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We developed a two-dimensional model of transport-dependent intracellular calcium signaling in endothelial cells (ECs). Our purpose was to evaluate the effects of spatial colocalization of endothelial nitric oxide synthase (eNOS) and capacitative calcium entry (CCE) channels in caveolae on eNOS activation in response to ATP. Caveolae are specialized microdomains of the plasma membrane that contain a variety of signaling molecules to optimize their interactions and regulate their activity. In ECs, these molecules include CCE channels and eNOS. To achieve a quantitative understanding of the mechanisms of microdomain calcium signaling and the preferential sensitivity of eNOS to calcium entering the cell through CCE channels, we constructed a mathematical model incorporating the cell morphology and cellular physiological processes. The model predicts that the spatial segregation of calcium channels in ECs can create transport-dependent sharp gradients in calcium concentration within the cell. The calcium concentration gradient is affected by channel density and cell geometry. This transport-dependent calcium signaling specificity effect is enhanced in ECs by increasing the spatial segregation of the caveolar signaling domains. Our simulation significantly advances the understanding of how [Ca.sup.2+], despite its many potential actions, can mediate selective activation of signaling pathways. We show that diffusion-limited calcium transport allows functional compartmentalization of signaling pathways based on the spatial arrangements of [Ca.sup.2+] sources and targets. capacitative calcium entry; caveolae; endothelial cells: endothelial nitric oxide synthase; mathematical model

The instability and low concentration of nitric oxide (NO) in specimens make it difficult to be detected directly. In this chapter, a method for imaging nitric oxide using laser scanning confocal microscopy (LSCM) is presented. A cultured hippocampal neuron is dyed with DAF-2 and observed under a Zeiss LSM 510 laser scanning confocal microscope. A 488-nm laser power is applied as excitation and the emission light from the labeled nitric oxide in the neuron is detected. In this way, nitric oxide is imaged and its intracellular kinetic change is monitored. Furthermore, image processing and visualization techniques are employed to help analyze the image data.