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We investigated changes in calcium concentration in cultured bovine aortic endothelial cells (BAECs) and rat adrenomedulary endothelial cells (RAMECs, microvascular) in response to different levels of shear stress. In BAECs, the onset of shear stress elicited a transient increase in intracellular calcium concentration that was spatially uniform, synchronous, and dose dependent. In contrast, the response of RAMECs was heterogeneous in time and space. Shear stress induced calcium waves that originated from one or several cells and propagated to neighboring cells. The number and size of the responding groups of cells did not depend on the magnitude of shear stress or the magnitude of the calcium change in the responding cells. The initiation and the propagation of calcium waves in RAMECs were significantly suppressed under conditions in which either purinergic receptors were blocked by suramin or extracellular ATP was degraded by apyrase. Exogenously applied ATP produced similarly heterogeneous responses. The number of responding cells was dependent on ATP concentration, but the magnitude of the calcium change was not. Our data suggest that shear stress stimulates RAMECs to release ATP, causing the increase in intracellular calcium concentration via purinergic receptors in cells that are heterogeneously sensitive to ATP. The propagation of the calcium signal is also mediated by ATP, and the spatial pattern suggests a locally elevated ATP concentration in the vicinity of the initially responding cells.

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(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) > or = WT with l-NIL or iNOS(-/-) > eNOS(-/-) > or = eNOS(-/-) 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(-/-) mice but not in eNOS(-/-) 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.

One of the most important functions of the blood circulation is O2 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 O2. We measured the intraluminal and perivascular pO2 in rat mesenteric arterioles in vivo by using noninvasive phosphorescence quenching microscopy. From these measurements, we calculated the rate at which O2 diffuses out of microvessels from the blood. The rate of O2 efflux and the O2 gradients found in the immediate vicinity of arterioles indicate the presence of a large O2 sink at the interface between blood and tissue, a region that includes smooth muscle and endothelium. Mass balance analyses show that the loss of O2 from the arterioles in this vascular bed primarily is caused by O2 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 O2 the microvasculature has other functions that require a significant amount of O2.

The influence of O2-Hb reaction kinetics and the Fåhraeus effect on steady state O2 and CO2 transport in cat brain microcirculation was investigated using our refined multicompartmental model. The most important model predictions include: 1) capillaries are the sites in the microcirculation where the effect of O2-Hb kinetics is most pronounced; 2) while there is only a small difference between equilibrium and actual oxygen saturation, this effect is not negligible; 3) O2-Hb kinetics tends to make the PO2 level at the venous entrance higher than in venules; 4) the influence of the Fåhraeus effect leads to a lower tissue PO2 level than in venules and the outlet vein. The resultant decline in tissue PO2 may lead to a decrease in O2 consumption rate and extraction ratio; 5) although the Fåhraeus effect changes the ratio between arteriolar flux and capillary flux, incorporating the Fåhraeus effect and O2-Hb kinetics into the simulation does not change our previous conclusion, that most of the O2 and CO2 exchange takes place at the capillary level; 6) in general, influences of O2-Hb kinetics and Fåhraeus effect are synergistic; 7) a model that excludes these two mechanisms might overestimate the tissue oxygenation level especially during severe hypoxia.

Purpose: The purpose of this study was to investigate whether blood flow in the cat optic nerve head (ONH) is related to increased neuronal activity elicited by diffuse luminance flickering light stimulation., Methods: ONH blood flow was measured by laser Doppler flowmetry in anesthetized cats during 1 to 3 minutes of flickering light stimulation at controlled luminance and frequency (n = 227 measurements in 18 cats) using either a conventional visual stimulator (repetitive short flashes) or a sinusoidally varying light stimulator. Potassium ion concentration ([K+]) changes in the vitreous humor immediately in front of the optic disk were measured with neutral carrier K+ ionophore liquid membrane microelectrodes. Effects of varying flicker frequency (2 to 80 Hz) at constant luminance were quantified. Effects of luminance were quantified by varying the modulation depth of the stimulus at constant frequency., Results: Both ONH blood flow and [K+] increased during flicker stimulus with an average slope of 0.305% +/- 0.064% (SE)/microM [K+] (257 measurements in 18 cats). The peak ONH blood flow increase was 59% +/- 11% above baseline at 33.3 +/- 3.1 Hz. The peak [K+] increase was 188 +/- 42 microM above baseline at 38.3 +/- 3.3 Hz. Both ONH blood flow and [K+] changes had similar bandpass characteristics with frequency, first increasing, then dropping off at higher frequencies (122 measurements in 10 cats). Both frequency responses were described by power law functions (y = af"). Luminance responses for both ONH blood flow and [K+] changes could be fit by a modified Hill model and were 50% of maximum at light modulation depths of 21.2% +/- 4.6% and 22.5% +/- 3.7%, respectively (53 measurements in 5 cats)., Conclusions: Increases in ONH blood flow were correlated with changes in [K+]. Both responses were remarkably similar, with no significant differences in the frequency for peak responses in ONH blood flow or [K+], in low- and high-frequency power law exponents of the two responses, or in the 50% response to light modulation. The results are consistent with close coupling of neuronal activity and ONH blood flow.

The activation energies were calculated from oxygen consumption rates obtained from oxygen partial pressure (PO2) profiles in olfactory cortical slices of guinea pig in vitro, assuming that the Krogh coefficient varied linearly over the range of investigated temperatures (19-39 degrees C).