Techniques For Invitro Pharmacology Lab Report Biology Essay

Techniques For Invitro Pharmacology Lab Report Biology EssaySchild piece Schild eyepatch is defined as pharmacological method of receptor classification. By employ schild plot dose-effect reduce for an agonist is determined in the mien of various concentrations of a matched antagonist for its receptor in the presence of agonist i.e. equilibrium disassociation constant is calculated. The experiment is carried out for series of dose ratios for a given effect. For example the ratio of the dose of agonist (A) to provoke a specific effect (e.g.,half maximal effect) in the presence of the antagonist (B) to the dose required in the absence of the antagonist (A) is calculated. This is determined for some(prenominal)(prenominal) doses of antagonist and thusly log ((A/A) -1) versus the negative log B is plotted. If the regression of log ((A/A) -1) on -log B is linear with a slope of -1, then this indicates that the dislike is matched and by rendering the agonist and antagonist act at the same recognition sites. If the slope of the regression is not -1, then by definition the antagonist is not competitive or some early(a) condition is in effect. This might include multiple bind sites or pharmacokinetic inter doings. friend Agonist is a medicate which has both affinity and efficacy.Antagonist Antagonist is a drug which has affinity and zero efficacy.AffinityAffinity is a property of a drug it measures how tight a drug binds to a receptor. To bind to a receptor a functional group of the drug should bind to the complementary receptor. The dressing capacity of the drug defines the action of the drug.Efficacy Efficacy of a drug bed be defined as ability of drug which activates the receptor to bring about desired effect afterward blanket.Affinity and efficacy are explained in the equation asK+1 A + R AR* receptionK-1 K+1B + R BR No receiptK-1Where A is agonist, B is antagonist, K+1 is association range constant for binding, K-1is dissociation rate c onstant for binding- Association rate constant for activation- Dissociation rate constant for activationBy using law of smoke action affinity is explained asB + R BRDrug free receptor drug-receptor complexAt equilibrium KB = R B KB = chemical equilibrium dissociation constantBRHill-Langmuir equation this equation explains drug occupancyRT = R + BRIf RT = Total numerate of receptors then by substituting this in law of mass action equationRB = BRT KB + BBy this equation it is determined that drug occupancy (affinity) depends on drug concentration and equilibrium dissociation constant.Equilibrium dissosciation constant proportion DISSOCIATION CONSTANT (Kd) It is the characteristic property of the drug and the receptors. It is defined as the concentration of the drug required to occupy 50 % of the receptors. The higher the affinity of the drug for the receptors lower is the Kd value. Mathematically Kd is k2/k1 where k2 is the rate of dissociation of the drug from the receptor and k1 is the rate of association of the drug for the receptor.Receptor (R) and Drug (D) interact in a bilateral manner to form a drug-receptor (RD) complex.Where R = ReceptorD = Drug (L for ligand is sometimes used in these equations)k1 = the association rate constant and has the units of M-1min-1k2 = the dissociation rate constant and has the units of min-1.k2 is sometimes written as k-1.If an agonist binds to the receptor, then the interaction of the agonist (D) and the receptor (R) results in a conformational change in the receptor ahead(p) to a response.If an antagonist binds to the receptor, then the interaction of antagonist (D) and receptor (R) does not result in the appropriate conformation change in the receptor and a response does not occur.For drugs that follow the law of uncomplicated mass action the rate of formation of the complex can be defined by the bordering equationdRD/dt refers to the change in the concentration of RD with time (t).Note the square brackets refer to concentration.This equation indicates that the rate at which the drug receptor complex (RD) is make is proportional to the concentration of both free receptor (R) and free drug (D). The proportionality constant is k1.The rate of dissociation can be defined by the following equation-dRD/dt is the decrease in drug-receptor complex with timeThis equation indicates that the rate at which the drug-receptor complex (RD) dissociates back to free drug and free receptor is proportional to the concentration of the drug receptor complex. The proportionality constant is k2.When the drug and the receptor are initially mixed together, the amount of drug-receptor complex formed go out exceed the dissociation of the drug-receptor complex. If the reaction is allowed to go for a long enough, the amount of drug-receptor complex formed per unit time get out be equal to the number of dissociations of drug-receptor complex per unit of time, and the system will be at equilibrium. That is equilibrium has occurred. Equilibrium can be defined asor k1RD = k2RDThis equation can be rearranged to giveBy definitionKd is the dissociation equilibrium constant. Kd has units of concentration as shown in the following equation.Simple competitive hostility simple competitive antagonism is the most important type of the antagonism. In this type of antagonism the antagonist will compete with available agonist for same receptor site. decent antagonist will displace agonist resulting in lower frequency of receptor activation. Presence of antagonist shifts agonist log dose response curve to right. A schild plot for a competitive antagonist will have a slope equal to 1 and the X-intercept and Y-intercept will each equal thedissociation constantof the antagonist.This can be explained in equation asOccupancy for agonistRA = A OR A/ KART KA+ A A/ KA +1In presence of competitive antagonist (B)RA = A/ KART A/ KA + B/ KB + 1Occupancy reduced according to B and KBTo obtain same occupancy, must increa se A to Ar = A / A = B / BSchild equationr = B / KB +1Where r depends on B and KBApplying log on both sideslog (r-1) = logB log KBAim The main aim of the experiment is to measure the equilibrium dissociation constant (KB) for atropine at acetylcholine muscuranic receptors and to determine the drug receptor interactions.ObjectivesThe main objectives of the experiment are as followsTo measure the equilibrium dissociation constant for atropine at acetylcholine muscuranic receptorsTo represent the reversible competitive antagonism of atropine at acetylcholine muscuranic receptorsTo determine the equilibrium dissociation constant (KB) for atropine at acetylcholine muscuranic receptors by using schild plot.MethodIsolation and mounting of Guinea-pig ileum in harmonium toiletGuinea-pig was first sacrificed and then the ileum was collected and transferred into physiological salt solution maintained at 370C. The food particles present in the ileum was expelled out finished running Krebs solution through the lumen. Then tissue was tied with a thread at both the ends where oneness was tied to the mounting hook and the other was attached to the transducer.Preparation of serial dilutions of drugThe drugs used in the experiment were acetylcholine (Ach) and atropine. To determine the simple competitive antagonism of atropine at Ach muscuranic receptors serial dilutions of Ach were carried out. Ach was given as 110-2M and from the higher up concentration of the drug the following concentrations were prepared to the organ bath concentration such as 110-6M, 310-6M, 110-7M, 310-7M, 110-8M, 310-8M, 110-9M and 310-9M Ach. Then atropine was diluted to 110-8M (organ bath) from the given 110-2M concentration.Determination of harmonium bath concentrationThe volume of physiological salt solution (pss) was 20 ml, and each time the volume of drug introduced into organ bath was 20l. thence if 20l of 110-2M drug was introduced into the organ bath then it gives 110-5M organ bath conc entration.Mathematical calculation of organ bath concentrationIn organ bath we have 20ml of pss which is equal to 20103 l of pss, if 20 l of 110-2 M Ach was introduced then the organ bath concentration20lXM20ml10-2M= 20 l x 10-2 M20x 103 l= 110-5M (organ bath concentration).The isolated guinea- pig ileum was mounted onto the organ bath and set up for recording isometric tension of the tissue using chart software in a Mac book.Step-1Calibration of the experimental apparatus The chart 5 software was calibrated and the sampling rate was adjust to 10 samples per second with a maximum input voltage to 10 mV. The baseline was set to zero and then darkness was started from the baseline zero then the force transducer was calibrated by placing 1 gram weight and after the calibration the trace produced was stopped for the moment to convert the units of tension into grams by selecting the trace produced previously.Step-2Sensitisation of preparation To check the viability of the tissue a resp onse of suitable height was obtained by adding a little high concentration of the drug. Here in the experiment an appreciable recording was noted at 110-7M Ach.Step-3The time round of drinks followed to construct a concentration- response curve was0 seconds to add the drug concentrations30 seconds to empty the organ bath and refill with fresh physiological salt solution180 seconds next drug concentration was added to the organ bath. submerging Response CurveBy making use of the above drug concentrations a concentration response curve was constructed according to the provided time cycle.20 l of 110-9M Ach was added into the organ bath at zero seconds at is allowed to stand for 30 seconds, then after 30 seconds the organ bath was emptied and refilled with pss. Pss was allowed to stand for 180 seconds. During the wash full stop if the peak does not return to the base then it was washed twice or thrice to make sure that all the drug dissociates from the receptors before the next ad dition of the other drug concentration. Each concentration was repeated twice or thrice until the two consecutive responses were reported with the same peak height.By following the procedure and time cycle, the concentration response curve was constructed with different concentrations of acetyl choline such as 110-9M,310-9M, 110-8M, 310-8M, 110-7M, 310-7M,110-6M and 310-6M Ach (organ bath concentration).Step-4Equilibration of Acetylcholine receptors with acetylcholineAfter step-2 the preparation was washed several times until the peak returned to the base line. Then atropine (110-8M organ bath concentration) was added to the preparation and then set aside for 40 minutes to allow atropine to equilibrate with acetylcholine muscuranic receptors.Step-5Concentration response curve in the presence of atropineThe concentration response curve with acetylcholine was repeated again in the presence of atropine by following the time cycle and procedure, which was same as same step 2.Therefore i n step 3 with each addition of acetylcholine concentration atropine was added simultaneously.Step-6AnalysisThe graphical record pad prism in the Mac book was used to plot concentration response curves in the absence and presence of atropine.Log concentration (acetylcholine) Vs response in gramsFrom the above plot EC 50 determine of acetylcholine in the presence and absence of atropine were obtained. Then the distance between the two curves control and response for the atropine presence was denoted by r, where r was called as shift.The shift was calculated mathematically asr= EC 50 of response in the presence of atropineEC 50 of Ach in the absence of atropineFrom the value of the shift, schild plot was plotted as log concentration of atropine presence against log(r-1).From the schild plot the dissociation constant KB for atropine at acetylcholine muscuranic receptors was determined.ResultsAs explained above in the procedure serial dilutions of acetylcholine was added to the organ ba th, where Ach has produced concentration dependent contractions of the guinea pig ileum as shown in the fig 1.Figure 1 fantasm display contractions produced by serial dilutions of acetylcholine at muscuranic receptors.As shown in Figure 1 the serial dilutions of acetylcholine are added into the organ bath from 110-7M to 310-6M Ach. Here in the trace it was clearly shown that contractions produced by the acetylcholine have been increased with respect to the concentrations.In step-2 the preparation was washed and added with 110-8M atropine and set aside for 40 minutes to equilibrate the acetylcholine receptors.Figure 2 Trace showing contractions produced by serial dilutions of acetylcholine at muscuranic receptors in the presence of atropine.In the trace it is clearly shown that, the contractions produced by serial dilutions of Ach from 110-8M to 310-4M in the presence of 110-8M atropine.When Trace 1 and Trace 2 are compared it is evident that the contractions produced by Ach unacc ompanied (trace 1) were greater than the contractions produced Ach in the presence of atropine (trace 2) which proves the simple competitive antagonism by atropine at muscuranic receptors.A graph is plotted to the log concentration response curve produced by Ach alone against Ach in the presence of atropine. (graph is attatched to the report)From the graph it is known that with the increase in the concentration of Ach, response have been increased when compared to Ach in the presence of atropine and also there is a shift towards right which shows the simple competitive antagonism produced by atropine.From the results produced by Ach alone against Ach in the presence of atropine the fractional difference which is called as shift can be obtained as followsMathematical Calculation shift r= EC50 of response after atropine (or) in the presence of atropineEC50 of control (or) Ach in the absence of atropine= 2.5110-6 = 8.363.0 x10-7r-1 =8.36 -1=7.36log(r-1)=log (7.36) =0.86Partial dissocia tion constant (PKB) or PA2 is measured to confirm the simple competitive antagonism, where pKB values play an important case in classifying receptors.Therefore PKB =log(r-1) -log atropine=0.86 -log (110-8)=0.86 (-8)=0.86+ 8=8.86From the above results log EC50 values for control (Ach alone) and Ach in the presence of atropine were given as 3.0e-007 and 2.51e-006 respectively.This shows the hoagie concentration of Ach which produces 50% of the maximal possible response is higher than the molar concentration response produced by Ach in the presence of atropine.Figure 5 (Graph2) Schild plotIf the antagonist is competitive, the dose ratio equals one plus the ratio of the concentration of antagonist divided by its Kd for the receptor. (The dissociation constant of the antagonist is sometimes called Kb and sometimes called Kd)A simple rearrangement givesHere we have plotted a graph with log (antagonist) on the X-axis and log (dose ratio -1) on the Y-axis. If the antagonist has shown sim ple competitive antagonism then the slope should be 1.0, X-intercept and Y-intercept values should be both equal the Kd of the antagonist obtained.If the agonist and antagonist are competitive, the Schild plot will have a slope of 1.0 and the X intercept will equal the logarithm of the Kd of the antagonist. If the X-axis of a Schild plot is plotted as log(molar), then minus one times the intercept is called the pA2 (p for logarithm, like pH A for antagonist 2 for the dose ratio when the concentration of antagonist equals the pA2). The pA2 (derived from functional experiments) will equal the Kd from binding experiments if antagonist and agonist compete for binding to a single class of receptor sites.Figure 6 (table 2) Results for Schild Plot.From Figure 5 and 6 it is evident that no concentrations of atropine have showed competitive antagonism perfectly. Therefore from the above results it is known that the concentrations of atropine has not shown simple competitive antagonism fairly .DiscussionReversible competitive antagonism The binding of drug to a receptor is fully reversible which produces a parallel shift of the dose response curve to the right in the presence of an antagonist.The tool of action of acetylcholine at muscuranic receptorsIn various gastrointestinal smooth sinews, acetylcholine and its derivatives produce contractions by activating muscuranic receptors. It is generally assumed that the M3 muscuranic receptor plays a key role in mediating this activity. The M3 receptor is coupled preferentially to Gq-type G proteins, resulting in the activation of phospholipase C (PLC) and the formation of ionositiol trisphosphate (IP3) and diacylglycerol (DAG) which are likely to participate in muscuranic receptor-mediated smooth muscle contractions. IP3 causes Ca2+ release from intracellular store and can also mobilize Ca2+ secondarily through Ca2+-sensitive or store-dependent mechanisms. DAG, via activation of protein kinase C, phosphorylates various prot eins and can directly activate non selective cationic channels.Figure 7 Diagrammatic representation of calcium and smooth muscle contraction.From the above results the value of shift obtained was 0.378 which denotes the simple competitive antagonism produced by the concentration of atropine used (110-8 M).From the value of shift the pKB value was calculated as 8.4.If atropine has shown simple competitive antagonism then the value of pKB should be equal to 1-X intercept.Therefore pKB=1-X intercept=1-(-8.86)=9.86We got value of pKB as 8.86.Therefore pKB is not equal to 1-X intercept.Therefore the concentration of atropine (110-8M organ bath concentration) used by our group has not shown simple competitive antagonism effectively. The literature value of pKB is given as approximately 9 and we have obtained the value of pKB as 8.86 which does not fit with literature value.Therefore from the above observations and results i can conclude that a little more high concentration of atropine ma y serve to produce complete simple competitive antagonism by atropine at acetylcholine muscuranic receptors.