Receptor Subtype-selective Agonists Research Articles

Sphingosine kinase 1 is involved in triglyceride breakdown by maintaining lysosomal integrity in brown adipocytes

Sphingosine 1-phosphate (S1P) has been implicated in brown adipose tissue (BAT) formation and energy consumption; however, the mechanistic role of sphingolipids, including S1P, in BAT remains unclear. Here, we showed that, in mice, BAT activation by cold exposure upregulated mRNA and protein expression of the S1P-synthesizing enzyme sphingosine kinase 1 (SphK1) and S1P production in BAT. Treatment of wild-type brown adipocytes with exogenous S1P or S1P receptor subtype-selective agonists stimulated triglyceride (TG) breakdown only marginally, compared with noradrenaline. However, genetic deletion of Sphk1 resulted in hypothermia and diminished body weight loss upon cold exposure, suggesting that SphK1 is involved in thermogenesis through mechanisms different from receptor-mediated, extracellular action of S1P. In BAT of wild-type mice, SphK1 was localized largely in the lysosomes of brown adipocytes. In the brown adipocytes of Sphk1−/− mice, the number of lysosomes was reduced and lysosomal function, including proteolytic activity, acid esterase activity, and motility, was impaired. Concordantly, nuclear translocation of transcription factor EB, a master transcriptional regulator of lysosome biogenesis, was reduced, leading to decreased mRNA expression of the lysosome-related genes in Sphk1−/− BAT. Moreover, BAT of Sphk1−/− mice showed greater TG accumulation with dominant larger lipid droplets in brown adipocytes. Inhibition of lysosomes with chloroquine resulted in a less extent of triglyceride accumulation in Sphk1−/− brown adipocytes compared with wild-type brown adipocytes, suggesting a reduced lysosome-mediated TG breakdown in Sphk1−/− mice. Our results indicate a novel role of SphK1 in lysosomal integrity, which is required for TG breakdown and thermogenesis in BAT.

Adenosine Attenuates Aortic Smooth Muscle Cell Calcification through A<sub>3</sub> Adenosine Receptor

Vascular calcification is a typical feature of atherosclerosis and is associated with adverse cardiovascular events such as myocardial infarction and stroke. Several studies have suggested that adenosine, an ATP metabolite may function as an endogenous regulator of arterial calcification. However, its effects on vascular smooth muscle cell calcification have not been clarified. In this study, we investigated the inhibitory effects of adenosine on vascular calcification in vitro by utilizing the culture of human aortic smooth muscle cells (HASMCs). Osteoblastic differentiation of HASMCs was induced by the treatment with oncostatin M and osteogenic differentiation medium. Adenosine and its metabolically stable analogue, 2-chloroadenosine (CADO) significantly reduced matrix mineralization and alkaline phosphatase (ALP) activities in HASMCs. The mRNA expression of tissue non-specific alkaline phosphatase (TNAP) was down-regulated by adenosine and CADO, but the mRNA expression of other osteoblastic differentiation markers, such as Runt-related transcription factor 2 (RUNX2) and bone sialoprotein (BSP)-II, was not significantly affected by these two reagents. Among the adenosine receptor (AR) subtype-selective agonists used, only IB-MECA (A3 AR-selective agonist) significantly decreased in vitro mineralization and ALP activities in HASMCs, but not with CCPA (A1 AR-selective agonist), CGS21680 (A2a AR-selective agonist), or BAY60-6583 (A2b AR-selective agonist). Importantly, IB-MECA also down-regulated expression of TNAP mRNA. Finally, knockdown of A3 AR, but not A1 AR, A2a AR, or A2b AR, significantly reversed the inhibitory actions of adenosine, CADO, or IB-MECA on in vitro calcification and ALP activities in HASMCs. These data suggest that adenosine attenuates HASMC calcification through A3 AR.

Effects of Exposure to the Endocrine-Disrupting Chemical Bisphenol A During Critical Windows of Murine Pituitary Development.

Critical windows of development are often more sensitive to endocrine disruption. The murine pituitary gland has two critical windows of development: embryonic gland establishment and neonatal hormone cell expansion. During embryonic development, one environmentally ubiquitous endocrine-disrupting chemical, bisphenol A (BPA), has been shown to alter pituitary development by increasing proliferation and gonadotrope number in females but not males. However, the effects of exposure during the neonatal period have not been examined. Therefore, we dosed pups from postnatal day (PND)0 to PND7 with 0.05, 0.5, and 50 μg/kg/d BPA, environmentally relevant doses, or 50 μg/kg/d estradiol (E2). Mice were collected after dosing at PND7 and at 5 weeks. Dosing mice neonatally with BPA caused sex-specific gene expression changes distinct from those observed with embryonic exposure. At PND7, pituitary Pit1 messenger RNA (mRNA) expression was decreased with BPA 0.05 and 0.5 μg/kg/d in males only. Expression of Pomc mRNA was decreased at 0.5 μg/kg/d BPA in males and at 0.5 and 50 μg/kg/d BPA in females. Similarly, E2 decreased Pomc mRNA in both males and females. However, no noticeable corresponding changes were found in protein expression. Both E2 and BPA suppressed Pomc mRNA in pituitary organ cultures; this repression appeared to be mediated by estrogen receptor-α and estrogen receptor-β in females and G protein-coupled estrogen receptor in males, as determined by estrogen receptor subtype-selective agonists. These data demonstrated that BPA exposure during neonatal pituitary development has unique sex-specific effects on gene expression and that Pomc repression in males and females can occur through different mechanisms.

Kinin-B1 and B2 receptor activity in proliferation and neural phenotype determination of mouse embryonic stem cells.

The kinins bradykinin and des-arg(9) -bradykinin cleaved from kininogen precursors by kallikreins exert their biological actions by stimulating kinin-B2 and B1 receptors, respectively. In vitro models of neural differentiation such as P19 embryonal carcinoma cells and neural progenitor cells have suggested the involvement of B2 receptors in neural differentiation and phenotype determination; however, the involvement of B1 receptors in these processes has not been established. Here, we show that B1 and B2 receptors are differentially expressed in mouse embryonic E14Tg2A stem cells undergoing neural differentiation. Proliferation and differentiation assays, performed in the presence of receptor subtype-selective agonists and antagonists, revealed that B1 receptor activity is required for the proliferation of embryonic and differentiating cells as well as for neuronal maturation at later stages of differentiation, while the B2 receptor acts on neural phenotype choice, promoting neurogenesis over gliogenesis. Besides the elucidation of bradykinin functions in an in vitro model reflecting early embryogenesis and neurogenesis, this study contributes to the understanding of B1 receptor functions in this process.

Structure-activity relationships of lysophosphatidylserine analogs as agonists of G-protein-coupled receptors GPR34, P2Y10, and GPR174.

Lysophosphatidylserine (LysoPS) is an endogenous lipid mediator generated by hydrolysis of membrane phospholipid phosphatidylserine. Recent ligand screening of orphan G-protein-coupled receptors (GPCRs) identified two LysoPS-specific human GPCRs, namely, P2Y10 (LPS2) and GPR174 (LPS3), which, together with previously reported GPR34 (LPS1), comprise a LysoPS receptor family. Herein, we examined the structure-activity relationships of a series of synthetic LysoPS analogues toward these recently deorphanized LysoPS receptors, based on the idea that LysoPS can be regarded as consisting of distinct modules (fatty acid, glycerol, and l-serine) connected by phosphodiester and ester linkages. Starting from the endogenous ligand (1-oleoyl-LysoPS, 1), we optimized the structure of each module and the ester linkage. Accordingly, we identified some structural requirements of each module for potency and for receptor subtype selectivity. Further assembly of individually structure-optimized modules yielded a series of potent and LysoPS receptor subtype-selective agonists, particularly for P2Y10 and GPR174.

Differential effects of dopamine receptor subtype-specific agonists with respect to operant behavior maintained on a differential reinforcement of low-rate responding (DRL) schedule

Previous studies have shown that d-amphetamine, a dopamine (DA) indirect agonist, alters operant responding with respect to the behavior maintained on a differential reinforcement of low-rate (DRL) schedule of reinforcement. These behavioral changes have been presumed to result from drug-induced hyperdopaminergia that leads to activation of DA receptors. This study investigated the acute dose effects of DA receptor subtype-selective agonists on the performance of DRL 10-sec behavior by rats. SKF38393 (a D1 receptor agonist) and quinpirole (a D2/D3 receptor agonist) were able to dose-dependently disrupt DRL 10-sec behavior by decreasing the total responses, the non-reinforced responses, and the peak rate of response. Bromocriptine (a D2/D3 receptor agonist) produced a significantly different pattern of behavioral changes when examined during two distinct time phases (15min and 3hr after the drug injection). DRL responding was only altered at higher doses of bromocriptine in the second phase as indicated by decreasing reinforced responses and peak rate, together with an increase of burst responses. In contrast to the D1 and D2/D3 receptor agonists, PD168077 (a D4 receptor agonist) did not affect DRL 10-sec behavior. None of these tested drugs affected DRL 10-sec behavior in a manner similar to that of d-amphetamine. These findings show that there are differential effects on the performance of DRL 10-sec behavior when drugs are able to preferentially activate D1, D2/D3 and D4 receptors, supporting the assertion that there is functional heterogeneity of the DA receptor subtypes.

Effects of intravenous administration of neurokinin receptor subtype-selective agonists on gonadotropin-releasing hormone pulse generator activity and luteinizing hormone secretion in goats.

Recent evidence suggests that neurokinin B (NKB), a member of the neurokinin (tachykinin) peptide family, plays a pivotal role in gonadotropin-releasing hormone (GnRH) pulse generation. Three types of neurokinin receptors (NKRs), NK1R, NK2R and NK3R, are found in the brain. Although NKB preferentially binds to NK3R, other NKRs are possibly also involved in NKB action. The present study examined the effects of intravenous administration of the NKR subtype-selective agonists GR73632 (NK1R), GR64349 (NK2R), and senktide (NK3R) on GnRH pulse generator activity and luteinizing hormone (LH) secretion. Multiple-unit activity (MUA) was monitored in ovariectomized goats (n = 5) implanted with recording electrodes. Characteristic increases in MUA (MUA volleys) were considered GnRH pulse generator activity. Although three NKR agonists dose-dependently induced an MUA volley and an accompanying increase in LH secretion, the efficacy in inducing the volley markedly differed. As little as 10 nmol of senktide induced an MUA volley in all goats, whereas a dose of 1000 nmol was only effective for the NK1R and NK2R agonists in two and four goats, respectively. When the treatment failed to evoke an MUA volley, no apparent change was observed in the MUA or LH secretion. Similar effects of the NK2R and NK3R agonists were observed in the presence of estradiol. The results demonstrated that NK3R plays a predominant role in GnRH pulse generation and suggested that the contributions of NK1R and NK2R to this mechanism may be few, if any, in goats.

Development of Small Molecule Non-peptide Formyl Peptide Receptor (FPR) Ligands and Molecular Modeling of Their Recognition

Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) expressed on a variety of cell types. These receptors play an important role in the regulation of inflammatory reactions and sensing cellular damage. They have also been implicated in the pathogenesis of various diseases, including neurodegenerative diseases, cataract formation, and atherogenesis. Thus, FPR ligands, both agonists and antagonists, may represent novel therapeutics for modulating host defense and innate immunity. A variety of molecules have been identified as receptor subtype-selective and mixed FPR agonists with potential therapeutic value during last decade. This review describes our efforts along with recent advances in the identification, optimization, biological evaluation, and structure-activity relationship (SAR) analysis of small molecule non-peptide FPR agonists and antagonists, including chiral molecules. Questions regarding the interaction at the molecular level of benzimidazoles, pyrazolones, pyridazin-3(2H)-ones, N-phenylureas and other derivatives with FPR1 and FPR2 are discussed. Application of computational models for virtual screening and design of FPR ligands is also considered.

How Sex Hormones Promote Skeletal Muscle Regeneration

Skeletal muscle regeneration efficiency declines with age for both men and women. This decline impacts on functional capabilities in the elderly and limits their ability to engage in regular physical activity and to maintain independence. Aging is associated with a decline in sex hormone production. Therefore, elucidating the effects of sex hormone substitution on skeletal muscle homeostasis and regeneration after injury or disuse is highly relevant for the aging population, where sarcopenia affects more than 30% of individuals over 60years of age. While the anabolic effects of androgens are well known, the effects of estrogens on skeletal muscle anabolism have only been uncovered in recent times. Hence, the purpose of this review is to provide a mechanistic insight into the regulation of skeletal muscle regenerative processes by both androgens and estrogens. Animal studies using estrogen receptor (ER) antagonists and receptor subtype selective agonists have revealed that estrogens act through both genomic and non-genomic pathways to reduce leukocyte invasion and increase satellite cell numbers in regenerating skeletal muscle tissue. Although animal studies have been more conclusive than human studies in establishing a role for sex hormones in the attenuation of muscle damage, data from a number of recent well controlled human studies is presented to support the notion that hormonal therapies and exercise induce added positive effects on functional measures and lean tissue mass. Based on the fact that aging human skeletal muscle retains the ability to adapt to exercise with enhanced satellite cell activation, combining sex hormone therapies with exercise may induce additive effects on satellite cell accretion. There is evidence to suggest that there is a 'window of opportunity' after the onset of a hypogonadal state such as menopause, to initiate a hormonal therapy in order to achieve maximal benefits for skeletal muscle health. Novel receptor subtype selective ligands and selective estrogen and androgen receptor modulators (SERMs, SARMs) promise to reduce health risks associated with classical hormonal therapies, whilst maintaining the positive effects on muscle repair. Dietary supplements containing compounds with structural similarity to estrogens (phytoestrogens) are increasingly used as alternatives to classical hormone-replacement therapies (HRT), but the effects on skeletal muscle are currently largely unknown. Research has started to investigate the combined effects of exercise and alternative HRTs, such as soy isoflavones, on skeletal muscle regenerative processes to provide safer and more efficient therapies to promote muscle regeneration and maintenance of muscle mass and strength in the aging population.

Sa1746 A1 and A2A Adenosine Receptor Subtype Selective Agonists Activate Splanchnic Colonic Afferents With a Clear Modulatory Role of Nav1.9

Sa1746 A1 and A2A Adenosine Receptor Subtype Selective Agonists Activate Splanchnic Colonic Afferents With a Clear Modulatory Role of Nav1.9

ChemInform Abstract: Neuropeptides and Neuropeptide Receptors: Drug Targets, and Peptide and Non‐Peptide Ligands: A Tribute to Prof. Dieter Seebach

AbstractReview: ca. 50 refs.

Inhibition of 50-kHz ultrasonic vocalizations by dopamine receptor subtype-selective agonists and antagonists in adult rats

Adult rats emit ultrasonic calls at around 22 and 50 kHz, which are often elicited by aversive and rewarding stimuli, respectively. Dopamine (DA) plays a role in aspects of both reward and aversion. The purpose of this study is to investigate the effects of DA receptor subtype-selective agonists on 22- and 50-kHz call rates. Ultrasonic calls were recorded in adult male rats that were initially screened with amphetamine to eliminate low 50-kHz callers. The remaining subjects were tested after acute intraperitoneal or subcutaneous injection of the following DA receptor-selective agonists and antagonists: A68930 (D1-like agonist), quinpirole (D2-like agonist), PD 128907 (D3 agonist), PD 168077 (D4 agonist), SCH 39166 (D1-like antagonist), L-741,626 (D2 antagonist), NGB 2904 (D3 antagonist), and L-745,870 (D4 antagonist). The indirect DA/noradrenaline agonist amphetamine served as a positive control. As expected, amphetamine strongly increased 50-kHz call rates. In contrast, D1-, D2-, and D3-selective DA receptor agonists, when given alone, inhibited calling; combinations of D1- and D2-like agonists also decreased call rate. Given alone, the D1-like and D3 antagonists significantly decreased call rate, with a similar trend for the D2 antagonist. Agonist-antagonist combinations also decreased calling. The D4 agonist and antagonist did not significantly affect 50-kHz call rates. Twenty-two-kilohertz calls occurred infrequently under all drug conditions. Following systemic drug administration, tonic pharmacological activation of D1-like or D2-like DA receptors, either alone or in combination, does not appear sufficient to induce 50-kHz calls. Dopaminergic transmission through D1, D2, and D3 receptors appears necessary for spontaneous calling.

Neuropeptides and Neuropeptide Receptors: Drug Targets, and Peptide and Non‐Peptide Ligands: a Tribute to Prof. Dieter Seebach

The number of neuropeptides and their corresponding receptors has increased steadily over the last fourty years: initially, peptides were isolated from gut or brain (e.g., Substance P, somatostatin), then by targeted mining in specific regions (e.g., cortistatin, orexin in the brain), or by deorphanization of G-protein-coupled receptors (GPCRs; orexin, ghrelin receptors) and through the completion the Human Genome Project. Neuropeptides (and their receptors) have regionally restricted distributions in the central and peripheral nervous system. The neuropeptide signaling is somewhat more distinct spatially than signaling with classical, low-molecular-weight neurotransmitters that are more widely expressed, and, therefore, one assumes that drugs acting at neuropeptide receptors may have more selective pharmacological actions with possibly fewer side effects than drugs acting on glutamatergic, GABAergic, monoaminergic, or cholinergic systems. Neuropeptide receptors, which may have a few or multiple subtypes and splice variants, belong almost exclusively to the GPCR family also known as seven-transmembrane receptors (7TM), a favorite class of drug targets in the pharmaceutical industry. Most neuropeptides are co-stored and co-released with classic neurotransmitters, albeit often only at higher frequencies of stimulation or at bursting activity, thus restricting the neuropeptide signaling to specific circumstances, another reason to assume that neuropeptide drug mimics may have less side effects. Neuropeptides possess a wide spectrum of functions from neurohormone, neurotransmitter to growth factor, but also as key inflammatory mediators. Neuropeptides become 'active' when the nervous system is challenged, e.g., by stress, injury, drug abuse, or neuropsychiatric disorders with genetic, epigenetic, and/or environmental components. The unsuspected number of true neuropeptides and their cognate receptors provides opportunities to identify novel targets for the treatment of both central and peripheral nervous system disorders. Both, receptor subtype-selective antagonists and agonists are being developed, as illustrated by the success of somatostatin agonists, angiotensin, and endothelin antagonists, and the expected clinical applications of NK-1/2/3 (substance P) receptor antagonists, CRF, vasopressin, NPY, neurotensin, orexin antagonists, or neuropeptide receptor modulators; such ligands have efficacy in preclinical or clinical models of pain and neuropsychiatric diseases, such as migraine, chronic/neuropathic pain, anxiety, sleep disorders, depression, and schizophrenia. In addition, both positive and negative allosteric modulators have been described with interesting in vivo activities (e.g., at galanin receptors). The field has become more complex now that an increasing number of heteromeric neuropeptide receptors are described, e.g., ghrelin receptors with 5-HT(2C) or dopamine D(1), D(2) receptors. At long last, structure-based drug discovery can now be envisaged with confidence, since crystal or solution structure of GPCRs and GPCR-ligand complexes, including peptide receptors, are published almost on a monthly basis. Finally, although most compounds acting at peptide receptors are still peptidomimetics, the last decade has seen the emergence of low-molecular-weight nonpeptide ligands (e.g., for orexin, ghrelin, or neurokinin receptors), and surprising progress has been made with β- and γ-peptides as very stable and potent mimetics of, e.g., somatostatin (SRIF), where the native SRIF has a half-life limited to 2-3 min. This last point will be illustrated more specifically, as we have had a long-standing collaboration with Prof. D. Seebach to whom this review is dedicated at the occasion of his 75th birthday.

5-Hydroxytryptamine potentiates neurogenic contractions of rat isolated urinary bladder through both 5-HT 7 and 5-HT 2C receptors

Serotonin (5-HT) enhances the neurogenic contractile response induced by electrical field stimulation (EFS) in the rat isolated urinary bladder. The aim of this study was to functionally characterize the receptors involved in this effect by using a range of 5-HT receptor subtype selective agonists and antagonists. 5-HT produced a concentration-dependent potentiation of contractile responses to EFS with a pEC 50 value of 6.86 ± 0.24. SB-269970 (0.01, 0.1 and 1 μM), a selective 5-HT 7 receptor antagonist, caused a concentration-dependent rightward shift of the 5-HT-induced response. The pA 2 value was 8.16 with a slope of 0.46 ± 0.08. Neither ketanserine nor SB-204741, 5-HT 2A and 5-HT 2B receptors antagonists, respectively, affected the concentration–response curve to 5-HT. However, 5-HT response was antagonized by the selective 5-HT 2C receptor antagonist SB-242084 (0.1 and 1 μM). In the presence of 1 μM of both antagonists SB-269970 and SB-242084, 5-HT response was almost fully inhibited. 5-CT, a 5-HT 7 receptor agonist, induced a biphasic concentration-dependent potentiation of neurogenic contractions. SB-269970 concentration-dependently antagonized the first phase of 5-CT response with a pA 2 value of 8.77 and a slope not significantly different from unity (0.91 ± 0.11) that suggests a competitive antagonism. WAY-161503, a 5-HT 2C receptor agonist (0.01–10 μM), induced a concentration-dependent potentiation of contractile response to EFS while DOI (a selective 5-HT 2A agonist) had no effect. SB-242084 (0.1 and 1 μM) antagonized the effect of WAY-161503 in a concentration-dependent manner. The current results demonstrate that 5-HT potentiates neurogenic contractions of rat isolated detrusor muscle through both 5-HT 7 and 5-HT 2c receptors.

Pharmacological tools for lysophospholipid GPCRs: development of agonists and antagonists for LPA and S1P receptors

Previous studies on lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) using various approaches have shown that both the molecules can act as intercellular signaling molecules. The discovery of the Edg subfamily of G-protein-coupled receptors (GPCRs) (later renamed LPA(1-3) and S1P(1-5)) for these molecules has opened up a new avenue for pathophysiological research on lysophospholipids. Genetic and molecular studies on lysophospholipid GPCRs have elucidated pathophysiological impacts and roles in cellular signaling pathways. Recently, lysophospholipid GPCR genes have been used to develop receptor subtype-selective agonists and antagonists. The discovery of FTY720, a novel immune modulator, along with other chemical tools, has provided a means of elucidating the functions of each lysophospholipid GPCR on an organ and the whole body level. This communication attempts to retrospectively review the development of agonists and antagonists for lysophospholipid GPCRs, provide integrated information on pharmacological tools for lysophospholipid GPCR signaling, and speculate on future drug development.

Estradiol and genistein antagonize the ovariectomy effects on skeletal muscle myosin heavy chain expression via ER-β mediated pathways

The age-related decline in ovarian sex hormone production following the onset of menopause alters skeletal muscle metabolic, structural and functional characteristics. The myosin heavy chain (MHC) expression pattern defines skeletal muscle contraction velocity and is therefore an important factor in skeletal muscle function. The present study was designed to examine the effects of 17β estradiol (E2), estrogen receptor (ER) subtype selective agonists (ERα, ERβ) or genistein (Gen) following ovary removal (OVX) in female Wistar rats in combination with a high intensity treadmill-based exercise protocol (Ex) or normal cage-based activity (NoEx) on MHC protein expression patterns in the slow fiber type m.Soleus (Sol) and the fast fiber type m.Gastrocnemius (Gas). Gen and E2 in the Sol significantly stimulated MHC-I expression relative to OVX only in the absence of exercise (NoEx). MHC-IIb expression in the Gas was significantly increased relative to OVX in Gen Ex and E2 Ex and NoEx groups. The estrogenic effects in the Sol and Gas were both predominantly mediated via ERβ pathways, since the ERβ agonist induced greater MHC increases than OVX or ERα. We therefore propose that high intensity exercise in combination with exposure to E2, Gen, ERα or ERβ agonists in OVX rats exerts differential effects on MHC expression in skeletal muscles composed of mainly slow type I MHC (Sol) or fast type II MHC (Gas). In summary, the data shows that MHC composition is affected by estrogens and exercise in a fiber type specific manner and that these effects are mainly mediated by ER-β. This is of great importance with respect to skeletal muscle health and potential treatment with ER selective agonists.

MRK-409 (MK-0343), a GABAA receptor subtype-selective partial agonist, is a non-sedating anxiolytic in preclinical species but causes sedation in humans

MRK-409 binds to α1-, α2-, α3- and α5-containing human recombinant GABA(A) receptors with comparable high affinity (0.21-0.40 nM). However, MRK-409 has greater agonist efficacy at the α3 compared with α1 subtypes (respective efficacies relative to the full agonist chlordiazepoxide of 0.45 and 0.18). This compound readily penetrates the brain in rats and occupies the benzodiazepine site of GABA(A) receptors, measured using an in vivo [(3)H]flumazenil binding assay, with an Occ(50) of 2.2 mg/kg p.o. and a corresponding plasma EC(50) of 115 ng/mL. Behaviourally, the α3-preferring agonist efficacy profile of MRK-409 produced anxiolytic-like activity in rodent and primate unconditioned and conditioned models of anxiety with minimum effective doses corresponding to occupancies, depending on the particular model, ranging from ∼35% to 65% yet there were minimal overt signs of sedation at occupancies greater than 90%. In humans, however, safety and tolerability studies showed that there was pronounced sedation at a dose of 2 mg, resulting in a maximal tolerated dose of 1 mg. This 2 mg dose corresponded to a C(max) plasma concentration of 28 ng/mL, which, based on the rodent plasma EC(50) for occupancy of 115 ng/mL, suggested that sedation in humans occurs at low levels of occupancy. This was confirmed in human positron emission tomography studies, in which [(11)C]flumazenil uptake following a single dose of 1 mg MRK-409 was comparable to that of placebo, indicating that occupancy of GABA(A) receptor benzodiazepine binding sites by MRK-409 was below the limits of detection (i.e. <10%). Taken together, these data show that MRK-409 causes sedation in humans at a dose (2 mg) corresponding to levels of occupancy considerably less than those predicted from rodent models to be required for anxiolytic efficacy (∼35-65%). Thus, the preclinical non-sedating anxiolytic profile of MRK-409 did not translate into humans and further development of this compound was halted.

Plasticity of GABAergic Mechanisms Within the Nucleus of the Solitary Tract in Hypertension

Adaptive changes have long been recognized to occur in the heart and vasculature in response to chronic hypertension. What might be less well-appreciated is the fact that chronically increased blood pressure is also associated with adaptive changes in neurons within the central nervous system (CNS). Changes in the properties of ligand-gated and voltage-gated channels have been described in a variety of neurons in a variety of central nuclei and in a variety of models of hypertension. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in virtually every region in the adult brain. Microinjections of GABA and GABA receptor subtype-selective agonists and antagonists have been performed within various cardiovascular-related regions of the CNS. In every cardiovascular-related region of the CNS tested, activation of GABA receptors alters cardiovascular function. This is likely attributable to the ubiquitous role of GABA within the CNS. GABAergic inhibition can be mediated by activation of receptors located in presynaptic and postsynaptic loci. Two major subtypes of the GABA receptor exist: the GABAA receptor is a pentameric, chloride ionophore that primarily mediates postsynaptic inhibition,1 whereas the GABAB receptor is a G-protein-coupled receptor that can induce reductions in calcium conductance to mediate presynaptic inhibition and increases in potassium conductance to mediate postsynaptic inhibition.2 GABAergic inhibition of central neurons can result in pressor or depressor responses depending on the central site being examined. Pressor responses are often assumed to be the result of GABAergic inhibition of neurons that reduce sympathetic discharge. Depending on the specific area being studied, GABA injections into the CNS can also alter vagal cardiac function and levels of vasoactive hormones such as vasopressin and angiotensin, in addition to changes in sympathetic outflow.3,4 In addition to microinjection studies, in vivo and in vitro electrophysiological analyses of functionally identified neurons in cardiovascular-related CNS …

Critical review of ropinirole and pramipexole - putative dopamine D3-receptor selective agonists - for the treatment of RLS

Ropinirole hydrochloride (REQUIP, ADARTREL) and pramipexole dihydrochloride (MIRAPEX, SIFROL) are two putative dopamine D(3) receptor subtype-selective agonists recently approved by the FDA for the treatment of 'restless legs syndrome' (RLS). RLS is a difficult to define condition that is possibly more prevalent than previously thought. Direct-to-consumer advertising has raised public and professional awareness of RLS, but questions, even skepticism about the very existence of the condition, persist. The drugs have adverse effects that can negatively impact on quality of life and thus, as true for all drugs, require consideration of the benefit : risk ratio. We review the definition, diagnostic criteria, pathophysiology, and treatment of RLS, and assess the clinical and preclinical evidence for a pharmacologic rationale for D(3) agonism in general and of the claimed D(3) selectivity of ropinirole and pramipexole in particular.

Characterization of Serotonin Receptors in Pregnant Human Myometrium

The monoamine, 5-hydroxytryptamine (5-HT), stimulates contraction of human uterine smooth muscle (myometrium), but the receptor subtypes involved have not been characterized. We studied the effects of a range of 5-HT receptor subtype-selective agonists and antagonists in isolated strips of myometrium obtained at the time of caesarean section. The 5-HT(1A) receptor agonist, 8-hydroxy-2-dipropylaminotetralin, produced an increase in contractions that was highly variable, of low potency, and was not significantly inhibited by the 5-HT(1A) antagonist WAY100635 [[O-methyl-3H]-N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide]. The 5-HT(2) receptor agonist, alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT), produced a strong, consistent, and concentration-dependent stimulation of contractions (pEC(50) = 7.60 +/- 0.10, n = 5). The 5-HT(2A) receptor antagonist, ketanserin [3-[2-[4-(4-fluoro benzoyl)-piperidin-1-yl]ethyl]-1H-quinazoline-2,4-dione], caused a parallel shift in the response to alpha-Me-5-HT, with a pK(B) value consistent with its known affinity for the 5-HT(2A) receptor (pK(B) = 8.47 +/- 0.16, n = 5), but it had no effect on the response to oxytocin. The 5-HT(2B) and 5-HT(2C) receptor agonists, BW723C86 [(alpha-methyl-5-(2-thienylmethoxy)-1H-indole-3-ethanamine)] and Ro-60-01-75 [(S)-2-(6-chloro-5-fluoro-indol-1-yl)-1-methyl-ethylamine fumarate], produced inconsistent responses at potencies that were lower than expected for activation of their cognate receptors. The response to alpha-Me-5-HT was unaffected by the 5-HT(2B) and 5-HT(2C) receptor antagonists, SB204741 [(N-(1-methyl-1H-indolyl-5-yl)-N-(3-methyl-5-isothiazolyl)urea)] and RS102221 [8-[5-(2,4-dimethoxy-5-(4-trifluoromethyl phenylsulphonamido)phenyl-5-oxopentyl]-1,3,8-triazaspiro[4.5]decane-2,4-dione]. The 5-HT(1B/1D) receptor agonist, sumatriptan [1-[3-(2-dimethylaminoethyl)-1H-indol-5-yl]-N-methyl-methanesulfonamide], the 5-HT(4) agonist, cisapride [4-amino-5-chloro-N-[1-[3-(4-fluorophenoxy)propyl]-3-methoxy-4-piperidyl]-2-methoxy-benzamide], and the 5-HT(7) agonist, AS19 [(2S)-(+)-5-(1,3,5-trimethylpyrazol-4-yl)-2-(dimethylamino)tetralin], all had no effect on myometrial contractility. 5-HT(2A) receptor mRNA and immunoreactivity were identified using reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry. Specific binding of [(3)H]ketanserin was demonstrated. This study provides strong evidence for the expression of contractile 5-HT(2A) receptors in pregnant human myometrium, and this receptor is a potential target for novel uterotonic therapies.