Identifying ligand-specific signalling within biased responses: focus on δ opioid receptor ligands.

UNLABELLED: Opioids activate GPCRs to produce powerful analgesic actions but at the same time induce side effects and generate tolerance, which restrict their clinical use. Reducing this undesired response profile has remained a major goal of opioid research and the notion of 'biased agonism' is raising increasing interest as a means of separating therapeutic responses from unwanted side effects. However, to fully exploit this opportunity, it is necessary to confidently identify biased signals and evaluate which type of bias may support analgesia and which may lead to undesired effects. The development of new computational tools has made it possible to quantify ligand-dependent signalling and discriminate this component from confounders that may also yield biased responses. Here, we analyse different approaches to identify and quantify ligand-dependent bias and review different types of confounders. Focus is on δ opioid receptor ligands, which are currently viewed as promising agents for chronic pain management. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Serotonin 4 receptor (5-HT4R) internalization is isoform-specific: effects of 5-HT and RS67333 on isoforms A and B.

Serotonin 4 receptors (5-HT4Rs) are particularly abundant within the limbic system, where they constitute potential targets for the development of novel, rapid acting antidepressants. However, the population of limbic 5-HT4Rs is not homogenous, comprising various isoforms of which 5-HT4(a) and 5-HT4(b) are among the most abundant variants. Sequence divergence at their C-termini is predictive of specificity in isoform signalling and regulation, but the differences, if any, remain ill-defined. The present study compared isoforms 5-HT4(a) and 5-HT4(b) in their ability to undergo endocytic regulation following exposure to 5-HT and to the putatively fast acting antidepressant RS67333. Both ligands differed in their ability to induce internalization of either isoform, 5-HT being more effective than RS67333 in HEK293 cells and in neurons. In contrast, trafficking induced by 5-HT was isoform-specific. In particular, while PKC, GRK2 and betaarrestin were necessary for 5-HT4(a)R internalization, sequestration of 5-HT4(b)Rs required PKC but not GRK2 and relied significantly less on betaarrestin. After endocytosis, isoform (b) appeared scattered throughout the intracellular compartment and efficiently recycled to the membrane upon agonist removal. Isoform (a) accumulated in the perinuclear compartment and displayed little recycling. Isoform-specific subcellular distribution was present in HEK293 cells and in neurons. In neurons, where internalization by RS67333 was more pronounced than in HEK293 cells, receptors internalized by this ligand followed the same distribution pattern as observed with 5-HT. These results point to isoform-related differences in the way that 5-HTRs respond to different ligands. Such diversity should be taken into account when developing therapeutic agents that target 5-HT4Rs.

Allosteric effects of G protein overexpression on the binding of beta-adrenergic ligands with distinct inverse efficacies.

Allosteric models of G protein-coupled receptors predict that G protein influences the spontaneous isomerization between inactive (R) and active (R*) conformations. Since inverse agonists have been proposed to preferentially bind to the inactive and uncoupled form(s), changes in the G protein content should influence the binding properties of these ligands. To test this hypothesis, we systematically assessed the effect of G proteins on the binding of beta(2)-adrenergic ligands with distinct levels of inverse efficacy. Recombinant baculoviruses encoding the human beta(2)-adrenoreceptor (beta(2)AR) were expressed alone or in combination with G protein subunits in Sf9 cells. Coexpression with the G protein alpha s beta 1 gamma 2 did not influence the relative efficacy of the ligands to inhibit the adenylyl cyclase but induced considerable decrease in number of sites detected by [(3)H]ICI 118551, [(3)H]propranolol, and (125)I-cyanopindolol. This loss was proportional to the inverse efficacy of the ligand used as the radiotracer in the assay. The addition of Gpp(NH)p inhibited the effects of G protein overexpression indicating that the G proteins acted allosterically. Consistent with this notion, Western blot analysis revealed that coexpression with the G proteins was not accompanied by a loss of immunoreactive beta(2)AR. Such allosteric effects of the G proteins were also observed in mammalian cells expressing endogenous level of G proteins indicating that the phenomenon is not unique to overexpression systems. Taken together, these results demonstrate that the apparent receptor number detected by radiolabeled inverse agonists is affected by the content in G proteins as a result of their influence on R/R* isomerization.

Short-term inverse-agonist treatment induces reciprocal changes in delta-opioid agonist and inverse-agonist binding capacity.

This study assessed the effects of short-term treatment (30-min) with inverse agonists on receptor protein levels and on the ability of agonists, inverse agonists, and neutral antagonists to bind to the human delta-opioid receptor (h delta OR). Incubation of human embryonic kidney 293s cells stably expressing h delta OR with the inverse agonist ICI174864 (1 microM) induced reciprocal changes in agonist and inverse-agonist binding. The total number of binding sites recognized by the agonists [(3)H]bremazocine and [(3)H][D-Pen(2),D-Pen(5)]-enkephalin was reduced by 33 and 57%, respectively, whereas binding capacity for the radiolabeled inverse-agonist [(3)H]Tyr-TicY[CH(2)NH]Cha-Phe-OH increased by 44%. In contrast, total receptor protein and sites labeled by neutral antagonists [(3)H]naltrindole and [(3)H]Tyr-D-Tic-Phe-Phe-OH remained unchanged. Pertussis toxin (PTX) and 5-guanylylimidodiphosphate (GppNHp) mimicked the outcome of ICI174864 pretreatment in promoting the loss of agonist binding sites. The lack of an additive effect on [(3)H]bremazocine binding when these three agents were combined indicates that inverse agonists may, in part, share the mechanism by which GppNHp and PTX reduce agonist binding capacity. Spontaneous recovery of maximal agonist binding capacity after inverse-agonist treatment was slow, suggesting a decrease in the isomerization rate between agonist- and inverse agonist-preferring conformations. Overall, the data presented are consistent with the idea that h delta ORs exist in multiple states capable of discriminating among ligands of different levels of efficacy and show that, after short-term treatment with an inverse agonist, the receptor ability to adopt conformations preferentially induced by agonist ligands is reduced.

Role of somatodendritic 5-HT autoreceptors in modulating 5-HT neurotransmission.

A very important element controlling serotonin (5-HT) release throughout the brain is the 5-HT1A autoreceptor present on the soma and dendrites of 5-HT neurons since it exerts a negative feedback influence on their firing activity. This 5-HT1A autoreceptor receives an increased activation by endogenous 5-HT at the beginning of a treatment with a selective 5-HT reuptake inhibitor (SSRI) and, consequently, a decreased 5-HT neuronal firing activity is obtained. As the SSRI treatment is prolonged, the 5-HT1A autoreceptor desensitizes and firing activity is restored in the presence of the SSRI. That this adaptive change underlies, at least in part, the delayed therapeutic effect of SSRI in major depression is supported by the acceleration of the antidepressant response by the concomitant administration of the 5-HT1A autoreceptor antagonist pindolol with SSRIs.

Tratamiento de la onicocriptosis. Matricectomía parcial con radiocirugía / Treatment of onychocryptosis. Partial matrixectomy with radio surgery

Presentamos un estudio propectivo, abierto, no comparativo, en 37 paciente con diagnóstico clínico de onicocriptosis, tratadas con radiocirugía en el Servicio de Dermatología del Hospital Universitario José E. González, en el periodo de julio de 1994 a septiembre de 1995. Nuestras conclusiones muestran a este método quirúrgico como una opción confiable y segura, donde se observan: pocas recidivas, baja morbilidad, disminución del sangrado y tiempo quirúrgico(SU)

Regulation of 5-hydroxytryptamine release from rat midbrain raphe nuclei by 5-hydroxytryptamine1D receptors: effect of tetrodotoxin, G protein inactivation and long-term antidepressant administration.

Our study was undertaken to characterize the functional properties of 5-hydroxytryptamine (5-HT)1D receptors in the rat midbrain raphe nuclei. In a first series of experiments, designed to assess whether 5-HT1D receptors are coupled to Gi/o proteins, the intracerebral injection of pertussis toxin into the dorsal raphe as well as incubation of midbrain raphe slices with the alkylating agent N-ethyl-maleimide (NEM) reduced the efficacy of the 5-HT1B/1D agonist sumatriptan to inhibit the electrically evoked overflow of [3H]5-HT from preloaded slices. Furthermore, preincubation with NEM also reduced the efficacy with which the 5-HT1B/1D antagonist GR 127935 enhanced evoked overflow of [3H]5-HT. These results indicate that, in rat midbrain raphe nuclei, 5-HT1D receptors are linked to Gi/o proteins. In an attempt to determine whether 5-HT1D receptors are located on 5-HT neurons, the inhibitory effect of sumatriptan and of the nonselective 5-HT agonist 5-carboxyamidotryptamine on K(+)-evoked overflow of [3H]5-HT was assessed in the presence of the Na+ channel blocker tetrodotoxin. Neither the inhibitory effect of sumatriptan nor that of 5-carboxyamidotryptamine were reduced by the addition of tetrodotoxin to the superfusion medium, suggesting that these 5-HT1D receptors are located on 5-HT neurons and may be considered autoreceptors. In a third series of experiments, rats were treated for 21 days either with the selective 5-HT reuptake inhibitor paroxetine (10 mg/kg/day, s.c.) or the reversible type A monoamine oxidase inhibitor befloxatone (0.75 mg/kg/day, s.c.) and superfusion experiments were performed after a 48-hr washout period. 5-HT1D receptors, similarly to 5-HT1A autoreceptors, desensitize after long-term treatment with a selective 5-HT reuptake inhibitor or a reversible type A monoamine oxidase inhibitor because the efficacy of sumatriptan and of 8-OH-DPAT to inhibit the electrically evoked overflow of [3H]5-HT was reduced after the administration of either drug.

Autoregulatory properties of dorsal raphe 5-HT neurons: possible role of electrotonic coupling and 5-HT1D receptors in the rat brain.

In the present study, the hypothesis that somatodendritic availability of 5-hydroxytryptamine (5-HT) could be regulated independently of the firing activity of dorsal raphe 5-HT neurons was tested. The 5-HT pathway was electrically stimulated at the level of the ventromedial tegmentum and the ensuing action potentials, recorded in the dorsal raphe, met all criteria for antidromic invasion of 5-HT neurons. The latency of antidromic spikes was current-dependent and the changes in latency were of quantal nature. This observation suggests an electrotonic coupling between 5-HT neurons. Stimulation of the ventromedial tegmentum also induced a decrease in the probability of firing of 5-HT neurons. This reduction in 5-HT neuron firing activity is a 5-HT-mediated response, due to an increased bioavailability of the neurotransmitter in the biophase of somatodendritic 5-HT1A autoreceptors. The intravenous administration of the 5-HT1 agonists TFMPP and RU 24969 reduced the duration of suppression of firing induced by the 5-HT-pathway stimulation, without altering the spontaneous firing rate of 5-HT neurons. The effect of TFMPP and RU 24969 on duration of suppression was blocked by (+-)mianserin, a drug with high affinity for the rat 5-HT1D, but not 5-HT1B, receptors. On the other hand, (-)propranolol, a mixed 5-HT antagonist also blocked the effect of TFMPP. However, the selective 5-HT1A antagonist (+)WAY 100135 did not alter the effect of TFMPP. These results, in keeping with previous anatomical studies, suggest the existence of electrotonic coupling of 5-HT neurons and indicate that 5-HT release in the rat dorsal raphe nucleus may be controlled independently of firing-regulating 5-HT1A autoreceptors. They also suggest that 5-HT1D receptors may play a role in this regulatory function of 5-HT neurons.

Regulation of [3H]5-HT release in raphe, frontal cortex and hippocampus of 5-HT1B knock-out mice.

Regulation of the electrically evoked release of [3H]5-HT was examined in midbrain, frontal cortex and hippocampus preloaded slices obtained from wild-type and 5-HT1B knock-out mice. In the absence of any drug [3H]5-HT released was increased in midbrain and hippocampus but not in frontal cortex slices of [3H]5-HT1B knock out mice. The selective 5-HT1B agonist CP 93129 and the 5-HT1B/1D agonist sumatriptan, inhibited [3H]5-HT release in hippocampus and cortical slices obtained from control mice but had no effect in mutants. In the two projection areas studied, the non-selective 5-HT agonist 5-carboxyamidotryptamine (5-CT) inhibited [3H]5-HT release in both groups of mice, indicating that additional 5-HT receptors, other than 5-HT1B, might be involved in the regulation of [3H]5-HT release from 5-HT terminals. In slices containing midbrain raphe nuclei, CP 93129 had no effect in either group. In contrast, sumatriptan inhibited [3H]5-HT release in controls and mutants. The latter effect was blocked by the 5-HT1D antagonist GR 127935, but not the 5-HT1A antagonist (+)WAY 100135, thus suggesting that a 5-HT1D-like receptor, possibly 5-HT1D alpha, negatively regulates 5-HT release in mouse midbrain raphe nuclei.

5-HT1D receptors regulate 5-HT release in the rat raphe nuclei. In vivo voltammetry and in vitro superfusion studies.

The aim of the present study was to characterize the pharmacological profile of 5-hydroxytryptamine (5-HT) receptors modulating 5-HT release in the mesencephalic raphe region. In a first series of experiments, differential normal pulse voltammetry and nafion-coated electrodes were used to measure extracellular 5-HT in the dorsal raphe of anesthesized rats. The intravenous administration of the selective 5-HT1A agonist 8-OH-DPAT (30 micrograms/kg) and the 5-HT1 agonist TFMPP (0.5 mg/kg) reduced the 5-hydroxyindole signal by 23% and 18%, respectively. Pretreatment with the 5-HT1A antagonist (+)WAY100135 (0.5 mg/kg IV) 30 minutes before the injection of the agonists, blocked the effect of 8-OH-DPAT but not that of TFMPP. The effect of TFMPP was blocked by (+/-)mianserin, a drug with high affinity for the rat 5-HT1D receptor, suggesting a role of this receptor subtype in the modulation of 5-HT release at the cell body level of 5-HT neurons. This was confirmed by in vitro superfusion experiments using mesencephalic raphe slices. The prototypical 5-HT1 agonist 5-carboxy-amiditryptamine (5-CT) and the 5-HT1B/1D agonist sumatriptan (1-1,000 nM) induced a concentration-dependent inhibition of the electrically evoked release of [3H]5-HT from preloaded raphe slices. 8-OH-DPAT (100 nM) produced an inhibitory effect similar to that of sumatriptan (100 nM). The selective 5-HT1B agonist CP 93129 (10-10,000 nM), had no effect in raphe slices, but it dose dependently inhibited [3H]5-HT release from hippocampal slices where autoreceptors are of the 5-HT1B subtype. The inhibitory effect of 5-CT was blocked by the 5-HT1/2 antagonist methiothepin (1 microM), the 5-HT1A antagonist S-UH-301 (1 microM), and the 5-HT1B/1D antagonist GR 127935 (1 microM). That of 8-OH-DPAT was blocked by S-UH-301 (1 microM) but not by GR 127935 (1 microM), and that of sumatriptan was blocked by GR 127935 (1 microM) but not by S-UH-301 (1 microM). These results show that, together with 5-HT1A autoreceptors, 5-HT1D receptors negatively modulate the somatodendritic release of 5-HT.

Effect of acute and prolonged tianeptine administration on the 5-HT transporter: electrophysiological, biochemical and radioligand binding studies in the rat brain.

In the present study, in vivo extracellular unitary recordings, in vitro [3H]5-HT uptake and [3H]cyanoimipramine binding assays were used to assess the effect of acute and prolonged administration of the putative antidepressant tianeptine, on the 5-hydroxytryptamine (5-HT) transporter. Microiontophoretic application of tianeptine onto dorsal hippocampus CA3 pyramidal neurons, as well as its intravenous administration (2 mg/kg), increased their firing frequency. Following intracerebroventricular administration of 5,7-dihydroxytryptamine, the activation induced by the microiontophoretic application of tianeptine remained unchanged, thus suggesting that the 5-HT carrier is not involved in this effect. Furthermore, in spite of its activating effect on CA3 pyramidal neuron firing frequency, the intravenous administration of tianeptine did not alter the time of recovery of these neurons from microiontophoretic applications of 5-HT, an index of 5-HT uptake activity. In keeping with this observation, the acute administration of tianeptine did not change the effectiveness of the 5-HT reuptake blocker paroxetine (1 mg/kg, i.v.) in prolonging the suppressant effect of microiontophoretically-applied 5-HT. However, in rats that had received tianeptine for 14 days (20 mg/kg/day, s.c.), the recovery time from the suppressant effect of microiontophoretic applications of 5-HT was reduced by 40% and the effectiveness of paroxetine (1 mg/kg, i.v.) was decreased. These effects were no longer observed following a 48 h washout period. In a second series of experiments, the ability of tianeptine to interfere with the uptake blocking capacity of paroxetine was assessed in vitro, using hippocampal slices obtained from rats that had been treated with tianeptine for 14 days (20 mg/kg/day, s.c.; by minipump).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of prolonged administration of tianeptine on 5-HT neurotransmission: an electrophysiological study in the rat hippocampus and dorsal raphe.

Extracellular unitary recordings of dorsal hippocampus CA3 pyramidal neurons and of dorsal raphe 5-hydroxytryptamine (5-HT) neurons were used to assess the effect of tianeptine, a putative antidepressant, on the efficacy of 5-HT neurotransmission. Sustained tianeptine administration (20 mg/kg/day, s.c. x 14 days) did not modify the firing activity of 5-HT neurons in the dorsal raphe. Their responsiveness to the intravenous injection of LSD, an agonist of the somatodendritic 5-HT autoreceptor, and of 8-OH-DPAT, a selective 5-HT1A agonist, was also unaffected by this treatment. The responsiveness of CA3 pyramidal neurons to microiontophoretic application of 5-HT remained unchanged after sustained tianeptine administration, but it was markedly enhanced in rats treated with repeated electroconvulsive shocks. Finally, the duration of suppression of firing activity of CA3 pyramidal neurons produced by electrical stimulation of the ascending 5-HT pathway, delivered at 1 Hz and 5 Hz, was not modified in rats treated with tianeptine. Methiothepin, an antagonist of the terminal autoreceptor enhanced the effectiveness of 5-HT pathway stimulation to the same extent in control and tianeptine-treated rats. The present results indicate that, administered at a dose known to stimulate 5-HT reuptake (20 mg/kg/day, s.c.; by minipump), and for a period of time (14 days) for which other antidepressant treatments have been shown to enhance 5-HT function, tianeptine does not modify the efficacy of 5-HT synaptic transmission in the rat hippocampus.

Desensitization of the neuronal 5-HT carrier following its long-term blockade.

In vivo extracellular unitary recordings, in vitro 3H-5-hydroxy-tryptamine (5-HT) uptake, and 3H-paroxetine binding assays were used to assess the effect of acute and long-term administration of the 5-HT reuptake inhibitor paroxetine on the neuronal 5-HT transporter in the rat dorsal hippocampus. Recovery time of the firing activity of CA3 hippocampus pyramidal neurons following microiontophoretic application of 5-HT was used as an index of in vivo reuptake activity. In a first series of experiments, the acute intravenous administration of paroxetine and 5-HT denervation with the neurotoxin 5,7-dihydroxytryptamine produced a marked prolongation of the suppressant effect of 5-HT, indicating that reuptake into 5-HT terminals plays a significant role in terminating the action of microiontophoretically applied 5-HT. In a second series of experiments, rats were treated with paroxetine (10 mg/kg/d, s.c.) for 2 or 21 d. In both treatment groups, there was a marked prolongation of the effect of microiontophoretically applied 5-HT; however, in rats treated for 2 d, the prolongation was significantly greater than that observed in rats treated for 21 d. After the 21 d treatment with paroxetine and a 48 hr washout, the prolongation of the effect of microiontophoretically applied 5-HT by acute intravenous paroxetine was significantly reduced, suggesting a decrease in the number of 5-HT carriers. In keeping with this interpretation, following the same treatment regimen, there was a 50% and 60% reduction of the in vitro 3H-5-HT uptake in hippocampal and dorsal raphe slices, respectively, and a reduced effectiveness of paroxetine in blocking 3H-5-HT uptake in both regions. The determination of the binding parameters of 3H-paroxetine revealed that, in rats treated for 21 d with paroxetine (10 mg/kg/d, s.c.), following a 48 hr washout Kd values were unchanged but Bmax values were reduced by 70% and 60% in hippocampal and cortical membranes, respectively.

Combined effects of midazolam and ethanol on sleep and on psychomotor performance in normal subjects.

A study was carried out on the effects of midazolam 15 mg in conjunction with ethanol 0.5 g/kg on objective and subjective sleep parameters and psychomotor performance in normal subjects. Midazolam significantly decreased total wake time. Total sleep time (TST) increase was related to larger amounts of stage 2 NREM sleep. Ethanol showed similar effects on sleep, although TST increase was associated with nonsignificant increments of NREM sleep and REM sleep. Ethanol slightly potentiated midazolam effects on sleep. Accordingly, total wake time, REM sleep time and number of wakes showed further depression than with midazolam alone. Subjective evaluations showed relatively good correlation with sleep laboratory findings. In addition, the different treatments did not impair subject's psychomotor performance the morning after their administration.