Reduction Of Desensitization Research Articles

Functional expression of P2 purinoceptors in a primary neuroglial cell culture of the rat arcuate nucleus

The arcuate nucleus (ARC) plays an important role in the hypothalamic control of energy homeostasis. Expression of various purinoceptor subtypes in the rat ARC and physiological studies suggest a modulatory function of P2 receptors within the neuroglial ARC circuitry. A differentiated mixed neuronal and glial microculture was therefore established from postnatal rat ARC, revealing neuronal expression of ARC-specific transmitters involved in food intake regulation (neuropeptide Y (NPY), proopiomelanocortin (POMC), tyrosine hydroxylase (TH)). Some NPYergic neurons cosynthesized TH, while POMC and TH expression proved to be mutually exclusive. Stimulation with the general purinoceptor agonists 2-methylthioadenosine-5′triphosphate (2-MeSATP) and ATP but not the P2X1/P2X3 receptor subtype agonist α,β-methyleneadenosine-5′triphosphate (α,β-meATP) induced intracellular calcium signals in ARC neurons and astrocytes. Some 5–10% each of 2-MeSATP responsive neurons expressed POMC, NYP or TH. Supporting the calcium imaging data, radioligand binding studies to hypothalamic membranes showed high affinity for 2-MeSATP, ATP but not α,β-meATP to displace [α-35S]deoxyadenosine-5′thiotriphosphate ([35S]dATPαS) from P2 receptors. Repetitive superfusion with equimolar 2-MeSATP allowed categorization of ARC cells into groups with a high or low (LDD) degree of purinoceptor desensitization, the latter allowing further receptor characterization. Calcium imaging experiments performed at 37°C vs. room temperature showed further reduction of desensitization. Agonist-mediated intracellular calcium signals were suppressed in all LDD neurons but only 25% of astrocytes in the absence of extracellular calcium, suggestive of metabotropic P2Y receptor expression in the majority of ARC astrocytes. The highly P2Y1-selective receptor agonists MRS2365 and 2-methylthioadenosine-5′diphosphate (2-MeSADP) activated 75–85% of all 2-MeSATP-responsive ARC astrocytes. Taking into consideration the high potency to dose-dependently stimulate ARC cells of the LDD group, the high affinity for rat P2X(1–3) and low affinity for rat P2X4, P2X7 and P2Y receptor subtypes except P2Y1 and P2Y13, the agonist 2-MeSATP primarily acted upon P2X2 and P2Y1 purinoceptors to trigger intracellular calcium signaling in ARC neurons and astrocytes.

Regulation of the Human Delta-Opioid Receptor by Alkaloids: Different Roles of Arrestins

Study background: We previously revealed that the arrestin 2 was differentially involved in the regulation of the human delta opioid receptor (hDOR) by peptidic and alkaloid agonists. In the present study, we examined whether i) hDOR regulation by two related alkaloid agonists (etorphine and morphine) involved the same arrestins ii) similar arrestin-dependent mechanisms occur upon short- and long-term agonist exposure. Methods: The human neuroblastoma SK-N-BE cells, endogenously expressing hDOR, were transfected with wild-type or mutant arrestins. Using shRNA, we also generated a clonal cell line depleted in arrestin 2, the only arrestin isoform expressed in this cell line. Cells were then exposed or not to either morphine ou etorphine for short- (1 h) or long-term (18 h), then receptor desensitization was examined on the cAMP pathway and internalization was visualized by confocal microscopy. Results: In arrestin 2-depleted cells, we observed a strong reduction of desensitization after 18 h of morphine exposure but not with etorphine. Over-expression of wild-type or mutant arrestins produced an opposite modulation of receptor desensitization induced by morphine and etorphine both upon short- and long-term exposure. Confocal fluorescence microscopy experiments did not reveal any strong impact on receptor internalization when different arrestins were over-expressed. Conclusion: Our results showed that i) two related opioid alkaloid agonists produce hDOR desensitization by different mechanisms ii) the involvement of arrestins in hDOR desensitization depends on the duration of agonist exposure.

Workıng Condıtıons And Control, Job Satısfactıon, Burnout, Depressıon Levels Among Anesthesıology-Reanımatıon And Internal Medıcıne Physıcıans

Aim: Investigation of working conditions and control, job satisfaction, burnout, and depression levels among anesthesiologyreanimation and internal medicine physicians.Material and Method: Resident and specialist physicians working in AR and IM branches in hospitals located at the central district of Ankara province, were included in the study. Maslach Burnout Inventory which analyzes emotional exhaustion, desensitization, and personal accomplishment; Minnesota Satisfaction Questionnaire which measures job satisfaction; Beck Depression Inventory which evaluates the level of depressive signs; adapted survey form of ‘Working Conditions and Control Questionnaire’ which examines the job conditions and control over those conditions; and the socio-demographic data collection form which assesses the individual characteristics, were delivered to the physicians along with the informed consent form. 412 forms received back from those physicians, were statistically evaluated with independent samples T, one-way variance analysis, Pearson chi-square, Yates chi-square, and Fischer exact tests. Results: In review of burnout in all the physicians, reduction in desensitization was found to occur with aging (p=0.001). Among residents, emotional exhaustion, desensitization and Beck Depression Inventory scores were high (p=0,020, p<0,001, p=0,013), whereas working conditions and control were low. In AR physicians, control was high, while desensitization was lower (p=0.006). Conclusion: We believe, working and personal conditions of physicians should be evaluated at certain intervals and amended when required to.

The Relationship between Agonist Potency and AMPA Receptor Kinetics

AMPA-type glutamate receptors are tetrameric ion channels that mediate fast excitatory synaptic transmission in the mammalian brain. When agonists occupy the binding domain of individual receptor subunits, this domain closes, triggering rearrangements that couple agonist binding to channel opening. Here we compare the kinetic behavior of GluR2 channels activated by four different ligands, glutamate, AMPA, quisqualate, and 2-Me-Tet-AMPA, full agonists that vary in potency by up to two orders of magnitude. After reduction of desensitization with cyclothiazide, deactivation decays were strongly agonist dependent. The time constants of decay increased with potency, and slow components in the multiexponential decays became more prominent. The desensitization decays of agonist-activated currents also contained multiple exponential components, but they were similar for the four agonists. The time course of recovery from desensitization produced by each agonist was described by two sigmoid components, and the speed of recovery varied substantially. Recovery was fastest for glutamate and slowest for 2-Me-Tet-AMPA, and the amplitude of the slow component of recovery increased with agonist potency. The multiple kinetic components appear to arise from closed-state transitions that precede channel gating. Stargazin increases the slow kinetic components, and they likely contribute to the biexponential decay of excitatory postsynaptic currents.

Impaired desensitization of a mutant adrenocorticotropin receptor associated with apparent constitutive activity.

A naturally occurring ACTH receptor [melanocortin 2 receptor (MC2R)] mutation (F278C) has been identified in a subject with ACTH-independent Cushing's syndrome. Functional characterization of this mutant receptor reveals that it is associated with elevated basal cAMP accumulation when compared with wild-type receptor-expressing cell lines. Dose responsiveness is similar between wild-type and mutant receptors in cell lines expressing similar numbers of binding sites. In view of the location of this mutation in the C-terminal tail of the MC2R, desensitization and internalization were investigated and found to be impaired. Inhibition of protein kinase A by H89 blocks wild-type MC2R desensitization and also results in increased basal activity, as does alanine substitution of Ser 280 in the C-terminal tail. Alanine substitution of Ser 208, the consensus protein kinase A phosphorylation target in the third cytoplasmic loop also results in a reduction in desensitization without significant change in basal activity or internalization. These findings suggest a novel mechanism is involved in the apparently constitutive activation of the MC2R in which failure of desensitization appears to be associated with enhanced basal receptor activity.

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.

Gadolinium reduces AMPA receptor desensitization and deactivation in hippocampal neurons.

The actions of the trivalent cation Gd(3+) on whole cell AMPA receptor-mediated currents were studied in isolated hippocampal neurons, in nucleated or outside-out patches taken from cultured hippocampal neurons, and on miniature excitatory postsynaptic currents (mEPSCs) recorded in cultured hippocampal neurons. Glutamate, AMPA, or kainate was employed to activate AMPA receptors. Applications of relatively low concentrations of Gd(3+) (0.1-10 microM) substantially enhanced steady-state whole cell glutamate and kainate-evoked currents without altering peak currents, suggesting that desensitization was reduced. However, higher concentrations (>30 microM) depressed steady-state currents, indicating an underlying inhibition of channel activity. Lower concentrations of Gd(3+) also increased the potency of peak glutamate-evoked currents without altering that of steady-state currents. An ultrafast perfusion system and nucleated patches were then used to better resolve peak glutamate-evoked currents. Low concentrations of Gd(3+) reduced peak currents, enhanced steady-state currents, and slowed the onset of desensitization, providing further evidence that this cation reduces desensitization. In the presence of cyclothiazide, a compound that blocks desensitization, a low concentration Gd(3+) inhibited both peak and steady-state currents, indicating that Gd(3+) both reduces desensitization and inhibits these currents. Gd(3+) reduced the probability of channel opening at the peak of the currents but did not alter the single channel conductance calculated using nonstationary variance analysis. Recovery from desensitization was enhanced, and glutamate-evoked current activation and deactivation were slowed by Gd(3+). The Gd(3+)-induced reduction in desensitization did not require the presence of the GluR2 subunit as this effect was seen in hippocampal neurons from GluR2 null-mutant mice. Gd(3+) reduced the time course of decay of mEPSCs perhaps as a consequence of its slowing of AMPA receptor deactivation although an increase in the frequency of mEPSCs also suggested enhanced presynaptic release of transmitter. These results demonstrate that Gd(3+) potently reduces AMPA receptor desensitization and mimics a number of the properties of the positive modulators of AMPA receptor desensitization such as cyclothiazide.

Allosteric interactions between cyclothiazide and AMPA/kainate receptor antagonists

1. Cyclothiazide blocks alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization and potentiates AMPA receptor gated currents. Interactions between cyclothiazide, and the non-competitive antagonist GYKI52466 (GYKI) and competitive antagonist 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo (F) quinoxaline (NBQX) were studied at native and recombinant AMPA/kainate receptors using whole-cell recording in order to characterize the modulation by cyclothiazide of these two antagonist sites. 2. GYKI 100 microM, which is sufficient to eliminate virtually hippocampal kainate (100 microM) currents, failed to prevent access of cyclothiazide to its site of potentiation, and was unable to enhance removal of cyclothiazide potentiation. However, cyclothiazide reduced GYKI (30 microM) block from 84 +/- 8.3% to 38 +/- 12%, and slowed the onset of the block with a time course much faster than the time course for onset and offset of potentiation induced by cyclothiazide. Cyclothiazide had qualitatively similar effects upon antagonism by NBQX 1 microM. 3. Kainate activated desensitizing currents in dorsal root ganglion (DRG) neurones, which were unaffected by cyclothiazide. GYKI blocked these kainate currents with lower affinity (IC50 > 120 microM) than for hippocampal neurones (IC50 < 30 microM), and cyclothiazide did not affect GYKI antagonism. 4. Steady-state AMPA currents from homomeric GluRA-Dflip receptors in HEK 293 cells were dramatically potentiated (up to 216 fold) by cyclothiazide via reduction of desensitization. In contrast, kainate-gated currents in HEK 293 cells expressing GluR6R receptors exhibited pronounced desensitization that was unaffected by cyclothiazide. GYKI retains its inhibition at both recombinant AMPA and kainate receptors. 5. These results indicate that cyclothiazide allosterically influences two important antagonist sites on AMPA receptors. In addition, AMPA/kainate receptor subunit composition influences the affinity of GYKI for the receptor.

Ca(2+)-permeable AMPA and NMDA receptor channels in basket cells of rat hippocampal dentate gyrus.

1. Glutamate receptor (GluR) channels were studied in basket cells in the dentate gyrus of rat hippocampal slices. Basket cells were identified by their location, dendritic morphology and high frequency of action potentials generated during sustained current injection. 2. Dual-component currents were activated by fast application of glutamate to outside-out membrane patches isolated from basket cell somata (10 microM glycine, no external Mg2+). The fast component was selectively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the slow component by D-2-amino-5-phosphonopentanoic acid (D-AP5). This suggests that the two components were mediated by alpha-amino-3- hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR)/kainate receptor and N-methyl-D-aspartate receptor (NMDAR) channels, respectively. The mean ratio of the peak current of the NMDAR component to that of the AMPAR/kainate receptor component was 0.22 (1 ms pulses of 10 mM glutamate). 3. The AMPAR/kainate receptor component, which was studied in isolation in the presence of D-AP5, was identified as AMPAR mediated on the basis of the preferential activation by AMPA as compared with kainate, the weak desensitization of kainate-activated currents, the cross-desensitization between AMPA and kainate, and the reduction of desensitization by cyclothiazide. 4. Deactivation of basket cell AMPARs following 1 ms pulses of glutamate occurred with a time constant (tau) of 1.2 +/- 0.1 ms (mean +/- S.E.M.). During 100 ms glutamate pulses AMPARs desensitized with a tau of 3.7 +/- 0.2ms. 5. The peak current-voltage (I-V) relation of AMPAR-mediated currents in Na(+)-rich extracellular solution showed a reversal potential of -4.0 +/- 2.6 mV and was characterized by a a doubly rectifying shape. The conductance of single AMPAR channels was estimated as 22.6 +/- 1.6 pS using non-stationary fluctuation analysis. AMPARs expressed in hippocampal basket cells were highly Ca2+ permeable (PCa/PK = 1.79). 6. NMDARs in hippocampal basket cells were studied in isolation in the presence of CNQX. Deactivation of NMDARs activated by glutamate pulses occurred bi-exponentially with mean tau values of 266 +/- 23 ms (76%) and 2620 +/- 383 ms (24%). 7. The peak I-V relation of the NMDAR-mediated component in Na(+)-rich extracellular solution showed a reversal potential of 1.5 +/- 0.6 mV and a region of negative slope at negative membrane potentials in the presence of external Mg2+, due to voltage-dependent block by these ions. The conductance of single NMDAR channels in the main open state was 50.2 +/- 1.8 pS.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of N‐methyl‐D‐aspartic acid receptor desensitization by glycine in mouse cultured hippocampal neurones.

1. Responses to N-methyl-D-aspartic acid (NMDA) were recorded from mouse embryonic hippocampal neurones in dissociated culture, using the tight-seal, whole-cell, patch-clamp technique for voltage clamp. A rapid perfusion system, with an exchange time constant of less than 10 ms, was used to apply NMDA under conditions which minimized slow, calcium-sensitive desensitization. With no added glycine, responses to 100 microM-NMDA applied for 1.5 s declined by greater than 90%, due to an additional component of desensitization of time constant 250 ms. 2. Adding glycine to the extracellular solution, over the range 30 nM to 3 microM, both potentiated responses to NMDA and to L-glutamate, and reduced fast desensitization. In the presence of 3 microM-glycine responses to NMDA declined by only 10%. Similar potentiation and reduction of desensitization was obtained with 3 microM concentrations of the glycine analogues D-alanine and D-serine. 3. Analysis of dose-response curves for the action of glycine on responses to 100 microM-NMDA revealed a 3-fold higher potency of glycine for potentiation of peak versus steady-state responses, with concentrations for half-activation of 134 and 382 nM, respectively. The competitive glycine antagonist 7-chlorokynurenic acid produced a similar shift of both the peak and steady-state dose-response curves for glycine, consistent with an equilibrium dissociation constant of 280 nM for interaction of 7-chlorokynurenic acid with the glycine binding site on NMDA receptors. 4. In the presence of 100 nM-glycine, 10 microM-7-chlorokynurenic acid produced nearly complete block of the response to 3 nM-NMDA, suggesting that glycine is absolutely required for activation of the NMDA receptor channel complex. 5. In some neurones responses to NMDA showed essentially no desensitization in the presence of 3 microM-glycine. Under these conditions, 7-chlorokynurenic acid produced a concentration-dependent block of both the initial and equilibrium response to NMDA, with a 4-fold greater sensitivity for block of the steady-state current (IC50 = 2.25 microM) than for block of the peak current (IC50 = 8.96 microM). As a result, in the presence of 7-chlorokynurenic acid, responses to NMDA showed strong desensitization, even in the presence of 3 microM-glycine. 6. Our results show that glycine-evoked potentiation of NMDA receptor activity is accompanied by reduced desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)

Glycine-insensitive desensitization of NMDA responses in cultured mouse embryonic neurons

The influence of glycine on the desensitization of NMDA-induced currents was studied using cultured embryonic mouse neurons. Although glycine often appeared to reduce desensitization in the whole-cell mode, it had no effect on desensitization in outside-out patches. Various interpretations can be proposed for this discrepancy, such as the presence in intact cells of an intracellular factor regulating desensitization, or the masking of desensitization in intact cells by restricted diffusion of the agonist in the extracellular space. The fact that glycine potentiates the NMDA responses under conditions where it does not regulate desensitization indicates that the potentiation cannot be explained by a reduction of desensitization.

Play desensitization of fear of darkness in preschool children

Systematic desensitization therapy (SDT) is considered to be a technique of established effectiveness in reducing monosymptomatic fears (Paul, 1969a, 1969b), yet little systematic research has been done to investigate the merit of desensitization procedures in modifying children's behavior. The few available controlled studies (Miller et al., 1972; Obler and Terwilliger, 1970; Oliver, 1971) on desensitization with children have produced equivocal results. Although imagery and relaxation have been reported of benefit when used separately (Graziano and Kean, 1971; Lazarus and Abramovitz, 1962), evidence suggests that the usual combination of imagery and relaxation is not effective in reducing children's fears (Miller et al., 1972). Investigators in this area have often modified traditional SDT to include alternatives to imagery such as pictures, drawings, play (Cassell, 1965; Lazarus, 1960; Obler and Terwilliger, 1970; Patterson, 1965), or in vivo exposure to the feared stimulus (Obler and Terwilliger, 1970). Relaxation has often been replaced with other responses thought to be incompatible with anxiety such as eating (Jones, 1924). Another deviation from traditional SDT has been the delivery of positive reinforcement (food, praise, or toys) contingent upon the child's approaching the feared object (Obler and Terwilliger, 1970; Patterson, 1965). On the basis of such varied techniques, it is impossible to determine which of these procedures constitutes an effective treatment method for children. In the following study, three desensitization procedures involving graded exposure to the feared stimulus (darkness) through symbolic play, were compared to no-treatment and placebo control groups under differing instructional sets. Treatments were designed to determine the therapeutic efficacy of play as a method of stimulus presentation with children, and to determine whether reciprocal inhibition, extinction, operant reinforcement, or instructional set contribute to fear reduction in desensitization with children.