Lubabegron fumarate acts as a ß-adrenergic receptor antagonist in cultured bovine intramuscular and subcutaneous adipocytes.

We hypothesized that lubabegron fumarate (LUB) (Experior, Elanco Animal Health, Greenfield, IN) would act as an antagonist to ß-adrenergic receptor (ß-AR) subtypes in primary bovine subcutaneous (s.c.) and intramuscular (i.m.) adipocytes differentiated in culture. This study employed LUB, dobutamine (DOB, a selective ß1-agonist), salbutamol (SAL, a selective ß2-agonist), and propranolol (PRO, a non-selective ß-AR antagonist). Preadipocytes were isolated by standard techniques from bovine longissimus muscle and overlying s.c. adipose tissue and differentiated to adipocytes for 14 d. The adipocyte source x stage of differentiation interaction was significant for ß-adrenergic receptors-1 (ADRB1) (P = 0.001) and ADRB2 (P = 0.01) in that expression of ADRB1 and ADRB2 was greater in s.c. adipocytes than in s.c. preadipocytes; expression of the ADRB1-3 did not change after differentiation of i.m. adipocytes. CCATT/enhancer-binding protein alpha (CEBPA) expression increased upon differentiation in both s.c. and i.m. adipocytes (P = 0.006). The source x stage of differentiation interaction was significant for peroxisome proliferator-activated receptor gamma (PPARG) (P ≤ 0.001) and fatty acid binding protein-4 (FABP4) (P = 0.004). Expression of PPARG increased after differentiation of s.c. preadipocytes to adipocytes, but PPARG expression did not change with differentiation of i.m. preadipocytes to adipocytes. FABP4 expression increased after differentiation of both s.c. and i.m. adipocytes, but FABP4 expression increased to a greater extent in s.c. adipocytes. In s.c. adipocytes, DOB elevated cAMP and glycerol production and protein kinase A (PKA) activity, and SAL increased PKA activity; these effects were abolished by LUB and PRO (P < 0.001). Incubation of i.m. adipocytes with SAL increased cAMP production and PKA activity, which was attenuated by LUB and PRO (P ≤ 0.006). In s.c. adipocytes, SAL, LUB + SAL, and LUB + DOB upregulated hormone sensitive lipase (HSL) (P < 0.001) and perilipin (P = 0.002) gene expression. In i.m. adipocytes, DOB and LUB + DOB increased HSL gene expression (P = 0.001) and LUB + SAL depressed adipose triglyceride lipase expression below control levels (P = 0.001). These results demonstrate that LUB is a ß-AR antagonist at the ß1-AR and ß2-AR subtypes in s.c. adipocytes, and that s.c. and i.m. exhibit different responses to ß-AA and LUB.

We hypothesized that lubabegron fumarate (Experior, Elanco, Greenfield, IN) would act as an antagonist to ß-adrenergic receptor subtypes in primary bovine backfat (subcutaneous) and marbling (intramuscular) adipocytes differentiated in culture. Fat cells were isolated from marbling of longissimus muscle and overlying backfat. In backfat cells, lubabegron fumarate downregulated genes associated with turnover of stored lipid, and lubabegron fumarate reversed the increase in cyclic AMP and protein kinase A caused by the ß1-adrenergic receptor agonist, dobutamine, and the ß2-adrenergic agonist, salbutamol. Increasing cyclic AMP amount and protein kinase A activity would lead to a decrease in backfat lipid stores (reducing backfat thickness), and this would be effectively blocked by lubabegron fumarate. Salbutamol but not dobutamine increased cyclic AMP amount and protein kinase A activity in marbling fat cells, and this effect was blocked by lubabegron fumarate. Taken together, the results of this study indicate that lubabegron fumarate antagonizes the effects of hormones that promote lipid loss from backfat and marbling. However, marbling fat cells are not as responsive as backfat fat cells to ß-adrenergic agonists, so ß-adrenergic agonists such as Zilmax and OptiFlex should have less effect on marbling scores than on backfat thickness.

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.

Stereoselective cell uptake of adrenergic agonists and antagonists by organic cation transporters.

Stereoselectivity is well described for receptor binding and enzyme catalysis, but so far has only been scarcely investigated in carrier-mediated membrane transport. We thus studied transport kinetics of racemic (anti)adrenergic drugs by the organic cation transporters OCT1 (wild-type and allelic variants), OCT2, OCT3, MATE1, and MATE2-K with a focus on stereospecificity. OCT1 showed stereoselective uptake with up to 2-fold higher vmax over their corresponding counterpart enantiomers for (R,R)-fenoterol, (R,R)-formoterol, (S)-salbutamol, (S)-acebutolol, and (S)-atenolol. Orciprenaline and etilefrine were also transported stereoselectively. The Km was 2.1-fold and 1.5-fold lower for the (S,S)-enantiomers of fenoterol and formoterol, while no significant difference in Km was seen for the other aforementioned drugs. Common OCT1 variants showed similar enantiopreference to wild-type OCT1, with a few notable exceptions (e.g. a switch in enantiospecificity for fenoterol in OCT1*2 compared to the wild-type). Other cation transporters showed strong differences to OCT1 in stereoselectivity and transport activity: The closely related OCT2 displayed a 20-fold higher vmax for (S,S)-fenoterol compared to (R,R)-fenoterol and OCT2 and OCT3 showed 3.5-fold and 4.6-fold higher vmax for the pharmacologically active (R)-salbutamol over (S)-salbutamol. MATE1 and MATE2-K generally mediated transport with a higher capacity but lower affinity compared to OCT1, with moderate stereoselectivity. Our kinetic studies showed that significant stereoselectivity exists in solute carrier-mediated membrane transport of racemic beta-adrenergic drugs with surprising, and in some instances even opposing, preferences between closely related organic cation transporters. This may be relevant for drug therapy, given the strong involvement of these transporters in hepatic and renal drug elimination.

The adrenergic receptor antagonist carvedilol interacts with serotonin 2A receptors both in vitro and in vivo.

There is increasing support for the potential clinical use of compounds that interact with serotonin 2A (5-HT2A) receptors. It is therefore of interest to discover novel compounds that interact with 5-HT2A receptors. In the present study, we used computational chemistry to identify critical ligand structural features of 5-HT2A receptor binding and function. Query of compound databases using those ligand features revealed the adrenergic receptor antagonist carvedilol as a high priority match. As carvedilol is used clinically for cardiovascular diseases, we conducted experiments to assess whether it has any interactions with 5-HT2A receptors. In vitro experiments demonstrated that carvedilol has high nanomolar affinity for 5-HT2A receptors. In vivo experiments demonstrated that carvedilol increases the ethanol-induced loss of the righting reflex and suppresses operant responding in mice, and that these effects are attenuated by pretreatment with the selective 5-HT2A receptor antagonist M100907. Moreover, carvedilol did not induce the head-twitch response in mice, suggesting a lack of psychedelic effects. However, carvedilol did not activate canonical 5-HT2A receptor signaling pathways and antagonized serotonin-mediated signaling. It also reduced the head-twitch response induced by 2,5-Dimethoxy-4-iodoamphetamine, suggesting potential in vivo antagonism, allosteric modulation, or functional bias. These data suggest that carvedilol has functionally relevant interactions with 5-HT2A receptors, providing a novel mechanism of action for a clinically used compound. However, our findings do not clearly delineate the precise mechanism of action of carvedilol at 5-HT2A receptors, and additional experiments are needed to elucidate the role of 5-HT2A receptors in the behavioral and clinical effects of carvedilol.

Design, synthesis and cardiovascular evaluation of some aminoisopropanoloxy derivatives of xanthone.

A series of aminoisopropanoloxy derivatives of xanthone has been synthesized and their pharmacological properties regarding the cardiovascular system has been evaluated. Radioligand binding and functional studies in isolated organs revealed that title compounds present high affinity and antagonistic potency for α1-(compound 2 and 8), ß-(compounds 1, 3, 4, 7), α1/ß-(compounds 5 and 6) adrenoceptors. Furthermore, compound 7, the structural analogue of verapamil, possesses calcium entry blocking activity. The title compounds showed hypotensive and antiarrhythmic properties due to their adrenoceptor blocking effect. Moreover, they did not affect QRS and QT intervals, and they did not have proarrhythmic potential at tested doses. In addition they exerted anti-aggregation effect. The results of this study suggest that new compounds with multidirectional activity in cardiovascular system might be found in the group of xanthone derivatives.

Effect of Cortisol on Plasma Lactate Levels following Cortisolinduced Hyperglycaemia in Common African Toad, Bufo regularis.

Previous studies in man have shown that cortisol induces hyperglycemia through gluconeogenesis. However,the metabolic substrates involved in cortisol-induced hyperglycemia and the role of adrenergic receptors in lactate productionin toads have not been well studied. This study investigated the effects of adrenergic receptor blockers in cortisol-inducedhyperglycemia and blood lactate levels in the common African toad (Bufo regularis). Each toad was fasted and anaesthetizedwith sodium thiopentone given intraperitoneally (50mg/kg/i.p). The animals (control) received 0.7% amphibian saline whileanimals (untreated) received cortisol intravenously (50µg/kg/i.v). In pre-treatment groups, animals received propanolol (0.5mg/kg/i.v), prazosin (0.2 mg/kg/i.v) and combination of propanolol (0.5mg/kg/i.v) and prazosin (0.2 mg/kg/i.v) respectivelyfollowed by administration of cortisol 50µg/kg/i.v. Thereafter, blood samples were collected for estimation of glucose andlactate using the modified glucose oxidase method and colorimetric method respectively. Cortisol caused significant increase in blood glucose level ((p<0.05) and reduction in blood lactate levels. Pre-treatment with Prazosin (0.2 mg/kg/i.v) causedsignificant (p<0.05) increase in blood glucose level and significant reduction in blood lactate levels while pre-treatment withPropanolol (0.5mg/kg/i.v) abolished cortisol-induced hyperglycemia and caused increase in blood lactate levels comparedwith the untreated group. The combination of both blockers abolished the hyperglycemic effect of cortisol and causedincrease in the blood lactate levels. The results of this study show that cortisol-induced hyperglycemia is a consequent ofgluconeogenesis and mediated through the beta-adrenergic receptors. The results also show that lactate is produced andused as a gluconeogenic substrate to induce cortisol hyperglycemia in the Common African toad bufo regularis. The betaadrenergic receptors are involved in the use of lactate to induce cortisol hyperglycemia in the Common African toad Buforegularis.

Adrenergic antagonists restrict replication of Legionella.

Legionella pneumophila is a facultative intracellular bacterium, which upon inhalation can cause a potentially fatal pneumonia termed Legionnaires' disease. The opportunistic pathogen grows in environmental amoebae and mammalian macrophages within a unique membrane-bound compartment, the 'Legionella-containing vacuole'. Bacteria are exposed to many environmental cues including small signalling molecules from eukaryotic cells. A number of pathogenic bacteria sense and respond to catecholamine hormones, such as adrenalin and noradrenalin, a process mediated via the QseBC two-component system in some bacteria. In this study, we examined the effect of adrenergic compounds on L. pneumophila, and discovered that the adrenergic receptor antagonists benoxathian, naftopidil, propranolol and labetalol, as well as the QseC sensor kinase inhibitor LED209, reduced the growth of L. pneumophila in broth or amoebae, while replication in macrophages was enhanced. Growth restriction was common to members of the genus Legionella and Mycobacterium, and was observed for L. pneumophila in the replicative but not stationary phase of the biphasic life cycle. Deletion of the L. pneumophila qseBC genes indicated that growth inhibition by adrenergics or LED209 is mediated only to a minor extent by this two-component system, implying the presence of other adrenergic sensing systems. This study identifies adrenergic molecules as novel inhibitors of extra- and intracellular growth of Legionella and reveals LED209 as a potential lead compound to combat infections with Legionella or Mycobacterium spp.

Differential effects of propranolol on conditioned hyperactivity and locomotor sensitization induced by morphine in rats.

According to memory reconsolidation theory, when long-term memory is reactivated by relevant clues, the memory traces become labile, which can be altered by pharmacological manipulations. Accumulating evidence reveals that memory related to drug abuse can be erased by disrupting reconsolidation process. We used an animal model that could simultaneously measure conditioned hyperactivity and locomotor sensitization induced by morphine. ß-Adrenoceptor antagonist propranolol or saline were administered following conditioned stimuli (CS) or a small dose of morphine reactivation. The results showed that the conditioned hyperactivity could be disrupted by propranolol treatment following CS reactivation. However, the expression of locomotor sensitization could not be disrupted by propranolol administration following CS or morphine reactivation. Furthermore, morphine injection and propranolol intervention enhanced the locomotor sensitization effect. These data suggest that blocking the reconsolidation process can disrupt the conditioned hyperactivity induced by environmental cues associated with morphine treatment, but not morphine-induced locomotor sensitization.

α1-Adrenoceptor and serotonin 5-HT(1A) receptor affinity of homobivalent 4-aminoquinoline compounds: an investigation of the effect of linker length.

α1-adrenoceptor (α1-AR) subtype-selective ligands lacking off-target affinity for the 5-HT(1A) receptor (5-HT(1A)-R) will provide therapeutic benefits in the treatment of urogenital conditions such as benign prostatic hyperplasia. In this study we determined the affinity of 4-aminoquinoline and eleven homobivalent 4-aminoquinoline ligands (diquinolines) with alkane linkers of 2-12 atoms (C2-C12) for α(1A), α(1B) and α(1D)-ARs and the 5-HT(1A)-R. These ligands are α(1A)-AR antagonists with nanomolar affinity for α(1A) and α(1B)-ARs. They display linker-length dependent selectivity for α(1A/B)-ARs over α(1D)-AR and the 5-HT(1A)-R. The C2 diquinoline has the highest affinity for α1A-AR (pKi 7.60±0.26) and greater than 30-fold and 600-fold selectivity for α(1A)-AR over α(1D)-AR and 5-HT(1A)-R respectively. A decrease in affinity for α1-ARs is observed as the linker length increases, reaching a nadir at 5 (α(1A/1B)-ARs) or 6 (α(1D)-AR) atoms; after which affinity increases as the linker is lengthened, peaking at 9 (α(1A/1B/1D)-ARs) or 8 (5-HT(1A)-R) atoms. Docking studies suggest that 4-aminoquinoline and C2 bind within the orthosteric binding site, while for C9 one end is situated within the orthosteric binding pocket, while the other 4-aminoquinoline moiety interacts with the extracellular surface. The limited α(1D)-AR and 5-HT(1A)-R affinity of these compounds makes them promising leads for future drug development of α(1A)-AR selective ligands without α(1D)-AR and the 5-HT(1A)-R off-target activity.

Molecular modeling study of chiral separation and recognition mechanism of ß-adrenergic antagonists by capillary electrophoresis.

Chiral separations of five ß-adrenergic antagonists (propranolol, esmolol, atenolol, metoprolol, and bisoprolol) were studied by capillary electrophoresis using six cyclodextrins (CDs) as the chiral selectors. Carboxymethylated-ß-cyclodextrin (CM-ß-CD) exhibited a higher enantioselectivity power compared to the other tested CDs. The influences of the concentration of CM-ß-CD, buffer pH, buffer concentration, temperature, and applied voltage were investigated. The good chiral separation of five ß-adrenergic antagonists was achieved using 50 mM Tris buffer at pH 4.0 containing 8 mM CM-ß-CD with an applied voltage of 24 kV at 20 °C. In order to understand possible chiral recognition mechanisms of these racemates with CM-ß-CD, host-guest binding procedures of CM-ß-CD and these racemates were studied using the molecular docking software Autodock. The binding free energy was calculated using the Autodock semi-empirical binding free energy function. The results showed that the phenyl or naphthyl ring inserted in the hydrophobic cavity of CM-ß-CD and the side chain was found to point out of the cyclodextrin rim. Hydrogen bonding between CM-ß-CD and these racemates played an important role in the process of enantionseparation and a model of the hydrogen bonding interaction positions was constructed. The difference in hydrogen bonding formed with the -OH next to the chiral center of the analytes may help to increase chiral discrimination and gave rise to a bigger separation factor. In addition, the longer side chain in the hydrophobic phenyl ring of the enantiomer was not beneficial for enantioseparation and the chiral selectivity factor was found to correspond to the difference in binding free energy.

Antiadrenergic and hemodynamic effects of ranolazine in conscious dogs.

Effects of ranolazine alone and in the presence of phenylephrine (PE) or isoproterenol (ISO) on hemodynamics, coronary blood flow and heart rate (HR) in the absence and presence of hexamethonium (a ganglionic blocker) were studied in conscious dogs. Ranolazine (0.4, 1.2, 3.6, and 6 mg/kg, intravenous) alone caused transient (<1 minute) and reversible hemodynamic changes. PE (0.3-10 µg/kg) caused a dose-dependent increase in blood pressure and decrease in HR. ISO (0.01-0.3 µg/kg) caused a dose-dependent decrease in blood pressure and an increase in HR. Ranolazine at high (11-13 mM), but not at moderate (4-5 mM) concentrations partially attenuated changes in mean arterial blood pressure and HR caused by either PE or ISO in normal conscious dogs. However, in dogs treated with hexamethonium (20 mg/kg) to cause autonomic blockade, ranolazine (both 4-5 and 11-13 µM) significantly attenuated both the PE- and ISO-induced changes in mean arterial blood pressure. The results suggest that a potential antiadrenergic effect of ranolazine was masked by autonomic control mechanisms in conscious dogs but could be observed when these mechanisms were inhibited (eg, in the hexamethonium-treated dog). Ranolazine, at plasma concentrations <10 µM and in conscious dogs with intact autonomic regulation, had minimal antiadrenergic (α and ß) effects.

Pharmacological interventions on asymmetric dimethylarginine, a clinical marker of vascular disease.

The aim of this paper is to review the latest data on the pharmacological modulation of asymmetric dimethylarginine in human disease. When the terminal nitrogens of the guanidine portion of an arginine become methylated through the action of N-methyl transferases, two chemically close, but physiologically different amino acids are synthesized: symmetric and asymmetric dimethylarginine. The vascular origin of asymmetric dimethylarginine and its inhibitory activity on endothelial nitric oxide synthase give it an important role in certain diseases in which microcirculation is compromised: hypertension, atherosclerosis, inflammatory bowel disease, and diabetes. This review discusses the role that asymmetric dimethylarginine plays in the development of vascular disease, and, wherever possible, evaluates its use in clinical diagnosis. The fact that a number of chemically unrelated drugs, such as angiotensin II antagonists, selective beta- 1 adrenergic antagonists, plant phenolics, statins, and farnesoid X receptor agonists have all been found to reduce dimethylarginine levels in plasma or tissue allows for an integrated study. Although it is difficult to determine exactly why these various agents all have the same effect on arginine metabolism, an explanation of their mechanisms of action is needed. We have thus analyzed the mechanisms involved and reviewed potential trends in the therapeutic use of these drugs.

Memory systems in the chick: regional and temporal control by noradrenaline.

Learning starts with the information about a situation or experience delivered to different brain areas in terms of visual, olfactory, auditory and tactile inputs. Memory processing occurs in different brain locations in a well-defined temporal sequence of physiologically based stages and biochemical cascades. Using neuropharmacological techniques in one species and a robust bead discrimination task, we have been able to chart the passage of memory from acquisition to consolidation in the chick and to dissect out the multiple roles for noradrenaline in consolidating this memory. Fortunately only a small fraction of sensory input is remembered and it is clear that modulatory neurotransmitters play a key role in determining what is remembered. We have identified roles for noradrenaline in the mesopallium or 'avian cortex', the hippocampus, medial striatum or basal ganglia and teased out the different effects of noradrenaline in each of these areas based on the receptor subtypes activated by the transmitter and the stages on which they act. Noradrenergic input from the locus coeruleus controls memory processing at two critical times after training-acquisition (0-2.5 min after training) and consolidation (25-30 min after training). We have also elucidated some of the cellular mechanisms whereby noradrenaline achieves memory modulation and finds that it has actions on both neurones and astrocytes with particularly important effects on energy metabolism in astrocytes. The memory system of the chick is very similar to that of mammals in terms of brain regions recruited in memory processing and in the ways memory is modulated by noradrenaline.

GPCR antitarget modeling: pharmacophore models for biogenic amine binding GPCRs to avoid GPCR-mediated side effects.

G protein-coupled receptors (GPCRs) form a large protein family that plays an important role in many physiological and pathophysiological processes. However, the central role that the biogenic amine binding GPCRs and their ligands play in cell signaling poses a risk in new drug candidates that reveal side affinities towards these receptor sites. These candidates have the potential to interfere with the physiological signaling processes and to cause undesired effects in preclinical or clinical studies. Here, we present 3D cross-chemotype pharmacophore models for three biogenic amine antitargets: the alpha(1A) adrenergic, the 5-HT(2A) serotonin, and the D2 dopamine receptors. These pharmacophores describe the key chemical features present within these biogenic amine antagonists and rationalize the biogenic amine side affinities found for numerous new drug candidates. First applications of the alpha(1A) adrenergic receptor model reveal that these in silico tools can be used to guide the chemical optimization towards development candidates with fewer alpha(1A)-mediated side effects (for example, orthostatic hypotension) and, thus, with an improved clinical safety profile.

Pharmacological characterisation of novel alpha 2-adrenoceptor antagonists as potential brain imaging agents.

Development of suitable imaging ligands to facilitate in vivo characterisation of alpha(2)-adrenoceptors has been limited in its success. In the present study, a series of iodinated derivatives and a fluorinated derivative of the classical alpha(2)-adrenoceptor antagonist, idazoxan, have been evaluated as potential imaging ligands. These compounds are based on the structure of idazoxan but more closely resemble the selective alpha(2)-adrenoceptor antagonists 2-methoxy-idazoxan (RX821002) and 2-ethoxy-idazoxan (RX811059). Preliminary studies, investigating their affinities at alpha(2)-adrenoceptors, using brain membranes prepared from a variety of species, and their ability to antagonise UK14, 304-induced inhibition of twitch in mouse vas deferens highlighted 2-iodopropoxy-idazoxan and 2-fluoroethoxy-idazoxan as the most promising candidates. Further characterisation of these two compounds showed they had a good selectivity for alpha(2)-adrenoceptors compared with imidazoline(2)-binding sites and beta-adrenoceptors. Additional functional studies also showed a lack of intrinsic activity at alpha(2)-adrenoceptors. Following intravenous injection, both compounds were able to cross the blood brain barrier when tested using an ex vivo binding assay. These data show that both 2-iodopropoxy-idazoxan and 2-fluoroethoxy-idazoxan have binding and functional properties suitable for imaging ligands. Further studies using radiolabelled forms of these ligands and a more extensive characterisation of their binding profiles are necessary but these initial evaluations demonstrate their potential.

Effect of alpha 2-adrenoceptor blockade on lithium action in the rat brain.

The inhibitory effect of different concentrations of lithium (0.15-10 x 10(-3) M) on cAMP production induced by isoprenaline (1 x 10(-4) M) after the blockade of alpha(2)-adrenoceptors in the rat cerebral cortex was investigated. Low lithium concentrations (0.3-0.6 x 10(-3) M) exerted a significant inhibitory effect after yohimbine (1 x 10(-5) M) addition, but had no effect when isoprenaline alone or prazosin (1 x 10(-7) M) was added. The recovery of [3H]yohimbine binding after irreversible inactivation by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was evaluated in cortical membranes to study how alpha(2)-adrenoceptor repopulation affects the action of lithium on the adenylyl cyclase-cAMP system. When the density of alpha(2)-adrenoceptors was lower than 21%, lithium showed a significant inhibitory effect at all concentrations tested. However, at higher densities, increased concentrations of lithium were required to inhibit cAMP production. Our results suggest that the inhibitory effect of lithium on cAMP levels in the rat brain is conditioned by alpha(2D)-adrenoceptors.

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.

Spinal gabapentin and antinociception: mechanisms of action.

Spinal gabapentin has been known to show the antinociceptive effect. Although several assumptions have been suggested, mechanisms of action of gabapentin have not been clearly established. The present study was undertaken to examine the action mechanisms of gabapentin at the spinal level. Male SD rats were prepared for intrathecal catheterization. The effect of gabapentin was assessed in the formalin test. After pretreatment with many classes of drugs, changes of effect of gabapentin were examined. General behaviors were also observed. Intrathecal gabapentin produced a suppression of the phase 2 flinching, but not phase 1 in the formalin test. The antinociceptive action of intrathecal gabapentin was reversed by intrathecal NMDA, AMPA, D-serine, CGS 15943, atropine, and naloxone. No antagonism was seen following administration of bicuculline, saclofen, prazosin, yohimbine, mecamylamine, L-leucine, dihydroergocristine, or thapsigargin. Taken together, intrathecal gabapentin attenuated only the facilitated state. At the spinal level, NMDA receptor, AMPA receptor, nonstrychnine site of NMDA receptor, adenosine receptor, muscarinic receptor, and opioid receptor may be involved in the antinociception of gabapentin, but GABA receptor, L-amino acid transporter, adrenergic receptor, nicotinic receptor, serotonin receptor, or calcium may not be involved.

Antiplatelet activity of synthetic pyrrolo-benzylisoquinolines.

Pyrrolo-benzylisoquinolines were prepared as target compounds and their antiplatelet aggregation activity, adreno-receptor affinity, and cytotoxicity were screened. Compounds 1d-9d showed specific antiplatelet aggregation activity induced by arachidonic acid and collagen. Among them, 8d and 9d exhibited better activity than the reference drug, aspirin and 9d also showed inhibition of platelet aggregation by all four inducers.