Anti-diarrheal activities of phytol along with its possible mechanism of action through in-vivo and in-silico models.

Phytol (PHY), a chlorophyll-derived diterpenoid, exhibits numerous pharmacological properties, including antioxidant, antimicrobial, and anticancer activities. This study evaluates the anti-diarrheal effect of phytol (PHY) along with its possible mechanism of action through in-vivo and in-silico models. The effect of PHY was investigated on castor oil-induced diarrhea in Swiss mice by using prazosin, propranolol, loperamide, and nifedipine as standards with or without PHY. PHY at 50 mg/kg (p.o.) and all other standards exhibit significant (p < 0.05) anti-diarrheal effect in mice. The effect was prominent in the loperamide and propranolol groups. PHY co-treated with prazosin and propranolol was found to increase in latent periods along with a significant reduction in diarrheal section during the observation period than other individual or combined groups. Furthermore, molecular docking studies also suggested that PHY showed better interactions with the α- and ß-adrenergic receptors, especially with α-ADR1a and ß-ADR1. In the former case, PHY showed interaction with hydroxyl group of Ser192 at a distance of 2.91Å, while in the latter it showed hydrogen bond interactions with Thr170 and Lys297 with a distance of 2.65 and 2.72Å, respectively. PHY exerted significant anti-diarrheal effect in Swiss mice, possibly through blocking α- and ß-adrenergic receptors.

A positive feedback loop between alpha1-adrenoceptors and inflammatory cytokines in keratinocytes.

A positive feedback loop between inflammatory cytokines and alpha1-adrenoceptors (α1-AR) (a target of the sympathetic nervous system neurotransmitter norepinephrine) influences inflammatory responses in immune cells. This cross-talk between the sympathetic nervous system and immune system may play a role in promoting chronic inflammation. Emerging evidence shows that α1-AR interact with inflammatory cytokines in keratinocytes, and this epidermal adrenergic signalling may contribute to skin inflammatory responses following injury, disease or stress. In this study, utilizing an in vitro approach, we hypothesized that α1-AR interact in a positive feedback loop with inflammatory mediators in keratinocytes. The pro-inflammatory cytokine tumor necrosis factor α (TNFα) was used to induce an inflammatory state in cultured keratinocytes. TNFα increased interleukin (IL)-1ß, IL-6, IL-8 and nerve growth factor (NGF) production and gene expression levels of α1-AR subtype B (α1B-AR). Additional stimulation of α1-AR further increased IL-6 levels, while maintaining high levels of IL-8 and decreasing levels of IL-1ß and NGF. Our results suggest that reciprocal influences between α1-ARs and inflammatory cytokines may play a role in normal inflammatory responses. However, if unchecked, this cycle could contribute to pathology (e.g. chronic inflammatory diseases, chronic pain conditions, and stress-induced cancer progression).

Coupling to Gq Signaling Is Required for Cardioprotection by an Alpha-1A-Adrenergic Receptor Agonist.

RATIONALE: Gq signaling in cardiac myocytes is classically considered toxic. Targeting Gq directly to test this is problematic, because cardiac myocytes have many Gq-coupled receptors. OBJECTIVE: Test whether Gq coupling is required for the cardioprotective effects of an alpha-1A-AR (adrenergic receptor) agonist. METHODS AND RESULTS: In recombinant cells, a mouse alpha-1A-AR with a 6-residue substitution in the third intracellular loop does not couple to Gq signaling. Here we studied a knockin mouse with this alpha-1A-AR mutation. Heart alpha-1A receptor levels and antagonist affinity in the knockin were identical to wild-type. In wild-type cardiac myocytes, the selective alpha-1A agonist A61603-stimulated phosphoinositide-phospholipase C and myocyte contraction. In myocytes with the alpha-1A knockin, both A61603 effects were absent, indicating that Gq coupling was absent. Surprisingly, A61603 activation of cardioprotective ERK (extracellular signal-regulated kinase) was markedly impaired in the KI mutant myocytes, and A61603 did not protect mutant myocytes from doxorubicin toxicity in vitro. Similarly, mice with the α1A KI mutation had increased mortality after transverse aortic constriction, and A61603 did not rescue cardiac function in mice with the Gq coupling-defective alpha-1A receptor. CONCLUSIONS: Gq coupling is required for cardioprotection by an alpha-1A-AR agonist. Gq signaling can be adaptive.

Impaired modulation of postjunctional α1 - but not α2 -adrenergic vasoconstriction in contracting forearm muscle of postmenopausal women.

KEY POINTS: Contraction-mediated blunting of postjunctional α-adrenergic vasoconstriction (functional sympatholysis) is attenuated in skeletal muscle of ageing males, brought on by altered postjunctional α1 - and α2 -adrenergic receptor sensitivity. The extent to which postjunctional α-adrenergic vasoconstriction occurs in the forearms at rest and during exercise in postmenopausal women remains unknown. The novel findings indicate that contraction-mediated blunting of α1 - (via intra-arterial infusion of phenylephrine) but not α2 -adrenergic (via intra-arterial infusion of dexmedetomidine) vasoconstriction was attenuated in postmenopausal women compared to young women. Additional important findings revealed that postjunctional α-adrenergic vasoconstrictor responsiveness at rest does not appear to be affected by age in women. Collectively, these results contribute to our understanding of local neurovascular control at rest and during exercise with age in women. ABSTRACT: Contraction-mediated blunting of postjunctional α-adrenergic vasoconstriction (functional sympatholysis) is attenuated in older males; however, direct confirmation of this effect remains unknown in postmenopausal women (PMW). The present study examined whether PMW exhibit augmented postjunctional α-adrenergic receptor vasoconstriction at rest and during forearm exercise compared to young women (YW). Eight YW (24 ± 1 years) and eight PMW (65 ± 1 years) completed a series of randomized experimental trials: (1) at rest, (2) under high flow (adenosine infusion) conditions and (3) during 6 min of forearm exercise at relative (20% of maximum) and absolute (7 kg) intensities. Phenylephrine (α1 -agonist) or dexmedetomidine (α2 -agonist) was administered during the last 3 min of each trial to elicit α-adrenergic vasoconstriction. Forearm vascular conductance (FVC) was calculated from blood flow and blood pressure. Vasoconstrictor responsiveness was identified as the change in FVC (%) during α-adrenergic agonist infusions from baseline (resting trial) or from steady-state conditions (high flow and exercise trials). During resting and high flow trials, the %FVC during α1 - and α2 -agonist stimulation was similar between YW and PMW. During exercise, α1 -mediated vasoconstriction was blunted in YW vs. PMW at relative (-6 ± 2% vs. -15 ± 3%) and absolute (-4 ± 2% vs. -14 ± 5%) workloads, such that blood flow and FVC were lower in PMW (P < 0.05 for all). Conversely, α2 -mediated vasoconstriction was similar between YW and PMW at relative (-22 ± 3% vs. -22 ± 4%; P > 0.05) and absolute (-19 ± 3% vs. -18 ± 4%; P > 0.05) workloads. Collectively, these findings demonstrate that despite similar α-adrenergic vasoconstrictor responsiveness at rest, PMW have a decreased ability to attenuate α1 -adrenergic vasoconstriction in contracting skeletal muscle.

CgA1AR-1 acts as an alpha-1 adrenergic receptor in oyster Crassostrea gigas mediating both cellular and humoral immune response.

We have now cloned an alpha-1 adrenergic receptor (A1AR) from the cDNA library of oyster Crassostrea gigas, designating as CgA1AR-1. The full length of CgA1AR-1 was 1149 bp and it encodes a protein of 382 amino acids containing a 7 transmembrane domain, whose putative topology was similar to the A1ARs in higher organisms and shared similarity of 19% with mammalian A1ARs according to the phylogenic analysis. After cell transfection of CgA1AR-1 into HEK293T cells and the incubation with its specific agonist norepinephrine (NE), the concentration of second messenger Ca2+ increased significantly (p < 0.05). But, this increasing of Ca2+ could be inhibited by adding A1AR antagonist DOX. Tissue distribution assays using qRT-PCR suggested that CgA1AR-1 mRNA was ubiquitously expressed in all the major tissues of oyster. LPS stimulation could induce the up-regulation of CgA1AR-1 mRNA in haemocytes from 12 h to 24 h post stimulation. Moreover, the blocking of CgA1AR-1 by DOX before LPS stimulation affected the mRNA expression of oyster TNF (CGI_10005109 and CGI_10006440) in haemocytes, resulting in the rise of haemocyte phagocytic rate and apoptosis index. In addition to cellular immunity, CgA1AR-1 was also involved in humoral immunity of oyster. Inhibition of CgA1AR-1 with DOX could repress the up-regulation of LZY and SOD activities caused by LPS stimulation. These results suggested that CgA1AR-1 acted as an α-1 adrenergic receptor in cetacholaminergic neuroendocrine-immune network mediating both cellular and humoral immune response.

Synthesis and structure-activity relationships of novel arylpiperazines as potent antagonists of α1-adrenoceptor.

Arylpiperazines 2-11 were synthesized, and their biological profiles at α1-adrenergic receptors (α1-ARs) assessed by binding assays in CHO cells expressing human cloned subtypes and by functional experiments in isolated rat vas deferens (α1A), spleen (α1B), and aorta (α1D). Modifications at the 1,3-benzodioxole and phenyl phamacophoric units resulted in the identification of a number of potent compounds (moderately selective with respect to the α1b-AR), in binding experiments. Notably, compound 7 (LDT451) showed a subnanomolar pKi of 9.41 towards α1a-AR. An encouragingly lower α1B-potency was a general trend for all the series of compounds, which showed α1A/D over α1B selectivity in functional assays. If adequately optimized, such peculiar selectivity could have relevance for a potential LUTS/BPH therapeutic application.

Discovery of the First Environment-Sensitive Near-Infrared (NIR) Fluorogenic Ligand for α1-Adrenergic Receptors Imaging in Vivo.

Fluorescent ligands are gaining popularity as tools to aid GPCR research. Nonetheless, in vivo application of such tools is hampered due to their short excitation wavelengths in the visible range and lack of fluorogenic switch. Here we report the discovery of fluorescent ligands (3a-f) for α1-adrenergic receptors (α1-ARs) by conjugating the environment-sensitive fluorophore cyane 5 (Cy5) with the quinazoline pharmacophore. Among them, the conjugated compound 3a, with acylated piperazine and the shortest carbon chain spacer, exhibited potent binding and remarkable changes in fluorescence (10-fold) upon binding to α1-AR. Furthermore, it could be employed to selectively and specifically label α1-ARs with no washing procedures in single cells, prostate tissue slices, intact tumor xenografts and organs in living mice. Especially, the slice imaging results gave direct and visual evidence that there is a close relationship between α1-ARs and pathological prostate. It is anticipated that our fluorescent tools will find broad applications in the study of α1-AR pharmacology and physiology to aid development of novel therapeutics.

The Increase in Signaling by Kisspeptin Neurons in the Preoptic Area and Associated Changes in Clock Gene Expression That Trigger the LH Surge in Female Rats Are Dependent on the Facilitatory Action of a Noradrenaline Input.

In rodents, kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3V) of the preoptic area are considered to provide a major stimulatory input to the GnRH neuronal network that is responsible for triggering the preovulatory LH surge. Noradrenaline (NA) is one of the main modulators of GnRH release, and NA fibers are found in close apposition to kisspeptin neurons in the RP3V. Our objective was to interrogate the role of NA signaling in the kisspeptin control of GnRH secretion during the estradiol induced LH surge in ovariectomized rats, using prazosin, an α1-adrenergic receptor antagonist. In control rats, the estradiol-induced LH surge at 17 hours was associated with a significant increase in GnRH and kisspeptin content in the median eminence with the increase in kisspeptin preceding that of GnRH and LH. Prazosin, administered 5 and 3 hours prior to the predicted time of the LH surge truncated the LH surge and abolished the rise in GnRH and kisspeptin in the median eminence. In the preoptic area, prazosin blocked the increases in Kiss1 gene expression and kisspeptin content in association with a disruption in the expression of the clock genes, Per1 and Bmal1. Together these findings demonstrate for the first time that NA modulates kisspeptin synthesis in the RP3V through the activation of α1-adrenergic receptors prior to the initiation of the LH surge and indicate a potential role of α1-adrenergic signaling in the circadian-controlled pathway timing of the preovulatory LH surge.

The eukaryotic translation initiation factor 3f (eIF3f) interacts physically with the alpha 1B-adrenergic receptor and stimulates adrenoceptor activity.

BACKGROUND: eIF3f is a multifunctional protein capable of interacting with proteins involved in different cellular processes, such as protein synthesis, DNA repair, and viral mRNA edition. In human cells, eIF3f is related to cell cycle and proliferation, and its deregulation compromises cell viability. RESULTS: We here report that, in native conditions, eIF3f physically interacts with the alpha 1B-adrenergic receptor, a plasma membrane protein considered as a proto-oncogene, and involved in vasoconstriction and cell proliferation. The complex formed by eIF3f and alpha 1B-ADR was found in human and mouse cell lines. Upon catecholamine stimulation, eIF3f promotes adrenoceptor activity in vitro, independently of the eIF3f proline- and alanine-rich N-terminal region. CONCLUSIONS: The eIF3f/alpha adrenergic receptor interaction opens new insights regarding adrenoceptor-related transduction pathways and proliferation control in human cells. The eIf3f/alpha 1B-ADR complex is found in mammals and is not tissue specific.

Identification of two novel α1-AR agonists using a high-throughput screening model.

α1-Adrenoceptors (ARs; 1A, 1B, and 1D) have been determined to perform different prominent functions in the physiological responses of the sympathetic nervous system. A high-throughput screening assay (HTS) was set up to detect α1-AR subtype-selective agonists by a dual-luciferase reporter assay in HEK293 cells. Using the HTS assay, two novel compounds, CHE3 and CHK3, were discovered as α1-ARs agonists in α1-ARs expressed in HEK293 cells. These compounds also showed moderate/weak anti-proliferative activities against tested cancer cell lines. The HTS assay proposed in this study represents a potential method for discovering more α1-AR subtype-selective ligands.

Alpha1a-adrenoceptor genetic variant induces cardiomyoblast-to-fibroblast-like cell transition via distinct signaling pathways.

The role of naturally occurring human α1a-Adrenergic Receptor (α1aAR) genetic variants associated with cardiovascular disorders is poorly understood. Here, we present the novel findings that expression of human α1aAR-247R (247R) genetic variant in cardiomyoblasts leads to transition of cardiomyoblasts into a fibroblast-like phenotype, evidenced by morphology and distinct de novo expression of characteristic genes. These fibroblast-like cells exhibit constitutive, high proliferative capacity and agonist-induced hypertrophy compared with cells prior to transition. We demonstrate that constitutive, synergistic activation of EGFR, Src and ERK kinases is the potential molecular mechanism of this transition. We also demonstrate that 247R triggers two distinct EGFR transactivation-dependent signaling pathways: 1) constitutive Gq-independent ß-arrestin-1/Src/MMP/EGFR/ERK-dependent hyperproliferation and 2) agonist-induced Gq- and EGFR/STAT-dependent hypertrophy. Interestingly, in cardiomyoblasts agonist-independent hyperproliferation is MMP-dependent, but in fibroblast-like cells it is MMP-independent, suggesting that expression of α1aAR genetic variant in cardiomyocytes may trigger extracellular matrix remodeling. Thus, these novel findings demonstrate that EGFR transactivation by α1aAR-247R leads to hyperproliferation, hypertrophy and alterations in cardiomyoblasts, suggesting that these unique genetically-mediated alterations in signaling pathways and cellular function may lead to myocardial fibrosis. Such extracellular matrix remodeling may contribute to the genesis of arrhythmias in certain types of heart failure.

Conopeptide ρ-TIA defines a new allosteric site on the extracellular surface of the α1B-adrenoceptor.

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey ß(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

Antiarrhythmic properties of phenylpiperazine derivatives of phenytoin with α1-adrenoceptor affinities.

An association between α(1)-adrenoceptor affinities, hERG K(+)-antagonistic properties and antiarrhythmic activities for a series of phenylpiperazine derivatives of hydantoin (2a-21a) was investigated. New compounds were synthesized and tested for their affinity for α(1)-adrenoceptors in radioligand binding assay using [(3)H]-prazosin as a selective radioligand. Antiarrhythmic activities in adrenaline- and barium chloride-induced arrhythmia models, an influence of the phenylpiperazine derivatives on the ECG-components and blood pressure were tested in vivo in normotensive rats. The hERG K(+)-antagonistic properties of the most potent antiarrhythmic agents were investigated in silico by the use of program QikProp. The highest α(1)-adrenoceptor affinity (K(i)=4.7 nM) and the strongest antiarrhythmic activity in adrenaline induced arrhythmia (ED(50)=0.1 mg/kg) was found for 1-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione hydrochloride (19a). The results indicated a significant correlation between α(1)-AR affinities (pK(i)) and antiarrhythmic activity (ED(50)) in adrenaline model (R(2)=0.92, p <0.005). Influence of the examined phenylpiperazine hydantoin derivatives on hERG K(+) channel, predicted by means of in silico methods, suggested their hERG K(+)-blocking properties.

Identification and profiling of novel α1A-adrenoceptor-CXC chemokine receptor 2 heteromer.

We have provided the first evidence for specific heteromerization between the α(1A)-adrenoceptor (α(1A)AR) and CXC chemokine receptor 2 (CXCR2) in live cells. α(1A)AR and CXCR2 are both expressed in areas such as the stromal smooth muscle layer of the prostate. By utilizing the G protein-coupled receptor (GPCR) heteromer identification technology on the live cell-based bioluminescence resonance energy transfer (BRET) assay platform, our studies in human embryonic kidney 293 cells have identified norepinephrine-dependent ß-arrestin recruitment that was in turn dependent upon co-expression of α(1A)AR with CXCR2. These findings have been supported by co-localization observed using confocal microscopy. This norepinephrine-dependent ß-arrestin recruitment was inhibited not only by the α(1)AR antagonist Terazosin but also by the CXCR2-specific allosteric inverse agonist SB265610. Furthermore, Labetalol, which is marketed for hypertension as a nonselective ß-adrenoceptor antagonist with α(1)AR antagonist properties, was identified as a heteromer-specific-biased agonist exhibiting partial agonism for inositol phosphate production but essentially full agonism for ß-arrestin recruitment at the α(1A)AR-CXCR2 heteromer. Finally, bioluminescence resonance energy transfer studies with both receptors tagged suggest that α(1A)AR-CXCR2 heteromerization occurs constitutively and is not modulated by ligand. These findings support the concept of GPCR heteromer complexes exhibiting distinct pharmacology, thereby providing additional mechanisms through which GPCRs can potentially achieve their diverse biological functions. This has important implications for the use and future development of pharmaceuticals targeting these receptors.

Effects of α-adrenoceptor antagonists on ABCG2/BCRP-mediated resistance and transport.

Acquired resistance of cancer cells to various chemotherapeutic agents is known as multidrug resistance, and remains a critical factor in the success of cancer treatment. It is necessary to develop the inhibitors for multidrug resistance. The aim of this study was to examine the effects of eight α-adrenoceptor antagonists on ABCG2/BCRP-mediated resistance and transport. Previously established HeLa/SN100 cells, which overexpress ABCG2/BCRP but not ABCB1/MDR1, were used. The effects of the antagonists on sensitivity to mitoxantrone and the transport activity of Hoehst33342, both substrates for ABCG2/BCRP, were evaluated using the WST-1 assay and cellular kinetics, respectively. ABCG2/BCRP mRNA expression and the cell cycle were also examined by real-time RT-PCR and flow cytometry, respectively. Sensitivity to mitoxantrone was reversed by the α-adrenoceptor antagonists in a concentration-dependent manner, although such effects were also found in the parental HeLa cells. Levels of ABCG2/BCRP mRNA expression were not influenced by the antagonists. The transport activity of Hoechst33342 was decreased by doxazosin and prazosin, but unaffected by the other antagonists. In addition, doxazosin and prazosin increased the proportion of S phase cells in the cultures treated with mitoxantrone, whereas the other α-adrenoceptor antagonists increased the percentage of cells in G(2)/M phase. These findings suggested that doxazosin and prazosin reversed resistance mainly by inhibiting ABCG2/BCRP-mediated transport, but the others affected sensitivity to mitoxantrone via a different mechanism.

Tamsulosin potently and selectively antagonizes human recombinant α(1A/1D)-adrenoceptors: slow dissociation from the α(1A)-adrenoceptor may account for selectivity for α(1A)-adrenoceptor over α(1B)-adrenoceptor subtype.

We determined the binding affinity of tamsulosin, a selective α(1)-adrenoceptor antagonist, for human α(1)-adrenoceptor subtypes in comparison with those of other α(1)-adrenoceptor antagonists including silodosin, prazosin, 5-methylurapidil, terazosin, alfuzosin, nafopidil, urapidil and BMY7378. The association and dissociation kinetics of [(3)H]tamsulosin for recombinant human α(1)-adrenoceptor subtypes were compared with those of [(3)H]prazosin. Tamsulosin competitively inhibited [(3)H]prazosin binding to human α(1A)-, α(1B)- and α(1D)-adrenoceptors (pK(i) values were 10.38, 9.33, 9.85) indicating 11 and 3.4-fold higher affinities for human α(1A)-adrenoceptor than those for α(1B)- and α(1D)-adrenoceptors, respectively. The affinity of tamsulosin for the human α(1A)-adrenoceptor was, respectively, 5, 9.9, 38, 120, 280, 400, 1200 and 10000 fold higher than those of silodosin, prazosin, 5-methylurapidil, terazosin, alfuzosin, naftopidil, urapidil and BMY7378, respectively. [(3)H]Tamsulosin dissociated from the α(1A)-adrenoceptor slower than from the α(1B)- and α(1D)-adrenoceptors (α(1B)>α(1D)>α(1A)). Moreover, [(3)H]tamsulosin dissociated slower than [(3)H]prazosin from the α(1A)-adrenoceptor and faster from the α(1B)- and α(1D)-adrenoceptors. In conclusion, tamsulosin potently and selectively antagonized α(1A/1D)-adrenoceptor ligand binding, and slowly dissociated from the α(1A)-adrenoceptor subtype.

NOX2 activated by α1-adrenoceptors modulates hepatic metabolic routes stimulated by ß-adrenoceptors.

The NADPH oxidase (NOX) family of enzymes oxidase catalyzes the transport of electrons from NADPH to molecular oxygen and generates O(2)(•-), which is rapidly converted into H(2)O(2). We aimed to identify in hepatocytes the protein NOX complex responsible for H(2)O(2) synthesis after α(1)-adrenoceptor (α(1)-AR) stimulation, its activation mechanism, and to explore H(2)O(2) as a potential modulator of hepatic metabolic routes, gluconeogenesis, and ureagenesis, stimulated by the ARs. The dormant NOX2 complex present in hepatocyte plasma membrane (HPM) contains gp91(phox), p22(phox), p40(phox), p47(phox), p67(phox) and Rac 1 proteins. In HPM incubated with NADPH and guanosine triphosphate (GTP), α(1)-AR-mediated H(2)O(2) synthesis required all of these proteins except for p40(phox). A functional link between α(1)-AR and NOX was identified as the Gα(13) protein. Alpha(1)-AR stimulation in hepatocytes promotes Rac1-GTP generation, a necessary step for H(2)O(2) synthesis. Negative cross talk between α(1)-/ß-ARs for H(2)O(2) synthesis was observed in HPM. In addition, negative cross talk of α(1)-AR via H(2)O(2) to ß-AR-mediated stimulation was recorded in hepatocyte gluconeogenesis and ureagenesis, probably involving aquaporine activity. Based on previous work we suggest that H(2)O(2), generated after NOX2 activation by α(1)-AR lightening in hepatocytes, reacts with cAMP-dependent protein kinase A (PKA) subunits to form an oxidized PKA, insensitive to cAMP activation that prevented any rise in the rate of gluconeogenesis and ureagenesis.

Effects of synephrine and beta-phenethylamine on human alpha-adrenoceptor subtypes.

Synephrine and beta-phenethylamine, two naturally occurring compounds, are structurally related to ephedrine. In this study, the effects of synephrine and beta-phenethylamine on alpha-adrenergic receptor (alpha-AR) subtypes are investigated in human embryonic kidney (HEK293) or Chinese hamster ovary (CHO) cells, and compared to that of 1R,2S-norephedrine. The rank order of binding affinities was found to be the same for the subtypes tested (alpha(1A)-, alpha(2A)-, and alpha(2C)-AR) viz, 1R,2S-norephedrine > beta-phenethylamine > synephrine. Functional studies on the alpha(1A)-AR subtype showed that synephrine was a partial agonist giving a maximal response at 100 microM that was equal to 55.3 % of the L-phenylephrine maximum. In contrast, neither 1R,2S-norephedrine nor beta-phenethylamine exhibited agonist activity at the highest concentration tested (300 microM). beta-Phenethylamine was more potent as an antagonist than 1R,2S-norephedrine and synephrine on the alpha(1A)-AR subtype. Functional studies on the alpha(2A)- and alpha(2C)-AR subtypes indicated that synephrine and beta-phenethylamine did not act as agonists. Similar to 1R,2S-norephedrine, both of these analogs reversed the effect of medetomidine against forskolin-induced cAMP elevations at 300 microM, and the rank order of antagonist potency was: 1R,2S-norephedrine = beta-phenethylamine > synephrine; and beta-phenethylamine > 1R,2S-norephedrine > synephrine, respectively. These differences suggest that the presence of a 4-hydroxy group, as in synephrine, reduced the potency in these subtypes. In conclusion, at the alpha(1A)-AR, synephrine acted as a partial agonist, while beta-phenethylamine did not exhibit any direct agonist activity. Both, synephrine and beta-phenethylamine, may act as antagonists of pre-synaptic alpha(2A/2C)-ARs present in nerve terminals.

[Comparative pharmacophore analysis of dual dopamine D2/5-HT(2A) receptor antagonists].

Dual dopamine D2/5-HT2A receptor antagonists have potent activity and are referred to atypical antipsychotics due to their lower propensity to elicit EPS and their moderate efficacy toward negative symptoms. However, an on-going challenge in developing atypical antipsychotics drugs is to maintain the favorable profiles and avoid of cardiovascular risk. In this paper, comparative pharmacophore analysis of dual dopamine D2/5-HT2A receptor antagonists, hERG K+ channel blockers, and alA adrenoceptor antagonists is carried out, and the results could give some insight into multi-target drug design.

Examination of alpha 1 adrenoceptor subtypes in the human ureter.

AIM: Urinary stone disease is a common condition affecting up to 12% of the population. Speedy elimination of ureterolithiasis in the lower part of ureters is reported with the alpha 1-blocker. This study was carried out to characterize the alpha 1 adrenoceptors (AR) subtype gene and protein expression in the proximal, medial, and distal regions of the human ureter with the aim of facilitating stone expulsion. METHODS: The study was carried out on 20 volunteers. Proximal, medial and distal ureter specimens were obtained as discarded tissues after nephroureterectomy for renal pelvic or ureteral carcinoma. Proximal ureter specimens were obtained as discarded tissues after nephrectomy for renal carcinoma. Using ureter samples, real-time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining were performed. RESULTS: The alpha 1d subtype mRNA was highly expressed in each ureteral region, accounting for about 54% of the total AR Messenger Ribonucleic Acid. In the proximal and medial ureter, the distribution of ARs was alpha 1d > or = alpha 1a > alpha 1b as well as that of the total ureter. The distal ureter expressed the highest amount of alpha 1d ARs. The alpha 1d subtype expression tended to be high compared with the alpha 1a subtype expression. In the distal ureter, the distribution of ARs was alpha 1d > alpha 1a > alpha 1b. Immunohistochemical staining revealed that expressions of alpha 1a-AR and alpha 1d-AR were higher than that of alpha 1b-AR. CONCLUSIONS: According to our results, we should try the alpha 1d AR antagonist for the conservative expulsive therapy of distal ureteral stones in the highest region of alpha 1d subtype distribution.