Physiological Temperature Changes Fine-Tune ß 2- Adrenergic Receptor-Induced Cytosolic cAMP Accumulation.

Norepinephrine (NE) controls many vital body functions by activating adrenergic receptors (ARs). Average core body temperature (CBT) in mice is 37°C. Of note, CBT fluctuates between 36 and 38°C within 24 hours, but little is known about the effects of CBT changes on the pharmacodynamics of NE. Here, we used Peltier element-controlled incubators and challenged murine hypothalamic mHypoA -2/10 cells with temperature changes of ±1°C. We observed enhanced NE-induced activation of a cAMP-dependent luciferase reporter at 36 compared with 38°C. mRNA analysis and subtype specific antagonists revealed that NE activates ß 2- and ß 3-AR in mHypoA-2/10 cells. Agonist binding to the ß 2-AR was temperature insensitive, but measurements of cytosolic cAMP accumulation revealed an increase in efficacy of 45% ± 27% for NE and of 62% ± 33% for the ß 2-AR-selective agonist salmeterol at 36°C. When monitoring NE-promoted cAMP efflux, we observed an increase in the absolute efflux at 36°C. However, the ratio of exported to cytosolic accumulated cAMP is higher at 38°C. We also stimulated cells with NE at 37°C and measured cAMP degradation at 36 and 38°C afterward. We observed increased cAMP degradation at 38°C, indicating enhanced phosphodiesterase activity at higher temperatures. In line with these data, NE-induced activation of the thyreoliberin promoter was found to be enhanced at 36°C. Overall, we show that physiologic temperature changes fine-tune NE-induced cAMP signaling in hypothalamic cells via ß 2-AR by modulating cAMP degradation and the ratio of intra- and extracellular cAMP. SIGNIFICANCE STATEMENT: Increasing cytosolic cAMP levels by activation of G protein-coupled receptors (GPCR) such as the ß 2-adrenergic receptor (AR) is essential for many body functions. Changes in core body temperature are fundamental and universal factors of mammalian life. This study provides the first data linking physiologically relevant temperature fluctuations to ß 2-AR-induced cAMP signaling, highlighting a so far unappreciated role of body temperature as a modulator of the prototypic class A GPCR.

Noradrenaline protects neurons against H2 O2 -induced death by increasing the supply of glutathione from astrocytes via ß3 -adrenoceptor stimulation.

Oxidative stress has been implicated in a variety of neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. Astrocytes play a significant role in maintaining survival of neurons by supplying antioxidants such as glutathione (GSH) to neurons. Recently, we found that noradrenaline increased the intracellular GSH concentration in astrocytes via ß3 -adrenoceptor stimulation. These observations suggest that noradrenaline protects neurons from oxidative stress-induced death by increasing the supply of GSH from astrocytes to neurons via the stimulation of ß3 -adrenoceptor in astrocytes. In the present study, we examined the protective effect of noradrenaline against H2 O2 -induced neurotoxicity using two different mixed cultures: the mixed culture of human astrocytoma U-251 MG cells and human neuroblastoma SH-SY5Y cells, and the mouse primary cerebrum mixed culture of neurons and astrocytes. H2 O2 -induced neuronal cell death was significantly attenuated by pretreatment with noradrenaline in both mixed cultures but not in single culture of SH-SY5Y cells or in mouse cerebrum neuron-rich culture. The neuroprotective effect of noradrenaline was inhibited by SR59230A, a selective ß3 -adrenoceptor antagonist, and CL316243, a selective ß3 -adrenoceptor agonist, mimicked the neuroprotective effect of noradrenaline. DL-buthionine-[S,R]-sulfoximine, a GSH synthesis inhibitor, negated the neuroprotective effect of noradrenaline in both mixed cultures. MK571, which inhibits the export of GSH from astrocytes mediated by multidrug resistance-associated protein 1, also prevented the neuroprotective effect of noradrenaline. These results suggest that noradrenaline protects neurons against H2 O2 -induced death by increasing the supply of GSH from astrocytes via ß3 -adrenoceptor stimulation.

ß3-Adrenoceptors as Putative Regulator of Immune Tolerance in Cancer and Pregnancy.

Understanding the mechanisms of immune tolerance is currently one of the most important challenges of scientific research. Pregnancy affects the immune system balance, leading the host to tolerate embryo alloantigens. Previous reports demonstrated that ß-adrenergic receptor (ß-AR) signaling promotes immune tolerance by modulation of NK and Treg, mainly through the activation of ß2-ARs, but recently we have demonstrated that also ß3-ARs induce an immune-tolerant phenotype in mice bearing melanoma. In this report, we demonstrate that ß3-ARs support host immune tolerance in the maternal microenvironment by modulating the same immune cells populations as recently demonstrated in cancer. Considering that ß3-ARs are modulated by oxygen levels, we hypothesize that hypoxia, through the upregulation of ß3-AR, promotes the biological shift toward a tolerant immunophenotype and that this is the same trick that embryo and cancer use to create an aura of immune-tolerance in a competent immune environment. This study confirms the analogies between fetal development and tumor progression and suggests that the expression of ß3-ARs represents one of the strategies to induce fetal and tumor immune tolerance.

Real-time monitoring of cAMP in brown adipocytes reveals differential compartmentation of ß1 and ß3-adrenoceptor signalling.

OBJECTIVE: 3',5'-Cyclic adenosine monophosphate (cAMP) is a central second messenger governing brown adipocyte differentiation and function. ß-adrenergic receptors (ß-ARs) stimulate adenylate cyclases which produce cAMP. Moreover, cyclic nucleotide levels are tightly controlled by phosphodiesterases (PDEs), which can generate subcellular microdomains of cAMP. Since the spatio-temporal organisation of the cAMP signalling pathway in adipocytes is still unclear, we sought to monitor real-time cAMP dynamics by live cell imaging in pre-mature and mature brown adipocytes. METHODS: We measured the real-time dynamics of cAMP in murine pre-mature and mature brown adipocytes during stimulation of individual ß-AR subtypes, as well as its regulation by PDEs using a Förster Resonance Energy Transfer based biosensor and pharmacological tools. We also correlated these data with ß-AR stimulated lipolysis and analysed the expression of ß-ARs and PDEs in brown adipocytes using qPCR and immunoblotting. Furthermore, subcellular distribution of PDEs was studied using cell fractionation and immunoblots. RESULTS: Using pre-mature and mature brown adipocytes isolated from transgenic mice expressing a highly sensitive cytosolic biosensor Epac1-camps, we established real-time measurements of cAMP responses. PDE4 turned out to be the major PDE regulating cytosolic cAMP in brown preadipocytes. Upon maturation, PDE3 gets upregulated and contributes with PDE4 to control ß1-AR-induced cAMP. Unexpectedly, ß3-AR initiated cAMP is resistant to increased PDE3 protein levels and simultaneously, the control of this microdomain by PDE4 is reduced upon brown adipocyte maturation. Therefore we postulate the existence of distinct cAMP pools in brown adipocytes. One cAMP pool is formed by ß1-AR associated with PDE3 and PDE4, while another pool is centred around ß3-AR and is much less controlled by these PDEs. Functionally, lower control of ß3-AR initiated cAMP by PDE3 and PDE4 facilitates brown adipocyte lipolysis, while lipolysis activated by ß1-AR and is under tight control of PDE3 and PDE4. CONCLUSIONS: We have established a real-time live cell imaging approach to analyse brown adipocyte cAMP dynamics in real-time using a cAMP biosensor. We showed that during the differentiation from pre-mature to mature murine brown adipocytes, there was a change in PDE-dependent compartmentation of ß1-and ß3-AR-initiated cAMP responses by PDE3 and PDE4 regulating lipolysis.

Dual cholinergic signals regulate daily migration of hematopoietic stem cells and leukocytes.

Hematopoietic stem and progenitor cells (HSPCs) and leukocytes circulate between the bone marrow (BM) and peripheral blood following circadian oscillations. Autonomic sympathetic noradrenergic signals have been shown to regulate HSPC and leukocyte trafficking, but the role of the cholinergic branch has remained unexplored. We have investigated the role of the cholinergic nervous system in the regulation of day/night traffic of HSPCs and leukocytes in mice. We show here that the autonomic cholinergic nervous system (including parasympathetic and sympathetic) dually regulates daily migration of HSPCs and leukocytes. At night, central parasympathetic cholinergic signals dampen sympathetic noradrenergic tone and decrease BM egress of HSPCs and leukocytes. However, during the daytime, derepressed sympathetic noradrenergic activity causes predominant BM egress of HSPCs and leukocytes via ß3-adrenergic receptor. This egress is locally supported by light-triggered sympathetic cholinergic activity, which inhibits BM vascular cell adhesion and homing. In summary, central (parasympathetic) and local (sympathetic) cholinergic signals regulate day/night oscillations of circulating HSPCs and leukocytes. This study shows how both branches of the autonomic nervous system cooperate to orchestrate daily traffic of HSPCs and leukocytes.

Sustained stimulation of ß2- and ß3-adrenergic receptors leads to persistent functional pain and neuroinflammation.

Functional pain syndromes, such as fibromyalgia and temporomandibular disorder, are associated with enhanced catecholamine tone and decreased levels of catechol-O-methyltransferase (COMT; an enzyme that metabolizes catecholamines). Consistent with clinical syndromes, our lab has shown that sustained 14-day delivery of the COMT inhibitor OR486 in rodents results in pain at multiple body sites and pain-related volitional behaviors. The onset of COMT-dependent functional pain is mediated by peripheral ß2- and ß3-adrenergic receptors (ß2- and ß3ARs) through the release of the pro-inflammatory cytokines tumor necrosis factor α (TNFα), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6). Here, we first sought to investigate the role of ß2- and ß3ARs and downstream mediators in the maintenance of persistent functional pain. We then aimed to characterize the resulting persistent inflammation in neural tissues (neuroinflammation), characterized by activated glial cells and phosphorylation of the mitogen-activated protein kinases (MAPKs) p38 and extracellular signal-regulated kinase (ERK). Separate groups of rats were implanted with subcutaneous osmotic mini-pumps to deliver OR486 (15 mg/kg/day) or vehicle for 14 days. The ß2AR antagonist ICI118551 and ß3AR antagonist SR59230A were co-administrated subcutaneously with OR486 or vehicle either on day 0 or day 7. The TNFα inhibitor Etanercept, the p38 inhibitor SB203580, or the ERK inhibitor U0126 were delivered intrathecally following OR486 cessation on day 14. Behavioral responses, pro-inflammatory cytokine levels, glial cell activation, and MAPK phosphorylation were measured over the course of 35 days. Our results demonstrate that systemic delivery of OR486 leads to mechanical hypersensitivity that persists for at least 3 weeks after OR486 cessation. Corresponding increases in spinal TNFα, IL-1ß, and IL-6 levels, microglia and astrocyte activation, and neuronal p38 and ERK phosphorylation were observed on days 14-35. Persistent functional pain was alleviated by systemic delivery of ICI118551 and SR59230A beginning on day 0, but not day 7, and by spinal delivery of Etanercept or SB203580 beginning on day 14. These results suggest that peripheral ß2- and ß3ARs drive persistent COMT-dependent functional pain via increased activation of immune cells and production of pro-inflammatory cytokines, which promote neuroinflammation and nociceptor activation. Thus, therapies that resolve neuroinflammation may prove useful in the management of functional pain syndromes.

Adrenergic receptor ß3 is involved in the memory consolidation process in mice

Attention and emotion have a positive impact on memory formation, which is related to the activation of the noradrenergic system in the brain. The hippocampus and amygdala are fundamental structures in memory acquisition, which is modulated by noradrenaline through the noradrenergic receptors. Pharmacological studies suggest that memory acquisition depends on the action of both the β3 (β3-AR) and β2 (β2-AR) receptor subtypes. However, the use of animal models with specific knockout for the β3-AR receptor only (β3-ARKO) allows researchers to more accurately assess its role in memory formation processes. In the present study, we evaluated short- and long-term memory acquisition capacity in β3-ARKO mice and wild-type mice at approximately 60 days of age. The animals were submitted to the open field test, the elevated plus maze, object recognition, and social preference. The results showed that the absence of the β3-AR receptor caused no impairment in locomotion and did not cause anxious behavior, but it caused significant impairment of short- and long-term memory compared to wild-type animals. We also evaluated the expression of genes involved in memory consolidation. The mRNA levels for GLUT3, a glucose transporter expressed in the central nervous system, were significantly reduced in the amygdala, but not in the hippocampus of the β3-ARKO animals. Our results showed that β3-AR was involved in the process of acquisition of declarative memory, and its action may be due to the facilitation of glucose absorption in the amygdala.

ß3 adrenergic receptor activation relaxes human corpus cavernosum and penile artery through a hydrogen sulfide/cGMP-dependent mechanism.

Erectile function is a widely accepted indicator of systemic endothelial activity since from a clinical standpoint erectile dysfunction (ED) often precedes cardiovascular events. Recently it has been described a potential role for ß3 adrenoceptor in cardiovascular diseases emphasizing a possible development of new drugs. ß3 adrenoceptor stimulation relaxes human corpus cavernosum (HCC) strips in cyclic guanosine monophosphate (cGMP)-dependent and endothelium/nitric oxide (NO)-independent manner. Hydrogen sulfide (H2S), along with NO, is another gaseous molecule involved in cardiovascular system and as a consequence also in penile erection. Cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE), the enzymes mainly responsible for H2S biosynthesis, are constitutively expressed in HCC. CSE rather than CBS is more abundant in human penile tissue. Herein we investigated the involvement of H2S pathway in ß3 adrenoceptor-induced relaxation in HCC and penile artery. Penile artery expresses both CSE and ß3 adrenoceptor. BRL37344, a ß3 selective agonist, relaxed HCC strips and penile artery rings and this effect was significantly reduced by CSE inhibition. Incubation of HCC and penile artery homogenate with BRL37344 significantly increased H2S production. This effect was significantly reduced by the inhibition of either CSE or ß3 adrenoceptor. Finally, the BRL37344-induced increase in cGMP was reduced by CSE inhibition in both tissues. Thus, BRL37344-induced relaxation in HCC and penile artery occurs in a H2S/cGMP-dependent manner. In conclusion, ß3/H2S/cGMP pathway can act as an alternative to NO. Since about 15% of patients do not respond to phosphodiesterase-5 inhibitors, ß3 agonists could represent a therapeutic alternative or a useful adjuvant therapy to treat these patients.

[Effects 'of ß3 adrenoceptors on the contractility of rat thoracic aorta smooth muscle and the mechanism].

OBJECTIVE: To observe the effect of ß3adrenoceptors (ß3-AR) activation on rat thoracic aorta smooth muscle contractility and the possible related mechanism. METHODS: The endothelium removed thoracic aorta was pre-contracted with 30 mmol/L KCl physiological saline solution (PSS). Then the tension of the thoracic aorta was recorded in presence of BRL37344 (BRL) to determine the action of ß3-AR. The tension of the thoracic aorta was also recorded in the presence of Propranolol (PRA), SR59230A (SR), L-NNA, H-89 and Iberiotoxin (IBTX) respectively to reveal the underling mechanism of ß3-AR activation on rat vascular smooth muscle. Immunohistochemistry was adopted to confirm the existence and the distribution of ß3-AR in rat thoracic aorta. RESULTS: The results showed that: (1) The thoracic aorta was relaxed by ß3-AR activation, with a relaxation percentage of (10.59 ± 0.79). (2) ß3-AR was expressed in both endothelial and smooth muscle layer in thoracic aorta sections of rats. (3) PRA did not block the effect of BRL on the thoracic aorta. The relaxation actions of BRL could be antagonized by pre-incubating the thoracic aorta with SR. (4) L-NNA (a NOS inhibitor) and H-89 (a PKA inhibitor) reversed the relaxation effect of BRL on vascular smooth muscle. (5) The effect of BRL was decreased after application of Ibriotoxin (IBTX), a large conductance calcium dependent potassium channel blocker. CONCLUSION: The results confirmed that activation of ß3-AR led to relaxation of thoracic aorta smooth muscle. The relaxation action of ß3-AR on smooth muscle of rat thoracic aorta was related to activation of NOS and PKA signaling pathway. Large conductance Ca²âº-K⁺ channels were involved in the relaxation action of ß3-AR activation on rat thoracic aorta smooth muscle.

ß3 adrenergic receptor in the kidney may be a new player in sympathetic regulation of renal function.

To date, the study of the sympathetic regulation of renal function has been restricted to the important contribution of ß1- and ß2-adrenergic receptors (ARs). Here we investigate the expression and the possible physiologic role of ß3-adrenergic receptor (ß3-AR) in mouse kidney. The ß3-AR is expressed in most of the nephron segments that also express the type 2 vasopressin receptor (AVPR2), including the thick ascending limb and the cortical and outer medullary collecting duct. Ex vivo experiments in mouse kidney tubules showed that ß3-AR stimulation with the selective agonist BRL37344 increased intracellular cAMP levels and promoted 2 key processes in the urine concentrating mechanism. These are accumulation of the water channel aquaporin 2 at the apical plasma membrane in the collecting duct and activation of the Na-K-2Cl symporter in the thick ascending limb. Both effects were prevented by the ß3-AR antagonist L748,337 or by the protein kinase A inhibitor H89. Interestingly, genetic inactivation of ß3-AR in mice was associated with significantly increased urine excretion of water, sodium, potassium, and chloride. Stimulation of ß3-AR significantly reduced urine excretion of water and the same electrolytes. Moreover, BRL37344 promoted a potent antidiuretic effect in AVPR2-null mice. Thus, our findings are of potential physiologic importance as they uncover the antidiuretic effect of ß3-AR stimulation in the kidney. Hence, ß3-AR agonism might be useful to bypass AVPR2-inactivating mutations.

Role of host ß1- and ß2-adrenergic receptors in a murine model of B16 melanoma: functional involvement of ß3-adrenergic receptors.

Complex interactions between tumor cells and their surrounding compartment are strongly influenced by the host in which the tumor grows. In melanoma, for instance, stress-associated norephinephrine (NE), acting at ß-adrenergic receptors (ß-ARs), stimulates melanoma cell proliferation and tumor angiogenesis. Among ß-ARs, ß3-ARs play a role acting not only at tumor cells but also at non-neoplastic stromal cells within the melanoma. In the present study, we used a murine model of B16 melanoma to evaluate the role of the host ß1- and ß2-ARs in melanoma growth and we determined whether the role of ß3-ARs can be influenced by the absence of stromal ß1- and ß2-ARs. As compared to wild-type mice, ß1/2-AR knockout mice displayed (i) increased intratumoral levels of both NE and ß3-ARs, as evidentiated at both messenger and protein levels; (ii) increased tumor vascularization; (iii) decreased tumor cell proliferation but increased tumor cell apoptosis; and (iv) increased responsiveness to intratumoral injection of the ß3-AR blocker L-748,337 in terms of decrease in tumor growth, tumor vascular response, tumor cell proliferation, and increase in tumor cell death. These findings together validate the role of ß-AR signaling in melanoma microenvironment suggesting that non-neoplastic stromal cells may be targeted by ß-AR-related drugs. The additional fact that ß3-ARs play an important role in melanoma growth suggests selective ß3-AR antagonists as important proapoptotic agents.

ß3-adrenergic receptor activity modulates melanoma cell proliferation and survival through nitric oxide signaling.

We have recently shown in B16F10 melanoma cells that blockade of ß3-adrenergic receptors (ß3-ARs) reduces cell proliferation and induces apoptosis, likely through the involvement of nitric oxide (NO) signaling. Here, we tested the hypothesis that the effects of ß3-AR blockade on melanoma cells are mainly mediated by a decrease in the activity of the NO pathway, possibly due to reduced expression of inducible NO synthase (iNOS). B16F10 cells were used. Nitrite production, iNOS expression, cell proliferation, and apoptosis were evaluated. ß3-AR blockade with L-748,337 reduced basal nitrite production, while ß3-AR stimulation with BRL37344 increased it. The effects of ß3-AR blockade were prevented by NOS activation, while the effects of ß3-AR activation were prevented by NOS inhibition. Treatments increasing nitrite production also increased iNOS expression, while treatments decreasing nitrite production reduced iNOS expression. Among the different NOS isoforms, experiments using L-748,337 or BRL37344 with activators or inhibitors targeting specific NOS isoforms demonstrated a prominent role of iNOS in nitrite production. ß3-AR blockade decreased cell proliferation and induced apoptosis, while ß3-AR activation had the opposite effects. The effects of ß3-AR blockade/activation were prevented by iNOS activation/inhibition, respectively. Taken together, these results demonstrate that iNOS-produced NO is a downstream effector of ß3-ARs and that the beneficial effects of ß3-AR blockade on melanoma B16F10 cell proliferation and apoptosis are functionally linked to reduced iNOS expression and NO production. Although it is difficult to extrapolate these data to the clinical setting, the targeted inhibition of the ß3-AR-NO axis may offer a new therapeutic perspective to treat melanomas.

Modulation of nerve-evoked contractions by ß3-adrenoceptor agonism in human and rat isolated urinary bladder.

Activation of ß3-adrenoceptors has been shown to have a direct relaxant effect on urinary bladder smooth muscle from both rats and humans, however there are very few studies investigating the effects of ß3-adrenoceptor agonists on nerve-evoked bladder contractions. Therefore in the current study, the role of ß3-adrenoceptors in modulating efferent neurotransmission was evaluated. The effects of ß3-adrenoceptor agonism on neurogenic contractions induced by electrical field stimulation (EFS) were compared with effects on contractions induced by exogenous acetylcholine (Ach) and αß-methylene adenosine triphosphate (αß-meATP) in order to determine the site of action. Isoproterenol inhibited EFS-induced neurogenic contractions of human bladder (pD2=6.79; Emax=65%). The effect of isoproterenol was selectively inhibited by the ß3-adrenoceptor antagonist L-748,337 (pKB=7.34). Contractions induced by exogenous Ach (0.5-1µM) were inhibited 25% by isoproterenol (3µM) while contractions to 10Hz in the same strip were inhibited 67%. The selective ß3-adrenoceptor agonist CL-316,243 inhibited EFS-induced neurogenic contractions of rat bladder (pD2=7.83; Emax=65%). The effects of CL-316,243 were inhibited in a concentration dependent manner by L-748,337 (pA2=6.42). Contractions induced by exogenous Ach and αß-meATP were significantly inhibited by CL-316,243, 29% and 40%, respectively. These results demonstrate that the activation of ß3-adrenoceptors inhibits neurogenic contractions of both rat and human urinary bladder. Contractions induced by exogenously applied parasympathetic neurotransmitters are also inhibited by ß3-agonism however the effect is clearly less than on neurogenic contractions (particularly in human), suggesting that in addition to a direct effect on smooth muscle, activation of prejunctional ß3-adrenoceptors may inhibit neurotransmitter release.

Pharmacological profile of the selective ß3-adrenoceptor agonist mirabegron in cynomolgus monkeys.

Mirabegron is a novel ß3-adrenoceptor agonist developed for the treatment of overactive bladder. To clarify the relationship between the pharmacological effects of mirabegron in monkeys and the clinical efficacy in patients with overactive bladder, the effect of mirabegron on bladder function was evaluated using cynomolgus monkeys. Quantitative PCR revealed that mRNA expression of ß3-adrenoceptors was most abundant (98 %) among ß-adrenoceptor subtypes in the bladder of cynomolgus monkeys. Mirabegron, which showed selective and potent agonistic activity on monkey ß3-adrenoceptors expressed in Chinese hamster ovary cells with EC50 value of 32 nmol/L and intrinsic activity of 0.8, induced concentration-dependent relaxation of bladder smooth muscle strips isolated from cynomolgus monkeys with EC50 values of 120 nmol/L in 20 mmol/L KCl stimulation and 43 nmol/L under 9.81 mN resting tension. In conscious cynomolgus monkeys, mirabegron decreased micturition frequency at oral doses of 1 and 3 mg/kg and increased mean volume voided per micturition at an oral dose of 3 mg/kg. Plasma concentration at which bladder function improved in the cynomolgus monkeys was similar to that at the clinically effective dose in patients with overactive bladder. These data suggest that the relaxant function in monkey bladder is mainly mediated by ß3-adrenoceptors similar to that in the human bladder and mirabegron showed efficacy on the bladder functions of the same parameters in clinical evaluation endpoints.

Expression of activin receptor-like kinase 7 in adipose tissues.

The tissue distribution of activin receptor-like kinase 7 (Alk7) expression, the signaling ability of Alk7 variants, and Alk7 expression in response to ß3-adrenergic receptor activation were examined. Expression levels of Alk7 varied greatly among tissues but were highest in white adipose tissue and brown adipose tissue. In addition to full-length Alk7 (Alk7-v1), Alk7-v3, an Alk7 variant, was expressed in adipose tissues, brain, and ovary. Nodal transmits signals via Alk7 in cooperation with its coreceptor, Cripto. Evaluation of the ability of Alk7 variants to confer Nodal signaling using luciferase-based reporter assays showed that Alk7-v3 does not transmit Nodal-Cripto-mediated signals. Expression of Alk7 was down-regulated in brown but not in white adipose tissue treated with CL316,243, a ß3-adrenergic receptor agonist. These results suggest involvement of Alk7 in modulation of metabolism in the adipose tissues in response to ß3-adrenergic receptor activation.

Cooperation of beta(2)- and beta(3)-adrenergic receptors in hematopoietic progenitor cell mobilization.

CXCL12/SDF-1 dynamically regulates hematopoietic stem cell (HSC) attraction in the bone marrow (BM). Circadian regulation of bone formation and HSC traffic is relayed in bone and BM by beta-adrenergic receptors (beta-AR) expressed on HSCs, osteoblasts, and mesenchymal stem/progenitor cells. Circadian HSC release from the BM follows rhythmic secretion of norepinephrine from nerve terminals, beta(3)-AR activation, and Cxcl12 downregulation, possibly from reduced Sp1 nuclear content. Here, we show that beta-AR stimulation in stromal cells causes Sp1 degradation, partially mediated by the 26S proteasome. Inverted trends of circulating hematopoietic progenitors and BM Cxcl12 mRNA levels change acutely after light onset, shown to induce sympathetic efferent activity. In BM stromal cells, activation of beta(3)-AR downregulates Cxcl12, whereas beta(2)-AR stimulation induces clock gene expression. Double deficiency in beta(2)- and beta(3)-ARs compromises enforced mobilization. Therefore, beta(2)- and beta(3)-ARs have specific roles in stromal cells and cooperate during progenitor mobilization.

Circadian rhythms, adrenergic hormones and trafficking of hematopoietic stem cells.

IMPORTANCE OF THE FIELD: Under homeostasis, small numbers of haematopoietic stem cells (HSCs) are detectable in the bloodstream of mammals, but the mechanisms of their trafficking are unknown. AREAS COVERED IN THIS REVIEW: It has been shown that circulating HSCs exhibit marked circadian fluctuations due to standard cycles of 12 h light/12 h darkness. Circadian HSCs oscillations are strongly altered when mice are subjected to continuous light for two weeks or to a jet lag. In addition, circulating HSCs fluctuate in antiphase with the expression of the chemokine CXCL12 in the bone marrow microenvironment. Circadian HSC trafficking and expression of CXCL12 are modulated by core genes of the central clock through rhythmic secretion of adrenergic hormones from nerve terminals of the sympathetic nervous system (SNS) in the bone marrow. WHAT THE READER WILL GAIN: This review summarizes recent findings on the circadian regulation of HSC release in the bone marrow examining the molecular mechanisms through which the central molecular clock regulates CXCL12 in bone marrow stromal cells through rhythmic secretion of adrenergic hormones locally delivered in the bone marrow by nerve terminals from the SNS. TAKE HOME MESSAGE: The circadian HSCs trafficking during steady-state conditions, may promote the maintenance of haematopoiesis through the life of individuals.

Role of beta3-adrenoceptors for intrahepatic resistance and portal hypertension in liver cirrhosis.

UNLABELLED: Increased intrahepatic resistance and splanchnic blood flow cause portal hypertension in liver cirrhosis. Nonselective beta-adrenoceptor (beta-AR) antagonists have beneficial effects on hyperdynamic circulation and are in clinical use. In this context, the role of the beta(3)-AR is undefined. Here we investigated their expression and role in portal hypertension in patients and rats with liver cirrhosis. We analyzed cirrhotic human and rat tissues (liver, splanchnic vessels) and primary rat cells. Protein expression of beta(3)-AR was determined by western blot and messenger RNA (mRNA) levels by reverse-transcription polymerase chain reaction (RT-PCR). Activities of Rho-kinase and the nitric oxide (NO) effector protein kinase G (PKG) were assessed by way of substrate phosphorylation (moesin, vasodilator-stimulated phosphoprotein [VASP]). Cyclic 3',5' adenosine monophosphate (cAMP) accumulation was determined by an enzyme-immunoassay kit. The effects of selective beta(3)-AR agonists (CGP12177A, BRL37344) and antagonist (SR59230A) were investigated by collagen matrix contraction of hepatic stellate cells (HSCs), in situ liver perfusions, and in vivo hemodynamic parameters in bile duct ligation and carbon tetrachloride intoxication in cirrhotic rats. In cirrhosis of humans and rats, beta(3)-AR expression is markedly increased in hepatic and in splanchnic tissues. Stimulation of beta(3)-AR leads to relaxation of HSCs by way of cAMP accumulation, and by inhibition of Rho-kinase activity; any role of NO and its effector PKG was not observed. beta(3)-AR agonists decrease intrahepatic resistance and portal pressure in cirrhotic rats. CONCLUSION: There is a marked hepatic and mesenteric up-regulation of beta(3)-ARs in human cirrhosis and in two different animal models of cirrhosis. The beta(3)-AR-agonists should be further evaluated for therapy of portal hypertension.

Cross-regulation between beta 1- and beta 3-adrenoceptors following chronic beta-adrenergic stimulation in neonatal rat cardiomyocytes.

BACKGROUND AND PURPOSE: We have previously shown that beta-adrenoceptors continuously stimulated with noradrenaline induces an increase in beta(3)-adrenoceptors (G alpha(i)PCRs) and a decrease in beta(1)-adrenoceptors (G alpha(s)PCRs) at functional, genomic and protein levels. This compensatory modification induced by noradrenaline is probably one of the consequences of cardiac depression observed in heart disease. Therefore, we investigated further the interaction between beta(1)- and beta(3)-adrenoceptors in neonatal rat cardiomyocytes. EXPERIMENTAL APPROACH: Functional studies were performed by cyclic adenosine monophosphate (cAMP) accumulation assays in cells untreated or treated with dobutamine and ICI 118551 (beta(1)-adrenoceptor) or CL-3162436243 (beta(3)-adrenoceptor) for 24 h in the presence or absence of protein kinase inhibitors. Beta-adrenoceptor and protein kinase expression was monitored by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and by Western blotting, respectively. KEY RESULTS: Chronic beta(1)- or beta(3)-adrenoceptor stimulation reduced beta(1)-adrenoceptor-mediated cAMP accumulation in association with a decrease in beta(1)-adrenoceptor mRNA and protein levels through protein kinase C (PKC), phosphoinositide 3-kinase (PI3K) and p38 mitogen-activated protein kinase (p38MAPK) activation. In contrast, both treatments induced an increase in beta(3)-adrenoceptor expression and beta(3)-adrenoceptor-inhibited forskolin response through PKC, extracellular-signal-regulated kinases 1 and 2 (ERK1/2) and p38MAPK phosphorylation, although no beta(3)-adrenoceptor response was observed in untreated cells. ERK1/2 and p38MAPK were activated by both treatments. The modulation of beta(1)- or beta(3)-adrenoceptor function did not require stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) although chronic beta(1)-adrenoceptor stimulation activated SAPK/JNK. Beta(3)-adrenoceptor treatment activated Akt although PI3K was not involved in beta(3)-adrenoceptor up-regulation. CONCLUSION AND IMPLICATIONS: We show for the first time that chronic beta(1)- or beta(3)-adrenoceptor stimulation leads to the modulation of beta(1)- and beta(3)-adrenoceptors by a cross-regulation involving PKC, PI3K p38MAPK and MEK/ERK1/2 pathway, and through protein kinase A when beta(1)-adrenoceptors are chronically activated.