Variation in pituitary expression of mRNAs encoding the putative inhibin co-receptor (betaglycan) and type-I and type-II activin receptors during the chicken ovulatory cycle

Secretion of LH and FSH from the anterior pituitary is regulated primarily by hypothalamic GnRH and ovarian steroid hormones. More recent evidence indicates regulatory roles for certain members of the transforming growth factor beta (TGF beta) superfamily including inhibin and activin. The aim of this study was to identify expression of mRNAs encoding key receptors and ligands of the inhibin/activin system in the hen pituitary gland and to monitor their expression throughout the 24-25-h ovulatory cycle. Hens maintained on long days (16 h light/8 h dark) were killed 20, 12, 6 and 2 h before predicted ovulation of a midsequence egg (n = 8 per group). Anterior pituitary glands were removed, RNA extracted and cDNA synthesized. Plasma concentrations of LH, FSH, progesterone and inhibin A were measured. Real-time quantitative PCR was used to quantify pituitary expression of mRNAs encoding betaglycan, activin receptor (ActR) subtypes (type I, IIA), GnRH receptor (GnP,H-R), LH beta subunit, FSH beta subunit and GAPDH. Levels of mRNA for inhibin/activin beta A and beta B subunits, inhibin alpha subunit, follistatin and ActRIIB mRNA in pituitary were undetectable by quantitative PCR (< 2 amol/reaction). Significant changes in expression (P < 0.05) of ActRIIA and betaglycan mRNA were found, both peaking 6 h before ovulation just prior to the preovulatory LH surge and reaching a nadir 2 h before ovulation, just after the LH surge. There were no significant changes in expression of ActRI mRNA throughout the cycle although values were correlated with mRNA levels for both ActRIIA (r=0.77; P < 0.001) and betaglycan (r=0.45; P < 0.01). Expression of GnRH-R mRNA was lowest 20 h before ovulation and highest (P < 0.05) 6 h before ovulation; values were weakly correlated with betaglycan (r=0.33; P=0.06) and ActRIIA (r=0.34; P=0.06) mRNA levels. Expression of mRNAs encoding LH beta and FSH beta subunit were both lowest (P < 0.05) after the LH surge, 2 h before ovulation. These results are consistent with an endocrine, but not a local intrapituitary, role of inhibin-related proteins in modulating gonadotroph function during the ovulatory cycle of the hen, potentially through interaction with betaglycan and ActRIIA. In contrast to mammals, intrapituitary expression of inhibin/activin subunits and follistatin appears to be extremely low or absent in the domestic fowl.

Differential expression of mRNAs encoding co-receptor (betaglycan) and activin type-I preovulatory and prehierarchical follicles of the putative inhibin and type-II receptors in the laying hen ovary

Ovarian follicle development is primarily regulated by an interplay between the pituitary gonadotrophins, LH and FSH, and ovary-derived steroids. Increasing evidence implicates regulatory roles of transforming growth factor-beta (TGF beta) superfamily members, including inhibins and activins. The aim of this study was to identify the expression of mRNAs encoding key receptors of the inhibin/activin system in ovarian follicles ranging from 4 mm in diameter to the dominant F1 follicle (similar to 40 turn). Ovaries were collected (n=16) from inid-sequence hens maintained on a long-day photoschedule (16h of light:8 h of darkness). All follicles removed were dissected into individual granulosa and thecal layers. RNA was extracted and cDNA synthesized. Real-time quantitative PCR was used to quantify the expression of niRNA encoding betaglycan, activin receptor (ActR) subtypes (type-I, -IIA and -IIB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH); receptor expression data were normalized to GAPDH expression. Detectable levels of ActRI, -IIA and -IIB and the inhibin co-receptor (betaglycan) expression were found in all granulosa and thecal layers analysed. Granulosa ActRI mRNA peaked (P < 0(.)05) in 8-9(.)9 mm follicles, whereas ActRIIA rose significantly from 6-7(.)9 mm to 8-9(.)9 nun, before filling to F3/2; levels then rose sharply (3-fold) to F1 levels. Granulosa betaglycan niRNA expression rose 3-fold from 4-5(.)9 min to 8-9(.)9 mm, before falling 4-fold to F3/2; levels then rose sharply (4-fold) to F1 levels. ActRIIB levels did not vary significantly during follicular development. Thecal ActRI mRNA expression was similar from 4-7(.)9 mm then decreased significantly to a nadir at the F4 position, before increasing 2-fold to the F1 (P < 0(.)05). Although thecal ActRIIB and -IIA expression did not vary significantly from 4 nim to F3, ActRIIB expression increased significantly (2-fold) from F3 to F1 and ActIIA, increased 22-fold from F2 to F1 (P < 0(.)05). Thecal betaglycan fell to a nadir at F6 after follicle selection; levels then increased significantly to F2, before filling similar to 50% in the F I. In all follicles studied expression of betaglycan and ActRI (granulosa: 1-0(.)65, P < 0-001, n=144/group; theca: r=0(.)49, P < 0-001, n=144/group) was well correlated. No significant correlations were identified between betaglycan and ActRIIA or -IIB. Considering all follicles analysed, granulosa mRNA expression of betaglycan, ActRI ActRIIA and ActRIIB were all significantly lower than in corresponding thecal tissue (betaglycan, 11(.)4-fold; ActRIIB, 5(.)1-fold; ActR(.) 3-8-fold: ActRIIA, 2(.)8-fold). The co-localization of type-I and -II activin receptors and betaglycan on granulosa and thecal cells are consistent with a local auto/paracrine role of inhibins and activins in modulating ovarian follicle development, selection and progression in the domestic fowl.

Agonist-selective, receptor-specific interaction of human P-2Y receptors with beta-arrestin-1 and-2

Interaction of G-protein-coupled receptors with beta-arrestins is an important step in receptor desensitization and in triggering "alternative" signals. By means of confocal microscopy and fluorescence resonance energy transfer, we have investigated the internalization of the human P2Y receptors 1, 2, 4, 6, 11, and 12 and their interaction with beta-arrestin-1 and -2. Co-transfection of each individual P2Y receptor with beta-arrestin-1-GFP or beta-arrestin-2-YFP into HEK-293 cells and stimulation with the corresponding agonists resulted in a receptor-specific interaction pattern. The P2Y(1) receptor stimulated with ADP strongly translocated beta-arrestin-2-YFP, whereas only a slight translocation was observed for beta-arrestin-1-GFP. The P2Y(4) receptor exhibited equally strong translocation for beta-arrestin-1-GFP and beta-arrestin-2YFP when stimulated with UTP. The P2Y(6), P2Y(11), and P2Y(12) receptor internalized only when GRK2 was additionally cotransfected, but beta-arrestin translocation was only visible for the P2Y(6) and P2Y(11) receptor. The P2Y(2) receptor showed a beta-arrestin translocation pattern that was dependent on the agonist used for stimulation. UTP translocated beta-arrestin-1-GFP and beta-arrestin-2-YFP equally well, whereas ATP translocated beta-arrestin-1-GFP to a much lower extent than beta-arrestin2- YFP. The same agonist-dependent pattern was seen in fluorescence resonance energy transfer experiments between the fluorescently labeled P2Y(2) receptor and beta-arrestins. Thus, the P2Y(2) receptor would be classified as a class A receptor when stimulated with ATP or as a class B receptor when stimulated with UTP. The ligand-specific recruitment of beta-arrestins by ATP and UTP stimulation of P2Y(2) receptors was further found to result in differential stimulation of ERK phosphorylation. This suggests that the two different agonists induce distinct active states of this receptor that show differential interactions with beta-arrestins.

Agonist-dependent internalization of D2 receptors: Imaging quantification by confocal microscopy

In positron emission tomography and single photon emission computed tomography studies using D2 dopamine (DA) receptor radiotracers, a decrease in radiotracer binding potential (BP) is usually interpreted in terms of increased competition with synaptic DA. However, some data suggest that this signal may also reflect agonist (DA)-induced increases in D2 receptor (D2R) internalization, a process which would presumably also decrease the population of receptors available for binding to hydrophilic radioligands. To advance interpretation of alterations in D2 radiotracer BP, direct methods of assessment of D2R internalization are required. Here, we describe a confocal microscopy-based approach for the quantification of agonist-dependent receptor internalization. The method relies upon double-labeling of the receptors with antibodies directed against intracellular as well as extracellular epitopes. Following agonist stimulation, DA D2R internalization was quantified by differentiating, in optical cell sections, the signal due to the staining of the extracellular from intracellular epitopes of D2Rs. Receptor internalization was increased in the presence of the D2 agonists DA and bromocriptine, but not the D1 agonist SKF38393. Pretreatment with either the D2 antagonist sulpiride, or inhibitors of internalization (phenylarsine oxide and high molarity sucrose), blocked D2-agonist induced receptor internalization, thus validating this method in vitro. This approach therefore provides a direct and streamlined methodology for investigating the pharmacological and mechanistic aspects of D2R internalization, and should inform the interpretation of results from in vivo receptor imaging studies.

Changes in expression of bone morphogenetic proteins (BMPs), their receptors and inhibin co-receptor betaglycan during bovine antral follicle development: inhibin can antagonize the suppressive effect of BMPs on thecal androgen production

We reported previously that bone morphogenetic proteins (BMPs) potently suppress CYP17 expression and androgen production by bovine theca interna cells (TC) in vitro. In this study, real-time PCR was used to analyse gene expression in TC and granulosa cell (GC) layers from developing bovine antral follicles (1-18 mm). Abundance of mRNA transcripts for four BMPs (BMP2, BMP4, BMP6, and BMP7) and associated type I (BMPR1A, BMPR1B, ACVR1 and ACVR1B) and type II (BMPR2, ACVR2A and ACVR2B) receptors showed relatively modest, though significant, changes during follicle development. BMP2 was selectively expressed in GC, while BMP6, BMP7 and betaglycan (TGFBR3) were more abundant in TC. Abundance of betaglycan mRNA (inhibin co-receptor) in TC increased progressively (fivefold; P<0.001) as follicles grew from 1-2 to 9-10 mm. This suggests a shift in thecal responsiveness to GC-derived inhibin, produced in increasing amounts as follicles achieve dominance. This prompted us to investigate whether inhibin can function as a physiological antagonist of BMP action on bovine TC in vitro, in a manner comparable to that for activin signalling. BMP4, BMP6 and BMP7 abolished LH-induced androstenedione secretion and suppressed CYP17 mRNA >200-fold (P<0.001), while co-treatment with inhibin-A reversed the suppressive action of BMP in each case (P<0.001). Results support a physiological role for granulosa-derived inhibin as an antagonist of BMP action on thecal androgen synthesis. A shift in intrafollicular balance between thecal BMP signalling (inhibitory for androgen synthesis) and betaglycan-dependent inhibin signalling (stimulatory for androgen synthesis) accords with the physiological requirement to deliver an adequate supply of aromatase substrate to GC of developing follicles.

Dimetallic complexes of macrocycles with two rigid dibenzofuran units as receptors for detection of anionic substrates

The hexaazamacrocycles [28](DBF)2N6 {cyclo[bis(4,6-dimethyldibenzo[b,d]furaniminoethyleneiminoethylene]} and [32](DBF)2N6 {cyclo[bis(4,6-dimethyldibenzo[b,d]furaniminopropyleneiminopropylene]} form stable dinuclear copper(II) complexes suitable to behave as receptors for several anionic substrates. These two receptors were used to study the binding interactions with several substrates, such as imidazole (Him) and some carboxylates [benzoate (bz−), oxalate (ox2−), malonate (mal2−), phthalate (ph2−), isophthalate (iph2−), and terephthalate (tph2−)] by spectrophotometric titrations and EPR spectroscopy in MeOH (or H2O):DMSO (1:1 v/v) solution. The largest association constant was found for ox2− with Cu2[32](DBF)2N64+, whereas for the aromatic dicarboxylate anions the binding constants follow the trend ph2− > iph2− > tph2−, i.e. decrease with the increase of the distance of the two binding sites of the substrate. On the other hand, the large blue shift of 68 nm observed by addition of Him to Cu2[32](DBF)2N64+ points out for the formation of the bridged CuimCu cascade complex, indicating this receptor as a potential sensor for the detection and determination of imidazole in solution. The X-band EPR spectra of the Cu2[28](DBF)2N64+ and Cu2[32](DBF)2N6]4+ complexes and the cascade complexes with the substrates, performed in H2O:DMSO (1:1 v/v) at 5 to 15 K, showed that the CuCu distance is slightly larger than the one found in crystal state and that this distance increases when the substrate is accommodated between the two copper centres. The crystal structure of [Cu2[28](DBF)2N6(ph)2]·CH3OH was determined by X-ray diffraction and revealed the two copper centres bridged by two ph2− anions at a Cu···Cu distance of 5.419(1) Å. Each copper centre is surrounded by three carboxylate oxygen atoms from two phthalate anions and three contiguous nitrogen atoms of the macrocycle in a pseudo octahedral coordination environment.

Nitric oxide suppresses cerebral vasomotion by sGC-independent effects on ryanodine receptors and voltage-gated calcium channels

Background/Aims: In cerebral arteries, nitric oxide (NO) release plays a key role in suppressing vasomotion. Our aim was to establish the pathways affected by NO in rat middle cerebral arteries. Methods: In isolated segments of artery, isometric tension and simultaneous measurements of either smooth muscle membrane potential or intracellular [Ca 2+ ] ([Ca 2+ ] SMC ) changes were recorded. Results: In the absence of L -NAME, asynchronous propagating Ca 2+ waves were recorded that were sensitive to block with ryanodine, but not nifedipine. L -NAME stimulated pronounced vasomotion and synchronous Ca 2+ oscillations with close temporal coupling between membrane potential, tone and [Ca 2+ ] SMC . If nifedipine was applied together with L -NAME, [Ca 2+ ] SMC decreased and synchronous Ca 2+ oscillations were lost, but asynchronous propagating Ca 2+ waves persisted. Vasomotion was similarly evoked by either iberiotoxin, or by ryanodine, and to a lesser extent by ODQ. Exogenous application of NONOate stimulated endothelium-independent hyperpolarization and relaxation of either L -NAME-induced or spontaneous arterial tone. NO-evoked hyperpolarization involved activation of BK Ca channels via ryanodine receptors (RYRs), with little involvement of sGC. Further, in whole cell mode, NO inhibited current through L-type voltage-gated Ca 2+ channels (VGCC), which was independent of both voltage and sGC. Conclusion: NO exerts sGC-independent actions at RYRs and at VGCC, both of which normally suppress cerebral artery myogenic tone.

Oxygen/Glucose deprivation induces a reduction in synaptic AMPA receptors on hippocampal CA3 neurons mediated by mGluR1 and adenosine A3 receptors

Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca2+, resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 min OGD protocol, a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by metabotropic glutamate receptor 1 (mGluR1) or A3 receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC, or chelation of intracellular Ca2+ also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalization of AMPARs after OGD. We also show that a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A3 receptor antagonists, indicating that AMPARs are degraded following internalization. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotection

The role of myeloid receptors on murine plasmacytoid dendritic cells in induction of type I interferon

This study tested the hypothesis that a set of predominantly myeloid restricted receptors (F4/80, CD36, Dectin-1, CD200 receptor and mannan binding lectins) and the broadly expressed CD200 played a role in a key function of plasmacytoid DC (pDC), virally induced type I interferon (IFN) production. The Dectin-1 ligands zymosan, glucan phosphate and the anti-Dectin-1 monoclonal antibody (mAb) 2A11 had no effect on influenza virus induced IFNα/β production by murine splenic pDC. However, mannan, a broad blocking reagent against mannose specific receptors, inhibited IFNα/β production by pDC in response to inactivated influenza virus. Moreover, viral glycoproteins (influenza virus haemagglutinin and HIV-1 gp120) stimulated IFNα/β production by splenocytes in a mannan-inhibitable manner, implicating the function of a lectin in glycoprotein induced IFN production. Lastly, the effect of CD200 on IFN induction was investigated. CD200 knock-out macrophages produced more IFNα than wild-type macrophages in response to polyI:C, a MyD88-independent stimulus, consistent with CD200's known inhibitory effect on myeloid cells. In contrast, blocking CD200 with an anti-CD200 mAb resulted in reduced IFNα production by pDC-containing splenocytes in response to CpG and influenza virus (MyD88-dependent stimuli). This suggests there could be a differential effect of CD200 on MyD88 dependent and independent IFN induction pathways in pDC and macrophages. This study supports the hypothesis that a mannan-inhibitable lectin and CD200 are involved in virally induced type I IFN induction.

Affinity purification of 61- and 65-kDa rat brain corticotropin-releasing factor receptors and receptor-associated G proteins.

Corticotropin-releasing factor (CRF) has been shown to have a central role in physiological adaptation to stress. It is recognized for stimulating the release of adrenocorticotropin from the anterior pituitary gland, and has more recently been implicated as a regulator of autonomic and immunological responses to stress. Much confusion has surrounded the characterization of CRF receptors, with proteins of varying molecular weights having been identified but never purified and characterized. Recently, two CRF receptors have been cloned from brain and pituitary gland, but evidence from in-situ hybridization studies suggests that further CRF receptor types exist. We therefore developed two techniques which enable the isolation of CRF receptors from whole rat brain. The use of a solid-phase CRF analogue affinity column and elution using a competing ligand resulted in the purification of a single protein of 61 kDa. A second technique was devised which allowed the co-isolation of associated signalling proteins and the identification of CRF bound species following purification. CRF was covalently cross-linked to receptors and the complex purified using antibodies specific for the ligand. This enabled the purification of a CRF receptor of approximately 65 kDa and associated alpha and beta gamma G protein subunits. This study demonstrates the successful isolation of CRF receptors which are of different molecular weights to those previously observed from affinity cross-linking studies or predicted from cloned genes. In addition, we confirm the involvement of G proteins in CRF stimulated cell signalling by demonstrating their association with purified CRF receptor.

Trafficking and signaling of G protein-coupled receptors in the nervous system: implications for disease and therapy

G protein-coupled receptors (GPCRs) are expressed throughout the nervous system where they regulate multiple physiological processes, participate in neurological diseases, and are major targets for therapy. Given that many GPCRs respond to neurotransmitters and hormones that are present in the extracellular fluid and which do not readily cross the plasma membrane, receptor trafficking to and from the plasma membrane is a critically important determinant of cellular responsiveness. Moreover, trafficking of GPCRs throughout the endosomal system can initiate signaling events that are mechanistically and functionally distinct from those operating at the plasma membrane. This review discusses recent advances in the relationship between signaling and trafficking of GPCRs in the nervous system. It summarizes how receptor modifications influence trafficking, discusses mechanisms that regulate GPCR trafficking to and from the plasma membrane, reviews the relationship between trafficking and signaling, and considers the implications of GPCR trafficking to drug development.

Trypsin IV or mesotrypsin and p23 cleave protease-activated receptors 1 and 2 to induce inflammation and hyperalgesia

Although principally produced by the pancreas to degrade dietary proteins in the intestine, trypsins are also expressed in the nervous system and in epithelial tissues, where they have diverse actions that could be mediated by protease-activated receptors (PARs). We examined the biological actions of human trypsin IV (or mesotrypsin) and rat p23, inhibitor-resistant forms of trypsin. The zymogens trypsinogen IV and pro-p23 were expressed in Escherichia coli and purified to apparent homogeneity. Enteropeptidase cleaved both zymogens, liberating active trypsin IV and p23, which were resistant to soybean trypsin inhibitor and aprotinin. Trypsin IV cleaved N-terminal fragments of PAR(1), PAR(2), and PAR(4) at sites that would expose the tethered ligand (PAR(1) = PAR(4) > PAR(2)). Trypsin IV increased [Ca(2+)](i) in transfected cells expressing human PAR(1) and PAR(2) with similar potencies (PAR(1), 0.5 microm; PAR(2), 0.6 microm). p23 also cleaved fragments of PAR(1) and PAR(2) and signaled to cells expressing these receptors. Trypsin IV and p23 increased [Ca(2+)](i) in rat dorsal root ganglion neurons that responded to capsaicin and which thus mediate neurogenic inflammation and nociception. Intraplantar injection of trypsin IV and p23 in mice induced edema and granulocyte infiltration, which were not observed in PAR (-/-)(1)(trypsin IV) and PAR (-/-)(2) (trypsin IV and p23) mice. Trypsin IV and p23 caused thermal hyperalgesia and mechanical allodynia and hyperalgesia in mice, and these effects were absent in PAR (-/-)(2) mice but maintained in PAR (-/-)(1) mice. Thus, trypsin IV and p23 are inhibitor-resistant trypsins that can cleave and activate PARs, causing PAR(1)- and PAR(2)-dependent inflammation and PAR(2)-dependent hyperalgesia.

Agonists of protease-activated receptors 1 and 2 stimulate electrolyte secretion from mouse gallbladder

Cholecystitis is one of the most common gastrointestinal diseases. Inflammation induces the activation of proteases that can signal to cells by cleaving protease-activated receptors (PARs) to induce hemostasis, inflammation, pain, and repair. However, the distribution of PARs in the gallbladder is unknown, and their effects on gallbladder function have not been fully investigated. We localized immunoreactive PAR(1) and PAR(2) to the epithelium, muscle, and serosa of mouse gallbladder. mRNA transcripts corresponding to PAR(1) and PAR(2), but not PAR(4), were detected by RT-PCR and sequencing. Addition of thrombin and a PAR(1)-selective activating peptide (TFLLRN-NH(2)) to the serosal surface of mouse gallbladder mounted in an Ussing chamber stimulated an increase in short-circuit current in wild-type but not PAR(1) knockout mice. Similarly, serosally applied trypsin and PAR(2) activating peptide (SLIGRL-NH(2)) increased short-circuit current in wild-type but not PAR(2) knockout mice. Proteases and activating peptides strongly inhibited electrogenic responses to subsequent stimulation with the same agonist, indicating homologous desensitization. Removal of HCO(3)(-) ions from the serosal buffer reduced responses to thrombin and trypsin by >80%. Agonists of PAR(1) and PAR(2) increase intracellular Ca(2+) concentration in isolated and cultured gallbladder epithelial cells. The COX-2 inhibitor meloxicam and an inhibitor of CFTR prevented the stimulatory effect of PAR(1) but not PAR(2). Thus PAR(1) and PAR(2) are expressed in the epithelium of the mouse gallbladder, and serosally applied proteases cause a HCO(3)(-) secretion. The effects of PAR(1) but not PAR(2) depend on generation of prostaglandins and activation of CFTR. These mechanisms may markedly influence fluid and electrolyte secretion of the inflamed gallbladder when multiple proteases are generated.