Techniques For Invitro Pharmacology Lab Report Biology Essay

Techniques For Invitro Pharmacology Lab Report Biology assaySchild fleck Schild plot is be as pharmacological method of sensory sensory sense organ classification. By using schild plot treat-effect booze-up for an agonist is determined in the social movement of various densitys of a warlike obstructor for its receptor in the nominal head of agonist i.e. equilibrium dis give birth perpetual is cipher. The experimentation is carried prohibited for series of dose ratios for a condition effect. For example the ratio of the dose of agonist (A) to produce a specific effect (e.g.,half supreme effect) in the heading of the antagonist (B) to the dose required in the absence seizure of the antagonist (A) is measured. This is determined for several doses of antagonist and because log ((A/A) -1) versus the negatively charged log B is plotted. If the regression of log ((A/A) -1) on -log B is elongated with a toss of -1, thusly this indicates that the opposition is w arring and by description the agonist and antagonist act at the same recognition sites. If the slope of the regression is non -1, hence by definition the antagonist is non competitive or some other condition is in effect. This energy include multiple covering sites or pharmacokinetic inter doings.Agonist Agonist is a medicate which has both chemical attraction and qualification.Antagonist Antagonist is a dose which has affinity and zero efficacy.AffinityAffinity is a property of a do doses it mea authentics how tight a medicate binds to a receptor. To bind to a receptor a functional assemblage of the drug should bind to the antonymous receptor. The binding capacity of the drug defines the action of the drug.Efficacy Efficacy of a drug mint be defined as ability of drug which activates the receptor to produce desired effect after(prenominal) binding.Affinity and efficacy ar explained in the equation asK+1 A + R AR* ResponseK-1 K+1B + R BR No ResponseK-1Where A is ag onist, B is antagonist, K+1 is association arrange continual for binding, K-1is disassociation rate invariable for binding- Association rate constant for activation- Dissociation rate constant for activationBy using law of mass action affinity is explained asB + R BRDrug complete receptor drug-receptor coordination compoundAt equilibrium KB = R B KB = Equilibrium dissociation constantBRHill-Langmuir equation this equation explains drug line of workRT = R + BRIf RT = Total tot 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 assiduousness and equilibrium dissociation constant.Equilibrium dissosciation constant EQUILIBRIUM DISSOCIATION CONSTANT (Kd) It is the characteristic property of the drug and the receptors. It is defined as the parsimony of the drug required to occupy 50 % of the receptors. The higher(prenominal) the affinity of the drug for the receptor s reduce is the Kd value. numerally 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) move in a reversible manner to form a drug-receptor (RD) abstruse.Where R = ReceptorD = Drug (L for ligand is some sentences used in these equations)k1 = the association rate constant and has the building blocks of M-1min-1k2 = the dissociation rate constant and has the social units of min-1.k2 is some measure scripted as k-1.If an agonist binds to the receptor, then the interaction of the agonist (D) and the receptor (R) results in a con make-upal change in the receptor leading to a solution.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 receipt does not occur.For drugs that follow the law of guileless mass action the rate of formation of the comple x can be defined by the undermenti wizardd equationdRD/dt refers to the change in the concentration of RD with epoch (t).Note the straight brackets refer to concentration.This equation indicates that the rate at which the drug receptor complex (RD) is formed is proportional to the concentration of both complete 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 blend together, the amount of drug-receptor complex formed pass on exceed the dissociation of the drug-receptor complex. If the reception is allowed to go for a long enough, the amount of drug-receptor complex form ed per unit time will be peer 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 hatred simple competitive detestation is the most important type of the hostility. In this type of antagonism the antagonist will compete with available agonist for same receptor site. Sufficient antagonist will displace agonist resulting in lower frequency of receptor activation. Presence of antagonist shifts agonist log dose resolution carouse to discipline. A schild plot for a competitive antagonist will study a slope personify to 1 and the X-intercept and Y-intercept will each play off thedissociation constantof the antagonist.This can be explained in equation asOccupancy f or agonistRA = A OR A/ KART KA+ A A/ KA +1In presence of competitive antagonist (B)RA = A/ KART A/ KA + B/ KB + 1Occupancy cut down according to B and KBTo obtain same occupancy, must increase 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 KB heraldic bearing 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 install 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 upgrade of Guinea-pig ileum in organ bathtubGuinea-pig was f irst sacrificed and then the ileum was collected and transferred into physiologic table salt solution maintained at 370C. The food particles present in the ileum was expelled out done running Krebs solution through the lumen. Then thread was tied with a thread at both the ends where angiotensin-converting enzyme was tied to the mounting hook and the other was attached to the transducer.Preparation of accompanying 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 devoted as 110-2M and from the higher up concentration of the drug the following concentrations were prepared to the organ bath concentration much(prenominal) 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 Organ bath concentrationThe volume of physiological salt solution (pss) was 20 ml, and each time the volume of drug introduced into organ bath was 20l.Therefore if 20l of 110-2M drug was introduced into the organ bath then it gives 110-5M organ bath concentration.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 attach onto the organ bath and set up for recording isometric tenseness of the tissue using chart software in a mac book.Step-1Calibration of the experimental apparatus The chart 5 software was calibrate and the sampling rate was adjusted to 10 samples per second with a level best input voltage to 10 mV. The abodeline was set to zero and then trace 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 halt for the moment to convert the units of tension into grams by selecting the trace produced previously.Step-2Sensitisation of education To check the viability of the tissue a response 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 cycle followed to lay down 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. immersion 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 base for 30 seconds, then after 30 seconds the organ bath was emptied and refille d with pss. Pss was allowed to stand for 180 seconds. During the wash period if the peak does not return to the base then it was process twice or thrice to make sure that all the drug dissociates from the receptors before the next addition of the other drug concentration. Each concentration was repeated twice or thrice until the cardinal consecutive responses were reported with the same peak height.By following the operation 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 measure-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.S tep-5 assimilation 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 mapping, which was same as same step 2.Therefore in step 3 with each addition of acetylcholine concentration atropine was added simultaneously.Step-6AnalysisThe graphical record plod 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 values of acetylcholine in the presence and absence of atropine were obtained. Then the distance between the two curves incorporate 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 atropin e 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 bath, where Ach has produced concentration dependent contractions of the guinea pig ileum as shown in the fig 1.Figure 1 Trace showing 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 change magnitude 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 atr opine.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 alone (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 a s 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) =0.86 overtone dissociation constant (PKB) or PA2 is measured to confirm the simple competitive antagonism, where pKB values play an important role 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 molar 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 dissociat ion 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 simple 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 wiz class of receptor sites.Figure 6 (table 2) Results for Schild Plot.From F igure 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.Discussion reversible competitive antagonism The binding of drug to a receptor is to the full reversible which produces a parallel shift of the dose response curve to the right in the presence of an antagonist.The mechanism of action of acetylcholine at muscuranic receptorsIn various gastrointestinal brush up muscles, acetylcholine and its derivatives produce contractions by activating muscuranic receptors. It is for the most part 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 mus curanic receptor-mediated smooth muscle contractions. IP3 causes Ca2+ let on 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 proteins and can directly activate non discriminating 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 eff ectively. 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 cease that a little more high concentration of atropine may exercise to produce complete simple competitive antagonism by atropine at acetylcholine muscuranic receptors.