Mechanisms of inverse agonist action at D-2 dopamine receptors

1 Mechanisms of inverse agonist action at the D-2(short) dopamine receptor have been examined. 2 Discrimination of G-protein-coupled and -uncoupled forms of the receptor by inverse agonists was examined in competition ligand-binding studies versus the agonist [H-3]NPA at a concentration labelling both G-protein-coupled and -uncoupled receptors. 3 Competition of inverse agonists versus [H-3] NPA gave data that were fitted best by a two-binding site model in the absence of GTP but by a one-binding site model in the presence of GTP. K-i values were derived from the competition data for binding of the inverse agonists to G-protein-uncoupled and -coupled receptors. K-coupled and K-uncoupled were statistically different for the set of compounds tested ( ANOVA) but the individual values were different in a post hoc test only for (+)-butaclamol. 4 These observations were supported by simulations of these competition experiments according to the extended ternary complex model. 5 Inverse agonist efficacy of the ligands was assessed from their ability to reduce agonist-independent [S-35]GTPγ S binding to varying degrees in concentration-response curves. Inverse agonism by (+)-butaclamol and spiperone occurred at higher potency when GDP was added to assays, whereas the potency of (-)-sulpiride was unaffected. 6 These data show that some inverse agonists ((+)-butaclamol, spiperone) achieve inverse agonism by stabilising the uncoupled form of the receptor at the expense of the coupled form. For other compounds tested, we were unable to define the mechanism.

Diverse signalling by different chemokines through the chemokine receptor CCR5

We have investigated the signalling properties of the chemokine receptor, CCR5, using several assays for agonism: stimulation of changes in intracellular Ca(2+) or CCR5 internalisation in CHO cells expressing CCR5 or stimulation of [(35)S]GTPgammaS binding in membranes of CHO cells expressing CCR5. Four isoforms of the chemokine CCL3 with different amino termini (CCL3, CCL3(2-70), CCL3(5-70), CCL3L1) were tested in these assays in order to probe structure/activity relationships. Each isoform exhibited agonism. The pattern of agonism (potency, maximal effect) was different in the three assays, although the rank order was the same with CCL3L1 being the most potent and efficacious. The data show that the amino terminus of the chemokine is important for signalling. A proline at position 2 (CCL3L1) provides for high potency and efficacy but the isoform with a serine at position 2 (CCL3(2-70)) is as efficacious in some assays showing that the proline is not the only determinant of high efficacy. We also increased the sensitivity of CCR5 signalling by treating cells with sodium butyrate, thus increasing the receptor/G protein ratio. This allowed the detection of a change in intracellular Ca(2+) after treatment with CCL7 and Met-RANTES showing that these ligands possess measurable but low efficacy. This study therefore shows that sodium butyrate treatment increases the sensitivity of signalling assays and enables the detection of efficacy in ligands previously considered as antagonists. The use of different assay systems, therefore, provides different estimates of efficacy for some ligands at this receptor.

A low delta-9-tetrahydrocannabinol cannabis extract induces hyperphagia in rats

Appetite stimulation via partial agonism of cannabinoid type 1 receptors by Δ9tetrahydrocannabinol (Δ9THC) is well documented and can be modulated by non-Δ9THC phytocannabinoids. Δ9THC concentrations sufficient to elicit hyperphagia induce changes to both appetitive (reduced latency to feed) and consummatory (increased meal one size and duration) behaviours. Here, we show that a cannabis extract containing too little Δ9THC to stimulate appetite can induce hyperphagia solely by increasing appetitive behaviours. Twelve, male Lister hooded rats were presatiated before treatment with a low-Δ9THC cannabis extract (0.5, 1.0, 2.0 and 4.0 mg/kg). Hourly intake and meal pattern data were recorded and analyzed using one-way analyses of variance followed by Bonferroni post-hoc tests. The cannabis extract significantly increased food intake during the first hour of testing (at 4.0 mg/kg) and significantly reduced the latency to feed versus vehicle treatments (at doses ≥1.0 mg/kg). Meal size and duration were unaffected. These results show only the increase in appetitive behaviours, which could be attributed to non-Δ9THC phytocannabinoids in the extract rather than Δ9THC. Although further study is required to determine the constituents responsible for these effects, these results support the presence of non-Δ9THC cannabis constituent(s) that exert a stimulatory effect on appetite and likely lack the detrimental psychoactive effects of Δ9THC.

Cannabinol and cannabidiol exert opposing effects on rat feeding patterns

Rationale: Increased food consumption following Δ9- tetrahydrocannabinol-induced cannabinoid type 1 receptor agonism is well documented. However, possible non-Δ9- tetrahydrocannabinol phytocannabinoid-induced feeding effects have yet to be fully investigated. Therefore, we have assessed the effects of the individual phytocannabinoids, cannabigerol, cannabidiol and cannabinol, upon feeding behaviors. Methods: Adult male rats were treated (p.o.) with cannabigerol, cannabidiol, cannabinol or cannabinol plus the CB1R antagonist, SR141716A. Prior to treatment, rats were satiated and food intake recorded following drug administration. Data were analyzed for hourly intake and meal microstructure. Results: Cannabinol induced a CB1R-mediated increase in appetitive behaviors via significant reductions in the latency to feed and increases in consummatory behaviors via increases in meal 1 size and duration. Cannabinol also significantly increased the intake during hour 1 and total chow consumed during the test. Conversely, cannabidiol significantly reduced total chow consumption over the test period. Cannabigerol administration induced no changes to feeding behavior. Conclusion: This is the first time cannabinol has been shown to increase feeding. Therefore, cannabinol could, in the future, provide an alternative to the currently used and psychotropic Δ9-tetrahydrocannabinol-based medicines since cannabinol is currently considered to be non-psychotropic. Furthermore, cannabidiol reduced food intake in line with some existing reports, supporting the need for further mechanistic and behavioral work examining possible anti-obesity effects of cannabidiol.

Adrenergic receptor stimulation of the mitogen-activated protein kinase cascade and cardiac hypertrophy.

Phenylephrine and noradrenaline (alpha-adrenergic agonism) or isoprenaline (beta-adrenergic agonism) stimulated protein synthesis rates, increased the activity of the atrial natriuretic factor gene promoter and activated mitogen-activated protein kinase (MAPK). The EC50 for MAPK activation by noradrenaline was 2-4 microM and that for isoprenaline was 0.2-0.3 microM. Maximal activation of MAPK by isoprenaline was inhibited by the beta-adrenergic antagonist, propranolol, whereas the activation by noradrenaline was inhibited by the alpha1-adrenergic antagonist, prazosin. FPLC on a Mono-Q column separated two peaks of MAPK (p42MAPK and p44MAPK) and two peaks of MAPK-activating activity (MEK) activated by isoprenaline or noradrenaline. Prolonged phorbol ester exposure partially down-regulated the activation of MAPK by noradrenaline but not by isoprenaline. This implies a role for protein kinase C in MAPK activation by noradrenaline but not isoprenaline. A role for cyclic AMP in activation of the MAPK pathway was eliminated when other agonists that elevate cyclic AMP in the cardiac myocyte did not activate MAPK. In contrast, MAPK was activated by exposure to ionomycin, Bay K8644 or thapsigargin that elevate intracellular Ca2+. Furthermore, depletion of extracellular Ca2+ concentrations with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid (BAPTA) or blocking of the L-type Ca2+ channel with nifepidine or verapamil inhibited the response to isoprenaline without inhibiting the responses to noradrenaline. We conclude that alpha- and beta-adrenergic agonists can activate the MEK/MAPK pathway in the heart by different signalling pathways. Elevation of intracellular Ca2+ rather than cyclic AMP appears important in the activation of MAPK by isoprenaline in the cardiac myocyte.

Contested dominance modifies the anovulatory consequences of social subordination in female marmosets

Dominance status among female marmosets is reflected in agonistic behavior and ovarian function. Socially dominant females receive submissive behavior from subordinates, while exhibiting normal ovulatory function. Subordinate females, however, receive agonistic behavior from dominants, while exhibiting reduced or absent ovulatory function. Such disparity in female fertility is not absolute, and groups with two breeding females have been described. The data reported here were obtained from 8 female-female pairs of captive female marmosets, each housed with a single unrelated male. Pairs were classified into two groups: “uncontested” dominance (UD) and “contested” dominance (CD), with 4 pairs each. Dominant females in UD pairs showed significantly higher frequencies (4.1) of agonism (piloerection, attack and chasing) than their subordinates (0.36), and agonistic behaviors were overall more frequently displayed by CD than by UD pairs. Subordinates in CD pairs exhibited more agonistic behavior (2.9) than subordinates in UD pairs (0.36), which displayed significantly more submissive (6.97) behaviors than their dominants (0.35). The data suggest that there is more than one kind of dominance relationship between female common marmosets. Assessment of progesterone levels showed that while subordinates in UD pairs appeared to be anovulatory, the degree of ovulatory disruption in subordinates of CD pairs was more varied and less complete. We suggest that such variation in female-female social dominance relationships and the associated variation in the degree and reliability of fertility suppression may explain variations of the reproductive condition of free-living groups of common marmosets

Caracterização comportamental endócrina das fases ontogenéticas de sagüi comum (Callithrix jacchus)

The use of animal models in biomedical research is ever increasing. Models that use primates might also have advantages in terms of low maintenance costs and availability of biological knowledge, thereby favoring their use in different experimental protocols. Many current stress studies use animal models at different developmental stages since biological response differs during ontogeny. The aims of this study were to perform a detailed characterization of the developmental stages of common marmosets (Callithrix jacchus), a very important animal model used in biomedical research. Ten subjects, 6 females and 4 males, were followed from birth to initial adult age (16 months). Behavioral and fecal collection for measurement of adrenal (cortisol) and sex (progesterone, estradiol and androgens) hormones took place twice a week during the first month of life and once a week for the remainder of the study. Behavior was observed for 30 minutes in the morning (0700-09:00h) and afternoon (12:00-14:00h). Behavioral profile showed changes during ontogeny, characterizing the 4 developmental stages and the respective phases proposed by Leão et al (2009).. Differentiation of developmental stages was considered using the onset, end, change and stabilization of the behavioral profile parental care (weaning and carrying), ingestion (solid food), affiliation (social grooming) and autogrooming, agonism (scent marking and piloerection) and play behavior and endocrine profile. Infant weaning and carrying terminated within the infantile stage and the peak of solid food ingestion was recorded in the infantile III phase. Receiving grooming was recorded earlier than grooming performed by the infant and autogrooming. The first episode of scent marking was recorded in the 4th week and it was the least variable behavior, in terms of its onset, which, in almost all animals, was between the 5th and 7th week of life. Solitary play and play with the twin started around the 7th week and play with other members of the group started 8 weeks later. Sex hormone secretion started to differ from basal levels between the 21st and 23rd week of life, in males and females, suggesting that puberty occurs simultaneously in both sexes. Basal cortisol, even at an early age, was higher in females than in males. However, cortisol was not correlated with the juvenile stage, as expected, since this stage corresponds to the transition between infancy and adult age and most behaviors are intensified by this time. The behavioral and endocrine profile of subadult animals did not differ from that of the adults. These results provide more detailed parameters for the developmental process of C. jacchus and open new perspectives for the use of experimental approaches focused on the intermediate ontogenetic phases of this species

As competições sociais em grupos de Callithrix jacchus (Callitrichidae, Primates) são influenciadas pelo parentesco e pela convivência?

The Callitrichidae family is characterized by flexibility in its mating system, being possible to find monogamous, polyandrid and polygynic groups. Its social organization and mating system can be defined by the interaction between ecological and demographic factors plus the degree of relatedness among the individuals in the population. The objective of this work was to demonstrate the influence of relatedness and coexistence on the establishment and maintenance of social relations between Callithrix jacchus adult males. Four pairs of related adult males (CP), 4 pairs with coexistence between the animals in the pair (CC) and 4 pairs with no relatedness (SP) were studied. The pairs in the group CC had been kept in the same cage for at least 8 months before the experiment and the pairs in the group SP were put together at the beginning of the study. Each animal was observed 3 times/week for 2 months, in 15 min. sessions, through focal time sampling with instantaneous record each minute. In the first month, only the pair of males was kept in the cage (Phase I) and in the second month, a female was introduced into the cage (Phase II). The affiliative, agonistic and sexual interactions were registered. Affiliative interactions showed similar frequencies for all groups in phases. There was also no significant difference in the agonistic interactions of the CP, CC and SP males in Pase I, even considering that group SP exhibited higher levels of agonism. In Phase II, there was a statistically significant increase comparing to the others. The results demonstrate that relatedness and coexistence are of great importance for maintaining reprodutive and social stability inside the group. Nonetheless, in this study, only one of the males in the pair had sexual interations with the females, except for 2 pairs. This was assured through mate guarding and agonism directed to the potencial competitor. The most probable mating system would be functional monogamy, where the males would present low levels of competition, if there is relatedness and coexistence among them; on the other hand, a strong competition if there is no relatedness between the individuals. Even so, a polyandric system would also have to be considered

Relações sociais de callithrix jacchus machos: competição ou cooperação?

The group Callithrichidae is characterized by the flexibility in its mating system. This fact generates questions about the mechanisms that Callithrix jacchus males reach the breeding position in a group and how they prevent the subordinates reproduction, since the competition apparently happens with less intensity when compared to the females. Studies have shown the importance of social interactions and kinship among individuals in the distribution of mating opportunities in a group. This work aimed to evaluate if dyads of Callithrix jacchus males acted socially different due to kinship. We observed the agonistic and affiliative behaviours made by the males when associated in dyads (Phase 1) and when these dyads received a female (Phase 2), which was introduced in the pair`s cage for 45 minutes. In the phase 2, we also accounted for the sexual interactions between males and female in each trio. We used 16 males, 8 related (brothers-R) and 8 nonrelated (NR), as well as 8 females. Each phase lasted 4 weeks, with 3 observations/week, during 15 min/male in phase 1 and 2. In the first phase, two pairs of R males had a contested dominance and the other two, just like in the NR males, had an uncontested dominance. The NR dominant males were more aggressive than the subordinates. With the female`s entrance in the cage, all male pairs began to have an uncontested dominance. The NR males aggression frequency rose along with the subordinates` submission. In the R males, the frequency of grooming and physical contact was higher than in the NR males. So, we conclude that the low levels of agonism presented by the R males, along with the higher tolerance shown amongst them, lead us to believe that kinship attenuate the competition among these males

Correlato hormonal do comportamento reprodutivo de machos de sagüi comum (Callithrix jacchus) em ambiente natural

Contrary to what is recorded for Callithrix jacchus females, the social interactions and hormonal profiles of males are less studied, and mainly in wild groups. The goal of this study was to investigate the behavioral and endocrine profiles of reproductive (RMs) and non-reproductive (NRs) common marmoset adult free-ranging males living in two natural groups (GC1 and GR2). The groups inhabited the area of the Escola Agrícola de Jundiaí/UFRN, located in Macaíba, Brazil. Fecal collection for cortisol and androgen measurement and behavioral monitoring was carried out during the active phase from April to September, 2005. For behavioral data collection the focal instantaneous method was used every 5 min, for a total of 11.563 records. Statistical analysis was performed using non parametric tests and p < 0.05. Besides showing diurnal variation, the frequency of affiliative behaviors was significantly higher for RMs toward reproductive females than for NRs. Affiliative interactions of RMs with both reproductive females and NRs were similar, probably related to pair bond formation and helper recruitment, respectively. Parental care was also similar for both RMs and NRs. Both androgen and cortisol levels increased after the birth of the infants, mainly in RMs. The longitudinal profile of androgens fluctuates more in response to agonistic encounters and sexual behavior than that of cortisol. The mean basal excretion of both hormones was significantly higher in RMs and seems to reflect their higher participation in territorial vigilance and mate guarding behaviors. Significant positive correlations were found between agonism and cortisol and androgen hormones. These results describe, for the first time, the behavioral and hormonal profiles of common marmosets living in free-ranging groups and suggest that reproductive males are more responsive both behaviorally and hormonally to social group dynamics

Mode of Peroxisome Proliferator-Activated Receptor gamma Activation by Luteolin

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

Preliminary Fusion Testing Between Whole Young Colonies of Coptotermes gestroi (Isoptera: Rhinotermitidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Comportamento intra-específico do cupim Heterotermes tenuis (Hagen) (Isoptera, Rhinotermitidae) em condições de laboratório

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Preliminary studies on agonistic behavior between colonies of Coptotermes havilandi (Isoptera, Rhinotermitidae) in laboratory bioassays

Intercolonial aggression is being used to delimit foraging territory in termite species of subterranean termites. The advance of the introduced pest termite Coptotermes havilandi Holmgren in the interior of São Paulo State is increasing its economic impact as well as its interspecific and intraspecific competition in Brazil. In order to evaluate the intraspecific agonism among different colonies collected in urban areas of Sȧo Paulo State were set up a series of preliminary bioassays. Different combination of nestmates from field colonies of C. havilandi of Rio Claro city showed lack of agonistic behavior. Nevertheless, encounters among individuals from São Paulo and Rio Claro cities showed agonistic behaviors. These preliminary results suggest that caution should be taken in using intercolonial aggression to delimit the foraging territory of C. havilandi colonies in São Paulo State.

Investigação de agonismo tendencioso em α1A- e α1B-adrenoceptores

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)