313 PHL Lab # 1 Effects and Sites of Action of Different Agonists Drugs (Stimulant or Relaxant)on The Isolated Rabbit Intestine

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 Normal time (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.

This increase or decrease in muscle contraction is observed as follows: ↑ amplitude ↓ amplitude

Or ↑ tone ↓ tone

Or ↑ tone & amplitude ↓ tone & amplitude

The nervous control of intestine: Is composed of(duodenum,jejunum,ileum) . It is a Smooth muscle has myogenic contraction (i.e. spontaneous rhythmic contraction). N.B this rhythmicity due to its pendular movement. Is involuntary muscle undergoes the control of nervous system(sympathetic and parasympathetic).

The intestine innervated by ANS : Parasympathetic Acetylcholine (Ach) . Cholinergic: Central: Nicotinic (Nn) . and peripheral: muscarinic (M3) excitatory (↑contraction) contraction Sympathetic Transmitter: noradrenalin mainly, Adrenaline . Receptors present in (intestine): Adrenergic: α1,B2 Action: inhibitory (↓contraction) relaxation .

Types of receptors present in the intestine: Adrenergic. Cholinergic. Histaminergic. Purinergic. Serotinergic.

Stimulant Drugs (Spasmogenic) A- Autonomic cholinergic stimulants , act on: 1-Nicotinic Neuronal receptor: e.g. Nicotine (small doses)= Diluted Nicotine Mechanism of Action: Diluted Nicotine activates Nn receptors in the cell membrane This will activate Na+/K+ channel  Na+ influx increase intracellular Na+  rapid depolarization  intestinal smooth muscle contraction .

2)Muscarinic Receptors (M3 subtypes): e.g. Cholinomimetic 1-Acetylcholine or its derivative : e.g. .Methacholine, Bethanecol, Carbachol. 2- Naturally occurring alkaloid : e.g. Pilocarpine.

Mechanism of Action : 1. Acetylcholine binds with M3 receptors  activation of PLC ( phospholipase C) . 2. PLC hydrolyzes PIP2(phosphotidyl inositol biphosphate) into DAG and IP3 . DAG=diacylglycerol. IP3= inositol triphosphate.

3. DAG activates Ca+ + channel  increase intracellular Ca+ +. 4.IP3 increase Ca+ + release from intracellular stores. 3,4  increase intracellular Ca+ +  intestinal smooth muscle contraction.

Stimulant Drugs (Spasmogenic) B-Direct smooth muscle stimulants : e.g. Barium Chloride Mechanism of Action: BaCl2 stimulates the muscle fiber  increase muscle contraction.

Inhibitory drugs (Spasmolytics) A-Autonomic adrenergic stimulants: e.g. Adrenaline, Noradrenaline Mechanism of Action: Adrenaline will stimulate α1 and ß 2 receptors in the intestinal smooth muscle  inhibits tone and motility  decrease intestinal contraction .

Inhibitory drugs (Spasmolytic) B-Direct Inhibitory Drugs : e.g. Mgcl2 Papaverine Mechanism of Action : Mgcl2 inhibits the contraction of muscle fiber  muscle relaxation .