The mammalian brain contains a regulatory center in the diencephalic region known as the hypothalamus that plays a critical role in physiological homeostasis, and contains a variety of centers for behavioral drives, such as hunger and thirst. Located deep within the hypothalamus is the suprachiasmatic nucleus (SCN), or the master biological clock, that organizes rhythmic physiology and behavior, such that critical events take place at the most appropriate time of the day or night and in the most appropriate temporal, phase relationships. Cell-to-cell communication is essential for conveying inputs to and outputs from the SCN. The goal of the present study was to use an immortalized neural cell line (SCN2.2), derived from the presumptive anlage of the rat suprachiasmatic nucleus, as an in vitro model system to study intercellular communication among SCN cells. I tested whether the pineal neurohormone melatonin could modulate cell-to-cell signaling, via both dye coupling (gap junctional communication) and calcium waves (ATP-dependent gliotransmission). I also tested whether extracellular ATP could influence the spread of calcium waves in SCN2.2 cells. Lastly, the ability of extracellular ATP to modulate SCN physiological responses to melatonin in SCN2.2 cells was examined. I show that melatonin at a physiological concentration (nM) reduced dye coupling (gap junctional communication) in SCN2.2 cells, as determined by a scrape loading procedure employing the fluorescent dye lucifer yellow. Melatonin caused a significant reduction in the spread of calcium waves in cycling SCN2.2 cultures as determined by ratiometric calcium imaging with Fura-2 AM, a calcium sensitive indicator dye. This reduction was greatest when an endogenous circadian rhythm in extracellular ATP accumulation, determined by luciferase assay, was at its trough or lowest extracellular concentration. In addition, melatonin and ATP interacted in the regulation of gliotransmission (calcium waves), and this interaction was also specific to particular phases of the endogenous SCN physiological rhythmicity. Thus, I have established that a complex interaction exists between established melatonin signaling pathways and this newly discovered ATP accumulation rhythm, with the mechanisms underlying this relationship linked to endogenous cycling of SCN cellular physiology.

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.