Effects Of Clonidine On Cardiovascular Function Biology Essay

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It is known that the antihypertensive drug clonidine acts on a2- adrenoceptors. This study was designed to see whether alpha 2-agonist, clonidine, binds to its a2- adrenoceptors receptors within the vascular smooth muscle and producing its effects on the cardiovascular system, affecting the contractility of the heart and the heart rate through its effect on the pacemaker. In the isolated heart, the highest concentration of clonidine used (1x10-5M) had produced the maximum effect. The heart of a rat and guinea pig had been isolated and used with langendorff apparatus. The effect of clonidine was compared to the effects of acetylcholine which decreased the heart rate and contractility whereas noradrenaline which increased the heart rate and contractility. The dose of drug administered to the isolated heart was important, as the concentration of drug increased the effect also increased. In conclusion, it is evident that the addition of clonidine to the isolated heart produced a bradycardiac effect lowering both the heart rate and contractility by activating a2- adrenoceptors.

Introduction

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The autonomic nervous (non-voluntary) consists of three divisions, sympathetic, parasympathetic and enteric, where it regulates the contraction and relaxation of vascular and visceral smooth muscle. The neurons that connect these pathways are the preganglionic and postganglionic. Acetylcholine and noradrenaline are the two main transmitters that operate in the autonomic system. Acetylcholine acts on the muscarinic receptors, whereas noradrenaline acts on both alpha- or beta-adrenoceptors.

The a2- adrenoceptors are distributed extensively in the cardiovascular system. There are three types of adrenoceptors that are present within the vascular smooth muscle cells of the heart, which are alpha 1-, alpha 2- and beta 1- adrenoceptors. Alpha-adrenoceptor agonists (a-agonist) such as clonidine bind to their complementary alpha receptors on vascular smooth muscle. When a-agonists bind to their receptors they produce sympathetic effects such as contraction of smooth muscle and vasoconstriction (Molin and Bendhack 2004).

There are two types of alpha-adrenoceptors on vascular smooth muscle, alpha 1 (a1) and alpha 2 (a2). The a1-adrenoceptors are located in the vascular smooth muscle whereas the a2- adrenoceptors are located both on the sympathetic nerve terminals and on the vascular smooth muscle.

Clonidine is known for its main use as an antihypertensive medication used to treat high blood pressure (hypertension), with its vasoconstrictive properties. Clonidine (antihypertensive and antisympathetic drug agent) is an alpha 2-agonist (Knaus, A. et al 2007) which acts centrally and stimulates receptors in the brain that are sensitive to levels of catecholamines (hormones such as adrenaline and noradrenaline released due to stress) in the blood.

The action of clonidine is regulated by a negative feedback loop. The brain controls the production of catecholamines via the descending sympathetic nerves in the adrenal medulla. Clonidine causes the brain to believe that the levels of catecholamines are very high; the brain then reduces its signals that are sent to the adrenal medulla which cause a decrease in the levels of catecholamines in the blood. This results in reduction in blood pressure and a lower heart rate.

When using clonidine there can be side effects on the cardiovascular system, such as bradycardia, tachycardia, congestive heart failure, palpitations, syncope, orthostatic symptoms, Raynaud’s phenomenon, also electrocardiographic abnormalities including atrioventricular block, sinus node arrest and arrhythmias). This will be taken note of when working with the isolated heart.

In this experiment clonidine and other drugs such as acetylcholine, noradrenaline and phentolamine will be used to assess the effects and the mechanisms of action on the heart rate and force of contraction. Isolated rat heart tissue preparations will be perfused with clonidine, using the Langendorff apparatus.

This langendorff isolated perfused heart allows the examination of the heart in a nutrient rich fluid with oxygen but without the hormonal and neuronal effects. This allows the contractility of the heart to be measured, along with the heart rate. The heart then can be observed for many hours.

The isolated heart in this experiment is denervated, so this can be an advantage when observing just the cardiac function, while the other sympathetic and vagal stimulation is ceased. The denervation of the heart may be compensated by the perfusates with addition of the neurotransmitters itself or by the addition of other peripheral factors.

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There are not many studies that show the direct effects of clonidine on the heart, but studies show that clonidine is able to act not only on its target receptors, a2- adrenoceptors but it is also able to work without acting on its specific receptors, as it can also act on a1-adrenoceptors (Molin, J. C. et al 2005). It has also been found that Clonidine has a partial agonist effect on the a1-adrenoceptors (Jie, K. et al 1984).

The activation of the alpha-2 adrenoceptors by clonidine may have other proteins that contribute to this effect of clonidine, but this has yet not been evident or proved. Clonidine can act on two different sites to lower blood pressure, such as one example, the activation of alpha-2 adrenoceptors in brain stem nuclei leads to a decrease in the sympathetic tone. In another example, it can also activate inhibitory alpha-2 adrenoceptors on postganglionic fibers reducing the release of noradrenaline.

A bradycardiac effect of clonidine has also been observed in mice in isolated atria. This effect occurred by the inhibition of the cardiac hyperpolarisation-activated pacemaker current. The cardiac hyperpolarisation pacemaker current plays an important part in generating pacemaker potentials in the sinoatrial node cells. The number of pacemaker potentials was also lowered in the sinotrial node cells of the heart (Knaus, A. et al 2007).

In addition to this bradycardiac effect, mice injected with clonidine after being injected with anaesthesia, the heart rate and arterial pressure was significantly reduced. The same effect was also seen in mice that were awake, as well as mice that had their alpha-2 adrenoceptor removed.

In a different study it has been noticed that, the effect of the selective drug clonidine on the alpha-2 adrenoceptor, can stimulate contractions, but this is totally dependent on the extracellular calcium.

In another study, it has been seen that if other drugs are absent (agonists and antagonists), clonidine is able to contract the denuded aortic cells, this effect of clonidine occurs in the alpha-1 adrenoceptors only.

Acetycholine generally causes vasodilation, an indirect effect, which acts on the vascular endothelial cells releasing nitric oxide causing relaxation of the smooth muscles. The muscarinic receptors which are G-protein coupled receptors have five sub-types (M1-M5).

Studying the effects of clonidine could be beneficial, as clonidine is used by some patients to treat hypertension, and so the amounts given to the patients can be either increased or decreased depending on the amount already taken (RM Boyar et al 1980).

Aims and Objectives

To investigate the effects of clonidine on isolated rat heart preparations. To also establish whether clonidine affects the heart rate and force through its effect on the pacemaker current, as well as myocardial contractility.

Different drugs (agonist such as acetylcholine and noradrenaline and antagonists such as phentolamine) will be used against clonidine to observe and assess the effects ptroduced. The experimental technique used to test this effect of clonidine, will be a langendorff apparatus using an isolated perfused heart where the heart will be kept alive.

MATERIALS AND METHODS:

Preparation of isolated Guinea pig and rat hearts

Male Dunkin-Hartley Guinea pigs were kept on a Harlan 2040 diet and Wistar rats were kept on a Harlan 2018 diet along with Ad lib filtered tap water (both weighing at 250g+). Animals came from Harlan UK animal research laboratory. The animals were housed in a group, with grade 6 woodchip and sizzle nest bedding for the rat and grade 6 woodchip and hay for the guinea pig, along with enrichments including plastic igloos, gnawing blocks, plastic and cardboard fun tunnels. They were exposed to 12 hours of light and 12 hours of dark, kept at a temperature between 19-23 oC with room humidity of 45-65%.

Animals were killed by cervical dislocation under the scientific procedures act (1986) and the heart was then isolated, removing its surrounding tissue. The set-up used in this experiment for perfusing the heart, was the Langendorff Apparatus (Havard Apparatus UK). This is so that the heart can be kept alive allowing nutrient rich, oxygenated fluid to be perfused through the coronary vasculature via a catheter that is inserted into the ascending aorta. Drugs to be used were prepared with Krebs salt solution gassed with 95% O2 and 5% CO2, kept at constant pressure, pH and temperature of 37 oC (see below for composition).

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The heart was kept in a beaker of Krebs on ice to halt its activity, just before the cannula was inserted into the aorta of the heart. Electrodes were also attached to the heart, to enable the heart to be field stimulated. After the cannula was inserted into the aorta and secured using a cotton tie, the heart was perfused with normal Krebs salt solution. The apex of the heart was clipped using a heart clip connected to a force transducer to measure the tension (Havard Apparatus UK and ADInstruments).

Contractile activity of the heart was allowed to stabilise before the addition of drugs. Drugs used was taken from a stock solution of 10mls and diluted to get the lower concentrations.

100ul of 1x10-3 M (0.1ml/10ml) of clonidine was pipetted into the first 100ml bath which gave 1x10-6 M, and was then allowed to run, up until a response was seen. Before the addition of then next drug, clonidine was washed out with the control solution (KSS). Next 100ul (0.1ml/10ml) of phentolamine, was added into the second 100ml bath again letting it to run and wait (for up to 1-2 minutes) to see effect, then both clonidine and phentolamine, again 100ul (0.1ml/10ml) of each drug was added to the third 100ml bath by instantly switching between physiological solutions to see this effect (see results).

After the testing of clonidine with its antagonist and control it was tested again by increasing its concentration. Clonidine was also added in a dose-dependent fashion alongside noradrenaline and acetylcholine. Low concentration of drugs was used starting from 1x10-8M to the highest of 1x10-5M, adding 0.1ml each time to the isolated heart. Every time a different drug was added the last drug was first washed out with control Krebs Salt Solution.

Solutions and drugs:

The drugs used were clonidine, phentolamine, noradrenaline, acetylcholine, and ascorbic acid. Ascorbic acid was added only to catecholamines to prevent oxidation. All drugs were ordered from Sigma-Aldrich UK address. Drugs were dissolved in distilled water to prepare stock solutions and then diluted in Krebs Salt Solution (accurate to ±0.2ml), all made up fresh everyday.

The highest concentration of drugs used was at 1x10-2 and the lowest concentration at 1x10-8. All drugs were kept on ice and had ascorbic acid added to them to prevent oxidation. The composition of Krebs salt solution (in mmol/L) was NaCl 118, NaHCO3 25, glucose 11, potassium chloride (10% solution) 4.7, CaCl2 10% solution 2.5, potassium phosphate 10% solution 1.18, magnesium sulphate 10% solution 1.18, gassed with 95% O2 and 5% CO2.

Statistical analysis:

Results are displayed as mean S.E.M (Labchart 7.0 Reader). The raw data was analysed by using a one sample t-test (using IBM SPSS PASW Statistics 18 software). P values less than 0.05 are taken to be statistically significant.

Results

The addition of clonidine to the isolated heart had always shown a decrease in the force of contraction and the heart rate, which had either taken effect within one minute or a couple of minutes after stabilisation depending on the tissue itself as both guinea pig and rat heart was used to test.

The traces that follow compare the effects of each drug used on the isolated heart. Clonidine was compared to acetylcholine and noradrenaline at concentrations starting from 1x10-8M to higher concentrations of 1x10-5M. Addition of acetylcholine to the isolated heart had shown that there is a decrease in the heart rate and contractility.

When the lowest concentration of clonidine was added to the heart, there was not much effect of the drug seen, but as the concentration was increased, there was a gradual decrease in the heart rate and contractility (figure 1b). Acetylcholine was first added to the isolated heart so that the results could be compared to the effect of clonidine, as acetylcholine also decreases the contractility and heart rate, and here acetylcholine can be used as a control clearly shown in figure 1a. The last drug to be tested on the heart was noradrenaline which increase the heart rate and contractility as seen in figure1c, this is also compared to the other two drugs (figure 3).

Figure 1a. The effect of acetylcholine 1x10-5M on the isolated heart

Figure 1b. The effect of clonidine 1x10-5M on the isolated heart

Figure 1c. The effect of noradrenaline 1x10-5M on the isolated heart

Figure 1d. The effect of both clonidine and phentolamine 1x10-5M on the isolated heart.

Higher concentrations were required for phentolamine to produce an effect that was detectable, as in this experiment 0.1ml of phentolamine (1x10-5M) did not produce a significant effect as seen on figure1d. Thereafter, the concentrations of all drugs were increased and the effect of dose administered to the isolated heart was taken into account (see below).

The trace (figure 2a) below shows the control compared to the clonidine drug (10-5M) administered (shown by arrow). After running the isolated heart on Krebs salt solution until the contractility had become stabilised and then adding clonidine. It can be clearly seen that once the clonidine is added to the isolated heart there is a rapid decrease in the heart rate and force of contraction in within one minute of administering the drug.

Figure 2a. A contractile response to clonidine (1x10-5M), showing the tension (g) vs. time (minutes).

To further test and verify the effect of clonidine on the isolated heart, the concentration of clonidine had been increase by 10 fold. This produced a trace that showed the rate of contraction and the force of the heart decrease significantly.

Next, the effect of alpha-2 agonist clonidine on heart was assessed. The next trace shows clonidine administered in a dose-response manner, from 1x10-8M to 1x10-5M final bath concentration.

Figure 2b. The addition of clonidine to the isolated heart in a dose-response manner, starting from adding 100ul of 1x10-8M clonidine.

Acetylcholine is a more potent drug than clonidine giving a higher curve compared to clonidine which has a lower curve and is less potent. However noradrenaline is further up than acetylcholine with a greater Emax percentage compared to clonidine and acetylcholine (figure3).

Figure 3. Dose-response curves showing clonidine, acetylcholine and noradrenaline in comparison to each other.

From figure 1 (above), the mean of the tension produced was taken into account. To statistically anaylse the data a one sample t-test was used to find the P value shown in figure 4 below. The P value was known to be statistically significant as the value obtained was less than 0.05.

One-Sample Statistics

Drug

Concentration

N

Mean

Std. Deviation

Clonidine

1x10-5M

Tension (g)

10

2.080110

1.3794321

1x10-6M

Tension (g)

10

6.334970

2.6142477

1x10-7M

Tension (g)

10

11.727820

3.6894815

1x10-8M

Tension (g)

10

11.923370

3.8369163

Acetylcholine

1x10-5M

Tension (g)

10

9.111360

3.3033855

1x10-6M

Tension (g)

10

10.114160

3.9386107

1x10-7M

Tension (g)

10

11.943510

2.7430438

1x10-8M

Tension (g)

10

11.778800

2.7727996

Noradrenaline

1x10-5M

Tension (g)

10

10.714420

3.4998647

1x10-6M

Tension (g)

10

11.004630

3.6337879

1x10-7M

Tension (g)

10

10.770750

3.6457193

1x10-8M

Tension (g)

10

11.177820

2.8014200

Above is figure 4a. Showing the one sample t-test statistics obtained, including the mean values, the standard error mean and the standard deviation values.

At the lowest concentration of clonidine (1 x 10-8 M) the mean response (11.923370) was close to the value obtained from the lowest concentration of acetylcholine (11.778800), noradrenaline also had a mean value that was similar (11.177820).

As seen from figure 4b, the significance value (P) is below 0.05, allowing the alternative hypothesis (H1) to be accepted, which shows that there is a significant difference between the drugs used, as the response from the drugs were all different.

One-Sample Test

Drug

Concentration

Test Value = 0

t

df

Sig. (2-tailed)

Mean Difference

95% Confidence Interval of the Difference

Lower

Clonidine

1x10-5M

Tension (g)

4.769

9

.001

2.0801100

1.093324

1x10-6M

Tension (g)

7.663

9

.000

6.3349700

4.464850

1x10-7M

Tension (g)

10.052

9

.000

11.7278200

9.088524

1x10-8M

Tension (g)

9.827

9

.000

11.9233700

9.178605

Acetylcholine

1x10-5M

Tension (g)

8.722

9

.000

9.1113600

6.748260

1x10-6M

Tension (g)

8.121

9

.000

10.1141600

7.296648

1x10-7M

Tension (g)

13.769

9

.000

11.9435100

9.981255

1x10-8M

Tension (g)

13.433

9

.000

11.7788000

9.795259

Noradrenaline

1x10-5M

Tension (g)

9.681

9

.000

10.7144200

8.210768

1x10-6M

Tension (g)

9.577

9

.000

11.0046300

8.405175

1x10-7M

Tension (g)

9.342

9

.000

10.7707500

8.162760

1x10-8M

Tension (g)

12.618

9

.000

11.1778200

9.173805

Above is figure 4b. The one sample t-test, showing the significance values and the test value for clonidine, acetylcholine and noradrenaline and the different concentrations. For the lowest concentration of each drug the values are very similar for each drug.

Discussion

The aim of this project was to find the effect of clonidine on cardiovascular function, affecting both myocardial contractility and heart rate through its effect on the pacemaker current.

The main finding of the study was to identify the effect of clonidine on the isolated heart, on the heart rate and contractility of the heart. To verify that clonidine had an effect on the heart, a number of experiments were performed on both guinea pig and rat hearts (Iriuchijima J. 1999).

The receptors found in the heart are alpha 1, alpha 2 and beta 1 adrenoceptors, and so clonidine, acetylcholine and noradrenaline drugs were used to assess the effects in comparison to each other and on they own.

The effect of all drugs used on the isolated heart was on was on spontaneous heart beat (without stimulation), where clonidine had decreased pacemaker potentials. Depending on the concentration of clonidine and the species the drug was used on, the stimulation of a2- adrenoceptors produced first an increase in heart rate which is known as the side effect of the drug, but then there was a rapid decrease in the heart rate (Young, T. M et al 2006).

Cardiac muscle is myogenic (it has the ability to contract itself via the SA Node) can generate action potentials on its own. The parasympathetic and sympathetic activity of the autonomic system has an influence on the heart rate and contractility.

Clonidine, the centrally acting vasodilator acts on the alpha 2-adrenoceptors. This activation of receptors causes a decrease in the heart rate and contractility. In figure 1b it can be seen that clonidine produces a bradycardiac effect on the heart, reducing the frequency of spontaneous SAN pacemaker potentials (Knaus, A. et al 2007). Tension decreased showing that less stress was produced therefore producing fewer contractions and heart beats.

The vascular smooth muscle and a2 â€" adrenoceptors are post-junctional linked to a G-q protein. This activates IP3 signal transduction pathway causing the contraction of the smooth muscle. Also the a2- adrenoceptors (pre-junctional) on the sympathetic nerve terminals can act as a negative feedback mechanism releasing norepinephrine (noradrenaline). Alpha-2 adrenoceptors inhibit the release of adenylyl cyclase which decreases cAMP formation causing a decrease in the force of contraction and heart rate.

Noradrenaline (an agonist) which is a sympathomimetic drug can act on both the alpha and beta receptors in the heart. In figure 3, it can be clearly seen that noradrenaline is a very potent drug that has taken effect at its highest concentration, showing that the effect is does-dependent.

The receptors for noradrenaline are G-protein-coupled receptors, associated with a specific second messenger system. The β1-adrenoceptors are found in the heart, here the receptors act by stimulating adenylyl cyclase. Therefore adding noradrenaline to the heart produces inotropic and chronotropic effects, which is an increase in this case.

Alpha-blockers such as phentolamine (usually used in treating hypertension) dilate arteries and veins. This is due to these blood vessels being innervated by the sympathetic adrenergic nerves.

Muscarinic receptors are G-protein-coupled receptors with five subtypes (M1-M5). The M1, M3 and M5 subtypes are coupled with Gq activating inositol phosphate pathway, while the M2 and M4 subtypes act through the Gi inhibiting adenylyl cyclase and reducing intracellular cAMP.

Acetylcholine a neurotransmitter acts on muscarinic receptors (M2) it decrease the heart rate and strength of contraction dilating blood vessels. It acts on muscarinic receptors which causes inhibition and or a decrease in force of contraction of the cardiac muscle.

Acetylcholine is usually produced naturally inside the body in response to a fight or flight situation, but in this study when acetylcholine was added to the isolated heart there was a decrease in the heart rate and contractility.

Addition of small amount (1x10-5M) of acetylcholine (ACh) produces a transient fall in blood pressure seen in figure 1a, this is due to arteriolar vasodilation and the slowing of the heart. Acetylcholine generally causes vasodilation, an indirect effect, which acts on the vascular endothelial cells releasing nitric oxide causing relaxation of the smooth muscles.

Here the amount of drug that is exposed to the isolated heart is important as, the higher the concentration the greater the effect seen in a short amount of time as seen in figure 3. The amount of time it takes for the drug to get to heart through the pipes was therefore timed, and added cumulatively (Knaus, A. et al 2007).

The efficacy of all drugs used was high. When clonidine was administered to the isolated heart, the effect moderate compared to noradrenaline and acetylcholine. Here acetylcholine was the drug that had the highest efficacy and due to this reason is the control.

Due to the nature of the heart and the amount of time that can be spent on one tissue is not enough to see any other effects. Further work that can be carried out is to see whether clonidine has an effect on other receptors such as a1-adrenoceptor. Here clonidine will be tested with its agonist and antagonist to see the effects that take place.

Concluding the study, the sympathomimetic drug, clonidine, does have an effect on cardiovascular system making the alpha 2 - adrenoceptors sensitive to clonidine. There is a greater effect when the concentration of clonidine is increased, however even in the presence of a low concentration of clonidine there is a small noticeable effect. The therapeutic effects of clonidine in humans can be determined to se whether the bradycardica effect still takes place as this study only looks at guinea pig and rats.