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[3H]-3-quinuclidinyl benzilate ([3H]-QNB) binds with high affinity and specificity to the receptors present in the homogenate of rat brain membrane. These receptors exhibit resemblance to muscarinic cholinergic receptors. Specific binding of QNB is saturable and depends on the concentration of the membrane homogenate and the time of incubation. The saturability and specificity of [3H]-QNB binding to receptors present in the rat membrane is demonstrated. There is concentration dependent displacement of the radiolabeled QNB from their binding sites in the presence of atropine. In this study, IC50, Bmax and Kd are calculated and compared with those published by Yamamura et al.
Keywords: binding affinity, dissociation constant, radioligand binding assay, [3H]-QNB, cholinergic receptors, quantification, QNB binding sites, IC50, BSA.
Neurons communicate with each other by releasing neurotransmitters. Binding of these neurotransmitters to specific receptors on adjacent neurons, regulate the expression of number of receptors at the cell surface. The binding of neurotransmitter to receptors plays a very crucial role and attempts have been made to quantify them by chemical measurements of direct binding.
Receptors for acetylcholine are present in many tissues and can be classified as muscarinic or nicotinic cholinergic receptors respectively. Substances which bind to the receptor and elicit a response are known as agonists whereas substances which bind but do not elicit a response are known as antagonists. The interaction between an agonist or an antagonist can be quantified in ligand binding assays.
Ligand binding studies of muscarinic [1-2] as well as nicotinic [3-5] cholinergic receptors have been reported in literature. Muscarinic antagonists have 3 to 5 times more affinity for QNB binding sites than do cholinergic agonists. The binding of [3H]-QNB appears closely similar to the binding of a benzilylcholine mustard homologue , and may resemble [3H]-atropine binding to a rat brain homogenate . Albanus  and Meyerhoffer  independently reported that QNB is a potent central muscarinic antagonist.
In the present investigation, radioactive [3H]-QNB was taken for receptor labelling studies as reported by Yamamura et al., due to its potency, specificity and persistence of action. In this report, we describe a quantitative approach for in-vitro measurement of receptor densities based on equilibrium distribution of radiolabeled ligand. The ultimate purpose of this experiment is to identify maximal receptor density (Bmax) and dissociation constant (Kd). Additional experiments indicating IC50 were also performed.
Materials and Methods
Sodium potassium phosphate (NaPK) 50mM, pH 7.4; [3H]-QNB (1.3nM and 6.5nM), specific activity 11.2 x 102 Bq /pmol; Atropine solution (10Âµm); Sucrose (0.32M); Phenylmethylsulphonyl fluoride (PMSF) (0.1mM); standard solution of bovine serum albumin (BSA) 1mg/ml.
Rat brains were homogenized in 10 volumes of ice-cold 0.32M sucrose /0.1mM PMSF with a Teflon-glass potter-Elvehjem glass homogeniser. The whole homogenate was centrifuged at 12000g for 10 minutes and the pellet was resuspended in original volume of sucrose and frozen in aliquots.
QNB was tritiated and purified by the standard procedure reported by Yamamura et al .
Lowry Assay for protein estimation
50Âµl of rat membrane homogenate was taken in a test tube and the volume was made upto 1.0ml with water. Various concentrations of BSA (in the range of 0-200Âµg) were prepared from the standard BSA solution. 1.5ml of reagent 1 [0.5ml copper tartarate (0.1g CuSO4.5H2O + 0.2g sodium potassium tartarate in 20ml water) and 50ml alkaline carbonate (2g NaOH in 20ml water + 10g Na2CO3 in 100ml water)] were added to the above test tubes containing BSA and rat membrane homogenate and allowed to stand for 10min at room temperature. Further, 0.3ml of reagent 2 (commercial Folin-ciocalteau reagent, 1:1 in water) was added and left for 30min with occasional swirling. The absorbance for all the samples was recorded at 660nm using UV-Visible spectrophotometer. The data were plotted from the standard BSA tubes and the protein concentration in the membrane was extrapolated.
In the present investigation, all assays have a final volume of 2.0ml, made up of 1.5ml [3H]-QNB assay mix and 0.3ml of either water or atropine.
Saturation binding studies of [3H]-QNB
Binding of [3H]-QNB to brain homogenates was determined as described by Yamamura et al, with slight modifications. In-vitro QNB binding to homogenates of rat brain was measured with a filtration assay. Incubation time for binding of [3H]-QNB was determined by incubating 4ml of rat membrane to 1.3nM [3H]-QNB mix and measuring radioactivity at various time intervals as shown in supplementary information (sheet 1 of MS Excel speadsheet), by removing 2.0ml aliquots to glass filter fibres (GF/B) positioned over a vacuum. Incubations were terminated by increasing the volume using 20ml NaPK, followed by rapid vacuum filtration and subsequent washing with 5ml NaPK. Every determination of binding was performed in triplicate. Filters were placed in scintillation vials containing 5ml of scintillation cocktail for atleast one hour at room temperature. Total radioactivity on the filters were counted by liquid scintillation spectrometry using a Beckman LS 9000 scintillation counter with external quench correction at a counting efficiency of 50% in the dps. The correction factor was introduced as the "counts" may not have registered all the disintegrations in the sample (the flashes of light in the scintillant may be quenched by colour in the solution or other artifacts). These values of radioactivity were plotted against time in the saturation time kinetics graph (Figure 1).
Determination of IC50 and Ki
IC50 is that atropine concentration (competing drug) which displaces 50% of [3H]-QNB binding. It was determined by incubating 200Âµl rat membrane preparation with various concentrations of atropine obtained by spiking solutions (serial dilution) of atropine as shown in supplementary information (sheet 2 of MS Excel speadsheet) in the presence of saturating (1.3nM) concentration of [3H]-QNB for 45min in triplicate. This incubation period was chosen since kinetic experiments shows that saturation is reached after 45min. The incubation was terminated by vacuum filtration over GF/B filters, followed by washing. The radioactivity was determined as described above. The average radioactivity bound to each triplicate set of filters was calculated in nanomoles or picomoles of QNB bound. Further, Lowry assay was performed to calculate the amount of bound QNB in femtomoles per mg of the protein. These values were plotted against log10 [atropine] and to determine the IC50 from the mid-point of the curve.
The experimentally determined IC50 was then used to calculate the affinity constant Ki of the atropine using the Cheng-Prusoff equation, Ki = IC50 /(1 + [L*] /Kd), where Ki is the apparent equilibrium affinity constant of atropine for QNB binding site, Kd is the equilibrium dissociation constant of QNB for the binding site and [L*] is the concentration of the radioactive QNB used.
Determination of Bmax and Kd for [3H]-QNB
To assay the total binding (Bmax) of [3H]-QNB, 200Âµl rat membrane preparation was incubated in triplicate with varying concentrations of [3H]-QNB supplementary information (sheet 3 of MS Excel spreadsheet). The incubation was terminated by filtration followed by washing. The radioactivity was determined as described above. [3H]-QNB can bind only non-specifically in the presence of atropine as all the receptors are occupied by the atropine. Therefore, non-specific binding of [3H]-QNB were determined in the presence of atropine (10Âµm). Linear regression was carried out to calculate corrected non-specific binding. Non-specific binding is proportional to the concentration of QNB (within the range it is used). The specific binding of [3H]-QNB is then calculated by taking the difference between the total binding and the corrected non-specific values.
Data analysis to calculate Kd and Bmax
The affinity (Kd) and maximal density (Bmax) of receptors in a sample were determined by saturation studies. In this, increasing concentrations of QNB were used and the bound (B) and free (F) ligand concentrations were measured at the time when the system is assumed to have reached an equilibrium state. Using the equation, B /F = (Bmax - B) /Kd, a scatter graph was plotted between B/F (Y-axis) and B (X-axis) and a straight line was fit. The slope of this straight line is equal to the negative inverse of the equilibrium dissociation constant (Kd) and whose intercept on X axis when Y is equal to zero gives the total receptor concentration (Bmax).
In order to overcome the limitation of scatchard plots in giving the most accurate analysis of Bmax and Kd because of the distortion of experimental errorby linear transformation, non-linear regression method was employed.
An algorithm "Solver" in Microsoft excel was used to optimise the parameters in order to minimize the sum of the squares of deviations of estimated and observed values of bound QNB. This was achieved by changing the values of previously calculated Bmax and Kd. This resulted in the reduced estimated values of bound which were then plotted to evaluate the fit along with the observed bound values.
Further, to calculate the standard deviation of Pt (Bmax) and Kd, various parameters, Fio, FPt, FKd, FL, FPL and Wi were calculated using the formulae mentioned in the supplementary information (MS Excel spreadsheet). Then, the matrix and the inverse matrix of the above mentioned parameters were created to calculate the Î”Pt and Î”Kd; and Ïƒ2Pt and Ïƒ2Kd respectively.
Radioactivity contamination check
Six different samples from the work bench, gloves and other items that might have come in contact with hot [3H]-QNB were taken using swabs for checking any radioactive contamination. To do so, each swab was put into a separate vial containing 5ml of scintillant, and its radioactivity was determined as describes above.
Results and Discussion
Incubation time for binding of [3H]-QNB to rat membrane homogenate was found to be 45 min using the saturation binding studies (Figure 1). In further experiments, rat membrane homogenate was incubated with [3H]-QNB for 45 min.
IC50 for atropine was determined by incubating atropine in the presence of saturating concentrations of [3H]-QNB. By plotting the graph between bound concentrations of [3H]-QNB and -log [Atropine], IC50 of atropine was found to be 1.778 nM (Figure 2). This is similar (1-2nM) to the one reported by Yamamura et al .
To differentiate between the specific and non-specific binding sites, a radioactive muscarinic antagonist QNB with high affinity and selectivity for binding site was incubated with a known quantity of rat membrane homogenate in vitro in the absence and presence of a concentration of atropine that will displace QNB from the receptors. Since both QNB and atropine are competing for the same receptor site, as the concentration of atropine increase the amount of bound QNB decreases.
In binding study with [3H]-QNB alone total binding was observed. And in the presence of 10ÂµM atropine, binding was reduced as shown in Figure 3. Results from incubation in the presence of atropine suggest that atropine displaces the QNB from its binding sites. At t = 0, more sites are available on the receptor to bind to the ligand but near saturation the number of free sites availability decreases. Specific binding is saturable with increasing concentrations of [3H]-QNB. Non-specific binding (weak interactions because of hydrophobic interactions) of QNB in the presence of atropine is not saturable and increases linearly with increasing concentration of [3H]-QNB (Figure 3). The displacement of QNB from its receptors by atropine in this study corroborated the fact that QNB binds to receptors that are similar to muscarinic cholinergic receptors (explained in Appendix 3).
Using the straight line that fits the results in the presence of atropine, the amount of non-specific binding for each QNB concentration was estimated (Figure 3). The specific binding was calculated as described in Materials and Methods section. The concentration of free QNB was calculated by taking specific bound QNB from the total concentration of QNB (supplementary information, sheet 3 of MS Excel speadsheet).
A binding curve is not a linear equation but a rectangular hyperbola. In rectangular hyperbola, it is difficult to get a saturation value. So, the data from this experiment was further analysed using scatchard plot (Figure 4). From this analysis, Kd and Bmax were calculated as 0.609nM and 0.259nM respectively. These values were used to calculate the estimated bound using the formula B = Bmax [F] /(Kd +[F]). The graph of bound vs free was plotted for both the observed and estimated values (Figure 5).
To overcome the limitation of scatchard plot non-linear regression analysis was performed. Use of "solver" algorithm in Microsoft Excel minimized the sum of the deviations which changes the Kd and Bmax value as 0.612 nM and 0.257 nM. The value reported by Yamamura et al for Kd is in the range of 0.4 -0.5 nM. Then with the help of matrix we solved for the change of our estimate of Kd and Pt, Î”Kd and Î”Pt. Finally, the standard deviation of the parameters which are square root of element (1,1) and (2,2) of the inverse matrix were calculated.
Further, a new Kd and Pt was calculated with the Î”Kd and Î”Pt values using the formula Î”Kd = (Kdo - Kd) and Î”Pt = (Pto - Pt), where Kdo is the initial estimate of Kd and Pto is the initial estimate of Pt (supplementary information, sheet 3 of MS Excel speadsheet). These are the most important results because compared to other techniques of curve fitting they gave us the estimates of variances.
In conclusion, a radioligand binding assay has been demonstrated. The method might be suitable for high throughput screening of drug interaction with muscarinic cholinergic receptors.
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Figure 2: The graph between concentration of QNB (nmol) and -Log[Atropine] to calculate the IC50 of atropine
Figure 1: The graph showing the saturation kinetics to calculate the incubation time
Figure 3: Graph showing raw counts per minute (CPM) bound against concentration of QNB. A straight line is fitted through the atropine values to obtain an equation that can be used to convert calculated non-specific values to corrected non-specific values.
Figure 4: Scatchard plot between bound/free and free [3H]-QNB. A straight line is fitted to calculate Bmax and Kd using the equation mentioned.
Figure 5: Graph of bound vs free for both the observed and estimated values of bound QNB.
3-quinuclidinyl benzilate (QNB)
QNB is an anticholinergic compound structurally similar to atropine. It acts as a competitive inhibitor of the neurotransmitter acetylcholine at postsynaptic and postjunctional muscarinic receptors in cardiac and smooth muscle, exocrine glands, autonomic ganglia, and the brain. It decreases the effective concentration of acetylcholine at these receptors sites as the proportion of receptors available for binding to acetylcholine decreases.
Atropine is a monocyclic tropane alkaloid which is found in plants of the Solanaceae family like deadly nightshade (Atropa belladonna), jimsonweed (Datura stramonium), mandrake (Mandragora officinarum). It is a secondary metabolite of these plants and is a competitive antagonist for the muscarinic acetylcholine receptor.
Atropine increases firing of the sinoatrial node and conduction through the atrioventricular node of the heart, opposes the actions of the vagus nerve, blocks acetylcholine receptor sites, and decreases bronchial secretions.