Nitric oxide (NO), a vasodilator molecule produced by the enzyme nitric oxide synthase (NOS), is implicated in vascular diseases such as hypertension and stroke. A complex control mechanism has evolved for the endothelial isoform of NOS to produce NO in a well-coordinated manner in accordance with physiological needs (fig 1). For example, sub-cellular trafficking of eNOS, including internalization of membrane pools of eNOS, is believed to contribute to the control of NO production in response to agonists such as bradykinin (BK). However, the mechanisms of eNOS internalization and their implications for NO generation are not well understood (fig 2). Thus, our overall objective is to dissect the pathway of eNOS internalization under agonist influence and to study its implication in NO production. It has been shown that specific cellular GTPases such as dynamin are involved in a vesicle scission and protein trafficking (fig 3). These concepts have propelled us to formulate the central hypothesis that GTPase proteins may play a pivotal role in the internalization of eNOS and ensuing NO generation.

Objective

The broad objective is to investigate the mechanistic aspects of sub-cellular translocation of eNOS and their implications in NO production with special reference to vascular diseases

Aims

1. BK mediated internalization of plasma membrane pools of eNOS triggers NO production.
2. BK mediated internalization of eNOS from the plasma membrane is an endocytic process in which dynamin-2 (dyn-2) is coupled to the formation of clathrin-independent caveolae.

Techniques

We use cell and molecular biology techniques including pseudo-confocal imaging, live cell imaging, sub-cellular fractionation and immuno techniques.