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Date of Award

2-1999

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Medical Sciences

Supervisor

Professor A.K. Grover

Abstract

Endothelin-1, the most potent endogenous vasoconstrictor peptide known, plays a key role in regulating coronary artery vascular tone. Reactive oxygen species generated during cardiac ischemia-reperfusion cause several types of damage to cardiovascular tissues, in particular to the intracellular Ca2+ -regulating mechanisms. This thesis explores the endothelin-1 receptor types in coronary artery smooth muscle and their signal transduction pathways and the effects of reactive oxygen on them . Endothelin-1 mediated contraction of de-endothelialized pig coronary artery rings. There are two types of endothelin receptors known: ETA and ETB . Using ETA and ETB receptors selective agonists and antagonists, the contraction mediated by ETB -receptors was approximately 20% and the remaining was due to ETA receptors. Ca2+ pools mobilized by the two receptors were similar except that the ETB receptor activation utilized more of the intracellular Ca2+ pool than the ETA activation. 125 I-ET-1 binding to microsomes isolated from smooth muscle of this artery also showed ETA and ETB binding sites with most binding occurring at the ETA sites. Thus, the endothelin-1 induced vasoconstrictor response in pig coronary artery smooth muscle involves both ETA and ETB -receptors with ETA being predominant. Pretreating the artery with hydrogen peroxide inhibited the subsequent contraction upon ETA or ETB receptor activation. However, the ETB -mediated contraction was significantly (p < 0.05) sensitive to peroxide (IC50 = 0.3 ± 0.08 mM) than the ETA receptor mediated contraction (IC50 = 1 ± 0.3 mM). Pretreating smooth muscle cells cultured from pig coronary artery with 0.3 mM hydrogen peroxide inhibited the endothelin-1 induced increase in [Ca2+ ] i by more than 95%. Thus the exposure to reactive oxygen damaged the ETB receptor mediated contractions preferentially, possibly as a result of a Ca2+ -independent component in ETA receptor mediated contractions. ATP-dependent azide-insensitive oxalate-stimulated Ca2+ -uptake in permeabilized smooth muscle cells cultured from pig coronary artery exhibited the expected kinetic properties of the sarcoplasmic reticulum (SR) Ca 2+ -pump. Under optimum conditions, inositol 1,4,5 trisphosphate (IP 3 ) released up to 65% of 45 Ca2+ -loaded into the SR. Pretreating the cells with hydrogen peroxide or superoxide did not affect the IP3 dependent Ca2+ -release but inhibited the Ca2+ -uptake in the SR. Peroxide was equipotent in inhibiting 45 Ca2+ -loading into IP3 -sensitive and IP 3 -insensitive Ca2+ pools but superoxide inhibited loading only into the IP3 -sensitive pool indicating that the SR Ca 2+ pump in vascular smooth muscle cells is heterogenous. These results indicate that both ETA and ETB receptors are involved in ET-1 mediated contraction in smooth muscle pig coronary artery, with similar Ca2+ utilization pathways but the ETA receptors may also induce contraction in part by a Ca2+ -independent mechanism. The peroxide pretreatment damages the SR Ca2+ pump and this leads to a diminished contraction by endothelin-1, with the exception of the Ca2+ -independent mechanism(s) associated with ET A receptor activation which may be resistant to peroxide. This Ca 2+ -independent mechanism provides a potential therapeutic target for diseases where ETA plays a major role. The second major finding is the heterogeneity of SR Ca2+ pool which can also be used in designing pharmacological agents specific to a distinct component of the SR Ca2+ pool.

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