Determination of intracellular calcium inhibition effect and cytotoxocity of engineered Bradykinin antagonists.

Abstract

Chronic pain is a physiological issue universally associated with many medical conditions, severe injuries and major surgeries. Bradykinin (BK) and kallidin (KD) are short-term peptidic mediators that double as potent endogenous pain inducers. Jointly known as kinins, their releases occur during tissue injury or injurious stimulation, and modulate pain by activating both B1 and B2 G-protein-coupled receptors (GPCR). Activated by the kinins’ carboxypeptidase metabolites, des-Arg9-BK and des-Arg10-KD, the B1 receptor stimulates the chronic phase of the inflammatory pain response while the acute phase is stimulated by the B2 receptor. Different chronic pain responses are mediated by the B1 receptor through phospholipase C activation, which triggers diacylglycerol and inositol triphosphate release, further activating protein kinase C and Ca2+ mobilization.

This report journals all relevant methods and protocols employed in the tests conducted on the engineered bradykinin peptidic antagonists.

The experiments in this final year project involve the loading of BK antagonists onto fluorescent HeLa cells and the subsequent addition of B1 agonists to trigger a transient increase in the level of intracellular Ca2+ ions, so this inhibition effect reflected in the gradual Ca2+ level decrease caused by the antagonists can then be measured. In addition, the degrees of toxicity of these BK antagonists towards cervical cancer cells are evaluated.