The ß2-adrenergic receptor agonist terbutaline upregulates T helper-17 cells in a protein kinase A-dependent manner.

BACKGROUND: T helper 17 (Th17) cells produce IL-17A cytokine and can exacerbate autoimmune diseases and asthma. The ß2 adrenergic receptor is a g protein-coupled receptor that induces cAMP second messenger pathways. We tested the hypothesis that terbutaline, a ß2-adrenergic receptor-specific agonist, promotes IL-17 secretion by memory Th17 cells in a cAMP and PKA-dependent manner. METHODS: Venous peripheral blood mononuclear cells (PBMC) from healthy human participants were activated with anti-CD3 and anti-CD28 antibodies. Secreted IL-17A was measured by enzyme linked immunosorbent assay, intracellular IL-17A, and RORγ were measured using flow cytometry, and RORC by qPCR. Memory CD3+CD4+CD45RA-CD45RO+ T cells were obtained by immunomagnetic negative selection and activated with tri-antibody complex CD3/CD28/CD2. Secreted IL-17A, intracellular IL-17A, RORC were measured, and phosphorylated-serine133-CREB was measured by western blotting memory Th cells. RESULTS: Terbutaline increased IL-17A (p < 0.001), IL-17A+ cells (p < 0.05), and RORC in activated PBMC and memory Th cells. The PKA inhibitors H89 (p < 0.001) and Rp-cAMP (p < 0.01) abrogated the effects of terbutaline on IL-17A secretion in PBMC and memory T cells. Rolipram increased IL-17A (p < 0.01) to a similar extent as terbutaline. P-Ser133-CREB was increased by terbutaline (p < 0.05) in memory T cells. CONCLUSION: Terbutaline augments memory Th17 cells in lymphocytes from healthy participants. This could exacerbate autoimmune diseases or asthma, in cases where Th17 cells are considered to be pro-inflammatory.

Drug Repurposing for Cystic Fibrosis: Identification of Drugs That Induce CFTR-Independent Fluid Secretion in Nasal Organoids.

Individuals with cystic fibrosis (CF) suffer from severe respiratory disease due to a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which impairs airway epithelial ion and fluid secretion. New CFTR modulators that restore mutant CFTR function have been recently approved for a large group of people with CF (pwCF), but ~19% of pwCF cannot benefit from CFTR modulators Restoration of epithelial fluid secretion through non-CFTR pathways might be an effective treatment for all pwCF. Here, we developed a medium-throughput 384-well screening assay using nasal CF airway epithelial organoids, with the aim to repurpose FDA-approved drugs as modulators of non-CFTR-dependent epithelial fluid secretion. From a ~1400 FDA-approved drug library, we identified and validated 12 FDA-approved drugs that induced CFTR-independent fluid secretion. Among the hits were several cAMP-mediating drugs, including ß2-adrenergic agonists. The hits displayed no effects on chloride conductance measured in the Ussing chamber, and fluid secretion was not affected by TMEM16A, as demonstrated by knockout (KO) experiments in primary nasal epithelial cells. Altogether, our results demonstrate the use of primary nasal airway cells for medium-scale drug screening, target validation with a highly efficient protocol for generating CRISPR-Cas9 KO cells and identification of compounds which induce fluid secretion in a CFTR- and TMEM16A-indepent manner.

Effects of adrenergic-stimulated lipolysis and cytokine production on in vitro mouse adipose tissue-islet interactions.

Inflammatory cytokines and non-esterified fatty acids (NEFAs) are obesity-linked factors that disturb insulin secretion. The aim of this study was to investigate whether pancreatic adipose tissue (pWAT) is able to generate a NEFA/cytokine overload within the pancreatic environment and as consequence to impact on insulin secretion. Pancreatic fat is a minor fat depot, therefore we used high-fat diet (HFD) feeding to induce pancreatic steatosis in mice. Relative Adipoq and Lep mRNA levels were higher in pWAT of HFD compared to chow diet mice. Regardless of HFD, Adipoq and Lep mRNA levels of pWAT were at least 10-times lower than those of epididymal fat (eWAT). Lipolysis stimulating receptors Adrb3 and Npr1 were expressed in pWAT and eWAT, and HFD reduced their expression in eWAT only. In accordance, HFD impaired lipolysis in eWAT but not in pWAT. Despite expression of Npr mRNA, lipolysis was stimulated solely by the adrenergic agonists, isoproterenol and adrenaline. Short term co-incubation of islets with CD/HFD pWAT did not alter insulin secretion. In the presence of CD/HFD eWAT, glucose stimulated insulin secretion only upon isoproterenol-induced lipolysis, i.e. in the presence of elevated NEFA. Isoproterenol augmented Il1b and Il6 mRNA levels both in pWAT and eWAT. These results suggest that an increased sympathetic activity enhances NEFA and cytokine load of the adipose microenvironment, including that of pancreatic fat, and by doing so it may alter beta-cell function.

The involvement of the adrenergic nervous system in activating human brown adipose tissue and browning.

Obesity is a chronic condition of multifactorial etiology characterized by excessive body fat due to a calorie intake higher than energy expenditure. Given the intrinsic limitations of surgical interventions and the difficulties associated with lifestyle changes, pharmacological manipulation is currently one of the main therapies for metabolic diseases. Approaches aiming to promote energy expenditure through induction of thermogenesis have been explored and, in this context, brown adipose tissue (BAT) activation and browning have been shown to be promising strategies. Although such processes are physiologically stimulated by the sympathetic nervous system, not all situations that are known to increase adrenergic signaling promote a concomitant increase in BAT activation or browning in humans. Thus, a better understanding of factors involved in the thermogenesis attributed to these tissues is needed to enable the development of future therapies against obesity. Herein we carry out a critical review of original articles in humans under conditions previously known to trigger adrenergic responses-namely, cold, catecholamine-secreting tumor (pheochromocytoma and paraganglioma), burn injury, and adrenergic agonists-and discuss which of them are associated with increased BAT activation and browning. BAT is clearly stimulated in individuals exposed to cold or treated with high doses of the ß3-adrenergic agonist mirabegron, whereas browning is certainly induced in patients after burn injury or with pheochromocytoma, as well as in individuals treated with ß3-adrenergic agonist mirabegron for at least 10 weeks. Given the potential effect of increasing energy expenditure, adrenergic stimuli are promising strategies in the treatment of metabolic diseases.

Adenosine receptor signalling: Probing the potential pathways for the ministration of neuropathic pain.

Neuropathic pain is a critical burdensome problem due to the complex interplay of several pathological mechanisms and lack of availability of effective therapeutic interventions. The available therapeutic options are associated with a variety of limitations, including severe side effects, and unmet medical needs, warranting further research to identify and validate potential targets. Adenosine receptors system is a widely studied target, which evidently was successful in alleviation of neuropathic pain in several experimental paradigms, and researchers are putting efforts in building its clinical roadmap. The adenosine receptors act by different mechanisms and targeting adenosine receptors for neuropathic pain includes several important pathways such as p38-mitogen-activated protein kinases (MAPK), extracellular signal-regulated kinases (ERK), brain-derived neurotrophic factor (BDNF) signalling, γ-aminobutyric acid (GABA) as well as the ion channel modulations. Various studies have also shown the relevance of targeting adenosine receptors in chemotherapy-induced neuropathic pain and diabetic neuropathy. Several drugs acting on adenosine receptors have undergone clinical trials for management of neuropathic pain, whereas many other drugs are yet to be studied to find a potential anti-nociceptive agent. In this review, we have discussed the roadmap of adenosine receptors as a potential target for the treatment of neuropathic pain.

Optimum topical delivery of adrenergic agonists to oral mucosa vasculature.

PURPOSE: Identify an orotopical vehicle to deliver an α-adrenergic vasoconstrictor to submucosal vasculature that is readily palatable to cancer/bone marrow transplant patients that suppresses chemo-radiotherapy-associated oral mucositis. METHODS: A [(3)H] norepinephrine ligand binding assay was developed to quantify receptor binding in hamster oral mucosa. Vehicle components (alcohols, polyols, cellulose, PVP) were tested versus [(3)H] norepinephrine binding. Vehicle refinement was also done to mask phenylephrine bitter taste and achieve human subject acceptance. The optimized vehicle was tested with α-adrenergic active agents to suppress radiation-induced oral mucositis in mice. RESULTS: The ligand binding assay quantified dose- and time-dependent, saturable binding of [(3)H] norepinephrine. An ethanol:glycerol:propylene glycol:water (6:6:8:80) vehicle provided the best delivery and binding. Further vehicle modification (flavoring and sucralose) yielded a vehicle with excellent taste scores in humans. Addition of phenylephrine, norepinephrine or epinephrine to the optimized vehicle and painting into mouse mouths 20 min before 19 Gy irradiation conferred significant suppression of the weight loss (P < 0.001) observed in mice who received oral vehicle. CONCLUSION: We identified a highly efficient vehicle for the topical delivery of phenylephrine to the oral mucosa of both hamster and human subjects. This will enable its testing to suppress oral mucositis in an upcoming human clinical trial.

Equilibrium between adenylyl cyclase and phosphodiesterase patterns adrenergic agonist dose-dependent spatiotemporal cAMP/protein kinase A activities in cardiomyocytes.

Beta-adrenergic receptor induces cAMP/Protein kinase A (PKA) activation to regulate cardiac contraction. Using real-time fluorescence resonance energy transfer imaging for highly sensitive detection of cAMP and PKA activities, we show two distinct phases in isoproterenol dose-dependent responses in cardiomyocytes: a transient and dose-dependent increase in cAMP and PKA activities at lower concentrations from 10(-12) to 10(-8) M; and a saturated initial increases at higher concentrations from 10(-8) to 10(-5) M followed by a rapid decrease to different levels that were later sustained in a dose-dependent manner. The dose-dependent temporal responses are patterned by equilibrium between receptor-activated adenylyl cyclase (AC) and phosphodiesterase (PDE). At lower concentrations, cAMP is produced in an agonist dose-dependent manner with AC as a rate-limiting factor. However, the cAMP activities are confined within local domains for phosphorylation of PDE isoforms in the receptor complex but not for phosphorylation of phospholamban and troponin I. At higher concentrations, isoproterenol promotes a dose-dependent selective dissociation of PDE4D but not ACVI from the receptor complex, which shifts the equilibrium between AC and PDE. This shifted balance leads to sustained cAMP accumulation and diffusion for PKA phosphorylation of phospholamban and troponin I, and for myocyte contraction. Pharmacological inhibition or overexpression of either ACVI or PDE4D8 disrupts the balance and shapes the temporal responses in cAMP accumulation. Together, our data reveal a new paradigm for adrenergic agonist dose-dependent cAMP/PKA activities for substrate-specific phosphorylation dictated by dual regulation of AC and PDE in cardiomyocytes.

Oleuropein, a phenolic compound in extra virgin olive oil, increases uncoupling protein 1 content in brown adipose tissue and enhances noradrenaline and adrenaline secretions in rats.

The effects of oleuropein, a phenolic compound in extra virgin olive oil (EV-olive oil), on triglyceride metabolism were investigated by measuring the degree of thermogenesis in interscapular brown adipose tissue (IBAT), and noradrenaline and adrenaline secretions in rats. In Experiment 1, rats were given a high-fat diet (control diet) with the oleuropein supplementation of 1, 2 or 4 mg/kg of diet (0.1, 0.2 or 0.4% oleuropein diet, respectively). After 28 d of feeding, body weight, perirenal adipose tissue, epididymal fat pad, and plasma triglyceride, free fatty acid and total cholesterol concentrations were reduced by the 0.1, 0.2 or 0.4% oleuropein diet and were significantly lowest in rats fed the 0.4% oleuropein diet, as compared with those of rats fed with the control diet. The content of uncoupling protein 1 (UCP1) in IBAT and urinary noradrenaline and adrenaline excretions were significantly higher in rats fed the 0.1 or 0.2% oleuropein diet, as compared with those of rats fed with the control diet, although there were no significant differences in rats fed the 0.4% oleuropein diet. In Experiment 2, the effects of oleuropein on noradrenaline and adrenaline secretion were evaluated. The intravenous administration of oleuropein and oleuropein aglycone significantly increased plasma noradrenaline and adrenaline concentrations. Furthermore, oleuropein aglycone induced the secretions of noradrenaline and adrenaline about ten fold more potently than oleuropein. These results suggest that the phenolic compound oleuropein in EV-olive oil enhances thermogenesis by increasing the UCP1 content in IBAT and noradrenaline and adrenaline secretions in rats.

Regulation of constitutive androstane receptor and its target genes by fasting, cAMP, hepatocyte nuclear factor alpha, and the coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha.

Animal studies reveal that fasting and caloric restriction produce increased activity of specific metabolic pathways involved in resistance to weight loss in liver. Evidence suggests that this phenomenon may in part occur through the action of the constitutive androstane receptor (CAR, NR1I3). Currently, the precise molecular mechanisms that activate CAR during fasting are unknown. We show that fasting coordinately induces expression of genes encoding peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), CAR, cytochrome P-450 2b10 (Cyp2b10), UDP-glucuronosyltransferase 1a1 (Ugt1a1), sulfotransferase 2a1 (Sult2a1), and organic anion-transporting polypeptide 2 (Oatp2) in liver in mice. Treatments that elevate intracellular cAMP levels also produce increased expression of these genes in cultured hepatocytes. Our data show that PGC-1alpha interaction with hepatocyte nuclear factor 4alpha (HNF4alpha, NR2A1) directly regulates CAR gene expression through a novel and evolutionarily conserved HNF4-response element (HNF4-RE) located in its proximal promoter. Expression of PGC-1alpha in cells increases CAR expression and ligand-independent CAR activity. Genetic studies reveal that hepatic expression of HNF4alpha is required to produce fasting-inducible CAR expression and activity. Taken together, our data show that fasting produces increased expression of genes encoding key metabolic enzymes and an uptake transporter protein through a network of interactions involving cAMP, PGC-1alpha, HNF4alpha, CAR, and CAR target genes in liver. Given the recent finding that mice lacking CAR exhibit a profound decrease in resistance to weight loss during extended periods of caloric restriction, our findings have important implications in the development of drugs for the treatment of obesity and related diseases.

Roles of transmembrane domain 2 and the first intracellular loop in human noradrenaline transporter function: pharmacological and SCAM analysis.

The aim was to investigate the roles of transmembrane domain 2 and the adjacent region of the first intracellular loop in determining human noradrenaline transporter (hNET) function by pharmacological and substituted-cysteine accessibility method (SCAM) analyses. It was first necessary to establish a suitable background NET for SCAM. Alanine mutants of endogenous hNET cysteines, hC86A, hC131A and hC339A, were examined and showed no marked effects on expression or function. hNET and the mutants were also resistant to methanethiosulfonate (MTS), ethylammonium (MTSEA) and MTStrimethylammonium (MTSET). Hence, wild-type hNET is an appropriate background for production of cysteine mutants for SCAM. Pharmacological investigation showed that all mutants except hT99C and hL109C showed reduced cell-surface expression, while all except hM107C showed a reduction in functional activity. The mutations did not markedly affect the apparent affinities of substrates, but apparent affinities of cocaine were decreased 7-fold for hP97C and 10-fold for hF101C and increased 12-fold for hY98C. [3H]Nisoxetine binding affinities were decreased 13-fold for hP97C and 5-fold for hF101C. SCAM analysis revealed that only hL102C was sensitive to 1.25 mm MTSEA, and this sensitivity was protected by noradrenaline, nisoxetine and cocaine. The results suggest that this region of hNET is important for interactions with antidepressants and cocaine, but it is probably not involved in substrate translocation mechanisms.

In vivo epinephrine-mediated regulation of gene expression in human skeletal muscle.

The stress hormone epinephrine produces major physiological effects on skeletal muscle. Here we determined skeletal muscle mRNA expression profiles before and during a 6-h epinephrine infusion performed in nine young men. Stringent statistical analysis of data obtained using 43000 cDNA element microarrays showed that 1206 and 474 genes were up- and down-regulated, respectively. Microarray data were validated using reverse transcription quantitative PCR. Gene classification was performed through data mining of Gene Ontology annotations, cluster analysis of regulated genes among 14 human tissues, and correlation analysis of mRNA and clinical parameter variations. Evidence of an autoregulatory control was provided by the regulation of key genes of the cAMP-dependent transcription pathway. Genes with known functional cAMP response elements were regulated by the hormone. The impact on metabolism was illustrated by coordinated regulations of genes involved in carbohydrate and protein metabolisms. Epinephrine had a profound effect on genes involved in immunity and inflammatory response, a previously unappreciated aspect of catecholamine action. Information on 526 mRNAs corresponded to genes of unknown function. These data define the molecular signatures of epinephrine action in human skeletal muscle. They may contribute to the understanding of skeletal muscle alterations observed in pathological conditions characterized by sympathetic nervous system overdrive.

Molecular mechanisms of ligand-receptor interactions in transmembrane domain V of the alpha2A-adrenoceptor.

1. The structural determinants of catechol hydroxyl interactions with adrenergic receptors were examined using 12 alpha2-adrenergic agonists and a panel of mutated human alpha2A-adrenoceptors. The alpha2ASer201 mutant had a Cys --> Ser201 (position 5.43) amino-acid substitution, and alpha2ASer201Cys200 and alpha2ASer201Cys204 had Ser --> Cys200 (5.42) and Ser --> Cys204 (5.46) substitutions, respectively, in addition to the Cys --> Ser201 substitution. 2. Automated docking methods were used to predict the receptor interactions of the ligands. Radioligand-binding assays and functional [35S]GTPgammaS-binding assays were performed using transfected Chinese hamster ovary cells to experimentally corroborate the predicted binding modes. 3. The hydroxyl groups of phenethylamines were found to have different effects on ligand affinity towards the activated and resting forms of the wild-type alpha2A-adrenoceptor. Substitution of Ser200 or Ser204 with cysteine caused a deterioration in the capability of catecholamines to activate the alpha2A-adrenoceptor. The findings indicate that (i) Cys201 plays a significant role in the binding of catecholamine ligands and UK14,304 (for the latter, by a hydrophobic interaction), but Cys201 is not essential for receptor activation; (ii) Ser200 interacts with the meta-hydroxyl group of phenethylamine ligands, affecting both catecholamine binding and receptor activation; while (iii) substituting Ser204 with a cysteine interferes both with the binding of catecholamine ligands and with receptor activation, due to an interaction between Ser204 and the para-hydroxyl group of the catecholic ring.

Influence of agonist efficacy and receptor phosphorylation on antagonist affinity measurements: differences between second messenger and reporter gene responses.

The ability of an antagonist to bind to a receptor is an innate property of that ligand-receptor chemical interaction. Provided no change in the antagonist or receptor chemical nature occurs, this affinity should remain constant for a given antagonist-receptor interaction, regardless of the agonists used. This fundamental assumption underpins the classification of receptors. Here, measurements of beta2-adrenoceptor-mediated cAMP accumulation and cAMP response-element (CRE)-mediated reporter-gene transcription revealed differences in antagonist affinity that depended upon agonist incubation time and the efficacy of the competing agonist. In cAMP accumulation studies (10-min agonist incubation), antagonist affinities were the same regardless of the agonist used. The CRE-reporter gene assay (5 h of incubation) antagonist affinities were 10-fold lower in the presence of isoprenaline and adrenaline than when salbutamol or terbutaline were present (e.g., log KD propranolol -8.65 +/- 0.08, n = 22, and -9.68 +/- 0.07, n = 17, for isoprenaline and salbutamol-induced responses, respectively). Isoprenaline and adrenaline were more efficacious in functional studies, and their ability to internalize GFP-tagged human beta2-adrenoceptors. Longer-term cAMP studies also showed significant differences in KD values moving toward that seen with gene transcription. Agonist-dependent differences in antagonist affinity were reduced for reporter-gene responses when a phosphorylation-deficient mutant of the beta2-adrenoceptor was used. This study suggests that high-efficacy agonists induce a chemical modification in beta2-adrenoceptors (via phosphorylation) that reduces antagonist affinities. Because reporter-gene assays are used for high-throughput screening in drug discovery, less efficacious or partial agonists may be more reliable than highly efficacious agonists when reporter-gene techniques are used to estimate antagonist affinity.

The effects of epiphyseal peptides on the release of immunoglobulins in Peyer's patches in rats in vitro.

The effects of epiphyseal peptides (1 microg/ml) on the release of immunoglobulins into the incubation medium by isolated Peyer's patches from non-immunized mice and mice immunized orally against ovalbumin were studied during 40-min incubations. The possibility that epiphyseal peptides act on adrenoreceptors of cells in secondary lymphoid organs in the small intestine was assessed using alpha- and beta-adrenoreceptor blockers, i.e., phentolamine HCl (0.02 mg/ml) and anaprilin (0.06 mg/ml) respectively. Basal levels of secretory activity in control Peyer's patches from immunized rats were 2.4 times (p < 0.01) greater than for the lymphoid organs of non-immunized animals. The effects of epiphyseal peptides on the secretory activity of antibody-forming cells depended on the functional state of Peyer's patches. Application of epiphyseal peptides led to a 35% increase (p < 0.05) in the release of immunoglobulins from Peyer's patches in non-immunized rats and a 25% decrease (p < 0.05) in the release of antibody into the incubation medium from the lymphoid organs of immunized animals. These data lead to the suggestion that the activatory effect of epiphyseal peptides on antibody-forming cells in Peyer's patches from non-immunized animals is associated with alpha-adrenoceptors, while their inhibitory action on immunoglobulin release by the small intestine lymphoid organs from immunized animals is not mediated via adrenoceptors.

Modulatory effect of the adrenergic system upon fibroblast proliferation: participation of beta 3-adrenoceptors.

1. The beta3-adrenoceptor agonist ZD 7114, like the non-selective beta-adrenoceptor agonist isoprenaline, but unlike the beta1-adrenoceptor agonist dobutamine and the beta2-adrenoceptor agonist salbutamol, produced an increment on mouse embryonic fibroblast proliferation. The half-maximal stimulation of cell growth occurred at substantially lower concentrations with the beta3-adrenoceptor agonist (EC50: 5.5 x 10(-8) m) than with isoprenaline (EC50: 1.25 x 10(-6) m). 2. The selective beta3-adrenoceptor antagonist SR 5923 OA prevented the beta3-stimulated fibroblast proliferation. Conversely, practolol and butoxamine did not prevent fibroblast growth. 3. Additionally, a decrease of cAMP was obtained in fibroblasts cells upon stimulation with isoprenaline and ZD 7114. 4. The expression of beta-adrenoreceptors on fibroblast cells was also studied by radioligand binding. The Ki values in the presence of beta1- and beta2-adrenoceptor antagonist was two-fold higher than the Ki values for beta3 adrenoceptor antagonist indicating the presence of A3-receptor subtype. 5. Inhibitors of different intracellular coupling pathways including phospholipase C (U 73122), protein kinase C (staurosporine), calcium/calmodulin (trifluoroperazine) and calcium channel (verapamil), prevented the stimulatory actions of the selective beta3-adrenoceptor agonist ZD 7114. 6. The presence of beta3-adrenoceptors on embryonic mouse fibroblast cells may play a role in the modulation of cell growth and biologic activity. The mechanism by which ZD 7114 triggers cell proliferation and function, involves the activation of phospholipase C, PKC, calcium/calmodulin and the influx of calcium.

Cultured stromal cells: an in vitro model of prostatic mesenchymal biology.

BACKGROUND: Initial efforts to develop in vitro models to study prostatic biology focused on the culture and characterization of epithelial cells. Recently, attention has turned towards inclusion of stromal cells in experimental systems. METHODS: Improved methods to isolate and culture stromal cells have been developed. An array of markers are employed to characterize subtypes of stromal cells, with particular interest in smooth muscle differentiation. RESULTS: Defined, serum-free media are available for certain experimental applications. Conditions that promote smooth muscle differentiation have been identified. Investigators have characterized hormonal and peptide factors that regulate the growth of prostatic stromal cells, and have also described paracrine factors produced by stromal cells that influence epithelial biology. CONCLUSIONS: Prostatic stromal-cell cultures are now widely employed by a large number of investigators for a diverse array of experimental purposes. While further refinement is required to obtain model systems that fully mimic in vivo processes, the availability of stromal- and epithelial-cell cultures provides a valuable resource for studying normal prostatic biology as well as benign prostatic hyperplasia (BPH) and cancer.

Thermogenic responses in brown fat cells are fully UCP1-dependent. UCP2 or UCP3 do not substitute for UCP1 in adrenergically or fatty scid-induced thermogenesis.

To examine the thermogenic significance of the classical uncoupling protein-1 (UCP1), the thermogenic potential of brown adipocytes isolated from UCP1-ablated mice was investigated. Ucp1(-/-) cells had a basal metabolic rate identical to wild-type; the mitochondria within them were coupled to the same degree. The response to norepinephrine in wild-type cells was robust ( approximately 10-fold increase in thermogenesis); Ucp1(-/-) cells only responded approximately 3% of this. Ucp1(-/-) cells were as potent as wild-type in norepinephrine-induced cAMP accumulation and lipolysis and had a similar mitochondrial respiratory complement. In wild-type cells, fatty acids induced a thermogenic response similar to norepinephrine, but fatty acids (and retinoate) were practically without effect in Ucp1(-/-) cells. It is concluded that no other adrenergically induced thermogenic mechanism exists in brown adipocytes except that mediated by UCP1 and that entopic expression of UCP1 does not lead to overt innate uncoupling, and it is suggested that fatty acids are transformed to an intracellular physiological activator of UCP1. High expression of UCP2 and UCP3 in the tissue was not associated with an overt innate highly uncoupled state of mitochondria within the cells, nor with an ability of norepinephrine or endo- or exogenous fatty acids to induce uncoupled respiration in the cells. Thus, UCP1 remains the only physiologically potent thermogenic uncoupling protein in these cells.

Activation of leukotriene synthesis in human neutrophils by exogenous arachidonic acid: inhibition by adenosine A(2a) receptor agonists and crucial role of autocrine activation by leukotriene B(4).

We report here that the apparent inability of isolated human polymorphonuclear leukocytes (PMNs) to efficiently transform arachidonic acid (AA) is the consequence of A(2a) receptor engagement by endogenous adenosine accumulating in incubation media. Indeed, when adenosine is eliminated from PMN suspensions by the addition of adenosine deaminase, or when cells are incubated with adenosine A(2a) receptor antagonists, important quantities (40-80 pmol/10(6) cells) of 5-lipoxygenase products are synthesized by PMN incubated with 1 to 5 microM exogenous AA. The selective A(2a) receptor agonist CGS21680 was a very potent inhibitor of the AA-induced leukotriene (LT) synthesis, showing an IC(50) of approximately 1 nM. The mechanism of AA-induced stimulation of LT synthesis observed in the absence of extracellular adenosine was investigated. In adenosine deaminase-treated PMN, exogenous AA induced Ca(2+) mobilization and the translocation of 5-lipoxygenase to nuclear structures. A time lag of 20 to 60 s (variable between PMN preparations) was observed consistently between the addition of AA and the elevation of intracellular Ca(2+) concentration (and LT synthesis), indicating that AA itself did not trigger the Ca(2+) mobilization in PMN. This AA-induced Ca(2+) mobilization, as well as the corresponding 5-lipoxygenase translocation and stimulation of LT synthesis, was blocked efficiently by the LT synthesis inhibitor MK0591, the LTB(4) receptor antagonists CP105696 and LY223982, and the LTA(4) hydrolase inhibitor SC57461A. These data demonstrate that AA is a highly potent and effective activator of LT synthesis and acts through a mechanism that requires an autocrine stimulatory loop by LTB(4).

Dopexamine hydrochloride in the human heart: receptor binding and effects on cAMP generation.

Dopexamine hydrochloride is a synthetic catecholamine proposed for the short-term treatment of heart failure and postoperative low cardiac output. The pharmacological profile and anatomical localization of dopexamine binding were investigated in sections of right and left ventricle using [3H]-dopexamine and ligand techniques associated with light microscope autoradiography. Its effects on the 3-5-cyclic adenosine monophosphate (cAMP) generating system in membrane particles of the human right or left ventricle were also studied. [3H]-Dopexamine was specifically bound to sections of human right or left ventricle. The binding was time-, temperature- and concentration-dependent and was dissociable. The apparent equilibrium constant of dissociation was 3.5 nM. A decreased [3H]-dopexamine binding capacity from the base to the apex and ventricles was noticeable. The pharmacological profile of [3H]-dopexamine binding to sections of right or left ventricle was consistent with the labelling of both beta 2-adrenoceptors and dopamine DA-2 receptors. The most potent displacer of [3H]-dopexamine was the beta 2-adrenoceptor antagonist ICI 118,551 followed by dopamine, noradrenaline and domperidone. The beta 1-adrenoceptor antagonist metoprolol or the dopamine DA-1 receptor antagonist SCH 23390 were ineffective as displacers of [3H]-dopexamine binding. Light microscope autoradiography revealed the localization of [3H]-dopexamine binding sites within the wall of the human right and left ventricle. The density of silver grains was slightly higher in the right than in the left ventricle and showed a uniform transmural distribution across the ventricular wall.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical, autoradiographic and pharmacological evidence for the involvement of tubular DA-1 receptors in the natriuretic response to dopexamine hydrochloride.

Dopexamine hydrochloride (DPX) is a dopamine analog and it possesses agonistic action at DA-1 receptors and beta 2-adrenoceptors. It also is a weak agonist at DA-2 receptors. In the present study, we have examined the anatomical localization of DPX binding sites in rat kidney and their functional significance in terms of the renal effects of this compound. In receptor-ligand binding studies, [3H]-DPX was found to bind specifically to sections of rat kidney in a time (maximum binding at 60 min), temperature (optimal temperature 25 degrees C) and concentration (highest specific/non-specific ratio at 2 nmol/l) dependent manner. Autoradiographic studies revealed the presence of [3H]-DPX binding sites in renal tubules, glomerulus and various layers of small and large blood vessels. Inhibition studies with SCH 23390, ICI 118.551 and 1-sulpiride showed that DPX binds primarily to DA-1 receptors in tubules, only to beta 2-adrenoceptors in glomerulus and to beta 2-adrenoceptors, DA-1 and DA-2 receptors in blood vessels. Also, DPX caused concentration related increases in cyclic AMP levels in rat kidney membrane particles, which could be completely abolished by a combined presence of SCH 23390 and propranolol suggesting that both binding sites of DPX are linked to adenylate cyclase. In functional studies DPX (1 microgram/kg.min for 30 min) produced a modest fall in blood pressure, pronounced tachycardia and slight but significant increase in renal blood flow (11%). These responses were accompanied by increases in urine output (97%), urinary sodium excretion (89%), and fractional excretion of sodium (132%). There was no change in glomerular filtration rate. Propranolol pretreatment abolished DPX-induced hypotension and tachycardia but seemed to potentiate the natriuretic responses to DPX. On the other hand, SCH 23390, a DA-1 receptor antagonist completely abolished DPX-induced hypotension, natriuresis and diuresis without affecting tachycardia. These results indicate that (1) DPX binds predominantly to DA-1 receptors in renal tubules, to beta 2-adrenoceptors in glomerulus and to beta 2-adrenoceptors, as well as DA-1 and DA-2 receptors in renal blood vessels (2) DPX stimulates cAMP formation in the kidney by activating both DA-1 and beta 2-adrenoceptors and (3) DPX produces natriuresis and diuresis by selectively activating DA-1 receptors located on renal tubules.