It may be possible to utilize other tissues instead than that of higher animate beings as an educational tool for the survey of pharmacological drugs. Previous experimental surveies were carried out in order to find how closely related the basic electrophysiology of daphnia bosom was to the human bosom. Work such as that of Krijgsman and Krijgsman – Berger ( 1950,1951 ) indicated that the automatism of the bosom of insects has a mechanism which is neurogenic. An alternate position, nevertheless, revealed that the Black Marias of water flea are infact myogenic ( Needham 1950 ) . A close relation to the human bosom would supply a utile theoretical account with which to analyze mechanism of cardioactive drugs. Since merely a few drugs have been tested on the daphnia bosom, it seemed utile to prove other drugs to see whether the responses of the daphnia bosom correlated with the responses of the drugs to the human bosom. Transporting out these experiments would demo how utile a daphnia bosom would be for instruction intents.
Stein and Richter carried out ultrastructural surveies on the bosom of the water flea in order to find the construction of it, specifically as to whether the bosom was striated. They found that the bosom wall was one cell midst ( occasional parts were five cells in thickness ) , the cells were thin and contained many chondriosomes, long striated sarcostyles and an copiousness of sacroplasm with an irregular indented cell surface.
From this research, it can be concluded that the structural characteristics of the water flea in many respects are rather similar to that of striated and cardiac musculus of other carnal species, hence water flea could be used for educational intents in undergraduate survey. Since surveies have non been carried out utilizing cardioactive drugs, specifically channel blockers, we set out to by experimentation find how closely related the daphnia bosom was to the mammalian bosom and whether the water flea could be used in undergraduate survey.
The advantages of utilizing a water flea in experimental surveies are the fact that it is crystalline and one cell midst, hence sing the bosom would be trouble-free. Using a water flea for experimental intents has the advantage of being non-invasive because the whipping insect bosom can be viewed easy through the exoskeleton, and is non-destructive Internet Explorer. dissections need non be carried out and so the being can be released back to nature after it has assisted us with our work.
The drugs required to be used in this experiment are cardiac ion channel blockers, these act on different ion channels of the bosom doing the cardiac potency to be affected.
Cardiac Action Potential
File: Action possible ventr myocyte.gif
Phase 4 as portrayed in the diagram shows the resting membrane potency.
Phase 0 is known as the rapid depolarisation stage. The incline as shown in the diagram portrays the rate of depolarisation of the cell and is known as dV/dtmax. At this stage the Sodium Channels unfastened which caused a rapid inflow of Sodium ions into the cell.
The ability of the cell to open the Sodium channels is to make with the membrane potency when it has become aroused. At baseline -85mV the Sodium ion channels are non unfastened, nevertheless when excited they open, doing an inflow of Na ions.
If the membrane is less negative, the fast Na ions become inactive and insensitive to gap, and so this leads to a little response to excitement of the cell membrane therefore taking to a lower Vmax. If the resting membrane becomes excessively positive so Na channels will non be able to flux through efficaciously and so will do the hold of conductivity through the bosom. This can do an addition in arrhythmias.
Phase 1 takes topographic point with the inactivation of fast Sodium Channels. Chloride ions move across the cell membrane and hence this causes a alteration in membrane potency, from the Potassium ions,
Phase 2 otherwise known as the ‘plateau ‘ stage of the cardiac action potency is maintained by an inward motion of Ca ions and an outward motion of Potassium ions.
Phase 3 known as the rapid repolarisation stage, the action potency of the Calcium channels near and the postassium channels at this point are still unfastened, this therefore enables a net outward current which causes a negative charge in membrane potency, this causes the K channel to open and the net outward of Potassium channels causes the cell to repolarise. The K channel near when the membrane potency is back to -85mV.
The drugs to be used on the daphnia bosom are channel blockers and therefore may impact the cardiac action potency due to the blocking of certain channels, the drugs that are to be applied to the daphnia bosom are:
Lidocaine is a Class Ib antiarrhythmic agents and are known as Na channel blockers. They have a fast oncoming and countervail kinetic which means the drug is more effectual on faster bosom rates. The drug has the ability to shorten the action possible continuance and cut down the furnace lining period. The Vmax decreases in depolarised cells with fast response action potencies due to the drug. The action possible continuance either goes unchanged it may diminish. File: Action possible Class Ib.svg
The consequence of Class Ib drugs on the cardiac action potency
Class Ib drugs are by and large more specific towards electromotive force gated Sodium Channels, Lidocaine blocks the Sodium channels in their unfastened and inactive provinces and does hold the ability to adhere in the resting province.
Lidocaine Acts of the Apostless by the suppression and conductivity of nervus urges by diminishing the neural membranes permeableness to sodium ions ; this prevents depolarization from happening and so consequences in a encirclement of conductivity.
Glibenclamide is a sulfonylurea that acts on Type II diabetes, at nanomolar concentrations, the drug binds to the sulfonylurea receptor ( SUR ) which are found on ? – Pancreatic cells, the drug causes the suppression of ATP sensitive Potassium channels and this causes insulin to be released and hence secreted into the blood.
Glibenclamide is besides used as a CFTR Chloride channel blocker, it is voltage dependent and the drug Acts of the Apostless by viing with Chloride ions for a common binding site within a big intercellular anteroom portion of the CFTR pore.
Verapamil is an L – type Calcium channel blocker. The drug Acts of the Apostless by barricading the electromotive force gated calcium channel in cardiac musculus and therefore causes a lessening in cardiac contractility.
In cardiac pharmacological medicine, Ca channel blockers can besides be classified as IV antiarrhythmic agents. As Ca channels are concentrated in the sinoatrial ( SA ) and atrio-ventricular nodes ( AV ) , these agents can be used to diminish impulse conductivity through the AV node, and hence decelerate the bosom rate down.
4 – Aminopridine is a Potassium channel blocker besides known as a Class III agent ; this therefore prolongs the procedure of depolarization.
File: Action possible Class III.svg
Consequence of Class III agent on cardiac potency
Since the Class III agent does non correlate with the Na channel so conductivity speed does non diminish. Prolongation of the action potency, continuance and stubborn period along with normal conductivity speed, causes re – entrant arrhythmias from being prevented.
Carbenoxelone is a spread junction blocker and Acts of the Apostless by barricading the enzyme 11?-hydroxysteroid dehydrogenase ( 11?-HSD ) they block the connection channels and spread junctions.
The usage of the drugs ( as mentioned above ) are to show whether the daphnia bosom will react to the drugs as we expect them to ie. how the mammalian bosom would react. Depending on how good the consequences from the daphnia bosom correlates with what we know from the mammalian bosom, determines the grade of utility of the water flea in educational intents.
Purposes and Aims
The intent of the experiment was to find the presence of the rule electromotive force gated ion channels within the daphnia bosom. The usage of cardioactive drugs to suppress assorted channels would bespeak the being of the receptor sites contained by the daphnia bosom.
Cardioactive drugs being used:
i‚· Lidocaine – Sodium Channel Blocker
i‚· Glibenclamine – Chloride Channel blocker,
ATP sensitive Potassium Channel Blocker
i‚· 4 – aminopyridine – Potassium Channel Blocker
i‚· Verapamil – Calcium Channel Blocker
Procedures for Safety
Since this was a laboratory experiment, we had to guarantee safety within the lab. The research lab that we carried out our experiment in was good ventilated, this was to understate any inspiration of the drug or its exhausts. To forestall consumption of any stuff, nutrient and drink was purely prohibited within the lab.
Lab coats, goggles and baseball mitts were worn in order to forestall contact of the cardioactive drugs with the tegument or the eyes. It was advised that we use the drugs quickly and when we no longer required it, to set it back in the packaging and to the side.
A COSHH and biological safety signifier was filled out for all of the four cardioactive drugs, this was to guarantee that the safety guidelines for each of the four drugs were known prior to the experiment.
Twenty water flea were pipette from a bag incorporating water flea, pool H2O and residue. These water fleas were placed into single, conelike bottomed multiwell home bases. Excess H2O was removed from each well utilizing a pipette with a thin tip, this was done to forestall the water flea from traveling overly and hence being able to see clearly the pulse of the water flea.
Once the procedure was completed with 20 water flea, a Lympus CK-40 light microscope was used to detect the pulse, scenes on the microscope required adjusting in order to see a precise image of the bosom round. An image analysis package known as ‘Mirovideo Capture ‘ was used in order to enter the pulse of single water flea 3 times for 10 seconds each. The 10 2nd pictures were played in slow gesture, in order to number the figure of beats per 10 seconds. An norm of the three values were calculated and so multiplied by 6 in order to happen the beats per minute.
This experiment was carried out in order to see whether the group average bosom rate varied with an increasing sample size.
Experiment on changing temperatures.
In order to see whether temperature had an consequence on the pulse we put the water flea at temperatures of:
6-8 & A ; deg ; C
21 & A ; deg ; C ( Room Temperature )
37 & A ; deg ; C
6-8 & A ; deg ; C
Daphnia were put in the electric refrigerator to accomplish a temperature of 6 – 8 & A ; deg ; C. Six water flea were so placed into single Wellss ( process as in Control Experiment ) . The microscope was used to detect the bosom beats and three recordings were made for each water flea. An norm of the three values were calculated and so multiplied by 6 in order to happen the beats per minute.
21 & A ; deg ; C ( Room Temperature )
The water flea were stored in room temperature and the experiment above ( 6-8 & A ; deg ; C ) was repeated for this temperature.
37 & A ; deg ; C
The water flea were stored in an brooder at 37 & A ; deg ; C and the experiment above ( 6-8 & A ; deg ; C ) was repeated for this temperature
Control experiment utilizing Ethanol
From the cardioactive drugs, Lidocaine and Glibenclamide were required to be dissolved in ethyl alcohol, before they could be made up into a stock solution, www.tocris.com was visited to happen the undermentioned information. Six water flea were hence pipette into single Wellss, a 10 % solution of ethyl alcohol was added to each water flea and put aside for 10 proceedingss, this was to give clip for the solution to hold its consequence, if any on the water flea. The mean pulse of each water flea was so recorded to see if there was any change in bosom rate.
The same experiment was repeated with a strength of 1 % ethyl alcohol.
Control experiment utilizing distilled H2O
An experiment was carried out in order to see whether the water flea were able to last in distilled H2O, foremost the water flea were placed in six Wellss and their hearbeat was recorded, the six water flea were taken from their original environment of pool H2O and replaced with distilled H2O. The water flea were left to swim in the distilled H2O over dark. The bosom beats of the water flea were so recorded to see if they were any different to that of the pool H2O, consequences showed that there was no difference and hence distilled H2O excessively could be used to do up stock solution.
Procedure for Application of drugs.
Prior to the drugs being added, bosom rates of the six single water flea were recorded three times.
Stock solutions were made up by weighing out xg on an accurate deliberation balance ( 4 denary topographic points ) , the drug was poured into a 10ml conelike flask, 1ml of ethyl alcohol was added to the solution ( for Lidocaine and Glibenclamide merely ) , this was so brought up to the base of the semilunar cartilage utilizing portion pool H2O and portion distilled H2O. Six water flea were placed in welled home bases, extra H2O was removed and the drug solution at x % was added, they were placed aside for 10 proceedingss, this was in order to guarantee adequate clip was given for pharmacological effects of the drug to take topographic point on the daphnia bosom.
Experiment utilizing Lidocaine
As there was uncertainness as to the strength that would hold an consequence on the daphnia bosom, a standard solution of 1g in 100ml ( 0.1 % ) was made up. Since the 0.1 % solution had a profound consequence on the bosom beat it was used as a stock solution and so it was diluted down to organize the remainder of the strengths in order to accomplish a dose response curve.
At a strength of 0.1 % it was found that the Lidocaine stopped the pulse of the water flea, therefore dilutants of the 0.1 % solution were made up:
0.05 % solution – 5ml of stock made up to 10ml ( with pool and distilled H2O )
0.02 % solution – 4ml of 0.05 % solution made up to 10ml
0.01 % solution – 5ml of 0.02 % solution made up to 10ml
0.001 % solution – 1ml of 0.01 % solution made up to 10ml
Each solution of changing concentrations were tested on the water flea utilizing the same process as above, each solution was left for 10 proceedingss in order to see the effects on the daphnia bosom. Dilutants were continually made until they had no consequence on the bosom.
Experiment utilizing Glibenclamide
A concentration of 0.1 % was made up in order to see the effects on the bosom, it was found at this strength there was small if no consequence to the bosom, hence with the assistance of test and mistake it was found that at a strength of 0.3 % the rate of the bosom decreased, nevertheless it did non restrict it wholly. We hence tested the water flea at drug concentrations of 0.5 % and it seemed to hold the same consequence as the strength at 0.3 % , we therefore used this strength as the stock solution and made dilutants from it:
0.3 % stock solution – 0.03g glibenclamide dissolved in 1ml of 5mM ethyl alcohol and made up to 10ml
0.25 % solution – 7.5ml of stock solution made up to 10ml
0.2 % solution – 8ml of 0.25 % solution made up to 10ml
0.1 % solution – 0.01g of glibenclamide made up to 10ml ( original )
The different strengths were applied to the water flea ( process as mentioned before ) and the consequences for each concentration were obtained.
Experiment utilizing Calan
The standard solution of 0.1 % was made, though this solution had an consequence on the bosom it was non a profound consequence, therefore the water flea were tested at drug concentrations of 0.2 % which finally stopped the bosom round wholly, therefore it was used as a stock solution and dilutants were made from it:
0.2 % stock solution – 0.02g Calan dissolved in 10ml
0.15 % – 7.5ml of stock solution ( 0.2 % ) made up to 10ml
0.1 % – 0.01g of Calan made up to 10ml ( original )
0.01 % – 1ml of original ( 0.1 % ) made up to 10ml
These changing strengths were added to the water flea ( process as mentioned before ) to see the consequence on the bosom.
Experiment utilizing 4-aminopyridine
The standard solution of 0.1 % was made and from it its dilutants. At 0.1 % the bosom rate was affected so a solution of 0.5 % was made to see if any farther alterations to the bosom occurred.
0.5 % solution – 0.05g of 4-aminopyridine made up to 10ml
0.1 % stock solution – 0.01g of 4 – aminopyridine made up to 10ml
0.01 % solution – 1ml of stock solution made up to 10ml
0.001 % solution – 1 milliliter of 0.01 % solution made up to 10ml
Each strength were added to the welled home bases incorporating water flea ( process as mentioned before ) .to see the consequence on the bosom.
Experiment utilizing Carbenoxelone
As researched we found that a little weight of carbenoxelone was required to consequence the bosom rate, we hence decided to lodge with the standard strength of 0.1 % , this had no consequence on the bosom and so we made up a strength of 0.2 % which excessively had no consequence on the bosom, we therefore decided that it would non be worthwhile to utilize more drug, since the concentrations had no impact on the bosom. The solutions made hence were:
0.2 % solution – 0.02g of carbenoxelone made up to 10ml
0.1 % solution – 0.01g of carbenoxelone made up to 10ml
Each changing strength of drug was added to single water flea ( process as mentioned before ) .
Figure 1 – A graph to demo the discrepancy of pulse in 20 water flea. ( Control Experiment )
Figure 2 – A dose response curve for Lidocaine.
Figure 3 – A dose response curve for Glibenclamide.
Figure 4 – A dose response curve for Verapamil
Figure 5 – A dose response curve for 4 – aminopyridine.
Figure 6 – A dose response curve for Carbenoxelone.
The control experiment was carried out in order to see whether the group average bosom rate varied with an increasing sample size, the consequences show that the standard divergence was within 10 % of the mean, 7.96 % to be precise, hence this method was acceptable for usage, since the bosom beats did non vary much.
We hence adopted this method and lowered the figure of water flea from 20 to 6, since 6 was a sufficient figure for accurate consequences.
Dose response curve of Lidocaine
The consequences show that the application of Lidocaine on the daphnia bosom caused a dramatic addition in per centum alteration in bosom rate at a strength of 0.1 % , by experimentation it was found that within 20 – 30 seconds the daphnia bosom wholly stopped crushing. This is since Lidocaine is a Sodium channel blocker and Acts of the Apostless by impairing the conductivity of Sodium ions through Na channels. Therefore at a strength of 0.1 % the drug was equal to do a 100 % per centum alteration in bosom round.
By thining the strength to 0.05 % the drug decreased the per centum alteration in bosom rate compared to 0.1 % nevertheless it did non wholly halt the bosom nor did it impact the bosom tissue, the per centum alteration was 38 % .
At a strength of 0.001 % the drug had small to no consequence on the bosom
The general tendency of the graph shows that when increasing the concentration of the drug, there is a general addition in the per centum alteration in bosom rate which means that the bosom rate is decelerating down.
Dose response curve of Glibenclamide
The graph shows that at a concentration of 0.3 % of Glibenclamide the drug had a mild consequence on the bosom, it caused a 30 per centum alteration in bosom rate and hence though this was a high concentration of drug it did hold a minimum consequence
At a strength of 0.25 % of Glibenclamide, the consequence of the drug fell by 50 % , demoing that the per centum alteration in bosom rate was about 15 % .
At a strength of 0.1 % of Glibenclamide there was small to no alteration in bosom rate, demoing that the drug had no consequence on the bosom.
The general tendency of the graph shows that with an addition of drug there was addition in per centum alteration of bosom rate, which means the more concentrated the drug the more consequence it had on the bosom in footings of decelerating the bosom rate down.
Since the drug inhibits the CFTR
Dose response curve of Verapamil
Verapamil was the most powerful drug out of all the cardioactive drugs. At a concentration of 0.2 % it caused a 100 % alteration in bosom rate which means it stopped the bosom round wholly.
At 0.1 % it still had a dramatic impact in footings of decelerating the bosom rate down, there was a 75 % alteration in bosom rate which shows at this concentration, though the Black Marias had non to the full stopped, though they did diminish dramatically.
At a concentration of 0.001 % , there was small to no alteration in bosom rate, demoing that the drug, at this strength, had no consequence on the bosom.
Experimentally, we found that the drug besides affected the manner the bosom contracted Internet Explorer. the motion, see images below:
( put in images and explain )
Dose response curve of 4 – amino pyridine
In order to accomplish a dose response curve for 4 – amino pyridine we required a big scope of values from 0.001 % to 0.5 %
The graph shows that at 0.5 % there was a 100 per centum alteration in bosom rate, this shows that at this concentration the drug was more powerful and stopped the bosom round wholly
At a concentration of 0.01 % the drug had a small to no consequence on the bosom rate.
The general tendency of the graph is – the greater the concentration of 4 – amino pyridine the greater the per centum alteration in bosom rate.
Dose response curve of Carbenoxelone
The add-on of carbenoxelone had no consequence on the daphnia bosom, this is portrayed from the graph as there is a consecutive line at concentrations 0.2 % and 0.1 % , hence the drug had no impact on the bosom rate.
This may be due to one of two grounds, either there are no spread junctions present in the daphnia bosom, or the carbenoxelone does non hold an consequence on the innexins ( found in daphnia bosom ) , hence merely connexins ( found in human bosom ) .
Upon analysis, it can be seen that the drugs Lidocaine, Verapamil and 4 – Aminopyridine slowed the bosom rate down, this shows that Sodium channels, Potassium channels and Calcium channels are present in the bosom of the water flea.
Though the bosom rate of the water flea did decelerate down when Glibenclamide was added to it, it did non diminish dramatically. There is no cogent evidence, hence, to propose that there are chloride ions present in the daphnia bosom, this is since glibenclamide can be used as a chloride channel blocker, but it besides inhibits ATP sensitive Potassium channels, hence farther research needs to be done in order to see whether the daphnia bosom contains chloride ions. The usage of another drug that specifically blocks chloride channels, seems like a good option.
The drug, carbenoxelone shows that there may non be any gap junctions present in the daphnia bosom, therefore an alternate method may be used for intercellular signal transmittal.
Overall the water flea could be used to demo the effects of the Lidocaine, Verapamil and 4 – Aminopyridine on the bosom, therefore the daphnia bosom is a utile educational tool, to demo the effects of drugs on the bosom. The fact the water flea is one cell midst and transparent makes it straightforward to happen the bosom under the microscope. Therefore the water flea can be used for educational intents in undergraduate survey.
An ECG survey can be carried out on the bosom of the water flea by utilizing investigations to touch the daphnia bosom. The ECG survey can be used in order to construe the electrical activity of the bosom. From the consequences we would be able to analyze the ion channels and at what point ions leave and enter the membrane, from this we are able to see how closely related the daphnia bosom is to the mammalian bosom.