Regulation of Ras.GTP loading and Ras-Raf association in neonatal rat ventricular myocytes by G protein-coupled receptor agonists and phorbol ester. Activation of the extracellular signal-regulated kinase cascade by phorbol ester is mediated by Ras.

The small G protein Ras has been implicated in hypertrophy of cardiac myocytes. We therefore examined the activation (GTP loading) of Ras by the following hypertrophic agonists: phorbol 12-myristate 13-acetate (PMA), endothelin-1 (ET-1), and phenylephrine (PE). All three increased Ras.GTP loading by 10-15-fold (maximal in 1-2 min), as did bradykinin. Other G protein-coupled receptor agonists (e.g. angiotensin II, carbachol, isoproterenol) were less effective. Activation of Ras by PMA, ET-1, or PE was reduced by inhibition of protein kinase C (PKC), and that induced by ET-1 or PE was partly sensitive to pertussis toxin. 8-(4-Chlorophenylthio)-cAMP (CPT-cAMP) did not inhibit Ras.GTP loading by PMA, ET-1, or PE. The association of Ras with c-Raf protein was increased by PMA, ET-1, or PE, and this was inhibited by CPT-cAMP. However, only PMA and ET-1 increased Ras-associated mitogen-activated protein kinase kinase 1-activating activity, and this was decreased by PKC inhibition, pertussis toxin, and CPT-cAMP. PMA caused the rapid appearance of phosphorylated (activated) extracellular signal-regulated kinase in the nucleus, which was inhibited by a microinjected neutralizing anti-Ras antibody. We conclude that PKC- and Gi-dependent mechanisms mediate the activation of Ras in myocytes and that Ras activation is required for stimulation of extracellular signal-regulated kinase by PMA.

Activation of protein kinase cascades in the heart by hypertrophic G protein-coupled receptor agonists.

Cardiac myocyte hypertrophy involves changes in cell structure and alterations in protein expression regulated at both the transcriptional and translational levels. Hypertrophic G protein-coupled receptor (GPCR) agonists such as endothelin-(ET-1) and phenylephrine stimulate a number of protein kinase cascades in the heart. Mitogen-activated protein kinase (MAPK) cascades stimulated include the extracellularly regulated kinase cascade, the stress-activated protein kinase/c-Jun N-terminal kinase cascade, and the p38 MAPK cascade. All 3 pathways have been implicated in hypertrophy, but recent ex vivo evidence also suggests that there may be additional effects on cell survival. ET-1 and phenylephrine also stimulate the protein kinase B pathway, and this may be involved in the regulation of protein synthesis by these agonists. Thus, protein kinase-mediated signaling may be important in the regulation of the development of myocyte hypertrophy.

Natriorexigenic effect of baclofen is reduced by AT(1) receptor blockade in the lateral parabrachial nucleus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Kinin-B2 Receptor Mediated Neuroprotection after NMDA Excitotoxicity Is Reversed in the Presence of Kinin-B1 Receptor Agonists

Background: Kinins, with bradykinin and des-Arg(9)-bradykinin being the most important ones, are pro-inflammatory peptides released after tissue injury including stroke. Although the actions of bradykinin are in general well characterized; it remains controversial whether the effects of bradykinin are beneficial or not. Kinin-B2 receptor activation participates in various physiological processes including hypotension, neurotransmission and neuronal differentiation. The bradykinin metabolite des-Arg(9)-bradykinin as well as Lys-des-Arg(9)-bradykinin activates the kinin-B1 receptor known to be expressed under inflammatory conditions. We have investigated the effects of kinin-B1 and B2 receptor activation on N-methyl-Daspartate (NMDA)-induced excitotoxicity measured as decreased capacity to produce synaptically evoked population spikes in the CA1 area of rat hippocampal slices. Principal Findings: Bradykinin at 10 nM and 1 mu M concentrations triggered a neuroprotective cascade via kinin-B2 receptor activation which conferred protection against NMDA-induced excitotoxicity. Recovery of population spikes induced by 10 nM bradykinin was completely abolished when the peptide was co-applied with the selective kinin-B2 receptor antagonist HOE-140. Kinin-B2 receptor activation promoted survival of hippocampal neurons via phosphatidylinositol 3-kinase, while MEK/MAPK signaling was not involved in protection against NMDA-evoked excitotoxic effects. However, 100 nM Lys-des-Arg(9)-bradykinin, a potent kinin-B1 receptor agonist, reversed bradykinin-induced population spike recovery. The inhibition of population spikes recovery was reversed by PD98059,showing that MEK/MAPK was involved in the induction of apoptosis mediated by the B1 receptor. Conclusions: Bradykinin exerted protection against NMDA-induced excitotoxicity which is reversed in the presence of a kinin-B1 receptor agonist. As bradykinin is converted to the kinin-B1 receptor metabolite des-Arg(9)-bradykinin by carboxypeptidases, present in different areas including in brain, our results provide a mechanism for the neuroprotective effect in vitro despite of the deleterious effect observed in vivo.

Molecular analysis of Sigma-1 receptor modulation of the dopamine transporter

Sigma (σ) receptors are well established as a non-opioid, non-phencyclidine, and haloperidol-sensitive receptor family with its own binding profile and a characteristic distribution in the central nervous system (CNS) as well as in endocrine, immune, and some peripheral tissues. Two σ receptors subtypes, termed σ1 and σ2, have been pharmacologically characterized, but, to date, only the σ1 has also been cloned. Activation of σ1 receptors alter several neurotransmitter systems and dopamine (DA) neurotrasmission has been often shown to constitute an important target of σ receptors in different experimental models; however the exact role of σ1 receptor in dopaminergic neurotransmission remains unclear. The DA transporter (DAT) modulates the spatial and temporal aspects of dopaminergic synaptic transmission and interprer the primary mechanism by wich dopaminergic neurons terminate the signal transmission. For this reason present studies have been focused in understanding whether, in cell models, the human subtype of σ1 (hσ1) receptor is able to directly modulate the human DA transporter (hDAT). In the first part of this thesis, HEK-293 and SH-SY5Y cells were permanently transfected with the hσ1 receptor. Subsequently, they were transfected with another plasmid for transiently expressing the hDAT. The hDAT activity was estimated using the described [3H]DA uptake assay and the effects of σ ligands were evaluated by measuring the uptaken [3H]DA after treating the cells with known σ agonists and antagonists. Results illustrated in this thesis demonstrate that activation of overexpressed hσ1 receptors by (+)-pentazocine, the σ1 agonist prototype, determines an increase of 40% of the extracellular [3H]DA uptake, in comparison to non-treated controls and the σ1 antagonists BD-1047 and NE-100 prevent the positive effect of (+)-pentazocine on DA reuptake DA is likely to be considered a neurotoxic molecule. In fact, when levels of intracellular DA abnormally invrease, vescicles can’t sequester the DA which is metabolized by MAO (A and B) and COMT with consequent overproduction of oxygen reactive species and toxic catabolites. Stress induced by these molecules leads cells to death. Thus, for the second part of this thesis, experiments have been performed in order to investigate functional alterations caused by the (+)-pentazocine-mediated increase of DA uptake; particularly it has been investigated if the increase of intracellular [DA] could affect cells viability. Results obtained from this study demonstrate that (+)-pentazocine alone increases DA cell toxicity in a concentration-dependent manner only in cells co-expressing hσ1 and hDAT and σ1 antagonists are able to revert the (+)-pentazocine-induced increase of cell susceptibility to DA toxicity. In the last part of this thesis, the functional cross-talking between hσ1 receptor and hDAT has been further investigated using confocal microscopy. From the acquired data it could be suggested that, following exposure to (+)-pentazocine, the hσ1 receptors massively translocate towards the plasma membrane and colocalize with the hDATs. However, any physical interaction between the two proteins remains to be proved. In conclusion, the presented study shows for the first time that, in cell models, hσ1 receptors directly modulate the hDAT activity. Facilitation of DA uptake induced by (+)-pentazocine is reflected on the increased cell susceptibility to DA toxicity; these effects are prevented by σ1 selective antagonists. Since numerous compounds, including several drugs of abuse, bind to σ1 receptors and activating them could facilitate the damage of dopaminergic neurons, the reported protective effect showed by σ1 antagonists would represent the pharmacological basis to test these compounds in experimental models of dopaminergic neurodegenerative diseases (i.e. Parkinson’s Disease).

Glucagonlike peptide-1 receptor: an example of translational research in insulinomas: a review

Glucagonlike peptide-1 receptors (GLP-1R) play an increasingly important role in endocrine gastrointestinal tumor management. In particular, virtually all benign insulinomas express GLP-1R in high density. Exendin-4 is a GLP-1 analog that has a longer half-life than GLP-1. Targeting GLP-1R by (111)In-DOTA-exendin-4 or (111)In-DPTA-exendin-4 offers a new approach that permits the successful localization of small benign insulinomas. It is likely that this new noninvasive technique has the potential to replace the invasive localization by selective arterial stimulation and venous sampling.

Glucagon-like peptide-1 versus somatostatin receptor targeting reveals 2 distinct forms of malignant insulinomas

Glucagon-like peptide-1 (GLP-1) receptor imaging is superior to somatostatin receptor subtype 2 (sst(2)) imaging in localizing benign insulinomas. Here, the role of GLP-1 and sst(2) receptor imaging in the management of malignant insulinoma patients was investigated.

Glucagon-like peptide-1 receptor overexpression in cancer and its impact on clinical applications

Peptide hormones of the glucagon-like peptide (GLP) family play an increasing clinical role, such as GLP-1 in diabetes therapy. Moreover, GLP receptors are overexpressed in various human tumor types and therefore represent molecular targets for important clinical applications. In particular, virtually all benign insulinomas highly overexpress GLP-1 receptors (GLP-1R). Targeting GLP-1R with the stable GLP-1 analogs (111)In-DOTA/DPTA-exendin-4 offers a new approach to successfully localize these small tumors. This non-invasive technique has the potential to replace the invasive localization of insulinomas by selective arterial stimulation and venous sampling. Malignant insulinomas, in contrast to their benign counterparts, express GLP-1R in only one-third of the cases, while they more often express the somatostatin type 2 receptors. Importantly, one of the two receptors appears to be always expressed in malignant insulinomas. The GLP-1R overexpression in selected cancers is worth to be kept in mind with regard to the increasing use of GLP-1 analogs for diabetes therapy. While the functional role of GLP-1R in neoplasia is not known yet, it may be safe to monitor patients undergoing GLP-1 therapy carefully.

[Lys40(Ahx-DTPA-111In)NH2]-Exendin-4 is a highly efficient radiotherapeutic for glucagon-like peptide-1 receptor-targeted therapy for insulinoma

PURPOSE: Although metabolic changes make diagnosis of insulinoma relatively easy, surgical removal is hampered by difficulties in locating it, and there is no efficient treatment for malignant insulinoma. We have previously shown that the high density of glucagon-like peptide-1 receptors (GLP-1R) in human insulinoma cells provides an attractive target for molecular imaging and internal radiotherapy. In this study, we investigated the therapeutic potential of [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4, an (111)In-labeled agonist of GLP-1, in a transgenic mouse model of human insulinoma. EXPERIMENTAL DESIGN: [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4 was assessed in the Rip1Tag2 mouse model of pancreatic beta-cell carcinogenesis, which exhibits a GLP-1R expression comparable with human insulinoma. Mice were injected with 1.1, 5.6, or 28 MBq of the radiopeptide and sacrificed 7 days after injection. Tumor uptake and response, the mechanism of action of the radiopeptide, and therapy toxicity were investigated. RESULTS: Tumor uptake was >200% injected activity per gram, with a dose deposition of 3 Gy/MBq at 40 pmol [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4. Other GLP-1R-positive organs showed > or =30 times lower dose deposition. A single injection of [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4 resulted in a reduction of the tumor volume by up to 94% in a dose-dependent manner without significant acute organ toxicity. The therapeutic effect was due to increased tumor cell apoptosis and necrosis and decreased proliferation. CONCLUSIONS: The results suggest that [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4 is a promising radiopeptide capable of selectively targeting insulinoma. Furthermore, Auger-emitting radiopharmaceuticals such as (111)In are able to produce a marked therapeutic effect if a high tumor uptake is achieved.

Nonpeptide somatostatin receptor agonists specifically target ocular neovascularization via the somatostatin type 2 receptor

PURPOSE: To define the molecular pharmacology underlying the antiangiogenic effects of nonpeptide imidazolidine-2,4-dione somatostatin receptor agonists (NISAs) and evaluate the efficacy of NISA in ocular versus systemic delivery routes in ocular disease models. METHODS: Functional inhibitory effects of the NISAs and the somatostatin peptide analogue octreotide were evaluated in vitro by chemotaxis, proliferation, and tube-formation assays. The oxygen-induced retinopathy (OIR) model and the laser model of choroidal neovascularization (CNV) were used to test the in vivo efficacy of NISAs. Transscleral permeability of a candidate NISA was also measured. RESULTS: NISAs inhibited growth factor-induced HREC proliferation, migration and tube formation with submicromolar potencies (IC(50), 0.1-1.0 microM) comparable to octreotide. In the OIR model, systemic administration of the NISAs RFE-007 and RFE-011 inhibited retinal neovascularization in a dose-dependent manner, comparable to octreotide. In the CNV model, intravitreal RFE-011 resulted in a 56% reduction (P < 0.01) in CNV lesion area, whereas systemic administration resulted in a 35% reduction (P < 0.05) in lesion area. RFE-011 demonstrated transscleral penetration. CONCLUSIONS: Micromolar concentrations of octreotide and NISAs are necessary for antiangiogenic effects, whereas nanomolar concentrations are effective for endocrine inhibition. This suggests that the antiangiogenic activity of NISAs and octreotide is mediated by an overall much less efficient downstream coupling mechanism than is growth hormone release. As a result, the intravitreal or transscleral route of administration should be seriously considered for future clinical studies of SSTR2 agonists used for treatment of ocular neovascularization to ensure efficacious concentrations in the target retinal and choroidal tissue.

Glucagon-like peptide-1 receptor imaging for localization of insulinomas

The surgical removal of insulinomas is hampered by difficulties to localize it using conventional radiological procedures. Recently these tumors were shown to exhibit a very high density of glucagon-like peptide-1 receptors (GLP-1R) in vitro that may be used as specific targets for in vivo receptor radiolabeling.

Beta2-adrenergic receptor agonists reduce proliferation but not protein synthesis of periodontal fibroblasts stimulated with platelet-derived growth factor-BB

OBJECTIVE Catecholamines released from β-adrenergic neurons upon stress can interfere with periodontal regeneration. The cellular mechanisms, however, are unclear. Here, we assessed the effect of catecholamines on proliferation of periodontal fibroblasts. METHODS Fibroblasts from the gingiva and the periodontal ligament were exposed to agonists of the β-adrenergic receptors; isoproterenol (ISO, non-selective β-adrenergic agonist), salbutamol (SAL, selective β2-adrenergic receptor agonist) and BRL 37344 (BRL selective β3-receptor agonist). Proliferation was stimulated with platelet-derived growth factor-BB (PDGF-BB). Pharmacological inhibitors and gene expression analysis further revealed β-adrenergic signalling. RESULTS Gingiva and periodontal ligament fibroblast express the β2-adrenergic receptor. ISO and SAL but not BRL decreased proliferation of fibroblasts in the presence of PDGF-BB. The inhibitory effect of β-adrenergic signalling on proliferation but not protein synthesis in response to PDGF-BB was reduced by propranolol, a non-selective β-adrenergic antagonist. CONCLUSIONS These results suggest that β2-receptor agonists can reduce the mitogenic response of periodontal fibroblasts. These data add to the compelling concept that blocking of β2-receptor signalling can support tissue maintenance and regeneration.

Preoperative Glucagon-like peptide-1 receptor imaging reduces surgical trauma and pancreatic tissue loss in insulinoma patients: a report of three cases

BACKGROUND Insulinomas are rare tumors, in the majority of cases best treated by surgical resection. Preoperative localization of insulinoma is challenging. The more precise the preoperative localization the less invasive and safer is the resection. The purpose of the study is to check the impact of a new technique to localize insulinoma on the surgical strategy. FINDINGS We present exact preoperative localization with Glucagon-like peptide-1 receptor (GLP-1R) imaging. This allows a more precise resection thereby reducing surgical access trauma, loss of healthy pancreatic tissue and increasing safety and quality of the surgical intervention. CONCLUSION With the help of precise preoperative localization of insulinoma with GLP-1R imaging the surgeon is able to minimize the amount of resected healthy pancreatic tissue. We hypothesize that GLP-1R imaging will become a preoperative diagnostic tool to be used for many patients scheduled for open or laparoscopic insulinoma resection.

Is liraglutide or exenatide better in type 2 diabetes?

Background: Subjects with type 2 diabetes have high circulating levels of glucose. Glucagon-like peptide-1 (GLP-1) is an intestinal hormone that has a major role in glucose homeostasis. Exenatide and liraglutide are both agonists at the GLP-1 receptor, and are effective at reducing circulating glucose levels (measured as HbA1c levels), but they have not been compared. Objectives/methods: This evaluation is of a clinical trial comparing liraglutide once a day with exenatide twice a day in subjects with type 2 diabetes. Results: In the Liraglutide Effect and Action in Diabetes (LEAD)-6 trial, subcutaneous liraglutide 1.8 mg once a day was compared with exenatide 10 μg twice a day. The primary efficacy outcome was change in HbA1c levels, and this was significantly greater with liraglutide (1.12%) than with exenatide (0.79%). Liraglutide and exenatide had similar small abilities to reduce body weight, blood pressure and LDL-cholesterol. Conclusions: Liraglutide was more effective than exenatide for overall glycaemic control in subjects with type 2 diabetes. However, this is only true for the preparations and doses tested, that is liraglutide 1.8 mg once weekly and exenatide 10 μg b.i.d., and may not apply when the comparison is undertaken with the new longer-lasting preparation of exenatide once weekly.

The neurokinin 1 receptor antagonist, ezlopitant, reduces appetitive responding for sucrose and ethanol

Abstract Background: The current obesity epidemic is thought to be partly driven by over-consumption of sugar-sweetened diets and soft drinks. Loss-of-control over eating and addiction to drugs of abuse share overlapping brain mechanisms including changes in motivational drive, such that stimuli that are often no longer ‘liked’ are still intensely ‘wanted’ [7,8]. The neurokinin 1 (NK1) receptor system has been implicated in both learned appetitive behaviors and addiction to alcohol and opioids; however, its role in natural reward seeking remains unknown. Methodology/Principal Findings: We sought to determine whether the NK1-receptor system plays a role in the reinforcing properties of sucrose using a novel selective and clinically safe NK1-receptor antagonist, ezlopitant (CJ-11,974), in three animal models of sucrose consumption and seeking. Furthermore, we compared the effect of ezlopitant on ethanol consumption and seeking in rodents. The NK1-receptor antagonist, ezlopitant decreased appetitive responding for sucrose more potently than for ethanol using an operant self-administration protocol without affecting general locomotor activity. To further evaluate the selectivity of the NK1-receptor antagonist in decreasing consumption of sweetened solutions, we compared the effects of ezlopitant on water, saccharin-, and sodium chloride (NaCl) solution consumption. Ezlopitant decreased intake of saccharin but had no effect on water or salty solution consumption. Conclusions/Significance: The present study indicates that the NK1-receptor may be a part of a common pathway regulating the self-administration, motivational and reinforcing aspects of sweetened solutions, regardless of caloric value, and those of substances of abuse. Additionally, these results indicate that the NK1-receptor system may serve as a therapeutic target for obesity induced by over-consumption of natural reinforcers.