Biomedical subjects
A Christopoulos
Publications and source records attributed to A Christopoulos.
Interaction of anandamide with the M(1) and M(4) muscarinic acetylcholine receptors.
The M(1) and M(4) muscarinic acetylcholine receptors are the most abundant muscarinic receptor subtypes in the brain, and are involved in learning and memory. Because cannabinoid receptors are also abundantly expressed in similar brain regions and mediate opposite effects to acetylcholine on cognition, the present study investigated whether the endocannabinoid agonist, anandamide, and its metabolically stable derivative, methanandamide, directly modified the binding properties of the human M(1) and M(4) receptors individually expressed in CHO cell membranes. Experiments utilized the antagonists, [(3)H]N-methylscopolamine and [(3)H]quinuclidinyl benzilate. When acetylcholine was used as the inhibiting ligand, shallow, biphasic isotherms were observed at both receptors, characterised by similar apparent dissociation constants for high and low affinity binding at each receptor but with a greater proportion of high affinity sites at the M(4) (40-45%) than at the M(1) receptor (17-20%). In contrast, anandamide and methanandamide inhibited the binding of both radioligands over a narrow (low micromolar) concentration range, with monophasic isotherms characterized by Hill coefficients significantly greater than 1 at both receptors. These effects were not due to the vehicle used. Further saturation binding analyses found anandamide able to significantly reduce the apparent affinity and maximal density of binding sites labeled by [(3)H]quinuclidinyl benzilate. Interestingly, no significant inhibition of radioligand binding was noted using the synthetic cannabinoid agonist, WIN55212-2, or the cannabinoid CB(1) receptor antagonist, SR141716A. These data thus provide evidence for a direct role of anandamides in modulating muscarinic receptor binding properties through a non-competitive mechanism that is unrelated to their actions on cannabinoid receptors.
The allosteric interaction of otenzepad (AF-DX 116) at muscarinic M2 receptors in guinea pig atria.
The effects of the muscarinic receptor antagonist, otenzepad, in combination with the competitive antagonists N-methylscopolamine, dexetimide and atropine, or the allosteric modulators, C(7)/3'-phth, gallamine and alcuronium, were measured in the guinea pig electrically driven left atrium using the agonists, carbachol or acetylcholine. Otenzepad, in combination with C(7)/3'-phth or gallamine, gave concentration-ratios close to additive and in agreement with theoretical model predictions for combination of two allosteric modulators acting at a common site. However, when otenzepad was combined with alcuronium, dexetimide or N-methylscopolamine, supra-additive effects were observed. For either competitive antagonist in combination with otenzepad, the degree of supra-additivity was more evident after 2-h equilibration than after 40 min. When otenzepad was combined with atropine, no supra-additivity was observed with carbachol as the agonist, but was evident with acetylcholine. Otenzepad was also unable to fully inhibit [3H]N-methylscopolamine binding when the radioligand was employed at a concentration of approximately 100 x K(D). It is concluded that the action of otenzepad involves an allosteric site and a number of possibilities are discussed for its location.
Insurmountable AT1 receptor antagonism: message in a model?
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Pharmacological analysis of cannabinoid receptor activity in the rat vas deferens.
1. The interaction between the cannabinoid agonists, WIN 55,212-2 or CP 55,940 with the CB(1) receptor-selective antagonists, SR141716A or LY320135 was investigated using the rat electrically-stimulated vas deferens bioassay. 2. Tissues were stimulated by single-field pulses (150 V, 0.5 ms) delivered every 30 mins. In the presence of nifedipine (3 microM), agonists elicited a concentration-dependent inhibition of the contractile response, with pEC(50) values of 7.93 and 6.84 for WIN 55,212-2 and CP 55,940, respectively. 3. SR141716A and LY320135 caused parallel dextral displacements of the agonist concentration-response curves. However, the shift of the agonist curves by either antagonist was accompanied by a concentration-dependent enhancement of basal (agonist-independent) tissue contraction. 4. Addition of the amidase inhibitor, phenylmethylsulphonylfluoride (200 microM), resulted in a significant reduction of the basal twitch response, an effect consistent with the presence of tonic receptor activation mediated by the endogenous cannabinoid, anandamide. 5. In light of these findings, we propose a theoretical model of competitive agonist-antagonist interaction in the presence of endogenous agonist tone that was used to derive an optimized analytical approach for the determination of antagonist potency estimates under conditions of tonic receptor activation. 6. This approach yielded pK(B) estimates for SR141716A and LY320135 that were in good agreement with their activity at cannabinoid CB(1) receptors. 7. It is concluded that the rat vas deferens contains prejunctional cannabinoid CB(1) receptors that are under tonic activation from endogenous substances; under these conditions our analytical approach is preferable to the standard methods for the determination of antagonist potency.
From 'captive' agonism to insurmountable antagonism: demonstrating the power of analytical pharmacology.
1. Mathematical modelling is useful in pharmacology, allowing the investigator to obtain insights into the biological processes under study that may not always be intuitively obvious. Examples are presented in this review using the pharmacology of the muscarinic acetylcholine receptor (mAChR) agonist xanomeline. 2. Xanomeline possesses a novel mode of action that involves persistent binding to the M1 mAChR, yielding a fraction of agonist in the receptor compartment that continually activates the receptor, despite extensive washout, as assessed in functional assays measuring the cumulative production of M1 mAChR-mediated L-[3H]-citrulline. This persistent effect was reversed by the antagonist atropine, but re-established upon the removal of atropine. Thus, xanomeline may represent the first 'captive' agonist of the mAChR. 3. Atropine was equally potent at reversing the effect of persistently bound xanomeline and preventing the effect of added xanomeline. Application of standard quantitative equilibrium models of agonist-antagonist interaction to these data suggested that the interaction between xanomeline and atropine satisfied the criteria of competitivity in each case. 4. Subsequent real-time assays of M1 mAChR-mediated intracellular calcium mobilization found that atropine inhibited the effects of xanomeline in an insurmountable manner. 5. The discrepancy between the modes of antagonism in the various functional assays could be reconciled in a dynamic receptor model of antagonism within a transient response system and subsequent Monte Carlo simulations allowed for the development of an optimized analytical procedure to quantify antagonist potency under such conditions of response fade. 6. These types of studies exemplify the diagnostic and integrative features of analytical pharmacology.
Synthesis and pharmacological evaluation of dimeric muscarinic acetylcholine receptor agonists.
Two dimeric analogs of the muscarinic acetylcholine receptor (mAChR) agonist phenylpropargyloxy-1,2,5-thiadiazole-quinuclidine (NNC 11-1314) were synthesized and pharmacologically evaluated. In radioligand binding assays on Chinese hamster ovary (CHO) cell membranes expressing the individual human M(1) to M(5) mAChR subtypes, both dimers [(3S)-1,4-bis-(3-[(3-azabicyclo[2.2.2]octanyl)-1,2,5-thiadiazol-4-yloxy]-1-propyn-1-yl)benzene,2-L-(+)-tartrate (NNC 11-1607) and (3S)-1,3-bis-(3-[(3-azabicyclo[2.2.2]octanyl)-1,2,5-thiadiazol-4-yloxy]-1-propyn-1-yl)benzene,2-L-(+)-tartrate (NNC 11-1585)] exhibited higher binding affinities than the monomeric NNC 11-1314. Only NNC 11-1585, however, displayed significant selectivity for the M(1) and M(2) mAChRs relative to the other subtypes. Although binding studies in rat brain homogenates supported the selectivity profile of NNC 11-1585 observed in the CHO membranes, rat heart membrane experiments revealed complex binding behavior for all three agonists that most likely reflected differences in species and host cell environment between the heart and CHO cells. Subsequent functional assays with phosphatidylinositol hydrolysis revealed that all three novel ligands were partial agonists relative to the full agonist oxotremorine-M at the CHO M(1), M(3), and M(5) mAChRs, with NNC 11-1607 displaying the highest functional selectivity. In the CHO M(2) and M(4) mAChR cells, agonist-mediated effects on forskolin-stimulated cAMP accumulation were characterized by bell-shaped concentration-response curves, with the exceptions of NNC 11-1607, which had no discernible effects at the M(2) mAChR, and NNC 11-1585, which could only inhibit cAMP accumulation at the M(4) mAChR. Thus, we identified NNC 11-1607 as a novel functionally selective M(1)/M(4) mAChR agonist. Our data suggest that dimerization of mAChR agonists is a viable approach in designing more potent and functionally selective agonists, as well as in providing novel tools with which to probe the nature of agonism at these receptors.
Further evidence for the heterogeneity of functional muscarinic receptors in guinea pig gallbladder.
Previous studies have suggested the presence of multiple muscarinic receptor subtypes in guinea pig gallbladder smooth muscle, although the relative abundance and functional role of these subtypes remains an area of significant research efforts. The present study utilized both radioligand kinetic and functional experiments to further probe the nature of the muscarinic receptors in gallbladder smooth muscle and their mode of coupling to intra- and extra-cellular Ca(2+) sources. Dissociation kinetic studies using [3H]N-methylscopolamine ([3H]NMS) indicated that the binding profile in guinea pig gallbladder smooth muscle could not be reconciled with that expected for a single muscarinic receptor subtype, the latter determined in parallel experiments conducted on the cloned muscarinic M(1)-M(5) subtypes in Chinese hamster ovary (CHO) cells. Furthermore, comparison of the gallbladder data with the dissociation characteristics of [3H]NMS in guinea pig urinary bladder revealed a significantly different kinetic profile, with the urinary bladder, but not the gallbladder, demonstrating biphasic radioligand dissociation kinetics. In functional experiments, carbachol caused a concentration-dependent contraction of guinea pig gallbladder smooth muscle strips in Ca(2+)-free or 5 mM Sr(2+)-substituted physiological salt solutions (PSS) with amplitudes of the maximal contractions corresponding to 45.8+/-8.0% and 33.2+/-6.6% of control responses in normal PSS, respectively. Furthermore, the stimulus-response characteristics of carbachol-mediated contraction appeared significantly altered in Ca(2+)-free PSS relative to normal or Sr(2+)-substituted PSS. The antagonist, methoctramine (1x10(-7)-3x10(-5) M), exerted only a slight inhibition of carbachol (10(-5) M)-induced contractions in 5 mM Sr(2+)-substituted medium, whereas it was significantly more potent in antagonizing gallbladder contractions in response to 10(-5) M carbachol in the absence of extracellular Ca(2+). Both atropine and tripitramine were equipotent in antagonizing carbachol-induced contractions in Ca(2+)-free (pIC(50): 6.85+/-0.11 for atropine and 5.75+/-0.32 for tripitramine) and Sr(2+)-substituted media (pIC(50): 6.88+/-0.25 for atropine and 5.70+/-0.16 for tripitramine), and pirenzepine was only slightly more potent in Ca(2+)-free PSS (pIC(50): 5.66+/-0.23) than in Sr(2+)-substituted PSS (pIC(50): 5.33+/-0.21). Taken together, our data indicate that carbachol contracts guinea pig gallbladder by stimulating two distinct muscarinic receptor subtypes linked to extracellular Ca(2+) influx and intracellular Ca(2+) release. These two subtypes may represent the muscarinic M(3) and M(4) receptors, although the presence of the muscarinic M(2) receptor subtype is also suggested from the binding data.
Dynamic mechanisms of non-classical antagonism by competitive AT(1) receptor antagonists.
Selective competitive angiotensin AT(1) receptor antagonists exhibit diverse patterns of antagonism of angiotensin-II-mediated responses in functional assays. These range from the classical parallel rightward shift of agonist concentration-response curves with no depression of the maximum response to an apparently straightforward insurmountable antagonism with complete depression of the maximum response and no rightward shift. This article reviews some earlier equilibrium-based models that have been used to explain the insurmountable antagonism, and suggests that a kinetic model might provide a more satisfactory account of the observations. Such a model might provide deeper insights into the pharmacology of G-protein-coupled receptors than the more popular equilibrium models.
Beyond eyeballing: fitting models to experimental data.
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Upper eyelid island orbicularis oculi myocutaneous flap for periorbital reconstruction.
PURPOSE: To describe the upper eyelid island orbicularis oculi myocutaneous flap, medially or laterally based, for reconstruction of periorbital defects. METHODS: During the past three years we have used the island orbicularis oculi myocutaneous flap in 62 patients with tumors of the periorbital area, with the following indications: (a) anterior lamellar defects of the medial aspect of the upper eyelid, when the peripheral arcade is intact; (b) up to 2/3 anterior lamellar lower eyelid defects; (c) inner and outer canthus defects; and (d) defects of the peripalpebral area (the lateral half of the eyebrow, bridge of the nose and suprazygomatic areas). RESULTS: The flap proved to be flexible, safe, relatively simple, and provided good functional and aesthetic results. Complications were minimal. CONCLUSIONS: The upper eyelid island orbicularis oculi myocutaneous flap may be a useful tool for periorbital reconstruction.
The assessment of antagonist potency under conditions of transient response kinetics.
The muscarinic acetylcholine receptor antagonists, atropine and pirenzepine, produced an apparent insurmountable antagonism of muscarinic M(1) receptor-mediated intracellular Ca(2+) mobilization in Chinese hamster ovary (CHO) cells when tested against the agonists carbachol or xanomeline. Each antagonist caused a dextral shift of the agonist concentration-response curves with depression of the maximum response that was incomplete (i.e., saturated) and which varied with the pairs of agonist and antagonist. Equilibrium competition binding assays found no deviation from simple, reversible competitive behavior for either antagonist. The relative rates of dissociation of unlabeled atropine and pirenzepine were also assessed in radioligand kinetic studies and it was found that atropine dissociated from the receptor approximately 8-fold slower than pirenzepine. Numerical dynamic simulations suggested that the insurmountability of antagonism observed in the present study was probably a kinetic artifact related to the measurement of transient responses to a non-equilibrated agonist in the presence of a slowly dissociating antagonist. Importantly, the patterns of antagonism observed included a saturable depression of agonist maximal response, a mode of antagonism that is incompatible with the previously described phenomenon of hemi-equilibrium states. Monte Carlo simulations indicated that reasonable, semi-quantitative estimates of antagonist potency could be determined by a minor modification of standard methods, where equieffective agonist concentrations, rather than EC(50) values, are compared in the absence and presence of antagonist. Application of the latter approach to the functional data yielded estimates of antagonist potency that were in excellent agreement with those derived from the equilibrium binding assays, thus indicating that the present method can be useful for quantifying antagonist potency under non-equilibrium conditions.
Qualitative and quantitative assessment of relative agonist efficacy.
Historically, the ability of a ligand to bind to its receptor and the ability to subsequently activate that receptor have been described as the properties of affinity and intrinsic efficacy, respectively. These properties were originally believed to be independent of one another; both are possessed by ligands classed as "agonists," and they have served as the quantitative foundation of the drug and receptor classification process. Although affinity has been interpreted readily in physicochemical terms, equivalent molecular models for efficacy remain elusive. In recent times, there has been a significant paradigm shift in our understanding of the interrelationship between affinity and intrinsic efficacy, particularly on theoretical grounds, yet the actual methods available to measure these parameters remain largely operational. Nevertheless, a number of approaches, based on both functional measurements and radioligand binding studies, are available to quantify agonist efficacy on a relative scale and, to date, these remain the most practical. This commentary discusses the most common of these methods, their advantages and limitations, the dependence of the expression of agonism on the chosen assay system, and the impact of recent biochemical and molecular biological advances on the study of efficacy. Additionally, some of the more contemporary theories regarding the molecular nature of efficacy are briefly discussed, as well as the caveats that always must be borne in mind when any determinations of relative agonist efficacy are made.
Regulation of acetylcholine binding by ATP at the muscarinic M(1) receptor in intact CHO cells.
ATP may have a modulatory effect on cholinergic transmission, as it is known that ATP is released as a co-transmitter with acetylcholine from nerve terminals. The ability of ATP to influence the binding of acetylcholine to the M(1) muscarinic acetylcholine receptor expressed in intact CHO cells was investigated. In competition binding experiments, acetylcholine completely inhibited the binding of [3H]N-methylscopolamine, but yielded a shallow competition isotherm that was best described in terms of two affinity states. When these experiments were repeated in the presence of 1 mM ATP, the acetylcholine competition curve was better described in terms of a single, low-affinity state with a Hill slope not significantly different from unity. This modulatory effect of ATP was completely reversed by the addition of the P(2) purinoceptor antagonist, suramin, to the assay medium. When the competition between the muscarinic receptor antagonist, atropine, and [3H]N-methylscopolamine was investigated, however, ATP was unable to modulate the binding of atropine, which was consistent with a one-site binding model in each instance. In contrast to the intact cell studies, ATP did not affect either affinity state of acetylcholine binding when studied in homogenate preparations. The results of the present study indicate that ATP, acting via endogenously expressed purinoceptors, is able to influence agonist binding to the M(1) muscarinic acetylcholine receptor via a cross-talk that requires the functional integrity of intact CHO cells.
Characterization of the subtype selectivity of the allosteric modulator heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl) ammonium bromide (C7/3-phth) at cloned muscarinic acetylcholine receptors.
The present study investigated the interaction between the muscarinic acetylcholine receptor (mAChR) allosteric modulator heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl) ammonium bromide (C(7)/3-phth) and the orthosteric antagonist [3H]N-methylscopolamine ([3H]NMS) at the five cloned human mAChRs expressed in Chinese hamster ovary cells. Equilibrium binding studies, using two different concentrations of radioligand, showed the interaction between C(7)/3-phth and [3H]NMS to be characterized by different degrees of negative cooperativity, depending on the receptor subtype. The modulator exhibited the highest affinity (85 nM) for the unoccupied M2 receptor and the lowest affinity for the unoccupied M5 receptor, the latter being approximately 100-fold lower. In contrast, the highest degree of negative cooperativity was observed at the M5 receptor, whereas lowest negative cooperativity was found at the M1 and M4 receptors. Non-equilibrium dissociation kinetic studies also confirmed the allosteric properties of C(7)/3-phth at all five mAChRs and yielded independent estimates of the modulator affinity for the occupied receptor. The latter estimates showed good agreement with those calculated using parameter values determined from the equilibrium experiments. The present results extend previous findings that C(7)/3-phth is a potent allosteric modulator at mAChRs, particularly the M2 subtype, and also highlight the effects of cooperativity on apparent drug-receptor subtype selectivity.
The generation of nitric oxide by G protein-coupled receptors.
The highly reactive free radical gas, nitric oxide, serves a variety of biomodulatory functions and has been implicated in a growing array of physiological and pathophysiological states. The striking differences between this labile substance and other, more conventional, signaling molecules highlight the tight degree of nitric oxide regulation that is required in order to maintain appropriate cellular homeostasis. The generation of nitric oxide represents a common component of the signal transduction pathways of a number of chemical signaling molecules that act via binding to G protein-coupled receptors. This review focuses on the relationship between this receptor superfamily, the generation of nitric oxide via the actions of the nitric oxide synthases and some of the inter- and intracellular roles of nitric oxide.
Head and neck soft-tissue reconstruction using the vertical trapezius musculocutaneous flap.
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Upper eyelid orbicularis oculi flap with tarsoconjunctival island for reconstruction of full-thickness lower lid defects.
The reconstruction of full-thickness defects of the lower eyelid after tumor resection is a challenging operation. The great number of techniques for these defects shows that none of them is the operation of choice. The method of reconstruction will be chosen according to the extent of the defect, the missing components of the eyelid structure, the age of the patient, and the quantity and the quality of the surrounding tissues. In this article, we propose, under specific indications, the use of a pedicled myocutaneous orbicularis oculi flap with a tarsoconjunctival island from the upper eyelid to reconstruct full-thickness lower lid defects. The more choices in a surgeon's armamentarium, the more easily the difficult problems of periorbital surgical oncology will be resolved.