CD73-generated adenosine restricts lymphocyte migration into draining lymph nodes.

After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naive T cells with Ag-loaded dendritic cells. In this study, we show that CD73 (ecto-5'-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73(-/-) mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared with wild-type mice 24 h after LPS administration. Migration rates of cd73(+/+) and cd73(-/-) lymphocytes into lymph nodes of wild-type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A(2B) receptor is a likely target of CD73-generated adenosine, because it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up-regulated by TNF-alpha. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73(-/-) mice is largely normalized by pretreatment with the selective A(2B) receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.

The Role of IL-6 in Adenosine-Mediated Pulmonary Fibrosis

Adenosine is a purinergic signaling molecule that regulates various aspects of inflammation and has been implicated in the pathogenesis of chronic lung diseases. Previous studies have demonstrated that adenosine up-regulates IL-6 production through the engagement of the A2B adenosine receptor in various cell types, including alveolar macrophages. IL-6 is elevated in mouse models and humans with chronic lung disease, suggesting a potential role in disease progression. Furthermore, chronic elevation of adenosine in the lungs of adenosine deaminase deficient (Ada-/-) mice leads to the development of pulmonary inflammation, alveolar destruction, and fibrosis, in conjunction with IL-6 elevation. Thus, it was hypothesized that IL-6 contributes to pulmonary inflammation and fibrosis in this model. To test this hypothesis, Ada/IL-6 double knockout mice (Ada/IL-6-/-) were generated to assess the consequences of genetically removing IL-6 on adenosine-dependent pulmonary injury. Ada/IL-6-/- mice exhibited a significant reduction in inflammation, alveolar destruction, and pulmonary fibrosis. Next, Ada-/- mice were treated systematically with IL-6 neutralizing antibodies to test the efficacy of blocking IL-6 on chronic lung disease. These treatments were associated with decreased pulmonary inflammation, alveolar destruction, and fibrosis. To determine the role of IL-6 in a second model of pulmonary fibrosis, wild type mice and IL-6-/- mice were subjected to intraperitoneal injections of bleomycin twice a week for four weeks. Results demonstrated that IL-6-/- mice developed reduced pulmonary fibrosis. To examine a therapeutic approach in this model, wild type mice exposed to bleomycin were treated with IL-6 neutralizing antibodies. Similar results were observed as with Ada-/- mice, namely diminished pulmonary inflammation and fibrosis. In both models, elevations in IL-6 were associated with increased phosphorylated STAT-3 in the nuclei of numerous cell types in the airways, including type II alveolar epithelial cells (AEC). Genetic removal and neutralization of IL-6 in both models was associated with decreased STAT-3 activation in type II AEC. The mechanism of activation in these cells that lack the membrane bound IL-6Ra suggests IL-6 trans-signaling may play a role in regulating fibrosis. Characterization of this mechanism demonstrated that the soluble IL-6Ra (sIL-6Ra) is upregulated in both models during chronic conditions. In vitro studies in MLE-12 alveolar epithelial cells confirmed that IL-6, in combination with the sIL-6Ra, activates STAT-3 and TWIST in association with enhancement of epithelial-to-mesenchymal transition, which can contribute to fibrosis. Similarly, patients with idiopathic pulmonary fibrosis demonstrated a similar pattern of increased IL-6 expression, STAT-3 activation, and sIL-6Ra increases. These findings demonstrate that adenosine-dependent elevations in IL-6 contribute to the development and progression of pulmonary inflammation and fibrosis. The implications from these studies are that adenosine and/or IL-6 neutralizing agents represent novel therapeutic targets for the treatment of pulmonary disorders where fibrosis is a detrimental component.

THE EFFECT OF GABAMIMETICS ON NEUROTRANSMITTER RECEPTOR BINDING AND FUNCTION IN RAT BRAIN

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and alterations in central GABAergic transmission may contribute to the symptoms of a number of neurological and psychiatric disorders. Because of this relationship, numerous laboratories are attempting to develop agents which will selectively enhance GABA neurotransmission in brain. Due to these efforts, several promising compounds have recently been discovered. Should these drugs prove to be clinically effective, they will be used to treat chronic neuropsychiatric disabilities and, therefore, will be administered for long periods of time. Accordingly, the present investigation was undertaken to determine the neurochemical consequences of chronic activation of brain GABA systems in order to better define the therapeutic potential and possible side-effect liability of GABAmimetic compounds.^ Chronic (15 day) administration to rats of low doses of amino-oxyacetic acid (AOAA, 10 mg/kg, once daily), isonicotinic acid hydrazide (20 mg/kg, b.i.d.), two non-specific inhibitors of GABA-T, the enzyme which catabolizes GABA in brain, or (gamma)-acetylenic GABA (10 mg/kg, b.i.d.) a catalytic inhibitor of this enzyme, resulted in a significant elevation of brain and CSF GABA content throughout the course of treatment. In addition, chronic administration of these drugs, as well as the direct acting GABA receptor agonists THIP (8 mg/kg, b.i.d.) or kojic amine (18 mg/kg, b.i.d.) resulted in a significant increase in dopamine receptor number and a significant decrease in GABA receptor number in the corpus striatum of treated animals as determined by standard in vitro receptor binding techniques. Changes in the GABA receptor were limited to the corpus striatum and occurred more rapidly than did alterations in the dopamine receptor. The finding that dopamine-mediated stereotypic behavior was enhanced in animals treated chronically with AOAA suggested that the receptor binding changes noted in vitro have some functional consequence in vitro.^ Coadministration of atropine (a muscarinic cholinergic receptor antagonist) blocked the GABA-T inhibitor-induced increase in striatal dopamine receptors but was without effect on receptor alterations seen following chronic administration of direct acting GABA receptor agonists. Atropine administration failed to influence the drug-induced decreases in striatal GABA receptors.^ Other findings included the discovery that synaptosomal high affinity ('3)H-choline uptake, an index of cholinergic neuronal activity, was significantly increased in the corpus striatum of animals treated acutely, but not chronically, with GABAmimetics.^ It is suggested that the dopamine receptor supersensitivity observed in the corpus striatum of animals following long-term treatment with GABAmimetics is a result of the chronic inhibition of the nigrostriatal dopamine system by these drugs. Changes in the GABA receptor, on the other hand, are more likely due to a homospecific regulation of these receptors. An hypothesis based on the different sites of action of GABA-T inhibitors vis-a-vis the direct acting GABA receptor agonists is proposed to account for the differential effect of atropine on the response to these drugs.^ The results of this investigation provide new insights into the functional interrelationships that exist in the basal ganglia and suggest that chronic treatment with GABAmimetics may produce extrapyramidal side-effects in man. In addition, the constellation of neurochemical changes observed following administration of these drugs may be a useful guide for determining the GABAmimetic properties of neuropharmacological agents. ^

The Antimalarial Drug Proguanil Is An Antagonist at 5-HT3 Receptors

Proguanil is an antimalarial prodrug that is metabolized to 4-chlorophenyl-1-biguanide (CPB) and the active metabolite cycloguanil (CG). These compounds are structurally related to meta-chlorophenyl biguanide (mCPBG), a 5-hydroxytryptamine 3 (5-HT3) receptor agonist. Here we examine the effects of proguanil and its metabolites on the electrophysiology and ligand-binding properties of human 5-HT3A receptors expressed in Xenopus oocytes and human embryonic kidney 293 cells, respectively. 5-HT3 receptor responses were reversibly inhibited by proguanil, with an IC50 of 1.81 μM. Competitive antagonism was shown by a lack of voltage-dependence, Schild plot (Kb = 1.70 μM), and radioligand competition (Ki = 2.61 μM) with the 5-HT3 receptor antagonist [3H]granisetron. Kinetic measurements (kon = 4.0 × 104 M−1 s−1; koff = 0.23 s−1) were consistent with a simple bimolecular reaction scheme with a Kb of 4.35 μM. The metabolites CG and CPB similarly inhibited 5-HT3 receptors as assessed by IC50 (1.48 and 4.36 μM, respectively), Schild plot (Kb = 2.97 and 11.4 μM), and radioligand competition (Ki = 4.89 and 0.41 μM). At higher concentrations, CPB was a partial agonist (EC50 = 14.1 μM; I/Imax = 0.013). These results demonstrate that proguanil competitively inhibits 5-HT3 receptors, with an IC50 that exceeds whole-blood concentrations following its oral administration. They may therefore be responsible for the occasional gastrointestinal side effects, nausea, and vomiting reported following its use. Clinical development of related compounds should therefore consider effects at 5-HT3 receptors as an early indication of possible unwanted gastrointestinal side effects.

Development of small molecular tools for the cellular study of adenosine receptors

The adenosine receptors are members of the G-protein coupled receptor (GPCR) family which represents the largest class of cell-surface proteins mediating cellular communication. As a result, GPCRs are formidable drug targets and it is estimated that approximately 30% of the marketed drugs act through members of this receptor class. There are four known subtypes of adenosine receptors: A1, A2A, A2B and A3. The adenosine A1 receptor, which is the subject of this presentation, mediates the physiological effects of adenosine in various tissues including the brain, heart, kidney and adipocytes. In the brain for instance, its role in epilepsy and ischemia has been the focus of many studies. Previous attempts to study the biosynthesis, trafficking and agonist-induced internalisation of the adenosine A1 receptor in neurons using fluorescent protein-receptor fusion constructs have been hampered by the sheer size of the fluorescent protein (GFP) that ultimately affected the function of the receptor. We have therefore initiated a research programme to develop small molecule fluorescent agonists that selectively activate the adenosine A1 receptor. Our probe design is based on the endogenous ligand adenosine and the known unselective adenosine receptor agonist NECA. We have synthesised a small library of non-fluorescent adenosine derivatives that have different cyclic and bicyclic moieties at the 6 position of the purine ring and have evaluated the pharmacology of these compounds using a yeast-based assay. This analysis revealed compounds with interesting behaviour, i.e. exhibiting subtype-selectivity and biased signalling, that can be potentially used as tool compounds in their own right for cellular studies of the adenosine A1 receptor. Furthermore, we have also linked fluorescent dyes to the purine ring and discovered fluorescent compounds that can activate the adenosine A1 receptor.

Evaluation of three different families of bombesin receptor radioantagonists for targeted imaging and therapy of gastrin releasing peptide receptor (GRP-R) positive tumors.

Two new classes of radiolabeled GRP receptor antagonists are studied and compared with the well-established statine-based receptor antagonist DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2 (RM2, 1; DOTA:1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; Sta:(3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid). The bombesin-based pseudopeptide DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leuψ(CHOH-CH2)-(CH2)2-CH3 (RM7, 2), and the methyl ester DOTA-4-amino-1-carboxymethylpiperidine-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-OCH3 (ARBA05, 3) analogues are labeled with (111)In and evaluated in vitro in PC-3 cell line and in vivo in PC-3 tumor-bearing nude mice. Antagonist potency was assessed by immunofluorescence-based receptor internalization and Ca(2+) mobilization assays. The conjugates showed good binding affinity, the IC50 value of 2 (3.2 ± 1.8 nM) being 2 and 10 times lower than 1 and 3. Compared to (111)In-1, (111)In-2 showed higher uptake in target tissues such as pancreas (1.5 ± 0.5%IA/g and 39.8 ± 9.3%IA/g at 4 h, respectively), whereas the compounds had similar tumor uptake (11.5 ± 2.4%IA/g and 11.8 ± 3.9%IA/g at 4h, respectively). The displacement of the radioligand in vivo was different in different receptor positive organs and depended on the displacing peptide.

Downregulation of sphingosine 1-phosphate (S1P) receptor 1 by dexamethasone inhibits S1P-induced mesangial cell migration

Sphingosine 1-phosphate (S1P) is generated by sphingosine kinase (SK)-1 and -2 and acts mainly as an extracellular ligand at five specific receptors, denoted S1P1-5. After activation, S1P receptors regulate important processes in the progression of renal diseases, such as mesangial cell migration and survival. Previously, we showed that dexamethasone enhances SK-1 activity and S1P formation, which protected mesangial cells from stress-induced apoptosis. Here we demonstrate that dexamethasone treatment lowered S1P1 mRNA and protein expression levels in rat mesangial cells. This effect was abolished in the presence of the glucocorticoid receptor antagonist RU-486. In addition, in vivo studies showed that dexamethasone downregulated S1P1 expression in glomeruli isolated from mice treated with dexamethasone (10 mg/kg body weight). Functionally, we identified S1P1 as a key player mediating S1P-induced mesangial cell migration. We show that dexamethasone treatment significantly lowered S1P-induced migration of mesangial cells, which was again reversed in the presence of RU-486. In summary, we suggest that dexamethasone inhibits S1P-induced mesangial cell migration via downregulation of S1P1. Overall, these results demonstrate that dexamethasone has functional important effects on sphingolipid metabolism and action in renal mesangial cells.

Lindane induced cellular dysfunction in MDCK cells: The role of the peripheral benzodiazepine receptor

The central nervous system GABAA/Benzodiazepine (GABAA/BZD) receptors are targets for many pharmaceutical agents and several classes of pesticides. Lindane is an organochlorine pesticide, although banned from production in the U.S. since 1977, still imported for use as an insecticide and pharmaceutically to control ectoparasites (ATSDR, 1994). Lindane functions as a GABA/BZD receptor antagonist within the central nervous system (CNS). Outside of the CNS, peripheral BZD receptors have been localized to the distal tubule of the kidney. Previous research in our laboratory has shown that incubation of renal cortical slices with lindane can produce an increase in kallikrein leakage, suggesting a distal tubular effect. In this study, Madin Darby Canine Kidney (MDCK) cells were used as an in vitro system to assess the toxicity of lindane. This purpose of this study was to determine if interactions between a renal distal tubular BZD-like receptor and lindane could lead to perturbations in renal distal cellular chloride (Cl−) transport and mitochondrial dysfunction and ultimately, cellular death. ^ Pertubations in renal chloride transport were measured indirectly by determining if lindane altered cell function responsiveness following osmotic stress. MDCK cells pre-treated with lindane and then subjected to osmotic stress remained swollen for up to 12 hours post-stress. Lindane-induced dysfunction was assessed through stress protein induction measured by Western Blot analysis. Lindane pretreatment delayed Heat Shock Protein 72 (HSP72) induction by 36 hours in osmotically stressed cells. Pretreatment with 1 × 10 −5 M LIN followed by osmotic stress elevated p38 and Stress Activated Protein Kinase (SAPK/JNK) at 15 minutes which declined at 30 minutes. Lindane appeared to have no effect on Endoplasmic Reticulum Related Kinase (ERK) induction. Lindane did not effect osmotically stressed LLC-PKI cells, a control cell line. ^ Lindane-treated MDCK cells did not exhibit necrosis. Instead, apoptosis was observed in lindane-treated MDCK cells in both time- and dose-dependent manners. LLC-PKI cells were not affected by LIN treatment. ^ To better understand the mechanism of lindane-induced apoptosis, mitochondrial function was measured. No changes in cytochrome c release or mitochondrial membrane potential were observed suggesting the mitochondrial pathway was not involved in lindane-induced apoptosis. ^ Further research will need to be conducted to determine the mechanism of lindane-induced adverse cellular effects. ^

Adenosine mediated lung mast cell degranulation in the mouse

Adenosine has been implicated to play a role in inflammatory processes associated with asthma. Most notable is adenosine's ability to potentiate mediator release from mast cells. Mast cells are bone marrow derived inflammatory cells that can release mediators that have both immediate and chronic effects on airway constriction and inflammation. Most physiological roles of adenosine are mediated through adenosine receptors. Four subtypes of adenosine receptors have been identified, A1, A2A, A2B and A 3. The mechanisms by which adenosine can influence the release of mediators from lung tissue mast cells is not understood due to lack of in vivo models. Mice deficient in the enzyme adenosine deaminase (ADA) have been generated. ADA controls the levels of adenosine in tissues and cells, and consequently, adenosine accumulates in the lungs of ADA-deficient mice. ADA-deficient mice develop features seen in asthmatics, including lung eosinophilia and mucus hypersecretion. In addition, lung tissue mast cell degranulation was associated with elevated adenosine in ADA-deficient lungs and can be prevented by ADA enzyme therapy. We established primary murine lung mast cell cultures, and used real time RT-PCR and immunofluorescence to demonstrate that A 2A, A2B and A3 receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists and A3 receptor deficient (A3−/−) mast cells suggested that activation of A3 receptors could induce mast cell mediator release in vitro. Furthermore, this mediator release was associated with increases in intracellular Ca++ that appeared to be mediated through a Gi and PI3K pathway. In addition, nebulized A3 receptor agonist directly induced lung mast cell degranulation in wild type mice while having no effect in A3−/− mice. These results demonstrate that the A3 receptor plays an important role in adenosine mediated murine lung mast cell degranulation. Therefore, the A3 adenosine receptor and its signaling pathways may represent novel therapeutic targets for the treatment and prevention of asthma. ^

The role of AT1 receptor autoantibodies in the pathophysiology of preeclampsia

Preeclampsia is a disease that affects 3–5% of all pregnancies. The cause is unknown and there is currently no treatment. The disease poses significant health risks to both the mother and the fetus. To date, research on the topic has not produced a convincing cause for the development of the hallmark symptoms of preeclampsia. The hypothesis of an agonistic autoimmune response to the AT1 receptor is presented. Immunoglobulin fractions from normotensive and preeclampsia patients were prepared for experimental tests. Model systems were tested in three categories to determine if AT 1 receptor specific activation and receptor-ligand interaction was caused by a suspected autoantibody. Activation was found in rat neonatal cardiornyocytes that caused an increased contraction rate. This activity was found in preeclampsia patients, absent in normotensive patients. The activation was antagonized by losartan, an AT1 receptor antagonist, and by epitope peptide competition of the receptor-ligand type interaction. This epitope was the 7 amino acid peptide fragment, AFHYESQ, a sequence present in the second extracellular loop of the AT1 receptor. The patterns of AT1 receptor activation were also found in a human trophoblast cell line, HTR8, with an effect on Pai-1 secretion, a factor that plays a role in preventing hypercoagulation. In human mesangial cells, the AT1 receptor autoantibody present in the immunoglobulin fraction from preeclampsia patients was found to stimulate the secretion of Pai-1, and IL-6, a factor that plays a role in the activation of an inflammatory response. This activity was found in samples from preeclampsia patients, but absent in normotensive patients. Tests including losartan, AFHYESQ, and a non-competitive peptide demonstrated that the secretion of Pai-1 and IL-6 met the criteria for AT1 receptor activation by the suspected agonistic autoantibody. These three model systems address relevant pathophysiology for preeclampsia patients, including increased cardiac output, abnormal placentation, and renal damage. The AT1 receptor agonistic autoantibody is potentially a key player in the development of the pathology and symptoms of preeclampsia. ^

Ultraviolet radiation modulates the immune system through the platelet -activating factor receptor

Ultraviolet radiation plays a critical role in the induction of non-melanoma skin cancer. UV radiation is also immune suppressive. Moreover, UV-induced systemic immune suppression is a major risk factor for skin cancer induction. Previous work had shown that UV exposure in vivo activates a cytokine cascade involving PGE2, IL-4, and IL-10 that induces immune suppression. However, the earliest molecular events that occur immediately after UV-exposure, especially those upstream of PGE2, were not well defined. To determine the initial events and mediators that lead to immune suppression after a pathological dose of UV, mouse keratinocytes were analyzed after sunlamp irradiation. It is known that UV-irradiated keratinocytes secrete the phospholipid mediator of inflammation, platelet-activating factor (PAF). Since PAF stimulates the production of immunomodulatory compounds, including PGE2, the hypothesis that UV-induced PAF activates cytokine production and initiates UV-induced immune suppression was tested. Both UV and PAF activated the transcription of cyclooxygenase (COX)-2 and IL-10 reporter gene constructs. A PAF receptor antagonist blocked UV-induced IL, 10 and COX-2 transcription. PAF mimicked the effects of UV in vivo and suppressed delayed-type hypersensitivity (DTH), and immune suppression was blocked when UV-irradiated mice were injected with a PAF receptor antagonist. This work shows that UV generates PAF-like oxidized lipids, that signal through the PAF receptor, activate cytokine transcription, and induce systemic immune suppression. ^

In vivo inhibition of rat stellate cell activation by soluble transforming growth factor β type II receptor: A potential new therapy for hepatic fibrosis

Transforming growth factor β (TGF-β) is a well characterized cytokine that appears to play a major role in directing the cellular response to injury, driving fibrogenesis, and, thus, potentially underlying the progression of chronic injury to fibrosis. In this study, we report the use of a novel TGF-β receptor antagonist to block fibrogenesis induced by ligation of the common bile duct in rats. The antagonist consisted of a chimeric IgG containing the extracellular portion of the TGF-β type II receptor. This “soluble receptor” was infused at the time of injury; in some experiments it was given at 4 days after injury, as a test of its ability to reverse fibrogenesis. The latter was assessed by expression of collagen, both as the mRNA in stellate cells isolated from control or injured liver and also by quantitative histochemistry of tissue sections. When the soluble receptor was administered at the time of injury, collagen I mRNA in stellate cells from the injured liver was 26% of that from animals receiving control IgG (P < 0.0002); when soluble receptor was given after injury induction, collagen I expression was 35% of that in control stellate cells (P < 0.0001). By quantitative histochemistry, hepatic fibrosis in treated animals was 55% of that in controls. We conclude that soluble TGF-β receptor is an effective inhibitor of experimental fibrogenesis in vivo and merits clinical evaluation as a novel agent for controlling hepatic fibrosis in chronic liver injury.

Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes

In cultured oligodendrocytes isolated from perinatal rat optic nerves, we have analyzed the expression of ionotropic glutamate receptor subunits as well as the effect of the activation of these receptors on oligodendrocyte viability. Reverse transcription–PCR, in combination with immunocytochemistry, demonstrated that most oligodendrocytes differentiated in vitro express the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR3 and GluR4 and the kainate receptor subunits GluR6, GluR7, KA1 and KA2. Acute and chronic exposure to kainate caused extensive oligodendrocyte death in culture. This effect was partially prevented by the AMPA receptor antagonist GYKI 52466 and was completely abolished by the non-N-methyl-d-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), suggesting that both AMPA and kainate receptors mediate the observed kainate toxicity. Furthermore, chronic application of kainate to optic nerves in vivo resulted in massive oligodendrocyte death which, as in vitro, could be prevented by coinfusion of the toxin with CNQX. These findings suggest that excessive activation of the ionotropic glutamate receptors expressed by oligodendrocytes may act as a negative regulator of the size of this cell population.

Mechanisms underlying kainate receptor-mediated disinhibition in the hippocampus

Kainate (KA) receptor activation depresses stimulus-evoked γ-aminobutyric acid (GABA-mediated) synaptic transmission onto CA1 pyramidal cells of the hippocampus and simultaneously increases the frequency of spontaneous GABA release through an increase in interneuronal spiking. To determine whether these two effects are independent, we examined the mechanism by which KA receptor activation depresses the stimulus-evoked, inhibitory postsynaptic current (IPSC). Bath application of the α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)/KA receptor agonist KA in the presence of the AMPA receptor antagonist GYKI 53655 caused a large increase in spontaneous GABA release and a coincident depression of the evoked IPSC. The depressant action on the evoked IPSC was reduced, but not abolished, by the GABAB receptor antagonist SCH 50911, suggesting that the KA-induced increase in spontaneous GABA release depresses the evoked IPSC through activation of presynaptic GABAB receptors. KA had no resolvable effect on the potassium-induced increase in miniature IPSC frequency, suggesting that KA does not act through a direct effect on the release machinery or presynaptic calcium influx. KA caused a decrease in pyramidal cell input resistance, which was reduced by GABAA receptor antagonists. KA also caused a reduction in the size of responses to iontophoretically applied GABA, which was indistinguishable from the SCH 50911-resistant, residual depression of the evoked IPSC. These results suggest that KA receptor activation depresses the evoked IPSC indirectly by increasing interneuronal spiking and GABA release, leading to activation of presynaptic GABAB receptors, which depress GABA release, and postsynaptic GABAA receptors, which increase passive shunting.

Estrogenic responses in estrogen receptor-α deficient mice reveal a distinct estrogen signaling pathway

Estrogens are thought to regulate female reproductive functions by altering gene transcription in target organs primarily via the nuclear estrogen receptor-α (ER-α). By using ER-α “knock-out” (ERKO) mice, we demonstrate herein that a catecholestrogen, 4-hydroxyestradiol-17β (4-OH-E2), and an environmental estrogen, chlordecone (kepone), up-regulate the uterine expression of an estrogen-responsive gene, lactoferrin (LF), independent of ER-α. A primary estrogen, estradiol-17β (E2), did not induce this LF response. An estrogen receptor antagonist, ICI-182,780, or E2 failed to inhibit uterine LF gene expression induced by 4-OH-E2 or kepone in ERKO mice, which suggests that this estrogen signaling pathway is independent of both ER-α and the recently cloned ER-β. 4-OH-E2, but not E2, also stimulated increases in uterine water imbibition and macromolecule uptake in ovariectomized ERKO mice. The results strongly imply the presence of a distinct estrogen-signaling pathway in the mouse uterus that mediates the effects of both physiological and environmental estrogens. This estrogen response pathway will have profound implications for our understanding of the physiology and pathophysiology of female sex steroid hormone actions in target organs.