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BIOASSAY OF HISTAMINE BIOASSAY OF HISTAMINE
GIRIJA MAGANTI
MPHARM (PHARMACOLOGY)
What is histamine?What is histamine?
• Histamine is a small ,water soluble molecule or amine autocoid or locally acting hormone which mediates its effects by binding to receptors H1,H2,H3,H4.
Histamine synthesisHistamine synthesis
Site of synthesis and storage:Site of synthesis and storage:
Histamine is synthesized and stored in the following sites:
1- Neurons in the brain
2- Entero chromaffin cells in the gastric mucosa
3- Mast cells
Non mast cell sources of histamine Non mast cell sources of histamine in the body in the body
1- Brain: (functions as neurotransmitter)
2- Entero chromaffin cells (EC) in the stomach
(function: stimulates HCL secretion by parietal cells of the stomach)
RELEASE OF HISTAMINE FROM RELEASE OF HISTAMINE FROM MAST CELLMAST CELL1.Immune mediated 1.Immune mediated
2.Non immune mediated 2.Non immune mediated
• Certain drugs such as morphine and tubocurarine, can displace histamine from mast cells. This type of release does not require prior exposure.
Pharmacological actions of Pharmacological actions of histaminehistamine
• The pharmacological actions of histamine depend on the tissue and type of receptor present at the area of release
Histamine receptorsHistamine receptorsR subtype Distribution Action Agonist Antagonist
H1 Sm. Muscle Contraction of GIT,bronchoconstricton.
Hist. Mepyramine
Cyproheptadine
H2 Gastric mucosa
Acid release Hist. Cimetidine
Ranitidine
H3 Presynaptic Autoregulation of histamine release
Hist. Thioperamide
H4 Eosinophils
Neutrophils
CD4T cells
Modulate the production of blood cells & cytokines
Hist. Thioperamide
Symptoms associated with histamine Symptoms associated with histamine release from mast cellsrelease from mast cells
• Mild cutaneous release
• Moderate release
• Severe release (anaphylactic)
• erythema, urticaria, and/or itching
• skin reactions, tachycardia, moderate hypotension, mild respiratory distress
• severe hypotension, ventricular fibrillations, cardiac arrest, bronchospasm, respiratory arrest
DefinitionDefinition• Estimation of the conc / potency of a
substance by measuring its biological
response in living systems
• i.e.Observation of pharmacological effects on
• [1] living tissues, or cells
• [2] microorganisms
• [3] animals
Bioassay of histamine can be done by recording
1. Contractions of isolated guinea pig ileum.
2. BP fall in anaesthetised cat or dog .
Bioassay using guinea pig ileumBioassay using guinea pig ileum
• Bioassay of histamine on isolated guinea pig ileum can be determined by
Matching bioassayInterpolation bioassayBracketing assayMultiple point assays
Ileum Ileum
• 3/5 of intestine
• Empties in the large intestine via ileocecal valve
• Bile salts, vitamin B12, water and electrolytes absorption
• Doesn’t have myogenic contraction
• More sensitive to histamine action
Histamine receptors in ileum Histamine receptors in ileum
• H1 receptors• Receptor type: G-protein-coupled receptor
• Agonist: Histamine
• MOA: ++ G-protein → ++phspholipase C → splitting of PIP2 into 1) DAG that increases the opening of calcium channels 2)IP3 which increases calcium mobilization from sarcoplasmic stores;
DAG & IP3 lead to increase in the intracellular concentration of calcium and smooth muscles contraction.
REQUIREMENTSREQUIREMENTS
• Instruments:thermo statically controlled organbath,chymograph,aerator
• Physiological solution:tyrode’s solution
• Temperature:32°c
• Animal :guinea pig
• Standard histamine solution(10µg/ml)
Preparing standardPreparing standard
• Take 10mg of histame + 10ml of water
(1000µg/ml)
• Take0.1ml and dilute with 10mlwater(10µg/ml)
THREE POINT BIOASSAYTHREE POINT BIOASSAY
• 2STANDARD
• 1 TEST
BIOASSAY USING BIOASSAY USING ANAESTHETISED CAT OR ANAESTHETISED CAT OR
DOGDOG• Cat or dog is anaesthetised with chloralose
or barbiturate and prepared for recording of BP
• Sensitivity is determined by injecting standard solution of 0.05,0.1,0.15µg of histamine base per kg bodyweight is given for 5min interval
• A fixed dose of standard producing a fall in BP about 20mmHg is injected with changing doses of test at regular intervals and matching assay is done
MECHANISM OF BP FALLMECHANISM OF BP FALL
• histamine binds wth histamineH1 receptor of endothelium causes release of EDRF (endothelium derived relaxing factor). This EDRF diffuses out and reaches the smooth muscle of the arteriole and causes generation of cyclic-GMP → causing reduction of Ca++ in smooth muscle relaxation of the smooth muscle→ arteriolar dilatation. Another possibility is that combination of H1 with the Histamine causes release of
PGI2 which causes vasodilatation.
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