Nitric oxide (O), identified as endothelium-derived relaxing factor (EDRF), plays a major role in modulation of vascular tone, vasoreactivity and regional blood flow. Experiments are proposed to test hypotheses on the basic cellular mechanisms by which endothelium-dependent relaxation, mediated by NO, is controlled by local vasoactive substances ATP and bradykinin. In endothelial cells, these substances may initiate inositol lipid hydrolysis a rise of [Ca2+]i and oscillatory or propagating changes in [Ca2+]i. The formation of NO from L-arginine is regulated by a Ca2+/calmodulin=activated cytoplasmic enzyme (NO synthase) which is fully activated at [Ca2+]i during agonist induced transients and oscillations. Complex spatio-temporal patterns of cytoplasmic Ca2+ may have a key role in regulating release of NO. The four major specific aims of the proposed are: 1) determine the cellular mechanisms that control endothelial cell Ca2+ in response to vasoactive substances, 2) direct measurement of NO released from single endothelial cells by means of a novel NO-microsensor, 3) determine quantitatively how the amount of NO released is related to the change in endothelial cell Ca2+, 4) determine how NO affects [Ca2+]i in vascular smooth muscle. [Ca2+]i in endothelial and smooth muscle cells will be determined, with spatial and temporal resolution, using intracellular fluorescent indicators and digital imaging microscopy.
For aim #1, hypotheses to be tested will usually take the form of a set of simultaneous differential equations (mathematical models of [Ca2+]i-dynamics and [Ca2+]i-oscillations) describing the biochemical reactions leading to release or entry of Ca2+, the diffusion, binding and transport of Ca2+ in the endothelial cells, and the activation by cytoplasmic Ca2+ of macromolecules (NO synthase, protein kinases). Advanced techniques of image restoration, based on mathematical deconvolution (""""""""de-blurring""""""""), will provide morphological data for athe models and improve the spatial resolution of [Ca2+]i-images. Experimental interventions that will distinguish the hypotheses include rapid changes in [Ca2+]i and IP3 as induced by flash photolysis, interference with phosphoinositide metabolism, and pharmacological perturbation of intracellular Ca2+-channels, Ca2+-pumps, and Ca2+-stores.
For aim #2 a novel microsensor, a porphyrin based NO microelectrode, will allow the continuous monitoring of NO released from single endothelial cells.
For aim #3 the simultaneous use of the NO microsensor and [Ca2+]i imaging will allow to correlate the amount of NO released with the changes of [Ca2+]i in endothelial cells. The hypothesis will be tested if the amplitude and/or the frequency (frequency vs. amplitude encoded signalling) of [Ca2+]i- transients transmit the signal for NO-release.
Aim #4, the effect of NO on [Ca2+]i-regulation in vascular smooth muscle, will be achieved through exposure of smooth muscle cells to native NO, NO releasing vasodilator drugs and caged NO. The effect of NO on Ca2+ entry and extrusion, release and up-take will be studied with the combined use of [Ca2+]i-imaging and the whole-cell patch clamp technique. The proposed research is intended to provide fundamental new information on endothelial - smooth muscle cell interactions in cardiovascular function.
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