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The role of the NMDA receptor in alcohol relapse: a pharmacological mapping study using the alcohol deprivation effect.

Modulators of glutamate receptors especially of the N-methyl-D-aspartate receptors (NMDARs) have recently been suggested as putative pharmacotherapeutic agents in the treatment of alcohol relapse. However, at present it is not clear, which binding and modulatory sites of the NMDAR are involved in relapse behavior. We, therefore, performed a pharmacological mapping study in long-term alcohol drinking rats using the alcohol deprivation effect (ADE) as a model for relapse behavior. In a comprehensive fashion, we studied dose-response curves, employing the following selective pharmacological agents: the NMDAR competitive antagonist CGP37849, the glycine binding site antagonist L-701.324, the NR2B subunit selective antagonist ifenprodil, which acts at the polyamine binding site, the NMDAR channel blocker neramexane, and ethanol, which acts as a functional antagonist at the NMDAR. Our data show that the animals' alcohol consumption inversely correlates with the dose of ethanol administered intraperitoneally. This indicates that under the present experimental conditions alcohol intake during an ADE is an entirely pharmacologically driven behavior that is not under the control of other factors such as taste or novelty of alcohol re-exposure. The effects of the administration of the aforementioned compounds were comparable to those of ethanol, suggesting a similar pharmacological impact on relapse behavior. Repeated administration of both competitive and uncompetitive NMDAR antagonist dose-dependently suppressed alcohol consumption during ADE. In addition, ifenprodil and L-701.324 dose-dependently reduced the expression of an ADE as well. In summary, the results suggest that an inhibition of NMDAR function in general, rather than a particular interference with a specific binding site of this receptor, is sufficient for the reduction of relapse behavior.

Alcohol Drinking↗

The excitatory amino acid transporters: pharmacological insights on substrate and inhibitor specificity of the EAAT subtypes.

L-glutamate serves as the primary excitatory neurotransmitter in the mammalian CNS, where it can contribute to either neuronal communication or neuropathological damage through the activation of a wide variety of excitatory amino acid (EAA) receptors. By regulating the levels of extracellular L-glutamate that have access to these receptors, glutamate uptake systems hold the potential to effect both normal synaptic signaling and the abnormal over-activation of the receptors that can trigger excitotoxic pathology. Among the various membrane transporters that are capable of translocating this dicarboxylic amino acid, the majority of glutamate transport in the CNS, particularly as related to excitatory transmission, is mediated by the high-affinity, sodium-dependent, excitatory amino acid transporters (EAATs). At least 5 subtypes of EAATs have been identified, each of which exhibits a distinct distribution and pharmacology. Our growing appreciation for the functional significance of the EAATs is closely linked to our understanding of their pharmacology and the consequent development of inhibitors and substrates with which to delineate their activity. As was the case with EAA receptors, conformationally constrained glutamate mimics have been especially valuable in this effort. The success of these compounds is based upon the concept that restricting the spatial positions that can be occupied by required functional groups can serve to enhance both the potency and selectivity of the analogues. In the instance of the transporters, useful pharmacological probes have emerged through the introduction of additional functional groups (e.g., methyl, hydroxyl, benzyloxy) onto the acyclic backbone of glutamate and aspartate, as well as through the exploitation of novel ring systems (e.g., pyrrolidine-, cyclopropyl-, azole-, oxazole-, and oxazoline-based analogues) to conformationally lock the position of the amino and carboxyl groups. The focus of the present review is on the pharmacology of the EAATs and, in particular, the potential to identify those chemical properties that differentiate the processes of binding and translocation (i.e., substrates from non-substrate inhibitors), as well as strategies to develop glutamate analogues that act selectively among the various EAAT subtypes.

Animals↗

The presence of natural human antibodies reactive against pharmacologically active pectic polysaccharides from herbal medicines.

Direct ELISA was performed using normal human sera and human colostrum, to analyse the presence of antibodies which react with pharmacologically active pectic polysaccharides isolated from plants used in traditional Japanese herbal (Kampo) medicine. All sera and colostrum were shown to contain IgM, IgG, IgA and secretory IgA class antibodies which react with the active pectic polysaccharides to different degrees. The reacting IgG antibody in normal human serum recognized the ramified regions (rhamnogalacturonan core with carbohydrate side-chains) of the pharmacologically active pectic polysaccharides as the active sites for complement-activating activity. Correlation analysis indicated that a significant and positive correlation was observed between reactivity with the reacting antibody of IgG class and the degree of complement-activating activity of the active polysaccharides. The reacting IgG class antibody, which was purified from normal human serum by affinity chromatography on bupleuran 2IIc (a pharmacologically active pectic polysaccharide from the roots of Bupleurum falcatum)-immobilized Sepharose, showed cross-reactivity not only with some other pharmacologically active pectic polysaccharides from other medicinal herbs but also with autoantigens such as single-strand DNA, myosin and tublin from mammals.

Angelica↗

Animal models of obsessive-compulsive disorder: rationale to understanding psychobiology and pharmacology.

Animal models have shown progressive development and have undoubtedly proven their supportive value in OCD research. Thus, various animal models have confirmed the importance of the 5-HT [72-74] and dopamine systems [104,111] in the neurobiology and treatment of OCD. Given the neurochemical, emotional, and cognitive complexity of the disorder, how-ever, animal models are being used to investigate more and more complicated neurochemical and behavioral theories purported to underlie OCD. The lever-press model, for example, has implicated deficient response feed-back in a neural system that regulates operant behavior [74]. Studies on stereotypic movement disorder [89] have opened a new avenue of investigation into the neurobiology of stereotypy that may be applicable to more complex syndromes such as OCD. Models that have focused on specific neuropsychologic aspects of OCD such as reward [74], displacement behavior[63,101], perseveration and indecisiveness [73,102], and spontaneous stereotypy [90,94] are important in their attempt to unify the diverse behavioral manifestations of this disorder. It is clear that for a deeper, more holistic understanding of OCD, multiple animal models will be needed to allow investigation of the various aspects of the disorder and to provide convergent validation of the research findings. The heterogeneous nature of OCD, the various subtypes that exist within the disorder, and the range of obsessive-compulsive spectrum disorders suggest that particular questions regarding OCD may be addressed best by us-ing a particular ethologic model, whereas other questions might require a pharmacologic model or a combination of both for meaningful results[62,115]. Genetic models will be extremely useful for studying the genetics of pathologic behavior and for relating these findings to neuroanatomic and neurochemical changes in the model (eg, DICT-7 mice as a model for Tourette's syndrome and OCD). Neither ethologic nor pharmacologic models, however, can assess whether the "compulsive" behavior is a response to an "obsessive" anxiety or fear. Perhaps the symptoms seen in patients who have OCD, which may be exacerbated by everyday stress, are analogous to displacement behaviors in animals and also reflect some form of anxiety or stress [98]. In this regard, the bank vole model [116]has provided evidence that previously developed stereotypies increase markedly after acute stress and argues that healthy individuals "habituate" to everyday stress, whereas patients who have OCD do not. Interindividual variation in behavioral response and attempts to replicate studies in different laboratories often is the nemesis of the behavioral scientist. Small within- and between-subject variability is usually desirable, how-ever, because there are cases in which the study of the variability of the model could lead to a better understanding of the disorder. Variability can-not always be considered an error; it is possible that previously disregarded neuronal systems may have a place in the observed variation and, indeed, in the pathophysiology of OCD. In this regard, SRIs are not always effective for OCD [6,29,30] such that a lack of effect in a model may reflect an un-known neurobiological basis for compulsive behavior in a sub-group of SRI refractory patients. Similarly, separating the afflicted (ie, working with animals that show greater behavioral change in a model and/or after drug treatment) would have distinct benefits. To increase successful implementation of an ethologic animal model, especially when reinforcement models or signal attenuation models are used,the laboratory must be equipped with the essential behavioral testing apparatus as well as the operant chambers/rooms in which to conduct the train-ing and data collection. Quantification of certain stereotypy behaviors also requires experienced or trained observers. An illustration of the difficulty in measuring behavioral changes is that in the rewarded alternation model,a good response to behavioral treatment (alternation training) may lead to a floor effect [73] which, after successful drug treatment of the animal,produces no residual persistence (ie, measurable behavioral change) on which a drug treatment can be tested. Clearly, the choice of ethologic, pharmacologic, or genetic models should be considered carefully. A well-validated model may quell many of the limitations and considerations described previously. Noninvasive neuroimaging(eg, the use of small-animal single-photon emission CT) to explore the neuroanatomic basis of OCD offers an exciting future challenge, especially if combined with pharmacologic or ethologic models, and could confirm or ex-tend knowledge of the neuroanatomy of OCD. Although studies to investigate further the interactive role of 5-HT, dopamine, GABA, and glutamate are still needed, the role of neuroactive peptides such as cholecystokinin, corticotrophin-releasing factor, neuropeptide Y, tachykinins (ie, substance P),and natriuretic peptides in OCD should also be considered. Genetically engineered animal models will become increasingly valuable in combination with new technologies such as gene-chip microarrays, RNA interference, and advanced proteomics that will help further the understanding of OCD. Animal models of OCD are poised to play a vital role in extending the knowledge of the disorder now and in the future.

Animals↗

Biochemistry and pharmacology of the myometrium and labor: regulation at the cellular and molecular levels.

This review presents the essentials of cell biology, biochemistry, and pharmacology of the myometrium and labor, especially as they relate to premature labor. The aim is to explore the neuroendocrine and pharmacologic regulation of myometrial muscle cells in the context of cell membrane function and contractile processes. In the first section, smooth muscle protein structure and interactions are described, the central roles of myosin light-chain kinase, calcium, and cyclic adenosine monophosphate (cAMP) are reviewed. Subsequently, events relating to hormone/drug receptor function and implications for pharmacologic applications are discussed. Finally, the processes of information transfer via hormones and drugs are incorporated into a model which explains how the hormones and drugs used in clinical practice may mediate myometrial contractility. The reader is introduced to current concepts of the cellular, molecular, and pharmacologic aspects of labor in order to facilitate the understanding of medical augmentation of labor and tocolytic therapy.

Actins↗

Human ether-a-gogo related gene (HERG) K+ channels as pharmacological targets: present and future implications.

Electrophysiological and molecular biology techniques have widely expanded our knowledge of the diverse functions where K+ channels are implicated as potential and proven pharmacological targets. The aim of the present commentary is to review the recent progress in the understanding of the functional role of the K+ channels encoded by the human ether-a-gogo related gene (HERG), with particular emphasis on their direct pharmacological modulation by drugs, or on their regulation by pharmacologically relevant phenomena. About 3 years have passed since the cloning, expression, and description of the pathophysiological role of HERG K+ channels in human cardiac repolarization. Despite this short lapse of time, these K+ channels have already gained considerable attention as pharmacological targets. In fact, interference with HERG K+ channels seems to be the main mechanism explaining both the therapeutic actions of the class III antiarrhythmics and the potential cardiotoxicity of second-generation H1 receptor antagonists such as terfenadine and astemizole, as well as of psychotropic drugs such as some antidepressants and neuroleptics. It seems possible to anticipate that the main tasks for future investigation will be, on the one side, the better understanding of the intimate mechanism of action of HERG K+ channel-blocking drugs in order to elucidate the conditions regulating the delicate balance between antiarrhythmic and proarrhythmic potential and, on the other, to unravel the pathophysiological role of this K+ channel in the function of the brain and of other excitable tissues.

Animals↗

Pharmacological delta1- and delta2-opioid receptor subtypes in the human neuroblastoma cell line SK-N-BE: no evidence for distinct molecular entities.

The two pharmacological delta-opioid receptor subtypes, delta1 and delta2, have been defined on the basis of pharmacological tools but remain to be characterized at the molecular level, since only a single cDNA has been cloned. The present study aimed to investigate the pharmacological properties of delta1- and delta2-opioid subtypes expressed in the human neuroblastoma cell line SK-N-BE and to characterize their putative corresponding mRNAs. Binding experiments using "selective" delta1- and delta2-opioid agonists and antagonists revealed the presence of two binding sites, demonstrating the presence of these delta1-opioid subtypes as they were previously described. The activation of these pharmacological subtypes by the selective agonists induced the incorporation of [alpha-(32)P]azidoanilide-GTP into Galpha(i2)/Galpha(0) subunits with the same efficiency and potency and inhibited adenosine 3', 5'-cyclic monophosphate (cAMP) accumulation with similar efficiency, while their sustained activation for 15 min induced a cross-desensitization. The "selective" delta1 and delta2 antagonists, 7-benzylidenenaltrexone and naltrindole benzofuran, respectively, were found to be as potent in blocking the inhibition of cAMP accumulation induced by both [D-Pen(2,5)]enkephalin and Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH(2). The possibility that delta-opioid subtypes could arise from alternative splicing was ruled out by reverse transcription-polymerase chain reaction (RT-PCR) experiments and the sequencing of PCR products, which revealed the presence of a single transcript encoding for the delta-opioid receptor. Different possibilities which could account for the delta-opioid receptor heterogeneity observed in the SN-N-BE cell line are discussed.

Adenylyl Cyclases↗

Effects of cellular pharmacology on drug distribution in tissues.

The efficacy of targeted therapeutics such as immunotoxins is directly related to both the extent of distribution achievable and the degree of drug internalization by individual cells in the tissue of interest. The factors that influence the tissue distribution of such drugs include drug transport; receptor/drug binding; and cellular pharmacology, the processing and routing of the drug within cells. To examine the importance of cellular pharmacology, previously treated only superficially, we have developed a mathematical model for drug transport in tissues that includes drug and receptor internalization, recycling, and degradation, as well as drug diffusion in the extracellular space and binding to cell surface receptors. We have applied this "cellular pharmacology model" to a model drug/cell system, specifically, transferrin and the well-defined transferrin cycle in CHO cells. We compare simulation results to models with extracellular diffusion only or diffusion with binding to cell surface receptors and present a parameter sensitivity analysis. The comparison of models illustrates that inclusion of intracellular trafficking significantly increases the total transferrin concentration throughout much of the tissue while decreasing the penetration depth. Increasing receptor affinity or tissue receptor density reduces permeation of extracellular drug while increasing the peak value of the intracellular drug concentration, resulting in "internal trapping" of transferrin near the source; this could account for heterogeneity of drug distributions observed in experimental systems. Other results indicate that the degree of drug internalization is not predicted by the total drug profile. Hence, when intracellular drug is required for a therapeutic effect, the optimal treatment may not result from conditions that produce the maximal total drug distribution. Examination of models that include cellular pharmacology may help guide rational drug design and provide useful information for whole body pharmacokinetic studies.

Animals↗

Nitric oxide as a mediator of delayed pharmacological (A(1) receptor triggered) preconditioning; is eNOS masquerading as iNOS?

BACKGROUND: Nitric oxide (NO), synthesised from the inducible isoform of nitric oxide synthase (iNOS), is implicated in mediating second window of protection (SWOP)/delayed ischemic preconditioning. However the role of NO and iNOS in delayed pharmacological protection remains unclear and is the subject of this investigation. METHODS: To test the hypothesis that iNOS is necessary for delayed pharmacological preconditioning, the adenosine A(1) receptor agonist, 2-chloro N(6) cyclopentyl adenosine (CCPA) (25 microg/kg i.v.) or saline was administered to wild type (WT) or iNOS gene knockout mice (KO). Twenty-four hours later, the hearts were isolated, Langendorff perfused and subjected to 35 min ischemia/30 min reperfusion prior to infarct size determination. RESULTS: WT and KO control hearts had identical infarct sizes of 37 +/- 3% and 37 +/- 2%, respectively. CCPA significantly reduced infarct size in WT hearts to 22 +/- 2% and also, unexpectedly, in KO hearts (27 +/- 2%). This protection was abrogated with the non-specific NOS inhibitor, N(omega) nitro L-arginine methyl ester (L-NAME, 100 microM), and could be mimicked in naïve hearts with the NO donor, donor S-nitroso N-acetyl DL penicillamine (SNAP, 1 microM). Delayed protection appeared to be mediated by NO synthesis in both WT and KO hearts. Additional studies using Western blot analysis demonstrated endothelial NOS (eNOS) upregulation and increased NO(x) release in both WT and KO hearts. CONCLUSIONS: This is the first study to demonstrate a role for eNOS in delayed A(1) receptor triggered (pharmacological) preconditioning, potentially representing a new pharmacological target for protecting the ischemic heart.

Analysis of Variance↗

Pharmacological and functional implications of developmentally-regulated changes in GABA(A) receptor subunit expression in the cerebellum.

The cerebellum undergoes many morphological, pharmacological, and electrophysiological changes during the first 3 weeks of postnatal development. The purpose of this review is to present the most up to date synopsis of the pharmacological and functional changes in, gamma-aminobutyric acid (GABA) type A receptors during this time of cerebellar maturation. Since most of the diversity in cerebellar, GABA(A) receptor pharmacology lies within the granule cell layer, research groups have focused on this area of the cerebellum to study the developmental changes in GABA(A) receptor subunit expression and the neurodifferentiating factors involved in regulating this expression. Thus, it is important to note that developmental changes in GABA(A) receptor composition and its corresponding pharmacology will be essential for determining the type of GABA-mediated transmission that occurs between neuronal contacts in the neonatal and subsequently in the mature cerebellum.

Animals↗

Pharmacological characterization of recombinant human and rat P2X receptor subtypes.

ATP functions as a fast neurotransmitter through the specific activation of a family of ligand-gated ion channels termed P2X receptors. In this report, six distinct recombinant P2X receptor subtypes were pharmacologically characterized in a heterologous expression system devoid of endogenous P2 receptor activity. cDNAs encoding four human P2X receptor subtypes (hP2X1, hP2X3, hP2X4, and hP2X7), and two rat P2X receptor subtypes (rP2X2 and rP2X3), were stably expressed in 1321N1 human astrocytoma cells. Furthermore, the rP2X2 and rP2X3 receptor subtypes were co-expressed in these same cells to form heteromultimeric receptors. Pharmacological profiles were determined for each receptor subtype, based on the activity of putative P2 ligands to stimulate Ca2+ influx. The observed potency and kinetics of each response was receptor subtype-specific and correlated with their respective electrophysiological properties. Each receptor subtype exhibited a distinct pharmacological profile, based on its respective sensitivity to nucleotide analogs, diadenosine polyphosphates and putative P2 receptor antagonists. Alphabeta-methylene ATP (alphabeta-meATP), a putative P2X receptor-selective agonist, was found to exhibit potent agonist activity only at the hP2X1, hP2X3 and rP2X3 receptor subtypes. Benzoylbenzoic ATP (BzATP, 2' and 3' mixed isomers), which has been reported to act as a P2X7 receptor-selective agonist, was least active at the rat and human P2X7 receptors, but was a potent (nM) agonist at hP2X1, rP2X3 and hP2X3 receptors. These data comprise a systematic examination of the functional pharmacology of P2X receptor activation.

Adenosine Triphosphate↗

Nocturnal penile tumescence predicting response to intracorporeal pharmacological erection testing.

Although response to intracorporeal pharmacological erection testing has been proposed to determine the etiology of impotence, physiological criteria predicting this response have not been established and the literature includes conflicting results regarding which patients respond to pharmacological erection therapy. In this study 37 impotent patients underwent a diagnostic nocturnal penile tumescence evaluation (including measurements of rigidity, pulsations and bulboischiocavernosus muscle activity) and the results were correlated with subsequent response to intracorporeal pharmacological testing. Most but not all patients with psychogenic impotence and all with neurogenic impotence responded with good erections. For vasculogenic impotence response rate depended upon impairment severity determined from nocturnal penile tumescence measurements; none of the severe cases versus 90 per cent of the milder cases responded. The results indicate that response to intracorporeal pharmacological testing does not accurately distinguish psychogenic from organic impotence, is best for neurogenic impotence and worst for severe vasculogenic impotence, and can be predicted accurately by nocturnal penile tumescence measurements.

Adult↗

Pharmacologic treatments of dementia.

The management of dementia patients encompasses pharmacologic, behavioral, and psychosocial intervention strategies. Before pharmacologic intervention is instituted, it is important that sources of excess disability and comorbidity be eliminated or reduced. Identification of comorbid medical and psychiatric conditions, such as depression and delirium, should be identified and appropriately treated. Providing caregivers with education, support, and practical advice is a critical component of the management of the demented patient. The current standard of care for pharmacologic management of the cognitive and functional disabilities of AD consists of the combination of a cholinesterase inhibitor and high-dose vitamin E. This standard is based on the results of large-scale, double-blind, placebo-controlled trials. Cholinesterase inhibitors are the only FDA-approved pharmacologic treatments for AD. Cholinesterase inhibitors have been shown to be effective in the treatment of the cognitive, behavioral, and functional deficits of AD. Large-scale placebo-controlled trials of tacrine, donepezil, rivastigmine, and galantamine have demonstrated moderate benefits in patients with mild to moderate AD. Donepezil, rivastigmine, and galantamine are the first-line choices in the treatment of AD because of their lack of hepatotoxicity, ease of administration, few significant drug-drug interactions, and mild to moderate side effects. There are few contraindications to the use of cholinesterase inhibitors. Known hypersensitivity to a specific drug or its derivatives is the only true contraindication. Cautious administration of cholinesterase inhibitors is advised in patients who have a previous history of allergy or adverse reactions to prior cholinesterase inhibitors, severe liver disease, preexisting bradycardia, peptic ulcer disease, current alcoholism, asthma, or chronic obstructive pulmonary disease. Nausea, vomiting, diarrhea, and anorexia are the most common side effects of cholinesterase inhibitors. These gastrointestinal side effects can be minimized by gradual dose increases, administration with food, adequate hydration, and judicious use of an antiemetic. Vitamin E has been demonstrated to slow the progression of AD in several small and one large placebo-controlled trials. Because of its low cost and safety, it is recommended in addition to a cholinesterase inhibitor for the treatment of AD. There are no FDA-approved treatments for DLB and VaD. One small placebo-controlled trial demonstrated that rivastigmine may be effective in the treatment of DLB. More large-scale placebo-controlled trials are needed to confirm the results of this study. Treatment of VaD focuses on the control, identification, and management of cerebrovascular disease and vascular risk factors. Although there are no peer-reviewed reports on the efficacy of cholinesterase inhibitors for VaD or mixed AD/VaD, early reports suggest that these agents may also be effective for mixed AD/VaD. The indications for the use of cholinesterase inhibitor drugs are eventually likely to broaden to include DLB, mixed AD/VaD, and AD in its more advanced stages.

Aged↗

Pharmacologic management of the critically ill patient in the perioperative period--emphasis on the sepsis syndrome.

The critically ill perioperative patient requires complex pharmacologic management. The ability to measure circulating drug concentrations has helped the clinician to properly modulate therapy, especially with potentially toxic agents. Postoperative sepsis remains a difficult therapeutic problem. Controversial therapies, such as the use of corticosteroids and the utility of newer pharmacologic approaches, are beginning to be properly tested in multicenter trials. Improvements in technology (e.g., development of monoclonal antibodies), biochemistry, and pharmacology have resulted in the development of exciting, new pharmacologic avenues for the management of the critically ill perioperative patient.

Bacterial Infections↗

Molecular modeling and pharmacological analysis of species-related histamine H(3) receptor heterogeneity.

Presynaptic histamine H(3) receptors (H(3)R) regulate neurotransmitter release in the central nervous system, suggesting an important role for H(3) ligands in human diseases such as cognitive disorders, sleep disturbances, epilepsy, or obesity. Drug development for many of these human diseases relies upon rodent-based models. Although there is significant sequence homology between the human and rat H(3)Rs, some compounds show distinct affinity profiles. To identify the amino acids responsible for these species disparities, various mutant receptors were generated and their pharmacology studied. The N-terminal portion was shown to determine the species differences in ligand binding since a chimeric H(3)R containing N-terminal human and C-terminal rat receptor sequences exhibited similar pharmacology to the human receptor. Sequence analysis and molecular modeling studies suggested key amino acids at positions 119 and 122 in transmembrane region 3 play important roles in ligand recognition. Mutant receptors changing amino acids 119 or 122 of the human receptor to those in the rat improved ligand binding affinities and functional potencies of antagonist ligands, confirming the significant role that these amino acids play in species-related pharmacological differences. A model has been developed to elucidate the ligand receptor interactions for H(3)Rs, and pharmacological aspects of this model are described.

Amino Acid Sequence↗

2-Alkylthio-substituted platelet P2Y12 receptor antagonists reveal pharmacological identity between the rat brain Gi-linked ADP receptors and P2Y12.

The Gi-linked platelet ADP receptor, now designated as P2Y12, accounts for ADP-induced inhibition of adenylyl cyclase in platelets and certain clonal rat cell lines. The pharmacology of this receptor is well characterized. Based on the functional approach of [35S]GTPgammaS autoradiography, we recently disclosed the widespread presence of Gi-linked ADP receptors in the rat nervous system. Based on initial pharmacological analysis, these receptors were strikingly similar with P2Y12. Here, we extend this analysis by comparing the potencies of six 2-alkylthio-substituted ATP analogues, including the adenosine-aspartate conjugate 2-hexylthio-AdoOC(O)Asp2 and five AR-C compounds (AR-C67085, AR-C69931, AR-C78511, AR-C69581, AR-C70300) with wide range of affinities towards P2Y12, in reversing 2-methylthio-ADP stimulated G protein activity in rat brain sections and human platelet membranes. Closely matching pIC50 values (r2=0.99) revealed pharmacological similarity between the two receptors with one exception: AR-C67085 more avidly recognized the platelet P2Y12. Further analysis of the rat brain pIC50 data against those available for three of the AR-C compounds in reversing P2Y12-mediated adenylyl cyclase inhibition in rat platelets (r2=0.96) and rat C6 glioma cells (r2=1.00) demonstrated that the three P2Y receptors are pharmacologically indistinguishable. We conclude that the rat brain Gi-linked ADP receptors, as revealed using [35S]GTPgammaS autoradiography, correspond to P2Y12.

Adenosine Triphosphate↗

Molecular cloning and pharmacological characterization of guinea pig 5-HT1B and 5-HT1D receptors.

Human 5-HT1B and 5-HT1D receptors have been implicated as molecular targets for the treatment of acute migraine based upon the pharmacological actions and clinical efficacy of sumatriptan, an agonist for human 5-HT1B/1D receptors. The guinea pig has served as an animal model to assess 5-HT1B/1D receptor function, most recently in evaluating 5-HT1B/1D receptor agonists as potential anti-migraine agents. Since two distinct, but closely-related receptors displaying "5-HT1D receptor pharmacology" have been cloned previously from most mammalian species, the genes encoding these receptors were isolated from a guinea pig liver genomic DNA library using oligonucleotide probes targeted to nonconserved regions of recombinant human 5-HT1B and 5-HT1D receptors. Sequence analysis indicates that guinea pig 5-HT1B and 5-HT1D receptors are comprised 390 and 378 amino acids, respectively. Comparison of the deduced amino acid sequences of guinea pig 5-HT1B and 5-HT1D receptor subtypes show that they display overall and transmembrane (TM) identities of 63% and 77%, respectively. Both clones contain a conserved threonine residue in TM7, a structural feature imparting "5-HT1D receptor pharmacology". Guinea pig 5-HT1B and 5-HT1D receptor genes were transiently expressed in Cos-7 cells and their binding properties were evaluated using [3H]5-HT. Both cloned receptor subtypes displayed "5-HT1D receptor pharmacology" with the following rank order of binding affinities: 5-CT > 5-HT > sumatriptan > 8-OH-DPAT > (-)-pindolol. Ketanserin displayed modest (five-fold) 5-HT1D receptor selectivity, while methiothepin exhibited a similar selectivity for the 5-HT1B subtype. In particular, ketanserin exhibits profound differences in 5-HT1D receptor affinity (and selectivity) across species. High correlations were observed between the binding affinities of serotonergic ligands for 5-HT1D binding sites measured in guinea pig cortical membranes and both cloned guinea pig 5-HT1B (r2 = 0.88) and 5-HT1D (r2 = 0.80) receptors, indicating that the development of subtype selective compounds (i.e. 5-HT1B versus 5-HT1D) using native tissues may be more difficult to achieve without the advantage of using recombinant receptor subtypes. Additionally, there is a good correspondence between binding profiles of recombinant guinea pig 5-HT1B and 5-HT1D receptor subtypes and to their respective cloned human homologs. However, species differences in binding affinities of a subset of compounds are evident. These data extend previous observations that subtype selective (i.e. 5-HT1D) compounds identified in one species may not discriminate between closely related receptors (i.e. 5-HT1B and 5-HT1D) in all animal model systems.

Amino Acid Sequence↗

Characterisation of the fish sst3 receptor, a member of the SRIF1 receptor family: atypical pharmacological features.

The first cloned non-mammalian somatostatin (somatostatin release-inhibiting factor = SRIF) receptor previously obtained from the teleost fish Apteronotus albifrons and generically named somatostatin receptor 3 (fsst3), was stably expressed and characterised in Chinese hamster lung fibroblast (CCL39) cells. Radioligand binding studies were performed with four radioligands selective for SRIF receptors in CCL39 cells expressing the fsst3 receptors; [125I]LTT-SRIF28 ([Leu8, D-Trp22, 125I-Tyr25]-SRIF28), [125I]Tyr10-cortistatin, [125I]CGP 23996, and [125I]Tyr3-octreotide labelled the fsst3 receptor with high affinity (pKd values: 10.47, 10.87, 9.59 and 9.57) and in a saturable manner, but defined different Bmax values; 4500, 4000, 3400 and 1500 fmol/mg, respectively. The affinities of SRIF peptides and analogues determined for fsst3 receptors displayed the following rank order of potency: seglitide = SRIF25 > SRIF14 = SRIF28 > cortistatin 14 > BIM 23014 > RC160 = L361,301 = octreotide > or = BIM 23052 > or = L362,855 > CGP23996 > BIM 23056 > BIM 23030 = cycloantagonist > SRIF22. The pharmacological profiles determined with [125I]LTT-SRIF28, [125I]CGP 23996 and [125I]Tyr10-cortistatin correlated highly significantly (r = 0.96-0.99), whereas [125I]Tyr3-octreotide binding was rather divergent (r = 0.78-0.81). Further, [125I]Tyr3-octreotide- and [125I]CGP 23996-labelled sites showed higher affinity for the various peptides than [125I]LTT-SRIF28 and [125I]Tyr10-cortistatin-labelled sites, although there were exceptions. [125I]LTT-SRIF28-binding to fsst3 receptors and human sst1-5 receptors was compared; the fsst3 binding profile correlated better with the hsst5- than with the hsst3 receptor profile. SRIF inhibited potently forskolin-stimulated adenylate cyclase activity in fsst3 transfected CCL39 cells; this effect was blocked by pertussis toxin, suggesting coupling of the fsst3 receptor to Gialpha and/or Goalpha. [125I]LTT-SRIF28 binding was detected in fish brain, liver, heart, spleen, and stomach, but not in gut. The pharmacological profile of [125I]LTT-SRIF28-labelled sites in brain, but not in liver, correlated significantly with the recombinant fsst3 receptor, in agreement with expression of the fsst3 receptor gene found by RT-PCR in the brain. However, biphasic binding curves obtained with two SRIF-analogues in brain, as well as the distinct pharmacological profile of the liver SRIF receptor, suggest the existence of several yet to be defined SRIF receptor subtypes in fish. The present data demonstrate that the recombinantly expressed fsst3 receptor has a pharmacological profile compatible with that of a SRIF1 receptor, although the rank order of affinity of fsst3 is closer to that of hsst5 than hsst3 receptors, as may be found when comparing very distantly related species. The fsst3 receptor expressed in CCL39 cells, is negatively coupled to adenylate cyclase activity via pertussis toxin-sensitive G-proteins, like mammalian sst3 receptors. Radioligand binding performed with fish tissue suggests the presence of a native sst3 receptor in brain as well as other yet to be defined SRIF receptor subtypes.

Adenylyl Cyclases↗