Endothelial and Adrenergic Control

Core Messages

¥In the recent years it has become clear that the vasular endothelium plays a major role in the regulation of vascular tone. Thus, intact blood ßow regulation is largely dependent on a functioning vascular endothelium and endothelium derived substances play a substantial role in regulation vascular tone in health and diseases. This chapter will summarize our knowledge on the role of the vascular endothelium in blood ßow regulation.

Within the last 20 years, it has become clear that the endothelium plays a key role in maintaining vascular tone within all vascular beds of the human body. Since the Þrst in vitro study showing the obligatory role of the endothelium in mediating acetylcholine-induced vasodilatation in 1980 [66], the study of endothelium-derived

G. Garhšfer, M.D. ( )

Department of Clinical Pharmacology, Medical University of Vienna,

Waehringer Guertel 18-20, Vienna A-1090, Austria e-mail: gerhard.garhoefer@meduniwien.ac.at

L. Schmetterer, Ph.D.

Department of Clinical Pharmacology, Center of Medical Physics and Biomedical Engineering, Medical University of Vienna,

Waehringer Guertel 18-20, Vienna A-1090, Austria e-mail: leopold.schmetterer@meduniwien.ac.at

vasoactive substances has become an important research area. Nowadays, it is assumed that the endothelium produces a large variety of vasodilators and vasoconstrictors. Only if there is a balance between the production of endotheliumderived vasodilators and endothelium-derived vasoconstrictors is a vessel under normal physiological tone (Fig. 13.1). This also holds true for the eye, where numerous in vitro animal and human studies have proven the concept of endothelial control of blood ßow in the ocular vascular systems.

13.1Nitric Oxide

In their original experiment, Furchgott and Zawadzki proposed the existence of a potent endothelium-derived relaxing factor (EDRF), which was, however, not identiÞed. In the following years, it became clear that this EDRF is nitric oxide (NO), produced from the amino acid l-arginine, with cyclic GMP as a second messenger [105, 184, 185, 198].

Three distinct isoforms of nitric oxide synthase (NOS), which are products of different genes, are used to produce NO. NOS is required to oxidize the guanidine group of l-arginine in a process involving Þve electrons. The three isoforms of NOS are termed NOS1, NOS2, and NOS3. In older textbooks and articles, NOS1 was termed neuronal NOS, NOS2 was termed inducible NOS, and NOS3 was termed endothelial NOS. NOS1 and NOS3 were characterized as constitutive and

Fig. 13.1 The endothelium plays a key role in maintaining basal vascular tone. A balance between the production of endothelium-derived vasodilators and vasoconstrictors is required

Fig. 13.2 Biosynthesis of nitric oxide. For details please see text

NOS2 as inducible. However, the notion that all NOS isoforms are regulated dynamically required a new nomenclature. When NOS1 and NOS3 are activated, NO is produced via the calcium/ calmodulin complex, when NOS2 is activated NO production is independent of calcium. Large amounts of NO via NOS-2 are produced in the presence of immunological and inßammatory stimuli (Fig. 13.2). The NOS gene family shares similar compositions with each other: All have two domains: N-terminal half of heme-oxygenase domain, with tetrahydrobiopterin, hemeand argi- nine-binding sites, and C-terminal half of P-450 reductase domain with the positions of recognition sites for NADPH, as well as for ßavin mononucleotide (FMN) and ßavin adenosine dinucleotide (FAD). The NOS1, NOS 2, and NOS3 genes have

been mapped on the chromosomes 12q24, 17q11.2, and 7q35-q36, respectively.

Much of our knowledge of the role of NO in the control of blood ßow is based on experiments using NOS inhibitors. NOS inhibition can be achieved by L-arginine analogs such as NG- monomethyl-L-arginine (L-NMMA), NG-nitro- L-arginine methyl ester (L-NAME) and NG-nitro-L-arginine (L-NA). They are competitive inhibitors of NOS and not speciÞc for any of the isoforms. Short-term effects of L-NMMA, L-NAME, or L-NA can be reversed by excess doses of l-arginine. To study the role of the different isoforms of NO, a number of speciÞc inhibitors were also employed. 7-Nitroindazole (7-NINA) is the most widely used inhibitor of NOS1, but at higher dosages, it also inhibits NOS3 in cerebral arteries [11]. In earlier studies, aminoguanidine was used as a speciÞc inhibitor of NOS2, although this drug exerts a variety of other pharmacological actions including inhibition of advanced glycation end products. Inhibitors of NOS are also produced endogenously. Among the identiÞed endogenous inhibitors, asymmetric dimethylarginine (ADMA) appears to be the most important. Plasma levels of ADMA are increased in a variety of vascular diseases including end-stage renal disease, hypertension, hypercholesterolemia, atherosclerosis, and diabetes [21]. In these diseases, it appears to contribute to endothelial dysfunction. Whether endogenous inhibitors also play a role in ocular vascular disease is not established.

Due to its small size and its speciÞc properties NO is an ubiquitous messenger throughout the human body. NO is soluble in tissues and can easily diffuse across membranes like other small molecule gases such as O2, CO2, or CO. Nitric oxide has a very short half life of only a few seconds. Accordingly, NO production is regulated at the level of biosynthesis because it cannot be stored in vivo. Also related to the small size of the molecule is its ability to diffuse over large distances up to several hundreds of micrometers. Hence, a single NO molecule can affect numerous cells adjacent to the location of its production despite the short half-life.

Nitric oxide has a key role in the maintenance of vascular tone in humans [246] and is a major regulator of systemic blood pressure [99]. NO also exerts a variety of other physiological and pathophysiological effects, which are not directly related to the control of vascular tone and blood ßow. In the eye, this includes processes related to signal transduction, neurotransmission, neurodegeneration, and oxidative stress. A more detailed discussion of these effects is, however, beyond the scope of this chapter.

All three types of NOS were identiÞed in the eye. As in other tissues, staining of cells for NADPH diaphorase activity has been widely used to determine the regional distribution of NOS because of its high sensitivity. The different isoforms of NOS can, however, not be distinguished. Immuncytochemistry and ßuorescence methods overcome this problem, employing speciÞc monoclonal and polyclonal antibodies. Molecular biology based methods including measurement of mRNA expression by reverse transcription polymerase chain reaction, measurement of protein expression using SDS-polyacrylamide gel electrophoresis and western blotting, or in situ hybridization were also used to characterize NOS in ocular tissues.

In the retina, NOS was found in amacrine and ganglion cells [37, 43, 128] retinal pigment epithelium [70, 71], MŸller cells [127], photoreceptors and nerve Þbers in the inner and outer plexiform layers [256]. As in other vascular beds, NOS is also present in the endothelium of retinal vessels [155, 240] and in retinal capillary endothelial cells and pericytes [31]. As expected, NOS3

was also identiÞed in the endothelium of optic nerve head and choroidal blood vessels [35, 61, 155, 165].

Under physiological conditions, NO is continuously produced in the endothelium to ensure that vessels are under constant vasodilator tone. Numerous in vitro, animal, and human experiments indicate that this is also the case in the ocular vasculature. In isolated porcine ophthalmic and ciliary arteries, inhibition of NOS with L-NMMA induces dose-dependent contraction [83, 260]. NO also relaxes the contractile tone of retinal bovine pericytes [85]. Given that the ratio of pericytes to endothelial cells in the retinal microvasculature is extremely high (approximately 1:1), this indicates a major role for NO also in the smaller retinal vessels, where most of the resistance to ßow occurs. In the isolated perfused porcine eye, NOS inhibition decreases ocular blood ßow and increases vascular resistance [152].

In animal and human studies, evidence for a reduction in ocular blood ßow after NOS inhibition has been accumulated for all vascular beds of the eye (Fig. 13.3). Using a variety of different techniques, unequivocal data have been presented for the choroid, the optic nerve head, the ciliary body, and the iris [47, 73, 118, 130, 132, 142, 148, 208, 232]. Data have also been generated that the effects of the NOS inhibitor L-NMMA in the human choroid is reversible by administration of high-dose l-arginine indicating the vasoconstrictor effect is speciÞc to the NO pathway. In contrast, some [45, 49, 50, 52, 211], but not all studies [47, 182, 183, 238] indicated that NOS inhibition also reduces retinal blood ßow. One human study reported a dose-dependent vasoconstrictor effect of L-NMMA on retinal arterial and arterial vessel diameters after systemic administration [52]. The negative results obtained were all collected with the radioactive microsphere technique and are most likely related to the limitations of this technique in assessing retinal blood ßow [204]. Only few data are available for speciÞc NOS inhibitors. In rats, L-NAME, but not 7-NINA, increased blood pressure. Both drugs, however, decreased ocular blood ßow, suggesting a role for NOS1 in the maintenance of basal vascular tone [117]. A variety of other studies reported, however, that

Fig. 13.3 Effect of the NO synthase inhibitor L-NMMA on ocular blood ßow parameters in young healthy volunteers. Percent change in fundus pulsation amplitude (FPA); blood ßow in the choroid, FLOW (Choroid); and blood ßow in the optic nerve head, FLOW (ONH) after administration of L-NMMA (hatched bars:

3 mg/kg over 5 minutes followed by 30 µg/kg per minute over 55 minutes; solid bars: 6 mg/kg over 5 minutes followed by 60 µg/kg per minute over 55 minutes) or placebo (hollow bars). Data are presented as mean ± SD (n = 12). Asterisks indicate signiÞcant effects of L-NMMA versus baseline as calculated from the absolute values

7-NINA does not affect retinal or choroidal blood ßow under physiological conditions in the cat [169], rat [132] or pigeon [263].

Nitric oxide is also a key candidate for regulating ocular blood ßow during changes in perfusion pressure. Obviously, vascular resistance decreases when blood ßow is kept constant during a decrease in perfusion pressure and increases during an increase in perfusion pressure. According to the work of [118] employing laser Doppler ßowmetry in the rabbit, NO is a key candidate to control vascular tone during changes in perfusion pressure (Fig. 13.4). On the other hand, Koss [133] failed

to detect an effect of a NO synthase inhibitor on the choroidal pressure-ßow relationship in the cat during changes in perfusion pressure. In humans, NO synthase inhibition alters the ocular perfusion pressure/choroidal ßow relationship during an increase in perfusion pressure induced by isometric exercise [143] (Fig. 13.5). Whether this truly indicates a role for NO in human choroidal autoregulation is, however, unclear because the neural input to the choroid cannot be eliminated in a human experiment. NOS inhibition also modulated the response of ONH blood ßow assessed with hydrogen clearance to an increase in IOP

1,000

Fig. 13.4 Effect of the nitric oxide synthase inhibitor L-NAME on choroidal pressure/ßow relationships in the rabbit. The left graph show tracings as obtained in a single

rabbit. The right graph shows means ± SDs as obtained from a group of animals

Fig. 13.5 Choroidal PressureÐßow relationship using the categorized ocular perfusion pressue (OPP) and choroidal blood ßow (CBF) values during isometric exercise. Relative data were sorted into groups of nine values each, according to ascending OPPs. The Þrst period of squatting was performed without drug administration (baseline; open down triangles). The second squatting period was performed during administration of placebo, L-NMMA, or PE (solid up triangles). The means and the lower limits of the 95% conÞdence intervals are shown (n = 12). The dotted line indicates 100% of baseline

CBF

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CBF

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130 Phenylephrine

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130 L-NMMA

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OPP (% of baseline)

% of baseline

[177]. Data that NO is involved in retinal and choroidal autoregulation have also been generated in newborn animals [89, 109]. Such data are of major relevance for the understanding of diseases such as retinopathy of permaturity, but most likely cannot be applied to adults.

Nitric oxide has also been shown to play a major role in mediating the ocular vasodilator effects to a variety of agonists as well as in response to changes in perfusion pressure. As in many other vascular beds, removal of the vascular endothelium in isolated bovine retinal as well as in human retinal and ophthalmic arteries is associated with a signiÞcant reduction of acetylcholine-induced relaxation [16, 83, 260]. In canine ophthalmic and retinal arteries, relaxations to acetylcholine were, however, endothe- lium-independent indicating signiÞcant species differences [240, 248]. Data for NO-dependent relaxation of bradykinin are more consistent and were collected for isolated ocular vessels from different species [83, 84, 260, 266] and in the isolated perfused porcine eye [152]. The list of agonists that appear to induce vasodilatation via NO-dependent mechanisms in the ocular vasculature is long and includes substance P [123], low-dose arginine [175], and a selective antidiuretic desmopressin [241], dipyridamole [154]. The evidence for histamineand insulin-induced vasodilatation as well as the NO dependence of these effects will be discussed later in this chapter.

Some, but not all, experiments indicate that NO also interacts with the changes in ocular blood ßow during changes in pO2. In retinal pericytes, hypoxia ampliÞed relaxations to the NO donor sodium nitroprusside, but hypoxia alone did not inßuence pericyte basal tone [86]. In vivo NOS inhibition did not affect choroidal blood ßow after hyperoxia in humans [206] but reduced the retinal blood ßow response to hypoxia in the cat [162], indicating that NO contributes to hypoxia-induced vasodilatation. As in the brain, the vasodilator response to CO2 is signiÞcantly reduced by NO-synthase inhibitors in the retina [203]andchoroid[206]indicatingNO-dependence of hypercapnia-induced effects. In the cat retina, this effect can also be achieved with 7-NINA, suggesting a role for neuronal NOS [203].

A major role for NO was discovered for vasodilator effects after neural stimulation. This topic is

discussed in some detail in another chapter of this book. In isolated dog ophthalmic arteries, relaxation by nicotine or electrical neural stimulation was abolished by NO-synthase inhibition and restored by adding high-dose l-arginine [239]. This is in keeping with data showing NO release from the autonomic system in the posterior ciliary arteries [225, 253]. The presence of nerves releasing NO was also shown for choroidal arterioles by measuring membrane potentials with the microelectrode technique [97]. This is in good agreement with a variety of in vivo studies. In the primate, electrical stimulation of the pterygopalatine or geniculate ganglion dilates the ophthalmic artery. This effect was abolished by L-NA and restored by high-dose l-arginine, proving NO-dependence [10]. Neurally derived NO also plays a major role in the choroidal blood ßow increase caused by stimulation of the Edinger-Westphal nucleus in pigeons [263] or facial nerve stimulation in the cat [169].

Flicker light stimulation in the miniature pig increases NO concentrations in preretinal vitreous humor [49]. This is in keeping with data in cats, showing increased NO levels in the vitreous humor near the optic nerve head [25]. In addition, these experiments revealed that the ßickerinduced increase in ONH blood ßow measured with laser Doppler ßowmetry is reduced but not abolished by NOS inhibition, which is in good agreement with microsphere experiments [130]. Data are also available for humans, indicating that L-NMMA blunts the retinal vasodilator response to ßicker stimulation [51]. Nitric oxide synthase inhibition also modulates the response of human choroidal blood ßow to a light/dark transition [103]. The physiological relevance of the blood ßow decrease in the choroid during a light/dark transition is largely unknown. It appears, however, that this is a neurally mediated effect [65] as it is in the pigeon [60].

The role of NO in the development of ocular perfusion abnormalities in vascular diseases of the eye is discussed in other articles of this book. Alterations in the l-arginine/NO system affecting blood ßow regulation have, however, been proven in patients with diabetes [205] and glaucoma [193]. This makes the l-arginine/NO systemamajorcandidatefortherapeuticinterventions in ocular vascular disease. Indeed, high-dose