Semester

Summer

Date of Graduation

2013

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

S. Jamal Mustafa.

Abstract

Cardiovascular diseases are one of the leading causes of morbidity and mortality worldwide. The regulation of vascular tone plays an important role in normal cardiovascular function. Adenosine, an autacoid has several physiological and pathophysiological roles, apart from the regulation of vascular tone. Adenosine receptor (AR) contracts and relaxes blood vessels through all four subtypes (A1, A2A, A2B, and A3) linked to different signaling mechanisms. Deciphering complex tissue responses remains difficult because relationships of individual receptor subtypes and various end-effectors (e.g., ion channels) are yet to be identified. Apart from adenosine, 20-HETE, a cytochrome P4504a (Cyp4a) metabolite of arachidonic acid (AA) is a potent vasoconstrictor.;We hypothesized that A1AR induced contraction of the smooth muscle involves Cyp4a, with Protein Kinase C (PKC)-alpha, extracellular regulated kinase (ERK) 1/2 contributing to the downstream signaling events. Another key question we addressed were the ion channel(s) contributing to smooth muscle contraction. Experiments included isometric tension recordings of aortic contraction and western blots. In addition, patch clamp experiments were done with freshly isolated smooth muscle cells from wild type (WT) and A1 knockout (A1KO) mice aortae. We found that inhibition of Cyp4a led to lesser contraction in the adenosine agonists' mediated responses. 20-HETE induced contraction in both WT and A1KO, but this response was lower in A1KO. Inhibition of PKC-alpha and ERK1/2 attenuated the 20-HETE-induced contraction in both WT and A1KO. These findings suggest that A1AR couples with 20-HETE and negatively modulates vascular tone through PKC-alpha and ERK1/2. Furthermore, electrophysiological experiments revealed that non-selective adenosine agonist increased the BK current in A1KO as compared to the WT. This suggests A1 receptors have a negative regulatory effect on BK current. On the other hand, A1 selective agonist decreased the BK current in WT, with no effect on A1KO. PKC-alpha inhibitor abolished the effect of the A 1 selective agonist on BK current. These findings suggest that A 1AR regulates contraction of the aortic smooth muscle through inhibition of BK channels in a PKC-alpha dependent manner. From these data, we conclude that A1AR negatively couples with 20-HETE and by inhibiting BK channels mediates smooth muscle contraction via PKC-alpha.

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