Pharmacology Lab Report
Introduction
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Methods
Tissue preparation:
Tracheal segments were isolated from male Wistar rats aged 6‐8 weeks from the Animal Resources Centre, Murdoch, and W.A. Tissues suspended in baths containing Krebs solution maintained at 37oC and bubbled with 5% CO2 in O2 throughout the experiments. Preparations were then equilibrated for 45 min to 500 mg of tension with Kerbs solution replaced after 15 min. The viability of each tissue was evaluated by examining the contractile responses to carbachol .0.2µM was added to produce a sub maximal contraction followed by 10 µM. Once a stable contractile tone had been attained, tissues were relaxed to baseline tension (500mg) for 20 min. However, tissues that did not contract or contraction was less than 500mg were deemed unviable and discarded. Receptor‐selective agonists and antagonists were used in conjunction with isometric tension recording techniques to characterize the muscarinic cholinoceptor subtype that mediate contraction in the preparation. The functional studies provided information regarding the presence of muscarinic cholinoceptors that mediate contraction only. Thus radioligand binding techniques were also employed to determine which muscarinic cholinoceptor subtypes were present and their relative densities.
In vitro functional studies:
Experimental protocol I: The intent of this experiment was to determine the relative potencies of selected muscurinic chloinoceptor agonists. The stock solution of each agonist was diluted 10-fold and the appropriate volume was added to the tissue which produced responses as changes in tension from baseline in mg hence establishing a concentration-response curves to carbachol, acetycholine, oxotremorine, pilocarpine and methacholine that were constructed in a cumulative fashion (CDRC) using concentrations of agoinst spaced 0.5 log intervals. After each treatment of agonist, the tissue was washed and left to rest for 20 min.
Insert text for Introduction here …
Methods
Tissue preparation:
Tracheal segments were isolated from male Wistar rats aged 6‐8 weeks from the Animal Resources Centre, Murdoch, and W.A. Tissues suspended in baths containing Krebs solution maintained at 37oC and bubbled with 5% CO2 in O2 throughout the experiments. Preparations were then equilibrated for 45 min to 500 mg of tension with Kerbs solution replaced after 15 min. The viability of each tissue was evaluated by examining the contractile responses to carbachol .0.2µM was added to produce a sub maximal contraction followed by 10 µM. Once a stable contractile tone had been attained, tissues were relaxed to baseline tension (500mg) for 20 min. However, tissues that did not contract or contraction was less than 500mg were deemed unviable and discarded. Receptor‐selective agonists and antagonists were used in conjunction with isometric tension recording techniques to characterize the muscarinic cholinoceptor subtype that mediate contraction in the preparation. The functional studies provided information regarding the presence of muscarinic cholinoceptors that mediate contraction only. Thus radioligand binding techniques were also employed to determine which muscarinic cholinoceptor subtypes were present and their relative densities.
In vitro functional studies:
Experimental protocol I: The intent of this experiment was to determine the relative potencies of selected muscurinic chloinoceptor agonists. The stock solution of each agonist was diluted 10-fold and the appropriate volume was added to the tissue which produced responses as changes in tension from baseline in mg hence establishing a concentration-response curves to carbachol, acetycholine, oxotremorine, pilocarpine and methacholine that were constructed in a cumulative fashion (CDRC) using concentrations of agoinst spaced 0.5 log intervals. After each treatment of agonist, the tissue was washed and left to rest for 20 min.
References: Faheema Adat Molecular Pharmacology 3301, School of Medicine and Pharmacology, University of Western Australia, Australia, 6009.