© 1998 American Heart Association, Inc.
Original Contributions |
Retinal Arterial Tone Is Controlled by a Retinal-Derived Relaxing Factor
From the Department of General Physiology and Human Physiology and Pathophysiology, University of Gent, Belgium.
Correspondence to Johan Van de Voorde, Laboratory of Physiology and Pathophysiology, De Pintelaan 185, B-9000 Gent, Belgium. E-mail johan.vandevoorde{at}rug.ac.be
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Abstract |
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Abstract—The present study provides evidence that retinal tissue may profoundly influence the retinal arterial smooth muscle cell tone by releasing an unknown retinal relaxing factor. Isolated bovine retinal arteries with and without adhering retinal tissue were mounted in a wire myograph for isometric tension recordings. The maximal contraction induced by prostaglandin F2
was 0.95±0.7 mN (n=6)
in the presence and 5.15±0.76 mN (n=6) in the absence of adhering
retinal tissue. The contractions induced by U-46619,
serotonin, and endothelin-1 were similarly blocked in the
presence of retinal tissue. The K+ 120 mmol/L-induced
contraction was not significantly affected (2.8±0.7 mN, n=6, in the
presence and 3.6±0.7 mN, n=6, in the absence of retinal tissue).
Placing a piece of bovine retinal tissue in the proximity of a
contracted (ie, with prostaglandin F2)
retinal artery induced a complete relaxation of the retinal vessel,
suggesting the involvement of a diffusible chemical vasorelaxant. Also
porcine, canine, and ovine retinal tissue completely relaxed the
contracted (with prostaglandin F2) bovine
retinal artery. Other smooth muscle preparations, including rat
mesenteric and renal arteries and rat main bronchi, also relaxed with
the application of a piece of bovine retinal tissue. Incubation of
bovine retinas in a Krebs-Ringer bicarbonate solution yielded a
solution that relaxed isolated precontracted bovine retinal arteries,
confirming the involvement of a diffusible chemical messenger. Hexane
extraction, heating the solution to 70°C, or treatment with trypsin
did not alter the relaxing properties of the incubation solution. The
characteristics of the retinal relaxing factor do not correspond with
those of nitric oxide, prostanoids, adenosine, acetylcholine,
or any other of the known vasoactive neurotransmitters released from
the retina. Our results suggest that retinal arterial tone
is controlled by a diffusible, hydrophilic, and heat-stable relaxing
factor that does not correspond with a known vasoactive molecule formed
within the retina.
Key Words: retina • bovine • arteries • vasodilation • relaxation
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Introduction |
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Vascular smooth muscle tone is the result of myogenic tone modulated by circulating humoral factors and by neurotransmitters released from the vascular nerve endings. The original observations of Furchgott and Zawadzki1 make it obvious that vascular smooth muscle tone is also modulated by adjacent cells. In a study on isolated bovine retinal arteries, we accidentally observed that retinal arteries incompletely cleaned of retinal tissue contracted strikingly weaker in response to prostaglandin F2
than retinal arteries without adhering
retinal tissue. This suggests that retinal tissue might profoundly
influence vascular smooth muscle tone in retinal arteries. The
regulation of retinal circulation by local factors might be very
important considering the absence in the retinal vessels of sympathetic
innervation that predominantly controls the arterial tone
in most other parts of the body. Indeed, sympathetic innervation of the
central retinal artery reaches only as far as the lamina
cribrosa.2 A concept previously put forth is that surrounding retinal tissue might control retinal arterial tone. Donati et al3 suggested that nitric oxide (NO) released from the retina could control arterial tone. Prostaglandins derived from retinal tissue also have been proposed as mediators of retinal arterial dilation.4 Here we report that bovine retinal arterial tone is influenced by a diffusible relaxing factor from the retina that does not correspond, however, to the characteristics of NO or prostaglandins. We examined the potential role of the endothelium, noted some characteristics of the retinal relaxing factor (RRF), and compared the effect of the RRF with that of known vasoactive molecules formed within the retina. In addition, we investigated whether this RRF is released from retinas of other species and whether this factor relaxes nonretinal blood vessels and nonvascular smooth muscle preparations (eg, rat main bronchi). This was done to determine whether the RRF might be a more general relaxant agent.
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Materials and Methods |
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Blood Vessels
Bovine eyes were obtained from the local slaughterhouse and transported to the laboratory in ice-cold Krebs-Ringer bicarbonate solution. Canine, porcine, and ovine eyes were enucleated from animals that were killed for other experiments. The extraocular muscles and connective tissues were dissected from the globe, the anterior segment and the vitreous were removed, and the eyecup was placed in cold Krebs-Ringer bicarbonate solution for further preparation. The retina was gently teased free from the choroid and cut loose at the optic nerve. The detached retina was then either placed in a cold Krebs-Ringer bicarbonate solution to isolate the retinal artery or incubated for 6 hours in a Warburg apparatus.
Under a dissecting microscope, arterial segments (209.9±2.5 µm, n=156) with surrounding retinal tissue were carefully excised from bovine retinas. Segments of the mesenteric arteries (328.6±12.1 µm, n=5), renal arteries (289.6±19.6 µm, n=5), and main bronchi (n=13) were isolated from male Wistar rats (300 g) that were killed by cervical dislocation. The animals were killed in accordance with a protocol that was approved by the local ethics committee.
Tension Measurements
The vessels were moved to an automated dual myograph (model 500
A, JP Trading, Aarhus, Denmark) containing 10 mL Krebs-Ringer
bicarbonate solution. Two stainless steel wires were guided through the
lumen of the vessels. One wire was fixed to a force displacement
transducer, and the other was connected to a micrometer.
The retinal tissue remained attached to the retinal arteries or, when
necessary, was removed carefully after the first wire was fixed.
The segments were allowed to equilibrate for half an hour in the Krebs-Ringer bicarbonate solution bubbled with 95% O2/5% CO2 and heated to 37°C. Then the passive wall tension–internal circumference characteristics of the vessels were determined. On the basis of this relationship, the circumference was set to a normalized internal circumference.5 6 In the experiments on rat main bronchi, the preparations were stretched to 5 mN.
After the normalization procedure, the vessels were repeatedly
activated with K+ 120 mmol/L.
Maximal contractility was assessed by stimulating the
arteries simultaneously with K+
120 mmol/L, prostaglandin F2
30 µmol/L, and serotonin 10 µmol/L. The main
bronchi were repeatedly contracted with K+
120 mmol/L and carbachol 1 mmol/L.
Removal of the Endothelium
For this procedure, the arteries were unstretched in the
myograph. An L-shaped micropipette was positioned at the proximal end
of the vessel, and 95% O2/5%
CO2 was bubbled through the lumen for 2 minutes.
Thereafter, the wires were reset to their original positions, and the
vessel was allowed to reequilibrate for half an hour. The absence of
endothelium was tested by the lack of
acetylcholine-induced (10 µmol/L)
relaxation.7
Incubation
Detached retinas were incubated in pairs (wet weight, ±650 mg
each) in 20 mL oxygenated Krebs-Ringer bicarbonate solution
heated at 37°C for 6 hours using a Warburg apparatus.
After incubation, the retinas were removed from the flask, and the
remaining solution was centrifuged at 3000 rpm for 10 minutes.
After centrifugation, the supernatant was frozen and
stored. When sufficient amounts were collected, the incubation fluid of
different flasks was pooled and divided into 10 mL fractions. To each
fraction, 10 mL of hexane was added; the fraction was then shaken for
20 minutes, and it was centrifuged at 3000 rpm for 10 minutes
(In the initial series of experiments, the solution was extracted 3
times with hexane [Figure 4a
]). After centrifugation,
the hexane fraction was discarded. This procedure allowed a partial
purification of the solution to occur. The hexane-extracted solution
was then directly tested or exposed to heating to 70°C for 1 hour or
treatment with trypsin (1 µg/mL during 2 hours at 37°C). A
Krebs-Ringer bicarbonate solution incubated without retinas but treated
in the same manner as the solution incubated with retinas served as a
control solution in these experiments.
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230 mm2) in close proximity to an isolated bovine
retinal artery contracted with prostaglandin
F2
Bioassay
The arteries were mounted, normalized, and activated as
described above. After these procedures, the vessels were superfused at
a rate of 0.25 mL/min with warmed (37°C) and bubbled (95%
O2/5% CO2) test
solutions.
Drugs
The experiments were performed using a Krebs-Ringer bicarbonate
solution of the following composition (mmol/L): NaCl 135, KCl 5,
NaHCO3 20, glucose 10,
CaCl2 2.5, MgSO4 7,
H2O 1.3,
KH2PO4, 1.2, and EDTA
0.026. A Krebs-Ringer bicarbonate solution containing 120 mmol/L
K+ was prepared by equimolar replacement of NaCl
with KCl.
U-46619, serotonin
(5-hydroxytryptamine), acetylcholine chloride,
indomethacin, L-NA, endothelin-1, L-glutamic
acid, adenosine, 8-phenyltheophylline, dopamine, melatonin, and
tetrodotoxin were obtained from Sigma Chemical Co;
prostaglandin F2 (dinoprostum
trometamolum, Dinolytic) from Upjohn (Puurs, Belgium); and
-aminobutyric acid (GABA) from Interlaboratoire (Wezenbeek,
Belgium). Methylene blue was purchased from Merck (Darmstadt, Germany).
Trypsin 10x [1:250] was obtained from Gibco (Merelbeke, Belgium).
Stock solutions were made in water, except for the
indomethacin, which was dissolved in ethanol, and
8-phenyltheophylline, which was dissolved in DMSO.
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Results |
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Influence of Retinal Tissue on Contractions Induced by Different Agonists
In the first series of experiments, the contractile responses of arteries that were dissected free from all retinal tissue (control preparations) and arteries to which a piece of retinal tissue remained attached were compared. An original tracing of such an experiment is represented in Figure 1
. In
response to increasing concentrations of prostaglandin
F2 (0.1 to 30 µmol/L), the
retinal artery with adhering retinal tissue showed only a weak
contraction compared with the preparation without retinal tissue;
however, both preparations (with and without retinal tissue) contracted
in a similar way in response to a standard activating solution
containing K+ 120 mmol/L,
serotonin 10 µmol/L, and prostaglandin
F2 30 µmol/L.
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Contractions induced by serotonin (1 nmol/L to
10 µmol/L), endothelin-1 (0.1 pmol/L to 0.1 µmol/L), and
the thromboxane A2 mimetic, U-46619
(1 nmol/L to 1 µmol/L) (Figure 2)
were also less pronounced in the presence of retinal tissue. Retinal
tissue does not appear to mechanically impair the contractions because
the contractile response to K+ 120 mmol/L
was similar in preparations with (2.8±0.7 mN, n=6) and without
(3.6±0.7 mN, n=6) adhering retinal tissue.
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) and
presence of adhering retinal tissue (
). Bars represent SEM;
n indicates the number of experiments. Unpaired Student
t test with vs without retinal tissue,
*P<0.05; **P<0.02;
***P<0.01; ****P<0.001.
Effect of L-NA and Indomethacin
The NO-synthase inhibitor L-NA and the
cyclooxygenase blocker indomethacin
were used to exclude the potential influence of NO or
cyclooxygenase metabolites synthesized by the
retinal tissue. Concentration-response curves for
prostaglandin F2 (10 nmol/L to
30 µmol/L) were constructed in the presence or absence of L-NA
(0.1 mmol/L, incubated for 10 minutes) or
indomethacin (10 µmol/L, incubated for 20
minutes) on retinal arteries with and without adhering retinal
tissue.
Indomethacin did not significantly alter the
inhibitory influence of the adhering retinal tissue. The
maximal contraction induced by prostaglandin
F2 on the retinal artery with adhering retinal
tissue was 0.22±0.09 mN (n=6) before the addition of
indomethacin and 0.30±0.16 mN after the addition of
indomethacin. L-NA enhanced (P<0.025) the
maximum contraction induced by prostaglandin
F2 of the preparation with adhering retinal
tissue (1.60±0.34 mN, n=6); however, L-NA also significantly
(P<0.05) enhanced the sensitivity of the preparation
without adhering retinal tissue (log EC50,
-4.841±0.301, n=6 before incubation with L-NA and -5.360±0.161
after incubation with L-NA).
Effect of Tetrodotoxin
Concentration-response curves for prostaglandin
F2 (10 nmol/L to 30 µmol/L) were
constructed in the absence or presence of tetrodotoxin ([TTX] 10
µmol/L) on retinal arteries with and without adhering retinal tissue.
TTX had no significant influence on the maximum contractions induced by
prostaglandin F2 on either
preparation. Prostaglandin-induced contractions of the
arteries without adhering retinal tissue (n=3) were 7±0.9 mN in the
absence of TTX and 5.9±1.2 mN in the presence of TTX. The maximal
contractions of the preparations with adhering retinal tissue (n=5)
were 0.1±0.1 mN and 0.3±0.2 mN, respectively, in the absence and
presence of TTX.
Effect of Detached Retinal Tissue of Different Species on
Contractions Induced by Prostaglandin F2 and
High Potassium
This series of experiments was performed to find out whether a
diffusible chemical messenger was responsible for the
inhibitory effect of the retinal tissue. A ring segment of
the retinal artery that was carefully cleaned of all retinal tissue was
mounted for isometric tension recording, contracted with
prostaglandin F2 (30
µmol/L), and then covered with a piece of retinal tissue (±230
mm2) (schematic representation, Figure 3). This elicited a complete and stable
relaxation (100±3.3% relaxation, n=8) of the vessel that rapidly
recovered when the retina was removed (Figure 4a
). The same arteries contracted with
K+ 120 mmol/L relaxed significantly
(P<0.001) weaker with application of the retinal tissue
(46.6±6.0% relaxation, n=8) (Figure 4a), confirming the initial
observations that the presence of retinal tissue had only a moderate
influence on K+ 120 mmol/L–induced
contractions. A similar-sized piece of choroidal tissue (Figure 4b) was
incapable of inducing such a pronounced relaxation (13±2.7%
relaxation, n=7) of the retinal artery contracted with
prostaglandin F2 (30
µmol/L).
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In additional experiments, we investigated whether application of
canine (n=2), porcine (n=4), or ovine (n=2) retinas would relax a
bovine retinal artery contracted with prostaglandin
F2 (30 µmol/L). All of the retinas from
these different species completely relaxed bovine retinal arteries
(Figure 4b).
Role of the Endothelium
During this series of experiments, we investigated whether the
presence of the endothelium was required for the
retina-induced relaxations. Retinal arteries without retinal tissue
were first contracted with prostaglandin
F2 (30 µmol/L). When the contraction
reached a steady state, the retinal artery was covered with a piece of
retinal tissue (±230 mm2), resulting in
pronounced relaxation. This procedure was repeated after the
endothelium was removed and after the absence of
functional endothelium was confirmed by the lack of
relaxation induced by acetylcholine (10 µmol/L). Removal of the
endothelium did not alter the relaxations induced by
the retinal tissue. The relaxation of the arteries before removal of
the endothelium was 99.3±2.8% and after removal of
the endothelium was 98.9±0.9% (n=6).
Influence of Methylene Blue on the Relaxations Induced by the
Application of a Piece of Retinal Tissue
Methylene blue, a blocker of guanylyl cyclase, was used to exclude
the involvement of cGMP-dependent mechanisms. Isolated bovine retinal
arteries without adhering retinal tissue were contracted with
prostaglandin F2 (30
µmol/L), after which the vessels were covered with a piece of bovine
retinal tissue. This resulted in a complete relaxation (100%, n=8) of
the bovine retinal arteries. The same procedure was repeated 20 minutes
after methylene blue (5 µmol/L) was added to the organ bath.
Methylene blue induced a marked contraction (1.4±0.4 mN, n=8) of the
retinal arteries. The relaxation induced by the application of the
retinal tissue, however, was not affected. The application of the
retinal tissue still induced a complete relaxation after incubation
with methylene blue.
Influence of Retinal Tissue on Rat Renal and Mesenteric Arteries
and on Rat Main Bronchi
Our investigation included whether other arteries and nonvascular
smooth muscle preparations would relax in the presence of retinal
tissue. Therefore, rat mesenteric and renal arteries were contracted
with prostaglandin F2 (30
µmol/L), and rat main bronchi were contracted with carbachol
(0.1 mmol/L). Application of a piece of retinal tissue (±230
mm2) relaxed both the rat mesenteric
(40.7±11.2% relaxation, n=5) and renal (68.0±13.3% relaxation, n=5)
arteries (Figure 4c). Also, the rat main bronchi relaxed when they were
brought in close contact with a piece of bovine retinal tissue
(52.8±10.6% relaxation, n=7).
Bioassay
Having established that the relaxing effect of the retinal
tissue was most likely caused by a relaxing factor released by the
retina, a bioassay setup was developed to analyze some
characteristics of this RRF. In this technique, an isolated bovine
retinal artery (detector preparation) was mounted for isometric tension
recording. The artery was continuously superfused with warmed
and bubbled Krebs-Ringer bicarbonate solution containing
prostaglandin F2 (30
µmol/L) to precontract the detector preparations. Changing the
superfusion from a solution without RRF to a solution with RRF
(obtained by incubation for 6 hours with bovine retina) elicited a
relaxation (32.5±13.2%, n=4) of the detector preparation. The
relaxing influence of the RRF-containing solution was not significantly
changed after extraction with hexane (10 mL solution extracted 3 times
with 10 mL hexane)(38.5±8.2% relaxation, n=4, Figure 5a). Heating the solution at 70°C for 1
hour did not alter the relaxing effect (33.2±6% relaxation, n=4,
Figure 5b). Treatment with trypsin (1 µg/mL incubated for 2 hours at
37°C) had no influence although the same treatment abolished the
contraction induced by an endothelin-1–containing (40 nmol/L) solution
(Figure 5c). The relaxation induced by the RRF-containing solution
before treatment with trypsin (24.9±10.5%) compared well with the
relaxation after treatment (24.5±8.7%, n=3).
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pH Measurements of the Medium and the RRF-Containing
Solution
In this series of experiments, the pH of the medium and the pH of
the incubation solution, which was obtained after 6 hours of
incubation, were checked. The pH was measured in a flask containing 10
mL of heated (37°C) and oxygenated (95%
O2/5% CO2) Krebs-Ringer
bicarbonate solution. Addition of a piece of retinal tissue (±230
mm2) did not affect the pH of the medium.
The pH measurements of the incubation solution revealed that after 6
hours of incubation, the pH had dropped from 7.32±0.02 to 6.90±0.03
(n=5, measurements at 37°C during bubbling with 95%
O2/5% CO2). To exclude the
possibility that this change in pH was responsible for the relaxations
observed during the bioassay experiments summarized in Figure 5, we
performed the following experiments. We prepared a Krebs-Ringer
bicarbonate solution in which the sodium bicarbonate concentration was
half the normal concentration. This reduced the pH of the Krebs-Ringer
bicarbonate solution to 6.93±0.02 (n=5, measurements at 37°C during
bubbling with 95% O2/5%
CO2). This reduction in pH, however, did not
induce a relaxation (0.5±2.4% increase in tone, n=5) of the retinal
artery contracted with prostaglandin
F2 during bioassay experiments. On the other
hand, the RRF-containing solution with a similar pH (6.9±0.03, n=5)
induced a 28.5±4.9% (n=5) relaxation of the artery.
Correcting the pH of the RRF-containing solution did not significantly change the relaxing properties of the RRF-containing solution. Sodium bicarbonate was added to the RRF-containing solution to correct pH to normal values (pH=7.31±0.015, n=5). The relaxation induced by the pH-corrected solution (24.5±4.8%, n=5) was not significantly different from the relaxation induced by the pH-uncorrected solution (28.5±4.9%, n=5).
Influence of Different Retinal Neurotransmitters on the Retinal
Arterial Tone
The retina releases various neurotransmitters that might
influence the retinal arterial tone. In this series of
experiments, the effect of various known retinal neurotransmitters on
retinal arteries cleaned from all surrounding retinal tissue was
investigated. High concentrations of glutamate (1 mmol/L, n=6),
glycine (1 mmol/L, n=6), and GABA (0.1 mmol/L, n=2) had no
influence on the retinal arteries contracted with
prostaglandin F2 (30
µmol/L). Increasing concentrations of melatonin (10 nmol to 10
µmol/L, n=2) and dopamine (10 nmol to 0.1 mmol/L, n=6) also did
not relax the retinal arteries contracted with
prostaglandin F2 (30
µmol/L). Adenosine (10 µmol/L to 0.1 mmol/L, n=6)
induced a concentration-dependent relaxation of the retinal artery
contracted with prostaglandin F2
(30 µmol/L). At a concentration of 0.1 mmol/L,
adenosine induced a 52.7±12.9% (n=7) relaxation. In
additional experiments, the effect of adenosine (10
µmol/L to 0.1 mmol/L) was tested on rat main bronchi contracted
with carbachol (0.1 mmol/L). Adenosine did not relax the
rat main bronchi and at high concentrations even showed a tendency to
increase the contraction induced by carbachol (an increase of
18.7±3.4%, n=7).
Effect of 8-Phenyltheophylline on the Relaxation Induced by the
Retinal Tissue
To further exclude the involvement of adenosine in the
relaxing effect of the retinal tissue, 8-phenyltheophylline (10
µmol/L), an aspecific adenosine receptor blocker, was used.
8-phenyltheophylline (10 µmol/L) significantly
(P<0.05) blocked the adenosine-induced (10
µmol/L) relaxation of the retinal artery contracted with
prostaglandin F2 (30
µmol/L). This is a 4.2±1% relaxation in the presence of
8-phenyltheophylline compared with a 34.5±11.6% relaxation in the
absence of 8-phenyltheophylline (n=6). The presence of
8-phenyltheophylline, however, did not alter significantly the
relaxations induced by the application of a piece of the retinal
tissue.
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Discussion |
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The main finding of the present study is that retinal tissue exerts a marked inhibitory influence on the contractile tone of isolated retinal arteries. These observations cannot be ascribed to mechanical hindrance, because the contractions induced by K+ 120 mmol/L or by the standard activating solution (containing K+ 120 mmol/L, prostaglandin F2
, and
serotonin) are not significantly affected by the presence
of retinal tissue.
The adhering retinal tissue probably influences the vascular smooth
muscle cell through a diffusible chemical messenger. The initial
demonstration of the involvement of RRF required the apposition
of detached retinal tissue to a detector preparation. The detector
preparation in the initial experiments was a bovine retinal artery
cleaned of all retinal tissue and contracted with
prostaglandin F2. In later
experiments, the bovine retinal artery was replaced by other arteries
(eg, rat mesenteric and renal arteries) and another smooth muscle
preparation (rat main bronchi). These different preparations also
relaxed on application of retinal tissue, which suggests that RRF can
be considered a more general smooth muscle relaxant. Nevertheless, the
retinal artery seemed to be the most responsive of all preparations
tested.
Besides changing the detector preparation, the source of the RRF was
also changed. Retinas of pigs, dogs, and sheep also seemed to release a
relaxing factor, because application of these tissues also fully
relaxed the bovine retinal arteries contracted with
prostaglandin F2. No such
relaxation was seen when a similar-sized piece of bovine choroidal
tissue was placed in the proximity of the artery, which excluded the
involvement of mechanical disturbances in this kind of
experiment.
Retinal arteries contracted with K+ 120 mmol/L relaxed significantly less on application of retinal tissue. The presence of retinal tissue had only a moderate influence on K+ 120 mmol/L–induced contractions. The moderate relaxation of the artery contracted with K+ 120 mmol/L might be explained by a decreased release of the RRF from the depolarized retina or a decreased sensitivity of the depolarized arterial smooth muscle cells to the RRF.
The existence of this tentative RRF was confirmed by incubating bovine retinas in Krebs-Ringer bicarbonate solution and testing the effect of the incubation solution on isolated and cleaned retinal arteries. This solution elicited a relaxation of the retinal artery. The relaxing influence of the RRF-containing solution was not changed after extraction with hexane. Heating the solution to 70°C for 1 hour did not alter the relaxing effect. Treatment with trypsin also had no influence although the same treatment abolished the contraction induced by an endothelin-1–containing solution. The relaxations induced by the RRF-containing solution could not be attributed to changes in pH, because the pH changes alone were not sufficient to influence the retinal arterial tone. These data suggest that RRF is hydrophilic, thermostable, and is not a polypeptide or protein.
That the retinal arterial tone may be controlled by mediators released from the surrounding retinal tissue has already been suggested. Both NO3 and prostaglandin E14 have been proposed as possible factors. However, both can be excluded from being the RRF.
NO has a very short half-life and would be destroyed after 1 hour at
70°C. In addition, NO induces only a moderate relaxation of the
bovine retinal artery.8 L-NA (0.1 mmol/L), a
NO synthesis blocker, was unable to abolish the inhibitory
influence of the adhering retinal tissue although its presence enhanced
the prostaglandin F2–induced
contractions. We think that this is caused by an inhibition of the
basal release of NO from the endothelium, because L-NA
also significantly enhanced the sensitivity of the preparation without
the adhering retinal tissue. Spontaneous release of NO from the
endothelium is also suggested by the contraction
induced by methylene blue,9 a blocker of guanylyl
cyclase. Methylene blue had no effect on the relaxations induced by the
application of a piece of retinal tissue on a bovine retinal artery
contracted with prostaglandin F2.
This is another argument against the involvement of NO in the
retina-induced relaxations.
Indomethacin (10 µmol/L), a
cyclooxygenase inhibitor, did not alter
the concentration-response curves to prostaglandin
F2 when used on preparations with adhering
retinal tissue, which argues against the RRF being
prostaglandin E1 or another
cyclooxygenase product.
Because the RRF did not correspond with already proposed relaxing
factors from the retina, the potential role of other substances formed
by the retina was investigated. The neural retina consists of 6 types
of neurons: photoreceptor, horizontal, bipolar, amacrine,
interplexiform, and ganglion cells.10
Müller cells and retinal pigment epithelium are the two principal
nonneuronal cells of the retina.11 Astrocytes are
present in the nerve-fiber layer but are absent from the deeper
retinal laminae. Each of these cells releases various molecules, of
which some have vasoactive properties and thus are potential candidates
for being the RRF. The neurotransmitter released by rod-and-cone
photoreceptor cells is generally thought to be
glutamate.12 Glutamate also appears to be the
major neurotransmitter of the cone bipolar cells and the ganglion
cells.12 Glycine and GABA are also present in
various retinal neuronal cells.12 However,
glutamate, glycine, or GABA does not induce relaxation of isolated
retinal arteries contracted with prostaglandin
F2. Dopamine is the principal
catecholamine transmitter of vertebrate
retina.13 Dopamine, however, is also unable to
induce a relaxation of the bovine retinal artery contracted with
prostaglandin F2. Melatonin, which
is released by the retina, is also incapable of relaxing these
arteries. From the various peptides that are released, calcitonin
gene-related peptide (CGRP) was the most likely candidate. Prieto et
al14 reported that CGRP invariably induced a
slow-acting but potent relaxation of isolated bovine retinal arteries
contracted with prostaglandin F2.
As mentioned above, treatment of the incubation solution with trypsin
did not alter the relaxation induced by the RRF, which not only makes
the involvement of CGRP unlikely but also excludes other peptides, such
as vasoactive intestinal peptide, substance P, and somatostatin.
Acetylcholine is a neurotransmitter of the starburst amacrine
cells.12 Acetylcholine induces a moderate
endothelium-dependent relaxation of the isolated
retinal artery.7 9 Because the relaxation induced
by the retinal tissue occurs independently of the
endothelium, acetylcholine and other
endothelium-dependent vasodilators, (eg,
histamine)15 are unlikely to be the RRF.
Adenosine is a stable, hydrophilic molecule and is capable of
relaxing the isolated retinal artery contracted with
prostaglandin F2. However,
considerably high concentrations (10 µmol/L to 100
µmol/L) are needed to induce a pronounced relaxation of the retinal
artery. The presence of 8-phenyltheophylline (10 µmol/L), an
aspecific adenosine receptor blocker, did not alter the
relaxations induced by application of the retinal tissue.
Adenosine is unable to relax isolated rat main bronchi
contracted with carbachol, whereas the retinal tissue induces a
relaxation of this same preparation. Moreover, adenosine even
slightly contracts isolated rat main bronchi. These data thus exclude
adenosine from being the RRF.
The identity of the RRF thus remains to be established. Also, the cell type releasing RRF is as yet unknown. The presence of tetrodotoxin (10 µmol/L) did not significantly alter the inhibitory effect of the retinal tissue, which suggests that glial rather than neuronal cells release this factor. Also, the conditions of release of this elusive factor and the way RRF relaxes smooth muscle cells remain to be determined. The RRF seems to be released continuously from the isolated retina, because the inhibitory influence exerted by the retinal tissue does not diminish during prolonged experiments.
Continuously released RRF might have a physiological role in regulating retinal blood flow, because atrophy of the retina decreases the retinal vessel diameter. Retinitis pigmentosa, panretinal photocoagulation, and descending optic atrophy are pathologies with very different origins but with two characteristics in common; namely, retinal cell loss and attenuated retinal vessels (ie, decreased diameters). Attenuation of the retinal vessels is attributed to vascular remodeling caused by decreased metabolic demand, which is secondary to the loss of retinal cells.16 Vascular remodeling arises from sustained changes of blood flow.16 Changes in retinal blood flow should be seen before vascular remodeling. In early stages of retinitis pigmentosa, the changes in retinal blood flow can be detected before ophthalmoscopic signs and atrophy of the retinal vessels.17 Destruction of the outer retina may allow choroidal oxygen to diffuse to the inner retina, resulting in increased oxygen levels and autoregulatory vasoconstriction.18 19 This hypothesis may explain the narrow vessels in retinitis pigmentosa and panretinal photocoagulation. In descending optic atrophy, the primary lesion is located in the optic nerve and affects the inner retina, which makes this hypothesis, at least in this disease, less probable. Furthermore, the vessels in retinitis pigmentosa show a normal regulatory response to hyperoxia, suggesting that there is no hyperoxia in the inner retina of patients with retinitis pigmentosa.19 The mechanisms responsible for the changes in blood flow are obscure but might be explained by a lack of RRF. However, the physiological role of RRF in the retinal circulation can be established only after the RRF is identified and selective antagonists become available.
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Acknowledgments |
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This work was supported by grants from the University of Gent (grant 011D0196) and the National Fund for Scientific Research Belgium (grant 31.5056.96). The authors thank E. Behaeghe, C. Mabilde, and E. Tack for their excellent technical assistance.
Received March 23, 1998; accepted July 14, 1998.
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