MEDICAL PHARMACOLOGY – PCOL 425 FALL 2009 – CHOLINERGIC AGONISM AND ANTAGONISM MODULE MARCELO G. BONINI Assistant Professor College of Medicine

Overview - Cholinergic action The junction, molecular mechanism, sites of action - Muscarinic Receptor Structure, types, coupling with G-Proteins - Cholinomimetic Drugs Structure, general features, organ effects - Cholinomimetic Drugs Therapeutic uses, toxicology - Muscarinic Antagonism Drugs, therapeutic uses, toxicity

MOVIE Neurons and neurotransmitters – general overview

Generalized Cholinergic Junction

Acetylcholine binding and receptor structural changes

Cholinergic action (molecular mechanism) Intracellular signaling triggered by acetylcholine in the smooth muscle Main molecular players: M3, Heterotrimeric protein Gq, PLC/IP3, Ca(2+), MLCK

Cholinergic action (molecular mechanism) Intracellular signaling triggered by acetylcholine in the Heart Main molecular players: M2, heterotrimeric G Protein Gi, Adenylyl cyclase

Intracellular signaling triggered by acetylcholine in the endothelium eNOS ● NO L-Arg L-Citruline Major molecular players: M3, heterotrimeric G Protein Gq, Ca(2+)-CaM, eNOS, NO Cholinergic action (molecular mechanism) eNOS Nitric oxide synthase

Cholinergic action (molecular mechanism) acetylcholine mediated endothelium-dependent vasodilation Start Here +ACh NO probe - ACh NO probe

Cholinergic action (molecular mechanism) (endothelium present)(endothelium absent) - + Endothelium dependence of Ach-induced vasodilation of pre-constricted arterial rings

Sites of Cholinergic Activity -Preganglionic synapses of both sympathetic and parasympathetic ganglia - Parasympathetic postganglionic neuroeffector junctions - All somatic motor end plates on skeletal muscles M2M4M5M3M1 Gi Go Adenylyl cyclase cAMP Hyperpolarization (heart) Cardiac inhibition Antagonism of smooth muscle relaxation RECEPTOR INTRACELLULAR TRANSDUCER ELECTRICAL MECHANICAL PHYSIOLOGICAL RESPONSES Gq Phospholipase C Diacyl-glycerol IP 3 Depolarization Smooth muscle contraction Glandular secretion

Muscarinic receptor types experiments that led to their discovery M1 - Neurotransmission in Cortex and Ganglia (-/-) mice - abrogation of pilocarpine – induced seizures M2 - Agonist-mediated bradycardia, tremor, autoinhibition of release in several brain regions (-/-) mice - loss of oxytremorine-induced tremors; loss of agonist-induced bradycardia; diminished hypothermia M3 - Smooth muscle contraction, gland secreation, pupil dilation, food intake and possibly weight gain (-/-) mice - loss of agonist-induced bronchoconstriction, higher basal pupil dilation, reduction of agonist-induced salivation M4 and M5 – Central Nervous System (CNS) roles.

Classes of cholinergic stimulants Direct-acting Receptor agonists Choline esters ACETYLCHOLINE BETHANECOL Alkaloids PILOCARPINE Cholinesterase inhibitors Carbamates PHYSOSTIGMINE NEOSTIGMINE PYRIDOSTIGMINE EDROPHONIUM Phosphates ISOFLUROPHATE Antidote PRALIDOXIMINE Indirect-acting

Chemical Structure of Cholinergic agonists Tertiary amine Quaternary ammonium

Pilocarpine – source/history Chewing pilocarpus caused salivation Amazon Experiments performed in Brazil in 1874, isolated in 1875, methacholine and carbachol studies in 1911

Absorption, metabolism, distribution - Absorption: polarity dependent (poor for ACh, quaternary ammonium), intravenous, subcutaneous and intramuscular for local effects (Ach) - Metabolism: Highly dependent on the susceptibility to acetylcholinesterase (AChE) Compound Susceptibility (AChE)Muscarinic Effect Acetylcholine chloride High (++++)High (limited by AChE) Methacholine chloride Low (+)Highest (++++) Carbachol chloride NegligibleMedium (++) Bethanechol chloride NegligibleMedium (++)

Organ effects – Eye/Cardiovascular - Eyes: contraction of ciliary muscle and smooth muscle of the iris sphincter (miosis) – aqueous humor outflow, drainage of the anterior chamber - Cardiovascular: Bradycardia (possibly preceded by tachycardia), vasodilation (all vascular beds including pulmonary and coronary – M3) and hypotension, reduction of the contraction strength (atrial and ventricular cells, I K+, I Ca2+ diastolic depolarization, NO- inhibitable ATP?), negative chronotropic effect (inhibition of adrenergic activation).

- GI - increases in tone, amplitude of contractions, and peristaltic activity of the stomach and intestines, enhances secretory activity of the gastrointestinal tract. - Urinary bladder - increase ureteral peristalsis, contract the detrusor muscle of the urinary bladder, increase the maximal voluntary voiding pressure, and decrease the capacity of the bladder. - Other effects – I ncreased secretion from all glands that receive parasymphatetic enervation (salivary, lacrimal, tracheobronchial, digestive and exocrine sweat glands) - IMPORTANT - BROCHOCONSTRICTION Organ effects – GI/urinary bladder

Therapeutic uses (BETHANECHOL) Bethanechol chloride (carbamylmethylcholine chloride; URECHOLINE) Stimulant of the smooth muscle of the GI tract and the urinary bladder. Postoperative abdominal distension and gastric retention or gastroparesis. Urinary retention and inadequate emptying of the bladder when organic obstruction is absent: - postoperative - postpartum urinary retention - certain cases of chronic hypotonic or neurogenic bladder. -alternative to pilocarpine to promote salivation Xerostomia (dryness of the mouth). -Sjogren syndrome (immunologic disorder with destruction of the exocrine glands) leading to mucosal dryness

Administration/Precaution/Toxicity Bethanechol should be administered only by the oral or subcutaneous route for systemic effects; they also are used locally in the eye. Antidote - atropine. Epinephrine may be used to overcome severe cardiovascular or bronchoconstrictor responses. Among the major contra-indications to the use of the choline esters are asthma, hyperthyroidism, coronary insufficiency, and acid-peptic disease. Bronchoconstrictor action could precipitate an asthmatic attack Hyperthyroid patients may develop atrial fibrillation. Hypotension induced by these agents can severely reduce coronary blood flow, especially if it is already compromised. The gastric acid secretion produced by the choline esters can aggravate the symptoms of acid-peptic disease. POSSIBLE SIDE EFFECTS : sweating (very common), abdominal cramps, a sensation of tightness in the urinary bladder, difficulty in visual accommodation for far vision, headache, and salivation.

Therapeutic use/toxicity (carbachol/methacholine) Carbachol usually is not employed for these purposes because of its relatively larger component of nicotinic action at autonomic ganglia. The unpredictability of the intensity of response has virtually eliminated the use of methacholine or other cholinergic agonists as vasodilators and cardiac vagomimetic agents. Methacholine chloride (acetyl-b-methylcholine chloride; PROVOCHOLINE) may be administered for diagnosis of bronchial hyperreactivity and asthmatic conditions.

Toxicity/Mycetism Exageration of all symptoms of muscarinic agonism Significance: Higher consumption of wild mushrooms (culinary) minutes, salivation, lacrimation, excessive sweating, nausea, vomiting diarrhea, bronchospasm, headache, visual disturbances, abdominal colic, bradychardia, hypotension, shock ALL SYMPTOMS REVERTED BY ATROPINE1 - 2 mg intramuscular A. muscaria

Mycetism/non muscarinic Amanita phalloides – deadly nightcap Inhibits mRNA synthesis – 24 h symptom free period followed by liver and kidney malfunction, death within 4-7 days A. phalloides A. muscaria

Muscarinic antagonism Attropa belladona

History/sources Atropa belladona - used in the renaissance Deadly nightshade - used in the middle ages to produce polonged poisoning Jimson plant leaves burned in India to treat Asthma (1800) purification of atropine (1831)

Muscarinic Antagonists ATROPINE SCOPOLAMINE

Muscarinic Antagonists ATROPINE SCOPOLAMINE Attropa belladona - Atropine and Scopolamine are belladona alkaloids (competitive inhibitors) -Drugs differ in their CNS effects, scopolamine permeates the blood-brain barrier -At therapeutic doses atropine has negligible effect upon the CNS, scopolamine even at low doses has prominent CNS effects.

Mechanism of drug action - Competitively block muscarinic receptors - Salivary, bronchial, and sweat glands are most sensitive to atropine - Smooth muscle and heart are intermediate in responsiveness -In the eye, causes pupil dilation and difficulty for far vision accomodation -Relaxation of the GI, slows peristalsis

Effect of muscarinic inhibitor in the eye Pupil dilation vs accomodation

Effect of muscarinic inhibition in the heart and salivary glands - Increases the heart rate after a transient bradychardia at the low dose - Diminishes gland excretory function

Graphic summary of atropine effects

Organ effect – drug review Antidotes ORGAN DRUGAPPLICATION CNS Benztropine Treat Parkinson’s disease Scopolamine Prevent/Reduce motion sickness Eye Atropine Pupil dilation Bronchi Ipatropium Bronchodilate in Asthma, COPD GI MethscopolamineReduce motility/cramps GU OxybutininTreat transient cystitis Postoperative bladder spasms

Toxicity of muscarinic antagonists “DRY AS BONE, RED AS A BEET, MAD AS HATTER.” Dry is a consequence of decreased sweating, salivation and lacrimation Red is a result of reflex peripheral (cutaneous) vasodilation to dissipate heat (hyperthermia) Mad is a result of the CNS effects of muscarinic inhibition which can lead to sedation, amnesia (hypersensitivity), or hallucination

Preview - Indirect cholinergic agonism (Inhibition of AChE) - Nicotine-acetylcholine agonism / antagonism - Therapeutic use and toxicology