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(Investigative Ophthalmology and Visual Science. 2000;41:3955-3962.)
© 2000 by The Association for Research in Vision and Ophthalmology, Inc.

Endothelin-3 Regulation of Retinal Hemodynamics in Nondiabetic and Diabetic Rats

Fumihiko Mori, George L. King, Allen C. Clermont, Dahlia K. Bursell and Sven-Erik Bursell

From the Research Division and Beetham Eye Institute, Joslin Diabetes Center, Harvard Medical School, Boston, Massachusetts.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate the mechanisms of action of endothelin (ET)-3 on the regulation of retinal hemodynamics in diabetic and nondiabetic rats.

METHODS. Retinal blood flow changes were measured using video fluorescein angiography. Measurements were made before and after intravitreal injections of different ET-3 concentrations in nondiabetic rats and rats with streptozotocin (STZ)-induced diabetes. The effect of ET-3 on retinal blood flow was also investigated in nondiabetic rats after pretreatment with NG-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase (NOS) inhibitor; BQ-788, an ET receptor B (ETB) antagonist; and BQ-123, an ET receptor A (ETA) antagonist. Control animals were injected intravitreally with vehicle alone.

RESULTS. In nondiabetic rats, ET-3 induced a dose-dependent rapid increase in retinal blood flow 2 minutes after intravitreal injection (maximal at 10-8 M, P < 0.01) followed 15 and 30 minutes after ET-3 injection by dose-dependent decreases in retinal blood flow (maximal effect at 10-6 M, P < 0.05). The ET-3–stimulated retinal blood flow increase was inhibited by 10-4 M BQ-788 (P < 0.01) and 10-3 M L-NMMA (P < 0.05). The ET-3–stimulated decrease in retinal blood flow at later times (15 minutes) was inhibited (P < 0.03) by 10-4 M BQ-123. In diabetic rats, baseline retinal blood flows were decreased compared with nondiabetic rats (P < 0.01), showed dose-dependent increases 2 minutes after ET-3 injection (P < 0.03), and at later times remained significantly increased (P < 0.05) in contrast to flows in nondiabetic rats.

CONCLUSIONS. The ET-3–induced initial rapid retinal blood flow increase in nondiabetic rats is mediated by the ET-3/ETB and NOS action. The subsequent retinal blood flow decrease is mediated by ET-3/ETA action. Diabetic rats showed comparable ET-3–induced retinal blood flow increases indicating normal ET-3/ETB action. However, at later times, retinal blood flow remained increased, suggesting an abnormal ET-3/ETA action.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
An understanding of the physiological regulation of retinal hemodynamics and the maintenance of vascular tone by endogenous vasoactive hormones and cytokines in association with the development of retinal hemodynamic changes in diabetes is important, especially because abnormal retinal hemodynamics in diabetes can manifest in the early stages of the disease, both in animals1 2 3 4 5 and in patients with no diabetic retinopathy,6 7 8 and may be associated with an increased risk for development of diabetic retinopathy. Factors responsible for regulating vascular tone in the retina include the endothelins (ETs),9 10 11 12 13 14 15 nitric oxide (NO),16 17 18 prostaglandins,19 20 and angiotensin.21 22 However, the role of these factors in the development of abnormal retinal hemodynamics in diabetes is not well defined.

ETs are potent vasoactive agents,23 and in the retina, ET-1 and ET-3 appear to play a role in vascular homeostasis. ET-1 is a potent retinal vasoconstrictor9 10 11 12 13 binding to the high-affinity ET receptor A (ETA)24 in retinal vascular smooth muscle cells and pericytes.25 26 ET-3 also binds to the ETA but with lower affinity and less vasoconstrictor action than in the ET-1/ETA action. ET-1 has a role in maintaining normal vascular tone, and, in the diabetic rat retina, increased ET-1 production contributes to measured retinal blood flow reduction.9 10 11 In contrast, ET-3 interacts primarily with the endothelial cell ET receptors type B (ETB), which have an equal affinity for both ET-1 and ET-3.27 28 ETB action initiates vasodilation through NO and/or prostacyclin.29 Changes in tissue ET-3/ETB interactions in diabetes have been reported30 31 ; however, the ET-3/ETA/ETB interactions in the retinal hemodynamics in diabetes are not well characterized, prompting the current investigation.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Instrumentation
The video fluorescein angiography (VFA) system used for these studies has been described previously1 2 3 4 and consists of a fundus camera (NFC-50; Nikon, Tokyo, Japan) interfaced to a video camera (SIT; Dage-MTI, Michigan City, IN). The video camera output was directly digitized (512 x 512 pixels by 8 bits) at 30 frames/sec and stored in a data storage system (Trapix Plus/DataStore; Recognition Concepts, Carson City, NV). The digitized angiogram images were analyzed on a frame-by-frame basis to determine the retinal circulatory parameters of interest.

Animals
One hundred three male Sprague–Dawley rats (Taconic Farms, Germantown, NY) with initial weights between 200 and 250 g were used. All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care and Use Committee of the Joslin Diabetes Center. Diabetes was induced in 34 rats with an intraperitoneal injection of 65 mg/kg of streptozocin (STZ; Sigma, St. Louis, MO) in 10 mM citrate buffer (pH 4.5) after a 12-hour fast. Diabetes was confirmed with blood glucose measurements (>250 mg/dl) 24 hours after STZ injection. The rats were housed under standard conditions with free access to water and standard food. All animals were maintained for 2 weeks before retinal blood flow measurements. Blood glucose levels and body weights were monitored every other day.

Twenty-four hours before retinal blood flow measurements, all animals (under anesthesia, 0.1 mg/kg amobarbital sodium; Eli Lily, Indianapolis, IN) underwent catheterization with a polyvinyl catheter inserted into the right jugular vein.5 The catheter was flushed with 0.1 ml of 1000 U sodium heparin before and after implantation. It was positioned subcutaneously along the shoulder, and the distal end was externalized to the back of the neck.

VFA Procedure
Immediately before VFA measurements, each rat was anesthetized, the left eye was dilated (1% tropicamide, Mydriacyl: Alcon, Fort Worth, TX), and a syringe (Hamilton, Reno, NV) containing 10% sodium fluorescein was connected to the externalized jugular vein catheter. The rats were positioned on a platform attached to the retinal fundus camera. The optic disc was centered and focused in the field of view, the VFA recording sequence was initiated, and a 5-µl bolus of fluorescein dye was rapidly injected into the jugular vein catheter.4 5 The injection time was marked on the video recording.

Baseline angiograms were recorded from each rat before intravitreal injection with the different agents under investigation. A further series of angiograms were then recorded at selected time points after the intravitreal injection.

Intravitreal injections were performed by inserting a 27-gauge needle, attached to a 10-µl syringe (Hamilton), into the vitreous from a site 1 mm posterior to the limbus. Infusion was performed directly over the optic disc region under direct visualization, and a timer was started. VFA recordings were obtained at selected times after injection. The effective vitreal concentrations of the injected agents were estimated knowing that the rat vitreous volume is approximately 120 µl.32 Thus the retina would be exposed to a 12-fold lower concentration than the injected concentration.

Time Course and Dose Response of ET-3 in Nondiabetic and Diabetic Rats
Intravitreal injections in 34 STZ-induced diabetic rats and 37 nondiabetic rats were performed using different concentrations (10-9 to 10-6 M) of ET-3 (Sigma, St. Louis, MO) dissolved in vehicle of 2.5% Emulphor EL-620 (GAF Chemical, Wayne, NJ) in phosphate-buffered saline (PBS). Rats injected intravitreally with vehicle alone served as control subjects.

VFA recordings were obtained before and at 2, 5, 15, and 30 minutes after intravitreal injection. Blood pressures and heart rates were monitored using a noninvasive tail-cuff sensor and monitoring system (Ueda Electronics, Tokyo, Japan). Animals were maintained on a heated pad during the course of the measurements.

NOS Inhibitor and ETB Antagonist Action
Thirty-two nondiabetic rats were used. Pretreatment was performed with intravitreal injections of either 10-3 M NG-monomethyl-L-arginine (L-NMMA; Sigma) a nitric oxide synthase (NOS) inhibitor, or 10-4 M BQ-788 (Sigma), a specific ETB antagonist. For both agents, the vehicle was 2.5% Emulphor EL-620 in PBS. Pretreatments with 10-3 M L-NMMA (8 x 10-5 M effective vitreous concentration) or 10-4 M BQ-788 (8 x 10-6 M effective vitreous concentration) were used to ensure maximal NOS33 and ETB inhibition.34 VFAs were recorded at baseline and 15 minutes after intravitreal pretreatment with L-NMMA, BQ-788, or vehicle. Each rat then underwent an intravitreal injection of 10-8 M ET-3 (maximal retinal hemodynamic response), and subsequent VFA recordings were obtained at 2 and 15 minutes after ET-3 injection.

ETA Antagonist Action
Nine nondiabetic rats were used. Pretreatment was performed with intravitreal injections of 10-4 M BQ-123 (American Peptide, Sunnyvale, CA), a specific ETA antagonist with vehicle consisting of 2.5% Emulphor EL-620 in PBS. At this concentration, prior results10 have shown a maximal retinal blood flow increase at 5 minutes after intravitreal injection with a return to baseline values by 15 minutes after injection. In five animals, baseline VFA recordings were obtained followed by an intravitreal injection of BQ-123. VFA recordings were repeated 2 minutes after the BQ-123 injections. Immediately after these recordings, an intravitreal injection of 10-7 M ET-3 was performed, and VFA recordings were repeated at 2, 5, 15, and 30 minutes after the ET-3 injection. In four animals, only BQ-123 was injected at baseline and VFA recordings were performed at 2, 5, 15, and 30 minutes after injection.

Data Analysis
The recorded fluorescein angiograms were digitized on a frame-by-frame basis and analyzed densitometrically to determine retinal vessel diameters and retinal mean circulation times (MCTs).4 5

Vessel diameters in units of pixels were determined from images recorded before fluorescein dye injection at defined vessel sample sites using a boundary-crossing algorithm. The average vessel diameters for each eye represent the average of the individual vessel diameters for that eye.

At the fixed vessel sites, the average vessel fluorescence within a sample area defined by the vessel width was measured on a frame-by-frame basis to generate temporal fluorescence intensity or dye dilution curves. The resultant artery and vein fluorescence data were fit to a log normal distribution function5 from which average arterial and venous circulation times were calculated. The arterial appearance time (AT) of the dye bolus, defined as the time between dye injection and the first detectable appearance of dye in the retinal artery, represents an assessment of systemic circulation times. The MCT was calculated as the difference between the retinal arterial and venous circulation times for corresponding artery and vein pairs, and the average retinal MCT for each rat represents the average of the individual artery–vein MCTs. Segmental retinal blood flows (in square pixels per second) were calculated from the individual MCTs and the corresponding vessel diameter determinations, assuming that blood flow was proportional to the sum of the squares of the arterial and venous diameters divided by the MCT.35 The average segmental retinal blood flow represented the average of the individual segmental flows.

Statistical Analysis
All values are reported as the mean ± SD. Statistical analysis software (SigmaStat; Jandel Scientific, San Rafael, CA) was used for statistical comparisons. One-way repeated-measures analysis of variance (ANOVA) was used to compare values for the same rats at the different measurement times. Group comparisons were performed using one-way ANOVA. Population normality and equality of variances were tested using the Kolmogorov–Smirnov test and the Levene median test, respectively. If either test failed, then the Kruskal-Wallis ANOVA on ranks was performed. All pair-wise multiple comparisons were performed using the Student–Newman–Keuls test. Values of P < 0.05 were considered to be statistically significant. Power analyses for retinal blood flow measurements, based on the measured variances in retinal blood flow, showed that a difference of 25 pixel2/sec in retinal blood flow could be detected at a significance of 0.05 with a power of 0.8, using six rats per group.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The animal characteristics at the time of retinal blood flow measurements are summarized in Table 1 for the 37 nondiabetic and the 34 STZ-induced diabetic rats used in the ET-3 dose–response and time course experiments. Group comparisons showed that although diabetic rats all gained weight, they gained significantly (P < 0.01) less weight than the nondiabetic rats and had significantly (P < 0.01) higher blood glucose levels. There were no significant differences in hematocrit, mean blood pressure, heart rate, and retinal ATs between diabetic and nondiabetic rats. Diabetic rats showed significantly prolonged MCTs (P < 0.01), significantly reduced retinal blood flow (P < 0.01), but no significant differences in primary retinal artery or vein diameters compared with nondiabetic rats.


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  		Table 1. Baseline Characteristics for Nondiabetic and Diabetic Rats

 
The Effect of ET-3 on Retinal Hemodynamics in Nondiabetic Rats
After intravitreal injections of ET-3 in the nondiabetic rats, there were no significant changes in heart rate (403.5 ± 67.5 vs. 377.8 ± 74.4 beats/min), mean blood pressure (78.2 ± 17.2 vs. 77.2 ± 15.5 mm Hg) or AT (2.2 ± 0.7 vs. 2.2 ± 0.3 seconds) compared with preinjection measurements, indicating that intravitreal injection of ET-3 had no significant effect on the systemic circulation.

Retinal MCT and blood flow responses to intravitreally injected ET-3 or vehicle alone are summarized in Figures 1A and 1B , respectively. The MCT response to ET-3 was characteristically biphasic in time with an initial rapid dose-dependent decrease (maximum 2 minutes after injection) followed at later times (15 and 30 minutes) by dose-dependent increases in MCTs compared with baseline or vehicle values. The maximum decrease in MCT at 2 minutes occurred at 10-8 M ET-3 (0.61 ± 0.17 seconds) and was significantly (P < 0.01) decreased compared with vehicle (0.95 ± 0.22 seconds). The primary retinal artery and vein diameters tended to dilate at this time and concentration but were not significantly different from baseline (arteries, 6.7 ± 0.7 vs. 6.9 ± 0.9 pixels; veins, 7.6 ± 0.5 vs. 8.1 ± 0.8 pixels) or vehicle (arteries, 6.6 ± 0.6 vs. 6.4 ± 0.8 pixels; veins, 7.7 ± 0.6 vs. 7.6 ± 0.8 pixels).



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  		Figure 1. (A) Effect of intravitreal injection of ET-3 (10-9 M, {circ}, n = 7; 10-8 M, {blacktriangledown}, n = 8; 10-7 M, {triangledown}, n = 6; 10-6 M, {blacksquare}, n = 6) and vehicle alone (•, n = 10) on retinal MCT in nondiabetic rats at different times after ET-3 injection. (B) Corresponding retinal blood flow responses to ET-3 in nondiabetic rats. (C) Effect of intravitreal injection of ET-3 (10-9 M, {circ}, n = 6; 10-8 M, {blacktriangledown}, n = 9; 10-7 M, {triangledown}, n = 6; 10-6 M, {blacksquare}, n = 5) and vehicle alone (•, n = 8) on retinal MCT in diabetic rats at different times after ET-3 injection. (D) Corresponding retinal blood flow responses to ET-3 in diabetic rats. *P < 0.05 compared with vehicle injection.

 
At later times after ET-3 intravitreal injection, there was a dose-dependent prolongation of the MCT at concentrations of 10-8 to 10-6 M ET-3; however, at 10-9 M ET-3, there was no prolongation of the MCT, with values reverting to baseline at 15 and 30 minutes. The vasoconstrictive response 30 minutes after injection of 10-6 M ET-3 was characterized by significant MCT prolongation (2.73 ± 0.34 seconds) compared with vehicle (1.03 ± 0.05 seconds; P < 0.02) and significant primary retinal vessel diameter constriction (arteries, 5.8 ± 0.5; veins, 6.6 ± 0.3 pixels) compared with vehicle (arteries, 6.9 ± 0.5; veins, 7.9 ± 0.6 pixels; P < 0.01).

In parallel with the MCT decrease, the retinal blood flow (Fig. 1B) was significantly increased 2 minutes after intravitreal injection of 10-9 to 10-7 M ET-3 (193.6 ± 33.2 pixel2/sec at 10-8 M ET-3;) compared with vehicle (105.6 ± 7.6 pixel2/sec; P < 0.03). At later times, retinal blood flow decreased, and 30 minutes after injections of 10-8 to 10-6 M ET-3 was significantly reduced (28.4 ± 3.3 pixel2/sec at 10-6 M) compared with vehicle (109.6 ± 4.3 pixel2/sec; P < 0.01). At 10-9 M ET-3 the retinal blood flow showed an initial rapid increase followed at later times with a reversion to baseline but no further decrease.

The Effect of ET-3 on Retinal Hemodynamics in Diabetic Rats
After intravitreal injections of ET-3 in diabetic rats, there were no significant changes compared with baseline in heart rate (370.0 ± 43.9 vs. 358.8 ± 41.7 beats/min), mean blood pressure (83.2 ± 22.7 vs. 77.7 ± 20.2 mm Hg), or retinal AT (2.3 ± 0.3 vs. 2.4 ± 0.5 seconds).

The diabetic rat retinal MCT responses to ET-3 are summarized in Figure 1C . The MCTs at 2 minutes after ET-3 injection showed an initial rapid decrease; however, baseline MCTs were prolonged compared with nondiabetic rats (P < 0.01), and at 2 minutes after ET-3 injection remained prolonged compared with corresponding MCTs in nondiabetic rats (P < 0.05; Fig. 1A ). In contrast to nondiabetic rats, the retinal MCT decrease in diabetic rats was sustained for a longer period, with the maximal response occurring 15 minutes after injection at 10-9 to 10-7 M ET-3. At 15 minutes after injection, MCTs were significantly decreased at 10-9 M (0.80 ± 0.33 seconds) and 10-8 M ET-3 (0.71 ± 0.41 seconds) compared with vehicle (1.52 ± 0.59 seconds; P < 0.01), and the decrease was sustained at 30 minutes after ET-3 injection (P < 0.05). At 10-6 M ET-3 the retinal MCT showed a time-attenuated biphasic response with an initial rapid decrease followed at later times by a prolongation that at 30 minutes after injection (2.81 ± 0.20 seconds) was significant compared with vehicle (1.55 ± 0.21 seconds; P < 0.01). There were no significant changes in the major retinal vessel diameters after ET-3 injections.

The baseline retinal blood flow in diabetic rats was decreased compared with that in nondiabetic rats and in response to 10-9 to 10-7 M ET-3 increased at 2, 5, and 15 minutes (Fig. 1D) with a significant maximal response at 15 minutes after injection of 10-9 M (127.1 ± 23.2 pixel2/sec) and 10-8 M (151.7 ± 52.0 pixel2/sec) compared with vehicle (62.4 ± 10.8 pixel2/sec; P < 0.01). By 30 minutes after injection, retinal blood flows reverted to baseline. At 10-6 M ET-3 the initial retinal blood flow increase was less pronounced than at lower ET-3 concentrations, reached a maximum effect at 5 minutes after injection, and was significantly decreased at 30 minutes (27.7 ± 2.3 pixel2/sec) compared with vehicle (58.4 ± 12.2 pixel2/sec; P < 0.01).

Retinal Hemodynamic Dose Responses to ET-3
Figure 2 summarizes the diabetic and nondiabetic rat retinal blood flow dose responses to ET-3 calculated as the percentage change from baseline at 2 and 15 minutes after injection. There was a dose-dependent increase in the percentage of retinal blood flow change 2 minutes after ET-3 injection, with the maximum at 10-8 M ET-3. There were no significant differences, however, in the magnitude of the percentage of retinal blood flow increase between diabetic (104.9% ± 85.4%, at 10-8 M) and nondiabetic rats (106.6% ± 61.7% at 10-8 M).



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  		Figure 2. The dose response characteristics of the percentage change from baseline of retinal blood flow at 2 and 15 minutes after ET-3 injections in diabetic and nondiabetic rats. *P < 0.05 and **P < 0.01 compared with vehicle injection.

 
Fifteen minutes after ET-3 injection, there was a dose-dependent decrease in the percentage of retinal blood flow change in nondiabetic rats that reached significance at 10-6 M compared with vehicle (P < 0.05). In diabetic rats, by contrast, the percentage of retinal blood flow change at 15 minutes dose dependently increased to a maximum at 10-8 M ET-3 (161.3% ± 84.7%; P < 0.01 compared with vehicle) and decreased at higher concentrations. Additionally, the magnitude of the percentage of retinal blood flow change in diabetic rats was significantly different compared with nondiabetic rats for the 10-9 to 10-7 M ET-3 (P < 0.01).

NOS Inhibitor and ETB Antagonist Action
The retinal hemodynamic responses to intravitreal injection of 10-3 M L-NMMA or 10-4 M BQ-788 alone in nondiabetic rats are presented in Table 2 . After NOS inhibition, there were no significant retinal hemodynamic changes at 15 minutes or 30 minutes; however, 40 minutes after injection, there was a significant decrease in retinal blood flow compared with baseline measurements (77.5 ± 18.2 pixel2/sec; P < 0.05; data not shown). After injection of the ETB antagonist, retinal blood flow was significantly decreased 15 minutes after injection (P < 0.05) and at 30 minutes reverted to flows comparable to baseline.


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  		Table 2. The Effects of L-NMMA and BQ-788 on Retinal Hemodynamics in Nondiabetic Rats

 
The retinal response to 10-8 M ET-3 in nondiabetic rats after a 15-minute pretreatment with 10-3 M L-NMMA (n = 6), 10-4 M BQ-788 (n = 6), or vehicle (n = 6) is summarized in Figure 3 . In the eyes pretreated with vehicle alone, the expected biphasic retinal blood flow response to ET-3 was measured with a rapid transient increase in retinal blood flow (173.6 ± 71.0 pixel2/sec compared with baseline 101.6 ± 29.4 pixel2/sec; P < 0.05) 2 minutes after ET-3 injection, followed by a reduction in retinal blood flow 15 minutes after ET-3 injection (86.1 ± 49.8 pixel2/sec). In the eyes pretreated with the NOS inhibitor L-NMMA, the characteristic blood flow response to ET-3 was completely inhibited. Blood flow at 2 minutes (98.3 ± 31.7 pixel2/sec) was significantly decreased compared with that in vehicle-pretreated eyes (173.6 ± 71.0 pixel2/sec; P < 0.05). Similarly, in the eyes pretreated the ETB antagonist (BQ-788) the characteristic retinal blood flow response to ET-3 was significantly inhibited (P < 0.02, compared with vehicle pretreated eyes). BQ-788 pretreatment also significantly reduced retinal blood flow (74.9 ± 20.3 pixel2/sec) compared with baseline (108.9 ± 38.4 pixel2/sec; P < 0.05) before intravitreal ET-3 injection.



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  		Figure 3. The effect of 10-8 M ET-3 on retinal blood flow after a 15-minute pretreatment with L-NMMA (10-3 M, n = 6), BQ-788 (10-4 M, n = 6), or vehicle alone (n = 6). Time 0 represents initiation of pretreatment with L-NMMA, BQ-788, or vehicle. ET-3 was injected after 15 minutes (arrow), and subsequent retinal blood flow measurements were made at 2 and 15 minutes after ET-3 injection. *P < 0.05 compared with vehicle pretreatment.

 
ETA Antagonist Action
The retinal response to 10-7 M ET-3 after a 2-minute pretreatment with 10-4 M BQ-123, an ETA antagonist, is summarized in Figure 4 . The results are plotted with respect to the percentage decrease in the retinal MCT from baseline. For comparison, the retinal MCT data shown in Figure 1A for the 10-7 M ET-3 concentration are presented also as the percentage of decrease in MCT from baseline. The retinal response to BQ-123 alone was comparable to that measured previously,10 with a maximal decrease in MCT occurring 5 minutes after injection and a return to baseline by 15 minutes after injection. In contrast, the retinal response to ET-3 after pretreatment with BQ-123 was characterized by a similar rapid decrease in MCT. However, the following increase in MCT at later times (15 minutes) was significantly attenuated compared with the response to BQ-123 alone and compared with the vasoconstrictive retinal response to ET-3 alone at this measurement time and concentration(P < 0.03; Fig. 1A )



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  		Figure 4. The effect of 10-7 M ET-3 on percentage decrease of retinal MCT after a 2-minute pretreatment with 10-4 M BQ-123, an ETA antagonist (n = 5), BQ-123 alone (10-4 M, n = 4), and ET-3 alone. Data taken from Figure 1A for 10-7 M ET-3 (n = 6) in nondiabetic rats. Arrow: Time of ET-3 injection. *P < 0.03 compared with BQ-123 and ET-3 alone.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In nondiabetic rats, there was a dose-dependent, biphasic retinal blood flow response to intravitreal injection of ET-3 with an initial transient rapid increase and subsequent decreased retinal blood flow. The initial retinal blood flow increase was completely inhibited by L-NMMA, a NOS inhibitor and by BQ-788, an ETB antagonist. The retinal blood flow decrease at later times was inhibited by BQ-123, an ETA antagonist. In the rats with diabetes of 2 weeks’ duration, there was an ET-3–stimulated dose-dependent initial retinal blood flow increase. However, compared with nondiabetic rats, this increase was sustained for longer times except at the highest ET-3 concentration at which a temporally attenuated biphasic response was observed. In addition, because baseline diabetic rat retinal blood flows were lower, the magnitude of the ET-3–induced blood flow increase was reduced compared with that in nondiabetic rats. However, the dose-dependent percentage change in retinal blood flow increases (Fig. 3) was comparable to that in nondiabetic rats, which indicates that the initial vasodilatory response to ET-3 was not impaired in diabetic rats.

The median effective concentration (EC50) for the initial retinal blood flow increase 2 minutes after ET-3 intravitreal injection was 8 x 10-11 M (effective vitreous concentration), consistent with results in other studies.36 This phenomenon appeared to be primarily associated with microcirculatory vasorelaxation, rather than with dilation of the primary retinal vessels, because the diameter changes in these vessels were not statistically significant. The ET-3–induced vasorelaxation appeared to be mediated through NO action, because the effect was abolished with NOS inhibitor pretreatment. Other preliminary results have shown that endothelium-independent NO action also induces a rapid (2-minute) transient retinal blood flow increase comparable in magnitude to the ET-3 responses.37 These results indicate that the initial ET-3–associated retinal vasorelaxation is mediated by ET-3 binding to the G-protein–coupled ETB, subsequent activation of NOS, and NO production,34 38 39 40 41 42 which causes vascular smooth muscle and pericyte cell relaxation through cyclic guanosine monophosphate increases.43 44

There was a characteristic dose-dependent ET-3–induced reduction in retinal blood flow in nondiabetic rats at the later measurement times, with an EC50 of 8 x 10-9 M effective vitreous concentration 15 minutes after injection, consistent with a prior study.45 The EC50 for the ET-3–mediated vasoconstriction was 100 times greater than that for the initial ET-3–mediated vasodilation. This difference was reflected in the temporally augmented vasodilatory response to 10-9 M ET-3, which was sustained for a longer period (15 minutes) than the responses at the higher ET-3 concentrations. Additional data showed that pretreatment with an ETA antagonist resulted in a significant attenuation of this later retinal vasoconstrictive response to ET-3. These data indicate that the later retinal blood flow reductions were related to ET-3/ETA interaction with a decreased vasoconstrictive action compared with ET-19 46 (ETA affinity for ET-3 is 1000 times less than for ET-124 25 ). Thus, the measured biphasic retinal hemodynamic response to ET-3 depends on a balance between the vasodilatory actions of ETB and the vasoconstrictive actions of ETA.

In diabetic rats at baseline, the MCT was prolonged, primary vessel diameters were not different, and retinal blood flow was decreased compared with nondiabetic rats consistent with prior studies.1 2 3 4 5 The absence of any changes in primary vessel diameters would indicate that the hemodynamic changes were associated with increased flow resistance in the microcirculation. Prior studies showed that the decreased retinal blood flow in diabetic rats was related to increased protein kinase C-ß activation3 4 and increased ET-1 expression.10 47

Diabetic rats also responded to ET-3 with a dose-dependent initial retinal blood flow increase. However, the maximal response occurred 15 minutes after injection, and the increase was sustained at 30 minutes after injection, which was characteristically different from the effect in nondiabetic rats. It was only at 10-6 M ET-3 that a temporally attenuated biphasic blood flow response was noted with the maximal vasodilatory effect at 5 minutes and a retinal blood flow decrease at 30 minutes after injection. The sustained ET-3–mediated retinal blood flow increase in the diabetic rats could be associated with a decrease in ET-3/ETA interaction associated with competition from increased ET-1 in the diabetic rat retina.10 Data from other studies48 suggest that chronically increased levels of ET-1 in diabetes may lead to decreased affinity or downregulation of ETAs. Thus a decrease in the ET-3/ETA action in diabetic rats would lead to an attenuation of the expected vasoconstriction and the resultant temporal augmentation of the ET-3 vasodilatory effect.

The sustained elevated retinal blood flow in diabetic rats may be associated with other vasodilatory actions. For example, in cultured endothelial cells vasodilatory prostacyclins are produced in a time-dependent manner after ET-3 stimulation,38 in human hepatic stellate cells ETBs are upregulated by ET-1/ETB–stimulated production of cyclic adenosine monophosphate and prostacyclin,49 and in diabetes NOS activation is increased by ETB action through calcium-calmodulin and protein tyrosine kinase–dependent pathways.50 Thus, upregulation of the ETB and/or activation of postreceptor intracellular signal transduction cascades could result in increased production and action of NO and/or prostacyclin, which could contribute to sustained ET-3–stimulated retinal blood flow increase in the diabetic rats.

Intravitreally introduced ET-3 caused an initial rapid increase in retinal blood flow followed at later times by reduced retinal blood flow in nondiabetic rats. The initial retinal blood flow increase was mediated through ET-3/ETB action and NO release. The subsequent retinal blood flow decrease was associated with ET-3/ETA action. Diabetic rats showed comparable early retinal blood flow increases, indicating that ET-3 action may not be impaired. However, the prolongation of this blood flow increase suggests a diminution in ET-3/ETA action.


   Footnotes
 
Supported by National Institutes of Health Grant EY09178 and by a grant from the Massachusetts Lions Eye Research Fund, Inc.

Submitted for publication January 20, 2000; revised July 6, 2000; accepted August 14, 2000.

Commercial relationships policy: N.

Corresponding author: Sven-Erik Bursell, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. sbursell{at}joslin.harvard.edu


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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