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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

Endothelial progenitor cells (EPCs) are essential in vasculogenesis and wound healing, but their circulating and wound level numbers are decreased in diabetes. This study aimed to determine mechanisms responsible for [...]

Nitric oxide (NO) is believed to play an important role in pancreatic islet physiology and pathophysiology. Research in this area has been hampered, however, by the use of indirect methods to measure islet NO. To investigate the role of NO in islet function, we positioned NOsensitive, recessed-tip microelectrodes in close proximity to individual islets and monitored oxidation current to detect subnanomolar NO in the bath. NO release from islets consisted of a series of rapid bursts lasting several seconds and/or slow oscillations with a period of 100-300 s. Average baseline NO near the islets in 2.8 mM glucose was 524 [+ or -] 59 nM (n = 12). Raising glucose from 2.8 to 11.1 mM augmented NO release by 429 [+ or -] 133 nM (n = 12, P < 0.05), an effect blocked by the NO synthase inhibitor L-NAME (n = 3). We also observed that glucose-stimulated increases in NO release were contemporaneous with changes in NAD(P)H and [O.sub.2] but occurred well before increases in calcium associated with glucose-stimulated insulin secretion. In summary, we demonstrate that NO release from islets is oscillatory and rapidly augmented by glucose, suggesting that NO release occurs early following an increase in glucose metabolism and may contribute to the stimulated insulin secretion triggered by suprathreshold glucose. calcium; oscillations; [N.sub.G]-nitro-L-arginine methyl ester; oxygen; reduced forms of islet nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate

We hypothesized that exposure to hyperbaric oxygen (HB[O.sub.2]) would mobilize stem/progenitor cells from the bone marrow by a nitric oxide (*NO) -dependent mechanism. The population of CD[34.sup.+] cells in the peripheral circulation of humans doubled in response to a single exposure to 2.0 atmospheres absolute (ATA) O2 for 2 h. Over a course of 20 treatments, circulating CD[34.sup.+] cells increased eightfold, although the overall circulating white cell count was not significantly increased. The number of colony-forming cells (CFCs) increased from 16 [+ or -] 2 to 26 [+ or -] 3 CFCs/100,000 monocytes plated. Elevations in CFCs were entirely due to the CD[34.sup.+] subpopulation, but increased cell growth only occurred in samples obtained immediately posttreatment. A high proportion of progeny cells express receptors for vascular endothelial growth factor-2 and for stromal-derived growth factor. In mice, HB[O.sub.2] increased circulating stem cell factor by 50%, increased the number of circulating cells expressing stem cell antigen-1 and CD34 by 3.4-fold, and doubled the number of CFCs. Bone marrow *NO concentration increased by 1,008 [+ or -] 255 nM in association with HB[O.sub.2]. Stem cell mobilization did not occur in knockout mice lacking genes for endothelial *NO synthase. Moreover, pretreatment of wild-type mice with a *NO synthase inhibitor prevented the HB[O.sub.2]-induced elevation in stem cell factor and circulating stem cells. We conclude that HB[O.sub.2] mobilizes stem/ progenitor cells by stimulating *NO synthesis. nitric oxide; CD34; CD133; CXCR4; cKit; colony-forming cells; progenitor cells

We tested the hypothesis that high-viscosity (HV) plasma in extreme hemodilution causes wall shear stress to be greater than low-viscosity (LV) plasma, leading to enhanced production of nitric oxide (NO). The perivascular concentration of NO was measured in arterioles and venules and the tissue of the hamster chamber window model, subjected to acute extreme hemodilution, with a hematocrit (Hct) of 11% using Dextran 500 (n = 6) or Dextran 70 (n = 5) with final plasma viscosities of 1.99 [+ or -] 0.11 and 1.33 [+ or -] 0.04 cp, respectively. HV plasma significantly increased the periarteriolar, perivenular, and tissue NO concentration by 2.0, 1.9, and 1.4 times the control (n = 7). The NO concentration with LV plasma was not statistically different from control. Arteriolar shear stress was significantly increased in HV plasma relative to LV plasma in arterioles but not in venules. Aortic endothelial NO synthase (eNOS) protein expression was increased with HV plasma but not with LV plasma. There was a weak correlation between perivascular NO concentration and the locally calculated shear stress induced by the procedures, when blood viscosity was corrected according to Hct values previously determined in studies of microvascular Hct distribution. The finding that the periarteriolar and venular NO concentration in HV plasma was the same although arteriolar shear stress was significantly greater than venular shear stress maybe be due to differences in vessel wall metabolism between arterioles and venules and the presence of NO transport through the blood stream in the microcirculation. Results support the concept that in extreme hemodilution HV plasma maintains functional capillary density through a NO-mediated vasodilatation. functional capillary density; microvascular flow; shear stress; plasma expanders; mechanotransduction

We hypothesized that elevated partial pressures of [0.sub.2] would increase perivascular nitric oxide (.NO) synthesis. Rodents with [0.sub.2-] and NO-specific microelectrodes implanted adjacent to the abdominal aorta were exposed to [0.sub.2] at partial pressures from 0.2 to 2.8 atmospheres absolute (ATA). Exposures to 2.0 and 2.8 ATA [0.sub.2] stimulated neuronal (type I) NO synthase (nNOS) and significantly increased steady-state *NO concentration, but the mechanism for enzyme activation differed at each partial pressure. At both pressures, elevations in *NO concentration were inhibited by the nNOS inhibitor 7-nitroindazole and the calcium channel blocker nimodipine. Enzyme activation at 2.0 ATA 02 appeared to be due to an altered cellular redox state. Exposure to 2.8 ATA [0.sub.2], but not 2.0 ATA [0.sub.2], increased nNOS activity by enhancing nNOS association with calmodulin, and an inhibitory effect of geldanamycin indicated that the association was facilitated by heat shock protein 90. Infusion of superoxide dismutase inhibited .NO elevation at 2.8 but not 2.0 ATA [0.sub.2]. Hyperoxia increased the concentration of .NO associated with hemoglobin. These findings highlight the complexity of oxidative stress responses and may help explain some of the dose responses associated with therapeutic applications of hyperbaric oxygen. neuronal nitric oxide synthase; heat shock protein 90; calmodulin; hyperbaric oxygen

One of the most important functions of the blood circulation is [O.sub.2] delivery to the tissue. This process occurs primarily in microvessels that also regulate blood flow and are the site of many metabolic processes that require [O.sub.2]. We measured the intraluminal and perivascular p[O.sub.2] in rat mesenteric arterioles in vivo by using noninvasive phosphorescence quenching microscopy. From these measurements, we calculated the rate at which [O.sub.2] diffuses out of microvessels from the blood. The rate of [O.sub.2] efflux and the [O.sub.2] gradients found in the immediate vicinity of arterioles indicate the presence of a large [O.sub.2] sink at the interface between blood and tissue, a region that includes smooth muscle and endothelium. Mass balance analyses show that the loss of [O.sub.2] from the arterioles in this vascular bed primarily is caused by [O.sub.2] consumption in the microvascular wall. The high metabolic rate of the vessel wall relative to parenchymal tissue in the rat mesentery suggests that in addition to serving as a conduit for the delivery of [O.sub.2] the microvasculature has other functions that require a significant amount of [O.sub.2].

Nitric oxide (NO) plays a critical role in vascular endothelial growth factor (VEGF)-induced angiogenesis and vascular hyperpermeability. However, the relative contribution of different NO synthase (NOS) isoforms to these processes is not known. Here, we evaluated the relative contributions of endothelial and inducible NOS (eNOS and iNOS, respectively) to angiogenesis and permeability of VEGF-induced angiogenic vessels. The contribution of eNOS was assessed by using an eNOS-deficient mouse, and iNOS contribution was assessed by using a selective inhibitor [L-[N.sup.6]-(1-iminoethyl) lysine, L-NIL] and an iNOS-deficient mouse. Angiogenesis was induced by VEGF in type I collagen gels placed in the mouse cranial window. Angiogenesis, vessel diameter, blood flow rate, and vascular permeability were proportional to NO levels measured with microelectrodes: Wild-type (WT) [is greater than or equal to] WT with L-NIL or [iNOS.sup.-/-] [is greater than] [eNOS.sup.-/-] [is greater than or equal to] [eNOS.sup.-/-] with L-NIL The role of NOS in VEGF-induced acute vascular permeability increase in quiescent vessels also was determined by using eNOS- and iNOS-deficient mice. VEGF superfusion significantly increased permeability in both WT and [iNOS.sup.-/-] mice but not in [eNOS.sup.-/-] mice. These findings suggest that eNOS plays a predominant role in VEGF-induced angiogenesis and vascular permeability. Thus, selective modulation of eNOS activity is a promising strategy for altering angiogenesis and vascular permeability in vivo.