Novel Vasodilator Released by Retinal Tissue

Correspondence to Gabor Kaley, PhD, Department of Physiology, New York Medical College, Valhalla, NY 10595. E-mail gabor_kaley{at}nymc.edu

Key Words: retina-derived relaxing factor • vasodilation • vascular regulation.

Aseries of classical experiments within the past 20 years, all of which used the incubation of donor tissues with detector blood vessels, have yielded a rich trove of vasoactive principles, culminating in the discovery of the physiologically relevant vasodilator mediators prostacyclin¹ and nitric oxide (NO).² In a like manner, Delaey and Van de Voorde³ report in this issue of Circulation Research, evidence for the existence of yet another tissue-derived vasoactive factor that may have broad implications for vascular research.

The salient finding of the study presented by Delaey and Van de Voorde³ is that adherent retinal tissue, but not a similar-sized piece of choroidal tissue, of a variety of species exerts an inhibitory effect on the contractile tone of isolated, endothelium-intact, or denuded bovine retinal arteries. Moreover, contractions induced by a variety of agents, ie, U-46619, serotonin, and endothelin-1, were also blocked in the presence of retinal tissue. Collectively, these experiments can be interpreted to mean that retinal tissue releases a diffusible vasorelaxant that not only relaxes the retinal artery but also other smooth muscle preparations such as rat mesenteric and renal arteries and rat main bronchi, as demonstrated by Delaey and Van de Voorde. Also shown is that incubation of retinal tissue yields a solution that relaxes isolated bovine retinal arteries, further confirming the involvement of a diffusible chemical substance.

Previous studies by other investigators have already shown that prostaglandin (PG) E₁ is capable of causing dilation of pig retinal arterioles and that administration of indomethacin into the retinal circulation reversibly inhibits retinal vasodilation induced by hypercapnia.⁴ In addition, in a more recent study, it was reported that intact retinal tissue of the miniature pig releases NO, as measured by an NO microprobe, most likely from glial cells and not vascular endothelial cells. These in vivo studies also showed that microinjections of nitro-L-arginine induced significant reversible constriction of retinal arterioles, suggesting that NO controls basal arteriolar tone in the inner retina.⁵ It is also likely that a close interaction between PGs and NO exists, because retinal vasoconstriction induced by inhibition of either cyclooxygenase⁶ or NO synthase³ is not maintained, suggesting that when either system is prevented from acting, the other can readily compensate to modulate retinal vascular tone.

In an attempt to demonstrate the existence of a novel retina-derived dilator agent, Delaey and Van de Voorde³ needed to demonstrate first that the characteristics of this agent do not correspond to those of NO or PGs or other known vasoactive mediators that might also be formed within the retina. The authors advance cogent arguments to suggest that the release of NO from retinal tissue is not responsible for these observations. NO induced only moderate relaxation of the isolated retinal artery, whereas application of a piece of retinal tissue caused complete relaxation. Moreover, although nitro-L-arginine enhanced the contraction induced by PGF₂ of the vessel with adhering retinal tissue, most likely due to inhibition of the basal release of NO from the endothelium, it did not abolish the inhibitory influence of retinal tissue on the prostanoid-induced contraction. Methylene blue, a blocker of guanylate cyclase, also failed to affect relaxation of the retinal artery, excluding the participation of NO in the response. In a similar fashion, the participation of PGs was excluded by the administration of indomethacin and adenosine by the use of 8-phenyltheophylline. The vasoactivity of the retina-derived relaxant, as measured by inhibition of the contraction induced by PGF₂ of retinal arteries with or without adherent retinal tissue, was also proven to be unaffected by the presence of tetrodotoxin. In addition, when retinal tissue was incubated for 6 hours and the incubation solution, to be used in bioassay experiments, was extracted with hexane and then, treated with trypsin or heated to 70°C, the relaxant properties of the solution still remained unaffected. These latter experiments provide additional evidence that the vasoactive factor is not NO, because NO would be destroyed after incubation for 1 hour at 70°C, and that it is thermostable and not a protein nor a polypeptide, excluding the possibility that neuropeptides, such as calcitonin gene-related peptide, melatonin, vasoactive intestinal peptide, substance P, or somatostatin could be responsible for the relaxation of vascular tissue. Other neurotransmitters, such as glutamate or -aminobutyric acid, were also excluded from being identified as the retina-derived relaxant factor, because their administration, even in high concentrations, had no influence on the precontracted retinal artery. Finally, Delaey and Van de Voorde provide convincing evidence that pH changes of the solution in which the retina was incubated could not account for the vasoactivity. This issue is of pertinence, because in a previous study, it was demonstrated that hypoxia-induced dilation of retinal arterioles is mimicked by injections of lactate.⁶

What then is the nature of this powerful vasoactive principle released by mammalian retinas? Assuming that the reported observations are not due to an unexplained artifact, it is worthwhile to speculate what other known vasoactive agent(s) might account for the activity of this continuously released vasorelaxant from the retina. Throughout the experiments of Delaey and Van de Voorde,³ retinal tissue and detector vessels were bathed in a solution containing 95% O₂, a condition conducive for the generation of radical O₂ species. Although a superoxide radical is unlikely to survive a 6-hour incubation, one cannot rule out the possibility that hydrogen peroxide may well do so, and could under favorable conditions account for the vasodilator property of the incubation solution,⁷ by causing smooth muscle relaxation directly, without the involvement of the generation of PGs or the activation of guanylate cyclase. Administration of superoxide dismutase and/or catalase could exclude or confirm the involvement of hydrogen peroxide. An alternative explanation may be that a high enough concentration of CO, a gas more stable than NO, may be generated by retinal tissue,⁸ via activation of heme oxygenase-2 or induction of heme oxygenase-1, to affect the tone of detector blood vessels directly, independent of other mediators or the synthesis of radical oxygen species. A more likely scenario, however, is one that implicates lipid mediators in the profound vascular effect exerted by retinal tissue.

Cytochrome P450 epoxygenase metabolites of arachidonic acid, namely epoxyeicosatrienoic acids (EETs), are potent vasodilators that may account for the activity of the not yet completely identified endothelium-derived hyperpolarizing factor.⁹ The four regioisomers 5,6-, 8,9-, 11,12-, and 14,15-EET have been shown to dilate blood vessels by activating large conductance Ca²⁺-activated K⁺ channels. Because endothelial cells of a variety of tissues have already been shown to produce and store EETs, it is plausible to assume that they can also be released from endothelial cells of retinal vessels. Recent reports indicate that EETs are synthesized by astrocytes on exposure to glutamate and may be responsible for the vasodilation and increase in cerebral blood flow in response to neuronal activity.¹⁰ In this context, astrocytes, present in the nerve fiber layer of the retina, may release vasoactive EETs via stimulation of P450 epoxygenase in response to glutamate receptor activation. This possibility is not contradicted by the experiments of Delaey and Van de Voorde,³ in which glutamate failed to dilate the retinal artery, because in this part of their studies no retinal tissue was attached to the vessel. Furthermore, epoxides are poorly extracted by hexane, for which a more polar solvent such as ethyl acetate would be required, and appear to be stable at 70°C. It is also of interest that EETs are rapidly converted by cellular epoxide hydrolases into their respective diol products, dihydroxyeicosatrienoic acids, which have recently been shown to be capable of modulating vascular function in a manner similar to their parent EETs.¹¹ Finally, retinal tissue is also quite rich in phospholipids containing docosahexaenoic acid, which could, via the action of phospholipase A₂ or spontaneous hydrolysis, be converted into vasoactive compounds.

Quite aside from the necessity of identifying the retina-derived relaxing agent, a number of important issues ought to be addressed. On the basis of the in vitro experiments reported, the retina releases this agent continuously without requiring a specific stimulus. If the findings have physiological relevance and implications for in vivo circumstances, one would have to ascertain that the vasoactive principle obtained is not simply a result of the artifactual conditions (eg, dissection of the retina or 95% O₂) attendant with the in vitro experiments. It would also be important to demonstrate that the retinal relaxing factor does not just "leak out" of the retina, but that it is released in a regulated manner in response to certain stimuli. Regardless of whether the retinal relaxing factor originates in retinal blood vessels or tissues, it would be of interest to learn if it can affect the retinal microcirculation, which is composed of vessels that are significantly more responsible than the retinal artery for the regulation of blood flow. It would also be fascinating to learn what the mechanism or mechanisms of action of the retina-derived factor might be. Does it affect K⁺ channels or Ca²⁺ movement in vascular and nonvascular smooth muscle? And is it perhaps synthesized by tissues and organs other than the retina and thus will be proven to be of more global significance in the regulation of blood vessel function? These are some of the many intriguing questions that remain to be answered. Clearly, there are also some clinically relevant issues emerging. It has long been known that regardless of origin, pathological conditions characterized by loss of retinal cells are accompanied by reductions in the diameter of retinal vessels and retinal blood flow. Whereas remodeling of the vessels is likely to be the result of the reduction in blood flow secondary to the loss of retinal tissue and/or decrease in retinal metabolism, the hypothesis that Delaey and Van de Voorde³ put forth, that this may be related to a diminished synthesis of the retina-derived relaxing factor, is quite consistent with their provocative findings and merits consideration.

Footnotes

The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

References

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