High Agonist Concentrations Research Articles

Allosteric Potentiators of Metabotropic Glutamate Receptor Subtype 5 Have Differential Effects on Different Signaling Pathways in Cortical Astrocytes

The metabotropic glutamate receptor subtype 5 (mGluR5) activates calcium mobilization and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in cortical astrocytes. These are independent signaling systems, and they can be differentially regulated. We recently discovered two novel selective allosteric potentiators of mGluR5, 3,3'-difluorobenzaldazine (DFB) and N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) methyl]phenyl}-2-hydroxybenzamide (CPPHA). In studies of mGluR5 activation of calcium transients in recombinant systems, both DFB and CPPHA are without effect on baseline calcium levels, but they induce parallel leftward shifts in the concentration-response curve to agonists. However, it is conceivable that these compounds will have differential effects on different signaling pathways in native systems. Here, we examined the effects of CPPHA and DFB on mGluR5-induced calcium transients and ERK1/2 phosphorylation in cultured rat cortical astrocytes. Both potentiators induced parallel leftward shifts of the concentration-response curves of DHPG- and glutamate-induced calcium transients in astrocytes. These effects are identical to their effects on mGluR5 expressed in human embryonic kidney 293 or Chinese hamster ovary cells. DFB induced a similar shift of concentration-response curve of DHPG-induced ERK1/2 phosphorylation. Interestingly, CPPHA induced an increase in basal mGluR5-mediated ERK1/2 phosphorylation and potentiated the effect of low concentrations of agonists. In contrast, CPPHA significantly decreased ERK1/2 phosphorylation induced by high concentrations of agonists. Thus, CPPHA has qualitatively different effects on mGluR5-mediated calcium responses and ERK1/2 phosphorylation. Together, these data provide evidence that different allosteric potentiators can differentially modulate coupling of a single receptor to different signaling pathways.

How Good Vibes Turn Bad: Rise of Ictal Events From Oscillatory Network Activity. Focus on “Transient Depression of Excitatory Synapses on Interneurons Contributes to Epileptogenesis During Gamma Oscillations in the Mouse Hippocampal Slice”

small alterations in network properties might destabilize the network. In the first, experimental, part of the study, Traub et al. used high concentrations (10 –100 M) of the metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) to evoke transitions from physiological to pathological oscillatory activity in the CA3 region of the hippocampus. Application of DHPG is known to be epileptogenic in vivo, and, under the in vitro conditions used in this study, DHPG resulted in oscillatory field potentials in the gamma range interspersed with epileptiform burst activity recordable from the CA3 pyramidal layer. In addition, very fast oscillations became apparent immediately before and during the bursts. Thus the DHPG-induced in vitro activity mimicked some of the defining features observed during the onset of ictal events in vivo. Subsequently, intracellular recordings showed that the transitions from gamma oscillations to burst activity were accompanied by an increase in excitatory input and a decrease in GABAergic input to CA3 pyramidal neurons. Conversely, the excitatory synaptic activity onto interneurons of the pyramidal layer sharply declined prior to the occurrence of a population burst, transiently weakening the interneuronal control of the pyramidal cells.

Agonist activation of arachidonate‐regulated Ca2+‐selective (ARC) channels in murine parotid and pancreatic acinar cells

ARC channels (arachidonate-regulated Ca(2+)-selective channels) are a novel type of highly Ca(2+)-selective channel that are specifically activated by low concentrations of agonist-induced arachidonic acid. This activation occurs in the absence of any depletion of internal Ca(2+) stores (i.e. they are 'non-capacitative'). Previous studies in HEK293 cells have shown that these channels provide the predominant pathway for the entry of Ca(2+) seen at low agonist concentrations where oscillatory [Ca(2+)](i) signals are typically produced. In contrast, activation of the more widely studied store-operated Ca(2+) channels (e.g. CRAC channels) is only seen at higher agonist concentrations where sustained 'plateau-type'[Ca(2+)](i) responses are observed. We have now demonstrated the presence of ARC channels in both parotid and pancreatic acinar cells and shown that, again, they are specifically activated by the low concentrations of appropriate agonists (carbachol in the parotid, and both carbachol and cholecystokinin in the pancreas) that are associated with oscillatory [Ca(2+)](i) signals in these cells. Uncoupling the receptor-mediated activation of cytosolic phospholipase A(2) (cPLA(2)) with isotetrandrine reduces the activation of the ARC channels by carbachol and, correspondingly, markedly inhibits the [Ca(2+)](i) signals induced by low carbachol concentrations, whilst those signals seen at high agonist concentrations are essentially unaffected. Interestingly, in the pancreatic acinar cells, activation by cholecystokinin induces a current through the ARC channels that is only approximately 60% of that seen with carbachol. This is consistent with previous reports indicating that carbachol-induced [Ca(2+)](i) signals in these cells are much more dependent on Ca(2+) entry than are the cholecystokinin-induced responses.

This Month in Anesthesiology

This Month in Anesthesiology

Classic Benzodiazepines Modulate the Open–Close Equilibrium in α1β2γ2Lγ-Aminobutyric Acid Type A Receptors

Classic benzodiazepine agonists induce their clinical effects by binding to a site on gamma-aminobutyric acid type A (GABAA) receptors and enhancing receptor activity. There are conflicting data regarding whether the benzodiazepine site is allosterically coupled to gamma-aminobutyric acid binding versus the channel open-close (gating) equilibrium. The authors tested the hypothesis that benzodiazepine site ligands modulate alpha1beta2gamma2L GABAA receptor gating both in the absence of orthosteric agonists and when the orthosteric sites are occupied. GABAA receptors were recombinantly expressed in Xenopus oocytes and studied using two-microelectrode voltage clamp electrophysiology. To test gating effects in the absence of orthosteric agonist, the authors used spontaneously active GABAA receptors containing a leucine-to-threonine mutation at residue 264 on the alpha1 subunit. To examine effects on gating when orthosteric sites were fully occupied, they activated wild-type receptors with high concentrations of a partial agonist, piperidine-4-sulfonic acid. In the absence of orthosteric agonists, the channel activity of alpha1L264Tbeta2gamma2L receptors was increased by diazepam and midazolam and reduced by the inverse benzodiazepine agonist FG7142. Flumazenil displayed very weak agonism and blocked midazolam from further activating mutant channels. In wild-type receptors activated with saturating concentrations of piperidine-4-sulfonic acid, midazolam increased maximal efficacy. Independent of orthosteric site occupancy, classic benzodiazepines modulate the gating equilibrium in alpha1beta2gamma2L GABAA receptors and are therefore allosteric coagonists. A Monod-Wyman-Changeux coagonist gating model quantitatively predicts these effects, suggesting that benzodiazepines minimally alter orthosteric ligand binding.

Amplification of Ca2+ Signaling by Diacylglycerol-mediated Inositol 1,4,5-Trisphosphate Production

Stimulation of various cell surface receptors leads to the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through phospholipase C (PLC) activation, and the IP3 and DAG in turn trigger Ca2+ release through IP3 receptors and protein kinase C activation, respectively. The amount of IP(3) produced is particularly critical to determining the spatio-temporally coordinated Ca(2+)-signaling patterns. In this paper, we report a novel signal cross-talk between DAG and the IP3-mediated Ca(2+)-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of protein kinase C activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was completely abolished by depletion of Ca2+ stores or inhibition of PLC and IP3 receptors, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IP3-induced Ca2+ release. Furthermore, intracellular accumulation of endogenous DAG by a DAG-lipase inhibitor greatly increased the number of cells responding to agonist stimulation at low doses. These results suggest a novel physiological function of DAG, i.e. amplification of Ca2+ signaling by enhancing IP3 production via its positive feedback effect on PLC activity.

ARC Channels: A Novel Pathway for Receptor-Activated Calcium Entry

In many nonexcitable cells, stimulation with low agonist concentrations specifically activates Ca2+ entry via arachidonic acid-regulated, highly Ca2+-selective ARC channels. Only at high agonist concentrations are the more widely studied store-operated channels activated, producing sustained elevated cytosolic Ca2+ concentration signals. These signals activate calcineurin, which in turn inhibits the ARC channels, resulting in a "reciprocal regulation" of these two distinct Ca2+-entry pathways that may have important functional implications for the cell.

Regulation of A2B adenosine receptor functioning by tumour necrosis factor a in human astroglial cells

Low-affinity A2B adenosine receptors (A2B ARs), which are expressed in astrocytes, are mainly activated during brain hypoxia and ischaemia, when large amounts of adenosine are released. Cytokines, which are also produced at high levels under these conditions, may regulate receptor responsiveness. In the present study, we detected A2B AR in human astrocytoma cells (ADF) by both immunoblotting and real-time PCR. Functional studies showed that the receptor stimulated adenylyl cyclase through Gs proteins. Moreover, A2B ARs were phosphorylated and desensitized following stimulation of the receptors with high agonist concentration. Tumour necrosis factor alpha (TNF-alpha) treatment (24- h) increased A2B AR functional response and receptor G protein coupling, without any changes in receptor protein and mRNA levels. TNF-alpha markedly reduced agonist-dependent receptor phosphorylation on threonine residues and attenuated agonist-mediated A2B ARs desensitization. In the presence of TNF-alpha, A2B AR stimulation in vitro induced the elongation of astrocytic processes, a typical morphological hallmark of in vivo reactive astrogliosis. This event was completely prevented by the selective A2B AR antagonist MRS 1706 and required the presence of TNF-alpha. These results suggest that, in ADF cells, TNF-alpha selectively modulates A2B AR coupling to G proteins and receptor functional response, providing new insights to clarify the pathophysiological role of A2B AR in response to brain damage.

Agonist Costimulation Increases Recruitment of Platelet Subpopulations to a Procoagulant Phenotype.

Previously we determined that platelets stimulated with PAR 1 agonists, even at levels eliciting maximal responses for support of factor Xa generation, showed only a subpopulation (~10%) that exposed binding sites for factor IXa. Now we show that the same subpopulation of platelets supported binding of both factor Xa and factor IXa. While stimulation of platelets with GPVI agonists collagen or convulxin also caused exposure of factor Xa binding sites on a small subpopulation (~10%) of platelets, simultaneous stimulation with PAR 1 and GPVI agonists resulted in synergistic recruitment of platelets to expose factor Xa binding sites (30–60%). In kinetic studies of platelet-supported thrombin generation, platelets preactivated with thrombin or collagen showed roughly equivalent initial rates of prothrombinase activity (5nM/min, 10nM/min, and 12nM/min for low, medium and high agonist concentrations) that increased with reaction time up to 20 minutes reflecting platelet stimulation by the thrombin generated. Platelets preactivated with both collagen and thrombin, even at high concentrations (collagen 50μg/ml and thrombin 2U/ml), supported additive rates of prothrombinase in contrast to the synergy achieved in exposure of factor Xa binding sites. These data indicate that although single strong agonists recruit only a small subpopulation of activated platelets to undergo procoagulant surface changes, co-stimulation acts synergistically to recruit a larger population. It was previously shown that treatment of platelets with 100μM of the protein kinase C inhibitor RO318220 permitted an increased (three-fold) PAR 1-stimulated platelet support of prothrombinase and an almost 20-fold increase in the size of the population exposing annexin V binding sites. We now show that agonist-stimulated platelets pretreated with 100μM RO318220, whether stimulated with PAR 1 or GPVI agonists separately or combined, recruited ~80% of platelets to bind factor Xa. These data suggest that exposure of a procoagulant phenotype is negatively regulated by a pathway containing a target of high concentrations of RO318220, and that both PAR 1 and GPVI pathways normally converge in relief of that regulation.

Fate of ah receptor agonists during biological treatment of an industrial sludge containing explosives and pharmaceutical residues

Sweden is meeting prohibition for deposition of organic waste from 2005. Since 1 million tons of sludge is produced every year in Sweden and the capacity for incineration does not fill the demands, other methods of sludge management have to be introduced to a higher degree. Two biological treatment alternatives are anaerobic digestion and composting. Different oxygen concentrations result in different microbial degradation pathways and, consequently, in a different quality of the digestion or composting residue, It is therefore necessary to study sludge treatment during different oxygen regimes in order to follow both degradation of compounds and change in toxicity. In this study, an industrial sludge containing explosives and pharmaceutical residues was treated with anaerobic digestion or composting, and the change in toxicity was studied. Nitroaromatic compounds, which are the main ingredients of both pharmaceutical and explosives, are well known to cause cytotoxicity and genotoxicity. However, little data are available concerning sludge with nitroaromatics and any associated dioxin-like activity. Therefore, we studied the sludge before and after the treatments in order to detect any changes in levels of Ah receptor (AhR) agonists using two bioassays for dioxin-like compounds. An industrial sludge was treated with anaerobic digestion or composting in small reactors in a semi-continuous manner. The same volume as the feeding volume was taken out daily and stored at -20 degrees C. Sample preparation for the bioassays was done by extraction using organic solvents, followed by clean up with silica gel or sulphuric acid, yielding two fractions. The fractions were dissolved in DMSO and tested in the bioassays. The dioxin-like activity was measured using the DR-CALUX assay with transfected H4IIE rat hepatoma pGudluc cells and an EROD induction assay with RTL-W1 rainbow trout liver cells. The bioassays showed that the sludge contained AhR agonists at levels of TCDD equivalents (TEQs) higher than other sludge types in Sweden. In addition, the TEQ values for the acid resistant fractions increased considerably after anaerobic digestion, resulting in an apparent formation of acid resistant TEQs in the anaerobic reactors. Similar results have been reported from studies of fermented household waste. There was a large difference in effects between the two bioassays, with higher TEQ levels in the RTL-W1 EROD assay than in the DR-CALUX assay. This is possibly due to a more rapid metabolism in rat hepatocytes than in trout hepatocytes or to differences in sensitivities for the AhR agonists in the sludge. It was also demonstrated by GC/FID analysis that the sludge contained high concentrations of nitroaromatics. It is suggested that nitroaromatic metabolites, such as aromatic amines and nitroanilines, are possible candidates for the observed bioassay effects. It was also found that the AhR agonists in the sludge samples were volatile. The sludge contained fairly high concentrations of volatile AhR agonists. The increase of acid resistant AhR agonist after anaerobic digestion warrants further investigations of the chemical and toxic properties of these compounds and of the mechanisms behind this observation. This study has pointed out the benefits of using different types of mechanism-specific bioassays when evaluating the change in toxicity by sludge treatment, in which measurement of dioxin-like activity can be a valuable tool. In order to study the recalcitrant properties of the compounds in the sludge using the DR-CALUX assay, the exposure time can be varied between 6 and 24 hours. The properties of the acid-resistant AhR agonists formed in the anaerobic treatment have to be investigated in order to choose the most appropriate method for sludge management.

Receptors, agonists and antagonists

Most drugs act by being either agonists or antagonists at receptors that respond to chemical messengers such as neurotransmitters. An agonist binds to the receptor and produces an effect within the cell. An antagonist may bind to the same receptor, but does not produce a response, instead it blocks that receptor to a natural agonist. A partial agonist can produce an effect within a cell that is not maximal and then block the receptor to a full agonist. Antagonism may be competitive and reversed by higher concentrations of agonist. Insurmountable antagonists bind strongly to the receptor and are not reversed by additional agonist. Pharmacological receptors can be divided into four superfamilies: ligand-gated ion channels, G-protein coupled receptor, direct enzyme-linked receptors, and intracellular receptors affecting gene transcription.

Imaging of muscarinic acetylcholine receptor signaling in hippocampal neurons: evidence for phosphorylation-dependent and -independent regulation by G-protein-coupled receptor kinases.

We used the inositol 1,4,5-trisphosphate (IP3) biosensor, the pleckstrin homology (PH) domain of PLCdelta1 (phospholipase C) tagged with enhanced green fluorescent protein (eGFP-PH(PLCdelta)), to examine muscarinic acetylcholine (mACh) receptor regulation of phospholipase C/IP3 signaling in intact single hippocampal neurons in "real time." Initial experiments produced a pharmacological profile consistent with the presence of a predominant M1 mACh receptor population coupled to the IP3 response. To investigate M1 mACh receptor regulation, neurons were stimulated with approximate EC50 concentrations of the mACh receptor agonist methacholine before (R1) and after (R2) a short (60 sec) exposure to a high concentration of agonist. This resulted in a marked attenuation in the R2 relative to R1 response. Inhibition of endogenous GRK6 (G-protein-coupled receptor kinase) activity, by the introduction of catalytically inactive (K215R)GRK6, partially reversed the attenuation of agonist-induced responsiveness, whereas overexpression of wild-type GRK6 increased receptor desensitization. Manipulation of endogenous GRK2 activity through introduction of either wild-type or catalytically inactive GRK2 ((K220R)GRK2) almost completely inhibited agonist-stimulated IP3 production, implying a phosphorylation-independent regulation of M1 mACh receptor signaling, most probably mediated by a GRK2 N-terminal RGS-like (regulator of G-protein signaling) domain interaction with GTP-bound Galpha(q/11). Together, our data suggest a role for both phosphorylation-dependent and -independent regulation of M1 mACh receptors in hippocampal neurons.

The Duration and Amplitude of the Plateau Phase of ATP- and ADP-Evoked Ca2+ Signals Are Modulated by Ectonucleotidases in in situ Endothelial Cells of Rat Aorta

ATP has a long-lasting vasodilatory effect, possibly due to its capability to induce a prolonged increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in endothelial cells (EC) and activate constitutive nitric oxide synthase. However, contradictory data have been reported regarding the time course of ATP-evoked Ca²⁺ signals in in situ EC. In particular, short-duration Ca²⁺ signals have been reported, which might be thought to be unable to sustain a prolonged, NO-induced vasodilation. The current experiments were therefore performed in in situ EC of rat aorta in order to more fully define the time course of ATP-evoked Ca²⁺ signals. 20 µM ATP evoked a short-lasting Ca²⁺ signal. However, medium stirring, high agonist concentrations, inhibition of ectonucleotidases and application of a poorly hydrolyzable agonist evoked long-lasting Ca²⁺ signals (up to 20 min at 37°C). These studies suggest that ATP is able to sustain a prolonged [Ca²⁺]_i increase, unless ectonucleotidase activity reduces the agonist concentration near the EC surface to subthreshold values, quickly cutting the Ca²⁺ signal. Furthermore, the amplitude of the long-lasting phase of the Ca²⁺ signal depended on the balance between agonist degradation by ectonucleotidases and agonist transport, by diffusion and convection, from bulk solution to the EC surface.

On the mechanism and significance of ligand-induced internalization of human neutrophil chemokine receptors CXCR1 and CXCR2.

It is well established that leukocyte chemotactic receptors, a subset of G protein-coupled receptors, undergo endocytosis after stimulation by ligand. However, the significance of this phenomenon to cell motility and other important leukocyte functions induced by chemoattractants has not been clearly defined. Here we show that in primary human neutrophils, the threshold levels of agonist required for endocytosis of the chemotactic receptors CXCR1 and CXCR2 were approximately 10-fold or higher than those needed for maximal chemotactic and calcium flux responses. Moreover, when stimulated by agonists at concentrations that are high enough for chemotaxis but too low for receptor endocytosis, neutrophil CXCR1 and CXCR2 could be reactivated in response to repeated application of the same agonist. Both receptors were excluded from Triton X-100-insoluble lipid rafts, and at high agonist concentrations were rapidly endocytosed by a clathrin/rab5/dynamin-dependent pathway. These data support the conclusion that neutrophil migration in response to CXCR1 or CXCR2 agonists is not dependent on endocytosis of CXCR1 or CXCR2. Rather than being integral to the process of cell migration, receptor endocytosis may be a terminal stop signal when cells reach the focus of inflammation where the chemoattractant concentrations are the highest.

The epidemic of asthma and allergy

In his treatise On Asthma: Its Pathology And Treatment first published in 1860, Henry Hyde Salter (Figure 1), a physician at the Charing Cross Hospital, London, differentiated asthma from other causes of breathlessness as `paroxysmal dyspnoea of a peculiar character with intervals of healthy respiration between attacks'.1 6 years later, from an analysis of 150 unpublished cases, he described many of the characteristic features of this disease including hyperresponsiveness to cold air and exercise and attacks provoked by exposure to chemical and mechanical irritants, to particular kinds of air as well as to certain foods and wine.2,3 His observations were further enhanced by the use of the spirograph, the earliest record of a water spirometer.4 In these publications Salter identified asthma as a spasmodic stricture occurring throughout the conducting airways, and differentiated the condition from bronchial catarrh, recent bronchitis and old bronchitis (Figure 2). He drew special attention to the musical rhonchus that characterized asthmatic bronchoconstriction and indicated that the sibilant bronchi could not be relieved by coughing. Also of great significance was his observation of cells in the asthmatic sputum, which he identified by the presence of a nucleus, nucleolus and cell wall. The identification of eosinophils in sputum had to await the development of eosin by Paul Ehrlich some 15 years later.5 Sir William Osler, in his first edition of Principles and Practice of Medicine,6 likewise drew attention to the factors that could exacerbate asthma including allergens, air pollutants, infections, exercise, weather, food and emotions. Figure 1 Henry Hyde Salter (1823–1871) Figure 2 Plate from Salter's Treatise (1860) illustrating the different causes of airflow obstruction Hyperresponsiveness of the conducting airways, a characteristic feature of all forms of asthma, can be quantified in the laboratory by use of inhalation bronchial provocation tests with such agents as methacholine and histamine. In asthma the dose–response curve to these agonists is displaced to the left in proportion to disease severity, and at high agonist concentrations there is loss of the normal protective plateau (Figure 3). As pointed out by both Salter and Osler, hyperresponsiveness is in part the result of a characteristic type of inflammation that affects the conducting airways and is accompanied by marked structural changes to the airways which include an increase in airway smooth muscle and deposition of matrix leading to an overall thickening of the airway wall (remodelling). The pathological features of asthma are vividly illustrated by Huber and Koessler in their classic paper of 1922.7 These combine to make the airways contract too much and too easily in response to exogenous and endogenous stimuli, as well as contributing to the diurnal variation in airway calibre that is characteristic of the disease. Figure 3 Typical dose–response curves in normal and asthmatic individuals on aerosol bronchial provocation with increasing concentrations of methacholine Today, fibreoptic bronchoscopy allows ready access to airways, and lavage and mucosal biopsy samples confirm the presence of a special type of inflammation characterized by infiltration of the airway wall with activated T lymphocytes, mast cells, basophils, eosinophils and macrophages. In addition, morphometric studies on airways from patients who have died from or with asthma have quantified the impressive increase in airway smooth muscle that occurs in this disease, along with structural changes that include shedding of epithelial cells and epithelial mucous metaplasia, deposition of collagen and other matrix proteins in the lamina reticularis beneath a normal epithelial basement membrane, increased deposition of proteoglycans and repair collagen throughout the airway wall, and an increase in submucosal microvessels and nerves—all changes tantamount to airway remodelling. The fact that these structural changes occur in early childhood, at the inception of asthma,8 indicates that they are fundamental to pathogenesis and occur parallel to, rather than as a consequence of, airway inflammation.9

Receptor Trafficking via the Perinuclear Recycling Compartment Accompanied by Cell Division Is Necessary for Permanent Neurotensin Cell Sensitization and Leads to Chronic Mitogen-activated Protein Kinase Activation

Most G protein-coupled receptors are internalized after interaction with their respective ligand, a process that subsequently contributes to cell desensitization, receptor endocytosis, trafficking, and finally cell resensitization. Although cellular mechanisms leading to cell desensitization have been widely studied, those responsible for cell resensitization are still poorly understood. We examined here the traffic of the high affinity neurotensin receptor (NT1 receptor) following prolonged exposure to high agonist concentration. Fluorescence and confocal microscopy of Chinese hamster ovary, human neuroblastoma (CHP 212), and murine neuroblastoma (N1E-115) cells expressing green fluorescent protein-tagged NT1 receptor revealed that under prolonged treatment with saturating concentrations of neurotensin (NT) agonist, NT1 receptor and NT transiently accumulated in the perinuclear recycling compartment (PNRC). During this cellular event, cell surface receptors remained markedly depleted as detected by both confocal microscopy and (125)I-NT binding assays. In dividing cells, we observed that following prolonged NT agonist stimulation, NT1 receptors were removed from the PNRC, accumulated in dispersed vesicles inside the cytoplasm, and subsequently reappeared at the cell surface. This NT binding recovery allowed for constant cell sensitization and led to a chronic activation of mitogen-activated protein kinases p42 and p44. Under these conditions, the constant activation of NT1 receptor generates an oncogenic regulation. These observations support the potent role for neuropeptides, such as NT, in cancer progression.

Nicotine is highly effective at producing desensitization of rat alpha4beta2 neuronal nicotinic receptors.

We examined desensitization by acetylcholine (ACh) and nicotine at the rat alpha4beta2 neuronal nicotinic receptor stably expressed in HEK cells. For both agonists, the decay in response due to desensitization ('onset') was best fitted by the sum of two exponentials with the fast component dominant at concentrations > 1 microM. The time constants for onset were similar for both agonists, and showed little concentration dependence over the range of 0.1-100 microM. Recovery from desensitization also showed two exponential components. In contrast to the similarity in onset, nicotine produced longer lasting desensitization, resulting from an increase in the proportion of receptors in the slowly recovering population and from an increase in the time constant for the slow recovery process. The proportion of receptors in the slowly recovering population increased as the duration of the desensitizing pulse increased. Desensitization was also induced by low concentrations of agonist, with no apparent macroscopic response. A 100 s application of 10 nM nicotine desensitized 70 % of the peak response, while 100 s of 10 nM ACh desensitized only 15 %. At higher concentrations of agonist, which result in a macroscopic response, desensitization in the absence of activation also can occur. Nicotine is a very potent and efficacious desensitizing agent at this neuronal nicotinic receptor.

Calcium entry and the control of calcium oscillations.

During oscillatory Ca(2+) signals, the agonist-induced enhanced entry of extracellular Ca(2+) plays a critical role in modulating the frequency of the oscillations. Although it was originally assumed that the entry of Ca(2+) under these conditions occurred via the well-known, and apparently ubiquitous, store-operated mechanism, subsequent studies suggested that this was unlikely. It is now known that, in many cell types, a novel non-capacitative Ca(2+)-selective pathway whose activation is dependent on arachidonic acid is responsible, and the channels involved [ARC channels (arachidonate-regulated Ca(2+) channels)] have been characterized. These ARC channels co-exist with the store-operated CRAC channels (Ca(2+)-release-activated Ca(2+) channel) in cells, but each plays a unique and non-overlapping role in Ca(2+) signalling. In particular, it is the ARC channels that are specifically activated at the low agonist concentrations that give rise to oscillatory Ca(2+) signals and provide the predominant mode of Ca(2+) entry under these conditions. The indications are that Ca(2+) entry through the ARC channels increases the likelihood that low concentrations of Ins(1,4,5)P(3) will trigger repetitive Ca(2+) release. At higher agonist concentrations, store-depletion is more complete and sustained resulting in the activation of CRAC channels. At the same time the ARC channels are turned off, resulting in what we have described as a reciprocal regulation of these two distinct Ca(2+) entry pathways.

Regulation of sustained actin dynamics by the TCR and costimulation as a mechanism of receptor localization.

The localization of receptors, signaling intermediates, and cytoskeletal components at the T cell/APC interface is thought to be a major determinant of efficient T cell activation. However, important questions remain open. What are the dynamics of the T cell cytoskeleton as a potential mediator of such localization? How are they regulated by the TCR and costimulatory receptors? Do they actually mediate receptor localization? In this study, we have addressed these questions. Even under limiting T cell activation conditions, actin accumulated immediately and transiently at the T cell/APC interface, the microtubule organizing center reoriented toward it. In contrast, sustained (>5 min) actin accumulation in highly dynamic patterns depended on an optimal T cell stimulus: high concentrations of the strong TCR ligand agonist peptide/MHC and engagement of the costimulatory receptors CD28 and LFA-1 were required in an overlapping, yet distinct, fashion. Intact sustained actin dynamics were required for interface accumulation of TCR/MHC in a central pattern and for efficient T cell proliferation, as established using a novel approach to selectively block only the sustained actin dynamics. These data suggest that control of specific elements of actin dynamics by TCR and costimulatory receptors is a mechanism to regulate the efficiency of T cell activation.

Agonist- and antagonist-induced sequestration/internalization of neuropeptide Y Y1 receptors in HEK293 cells.

1 Neuropeptide Y Y(1) receptors are known to internalize following the binding of agonists. In the present study, a pseudopeptide Y(1) receptor antagonist, homodimeric Ile-Glu-Pro-Dpr-Tyr-Arg-Leu-Arg-Tyr-CONH(2) (GR231118), also induced Y(1) receptor internalization in human embryonic kidney (HEK293) cells. 2 We demonstrated first that both specifically bound radiolabeled antagonist ([(125)I]GR231118) and agonist ([(125)I][Leu(31), Pro(34)]PYY) underwent receptor-mediated sequestration/internalization in transfected HEK293 cells. 3 Agonist-induced Y(1) receptor internalization was dependent on clathrin-coated pits and was regulated in part by Gi/o-protein activation as revealed by pertussin toxin sensitivity. In contrast, antagonist-induced sequestration of Y(1) receptors was partly dependent on clathrin-coated pits, but independent from Gi/o-protein activation. 4 Exposure to high concentrations of agonist or antagonist caused a 50 and 75% loss of cell surface binding, respectively. The loss caused by the agonist rapidly recovered. This phenomenon was blocked by monensin, an inhibitor of endosome acidification, suggesting that cell surface receptor recovery is due to recycling. In contrast to the agonist, GR231118 induced a long-lasting sequestration of Y(1) receptors in HEK293 cells. 5 Immunofluorescence labeling indicated that following 40 min of incubation with either the agonist or the antagonist, Y(1) receptors followed markedly different intercellular trafficking pathways. 6 Taken together, these findings provided evidence that a pseudopeptide Y(1) receptor antagonist can induce long-lasting disappearance of cell surface receptors through a pathway distinct from the classical endocytic/recycling pathway followed by stimulation with an agonist.