An Intracellular Tripeptide Arg-His-Trp of Serum Origin Detected in MCF-7 Cells is a Possible Agonist to ß2 Adrenoceptor.

BACKGROUND: The need for agonists and antagonists of ß2 adrenoceptor (ß2AR) is warranted in various human disease conditions, including cancer, cardiovascular and other metabolic disorders. However, the sources of agonists of ß2AR are diverse in nature. Interestingly, there is a complete gap in the exploration of agonists of ß2AR from serum that is a well-known component of culture media that supports growth and proliferation of normal and cancer cells in vitro. METHODS: In this paper, we employed a novel vertical tube gel electrophoresis (VTGE)-assisted purification of intracellular metabolites of MCF-7 cells grown in vitro in complete media with fetal bovine serum (FBS). Intracellular metabolites of MCF-7 cells were then analyzed by LC-HRMS. Identified intracellular tripeptides of FBS origin were evaluated for their molecular interactions with various extracellular and intracellular receptors, including ß2AR (PDB ID: 2RH1) by employing molecular docking and molecular dynamics simulations (MDS). A known agonist of ß2AR, isoproterenol was used as a positive control in molecular docking and MDS analyses. RESULTS: We report here the identification of a few novel intracellular tripeptides, namely Arg-His- Trp, (PubChem CID-145453842), Pro-Ile-Glu, (PubChem CID-145457492), Cys-Gln-Gln, (PubChem CID-71471965), Glu-Glu-Lys, (PubChem CID-11441068) and Gly-Cys-Leu (PubChem CID-145455600) of FBS origin in MCF-7 cells. Molecular docking and MDS analyses revealed that among these molecules, the tripeptide Arg-His-Trp shows a favorable binding affinity with ß2AR (-9.8 Kcal/mol). The agonistic effect of Arg-His-Trp is significant and comparable with that of a known agonist of ß2AR, isoproterenol. CONCLUSION: In conclusion, we identified a unique Arg-His-Trp tripeptide of FBS origin in MCF-7 cells by employing a novel approach. This unique tripeptide Arg-His-Trp is suggested to be a potential agonist of ß2AR and it may have applications in the context of various human diseases like bronchial asthma and chronic obstructive pulmonary disease (COPD).

Ligands of Adrenergic Receptors: A Structural Point of View.

Adrenergic receptors are G protein-coupled receptors for epinephrine and norepinephrine. They are targets of many drugs for various conditions, including treatment of hypertension, hypotension, and asthma. Adrenergic receptors are intensively studied in structural biology, displayed for binding poses of different types of ligands. Here, we summarized molecular mechanisms of ligand recognition and receptor activation exhibited by structure. We also reviewed recent advances in structure-based ligand discovery against adrenergic receptors.

Mutual Enhancement of Opioid and Adrenergic Receptors by Combinations of Opioids and Adrenergic Ligands Is Reflected in Molecular Complementarity of Ligands: Drug Development Possibilities.

Crosstalk between opioid and adrenergic receptors is well characterized and due to interactions between second messenger systems, formation of receptor heterodimers, and extracellular allosteric binding regions. Both classes of receptors bind both sets of ligands. We propose here that receptor crosstalk may be mirrored in ligand complementarity. We demonstrate that opioids bind to adrenergic compounds with micromolar affinities. Additionally, adrenergic compounds bind with micromolar affinities to extracellular loops of opioid receptors while opioids bind to extracellular loops of adrenergic receptors. Thus, each compound type can bind to the complementary receptor, enhancing the activity of the other compound type through an allosteric mechanism. Screening for ligand complementarity may permit the identification of other mutually-enhancing sets of compounds as well as the design of novel combination drugs or tethered compounds with improved duration and specificity of action.

Micro-pharmacokinetics: Quantifying local drug concentration at live cell membranes.

Fundamental equations for determining pharmacological parameters, such as the binding affinity of a ligand for its target receptor, assume a homogeneous distribution of ligand, with concentrations in the immediate vicinity of the receptor being the same as those in the bulk aqueous phase. It is, however, known that drugs are able to interact directly with the plasma membrane, potentially increasing local ligand concentrations around the receptor. We have previously reported an influence of ligand-phospholipid interactions on ligand binding kinetics at the ß2-adrenoceptor, which resulted in distinct "micro-pharmacokinetic" ligand profiles. Here, we directly quantified the local concentration of BODIPY630/650-PEG8-S-propranolol (BY-propranolol), a fluorescent derivative of the classical ß-blocker propranolol, at various distances above membranes of single living cells using fluorescence correlation spectroscopy. We show for the first time a significantly increased ligand concentration immediately adjacent to the cell membrane compared to the bulk aqueous phase. We further show a clear role of both the cell membrane and the ß2-adrenoceptor in determining high local BY-propranolol concentrations at the cell surface. These data suggest that the true binding affinity of BY-propranolol for the ß2-adrenoceptor is likely far lower than previously reported and highlights the critical importance of understanding the "micro-pharmacokinetic" profiles of ligands for membrane-associated proteins.

Molecular Recognition by a Short Partial Peptide of the Adrenergic Receptor: A Bottom-Up Approach.

Receptor-neurotransmitter molecular recognition is key for neurotransmission. Although crystal structures of the receptors are known, the mechanism for recognition is not clear. Reported here is the ultraviolet (UV) and infrared (IR) spectra of complexes between a partial peptide (SIVSF), mimicking the binding motif of a catechol ring in the adrenergic receptor, and various ligands. The UV spectra show that two isomers coexist in the complex of SIVSF with properly recognized ligands, such as protonated adrenaline (adrenalineH+ ). From IR spectra, they are assigned to catechol- and amino-bound structures. The catechol-bound structure is not observed when the ligand is replaced by nonproper molecules, such as noradrenalineH+ . The results suggest that SIVSF not only recognizes the catechol ring but can distinguish differences in the amine side chain. The method provides a new possibility for screening molecules as potential therapeutics for activating the receptor.

Bioactive compounds of Shuang-Huang-Lian prescription and an insight into its binding mechanism by ß2 -adrenoceptor chromatography coupled with site-directed molecular docking.

Owing to the promising clinical efficacy and relatively simple composition, Shuang-Huang-Lian prescription is widely prescribed for the treatment of acute upper respiratory tract infection and acute bronchitis in practice. This necessitates the understanding of the bioactive compounds of the prescription and their binding mechanism to ß2 -adrenoceptor, which mediates the aforementioned ailments. In this work, a column containing immobilized ß2 -adrenoceptor was prepared using a diazonium salt reaction. The bioactive compound collected from the ß2 -adrenoceptor column was identified as chlorogenic acid by using high-performance liquid chromatography coupled with ion trap mass spectrometry. Using an injection amount dependent method, chlorogenic acid proved the binding to ß2 -adrenoceptor through two kinds of sites. The numbers of the sites were (1.42 ± 0.03) × 10-8 and (9.06 ± 0.49) × 10-8  M. The association constants were (2.72 ± 0.01) × 105 and (2.80 ± 0.01) × 104  M-1 , respectively. Molecular docking analysis of the interaction between chlorogenic acid and ß2 -adrenoceptor indicated that the binding mainly occurred on Ser169 , Ser173 , and Phe287 of ß2 -adrenoceptor. These results paved the way to screen bioactive compounds of other traditional medicines by receptor chromatography.

An Ensemble-Based Protocol for the Computational Prediction of Helix-Helix Interactions in G Protein-Coupled Receptors using Coarse-Grained Molecular Dynamics.

The accurate identification of the specific points of interaction between G protein-coupled receptor (GPCR) oligomers is essential for the design of receptor ligands targeting oligomeric receptor targets. A coarse-grained molecular dynamics computer simulation approach would provide a compelling means of identifying these specific protein-protein interactions and could be applied both for known oligomers of interest and as a high-throughput screen to identify novel oligomeric targets. However, to be effective, this in silico modeling must provide accurate, precise, and reproducible information. This has been achieved recently in numerous biological systems using an ensemble-based all-atom molecular dynamics approach. In this study, we describe an equivalent methodology for ensemble-based coarse-grained simulations. We report the performance of this method when applied to four different GPCRs known to oligomerize using error analysis to determine the ensemble size and individual replica simulation time required. Our measurements of distance between residues shown to be involved in oligomerization of the fifth transmembrane domain from the adenosine A2A receptor are in very good agreement with the existing biophysical data and provide information about the nature of the contact interface that cannot be determined experimentally. Calculations of distance between rhodopsin, CXCR4, and ß1AR transmembrane domains reported to form contact points in homodimers correlate well with the corresponding measurements obtained from experimental structural data, providing an ability to predict contact interfaces computationally. Interestingly, error analysis enables identification of noninteracting regions. Our results confirm that GPCR interactions can be reliably predicted using this novel methodology.

Mitochondrial Superoxide Signaling Contributes to Norepinephrine-Mediated T-Lymphocyte Cytokine Profiles.

Norepinephrine (NE) produces multifaceted regulatory patterns in T-lymphocytes. Recently, we have shown that NE utilizes redox signaling as evidenced by increased superoxide (O2●-) causally linked to the observed changes in these cells; however, the source of this reactive oxygen species (ROS) remains elusive. Herein, we hypothesized that the source of increased O2●- in NE-stimulated T-lymphocytes is due to disruption of mitochondrial bioenergetics. To address this hypothesis, we utilized purified mouse splenic CD4+ and CD8+ T-lymphocytes stimulated with NE and assessed O2●- levels, mitochondrial metabolism, cellular proliferation, and cytokine profiles. We demonstrate that the increase in O2●- levels in response to NE is time-dependent and occurs at later points of T-lymphocyte activation. Moreover, the source of O2●- was indeed the mitochondria as evidenced by enhanced MitoSOX Red oxidation as well as abrogation of this signal by the addition of the mitochondrial-targeted O2●--scavenging antioxidant MitoTempol. NE-stimulated T-lymphocytes also demonstrated decreased mitochondrial respiratory capacity, which suggests disruption of mitochondrial metabolism and the potential source of increased mitochondrial O2●-. The effects of NE in regards to redox signaling appear to be adrenergic receptor-dependent as specific receptor antagonists could reverse the increase in O2●-; however, differential receptors regulating these processes were observed in CD4+ versus CD8+ T-lymphocytes. Finally, mitochondrial O2●- was shown to be mechanistic to the NE-mediated T-lymphocyte phenotype as supplementation of MitoTempol could reverse specific changes in cytokine expression observed with NE treatment. Overall, these studies indicate that mitochondrial metabolism and O2●--mediated redox signaling play a regulatory role in the T-lymphocyte response to NE.

Current Trends on Antipsychotics: Focus on Asenapine.

Over the years, both first- (FGAs) and second-generation antipsychotics (SGAs), continue to gain increasing evidence of being effective in the treatment of psychotic symptoms. Currently, they represent the first-line treatment of schizophrenia and bipolar disorder, although they are widely used in psychotic depression and other clinical conditions, such as agitation and/or behavioural disturbances. Despite representing an indispensable tool for the treatment of severe psychotic disorders, they are widely known to have a number of unwanted side effects that the clinician must be aware of, and handle carefully to provide the patient the best available treatment in the short and long-term. However, even with respect to the long-term use of some of the most effective SGAs, it is imperative for clinicians not to overlook the risk linked to the onset of potentially severe metabolic side effects such as weight gain, dyslipidaemia, insulinresistance and type II diabetes. Asenapine is one of the newest SGAs licenced in Europe for the treatment of manic episodes and in the US for schizophrenia. It belongs to the same class of clozapine, olanzapine and quetiapine, sharing with them a rather complex pharmacological binding profile. In fact, asenapine shows a high affinity for the serotonin (5HT) receptor of the type 2A (5HT2A) and to a lesser extent for the dopamine receptor of the type 2 (D2), similar to other SGAs. Asenapine behaves also as an antagonist at the level of 5HT2C, H1 and α2-receptors. Asenapine has been reported to be effective either in monotherapy or in combination with mood stabilers (lithium and valproate) in the treatment of manic or mixed episodes, with a lower propensity to induce, or being followed by, depressive symptoms, when compared to other SGAs. These unique properties may explain the increasing interest towards the use of this drug in mixed states, besides schizophrenia and acute mania. The aim of this paper was at reviewing current data on pharmacological properties and clinical use of asenapine, as well as on possible future indication of this SGA.

Mapping HA-tagged protein at the surface of living cells by atomic force microscopy.

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the ß2-adrenergic receptor (ß2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of ß2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process.

Single-molecule imaging revealed dynamic GPCR dimerization.

Single fluorescent-molecule video imaging and tracking in living cells are revolutionizing our understanding of molecular interactions in the plasma membrane and intracellular membrane systems. They have revealed that molecular interactions occur surprisingly dynamically on much shorter time scales (≪1s) than those expected from the results by conventional techniques, such as pull-down assays (minutes to hours). Single-molecule imaging has unequivocally showed that G-protein-coupled receptors (GPCRs) undergo dynamic equilibrium between monomers and dimers, by enabling the determination of the 2D monomer-dimer equilibrium constant, the dimer dissociation rate constant (typically ∼10s(-1)), and the formation rate constant. Within one second, GPCRs typically undergo several cycles of monomer and homo-dimer formation with different partners.

Evidence for a critical role of catecholamines for cardiomyocyte lineage commitment in murine embryonic stem cells.

Catecholamine release is known to modulate cardiac output by increasing heart rate. Although much is known about catecholamine function and regulation in adults, little is known about the presence and role of catecholamines during heart development. The present study aimed therefore to evaluate the effects of different catecholamines on early heart development in an in vitro setting using embryonic stem (ES) cell-derived cardiomyocytes. Effects of catecholamine depletion induced by reserpine were examined in murine ES cells (line D3, αPIG44) during differentiation. Cardiac differentiation was assessed by immunocytochemistry, qRT-PCR, quantification of beating clusters, flow cytometry and pharmacological approaches. Proliferation was analyzed by EB cross-section measurements, while functionality of cardiomyocytes was studied by extracellular field potential (FP) measurements using microelectrode arrays (MEAs). To further differentiate between substance-specific effects of reserpine and catecholamine action via α- and ß-receptors we proved the involvement of adrenergic receptors by application of unspecific α- and ß-receptor antagonists. Reserpine treatment led to remarkable down-regulation of cardiac-specific genes, proteins and mesodermal marker genes. In more detail, the average ratio of ∼40% spontaneously beating control clusters was significantly reduced by 100%, 91.1% and 20.0% on days 10, 12, and 14, respectively. Flow cytometry revealed a significant reduction (by 71.6%, n = 11) of eGFP positive CMs after reserpine treatment. By contrast, reserpine did not reduce EB growth while number of neuronal cells in reserpine-treated EBs was significantly increased. MEA measurements of reserpine-treated EBs showed lower FP frequencies and weak responsiveness to adrenergic and muscarinic stimulation. Interestingly we found that developmental inhibition after α- and ß-adrenergic blocker application mimicked developmental changes with reserpine. Using several methodological approaches our data suggest that reserpine inhibits cardiac differentiation. Thus catecholamines play a critical role during development.

Fluorescence correlation spectroscopy analysis of serotonin, adrenergic, muscarinic, and dopamine receptor dimerization: the oligomer number puzzle.

The issue of G protein-coupled receptor (GPCR) oligomer status has not been resolved. Although many studies have provided evidence in favor of receptor-receptor interactions, there is no consensus as to the exact oligomer size of class A GPCRs. Previous studies have reported monomers, dimers, tetramers, and higher-order oligomers. In the present study, this issue was examined using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH) analysis, a sensitive method for monitoring diffusion and oligomer size of plasma membrane proteins. Six different class A GPCRs were selected from the serotonin (5-HT2A), adrenergic (α1b-AR and ß2-AR), muscarinic (M1 and M2), and dopamine (D1) receptor families. Each GPCR was C-terminally labeled with green fluorescent protein (GFP) or yellow fluorescent protein (YFP) and expressed in human embryonic kidney 293 cells. FCS provided plasma membrane diffusion coefficients on the order of 7.5 × 10(-9) cm(2)/s. PCH molecular brightness analysis was used to determine the GPCR oligomer size. Known monomeric (CD-86) and dimeric (CD-28) receptors with GFP and YFP tags were used as controls to determine the molecular brightness of monomers and dimers. PCH analysis of fluorescence-tagged GPCRs revealed molecular brightness values that were twice the monomeric controls and similar to the dimeric controls. Reduced χ(2) analyses of the PCH data best fit a model for a homogeneous population of homodimers, without tetramers or higher-order oligomers. The homodimer configuration was unaltered by agonist treatment and was stable over a 10-fold range of receptor expression level. The results of this study demonstrate that biogenic amine receptors freely diffusing within the plasma membrane are predominantly homodimers.

Early history of the recognition of molecular biochirality.

This opening chapter recalls the history of the discoveries that led to the appreciation of the nature and importance of molecular chirality in biology, as well as the development of stereochemistry as an interdisciplinary field connecting chemistry and biology. The discoveries described cover roughly the period of ca. 1840-1940, although certain relevant events of earlier or later times are also addressed. A large number of chiral substances occur in nature in unichiral (i.e., single-enantiomer) form, and for centuries many such substances were used in crude extracts for relief from diseases. For the science of biochirality, the first milestone was the discovery of molecular chirality by Louis Pasteur in 1848. Thereafter, fundamental advances were made, beginning in 1857 with Pasteur's discovery of biological enantioselectivity, in the metabolism of (±)-tartaric acid. With the advances in organic chemistry during the second half of the nineteenth century, the structures of many organic molecules were elucidated and new chiral compounds synthesized, and by the turn of the twentieth century studies of stereoselectivity in the biological activity or enzymatic transformations of natural or synthetic substances were proliferating, and chiroselectivity was often found. Among the names associated with important discoveries in biochirality appear Pasteur, Piutti, Fischer, Cushny, Easson and Stedman, and others. The findings soon prompted attempts to explain the phenomenon of enantioselectivity in biological action, beginning with Pasteur's proposal to account for enantioselectivity in the metabolism of tartaric acid. In 1894 Fischer announced his "lock-and-key" metaphor to explain enantioselectivity in enzyme-substrate interactions and in 1933 Easson and Stedman advanced the first chemical-structure-based model, the three-point-attachment paradigm, to rationalize enantioselectivity at adrenergic receptors. This model has been generalized as the simplest basis for enantioselectivity in biological activity. Today molecular chirality is widely recognized as an important modulator of the effects of chiral substances in a variety of branches of biology and medicine.

Synthesis and preliminary evaluation of pharmacological properties of some piperazine derivatives of xanthone.

A series of 9 piperazine derivatives of xanthone were synthesized and evaluated for cardiovascular activity. The following pharmacological experiments were conducted: the binding affinity for adrenoceptors, the influence on the normal electrocardiogram, the effect on the arterial blood pressure and prophylactic antiarrhythmic activity in adrenaline induced model of arrhythmia (rats, iv). Three compounds revealed nanomolar affinity for α(1)-adrenoceptor which was correlated with the strongest cardiovascular (antiarrhythmic and hypotensive) activity in animals' models. The most promising compound was 4-(3-(4-(2-methoxyphenyl)piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride (12) which revealed antiarrhythmic activity with ED(50) value of 0.69 mg/kg in adrenaline induced arrhythmia (rats, iv). Other synthesized xanthone derivatives, that is, (R,S)-4-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride (10) and (R,S)-4-(2-acetoxy-3-(4-(2-methoxyphenyl)piperazine-1-yl)propoxy)-9H-xanthen-9-one hydrochloride (11) also acted as potential antiarrhythmics in adrenaline induced model of arrhythmia in rats after intravenous injection (ED(50) = 0.88 mg/kg and 0.89 mg/kg, respectively). These values were lower than values obtained for reference drugs such as propranolol and urapidil, but not carvedilol. Results were quite promising and suggested that in the group of xanthone derivatives new potential antiarrhythmics and hypotensives might be found.

Membrane lipids in the function of serotonin and adrenergic receptors.

G-protein coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major targets in the development of novel drug candidates in all clinical areas. Since GPCRs are integral membrane proteins, interaction of membrane lipids such as cholesterol and sphingolipids with GPCRs constitutes an emerging area of research in contemporary biology. Cholesterol and sphingolipids represent important lipid components of eukaryotic membranes and play a crucial role in a variety of cellular functions. In this review, we highlight the role of these vital lipids in the function of two representative GPCRs, the serotonin(1A) receptor and the adrenergic receptor. We believe that development in deciphering molecular details of the nature of GPCR-lipid interaction would lead to better insight into our overall understanding of GPCR function in health and disease.

Regulatory effect of connexin 43 on basal Ca2+ signaling in rat ventricular myocytes.

BACKGROUND: It has been found that gap junction-associated intracellular Ca(2+) [Ca(2+)](i) disturbance contributes to the arrhythmogenesis and hyperconstriction in diseased heart. However, whether functional gaps are also involved in the regulation of normal Ca(2+) signaling, in particular the basal [Ca(2+)](i) activities, is unclear. METHODS AND RESULTS: Global and local Ca(2+) signaling and gap permeability were monitored in cultured neonatal rat ventricular myocytes (NRVMs) and freshly isolated mouse ventricular myocytes by Fluo4/AM and Lucifer yellow (LY), respectively. The results showed that inhibition of gap communication by heptanol, Gap 27 and flufenamic acid or interference of connexin 43 (Cx43) with siRNA led to a significant suppression of LY uptake and, importantly, attenuations of global Ca(2+) transients and local Ca(2+) sparks in monolayer NRVMs and Ca(2+) sparks in adult ventricular myocytes. In contrast, overexpression of rat-Cx43 in NRVMs induced enhancements in the above measurements, and so did in HEK293 cells expressing rat Cx43. Additionally, membrane-permeable inositol 1,4,5-trisphosphate (IP(3) butyryloxymethyl ester) and phenylephrine, an agonist of adrenergic receptor, could relieve the inhibited Ca(2+) signal and LY uptake by gap uncouplers, whereas blockade of IP(3) receptor with xestospongin C or 2-aminoethoxydiphenylborate mimicked the effects of gap inhibitors. More importantly, all these gap-associated effects on Ca(2+) signaling were also found in single NRVMs that only have hemichannels instead of gap junctions. Further immunostaining/immunoblotting single myocytes with antibody against Cx43 demonstrated apparent increases in membrane labeling of Cx43 and non-junctional Cx43 in overexpressed cells, suggesting functional hemichannels exist and also contribute to the Ca(2+) signaling regulation in cardiomyocytes. CONCLUSIONS: These data demonstrate that Cx43-associated gap coupling plays a role in the regulation of resting Ca(2+) signaling in normal ventricular myocytes, in which IP(3)/IP(3) receptor coupling is involved. This finding may provide a novel regulatory pathway for mediation of spontaneous global and local Ca(2+) activities in cardiomyocytes.

Stress is a critical player in CYP3A, CYP2C, and CYP2D regulation: role of adrenergic receptor signaling pathways.

Stress is a critical player in the regulation of the major cytochrome P-450s (CYPs) that metabolize the majority of the prescribed drugs. Early in life, maternal deprivation (MD) stress and repeated restraint stress (RS) modified CYP expression in a stress-specific manner. In particular, the expression of CYP3A1 and CYP2C11 was increased in the liver of MD rats, whereas RS had no significant effect. In contrast, hepatic CYP2D1/2 activity was increased by RS, whereas MD did not affect it. The primary effectors of the stress system, glucocorticoids and epinephrine, highly induced CYP3A1/2. Epinephrine also induced the expression of CYP2C11 and CYP2D1/2. Further investigation indicated that AR-agonists may modify CYP regulation. In vitro experiments using primary hepatocyte cultures treated with the AR-agonists phenylephrine, dexmedetomidine, and isoprenaline indicated an AR-induced upregulating effect on the above-mentioned CYPs mediated by the cAMP/protein kinase A and c-Jun NH2-terminal kinase signaling pathways. Interestingly though, in vivo pharmacological manipulations of ARs using the same AR-agonists led to a suppressed hepatic CYP expression profile, indicating that the effect of the complex network of central and peripheral AR-linked pathways overrides that of the hepatic ARs. The AR-mediated alterations in CYP3A1/2, CYP2C11, and CYP2D1/2 expressions are potentially connected with those observed in the activation of signal transducer and activator of transcription 5b. In conclusion, stress and AR-agonists may modify the expression of the major CYP genes involved in the metabolism of drugs used in a wide range of diseases, thus affecting drug efficacy and toxicity.