Cholinergic relates to the responses in various systems to the neuro-transmitter molecule Acetycholine (ACh). Â They are the protein that are permanently attached to theÂ biological membrane or the integral membrane proteinÂ (IMP). If the set of response is seen where Ach is a normal transmitter it is seen that they are grouped based on nicotinic acetylcholine receptors (nAChR) that respond to nicotine, and muscarinic acetylcholine receptors (mAChR) that bind muscarine. These Nicotine and muscarine are extrinsic molecules that get the same response but with different sensitivity. Drugs that bind to muscarinic receptors are classified based on
1) Agonists (which activate the neuronal receptor and produce a response)
2) Antagonists (which do not activate the receptor and block the agonist binding site)
Antagonists are now used to study the drug-receptor binding as they bind with a higher affinity (i.e lower dissociation constant kd) when compared with agonists
Pharmacology studies have shown that antagonists have higher affinity but no efficacy to their cognate receptors. They intervene their effect by going and binding to the active site or to allosteric sites on the receptor. They can also go and bind to unique binding sites that do not participate in biological regulation of any receptor activity. The activity that antagonist causes may be reversible or irreversible, depending on the long life of the antagonist-receptor complex.
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Studies have shown that 3-Quinuclinidyl benzilate (QNB) is a potent muscarinic antagonist in CNS (central nervous system) and peripheral tissues. QNB shows specific binding to the receptor of interest it binds. It can also bind to other sites of the membrane and these can cause changes. We can measure specific binding by filtering radioactive 3H-QNB and then measuring the amount of QNB. To measure non-specific binding, Atropine is used to displace QNB from the specific sites, while the non-specifically bound QNB remains and can be quantified by measuring radioactivity.
(Source:Yamamura et al. May 1974)
Overview of the experiment
QNB is carried out in a radioactive binding assay where the concentration of QNB that is specific bound without atropine and QNB that is non-specifically bound with atropine is measured over successive interval of time. It is allowed to incubate so as for binding site to reach saturation is allowed where the equilibrium is reached. After this any further increase incubation time does not cause the amount of QNB bound to change. This QNB bound to the membrane is measured. By calculating the incubation time, IC50 of atropine is measured by measuring the atropine at which 50% of bound QNB is displaced. Amount of free QNB when 50% of bound QNB is displaced is used to measure the dissociation constant (Kd).
Materials and Methods
Determination of QNB specific and non-specific binding
Two bulk assays was carried out
To measure QNB binding (in the presence of water)
To measure non specific binding (with the presence of atropine)
There were two conical flask taken A and B. Tube A was added with 30 ml of 1.3 nM 3H-QNB and 6ml water. And to the flask B flask B, 30 ml 3H-QNB and 6ml atropine was added. S filter tower is then set with 6 GF/C filters and 4.0 ml of rat membrane was added to each flask and the flask were swirled to mix well. 2ml aliquots from A flask (A1, A2, A3) and (B1, B2, B3) from the B flask were produced and were run through fresh GF/C filters. Each of the filters was then washed to remove mini-vials, and then 5 ml scintillant was added and was left for at least an hour. After a hour the radioactivity was counted in the scintilliant counter. This protocol was repeated for a couple of more time to produce triplicates at the time interval of 10, 20, 30, 45 and 60 min.
Determination of IC50 for atropine
Five glass test tubes having 1200 Î¼l of distilled water in each was taken. To the test tube 1, 300 Î¼l of 10 10 Î¼M atropine was added and was mixed well. 300 Î¼l of the solution was added to tube 2 and mixed well. The same method is carried out for a series of dilutions to be done in tube 3 to 5. Atropine concentration in each tube is calculated.
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Seven triplicate tubes (A1, A2, A3â€¦G1, G2, G3) are made each containing 1500 Î¼l of 1.3nM QNB assay and the tubes are mixed well. 300 Î¼l of 10 Î¼M atropine was added to the three tubes of A and three B tubes were added with 300 Î¼l of solution from tube 1. The dilution process was carried out for tubes C, D, E, F from tube 2, tube 3, tube 4 and tube 5 respectively. To tubes G, 300 Î¼l of distilled water was added instead. 200 Î¼l membrane was then added quickly to all the tubes. The 21 tubes were then left for incubation for 45 min and the radioactivity was then measured.
Determination of concentration of protein using Lowry Assay
Test tubes were prepared that contained 0, 50, 100, 150 and 200 Î¼g BSA (Bovine serum albumin) made up to 1 ml with water. A 6th tube was taken that had 50 Î¼l of membrane that was made up to 1ml with water. 1.5ml of reagent 1 that contains 0.5 ml copper tartrate + 50ml alkaline carbonate was added and mixed well and let to stand for 10 min at room temperature. Then 0.3 ml of reagent 2 that contains Commercial Folin-Ciocalteau reagent was added to the tubes and mixed well. The tubes were then left for incubation for 30 min. Absorbance or optical density was read at 660nm.
Determination of kd for QNB
Eight test tube was taken, four containing low QNB concentration (1.3nM QNB mix) and four tubes containing high QNB concentration (6.5nM QNB mix). Tubes 1 to 4 were added with 7.50 ml, 2.50 ml, 5 ml and 3.2 ml of 6.5 nM QNB mix respectively. Lower concentration of QNB is made by diluting the standard QNB assay mix with NaKP solution. These tubes are labelled 1-8. The solution of tube 1-8, of about 1500 Î¼l each was added to the triplicate tubes (A1, A2, A3, ...H1, H2, H3) respectively. Solution of tube 1 is added to tubes A, Tube 2 to B tubes till tube 8 to tubes H. 300 Î¼l water + 200 Î¼l membrane was then added to all tubes. For tubes A4-H4, 300 Î¼l Atropine plus (Tube 1-8) respectively plus 200 Î¼l membranes was added. Radioactivity was measured in all tube. A lowry assay was also carried out.
RESULT AND DISCCUSION
Figure 1: graph plotting cpm bound vs time
Here in the graph the values are plotted for QNB bound with atropine (with as show in the graph), QNB bound without atropine (Without as shown in the graph) and Corrected vales are obtained by subtracting QNB bound with atropine from the QNB bound without atropine (corrected as shown in the graph ) against time. Here QNB bound without atropine is total amount of QNB bound to the receptor; QNB bound with atropine is the Non-specific binding of QNB to the receptor and corrected is the specific binding of QNB to the receptor.
After a particular time of incubation receptors reach equilibrium, where no more binding of QNB takes place to the binding sites. At this point when no more binding of QNB takes place the plateau is formed in the graph showing saturation. This incubation time is approximately "45 min" as shown by the graph reaching the plateau.
The graph shows us that with and corrected points of the graph forms a plateau after reaching incubation time of approximately 45 min. If an addition incubation time was taken after 60 min we would have got a plateau for without graph also showing us a plateau.
The graph shows that the cmp value increase over time after which when reaching a particular time no more binding occurs thus forming a plateau showing the saturation or equilibrium has reached. Small decline in the graph can be seen at time 30 to 45 min, this could have been due to experimental errors. The errors could have been caused during pipetting, in proper vacuum, formation of bubbles, adding samples properly between time intervals etc. This can be avoided by more careful handling of the instrument and doing a initial check up for errors so as to not cause changes in the experiment's result.
Taking the above data into consideration we have chosen 45 min as incubation time for determining IC50 of atropine. This is because, saturation of binding sites is achieved and no further unbinding of QNB also occurs, as the 'off-rate' or reaction constant of QNB unbinding is very low. So there is no further change in the amount of bound QNB and hence this incubation time is considered appropriate.
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Figure 2: graph showing QNB bound Vs Log [atropine] conc.
By serial dilution different concentration of atropine was prepared. The graph shows us that the amount to QNB bound to the receptor of the membrane reduces with increase in concentration. This happens because atropine is a competitive binder and binds competitively with specific sites to the receptor. The amount of QNB specifically bound will be inversely proportional with atropine concentration.
Half maximal inhibitory concentration (IC50)Â is a measure of how effective a compound is in inhibiting biological or biochemical function. This is a quantitative measure that let us know how much concentration of the drug or biological substance (inhibitor) is required to inhibit a given biological process by half. So we are calculating the IC50 of atropine to determine its potency. It is calculated by taking atropine concentration at which 50% QNB is displaced. The IC 50 value was found to be 0.0008912 Î¼M. This shows that atropine is a drug with good potency. Ic 50 does not directly discuss the binding constant so we cannot compare the binding affinity of QNB and receptor.
Figure3: Lowry's assay is used to determine concentration of membrane protein.
Lowry's assay was carried out for determining the concentration of membrane protein. First different concentration of BSA was used and we generated a graph for it, taking concentration and OD. The membrane protein was then checked for absorbance and was found to be 0.322. Using the linear regression equation and the absorbance, concentration of the membrane protein was found to be 0.803 mg/ml.
Figure4: Lowry's assay is used to determine concentration of membrane protein.
This test was done for another membrane protein sample. The absorbance of the membrane was 0.27. Again using the regression equation and the absorbance, concentration of the membrane protein was found to be 0.293529412 mg/ml.
Determination of Kd:
Kd is -1/m and was the equation was used is y = -8499.6x - 1.3669. the kd is used to define the affinity between the drug and the protein . the value of Bmax was 0.001161 nm.