pKD Values Research Articles

Thermodynamic studies on the formation of ion-pair cobalt complexes in acetone–water mixtures

Dicarboxylic acids are stronoger acids than moncraboxylic acids and more soluble in water due to the presence of two carboxylic (–COOH) groups which are industrially important in producing polyester, polyamides, and polyols and as a pioneer to pharmaceutical additives. Succinic acid is one of the most prosperous chemicals that can be generated from renewable resources which can be used in the industries of antiseptics, cosmetics, sutures, drugs, solvents, detergents, herbicides, fungicides, synthetic resins, and inks. The ion–pair formation is very important in diferent applications, used widely for inorganic analysis by liquid chromatography and used for the heavy metal removal from waste water, so studing the ion-pair association constant (KA) formed between chloropentamminecobalt(III) cation [CpX2+] and a different ligands of dicarboxylate anions (AC2-), have been determined by EMF technique in different compositions of acetone aqueous media (0–44.20 wt% acetone) for succinic acid and for the other dicarboxylic acids (Maleic, Malonic and Malic) in the same solvent composition (40 wt% acetone) with in temperature range (30 – 60 °C). The thermodynamic association properties (ΔGA°, ΔHA° and ΔSA°) have been estimated and discussed. Extra thermodynamic correlations like ΔHA°- ΔSA° and ΔGA°- ΔGI(orII)° (where I and II are mentioned for the first and second dissociation constant of the dicarboxylic acids) in different compositions of acetone aqueous media for succinic acid and in fixed solvent composition for the other dicarboxylic acids were examined in order to give more information about the behavior of solvent molecules and its effect on formation of the ion-pair molecules. In addition, The Gibbs free energy of transfer for the association process ΔGA°(t) from water to acetone mixed solvents were calculated for succinic acid in order to indicate the spontaneous nature and the stabilization of the ion-pair by increasing the acetone weight percent in the mixed acetone – aqueous media. The linear correlation between pKD values and the relative permittivity of acetone aqueous medium is in accordance to Born’s equation.

Effect of ligand torsion number on the AutoDock mediated prediction of protein-ligand binding affinity

Molecular docking simulation is a useful tool in the prediction of protein-ligand binding affinity on a large scale and has great potential in various application fields such as virtual screening of potential drug molecules. However, the reliability of molecular docking is still weak in the estimation of ligand-binding free energy, which limits the applicability of molecular docking simulation. Ligand torsion number is related to the flexibility of ligand and generally incorporated as a crucial variable in the thermodynamic function of binding free energy. In this study, we investigated how the ligand torsion number has influence on the binding affinity prediction of AutoDock, a popular molecular docking simulation tool. The pKd values of various protein-ligands were estimated by using the binding free energy function of AutoDock and compared with their experimental pKd values. The torsion number dependent comparison showed that the predicted binding affinities were mostly underestimated in the complexes of higher torsion numbers, whereas the underestimated and overestimated cases were relatively balanced at relatively lower torsion numbers. A new weight factor for torsion-free energy term of binding energy function was determined and introduced to make correction to the underestimation of binding affinity of ligands with high torsion numbers. It is expected that the torsion number dependent deviation pattern of AutoDock and its correction strategy are useful in the large-scale validation of protein-ligand binding affinity.

5-HT2A receptor is the predominant receptor mediating contraction of the isolated porcine distal ureter to 5-HT in young and old animals

Isolated ureteral strips develop spontaneous phasic contractile activity which is enhanced by 5-hydroytryptamine (5-HT). The aim of this study was to identify the receptor subtype mediating these responses and to determine whether responses to 5-HT change with age. The frequency of contractions and the overall contractile activity (measured as the area under the curve, AUC) were recorded in strips of porcine distal ureter isolated from young (3 months) and old (2 years) pigs. Responses to 5-HT were examined in the absence and presence of selective 5-HT receptor subtype antagonists. Tissues from the younger animals elicited larger contractile responses to 5-HT (5885 ± 335g−1 s) than tissues from the older animals (2787 ± 317g−1 s, P < 0.001). The 5-HT2A receptor antagonist ketanserin (10–100nM) elicited rightward shifts of 5-HT concentration-response curves, antagonising AUC and frequency responses with high affinity in tissues from both age groups (pKD values 8.4–8.8). The slopes of the corresponding Schild plots were not significantly different from unity, suggesting a competitive antagonism at a single receptor, except for frequency responses to 5-HT in the older animals. Antagonists selective for other 5-HT receptor subtypes: methiothepin (non-selective), RS-10221 (5-HT2C), ondansetron (5-HT3), GR-113808 (5-HT4), SB699551 (5-HT5), SB399885 (5-HT6), SB269970 (5-HT7)) had no effect on 5-HT-induced responses. The results suggest that the 5-HT2A receptor subtype is the predominant receptor mediating 5-HT responses in ureteral tissues, being the sole mediator of responses in tissues from young animals, but with another receptor subtype also playing a minor role in the older animals.

Functional and radioligand binding characterization of the α1L-adrenoceptor subtype of the human vas deferens.

Alpha1 -adrenoceptor antagonists can cause ejaculatory dysfunction as an adverse effect. Contractions of the human vas deferens are mediated via α1A -adrenoceptors, and this study investigated whether the low affinity state of this receptor (α1L -adrenoceptor) is involved in mediating contractions of this tissue. The potency of agonists and the affinity of receptor subtype selective antagonists were determined in functional experiments and in [(3) H]tamsulosin binding experiments to identify the α1 -adrenoceptor subtype population present in the human vas deferens. The α1A -adrenoceptor selective agonist A61603 was a full agonist and was 250-fold more potent than noradrenaline. Prazosin antagonized contractile responses to phenylephrine with a low affinity (pKd = 8.6). Only high concentrations of RS17053 antagonized responses to phenylephrine and yielded a relatively low affinity estimate of 7.0. BMY7378 (α1D -adrenoceptor selective) gave a low affinity estimate (pKd = 6.7), whilst tamsulosin (α1A - and α1D -adrenoceptor selective) had a high affinity (pKd = 9.9). [(3) H]Tamsulosin bound to human vas deferens membranes with a high affinity (pKd = 10.0). Prazosin, RS17053 and BMY7378 competed with [(3) H]tamsulosin with low affinities for a single population of binding sites (pKd values of 8.5, 7.2 and 6.3, respectively). These functional and radioligand binding data indicate that the human vas deferens possesses a homogeneous population of α1 -adrenoceptors which have the pharmacological properties of the putative α1L -adrenoceptor, the same functional receptor previously identified in the human prostate.

Identification of protein-ligand binding sites by the level-set variational implicit-solvent approach.

Protein–ligand binding is a key biological process at the molecular level. The identification and characterization of small-molecule binding sites on therapeutically relevant proteins have tremendous implications for target evaluation and rational drug design. In this work, we used the recently developed level-set variational implicit-solvent model (VISM) with the Coulomb field approximation (CFA) to locate and characterize potential protein–small-molecule binding sites. We applied our method to a data set of 515 protein–ligand complexes and found that 96.9% of the cocrystallized ligands bind to the VISM-CFA-identified pockets and that 71.8% of the identified pockets are occupied by cocrystallized ligands. For 228 tight-binding protein–ligand complexes (i.e, complexes with experimental pKd values larger than 6), 99.1% of the cocrystallized ligands are in the VISM-CFA-identified pockets. In addition, it was found that the ligand binding orientations are consistent with the hydrophilic and hydrophobic descriptions provided by VISM. Quantitative characterization of binding pockets with topological and physicochemical parameters was used to assess the “ligandability” of the pockets. The results illustrate the key interactions between ligands and receptors and can be very informative for rational drug design.

Electrochemical Behavior of Dopamine with 2-Hydroxipropil-β-Cyclodextrin for the Determination of the Complexation Constant

The complexation constant was evaluated, pKd, between dopamine and 2-hydroxypropilbetaCD through two methods at pH 1: by cyclic voltammetry and with spectrophotometry using the software SQUAD, which requires absorbance data and a chemical model that considers the inclusion complex that is likely to be formed. Both methods produced very similar pKd values, namely: spectrophotometry gave 3.69 whereas electrochemistry gave 3.75. The composition ratio for the complex was dopamine-hydroxypropil−β−CD 1:1.

An Activity Model for Novel Antidepressants that Interact with the Serotonin Transporter (SERT)

Dysfunction of serotoninergic neurotransmission is known to be involved in the pathophysiology of major depression. The molecules that enhance the level of serotonin either via blocking serotonin reuptake or through inhibition of its metabolism are effective antidepressants. With this as the basis, a group of new molecules that supposedly effect serotoninergic neurotransmission were designed and tested. The new molecular entities (NME-2, NME-5, NME-16 and NME- 24) are active in animal models of behavioral despair. In the present study, the binding of these new NMEs to the serotonin transporter protein (SERT) has been modeled and their activity correlated with the behavioral pattern observed in the mouse forced swim test (FST) model. The putative binding orientations of the NMEs have been identified by docking the molecules into the active site of the SERT. A 3D model of the SERT active site was constructed using comparative protein modeling principles with the X-ray structure of the leucine transporter (LeuT) as template. 3D-QSAR models based on the CoRIA formalism were generated from the experimental data and docking scores for eight novel SERT inhibitors. The CoRIA models highlight the salient features for effective binding of NMEs to the serotonin transporter and are also able to predict pKd values. Based on the significant correlation between the anti-immobility effect seen in the mouse FST study and the binding energies obtained from the docking study along with insights from the interaction patterns with the receptor obtained from the docking, the CoRIA models can be used to suggest structural modifications that can help in optimization of the SERT inhibition. Experimental evidence shows that the NMEs are highly efficacious and could be developed into potential antidepressants.

Determination of the surface pK of carboxylic- and amine-terminated alkanethiols using surface plasmon resonance spectroscopy.

When using self-assembled monolayers (SAMs) with ionizable functional groups, such as COOH and NH2, the dissociation constant (pKd) of the surface is an important property to know, since it defines the charge density of the surface for a given bulk solution pH. In this study, we developed a method using surface plasmon resonance (SPR) spectroscopy for the direct measurement of the pKd of a SAM surface by combining the ability of SPR to detect the change in mass concentration close to a surface and the shift in ion concentration over the surface as a function of surface charge density. This method was then applied to measure the pKd values of both COOH- and NH2-functionalized SAM surfaces using solutions of CsCl and NaBr salts, respectively, which provided pKd values of 7.4 and 6.5, respectively, based on the bulk solution pH. An analytical study was also performed to theoretically predict the shape of the SPR plots by calculating the excess mass of salt ions over a surface as a function of the difference between the solution pH and surface pKd. The analytical relationships show that the state of surface charge also influences the local hydrogen ion concentration, thus resulting in a substantial local shift in pH at the surface compared to the bulk solution as a function of the difference between the bulk solution pH and the pKd of the surface.

3H]GR205171 displays similar NK1 receptor binding profile in gerbil and human brain

1 In this study, [(3)H]GR205171 (3(S)-(2-methoxy-5-(5-trifluoromethyltetrazol-1-yl)-phenylmethylamino)-2(S)-phenylpiperidine), a potent and selective NK1 receptor antagonist, was characterised in autoradiographic studies in gerbil brain and in binding experiments on homogenates from gerbil and human brain cortex and striatum. 2 In autoradiographic studies in gerbil brain, highest levels of [(3)H]GR205171 binding sites were observed in caudate putamen, nucleus accumbens, medial and cortical nuclei of the amygdala and intermediate levels were detected in the hypothalamus, basolateral amygdala, septum, and cortex. 3 Saturation experiments in homogenates of brain striatum from gerbil showed that [(3)H]GR205171 binds to a single receptor population with a pK(d) value of 10.8+/-0.2 and a B(max) value of 607+/-40 fmol mg(-1). A lower number of NK1 receptor sites was found in cortex, where a B(max) of 94+/-6 fmol mg(-1) protein was obtained. Saturation experiments performed on homogenates from brain striatum of two human subjects and brain cortex of three human subjects showed that [(3)H]GR205171 binds with pK(d) values not different from gerbil and B(max) values ranging from 318+/-51 to 432+/-27 fmol mg(-1) protein in striatum and from 59+/-1 to 74+/-21 fmol mg(-1) protein in cortex. The natural ligand [(3)H]Substance P (SP) bound with sub-nanomolar affinity to 15 and 6% sites compared to [(3)H]GR205171 in gerbil and human striatum, respectively. 4 In competition binding experiments, GR205171 and the NK1 receptor antagonists aprepitant (MK-869), L-733,060 and NKP-608 bound with similar pK(i) values in gerbil and human striatum, irrespective of the use of [(3)H]GR205171 or [(3)H]SP as radioligand. The following rank order was found in terms of pK(i) values: GR205171>aprepitant> or =L-733,060>NKP-608. In homologous displacement experiments in gerbil and human striatum, SP showed nanomolar affinity, whereas in [(3)H]GR205171 competition experiments SP bound with pIC(50) values in the micromolar range and Hill slopes significantly lower than one. 5 It is concluded that the similarities of [(3)H]GR205171 binding characteristics and pharmacology between gerbil and human in cortex and striatum support the use of gerbil in preclinical models to study the effects of NK1 receptor antagonists in the central nervous system.

The upper limit pH in the dye-binding method for the determination of serum protein via measurements of the absorbance increase produced by protein error.

In the dye-binding method for determining the albumin concentration, the absorbance increase due to the change of the color shade by protein error of a pH indicator can be measured by a spectrophotometer. This absorbance increase is observed only in a restricted pH region, but this pH region is not theoretically studied yet. Thus, the author investigated the upper limit pH (pHUL) at which the absorbance increase occurs by the theoretical calculation, and compared these results with those obtained experimentally using four pH indicators. The pHUL is not affected by the dye or protein concentrations, or by the formation constant of the dye-protein complex; but the value changes according to the acid-dissociation constant of the dye (KD) and the ratio of the molar absorptivities of the proton-dissociated dye anion (epsilonD) and the dye-protein complex (epsilonPD). The pHUL value can be calculated by the equation, found theoretically. The calculated pHUL values of BPB, BCG, BCP and BTB were 5.1, 4.8, 6.2 and 5.5, respectively. These values correlated with the experimental results of 4.5 for BPB, 4.7 for BCG, 5.9 for BCP and 5.2 for BTB, but were not associated with the pKD values of each dye. The pHUL of these dyes did not change significantly for various dye and protein concentrations, as was expected from the thoretical calculation.

Comparison of the binding of [3H]nociceptin/orphaninFQ(1–13)NH2, [3H]nociceptin/orphaninFQ(1–17)OH and [125I]Tyr14nociceptin/orphaninFQ(1–17)OH to recombinant human and native rat cerebrocortical nociceptin/orphanin FQ receptors

Nociceptin/orphanin FQ (N/OFQ) is a 17-amino acid endogenous neuropeptide ligand for the nociceptin receptor (NOP). We have prepared a [3H]-labelled truncated N/OFQ peptide, [3H]N/OFQ(1–13)NH2 and compared its binding characteristics with [3H]N/OFQ(1–17)OH and [125I]Y14N/OFQ(1–17)OH in membranes prepared from Chinese hamster ovary cells expressing the recombinant human NOP (CHOhNOP) and the rat cerebrocortex. [3H]N/OFQ(1–13)NH2, [3H]N/OFQ(1–17)OH and [125I]Y14N/OFQ(1–17)OH binding to CHOhNOP was concentration dependent and saturable with receptor density (Bmax) and radioligand equilibrium dissociation constant (pKd) values (mean±SEM) of 1043±58 fmol/mg protein and 10.35±0.03, 1348±44 fmol/mg protein and 10.06±0.04, and 1169±76 fmol/mg protein and 10.45±0.06, respectively. In the rat, Bmax and pKd values for [3H]N/OFQ(1–13)NH2 and [3H]N/OFQ(1–17)OH were 130±1 fmol/mg protein and 10.70±0.03, and 157±4 fmol/mg protein and 10.34±0.02, respectively. The binding of all radioligands was displaced by a range of peptide and non-peptide ligands. There was a strong correlation (r2=0.92, P=0.002) between pKi values estimated with [3H]N/OFQ(1–13)NH2 and [3H]N/OFQ(1–17)OH. No such correlation was observed in comparison with the [125I]-labelled peptide (poor agreement with low affinity N/OFQ(1–9)NH2, Dynorphin-A and Naloxone benzoylhydrazone). We suggest that [3H]N/OFQ(1–13)NH2 may be a useful alternative to [3H]N/OFQ(1–17)OH.

3H]-Mesulergine labels 5-HT7 sites in rat brain and guinea-pig ileum but not rat jejunum.

1. The primary aim of this investigation was to determine whether binding sites corresponding to the 5-HT7 receptor could be detected in smooth muscle of the rat jejunum. Binding studies in rat brain (whole brain minus cerebellum) and guinea-pig ileal longitudinal muscle were also undertaken in order to compare the binding characteristics of these tissues. Studies were performed using [3H]-mesulergine, as it has a high affinity for 5-HT7 receptors. 2. In the rat brain and guinea-pig ileum, pKD values for [3H]-mesulergine of 8.0 +/- 0.04 and 7.9 +/- 0.11 (n = 3) and Bmax values of 9.9 +/- 0.3 and 21.5 +/- 4.9 fmol mg(-1) protein were obtained respectively, but no binding was detected in the rat jejunum. [3H]-mesulergine binding in the rat brain and guinea-pig ileum was displaced with the agonists 5-carboxamidotryptamine (5-CT) > 5-hydroxytryptamine (5-HT) > or = 5-methoxytryptamine (5-MeOT) > sumatriptan and the antagonists risperidone > or = LSD > or = metergoline > ritanserin > > pindolol. 3. Despite the lack of [3H]-mesulergine binding in the rat jejunum, functional studies undertaken revealed a biphasic contractile response to 5-HT which was partly blocked by ondansetron (1 microM). The residual response was present in over 50% of tissues studied and was found to be inhibited by risperidone > LSD > metergoline > mesulergine = ritanserin > pindolol, but was unaffected by RS 102221 (3 microM), cinanserin (30 nM), yohimbine (0.1 microM) and GR 113808 (1 microM). In addition, the agonist order of potency was 5-CT > 5-HT > 5-MeOT > sumatriptan. 4. In conclusion, binding studies performed with [3H]-mesulergine were able to detect 5-HT7 sites in rat brain and guinea-pig ileum, but not in rat jejunum, where a functional 5-HT7-like receptor was present.

A re-investigation of questionable subclassifications of presynaptic alpha2-autoreceptors: rat vena cava, rat atria, human kidney and guinea-pig urethra.

It has been suggested that at least the majority of mammalian presynaptic alpha2-autoreceptors belong to the genetic alpha2A/D-adrenoceptor subtype. The aim of the present study was to re-examine the alpha2-autoreceptors in tissues in which previous assignments conflicted with this alpha2A/D rule: in the rat vena cava and rat heart atria, where the autoreceptors were classified as alpha2B or similar to, but not identical with, alpha2D, and in the human kidney, where they were classified as alpha2C. Also re-examined were the autoreceptors in the guinea-pig urethra, where they were suggested to be alpha2A, in agreement with the rule, but in contrast to indications that the alpha2A/D-adrenoceptor of the guinea pig possesses alpha2D pharmacological properties. Tissue pieces were preincubated with 3H-noradrenaline and then superfused and stimulated electrically under autoinhibition-free or almost autoinhibition-free conditions. The Kd values of up to 14 antagonists (including the partial agonist oxymetazoline) against the release-inhibiting effect of the alpha2 agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined. UK 14,304 reduced the evoked overflow of tritium with an EC50 between 6.3 and 13.2 nM. All antagonists (except prazosin in one case) shifted the concentration-inhibition curve of UK 14,304 to the right. Comparison of the Kd values thus obtained with Kd values at known alpha2 subtypes indicated that the autoreceptors in the rat vena cava, rat atria and the guinea-pig urethra were alpha2D and those in the human kidney alpha2A. For example, the pKd values of the antagonists in the rat vena cava, in rat atria and in the guinea-pig urethra were closely correlated with pKd values at the prototypic alpha2D radioligand binding sites in the bovine pineal gland (r = 0.96, P < 0.001; r = 0.92, P < 0.01; and r = 0.95; P < 0.001) and with the pKd values at the alpha2D-autoreceptors of guinea-pig atria (r = 0.99, P < 0.001; r = 0.95, P < 0.001; and r = 0.98, P < 0.001). The pKd values at the autoreceptors in rat vena cava, rat atria and guinea-pig urethra were not significantly or more loosely correlated with pKd values at alpha2A, alpha2B and alpha2C binding sites and alpha2A-autoreceptors. On the other hand, the pKd values of the antagonists in the human kidney were closely correlated with pKd values at the prototypic alpha2A radioligand binding sites in HT29 cells (r = 0.95; P < 0.001) and with pKd values at the alpha2A-autoreceptors of the pig brain cortex (r = 0.97; P < 0.001), but were not significantly or more loosely correlated with pKd values at alpha2B, alpha2C and alpha2D binding sites and alpha2D-autoreceptors. In contrast to previous suggestions, the autoreceptors in rat vena cava and atria are alpha2D, those in the human kidney alpha2A, and those in the guinea-pig urethra equally alpha2D. All, therefore, conform to the rule that alpha2-autoreceptors belong at least predominantly to the genetic alpha2A/D subtype of the alpha2-adrenoceptor. The apparent paradox of an alpha2A-autoreceptor in the urethra of the guinea pig, a species in which the genetic alpha2A/D-adrenoceptor otherwise has alpha2D pharmacological properties, is removed.

Species differences in brain adenosine A1 receptor pharmacology revealed by use of xanthine and pyrazolopyridine based antagonists.

1. The pharmacological profile of adenosine A1 receptors in human, guinea-pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). 2. The affinity of [3H]-DPCPX binding sites in rat cortical and hippocampal membranes was similar. Binding site affinity was higher in rat cortical membranes than in membranes prepared from guinea-pig cortex and hippocampus, mouse cortex and human cortex. pKD values (M) were 9.55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (Bmax) was lower in rat cortical membranes than in guinea-pig or human cortical membranes. 3. The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5'-N-ethylcarboxamidoadenosine (NECA), agonist affinity was 3.5-26.2 fold higher in rat cortical membranes than in human and guinea-pig brain membranes; affinity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher affinity in rat compared to human cortical membranes, affinity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 microM) reduced 2-chloro-N6-cyclopentyladenosine (CCPA) affinity 7-13.9 fold, whereas the affinity of DPCPX was unaffected. 4. The affinity of six xanthine-based adenosine receptor antagonists was 2.2-15.9 fold higher in rat cortical membranes compared with human or guinea-pig membranes. The rank order of potency was species-independent. In contrast, three pyrazolopyridine derivatives, (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-2-piperidine ethanol (FK453), (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352) and 6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-pyridazinebutyric acid (FK838) exhibited similar affinity in human, guinea-pig, rat and mouse brain membranes. pKi values (M) for [3H]-DPCPX binding sites in human cortical membranes were 9.31, 7.52 and 7.92, respectively. 5. Drug affinity for adenosine A2A receptors was determined in a [3H]-2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido ade nosine ([3H]-CGS 21680) binding assay in rat striatal membranes. The pyrazolopyridine derivatives, FK453, FK838 and FK352 exhibited pKi values (M) of 5.90, 5.92 and 4.31, respectively, compared with pKi values of 9.31, 8.18 and 7.57 determined in the [3H]-DPCPX binding assay in rat cortical membranes. These novel pyrazolopyridine derivatives therefore represent high affinity, adenosine A1 receptor selective drugs that, in contrast to xanthine based antagonists, exhibit similar affinity for [3H]-DPCPX binding sites in human, rat, mouse and guinea-pig brain membranes.

Presynaptic alpha 2-autoreceptors in mouse heart atria: evidence for the alpha 2D subtype.

Presynaptic alpha 2-autoreceptors in mouse atria were characterized in terms of the alpha 2A, alpha 2B, alpha 2C and alpha 2D subtypes. Segments of the atria were preincubated with 3H-noradrenaline and then superfused and stimulated electrically. The affinity of up to 16 antagonists for the autoreceptors was assessed as (1) pEC30% values. i.e. concentrations that increased previously autoinhibited release of 3H-noradrenaline (120 pulses, 3 Hz) by 30%, and (2) pKd values against the release-inhibiting effect of 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) under conditions of no or little autoinhibition (2 trains of 20 pulses, 50 Hz, train interval 120 s). The pKd values correlated well with the pEC30% values (r = 0.98; P < 0.001; slope of regression line 0.93), indicating that UK 14,304 and released noradrenaline modulated the release of noradrenaline through pharmacologically identical receptors. Comparison with antagonist affinities for (1) prototypic native alpha 2 radioligand binding sites, (2) radioligand binding sites in COS cells transfected with alpha 2 subtype genes, and (3) previously classified presynaptic alpha 2-autoreceptors-all taken from the literature-indicated that the mouse atrial autoreceptors corresponded to the alpha 2D subtype. For example, the pKd values at mouse atrial auto-receptors correlated closely with pKd values at native alpha 2D binding sites in the bovine pineal gland (r = 0.96; P < 0.001); with pKd values at alpha 2D binding sites in COS cells transfected with the rat alpha 2D gene (r = 0.85; P < 0.01); and with pKd values at guinea-pig cerebral and atrial and mouse cerebral alpha 2D-autoreceptors (r = 0.96-0.98; P < 0.001). The antagonist pKd values at mouse atrial autoreceptors correlated less with pKd values at alpha 2A, alpha 2B and alpha 2C sites. It is concluded that the presynaptic alpha 2-autoreceptors in mouse atria are alpha 2D. This identification supports the hypothesis that at least the majority of alpha 2-autoreceptors belong to the alpha 2A/D pair of orthologous alpha 2-adrenoceptors.

Binding of aliphatic ketones to cyclodextrins in aqueous solution

Dissociation constants (Kd) for the complexation of 22 simple ketones with α-, β- and hydroxypropyl-β- cyclodextrin (α-CD, β-CD, and HP-β-CD) in aqueous solution have been determined. For these constants, there are various correlation involving pKd(=–log Kd) which have the form of linear free energy relationships. In particular, there are strong correlation between the pKd values of ketones (RCOR′) and related secondary alcohols [RCH(OH)R′], including cases where R and R′ form a ring. As with other alkyl derivatives, pKd values for alkan-2-ones and alkan-3-ones increase monotomcally with chain length, with slopes about 0.4, corresponding to Gibbs energy increments of ca. 2.3 kJ mol–1 for each CH2 group that is sequestered by the CD. The strengths of binding of linear derivatives to α-CD and β-CD correlate well, but bulky and cyclic ketones bind more weakly to α-CD, due to its smaller cavity. The pKd values for complexation of 18 of the 22 ketones by HP-β-CD and β-CD are fairly close and linearly related with a slope of 0.96 ± 0.03. These data are a subset of a larger set for 68 aliphatic compounds for which the slope is 0.99 ± 0.02. Thus, the strength of binding of such aliphatics to HP-β-CD and β-CD is generally close, although the penetration of the CD cavity by the guests is not necessarily the same for these two CDs.

α2c‐Adrenoceptor‐modulated release of noradrenaline in human right atrium

1. The aim of the present study was to characterize the presynaptic alpha 2-autoreceptors in human right atrium in terms of the alpha 2A-D system. Segments of atrial appendages were preincubated with [3H]-noradrenaline and then superfused in the presence of cocaine and stimulated electrically. pEC30% values of eight alpha-adrenoceptor antagonists with discriminatory power were determined. pEC30% is the negative logarithm of the antagonist concentration that increased the stimulation-induced overflow of tritium by 30%. For four antagonists, the dissociation constant KD was determined, in addition to pEC30%, against the overflow-inhibiting effect of 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) under autoinhibition-free conditions. 2. pEC30% and KD values yielded identical rank orders of antagonist affinity (rauwolscine > WB 4101 > phentolamine > prazosin) suggesting that both released noradrenaline and the exogenous agonist UK 14,304 activated the same receptor to inhibit release. 3. The eight antagonist pEC30% values obtained in right atrium correlated significantly with their pEC30% values, reported in the literature, at the presynaptic alpha 2C-autoreceptors in human kidney (r = 0.817; slope of the regression line 1.03). No significant correlation was obtained between pEC30% values at atrial autoreceptors and pKD values at previously characterized alpha 2A-autoreceptors in rabbit and alpha 2D-autoreceptors in rat, mouse and guinea-pig tissues. 4. Comparison of antagonist pEC30% values with their pKD values at native alpha 2 binding sites in cells or tissues that express a single subtype only, and with pKD values at alpha 2 binding sites in membranes of COS cells transfected with human alpha 2 subtype genes confirms the alpha 2C character of the atrial autoreceptors: significant correlations were obtained exclusively with the alpha 2C binding sites. 5. Ratios of KD values were computed for alpha 2-autoreceptors in human right atrium and for binding sites in COS cells transfected with human alpha 2 subtype genes. The autoreceptor ratios corresponded well with the respective ratios for the alpha 2C binding sites (maximal three fold deviation) but were, in part, markedly different from ratios calculated for alpha 2A and alpha 2B binding sites (up to 166 fold deviation). This outcome supports the alpha 2C designation of the autoreceptors. 6. In conclusion, the presynaptic alpha 2-autoreceptors in human right atrium are alpha 2C. In this they agree with the previously characterized alpha 2-autoreceptors in human kidney. The alpha 2C classification possibly separates, in general, human alpha 2-autoreceptors from those in lagomorph (rabbit) and rodent (rat, mouse, guinea pig) species that have been proposed to be predominantly alpha 2A or alpha 2D.

Inhibitory effect of 5-hydroxytryptamine on rat stomach fundus: mediated indirectly by activation of noradrenaline release.

Biphasic cumulative concentration-response curves to 5-hydroxytryptamine (5-HT) and alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT) using rat stomach fundus in the presence of 50 microM pargyline suggested two sites of interaction (high and low affinity). The order of agonist potencies of 5-HT agonists for the high-affinity (contractile) response confirmed reports of a strong correlation of the 5-HT2B (contractile) receptor on fundus with reported rat brain (radioligand binding) pKd values at the 5-HT2C receptor (r = 0.94, b = 0.93 P < 0.01). 1(1-Naphthyl)-piperazine and ketanserin antagonized the high-affinity responses. The order of potencies for the low affinity (inhibitory) response was: alpha-Me-5-HT > 5-HT. Preincubation with mianserin or S-(-)propranolol, pretreatment with reserpine and removal of the mucosa layer resulted in amplification of the contractile effects and disappearance or reversal of the inhibitory effects of 5-HT. Activation of a second receptor site inducing catecholamine release may explain this effect of 5-HT on rat stomach fundus.

Subclassification of presynaptic alpha 2-adrenoceptors: alpha 2A-autoreceptors in rabbit atria and kidney.

The study was devised to classify, by means of antagonist affinities, the presynaptic alpha 2-autoreceptors of rabbit atria and kidney in terms of alpha 2A, alpha 2B, alpha 2C and alpha 2D. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the left atrium and slices of the kidney cortex were preincubated with 3H-noradrenaline and then superfused and stimulated electrically. In one series of experiments, tissue pieces were stimulated by relatively long pulse trains (1 or 2 min) leading to alpha 2-autoinhibition. All 11 (atria) or 10 (kidney) antagonists increased the evoked overflow of tritium. pEC30% values (concentrations causing 30% increase) were interpolated from concentration-response curves. In a second series of experiments, tissue pieces were stimulated by brief pulse trains (0.4 s) that did not lead to alpha 2-autoinhibition, and concentration-inhibition curves of the alpha 2-selective agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) were determined. Most of the 11 (atria) or 8 (kidney) antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. pKd values of the antagonists were calculated from the shifts. pEC30% values correlated with pKd values, both in atria (r = 0.728) and in the kidney (r = 0.930). pEC30% values in atria correlated with pEC30% values in the kidney (r = 0.988) and pKd values in atria correlated with pKd values in kidney (r = 0.923). It is concluded that the alpha 2-autoreceptors in atria and the kidney are the same. Comparison with antagonist affinities for prototypic native alpha 2 binding sites, alpha 2 binding sites in cells transfected with alpha 2 subtype genes, and previously classified presynaptic alpha 2-adrenoceptors--all taken from the literature--indicates that both autoreceptors are alpha 2A. This conclusion is reached with either of the two independent estimates of autoreceptor affinity, pEC30% and pKd. The results are compatible with the hypothesis that at least the majority of alpha 2-autoreceptors belong to the alpha 2A/D branch of the alpha 2-adrenoceptor tree, across mammalian or at least rodent and lagomorph species.

Subclassification of presynaptic ?2-adrenoceptors: ?2D-autoreceptors and ?2D-adrenoceptors modulating release of acetylcholine in guinea-pig ileum

The study was designed to classify in terms of alpha 2A, alpha 2B, alpha 2C and alpha 2D the presynaptic alpha 2-autoreceptors, as well as the alpha 2-receptors modulating the release of acetylcholine, in the myenteric plexus-longitudinal muscle (MPLM) preparation of the guinea-pig ileum. A set of antagonists was chosen that was able to discriminate between the four subtypes. Small pieces of the MPLM preparation were preincubated with 3H-noradrenaline or 3H-choline and then superfused and stimulated electrically. The stimulation periods used (3H-noradrenaline: 3 trains of 20 pulses, 50 Hz, train interval 60 s; 3H-choline: single trains of 30 pulses, 0.2 Hz) did not lead to alpha 2-autoinhibition or inhibition of 3H-acetylcholine release by endogenous noradrenaline. The alpha 2-selective agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) reduced the evoked overflow of tritium in both 3H-noradrenaline and 3H-choline experiments. Most (3H-noradrenaline) or all (3H-choline) of the 10 antagonists shifted the concentration-inhibition curves of UK 14,304 to the right. pKd values of the antagonists were calculated from the shifts. pKd values from 3H-noradrenaline experiments correlated with pKd values from 3H-choline experiments (r = 0.981). It is concluded that alpha 2-autoreceptors and alpha 2-heteroreceptors modulating the release of acetylcholine in the MPLM preparation are of the same subtype. Comparison with antagonist affinities for prototypic native alpha 2 binding sites, binding sites in cells transfected with alpha 2 subtype genes, and previously classified presynaptic alpha 2-adrenoceptors--all taken from the literature--indicates that both are alpha 2D.(ABSTRACT TRUNCATED AT 250 WORDS)