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Answers written by a student to the TRIPSE problem 'Nuts about Grapes' (example 1)
Student name: N.M.
LIST 1 -3 POSSIBLE "EXPLANATIONS" FOR THE
OBSERVATIONS MADE.
You have 20 MINUTES to complete your answer. Hand your answer to the instructor when you
are finished.
1. RWH is not a reversible, competitive antagonist. It competes with PE for the binding to receptors involved in the contraction of the aorta. These receptors are located in the endothelium of the aortic ring. A second typical receptor is exposed when the endothelium is removed. This receptor also induces contraction with agonist but is not specific for RHW.
2. RWH extinguishes the effect of agonist (again, not reversible, competitive) by binding to receptors on the endothelium. Once the sub-endothelium is exposed, a new type of receptor is exposed that allows for reversible, competitive binding.
3. Receptors were exposed when the endothelium was removed that were less specific for RWH.
Explanation #2 can be tested by repeating the experiment as described; with the endothelium removed. Greater concentrations of antagonist would have to be added and the agonist dosing repeated. If the antagonist was reversible and competitive, the maximum response of the agonist would not change, and neither would the affinity of the receptor for the agonist. The curve would simply shift, parallel to the first curve, rightwards.
The EC50's with and without the presence of the antagonist can be calculated from each curve generated. The dose ratio (with antagonist / without antagonist) can be calculated and a Schild Plot, DR-1 vs log [antagonist] can be done. The slope of the Schild plot should equal 1 if all assumptions of the Schild Analysis where valid. m>1 less potent, m<1 more.
The type of antagonism seen in A can be determined. Competitive, reversible has already been ruled out because the maximum response was not regenerated.
Binding studies can determine whether it is chemical antagonism - the drugs wouldn't bind to the receptors when placed in solution together.
Pharmacokinetic antagonism can be ruled out by adding more antagonist and seeing if the metabolism is sped up or the absorption decreased.
Irreversible competitive antagonism can be determined again by binding studies. Agonist addition would not remove the antagonist.
Pharmacological antagonism can be studied by determining which point in the pathway is blocked and providing it from an external source.
Functional antagonism can be studied by examining the effects of the antagonist alone.
The relaxations produced by grape products appear to involve the endothelium derived relaxing factor (EDRF), NO or an NO derivative. In the presence of NO, the intracellular messenger adenylate cyclose catalyses the formation of cGMP. Increases in cGMP lead to protein kinase G phosphorylation and smooth muscle relaxation. Endothelial cells stimulate the synthesis of NO. Grape Skin Extracts (GSE) would decrease degradation of NO or cGMP. This, however, is not likely because of their already low levels (therefore not pharmacokinetic).
GSE added before PE reduced maximal contraction, 8EC50 of an adrenergic agonist, but had less response to KCl. This may suggest ligand gated Ca2+ channels.
Thus, some component of GSE is likely an agonist of a receptor responsible for the synthesis of nitric oxide from the endothelium. This receptor is independent of the one PE bound to for induced contraction (because PE induced contraction and no endothelium) and NO is endothelium-synthesized.
Thus, my hypothesis is not a possible explanation because the antagonists that can possibly be ruled out are a pharmacokinetic antagonist responsible for increased metabolism and any competitive antagonist.
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