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1The John B. Pierce Laboratory and Departments of 2Cellular and Molecular Physiology, 3Pathology, and 4Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06519
Submitted 31 January 2003 ; accepted in final form 4 April 2003
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ABSTRACT |
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5 ± 1 µm) to elevating superfusate oxygen
from 0 to 21%. For arterioles of C57BL6 and PECAM-1–/–
mice, cumulative addition of histamine to the superfusate produced
vasodilation (1 nM–1 µM; peak response, 9 ± 1 µm) and then
vasoconstriction (10–100 µM; peak response, 12 ± 2 µm). In
eNOS–/– mice, histamine produced
only vasoconstriction. In C57BL6 and
PECAM-1–/– mice, vasodilation was
abolished with N
-nitro-L-arginine (30
µM); in all mice, vasoconstriction was abolished with nifedipine (1 µM).
Vasomotor responses were eliminated with pyrilamine (1 µM; H1
receptor antagonist) yet remained intact with cimetidine (1 µM;
H2 receptor antagonist). These findings illustrate that the
biphasic vasomotor response of mouse cremaster arterioles to histamine is
mediated through H1 receptors on endothelium (NO-dependent
vasodilation) as well as smooth muscle (Ca2+ entry and
constriction). Thus histamine can increase as well as decrease muscle blood
flow, according to local concentration. However, when NO production is
compromised, only vasoconstriction and flow reduction occur.
microcirculation; blood flow control; endothelial nitric oxide synthase
The initiation of vasomotor responses to histamine is mediated through H1 and H2 receptor subtypes (10). However, despite the availability of selective pharmacological reagents, neither the presence of respective receptor subtypes nor the signaling pathways activated have been well characterized in the resistance vessels that control tissue blood flow. Whereas transgenic technology has enabled selective elimination of molecules that are integral to vascular cell signaling and adhesion [e.g., nitric oxide (NO) synthase (NOS) and platelet endothelial cell adhesion molecule-1 (PECAM-1; i.e., CD31)], little is known of whether gene ablation may influence the vasomotor effects of histamine in the microcirculation.
In the present study, experiments were performed in null PECAM-1 (PECAM-1–/–) and null endothelial NOS (eNOS–/–) mice with reference to C57BL6 mice to determine whether genetically ablating these molecules would influence the actions of histamine on arterioles controlling blood flow in skeletal muscle. We tested the hypothesis that histamine would increase muscle blood flow through stimulating NO release from microvascular endothelium. Complimentary experiments used pharmacological interventions to resolve the receptor subtype and signaling pathways that mediate vasomotor responses through a range of histamine concentrations spanning six orders of magnitude and which encompass the initiation of vasodilation through closure of the arteriolar lumen.
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MATERIALS AND METHODS |
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The anesthetized mouse was placed supine on a clear acrylic platform. Esophageal temperature was maintained at 37–38°C by radiant heat. The left cremaster muscle was prepared as described (12). Briefly, a midline incision was made in the left scrotal sac. Connective tissue was cleared, and the exposed muscle was opened longitudinally and separated from the testis, which was repositioned in the abdominal cavity. The muscle was spread evenly and pinned onto a pedestal of transparent Sylgard (Dow Corning; Midland, MI) while being superfused (5 ml/min) with bicarbonate-buffered physiological salt solution (PSS; 34°C, pH 7.4) of the following composition (in mM): 137 NaCl, 4.7 KCl, 1.2 MgSO4, 2 CaCl2, and 18 NaHCO3. The PSS reservoir was equilibrated with 5% CO2-95% N2 unless noted otherwise.
Intravital microscopy. The completed preparation was transferred
to the stage of an intravital microscope (modified model 20T, Zeiss),
equilibrated for 60 min and viewed with brightfield illumination (condensor
numerical aperture 0.32). A drawing of the arteriolar network was made during
this period. During experiments, arterioles were observed using a Zeiss UD 40
objective (numerical aperture 0.41) coupled to a video camera (model NC 70X,
Dage-MTI; Michigan City, IN); total magnification on the video monitor (model
PVM-132, Sony) was x620. Vessel diameter was determined from the edges
of the lumen with the use of a video caliper (modified model 321, Colorado
Video; Boulder, CO) with spatial resolution 
2 µm. Data were acquired at
40 Hz with the use of a PowerLab system (model 8S, ADInstruments; Castle Hill,
Australia) coupled to a personal computer.
Chemicals and reagents. Chemicals and reagents were purchased from
Sigma Aldrich (St. Louis, MO). Final working concentrations are given after
diluting stock solutions at least 100-fold in fresh PSS: histamine (1 nM to
100 µM), pyrilamine (1 µM; H1 receptor antagonist),
cimetidine (1 µM; H2 receptor antagonist),
N-nitro-L-arginine (L-NNA, 30
µM; competitive inhibitor of NOS), L-arginine (L-Arg,
1 mM; biological substrate for NOS), sodium nitroprusside (SNP, 10 µM; NO
donor), and nifedipine (1 µM; antagonist of L-type
Ca2+ channels). Nifedipine was first dissolved in
ethanol (0.01% final concentration; vehicle controls had no effect on
arteriolar diameter or reactivity).
Experimental protocols. Experimental manipulations were performed while observing second-order arterioles that were located in the central region of the cremaster muscle preparation and away from adjacent venules (12, 17). One arteriole was studied in each mouse. Resting internal diameter was measured under control conditions and after elevating superfusate oxygen to 21% (with 5% CO2, balance N2) for 10 min. At the conclusion of each experiment, maximal diameter was recorded during topical application of SNP. The actions of histamine (concentration response, receptor subtype, and signaling pathways) were initially evaluated in arterioles from C57BL6 mice. The mouse genotype was then varied across experiments to avoid an order effect.
Vasomotor responses to histamine. The effect of changing histamine
concentration was determined by cumulative addition (1 nM to 100 µM) to the
superfusion solution. At each concentration, arteriolar diameter was allowed
to stabilize for at least 2 min and then recorded before the next increment.
After the response to 100 µM histamine, superfusion with control PSS was
restored, and resting diameter typically recovered within 25 min.
Histamine receptor antagonists. After the initial evaluation of
and recovery from responses to histamine, pyrilamine was equilibrated for 20
min and the concentration-response relationship to histamine was reevaluated.
Superfusion with control PSS was restored (30 min) and the histamine
concentration-response relationship reevaluated (not different from control;
data not shown). Cimetidine was then equilibrated for 20 min and the
concentration-response relationship to histamine evaluated a final time.
Signaling pathways. Concentration-response relationships were
first obtained under control conditions and arterioles recovered as described
above. To investigate a role for NO in arteriolar responses, L-NNA
was equilibrated for at least 30 min and vasomotor responses to incremental
histamine concentrations were reevaluated. The specificity of NOS inhibition
was determined by the addition of L-Arg to the PSS and reevaluation
of the histamine concentration-response relationship. To investigate a role
for Ca2+ influx in vasoconstriction
(10), the response to 100
µM histamine was recorded, control superfusion was restored until diameter
recovered (20 min), and then histamine (100 µM) and nifedipine were
applied simultaneously. Superfusion with control PSS was then resumed for
10 min and the response to 100 µM histamine was reevaluated.
Data analysis. Data were analyzed using one-way repeated-measures ANOVA with Tukey post hoc comparisons (SigmaStat version 2.03; SPSS, Chicago, IL). Summary data are presented as means ± SE. Values for n refer to the number of arterioles studied in as many mice. Differences between groups were accepted as statistically significant with P < 0.05.
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RESULTS |
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Vasomotor responses to histamine. In C57BL6 mice, arterioles
dilated progressively as histamine concentration increased from 1 nM to 1
µM, reaching a peak diameter (26 ± 2 µm) that was 58% of the
maximum diameter obtained with SNP (Fig.
1). Arterioles then constricted progressively as histamine
concentration increased to 10 and 100 µM. Peak constriction was typically
accompanied by occlusion of the vessel lumen and blood flow ceased in 50%
of arterioles. In PECAM-1–/–
mice, arteriolar responses to histamine were similar to those of C57BL6 mice
(Fig. 1). Remarkably, only
vasoconstriction was observed in
eNOS–/– mice. For example, at 1
µM histamine, arterioles of
eNOS–/– mice constricted by
10 µm, whereas those from C57BL6 and
PECAM-1–/– mice dilated by a
similar amount (Fig. 1). Across
all three strains of mice, there was no difference in the peak amplitude of
vasoconstriction with 100 µM histamine.
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Histamine receptor antagonists. Neither pyrilamine nor cimetidine had a significant effect on resting diameter. In C57BL6 mice, both dilation and constriction of arterioles to histamine were inhibited by pyrilamine (Fig. 2A). In contrast, cimetidine had no effect on arteriolar dilations from 1 nM to 1 µM histamine or constriction to 100 µM histamine. However, cimetidine attenuated vasoconstriction to 100 µM histamine. In PECAM-1–/– and eNOS–/– mice, arteriolar responses to histamine were also inhibited completely by pyrilamine (Fig. 2B).
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Signaling pathways. In C57BL6 mice, arteriolar dilations at histamine concentrations of 1 nM to 1 µM were reversed to constrictions in the presence of L-NNA (Fig. 3), which otherwise had no significant affect on vessel diameter (control, 20 ± 2 µm; L-NNA, 21 ± 4 µm; L-NNA + L-Arg, 22 ± 4 µm; n = 4). Arteriolar constrictions at 10 and 100 µM histamine were unaffected by L-NNA. The addition of L-Arg completely reversed the actions of L-NNA and restored vasodilation from 1 nM to 1 µM histamine (Fig. 3). In C57BL6 mice, nifedipine reversed vasoconstriction to vasodilation at 10–4 M histamine (Fig. 4). The vasoconstriction to 100 µM histamine was restored after washout of nifedipine and return to resting diameter (Fig. 4). In PECAM-1–/– mice, arteriolar dilation and constriction to histamine were abolished by L-NNA and nifedipine, respectively (n = 4; P < 0.05 vs. control). In eNOS–/– mice, arteriolar constriction was abolished by nifedipine (n = 4; P < 0.05 vs. control).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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The absence of vasodilation in eNOS–/– mice and its reversible abolition with L-NNA in C57BL6 and PECAM-1–/– mice indicates that NO release fully accounts for the actions of histamine as a vasodilator in our experiments. In conduit arteries, smooth muscle relaxation in response to histamine is mediated through H1 receptors on endothelial cells (4, 10). In C57BL6 and PECAM-1–/– mice, the inhibition of arteriolar dilation with pyrilamine and the lack of effect of cimetidine indicate that NO production is coupled to the activation of H1 receptors on microvascular endothelium. Our finding that dilatory responses to SNP (an NO donor) were similar for arterioles of eNOS–/–, C57BL6, and PECAM-1–/– mice indicates that the biological actions of NO were not different across these strains. Furthermore, it is unlikely that the vasodilation observed here is mediated through the release of prostaglandins or other endothelium-derived autacoids. We therefore conclude that eNOS is central to the ability of histamine to dilate arterioles controlling blood flow to the mouse cremaster muscle.
In conduit arteries, H1 receptors on smooth muscle cells are coupled to the G protein-phospholipase C pathway, leading to the activation of L-type Ca2+ channels and an increase in intracellular Ca2+ concentration (9, 10, 14). Alternatively, the activation of H2 receptors on smooth muscle stimulates the cAMP-adenylate cyclase pathway to promote relaxation (4, 10, 14). Our finding that arteriolar constrictions to histamine were abolished by pyrilamine, and that cimetidine had negligible effect, indicates that histamine acted on arteriolar smooth muscle through the classic G protein, H1 receptor-coupled pathway. We conclude that both constriction and dilation of arterioles are promoted through H1 receptors and that H2 receptors have little or no role in blood flow control in the microcirculation of mouse skeletal muscle.
Arteriolar responses to increasing histamine concentration were biphasic,
with dilation at 1 µM, followed by constriction at >1 µM.
However, inhibiting constriction with nifedipine unmasked a dilation that was
otherwise overshadowed (Fig.
4). In a reciprocal manner, genetic ablation of eNOS or
pharmacological inhibition of NOS revealed constriction of arterioles at
histamine concentrations that otherwise caused dilation (Figs.
1 and
3). These findings illustrate
that competing responses of endothelium and smooth muscle to histamine can be
active simultaneously with the prevailing histamine concentration determining
which signaling pathway dominates the arteriolar vasomotor response. Whereas
NO has been implicated as a key mediator of the integrity of capillary and
venular endothelium (3,
13), the present data
illustrate that precapillary actions of histamine have pronounced effects on
the control of tissue blood flow.
PECAM-1 is found predominantly in the lateral junctions between endothelial cells, where it acts in signal transduction and as a stabilizing link between endothelial cells (1, 16). Activating H1 receptors can lead to changes in vascular permeability (15). Indeed, recent studies have shown that histamine promotes greater leakiness in endothelium of PECAM-1–/– mice compared with C57BL6 mice (8). However, the lack of PECAM-1 had no apparent effect on the vasomotor actions of histamine, because arteriolar responses in PECAM-1–/– mice were similar to those observed in C57BL6 mice (Fig. 1). This finding argues that genetic ablation in and of itself does not affect arteriolar responses to histamine in the mouse cremaster muscle. Furthermore, given that the vasomotor responses to histamine in eNOS–/– mice were indistinguishable from those in C57BL6 and PECAM-1–/– mice in which NO production was inhibited with L-NNA, the present data provide a novel demonstration of how genetic ablation of eNOS recapitulates pharmacological inhibition of NOS activity.
In summary, arterioles from C57BL6 mice display a biphasic response to changes in histamine concentration, with vasodilation manifest at or below 1 µM and vasoconstriction >1 µM. Both dilation and constriction are mediated via H1 receptors, with vasodilation dependent on NO production by endothelium and vasoconstriction promoted through the activation of L-type calcium channels in smooth muscle. With both cell layers activated simultaneously, the local concentration of histamine will dictate the actual vasomotor response. Thus moderate release of histamine (e.g., nM) would promote vasodilation and increase muscle blood flow. However, during impaired NO production (e.g., with endothelium dysfunction), the hyperemia associated with an inflammatory response will be abrogated. When histamine release is robust (e.g., exceeding µM), arteriolar constriction will serve to reduce capillary hydrostatic pressure along with muscle blood flow, thereby minimizing fluid extravasation from capillaries and venules. In such a manner, histamine can serve as a key determinant of muscle blood flow and fluid filtration during injury and inflammation.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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