Mechanisms of heterogeneous endothelial cytoplasmic calcium increases in venular microvessels

Objective: Localized inflammatory leaky sites form at regions of the microvessel wall with the largest increase in endothelial cell cytoplasmic calcium concentration. [Ca²⁺]_i. We investigated the mechanisms that modulate localized increases in [Ca²⁺]_i in individual endothelial cells of microvessels after exposure to ATP. Methods: [Ca²⁺]_i was measured by using digital fluorescence microscopy and fura-2 in the endothelial cells forming the walls of individually perfused frog mesenteric microvessels. The spread of [Ca²⁺]_i from a localized mechanical stimulus was also measured. Results: The peak [Ca²⁺]_i after ATP showed marked heterogeneity, ranging from 227 to 1469 nM from resting values of 69 ± 5nM. After depolarization with high-potassium solutions, the endothelial cells with the largest peak increase in [Ca²⁺]_i had the largest fractional reduction. Localized increases in [Ca²⁺]_i due to mechanical stimulus did not spread. Conclusion: The key mechanism regulating the heterogeneity in initial peak increase in [Ca²⁺]_i is a calcium-dependent process regulated by the calcium influx itself. One such mechanism, the calcium-dependent opening of additional potassium channels leading to membrane hyperpolarization and increased driving force for calcium entry through passive conductance pathways, accounts for a significant amount of the heterogeneity of [Ca²⁺]_i in our experiments. Further investigations of both localized calcium influx and membrane potentials in the endothelial cells of intact microvessels in both frog and mammals using the imaging methods developed for these investigations are needed to understand the formation of localized leaky sites in inflamed microvessels.