Striatal GABA Receptor Alterations in Hypoxic Neonatal Rats: Role of Glucose,Oxygen and Epinephrine Treatment

Hypoxia in neonates disrupts the oxygen flow to the brain, essentially starving the brain and preventing it from performing vital biochemical processes important for central nervous system development. Hypoxia results in a permanent brain damage by gene and receptor level alterations mediated through neurotransmitters. The present study evaluated GABA, GABAA, GABAB receptor functions and gene expression changes in glutamate decarboxylase in the corpus striatum of hypoxic neonatal rats and the treatment groups with glucose, oxygen and epinephrine. Since GABA is the principal neurotransmitter involved in hypoxic ventilatory decline, the alterations in its level under hypoxic stress points to an important aspect of respiratory control. Following hypoxic stress, a significant decrease in total GABA, GABAA and GABAB receptors function and GAD expression was observed in the striatum, which accounts for the ventilator decline. Hypoxic rats treated with glucose alone and with oxygen showed a reversal of the receptor alterations and changes in GAD to near control. Being a source of immediate energy, glucose can reduce the ATP-depletion-induced changes in GABA and oxygenation helps in overcoming reduction in oxygen supply. Treatment with oxygen alone and epinephrine was not effective in reversing the altered receptor functions. Thus, our study point to the functional role of GABA receptors in mediating ventilatory response to hypoxia and the neuroprotective role of glucose treatment. This has immense significance in the proper management of neonatal hypoxia for a better intellect in the later stages of life.     

δ-Opioid Receptor Activation Modified MicroRNA Expression in the Rat Kidney under Prolonged Hypoxia

Hypoxic/ischemic injury to kidney is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs are differentially involved in hypoxic/ischemic events and δ-opioid receptor (DOR) activation is known to protect against hypoxic/ischemic injury, we speculated on the involvement of DOR activation in altering the microRNA (miRNA) expression in kidney under hypoxic condition. We selected 31 miRNAs based on microarray data for quantitative PCR analysis. Among them, 14 miRNAs were significantly altered after prolonged hypoxia, DOR activation or a combination of both. We found that 1) DOR activation alters miRNA expression profiles in normoxic conditions; 2) hypoxia differentially alters miRNA expression depending on the duration of hypoxia; and 3) DOR activation can modify hypoxia-induced changes in miRNA expression. For example, 10-day hypoxia reduced the level of miR-212 by over 70%, while DOR activation could mimic such reduction even in normoxic kidney. In contrast, the same stress increased miR-29a by >100%, which was reversed following DOR activation. These first data suggest that hypoxia comprehensively modifies the miRNA profile within the kidney, which can be mimicked or modified by DOR activation. Ascertaining the targeted pathways that regulate the diverse cellular and molecular functions of miRNA may provide new insights into potential therapies for hypoxic/ischemic injury of the kidney.     

δ-Opioid Receptor Activation and MicroRNA Expression of the Rat Cortex in Hypoxia

Prolonged hypoxic/ischemic stress may cause cortical injury and clinically manifest as a neurological disability. Activation of the δ-opioid receptor (DOR) may induce cortical protection against hypoxic/ischemic insults. However, the mechanisms underlying DOR protection are not clearly understood. We have recently found that DOR activation modulates the expression of microRNAs (miRNAs) in the kidney exposed to hypoxia, suggesting that DOR protection may involve a miRNA mechanism. To determine if the miRNAs expressed in the cortex mediated DOR neuroprotection, we examined 19 miRNAs that were previously identified as hypoxia- and DOR-regulated miRNAs in the kidney, in the rat cortex treated with UFP-512, a potent and specific DOR agonist under hypoxic condition. Of the 19 miRNAs tested, 17 were significantly altered by hypoxia and/or DOR activation with the direction and amplitude varying depending on hypoxic duration and times of DOR treatment. Expression of several miRNAs such as miR-29b, -101b, -298, 324-3p, -347 and 466b was significantly depressed after 24 hours of hypoxia. Similar changes were seen in normoxic condition 24 hours after DOR activation with one-time treatment of UFP-512. In contrast, some miRNAs were more tolerant to hypoxic stress and showed significant reduction only with 5-day (e.g., miR-31 and -186) or 10-day (e.g., miR-29a, let-7f and -511) exposures. In addition, these miRNAs had differential responses to DOR activation. Other miRNAs like miRs-363* and -370 responded only to the combined exposure to hypoxia and DOR treatment, with a notable reduction of >70% in the 5-day group. These data suggest that cortical miRNAs are highly yet differentially sensitive to hypoxia. DOR activation can modify, enhance or resolve the changes in miRNAs that target HIF, ion transport, axonal guidance, free radical signaling, apoptosis and many other functions.     

gamma-Aminobutyric acid (GABA) induces a receptor-mediated reduction in GABAA receptor alpha subunit messenger RNAs in embryonic chick neurons in culture.

gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in brain, is known to interact with a subclass of receptors that activate a ligand-gated chloride ion channel. Exposure of cultured embryonic chick neurons to physiological concentrations of GABA results in a time-dependent down-regulation of these GABAA receptors. To delineate the cellular mechanism(s) responsible for agonist-induced down-regulation of GABAA receptors we quantified the levels of GABAA receptor alpha subunit messenger RNAs, which encode the subunit(s) containing agonist recognition site(s), and observed a marked reduction in alpha subunit mRNAs following exposure of embryonic chick neurons to GABA. Both the down-regulation of GABAA receptors and the reduction in alpha subunit mRNAs induced by GABA were completely antagonized by the specific GABAA receptor antagonist SR-95531. These data demonstrate the presence of an agonist-induced receptor-mediated mechanism for regulating the expression of receptor subunit-encoding mRNAs that may be involved in the development of tolerance to the pharmacological actions of drugs known to act via GABAA receptors.     

Cerebellar GABA(A) receptor alterations in hypoxic neonatal rats: Role of glucose, oxygen and epinephrine supplementation

Hypoxia in neonates causes dysfunction of excitatory and inhibitory neurotransmission resulting in permanent brain damage. The present study is to understand the cerebellar GABA(A) receptor alterations and neuroprotective effect of glucose supplementation prior to current sequence of resuscitation - oxygen and epinephrine supplementation in hypoxic neonatal rats. Hypoxic insult caused a significant decrease in GABA(A) receptor number along with down regulated expression of GABA(Aα1,) GABA(Aα5), GABA(Aδ) and GABA(Aγ3) receptor subunits in the cerebellum which accounts for the respiratory inhibition. Hypoxic rats supplemented with glucose alone and with oxygen showed a reversal of the receptor alterations and changes in GABA(A) receptor subunits expression to near control. Glucose can reduce ATP-depletion-induced alterations in GABA receptors, thereby assisting in overcoming the neuronal damage caused by hypoxia. Resuscitation with oxygen alone and epinephrine was less effective in reversing the receptor alterations. The reduction in the GABA(A) receptors functional regulation during hypoxia plays an important role in cerebellar damage. Resuscitation with glucose alone and glucose with oxygenation to hypoxic neonatal rats helps in protecting the brain from severe hypoxic damage.     

Neuroprotective role of delta-opioid receptors in cortical neurons

We recently demonstrated that delta-opioid receptor (DOR) activation protects cortical neurons against glutamate-induced injury. Because glutamate is a mediator of hypoxic injury in neurons, we hypothesized that DOR is involved in neuroprotection during O2 deprivation and that its activation/inhibition may alter neuronal susceptibility to hypoxic stress. In this work, we tested the effect of opioid receptor activation and inhibition on cultured cortical neurons in hypoxia (1% O2). Cell injury was assessed by lactate dehydrogenase release, morphology-based quantification, and live/dead staining. Our results show that 1) immature neurons (days 4 and 6) were not significantly injured by hypoxia until 72 h of exposure, whereas day 8 neurons were injured after only 24-h hypoxia; 2) DOR inhibition (naltrindole) caused neuronal injury in both day 4 and day 8 normoxic cultures and further augmented hypoxic injury in these neurons; 3) DOR activation ([D-Ala2,D-Leu5]enkephalin) reduced neuronal injury in day 8 cultures after 24 h of normoxic or hypoxic exposure and attenuated naltrindole-induced injury with prolonged exposure; and 4) mu- or kappa-opioid receptor inhibition (beta-funaltrexamine or nor-binaltorphimine) had little effect on neurons in either normoxic or hypoxic conditions. Collectively, these data suggest that DOR plays a crucial role in neuroprotection in normoxic and hypoxic environments.     

Decreased GABAB receptor function in the cerebellum and brain stem of hypoxic neonatal rats: Role of glucose,oxygen and epinephrine resuscitation

Background-

Hypoxia during the first week of life can induce neuronal death in vulnerable brain regions usually associated with an impairment of cognitive function that can be detected later in life. The neurobiological changes mediated through neurotransmitters and other signaling molecules associated with neonatal hypoxia are an important aspect in establishing a proper neonatal care.

Methods-

The present study evaluated total GABA, GABA_B receptor alterations, gene expression changes in GABA_B receptor and glutamate decarboxylase in the cerebellum and brain stem of hypoxic neonatal rats and the resuscitation groups with glucose, oxygen and epinephrine. Radiolabelled GABA and baclofen were used for receptor studies of GABA and GABA_B receptors respectively and Real Time PCR analysis using specific probes for GABA_B receptor and GAD mRNA was done for gene expression studies.

Results-

The adaptive response of the body to hypoxic stress resulted in a reduction in total GABA and GABA_B receptors along with decreased GABA_B receptor and GAD gene expression in the cerebellum and brain stem. Hypoxic rats supplemented with glucose alone and with oxygen showed a reversal of the receptor alterations and changes in GAD. Resuscitation with oxygen alone and epinephrine was less effective in reversing the receptor alterations.

Conclusions-

Being a source of immediate energy, glucose can reduce the ATP-depletion-induced changes in GABA and oxygenation, which helps in encountering hypoxia. The present study suggests that reduction in the GABA_B receptors functional regulation during hypoxia plays an important role in central nervous system damage. Resuscitation with glucose alone and glucose and oxygen to hypoxic neonatal rats helps in protecting the brain from severe hypoxic damage.     

Constitutive GABAA receptor endocytosis is dynamin-mediated and dependent on a dileucine AP2 adaptin-binding motif within the beta 2 subunit of the receptor

Receptor endocytosis is an important mechanism for regulating the synaptic efficacy of neurotransmitters. There is strong evidence that GABA(A) receptor endocytosis is clathrin-dependent; however, this process is not well understood. Here we demonstrate that in HEK 293 cells, endocytosis of GABA(A) receptors composed of either alpha1beta2gamma2Lor alpha1beta2 subunits is blocked by the dominant negative dynamin construct K44A. Furthermore, we identify a dileucine AP2 adaptin-binding motif within the receptor beta2 subunit that is critical for endocytosis. Internalization of GABAA receptors lacking this motif is dramatically inhibited, and the receptors appear to accumulate on the cell surface. Patch clamp analysis of receptors lacking the dileucine motif show that there is an increase in the peak amplitude of GABA-gated chloride currents compared with wild-type receptors. Additionally, GABA-gated chloride currents in HEK 293 cells expressing wild-type receptors are increased by introduction of a peptide corresponding to the dileucine motif region of the receptor beta2 subunit but not by a control peptide containing alanine substitutions for the dileucine motif. In mouse brain cerebral cortical neurons, the dileucine motif peptide increases GABA-gated chloride currents of native GABA(A) receptors. This is the first report to our knowledge that an AP2 adaptin dileucine recognition motif is critical for the endocytosis of ligand-gated ion channels belonging to this superfamily.     

delta-, but not mu-, opioid receptor stabilizes K(+) homeostasis by reducing Ca(2+) influx in the cortex during acute hypoxia

Past work has shown that delta-opioid receptor (DOR) activation by [D-Ala(2),D-Leu(5)]-enkephalin (DADLE) attenuated the disruption of K(+) homeostasis induced by hypoxia or oxygen-glucose deprivation (OGD) in the cortex, while naltrindole, a DOR antagonist blocked this effect, suggesting that DOR activity stabilizes K(+) homeostasis in the cortex during hypoxic/ischemic stress. However, several important issues remain unclear regarding this new observation, especially the difference between DOR and other opioid receptors in the stabilization of K(+) homeostasis and the underlying mechanism. In this study, we asked whether DOR is different from micro-opioid receptors (MOR) in stabilizing K(+) homeostasis and which membrane channel(s) is critically involved in the DOR effect. The main findings are that (1) similar to DADLE (10 microM), H-Dmt-Tic-NH-CH (CH(2)--COOH)-Bid (1-10 microM), a more specific and potent DOR agonist significantly attenuated anoxic K(+) derangement in cortical slice; (2) [D-Ala(2), N-Me-Phe(4), glycinol(5)]-enkephalin (DAGO; 10 microM), a MOR agonist, did not produce any appreciable change in anoxic disruption of K(+) homeostasis; (3) absence of Ca(2+) greatly attenuated anoxic K(+) derangement; (4) inhibition of Ca(2+)-activated K(+) (BK) channels with paxilline (10 microM) reduced anoxic K(+) derangement; (5) DADLE (10 microM) could not further reduce anoxic K(+) derangement in the Ca(2+)-free perfused slices or in the presence of paxilline; and (6) glybenclamide (20 microM), a K(ATP) channel blocker, decreased anoxia-induced K(+) derangement, but DADLE (10 microM) could further attenuate anoxic K(+) derangement in the glybenclamide-perfused slices. These data suggest that DOR, but not MOR, activation is protective against anoxic K(+) derangement in the cortex, at least partially via an inhibition of hypoxia-induced increase in Ca(2+) entry-BK channel activity.     

Impaired reproduction in transgenic mice overexpressing γ-aminobutyric acid transporter I （GAT1）

It is well documented that 7-aminobutyric acid (GABA) system existed in reproductive organs. Recent researches showed that GABAA and GABAB receptors were present in testis and sperm, and might mediate the acrosome reaction induced by GABA and progesterone. GABA transporter I (GAT1) also existed in testis and sperm, but its physiological function was unknown. In the present study, we used GAT1 overexpressing mice to explore GAT1 function in male reproductive system. We found that the expression level of GAT1 continuously increased in wild-type mouse testis from 1 month to 2 months after birth. GAT1 overexpression in mouse affected testis development, which embodied reduced testis mass and slowed spermatogenesis in transgenic mice. Moreover, transgenic mice showed increase of the percentage of broken sperm. The further study revealed that the reproductive capacity was impaired in GAT1 overexpressing mice. In addition, testosterone level was significantly low in transgenic mice compared with that in wild-type mice. Our findings provided the first evidence that abnormal expression of GAT1 could result in dysgenesis,and indicated that GAT1 might be therapeutically targeted for contraception or dysgenesis treatment.     

An asymmetric contribution to gamma-aminobutyric type A receptor function of a conserved lysine within TM2-3 of alpha1, beta2, and gamma2 subunits

Mutations that impair the expression and/or function of gamma-aminobutyric acid type A (GABAA) receptors can lead to epilepsy. The familial epilepsy gamma2(K289M) mutation affects a basic residue conserved in the TM2-3 linker of most GABAA subunits. We investigated the effect on expression and function of the Lys --> Met mutation in mouse alpha1(K278M), beta2(K274M), and gamma2(K289M) subunits. Compared with cells expressing wild-type and alpha1beta2gamma2(K289M) receptors, cells expressing alpha1(K278M)beta2gamma2 and alpha1beta2(K274M)gamma2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of alpha1beta2(K274M)gamma2 receptors coincided with reduced surface expression. By contrast the surface expression of alpha1(K278M)beta2gamma2 receptors was similar to wild-type and alpha1beta2gamma2(K289M) receptors suggesting that the alpha1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by alpha1(K278M)beta2gamma2 receptors had brief open times. To a lesser extent gamma2(K289M) also reduced mean open time, whereas beta2(K274M) had no effect. We used propofol as an alternative GABAA receptor agonist to test whether the functional deficits of mutant subunits were specific to GABA activation. Propofol was less potent as an activator of alpha1(K278M)beta2gamma2 receptors. By contrast, neither beta2(K274M) nor gamma2(K289M) affected the potency of propofol. The beta2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the alpha1 subunit Lys-278 residue plays a pivotal role in channel gating that is not dependent on occupancy of the GABA binding site. Moreover, the conserved TM2-3 loop lysine has an asymmetric function in different GABAA subunits.     

GABA-stimulated 36Cl- influx into reconstituted vesicles with purified GABAA/benzodiazepine receptor complex

Solubilized and Purified gamma-aminobutyric acid (GABA)A receptors from membrane vesicles of the bovine cerebral cortex were reconstituted into phospholipid vesicles and 36Cl- influx into the vesicles was examined. GABA induced a significant stimulation of the 36Cl- influx into reconstituted vesicles with 1.5% CHAPS/0.15% asolectin solubilized receptor and flunitrazepam further enhanced the GABA-stimulated influx. The purification of GABAA/benzodiazepine receptor complex and Cl- channel solubilized by 1.5% CHAPS/0.15% asolectin from membrane vesicles was achieved by 1012-S affinity column chromatography. The reconstituted vesicles with the purified receptor complex and Cl- channel also exhibited GABA-stimulated 36Cl- influx. This GABA-stimulated influx of 36Cl- was also enhanced by flunitrazepam, while suppressed by bicuculline, a GABAA receptor antagonist. These results strongly suggest that GABAA receptor is directly coupled with Cl- channel, whereas benzodiazepine receptor may be functionally coupled with GABAA receptor and modulates the GABA-stimulated Cl- influx through GABAA receptor. The present results also indicate that the purified GABAA receptor complex is coupled with Cl- channel and possesses functional characteristics as GABAA receptor.     

Decreased GABA<Subscript>A</Subscript> Receptors Functional Regulation in the Cerebral Cortex and Brainstem of Hypoxic Neonatal Rats: Effect of Glucose and Oxygen Supplementation

Hypoxia in neonates can lead to biochemical and molecular alterations mediated through changes in neurotransmitters resulting in permanent damage to brain. In this study, we evaluated the changes in the receptor status of GABA_A in the cerebral cortex and brainstem of hypoxic neonatal rats and hypoxic rats supplemented with glucose and oxygen using binding assays and gene expression of GABA_Aα1 and GABA_Aγ5. In the cerebral cortex and brainstem of hypoxic neonatal rats, a significant decrease in GABA_A receptors was observed, which accounts for the respiratory inhibition. Hypoxic rats supplemented with glucose alone and with glucose and oxygen showed a reversal of the GABA_A receptors, andGABA_Aα1 and GABA_Aγ5 gene expression to control. Glucose acts as an immediate energy source thereby reducing the ATP-depletion-induced increase in GABA and oxygenation, which helps in encountering anoxia. Resuscitation with oxygen alone was less effective in reversing the receptor alterations. Thus, the results of this study suggest that reduction in the GABA_A receptors functional regulation during hypoxia plays an important role in mediating the brain damage. Glucose alone and glucose and oxygen supplementation to hypoxic neonatal rats helps in protecting the brain from severe hypoxic damage.     

Developmental changes in the expression of GABAA receptor subunits alpha1, alpha2, and alpha3 in the rat pre-Botzinger complex.

Previously, we reported that the pre-B?tzinger complex (PBC) exhibited a dramatic reduction in cytochrome oxidase activity at postnatal day (P) 12. This coincided in time with decreases in glutamate and NMDA receptor subunit 1 and increases in GABA, GABAB, glycine receptor, and glutamate receptor GluR2. To test our hypothesis that various alpha-subunits of GABAA receptors also undergo changes in their expression during postnatal development, as they do in other brain regions, we undertook an in-depth immunohistochemical study of GABAA receptor subunits alpha1, alpha2, and alpha3 in the PBC of P0 to P21 rats. We found that 1) GABAA alpha3-subunit was expressed at relatively high levels at P0, which then declined with age; 2) GABAA alpha1-subunit was expressed at relatively low levels at P0 but increased with age; 3) the developmental trends of subunits alpha1 and alpha3 intersected at P12; and 4) GABAA alpha2-subunit expression was moderate to light at P0 and remained quite constant during development, being lowest at P21. These findings suggest that the apparent switch in relative expressions of subunits alpha3 and alpha1 during development and the intersection of slopes around P12 may be associated with possible changes in GABAA receptor subtypes that would mediate different functional properties of GABA transmission, such as primarily a less efficient inhibitory transmission before P12 and a more mature inhibitory effect at P12 and thereafter, as suggested by the kinetics of distinct postsynaptic potentials. This mechanism may contribute partially to the dramatic reduction in cytochrome oxidase activity within the PBC at P12, as shown previously.     

IL-13 receptor alpha2 selectively inhibits IL-13-induced responses in the murine lung

IL-13 is a critical cytokine at sites of Th2 inflammation. In these locations it mediates its effects via a receptor complex, which contains IL-4Ralpha and IL-13Ralpha1. A third, high-affinity IL-13 receptor, IL-13Ralpha2, also exists. Although it was initially felt to be a decoy receptor, this has not been formally demonstrated and the role(s) of this receptor has recently become controversial. To define the role(s) of IL-13Ralpha2 in IL-13-induced pulmonary inflammation and remodeling, we compared the effects of lung-targeted transgenic IL-13 in mice with wild-type and null IL-13Ralpha2 loci. We also investigated the effect of IL-13Ralpha2 deficiency on the OVA-induced inflammatory response. In this study, we show that in the absence of IL-13Ralpha2, IL-13-induced pulmonary inflammation, mucus metaplasia, subepithelial fibrosis, and airway remodeling are significantly augmented. These changes were accompanied by increased expression and production of chemokines, proteases, mucin genes, and TGF-beta1. Similarly, an enhanced inflammatory response was observed in an OVA-induced phenotype. In contrast, disruption of IL-13Ralpha2 had no effect on the tissue effects of lung-targeted transgenic IL-4. Thus, IL-13Ralpha2 is a selective and powerful inhibitor of IL-13-induced inflammatory, remodeling, and physiologic responses in the murine lung.     

The rat beta 1-subunit of the GABAA receptor forms a picrotoxin-sensitive anion channel open in the absence of GABA

The structural basis of GABA-gated chloride channels in mammalian brain is presently explored by the functional expression of cDNAs coding for the alpha, beta or gamma-subunits of the receptor and their isoforms. In this context, we expressed the cloned cDNA coding for the rat beta 1-subunit of the GABAA receptor in the Xenopus oocyte. Surprisingly, efficient expression of a functional ion channel was found. The channel was anion-selective, and able to open in the absence of GABA. Since this channel could be shunt by the GABA-channel blocker picrotoxin, we conclude that the beta 1-subunit of the GABAA receptor is sufficient to form binding sites for picrotoxin.     

Expression of the GABAA receptor/chloride channel in murine spermatogenic cells

Previous studies from our laboratory have demonstrated that γ-aminobutyric acid (GABA) and GABAB receptor subunits are expressed within the acrosome of spermatids during spermiogenesis. Furthermore, our previous study with the glutamate decarboxylase (GAD) 67-GFP knock-in mouse demonstrated that GFP-positive cells were localized to the epithelium of the caput of epididymis. In the present study, we detected GABAA subunits, including α1, α5, β1-3 and γ3, and both isoforms of GAD, GAD65 and GAD67, in mouse spermatogenic cells using RT-PCR. The expression of these proteins was subsequently confirmed by western blot analysis. Immunohistochemistry also revealed that GABA, GAD65, and α5, β1 and γ3 subunits of the GABAA receptor were localized in the membrane of spermatogenic cells, including spermatocytes and spermatids. The whole-cell patch-clamp analysis demonstrated that GABA application induced an inward chloride current in some of the large and round spermatogenic cells. Our findings show that spermatogenic cells have a GABA producing system by themselves, and that GABA may function via the ionotropic GABAA receptor. This data suggests that the GABAergic system may play important roles in the male reproductive system.     

Oxygen-sensitive {delta}-opioid receptor-regulated survival and death signals: novel insights into neuronal preconditioning and protection

The detrimental effect of severe hypoxia (SH) on neurons can be mitigated by hypoxic preconditioning (HPC), but the molecular mechanisms involved remain unclear, and an understanding of these may provide novel solutions for hypoxic/ischemic disorders (e.g. stroke). Here, we show that the delta-opioid receptor (DOR), an oxygen-sensitive membrane protein, mediates the HPC protection through specific signaling pathways. Although SH caused a decrease in DOR expression and neuronal injury, HPC induced an increase in DOR mRNA and protein levels and reversed the reduction in levels of the endogenous DOR peptide, leucine enkephalin, normally seen during SH, thus protecting the neurons from SH insult. The HPC-induced protection could be blocked by DOR antagonists. The DOR-mediated HPC protection depended on an increase in ERK and Bcl 2 activity, which counteracted the SH-induced increase in p38 MAPK activities and cytochrome c release. The cross-talk between ERK and p38 MAPKs displays a "yinyang" antagonism under the control of the DOR-G protein-protein kinase C pathway. Our findings demonstrate a novel mechanism of HPC neuroprotection (i.e. the intracellular up-regulation of DOR-regulated survival signals).     

Hypoxia induces the expression and release of interleukin 1 receptor antagonist in mitogen-activated mononuclear cells

Hypoxia modulates the expression of inflammatory mediators in a variety of cell types. Since interleukin (IL-)1 receptor antagonist (Ra) is a cytokine widely associated with an inflammatory state and is expressed by activated mononuclear cells, we investigated whether hypoxia induces IL-1Ra expression in human peripheral blood mononuclear cells (PBMC) activated by phytohaemagglutinin (PHA). RNase protection assay, conducted on PHA-activated PBMC cultured under hypoxic conditions (2% O(2)) for 16-40 h, revealed that hypoxia enhances IL-1Ra mRNA expression. Further, IL-1Ra release was significantly affected by hypoxia, as determined by ELISA. Concomitantly, hypoxia enhanced, even though at a lesser extent, both IL-1alpha and IL-1beta mRNA expression and release, as determined by RPA and ELISA. However, at 40 h of treatment, hypoxia did not affect cell viability and DNA fragmentation, but caused an inhibition of the proliferation index after PHA stimulation, obtained by MTT assay. These results suggest that activated mononuclear cells tend to respond to hypoxic stress by modulating the expression of IL-1Ra and IL-1-related molecules and their release in the surrounding microenvironment.