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Transcript
313 PHL Lab #1 Introduction & Dose Response Curve of Different Doses of Acetylcholine Purpose of Pharmacological Experiments : The pharmacological experiments largely divided in to : Qualitative : - To determine ( analyze) the activity of drug (i.e. analgesic, hypotensive and hypoglycemic). -To determine mechanism of drugs action. Quantitative: To assay (measure) the potency of drugs (e.g. toxic level, therapeutic level, LD50). Types of pharmacological experiments : A- In vitro experiments - Study the action of drugs on isolated preparation: 1) Tissue e.g. (intestine, ileum) . 2) Organ e.g. (heart) . • Advantages: - Easier to study MOA (qualitative) . - Easier to study its potency (quantitative) e.g. DRC on rectus abdominis. • Disadvantages : - There is no interference by compensatory mechanisms of body that may affect the direct action of drug not easy to study the chemical and biological effect of the drug. B- In vivo experiments - Study the action of drugs on the whole body (intact) . • Advantages: - Observe the direct drug action(actual effect) . - Easier to study the chemical and biological effect . • Disadvantages : - Difficulty to determine the exact mechanism of action . In vitro experiments 1) Experimental conditions: An isolated organ or muscle should be bathed in a suitable media that is resemble to the normal condition in the body regarding (pH,osmolarity,temperature) . There fore, tissue is bathed in solution containing: 1- Electrolytes . 2- Nutrients . 3- Adjusted Physiological pH . 4- Constant temperature . 5- Aeration . 2) Physiological Salt Solution : • According to different tissue to be used, there is a different physiological solution prepared. • But all or most of them contain the following ingredients at different concentrations: 1- Nacl → to adjust the isotonicity. 2- Glucose → nutrient . 3- Ca+2, K+, sometimes Mg+2 as CL salt → to maintain normal muscle contraction. 3- Sodium bicarbonate → to adjust pH similar to that of blood . 4- NaH2PO4 (sodium di hydrogen phosphate) buffer→ to compensate for any change in pH . The two most commonly used physiological solutions are Tyrode’s and Krebs . 3) Aeration: Physiological salt solutions should be aerated by a suitable gas : 1- Pure oxygen → for heart tissues. 2- Normal air → for intestine. 3- Carbogen (95% O2, 5% CO2) → used for uterus . 3)Temperature: • Mammalian tissue must be bathed in a warmed solution adjusted at 37ºC e.g. Isolated rabbit intestine . • The temperature should be decreased in some experiments to reduce the myogenic contraction, the warmed solution adjusted at 32ºC e.g. Guinea-pig ileum. • Amphibian tissues survive at room temperature (25ºC) e.g. frog rectus abdominis . Dose cycle & contact time Dose cycle (NT): the time between different drugs addition. Contact time (CT): the duration of drug presence in the organ bath until its removal by washing . • CT= 30 sec, NT=60 sec, washing 3 times 30 sec for each total cycle time = 3 min. Apparatus for experiments with isolated amphibian and mammalian tissue: Physiograph and thermostatically controlled organ bath. Drugs acting on the isolated tissue: • isolated rabbit intestine has normal myogenic contraction which is under the control of the Autonomic nervous system (ANS). • The effect of drugs that act on the intestine could be a stimulatory i.e. increase in the intestinal contraction or inhibitory i.e. decrease in the intestinal contraction. The intestine innervated by ANS : Sympathetic Transmitter: noradrenalin mainly, Adrenaline . Receptors present in (intestine): Adrenergic: α1,B2 Action: inhibitory (↓contraction) relaxation . Parasympathetic Acetylcholine (Ach) . Cholinergic: Central: Nicotinic (Nn) . and peripheral: muscarinic (M3) excitatory (↑contraction) contraction • This increase or decrease in muscle contraction is observed as follows: ↑ amplitude ↓ amplitude • Or ↑ tone ↓ tone • Or ↑ tone & amplitude ↓ tone & amplitude The effect of different doses of acetylcholine on smooth muscle of intestine: N 0.05 0.1 0.2 0.3 0.4 Ach Ach Ach Ach Ach