September 2016
Volume 57, Issue 11
Open Access
Retina  |   September 2016
Histamine-Induced Dilation of Isolated Porcine Retinal Arterioles: Role of Endothelium-Derived Hyperpolarizing Factor
Author Affiliations & Notes
  • Shinichi Otani
    Department of Ophthalmology Asahikawa Medical University, Asahikawa, Japan
  • Taiji Nagaoka
    Department of Ophthalmology Asahikawa Medical University, Asahikawa, Japan
  • Tsuneaki Omae
    Department of Ophthalmology Asahikawa Medical University, Asahikawa, Japan
  • Ichiro Tanano
    Department of Ophthalmology Asahikawa Medical University, Asahikawa, Japan
  • Takayuki Kamiya
    Department of Ophthalmology Asahikawa Medical University, Asahikawa, Japan
  • Shinji Ono
    Department of Ophthalmology Asahikawa Medical University, Asahikawa, Japan
  • Travis W. Hein
    Department of Ophthalmology, Scott & White Eye Institute, Temple, Texas, United States
    Department of Surgery, College of Medicine, Texas A&M Health Science Center, Temple, Texas, United States
  • Lih Kuo
    Department of Ophthalmology, Scott & White Eye Institute, Temple, Texas, United States
    Department of Surgery, College of Medicine, Texas A&M Health Science Center, Temple, Texas, United States
    Department of Medical Physiology, College of Medicine, Texas A&M Health Science Center, Temple, Texas, United States
  • Akitoshi Yoshida
    Department of Ophthalmology Asahikawa Medical University, Asahikawa, Japan
  • Correspondence: Taiji Nagaoka, Department of Ophthalmology, Asahikawa Medical University, Midorigaoka Higashi 2-1-1-1, Asahikawa, 078-8510, Japan; nagaoka@asahikawa-med.ac.jp
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 4791-4798. doi:10.1167/iovs.15-19038
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      Shinichi Otani, Taiji Nagaoka, Tsuneaki Omae, Ichiro Tanano, Takayuki Kamiya, Shinji Ono, Travis W. Hein, Lih Kuo, Akitoshi Yoshida; Histamine-Induced Dilation of Isolated Porcine Retinal Arterioles: Role of Endothelium-Derived Hyperpolarizing Factor. Invest. Ophthalmol. Vis. Sci. 2016;57(11):4791-4798. doi: 10.1167/iovs.15-19038.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Although endothelium-dependent nitric oxide (NO)–mediated dilation of retinal arterioles has been well described, the role of endothelium-derived hyperpolarizing factor (EDHF) in the retinal arteriolar response remains unclear. In the current study, we examined the contribution of EDHF to the retinal arteriolar dilation to the inflammatory agent histamine and investigated the signaling mechanisms underlying this vasomotor activity.

Methods: Porcine retinal arterioles were isolated, cannulated, and pressurized without flow for functional study by using video microscopic techniques. The immunohistochemical staining was performed to determine histamine receptor subtypes.

Results: Histamine (0.1–30 μM) produced concentration-dependent dilation of retinal arterioles in a manner sensitive to H1- and H2-receptor antagonists chlorpheniramine and famotidine, respectively. Histamine-induced vasodilation was almost abolished after endothelial removal. In the intact vessels, vasodilation to histamine was partially inhibited by the inhibitors of cyclooxygenase (indomethacin), NO synthase (NG-nitro-L-arginine methyl ester, L-NAME), or Ca2+ -activated K+ (KCa) channels (apamin plus charybdotoxin). Combination of the above inhibitors abolished histamine-induced vasodilation. Residual vasodilation in the presence of indomethacin and L-NAME was further reduced by the cytochrome P450 enzyme inhibitor sulfaphenazole but not by the gap junction inhibitor carbenoxolone or the hydrogen peroxide scavenger catalase. Immunohistochemical signals for H1- and H2-receptor expression were found only in the endothelium.

Conclusions: The endothelium plays an essential role in the dilation of porcine retinal arterioles to histamine via H1- and H2-receptor activation. The EDHF derived from cytochrome P450 contributed in part to this vasodilation via KCa channel activation, in addition to the endothelial release of NO and prostanoids.

The vascular endothelium consists of a monolayer of cells lining the inner wall of the vasculature. It plays a critical role in many homeostatic processes, including the trafficking of nutrients and immune cells between blood and underlying tissue, maintenance of blood fluidity, regulation of permeability, formation of new vasculature, and the control of vascular tone for blood flow regulation.1 The retinal blood flow is regulated by the activity of smooth muscle cells (i.e., contraction or relaxation) in small resistance arterioles. This vasomotor activity can be governed by the release of vasoactive factors from the endothelium.2 The release of endothelium-derived relaxing factors (EDRFs) such as nitric oxide (NO) and prostacyclin (PGI2) in response to various neurohumoral and shear stress stimulations3 has been well described in different organs and tissues,4 including the retina.5 However, some of the endothelium-dependent vasodilations are resistant to the blockade of NO synthase and cyclooxygenase but sensitive to agents that prevent membrane hyperpolarization.6,7 Therefore, the existence of an additional pathway that involves the release of endothelium-derived hyperpolarizing factor (EDHF) has been suggested.8,9 
The endothelial hyperpolarization initiated by the activation of Ca2+-activated K+ (KCa) channels9 or gap junctions10 has been suggested to play a critical role in mediating EDHF-induced vasodilatory responses. It has been shown that an EDHF-induced vasodilation could be prevented by a combined administration of apamin and charybdotoxin (ChTX), which selectively inhibit small- and intermediate/large-conductance KCa channels, respectively, in a number of vascular beds.11 Although the chemical nature of EDHF and its signaling pathways are not fully understood, hydrogen peroxide (H2O2) and epoxyeicostrienoic acids (EETs), which are synthesized by superoxide dismutase and cytochrome P450 enzyme CYP2C9, respectively, have been suggested to be involved in the signaling of EDHF in some vascular beds.9 While previous studies have reported that EDHF is involved in agonist-induced relaxation of rat mesenteric arterioles,12 the direct role of EDHF in regulation of retinal arteriolar tone remains unknown.13,14 
Histamine is known to be an important mediator for inflammatory reactions15 exerted by four different subtypes of histamine receptors, namely, H1, H2, H3, and H4.16 Histamine is present in the retina,17 and the enzymes responsible for its formation and inactivation also have been found in the retinal tissue.18 Histamine causes dilation of bovine large retinal arteries19 and increases retinal blood flow in rats20 and humans.2124 Plasma histamine level has been found to increase by 5-fold in the patients with diabetes.25 It is likely that the increased histamine may contribute to the regulation of blood flow, activation of immune and inflammation systems, and pathogenesis of diabetic complication and retinopathy. However, the histamine-induced vasomotor activity in retinal arterioles that control blood flow and its distribution has not been characterized. Although EDHF has been suggested to play a considerable role in mesenteric vasodilation to histamine,26 the importance of this dilation in retinal arterioles remains undetermined. Herein, we examined the contribution of EDHF to retinal arteriolar dilation to histamine and investigated the signaling mechanisms involved in this vasomotor activity. 
Materials and Methods
Animal Preparation
The Animal Care Committee of Asahikawa Medical University approved all animal procedures, which were performed according to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. After the pigs (age, 16–24 weeks either sex; weight, 25–35 kg) were killed in a local slaughterhouse, one of the eyes was enucleated immediately and transported to the laboratory in a moist chamber on ice. In each eyeball, only one retinal arteriole was isolated and a total of 75 pigs (i.e., vessels) were used for this study. 
Isolation and Cannulation of Microvessels
The techniques for visualization, identification, isolation, cannulation, and pressurization of the retinal arterioles have been described previously.2730 Briefly, the isolated retinal arterioles (90–110 μm in situ) were cannulated with a pair of glass micropipettes and pressurized to 55 cm H2O intraluminal pressure without flow by using two independent pressure reservoir systems.31 The internal diameter of the isolated vessels was recorded continuously by using videomicroscopic techniques throughout the experiments.27 
Control Experiment
The cannulated and pressurized arterioles were bathed in the physiologic saline solution (PSS; 36°C–37°C) containing albumin (0.1%). After the development of stable basal tone (∼30–40 minutes), the concentration-dependent response of retinal arterioles to histamine (0.1–30 μM) was examined. The vessels were exposed to each concentration of histamine until a stable diameter was established (∼3 minutes). After completing the concentration-dependent response, the vessels were washed with PSS and allowed to reestablish the basal tone (∼30–40 minutes). The reproducibility of the vascular response to histamine was then examined in the same vessels (n = 6). 
Role of Histamine Receptors and the Endothelium in Histamine-Induced Dilation
To determine the involvement of histamine receptor subtypes, the vessels were pretreated with respective H1, H2, H3, or H4 histamine receptor antagonist chlorpheniramine (H1; 1 μM),32 famotidine (H2; 10 μM),33 and thioperamide (H3/H4; 1 μM),34 for 30 minutes, and the concentration-dependent responses to histamine were reexamined. In another set of experiments, the endothelium of the isolated arterioles was disrupted by intraluminal perfusion of the nonionic detergent CHAPS (0.4%) for 1 to 2 minutes35 and the vascular response to histamine was reevaluated. To ensure that the vascular smooth muscle function was uncompromised by CHAPS treatment during denudation, the concentration-dependent dilation of the vessel in response to the endothelium-independent vasodilator sodium nitroprusside (SNP; 0.1–100 μM) before and after denudation was examined. Only vessels that exhibited normal basal tone, showed no vasodilation in response to endothelium-dependent vasodilator bradykinin (10 nM),35 and maintained unaltered vasodilation in response to SNP after removal of the endothelium were accepted for further study. 
Role of EDRF and KCa in Retinal Arteriolar Dilation
We assessed the involvement of PGI2, NO, and KCa in mediating the vascular response to histamine by using the known effective concentration of the specific inhibitors of cyclooxygenase (indomethacin, 10 μM),28,36 NO synthase (L-NAME, 10 μM),27,28 and KCa channels (combination of apamin 0.1 μM and ChTX 0.1 μM),3739 respectively. 
Chemical Nature of EDHF and the Role of Gap Junctions
Since H2O2 and the CYP enzyme metabolites EETs have been suggested to be involved in the EDHF-induced vasodilation in some vascular beds,9 we tested this possibility by pretreating the vessels with the effective concentration of H2O2 scavenger catalase (1000 U/mL)40,41 and the CYP2C9 enzyme inhibitor sulfaphenazole (10 μM),42 respectively. The role of gap junctions in histamine-induced responses was tested by the gap junction inhibitor carbenoxolone (100 μM).43 In these studies, the cyclooxygenase inhibitor (indomethacin, 10 μM) and NO synthase inhibitor (L-NAME, 10 μM) were present throughout the experiment to eliminate the confounding effects from prostaglandins and NO.44 
Response to Sodium Nitroprusside
The smooth muscle relaxing agent SNP (0.1–100 μM) was used to probe endothelium-independent vasodilation. The vascular response to SNP was examined in the presence of various pharmacologic antagonists, as mentioned previously. 
All drugs were administered extraluminally unless otherwise stated. The vessels were incubated with each pharmacologic inhibitor for a minimum of 30 minutes. 
Immunohistochemistry
Immunohistochemical detection of vascular histamine receptors was performed after preparation of cryomicrotome sections of the retinal arterioles as described previously.30 The following specific primary antibodies were used: anti–H1 receptor antibody (1:100; Aviva Systems Biology, San Diego, CA, USA), anti–H2 receptor antibody (1:100; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti–endothelial NO synthase (eNOS) antibody (1:100; Santa Cruz Biotechnology), and the anti–α-smooth muscle actin antibody (1:100; Sigma-Aldrich Corp., St. Louis, MO, USA). The slides then were incubated with fluorescein isothiocyanate (FITC)–conjugated antibody (1:100; Santa Cruz Biotechnology), Alexa Fluor 647–conjugated antibody (1:200; Abcam Ltd., Cambridge, UK), and Cy3-conjugated antibody (1:100; GE Healthcare Life Sciences, Piscataway, NJ, USA), observed for green (FITC) and red (Alexa Fluor 647 and Cy3) staining, and analyzed with a confocal microscope (Fluoview FV 1000, Olympus, Tokyo, Japan). Merged images were created by using ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). 
Chemicals
All drugs were purchased from Sigma-Aldrich Corp. Famotidine was dissolved in dimethyl sulfoxide (DMSO). Indomethacin and sulfaphenazole were dissolved in ethanol; other drugs were dissolved in PSS. All subsequent dilutions of these drugs were prepared in PSS. The final concentrations of DMSO and ethanol in the vessel bath were less than 0.1%. Vehicle-controlled studies indicated that these final solvent concentrations did not affect the arteriolar diameter.28 
Data Analysis
At the end of each experiment, the vessels were relaxed in ethylenediaminetetraacetic acid (1 mM) calcium-free PSS to obtain its maximal diameter at 55 cm H2O intraluminal pressure.27 The diameter changes in response to histamine and SNP were normalized to this maximal vasodilation and expressed as percentage maximal dilation.27 Data are reported as the mean ± standard error of the mean (SEM); n represents the number of vessels studied. Statistical comparisons of the changes in resting tone caused by antagonists were performed by using the paired t-test. Statistical comparisons of vasomotor responses to the same agonist under various treatments were performed by using 2-way repeated measures analysis of variance (ANOVA) followed by Bonferroni multiple-range test when appropriate. One-way ANOVA followed by Dunnett's post hoc comparison was used to determine the significance of changes in the baseline diameter in response to different concentrations of agonists. 
Results
Dilation of Retinal Arterioles to Histamine
In this study, all vessels (n = 75) developed a similar level of basal tone (i.e., constricted to 61% ± 1% of maximal diameter). The average resting and maximal vessel diameters were 62 ± 1 and 102 ± 1 μm, respectively. Histamine-induced concentration-dependent dilation of the retinal arterioles and the highest concentration (30 μM) elicited approximately 85% of maximal dilation (Fig. 1). The histamine-induced dilation was reproducible and did not deteriorate at ∼30 to 40 minutes after the first application (Fig. 1). 
Figure 1
 
Response of isolated retinal arterioles to histamine. There was no significant difference between the two trials (n = 6).
Figure 1
 
Response of isolated retinal arterioles to histamine. There was no significant difference between the two trials (n = 6).
Role of Endothelium
In this series of studies, 10 vessels were subjected to the denudation protocol. After perfusion with CHAPS, 3 of the 10 vessels lost basal tone and two showed partial inhibition by the endothelium-dependent vasodilator bradykinin. These apparently damaged or partially denuded vessels were excluded from further study. The remaining five vessels maintained basal tone (control 64% ± 2% versus denudation 64% ± 2%; P = 0.18) and the vasodilation induced by bradykinin (10 nM) was abolished (control 83% ± 2% versus denudation: 1% ± 1%). In addition, these vessels exhibited normal vasodilation in response to SNP (Table). In these five denuded vessels, the dilation in response to the lower concentrations of histamine (0.1–10 μM) was almost abolished, and the response to the highest histamine concentration (30 μM) was reduced from 87% to 6% (P < 0.001, Fig. 2). 
Table.
 
Resting Diameters and Dilation of Retinal Arterioles to SNP
Table.
 
Resting Diameters and Dilation of Retinal Arterioles to SNP
Figure 2
 
Effect of the removal of the endothelium by perfusion with 0.4% CHAPS. *P < 0.05 versus control.
Figure 2
 
Effect of the removal of the endothelium by perfusion with 0.4% CHAPS. *P < 0.05 versus control.
Role of Histamine Receptors
Blockage of the H1 receptor by chlorpheniramine significantly reduced histamine-induced vasodilation (Fig. 3). The H2-receptor antagonist famotidine exhibited a stronger inhibitory effect than chlorpheniramine on the histamine-induced vasodilation. Combined administration of chlorpheniramine and famotidine abolished histamine-induced vasodilation (Fig. 2). On the other hand, H3- and H4-receptor antagonist thioperamide did not have any effect on the vasodilator response to histamine. These histamine receptor antagonists did not alter the basal tone of retinal arterioles. 
Figure 3
 
Effect of incubation with H1 antagonist chlorpheniramine (1 μM), H2 antagonist famotidine (10 μM), or H3 and H4 antagonist thioperamide (1 μM). *P < 0.05 versus control.
Figure 3
 
Effect of incubation with H1 antagonist chlorpheniramine (1 μM), H2 antagonist famotidine (10 μM), or H3 and H4 antagonist thioperamide (1 μM). *P < 0.05 versus control.
Localization of the Histamine Receptor in the Retinal Arterioles
Immunohistologic stainings showed that the endothelium of retinal arterioles expresses eNOS and both histamine H1 and H2 receptors (Figs. 4A, 4B). The H1 and H2 receptors were not detectable in the vascular smooth muscle cells (Figs. 4C, 4D). No staining signals were detected in the control experiments in which the specific histamine receptor antibodies were omitted (data not shown). 
Figure 4
 
Immunohistochemical analysis of H1 and H2 receptors in retinal arterioles. (A) Staining with anti–H1 receptor (green) and anti-eNOS (red) antibodies shows expression of the H1 receptor and eNOS. The merged image shows overlapping staining (yellow) of the H1 receptor with eNOS. (B) Staining with anti–H2 receptor (green) and anti-eNOS (red) antibodies shows expression of the H2 receptor and eNOS. The merged image shows overlapping staining (yellow) of the H2 receptor and eNOS. (C) Staining with anti–H1 receptor (green) and anti–α-SMA (red) antibodies shows expression of the H1 receptor and SMA. The merged image shows no overlapping staining of the H1 receptor and SMA. (D) Staining with anti–H2 receptor (green) and anti–α-SMA (red) antibodies shows expression of the H2 receptor and SMA. The merged image shows no overlapping staining of the H2 receptor and SMA. The images are representative of three separate experiments.
Figure 4
 
Immunohistochemical analysis of H1 and H2 receptors in retinal arterioles. (A) Staining with anti–H1 receptor (green) and anti-eNOS (red) antibodies shows expression of the H1 receptor and eNOS. The merged image shows overlapping staining (yellow) of the H1 receptor with eNOS. (B) Staining with anti–H2 receptor (green) and anti-eNOS (red) antibodies shows expression of the H2 receptor and eNOS. The merged image shows overlapping staining (yellow) of the H2 receptor and eNOS. (C) Staining with anti–H1 receptor (green) and anti–α-SMA (red) antibodies shows expression of the H1 receptor and SMA. The merged image shows no overlapping staining of the H1 receptor and SMA. (D) Staining with anti–H2 receptor (green) and anti–α-SMA (red) antibodies shows expression of the H2 receptor and SMA. The merged image shows no overlapping staining of the H2 receptor and SMA. The images are representative of three separate experiments.
Role of EDRFs and KCa Channels
Indomethacin, L-NAME, and KCa channel inhibitors (apamin plus ChTX) partially inhibited the maximal histamine-induced vasodilation from 89% to 42%, 38%, and 66%, respectively (Fig. 5). Indomethacin and L-NAME exhibited equal inhibition (∼50%) on vasodilation to histamine and ChTX plus apamin produced approximately 30% inhibition. Combined inhibition of cyclooxygenase, NO synthase, and KCa channels almost completely blocked the histamine-induced relaxation (Fig. 5). These pharmacologic inhibitors did not significantly alter resting vascular tone. 
Figure 5
 
Effect of incubation with cyclooxygenase inhibitor indomethacin (IM, 10 μM), NO synthase inhibitor L-NAME (10 μM), or SKCa inhibitor apamin (Apa, 0.1 μM) plus IKCa inhibitor ChTX (0.1 μM), or incubation with all four inhibitors. *P < 0.05 versus control.
Figure 5
 
Effect of incubation with cyclooxygenase inhibitor indomethacin (IM, 10 μM), NO synthase inhibitor L-NAME (10 μM), or SKCa inhibitor apamin (Apa, 0.1 μM) plus IKCa inhibitor ChTX (0.1 μM), or incubation with all four inhibitors. *P < 0.05 versus control.
Role of EDHF and Gap Junction
The residual vasodilation to histamine in the presence of indomethacin and L-NAME was further reduced after treating the vessels with CYP450 enzyme inhibitor sulfaphenazole but not with the H2O2 scavenger catalase or the gap junction inhibitor carbenoxolone (Fig. 6). 
Figure 6
 
Effect of incubation with gap junction inhibitor carbenoxolone (CBX, 100 μM), H2O2 scavenger catalase (CAT, 1000 U/mL), and CYP450 enzyme inhibitor sulfaphenazole (SUL, 10 μM). To isolate the EDHF component of the histamine-induced vasodilator response, IM (10 μM) and L-NAME (10 μM) were added throughout the experiment. *P < 0.05 between groups.
Figure 6
 
Effect of incubation with gap junction inhibitor carbenoxolone (CBX, 100 μM), H2O2 scavenger catalase (CAT, 1000 U/mL), and CYP450 enzyme inhibitor sulfaphenazole (SUL, 10 μM). To isolate the EDHF component of the histamine-induced vasodilator response, IM (10 μM) and L-NAME (10 μM) were added throughout the experiment. *P < 0.05 between groups.
Response to SNP
The resting diameters and dilations of retinal arterioles to SNP in the absence of endothelial cells (i.e., denudation) and in the presence of various pharmacologic inhibitors were not significantly altered (Table). 
Discussion
The present study is the first to show that the endothelium plays an essential role in the dilation of porcine retinal arterioles to histamine via H1- and H2-receptor activation. In addition, the endothelium-derived vasodilators prostanoid and NO partially contributed to the retinal vasodilatation induced by histamine. The CYP450 enzyme metabolites, which act as EDHF, also contributed to histamine-induced vasodilation via KCa channel activation. 
In isolated bovine retinal arteries, histamine at a concentration of 1 mM elicits 60% vasodilation.19 On the other hand, intravitreal administration of 1 mM histamine induces approximately 10% vasodilation in rat retinal arteries, but the actual concentration of histamine acting on the retinal arteries is unclear.20 In the current study, 30 μM histamine elicited approximately 85% dilation of pressurized porcine retinal arterioles (Fig. 1). Although the experimental settings are different, the pig retinal arterioles appear to be more sensitive to histamine as compared with other species. In humans, the basal plasma concentration of histamine is approximately 0.3 to 1.0 ng/mL (2.7–9.0 nM),45 and a 5-fold increase is reported in patients with diabetes.25 Although these plasma levels of histamine are not sufficient to cause retinal arteriolar dilation, the elevation of histamine concentration in the local retinal tissue under disease states may have an impact on the retinal circulation.17,18 Since histamine can be synthesized in the neuroretina, its release is likely to influence nearby vasculature to exert vasodilation, as retinal arterioles are sensitive to extraluminal administration of histamine as demonstrated in the present study. 
Histamine tachyphylaxis has been reported in human airway smooth muscle.46 Benedito et al.19 also have reported histamine tachyphylaxis in bovine retinal arteries that are preconstricted with 10 μM prostaglandin PGF. In the current study, no apparent tachyphylaxis was observed in porcine retinal arterioles (Fig. 1). Although the reasons for these contradictory findings are unclear, the differences in species (bovine versus porcine), experimental setup (vascular ring wired myograph versus pressurized isolated vessel), and vessel size (∼240 vs. ∼100 μm internal diameter) may contribute to the observed discrepancy. The use of preconstrictor PGF might also have altered the vascular tone and triggered the histamine desensitization mechanism in the previous bovine vessel study19 as seen in other vascular preparations.47,48 
Histamine exerts its effects by binding to its four receptors, H1, H2, H3, and H4, on target cells in various tissues.16 In vasculatures, depending upon species and tissue types, Ottosson et al.49 have reported that histamine-induced dilation in small human temporal arteries is mediated by both H1 and H2 receptors. In guinea-pig pulmonary arteries, the dilation to histamine is mediated by H1 receptors.50 On the other hand, H2 receptors mediate the dilation of canine spinal arteries to histamine.51 In bovine retinal arteries, the dilation is reported to be mediated mainly by H1 with small contribution from H2 receptors.19 However, in the current study, we found that the H2-receptor antagonist famotidine was more effective than H1-blocker chlorpheniramine in inhibiting histamine-induced vasodilation (Fig. 3). In addition, combined administration of chlorpheniramine and famotidine abolished the histamine-induced response. Our data suggest that H2 receptors play a dominant role in the dilation of small porcine retinal arterioles to histamine. The role of H3 and H4 receptors appears to be minimal because inhibition of H3 and H4 receptors by thioperamide had no effect on the vasodilation to histamine (Fig. 3). The discrepancy between previous bovine (H1 dominant)19 and current porcine (H2 dominant) study on retinal vascular dilation to histamine is unclear. The different approaches (the pressurized vascular segment versus stretched vascular ring), the development of vascular tone (spontaneous basal tone versus external preconstrictor), vessel size (∼100 vs. 250 μm), and species difference (porcine versus bovine) may contribute to the observed inconsistent results. Interestingly, in healthy humans, histamine dilates retinal arteries and veins in a manner sensitive to H1-receptor antagonist diphenhydramine24 but not to H2-blocker cimetidine.23 However, the role of H2 receptors cannot be completely excluded because the efficacy of cimetidine in blocking H2 receptors in the above human study has not been tested.23 Our finding on the prominent role of H2 receptors in mediating retinal arteriolar dilation is consistent with the finding in human ophthalmic arteries.52 
The role of endothelium in mediating the histamine response is inconsistent across different tissues and species. The relaxation of rat aorta to histamine can be endothelium-dependent via H1-receptor activation.53 However, the relaxation of human dorsal penile arteries to histamine is endothelium-independent through activation of H2 receptors.54 In guinea-pig pulmonary arteries, the H1 receptor–mediated vasodilation to histamine is converted to vasoconstriction after endothelial removal.50 In the current study, we found that the intact endothelium is required for the dilation of retinal arterioles to histamine (Fig. 2), and this finding is consistent with that reported in bovine retinal arteries.19 The immunohistochemical results indicated that the histamine receptors H1 and H2 are expressed in the endothelium exclusively (Fig. 4), further supporting the functional role of H1 and H2 receptors in mediating endothelium-dependent vasodilation to histamine. 
The potential vasodilators derived from the endothelium can be PGI2 and NO, as well as EDHF, which activates KCa channels. Blocking these pathways independently by indomethacin, L-NAME, and apamin plus ChTX only produced a partial inhibition on histamine-induced vasodilation (Fig. 5). The residual histamine response in the presence of indomethacin and L-NAME was blocked almost completely by the addition of apamin and ChTX, suggesting that histamine-induced dilation is mediated by not only the release of PGI2 and NO but also the KCa channel activation. 
Since L-NAME can block both constitutive endothelial (eNOS) and inducible (iNOS) NOS isoforms, the contribution of iNOS to histamine-induced vasodilation cannot be completely excluded in the present study. Although the gene that encodes iNOS is not thought to be expressed in normal vessels,55 a recent study56 has reported that hypoxia stimulation for 5 to 10 minutes can relax preconstricted porcine retinal arterial rings with preserved perivascular retinal tissue in a manner sensitive to an iNOS inhibitor. The source of NO appears to be from the iNOS expressed in the perivascular retinal tissues during hypoxia. It is worth noting that our studies were performed in the arterioles devoid of retinal tissue and that the vasodilation to histamine was observed within a few minutes in a manner dependent upon the intact endothelium. It has been shown that histamine can stimulate rapid eNOS phosphorylation/activity57 and NO release58 from cultured endothelial cells in a matter of minutes. Based on the signaling characteristics in response to histamine, it is likely that eNOS contributes to such a rapid response in our studies. We found that blockade of prostanoids by indomethacin partly inhibited the histamine-induced vasodilation. Since both vasoconstrictor and vasodilator prostanoids59 can be blocked by indomethacin, the reduced histamine-induced vasodilation by indomethacin suggests the blockage of vasodilator prostanoids in the present study. It has been reported that the vessel relaxation to histamine can be attenuated by cyclooxygenase blockade in bovine retinal arteries19 and that histamine can induce the release of vasodilator prostanoids,53 possibly PGI2,60 from the arterial wall. Since indomethacin did not alter resting vascular tone, it is speculated that histamine activates cyclooxygenase for PGI2 release and subsequently leads to dilation of retinal arterioles in our study. 
This is the first report on the involvement of KCa channels in mediating histamine-induced vasodilation in small retinal arterioles. The vasodilations through the activation of KCa channels have been shown to be the main mechanism of EDHF-induced vasodilation in various tissues,11 especially the vasodilators related to CYP450 metabolites such as EETs.61 Similar to the finding of KCa channel inhibition, administration of CYP450 enzyme inhibitor sulfaphenazole in the presence of indomethacin and L-NAME abolished vasodilation to histamine (Fig. 6), suggesting the involvement of CYP450 enzymes in this vasodilation. Although some evidence has suggested that activation of myoendothelial gap junctions and release of H2O2 can contribute to the EDHF-induced vasodilation in certain vascular beds,9 these proposed mechanisms appear not to apply to the retinal arterioles, since H2O2 scavenger catalase or the gap junction inhibitor carbenoxolone had no effect on histamine-induced vasodilation (Fig. 6). Under the inhibition of cyclooxygenase and NOS pathways, sulfaphenazole and KCa channel inhibitors (apamin plus ChTX) produced comparable inhibition of histamine-induced vasodilation (Figs. 5, 6), suggesting that EETs may contribute to the dilation of the retinal arterioles by activating KCa channels. Interestingly, an earlier study19 has reported that the dilation of large bovine retinal arterioles (∼240 μm) to histamine was mediated solely by prostanoids and NO. This discrepancy may be related to the use of smaller sizes of arterioles in our study because the importance of EDHF was found to increase with decreasing vessel size.12,62 
Histamine might be a useful agent to facilitate studies on the physiologic and pathophysiologic regulation of vasomotor function by endothelium-derived vasodilators such as NO, prostanoids, and EDHF in the retinal microcirculation because it activates three major endothelium-dependent vasodilators from the retinal arteriolar wall. It has been shown that the EETs/EDHF-mediated vasodilation is sensitive to high glucose exposure by inhibiting CYP450 activity under oxidative stress.63 We previously have found that retinal arterial blood flow is reduced in patients with type 2 diabetes mellitus with minimal retinopathy,64 suggesting that the early blood flow dysregulation leads to the subsequent diabetic retinopathy. Since EDHF can play compensatory roles in vasoregulation, especially under conditions with compromised function of NO and prostanoids,65 the selective EDHF deficiency might have a significant impact on retinal blood flow regulation during disease development. Further study is needed to examine the effect of diabetes on retinal arteriolar dilation to histamine in relation to the pathogenesis of diabetic retinopathy in humans. 
In conclusion, we showed that histamine elicits potent dilation of the small retinal arterioles through activation of the H1 and H2 receptors in the retinal endothelial cells. The endothelium-dependent dilation is mediated by the activation of cyclooxygenase, NO synthase, and CYP450 enzymes. The EETs, derived from CYP450, might exert EDHF properties by activating KCa channels for vasodilation. 
Acknowledgments
Disclosure: S. Otani, None; T. Nagaoka, None; T. Omae, None; I. Tanano, None; T. Kamiya, None; S. Ono, None; T.W. Hein, None; L. Kuo, None; A. Yoshida, None 
References
Yu DY, Yu PK, Cringle SJ, Kang MH, Su EN. Functional and morphological characteristics of the retinal and choroidal vasculature. Prog Retin Eye Res. 2014; 40: 53–93.
Haefliger IO, Flammer J, Beny JL, Luscher TF. Endothelium-dependent vasoactive modulation in the ophthalmic circulation. Prog Retin Eye Res. 2001; 20: 209–225.
Ando J, Yamamoto K. Vascular mechanobiology: endothelial cell responses to fluid shear stress. Circ J. 2009; 73: 1983–1992.
Vanhoutte PM, Eber B. Endothelium-derived relaxing and contracting factors. Wien Klin Wochenschr. 1991; 103: 405–411.
Hein TW, Rosa RHJr, Ren Y, Xu W, Kuo L. VEGF receptor-2-linked PI3K/Calpain/SIRT1 activation mediates retinal arteriolar dilations to VEGF and shear stress. Invest Ophthalmol Vis Sci. 2015; 56: 5381–5389.
Feletou M, Vanhoutte PM. Endothelium-dependent hyperpolarization of canine coronary smooth muscle. Br J Pharmacol. 1988; 93: 515–524.
Huang AH, Busse R, Bassenge E. Endothelium-dependent hyperpolarization of smooth muscle cells in rabbit femoral arteries is not mediated by EDRF (nitric oxide). Naunyn Schmiedebergs Arch Pharmacol. 1988; 338: 438–442.
Taylor SG, Weston AH. Endothelium-derived hyperpolarizing factor: a new endogenous inhibitor from the vascular endothelium. Trends Pharmacol Sci. 1988; 9: 272–274.
Feletou M, Vanhoutte PM. EDHF: an update. Clinical Sci (Lond). 2009; 117: 139–155.
Griffith TM. Endothelium-dependent smooth muscle hyperpolarization: do gap junctions provide a unifying hypothesis? Br J Pharmacol. 2004; 141: 881–903.
Grgic I, Kaistha BP, Hoyer J, Kohler R. Endothelial Ca2+-activated K+ channels in normal and impaired EDHF-dilator responses--relevance to cardiovascular pathologies and drug discovery. Br J Pharmacol. 2009; 157: 509–526.
Shimokawa H, Yasutake H, Fujii K, et al. The importance of the hyperpolarizing mechanism increases as the vessel size decreases in endothelium-dependent relaxations in rat mesenteric circulation. J Cardiovasc Pharmacol. 1996; 28: 703–711.
Nakazawa T, Kaneko Y, Mori A, et al. Attenuation of nitric oxide- and prostaglandin-independent vasodilation of retinal arterioles induced by acetylcholine in streptozotocin-treated rats. Vascul Pharmacol. 2007; 46: 153–159.
Mori A, Suzuki S, Sakamoto K, Nakahara T, Ishii K. Role of calcium-activated potassium channels in acetylcholine-induced vasodilation of rat retinal arterioles in vivo. Naunyn Schmiedebergs Arch Pharmacol. 2011; 383: 27–34.
MacGlashan DJr. Histamine: a mediator of inflammation. J Allergy Clin Immunol. 2003; 112: S53–S59.
Maintz L, Novak N. Histamine and histamine intolerance. Am J Clin Nutr. 2007; 85: 1185–1196.
Nowak JZ, Nawrocki J. Histamine in the human eye. Ophthalmic Res. 1987; 19: 72–75.
Nowak JZ. Histamine in the retina: recent progress and perspectives. Agents Actions. 1990; 30: 202–205.
Benedito S, Prieto D, Nielsen PJ, Nyborg NC. Histamine induces endothelium-dependent relaxation of bovine retinal arteries. Invest Ophthalmol Vis Sci. 1991; 32: 32–38.
Clermont AC, Brittis M, Shiba T, McGovern T, King GL, Bursell SE. Normalization of retinal blood flow in diabetic rats with primary intervention using insulin pumps. Invest Ophthalmol Vis Sci. 1994; 35: 981–990.
Schmetterer L, Wolzt M, Graselli U, et al. Nitric oxide synthase inhibition in the histamine headache model. Cephalalgia. 1997; 17: 175–182.
Zawinka C, Resch H, Schmetterer L, Dorner GT, Garhofer G. Intravenously administered histamine increases choroidal but not retinal blood flow. Invest Ophthalmol Vis Sci. 2004; 45: 2337–2341.
Resch H, Zawinka C, Lung S, Weigert G, Schmetterer L, Garhofer G. Effect of histamine and cimetidine on retinal and choroidal blood flow in humans. Am J Physiol Regul Integr Comp Physiol. 2005; 289: R1387–R1391.
Weigert G, Zawinka C, Resch H, Schmetterer L, Garhofer G. Intravenous administration of diphenhydramine reduces histamine-induced vasodilator effects in the retina and choroid. Invest Ophthalmol Vis Sci. 2006; 47: 1096–1100.
Gill DS, Barradas MA, Fonseca VA, Dandona P. Plasma histamine concentrations are elevated in patients with diabetes mellitus and peripheral vascular disease. Metabolism. 1989; 38: 243–247.
Yousif MH, Oriowo MA, Cherian A, Adeagbo AS. Histamine-induced vasodilatation in the perfused mesenteric arterial bed of diabetic rats. Vascul Pharmacol. 2002; 39: 287–292.
Hein TW, Yuan Z, Rosa RHJr, Kuo L. Requisite roles of A2A receptors, nitric oxide, and KATP channels in retinal arteriolar dilation in response to adenosine. Invest Ophthalmol Vis Sci. 2005; 46: 2113–2119.
Hein TW, Xu W, Kuo L. Dilation of retinal arterioles in response to lactate: role of nitric oxide guanylyl cyclase and ATP-sensitive potassium channels. Invest Ophthalmol Vis Sci. 2006; 47: 693–699.
Nagaoka T, Hein TW, Yoshida A, Kuo L. Simvastatin elicits dilation of isolated porcine retinal arterioles: role of nitric oxide and mevalonate-rho kinase pathways. Invest Ophthalmol Vis Sci. 2007; 48: 825–832.
Omae T, Nagaoka T, Tanano I, Yoshida A. Pioglitazone, a peroxisome proliferator-activated receptor-gamma agonist, induces dilation of isolated porcine retinal arterioles: role of nitric oxide and potassium channels. Invest Ophthalmol Vis Sci. 2011; 52: 6749–6756.
Kuo L, Davis MJ, Chilian WM. Endothelium-dependent flow-induced dilation of isolated coronary arterioles. Am J Physiol. 1990; 259: H1063–H1070.
Tayo FM, Bevan JA. Pharmacological characterization of histamine receptors in the rabbit renal artery. Eur J Pharmacol. 1986; 121: 129–133.
Lin JH. Pharmacokinetic and pharmacodynamic properties of histamine H2-receptor antagonists: relationship between intrinsic potency and effective plasma concentrations. Clin Pharmacokinet. 1991; 20: 218–236.
Arrang JM, Garbarg M, Lancelot JC, et al. Highly potent and selective ligands for histamine H3-receptors. Nature. 1987; 327: 117–123.
Hein TW, Kuo L. cAMP-independent dilation of coronary arterioles to adenosine: role of nitric oxide G proteins and KATP channels. Cir Res. 1999; 85: 634–642.
Moncada S, Vane JR. Pharmacology and endogenous roles of prostaglandin endoperoxides thromboxane A2 and prostacyclin. Pharmacol Rev. 1978; 30: 293–331.
Burnham MP, Bychkov R, Feletou M, et al. Characterization of an apamin-sensitive small-conductance Ca(2+)-activated K(+) channel in porcine coronary artery endothelium: relevance to EDHF. Br J Pharmacol. 2002; 135: 1133–1143.
Bychkov R, Burnham MP, Richards GR, et al. Characterization of a charybdotoxin-sensitive intermediate conductance Ca2+-activated K+ channel in porcine coronary endothelium: relevance to EDHF. Br J Pharmacol. 2002; 137: 1346–1354.
Stankevicius E, Dalsgaard T, Kroigaard C, et al. Opening of small and intermediate calcium-activated potassium channels induces relaxation mainly mediated by nitric-oxide release in large arteries and endothelium-derived hyperpolarizing factor in small arteries from rat. J Pharmacol Exp Ther. 2011; 339: 842–850.
Shimokawa H, Morikawa K. Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in animals and humans. J Mol Cell Cardiol. 2005; 39: 725–732.
Thengchaisri N, Shipley R, Ren Y, Parker J, Kuo L. Exercise training restores coronary arteriolar dilation to NOS activation distal to coronary artery occlusion: role of hydrogen peroxide. Arterioscler Thromb Vasc Biol. 2007; 27: 791–798.
Earley S, Pastuszyn A, Walker BR. Cytochrome p-450 epoxygenase products contribute to attenuated vasoconstriction after chronic hypoxia. Am J Physiol Heart Circ Physiol. 2003; 285: H127–H136.
Kenny LC, Baker PN, Kendall DA, Randall MD, Dunn WR. The role of gap junctions in mediating endothelium-dependent responses to bradykinin in myometrial small arteries isolated from pregnant women. Br J Pharmacol. 2002; 136: 1085–1088.
Sandow SL. Factors fiction and endothelium-derived hyperpolarizing factor. Clin Exp Pharmacol Physiol. 2004; 31: 563–570.
Dyer J, Warren K, Merlin S, Metcalfe DD, Kaliner M. Measurement of plasma histamine: description of an improved method and normal values. J Allergy Clin Immunol. 1982; 70: 82–87.
Knight DA, Stewart GA, Thompson PJ. Histamine tachyphylaxis in human airway smooth muscle: the role of H2-receptors and the bronchial epithelium. Am Rev Respir Dis. 1992; 146: 137–140.
Rosenblum WI, Nelson GH. Tone regulates opposing endothelium-dependent and -independent forces: resistance brain vessels in vivo. Am J Physiol. 1990; 259: H243–H247.
Falloon BJ, Stephens N, Tulip JR, Heagerty AM. Comparison of small artery sensitivity and morphology in pressurized and wire-mounted preparations. Am J Physiol. 1995; 268: H670–H678.
Ottosson A, Jansen I, Edvinsson L. Pharmacological characterization of histamine receptors in the human temporal artery. Br J Clin Pharmacol. 1989; 27: 139–145.
Satoh H, Inui J. Endothelial cell-dependent relaxation and contraction induced by histamine in the isolated guinea-pig pulmonary artery. Eur J Pharmacol. 1984; 97: 321–324.
Kawai Y, Ohhashi T. Histamine H2 receptor-mediated endothelium-dependent relaxation in canine spinal artery. Jpn J Physiol. 1995; 45: 607–618.
Haefliger IO, Flammer J, Luscher TF. Nitric oxide and endothelin-1 are important regulators of human ophthalmic artery. Invest Ophthalmol Vis Sci. 1992; 33: 2340–2343.
Van de Voorde J, Leusen I. Role of the endothelium in the vasodilator response of rat thoracic aorta to histamine. Eur J Pharmacol. 1983; 87: 113–120.
Martinez AC, Prieto D, Raposo R, et al. Endothelium-independent relaxation induced by histamine in human dorsal penile artery. Clin Exp Pharmacol Physiol. 2000; 27: 500–507.
Kibbe M, Billiar T, Tzeng E. Inducible nitric oxide synthase and vascular injury. Cardiovasc Res. 1999; 43: 650–657.
Overso Hansen P, Kringelholt S, Simonsen U, Bek T. Hypoxia-induced relaxation of porcine retinal arterioles in vitro depends on inducible NO synthase and EP4 receptor stimulation in the perivascular retina. Acta Ophthalmol. 2015; 93: 457–463.
Thors B, Halldorsson H, Thorgeirsson G. Thrombin and histamine stimulate endothelial nitric-oxide synthase phosphorylation at Ser1177 via an AMPK mediated pathway independent of PI3K-Akt. FEBS Lett. 2004; 573: 175–180.
Lantoine F, Iouzalen L, Devynck MA, Millanvoye-Van Brussel E, David-Dufilho M. Nitric oxide production in human endothelial cells stimulated by histamine requires Ca2+ influx. Biochem J. 1998; 330 (pt 2): 695–699.
Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors: structures properties, and functions. Physiol Rev. 1999; 79: 1193–1226.
Toda N, Konishi M, Miyazaki M. Involvement of endogenous prostaglandin I2 in the vascular action of histamine in dogs. J Pharmacol Exp Ther. 1982; 223: 257–262.
Campbell WB, Gebremedhin D, Pratt PF, Harder DR. Identification of epoxyeicosatrienoic acids as endothelium-derived hyperpolarizing factors. Circ Res. 1996; 78: 415–423.
Busse R, Edwards G, Feletou M, Fleming I, Vanhoutte PM, Weston AH. EDHF: bringing the concepts together. Trends Pharmacol Sci. 2002; 23: 374–380.
Tsai SH, Hein TW, Kuo L, Yang VC. High glucose impairs EDHF-mediated dilation of coronary arterioles via reduced cytochrome P450 activity. Microvasc Res. 2011; 82: 356–363.
Nagaoka T, Sato E, Takahashi A, Yokota H, Sogawa K, Yoshida A. Impaired retinal circulation in patients with type 2 diabetes mellitus: retinal laser Doppler velocimetry study. Investi Ophthalmol Vis Sci. 2010; 51: 6729–6734.
Scotland RS, Madhani M, Chauhan S, et al. Investigation of vascular responses in endothelial nitric oxide synthase/cyclooxygenase-1 double-knockout mice: key role for endothelium-derived hyperpolarizing factor in the regulation of blood pressure in vivo. Circulation. 2005; 111: 796–803.
Figure 1
 
Response of isolated retinal arterioles to histamine. There was no significant difference between the two trials (n = 6).
Figure 1
 
Response of isolated retinal arterioles to histamine. There was no significant difference between the two trials (n = 6).
Figure 2
 
Effect of the removal of the endothelium by perfusion with 0.4% CHAPS. *P < 0.05 versus control.
Figure 2
 
Effect of the removal of the endothelium by perfusion with 0.4% CHAPS. *P < 0.05 versus control.
Figure 3
 
Effect of incubation with H1 antagonist chlorpheniramine (1 μM), H2 antagonist famotidine (10 μM), or H3 and H4 antagonist thioperamide (1 μM). *P < 0.05 versus control.
Figure 3
 
Effect of incubation with H1 antagonist chlorpheniramine (1 μM), H2 antagonist famotidine (10 μM), or H3 and H4 antagonist thioperamide (1 μM). *P < 0.05 versus control.
Figure 4
 
Immunohistochemical analysis of H1 and H2 receptors in retinal arterioles. (A) Staining with anti–H1 receptor (green) and anti-eNOS (red) antibodies shows expression of the H1 receptor and eNOS. The merged image shows overlapping staining (yellow) of the H1 receptor with eNOS. (B) Staining with anti–H2 receptor (green) and anti-eNOS (red) antibodies shows expression of the H2 receptor and eNOS. The merged image shows overlapping staining (yellow) of the H2 receptor and eNOS. (C) Staining with anti–H1 receptor (green) and anti–α-SMA (red) antibodies shows expression of the H1 receptor and SMA. The merged image shows no overlapping staining of the H1 receptor and SMA. (D) Staining with anti–H2 receptor (green) and anti–α-SMA (red) antibodies shows expression of the H2 receptor and SMA. The merged image shows no overlapping staining of the H2 receptor and SMA. The images are representative of three separate experiments.
Figure 4
 
Immunohistochemical analysis of H1 and H2 receptors in retinal arterioles. (A) Staining with anti–H1 receptor (green) and anti-eNOS (red) antibodies shows expression of the H1 receptor and eNOS. The merged image shows overlapping staining (yellow) of the H1 receptor with eNOS. (B) Staining with anti–H2 receptor (green) and anti-eNOS (red) antibodies shows expression of the H2 receptor and eNOS. The merged image shows overlapping staining (yellow) of the H2 receptor and eNOS. (C) Staining with anti–H1 receptor (green) and anti–α-SMA (red) antibodies shows expression of the H1 receptor and SMA. The merged image shows no overlapping staining of the H1 receptor and SMA. (D) Staining with anti–H2 receptor (green) and anti–α-SMA (red) antibodies shows expression of the H2 receptor and SMA. The merged image shows no overlapping staining of the H2 receptor and SMA. The images are representative of three separate experiments.
Figure 5
 
Effect of incubation with cyclooxygenase inhibitor indomethacin (IM, 10 μM), NO synthase inhibitor L-NAME (10 μM), or SKCa inhibitor apamin (Apa, 0.1 μM) plus IKCa inhibitor ChTX (0.1 μM), or incubation with all four inhibitors. *P < 0.05 versus control.
Figure 5
 
Effect of incubation with cyclooxygenase inhibitor indomethacin (IM, 10 μM), NO synthase inhibitor L-NAME (10 μM), or SKCa inhibitor apamin (Apa, 0.1 μM) plus IKCa inhibitor ChTX (0.1 μM), or incubation with all four inhibitors. *P < 0.05 versus control.
Figure 6
 
Effect of incubation with gap junction inhibitor carbenoxolone (CBX, 100 μM), H2O2 scavenger catalase (CAT, 1000 U/mL), and CYP450 enzyme inhibitor sulfaphenazole (SUL, 10 μM). To isolate the EDHF component of the histamine-induced vasodilator response, IM (10 μM) and L-NAME (10 μM) were added throughout the experiment. *P < 0.05 between groups.
Figure 6
 
Effect of incubation with gap junction inhibitor carbenoxolone (CBX, 100 μM), H2O2 scavenger catalase (CAT, 1000 U/mL), and CYP450 enzyme inhibitor sulfaphenazole (SUL, 10 μM). To isolate the EDHF component of the histamine-induced vasodilator response, IM (10 μM) and L-NAME (10 μM) were added throughout the experiment. *P < 0.05 between groups.
Table.
 
Resting Diameters and Dilation of Retinal Arterioles to SNP
Table.
 
Resting Diameters and Dilation of Retinal Arterioles to SNP
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