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Am J Physiol Heart Circ Physiol 292: H360-H368, 2007. First published August 18, 2006; doi:10.1152/ajpheart.00630.2006
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Characteristics of myogenic tone in the rat ophthalmic artery

Isamu Ito,1,3 Yagna P. R. Jarajapu,1,2 Maria B Grant,1 and Harm J Knot1,2,4

1Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida; 2Wake Forest Institute of Regenerative Medicine, Wake Forest University, Winston-Salem, North Carolina; 3Department of Ophthalmology, College of Medicine, Showa University, Tokyo, Japan; and 4Danish MyoTechnology, Aarhus, Denmark

Submitted 13 June 2006 ; accepted in final form 15 August 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The pressure-induced constriction in the rat ophthalmic artery was characterized. Ophthalmic arteries were isolated, cannulated in an arteriograph, and pressurized. Arteries developed 25% constriction at 70 mmHg of intraluminal pressure. Arteries maintained almost similar diameter over the range of pressures 50–210 mmHg, and forced dilatation was observed at pressures >210 mmHg. Denudation of endothelium increased the sensitivity of arteries to pressure-induced constriction, and significantly higher myogenic tone was observed in the pressure range of 10–100 mmHg. Indomethacin and cyclooxygenase-2 inhibition by SC-236 decreased myogenic tone, whereas cyclooxygenase-1 inhibition by SC-560 potentiated myogenic tone in a lower concentration range and decreased at a higher concentration. Pressure-induced constriction was completely blocked by 1 µM nifedipine. Phospholipase C inhibition by 6 µM U-73122 decreased myogenic tone by 39%, whereas PKC inhibitor GF-109203X (3 µM) had no effect. Constriction to phenylephrine was significantly decreased by U-73122 (1 µM) and GF-109203X (3 µM) at an intraluminal pressure of 10 mmHg. Rho-kinase inhibition by Y-27632 (30 µM) and HA-1077 (30 µM) decreased myogenic tone by 75% and 73%, respectively, and 1 µM Y-27632 significantly decreased myogenic tone developed in response to graded increases in pressure. These results suggest that rat ophthalmic artery has an efficient pressure-dependent autoregulatory function that is modulated by endothelium. Contribution of phospholipase C-activation to myogenic tone is minimal, whereas Rho-kinase activation plays a predominant role in the myogenic reactivity in this artery.

endothelium; phospholipase C; rho kinase

BY THE VIRTUE OF THEIR ABILITY to constrict in response to transmural pressure, small arteries and arterioles exist in a partially constricted state and offer resistance to blood flow. Pressure-induced constriction or myogenic tone, a phenomenon discovered by Bayliss (3), originates from vascular smooth muscle and is not dependent on but modulated by endothelium, endogenous vasoactive substances, neurohumoral factors, and tissue needs (metabolic regulation) (9). Myogenic tone is a major determinant of peripheral vascular resistance and ensures constant tissue perfusion despite continuously varying systemic blood pressure, termed as pressure-mediated autoregulation of blood flow.

In ocular circulation, consisting of blood flow from the ophthalmic artery to choroidal-uveal and cilioretinal circulation, stringent regulation of blood flow is essential for normal function of the retina. The unique anatomy of the ocular vascular bed (40), particularly the sudden transition of the ophthalmic artery (~200 µm in rat) to several small retinal and choroidal arterioles (<30 µm in rat), requires physiologically efficient regulation of blood flow within ophthalmic artery than in any other vascular bed. Consistent with this supposition, Knot's laboratory (28) recently observed that rat ophthalmic artery can autoregulate the diameter and maintain myogenic constriction over a wide range of intraluminal pressures; i.e., up to at least 199 mmHg, ophthalmic artery did not show forced dilatation. These observations suggested an efficient pressure-dependent autoregulation of blood flow to retinal and choroidal vasculature by ophthalmic artery and protection of these microvessels from exposure to higher systemic pressures that would otherwise result in potential hemorrhagic blood flow.

The nature of pressure or mechanical sensor in the arterial smooth muscle is still not known, although wall tension exerted by intravascular pressure was proposed to be the stimulus for activation of intracellular signaling events that elicit myogenic constriction. Recently, some studies (10) provided indirect evidence for an obligatory role of integrins that mechanically link extracellular matrix to cytoskeleton, implicating an extracellular matrix-integrin-cytoskeletal axis in pressure transduction (10). A recent report by Scotland et al. (49) provided evidence for a novel neurovascular pathway in the development of myogenic response involving 20-HETE in rat mesenteric arteries: 20-HETE activates transient receptor potential (TRP) vanilloid type 1 receptors on sensory C fibers, resulting in neuronal depolarization and the release of neuropeptide substance P, which in turn activates postjunctional tachykinin neurokinin-1 receptors on the smooth muscle producing myogenic response.

Several studies (22, 33, 60) showed evidence for the activation of different intracellular signaling mechanisms involved in myogenic constriction. Ca2+ influx secondary to smooth muscle membrane potential depolarization is evidently a central mechanism involved in myogenic constriction. Recently, evidence (11, 50, 59) was provided for a major role for nonselective cation channels, most likely TRP channels, in increased wall [Ca2+] associated with myogenic response. Several lines of evidence have been provided for the involvement of constrictor agonist-activated intracellular signaling mechanisms in pressure-evoked constriction. The activation of phosphatidylinositol-specific phospholipase C (PI-PLC) in response to transmural pressure stimulus was shown in studies (2, 7, 9, 29, 36) using pharmacological and biochemical approaches in arteries from different vascular beds. Myogenic tone accompanies membrane depolarization and increased arterial wall [Ca2+], and pharmacological inhibition of PI-PLC by U-73122 (41, 52) was shown to decrease arterial wall [Ca2+] and reduction in membrane potential depolarization in cerebral arteries, suggesting a role of PI-PLC in the myogenic depolarization of vascular smooth muscle (29). Furthermore, in recent studies, a major contribution of calcium-sensitization mechanisms was also observed. PKC-mediated sensitization was reported in rat cerebral, cremaster, and renal afferent arterioles (19, 24, 32), whereas Rho-mediated sensitization was observed in rat cerebral, cremaster, mesenteric, and renal arteries (20, 30, 36, 44, 48, 58) using different pharmacological inhibitors. MAPK was also implicated in the myogenic constrction in a few studies (31, 37) involving rat skeletal muscle arterioles and human coronary arterioles. Finally, cellular physical deformation was known to activate phospholipase A2-mediated signaling cascade leading to the formation of prostanoids and eicosanoids from arachidonic acid. Recently, cytochrome P-450 metabolite of arachidonic acid, 20-HETE, was found to have a significant role in myogenic constriction in renal, cerebral, skeletal muscle, and mesenteric arteries (15, 16, 18, 26).

Characteristics of pressure-dependent autoregulation of blood flow in ocular circulation are poorly studied, and mechanisms of myogenic tone in rat ophthalmic artery are not known. Therefore, the present study is aimed at 1) evaluating pressure-dependent autoregulation, 2) studying the modulatory role of endothelium in the development of myogenic tone, and 3) identifying the major intracellular mechanism(s) involved in pressure-induced constriction in the rat ophthalmic artery. In the context of the surprising finding that this artery can withstand higher intraluminal pressures (28), we compared the characteristics of this artery with that of the myogenic tone of the well-studied rat cerebral arteries.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Preparation of rat ophthalmic artery and measurement of diameter. Animal procedures have been reviewed and approved by the Institutional Animal Care and Use Committee of the University of Florida. Male Sprague-Dawley rats (250–300 g) were anesthetized by an intraperitoneal injection of pentobarbital sodium (160 mg/kg) and euthanized by decapitation. The brain was removed, and the skull with intact eyes was placed in an ice-cold oxygenated physiological saline solution (PSS, see Drugs, chemicals, and solutions for composition). The main ophthalmic artery was dissected out, cannulated with glass pipettes in an arteriograph [Danish Myotechnology (DMT), Aarhus, Denmark] as described earlier (28). In our earlier study, to pressurize cannulated ophthalmic artery, we used pressure servonull system (Living Systems, Waterbury, VT), and this system could apply intraluminal pressure up to only 199 mmHg; therefore, we could not identify the pressure range at which this artery would lose myogenic tone exhibiting forced dilatation. This problem was overcome in the present study by using the pressure myograph system from DMT that could apply pressures up to 250 mmHg.

Arteriograph was placed on the stage of an inverted microscope, and arteries were visualized by a charge-coupled device camera for continuous monitoring of changes in artery diameter, and the data were acquired by Myoview software (DMT). Arteries were slowly pressurized to 70 mmHg under no flow conditions, and arteriograph was warmed to 37°C with PSS, continuously bubbled with a gas mixture containing 21% O2-5% CO2-74% N2 (pH 7.3–7.4 in the bath). The working pressure of 70 mmHg was chosen based on the earlier observations by Riva et al. (46) in human ocular circulation and McCarron and Halpern (39) in rat cerebral circulation.

Experimental protocol. After an equilibration period of ~20 min, arteries showed stable myogenic tone at 70 mmHg. Concentration-response curves (CRCs) to different pharmacological agents were obtained by cumulative addition to PSS. CRCs were generated in half log order concentrations, and the arteries were exposed to different concentrations for at least 5 min or until the observed effect reached steady state. In some experiments, endothelium was denuded by introducing human hair into the lumen, followed by perfusing with PSS. The presence of functional endothelium was checked by applying 1 µmol/l carbachol. Endothelium-intact arteries showed almost complete relaxation or loss of myogenic tone, whereas in endothelium-denuded arteries, no relaxation was observed. Obtaining the maximum possible diameter or passive diameter in Ca2+-free PSS (see Drugs, chemicals, and solutions for composition) concluded all experiments. In some experiments, pressure-dependent changes in the diameter were evaluated by increasing the intraluminal pressure in 10 mmHg steps from 1–250 mmHg in PSS. Effects of different pharmacological inhibitors on pressure-dependent changes in myogenic tone were evaluated in the pressure range of 1–160 mmHg. Pressure was slowly returned back to the lowest and allowed to equilibrate for 20 min and then exposed to a pharmacological inhibitor for 15 min before subjecting the artery to graded increases in the intraluminal pressure. Pressure-dependent changes in diameter were reevaluated in the presence of Ca2+-free PSS. The myogenic tone was calculated according to the following equation:

Formula 1(1)
where Da is the internal diameter of the arterial segment with active myogenic tone in the presence of PSS at a particular intraluminal pressure and Dp is the passive diameter obtained at the same pressure in the presence of Ca2+-free PSS.

Pressure-diameter experiments, focused at identifying the pressure that results in forced dilatation and experiments with pharmacological inhibitors, were evaluated in different group of arteries so that the reactivity of the artery in other experimental protocols was not affected by exposure to a range of high pressures and vice versa.

Data analysis and statistics. Results are expressed as means ± SE; n indicates the number of independent experiments, which equals the number of animals used. Results were compared by either ANOVA or Student's t-test as applicable using the software program GraphPad prism, and a P value < 0.05 was considered statistically significant.

The efficacy of different agonists was expressed as the maximum response, constriction or dilation, produced. The potency was expressed as the negative logarithm of the concentration of the agonist that produces 50% of the maximum effect (pEC50). pEC50 values were calculated by analyzing CRCs using GraphPad Prism that fits the data to a four-parameter logistic equation given below:

Formula 2(2)
where minimum and maximum indicate the lowest 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.

Drugs, chemicals, and solutions. Phenylephrine, carbachol, U-73122, U-73343, HA-1077, nifiedipine, and GF-109203X were obtained from Sigma (St. Louis, MO), and Y-27632 was obtained from Tocris Cookson (Ellisville, MO). Stock solutions (10 mmol/l) of phenylephrine, carbachol, Y-27632, and HA-1077 were prepared in distilled water, whereas that of U-73122, U-73343 (2 mmol/l), nifedipine (10 mmol/l), and GF-109203X (10 mmol/l) were prepared in dimethylsulfoxide. The composition of PSS (in mmol/l) contained 120 NaCl, 3 KCl, 24 NaHCO3, 1.2 NaH2PO4.H2O, 2.5 CaCl2, 1.2 MgSO4.7H2O, and 4 glucose. Ca2+-free PSS was prepared by replacing CaCl2 with 2 mmol/l EGTA.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The outer diameter of ophthalmic arteries at an intraluminal pressure of 70 mmHg with stable myogenic tone was 239 ± 4 µm (n = 42), and the passive diameter, as obtained in the presence of Ca2+-free PSS, was 320 ± 6 µm, thus giving a myogenic tone of 25 ± 0.4%.

The pressure-induced constriction in the rat ophthalmic artery. Fig. 1A shows a representative tracing of changes in the outer diameter of a rat ophthalmic artery with an increase in the intraluminal pressure in the presence of PSS and a passive increase in the diameter in the presence of Ca2+-free PSS. An increase in intraluminal pressure resulted in an increase in diameter of ophthalmic arteries up to a pressure of 30 to 40 mmHg, 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 mmHg, was 27 ± 2% (n = 6), which continued to rise up to a maximum of 32 ± 3% with an increase in intraluminal pressure (Fig. 1B), revealing the autoregulatory function of this artery. Arteries maintained an almost similar diameter with increases in pressures ranging from 50 to 210 mmHg. Forced dilatation was observed at pressures >210 mmHg, and complete loss of myogneic tone was observed at 240 mmHg.


Figure 1
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  		Fig. 1. A: representative recording of outer diameter of the rat ophthalmic artery with increase in the intraluminal pressure in the presence of physiological saline solution (PSS) and Ca2+-free PSS. B: summary of changes in diameter of rat ophthalmic artery in response to graded increases in intraluminal pressure in the presence of PSS and Ca2+-free PSS (n = 6). Also shown is the percentage of myogenic tone at different pressures calculated from active and passive diameters.

 
The influence of endothelium on myogenic tone in the rat ophthalmic artery. As shown in Fig. 2A, the absence of endothelium resulted in an increased sensitivity of pressure-induced constriction. Significantly higher myogenic tone was observed at lower pressures starting from 10 mmHg and remained significantly higher up to the pressure of 100 mmHg (P < 0.05–0.0001, n = 6), suggesting that endothelium-derived dilatory factors tend to oppose the development of myogenic tone in normal conditions. At pressures >110 mmHg, no significant difference was observed compared with that in arteries with intact endothelium.


Figure 2
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  		Fig. 2. A: the myogenic tone developed in response to graded increases in intraluminal pressures up to 160 mmHg in the rat ophthalmic artery in the presence and absence of endothelium. Myogenic tone in the endothelium-denuded arteries was significantly higher than that in the control in the pressure range of 10–100 mmHg (P < 0.05–0.0001, n = 6). B: concentration-dependent effects of indomethacin, SC-560, and SC-236 on myogenic tone in rat ophthalmic artery pressurized at 70 mmHg. Maximum decrease in myogenic tone produced by indomethacin and SC-236 was 40 ± 7% (n = 6) and 42 ± 10% (n = 5), respectively. Increase and decrease in myogenic tone produced by SC-560 were 28 ± 4% (n = 6) and 23 ± 3% (n = 3), respectively.

 
Consistent with this observation, NG-nitro-L-arginine methyl ester (L-NAME), a selective nitric oxide synthase (NOS) inhibitor, constricted this artery in the presence of endothelium, but indomethacin, a COX inhibitor, decreased myogenic tone at 70 mmHg in a concentration-dependent fashion, as observed earlier (Fig. 2B) (28). In the present study, we further examined the role of COX-1 and COX-2 in this dilatory response. Selective inhibitors of COX-1 and COX-2, SC-560 (51) and SC-236 (43), respectively, were evaluated on the myogenic tone of ophthalmic artery pressurized at 70 mmHg. SC-236 mimicked the actions of indomethacin by decreasing the myogenic tone in a concentration-dependent manner in the range of 10 nM–30 µM concentration (n = 5) (Fig. 2B). Maximum decrease produced by 30 µM SC-236 in myogenic tone was 42 ± 10% (n = 5), which was similar to that produced by 100 µM indomethacin (40 ± 7%, n = 6). SC-560 showed a concentration-dependent biphasic effect on myogenic tone. Concentration-dependent increase in tone was observed in the range of 1–10 µM concentrations (increase in myogenic tone, 28 ± 4%, n = 6), and further higher concentration decreased myogenic tone by 23 ± 3% in three of the six arteries studied (Fig. 2B). Effects of these three compounds were readily reversible upon washout.

The effect of nifedipine on the pressure-induced constriction in the rat ophthalmic artery. The activation of voltage-gated Ca2+ channels by intraluminal pressure in this artery was verified by evaluating the pressure-dependent increase in the myogenic tone in the presence of 1 µM nifedipine, a selective blocker of voltage-gated Ca2+ channels. Myogenic tone, developed in response to intraluminal pressures up to 160 mmHg, was highly sensitive to this antagonist, and myogenic tone was almost completely blocked (n = 5) (Fig. 3). This confirms that the graded elevations in intraluminal pressure increase arterial wall [Ca2+], resulting in myogenic constriction by allowing Ca2+ influx via voltage-gated Ca2+ channels in this artery.


Figure 3
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  		Fig. 3. The myogenic tone developed in response to graded increases in intraluminal pressure in the rat ophthalmic artery in the presence and absence of 1 µM nifedipine (n = 5).

 
The effect of U-73122 on the pressure-induced constriction in the rat ophthalmic artery. In arterial segments pressurized at 70 mmHg, U-73122, a selective PI-PLC, decreased myogenic tone only at higher concentrations, i.e., a 10 ± 3% and 38 ± 8% (n = 5) decrease at 3 and 6 µM concentrations (Fig. 4A). U-73343, a negative control of U-73122, did not alter the myogenic tone in the concentration range of 100 nM–6 µM (n = 4, Fig. 4A). Concentrations of U-73122, higher than 6 µM, were not tried since we observed nonspecific effects in other preparations, such as rat cerebral arteries (29). Effects of U-73122 observed in the concentration range used were readily reversible upon washout of the inhibitor. Pressure-evoked constriction was also evaluated in this artery over a range of intraluminal pressures in the presence of U-73122. Development of myogenic tone was not affected in the presence of 3 µM up to pressures of 120 mmHg. A significant decrease was observed only at pressures of 140 (P < 0.05) and 160 (P < 0.02) mmHg (n = 5) (Fig. 4B).


Figure 4
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  		Fig. 4. A: effects of U-73122 (n = 5) and U-73343 (inactive analog of U-73122, n = 4) on myogenic tone of the rat ophthalmic artery pressurized at an intraluminal pressure of 70 mmHg. B: the effect of 3 µM U-73122 on the development of myogenic tone in response to graded increases in the intraluminal pressure. Myogenic tone was significantly decreased by U-73122 at pressures 140 (P < 0.05) and 160 (P < 0.02) mmHg (n = 5). C: the effect of 1 µM U-73122 on phenylephrine-induced constriction in the rat ophthalmic artery pressurized at 10 mmHg. The constriction at 1–30 µM phenylephrine was significantly decreased by U-73122 (*P < 0.01 and **P < 0.003, n = 5).

 
The involvement of cellular mechanisms triggered by PI-PLC in producing contraction in this artery was verified by studying constrictor responses to phenylephrine, a selective {alpha}1-adrenoceptor agonist. In this set of experiments, arteries were pressurized at 10 mmHg at which cellular mechanisms involved in pressure-induced constriction are minimally or not activated. Phenylephrine produced concentration-dependent constriction of this artery, and this response was evaluated in the presence of 1 µM U-73122, a concentration lower than that used (3 µM) to evaluate its effect on pressure-dependent increase in myogenic tone. A significant decrease in constrictor responses to 1, 3, 10, and 30 µM (P < 0.01–0.003, n = 5) was observed in the presence of U-73122, confirming the existence of PI-PLC-mediated cellular mechanisms in this artery that could be activated by {alpha}1-adrenoceptor stimulation but not by intravascular pressure.

Effects of Rho-kinase inhibitors, Y-27632 and HA-1077, on the pressure-induced constriction in the rat ophthalmic artery. calcium sensitization, a phenomenon by which a higher contraction is elicited in smooth muscle for a given intracellular [Ca2+] than for depolarization, involves activation of protein kinase C and Rho kinase (53, 54). Several studies, as mentioned earlier, reported that calcium sensitization is associated with the pressure-induced constriction in different arteries involving PKC and Rho kinase. Therefore, we attempted to evaluate the involvement of Rho-kinase and PKC activation in the pressure-induced constriction in this artery. Structurally different inhibitors of Rho kinase, Y-27632 and HA-1077 (8, 56), concentration dependently decreased myogenic tone at 70 mmHg (Fig. 5A) with a similar potency (pEC50) (Y-27632, 5.3 ± 0.1, n = 5; and HA-1077, 5.5 ± 0.1, n = 5). The maximum decrease observed in myogenic tone was 75 ± 3% and 73 ± 4% with Y-27632 and HA-1077, respectively. The effects of these two agents were readily reversible upon washout. The development of myogenic tone over a range of intraluminal pressures was also evaluated in the presence of 1 µM Y-27632 (a concentration, lower than that of its EC50, 4.70 µM, observed in the above experiments) in a different set of arteries. Myogenic tone, developed at pressures 60–160 mmHg, was significantly lower than that observed in control artery (P < 0.001 to 0.0001, n = 4) (Fig. 5B). These observations suggest significant contribution of Rho-kinase activation in the pressure-evoked constriction in this artery.


Figure 5
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  		Fig. 5. A: effects of Rho-kinase inhibitors Y-27632 and HA-1077 on myogenic tone in the rat ophthalmic artery pressurized at an intraluminal pressure of 70 mmHg. The maximum decrease observed in myogenic tone by Y-27632 and HA-1077 was 75 ± 3% (n = 5) and 73 ± 4% (n = 5), respectively, with pEC50 of 5.3 ± 0.1 and 5.5 ± 0.1, respectively. B: the effect of 1 µM Y-27632 on myogenic tone developed in response to graded increase in intraluminal pressure. The myogenic tone was significantly decreased by Y-27632 at pressures 60–160 mmHg (*P < 0.001–0.0001, n = 4).

 
The effect of PKC inhibitor GF-109203X on the pressure-induced constriction in the rat ophthalmic artery. In arterial segments pressurized at 70 mmHg, a selective protein kinase C inhibitor GF-109203X, bisindolylmaleimide I (8, 55), had no effect on the myogenic tone in the concentration used (1 nM to 3 µM, n = 4, data not shown). In the presence of 3 µM GF-109203X, a pressure-dependent increase in myogenic tone was evaluated at intraluminal pressures ranging from 1 to 160 mmHg (n = 5, Fig. 6A). Myogenic tone in response to pressures up to 150 mmHg was unaffected by 3 µM GF-109203X, but a significant decrease was observed only at 160 mmHg (P < 0.03), suggesting a weak PKC activation in response to pressure in this artery. Further experiments were performed to verify the involvement of PKC-activation in contractile mechanisms in this artery. The effect of this inhibitor on phenylephrine-induced constriction was evaluated, as described above, in arteries pressurized at 10 mmHg, in the presence of 1 µM GF-109203X (a concentration lower than that used to evaluate its effect on myogenic tone in response to graded increased in intraluminal pressure) (Fig. 6B). GF-109203X significantly decreased phenylephrine-mediated constriction in the concentration range of 1–30 µM (P < 0.02–0.0001, n = 5), suggesting that PKC could be activated by {alpha}1-adrenoceptor stimulation via Gq protein-PLC-diacylglycerol (DAG) pathway but not by intraluminal pressure in this artery.


Figure 6
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  		Fig. 6. A: the effect of 3 µM GF-109203X on myogenic tone developed in response to graded increase in intraluminal pressure. The myogenic tone was significantly decreased only at the pressure of 160 mmHg (*P < 0.003, n = 5). B: the effect of 1 µM GF-109203X on phenylephrine-induced constriction in the rat ophthalmic artery pressurized at 10 mmHg. A significant decrease in constriction was observed in the concentration range of 1–30 µM (*P < 0.02, **P < 0.001, and ***P < 0.0001, n = 5).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study reports for the first time the characteristics of myogenic tone in rat ophthalmic artery. Major findings of this study that show the characteristics of myogenic tone are as follows: 1) rat ophthalmic artery can withstand pressures up to as high as 210 mmHg; 2) myogenic tone in this artery is modulated by endothelium; 3) intraluminal pressure activates voltage-dependent Ca2+ channels, and pressure-evoked constriction is dependent on extracellular Ca2+; and 4) myogenic reactivity is associated with predominant activation of Rho/Rho-kinase pathway, and activation of PI-PLC and PKC has a minor contribution. This is purely a functional study, and conclusions were drawn based solely on the functional effects of selective pharmacological inhibitors. Lack of support for these findings from biochemical or molecular biological experiments is a limitation of the present study.

The pressure-dependent autoregulation in the rat ophthalmic artery. Autoregulation of arterial caliber ensures constancy of perfusion despite changes in systemic arterial pressure. Unique anatomy of ocular vascular bed (40), as mentioned above, demands physiologically efficient autoregulation of blood flow compared with many other vascular beds. Studies by Osol et al. (42) elegantly described the phenomenon of pressure-dependent autoregulation in rat cerebral arteries in a three-phase model: phase 1, initial development of myogenic tone with rapid increases in arterial wall [Ca2+]; phase 2, increased tone or myogenic reactivity with a small but consistent increase in the wall [Ca2+] (38) with subsequent increase in pressure; and phase 3, forced dilatation at further higher pressures yet accompanying increases in the wall [Ca2+]. The pressure range, over which phase 2 exists, determines the efficiency of pressure-dependent autoregulation in a particular artery. Results from the present study are consistent with this proposed model. Three different phases of autoregulation in rat ophthalmic artery were revealed at pressures ~30, 40–200, and >200 mmHg, respectively. These observations clearly suggest an efficient pressure-dependent autoregulation over a wide range of pressures in this artery, which is essential to protect small retinal and choroid arteries from exposure to potential haemorrhagic blood flow that would otherwise result in an irreversible damage to sensitive ocular structures, particularly the retina.

The myogenically active pressure range (corresponding to phase 2) in other vascular beds was reported to be quite smaller than that observed in the rat ophthalmic artery, and forced dilatation was reported to occur in the range of 150–170 mmHg in cerebral, mesenteric, and skeletal muscle arteries from Wistar-Kyoto rats (25, 30) and in cerebral arteries from Sprague-Dawley rats (6, 42) with myogenic tone being developed in the range of 30–40 mmHg. In contrast, cerebral and mesenteric arteries from newborn pigs and cerebral arteries from neonatal mice develop myogenic tone at pressure as low as 10 mmHg (1, 17, 45), and newborn porcine cerebral arteries exhibit forced dilatation at 90 mmHg (1). A wider range of pressures with myogenic reactivity or an ability of an artery to withstand higher pressures was observed only in the present study in rat ophthalmic artery, which could be attributed to the unique anatomy of the ocular vascular bed.

The modulatory effect of endothelium on myogenic tone in the rat ophthalmic artery. Pressure-induced constriction is known to originate from, and independent of, smooth muscle but could be modulated by endothelium (9, 12, 35). The present study clearly shows that endothelium-derived factors play a differential role in the modulation of myogenic tone with an overall opposing effect on pressure-induced constriction. This contrasting observation could be attributed, although speculatively, to the anatomical difference in the organization of the vascular bed. The sudden transition of the ophthalmic artery to retinal and choroidal arterioles that demands an efficient pressure-dependent autoregulation of blood flow in this vascular bed is associated with significant endothelial modulation of tone at physiological pressures.

The absence of endothelium significantly increased myogenic sensitivity to intraluminal pressures ranging from 10–100 mmHg. This is in agreement with our earlier studies, showing a concentration-dependent increase in myogenic tone to L-NAME, a NOS inhibitor, at an intraluminal pressure of 70 mmHg (28). According to this finding, it is likely that myogenic constriction or pressure-mediated circumferential stress on arterial wall activates NOS.

On the other hand, inhibition of COX by indomethacin resulted in a concentration-dependent decrease in myogenic tone (Ref. 28 and present study), suggesting a role of COX products in the regulation of myogenic tone and no role for the dilatory prostanoids, such as prostacyclin. Further experiments showed that the effect of COX-2 inhibition by SC-236 was similar to that of indomethacin, whereas COX-1 inhibition by SC-560 resulted in loss of myogenic tone, suggesting differential roles of COX metabolites in the regulation of arterial tone. SC-560 showed dilatory response at higher concentrations, i.e., 30 µM, that could be explained by its inhibitory effect on COX-2 enzyme at higher concentrations (IC50: COX-1, 9 nM; and COX-2, 10 µM) (51). In summary, endothelium potentiates myogenic tone via COX-2 metabolites and attenuates via NOS and COX-1 metabolites. Similar observations were reported earlier that provided evidence for a differential role of endothelium-derived factors on arterial tone in different pathophysiological conditions. Endothelial COX-mediated constrictor, likely to be thromboxane, was shown to be involved in hypoxic pulmonary constriction in newborn pigs (13). An elegant study by Ungvari and Koller (57) showed that endothelin and prostaglandin H2/thromboxane A2 mediate increased myogenic tone by potentiating Ca2+ sensitivity of smooth muscle in skeletal muscle arterioles of spontaneously hypertensive rats. COX products were shown to augment arterial tone in mesenteric arteries by inhibiting nitric oxide synthesis that is decreased in L-NAME-mediated hypertension in rats (4). Furthermore, endothelial COX-2 was shown to be upregulated in mesenteric arteries from rats with high NaCl intake, mediating myogenic tone suggesting a compensatory or protective role of COX-2 in pressure-mechanotransduction (38).

We recently reported the pathophysiological significance of myogenic tone and its modulation by endothelium in the rat ophthalmic artery. An acute exposure to high-glucose concentration dependently altered myogenic tone in this artery i.e., potentiated or attenuated, which was endothelium dependent (27). Furthermore, ophthalmic arteries from Type 2 diabetic BBZDR/Wor rats showed decreased myogenic tone compared with that in arteries from age-matched control rats, and this decrease was endothelium dependent (27). A decrease in myogenic constriction could be the underlying mechanism of reduced arterial resistance in ophthalmic vascular bed that would explain the increased ocular blood flow and the increased risk of retinal hemorrhage associated with diabetes (14, 21).

The intraluminal pressure activates voltage-dependent Ca2+ channels and produces constriction in the rat ophthalmic artery. The intraluminal pressure is known to induce membrane depolarization (23, 33, 34, 42) that activates voltage-gated Ca2+ channels, resulting in Ca2+ influx and smooth muscle contraction. In the present study we have not evaluated arterial wall [Ca2+] and membrane potential; however, we observed that nifedipine, a selective blocker of voltage-gated potassium channel, abolished the development of myogenic tone, and exposure to Ca2+-free PSS also resulted in a passive increase in diameter with increases in pressure. These findings suggest that activation of voltage-gated Ca2+ channels is an essential step involved in the development of myogenic tone in this artery most likely mediated by pressure-mediated smooth muscle membrane depolarization. Recent studies (11, 50, 59) proposed alternative mechanisms for the pressure-induced increase in the arterial wall [Ca2+], including DAG- and PKC-activated cation channels, most likely TRP channels. Involvement of these mechanisms in myogenic tone in the rat ophthalmic artery needs further investigation.

The involvement of PI-PLC in the development of myogenic tone in the rat ophthalmic artery. PI-PLC activation has been shown to be involved in pressure-induced constriction in several arteries as mentioned before. In rat cerebral arteries we evaluated the sensitivity of myogenic tone to a PI-PLC inhibitor U-73122 and found that myogenic tone was readily reversed by this compound with high potency (EC50 of 0.6 µM) (29), and the maximum decrease observed in myogenic tone was 87% at 3 µM U-73122. In contrast, in the rat ophthalmic artery in the present study, this inhibitor showed very low efficacy; i.e., maximum decrease observed was 39% with 6 µM U-73122. In addition, in experiments with graded increases in intraluminal pressures in rat cerebral arteries, 124 nM U-73122 significantly decreased and 500 nM completely reversed the development of myogenic tone in response to pressures ranging from 50–110 mmHg (29). In contrast, 3 µM U-73122 could produce significant decrease in myogenic tone only at pressures 140 and 160 mmHg in rat ophthalmic artery. These findings suggest a minor role of PLC activation in the pressure-induced constriction in this artery. Significant decrease in the contractile response to phenylephrine, an {alpha}1-adrenoceptor agonist, which is known to activate PI-PLC via receptor-coupled Gq protein activation, confirmed the availability of PI-PLC-activated contractile pathway in this arterial smooth muscle. A lack of prominent PLC activation is quite contrary to that observed in several other arterial preparations showing an important role of PI-PLC activation in pressure-mediated constriction, and the present study suggests vascular bed-dependent differences in the cellular signaling mechanisms involved in pressure-induced constriction. Different isoforms of PI-PLC and their biochemical characteristics in ophthalmic arteries are not known, and this information is essential to understand the relatively lower pressure-mediated activation PI-PLC in rat ophthalmic artery.

PI-PLC activation results in the production of phosphatidylinositol 4,5-bisphosphate metabolites DAG and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] that were known to be involved in amplifying or coupling myogenic stimulus to smooth muscle contraction. DAG, by activating PKC that causes calcium sensitization (19, 24), and Ins(1,4,5)P3, by releasing Ca2+ from sarcoplasmic stores, enhance smooth muscle contraction. Furthermore, as mentioned earlier, DAG-PKC pathway is known to activate TRP channels that were shown to participate in the myogenic constriction of arteries. In the light of the present finding that the activation of PI-PLC by pressure stimulus is minimal in this artery, the role of DAG-PKC and Ins(1,4,5)P3-mediated mechanisms may not play a significant role in the myogenic reactivity in this artery. We extended our observations to evaluate the role of PKC that further supported our findings that the role of PLC activation in myogenic constriction is insignificant (see below).

The involvement of Rho-kinase activation but not PKC activation in the myogenic tone in the rat ophthalmic artery. As mentioned before, calcium sensitization is another intracellular signaling mechanism known to play a predominant role in the myogenic constriction that could be mainly mediated by PKC and Rho kinase. In this present study, we have not used permeabilized arterial preparations to evaluate calcium sensitization, instead we used pharmacological inhibitors to evaluate the role of major kinases PKC and Rho-kinase in myogenic constriction in this artery. Our experiments, using GF-109203X in arteries with myogenic tone at 70 mmHg and myogenic response to graded increases in intraluminal pressure, ruled out a significant role of PKC in the pressure-mediated constriction, which agrees with what we observed with PI-PLC inhibitor as described above, whereas a constrictor agonist could activate PKC significantly.

In contrast to the pharmacological evidence against the involvement of PKC, we observed a predominant role of Rho-kinase activation in the myogenic constriction. Two structurally different Rho-kinase inhibitors, Y-27632 and HA-1077, reversed myogenic tone at 70 mmHg, and Y-27632 significantly antagonized the development of myogenic tone in response to graded increases in intraluminal pressure, suggesting a predominant involvement of Rho kinase in mygenic reactivity. It is clear from the data that in the lower range of pressures or the phase 1 of autoregulation curve, Y-27632 did not show significant decrease, whereas nifedipine antagonized myogenic constriction in this range of pressures as well, suggesting that Rho-kinase activation is not involved in the initiation of myogenic tone but plays a significant role in the maintenance of myogenic constriction (phase 2). It could be possible that a threshold level of Ca2+ influx is needed for the activation of Rho-Rho-kinase pathway. This was recently shown by Sakurada et al. (47) in rabbit aortic smooth muscle, suggesting that both depolarization and agonist stimulation leads to Rho activation and that depolarization-induced Rho activation could be possibly mediated by CaMK II (5). It is likely that the initial event in myogenic constriction, a Ca2+ influx-dependent process, leads to Rho activation that maintains myogenic reactivity with the subsequent increases in the pressure or the phase 2 of the autoregulatory curve.

In conclusion, myogenic tone in rat ophthalmic artery has different characteristics compared with that of arteries from other vascular beds. This study also suggests that this artery could be a suitable preparation to study to understand ocular hemodynamics and pressure-mediated regulation of blood flow to retinal vascular bed. This also offers a good model to study pressure transduction in the ocular vascular bed in rats. Our present studies are focused at understanding ionic regulation of myogenic tone in ophthalmic artery and, therefore, the regulation of blood flow to the retina.


   GRANTS
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 ABSTRACT
 METHODS
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 DISCUSSION
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REFERENCES
 
This work was supported by National Eye Institute Grants EY-12601 and EY-007739 and the Juvenile Diabetes Research Foundation International.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. P. R. Jarajapu, Wake Forest Institute of Regenerative Medicine, Wake Forest Univ. Baptist Medical Hospital, Medical Center Blvd., Winston-Salem, NC 27157 (e-mail: yjarajap{at}wfubmc.edu)

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.


   REFERENCES
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