Affinity Component Research Articles

Degradation of Phospholipid Transfer Protein (PLTP) and PLTP-generated Pre-β-high Density Lipoprotein by Mast Cell Chymase Impairs High Affinity Efflux of Cholesterol from Macrophage Foam Cells

Human atherosclerotic lesions contain mast cells filled with the neutral protease chymase. Here we studied the effect of human chymase on (i) phospholipid transfer protein (PLTP)-mediated phospholipid (PL) transfer activity, and (ii) the ability of PLTP to generate pre-beta-high density lipoprotein (HDL). Immunoblot analysis of PLTP after incubation with chymase for 6 h revealed, in addition to the original 80-kDa band, four specific proteolytic fragments of PLTP with approximate molecular masses of 70, 52, 48, and 31 kDa. This specific pattern of PLTP degradation remained stable for at least 24 h of incubation with chymase. Such proteolyzed PLTP had reduced ability (i) to transfer PL from liposome donor particles to acceptor HDL(3) particles, and (ii) to facilitate the formation of pre-beta-HDL. However, when PLTP was incubated with chymase in the presence of HDL(3), only one major cleavage product of PLTP (48 kDa) was generated, and PL transfer activity was almost fully preserved. Moreover, chymase effectively depleted the pre-beta-HDL particles generated from HDL(3) by PLTP and significantly inhibited the high affinity component of cholesterol efflux from macrophage foam cells. These results suggest that the mast cells in human atherosclerotic lesions, by secreting chymase, may prevent PLTP-dependent formation of pre-beta-HDL particles from HDL(3) and so impair the anti-atherogenic function of PLTP.

Characterization of tryptophan high affinity transport system in pinealocytes of the rat. Day-night modulation.

Tryptophan is required in the pineal gland for the formation of serotonin, precursor of melatonin biosynthesis. The level of this amino acid in the serum and in the pineal gland of the rat undergoes a circadian rhythm, and reduced plasma tryptophan concentration decreases secretion of melatonin in humans. Tryptophan is transported into the cells by the long chain neutral amine acid system T and by the aromatic amino acid system T. The high affinity component of [(3)H]tryptophan uptake was studied in pinealocytes of the rat. Inhibition was observed in the presence of phenylalanine or tyrosine, but not in the presence of neutral amino acids, alanine, glycine, serine, lysine or by 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid, a substrate specific for system L. The transport of tryptophan was temperature-dependent and trans-stimulated by phenylalanine and tyrosine, but was energy-, sodium-, chloride-, and pH-independent. In addition, the sulphydryl agent N-ethylmaleimide did not modify the high affinity transport of tryptophan in pinealocytes. The kinetic parameters were not significantly different at 12:00 as compared to 24:00 h. The treatment with the inhibitor of tryptophan hydroxylase, p-chlorophenylalanine, produced an increase in the maximal velocity of the uptake and a reduction in the affinity at 12:00, but not at 24:00 h, probably indicating that during the day, the formation of serotonin in the pineal gland is favoured by elevating the uptake of tryptophan, whereas at 24:00 h other mechanisms, such as induction of enzymes are taking place. High affinity tryptophan uptake in the rat pineal gland occurs through system T and is upregulated during the day when the availability of serotonin is reduced.

Developmental Pb 2+ exposure alters NMDAR subtypes and reduces CREB phosphorylation in the rat brain

In the present study we show that chronic exposure to low levels of lead (Pb 2+) during development alters the type of N-methyl- d-aspartate receptor (NMDAR) expressed in the developing and young adult rat brain. Ifenprodil inhibition of [ 3H]MK-801 binding to the NMDAR channel in cortical and hippocampal neuronal membranes expressed high and low affinity components. Previous studies have shown that the high affinity component is associated with NR1/NR2B receptor complexes while the low affinity component is associated with the appearance and insertion of the NR2A subunit to NMDAR complexes. Pb 2+-exposed rats express a greater number of [ 3H]MK-801 binding sites associated with the high affinity and low affinity components of ifenprodil inhibition. Further, [ 3H]ifenprodil saturation isotherms and Scatchard analysis in cortical and hippocampal membranes showed a higher number of binding sites ( B max) with no change in binding affinity ( K d) in Pb 2+-exposed animals relative to controls. Quantitative [ 3H]MK-801 autoradiography in response to glutamate and glycine provided evidence that NMDAR complexes in Pb 2+-exposed rat brain were maximally activated in situ. Higher levels of ifenprodil-sensitive binding sites and increased sensitivity to agonists are properties characteristic of NR1/NR2B recombinant receptors. Thus, our results strongly suggest that a greater proportion of the total number of NMDAR are NR1/NR2B receptors in the Pb 2+-exposed rat brain. This Pb 2+-induced change in NMDAR subtypes in the rat brain was associated with reduced CREB phosphorylation in cortical and hippocampal nuclear extracts. These findings demonstrate that chronic exposure to environmentally relevant levels of Pb 2+ altered the subunit composition of NMDAR complexes with subsequent effects on calcium-sensitive signaling pathways involved in CREB phosphorylation.

Evidence that the novel imidazoline compound FT005 is an alpha(2)-adrenoceptor agonist.

1: The aim of this study was to determine whether the hyperglycaemic action of the novel imidazoline compound FT005 could be mediated by activation of alpha(2)-adrenoceptors, using a variety of in vivo and in vitro methods including radioligand binding. 2: FT005 produced a dose-dependent increase in blood glucose levels of CBA/Ca mice (0.125-25 mg kg(-1), i.p.). The time course of this hyperglycaemic effect matched that of adrenaline (1 mg kg(-1)) more closely than glucagon (1 mg kg(-1)) or the K(ATP) channel opener diazoxide (25 mg kg(-1)). The hyperglycaemic effect of FT005 (1 mg kg(-1)) was significantly reduced by the alpha(2)-adrenoceptor antagonist rauwolscine (0.5 mg kg(-1)). 3: FT005 produced a significant reduction in plasma insulin levels of mice 30 min after administration. The hyperglycaemic effect of FT005 (25 mg kg(-1)), although still present, was significantly less in fasted mice in which insulin levels are lower, suggesting that a reduction of insulin secretion contributes to the action of FT005. 4: When studied in the mouse isolated vas deferens preparation, FT005 produced a complete inhibition of neurogenic contractions, which was blocked by rauwolscine. This is consistent with activation of pre-synaptic alpha(2)-adrenoceptors. 5: In radioligand binding studies FT005 completely displaced the alpha(2)-adrenoceptor antagonist [(3)H]-RX821002 from mouse whole brain homogenates. The displacement was best described by a two-site model of interaction comprising high and low affinity components. 6: The results indicate that FT005 is an agonist at alpha(2)-adrenoceptors. A reduction in insulin secretion contributes to the hyperglycaemic action of FT005, although an additional mechanism can not be excluded.

Renal action of progesterone: effect on calcium reabsorption

Recently, the kidney has been reported to be the site of receptors for progesterone. Although the exact segment of the nephron has not been precisely determined, the cortical collecting tubule was suspected, since the hormone displaces bound 3H aldosterone. The aim of the present study was to investigate the effect of progesterone on calcium (Ca 2+) transport by the renal luminal membranes and to determine the site and mechanisms of action. Incubation of proximal tubules from rabbit kidney with progesterone did not influence Ca 2+ or Na + transport by the luminal membranes. In the distal tubules (DT), a 5 min treatment with 10 −11 M of the hormone enhanced 0.5 mM 45Ca uptake from 0.60±0.02 to 0.84±0.08 pmol/μg per 10 s ( P<0.05) in the absence of Na + and from 0.26±0.02 to 0.41±0.02 pmol/μg per 10 s ( P<0.01) in the presence of 100 mM Na +. The dose–response curve showed a biphasic action with a peak at 10 −11 M. Ca 2+ uptake by DT membranes presents dual kinetics. The hormone enhanced the Vmax value of the high affinity component from 0.41±0.05 to 0.57±0.06 pmol/μg per 10 s ( P<0.05). In contrast, incubation of DT with 10 −8 M progesterone decreased 1 mM Na + uptake from 0.68±0.03 to 0.53±0.07 pmol/μg per 10 s ( P<0.05). Finally, 10 −11 M progesterone also enhanced Ca 2+ uptake by the DT membranes through a direct nongenomic mechanism.

Comparison of contractile responses to donitriptan and sumatriptan in the human middle meningeal and coronary arteries

Donitriptan is a potent, high efficacy agonist at 5-HT 1B/1D receptors. We investigated the contractile effects of donitriptan and sumatriptan on human isolated blood vessels of relevance to therapeutic efficacy in migraine (middle meningeal artery) and coronary adverse events (coronary artery). Furthermore, using the concentration–response curves in the middle meningeal artery, we predicted the plasma concentration needed for the therapeutic effect of donitriptan. Both donitriptan and sumatriptan contracted the middle meningeal artery with similar apparent efficacy ( E max: 103±8% and 110±12%, respectively), but the potency of donitriptan (pEC 50: 9.07±0.14) was significantly higher than that of sumatriptan (pEC 50: 7.41±0.08). In the coronary artery, the contraction to donitriptan was biphasic with a significantly higher maximal response ( E max: 29±6%) than sumatriptan ( E max: 14±2%; pEC 50: 5.71±0.16), yielding two distinct pEC 50 values (8.25±0.16 and 5.60±0.24). Incubation with the 5-HT 2 receptor antagonist ketanserin (10 μM) eliminated the low affinity component of the concentration–response curve of donitriptan and the resultant E max and pEC 50 were 9±2% and 7.33±0.21, respectively. Ketanserin was without effect on the sumatriptan-induced contraction. Based on the middle meningeal artery contraction, concentrations ( C max) of donitriptan that may be expected to have a therapeutic efficacy equivalent to that of 50 and 100 mg sumatriptan are predicted to be around 2.5 and 4.3 nM, respectively. Such concentrations are likely to induce only a small coronary artery contraction of 2.9±1.5% and 3.8±2.0%, respectively; these are not different from those by C max concentrations of sumatriptan (1.7±0.4% or 2.2±0.4%). The present results suggest that, like sumatriptan, donitriptan exhibits cranioselectivity and would be effective in aborting migraine attacks with a similar coronary side-effect profile as sumatriptan.

Role of Ca2+ and calmodulin-dependent enzymes in the regulation of glycine transport in Müller glia.

Glycine (Gly) is considered an obligatory co-agonist at NMDA receptors. Müller glia from the retina harbor functional NMDA receptors, as well as low and high affinity Gly transporters, the later identified as GLYT1. We here studied the regulation of Gly transport in primary cultures of Müller glia, as this process could contribute to the modulation of NMDA receptor activity at glutamatergic synapses in the retina. We demonstrate that neither glutamate stimulation nor the activation or inhibition of protein kinases A or C modify transport. In order to assess a function for Ca2+ and calmodulin (CaM)-dependent processes in the regulation of Gly transport, we explored the participation of Ca2+ concentration, CaM and Ca2+/CaM-dependent enzymes on Gly transporter activity. ATP and carbachol, known to induce Ca2+ waves in Müller cells, as well as caffeine-induced Ca2+ release from intracellular stores stimulated transport, whereas Ca2+ chelation by BAPTA-AM markedly reduced transport. CaM inhibitors W-7, ophiobolin A, R-24571 and trifluoperazine, induced a specific dose-dependent inhibition of transport. The inhibition of CaMKII by the autocamtide-2-related inhibitory peptide or by KN62 caused a decrease in transport which, in the case of KN62, was due to the abolition of the high affinity component, ascribed to GLYT1. Our results further suggest that Gly transport is under cytoskeletal control, as activation of calpain by major increases in [Ca2+]i induced by ionophores, as well as actin destabilization clearly inhibit uptake. We here demonstrate for the first time the participation of CaM, CaMKII and the actin cytoskeleton in the regulation of Gly transport in glia. Ca2+ waves are induced in Müller cells by distinct neuroactive compounds released by neurons and glia, hence the regulation of [Gly] by this system may be of physiological relevance in the control of retinal excitability.

Solubilization of high affinity G-protein-coupled serotonin1A receptors from bovine hippocampus using pre-micellar CHAPS at low concentration.

The serotonin1A (5-HT1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioural and cognitive functions. This paper reports an efficient strategy to solubilize 5-HT1A receptors from bovine hippocampal membranes using the zwitterionic detergent CHAPS which is mild and non-denaturing. Since high concentration of CHAPS has earlier been shown to induce dissociation and depletion of G-protein sub-units, a low (pre-micellar) concentration of CHAPS was used for solubilizing 5-HT1A receptors in the presence of NaCl followed by PEG precipitation. This results in solubilization of 5-HT1A receptors with a high degree of efficiency and gives rise to high affinity, functionally active G-protein-sensitive solubilized receptors. Optimal solubilization of the receptor from the native source with high ligand binding affinity and intact signal transduction components may constitute the first step in the molecular characterization of the 5-HT1A receptor in particular, and G-protein-coupled receptors in general.

H1 and H2 histamine receptors mediate the production of inositol phosphates but not cAMP in human breast epithelial cells.

In the present work we studied the H1 and H2 histamine receptor expression and function in HBL-100 and MCF-10A cells, derived from non-tumorigenic human breast epithelia, and in MCF-10T, the H-ras-transfected MCF-10A counterpart. The signal transduction pathways associated with these receptors, and the expression of proto-oncogenes c-fos, c-myc and c-jun at the mRNA and protein levels, were examined. Saturation analysis using intact cells, showed two binding sites for [3H]tiotidine and [3H]mepyramine. Pretreatment of purified membrane with guanosine 5'-ythiotriphosphate resulted in the loss of the low affinity component for [3H]tiotidine binding, and of the high affinity component for [3H]mepyramine. In both cases, there was no modification in the total number of sites for both ligands. Neither H1 nor H2 agonists stimulated cyclic AMP production, though this pathway is functional in these cells. On the other hand, both H1 and H2 agonists enhanced phosphoinositide turnover in a dose-dependent fashion, and this induction is pertussis toxin-insensitive. H1 and H2 agonists did not influence the expression of c-myc or c-fos mRNA, nor their encoded proteins. These results indicate that the three cell lines examined showed functional H1 and H2 histamine receptors, which are involved in the metabolic turnover of inositol phosphates but are ineffective in the modulation of the cyclic AMP response. The fact that H2 receptors have lost their ability to stimulate cyclic AMP production would imply the loss of a regulatory mechanism of cell growth.

Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate

BackgroundThiamine diphosphate (ThDP) is the active form of thiamine, and it serves as a cofactor for several enzymes, both cytosolic and mitochondrial. Isolated mitochondria have been shown to take up thiamine yet thiamine diphosphokinase is cytosolic and not present in mitochondria. Previous reports indicate that ThDP can also be taken up by rat mitochondria, but the kinetic constants associated with such uptake seemed not to be physiologically relevant.ResultsHere we examine ThDP uptake by mitochondria from several human cell types, including cells from patients with thiamine-responsive megaloblastic anemia (TRMA) that lack a functional thiamine transporter of the plasma membrane. Although mitochondria from normal lymphoblasts took up thiamine in the low micromolar range, surprisingly mitochondria from TRMA lymphoblasts lacked this uptake component. ThDP was taken up efficiently by mitochondria isolated from either normal or TRMA lymphoblasts. Uptake was saturable and biphasic with a high affinity component characterized by a Km of 0.4 to 0.6 μM. Mitochondria from other cell types possessed a similar high affinity uptake component with variation seen in uptake capacity as revealed by differences in Vmax values.ConclusionsThe results suggest a shared thiamine transporter for mitochondria and the plasma membrane. Additionally, a high affinity component of ThDP uptake by mitochondria was identified with the apparent affinity constant less than the estimates of the cytosolic concentration of free ThDP. This finding indicates that the high affinity uptake is physiologically significant and may represent the main mechanism for supplying phosphorylated thiamine for mitochondrial enzymes.

Amino acid transport by intestinal brush border vesicles of a marine fish, Boops salpa

The transport of glycine, alanine, methionine and alpha amino-isobutyric acid (AIB) was studied on brush border membrane vesicles of Boops salpa, a marine fish. This transport was Na +-, Cl −- and pH-dependent. In the presence of NaCl, the uptake decreased as the pH increased from 5.5 to 8.5. With Na 2SO 4, the transport of the four amino acids was strongly reduced and the pH optimum was 7–8. In the presence of NaCl, amino acid transport was described by high and low affinity kinetics. The K t of the high-affinity component was comparable for glycine, alanine and methionine (0.1 mM), and was significantly enhanced for AIB (0.6 mM). The J max of the low affinity component was significantly lower for methionine and AIB than for glycine and alanine. Lowering the sodium concentration from 80 to 20 mM significantly increased K t and J max of the high-affinity component of glycine transport. Moreover, the kinetics of AIB transport under 100 mM Na + were similar to glycine kinetics under 40 mM Na + and the two amino acids competed for the same carrier(s). These results suggest that chloride ions are essential in neutral amino acid transport in Boops, that multiple saturable components are involved in this process, and that sodium plays an important role in the differences between the transport kinetics of amino acids.

Role of neuronal nicotinic receptors in the transmission and processing of information in neurons of the central nervous system

The properties of nicotinic acetylcholine receptors (nAChRs) were studied following exogenous expression in a host system or using whole-cell recordings in brain slices, autoradiography and immunohistochemistry. When expressed in HEK-293 cells, α4β2 nAChRs displayed both a high and a low affinity component. The ratio of these two states was modified by chronic nicotine exposure, resulting in an enhanced sensitivity and a marked reduction in desensitization. Mutations in the gene coding for the α4 subunit are responsible for a particular form of nocturnal epilepsy. When expressed in Xenopus oocytes, α4β2 nAChRs containing these mutations displayed distinct alterations in agonist affinity, desensitization and calcium permeability. Magnocellular endocrine neurons in the supraoptic (SO) nucleus of the hypothalamus were found to express functional α7-containing nAChRs, which could play a role in regulating neurohypophysial peptide secretion. Facial (VII), hypoglossal (XII) and vagal (X) motoneurons of young rats responded to ACh by a fast inward current. The nAChRs present in VII and XII nuclei were of the non-α7-containing type, whereas those present in the X nucleus contained the α7 subunit. In Bcl-2 transgenic mice, facial nerve axotomy caused nAChRs downregulation by interfering negatively with the expression of the α4 subunit. Binding sites corresponding to α7-containing nAChRs were also detected in spinal motor nuclei and axotomy provoked a reduction of the binding. Together, these data indicate that long-term exposure to nicotine can promote neuroadaptive changes in nAChRs and that genetic alterations of neuronal nAChRs can result in transmissible neurological diseases. They also suggest that these receptors probably play a role in the central regulation of autonomic functions, as well as in motor control.

Turbidite provenance in the Lower Palaeozoic Manx Group, Isle of Man: implications for the tectonic setting of Eastern Avalonia

The provenance of turbidites from the predominantly Arenig-aged Manx Group has been constrained using petrographical and geochemical techniques. Petrographical analysis of sandstones reveals that mono- and poly-crystalline quartz grains of cratonic origin are dominant. Major, trace and rare earth element concentrations in both sandstones and mudstones complement the petrographical data indicating that the source of the sediments consisted of quartzose basement and mature sedimentary rocks. There is little evidence of a juvenile volcanic input but a minor palaeovolcanic component of active continental margin affinity is present. There are indications of a more immature character in the oldest units but, on the whole, geochemical data from the Manx Group suggest an overall passive margin setting. These results suggest that Eastern Avalonia was attached to Gondwana during deposition of the Manx Group and that Iapetus oceanic crust did not subduct beneath the region before the end of the Arenig.

Regulation of the expression of low affinity GABAA receptors in rat cerebellar granule cells.

GABAA receptors of cerebellar granule cells obtained from neonatal rats and kept in culture were studied by labelled muscimol binding. The data show that, according to the maturational state of those cells in vivo, one or two binding components appear. The low affinity component seems to be the one appearing later. The expression of this component seems to be regulated by protein tyrosine phosphorylation. In fact, its expression is down regulated by the protein tyrosine kinase (PTK) inhibitor, genistein. Viceversa, its expression is upregulated by insulin like growth factor I (IGF-I), most probably via PTK activation. A possible interpretation of the data is that in vivo IGF-I is one of the endogenous messages leading to the expression of this component during development. Another endogenous factor involved may be GABA itself. Low affinity GABAA receptors appear to be the ones involved in inhibitory synaptic transmission at glomeruli. Whereas the high affinity ones probably correspond to extrasynaptic GABAA receptors mediating the tonic form of inhibition in cerebellar granules.

Allosteric modulation of [3H]-CGP39653 binding through the glycine site of the NMDA receptor: further studies in rat and human brain.

Binding of D,L-(E)-2-amino-4-[(3)H]-propyl-5-phosphono-3-pentenoic acid ([(3)H]-CGP39653), a selective antagonist at the glutamate site of the NMDA receptor, is modulated by glycine in rat brain tissue. We have further investigated this phenomenon in rodent and human brain by means of receptor binding and quantitative autoradiography techniques. In rat cerebral cortical membranes the glycine antagonist 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloro-indole-2-carboxylic acid sodium salt (GV150526A) did not change basal [(3)H]-CGP39653 binding, but competitively reversed the high affinity component of [(3)H]-CGP39653 binding inhibition by glycine, with a pK(B) value of 8.38, in line with its affinity for the glycine site (pK(i)=8.49 vs. [(3)H]-glycine). Glycine (10 microM) significantly decreased [(3)H]-CGP39653 affinity for the NMDA receptor (with no change in the B(max)), whereas enhanced L-glutamate affinity (P<0.05, paired-samples Student's t-test). In rat brain sections the addition of GV150526A (30 microM) to the incubation medium increased [(3)H]-CGP39653 binding to 208% of control (average between areas), indicating the presence of endogenous glycine. The enhancement presented significant regional differences (P<0.05, two-way ANOVA), with striatum higher than cerebral cortex (282 and 187% of control, respectively; P<0.05, Fisher's LSD). On the contrary, there was not any significant variation in affinity values of [(3)H]-CGP39653, L-glutamate, glycine and GV150526A in striatal and cortical membranes. These results confirmed the existence of regionally distinct NMDA receptors subtypes with different glycine/glutamate allosteric modulation. Whole brain autoradiography revealed an uneven distribution of [(3)H]-CGP39653 binding sites in human brain. High levels of binding were determined in hippocampus and in cingulate, frontoparietal and insular cortex. Intermediate to low levels of binding were found in diencephalic nuclei and basal ganglia. [(3)H]-CGP39653 binding was increased to 216% of control (mean between areas) by 30 microM GV150526A. The enhancement, however, did not present significant regional differences. These results introduce GV150526A as a useful tool to identify NMDA receptor subtypes by means of receptor autoradiography; moreover, they demonstrate that the allosteric inhibition of [(3)H]-CGP39653 binding by glycine parallels an increase in receptor affinity to the endogenous ligand L-glutamate. Finally, this study provides the first detailed anatomical description of the regional distribution of [(3)H]-CGP39653 binding sites in human brain.

( S)-3,4-DCPG, a potent and selective mGlu8a receptor agonist, activates metabotropic glutamate receptors on primary afferent terminals in the neonatal rat spinal cord

( S)-3,4-Dicarboxyphenylglycine (DCPG) has been tested on cloned human mGlu1–8 receptors individually expressed in AV12-664 cells co-expressing a rat glutamate/aspartate transporter and shown to be a potent and selective mGlu8a receptor agonist (EC 50 value 31±2 nM, n=3) with weaker effects on the other cloned mGlu receptors (EC 50 or IC 50 values >3.5 μM on mGlu1–7). Electrophysiological characterisation on the neonatal rat spinal cord preparation revealed that ( S)-3,4-DCPG depressed the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) giving a biphasic concentration–response curve showing EC 50 values of 1.3±0.2 μM ( n=17) and 391±81 μM ( n=17) for the higher and lower affinity components, respectively. The receptor mediating the high-affinity component was antagonised by 200 μM ( S)-α-methyl-2-amino-4-phosphonobutyrate (MAP4, K D value 5.4±1.5 μM ( n=3)), a group III metabotropic glutamate (mGlu) receptor antagonist. The α-methyl substituted analogue of ( S)-3,4-DCPG, ( RS)-3,4-MDCPG (100 μM), antagonised the effects of ( S)-3,4-DCPG ( K D value 5.0±0.4 μM, n=3) in a similar manner to MAP4. ( S)-3,4-DCPG-induced depressions of the fDR-VRP in the low-affinity range of the concentration–response curve were potentiated by 200 μM ( S)-α-ethylglutamate (EGLU), a group II mGlu receptor antagonist, and were relatively unaffected by MAP4 (200 μM). However, depressions of the fDR-VRP mediated by the AMPA selective antagonist ( R)-3,4-DCPG were not potentiated by EGLU, suggesting that the low-affinity component of the concentration–response curve for ( S)-3,4-DCPG is not due to antagonism of postsynaptic AMPA receptors. It is suggested that the receptor responsible for mediating the high-affinity component is mGlu8. The receptor responsible for mediating the low-affinity effect of ( S)-3,4-DCPG has yet to be identified but it is unlikely to be one of the known mGlu receptors present on primary afferent terminals or an ionotropic glutamate receptor of the AMPA or NMDA subtype.

Pharmacological characterization of muscarinic receptors in dog isolated ciliary and urinary bladder smooth muscle.

1. The pharmacological characteristics of muscarinic receptors mediating contraction of dog isolated ciliary muscle were determined and compared to those mediating contraction of dog urinary bladder smooth muscle. 2. (+)-Cis-dioxolane induced concentration-dependent contractions of ciliary muscle (pEC50=7.18+/-0.07, Emax=453+/-64 mg, n=19) and urinary bladder isolated smooth muscle (pEC50=6.55+/-0.07, Emax=11+/-1 g, n=19). These responses were antagonized by several muscarinic receptor antagonists (pKb values for the ciliary muscle and the bladder smooth muscle, respectively): atropine (8.25+/-0.14 and 9.21+/-0.09), pirenzepine (6.31+/-0.13 and 6.70+/-0.25), tolterodine (7.97+/-0.14 and 8.68+/-0.12), oxybutynin (7.40+/-0.08 and 7.88+/-0.12), zamifenacin (6.46+/-0.19 and 7.69+/-0.11), S-secoverine (6.66+/-0.14 and 8.13+/-0.07), AQ-RA 741 (6.16+/-0.15 and 7.08+/-0.23), p-F-HHSiD (7.10+/-0.27 and 7.35+/-0.07) and responses were not antagonized by PD 102807 (up to 100 nM). 3. In urinary bladder smooth muscle, the profile of antagonist pKB values correlated significantly with pK(i) values at human recombinant m3 muscarinic receptors, suggesting that M3 muscarinic receptors mediated the response. In the ciliary muscle, a significant (P<0.01) correlation was obtained with human recombinant m3 and m5 receptors. 4. Darifenacin displayed insurmountable antagonism at receptors in the bladder. At receptors in the ciliary muscle, it exhibited two phases of antagonism, comprising an initial low affinity (pKB<6) component and a high affinity phase (pKB>8). 5. The role of pigmentation in the atypical behaviour of darifenacin was examined. In blue coloured eyes, darifenacin produced apparent surmountable, competitive antagonism of the responses to (+)-cis-dioxolane (pKB=8.76+/-0.07). The antagonist profile obtained in this tissue suggested the involvement of a site which has the pharmacological attributes of the M5 receptor. 6. We suggest that the dog urinary bladder contracts in response to M3 muscarinic receptor activation. Contraction of the brown-eyed dog ciliary muscle is more complex and may include involvement of at least two receptors, possibly the M5 and M3 receptor, whereas blue-eyed dog ciliary muscle may involve a single population of M5 muscarinic receptors.

Dexamethasone reduces the expression of p75 neurotrophin receptor and apoptosis in contused spinal cord

Apoptosis is an important cause of secondary cell death in spinal cord injury (SCI). SCI induces the expression of the low affinity neurotrophin receptor p75 (p75NTR), that in the absence of the high affinity component, TrkA, can promote cell death by apoptosis. We therefore hypothesized that a reduction of p75NTR expression in SCI may increase tissue sparing and therefore improve recovery of function. As a tool to test our hypothesis we used the synthetic glucocorticoid dexamethasone (DEX) to down-regulate p75NTR expression. A standardized thoracic spinal cord contusion injury was produced in female rats. Laminectomized and SCI rats received various doses of DEX immediately after injury and the treatment was continued daily for 7 days. DEX, given at high doses (20 mg/kg, s.c.) but not at low doses (1 or 8 mg/kg) prevented the increase in p75NTR mRNA and protein in SCI rats, without affecting the expression of TrkA. High doses of DEX also reduced cellular apoptosis both in white and gray matters. This effect correlated with the ability of DEX to accelerate behavioral recovery of function measured by a combined behavioral score. These data suggest that reduction of p75NTR in SCI may be a therapeutic strategy to limit cell and tissue damage and therefore to improve recovery of function in SCI patients.

Two Active Molecular Phenotypes of the Tachykinin NK1 Receptor Revealed by G-protein Fusions and Mutagenesis

The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between either Galpha(s) or Galpha(q) and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P and neurokinin A was observed in the tail-truncated Galpha(s) fusion construct, whereas the lower affinity component was displayed by the tail-truncated Galpha(q) fusion. The elusive difference between the affinity determined in heterologous versus homologous binding assays for substance P and especially for neurokinin A was eliminated in the G-protein fusions. An NK1 receptor mutant with a single substitution at the extracellular end of TM-III-(F111S), which totally uncoupled the receptor from Galpha(s) signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated Galpha(q) fusion construct. Thus, the heterogenous pharmacological phenotype displayed by the NK1 receptor is a reflection of the occurrence of two active conformations or molecular phenotypes representing complexes with the Galpha(s) and Galpha(q) species, respectively. We propose that these molecular forms do not interchange readily, conceivably because of the occurrence of microdomains or "signal-transductosomes" within the cell membrane.

Constitutive activity at serotonin 5-HT 1D receptors: detection by homologous GTPγS versus [ 35S]-GTPγS binding isotherms

Although many G-protein-coupled receptors (GPCRs) may display constitutive activity, their detection has, to date, depended on the use of inverse agonists. The present study exploited a novel procedure to investigate constitutive activity at recombinant human (h) serotonin (5-HT) 5-HT 1D receptors stably expressed in Chinese hamster ovary (CHO) cells. 5-HT modestly stimulated guanosine-5′-O-(3-[ 35S]thio)-triphosphate ([ 35S]-GTPγS) binding to CHO-h5-HT 1D membranes whereas methiothepin and the 5-HT 1B/1D-selective ligand, SB224,289, exerted robust inhibition of basal [ 35S]-GTPγS binding (inverse agonism). These actions were specific inasmuch as they were reversed by the novel, selective 5-HT 1B/1D ligand, S18127. Constitutive activity was investigated by homologous inhibition of [ 35S]-GTPγS binding to CHO-h5-HT 1D membranes with unlabelled GTPγS. Under ‘basal’ conditions (absence of receptor ligand), biphasic isotherms were observed. Most (80%) [ 35S]-GTPγS binding sites were in the high affinity (HA) versus low affinity (LA) component of the isotherms. HA binding was augmented by 5-HT (to 155%; relative to basal values=100%), but decreased by methiothepin (to 23%) and by SB224,289 (to 67%). In contrast, LA binding was not altered. Further, membranes of untransfected CHO cells exhibited only LA binding sites, indicating that the latter are not related to h5-HT 1D receptor-G-protein coupling. Thus, at 5-HT 1D receptors expressed in this CHO cell line, HA binding detected in homologous inhibition experiments (GTPγS versus [ 35S]-GTPγS) under basal conditions provides a measure of constitutive G-protein activation. Thus, it is suggested that for h5-HT 1D receptors and, possibly, other GPCRs, inverse agonists will be detectable by [ 35S]-GTPγS binding if a HA component is present under basal conditions.