Developmental changes in presynaptic muscarinic modulation of excitatory and inhibitory neurotransmission in rat piriform cortex in vitro: relevance to epileptiform bursting susceptibility

Suppression of depolarizing postsynaptic potentials and isolated GABA-A receptor-mediated fast inhibitory postsynaptic potentials by the muscarinic acetylcholine receptor agonist, oxotremorine-M (10 microM), was investigated in adult and immature (P14-P30) rat piriform cortical (PC) slices using intracellular recording. Depolarizing postsynaptic potentials evoked by layers II-III stimulation underwent concentration-dependent inhibition in oxotremorine-M that was most likely presynaptic and M2 muscarinic acetylcholine receptor-mediated in immature, but M1-mediated in adult (P40-P80) slices; percentage inhibition was smaller in immature than in adult piriform cortex. In contrast, compared with adults, layer Ia-evoked depolarizing postsynaptic potentials in immature piriform cortex slices in oxotremorine-M, showed a prolonged multiphasic depolarization with superimposed fast transients and spikes, and an increased 'all-or-nothing' character. Isolated N-methyl-d-aspartate receptor-mediated layer Ia depolarizing postsynaptic potentials (although significantly larger in immature slices) were however, unaffected by oxotremorine-M, but blocked by dl-2-amino-5-phosphonovaleric acid. Fast inhibitory postsynaptic potentials evoked by layer Ib or layers II-III-fiber stimulation in immature slices were significantly smaller than in adults, despite similar estimated mean reversal potentials ( approximately -69 and -70 mV respectively). In oxotremorine-M, only layer Ib-fast inhibitory postsynaptic potentials were suppressed; suppression was again most likely presynaptic M2-mediated in immature slices, but M1-mediated in adults. The degree of fast inhibitory postsynaptic potential suppression was however, greater in immature than in adult piriform cortex. Our results demonstrate some important physiological and pharmacological differences between excitatory and inhibitory synaptic systems in adult and immature piriform cortex that could contribute toward the increased susceptibility of this region to muscarinic agonist-induced epileptiform activity in immature brain slices.

Further characterization of muscarinic agonist-induced epileptiform bursting activity in immature rat piriform cortex, in vitro

The characteristics of muscarinic acetylcholine receptor agonist-induced epileptiform bursting seen in immature rat piriform cortex slices in vitro were further investigated using intracellular recording, with particular focus on its postnatal age-dependence (P+14-P+30), pharmacology, site(s) of origin and the likely contribution of the muscarinic acetylcholine receptor agonist-induced post-stimulus slow afterdepolarization and gap junction functionality toward its generation. The muscarinic agonist, oxotremorine-M (10 microM), induced rhythmic bursting only in immature piriform cortex slices; however, paroxysmal depolarizing shift amplitude, burst duration and burst incidence were inversely related to postnatal age. No significant age-dependent changes in neuronal membrane properties or postsynaptic muscarinic responsiveness accounted for this decline. Burst incidence was higher when recorded in anterior and posterior regions of the immature piriform cortex. In adult and immature neurones, oxotremorine-M effects were abolished by M1-, but not M2-muscarinic acetylcholine receptor-selective antagonists. Rostrocaudal lesions, between piriform cortex layers I and II, or layer III and endopiriform nucleus in adult or immature slices did not influence oxotremorine-M effects; however, the slow afterdepolarization in adult (but not immature) lesioned slices was abolished. Gap junction blockers (carbenoxolone or octanol) disrupted muscarinic bursting and diminished the slow afterdepolarization in immature slices, suggesting that gap junction connectivity was important for bursting. Our data show that neural networks within layers II-III function as primary oscillatory circuits for burst initiation in immature rat piriform cortex during persistent muscarinic receptor activation. Furthermore, we propose that muscarinic slow afterdepolarization induction and gap junction communication could contribute towards the increased epileptiform susceptibility of this brain area.

A novel component of cannabis extract potentiates excitatory synaptic transmission in rat olfactory cortex in vitro

Cannabis is a potential treatment for epilepsy, although the few human studies supporting this use have proved inconclusive. Previously, we showed that a standardized cannabis extract (SCE), isolated Delta(9)-tetrahydrocannabinol (Delta(9)-THC), and even Delta(9)-THC-free SCE inhibited muscarinic agonist-induced epileptiform bursting in rat olfactory cortical brain slices, acting via CB1 receptors. The present work demonstrates that although Delta(9)-THC (1microM) significantly depressed evoked depolarizing postsynaptic potentials (PSPs) in rat olfactory cortex neurones, both SCE and Delta(9)-THC-free SCE significantly potentiated evoked PSPs (all results were fully reversed by the CB1 receptor antagonist SR141716A, 1microM); interestingly, the potentiation by Delta(9)-THC-free SCE was greater than that produced by SCE. On comparing the effects of Delta(9)-THC-free SCE upon evoked PSPs and artificial PSPs (aPSPs; evoked electrotonically following brief intracellular current injection), PSPs were enhanced, whereas aPSPs were unaffected, suggesting that the effect was not due to changes in background input resistance. Similar recordings made using CB1 receptor-deficient knockout mice (CB1(-/-)) and wild-type littermate controls revealed cannabinoid or extract-induced changes in membrane resistance, cell excitability and synaptic transmission in wild-type mice that were similar to those seen in rat neurones, but no effect on these properties were seen in CB1(-/-) cells. It appears that the unknown extract constituent(s) effects over-rode the suppressive effects of Delta(9)-THC on excitatory neurotransmitter release, which may explain some patients' preference for herbal cannabis rather than isolated Delta(9)-THC (due to attenuation of some of the central Delta(9)-THC side effects) and possibly account for the rare incidence of seizures in some individuals taking cannabis recreationally

Nerve terminal GABAA receptors activate Ca2+/calmodulin-dependent signaling to inhibit voltage-gated Ca2+ influx and glutamate release

-Aminobutyric acid type A (GABAA) receptors, a family of Cl-permeable ion channels, mediate fast synaptic inhibition as postsynaptically enriched receptors for -aminobutyric acid at GABAergic synapses. Here we describe an alternative type of inhibition mediated byGABAA receptors present on neocortical glutamatergic nerve terminals and examine the underlying signaling mechanism(s). By monitoring the activity of the presynaptic CaM kinase II/synapsin I signaling pathway in isolated nerve terminals, we demonstrate that GABAA receptor activation correlated with an increase in basal intraterminal [Ca2]i. Interestingly, this activation of GABAA receptors resulted in a reduction of subsequent depolarization-evoked Ca2 influx, which thereby led to an inhibition of glutamate release. To investigate how the observed GABAA receptor-mediated modulation operates, we determined the sensitivity of this process to the Na-K-2Cl cotransporter 1 antagonist bumetanide, as well as substitution of Ca2 with Ba2, or Ca2/calmodulin inhibition by W7. All of these treatments abolished the modulation by GABAA receptors. Application of selective antagonists of voltage-gated Ca2 channels (VGCCs) revealed that the GABAA receptor-mediated modulation of glutamate release required the specific activity of L- and R-type VGCCs. Crucially, the inhibition of release by these receptors was abolished in terminals isolated from R-type VGCC knock-out mice. Together, our results indicate that a functional coupling between nerve terminal GABAA receptors and L- or R-type VGCCs is mediated by Ca2/calmodulin-dependent signaling. This mechanism provides a GABA-mediated control of glutamatergic synaptic activity by a direct inhibition of glutamate release.

Bone morphogenetic protein-6 enhances gonadotrophin-dependent progesterone and inhibin secretion and expression of rnRNA transcripts encoding gonadotrophin receptors and inhibin/activin subunits in chicken granulosa cells

The aims were to examine ovarian expression of bone morphogenetic protein (BMP) ligands/receptor mRNAs in the chicken and to test the hypothesis that theca-derived BMP(s) modulates granulosa cell function in a paracrine manner. RT-PCR revealed expression of multiple BMPs in granulosa and theca cells from prehierarchical and preovulatory follicles with greater expression in theca cells; both cell types expressed BMP receptors-1A, -1B and -II consistent with tissue responsiveness. Preovulatory granulosa cells F1, F2 and F3/4) were cultured with BMP-6 (expressed by theca but not granulosa) in the presence/absence of LH, FSH or 8-Br-cAMP. RMP-6 increased 'basal' and gonadotrophin-induced inhibin-A and progesterone secretion by each cell type but did not enhance the effect of 8-Br-cAMP. This indicates that the observed synergism between BMP-6 and gonadotrophin might involve BMP-induced up-regulation of gonadotrophin receptors. In support of this, BMP-6 alone increased LH-receptor (LHR) mRNA in F1 cells and FSH-receptor (FSHR) mRNA in F1, F2 and F3/4 cells. RMP-6 also enhanced LH/FSH-induced LHR transcript amount in each cell type but did not raise FSHR transcript amounts above those induced by BMP-6 alone. To further explore BMP6 action on inhibin-A secretion, we quantified inhibin/activin subunits (alpha, beta(A), beta(B)) mRNAs. Consistent with its effect on inhibin-A secretion, BMP-6 enhanced 'basal' expression of alpha- and beta(A)-Subunit mRNA in F1, F2 and F3/4 cells, and beta(B)-subunit mRNA in F3/4 cells. BMP-6 markedly enhanced FSH/LH-induced expression of alpha-subunit in all follicles and FSH-induced beta(A)-subunit in F2 and F3/4 follicles but not in F1 follicles. Neither BMP-6 alone, nor FSH/LH alone, affected 'basal' OB mRNA abundance. However, co-treatment with gonadotrophin and BMP-6 greatly increased beta(B)-subunit expression, the response being lowest in F1 follicles and greatest in F3/4 follicles. Collectively, these results support the hypothesis that intra-ovarian OMPs of thecal origin have a paracrine role in modulating granulosa cell function in the chicken in a preovulatory stage-dependent manner.

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.

Flavonoids inhibit the platelet TxA(2) signalling pathway and antagonize TxA(2) receptors (TP) in platelets and smooth muscle cells

What is already known about this subject center dot Flavonoids are largely recognized as potential inhibitors of platelet function, through nonspecific mechanisms such as antioxidant activity and/or inhibition of several enzymes and signalling proteins. center dot In addition, we, and few others, have shown that certain antiaggregant flavonoids may behave as specific TXA2 receptor (TP) ligands in platelets. center dot Whether flavonoids interact with TP isoforms in other cell types is not known, and direct evidence that flavonoid-TP interaction inhibits signalling downstream TP has not been shown. What this study adds center dot This study first demonstrates that certain flavonoids behave as ligands for both TP isoforms, not only in platelets, but also in human myometrium and in TP-transfected HEK 293T cells. center dot Differences in the effect of certain flavonoids in platelet signalling, induced by either U46619 or thrombin, suggest that abrogation of downstream TP signalling is related to their specific blockage of the TP, rather than to a nonspecific effect on tyrosine kinases or other signalling proteins. Flavonoids may affect platelet function by several mechanisms, including antagonism of TxA(2) receptors (TP). These TP are present in many tissues and modulate different signalling cascades. We explored whether flavonoids affect platelet TP signalling, and if they bind to TP expressed in other cell types. Platelets were treated with flavonoids, or other selected inhibitors, and then stimulated with U46619. Similar assays were performed in aspirinized platelets activated with thrombin. Effects on calcium release were analysed by fluorometry and changes in whole protein tyrosine phosphorylation and activation of ERK 1/2 by Western blot analysis. The binding of flavonoids to TP in platelets, human myometrium and TP alpha- and TP beta-transfected HEK 293T cells was explored using binding assays and the TP antagonist H-3-SQ29548. Apigenin, genistein, luteolin and quercetin impaired U46619-induced calcium mobilization in a concentration-dependent manner (IC50 10-30 mu M). These flavonoids caused a significant impairment of U46619-induced platelet tyrosine phosphorylation and of ERK 1/2 activation. By contrast, in aspirin-treated platelets all these flavonoids, except quercetin, displayed minor effects on thrombin-induced calcium mobilization, ERK 1/2 and total tyrosine phosphorylation. Finally, apigenin, genistein and luteolin inhibited by > 50% H-3-SQ29548 binding to different cell types. These data further suggest that flavonoids may inhibit platelet function by binding to TP and by subsequent abrogation of downstream signalling. Binding of these compounds to TP occurs in human myometrium and in TP-transfected HEK 293T cells and suggests that antagonism of TP might mediate the effects of flavonoids in different tissues.

Gonadotrophins modulate hormone secretion and steady-state mRNA levels for activin receptors (type I, IIA, IIB) and inhibin co-receptor (betaglycan) in granulosa and theca cells from chicken prehierarchical and preovulatory follicles

Ovarian follicle development is regulated through endocrine and local mechanisms. Increasing evidence indicates roles for transforming growth factor beta superfamily members, including inhibins and activins. We recently identified divergent expression of mRNAs encoding activin receptors (ActR) and inhibin co-receptor betaglycan in chicken follicles at different stages of maturation. Here, we compare the actions of LH and FSH (0, 1, 10, 100 ng/ml) on levels of mRNA for ActRI, ActRIIA, ActRIIB and betaglycan in chicken granulosa and theca cells (GC and TC) from preovulatory (F1) and prehierarchical (6-8 mm) follicles. The expression of mRNAs for LH-R and FSH-R and production of inhibin A, oestradiol and progesterone were also quantified. FSH decreased ActRIIB and ActRI mRNA levels in 6-8 mm GC, whereas LH increased the mRNA levels. Both LH and FSH enhanced ActRIIA (5- and 8.5-fold) and betaglycan mRNA expression (2- and 3.5-fold) in 6-8 mm GC. In 6-8 mm TC, LH and FSH both increased the betaglycan mRNA level (7- and 3.5-fold respectively) but did not affect ActRI, ActRIIA and ActRIIB transcript levels. In F1 GC, both LH and FSH stimulated ActRI (2- and 2.4-fold), ActRIIB (3.2- and 2.7-fold) and betaglycan (7- and 4-fold) mRNA levels, while ActRIIA mRNA was unaffected. In F1 TC, LH and FSH reduced ActRIIA (35-50%) and increased (4.5- and 7.6-fold) betaglycan mRNA, but had no effect on ActRI and ActRIIB transcript levels. Results support the hypothesis that expression of ActR and betaglycan are differentially regulated by gonadotrophins during follicle maturation in the hen. This may represent an important mechanism for fine-tuning follicle responsiveness to local and systemic activins and inhibins.

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.

Inhibition of coxsackie B virus infection by soluble forms of its receptors: Binding affinities, altered particle formation, and competition with cellular receptors

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus 133 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.

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.

Functional and molecular characterization of Tachykinins and tachykinin receptors in the mouse uterus

The aim of this study was to analyze the function and expression of tachykinins, tachykinin receptors, and neprilysin (NEP) in the mouse uterus. A previous study showed that the uterotonic effects of substance P (SP), neurokinin A (NKA), and neurokinin B (NKB) in estrogen-treated mice were mainly mediated by the tachykinin NK, receptor. In the present work, further contractility studies were undertaken to determine the nature of the receptors mediating responses to tachykinins in uteri of late pregnant mice. Endpoint and real-time quantitative RTPCR were used to analyze the expression of the genes that encode the tachykinins SP/NKA, NKB, and hemokinin-1 (HK-1) (Tac1, Tac2, and Tac4); and the genes that encode tachykinin NK1 (Tacr1), NK2 (Tacr2), and NK3 (Tacr3) receptors in uteri from pregnant and nonpregnant mice. The data show that the mRNAs of tachykinins (particularly NKB and HK-1), tachykinin receptors, and NEP are locally expressed in the mouse uterus, and their expression changes during the estrous cycle and during pregnancy. The tachykinin INK, receptor is the predominant tachykinin receptor in the nonpregnant and early pregnant mouse and may mediate tachykinin-induced uterine contractions in the nonpregnant mouse. The tachykinin NK, receptor is predominant in the late pregnant mouse and is the main receptor mediating uterotonic responses to tachykinins at late pregnancy. The tachykinin NK, receptor is expressed in considerable amounts only in uteri from nonpregnant diestrous animals, and its physiological significance remains to be clarified.

Mechanisms of inverse agonist action at D-2 dopamine receptors

1 Mechanisms of inverse agonist action at the D-2(short) dopamine receptor have been examined. 2 Discrimination of G-protein-coupled and -uncoupled forms of the receptor by inverse agonists was examined in competition ligand-binding studies versus the agonist [H-3]NPA at a concentration labelling both G-protein-coupled and -uncoupled receptors. 3 Competition of inverse agonists versus [H-3] NPA gave data that were fitted best by a two-binding site model in the absence of GTP but by a one-binding site model in the presence of GTP. K-i values were derived from the competition data for binding of the inverse agonists to G-protein-uncoupled and -coupled receptors. K-coupled and K-uncoupled were statistically different for the set of compounds tested ( ANOVA) but the individual values were different in a post hoc test only for (+)-butaclamol. 4 These observations were supported by simulations of these competition experiments according to the extended ternary complex model. 5 Inverse agonist efficacy of the ligands was assessed from their ability to reduce agonist-independent [S-35]GTPγ S binding to varying degrees in concentration-response curves. Inverse agonism by (+)-butaclamol and spiperone occurred at higher potency when GDP was added to assays, whereas the potency of (-)-sulpiride was unaffected. 6 These data show that some inverse agonists ((+)-butaclamol, spiperone) achieve inverse agonism by stabilising the uncoupled form of the receptor at the expense of the coupled form. For other compounds tested, we were unable to define the mechanism.

Oligomers of D-2 dopamine receptors - Evidence from ligand binding

There is increasing evidence that G protein-coupled receptors form oligomers and that this might be important for their function. We have studied this phenomenon for the D-2 dopamine receptor and have shown-using a variety of biochemical and biophysical techniques-that this receptor forms dimers or higher-order oligomers. Using ligand-binding studies, we have also found evidence that this oligomer formation has functional relevance. Thus, for the receptor expressed in either CHO cells or Sf 9 insect cells, the binding properties of several radioligands (in saturation, competition, and dissociation assays) do not conform to those expected for a monomeric receptor with a single binding site. We propose that the receptors exist in oligomers with homotropic and heterotropic negatively cooperative interactions between ligands.

Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4,-6 and-7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin

Given the paucity of information on the potential roles of bone morphogenetic proteins (BMPs) in the ruminant ovary we conducted immunolocalization and functional studies on cells isolated from bovine antral follicles. Immunocytochemistry revealed expression of BMP-4 and -7 in isolated theca cells whereas granulosa cells and oocytes selectively expressed RMP-6. All three cell types expressed a range of BMP-responsive type-I (BMPRIB, ActRI) and type-II (BMPRII, ActRII, ActRIIB) receptors supporting autocrine/paracrine roles within the follicle. This was reinforced by functional experiments on granulosa cells which showed that BMP-4, -6 and -7 promoted cellular accumulation of phosphorylated Smad-1 but not Smad-2 and enhanced 'basal' and IGF-stimulated secretion of oestradiol (E2), inhibin-A, activin-A and follistatin (FS). Concomitantly, each BMP suppressed 'basal' and IGF-stimulated progesterone secretion, consistent with an action to prevent or delay atresia and/or luteinization. BMPs also increased viable cell number under 'basal' (BMP-4 and -7) and IGF-stimulated (BMP-4, -6 and -7) conditions. Since FS, a product of bovine granulosa cells, has been shown to bind several BMPs, we used the Biacore technique to compare its binding affinities for activin-A (prototype FS ligand) and BMP-4, -6 and -7. Compared with activin-A (K-d 0.28 +/- 0.02 nM; 100%), the relative affinities of FS for BMP-4, -6 and -7 were 10, 5 and 1% respectively. Moreover, studies on granulosa cells showed that preincubation of ligand with excess FS abolished activin-A-induced phosphorylation of Smad-2 and BMP-4-induced phosphorylation of Smad-1. However, FS only partially reversed BMP-6-induced Smad-1 phosphorylation and had no inhibitory effect on BMP-7-induced Smad-1 phosphorylation. These findings support functional roles for BMP-4, -6 and -7 as paracrine/autocrine modulators of granulosa cell steroidogenesis, peptide secretion and proliferation in bovine antral follicles. The finding that FS can differentially modulate BMP-induced receptor activation and that this correlates with the relative binding affinity of FS for each BMP type implicates FS as a potential modulator of BMP action in the ovary.