deep DH neurons in vitro to both 5-HT and the NK-1R agonist [Sar9,Met(O2)11]-substance P (SP). Subsequently a combination of immunocytochemistry and confocal imaging was applied to biocytin-filled laminae III-VI neurons to reveal putative 5-HT innervation in this neuronal sample. A population of neurons was identified in which 5-HT (50 microM) significantly attenuated the dorsal root-evoked excitatory postsynaptic potential and [Sar9,Met(O2)11]-SP (2 microM) induced a direct tetrodotoxin-resistant depolarisation. Immunolabelling revealed that all of these neurons were inhibited by 5-HT, including those that were excited by [Sar9,Met(O2)11]-SP, were overlaid by a plexus of 5-HT immunoreactive fibres and in some instances, closely apposed putative contacts with somata and proximal dendrites identified although their incidence was low. Inhibition by 5-HT of deep DH neurons directly responsive to SP may account at least in part for monoamine-induced modulation of nociceptive processing in the spinal cord.