The Endogenous Retinoic Acid Receptor Pathway Is Exploited by Mycobacterium tuberculosis during Infection, Both In Vitro and In Vivo.

Retinoic acid (RA) is a fundamental vitamin A metabolite involved in regulating immune responses through the nuclear RA receptor (RAR) and retinoid X receptor. While performing experiments using THP-1 cells as a model for Mycobacterium tuberculosis infection, we observed that serum-supplemented cultures displayed high levels of baseline RAR activation in the presence of live, but not heat-killed, bacteria, suggesting that M. tuberculosis robustly induces the endogenous RAR pathway. Using in vitro and in vivo models, we have further explored the role of endogenous RAR activity in M. tuberculosis infection through pharmacological inhibition of RARs. We found that M. tuberculosis induces classical RA response element genes such as CD38 and DHRS3 in both THP-1 cells and human primary CD14+ monocytes via a RAR-dependent pathway. M. tuberculosis-stimulated RAR activation was observed with conditioned media and required nonproteinaceous factor(s) present in FBS. Importantly, RAR blockade by (4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphthalen-2-yl]ethenyl]benzoic acid), a specific pan-RAR inverse agonist, in a low-dose murine model of tuberculosis significantly reduced SIGLEC-F+CD64+CD11c+high alveolar macrophages in the lungs, which correlated with 2× reduction in tissue mycobacterial burden. These results suggest that the endogenous RAR activation axis contributes to M. tuberculosis infection both in vitro and in vivo and reveal an opportunity for further investigation of new antituberculosis therapies.

Monoacylglycerol Lipase Inhibition Prevents Short-Term Mitochondrial Dysfunction and Oxidative Damage in Rat Brain Synaptosomal/Mitochondrial Fractions and Cortical Slices: Role of Cannabinoid Receptors.

Inhibition of enzymes responsible for endocannabinoid hydrolysis represents an invaluable emerging tool for the potential treatment of neurodegenerative disorders. Monoacylglycerol lipase (MAGL) is the enzyme responsible for degrading 2-arachydonoylglycerol (2-AG), the most abundant endocannabinoid in the central nervous system (CNS). Here, we tested the effects of the selective MAGL inhibitor JZL184 on the 3-nitropropinic acid (3-NP)-induced short-term loss of mitochondrial reductive capacity/viability and oxidative damage in rat brain synaptosomal/mitochondrial fractions and cortical slices. In synaptosomes, while 3-NP decreased mitochondrial function and increased lipid peroxidation, JZL184 attenuated both markers. The protective effects evoked by JZL184 on the 3-NP-induced mitochondrial dysfunction were primarily mediated by activation of cannabinoid receptor 2 (CB2R), as evidenced by their inhibition by the selective CB2R inverse agonist JTE907. The cannabinoid receptor 1 (CB1R) also participated in this effect in a lesser extent, as evidenced by the CB1R antagonist/inverse agonist AM281. In contrast, activation of CB1R, but not CB2R, was responsible for the protective effects of JZL184 on the 3-NP-iduced lipid peroxidation. Protective effects of JZL184 were confirmed in other toxic models involving excitotoxicity and oxidative damage as internal controls. In cortical slices, JZL184 ameliorated the 3-NP-induced loss of mitochondrial function, the increase in lipid peroxidation, and the inhibition of succinate dehydrogenase (mitochondrial complex II) activity, and these effects were independent on CB1R and CB2R, as evidenced by the lack of effects of AM281 and JTE907, respectively. Our novel results provide experimental evidence that the differential protective effects exerted by JZL184 on the early toxic effects induced by 3-NP in brain synaptosomes and cortical slices involve MAGL inhibition, and possibly the subsequent accumulation of 2-AG. These effects involve pro-energetic and redox modulatory mechanisms that may be either dependent or independent of cannabinoid receptors' activation.

The chronic pharmacological antagonism of the CB1 receptor is not involved in the behavioral effects of antidepressants administered in mice submitted to chronic unpredictable stress.

Several pieces of evidence suggest that the monoaminergic theory of depression cannot fully explain all behavioral and neuroplastic changes observed after antidepressant chronic treatment. Other molecular targets, such as the endocannabinoid system, have been associated with the chronic effects of these drugs. In the present study, we hypothesized that the behavioral and neuroplastic effects observed after repeated treatment with the antidepressants (AD) Escitalopram (ESC) or venlafaxine (VFX) in chronically stressed mice depend on CB1 receptor activation. Male mice submitted to the chronic unpredictable stress (CUS) paradigm for 21 days were treated with Esc (10 mg/kg) or VFX (20 mg/kg) once a day in the presence or not of AM251 (0.3 mg/kg), a CB1 receptor antagonist/inverse agonist. At the end of the CUS paradigm, we conducted behavior tests to evaluate depressive- and anxiety-like behaviors. Our results demonstrated that chronic blockade of the CB1 receptor does not attenuate the antidepressant- or the anxiolytic-like effects of ESC nor VFX. ESC increased the expression of CB1 in the hippocampus, but AM251 did not change the pro-proliferative effects of ESC in the dentate gyrus or the increased expression of synaptophysin induced by this AD in the hippocampus. Our results suggest that CB1 receptors are not involved in behavioral and hippocampal neuroplastic effects observed after repeated antidepressant treatment in mice submitted to CUS.

Evidence for Endogenous Opioid Dependence Related to Latent Sensitization in a Rat Model of Chronic Inflammatory Pain.

Studies performed in a mouse model of chronic inflammatory pain induced by intraplantar injection of complete Freund's adjuvant (CFA) have shown that constitutive activation of the endogenous opioid signaling, besides serving as a mechanism of endogenous analgesia that tonically represses pain sensitization, also generates a state of endogenous opioid dependence. Since species-related differences concerning pain biology and addictive behaviors occur between mice and rats, the present study explored whether the coexistence of endogenous opioid analgesia and endogenous opioid dependence also characterizes a homologous rat model. To this aim, CFA-injured Wistar rats were treated with either 3 mg/kg or 10 mg/kg of the opioid receptor inverse agonist naltrexone (NTX) during the pain remission phase and monitored for 60 min for possible withdrawal behaviors. At 3 mg/kg, NTX, besides inducing the reinstatement of mechanical allodynia, also caused a distinct appearance of ptosis, with slight but nonsignificant changes to the occurrence of teeth chatters and rearing. On the other hand, 10 mg/kg of NTX failed to unmask pain sensitization and induced significantly lower levels of ptosis than 3 mg/kg. Such an NTX-related response pattern observed in the rat CFA model seems to differ substantially from the pattern previously described in the mouse CFA model. This supports the knowledge that mice and rats are not identical in terms of pharmacological response and stresses the importance of choosing the appropriate species for preclinical pain research purposes depending on the scientific question being asked.

Chlorpromazine, an Inverse Agonist of D1R-Like, Differentially Targets Voltage-Gated Calcium Channel (CaV) Subtypes in mPFC Neurons.

The dopamine receptor type 1 (D1R) and the dopamine receptor type 5 (D5R), which are often grouped as D1R-like due to their sequence and signaling similarities, exhibit high levels of constitutive activity. The molecular basis for this agonist-independent activation has been well characterized through biochemical and mutagenesis in vitro studies. In this regard, it was reported that many antipsychotic drugs act as inverse agonists of D1R-like constitutive activity. On the other hand, D1R is highly expressed in the medial prefrontal cortex (mPFC), a brain area with important functions such as working memory. Here, we studied the impact of D1R-like constitutive activity and chlorpromazine (CPZ), an antipsychotic drug and D1R-like inverse agonist, on various neuronal CaV conductances, and we explored its effect on calcium-dependent neuronal functions in the mouse medial mPFC. Using ex vivo brain slices containing the mPFC and transfected HEK293T cells, we found that CPZ reduces CaV2.2 currents by occluding D1R-like constitutive activity, in agreement with a mechanism previously reported by our lab, whereas CPZ directly inhibits CaV1 currents in a D1R-like activity independent manner. In contrast, CPZ and D1R constitutive activity did not affect CaV2.1, CaV2.3, or CaV3 currents. Finally, we found that CPZ reduces excitatory postsynaptic responses in mPFC neurons. Our results contribute to understanding CPZ molecular targets in neurons and describe a novel physiological consequence of CPZ non-canonical action as a D1R-like inverse agonist in the mouse brain.

Constitutive activity of the dopamine (D5 ) receptor, highly expressed in CA1 hippocampal neurons, selectively reduces CaV 3.2 and CaV 3.3 currents.

BACKGROUND AND PURPOSE: CaV 3.1-3 currents differentially contribute to neuronal firing patterns. CaV 3 are regulated by G protein-coupled receptors (GPCRs) activity, but information about CaV 3 as targets of the constitutive activity of GPCRs is scarce. We investigate the impact of D5 recpetor constitutive activity, a GPCR with high levels of basal activity, on CaV 3 functionality. D5 recpetor and CaV 3 are expressed in the hippocampus and have been independently linked to pathophysiological states associated with epilepsy. EXPERIMENTAL APPROACH: Our study models were HEK293T cells heterologously expressing D1 or D5 receptor and CaV 3.1-3, and mouse brain slices containing the hippocampus. We used chlorpromazine (D1 /D5 inverse agonist) and a D5 receptor mutant lacking constitutive activity as experimental tools. We measured CaV 3 currents and excitability parameters using the patch-clamp technique. We completed our study with computational modelling and imaging technique. KEY RESULTS: We found a higher sensitivity to TTA-P2 (CaV 3 blocker) in CA1 pyramidal neurons obtained from chlorpromazine-treated animals compared with vehicle-treated animals. We found that CaV 3.2 and CaV 3.3-but not CaV 3.1-are targets of D5 receptor constitutive activity in HEK293T cells. Finally, we found an increased firing rate in CA1 pyramidal neurons from chlorpromazine-treated animals in comparison with vehicle-treated animals. Similar changes in firing rate were observed on a neuronal model with controlled CaV 3 currents levels. CONCLUSIONS AND IMPLICATIONS: Native hippocampal CaV 3 and recombinant CaV 3.2-3 are sensitive to D5 receptor constitutive activity. Manipulation of D5 receptor constitutive activity could be a valuable strategy to control neuronal excitability, especially in exacerbated conditions such as epilepsy.

Toward a consensus nomenclature for ghrelin, its non-acylated form, liver expressed antimicrobial peptide 2 and growth hormone secretagogue receptor.

The stomach-derived octanoylated peptide ghrelin was discovered in 1999 and recognized as an endogenous agonist of the growth hormone secretagogue receptor (GHSR). Subsequently, ghrelin has been shown to play key roles in controlling not only growth hormone secretion, but also a variety of other physiological functions including, but not limited to, food intake, reward-related behaviors, glucose homeostasis and gastrointestinal tract motility. Importantly, a non-acylated form of ghrelin, desacyl-ghrelin, can also be detected in biological samples. Desacyl-ghrelin, however, does not bind to GHSR at physiological levels, and its physiological role has remained less well-characterized than that of ghrelin. Ghrelin and desacyl-ghrelin are currently referred to in the literature using many different terms, highlighting the need for a consistent nomenclature. The variability of terms used to designate ghrelin can lead not only to confusion, but also to miscommunication, especially for those who are less familiar with the ghrelin literature. Thus, we conducted a survey among experts who have contributed to the ghrelin literature aiming to identify whether a consensus may be reached. Based on the results of this consensus, we propose using the terms "ghrelin" and "desacyl-ghrelin" to refer to the hormone itself and its non-acylated form, respectively. Based on the results of this consensus, we further propose using the terms "GHSR" for the receptor, and "LEAP2" for liver-expressed antimicrobial peptide 2, a recently recognized endogenous GHSR antagonist/inverse agonist.

Biased agonism at histamine H1 receptor: Desensitization, internalization and MAPK activation triggered by antihistamines.

Histamine H1 receptor ligands used clinically as antiallergics rank among the most widely prescribed and over-the-counter drugs in the world. They exert the therapeutic actions by blocking the effects of histamine, due to null or negative efficacy towards Gαq-phospholipase C (PLC)-inositol triphosphates (IP3)-Ca2+ and nuclear factor-kappa B cascades. However, there is no information regarding their ability to modulate other receptor responses. The aim of the present study was to investigate whether histamine H1 receptor ligands could display positive efficacy concerning receptor desensitization, internalization, signaling through Gαq independent pathways or even transcriptional regulation of proinflammatory genes. While diphenhydramine, triprolidine and chlorpheniramine activate ERK1/2 (extracellular signal-regulated kinase 1/2) pathway in A549 cells, pre-treatment with chlorpheniramine or triprolidine completely desensitize histamine H1 receptor mediated Ca2+ response, and both diphenhydramine and triprolidine lead to receptor internalization. Unlike histamine, histamine H1 receptor desensitization and internalization induced by antihistamines prove to be independent of G protein-coupled receptor kinase 2 (GRK2) phosphorylation. Also, unlike the reference agonist, the recovery of the number of cell-surface histamine H1 receptors is a consequence of de novo synthesis. On the other hand, all of the ligands lack efficacy regarding cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) mRNA regulation. However, a prolonged exposure with each of the antihistamines impaires the increase in COX-2 and IL-8 mRNA levels induced by histamine, even after ligand removal. Altogether, these findings demonstrate the biased nature of histamine H1 receptor ligands contributing to a more accurate classification, and providing evidence for a more rational and safe use of them.

Advances in the Understanding of the Cannabinoid Receptor 1 - Focusing on the Inverse Agonists Interactions.

BACKGROUND: Cannabinoid Receptor 1 (CB1) is a membrane protein prevalent in the central nervous system, whose crystallographic structure has recently been solved. Studies will be needed to investigate CB1 complexes with its ligands and its role in the development of new drugs. OBJECTIVE: Our goal here is to review the studies on CB1, starting with general aspects and focusing on the recent structural studies, with emphasis on the inverse agonists bound structures. METHODS: We start with a literature review, and then we describe recent studies on CB 1 crystallographic structure and docking simulations. We use this structural information to depict protein-ligand interactions. We also describe the molecular docking method to obtain complex structures of CB 1 with inverse agonists. RESULTS: Analysis of the crystallographic structure and docking results revealed the residues responsible for the specificity of the inverse agonists for CB 1. Most of the intermolecular interactions involve hydrophobic residues, with the participation of the residues Phe 170 and Leu 359 in all complex structures investigated in the present study. For the complexes with otenabant and taranabant, we observed intermolecular hydrogen bonds involving residues His 178 (otenabant) and Thr 197 and Ser 383 (taranabant). CONCLUSION: Analysis of the structures involving inverse agonists and CB 1 revealed the pivotal role played by residues Phe 170 and Leu 359 in their interactions and the strong intermolecular hydrogen bonds highlighting the importance of the exploration of intermolecular interactions in the development of novel inverse agonists.

Alpha 1 adrenergic receptor-mediated inflammatory responses in human testicular peritubular cells.

Stress activates the sympathetic nervous system and is linked to impaired fertility in man. We hypothesized that catecholamines by acting on testicular cells have a role in these events, possibly by fostering an inflammatory environment. The cells of the wall of seminiferous tubules, human testicular peritubular cells (HTPCs), express adrenergic receptors (ADRs) α1B, α1D, ß1 and ß2. A selective α1-ADR agonist, phenylephrine, increased intracellular Ca2+-levels in cultured HTPCs and induced COX-2, IL-6 and MCP-1 mRNA expression without affecting IL-1ß mRNA. These changes were paralleled by a significant increase in the secretion of IL-6 and MCP-1. Epinephrine was also effective, but salbutamol, a selective ß2-ADR agonist was not. Our results suggest that stress-associated elevation of catecholamines may be able to promote inflammatory events by targeting peritubular cells in the human testis. Blockage of α1-ADRs may therefore be a novel way to interfere with stress-related impairment of male reproductive functions.

Calcium-sensing-receptor (CaSR) controls IL-6 secretion in metastatic breast cancer MDA-MB-231 cells by a dual mechanism revealed by agonist and inverse-agonist modulators.

IL-6 is a tightly controlled pleiotropic cytokine with hormone-like properties whose levels are frequently altered in cancer and inflammatory diseases. In highly invasive MDA-MB-231 breast cancer cells, basal activity of endogenously expressed calcium sensing receptor (CaSR) promotes IL-6 secretion. Interestingly, upon agonist stimulation, CaSR reduces IL-6 levels whereas it promotes secretion of various other cytokines and growth factors, raising intriguing questions about how CaSR signaling modulates IL-6 secretion. Here, using NPS-2143, which acted as an inverse agonist, we show that IL-6 secretion promoted by constitutive activity of CaSR is mechanistically linked to Gαs/PKC, MEK1/2 and mTORC1 signaling pathways, integrated by transactivated EGFR. On the other hand, agonist-stimulated CaSR engages in a Rab11a-dependent trafficking pathway critical to inhibit constitutive IL-6 secretion via the PI3K/AKT and PKC signaling pathways. These results support the emerging potential of CaSR as a therapeutic target in metastatic breast cancer whose pharmacological modulation would reduce IL-6.

Toxicological Evaluation of a Potential Immunosensitizer for Use as a Mucosal Adjuvant--Bacillus thuringiensis Cry1Ac Spore-Crystals: A Possible Inverse Agonist that Deserves Further Investigation.

In addition to their applicability as biopesticides, Bacillus thuringiensis (Bt) Cry1Ac spore-crystals are being researched in the immunology field for their potential as adjuvants in mucosal and parenteral immunizations. We aimed to investigate the hematotoxicity and genotoxicity of Bt spore-crystals genetically modified to express Cry1Ac individually, administered orally (p.o.) or with a single intraperitoneal (i.p.) injection 24 h before euthanasia, to simulate the routes of mucosal and parenteral immunizations in Swiss mice. Blood samples were used to perform hemogram, and bone marrow was used for the micronucleus test. Cry1Ac presented cytotoxic effects on erythroid lineage in both routes, being more severe in the i.p. route, which also showed genotoxic effects. The greater severity noted in this route, mainly at 6.75 mg/kg, as well as the intermediate effects at 13.5 mg/kg, and the very low hematotoxicity at 27 mg/kg, suggested a possible inverse agonism. The higher immunogenicity for the p.o. route, particularly at 27 mg/kg, suggested that at this dose, Cry 1Ac could potentially be used as a mucosal adjuvant (but not in parenteral immunizations, due to the genotoxic effects observed). This potential should be investigated further, including making an evaluation of the proposed inverse agonism and carrying out cytokine profiling.

Antihistaminergics and inverse agonism: potential therapeutic applications.

The accurate characterization of the molecular mechanisms involved in the action of receptor ligands is important for their appropriate therapeutic use and safety. It is well established that ligands acting at the histamine system currently used in the clinic exert their actions by specifically antagonizing G-protein coupled H1 and H2 receptors. However, most of these ligands, assumed to be neutral antagonists, behave as inverse agonists displaying negative efficacy in experimental systems. This suggests that their therapeutic actions may involve not only receptor blockade, but also the decrease of spontaneous receptor activity. The mechanisms whereby inverse agonists achieve negative efficacy are diverse. Theoretical models predict at least three possible mechanisms, all of which are supported by experimental observations. Depending on the mechanism of action engaged, the inverse agonist could interfere specifically with signaling events triggered by unrelated receptors. This possibility opens up new venues to explain the therapeutic actions of inverse agonists of the histamine receptor and perhaps new therapeutic applications.

Inflammation triggers constitutive activity and agonist-induced negative responses at M(3) muscarinic receptor in dental pulp.

The purpose of this study was to investigate whether the inflammation of rat dental pulp induces the muscarinic acetylcholine receptor (mAChR) constitutive receptor activity. Pulpitis was induced with bacterial lipolysaccharide in rat incisors dental pulp. Saturation assay with [(3)H]-quinuclidinyl benzilate ([(3)H] QNB), competitive binding with different mAChR antagonist subtypes, and nitric oxide synthase (NOS) activity were performed. A drastic change in expression and response to mAChR subtypes was observed in pulpitis. Inflamed pulp expressed high number of M(3) mAChR of high affinity, whereas the M(1) mAChR is the main subtype displayed in normal pulp. Consistent with the identification of the affinity constant (Ki) of M(3) and Ki of M(1) in both pulpitis and in normal pulps are the differences in the subtype functionality of these cells. In pulpitis, pilocarpine (1 × 10(-11) mol/L to 5 × 10(-9) mol/L) exerted an inhibitory action on NOS activity that was blocked by J 104129 fumarate (highest selective affinity to M(3) mAChR). In normal pulps, pilocarpine (1 × 10(-11) mol/L to 5 × 10(-9) mol/L) has no effect. NOS basal activity was 5.9 times as high in pulpitis as in the normal pulp as a result of the activation of inducible NOS. The irreversible pulpitis could induce a mAChR alteration, increasing the high-affinity receptor density and transduction-coupling efficiency of inducible NOS activity, leading to a spontaneously active conformation of the receptor. Pilocarpine acting as an inverse agonist might be useful therapeutically to prevent necrosis and subsequent loss of dental pulp.

Measurement of inverse agonism in ß-adrenoceptors.

Increasing numbers of compounds, previously classified as antagonists, were shown to inhibit this spontaneous or constitutive receptor activity, instead of leave it unaffected as expected for a formal antagonist. In addition, some other antagonists did not have any effect by themselves, but prevented the inhibition of constitutive activity induced by thought-to-be antagonists. These thought-to-be antagonists with negative efficacy are now known as "inverse agonists." Inverse agonism at ßAR has been evidenced for both subtypes in wild-type GPCRs systems and in engineered systems with high constitutive activity. It is important to mention that native systems are of particular importance for analyzing the in vivo relevance of constitutive activity because these systems have physiological expression levels of target receptors. Studies of inverse agonism of ß blockers in physiological setting have also evidenced that pathophysiological conditions can affect pharmacodynamic properties of these ligands. To date, hundreds of clinically well-known drugs have been tested and classified for this property. Prominent examples include the beta-blockers propranolol, alprenolol, pindolol, and timolol used for treating hypertension, angina pectoris, and arrhythmia that act on the ß2ARs, metoprolol, and bisoprolol used for treating hypertension, coronary heart disease, and arrhythmias by acting on ß1ARs. Inverse agonists seem to be useful in the treatment of chronic disease characterized by harmful effects resulting from ß1AR and ß2AR overactivation, such as heart failure and asthma, respectively.

Inverse benzodiazepine agonist beta-CCM does not reverse learning deficit induced by sleep deprivation.

Increasing evidence indicates that sleep deprivation (SD) alters responses to pharmacological agents by affecting specific transmitter systems. The present work addressed deficits in passive avoidance (PA) performance that are seen after SD, and investigated whether treatment with the inverse benzodiazepine agonist beta-CCM could prevent such deficits. Male Wistar rats were deprived of sleep for 96 h using the platform method (SD group), or were sleep deprived and then allowed to recover sleep for 24h (SR group). Animals were treated with saline or 0.5mg/kg beta-CCM before PA training, and were tested 30 min or 24h later. A separate set of animals was sacrificed for [(3)H]Ro 15-4513 binding analysis. beta-CCM increased PA performance in control animals in both short and long term retention tests, whereas SD and SR animals were unaffected by the drug treatment. Interestingly, [(3)H]Ro 15-4513 binding was reduced in the entorhinal cortex in both SD and SR groups. These findings suggest that the lack of promnesic effects of beta-CCM after SD and SR may be associated with benzodiazepine receptor downregulation in specific brain regions related to memory formation.

Therapeutic implications of beta-adrenergic receptor pharmacodynamic properties.

In the last decades new pharmacodynamic properties of beta-adrenoceptors have been discovered that could greatly impact in the use of beta-adrenergic agents in the clinical practice. Concepts such as multiple binding sites, constitutive activity, polymorphism and intracellular signaling of betaadrenoceptors may contribute in the discovery of more efficacious pharmacological agents for treatment of heart failure and asthma. beta-Adrenoceptors show a relative high constitutive activity in both cardiac and pulmonar tissues. Most beta-blockers exert an inverse agonist action that could contribute to their beneficial effects in the treatment of heart failure. Recently, the existence of multiple affinity sites has been described for beta1-adrenoceptor. It was proposed that beta-blockers that show agonist properties at the beta1L-adrenoceptors binding site may exert neutral or harmful effects when used for treatment of heart failure. Considering the cardiac effect of beta1L-adrenoceptors, activation of the low-affinity state of beta1-adrenoceptor could be deleterous in cardiovascular pharmacology. The ability of beta2-adrenoceptor to couple to Gs or Gi-protein gives the possibility that different agonists can activate different signaling cascades. Full beta2-adrenoceptor agonists would be highly useful for improvement bronchodilatation in the acute treatment of asthma. Polymorphic variants of beta-adrenoceptors have profound impact in the understanding of normal physiology and pathophysiology. Genotypic characterization of patients could improve selection of patients during beta-adrenergic pharmacotherapies. The aim of the present review is to describe new insights in pharmacological and biochemical properties of beta-adrenoceptors and their impact on the use of beta-adrenergic agents in the treatment of cardiovascular and respiratory diseases.

Alpha2-adrenoceptor action on cell proliferation and mammary tumour growth in mice.

BACKGROUND AND PURPOSE: Breast cancer, the most common cancer in women in most countries, is a highly stressful disease. Catecholamines released during stress bind to adrenoceptors and we have recently described alpha(2)-adrenoceptors in human breast cell lines, linked to enhanced cell proliferation. The purpose was to assess the in vivo effects of compounds acting on alpha(2)-adrenoceptors in a reliable model of breast cancer. EXPERIMENTAL APPROACH: The expression of alpha(2)-adrenoceptors was confirmed by immunocytochemistry, immunofluorescence and reverse transcription-PCR in the mouse mammary tumour cell line MC4-L5. Proliferation was assessed by [(3)H]thymidine incorporation and tumours were measured daily. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick-end labelling. KEY RESULTS: Incubation for 2 days with alpha(2)-adrenoceptor agonists (clonidine and dexmedetomidine) significantly enhanced proliferation of the mouse mammary tumour cell line MC4-L5. These agonists also significantly stimulated tumour growth of the progestin-dependent tumour C4-HD even in the presence of medroxyprogesterone acetate (MPA). In every tumour tested (C4-HD, CC4-2-HD and CC4-3-HI), regardless of MPA sensitivity, clonidine significantly enhanced tumour growth in the absence of MPA. The alpha(2)-adrenoceptor antagonists, yohimbine and rauwolscine, completely reversed the effects of clonidine. However, the group receiving yohimbine alone showed a nonsignificant but constant increase in tumour growth, whereas rauwolscine alone diminished tumour growth significantly, behaving as a reverse agonist. In CC4-3-HI tumours, rauwolscine treatment enhanced apoptosis and diminished the mitotic index, whereas clonidine had the inverse effect. CONCLUSIONS AND IMPLICATIONS: Alpha(2)-adrenoceptor agonists enhanced tumour growth and rauwolscine behaved in vivo as a reverse agonist, suggesting that it may be tested for adjuvant treatment.

Hemopressin is an inverse agonist of CB1 cannabinoid receptors.

To date, the endogenous ligands described for cannabinoid receptors have been derived from membrane lipids. To identify a peptide ligand for CB(1) cannabinoid receptors, we used the recently described conformation-state sensitive antibodies and screened a panel of endogenous peptides from rodent brain or adipose tissue. This led to the identification of hemopressin (PVNFKFLSH) as a peptide ligand that selectively binds CB(1) cannabinoid receptors. We find that hemopressin is a CB(1) receptor-selective antagonist, because it is able to efficiently block signaling by CB(1) receptors but not by other members of family A G protein-coupled receptors (including the closely related CB(2) receptors). Hemopressin also behaves as an inverse agonist of CB(1) receptors, because it is able to block the constitutive activity of these receptors to the same extent as its well characterized antagonist, rimonabant. Finally, we examine the activity of hemopressin in vivo using different models of pain and find that it exhibits antinociceptive effects when administered by either intrathecal, intraplantar, or oral routes, underscoring hemopressin's therapeutic potential. These results represent a demonstration of a peptide ligand for CB(1) cannabinoid receptors that also exhibits analgesic properties. These findings are likely to have a profound impact on the development of novel therapeutics targeting CB(1) receptors.