An experimental and theoretical analysis of nitric oxide availability in the microcirculation

Nitric Oxide (NO) is produced in the vascular endothelium where it then diffuses to the adjacent smooth muscle cells (SMC) activating agents known to regulate vascular tone. The close proximity of the site of NO production to the red blood cells (RBC) and its known fast consumption by hemoglobin, suggests that the blood will scavenge most of the NO produced. Therefore, it is unclear how NO is able to play its role in accomplishing vasodilation. Investigation of NO production and consumption rates will allow insight into this paradox. DAF-FM is a sensitive NO fluorescence probe widely used for qualitative assessment of cellular NO production. With the aid of a mathematical model of NO/DAF-FM reaction kinetics, experimental studies were conducted to calibrate the fluorescence signal showing that the slope of fluorescent intensity is proportional to [NO]2 and exhibits a saturation dependence on [DAF-FM]. In addition, experimental data exhibited a Km dependence on [NO]. This finding was incorporated into the model elucidating NO 2 as the possible activating agent of DAF-FM. A calibration procedure was formed and applied to agonist stimulated cells, providing an estimated NO release rate of 0.418 ± 0.18 pmol/cm2s. To assess NO consumption by RBCs, measurements of the rate of NO consumption in a gas stream flowing on top of an RBC solution of specified Hematocrit (Hct) was performed. The consumption rate constant (kbl)in porcine RBCs at 25°C and 45% Hct was estimated to be 3500 + 700 s-1. kbl is highly dependent on Hct and can reach up to 9900 + 4000 s-1 for 60% Hct. The nonlinear dependence of kbl on Hct suggests a predominant role for extracellular diffusion in limiting NO uptake. Further simulations showed a linear relationship between varying NO production rates and NO availability in the SMCs utilizing the estimated NO consumption rate. The corresponding SMC [NO] level for the average NO production rate estimated was approximately 15.1 nM. With the aid of experimental and theoretical methods we were able to examine the NO paradox and exhibit that endothelial derived NO is able to escape scavenging by RBCs to diffuse to the SMCs.

Theoretical Investigation of Intra- and Inter-cellular Spatiotemporal Calcium Patterns in Microcirculation

Microcirculatory vessels are lined by endothelial cells (ECs) which are surrounded by a single or multiple layer of smooth muscle cells (SMCs). Spontaneous and agonist induced spatiotemporal calcium (Ca2+) events are generated in ECs and SMCs, and regulated by complex bi-directional signaling between the two layers which ultimately determines the vessel tone. The contractile state of microcirculatory vessels is an important factor in the determination of vascular resistance, blood flow and blood pressure. This dissertation presents theoretical insights into some of the important and currently unresolved phenomena in microvascular tone regulation. Compartmental and continuum models of isolated EC and SMC, coupled EC-SMC and a multi-cellular vessel segment with deterministic and stochastic descriptions of the cellular components were developed, and the intra- and inter-cellular spatiotemporal Ca2+ mobilization was examined. Coupled EC-SMC model simulations captured the experimentally observed localized subcellular EC Ca2+ events arising from the opening of EC transient receptor vanilloid 4 (TRPV4) channels and inositol triphosphate receptors (IP3Rs). These localized EC Ca2+ events result in endothelium-derived hyperpolarization (EDH) and Nitric Oxide (NO) production which transmit to the adjacent SMCs to ultimately result in vasodilation. The model examined the effect of heterogeneous distribution of cellular components and channel gating kinetics in determination of the amplitude and spread of the Ca2+ events. The simulations suggested the necessity of co-localization of certain cellular components for modulation of EDH and NO responses. Isolated EC and SMC models captured intracellular Ca2+ wave like activity and predicted the necessity of non-uniform distribution of cellular components for the generation of Ca2+ waves. The simulations also suggested the role of membrane potential dynamics in regulating Ca2+ wave velocity. The multi-cellular vessel segment model examined the underlying mechanisms for the intercellular synchronization of spontaneous oscillatory Ca2+ waves in individual SMC. From local subcellular events to integrated macro-scale behavior at the vessel level, the developed multi-scale models captured basic features of vascular Ca2+ signaling and provide insights for their physiological relevance. The models provide a theoretical framework for assisting investigations on the regulation of vascular tone in health and disease.

Theoretical investigation of intra- and inter-cellular spatiotemporal calcium patterns in microcirculation

Microcirculatory vessels are lined by endothelial cells (ECs) which are surrounded by a single or multiple layer of smooth muscle cells (SMCs). Spontaneous and agonist induced spatiotemporal calcium (Ca2+) events are generated in ECs and SMCs, and regulated by complex bi-directional signaling between the two layers which ultimately determines the vessel tone. The contractile state of microcirculatory vessels is an important factor in the determination of vascular resistance, blood flow and blood pressure. This dissertation presents theoretical insights into some of the important and currently unresolved phenomena in microvascular tone regulation. Compartmental and continuum models of isolated EC and SMC, coupled EC-SMC and a multi-cellular vessel segment with deterministic and stochastic descriptions of the cellular components were developed, and the intra- and inter-cellular spatiotemporal Ca2+ mobilization was examined.^ Coupled EC-SMC model simulations captured the experimentally observed localized subcellular EC Ca2+ events arising from the opening of EC transient receptor vanilloid 4 (TRPV4) channels and inositol triphosphate receptors (IP3Rs). These localized EC Ca2+ events result in endothelium-derived hyperpolarization (EDH) and Nitric Oxide (NO) production which transmit to the adjacent SMCs to ultimately result in vasodilation. The model examined the effect of heterogeneous distribution of cellular components and channel gating kinetics in determination of the amplitude and spread of the Ca2+ events. The simulations suggested the necessity of co-localization of certain cellular components for modulation of EDH and NO responses. Isolated EC and SMC models captured intracellular Ca2+ wave like activity and predicted the necessity of non-uniform distribution of cellular components for the generation of Ca2+ waves. The simulations also suggested the role of membrane potential dynamics in regulating Ca2+ wave velocity. The multi-cellular vessel segment model examined the underlying mechanisms for the intercellular synchronization of spontaneous oscillatory Ca2+ waves in individual SMC. ^ From local subcellular events to integrated macro-scale behavior at the vessel level, the developed multi-scale models captured basic features of vascular Ca2+ signaling and provide insights for their physiological relevance. The models provide a theoretical framework for assisting investigations on the regulation of vascular tone in health and disease.^