Dopaminergic Vasodilation in the Choroidal Circulation by D1/D5 Receptor Activation

Abstract

purpose. To test whether exogenous dopamine can cause choroidal vasodilation and to identify the mediating receptors in anesthetized rabbits.

methods. Mean arterial pressure (MAP), intraocular pressure (IOP), and orbital venous pressure (OVP) were measured by direct cannulation of the central ear artery, the vitreous, and the orbital venous sinus, respectively. Laser Doppler flowmetry was used to measure choroidal blood flow (ChorBF) while MAP was manipulated mechanically with occluders on the aorta and vena cava, thus changing perfusion pressure (PP) over a wide range. In the first group of animals (n = 11), pressure–flow (PF) relationships were performed at control and in response to 40 μg/kg per minute intravenous (IV) dopamine (D40) and D40+SCH-23390 (0.5 mg/kg, bolus injection IV). In the second group of animals (n = 6), PF relationships were recorded at control and during infusion of SKF-38393 (80 μg/kg per minute).

results. D40 lowered IOP and caused an upward shift in the choroidal PF relationship, which was blocked by the D1/D5 antagonist SCH-23390 suggesting the involvement of the dopamine D1/D5 receptors. Stimulation of the D1/D5 receptors by infusion of the selective agonist SKF-38393 also lowered IOP and caused an upward shift in the PF relationship. Dopamine and SKF-38393 tended to decrease OVP, but the effect was not significant.

conclusions. Dopamine can cause choroidal vasodilation in anesthetized rabbits. Because SCH-23390 was able to block the response and SKF-38393 caused a similar vasodilation, we conclude that the vasodilation is caused by a D1/D5-receptor–mediated mechanism.

Dopamine is an endogenous catecholamine with a variety of direct and indirect actions. In the cardiovascular system, dopamine’s effects have been studied extensively and are well characterized. A unique property of dopamine is that low doses cause vasodilation and decrease systemic blood pressure, whereas high doses cause vasoconstriction and increase systemic blood pressure. ¹ In the eye, numerous publications in the past have focused on the neuromodulatory actions of dopamine in the sensory retina ² and its role in the regulation of intraocular pressure (IOP). ³ In contrast to the extensive research on dopamine’s action in the neural retina and on IOP homeostasis, little information is available about its role in ocular blood flow. In a recent study by Reitsamer and Kiel, ⁴ short-term infusions of dopamine were shown to affect ciliary vascular resistance in a dose-dependent manner. The lowest infusion rate used in that study (40 μg/kg per minute) caused ciliary vasodilation, whereas the higher infusion rates (600 and 1800 μg/kg per minute) caused substantial ciliary vasoconstrictions. At the midrange dose tested, the vasoconstrictor and vasodilator actions canceled each other out. The receptors causing these effects have not been identified yet. ⁴ Chiou and Chen ⁵ and Hong and Chiou ⁶ tested various dopaminergic agents and found no effect of dopamine on choroidal blood flow (ChorBF); however, their results are difficult to interpret because the experiments were performed at nonphysiologic IOP, and choroidal perfusion pressure (PP) was not measured. ^{5 6}  

Based on the findings in the ciliary body it seemed possible that dopamine could also cause a vasodilation in the choroid in addition to lowering IOP, which would make it an interesting drug from a clinical point of view. Thus, in the present study, we examined whether exogenous dopamine can cause choroidal vasodilation and then sought to identify the receptors involved in mediating the effect. 

Methods

All animal procedures were approved by the Institutional Animal Care and Use Committee and conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. At the end of the experiment, all animals were killed with an overdose of anesthetic without regaining consciousness. 

Animal Preparation

Locally obtained New Zealand albino rabbits (2–3 kg) of both sexes were housed for 2 weeks before the experiments in the institutional vivarium with food and water ad libitum. All animals were anesthetized with pentobarbital sodium (30 mg/kg, IV, supplemented as needed), and paralyzed with gallamine triethiodide (1 mg/kg) to eliminate eye movement. Intravenous injections and infusions were performed through cannulae inserted into the left and right marginal ear veins. The animals were intubated through a tracheotomy and respired with room air and supplemental oxygen. Expired Pco ₂ was monitored (NPB-75 Capnograph; Nellcor, Pleasanton, CA) and maintained at 40 to 45 mm Hg. Normal body temperature (38–39°C) was maintained with a heating pad. A catheter was inserted into the right central ear artery and advanced to the ear base, to estimate the ocular arterial pressure. The catheter was connected to a pressure transducer that was positioned at the same height above the heart as the eye. Blood pressure was controlled mechanically with hydraulic occluders placed around the thoracic descending aorta and inferior vena cava through a right thoracotomy. The aortic occluder was used to redirect the cardiac output to the upper body, thus increasing the blood pressure at the eye. The caval occluder was used to impede venous return, thus lowering cardiac output and reducing blood pressure throughout the circulation. 

After the surgical preparation, the animals were mounted in a stereotaxic head holder, the right eye anesthetized topically with lidocaine, and the eye cannulated with a 23-gauge needle inserted into the vitreous through the pars plana to measure the IOP with a pressure transducer (MLT844; ADInstruments, Colorado Springs, CO). To prevent an ocular trauma response, the right eye was anesthetized topically with 1% lidocaine before cannulation, ^{7 8} and care was taken not to disturb the cornea and anterior chamber. Orbital venous pressure (OVP) was measured through direct cannulation of the orbital venous sinus through the posterior supraorbital foramen. A detailed description of OVP measurement and its significance to ocular blood flow and IOP homeostasis has been published elsewhere. ⁹  

Measurement of ChorBF

Laser Doppler flowmetry (LDF) was used to measure ChorBF. LDF provides three indexes of perfusion derived from the frequency spectra collected from tissue illuminated with laser light: the number of moving blood cells, their mean velocity, and the flux, which is the product of the velocity and number of moving blood cells. The flux has been shown to correlate linearly with independent measures of blood flow in a variety of tissues. An in-depth description of LDF and its validation is provided in detail elsewhere. ¹⁰ The flowmeter (PF4000; Perimed, Stockholm, Sweden) was coupled to a needle probe (PF403; Perimed). The flowmeter was calibrated so that the flux registered 250 perfusion units (PU) when the probe was placed in a suspension of latex particles at 22°C. During the experiments, the total backscatter (i.e., the DC voltage at the photodetector) was maintained constant at 1.1 ± 0.05 V to ensure a consistent probe-to-tissue distance between and within experiments. ¹¹ To measure choroidal perfusion, the probe was advanced through the pars plana with a micromanipulator so that the probe tip was positioned in the vitreous, near the retinal surface over the posterior pole. For the wavelength (780 nm) and fiber separation (0.25 mm) used in this study, the volume of tissue sampled by the flowmeter was approximately 1 mm³, which is sufficient to measure perfusion in both the retina and the choriocapillaris. As the rabbit retina is mostly avascular and the probe was directed away from the few extant retinal vessels, the flux signal in this preparation originated solely from the choroid, under the visual streak. 

Carotid blood flow (CarBF) was measured at the left carotid artery with a bidirectional ultrasonic flow probe (Transonic Systems Inc., Ithaca, NY). Heart rate (HR) was calculated simultaneously from the carotid flow signal. 

Experimental Protocols

The measurement protocols were performed in three independent groups of animals (total n = 22). In groups 1 and 2, the goal was to vary MAP over a wide range using the hydraulic occluders placed around the thoracic aorta and the inferior vena cava. In group 1, baseline measurements and pressure–flow (PF) relationships were measured in a three-step protocol at control (step 1), during dopamine infusion (step 2), and during dopamine infusion and subsequent D1/D5 receptor blockade by SCH-23390 (step 3). For step 2, dopamine was given at 40 μg/kg per minute (D40). After 4 to 6 minutes, the vasodilation was robust and constant, and then the occlusions were performed. The dopamine infusion was continued until the end of step 3. In group 2, baseline readings and PF relationships were measured in a two-step protocol at control (step 1) and during the infusion of the D1/D5 receptor agonist SKF-38393 (step 2). To illustrate the protocol, representative traces of an experiment in group 1 are shown in Figure 1 . Except for the administration of one drug only, the traces of group 2 were similar to those in Figure 1 . In group 3 (n = 5) free plasma dopamine concentration was determined at control and during D40 infusion. For the baseline level determination, the first blood sample was obtained from the left ear artery before the first occlusion occurred. Blood was collected again during D40 infusion after a robust level of vasodilation was reached. Shortly before the second pair of occlusions was performed, blood samples were drawn into chilled tubes containing EGTA and reduced glutathione (RPN532; Amersham Biosciences Europe GmbH, Vienna, Austria) and the plasma was separated immediately and stored at −80°C until it was processed for HPLC analysis. Plasma concentrations of dopamine were determined by HPLC with electrochemical detection, using a reagent kit for routine analysis of plasma catecholamines (Chromsystems Instruments & Chemicals GmbH, Munich, Germany). All drugs were purchased from Sigma-Aldrich Handels GmbH (Vienna, Austria). 

Data Analysis

All variables were recorded with a data-acquisition system (PowerLab; ADInstruments). The data were later reduced off-line by averaging the measured variables in 5-mm Hg bins of PP (i.e., all readings temporally associated with PP between 80 and 75 mm Hg were averaged, then between 75 to 70 mm Hg, and so forth). PP was calculated by subtracting IOP from MAP, and choroidal vascular resistance (R) was calculated as R = MAP - IOP/ChorFlux. Differences in PF curves were identified by repeated-measures analysis of variance with PP and treatment as within-subject factors. Pair-wise comparisons of different treatments were performed with the Bonferroni adjustment. Within subject effects were calculated by using the Huynh-Feldt adjustment, and pair-wise group comparisons were performed with the Bonferroni adjustment. In group 2, paired t-tests were performed on the baseline values. All results are expressed as the mean ± SE (SPSS; SPSS Science, Chicago, IL). 

Results

Baseline Changes

The plasma dopamine levels determined by HPLC were 0.13 ± 0.07 ng/mL at baseline before the first pair of occlusions was performed and 175 ± 19 ng/mL during the dopamine infusion when the second pair of occlusions was performed. The baseline readings are comparable to data from rabbits published elsewhere. ^{12 13} Figure 2 shows the baseline effects of dopamine, and dopamine+SCH-23390 (0.5 mg/kg, IV bolus). Dopamine caused significant parallel decreases of MAP by 6% ± 2% and IOP by 15% ± 3% (Figs. 2A 2B) . While PP (MAP − IOP) was not changed significantly (Fig. 2D) , ChorBF was increased by 21% ± 3% (Fig. 2C) so that choroidal vascular resistance was decreased by 19% ± 2% (Fig. 2E) . Dopamine given at this infusion rate had no significant effects on CarBF, OVP, and HR (Fig. 2F 2G 2H) although there was the tendency for a decrease in CarBF and OVP. A bolus injection of the D1/D5 receptor antagonist SCH-23390 while dopamine was infused caused a significant increase in MAP, whereas the increase in IOP was not significant (Figs. 2A 2B) . Although PP increased significantly (29% ± 5%; Fig. 2D ), choroidal flux decreased by 7% ± 5% (Fig. 2C) ; however, the effect was not significant. As shown in Figure 2E , the decrease in vascular resistance caused by dopamine was successfully blocked by the D1/D5 receptor antagonist SCH-23390. Baseline changes in response to SKF-38393 (80 μg/kg per minute) in group 2 are similar to the effects caused by the dopamine infusion in group 1. The D1/D5 receptor agonist SKF-38393 caused a 28% ± 3% decrease in MAP and a 41% ± 16% decrease in IOP (Figs. 3A 3B) resulting in a 28% ± 4% decrease in PP (Fig. 3D) . ChorBF increased by 6% ± 1% (Fig. 3C) , and consequently choroidal vascular resistance was reduced by 33% ± 3% (Fig. 3E) . The effects of SKF-38393 on CarBF, OVP, and HR were not significant; however, there was again the tendency for a decrease in CarBF and OVP (Figs. 3F 3G 3H) . 

PF Relationships

Dopamine caused a significant upward shift of the choroidal PF relationship at PP greater than 20 mm Hg (P < 0.001) which was reversed (P < 0.01) by coadministration of SCH-23390 (Fig. 4A) . The changes in vascular resistance are also illustrated in the tracings of Figure 1 (upward arrows). SKF-38393 caused a similar and significant upward shift in the PF relationship at PP greater than 15 mm Hg (P < 0.001), also indicating choroidal vasodilation (Fig. 4B) . 

Discussion

Catecholamines are an important class of compounds that include dopamine, norepinephrine, and epinephrine. Although there is extensive information about catecholamine’s effects on ocular blood flow, most studies have focused on norepinephrine and epinephrine. By contrast, there is relatively little information available about dopamine’s role in the eye, aside from its well-recognized function as a neuromodulator in the sensory retina. However, there is evidence of dopaminergic innervation and dopamine receptors in nonretinal ocular tissues and evidence of dopamine’s effects on IOP and ocular blood flow. ³  

Our understanding of glaucoma is changing, and several groups have reported decreased choroidal and optic nerve head blood flow in patients with glaucoma. ^{14 15} Because of the possible relevance to glaucoma, the present study sought to determine whether dopamine can increase ChorBF and at the same time lower IOP. As a second goal, we sought to identify the receptors that are involved in the modulation of choroidal vascular resistance. The results show that exogenous dopamine can increase ChorBF at plasma concentrations of 175 ± 19 ng/mL by decreasing choroidal vascular resistance and that choroidal vasodilation is mediated through a D1/D5-receptor–coupled mechanism. In addition, the findings confirm the IOP-lowering effect of dopamine, as reported previously by Reitsamer and Kiel. ⁴  

Dopamine Receptors

Five different types of dopamine receptors have been identified by molecular cloning, D1-5, which can be divided into D1-like receptors (D1/D5) and D2-like receptors (D2/D3/D4). The selectivity of the dopaminergic agonist SKF-38393 for D1/D5 receptors is roughly 100 times higher than for the D2, -3, and -4 receptors. The selectivity of the dopaminergic antagonist SCH-23390 is roughly 1000 times higher. Therefore, both compounds are fairly selective for D1/D5; however, it is not possible to distinguish between D1 and D5 receptor stimulation because SCH-23390 and SKF-38393 are almost equally selective for both D1-like receptor subtypes. To the best of our knowledge, there are no drugs currently available to distinguish between the D1 and D5 receptor subtypes. ¹⁶  

Effects of Dopamine on IOP, OVP, CarBF, and HR

The present study confirms the findings reported by Reitsamer and Kiel ⁴ that short-term infusions of dopamine D40 cause a decrease in IOP. Blockage of the D1/D5 receptors by SCH-23390 during the dopamine infusion caused a marked increase in IOP, whereas the D1/D5 agonist SKF-38393 lowered IOP. It should be pointed out that these are short-term effects that should be compared only cautiously with long-term effects. Results published by others indicate that topical and intravenous administration of the D1/D5 agonist fenoldopam increases IOP in normal and hypertonic human eyes. ^{17 18 19} It is not known why fenoldopam causes an increase in IOP as opposed to the SKF-38393 used in the present study. The opposite findings may be caused by the differences between species that were studied (humans versus rabbits) or, as mentioned earlier, the differences in experimental timing protocols. Potter et al. ²⁰ also showed that the hypertensive effect could be caused by contracting eye muscles, which may not occur in our model because the muscles are paralyzed with gallamine. 

No significant changes in OVP and HR were found, although D1/D5 receptor activation tended to decrease OVP (Figs. 2G 3G) . D1/D5 activation had no significant effect on CarBF (Figs. 2F 3F) ; however, additional D1/D5 receptor blockage reduced CarBF to a significantly lower level compared with baseline (Fig. 2F) . 

Dopamine and ChorBF

Previous reports of dopamine’s effects on ChorBF are difficult to interpret, because the PP was not measured, and the IOP was often held at a nonphysiologic pressure of 40 mm Hg. Nonetheless, acute, topical application of dopamine antagonists in ocular hypertensive rabbits increased ChorBF, as estimated by microsphere entrapment ⁵ and pulsatile blood flow calculated from IOP pulse amplitude. ⁶ In the same studies, topical bromocriptine (D2/D3 antagonist) and dopamine tended to increase ocular blood flow at the single dose used, but the effect was not statistically significant. By contrast, in rats, subcutaneous injection of SDZ GLC-756 (a D1 antagonist and D2 agonist) increased anterior optic nerve head blood flow when measured by magnetic resonance imaging, but it is unclear whether this was an ocular effect, because neither blood pressure nor IOP was measured. ²¹  

In the present study, D40 was tested for its effects on ChorBF based on the earlier finding that short-term infusions of dopamine D40 and D600 have opposite effects on ciliary blood flow—that is, D40 causing ciliary vasodilation and D600 causing ciliary vasoconstriction. We hypothesized that D40 ciliary vasodilation is mediated by D1 receptor subtypes as opposed to higher infusion rates where the effect is probably comediated by dopaminergic, adrenergic, and possibly serotoninergic receptor subtypes. ^{1 22} For the present study, we focused on the possibility of a D1-mediated vasodilatory response in the choroid, leaving the more complex vasoconstrictor response for subsequent study. 

The baseline values shown in Figures 2 and 3 are similar to those reported previously for this preparation, ^{9 23} and the MAP and IOP are similar to those in conscious rabbits. ²⁴ As hypothesized, D40 caused choroidal vasodilation with a 19% ± 2% decrease in baseline vascular resistance occurring at a plasma dopamine concentration of 175 ± 19 ng/mL. This is a slightly greater decrease in vascular resistance than occurred for the ciliary circulation (−14% ± 4%) in the same rabbit preparation at the same rate of dopamine infusion. 

In addition to the baseline changes, ChorFlux was measured over a wide range of PPs (MAP - IOP). The choroidal PF relationship was shifted upward (Fig. 4A) , indicating a significant decrease in vascular resistance similar to that reported in the ciliary circulation. ⁴ Because dopamine acts on D1-like (D1/D5) and D2-like (D2/D3/D4) receptors, it was not clear which of the dopamine receptor subtypes caused the effect. Thus, after the second pair of occlusions and recovery of baseline stability, D1/D5 receptors were blocked using the selective D1/D5 receptor antagonist SCH-23390, while the dopamine infusion was continued. SCH-23390 blocked the effects of dopamine on choroidal vascular resistance and the choroidal PF relationship, indicating that the effects of D40 on choroidal hemodynamics were mediated by D1/D5 receptor stimulation, a conclusion that is further supported by the response to infusion of the D1/D5 receptor agonist SKF-38393 in a second group of animals. SKF-38393 caused a similar decrease in choroidal vascular resistance (Fig. 3E) and an upward shift of the choroidal pressure flow relationship (Fig. 4B) . 

As opposed to the ciliary body and the retina, distribution studies of dopaminergic receptor subtypes are not available for the choroid. Recent work by Schrödl et al. ²⁵ has shown colocalization of tyrosin-hydroxylase and dopamine-β-hydroxylase in avian choroidal nerve fibers and ganglion cells, which indicates that dopamine is at least synthesized as an intermediate product in the neuronal biosynthesis of norepinephrine, a well-known modulator of ChorBF. ²⁵ These findings do not imply a role for dopamine in ChorBF regulation; however, they are in accordance with the findings presented in this study. 

Because of the lack of literature on the distribution of dopamine receptors in the choroid, it is not known whether dopaminergic effects are mediated by pre- or postjunctional receptors. Dopamine can cause vasodilation by reducing the liberation of norepinephrine through prejunctional D2 and adrenergic α-2 receptors, but our data support a different interpretation. Because the vasodilation caused by dopamine was blocked by the D1/D5 receptor antagonist SCH-23390 (Fig. 4A) and because administration of the D1/D5 agonist SKF-38393 caused a similar sized upward shift of the choroidal PF relationship—that is, vasodilation (Fig. 4B) —it seems more likely that the vasodilation was caused by stimulation of postjunctional vascular D1/D5 receptors. ^{1 26}  

Conclusion

D1/D5 receptor stimulation decreases choroidal vascular resistance and IOP. These findings point to the dopaminergic system as a potential target for future drug developments that may be beneficial for the treatment of diseases with impaired ChorBF and elevated IOP. 

 Supported by institutional funds from the University of Vienna.

 Submitted for publication September 9, 2003; revised November 18, 2003; accepted November 24, 2003.

 Disclosure: H.A. Reitsamer, None; C. Zawinka, None; M. Branka, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Representative experimental tracings in group 1. Tracings of AP, IOP, choroidal flux, choroidal vascular resistance (R), and OVP during an experimental protocol in a single animal. Aortic and caval occlusions were performed before and after drug application. Dopamine was given as a continuous IV infusion at a rate of 40 μg/kg per minute (D40) from the first dashed line until the end of the tracing. After the second pair of occlusions, SCH-23390 was given (second dashed line) as a short IV bolus injection (0.5 mg/kg). Vasodilation occurred in response to the dopamine infusion (downward arrows), whereas SCH-23390 caused a marked increase in choroidal vascular resistance and blocked the effect of dopamine (upward arrows). Plasma dopamine levels were determined before the first (0.13 ± 0.07 ng/mL) and again before the second pair of occlusions (175 ± 19 ng/mL) were performed.

Figure 2.

 

Baseline data from group 1 (n = 11). The histograms in each plot show measurements under control conditions (Ctrl 1), in response to dopamine (Dop), and in response to treatment with dopamine and SCH-23390 (Dop+SCH). (A) MAP, (B) IOP, (C) ChorBF, (D) PF, (E) choroidal vascular resistance, (F) CarBF, (G) OVP, and (H) HR. n.s. not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 3.

 

Baseline data from group 2 (n = 6). The histograms in each plot show measurements under control conditions (Ctrl 2) and in response to SKF-38393 treatment (SKF). (A) MAP, (B) IOP, (C) ChorBF, (D) PF, (E) choroidal vascular resistance, (F) CarBF, (G) OVP, and (H) HR. n.s., not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Figure 4.

 

Choroidal PF relationships. (A) In group 1, the choroidal PF relationship was shifted upward by dopamine at PP greater than 20 mm Hg (P < 0.001), an effect that was completely blocked after coadministration of the D1/D5 receptor antagonist SCH-23390 (P < 0.01). (B) Application of the D1/D5 receptor agonist SKF-38393 in group 2 also caused a significant upward shift of the PF relationship, which was significant at PP greater than 15 mm Hg.

The authors thank the staff of the Department of Biomedical Research, University of Vienna Medical School, for technical assistance; Christian Bieglmayer at the Department of Medical and Clinical Diagnostics, University of Vienna Medical School, for performing the HPLC analysis; and the staff of the Department of Clinical Pharmacology, University of Vienna Medical School, for assistance during the process of setting up the new laboratory in Austria.