Structure activity relationships for central and peripheral alpha adrenergic activities of imidazoline derivatives.

The α adrenergic agonistic activity of a number of imidazolines was measured on the α adrenergic receptors of the isolated rabbit intestine. A number of imidazolines were full agonists having an intrinsic activity constant equal to 1 in relation to noradrenaline as a standard. Some other derivatives had only partial agonist activity. The affinities of the imidazolines for these adrenergic receptors was assessed using pD₂ values. The pD₂ values were correlated with a number of physicochemical parameters of the drugs, i.e. pK_a, the molar volume and the apparent and true partition coefficients (APC and TPC respectively) by stepwise regression analysis.Results indicate that pD₂ was correlated with pK_a primarily, whereas molar volume also was a factor determining agonistic activity at the peripheral α adrenergic receptor.Central α adrenergic receptor stimulating properties of the imidazolines were determined by measuring bradycardia induced by these agents after intrahypothalamic injection into rats. Although it was not possible to correlate central α adrenergic activity with any of the physicochemical parameters due to the lack of action of a number of imidazolines, results show that structural requirements for central α adrenergic activity clearly differ from those for peripheral α adrenergic activity.These data are discussed in view of the mechanism of action of the antihypertensive imidazoline clonidine.     

Characterisation of [3H]-Darifenacin as a Novel Radioligand for the Study of Muscarinic M3 Receptors

Abstract

Darifenacin, (S)-2-[1-[2,3-dihydrobenzofuran-5-yl]-3-pyrrolidinyl]-2,2-diphenylacetamide, is a novel muscarinic M₃ antagonist. In this study we have compared the binding of [³H]-darifenacin to the five cloned human muscarinic receptors (m₁ - m₅) expressed in CHO cells. [³H]-darifenacin binds with 6 fold higher affinity to m₃ (K_D = 0.33 nmol/l) over m₁ (K_D = 1.6 nmol/l) receptors. There was no specific binding of [³H]-darifenacin to m₂ receptors and specific binding to m₄ and m₅ receptors was insufficient to determine a K_D. Binding of [³H]-darifenacin to m₁ and m₃ was displaced by atropine (m₁ pK_i = 9.36, m₃ pK_i = 9.4), 4-DAMP (m₁ pK_i = 9.04, m₃ pK_i = 9.19), pirenzepine (m₁ pK_i = 8.63, m₃ pK_i = 6.85), methoctramine (m₁ pK_i = 7.28, m₃ pK_i = 6.63), and darifenacin (m₁ pK_i = 8.36, m₃ pK_i = 9.14), demonstrating that [³H]-darifenacin represents the first selective m₃ radioligand.     

Similarity of Central and Peripheral Alpha-1 Adrenoceptors in Rat and Rabbit

Abstract

In order to examine species and tissue differences in α₁ adrenoceptors, binding experiments were performed using ³H-prazosin and membrane homogenates of central nervous and peripheral tissues of rabbit (cortex and spleen), and rat (cortex, spleen, and liver). Saturation studies indicated one binding site for ³H-prazosin, with apparent log molar dissociation constants (pK_D) ranging from 9.43 to 10.20. The rank orders of affinities of three competing antagonists (prazosin ? idazoxan > rauwolscine) and five agonists (cirazoline > clonidine ～ (-)-norepinephrine > (-)-phenylephrine > (+)-norepinephrine) were typical of α₁ receptors in all tissues. There were small but significant differences in the mean affinities of rauwolscine, idazoxan and cirazoline among the five tissues. No significant differences in pseudo-Hill coefficients were observed among tissues, although agonist binding curves were shallow (.7 to.85) and prazosin competition curves were significantly steeper (>.85). Guanine nucleotide did not affect the position or slope of the (-)-norepinephrine competition profile in rat cortex. These results demonstrate a qualitative similarity among central and peripheral α₁ receptors of the rat and rabbit, with small differences observed between central and peripheral sites in both species.     

Identification of 5HT2-Receptors on Longitudinal Muscle of the Guinea Pig Ileum

Abstract

In binding experiments with the radioligands [³H]Ketanserin (HKet) and [¹²⁵I]LSD (ILSD) 21 compounds were investigated using rat brain cortex membranes. The pK_D-values of the compounds were virtually independent of the radioligand used and their rank order was consistent with classification of the binding sites as being of the 5-HT₂-type. In contrast, in the longitudinal muscle of the guinea pig ileum in the presence of 0.3 μM cinanserin, ILSD labelled sites which were quite different to those in the cortex. In a functional test antagonism of the 5HT induced contraction of the guinea-pig ileum was measured in the presence of 1 μM atropine. The pharmacological inhibition constants (IC₅₀-values) of 8 compounds correlated well with the dissociation constants for HKet binding in the cortex and did not correlate with the data from ILSD binding in the guinea pig ileum. It is concluded that the ileum contains postjunctional 5HT₂-receptors which mediate contraction. The nature of the ILSD binding sites in the ileum remains to be elucidated.     

A comparison of the effects of morphine withdrawal, thyroxine or thyroidectomy on the sensitivity of the anococcygeus muscle to agonists and on serum thyroxine levels in the rat

The effects of morphine withdrawal, thyroxine or thyroidectomy on the plasma levels of thyroxine and on the sensitivity of the anococcygeus muscle to acetylcholine and noradrenaline were investigated in the rat. Morphine withdrawal or thyroidectomy increased the maximum response of both agonists and morphine withdrawal increased the pD₂ value for acetylcholine. Thyroxine did not affect the maximum responses but increased the pD₂ values for both agonists. Plasma thyroxine levels were raised by thyroxine administration and were reduced by thyroidectomy and also during morphine administration and withdrawal.     

Potent dihydroquinolinone dopamine D2 partial agonist/serotonin reuptake inhibitors for the treatment of schizophrenia

A dihydroquinolinone moiety was found to be a potent serotonin reuptake inhibitor pharmacophore when combined with certain amines. This fragment was coupled with selected D₂ ligands to prepare a series of dual acting compounds with attractive in vitro profiles as dopamine D₂ partial agonists and serotonin reuptake inhibitors. Structure–activity studies revealed that the linker plays a key role in contributing to D₂ affinity, function, and SRI activity.     

[3H]5,7-Dichlorokynurenic Acid Recognizes two Binding Sites in Rat Cerebral Cortex Membranes

Abstract

Binding of [³H]5,7-dichlorokynurenic acid ([³H]DCKA), a competitive antagonist of the strychnine-insensitive glycine site of the N-methyl-D-aspartate (NMDA) receptor channel complex, was characterized in synaptic plasma membranes from rat cerebral cortex. Non linear curve fitting of [³H]DCKA saturation and homologous displacement isotherms indicated the existence of two binding sites: a specific, saturable, high affinity site, with a pK_D value of 7.24 (K_D = 57.5 nmol/1) and a maximum binding value (B_max) of 6.9 pmol/mg of protein and a second site, with micromolar affinity. The pharmacological profile of both binding components was determined by studying the effect on [³H]DCKA and [³H]glycine binding of a series of compounds known to interact with different excitatory and inhibitory amino acid receptors. These studies confirmed the identity of the high affinity site of [³H]DCKA binding with the strychnine-insensitive glycine site of the NMDA receptor channel complex. 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic acid sodium salt (GV 150526A), a new, high affinity, selective glycine site antagonist (1), was the most potent inhibitor of this component of binding (pK_i = 8.24, K_i = 5.6 nmol/1). The low affinity component of [³H]DCKA binding was insensitive to the agonists glycine and D-serine and the partial agonist (±)-3-amino-1-hydroxy-2-pyrrolidone (HA 966), though recognised by glycine site antagonists. The precise nature of this second, low affinity [³H]DCKA binding site remains to be elucidated.     

Investigation of Drug-Receptor Interactions by Means of Irreversible Receptor Inhibitors: Binding of Oxytocin and Angiotensin II to Their Receptors in Rat Uterus

Abstract

A stimulus-response coupling model in which individual steps follow hyperbolic or Hill-type laws has been employed to mimic phenomena associated with irreversible receptor inhibition (receptor reserve) in a responding biological system. Two methods for computation of ligand-receptor dissociation constant (K_A) have been derived from this model: 1) The relation between pD₂ and maximal attainable response allows a rough estimate of K_A; 2) A generalization of the earlier Furchgott-Bursztyn procedure employing equipotent doses for noninhibited and partially inhibited systems, has been achieved by introduction of Hill equation into the model. Applied to oxytocin and angiotensin II receptors in rat uterus, these pharmacological methods indicate rather low affinity of the two receptors for the respective peptides (around 2x10^?8 for angiotensin II and 2x10^?7 mol/1 for oxytocin), in case of oxytocin much lower than values reported from binding studies. Apparently, several binding sites are present on the target tissue cells from which the methods based on cellular response can pick up those corresponding to the receptors. Biophysical methods are lacking this ability. Single pD₂ values in noninhibited systems cannot themselves deliver any relevant information about receptor binding.     

Dopamine Receptor Subtype Agonists and Feeding Behavior

Stimulation or blockade of various dopamine receptor subtypes is associated with reduced feeding. For example, D₂ receptor agonists suppress feeding in food-deprived and free-feeding rats, and in rats given access to a highly palatable diet Similarly, reduced food intake is associated with the actions of diverse D₁ receptor agonists, and these compounds can interact synergistically with D₂ receptor agonists to potentiate reductions in feeding. Using micro-structural analysis to compare D₁ and D₂ agonist effects, specific differences emerge in their modes of action. D₁ agonists reduce the duration of feeding, primarily by decreasing the frequency of feeding bouts, whereas D₂ agonists reduce the local rate of eating. However, since D₁ agonists uniquely reduce feeding in the absence of other behavioral impairments and are less disruptive of the pattern of feeding behavior, it has been suggested that D₁ agonists are more likely than D₂ agonists to act on central mechanisms regulating food intake. Moreover, only D₁ agonists are effective in suppressing sucrose sham-feeding, suggesting that D₁ receptor stimulation may promote satiety. Nevertheless, many questions remain. For example, antagonist studies have implicated 5-HT receptor stimulation in the anorectic effects of D₁ agonists, suggesting that further pharmacological and behavioral analyses of receptor-subtype agonist effects are required. Above all, recent developments in the classification of dopamine receptor subtypes reveal the need for new studies examining the involvement of D₃, D₄ and D₅ receptors in feeding.     

Advances and challenges in the search for D2 and D3 dopamine receptor-selective compounds

Compounds that target D2-like dopamine receptors (DRs) are currently used as therapeutics for several neuropsychiatric disorders including schizophrenia (antagonists) and Parkinson's disease (agonists). However, as the D₂R and D₃R subtypes are highly homologous, creating compounds with sufficient subtype-selectivity as well as drug-like properties for therapeutic use has proved challenging. This review summarizes the progress that has been made in developing D₂R- or D₃R-selective antagonists and agonists, and also describes the experimental conditions that need to be considered when determining the selectivity of a given compound, as apparent selectivity can vary widely depending on assay conditions. Future advances in this field may take advantage of currently available structural data to target alternative secondary binding sites through creating bivalent or bitopic chemical structures. Alternatively, the use of high-throughput screening techniques to identify novel scaffolds that might bind to the D₂R or D₃R in areas other than the highly conserved orthosteric site, such as allosteric sites, followed by iterative medicinal chemistry will likely lead to exceptionally selective compounds in the future. More selective compounds will provide a better understanding of the normal and pathological functioning of each receptor subtype, as well as offer the potential for improved therapeutics.     

Evaluation of the Kinetics of Hydrolysis of Monoamino Analogues of 2′- or 3′-Deoxyadenosine and of 9-(2-Deoxy-β-D-Threo-pentofuranosyl)Adenine or 9-(3-Deoxy-β-D-threo-pentofuranosyl)Adenine by Liquid Chromatography

Abstract

Liquid chromatography was used to follow the degradation of monoamino analogues of 2′- or 3′-deoxyadenosine and of 9-(2-deoxy-β-D-threo-pentofuranosyl) adenine or 9-(3-deoxy-β-D-threo-pentofuranosyl) adenine in buffers of different pH and constant ionic strength (μ). Comparison of stabilities of some of the compounds under study with those of corresponding hydroxyl analogues showed that at acid pH the aminated compounds are more stable than the corresponding hydroxyl compounds. The higher stability associated with the presence of an amino group in the sugar is explained in function of pK_a values, which were determined by ¹³C NMR.     

Design,synthesis and evaluation of benzo[a]thieno[3,2-g]quinolizines as novel l-SPD derivatives possessing dopamine D1, D2 and serotonin 5-HT1A multiple action profiles

A novel scaffold derived from l-SPD with a substituted thiophene group in the D ring were designed, synthesized, and evaluated for their binding affinities at dopamine (D₁, D₂ and D₃) and serotonin (5-HT_1A and 5-HT_2A) receptors. Most of the tetracyclic compounds exhibited higher affinities for D₂ and 5-HT_1A receptors than l-SPD, while compound 23e showed the highest K_i value of 7.54 nM at D₂ receptor which was 14 times more potent than l-SPD. Additionally, compounds 23d and 23e were more potent than l-SPD at D₃ receptor. According to the functional assays, 23d and 23e were demonstrated as full antagonists at D₁ and D₂ receptors and full agonists at 5-HT_1A receptor. Since the combination of D₂ antagonism and 5-HT_1A agonism is considered effective in treating both the positive and negative symptoms of schizophrenia, these novel compounds are implicated as potential therapeutic agents.     

1-substituted apomorphines as potent dopamine agonists

A novel set of 1-substituted apomorphines as dopaminergic agonists were synthesized according to our new strategy employing the acid-catalyzed rearrangement of diversely functionalized 5β-substituted-6-demethoxythebaines. The activities of new compounds for dopamine receptors subtypes were evaluated using HEK293 based stable cell lines expressing D₁, D_2L or D₃ receptor subtypes. All studied compounds had affinities in nanomolar range for D_2L and D₃ receptors and the change of the nature of substituent in position 1 had only moderate effect. D₁ receptors were sensitive to the introduction of the 4-OH-benzyl function resulting in an increased affinity. The small hydrophilic group (hydroxymethyl) highly reduced the agonist affinity and potency thereby increasing subtype selectivity. This strategy for selective modulation of affinities and potencies of 1-substituted apomorphines gives essential hints for future design of subtype selective dopaminergic ligands.     

Pharmacological Characterisation of [35S]-GTPγS Binding to Chinese Hamster Ovary Cell Membranes Stably Expressing Cloned Human 5-HT1D Receptor Subtypes

Abstract

[³⁵S]-GTPγS binding has been used to study the function of cloned human 5-HT_1D receptor subtypes stably expressed in chinese hamster ovary (CHO) cells. 5-HT stimulated [³⁵S]-GTPγS binding to membranes from cells expressing 5-HT_1Dα or 5-HT_1Dβ receptors. In membranes containing 5-HT_1Dβ receptors, 5-CT and sumatriptan stimulated binding to a similar extent as 5-HT while yohimbine, metergoline and 8-OHDPAT were partial agonists. The order of potency for agonists was 5-CT > 5-HT > metergoline > sumatriptan > yohimbine > 8-OHDPAT. The stimulation of binding by 5-HT in membranes containing 5-HT_1Dβ receptors was potently antagonised by methiothepin (pA₂ 8.9 ± 0.1). The overall pharmacological profile for the human 5-HT_1Dβ receptor, defined using [³⁵S]-GTPγS binding, agreed well with that reported for inhibition of forskolin-stimulated adenylyl cyclase. In addition, methiothepin and ketanserin inhibited basal [³⁵S]-GTPγS binding to membranes containing 5-HT_1Dα or 5-HT_1Dβ receptors, suggesting that these compounds show negative efficacy at 5-HT_1D receptor subtypes. The data show that [³⁵S]-GTPγS binding is a suitable method for studying the interaction between cloned human 5-HT_1D receptors and G-proteins.     

Arylpiperazinylalkyl derivatives of 8-amino-1,3-dimethylpurine-2,6-dione as novel multitarget 5-HT/D receptor agents with potential antipsychotic activity

A series of new 7-arylpiperazinylalkyl-1,3-dimethyl-purine-2,6-dione derivatives with diversified 8-amino substituent in 8 position was synthesized and their 5-HT_1A, 5-HT_2A, 5-HT₆, 5-HT₇, and D₂ receptor affinities were determined. The binding study allowed identifying some potent 5-HT_1A/5-HT_2A/5-HT₇/D₂ ligands. The most interesting because of their multireceptor profile were 8-piperidine (30–35) and 8-dipropylamine (45–47) analogs with four and five carbon aliphatic linkers. The selected compounds 24, 31, 34, 39, 41, 43, 45, and 46 in the functional in vitro evaluation for all targeted receptors showed significant partial D₂ agonist, partial 5-HT_1A agonist, and 5-HT_2A antagonist properties. The advantageous in vitro affinity of compound 34 for 5-HT_1A and D₂ receptors has been explained by means of molecular modeling, taking into consideration its partial agonist activity towards the latter one. In behavioral studies, compounds 32 and 34 revealed antipsychotic-like properties, significantly decreasing d-amphetamine-induced hyperactivity in mice.     

Derivatives of (R)-2-amino-5-methoxytetralin: Antagonists and inverse agonists at the dopamine D2a receptor

A series of N-arylmethyl substituted (R)-5-methoxy-2-(propylamino)tetralins has been prepared and evaluated for affinity and efficacy at dopamine (DA) D_2a receptors. The novel compounds appeared to be antagonists or inverse agonists. (R)-2-[(Benzyl)propylamino]-5-methoxytetralin (7) was characterized as a potent inverse agonists at DA D_2a receptors in a [³⁵S]GTPγS binding assay.     

Effects of nandrolone and resistance training on the blood pressure,cardiac electrophysiology,and expression of atrial β-adrenergic receptors

AimsThis study was performed to assess isolated and combined effects of nandrolone and resistance training on the blood pressure, cardiac electrophysiology, and expression of the β₁- and β₂-adrenergic receptors in the heart of rats.Main methodsWistar rats were randomly divided into four groups and submitted to a 6-week treatment with nandrolone and/or resistance training. Cardiac hypertrophy was accessed by the ratio of heart weight to the final body weight. Blood pressure was determined by a computerized tail-cuff system. Electrocardiography analyses were performed. Western blotting was used to access the protein levels of the β₁- and β₂-adrenergic receptors in the right atrium and left ventricle.Key findingsBoth resistance training and nandrolone induced cardiac hypertrophy. Nandrolone increased systolic blood pressure depending on the treatment time. Resistance training decreased systolic, diastolic and mean arterial blood pressure, as well as induced resting bradycardia. Nandrolone prolonged the QTc interval for both trained and non-trained groups when they were compared to their respective vehicle-treated one. Nandrolone increased the expression of β₁- and β₂-adrenergic receptors in the right atrium for both trained and non-trained groups when they were compared to their respective vehicle-treated one.SignificanceThis study indicated that nandrolone, associated or not with resistance training increases blood pressure depending on the treatment time, induces prolongation of the QTc interval, and increases the expression of β₁- and β₂-adrenergic receptors in the cardiac right atrium, but not in the left ventricle.     

Molecular and pharmacological characterization of two D(1)-like dopamine receptors in the Lyme disease vector, Ixodes scapularis

Advancements in tick neurobiology may impact the development of acaricides to control those species that transmit human and animal diseases. Here, we report the first cloning and pharmacological characterization of two neurotransmitter binding G protein-coupled receptors in the Lyme disease (blacklegged) tick, Ixodes scapularis. The genes IscaGPRdop1 and IscaGPRdop2 were identified in the I. scapularis genome assembly and predicted as orthologs of previously characterized D₁-like dopamine receptors in the fruit fly Drosophila melanogaster and honeybee Apis mellifera. Heterologous expression in HEK 293 cells demonstrated that each receptor functioned as a D₁-like dopamine receptor because significant increases in levels of intracellular cyclic adenosine monophosphate (cAMP) were detected following dopamine treatment. Importantly, the receptors were distinct in their pharmacological properties regarding concentration-dependent response to dopamine, constitutive activity, and response to other biogenic amines. Exposure to a variety of dopamine receptor agonists and antagonists further demonstrated a D₁-like pharmacology of these dopamine receptors and highlighted their differential activities in vitro.     

Local Control Model of Excitation–Contraction Coupling in Skeletal Muscle

The voltage-activated H⁺ selective conductance of rat alveolar epithelial cells was studied using whole-cell and excised-patch voltage-clamp techniques. The effects of substituting deuterium oxide, D₂O, for water, H₂O, on both the conductance and the pH dependence of gating were explored. D⁺ was able to permeate proton channels, but with a conductance only about 50% that of H⁺. The conductance in D₂O was reduced more than could be accounted for by bulk solvent isotope effects (i.e., the lower mobility of D⁺ than H⁺), suggesting that D⁺ interacts specifically with the channel during permeation. Evidently the H⁺ or D⁺ current is not diffusion limited, and the H⁺ channel does not behave like a water-filled pore. This result indirectly strengthens the hypothesis that H⁺ (or D⁺) and not OH⁻ is the ionic species carrying current. The voltage dependence of H⁺ channel gating characteristically is sensitive to pH_o and pH_i and was regulated by pD_o and pD_i in an analogous manner, shifting 40 mV/U change in the pD gradient. The time constant of H⁺ current activation was about three times slower (τ_act was larger) in D₂O than in H₂O. The size of the isotope effect is consistent with deuterium isotope effects for proton abstraction reactions, suggesting that H⁺ channel activation requires deprotonation of the channel. In contrast, deactivation (τ_tail) was slowed only by a factor ≤1.5 in D₂O. The results are interpreted within the context of a model for the regulation of H⁺ channel gating by mutually exclusive protonation at internal and external sites (Cherny, V.V., V.S. Markin, and T.E. DeCoursey. 1995. J. Gen. Physiol. 105:861–896). Most of the kinetic effects of D₂O can be explained if the pK _a of the external regulatory site is ∼0.5 pH U higher in D₂O.     

Effects of Dopamine D2 Receptor Partial Agonist Antipsychotic Aripiprazole on Dopamine Synthesis in Human Brain Measured by PET with L-[β-11C]DOPA

Dopamine D₂ receptor partial agonist antipsychotic drugs can modulate dopaminergic neurotransmission as functional agonists or functional antagonists. The effects of antipsychotics on presynaptic dopaminergic functions, such as dopamine synthesis capacity, might also be related to their therapeutic efficacy. Positron emission tomography (PET) was used to examine the effects of the partial agonist antipsychotic drug aripiprazole on presynaptic dopamine synthesis in relation to dopamine D₂ receptor occupancy and the resulting changes in dopamine synthesis capacity in healthy men. On separate days, PET studies with [¹¹C]raclopride and L-[β-¹¹C]DOPA were performed under resting condition and with single doses of aripiprazole given orally. Occupancy of dopamine D₂ receptors corresponded to the doses of aripiprazole, but the changes in dopamine synthesis capacity were not significant, nor was the relation between dopamine D₂ receptor occupancy and these changes. A significant negative correlation was observed between baseline dopamine synthesis capacity and changes in dopamine synthesis capacity by aripiprazole, indicating that this antipsychotic appears to stabilize dopamine synthesis capacity. The therapeutic effects of aripiprazole in schizophrenia might be related to such stabilizing effects on dopaminergic neurotransmission responsivity.