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Biomedical subjects

Ivone Gomes

Publications and source records attributed to Ivone Gomes.

12 recordsLinked to original sources
PubMed2006

Conformation state-sensitive antibodies to G-protein-coupled receptors.

A growing body of evidence indicates that G-protein-coupled receptors undergo complex conformational changes upon agonist activation. It is likely that the extracellular region, including the N terminus, undergoes activation-dependent conformational changes. We examined this by generating antibodies to regions within the N terminus of micro-opioid receptors. We find that antibodies to the midportion of the N-terminal tail exhibit enhanced recognition of activated receptors, whereas those to the distal regions do not. The enhanced recognition is abolished upon treatment with agents that block G-protein coupling or deglycosylate the receptor. This suggests that the N-terminal region of mu receptors undergoes conformational changes following receptor activation that can be selectively detected by these region-specific antibodies. We used these antibodies to characterize micro receptor type-specific ligands and find that the antibodies accurately differentiate ligands with varying efficacies. Next, we examined if these antibodies can be used to investigate the extent and duration of activation of endogenous receptors. We find that peripheral morphine administration leads to a time-dependent increase in antibody binding in the striatum and prefrontal cortex with a peak at about 30 min, indicating that these antibodies can be used to probe the spatio-temporal dynamics of native mu receptors. Finally, we show that this strategy of targeting the N-terminal region to generate receptor conformation-specific antisera can be applied to other G(alpha)(i)-coupled (delta-opioid, CB1 cannabinoid, alpha(2A)-adrenergic) as well as G(alpha)(s)-(beta(2)-adrenergic) and G(alpha)(q)-coupled (AT1 angiotensin) receptors. Taken together, these studies describe antisera as tools that allow, for the first time, studies probing differential conformation states of G-protein-coupled receptors, which could be used to identify molecules of therapeutic interest.

Analgesics, Opioid↗
PubMed2006

mu opioid and CB1 cannabinoid receptor interactions: reciprocal inhibition of receptor signaling and neuritogenesis.

Several studies have described functional interactions between opioid and cannabinoid receptors; the underlying mechanism(s) have not been well explored. One possible mechanism is direct receptor-receptor interactions, as has been demonstrated for a number of G-protein-coupled receptors. In order to investigate interactions between opioid and cannabinoid receptors, we epitope tagged mu, delta and kappa opioid receptors with Renilla luciferase and CB1 cannabinoid or CCR5 chemokine receptors with yellow fluorescent protein and examined the extent of substrate hydrolysis induced bioluminescence resonance energy transfer (BRET) signal. We find that coexpression of opioid receptors with cannabinoid receptors, but not with chemokine receptors, leads to a significant increase in the level of BRET signal, suggesting that the opioid-cannabinoid interactions are receptor specific. In order to examine the implications of these interactions to signaling, we used GTPgammaS binding and mitogen-activated protein kinase (MAPK) phosphorylation assays and examined the effect of receptor activation on signaling. We find that the mu receptor-mediated signaling is attenuated by the CB1 receptor agonist; this effect is reciprocal and is seen in heterologous cells and endogenous tissue expressing both receptors. In order to explore the physiological consequences of this interaction, we examined the effect of receptor activation on the extent of Src and STAT3 phosphorylation and neuritogenesis in Neuro-2A cells. We find that the simultaneous activation of mu opioid and CB1 cannabinoid receptors leads to a significant attenuation of the response seen upon activation of individual receptors, implicating a role for receptor-receptor interactions in modulating neuritogenesis.

Cells, Cultured↗
PubMed2005

Serotonin receptor activation leads to neurite outgrowth and neuronal survival.

Serotonin 5-HT1 receptors are implicated in anxiety and depression. These receptors belong to the family A of G-protein-coupled receptors and couple to inhibitory G-proteins. Recent studies show that chronic activation of 5-HT1A receptors leads to proliferation of hippocampal neurons suggesting that neurogenesis contributes to the effects of antidepressants. However, the molecular mechanisms and pathways involved are not understood. We used Neuro 2A cells transfected with 5-HT1A receptors and SK-N-SH cells endogenously expressing the receptor to examine the effect of receptor activation on neuronal survival and neurite outgrowth. We find that receptor activation leads to increased neurite outgrowth that can be blocked by the receptor selective antagonist and by treatment with pertussis toxin or lactacystin implicating inhibitory G-proteins and proteasomal degradation in this process. Interestingly, the small G-protein Rap and the transcription factor STAT-3 are also involved since reducing the levels of Rap1 (using small interfering RNA) or STAT-3 (using dominant negative STAT3) significantly blocks 5-HT1A-receptor-mediated neurite outgrowth. The observed increase in the phosphorylation of Src and STAT-3, at sites leading to their activation, further supports a crucial role for these proteins in neurite outgrowth. We also find that prolonged activation of endogenous 5-HT1A receptors leads to increased cell survival even under starving conditions; this is completely blocked by co-treatment with the antagonist. Taken together, these findings indicate that activation of the 5-HT1A receptor leads to a number of neurotropic events by activating a series of signal transduction molecules leading to long-term changes required for neurogenesis.

Animals↗
PubMed2005

The G alpha(o/i)-coupled cannabinoid receptor-mediated neurite outgrowth involves Rap regulation of Src and Stat3.

The study of the signaling pathways regulating neurite outgrowth in culture is important because of their potential role in neuronal differentiation in vivo. We have previously shown that the G alpha(o/i)-coupled CB1 cannabinoid receptor (CB1R) activates Rap1 to induce neurite outgrowth. G alpha(o/i) also activates the Src-Stat3 pathway. Here, we studied the relationship between the G alpha(o/i)-Rap1 and Src-Stat3 pathways and the role of these signaling pathways in CB1R-mediated neurite outgrowth in Neuro-2A cells. The CB1 agonist HU-210 induced pertussis toxin-sensitive Src and Stat3 phosphorylation. Dominant negative (DN) mutants of Src and Stat3 blocked CB1R-induced neurite outgrowth. Constitutively active Rap 1B and Ral-activated Src and CB1R-induced Src phosphorylation was inhibited by Rap1-DN and Ral-DN, indicating that both Rap1 and Ral mediate downstream signaling from G alpha(o/i) for Src activation. Rap1-activated Ral and Ral-DN blocked Rap-induced Src phosphorylation. G alpha(o)-induced Stat3 activation was blocked by Ral-DN, whereas v-Src-induced Stat3 activation was not inhibited by Ral-DN, indicating that the CB1R, through G alpha(o), mediates the sequential activation of Rap1 to Ral to Src to Stat3 in Neuro-2A cells. Downstream of Src, the CB1R also activated Rac1 and JNK, which enhanced CBR1-mediated Stat3 activation. Rac-DN blocked CB1R-induced activation of JNK. Pharmacological inhibition of JNK blocked Src and CB1R activation of Stat3, indicating that Rac and JNK are also involved in CB1R-mediated neurite outgrowth. Overall, this study demonstrated that G alpha(o/i)-coupled CB1R triggers neurite outgrowth in Neuro-2A through the activation of a signaling network containing two pathways that bifurcate at Src and converge at Stat3.

Animals↗
PubMed2005

Cannabinoid receptor-induced neurite outgrowth is mediated by Rap1 activation through G(alpha)o/i-triggered proteasomal degradation of Rap1GAPII.

The G(alpha)o/i-coupled CB1 cannabionoid receptor induces neurite outgrowth in Neuro-2A cells. The mechanisms of signaling through G(alpha)o/i to induce neurite outgrowth were studied. The expression of G(alpha)o/i reduces the stability of its direct interactor protein, Rap1GAPII, by targeting it for ubiquitination and proteasomal degradation. This results in the activation of Rap1. G(alpha)o/i-induced activation of endogenous Rap1 in Neuro-2A cells is blocked by the proteasomal inhibitor lactacystin. G(alpha)o/i stimulates neurite outgrowth that is blocked by the expression of dominant negative Rap1. Expression of Rap1GAPII also blocks the G(alpha)o/i-induced neurite outgrowth and treatment with proteasomal inhibitors potentiates this inhibition. The endogenous G(alpha)o/i-coupled cannabinoid (CB1) receptor in Neuro-2A cells stimulates the degradation of Rap1GAPII; activation of Rap1 and treatment with pertussis toxin or lactacystin blocks these effects. The CB1 receptor-stimulated neurite outgrowth is blocked by treatment with pertussis toxin, small interfering RNA for Rap, lactacystin, and expression of Rap1GAPII. Thus, the G(alpha)o/i-coupled cannabinoid receptor, by regulating the proteasomal degradation of Rap1GAPII, activates Rap1 to induce neurite outgrowth.

Amino Acid Sequence↗
PubMed2005

Mechanisms of action of antidepressants: from neurotransmitter systems to signaling pathways.

Antidepressants are commonly used in the treatment of anxiety and depression, medical conditions that affect approximately 17-20% of the population. The clinical effects of antidepressants take several weeks to manifest, suggesting that these drugs induce adaptive changes in brain structures affected by anxiety and depression. In order to develop shorter-acting and more effective drugs for the treatment of anxiety and depression, it is important to understand how antidepressants bring about their beneficial effects. Recent reports suggest that antidepressants can induce neurogenesis in the adult brain, although the mechanisms involved are not clearly understood. In this review, we describe the different neurotransmitter systems that are affected by anxiety and depression and how they are modulated by antidepressant treatment with a focus on signaling molecules and pathways that are activated during neurotransmitter receptor induced neurogenesis.

Animals↗
PubMed2004

A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia.

Opiates such as morphine are the choice analgesic in the treatment of chronic pain. However their long-term use is limited because of the development of tolerance and dependence. Due to its importance in therapy, different strategies have been considered for making opiates such as morphine more effective, while curbing its liability to be abused. One such strategy has been to use a combination of drugs to improve the effectiveness of morphine. In particular, delta opioid receptor ligands have been useful in enhancing morphine's potency. The underlying molecular basis for these observations is not understood. We propose the modulation of receptor function by physical association between mu and delta opioid receptors as a potential mechanism. In support of this hypothesis, we show that mu-delta interacting complexes exist in live cells and native membranes and that the occupancy of delta receptors (by antagonists) is sufficient to enhance mu opioid receptor binding and signaling activity. Furthermore, delta receptor antagonists enhance morphine-mediated intrathecal analgesia. Thus, heterodimeric associations between mu-delta opioid receptors can be used as a model for the development of novel combination therapies for the treatment of chronic pain and other pathologies.

Analgesia↗
PubMed2004

Uptake of liposomally entrapped adenosine-3'-5'-cyclic monophosphate in mouse brain.

[3H] Adenosine-3',5'-cyclic monophosphate (cAMP) could be entrapped efficiently into small unilamellar vesicles when bound to cAMP-dependent protein kinase. The leakage of [3H]cAMP protein kinase complex from liposomes was reduced by more than 60% as compared to free [3H]cAMP. Hyperosmolar mannitol increased the delivery of liposomally entrapped [3H]cAMP protein kinase to the brain with maximum uptake occurring at 10 min after mannitol administration. Optimal delivery to the brain was observed when vesicles composed of total brain lipids or phosphatidylcholine:cholesterol:sulfatides (7:2:1) were used. A slower clearance of liposomally entrapped material from brain tissue was seen under hyperosmolar conditions.

Animals↗
PubMed2004

Interactions between delta opioid receptors and alpha-adrenoceptors.

1. Several studies have reported functional interactions between different subtypes of opioid and alpha2A-adrenoceptors in the induction of spinal cord analgesia. The mechanisms underlying this phenomenon are not well characterized. We propose that direct receptor-receptor associations could account for some of the observed functional interactions. In the present study, we examined the presence of delta opioid receptors and alpha2A-adrenoceptors in interacting complexes and the functional implications of such interactions on receptor activity. 2. Using the proximity based bioluminescence resonance energy transfer (BRET) assay, we found that the delta opioid receptors and alpha2A-adrenoceptors are in close enough proximity (< 100 A) in live cells that can foster physical interactions. 3. Using coimmunoprecipitation of differentially epitope-tagged receptors, we found that delta opiate receptors exist in interacting complexes with alpha2A-adrenoceptors in heterologous cells. 4. Finally, using receptor activity mediated neurite outgrowth in Neuro 2A cells as a physiological readout, we found that interactions between delta opiate receptors and alpha2A-adrenoceptors have functional consequences. The expression of alpha2A-adrenoceptors is sufficient to promote delta opiate receptor-mediated neurite outgrowth, suggesting that the presence of inactive alpha2A-adrenoceptors can enhance delta opiate receptor-mediated signalling. 5. Taken together, these findings suggest that modulation of receptor function as a result of physical associations between delta opiate receptors and alpha2A-adrenoceptors may account for the observed synergy between opiate and adrenergic agonists in spinal analgesia.

Blotting, Western↗
PubMed2002

Oligomerization of opioid receptors.

Opioid receptors belong to the family of G-protein-coupled receptors characterized by their seven transmembrane domains. The activation of these receptors by agonists such as morphine and endogenous opioid peptides leads to the activation of inhibitory G-proteins followed by a decrease in the levels of intracellular cAMP. Opioid receptor activation is also associated with the opening of K(+) channels and the inhibition of Ca(2+) channels. A number of investigations, prior to the development of opioid receptor cDNAs, suggested that opioid receptor types interacted with each other. Early pharmacological studies provided evidence for the probable interaction between opioid receptors. More recent studies using receptor selective antagonists, antisense oligonucleotides, or animals lacking opioid receptors further suggested that interactions between opioid receptor types could modulate their activity. We examined opioid receptor interactions using biochemical, biophysical, and pharmacological techniques. We used differential epitope tagging and selective immunoisolation of receptor complexes to demonstrate homotypic and heterotypic interactions between opioid receptor types. We also used the proximity-based bioluminescence resonance energy transfer assay to explore opioid receptor-receptor interactions in living cells. In this article we describe the biochemical and biophysical methods involved in the detection of receptor dimers. We also address some of the concerns and suggest precautions to be taken in studies examining receptor-receptor interactions.

Animals↗