In vivo assessment of microvascular nitric oxide production and its relation with blood flow.

To assess the hypothesis that microvascular nitric oxide (NO) is critical to maintain blood flow and solute exchange, we quantified NO production in the hamster cheek pouch in vivo, correlating it with vascular dynamics. Hamsters (100-120 g) were anesthetized and prepared for measurement of microvessel diameters by intravital microscopy, of plasma flow by isotopic sodium clearance, and of NO production by chemiluminescence. Analysis of endothelial NO synthase (eNOS) location by immunocytochemistry and subcellular fractionation revealed that eNOS was present in arterioles and venules and was 67 +/- 7% membrane bound. Basal NO release was 60.1 +/- 5.1 pM/min (n = 35), and plasma flow was 2.95 +/- 0.27 microl/min (n = 29). Local NO synthase inhibition with 30 microM N(omega)-nitro-L-arginine reduced NO production to 8.6 +/- 2.6 pmol/min (-83 +/- 5%, n = 9) and plasma flow to 1.95 +/- 0.15 microl/min (-28 +/- 12%, n = 17) within 30-45 min, in parallel with constriction of arterioles (9-14%) and venules (19-25%). The effects of N(omega)-nitro-L-arginine (10-30 microM) were proportional to basal microvascular conductance (r = 0.7, P < 0.05) and fully prevented by 1 mM L-arginine. We conclude that in this tissue, NO production contributes to 35-50% of resting microvascular conductance and plasma-tissue exchange.

Stimulation of NO production and of eNOS phosphorylation in the microcirculation in vivo.

The role of nitric oxide (NO) in microvascular permeability is controversial, in part because the regulation of its endothelial constitutive synthase, eNOS, has been studied in vitro but not in vivo. Our study was designed to detect the morphologic and functional presence of eNOS and to test whether eNOS could be phosphorylated by platelet-activating factor (PAF), an agent that induces hyperpermeability. Immunocytochemistry was applied using human anti-eNOS antibodies in the hamster cheek pouch (hcp). The hcp microvessels demonstrated positive reaction products in the endothelium. The functional presence of eNOS in hcp was investigated by topical application of 10(-7) M PAF to the hcp and by measuring NO production by chemiluminescence. The mean baseline value of NO release was 63.3 +/- 6.9 pmol/ml (mean +/- SE). Application of PAF led to an increase in mean NO release to 120.8 +/- 31.2 pmol/ml (P < 0.05). In another series of experiments, 10(-7) M PAF was applied topically to hcp preincubated with [(32)P]orthophosphoric acid. Immunoprecipitation and Western blots detected (32)P-labeled bands that migrated with the mobility of positive eNOS indicating phosphorylated eNOS protein. The intensity of the radioactive bands was evaluated by computer-assisted image analysis. Comparison of the net band intensities yielded a mean PAF-treated/control ratio of 1.6 +/- 0.1. Our data demonstrate the morphologic and functional presence of eNOS in the microcirculation. The data also provide evidence that the function of microvascular eNOS is subject to regulation by phosphorylation.

Leukocytes express connexin 43 after activation with lipopolysaccharide and appear to form gap junctions with endothelial cells after ischemia-reperfusion.

Levels and subcellular distribution of connexin 43 (Cx43), a gap junction protein, were studied in hamster leukocytes before and after activation with endotoxin (lipopolysaccharide, LPS) both in vitro and in vivo. Untreated leukocytes did not express Cx43. However, Cx43 was clearly detectable by indirect immunofluorescence in cells treated in vitro with LPS (1 micrograms/ml, 3 hr). Cx43 was also detected in leukocytes obtained from the peritoneal cavity 5-7 days after LPS-induced inflammation. In some leukocytes that formed clusters Cx43 immunoreactivity was present at appositional membranes, suggesting formation of homotypic gap junctions. In cell homogenates of activated peritoneal macrophages, Cx43, detected by Western blot analysis, was mostly unphosphorylated. A second in vivo inflammatory condition studied was that induced by ischemia-reperfusion of the hamster cheek pouch. In this system, leukocytes that adhered to venular endothelial cells after 1 hr of ischemia, followed by 1 hr of reperfusion, expressed Cx43. Electron microscope observations revealed small close appositions, putative gap junctions, at leukocyte-endothelial cell and leukocyte-leukocyte contacts. These results indicate that the expression of Cx43 can be induced in leukocytes during an inflammatory response which might allow for heterotypic or homotypic intercellular gap junctional communication. Gap junctions may play a role in leukocyte extravasation.