Leukocyte adherence inhibits adenosine-dependent venular control of arteriolar diameter and nitric oxide

Venular control of arteriolar perfusion has been the focus of several investigations in recent years. This study investigated 1) whether endogenous adenosine helps control venule-dependent arteriolar dilation and 2) whether venular leukocyte adherence limits this response via an oxidant-dependent mechanism in which nitric oxide (NO) levels are decreased. Intravital microscopy was used to assess changes in arteriolar diameters and NO levels in rat mesentery. The average resting diameter of arterioles (27.5 ± 1.0 μm) paired with venules with minimal leukocyte adherence (2.1 ± 0.3 per 100-μm length) was significantly larger than that of unpaired arterioles (24.5 ± 0.8 μm) and arterioles (23.3 ± 1.3 μm) paired with venules with higher leukocyte adherence (9.0 ± 0.5 per 100-μm length). Local superfusion of adenosine deaminase (ADA) induced significant decreases in diameter and perivascular NO concentration in arterioles closely paired to venules with minimal leukocyte adherence. However, ADA had little effect on arterioles closely paired to venules with high leukocyte adherence or on unpaired arterioles. To determine whether the attenuated response to ADA for the high-adherence group was oxidant dependent, the responses were also observed in arterioles treated with 10−4 M Tempol. In the high-adherence group, Tempol fully restored NO levels to those of the low-adherence group; however, the ADA-induced constriction remained attenuated, suggesting a possible role for an oxidant-independent vasoconstrictor released from the inflamed venules. These findings suggest that adenosine- and venule-dependent dilation of paired arterioles may be mediated, in part, by NO and inhibited by venular leukocyte adherence.

Carbon Monoxide Modulates Endotoxin-induced Microvascular Leukocyte Adhesion through Platelet-dependent Mechanisms

Background Although precise mechanisms remain to be determined, recent studies show that heme oxygenase-1 (HO-1), providing endogenous carbon monoxide (CO) and bilirubin, serves as an antiinflammatory enzyme. This study aimed to clarify roles of CO in regulation of microvascular adhesion of platelets and leukocytes in endotoxemia. Methods Rats pretreated with or without hemin were anesthetized with pentobarbital and received continuous infusion of endotoxin. Platelets labeled with carboxyfluorescein diacetate succinimidyl ester and leukocyte behavior in mesenteric venules were visualized using intravital ultra-high-speed intensified fluorescence videomicroscopy. To examine the mechanisms for the effects of HO-1 on platelet and leukocyte behavior during endotoxemia, these studies were repeated with superfusion of either CO, bilirubin, or zinc protoporphyrine-IX. Results Endotoxin caused a marked depression of platelet velocity traversing along periendothelial regions, accompanied by augmented rolling and adhesion of leukocytes in venules. The endotoxin-elicited changes were attenuated by the HO-1 induction with hemin and restored by blockade of the enzyme activity with zinc protoporphyrine-IX, a potent inhibitor of HO-1. Such an inhibitory action of HO-1 on microvascular cell adhesion was reproduced by local superfusion of the buffer containing CO at micromolar concentrations. Such antiadhesive actions of CO on leukocytes disappeared under immunoneutralization of glycoprotein Ibalpha, an adhesion molecule against platelets, but not against leukocytes. Platelets isolated from hemin-treated rats increased their ability to generate CO and displayed lesser sensitivity of agonist-induced aggregation than those from controls. Conclusions These results suggest that CO desensitizes endotoxin-induced adhesive responses of leukocytes, mainly through its ability to ameliorate platelet activation.

Leukocyte recruitment in the airways: an intravital microscopic study of rat tracheal microcirculation

Because of its relative inaccessibility, inflammatory cell extravasation within the airway circulation in vivo has been difficult to investigate in real time. A new method has been established using intravital microscopy in the anesthetized rat to visualize leukocytes in superficial postcapillary venules of the trachea. This technique has been validated using local superfusion of lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (FMLP). Basal leukocyte rolling velocity (55.4 ± 9.3 μm/s) and adhesion (1.4 ± 0.3 cells/100 μm) were monitored in postcapillary venules (33.9 ± 1.3 μm diameter). At all time points up to 90 min, these parameters were unaltered in control rats ( n= 7). In contrast, vessels exposed to 1 μg/ml of LPS ( n = 6) exhibited a 57% reduction in leukocyte rolling velocity and an increase in the number of adherent cells (4.7 ± 1 cells/100 μm, P < 0.05). Superfusion with 0.1 μM of FMLP ( n = 6) also resulted in a 45% reduction in rolling velocity and an increase in adherent cells (4 ± 0.7 cells/100 μm, P < 0.05). Histological evaluation confirmed local stimulus-induced leukocyte extravasation. These results demonstrate leukocyte recruitment in the airway microvasculature and provide an important new method to study airway inflammation in real time.

On bipolar cell responses in the teleost retina are generated by two distinct mechanisms

1. ON Bipolar cells were recorded in slices obtained from hybrid bass retinas. Cells were identified as bipolar cells by position in the slice, by characteristic voltage- and ligand-gated currents, and by filling with the fluorescent dye Lucifer yellow. Cells were recorded with the use of either whole cell or perforated-patch techniques. Standard electrophysiological protocols were used. Drugs were applied by puffing and by local superfusion. 2. Application of exogenous glutamate to ON bipolar cells generated two characteristic responses. One effect of glutamate was to open a conductance with a reversal potential close to the chloride equilibrium potential. The other effect of glutamate was to close a conductance with a reversal potential near 0 mV. These two effects of glutamate on ON bipolar cells match the effects of light described previously with the use of intracellular recordings. Thus the effects of glutamate that we report here appear to underlie the rod and cone inputs to these cells. 3. Many of the ON bipolar cells recorded demonstrated both classes of responses to glutamate. To isolate the two responses, 500 microM glutamate was first applied, and then glutamate in the presence of 5 microM 2-amino-4-phosphonobutyric acid (APB). APB specifically blocks the effects of glutamate on the putative roddriven glutamate receptor (the glutamate-elicited conductance decrease), allowing us to study in isolation the effects of glutamate on the cone component, the glutamate-activated chloride current (IGlu). 4. By isolating IGlu as described above, and taking advantage of the fact that amphotericin-perforated-patch recordings limit the diffusion of chloride ions between the patch pipette and the cell body, we found the physiological reversal potential of IGlu to be -58.9 +/- 7.7 (SD) mV. 5. Both the putative rod- and cone-mediated glutamatergic inputs to these bipolar cells could be activated by driving the photoreceptors with puffs of potassium. The currents recorded with this technique were very similar to those seen with direct application of glutamate.

Local superfusion modifies the inward rectifying potassium conductance of isolated retinal horizontal cells

1. Horizontal cells were enzymatically and mechanically dissociated from the white perch (Roccus americana) retina and voltage clamped using patch electrodes. Steady-state current-voltage (I-V) relationships of solitary horizontal cells were determined by changing the membrane potential in a rampwise fashion. 2. The I-V curve of cells bathed in normal Ringer solution exhibited a large conductance increase at negative membrane potentials. This conductance activated near the K+ equilibrium potential, had no clear reversal potential, was enhanced by raising the extracellular concentration of K+, and was suppressed by external Cs+. These properties identify the conductance as the inward (anomalous) rectifier. 3. Continuous superfusion of the cells' local environment with drug-free Ringer reduced the magnitude of the inward rectifier current and shifted its activation point to more negative potentials. This effect developed over approximately 30 s, lasted as long as superfusion continued and was reversible upon cessation of superfusion. 4. Pressure ejection of drug-free Ringer solution onto cells bathed in the identical solution also reduced the magnitude of the inward rectifier current, although the effects were more rapid and more transient than those exerted by superfusion. Pressure ejection had little effect when cells were simultaneously superfused with Ringer, suggesting a common mode of action on the inward rectifier. 5. In the absence of superfusion, pressure ejection of Ringer containing 200 microM L-glutamate had a biphasic effect on membrane conductance. At potentials above -60 mV, glutamate caused a conductance increase with a reversal potential near +10 mV. At potentials below -60 mV, glutamate caused a conductance decrease whose reversal potential could not reliably be determined. The latter effect was similar to the suppression of the inward rectifier by application of Ringer alone, suggesting that it may represent an artifact of pressure ejection rather than a direct effect of glutamate. 6. In support of this interpretation, we found that pressure ejection of glutamate in the presence of external Cs+ (which blocks the inward rectifier) or during local superfusion with Ringer (which prevents attenuation of the inward rectifier by pressure ejection) did not cause a conductance decrease at negative potentials. Under these conditions, glutamate caused primarily a conductance increase with a reversal potential near +10 mV.(ABSTRACT TRUNCATED AT 400 WORDS)