POSTSYNAPTIC ALPHA-2 RECEPTOR STIMULATION IMPROVES MEMORY IN AGED MONKEYS - INDIRECT EFFECTS OF YOHIMBINE VERSUS DIRECT EFFECTS OF CLONIDINE

Very low doses (0.00001 mg/kg) of the alpha-2 adrenergic antagonist, yohimbine, improved working memory performance in a subset of aged monkeys. Improvement appeared to result from increased norepinephrine (NE) release onto postsynaptic alpha-2 adrenoceptors, as the response was blocked by the ''postsynaptic'' alpha-2 antagonist, SKF104078. Cognitive-enhancing effects of low dose yohimbine treatment may depend on aged animals retaining an intact, endogenous NE system. In contrast to yohimbine, the alpha-2 agonist, clonidine, has improved working memory in air aged animals examined. In the present study, clonidine's beneficial effects were also blocked by the postsynaptic antagonists SKF104078 and SKF104856, suggesting that clonidine acts by directly stimulating postsynaptic alpha-2 adrenoceptors. Beneficial doses of clonidine (0.01 mg/kg) and yohimbine (0.00001 mg/kg) were combined to see if they would produce additive effects on memory enhancement. This strategy was successful in young monkeys with intact NE systems but was not effective in the aged monkeys. These findings demonstrate that drugs that indirectly stimulate postsynaptic alpha-2 receptors by increasing NE release are not as reliable in aged monkeys as directly acting agonists that can replace NE at postsynaptic alpha-2 receptors.

Molecular modeling on some human interleukins sharing gamma c of interleukin-2 receptor, and structure-function relationships

Five models for human interleukin-7 (HIL-7), HIL-9, HIL-13, HIL-15 and HIL-17 have been generated by SYBYL software package. The primary models were optimized using molecular dynamics and molecular mechanics methods. The final models were optimized using a steepest descent algorithm and a subsequent conjugate gradient method. The complexes with these interleukins and the common gamma chain of interleukin-2 receptor (IL-2R) were constructed and subjected to energy minimization. We found residues, such as Gln127 and Tyr103, of the common gamma chain of IL-2R are very important. Other residues, e.g. Lys70, Asn128 and Glu162, are also significant. Four hydrophobic grooves and two hydrophilic sites converge at the active site triad of the gamma chain. The binding sites of these interleukins interaction with the common gamma chain exist in the first helical and/or the fourth helical domains. Therefore, we conclude that these interleukins binds to the common gamma chain of IL-2R by the first and the fourth helix domain. Especially at the binding sites of some residues (lysine, arginine, asparagine, glutamic acid and aspartic acid), with a discontinuous region of the common gamma chain of IL-2R, termed the interleukins binding sites (103-210). The study of these sites can be important for the development of new drugs. (C) 2000 Elsevier Science B.V. All rights reserved.

Evidence for a Direct and Functional Interaction between the Regulators of G Protein Signaling-2 and Phosphorylated C Terminus of Cholecystokinin-2 Receptor

Signaling of G protein-coupled receptors (GPCRs) is regulated by different mechanisms. One of these involves regulators of G protein signaling (RGS), which are diverse and multifunctional proteins that bind to active G alpha subunits of G proteins and act as GTPase-activating proteins. Little is known about the molecular mechanisms that govern the selective use of RGS proteins in living cells. We first demonstrated that CCK2R-mediated inositol phosphate production, known to be G(q-)dependent, is more sensitive to RGS2 than to RGS4 and is insensitive to RGS8. Both basal and agonist-stimulated activities of the CCK2R are regulated by RGS2. By combining biochemical functional, and in silico structural approaches, we demonstrate that a direct and functional interaction occurs between RGS2 and agonist-stimulated cholecystokinin receptor-2 (CCK2R) and identified the precise residues involved: phosphorylated Ser434 and Thr439 located in the C-terminal tail of CCK2R and Lys62, Lys63, and Gln67, located in the N-terminal domain of RGS2. These findings confirm previous reports that RGS proteins can interact with GPCRs to modulate their signaling and provide a molecular basis for RGS2 recognition by the CCK2R.

Mechanism of Activation of a G Protein-coupled Receptor, the Human Cholecystokinin-2 Receptor

G protein-coupled receptors (GPCRs) represent a major focus in functional genomics programs and drug development research, but their important potential as drug targets contrasts with the still limited data available concerning their activation mechanism. Here, we investigated the activation mechanism of the cholecystokinin-2 receptor (CCK2R). The three-dimensional structure of inactive CCK2R was homology-modeled on the basis of crystal coordinates of inactive rhodopsin. Starting from the inactive CCK2R modeled structure, active CCK2R (namely cholecystokinin-occupied CCK2R) was modeled by means of steered molecular dynamics in a lipid bilayer and by using available data from other GPCRs, including rhodopsin. By comparing the modeled structures of the inactive and active CCK2R, we identified changes in the relative position of helices and networks of interacting residues, which were expected to stabilize either the active or inactive states of CCK2R. Using targeted molecular dynamics simulations capable of converting CCK2R from the inactive to the active state, we delineated structural changes at the atomic level. The activation mechanism involved significant movements of helices VI and V, a slight movement of helices IV and VII, and changes in the position of critical residues within or near the binding site. The mutation of key amino acids yielded inactive or constitutively active CCK2R mutants, supporting this proposed mechanism. Such progress in the refinement of the CCK2R binding site structure and in knowledge of CCK2R activation mechanisms will enable target-based optimization of nonpeptide ligands.

Constitutive activation of the Raf-MAPK pathway causes negative feedback inhibition of Ras-PI3K-AKT and cellular arrest through the EphA(2) receptor

The Raf-mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinase (PI3K)-AKT pathways are two downstream effectors of the small GTPase Ras. Although both pathways are positively regulated by Ras, the Raf-MAPK and PI3K-AKT pathways have been shown to control opposing functions within the cell, suggesting a need for cross-talk regulation. The PI3K -AKT pathway can inhibit the Raf-MAPK pathway directly during processes such as muscle differentiation. Here we describe the ability of the Raf-MAPK pathway to negatively regulate the PI3K-AKT pathway during cellular arrest. Constitutive activation of Raf or methyl ethyl ketone 1 (MEK1) leads to inhibition of AKT and cellular arrest. Furthermore, we show that activation of Raf-MEK1 signaling causes negative feedback inhibition of Ras through the ephrin receptor EphA(2). EphA(2)-mediated negative feedback inhibition is required for Raf-induced AKT inhibition and cell cycle arrest, therefore establishing the inhibition of the Ras-PI3K-AKT pathway as a necessary event for the Raf-MEK1-regulated cellular arrest.

Proton pump inhibitors and histamine-2-receptor antagonists and pancreatic cancer risk: a nested case-control study

Background: The relationship between use of proton pump inhibitors (PPIs) and histamine-2-receptor antagonists (H₂RAs) and pancreatic cancer risk has yet to be examined. Data from a range of studies suggest biologically plausible mechanisms, whereby these drugs (or the conditions for which they are prescribed) may affect pancreatic cancer risk. The objective of this study was to investigate the relationship between use of PPIs/H₂RAs and pancreatic cancer risk.

Methods: A nested case – control study was conducted within the UK general practice research database (GPRD). Cases had a diagnosis of exocrine pancreatic cancer and controls were matched to cases on general practice site, sex and year of birth. Exposure to PPIs and to H₂RAs since entry into GPRD until 2 years before the diagnosis date (corresponding date in controls) and in the 5 years before the diagnosis date were separately assessed. Conditional logistic regression analyses were used to generate odds ratios (ORs) and 95% confidence intervals (CIs) associated with PPI or H₂RA use compared with nonuse.

Results: Ever use of PPIs since entry into the GPRD (excluding the 2 years prior to diagnosis) was not associated with risk of pancreatic cancer; OR (95% CI) 1.02 (0.85 – 1.22). Neither the dose nor the duration of PPI or H₂RA use was associated with pancreatic cancer risk. No consistent patterns of association were seen when cumulative exposure (dose and duration) to these drugs was examined separately or together.

Conclusion: PPI/H₂RA use, in a UK population, was not associated with pancreatic cancer risk.

Changes in lymphocyte subsets, interleukin 2, and soluble interleukin 2 receptor in old and very old age

In this study, the changes in some of the cellular components of the immune system and the activity of the cytokine interleukin 2, important for immune activation and lymphocyte proliferation, were measured in a large cross-sectional study of all age groups including octogenarian and nonagenarian subjects. In 206 apparently well community-living subjects, the absolute lymphocyte count and T and B cell numbers fell a little in old and very old subjects. Within the T cell compartment, helper/inducer CD4+ T cells, together with their subsets identified as 'naive' (CD4+/CD45RA+) and 'memory' (CD4+/CD45RO+) cells, also showed a decline with increased age. The suppressor/cytotoxic CD8+ subset showed no age-related change. The levels of the cytokine interleukin 2 were very low in octogenarian and nonagenarian subjects, while the soluble interleukin 2 receptor levels increased with increasing age. The interleukin 2 levels were associated with number and percentage of the 'memory' (CD4+/CD45RO+) subset of T cells which mediates the host response to previously met antigens. Since the interleukin 2 values were very low in the oldest groups and were associated with a reduced 'memory' (CD4+/CD45RO+) compartment, this suggests a possible mechanism of why the very elderly subject is more susceptible to morbidity and mortality from infectious or other agents.

Distinct CCK-2 Receptor Conformations Associated with β-Arrestin-2 Recruitment or Phospholipase-C Activation Revealed by a Biased Antagonist

Seven-transmembrane receptors (7TMRs), also termed G protein-coupled receptors (GPCRs), form the largest class of cell surface membrane receptors, involving several hundred members in the human genome. Near 30% of marketed pharmacological agents target 7TMRs. 7TMRs adopt multiple conformations upon agonist binding. Biased agonists, in contrast to non-biased agonists, are believed to stabilize conformations preferentially activating either G-protein- or ß-arrestin-dependent signalling pathways. However, proof that cognate conformations of receptors display structural differences within their binding site where biased agonism initiates, are still lacking. Here, we show that a non-biased agonist, cholecystokinin (CCK) induces conformational states of the CCK2R activating Gq-protein-dependent pathway (CCK2RG) or recruiting ß-arrestin2 (CCK2Rß) that are pharmacologically and structurally distinct. Two structurally unrelated antagonists competitively inhibited both pathways. A third ligand (GV150,013X), acted as a high affinity competitive antagonist on CCK2RG but was nearly inefficient as inhibitor of CCK2Rß. Several structural elements on both GV150,013X and in CCK2R binding cavity, which hinder binding of GV150,013X only to the CCK2Rß were identified. At last, proximity between two conserved amino acids from transmembrane helices 3 and 7 interacting through sulphur-aromatic interaction was shown to be crucial for selective stabilization of the CCK2Rß state. These data establish structural evidences for distinct conformations of a 7TMR associated with ß-arrestin-2 recruitment or G-protein coupling and validate relevance of the design of biased ligands able to selectively target each functional conformation of 7TMRs.

Functional coupling of the human dopamine D-2 receptor with G alpha(i1), G alpha(i2), G alpha(i3) and G alpha(o) G proteins: evidence for agonist regulation of G protein selectivity

1 The human dopamine D-2long (D-2L) receptor was expressed with four different G proteins in Sf9 cells using the baculovirus expression system. When co-expressed with G(i)/G(o) G proteins (G(i1)alpha, G(i2)alpha, G(i3)alpha, or G(o)alpha, plus Gbeta(1) and Ggamma(2)) the receptor displayed a high-affinity binding site for the agonists (dopamine and NPA), which was sensitive to GTP (100 mum), demonstrating interaction between the receptor and the different G proteins. 2 The receptor to G protein ratio (R: G ratio) was evaluated using [H-3]-spiperone saturation binding (R) and [S-35]-GTPgammaS saturation binding (G). R: G ratios of 1: 12, 1: 3, 1: 14 and 1: 5 were found for G(i1), G(i2), G(i3), and Go preparations, respectively. However, when R:G ratios of 1:2 and 1: 12 were compared for G(i2) and G(o), no difference was found for the stimulation of [S-35]-GTPgammaS binding. 3 Several agonists were tested for their ability to stimulate [S-35]-GTPgammaS binding to membranes co-expressing the receptor and various G proteins. All the compounds tested showed agonist activity in preparations expressing G(i3) and G(o). However, for G(i2) and G(i1) preparations, compounds such as S-(-)-3-PPP and p-tyramine were unable to stimulate [S-35]-GTPyS binding. 4 Most of the compounds showed higher relative efficacies (compared to dopamine) and higher potencies in the preparation expressing G(o). Comparison of the effects of different agonists in the different preparations showed that each agonist differentially activates the four G proteins. 5 We conclude that the degree of selectivity of G protein activation by the D-2L receptor can depend on the conformation of the receptor stabilised by an agonist.

Blockage of Angiotensin II type 2 receptor prevents thyroxine-mediated cardiac hypertrophy by blocking Akt activation

Although most of effects of Angiotensin II (Ang II) related to cardiac remodelling can be attributed to type 1 Ang II receptor (AT(1)R), the type 2 receptor (AT(2)R) has been shown to be involved in the development of some cardiac hypertrophy models. In the present study, we investigated whether the thyroid hormone (TH) action leading to cardiac hypertrophy is also mediated by increased Ang II levels or by change on AT(1)R and AT(2)R expression, which could contribute to this effect. In addition, we also evaluated the possible contribution of AT(2)R in the activation of Akt and in the development of TH-induced cardiac hypertrophy. To address these questions, Wistar rats were treated with thyroxine (T(4), 0.1 mg/kg BW/day, i.p.), with or without AT(2)R blocker (PD123319), for 14 days. Cardiac hypertrophy was identified based on heart/body weight ratio and confirmed by analysis of atrial natriuretic factor mRNA expression. Cardiomyocyte cultures were used to exclude the influence of TH-related hemodynamic effects. Our results demonstrate that the cardiac Ang II levels were significantly increased (80%, P < 0.001) as well as the AT(2)R expression (50%, P < 0.05) in TH-induced cardiac hypertrophy. The critical involvement of AT(2)R to the development of this cardiac hypertrophy in vivo was evidenced after administration of AT(2) blocker, which was able to prevent in 40% (P < 0.01) the cardiac mass gain and the Akt activation induced by TH. The role of AT(2)R to the TH-induced cardiomyocyte hypertrophy was also confirmed after using PD123319 in the in vitro studies. These findings improve understanding of the cardiac hypertrophy observed in hyperthyroidism and provide new insights into the generation of future therapeutic strategies.

Effects of Ischemia and Reperfusion on P2X(2) Receptor Expressing Neurons of the Rat Ileum Enteric Nervous System

Purpose We investigated the effects of ischemia/reperfusion in the intestine (I/R-i) on purine receptor P2X(2)-immunoreactive (IR) neurons of the rat ileum. Methods The superior mesenteric artery was occluded for 45 min with an atraumatic vascular clamp and animals were sacrificed 4 h later. Neurons of the myenteric and submucosal plexuses were evaluated for immunoreactivity against the P2X(2) receptor, nitric oxide synthase (NOS), choline acetyl transferase (ChAT), calbindin, and calretinin. Results Following I/R-i, we observed a decrease in P2X(2) receptor immunoreactivity in the cytoplasm and surface membranes of neurons of the myenteric and submucosal plexuses. These studies also revealed an absence of calbindin-positive neurons in the I/R-i group. In addition, the colocalization of the P2X(2) receptor with NOS, ChAT, and calretinin immunoreactivity in the myenteric plexus was decreased following I/R-i. Likewise, the colocalization between P2X(2) and calretinin in neurons of the submucosal plexus was also reduced. In the I/R-i group, there was a 55.8% decrease in the density of neurons immunoreactive (IR) for the P2X(2) receptor, a 26.4% reduction in NOS-IR neuron, a 25% reduction in ChAT-IR neuron, and a 47% reduction in calretinin-IR neuron. The density of P2X(2) receptor and calretinin-IR neurons also decreased in the submucosal plexus of the I/R-i group. In the myenteric plexus, P2X(2)-IR, NOS-IR, ChAT-IR and calretinin-IR neurons were reduced in size by 50%, 49.7%, 42%, and 33%, respectively, in the I/R-i group; in the submucosal plexus, P2X(2)-IR and calretinin-IR neurons were reduced in size by 56% and 72.6%, respectively. Conclusions These data demonstrate that ischemia/reperfusion of the intestine affects the expression of the P2X(2) receptor in neurons of the myenteric and submucosal plexus, as well as density and size of neurons in this population. Our findings indicate that I/R-i induces changes in P2X(2)-IR enteric neurons that could result in alterations in intestinal motility.

Regulation of angiotensin type 2 receptor in bovine granulosa cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The role of interleukin-10 in the differential expression of interleukin-12p70 and its beta 2 receptor on patients with active or treated paracoccidioidomycosis and healthy infected subjects

Paracoccidioidomycosis patients present an antigen-specific Th1 immunosuppression. To better understand this phenomenon, we evaluated the interleukin (IL)-12 pathway by measuring IL-12p70 production and CD3(+) T cell expression of the IL-12 receptor (IL-12R)beta1/beta2 chains, induced with the main fungus antigen (gp43) and a control antigen, from Candida albicans (CMA). We showed that gp43-induced IL-12p70 production and IL-12Rbeta2 expression were significantly decreased in acute and chronic patients as compared to healthy subjects cured from PCM or healthy infected subjects from endemic areas. Interestingly, the healthy infected Subjects had higher gp43-induced IL12p70 production and beta2 expression than the cured subjects. The addition of a neutralizing anti-IL-10 antibody to the cultures increased IL12p70 levels and beta2 expression in acute and chronic patients to levels observed in Cured subjects. Conversely, addition of the cytokine IL-10 strongly inhibited both parameters in the latter group. In conclusion, we have shown that paracoccidioidomycosis-related Th1 immunosuppression is associated with down-modulation of the IL-12 pathway, that IL-10 may participate in this process, and that patients cured from paracoccidioidomycosis may not fully recover their immune responsiveness. (C) 2004 Elsevier B.V. All rights reserved.