CHOLINERGIC AGONISTS AND ANTAGONISTS Welcome to Pharmacology Lab 2! You should have completed Lab 1 before proceeding with this material. It would be very helpful if you review your lecture notes and/or your text concerning the subject material to be covered in this lab experiment. The purpose of this lab is to demonstrate some of the effects of drugs which influence the parasympathetic nervous system and its effector organs. In the last experiment, we anesthetized a cat and prepared it for recording blood pressure, heart rate, and a few other measurements. We'll use the same system here too. We were lucky to obtain a cat which again weighs exactly 2 kg. It was anesthetized with pentobarbital 35 mg/kg. This cat should be adequately anesthetized for about 6 hours. We cannulated the animal as we did in the first experiment, so you should have no problems proceeding with the experiment. 1. Which of the following is true about acetylcholine? stimulates the muscarinic receptors stimulates the adrenal medulla is released from parasympathetic pre- and postganglionic nerve endings is a mediator at both sympathetic and parasympathetic ganglia Excellent. No. Many different sites are affected by acetylcholine. All foils are correct! 2. Acetylcholine would directly produce which of the following? miosis increased G.I. motility bronchoconstriction glycogenolysis That's right. Nope, the first three foils are correct! Finally, we are ready to see what happens when we give a low dose of 0.1 microgram/kg dose of acetylcholine. Effect of 0.1 microgram/kg acetylcholine. 3. Why would there be minimal effects on the cardiovascular system with this dose of acetylcholine? rapid destruction of the compound cardiovascular compensatory reflexes the dose is small tolerance to acetylcholine Very good. No. Why wouldn't there be reflexes to correct the change in BP? The first three foils are correct! No. There is no tolerance to acetylcholine. The first three foils are correct! No. The first three foils are correct! 4. Which enzyme(s) is/are responsible for the rapid termination of the actions of acetylcholine? acetylcholinesterase MAO anticholinesterase pseudocholinesterase methylcholinesterase That's right! No. Remember which enzymes may inactivate acetylcholine? The first and fourth foils are correct! Let's see what happens when we increase the dose of acetylcholine to 0.5 micrograms/kg. Effect of 0.5 micrograms/kg acetylcholine. 5. The increase in the heart rate seen after the administration of the drug is due to direct action of the drug on the heart sympathetic reflex stimulation complete metabolism of the drug by acetylcholinesterase vasoconstriction of the splanchnic vessels none of the above That's right. No. The answer is B. Remember what happens to the blood pressure with ACh: The HR and BP initially decrease due to a direct action of the drug on the muscarinic receptors of the heart and the endothelium lining the blood vessels. Because of reflex mechanisms, there is an increased sympathetic discharge producing an increase in the heart rate and returning the peripheral resistance toward normal. This, along with rapid destruction of acetylcholine by cholinesterase restored the mean blood pressure. We must also remember that still larger doses of acetylcholine also stimulate nicotinic receptors and cause the release of epinephrine and norepinephrine from both the adrenal medulla and the sympathetic postganglionic nerves. 6. The next drug we will administer to this cat is neostigmine methylsulfate (0.05 mg/kg). Which of the following is a property of neostigmine? anticholinesterase blocks muscarinic receptors acts on skeletal muscle irreversibly binds to cholinesterase Hooray! You got a hard question right. Nope, The first annd third foils are correct! ,7. Now that we know that neostigmine is an anticholinesterase, let's discuss what happens at some of the organs affected by this drug. When neostigmine is applied locally to the eye, which of the following changes can take place miosis decreased intraocular pressure spasm of accommodation constriction of the intraocular vessels That's right. No. This one is a hard one. The first three foils are correct. Neostigmine prolongs the action of acetylcholine by preventing its hydrolysis by acetylcholinesterase. 8. If neostigmine prolongs the action of endogenous acetylcholine, what would we expect neostigmine's effect on the BP to be? no change because there is little cholinergic innervation in the blood vessels the BP would markedly decrease due to enhanced effects of endogenous acetylcholine the BP would markedly decrease because of the decreased HR the BP would increase because of sympathetic stimulation all of the above could occur Very good. No. What kind of innervation is present in the vessels? The answer is A. We won't show you on a graph what happens to the blood pressure tracing after the administration of neostigmine because nothing usually happens except some miosis, but just remember that we gave neostigmine and its effects will last for the duration of the experiment. 9. What will happen now if we give a small dose of ACh (0.1 microg/kg)? the BP will show the same effect as before the administration of neostigmine the response will be similar to a large dose of acetylcholine the BP will remain the same there will be a prolonged increase in the BP there will be a decrease in the BP but less than before neostigmine I see that you were paying attention! No. The answer is B. The effect of this small dose is shown in the next figure. If we give a small dose of acetylcholine (0.1 microgram/kg) after neostigmine, this is what happens: It produces the same action as a large dose. 10. What would happen if we administered acetylcholine in a large dose (0.5 microgram/kg) after neostigmine? there would be a parasympathomimetic effect there would be no change in the BP there would be a prolonged effect the BP would show a biphasic response characteristic of large doses of acetylcholine Very good. A large dose effect would occur. No. The first and third foils are correct. If a large dose of acetylcholine is given after neostigmine you would still get the same response except that the magnitude and duration of the response would be greater. 11. The next drug to be administered will be atropine sulfate 1 mg/kg. What would you expect the effects to be? bronchoconstriction increase in the heart rate urinary incontinence miosis Marvelous. Nope, the answer is B. Atropine will block the muscarinic receptors so you would expect it to block those in the heart. Recall that the predominant innervation to the heart is parasympathetic, therefore, an antagonist of endogenous acetylcholine would increase heart rate. 12. The following can be attributed to atropine administration. constipation mydriasis dry mouth increased urination Excellent deduction. Since nothing of any significance happens to the BP after the administration of atropine, let's proceed to the next drug. Acetylcholine will be given in a dose of 0.5 microgram/kg. No. The first three foils are correct! Since nothing of any significance happens to the BP after the administration of atropine, let's proceed to the next drug. Acetylcholine will be given in a dose of 0.5 microgram/kg. 13. After administration of this dose (0.5 microg/kg) of acetylcholine, which of the following would be true? the muscarinic receptors are blocked so there will be an initial fall in the BP there will be a decrease in the mean BP there will be an increase in the mean BP there will be no change in the mean BP That's fine. No. What happens when we block the muscarinic receptors? Only the fourth foil is correct! Let's refer to our pressure tracing to confirm what is happening: 14. What would you expect if we gave acetylcholine in an extremely large dose of 0.5 mg/kg. Note that this is 1000X larger than our previous dose of 0.5 microgram/kg. the BP would increase the BP would decrease then increase in a biphasic response the BP would decrease the BP would not significantly change none of the above That's right. No. Think about which receptors are being stimulated! The answer is A. This is what happens with a very large dose of acetylcholine. 15. Why, when we administered a dose of acetylcholine which is extremely large, did we get an increase in BP? the vessels do not have any cholinergic receptors the muscarinic effect of acetylcholine is effectively blocked by atropine so nicotinic effects become apparent this dose blocks the adrenergic receptors this dose stimulates the adrenergic receptors none of the above Correct. No. Think about other kinds of receptors acetylcholine stimulates! The answer is B. As you can see, there is a pressor response to the administration of an extremely large dose of acetylcholine. This is primarily due to the effects of acetylcholine on nicotinic receptors stimulating the sympathetic ganglia and the adrenal medulla with a resultant release of catecholamines. 16. Hexamethonium is the next drug which our animal will receive. Do you remember what the actions of hexamethonium are? Let's see what you can recall. Hexamethonium has the following action(s) antagonizes the effects of nicotine antagonizes the effects of DMPP is a ganglionic blocker inhibits muscarinic receptors Very sharp. That's wrong, but don't feel bad, the first three foils are correct. You may not have heard of DMPP. So let's discuss it. DMPP is short for dimethylphenylpiperazinium. (Now you know why most people call it DMPP). Anyway, DMPP is a ganglionic stimulant which acts in a manner similar to nicotine. Remember it, because it may be an unknown in a later lab. Getting back to the ganglionic blocker, hexamethonium, let's see what happens when we administer a dose of 2 mg/kg.: 17. Why did hexamethonium decrease the HR? hexamethonium blocks beta receptors hexamethonium stimulates muscarinic receptors hexamethonium blocks sympathetic ganglia vagal stimulation none of the above Very good. No. The answer is C. We should note that hexamethonium also blocks transmission at parasympathetic ganglia. However, para- sympathetic activity in this animal has already been blocked because of the administration of atropine earlier. Therefore, blockade of parasympathetic ganglia at this point of the experiment is of no consequence as far as the heart rate is concerned. On the other hand, sympathetic activity was still intact in this animal and as we saw, hexamethonium, by blocking sympathetic ganglionic transmission, produced a slowing in heart rate. 18. We are now ready to administer a super large dose of acetylcholine once again. The dose will be 0.5 mg/kg (1000 X normal). Administration of acetylcholine will now produce which of the following? no change in the mean BP no change in the systolic BP no change in the heart rate decreased BP Very good. No. Only the fourth foil is correct! Hexamethonium has blocked the nicotinic actions of the super large dose of acetylcholine. The latter now produces a small fall in blood pressure as a manifestation of a residual effect on muscarinic receptors (which were incompletely blocked by the dose of atropine employed). Let's confirm our prediction, that a large dose of acetylcholine in the presence of atropine and hexamethonium will cause a fall in blood pressure.