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Adrenoceptor Types And Subtypes Of Essay

The adrenergic receptors (or adrenoceptors) are a class of G protein-coupled receptors that are targets of the catecholamines, especially norepinephrine (noradrenaline) and epinephrine (adrenaline).

Many cells possess these receptors, and the binding of a catecholamine to the receptor will generally stimulate the sympathetic nervous system. The sympathetic nervous system is responsible for the fight-or-flight response, which includes dilating the pupil, increasing heart rate, mobilizing energy, and diverting blood flow from non-essential organs to skeletal muscle.

History[edit]

Main article: History of catecholamine research

By the turn of the 19th century, it was agreed that the stimulation of sympathetic nerves could cause different effects on body tissues, depending on the conditions of stimulation (such as the presence or absence of some toxin). Over the first half of the 20th century, two main proposals were made to explain this phenomenon:

  1. There were (at least) two different types of neurotransmitter released from sympathetic nerve terminals, or
  2. There were (at least) two different types of detector mechanisms for a single neurotransmitter.

The first hypothesis was championed by Walter Cannon and Arturo Rosenblueth,[1] who interpreted many experiments to then propose that there were two neurotransmitter substances, which they called sympathin E (for 'excitation') and sympathin I (for 'inhibition').

The second hypothesis found support from 1906 to 1913, when Henry Dale explored the effects of adrenaline (which he called adrenine at the time), injected into animals, on blood pressure. Usually, adrenaline would increase the blood pressure of these animals. Although, if the animal had been exposed to ergotoxine, the blood pressure decreased.[2][3] He proposed that the ergotoxine caused "selective paralysis of motor myoneural junctions" (i.e. those tending to increase the blood pressure) hence revealing that under normal conditions that there was a "mixed response", including a mechanism that would relax smooth muscle and cause a fall in blood pressure. This "mixed response", with the same compound causing either contraction or relaxation, was conceived of as the response of different types of junctions to the same compound.

This line of experiments were developed by several groups, including Marsh and colleagues,[4] who in February 1948 showed that a series of compounds structurally related to adrenaline could also show either contracting or relaxing effects, depending on whether or not other toxins were present. This again supported the argument that the muscles had two different mechanisms by which they could respond to the same compound. In June of that year, Raymond Ahlquist, Professor of Pharmacology at Medical College of Georgia, published a paper concerning adrenergic nervous transmission.[5] In it, he explicitly named the different responses as due to what he called α receptors and β receptors, and that the only sympathetic transmitter was adrenaline. While the latter conclusion was subsequently shown to be incorrect (it is now known to be noradrenaline), his receptor nomenclature and concept of two different types of dectors mechanisms for a single neurotransmitter, remains. In 1954, he was able to incorporate his findings in a textbook, Drill's Pharmacology in Medicine,[6] and thereby promulgate the role played by α and β receptor sites in the adrenaline/noradrenaline cellular mechanism. These concepts would revolutionise advances in pharmacotherapeutic research, allowing the selective design of specific molecules to target medical ailments rather than rely upon traditional research into the efficacy of pre-existing herbal medicines.

Categories[edit]

There are two main groups of adrenergic receptors, α and β, with several subtypes.

  • α receptors have the subtypes α1 (a Gq coupled receptor) and α2 (a Gi coupled receptor[7]). Phenylephrine is a selective agonist of the α receptor.
  • β receptors have the subtypes β1, β2 and β3. All three are linked to Gs proteins (although β2 also couples to Gi),[8] which in turn are linked to adenylate cyclase. Agonist binding thus causes a rise in the intracellular concentration of the second messenger cAMP. Downstream effectors of cAMP include cAMP-dependent protein kinase (PKA), which mediates some of the intracellular events following hormone binding. Isoprenaline is a non-selective agonist.

Roles in circulation[edit]

Epinephrine (adrenaline) reacts with both α- and β-adrenoreceptors, causing vasoconstriction and vasodilation, respectively. Although α receptors are less sensitive to epinephrine, when activated at pharmacologic doses, they override the vasodilation mediated by β-adrenoreceptors because there are more peripheral α1 receptors than β-adrenoreceptors. The result is that high levels of circulating epinephrine cause vasoconstriction. However, the opposite is true in the coronary arteries, where β2 response is greater than that of α1, resulting in overall dilatation with increased sympathetic stimulation. At lower levels of circulating epinephrine (physiologic epinephrine secretion), β-adrenoreceptor stimulation dominates since epinephrine has a higher affinity for the β2 adrenoreceptor than the α1 adrenoreceptor, producing vasodilation followed by decrease of peripheral vascular resistance.

Subtypes[edit]

Smooth muscle behavior is variable depending on anatomical location. Smooth muscle contraction/relaxation is generalized below. One important note is the differential effects of increased cAMP in smooth muscle compared to cardiac muscle. Increased cAMP will promote relaxation in smooth muscle, while promoting increased contractility and pulse rate in cardiac muscle.

ReceptorAgonist potency orderSelected action of agonistMechanismAgonistsAntagonists
α1: A, B, DNorepinephrine > epinephrine >> isoprenalineSmooth muscle contraction, mydriasis, vasoconstriction in the skin, mucosa and abdominal viscera & sphincter contraction of the GI tract and urinary bladderGq: phospholipase C (PLC) activated, IP3,and DAG, rise in calcium

(Alpha-1 agonists)

(Alpha-1 blockers)

(TCAs)

Antihistamines (H1 antagonists)

α2: A, B, CEpinephrine = norepinephrine >> isoprenaline (however do consider that a bit of Norepinephrine was already used in a1, thus giving Epinephrine a relatively stronger affinity in a2)Smooth muscle mixed effects, norepinephrine (noradrenaline) inhibition, platelet activationGi: adenylate cyclase inactivated, cAMP down

(Alpha-2 agonists)

(Alpha-2 blockers)
β1Isoprenaline > epinephrine = norepinephrinePositive Chronotropic, Dromotropic and inotropic effects, increased amylase secretionGs: adenylate cyclase activated, cAMP up1-adrenergic agonist)(Beta blockers)
β2Isoprenaline > epinephrine >> norepinephrineSmooth muscle relaxation (Ex. Bronchodilation)Gs: adenylate cyclase activated, cAMP up (also Gi, see α2)2-adrenergic agonist)(Beta blockers)
β3Isoprenaline = norepinephrine > epinephrineEnhance lipolysis, promotes relaxation of detrusor muscle in the bladderGs: adenylate cyclase activated, cAMP up3-adrenergic agonist)(Beta blockers)

There is no α1C receptor. At one time, there was a subtype known as C, but was found to be identical to one of the previously discovered subtypes. To avoid confusion, naming was continued with the letter D.

α receptors[edit]

α receptors have several functions in common, but also individual effects. Common (or still unspecified) effects include:

α1 receptor[edit]

Main article: Alpha-1 adrenergic receptor

α1-adrenergic receptors are members of the Gq protein-coupled receptor superfamily. Upon activation, a heterotrimeric G protein, Gq, activates phospholipase C (PLC). The PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2), which in turn causes an increase in inositol triphosphate (IP3) and diacylglycerol (DAG). The former interacts with calcium channels of endoplasmic and sarcoplasmic reticulum, thus changing the calcium content in a cell. This triggers all other effects, including a prominent slow after depolarizing current (sADP) in neurons [12]

Specific actions of the α1 receptor mainly involve smooth muscle contraction. It causes vasoconstriction in many blood vessels, including those of the skin, gastrointestinal system, kidney (renal artery)[13] and brain.[14] Other areas of smooth muscle contraction are:

Further effects include glycogenolysis and gluconeogenesis from adipose tissue[16] and liver, as well as secretion from sweat glands[16] and Na+ reabsorption from kidney.[16]

Antagonists may be used primarily in hypertension, anxiety disorder, and panic attacks.

α2 receptor[edit]

Main article: Alpha-2 adrenergic receptor

The α2 receptor couples to the Gi/o protein.[7] It is a presynaptic receptor, causing negative feedback on, for example, norepinephrine (NE). When NE is released into the synapse, it feeds back on the α2 receptor, causing less NE release from the presynaptic neuron. This decreases the effect of NE. There are also α2 receptors on the nerve terminal membrane of the post-synaptic adrenergic neuron.

There are 3 highly homologous subtypes of α2 receptors: α2A, α, and α2C.

Specific actions of the α2 receptor include:

  • inhibition of insulin release in the pancreas.[16]
  • induction of glucagon release from the pancreas.
  • contraction of sphincters of the gastrointestinal tract
  • negative feedback in the neuronal synapses - presynaptic inhibition of norepinephrine (NE) release in CNS
  • increased thrombocyte aggregation

β receptors[edit]

β1 receptor[edit]

Main article: Beta-1 adrenergic receptor

Specific actions of the β1 receptor include:

  • Increase cardiac output by increasing heart rate (positive chronotropic effect), conduction velocity (positive dromotropic effect), stroke volume (by enhancing contractility—positive inotropic effect), and rate of relaxation of the myocardium, by increasing calcium ion sequestration rate (positive lusitropic effect), which aids in increasing heart rate.
  • Increase renin secretion from juxtaglomerular cells of the kidney.
  • Increase ghrelin secretion from the stomach.[17]

β2 receptor[edit]

Main article: Beta-2 adrenergic receptor

The β2 receptor "binds epinephrine and is involved in the fight or flight response".[18]

Specific actions of the β2 receptor include the following:

β3 receptor[edit]

Main article: Beta-3 adrenergic receptor

Specific actions of the β3 receptor include:

  • Enhancement of lipolysis in adipose tissue. β3 activating drugs could theoretically be used as weight-loss agents, but are limited by the side effect of tremors.

See also[edit]

References[edit]

  1. ^Cannon WB, Rosenbluth A (31 May 1933). "Studies On Conditions Of Activity In Endocrine Organs XXVI: Sympathin E and Sympathin I". American Journal of Physiology. 104 (3): 557–574. 
  2. ^Dale HH (May 1906). "On some physiological actions of ergot". The Journal of Physiology. 34 (3): 163–206. doi:10.1113/jphysiol.1906.sp001148. PMC 1465771. PMID 16992821. 
  3. ^Dale HH (Jun 1913). "On the action of ergotoxine; with special reference to the existence of sympathetic vasodilators". The Journal of Physiology. 46 (3): 291–300. doi:10.1113/jphysiol.1913.sp001592. PMC 1420444. PMID 16993202. 
  4. ^Marsh DT, Pelletier MH, Rose CA (Feb 1948). "The comparative pharmacology of the N-alkyl-arterenols". The Journal of Pharmacology and Experimental Therapeutics. 92 (2): 108–20. PMID 18903395. 
  5. ^Ahlquist RP (Jun 1948). "A study of the adrenotropic receptors". The American Journal of Physiology. 153 (3): 586–600. PMID 18882199. 
  6. ^Drill VA (1954). Pharmacology in medicine: a collaborative textbook. New York: McGraw-Hill. 
  7. ^ abQin K, Sethi PR, Lambert NA (Aug 2008). "Abundance and stability of complexes containing inactive G protein-coupled receptors and G proteins". FASEB Journal. 22 (8): 2920–7. doi:10.1096/fj.08-105775. PMC 2493464. PMID 18434433. 
  8. ^Chen-Izu Y, Xiao RP, Izu LT, Cheng H, Kuschel M, Spurgeon H, Lakatta EG (Nov 2000). "G(i)-dependent localization of beta(2)-adrenergic receptor signaling to L-type Ca(2+) channels". Biophysical Journal. 79 (5): 2547–56. Bibcode:2000BpJ....79.2547C. doi:10.1016/S0006-3495(00)76495-2. PMC 1301137. PMID 11053129. 
  9. ^Nisoli E, Tonello C, Landi M, Carruba MO (Jan 1996). "Functional studies of the first selective beta 3-adrenergic receptor antagonist SR 59230A in rat brown adipocytes". Molecular Pharmacology. 49 (1): 7–14. PMID 8569714. 
  10. ^Elliott J (Aug 1997). "Alpha-adrenoceptors in equine digital veins: evidence for the presence of both alpha1 and alpha2-receptors mediating vasoconstriction". Journal of Veterinary Pharmacology and Therapeutics. 20 (4): 308–17. doi:10.1046/j.1365-2885.1997.00078.x. PMID 9280371. 
  11. ^Sagrada A, Fargeas MJ, Bueno L (Aug 1987). "Involvement of alpha-1 and alpha-2 adrenoceptors in the postlaparotomy intestinal motor disturbances in the rat". Gut. 28 (8): 955–9. doi:10.1136/gut.28.8.955. PMC 1433140. PMID 2889649. 
  12. ^Smith RS, Weitz CJ, Araneda RC (Aug 2009). "Excitatory actions of noradrenaline and metabotropic glutamate receptor activation in granule cells of the accessory olfactory bulb". Journal of Neurophysiology. 102 (2): 1103–14. doi:10.1152/jn.91093.2008. PMC 2724365. PMID 19474170. 
  13. ^Schmitz JM, Graham RM, Sagalowsky A, Pettinger WA (Nov 1981). "Renal alpha-1 and alpha-2 adrenergic receptors: biochemical and pharmacological correlations". The Journal of Pharmacology and Experimental Therapeutics. 219 (2): 400–6. PMID 6270306. 
  14. ^Circulation & Lung Physiology I M.A.S.T.E.R. Learning Program, UC Davis School of Medicine
  15. ^Moro C, Tajouri L, Chess-Williams R (Jan 2013). "Adrenoceptor function and expression in bladder urothelium and lamina propria". Urology. 81 (1): 211.e1–7. doi:10.1016/j.urology.2012.09.011. PMID 23200975. 
  16. ^ abcdefFitzpatrick D, Purves D, Augustine G (2004). "Table 20:2". Neuroscience (Third ed.). Sunderland, Mass: Sinauer. ISBN 0-87893-725-0. 
  17. ^Zhao TJ, Sakata I, Li RL, Liang G, Richardson JA, Brown MS, et al. (Sep 2010). "Ghrelin secretion stimulated by {beta}1-adrenergic receptors in cultured ghrelinoma cells and in fasted mice". Proceedings of the National Academy of Sciences of the United States of America. 107 (36): 15868–73. Bibcode:2010PNAS..10715868Z. doi:10.1073/pnas.1011116107. PMC 2936616. PMID 20713709. 
  18. ^Liszewski, Kathy (1 October 2015). "Dissecting the Structure of Membrane Proteins". Genetic Engineering & Biotechnology News (paper). 35 (17): 16. (subscription required)
  19. ^"Adrenergic and Cholinergic Receptors in Blood Vessels". Cardiovascular Physiology. Retrieved 5 May 2015. 
  20. ^Large V, Hellström L, Reynisdottir S, et al. (Dec 1997). "Human beta-2 adrenoceptor gene polymorphisms are highly frequent in obesity and associate with altered adipocyte beta-2 adrenoceptor function". The Journal of Clinical Investigation. 100 (12): 3005–13. doi:10.1172/JCI119854. PMC 508512. PMID 9399946. 
  21. ^Kline WO, Panaro FJ, Yang H, Bodine SC (Feb 2007). "Rapamycin inhibits the growth and muscle-sparing effects of clenbuterol". Journal of Applied Physiology. 102 (2): 740–7. doi:10.1152/japplphysiol.00873.2006. PMID 17068216. 
  22. ^Kamalakkannan G, Petrilli CM, George I, et al. (Apr 2008). "Clenbuterol increases lean muscle mass but not endurance in patients with chronic heart failure". The Journal of Heart and Lung Transplantation. 27 (4): 457–61. doi:10.1016/j.healun.2008.01.013. PMID 18374884. 
  23. ^Santulli, G.; Lombardi, A.; Sorriento, D.; Anastasio, A.; Del Giudice, C.; Formisano, P.; Beguinot, F.; Trimarco, B.; Miele, C.; Iaccarino, G. (2012). "Age-Related Impairment in Insulin Release: The Essential Role of 2-Adrenergic Receptor". Diabetes. 61 (3): 692–701. doi:10.2337/db11-1027. ISSN 0012-1797. PMC 3282797. PMID 22315324. 
  24. ^Kim, Soo Mi; Briggs, Josephine P.; Schnermann, Jurgen (2011). "Convergence of major physiological stimuli for renin release on the Gs-alpha/cyclic adenosine monophosphate signaling pathway". Clinical and Experimental Nephrology. 16 (1): 17–24. doi:10.1007/s10157-011-0494-1. ISSN 1342-1751. 
  25. ^Elenkov IJ, Wilder RL, Chrousos GP, et al. (Dec 2000). "The sympathetic nerve--an integrative interface between two supersystems: the brain and the immune system". Pharmacological Reviews. 52 (4): 595–638. PMID 11121511. 

Further reading[edit]

  • Rang HP, Dale MM, Ritter JM, Moore PK (2003). "Chapter 11: Noradrenergic transmission". Pharmacology (5th ed.). Elsevier Churchill Livingstone. ISBN 0-443-07145-4. 
  • Rang HP, Dale MM, Ritter JM, Flower RJ (2007). "Chapter 11: Noradrenergic transmission". Rang and Dale's Pharmacology (6th ed.). Elsevier Churchill Livingstone. pp. 169–170. ISBN 0-443-06911-5. 

External links[edit]

Adrenergic receptormodulators

α1
  • Antagonists:Abanoquil
  • Adimolol
  • Ajmalicine
  • Alfuzosin
  • Amosulalol
  • Anisodamine
  • Arotinolol
  • Atiprosin
  • Atypical antipsychotics (e.g., brexpiprazole, clozapine, olanzapine, quetiapine, risperidone)
  • Benoxathian
  • Buflomedil
  • Bunazosin
  • Carvedilol
  • Corynanthine
  • Dapiprazole
  • Domesticine
  • Doxazosin
  • Ergolines (e.g., ergotamine, dihydroergotamine, lisuride, terguride)
  • Etoperidone
  • Eugenodilol
  • Fenspiride
  • Hydroxyzine
  • Indoramin
  • Ketanserin
  • L-765,314
  • Labetalol
  • mCPP
  • Mepiprazole
  • Metazosin
  • Monatepil
  • Moxisylyte
  • Naftopidil
  • Nantenine
  • Neldazosin
  • Niaprazine
  • Nicergoline
  • Niguldipine
  • Pardoprunox
  • Pelanserin
  • Perlapine
  • Phendioxan
  • Phenoxybenzamine
  • Phentolamine
  • Phenylpiperazineantidepressants (e.g., hydroxynefazodone
The mechanism of adrenergic receptors. Adrenaline or noradrenaline are receptor ligands to either α1, α2 or β-adrenergic receptors. α1 couples to Gq, which results in increased intracellular Ca2+ and subsequent smooth muscle contraction. α2, on the other hand, couples to Gi, which causes a decrease in neurotransmitter release, as well as a decrease of cAMP activity resulting in smooth muscle contraction. β receptors couple to Gs, and increases intracellular cAMP activity, resulting in e.g. heart muscle contraction, smooth muscle relaxation and glycogenolysis.
Beta-2 adrenergic receptor (PDB: 2rh1​), which stimulates cells to increase energy production and utilization. The membrane is shown schematically with a gray stripe.

Introduction Adrenoceptors are membrane bound receptors located throughout the body neuronal and nonneuronal tissues where they mediate a diverse range of responses to endogenous catecholamines noradrenaline and adrenaline .Adrenoceptors are first divided into two subtypes; α and β adrenoceptors as determined by pharmacological studies in isolated tissues. α and β adrenoceptors are further subdivided into α1, α2 and β1, β2 and β3 respectively. β-adrenoceptors are defined by the catecholamine potency series of Isoprenaline > Adrenaline > Noradrenaline, whereas α-adrenoceptors are defined by the series of Noradrenaline > Adrenaline > Isoprenaline. (Starke et al., 1975) .The binding of catecholamines to the adrenoreceptors causes stimulation of the sympathetic nervous system. Adrenoreceptor agonists are able to cause either relaxation or contraction of a tissue depending on the presence of different subtypes of adrenoreceptors in the organ. Noradrenaline has high potency for α-adrenoceptors causing smooth muscle contractions whereas Isoprenaline has high potency for β-adrenoceptors causing smooth muscle relaxation. Therefore, subtype of adrenoreceptors present on an isolated smooth muscle tissue can be identified by determining the potency of isoprenaline, adrenaline and noradrenaline through measuring the magnitude of contraction. Designation of receptor subtypes on a particular isolated tissue is also possible with the usage of adrenoreceptors antagonists such as propranolol that specifically block β adrenoceptors and phentalamine which blocks α-adrenoceptors (Kenny et al., 1996). Drugs that mimic the action of adrenaline or noradrenaline are termed sympathomimetic drugs. Sympathomimetic are divided into direct-acting agents and indirect-acting agents. Direct-acting agents such as noradrenaline are drugs whose sympathomimetic actions result from direct binding to α- or β-adrenoceptors. On the other hand, actions of indirect-acting agents are dependent on the release of endogenous catecholamines. Indirect-acting agents act indirectly by preventing reuptake of a neurotransmitter or stimulating the production and release of cytoplasmic noradrenaline(Von Euler and Lishajko, 1968). Both types of sympathomimetics, direct and indirect, ultimately cause activation of adrenoceptors, leading to some or all of the characteristic effects of endogenous catecholamines. Isolated rat vas deferens preparation is a very useful tool to observe the actions of α and βadrenoreceptor agonists and antagonists as it is rich in sympathetic innervation which helps in demonstrating how different drugs interact with each other and adrenoreceptors in causing tissue response. Aims This practical aims to identify the subtypes of adrenoreceptors present in the rat vas deferens by ranking the potency of Noradrenaline, adrenaline and isoprenaline in order to investigate how chemical structures of these catecholamine affect their potency. This practical also aims to confirm the subtypes of adrenoreceptors through actions of antagonists such as propranolol and phentolamine. Besides that, it also aims to investigate the action of indirectly acting sympathomimetic amines such as tyramine and the effects of uptake blockers such as Desipramine on tyramine and noradrenaline. Materials and Methods An isolated rat vas deferens tissue was bathed in a 20 ml of Holman’s solution supplied with carbogen and maintained at 37°C under 1g resting tension. Table 1: Volume (ml) of prepared drug concentration (M) to be added to the isolated rat vas deferens tissue bath to make up final drug bath concentration (M) of each drug added; isoprenaline, adrenaline, noradrenaline, tyramine, desmethylimipramine, propranolol and phentolamine used in Part A, B and C of the experiment The respective final bath concentrations (M) which produced a sub-maximal response are also shown. Drug to be added to the Volume of prepared drug Final drug bath concentration tissue bath concentration to be added to (M) Noradrenaline the tissue bath 0.2mL of 1 x 10-5 M 1 x 10-7 0.2mL of 1 x 10-4 M 1 x 10-6 0.2mL of 1 x 10-3 M 1 x 10-5 0.2mL of 1 x 10-2 M 1 x 10-4 (sub-maximal 0.2mL of 1 x 10-5 M 0.2mL of 1 x 10-4 M 0.2mL of 1 x 10-3 M concentration ) 1 x 10-7 1 x 10-6 1 x 10-5 Adrenaline 0.2mL of 1 x 10-2 M 1 x 10-4 (sub-maximal concentration ) 0.2mL of 1 x 10-3 M 1 x 10-5 0.2mL of 1 x 10-2 M 1 x 10-4 0.2mL of 1 x 10-1 M 1 x 10-3 2.0mL of 1 x 10-1 M 1 x 10-2(sub-maximal Tyramine 0.2mlof 2x 10-1M concentration ) 2x10-5 (sub-maximal Desmethylimipramine Propranolol Phentolamine 0.2mlof 2x 10-1M 0.2 ml of 1×10-4M 0.2 ml of 1×10-4M concentration ) 1 x 10-6 1×10-6 1×10-6 Isoprenaline Part A: Rank order of potency From 10-2 M stock solutions for each noradrenaline and adrenaline, serial dilutions ranging from 10-2 M to 10-5 were prepared. Whereas, from 10-1 M stock solution for isoprenaline, serial dilutions ranging from 10-1 M to 10-3 were prepared. Volume (ml) of each prepared concentration (M) for each of the drug was used to prepare final bath drug concentration (M) according to table (1). For each drug, following the addition of the first final bath concentration(M) , the response ( tension of contraction of smooth muscle in g) of the isolated rat vas deferens was observed on the PowerLab chart program for 30 seconds. After which, the tissue bath solution was refilled again with 20 ml of Holman’s solution after draining to wash the isolated rat vas deferens tissue before the addition of the second final bath concentration(M) of same drug. This process was repeated until the final bath concentration (M) of same drug and the tissue response tension in g) was recorded. The same process repeated for each of Noradrenaline, Adrenaline and Isoprenaline. The submaximal concentration (M) which produced the sub-maximal response (tension in g) for was recorded for each drug. The resulting four point concentration response of each drug was plotted in a graph whereby the response (tension g) is plotted against the logarithm of the final bath concentration (M) of drug used. Part B: Direct and indirectly – acting amines Volume (ml) of 2.0 x 10-2 M tyramine stock solution was used to prepare submaximal final bath drug concentration (M) according to table (1). From 10-3 M stock solution for desmethylimipramine (DMI), a serial dilution from 10-3 M to 10-2 was prepared and volume (ml) of prepared diluted concentration (M) of the drug was used to prepare final bath drug concentration (M) according to table (1).The sub-maximal final bath concentration (M) of noradrenaline determined from part A was added to the isolated rat vas deferens tissue in 20ml of Holman’s solution, and the response in tissue contraction (tension in g) was observed. After which, the tissue bath solution was refilled again with 20 ml of Holman’s solution after draining to wash the isolated rat vas deferens tissue before the addition of submaximal final bath concentration (M) of tyramine observing if a 30% of previous tissue’s response(tension in g) obtained for 30 seconds. Desipramine (DMI) was added to the tissue bath after washing and refilling with 20ml of Holman solution, and was left for incubation for 10 minutes. After which, the sub-maximal final bath concentration (M) of noradrenaline was added into the tissue bath together with DMI, and the tissue’s response (tension in g) was observed. The exact same final bath concentration (M) of DMI was added again to the tissue bath and incubated for 3 minutes. After which, the sub-maximal final bath concentration (M) of tyramine was added into the tissue bath together with DMI , and the tissue’s response (tension in g) was observed for 30 seconds . Part C: α- and β- adrenoreceptor antagonists From 10-3 M stock solutions for each propranolol and phentolamine, serial dilutions ranging from 10-3 M to 10-4 were prepared. Volume (ml) of each prepared concentration (M) for each drug was used to prepare final bath drug concentration (M) according to table (1).The sub-maximal concentrations (M) for noradrenaline and isoprenaline were used from part A and isolated rat vas deferens tissue’s response(tension in g) was recorded for each of these drugs. After which, the tissue bath solution was refilled again with 20 ml of Holman’s solution after draining to wash the tissue before the addition of final bath concentration (M) of propranolol and left for incubation for 10 minutes. The final bath sub-maximal concentrations (M) of noradrenaline or isoprenaline was each then added, and the tissue’s response (tension in g) to each of these agonists in the presence of propranolol was observed for 30 seconds and recorded. The tissue bath was drained washing the tissue and refilling with 20 ml of Holman’s solution between the addition of noradrenaline and isoprenaline. Same process was repeated for both final bath sub-maximal concentrations (M) of noradrenaline and isoprenaline each in the presence of final bath concentration (M) of phentolamine , and the tissue’s response (tension in g) to each of these agonists in the presence of phentolamine was observed for 30 seconds and recorded. RESULTS Part A: Rank order of potency Table 1 : Final bath concentrations (M) of Noradrenaline, Adrenaline and Isoprenaline added to Holman bath solution and observed magnitude of contractions (tension in g) of rat isolated vas deferens tissue bathed in the solution. Description of expected responses (tension in g) listed as well. Drug added Noradrenaline Adrenaline Isoprenaline Final bath drug concentration (M) Gm tension of Expected description 1x10 contractions 0.18 of response/effect Gradual increase as 1x10-6 1x10-5 1x10-4(submaximal concentration) 0.67 1.19 1.55 the final bath M of 1x10-7 1x10-6 1x10-5 0.03 0.39 1.00 1x10-4 (submaximal concentration) 1.24 1x10-5 0.06 1x10-4 1x10-3 0.00 0.00 1x10-2(submaximal concentration) 0.68 -7 Noradrenaline increases Gradual increase as the final bath M of adrenaline increases No response Response(gm tension of contraction ) 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 -8 -7 -6 -5 -4 -3 -2 0 -1 Log (Final bath drug concentration M) Adrenaline Isoprenaline Noradrenaline Figure 1: Graph of gm tension of smooth muscle contraction of the isolated rat vas deferens tissue bathed in Holman solution versus Log (Final bath drug concentration M) for adrenaline, noradrenaline and isoprenaline. Part B: Direct and indirectly-acting amines Table 2: Submaximal final bath concentrations (M) of each noradrenaline and tyramine added alone and each in presence of desmethylimipramine(DMI) in Holman bath solution ,and observed magnitude of contractions (tension in g) of rat isolated vas deferens tissue bathed in the solution in response to each of these drug additions. Description of expected responses (tension in g) listed as well. Drug added Final bath drug concentration Tension(g) Expected (M) of description of contractions 2.08 response/effect Increase -4 Noradrenaline Submaximal concentration 1x10 Tyramine Submaximal concentration 2x10-5 1.27 Noradrenaline in the Submaximal concentration 1x10-4 1.94 Increase 0.00 No change presence of desmethylimipramine (DMI) Tyramine in the presence of Increase of Noradrenaline in presence of 1x10-6 of DMI Submaximal concentration 2x10-5 of Tyramine in presence of 1x10- 6 desmethylimipramine of DMI (DMI) Part C: α- and β- adrenoreceptor antagonist Table 3: Submaximal final bath concentrations (M) of each noradrenaline and isoprenaline each added in presence of propranolol or phentalamine in Holman bath solution ,and observed magnitude of contractions (tension in g) of rat isolated vas deferens tissue bathed in the solution in response to each of these drug additions. Description of expected responses (tension in g) listed as well. Drug added Final bath drug Tension(g) of Expected concentration (M) contractions description of Noradrenaline 1x10 (submaximal 0.17 response/effect Increase Isoprenaline concentration 1x10-4(Submaximal 0.69 Decrease Noradrenaline in the concentration) Submaximal 0.66 Increase presence of concentration 1x10-6 of Propranolol Noradrenaline in -6 presence of 1x10-6 of Propranolol Isoprenaline in the Submaximal presence of concentration 1x10-4 of Propranolol Isoprenaline in presence 0.01 Increase of 1x10-6 of Propranolol Noradrenaline in the Submaximal presence of concentration 1x10-6 of Phentolamine Noradrenaline in presence of 1x10-6 of 1.46 Decrease Isoprenaline in the Phentolamine Submaximal presence of concentration 1x10-4 of Phentolamine Isoprenaline in presence 0.01 Decrease of 1x10-6 of Phentolamine DISCUSSION The concentration-response curve in figure (1) show that at the same concentration (M), noradrenaline produces larger response (tension in g) of the isolated rat vas deferens tissue as compared to adrenaline while isoprenaline needs higher concentration (M) for a response. Drugs with higher potency evoke a larger response at low concentration, while drugs of lower potency produce a small response at low concentrations .This indicates that noradrenaline has the highest potency, followed by adrenaline and lastly isoprenaline. Therefore, the rank of potency for the agonists at the adrenoceptors in this tissue preparation is noradrenaline > adrenaline > isoprenaline. Potency of an agonist depends on both the affinity (the ability of a ligand to bind to a receptor) and efficacy is (the ability of the ligand to initiate a response). Noradrenaline has the highest ability to bind to α-adrenoceptors available initiating the greatest response even at low concentrations compared to isoprenaline. Whereas, isoprenaline has the highest ability to bind to β-adrenoceptors available initiating the greatest response at low concentrations compared to noradrenaline (Starke et al., 1975). Based on the rank order of potency, it can be concluded that the subtype of adrenoceptors present in the rat vas deferens are α-adrenoceptors. β- adrenoceptors are defined by the catecholamine potency series of Isoprenaline > Adrenaline > Noradrenaline. However, α-adrenoceptors are defined by the series of Noradrenaline > Adrenaline > Isoprenaline. The rank order of potency of the three catecholamines corresponds with the rank order of potency for α-adrenoceptors where noradrenaline is found to be the most potent and isoprenaline the least potent which means that α-adrenoceptors are present in the rat vas deferens. Differences in the chemical structures of catecholamines will results in differences in potencies. Noradrenaline, adrenaline and isoprenaline all contain catechols, which are chemicals that have two hydroxyl groups adjacent to a benzene ring and a side chain amine (HORN et al., 1971). The three differ in terms of substituent on the amino group. It is this change in the substituent on the amino group that lead to shifting of potency of the catecholamine. Increasing the size of alkyl substituents on the amino group tends to increase catecholamines potency upon β-adrenoceptors but reduces potency at α-adrenoceptors (HORN et al., 1971).For example, methyl substitution on noradrenaline, yielding adrenaline enhances activity at β-adrenoceptors. Isoprenaline potency on β-adrenoceptors is further enhanced with isopropyl substitution at the amino nitrogen. Hence, βadrenoceptors selective agonists generally require a large amino substituent group. In contrast, the larger the substituent on the amino group, the lower the activity at α-adrenoceptors and hence lower potency. Noradrenaline has no methyl substitution at the end of the N-terminal, making it the most potent for α-adrenoreceptor .Addition of more methyl substituent to noradrenaline as seen in adrenaline and isoprenaline shows a decrease in potency for α-adrenoreceptors (HORN et al., 1971). Part C is done to further clarify the rank of potency of the catecholamines and the distribution of receptors in rat vas deferens. The identification of adrenoceptor subtype is further confirmed by the use of antagonists such as phentolamine and propranolol in part C. Propranolol blocks βadrenoceptors while phentolamine blocks α-adrenoceptors reversibly(Kenny et al., 1996). Based on table 3, it is shown that in the presence of phentolamine, both sub-maximal tensions (g) for noradrenaline and isoprenaline increase and increase slightly respectively. For same final bath concentrations of both phentolamine and noradrenaline, the increase in sub-maximal tension (g) seen after addition of noradrenaline in presence of phentolamine confirms the high potency of noradrenaline at the α-adrenoceptors. Since noradrenaline has a high affinity to α-adrenoceptors compared to the antagonist phentolamine, then noradrenaline replace phentolamine at the αadrenoceptors reducing the blocking effect of binding of the antagonist to the α-adrenoceptors and thus increasing adrenergic transmission instead. This confirms noradrenaline is the most potent catecholamine for α-adrenoceptors present on rat vas deferens tissue. On the other hand, there is slight contraction (tension in g) with the addition of sub-maximal concentration of isoprenaline in the presence of phentalamine. Isoprenaline is the most potent catecholamine for β-adrenoceptors leading the smooth muscle of isolated tissue to relax thus no observed tension of contraction (g) should be expected. However, tension (g) increases slightly even in the presence of Isoprenaline indicating absence of β-adrenoceptors in the isolated tissue to bind to isoprenaline. Moreover, same submaximal tension (g) is observed for Isoprenaline in presence of propranolol (β-adrenoceptors antagonist) and in presence of phentolamine which suggests that with or without the presence of β-adrenoceptors, there is no difference in tissue response observed even with the presence of isoprenaline as there is not enough β-adrenoceptors in the isolated tissue to bind to isoprenaline. In part B of the experiment, the magnitude of tension (in g) for noradrenaline in the presence of desmethylimipramine (DMI) increases. DMI is a tricyclic antidepressant agent that inhibits noradrenaline reuptake by blocking noradrenaline transporter (NET). This interferes with the major mechanism for terminating the action of noradrenaline, causing accumulation of noradrenaline in the synaptic cleft(Von Euler and Lishajko, 1968)Thus, increasing stimulation at the postsynaptic membrane and hence resulting in the increase in magnitude of contraction (g) as seen. In contrast, the magnitude of tension (g) of the isolated tissue for tyramine remains unchanged in the presence of DMI. Tyramine is an indirectly acting sympathomimetic amine, exerting its pharmacological actions by the release of noradrenaline from presynaptic stores. Due to chemical resemblance to noradrenaline, tyramine is taken up into nerve terminal by NET .It causes displacement of vesicular noradrenaline when it is taken into vesicles, thereby resulting in non-vesicular release of noradrenaline from the nerve terminal(Von Euler and Lishajko, 1968). DMI antagonizes the action of tyramine by blocking noradrenaline transporter, preventing access of tyramine to vesicles storing noradrenaline and thereby preventing pharmacological response .Hence, DMI inhibits tyramine-evoked contractile response and results in the unchanged magnitude of tension(g) seen for tyramine in the presence of DMI. DMI will shift the concentration-response curve of noradrenaline to the left (Von Euler and Lishajko, 1968).This is because at any concentration, the amount of noradrenaline would be higher as a result of the molecules not being taken up and therefore the sympathetic innervation is increased. The potency of noradrenaline is increased in the presence of DMI. Therefore, the concentration response curve shifts to the left. Conclusion α-adrenoceptors are present in the rat vas deferens as the rank order of potency of the catecholamine was noradrenaline > adrenaline > isoprenaline. Increasing the size of alkyl substituents on the amino group increases catecholamines potency upon β-adrenoceptors but reduces potency at α-adrenoceptors. Presence of inhibitory action of phentolamine on noradrenaline confirms the presence of α-adrenoceptors on rat vas deferens. Tyramine causes release of noradrenaline. Desipramine inhibits uptake of noradrenaline and antagonizes the action of tyramine, increasing contraction tension (g) for noradrenaline from while tension (g) for tyramine remains unchanged. Desipramine causes a left shift of the concentration-response curve for noradrenaline. References HORN, A. S., COYLE, J. T. & SNYDER, S. H. 1971. Catecholamine Uptake by Synaptosomes from Rat Brain Structure-Activity Relationships of Drugs with Differential Effects on Dopamine and Norepinephrine Neurons. Molecular pharmacology, 7, 66-80. KENNY, B., MILLER, A., WILLIAMSON, I., O'CONNELL, J., CHALMERS, D. & NAYLOR, A. 1996. Evaluation of the pharmacological selectivity profile of α1 adrenoceptor antagonists at prostatic α1 adrenoceptors: binding, functional and in vivo studies. British journal of pharmacology, 118, 871-878. STARKE, K., ENDO, T. & TAUBE, H. 1975. Relative pre-and postsynaptic potencies of αadrenoceptor agonists in the rabbit pulmonary artery. Naunyn-Schmiedeberg's archives of pharmacology, 291, 55-78. VON EULER, U. & LISHAJKO, F. 1968. Effect of directly and indirectly acting sympathomimetic amines on adrenergic transmitter granules. Acta Physiologica Scandinavica, 73, 78-92.

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