January 2004
Volume 45, Issue 1
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Physiology and Pharmacology  |   January 2004
Myogenic Tone and Reactivity of the Rat Ophthalmic Artery
Author Affiliations
  • Yagna P. R. Jarajapu
    From the Department of Pharmacology and Therapeutics, Vascular Biology and Cell Physiology Group, College of Medicine, and McKnight Brain Institute, University of Florida, Gainesville, Florida.
  • Maria B. Grant
    From the Department of Pharmacology and Therapeutics, Vascular Biology and Cell Physiology Group, College of Medicine, and McKnight Brain Institute, University of Florida, Gainesville, Florida.
  • Harm J. Knot
    From the Department of Pharmacology and Therapeutics, Vascular Biology and Cell Physiology Group, College of Medicine, and McKnight Brain Institute, University of Florida, Gainesville, Florida.
Investigative Ophthalmology & Visual Science January 2004, Vol.45, 253-259. doi:https://doi.org/10.1167/iovs.03-0546
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      Yagna P. R. Jarajapu, Maria B. Grant, Harm J. Knot; Myogenic Tone and Reactivity of the Rat Ophthalmic Artery. Invest. Ophthalmol. Vis. Sci. 2004;45(1):253-259. https://doi.org/10.1167/iovs.03-0546.

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

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Abstract

purpose. To study and quantify myogenic behavior and reactivity of the rat ophthalmic artery to pressure and different vasoactive substances in vitro.

methods. Rat ophthalmic arteries (diameter of 217 ± 6 μm, n = 22) were isolated, cannulated with glass pipettes in an arteriograph and pressurized in a physiological buffer. Internal diameter was continuously monitored. The effect of intraluminal pressure on the diameter was assessed and concentration–response curves to different constrictor and dilator agonists were obtained at an intraluminal pressure of 70 mm Hg.

results. Myogenic tone developed at an intraluminal pressure of 30 to 40 mm Hg, continued to increase, and was maintained up to a pressure of 199 mm Hg in these arteries. Arteries dilated and constricted in response to 16 and 60 mM potassium, respectively. Endothelin-1 was the most potent and efficacious constrictor, with a biphasic concentration–response curve, followed by vasopressin, serotonin, U-46619 and phenylephrine. Carbachol was the most efficacious dilator, followed by isoprenaline. The peptide dilators calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) were potent but less efficacious than carbachol and isoprenaline. Histamine and adenosine were even less potent and less efficacious dilators. N G-nitro-l-arginine methyl ester (l-NAME) constricted and indomethacin dilated the arteries.

conclusions. This study provides the first direct evidence for myogenic autoregulatory properties and pharmacological heterogeneity in the rat ophthalmic artery.

The ophthalmic circulation consists of blood flow through the ophthalmic artery into ciliary arteries feeding the choroidal–uveal circulation and a central retinal artery or cilioretinal arteries feeding the retinal circulation. Regulatory control of blood flow in this vascular bed is essential for the normal functioning of the retina. Knowledge about hemodynamic responses to different endogenous vasoactive stimuli is necessary to understand the physiology and pathophysiology of ophthalmic circulation in diseases affecting the retinal circulation such as diabetes. The hemodynamics of the ophthalmic circulation was reported altered in proliferative diabetic retinopathy. 1  
The rat model is extensively used for different types of diabetes, but these models are yet to be exploited to understand diabetes-induced functional changes in the ocular circulation, even though several biochemical and molecular–biological studies have been undertaken. Before the mechanisms involved in functional alterations in pathologic states such as diabetes can be examined, the normal ophthalmic circulation must be understood, and this was the purpose of this study. 
Various isolated ocular preparations have been used in studies of ocular vascular biology, physiology, and pharmacology. 2 There is no single system (preparation) that is capable of addressing all the questions that must be answered if a complete understanding of mechanisms of vascular regulation in the eye is to be achieved. Functional studies in ophthalmic circulation can be performed by perfusing the whole eye or by studying isolated arterial segments—ophthalmic, ciliary, choroid, or retinal arterial segments—in vitro. Rat ciliary, choroid or retinal arterial segments may be too small to perform functional studies in which constrictor–contractile and dilator–relaxant responses are recorded; however, it is possible to study the feeding ophthalmic artery, as a pressurized or wire-mounted preparation. Clinical studies use hemodynamics in this artery as a measure of overall function of the ophthalmic circulation, 1 3 4 indicating that the ophthalmic artery is considered a valid preparation to study and understand pathophysiological changes in the dynamics of the ocular circulation. 
In this study we characterized the reactivity of rat ophthalmic artery to intraluminal pressure and quantified the responses to different vasoactive agents by using intact pressurized arterial segments of the ophthalmic artery taken just before entry into the optic nerve head. 
Methods
Preparation of Ophthalmic Arteries and Measurement of Luminal Diameter
Male Sprague-Dawley rats (250 to 300 g) were anesthetized by an intraperitoneal injection of pentobarbital sodium (160 mg/kg) and killed by decapitation. The brain was removed, and the skull with intact eyes was placed in an ice-cold oxygenated physiological cerebrospinal fluid (PCSF, see below for composition). The main ophthalmic artery running along the optic nerve to the eye was dissected out and cleaned from adherent tissues. Arterial segments were transferred to a custom-built arteriograph filled with ice-cold PCSF, cannulated with a glass pipette, and secured with nylon thread, as described earlier for rat cerebral arteries. 5 The arteriograph was then placed on the stage of an inverted microscope and visualized with a charge-coupled device (CCD) camera coupled to a calibrated video caliper system to measure arterial diameter. Arteries were slowly pressurized to 70 mm Hg under no-flow conditions with a pressure servonull system (Living Systems, Inc., Waterbury, VT), and warmed to 37°C while being continuously superfused (3 mL/min) with PCSF bubbled with 21% O2, 5% CO2, and 74% N2 (pH 7.3–7.4 in the bath). The working pressure of 70 mm Hg was chosen based on earlier observations by Riva et al. 6 in human ocular circulation and McCarron et al. 7 in rat cerebral circulation. This research was conducted in accord with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Florida. 
Experimental Protocol
After an equilibration period of approximately 20 minutes, arteries showed stable myogenic tone at 70 mm Hg. Afterward, the effect of different pharmacological agents on myogenic tone was evaluated. Concentration–response curves (CRCs) for these agents were obtained by cumulative addition to the superfusate. CRCs were generated in 0.5-log order concentrations, and the arteries were exposed to different concentrations for at least 8 minutes or until the observed effect reached steady state. Receptor-independent contraction and relaxation responses to 60 and 16 mM KCl, respectively, were also assessed. The presence of functional endothelium in the arteries was tested by applying carbachol. All arteries that showed at least 80% dilation (decrease in myogenic tone by 80%) after 300 nM carbachol were considered to have intact endothelia. Obtaining the maximum possible diameter or passive diameter in calcium-free PCSF (composition described later) concluded all experiments. 
Pressure-dependent changes in diameter were evaluated by increasing the intraluminal pressure in 10-mm Hg steps from 1 to 199 mm Hg in PCSF. Pressure-diameter curves were repeated in the presence of calcium-free PCSF as superfusate and the myogenic tone was calculated according to the following equation:  
\[\mathrm{Myogenic\ tone}{=}(D_{\mathrm{p}}{-}D_{\mathrm{a}})/D_{\mathrm{p}}\ {\cdot}\ 100\]
where D a is the internal diameter of the arterial segment with active myogenic tone in the presence of PCSF at a particular intraluminal pressure, and D p is the passive diameter obtained at the same pressure in the presence of calcium-free PCSF. 
Data Analysis and Statistics
The efficacy of different agonists was expressed as the maximum response, whether it be constriction or dilation, produced. The potency of different agonists was expressed as pEC50 (negative logarithm of the concentration of the agonist to produce 50% of the maximum effect). pEC50 values were calculated by analyzing CRCs with a program (Prism; GraphPad, San Diego, CA) that fits the data to a four-parameter logistic equation:  
\[Y{=}\mathrm{minimum}{+}\frac{(\mathrm{maximum}\ {-}\ \mathrm{minimum})}{1{+}10^{(\mathrm{log\ EC}_{50}{-}X)P}}\]
where minimum and maximum indicate the smallest and the highest responses produced by an agonist, X is the logarithm of the molar concentration of an agonist, Y is the response at a concentration of X, and P is the Hill slope. In one instance, a four-parameter logistic equation for a two-site fit was used:  
\[Y{=}\mathrm{minimum}1{+}\frac{(\mathrm{maximum}1\ {-}\ \mathrm{minimum}1)}{1{+}10^{(\mathrm{log\ EC}_{50}1{-}X)P1}}{+}\frac{(\mathrm{maximum}2\ {-}\ \mathrm{minimum}2)}{1{+}10^{(\mathrm{log\ EC}_{50}2{-}X)P2}}\]
where 1 and 2 refer to the first and second sites. 
Results are expressed as the mean ± SEM; n indicates the number of independent experiments, which equals the number of animals used. Means were compared by Student’s t-test and P < 0.05 was considered statistically significant. 
Drugs, Chemicals, and Solutions
Phenylephrine, vasopressin, angiotensin II, UK-14304, endothelin-1, U-46619, carbachol, histamine, isoprenaline, histamine, calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), indomethacin, and N G-nitro-l-arginine methyl ester (l-NAME) were purchased from Sigma-Aldrich (St. Louis, MO); octreotide from Novartis (East Hanover, NJ); and serotonin (serotonin HCl), bradykinin, and substance P from Fluka (Steinheim, Germany). Stock solutions of phenylephrine (10 mM), vasopressin (0.1 mM), angiotensin II (10 mM), serotonin (10 mM), carbachol (10 mM), isoprenaline (10 mM), histamine (10 mM), CGRP (0.1 mM), octreotide (1 mg/mL), substance P (1 mM), bradykinin (10 mM), and l-NAME (100 mM) were prepared in distilled water. UK-14304 (10 mM) was dissolved in dimethylsulfoxide (DMSO). Indomethacin was dissolved in aqueous solution of equimolar Na2CO3. U-46619 (supplied as 10 mg/mL solution in methyl acetate) was diluted to 0.1 mM with absolute ethanol. 
The composition of PCSF (mM): NaCl, 120; KCl, 3; NaHCO3, 24; NaH2PO4.H2O, 1.2; CaCl2, 2.5; MgSO4.7H2O, 1.2; and glucose, 4. PCSF with 16 and 60 mM KCl was prepared by replacing NaCl with an equimolar quantity of KCl. Calcium-free PCSF was prepared by replacing CaCl2 with 2 mM EGTA. 
Results
The average passive diameter (fully dilated, considered to be 0% constriction) of the rat ophthalmic arteries used in this study was 217 ± 6 (n = 22) μm. At an intraluminal pressure of 70 mm Hg, arteries spontaneously constricted to an average diameter of 159 ± 4 μm, representing an average myogenic tone of 28% ± 1%. 
Pressure-Dependent Changes in the Diameter of Rat Ophthalmic Artery
Figure 1a shows a representative tracing of changes in the internal diameter of a rat ophthalmic artery with an increase in the intraluminal pressure in the presence of PCSF and calcium-free PCSF. An increase in intraluminal pressure resulted in an increase in diameter of ophthalmic arteries up to a pressure of 30 to 40 mm Hg, after which the arteries showed no increase or a sustained decrease in diameter, indicating the development of myogenic tone (Fig. 1b) . Myogenic tone developed at 70 mm Hg was 26%, which continued to rise and remained the same in the pressure range of 120 to 199 mm Hg (34%–36% myogenic tone; Fig. 1b ), indicating the autoregulatory function of this artery. Pressures beyond 199 mm Hg were not possible to achieve with the instrumentation used (Living Systems Inc.), and so the pressure at which arteries tend to lose tone could not be obtained. 
Effect of Extracellular Potassium on Myogenic Tone of Rat Ophthalmic Artery
Elevation of extracellular potassium to 16 mM caused a near maximum dilation of the arterial segments to 211 ± 9 μm, or the arterial tone was reduced to 15% ± 4% of the initial myogenic constriction (n = 13). Elevation of extracellular potassium to 60 mM caused constriction of the arterial segments to 83 ± 4 μm or increased the arterial tone to 225% ± 8% of myogenic constriction (130% constriction, n = 22). Arterial segments with no myogenic tone did not dilate to 16 mM potassium but constricted to 60 mM potassium (data not shown). 
Studies with Vasoconstrictors
The constriction produced by different agonists in these arteries was expressed as the percent of constriction produced by 60 mM KCl. Efficacy or maximum responses produced by different agonists and their potency or pEC50 values are given in Table 1 . Serotonin, vasopressin, phenylephrine, U-46619 and UK-14304 produced concentration-dependent increases in arterial tone (reduction in the diameter). In the lower concentration range (1 fM to 30 pM), the constriction response to U-46619 was inconsistent and not sustained, but sustained constriction was observed at concentrations of more than 30 pM. Serotonin, vasopressin, and phenylephrine produced similar maximum constriction that was significantly higher than that produced by U-46619 and UK-14304 (Fig. 2a)
Endothelin-1 produced biphasic CRCs showing involvement of two different receptor subtypes in endothelin-1–mediated contractile responses. These curves were analyzed by a two-site model for nonlinear regression, which yielded pEC50 values of 12.2 ± 0.8 and 8.3 ± 0.1 for high- and low-affinity receptor subtypes of endothelin-1, respectively, with a contribution of 19% and 81%, respectively, to the total contractile response, which was 130% ± 7% of the response to 60 mM KCl. Contractile responses to endothelin-1 were significantly higher (P < 0.05, n = 5) than the other constrictors used. The order of efficacy of the constrictors was endothelin-1 > vasopressin = serotonin = phenylephrine > U-46619 > UK-14304. The order of potency of the constrictors was: endothelin-1 > vasopressin = serotonin > U-46619 > UK-14304 > phenylephrine. 
Angiotensin II did not produce constriction in the concentration range of 0.1 nM to 1 μM in three of the four arteries tried. In one of these four arteries, a small dilation was observed at high concentrations (0.1–1 μM; data not shown). 
Studies with Vasodilators
Decrease in the myogenic tone or vasodilator response to different agonists was expressed as a percentage of myogenic tone, as shown in Figure 3 . Maximum response and pEC50 of the vasodilators are shown in Table 1 . Carbachol produced the maximum vasodilation and completely reversed myogenic tone. Isoprenaline produced dilation with the same potency as carbachol, but the maximum dilation was significantly smaller than that produced by carbachol. Maximum dilation produced by CGRP, VIP, adenosine, and histamine were similar but significantly smaller than that produced by carbachol and isoprenaline. The potency of VIP and CGRP were significantly higher than all the other dilators tested. Adenosine and histamine were equipotent and significantly less potent than all the other dilators. The order of efficacy of the dilators was carbachol > isoprenaline > histamine = CGRP = VIP = adenosine. The order of potency of different dilators was CGRP > VIP > carbachol = isoprenaline > adenosine = histamine. Bradykinin, substance P, and octreotide did not produce sustained vasodilation responses in the ophthalmic arteries with myogenic tone. 
Studies with l-NAME and Indomethacin
The nitric oxide synthase (NOS) inhibitor l-NAME produced sustained, reversible, and concentration-dependent constriction of the arterial segments with myogenic tone in the concentration range of 10 nM to 100 μM in the present study (Fig. 4) . The maximum concentration used to obtain CRCs was 100 μM, at which the observed constriction was 148% ± 9% of myogenic tone (48% constriction). The cyclooxygenase (COX) inhibitor indomethacin produced a sustained, reversible, and concentration-dependent (10 nM to 100 μM) decrease in the myogenic tone of the arterial segments (Fig. 4) . At a maximum concentration of 100 μM, indomethacin decreased myogenic tone to 56% ± 7% of initial tone (representing a 44% dilation). 
Discussion
Myogenic Autoregulation in the Rat Ophthalmic Artery
The present study for the first time shows quantitative evidence of the extensive myogenic properties of the rat ophthalmic artery. The ophthalmic artery can withstand higher pressures (≥199 mm Hg) without losing myogenic tone. Autoregulation is the intrinsic ability of an organ or tissue to maintain its blood flow in response to changes in perfusion pressure over a relatively wide range. This response primarily resides in the smooth muscle cells of the vascular wall (myogenic autoregulation) and is further modulated by tissue needs (metabolic autoregulation) and neurohumoral factors. Autoregulation ensures constancy of perfusion despite pressure changes. The present study focused on myogenic autoregulation in the ophthalmic artery and shows evidence for a constant perfusion of ophthalmic circulation as long as mean arterial blood pressure remains in the range of 40 to 199 mm Hg. This property of the ophthalmic artery offers protection for choroidal and retinal vasculature from exposure to higher systemic pressures that could result in hemorrhage. The unique anatomy of the ocular vascular bed, 8 particularly the sudden transition of the ophthalmic artery (diameter of 150–225 μm in rat) to several small retinal and choroidal arterioles (diameter, ≤50 μm in rat), requires more physiologically efficient autoregulation of blood flow within the ophthalmic artery than in any other vascular bed. Myogenic autoregulation in the ophthalmic artery is essential for the constant and smooth perfusion of retinal and choroidal vasculature but is not necessarily indicative of their autoregulatory properties, which may vary or operate at a higher level, owing to their smaller caliber and that receive only a portion of the ophthalmic blood flow. 3 9  
Effect of Elevated External Potassium on Myogenic Tone
An increase in the extracellular potassium concentration altered the tone of the ophthalmic artery in a concentration-dependent manner. An increase to 16 mM potassium caused dilation of the arterial segments, whereas a further increase to 60 mM resulted in a strong constriction response. Elevation of extracellular potassium to 16 mM causes membrane hyperpolarization in smooth muscle, resulting in sustained dilation in rat cerebral, renal, and coronary arteries through the activation of inward rectifier potassium channels (KIR) that mediate this vasodilation response. 10 11 Our observation is the first evidence to show the presence of functional KIR channels in the rat ophthalmic artery. This response has important physiological significance, as extracellular potassium increases in stress conditions such as ischemia 12 and high neuronal activity. 13 14 In ischemia, extracellular potassium can reach concentrations of 10 to 16 mM in heart and brain 12 15 that activate KIR channels resulting in dilation of arterioles, thus allowing increased blood flow. Absence of KIR channels results in more constriction of arteries by closing of voltage-dependent potassium channels (KV) and inability to respond to ischemic insults. Higher concentrations of extracellular potassium (>20 mM) even in the presence of functional KIR channels evokes a strong constrictor response resulting from depolarization and activation of voltage-dependent calcium channels as observed in the present study at 60 mM of potassium in rat ophthalmic artery. 16  
Vasoconstrictor Responses
A range of vasoconstrictors were used in this study that showed heterogeneous regulation of tone in this artery under normal physiological conditions. Responses to endothelin-1, the most potent and efficacious vasoconstrictor, clearly showed two phases of constriction, similar to observations with other vascular preparations. This observation suggests involvement of two different subtypes in endothelin-1–mediated constriction. The affinity of endothelin-1 with these two subtypes differs by approximately 10,000-fold. Earlier studies in mesenteric arteries 17 and the porcine ophthalmic artery 18 showed evidence for the involvement of two different subtypes in the endothelin-1–mediated contractile responses, but clear separation of responses was not observed. Characterization of different subtypes involved in this biphasic response to endothelin-1 remains to be undertaken. Observations from the present study are consistent with the earlier findings in human ophthalmic artery 19 in vitro and human ocular circulation 20 and rabbit choroidal circulation 21 in vivo that were shown to be highly sensitive to endothelin-1. 
Vasopressin and serotonin were found to be as potent as endothelin-1, with relatively smaller efficacy, showing a major role of vasoconstrictor peptides and serotonin in the regulation of tone in the ophthalmic artery. Phenylephrine, a selective α1-adrenoceptor agonist produced a less potent response, but was equi-efficacious compared with serotonin and vasopressin, whereas the selective α2-adrenoceptor agonist UK-14304 produced a significantly smaller response. This evidently shows dominant postjunctional α1-adrenoceptor–mediated control of tone in this artery with a minor role of α2-adrenoceptors. The sustained constriction response to U-46619 is evidence for a significant population of thromboxane A2 (TXA2) receptors in this artery but inconsistent responses to this agonist in the lower concentration range suggest that it elicits a nonclassic pharmacological response. Lack of a constriction response to angiotensin II indicates insensitivity of the ophthalmic circulation to angiotensin II probably because of the absence of expression of receptors in this vascular bed. This is in agreement with findings by Nyborg et al. 22 in bovine retinal arteries, showing no contraction response to angiotensin II and with clinical findings by Krejcy et al. 23 showing no changes in the hemodynamics of ocular circulation by infusion of angiotensin II. 
Vasodilator Responses
Carbachol, a stable analogue of acetylcholine produced a robust concentration-dependent relaxation of the ophthalmic artery resulting in a complete reversal of myogenic tone. Carbachol-mediated relaxation is known to be mediated by endothelium with the involvement of different relaxing factors, such as nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF), the contribution of which varies with the vascular bed. 24 Further studies are needed to characterize carbachol-mediated relaxation in this artery. 
Isoprenaline, a nonselective β-adrenoceptor agonist, was unexpectedly shown to be as efficacious and potent as carbachol in relaxing the ophthalmic artery in the present study suggesting an important role of circulating catecholamines in controlling blood flow in the ophthalmic circulation. These data are in agreement with clinical reports showing evidence of decreased ocular blood flow caused by β- and β1-adrenoceptor selective antagonists 25 26 and support the cautious use of β-adrenoceptor antagonists in diabetic patients with ocular complications. 
The effect of histamine in the ocular vasculature was shown to vary among species. Relaxation in the bovine retinal artery, 27 contraction in feline ophthalmociliary artery, 28 potent NO-dependent relaxation in the human ophthalmic artery, 19 and biphasic responses (contraction and relaxation) in the human ciliary artery 29 were reported. However, in the present study, histamine was relatively a less efficacious and less potent vasodilator in rat ophthalmic artery. Similar responses were also observed for adenosine. Like histamine, adenosine is released during ischemia or hypoxia and conveys a protective effect by dilating the arteries to increase blood flow. The smaller vasodilation responses observed in the present study may result in a significant increase in the total blood flow to the eye, as shown in human ocular circulation by using the uptake inhibitors of adenosine. 30 In contrast, Portellos et al. 31 observed in cat, a significant increase in the choroidal blood flow, but not in the optic nerve head or retinal blood flow. 
Different peptide vasodilators were also studied. Earlier studies showed evidence for peptidergic innervation in rat ophthalmic vasculature, 32 and potent vasodilator responses were induced by CGRP in bovine retinal artery 33 and by VIP and substance P in porcine ophthalmic artery. 34 35 However, in the present study, bradykinin, substance P, and octreotide, a stable analogue of somatostatin, did not produce dilation in the rat ophthalmic artery. VIP and CGRP induced potent vasodilation, but the maximum dilation observed was significantly smaller than that of the nonpeptide vasodilators carbachol and isoprenaline. The direct vasodilation effect may not be the major physiological response of these peptides in this vascular bed, because some of them produce significant effect on vascular smooth muscle proliferation and angiogenesis. 36 37  
Responses to Inhibitors of the NOS and COX Enzymes
The effect of l-NAME, an inhibitor of constitutive nitric oxide synthase (cNOS), and indomethacin, a nonselective inhibitor of cyclooxygenase, on myogenic tone were assessed, to evaluate the role of basal NO and prostanoid production in the maintenance of basal tone in the ophthalmic artery. The sustained constriction in response to l-NAME suggests basal activation of NOS resulting in release of nitric oxide that would regulate myogenic tone under physiological conditions. Pressure-induced myogenic constriction itself could be a strong stimulus for the activation of NOS and release of NO. 38 In contrast, indomethacin resulted in concentration-dependent loss of myogenic tone, suggesting involvement of cyclooxygenase metabolites in its regulation. Vasodilator prostanoids may not be released in response to myogenic constriction or may not have the capacity to overcome the effect of vasoconstrictor prostanoids. Future studies will focus at characterizing different prostanoids involved in the development and control of myogenic tone in this artery. 
Conclusions and Limitations of this Study
This study provides the first evidence that the rat ophthalmic artery exhibits profound myogenic autoregulation in response to pressure. Responses to elevated external potassium suggest also for the first time a functional role for inward rectifier potassium channels in the ocular circulation. KIR channels have been identified as key modulators of metabolic autoregulation in brain, heart, and kidney resistance vessels. Responses to a spectrum of peptide and nonpeptide endogenous vasoactive agents, suggest an intriguing pharmacological heterogeneity in the regulation of the ophthalmic circulation. 
Our results are limited to the ophthalmic artery. The distal retinal and choroidal arteries of the rat are smaller than the ophthalmic artery and may respond differently to vasoactive agents, which is the subject of current investigation. Also, our results were obtained under no-flow conditions. Normal flow would be expected to modulate arterial diameter further through endothelial mechanisms. 
In conclusion, we have provided a quantitative physiological and pharmacological baseline study of the rat ophthalmic artery that should serve as a reference for future mechanistic studies and studies in diseases with profound ocular impact, such as diabetes. 
 
Figure 1.
 
(a) Representative tracing of changes in internal diameter of rat ophthalmic artery in the presence of PCSF and calcium-free PCSF. Intraluminal pressure was increased from 1 to 199 mm Hg. (b) Internal diameter of rat ophthalmic artery at different pressures in the presence of PCSF and calcium-free PCSF. Myogenic tone developed at different pressures and was calculated according to equation 1 .
Figure 1.
 
(a) Representative tracing of changes in internal diameter of rat ophthalmic artery in the presence of PCSF and calcium-free PCSF. Intraluminal pressure was increased from 1 to 199 mm Hg. (b) Internal diameter of rat ophthalmic artery at different pressures in the presence of PCSF and calcium-free PCSF. Myogenic tone developed at different pressures and was calculated according to equation 1 .
Table 1.
 
Maximum Responses and pEC50 Values of the Agonists
Table 1.
 
Maximum Responses and pEC50 Values of the Agonists
Agonist n pEC50 Maximum Responses
Constrictors*
 Endothelin-1 5 8.3 ± 0.1 130 ± 7
12.2 ± 0.8
 Vasopressin 4 9.3 ± 0.1 91 ± 6
 Serotonin 6 9.0 ± 0.1 110 ± 6
 U-46619 4 7.8 ± 0.5 93 ± 8
 Phenylephrine 5 6.0 ± 0.1 100 ± 7
 UK 14304 4 6.9 ± 0.2 22 ± 2
Dilators, †
 CGRP 4 8.6 ± 0.1 35 ± 3
 VIP 4 7.8 ± 0.6 35 ± 9
 Carbachol 5 7.6 ± 0.0 100 ± 0
 Isoproterinol 4 7.5 ± 0.1 79 ± 9
 Histamine 6 5.5 ± 0.2 38 ± 7
 Adenosine 4 5.9 ± 0.2 26 ± 5
Figure 2.
 
(a) Responses to different vasoconstrictors in rat ophthalmic artery pressurized at 70 mm Hg. Constrictor responses were expressed as a percent of the response to 60 mM KCl. (b) Constrictor responses to endothelin-1 showing a biphasic concentration–response curve.
Figure 2.
 
(a) Responses to different vasoconstrictors in rat ophthalmic artery pressurized at 70 mm Hg. Constrictor responses were expressed as a percent of the response to 60 mM KCl. (b) Constrictor responses to endothelin-1 showing a biphasic concentration–response curve.
Figure 3.
 
Vasodilation responses to different agonists resulting in a decrease in myogenic tone in rat ophthalmic artery pressurized at 70 mm Hg. Basal myogenic tone was taken to be 100%, and 0% represented full dilation of the artery.
Figure 3.
 
Vasodilation responses to different agonists resulting in a decrease in myogenic tone in rat ophthalmic artery pressurized at 70 mm Hg. Basal myogenic tone was taken to be 100%, and 0% represented full dilation of the artery.
Figure 4.
 
Effect of l-NAME and indomethacin on myogenic tone of rat ophthalmic artery pressurized at 70 mm Hg. Basal myogenic tone was taken to be 100%.
Figure 4.
 
Effect of l-NAME and indomethacin on myogenic tone of rat ophthalmic artery pressurized at 70 mm Hg. Basal myogenic tone was taken to be 100%.
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Figure 1.
 
(a) Representative tracing of changes in internal diameter of rat ophthalmic artery in the presence of PCSF and calcium-free PCSF. Intraluminal pressure was increased from 1 to 199 mm Hg. (b) Internal diameter of rat ophthalmic artery at different pressures in the presence of PCSF and calcium-free PCSF. Myogenic tone developed at different pressures and was calculated according to equation 1 .
Figure 1.
 
(a) Representative tracing of changes in internal diameter of rat ophthalmic artery in the presence of PCSF and calcium-free PCSF. Intraluminal pressure was increased from 1 to 199 mm Hg. (b) Internal diameter of rat ophthalmic artery at different pressures in the presence of PCSF and calcium-free PCSF. Myogenic tone developed at different pressures and was calculated according to equation 1 .
Figure 2.
 
(a) Responses to different vasoconstrictors in rat ophthalmic artery pressurized at 70 mm Hg. Constrictor responses were expressed as a percent of the response to 60 mM KCl. (b) Constrictor responses to endothelin-1 showing a biphasic concentration–response curve.
Figure 2.
 
(a) Responses to different vasoconstrictors in rat ophthalmic artery pressurized at 70 mm Hg. Constrictor responses were expressed as a percent of the response to 60 mM KCl. (b) Constrictor responses to endothelin-1 showing a biphasic concentration–response curve.
Figure 3.
 
Vasodilation responses to different agonists resulting in a decrease in myogenic tone in rat ophthalmic artery pressurized at 70 mm Hg. Basal myogenic tone was taken to be 100%, and 0% represented full dilation of the artery.
Figure 3.
 
Vasodilation responses to different agonists resulting in a decrease in myogenic tone in rat ophthalmic artery pressurized at 70 mm Hg. Basal myogenic tone was taken to be 100%, and 0% represented full dilation of the artery.
Figure 4.
 
Effect of l-NAME and indomethacin on myogenic tone of rat ophthalmic artery pressurized at 70 mm Hg. Basal myogenic tone was taken to be 100%.
Figure 4.
 
Effect of l-NAME and indomethacin on myogenic tone of rat ophthalmic artery pressurized at 70 mm Hg. Basal myogenic tone was taken to be 100%.
Table 1.
 
Maximum Responses and pEC50 Values of the Agonists
Table 1.
 
Maximum Responses and pEC50 Values of the Agonists
Agonist n pEC50 Maximum Responses
Constrictors*
 Endothelin-1 5 8.3 ± 0.1 130 ± 7
12.2 ± 0.8
 Vasopressin 4 9.3 ± 0.1 91 ± 6
 Serotonin 6 9.0 ± 0.1 110 ± 6
 U-46619 4 7.8 ± 0.5 93 ± 8
 Phenylephrine 5 6.0 ± 0.1 100 ± 7
 UK 14304 4 6.9 ± 0.2 22 ± 2
Dilators, †
 CGRP 4 8.6 ± 0.1 35 ± 3
 VIP 4 7.8 ± 0.6 35 ± 9
 Carbachol 5 7.6 ± 0.0 100 ± 0
 Isoproterinol 4 7.5 ± 0.1 79 ± 9
 Histamine 6 5.5 ± 0.2 38 ± 7
 Adenosine 4 5.9 ± 0.2 26 ± 5
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