Introduction
Noradrenaline is a hormone produced as a catecholamine by the sympathetic neurons from the heart; it is mainly used as a neurotransmitter. An increase in the levels of this hormone leads to contractions. The adrenal medulla is responsible in the production of this catecholamine that is further released by the synapses.
Noradrenaline lacks the methyl group present in adrenaline (Bailey, Schwieger & Hug 1993). It functions mainly in stress conditions whereby it suppresses the adrenoreceptors within the walls of blood vessels causing the muscles to contract. When the blood vessels narrow, the blood gets redirected to essential organs like the brain and heart. In this practical, the effects of noradrenaline towards vascular rings with and without endothelium will be studied and enable us to find out the difference (Aldasoro, Martínez, Vila, Flor & Lluch1993, p.106).
Principle
Isolated arterial rings were obtained based on the presence and absence of endothelial cells show different vascular dilation and constriction. When the arterial rings are incubated into the bath chambers containing different drugs, enzyme reactions occur to lead to an endothelium dependent reaction to noradrenaline. Acetylcholine is a vasoconstrictor (Aldasoro, Martínez, Vila, Flor & Lluch1993, p.105). Acetylcholine and prazosin accelerates the release of a certain factors that causes vasodilation such as nitric oxide. Prazosin relaxes both artery and vein. Noradrenaline reduces the output of acetylcholine. Higher concentration of acetylcholine produces a relaxation effect on the arteries (Edvinsson, Emson, McCulloch, Tatemoto & Uddman 1984).
Aims
- To obtain a cumulative concentration response curve to noradrenaline on arterial rings with intact endothelium.
- To obtain a cumulative concentration response curve to noradrenaline to arterial ring with endothelium removed.
- To obtain a cumulative concentration response curve to noradrenaline to arterial ring with prior endotoxin treatment to remove endothelium.
- To examine the effects of acetylcholine (1×10-6 mol.l-1) on the tissue
- To examine the effects of acetylcholine (at 2 concentrations, e.g. 1×10-9 and 1×10-6 mol.l-1) in the 3 arterial rings.
- To test the effect of acetylcholine when the tissue’s response to noradrenaline is at its maximum.
Materials
- A Computer programme that is developed to simulate the effects of drugs on isolated tissue preparations such as vascular rings. This program will investigate the effects of drugs and look at the pharmacological properties of vasodilator and vasoconstrictor agents.
- An arterial ring with either an intact endothelium or denuded of endothelium
- An endothelium denuded arterial ring removed from an animal treated four hours previously with E. coli endotoxin had been simulated.
- Noradrenaline (Concentrations of 1 x 10-9 to 1 x 10-6 mol.l-1)
- Acetylcholine (Concentrations of 1 x 10-9 to 1 x 10-6 mol.l-1)
- Prazosin (Concentration of 1 x 10-6 and 1 x 10-8 mol.l-1 )
Method
The simulation used in this practical was provided by a computer program known as vascular rings resource package. The programme has every option for the procedure to attain results on the effects of noradrenaline on vascular rings.
Noradrenaline was added to the vascular rings that were divided into three (arterial rings with intact endothelium, arterial ring with endothelium removed and arterial ring with prior endotoxin treatment to remove endothelium).A cumulative curve was then obtained on the different arterial rings.More noradrenaline was added progressively doubling the concentration of drug each time, i.e. add 5×10-9 mol.l-1, then 8×10-9 mol.l-1, 2×10-8mol.l-1, 5×10-8 mol.l-1, 1×10-7 mol.l-1, 5×10-7mol.l-1, 1×10-6 mol.l-1.This was done until adding more noradrenaline caused no further change or increase in the size of the response.
When trying to obtain the cumulative concentration response curve the wash out bath command was not selected. Effects of acetylcholine (1×10-6 mol.l-1) were observed on each preparation. The effect of acetylcholine was tested when the tissue’s response to noradrenaline was at its maximum (i.e. after pre-contraction of the arterial ring with noradrenaline). The effects of acetylcholine at two concentrations (1×10-9 and 1×10-6 mol.l-1) was investigated on the three arterial rings and the responses recorded. The data obtained was then tabulated on an assessment sheet and a semi log graph plotted to produce a log concentration /response curve.
Results
Using a concentration of 1×10-9 mol.l-1 (0.001 µmol.l-1) it was found out that a very small ‘response’ is produced in some preparations (e.g. using a concentration of 1×10-9 mol.l-1 (0.001 µmol.l-1) may only produce a small response). In any event this response was similar in size and shape to the kind of response you would produce if you had a real piece of arterial ring set up in an organ bath.
Statistics
The data obtained from the experiment was calculated as mean + standard error of the mean (S.E.M.).P < 0-050 was taken as the level of significance and the Student’s t test was then used to compare results.
The half-maximal effective concentration (EC50) was calculated by linear interpolation between two points on either side of the 500% of the maximal on each concentration-response curve and by reading the corresponding concentration on the logarithmic scale (Edvinsson, Emson, McCulloch, Tatemoto & Uddman 1984, p.160). Calculation of the mean of the readings was the obtained (Pepine 1998, 795).
The contractile response produced to each artery according to their vasoconstriction in the presence of noradrenaline was compared with the antagonist k2, where k2 is the mean of contractions before and after the contraction in the presence of the drug. The values obtained were of standard deviation (González & Estrada 1991, 370).
It was noted that there was no significant difference between arterial ring with endothelium removed and arterial ring with prior endotoxin treatment to remove endothelium (Chen, Suzuki & Weston 1988, 1172).
Definition of the EC50
The half maximal effective concentration (EC50) denotes the amount of a substance be it a drug, or a toxicant which brings about an effect that is refers to the concentration of a drug, antibody or toxicant which induces a response intermediate between the utmost and baseline observable effect after a particular period of exposure. It is commonly used as a measure of drug’s potency. The EC50 usually produces a curve as an expression of the response. This response signifies the amount of a substance at which 50% of the utmost response is obtained (Bredt, Hwang &Snyder 1990, p. 770).
Questions
EC50 for noradrenaline in the presence
- EC50 for noradrenaline in the presence of Prazosin 1 x 10-6
- 3 x 10-8
- EC50 for noradrenaline in the presence of Prazosin 1 x 10-8
- 1 x 10-7
- EC50 for noradrenaline in the presence of Prazosin 1 x 10-8
- 1 x 10-8
Emax for noradrenaline in the presence and Absence of Antagonist
- Emax for noradrenaline in the presence of Prazosin 1 x 10-6
- 1x 10-6
- Emax for noradrenaline in the presence of Prazosin 1 x 10-8
- 1 x 10-6
- Emax for noradrenaline in the presence of Prazosin 1 x 10-8
- 1 x 10-6
Arterial ring with endothelium: Presence of the endothelium on the artery regulated the contracting effect of acetylcholine and noradrenaline. The artery contracted to a lower dimension.
Arterial ring with endothelium removed: Absence of the endothelium allowed the artery to contract easily. An increase in the levels of noradrenaline also caused an increase in the rate of contraction of the artery.
Arterial ring with prior endotoxin treatment to remove endothelium: The endotoxin increased the constrictor effect of noradrenaline. Thus the diameter of the artery was smaller.
Briefly explain the differences in response to noradrenaline in the 3 preparations: The arterial ring with an endothelium showed minimum contraction due to the presence of the muscles on the artery. When the endothelium was removed the artery was smooth enough for to relax. Addition of E.coli strain on the artery led to an attenuation of the endothelium, thus the constriction observed was minimal (González, Fernandez, Martín, Moncada & Estrada 1992, p.155).
Discussion
The arteries without the endothelium were more responsive to noradrenaline than those with the endothelium. There was a relaxation –contraction response on the arteries that were already contracted with noradrenaline. The removal of the endothelium decreased the contractile response of the arteries towards noradrenaline (Caplan & Schwartz 1973, p. 719). However, contractile properties of noradrenaline were attenuated when the antagonist was added. The results presented above show that, low levels of acetylcholine induce contraction of the arterial rings. The response of the muscles on the aorta was recorded. It was noted that acetylcholine had two effects depending on the concentrations. It tends to show persistent endothelium-dependent relaxation when at low concentrations and at a higher concentration, the contraction was not dependant on the endothelium (Bredt, Hwang &Snyder 1990, p. 770).
From the results obtained, it is noted that prazosin produces a relaxation effect on the arteries depending on the content of the endothelium.
Vasodilations of the arteries are caused by the paracrine effect of the endothelial acetylcholine (Faraci 1991, p.40).
Conclusion
Acetylcholine is a vasoconstrictor. Acetylcholine and prazosin accelerates the release of a certain factor that causes vasodilation. Presence of the endothelium is plays a very important role in regulating the extent of contraction on the arteries.
Noradrenaline induced contraction, as acetylcholine had a relaxation effect on the arterial ring. The endothelium contributes independently to the mechanism of relaxation of the artery.
List of References
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