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,1
* The John B. Pierce Laboratory and Departments of
Cellular and Molecular Physiology,
Pathology, and
Pharmacology, Yale University School of Medicine, New Haven, Connecticut, USA
1 Correspondence: The John B. Pierce Laboratory, Yale University School of Medicine, 290 Congress Ave., New Haven, CT 06519, USA. E-mail: steven.segal{at}yale.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: microcirculation • nitric oxide • gap junctions • cell adhesion
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. For example, vasoactive signals originating from discrete sites within a tissue can be conducted rapidly along arterioles via cell-to-cell coupling through gap junction channels (2)
. In response to acetylcholine (ACh), conduction of vasodilation reflects the initiation and spread of hyperpolarization along endothelium (3
4
5)
and the ensuing relaxation of smooth muscle cells. Indeed, disruption of endothelium integrity eliminates this cellular pathway for signal transmission along the vessel wall (3
, 6
, 7)
.
The structural and functional integrity of endothelium is promoted through the complex interaction of molecules that comprise and regulate cell adhesion. For example, platelet endothelial cell adhesion molecule-1 (PECAM-1; i.e., CD31) and VE-cadherin are found predominantly in the lateral junctions among endothelial cells (8)
and are key regulators of endothelial cell adhesion and junctional integrity (9
, 10)
. Histamine is a potent mediator of inflammation and readily increases permeability in capillaries and postcapillary venules (11
12
13
14)
in part through the generation of nitric oxide (NO) (15)
. Although the increase in permeability has been attributed to the formation of gaps among neighboring endothelial cells (16
17
18)
, histamine can also disrupt the integrity independent of endothelial cell retraction (19
20
21
22
23)
, for example, through the phosphorylation and disassembly of junctional proteins. Remarkably, only limited information exists concerning precapillary actions of histamine in the microcirculation (24)
.
In the present study, we tested the hypothesis that histamine could disrupt the integrity of arteriolar endothelium as reflected by the inhibition of conducted vasodilation. Experiments were performed in control (C57BL6) mice and in mice genetically deficient in PECAM-1 or endothelial nitric oxide synthase (eNOS). Our findings reveal that histamine can rapidly and reversibly inhibit the conduction of vasodilation and that this action is mediated through stimulating the production of NO. This novel action of histamine implies that endothelium-derived NO serves as a second messenger to trigger a reduction in coupling among arteriolar endothelial cells. Further, PECAM-1 is integral to the restoration of intercellular coupling and blood flow control after histamine exposure.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(n=10; bred at Yale University) and eNOS-/- mice (26)
(n=6; Jackson Laboratories). Both strains of knockout mice were bred from C57BL6 mice (backcrossed 12 times). Mice were housed at 
24°C and maintained on a 12 h:12 h (light/dark) cycle with free access to food and water. On the morning of an experiment, a mouse was anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneal injection), supplemented as needed to prevent withdrawal from toe pinch. At the end of an experiment, the mouse was euthanized with an overdose of pentobarbital (intraperitoneal injection).
The anesthetized mouse was placed supine on a transparent acrylic platform. Esophageal temperature was maintained at 37–38°C using radiant heat. The left cremaster was prepared as described (27)
. A midline incision was made along the ventral surface of the left scrotal sac. Connective tissue was cleared and the exposed cremaster muscle was opened longitudinally and separated from the testis, which was repositioned in the abdominal cavity. The muscle was spread radially and pinned onto a pedestal of transparent Sylgard 184 (Dow Corning, Midland, MI, USA) while continuously superfused (5 mL/min) with a bicarbonate-buffered physiological salt solution (PSS) equilibrated with 5% CO2/95% N2 (pH 7.4, 34°C) of the following composition (in mM): 137 NaCl, 4.7 KCl, 1.2 MgSO4, 2 CaCl2, and 18 NaHCO3. These chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and J. T. Baker (Phillipsburg, NJ, USA).
Intravital microscopy
The completed preparation was transferred to a fixed stage of an intravital microscope (modified model 20T, Zeiss) mounted on an X-Y translator platform, allowing movement of the microscope independent of the preparation. The cremaster muscle was equilibrated for at least 60 min and viewed using bright-field illumination [Zeiss ACH/APL condenser, numerical aperture (NA) = 0.32]. A drawing of the arteriolar network was made during the equilibration period. For experiments, a second-order arteriole (2A) was located in the central region of the tissue and observed using a Zeiss UD 40 objective (NA=0.41) coupled a to a video camera (NC 70X, Dage-MTI, Michigan City, IN, USA); total magnification on the video monitor (model PVM-132, Sony, Japan) was 620x. Vessel diameter was determined from the edges of the lumen using a video caliper (modified model 321, Colorado Video Inc., Boulder, CO, USA) with spatial resolution 
2 µm. Data were acquired at 40 Hz using a PowerLab system (model 8S, ADI Instruments, Castle Hill, Australia) coupled to a personal computer.
Microiontophoresis
Micropipettes with internal tip diameters of 1–2 µm were pulled (P-97; Sutter Instruments; Novato, CA, USA) from borosilicate glass capillary tubes (GC120F-10; Warner Instruments; Hamden, CT, USA) and filled with 1 M ACh. A micropipette was placed in a holder that was mounted to the acrylic platform and positioned with its tip adjacent to the downstream end of a 2A using a remote-controlled hydraulic micromanipulator (MX610R; Siskiyou Design Instruments; Grants Pass, OR, USA). A pair of Ag/AgCl wires, secured in the micropipette holder and at the edge of the preparation, completed the electrical circuit. Acetylcholine was delivered as a brief pulse (500 ms, 1 µA) using microiontophoresis (model 260; World Precision Instruments; Sarasosta, FL, USA); retain current (200 nA) prevented leakage of ACh from the micropipette (27)
.
Vasoactive reagents
Vasoactive reagents were purchased from Sigma-Aldrich. Final working concentrations are given after diluting at least 100-fold in fresh PSS: histamine (10 µM), N
-nitro-L-arginine (L-NA, 30 µM; competitive inhibitor of NOS), sodium nitroprusside (SNP, 10 µM; NO donor), and 1-H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1 (ODQ, 10 µM; antagonist of soluble guanylate cyclase). The ODQ was first dissolved in EtOH (0.01% final concentration); vehicle controls had no effect on arteriolar diameter.
Experimental protocols
One 2A was studied in each mouse; the order in which C57BL6, PECAM-1-/-, and eNOS-/- mice were studied was varied across experiments. Resting internal diameter was measured under control conditions and after elevating superfusate oxygen to 21% (with 5% CO2, balance N2) for 10 min; the PSS was then reequilibrated with 5% CO2/95% N2. At the conclusion of each experiment, maximal diameter was recorded during superfusion with 10 µM SNP.
To evaluate conducted vasodilation in respective mice under control conditions, the vasomotor response to ACh was measured at the site of delivery ("local," i.e., distance=0) and at remote sites located 350, 700, 1050, and 1400 µm upstream from the site of stimulation; distances were defined using a calibrated eyepiece reticule with reference to anatomical landmarks (27)
. For each site observed, a separate ACh stimulus was delivered at the local site. To ensure that baseline conditions remained constant, the vessel was allowed to recover for 2–3 min before another ACh stimulus was delivered. Control experiments verified negligible tachyphylaxis to ACh and that the direct action of ACh released from a micropipette was confined to <100 µm.
Because the amplitude of conducted vasodilation decays with distance along the arteriole (27)
, the effect of histamine on conduction was evaluated 500 µm upstream in order to optimize resolution of conducted responses. After measurements were obtained under control conditions, histamine (5 µM) was added to the superfusion solution. At this concentration, histamine has a negligible effect on mean resting diameter of 2A in the mouse cremaster muscle (24)
. Local and conducted vasodilation were recorded at 10 min intervals during histamine exposure for 30 min, then superfusion with fresh PSS was resumed; conducted responses to ACh were reevaluated 15 and 25 min later.
In additional C57BL6 and PECAM-1-/- mice, conducted vasodilation to ACh in the presence of histamine (5 µM) was evaluated before and during NOS inhibition. For these experiments, local and conducted responses to ACh were recorded at 10 min intervals throughout 70 min of exposure to histamine. At 50 min of exposure, L-NA was added to the superfusion solution, and local and conducted vasodilations were evaluated at 60 and 70 min.
To further investigate a role for NO signaling in mediating the effect of histamine on responses to ACh, the effect of inhibiting soluble guanylate cyclase was investigated in C57BL6 mice. In one group, local and conducted vasodilations were evaluated in the presence of histamine for 30 min. After washout of histamine for 15 min, local and conducted responses were reevaluated and histamine was reapplied to test for reproducibility of its actions on conducted vasodilation. In a separate group, the effect of the second exposure to histamine was evaluated in the presence of ODQ.
Data analysis
At each site observed, the response to ACh was calculated as diameter change = peak response diameter minus resting diameter. Data were analyzed using one-way repeated measures analysis of variance with Tukey post hoc comparisons (Sigma Stat 2.03; SPSS, Chicago, IL, USA). Summary data are presented as means ± SE. Values for n refer to the number of arterioles studied in as many mice. Differences among groups were accepted as statistically significant with P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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5–10 µm) in response to raising superfusate PO2 from 0 to 21%. Cremaster muscle preparations were stable for 4–5 h and arterioles were evaluated during hours 2–4. Across strains of mice, equilibration with histamine for >30 min had no significant effect on resting diameter (Table 1
) or on the local response to ACh.
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Evaluation of conducted vasodilation
Microiontophoresis of ACh evoked robust vasodilation at the local site of stimulation, which rapidly (<1 s) conducted along the arteriole. Conducted vasodilation was consistently less (P<0.05) than local responses (Fig. 1
). Under control conditions, there was no significant difference in local or conducted vasodilation among strains of mice (Fig. 1)
.
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Effect of histamine
Representative traces of local and conducted vasodilation from arterioles of C57BL6, PECAM-1-/-, and eNOS-/- mice before and during histamine treatment are shown in Fig. 2
A. In the presence of histamine, conducted vasodilation was inhibited by >50% in arterioles of C57BL6 and PECAM-1-/- mice. In contrast, histamine had no effect on conduction in arterioles from eNOS-/- mice. Attenuation of conduction occurred within 10 min of histamine exposure and maximal attenuation was observed at 30 min (Fig. 2B
). Washout of histamine restored the amplitude of conducted vasodilation to control levels within 15 min in C57BL6 mice. In contrast, the recovery of conducted vasodilation took significantly longer (P<0.05) in PECAM-1-/- mice. In a separate group of C57BL6 mice (n=4), control experiments verified that conducted vasodilation was inhibited stably for as long as histamine was present (e.g., for 70 min) and reversed completely upon washout of histamine.
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Effect of NOS inhibition in C57BL6 and PECAM-1-/- mice
The addition of L-NA to the superfusion solution after 50 min of histamine exposure reversed the inhibition of conducted vasodilation (Fig. 3
). This restoration of conduction occurred within 10 min in arterioles from both strains of mice. In PECAM-1-/- mice, the conducted response was enhanced (P<0.05) after addition of L-NA.
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Effect of inhibiting soluble guanylate cyclase
In C57BL6 mice, the inhibition of conducted vasodilation by histamine for 30 min was fully reversed upon washout, and reapplication of histamine reproducibly inhibited conduction. In a separate group of C57BL6 mice, after the initial 30 min inhibition of conduction and its recovery during washout, reexposure to histamine in the presence of ODQ prevented the inhibition of conducted vasodilation (Fig. 4
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hyperpolarization traveling along endothelium through gap junction channels underlies the conduction of vasodilation (3
4
5)
. Because histamine inhibited conducted but not local vasodilation, the action of histamine on conduction is independent of initiating the vasodilatory response to ACh. With air or photodamage to disrupt continuity along endothelium, the loss of conduction is irreversible (3
, 6)
. During inflammation, histamine increases the permeability of capillaries and venules through compromising cell-to-cell adhesion, thereby reducing the integrity of the endothelial cell barrier (19
20
21
22
23
, 28)
. The present data are the first to indicate that histamine can have a similar (and reversible) effect on precapillary endothelial cells. Our finding that conduction was inhibited but not abolished at a concentration that was not vasoactive (5 µM) suggests that the effect of histamine on the coupling of arteriolar endothelium may vary with local concentration. Indeed, 2A of the mouse cremaster muscle dilate and constrict, respectively, to concentrations of histamine that are lower and higher than used here (24)
. Further, the ability of L-NA to interfere with these responses is fully reversed with an excess L-arginine (24)
, confirming the specificity of NOS inhibition.
Genetic ablation of either PECAM-1 or eNOS had no effect on local or conducted vasodilation, as responses under control conditions were similar to responses in C57BL6 mice. However, striking phenotypic differences in the response to ACh appeared during histamine exposure. Remarkably, histamine had no effect on conduction in eNOS-/- mice. In arterioles of PECAM-1-/- mice, histamine inhibited conduction to an extent similar to that in C57BL6 mice, but the recovery of conduction after washout of histamine was delayed significantly by gene ablation (Fig. 2)
. This behavior is consistent with previous studies of endothelial cell permeability in PECAM-1-/- mice, where in vivo and in vitro experiments both demonstrate enhanced permeability and delayed recovery of the endothelium after histamine exposure compared with wild-type C57BL6 mice (29)
. The consistency in the delayed recovery from pre- and postcapillary actions of histamine in PECAM-1-/- mice indicates that PECAM-1 helps to maintain the integrity of endothelium during inflammation and to restore its function after disruption by inflammatory mediators. The delay in recovery of conduction was not observed with L-NA, where arterioles of PECAM-1-/- and C57BL6 mice recovered with a similar time course. This temporal difference between histamine washout and NOS inhibition suggests that in the absence of PECAM-1-/-, the downstream actions of H1 receptor activation on arteriolar endothelium (24)
take longer to wane than does the direct inhibition of NO production. Remarkably, conduction was enhanced after NOS inhibition in PECAM-1-/- mice but not C57BL6 mice (Fig. 2)
, though the basis of this difference remains to be determined.
Nitric oxide plays a key role in mediating the permeability of capillary and postcapillary endothelium in response to inflammatory mediators, including histamine (14
, 30
31
32
33)
. Our present findings demonstrate that histamine has important and previously unrecognized actions on precapillary endothelium. Moreover, the activity of eNOS has been linked to its localization at cell borders in a manner that resembles localization of PECAM-1 and caveolin-1 (34)
and of the connexin molecules that form gap junctions in arteriolar endothelium (R. Looft-Wilson, G. Payne, and S. Segal, unpublished observations). As shown here by the lack of effect of histamine in eNOS-/- mice and by the blockade of its effect by L-NA in C57BL6 and PECAM-1-/- mice, activation of the NO signaling pathway is integral to promoting histamine-induced attenuation of conducted vasodilation in arterioles.
A model to explain the ability of histamine to disrupt the integrity of arteriolar endothelium is shown in Fig. 5
. Our finding that L-NA and ODQ were effective in reversing and preventing the inhibition of conducted vasodilation by histamine (Figs 3
, 4)
indicates that NO acts through guanylate cyclase to activate protein kinase G (PKG), which leads to disruption of endothelial cell integrity. We suggest that PKG (as well as protein kinase C and Src family members), perhaps through intermediate kinases and/or phosphatases, alters the phosphorylation states of cell adhesion molecules that comprise and modulate adherens junction formation along with connexin molecules that form gap junctions. Phosphorylation of VE-cadherin, along with ß and gamma catenin, reduce inter-endothelial cell adhesion (19
, 20)
whereas phosphorylation of connexins can reduce coupling among cells (35)
. The delay in return to baseline for conducted vasodilation after histamine washout observed in PECAM-1-/- mice may be due in part to the reduced ability of SHP-2 to dephosphorylate tyrosine-phosphorylated ß-catenin in the absence of the scaffolding and activating function of the PECAM-1 cytoplasmic domain (36
, 37)
. In contrast, in eNOS-/- mice, histamine does not stimulate the NO production and therefore is unable to activate PKG (or the subsequent phosphorylation and/or disassembly of adherens and gap junctions), which explains the lack of effect of histamine after genetic ablation of eNOS.
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In summary, the conduction of vasodilation in response to ACh is not different in arterioles from C57BL6, PECAM-1-/-, and eNOS-/- mice under control conditions. However, during exposure to histamine, conducted vasodilation is inhibited in C57BL6 and PECAM-1-/-mice but not in eNOS-/- mice. Pharmacological blockade of NOS and soluble guanylate cyclase, both integral to NO signaling, effectively mimic the behavior of genetic ablation of eNOS. Collectively, these observations suggest that histamine exerts its effect on conducted vasodilation entirely through the production of NO. In PECAM-1-/- mice, the delay in recovery for conduction after histamine exposure points to a key role for adhesion molecules in restoring barrier function and cell-to-cell coupling after endothelium disruption. The present findings uniquely demonstrate that cell-to-cell coordination of blood flow control in resistance networks is susceptible to modulation by inflammatory mediators.
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ACKNOWLEDGMENTS |
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Received for publication July 24, 2003. Accepted for publication September 19, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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