Arq. NeuroPsiquiatr. vol.46 número1

THE ROLE OF THE ENDOTHELIAL DEPENDENT

RELAXING FACTOR IN THE REGULATION

OF CEREBRAL CIRCULATION

ANTONIO A. F. DE SALLES *

S U M M A R Y — I t has r e c e n t l y b e e n d e m o n s t r a t e d t h a t v e s s e l d i l a t i o n i n d u c e d b y several p h y s i o l o g i c a l a g e n t s is d e p e n d e n t on an i n t a c t v a s c u l a r e n d o t h e l i u m . I n o r d e r t o e x p l a i n this e n d o t h e l i u m d e p e n d e n c e , i t has b e e n h y p o t h e s i z e d t h a t a still u n k n o w n chemical substance, g e n e r i c a l l y n a m e d E n d o t h e l i u m D e p e n d e n t R e l a x i n g F a c t o r ( E D R F ) i s necessary f o r p h y s i o l o g i c a l v a s o d i l a t i o n . T h e r o l e o f this E D R F in the c e r e b r o v a s c u l a r p h y s i o l o g y is not y e t w e l l u n d e r s t o o d . I n this a r t i c l e t h e c e r e b r o v a s c u l a r p h y s i o l o g i c a l c o n t r o l is r e v i e w e d in r e l a t i o n s h i p w i t h p o s s i b l e E D R F actions. T h e i m p o r t a n c e o f e n d o t h e l i a l l e s i o n s i n the c e r e b r o v a s c u l a r r e s p o n s e s is discussed.

Papel do fator relaxante endotelial na circulação sanguínea cerebral.

R E S U M O — R e c e n t e m e n t e f o i d e s c o b e r t o q u e o e n d o t é l i o v a s c u l a r d e v e estar i n t a c t o para que v a s o s s a n g u í n e o s d i l a t e m q u a n d o e s t i m u l a d o s p o r a g e n t e s f i s i o l ó g i c o s . A c r e d i t a - s e q u e u m a substância q u í m i c a a i n d a desconhecida, g e n e r i c a m e n t e c h a m a d a F a t o r R e l a x a n t e E n d o -telial ( F R E ) , p r o d u z i d a p e l o e n d o t é l i o , é i n d i s p e n s á v e l p a r a o r e l a x a m e n t o vascular. N e s t e t r a b a l h o é r e v i s t a a f i s i o l o g i a c i r c u l a t ó r i a c e r e b r a l e p o s s í v e i s a ç õ e s d o F R E . D i s c u t e - s e t a m b é m a i m p o r t â n c i a d e l e s õ e s e n d o t e l i a i s e m r e l a ç ã o a o c o n t r o l e c i r c u l a t ó r i o c e r e b r a l .

Arteries relax in response to various vasodilators: acetylcholine ( A c h ) , sub-stance P, adenosine triphosphate ( A T P ) , adenosine diphosphate ( A D P ) , ionophore A23187, eleidoisin, thrombin, arachidonic acid, histamine, hydralazine, platelet acti-vating factor ( P A F ) , and bradykinin ( B K N ) . H o w e v e r , this will only occur if the endothelium is present 4,19,43. One hypothesis which would explain this phenomenon is that these compounds stimulate the endothelial cells to release a vasodilator sub-stance which in turn relaxes the underlying smooth muscle 20. T h e endothelium depen-dence of vessel dilation w a s first described for the dilator effect of A c h in isolated rabbit thoracic aorta 20, T h i s phenomenon has been extensively studied in several vascular beds of various species. Production and release of an endothelium relaxant factor is thought to be primarily a property of arterial endothelium, although it has been documented in some venous preparations 40,43. Endothelium dependent relaxing factor ( E D R F ) appears to be unrelated to the arterial segment and mammalian species studied 43,44. Ach-induced relaxation of large cerebral arteries of cats and rabbits is also dependent upon an intact endothelium 35,54. T h e r e is not, to date, an agreement as to the chemical nature of the E D R F 19,22,31,44. p r i o r to Furchgott's finding of the o b l i g a t o r y role of endothelial cells for relaxation of arteries by A c h 20, it w a s known that cultured endothelial cells could produce prostaglandins ( P G ) , including pros-tacyclin (PGl2), a potent vasodilator of vascular beds and a relaxant of many

arteries 41. Extensive research has been carried out regarding the possibility of a P G derivative as the actual E D R F 16,21,22,31,39,43,53,54. Since the relaxing effect of mus-carinic agonists on many arteries can be reversed by inhibitors of phospholipase A2

D e p a r t m e n t o f P h y s i o l o g y , M e d i c a l C o l l e g e o f V i r g i n i a : * M . D . , P h . D .

and of lipoxygenase, but not of c y c l o o x y g e n a s e , it has been proposed that E D R F might be an unstable oxidation product of the arachidonic acid formed via the lipoxygenase pathway 3,16. H o w e v e r , in a recent study using aortic preparation from rabbit, alone and in cascade experiments with isolated perfused coronary preparations, Griffith et a l .2 2

demonstrated that E D R F is not a lipoxygenase derivative or a free radical. T h e y suggested that E D R F is an unstable compound with a carbonyl group at or near its active site. M o r e o v e r , by changing the length of the intervening tubing between aortic and coronary preparations, they w e r e able to calculate the half-life of

E D R F as 6.3 ± 0.6 sec. T h e s e authors concluded that E D R F is probably an alde-hyde, ketone or lactone.

T h e regulation of cerebrovascular tonus is closely related to the brain tissue metabolic demands 34. T h e adequacy of cerebral blood flow ( C B F ) is maintained despite large variations in systemic parameters such as arterial blood pressure, blood viscosity, and cardiac output 5,27,34,42. T h e mechanism of this strict control is still poorly understood 5,28. in pathological situations where cerebrovascular endothelium is affected ( e . g . , arterial hypertension, head i n j u r y ) , the coupling of C B F to brain metabolism is disturbed 21,30,31,49,50,60. Thus, it seems likely that, in addition to its barrier role, the endothelial layer may play an active role in modulating cerebro-vascular reactivity in normal and abnormal circumstances 54. T h e f o l l o w i n g sections will discuss the neurogenic, chemical and metabolic regulation of C B F and their possible relationship to the endothelium dependent relaxing factor.

E D R F A N D T H E C E R E B R O V A S C U L A R N E U R O G E N I C C O N T R O L .

T w o distinct types of nerve ending can be distinguished in the w a l l s of pial vessels by the use of ultrastructural techniques. One type is derived from fibers that originate in the superior cervical ganglion. It contains dense granular vesicles and is assumed to be adrenergic. T h e other, observed even in animals pretreated with 6-hydroxydopamine or 5-hydroxydopamine, contains agranular vesicles and is generally assumed to be cholinergic 36. Corresponding with this ultrastructural evidence, functional studies of cerebral vessels in vitro have distinguished t w o types of response to transmural nerve stimulation ( T N S ) . Stimulation of sympathetic nerves produces a contractile response in the pial vessels of rabbit, d o g , sheep and monkey- After sympathetic denervation or treatment with guanethidine, nerve stimu-lation produces dilator response in pial vessels of several species. T h i s dilator response is presumably cholinergically mediated 7. In addition to the sympathetic and parasympathetic innervation, other systems can be visualized by immunohistochemical techniques. T h e s e fibers contain peptides such as vasoactive intestinal polypeptide

( V I P ) 3 3 and substance P 1 0 , or monoamines such as serotonin 62

.

Adrenergic innervation — A d r e n e r g i c fibers originating in the ipsilateral

supe-rior cervical g a n g l i o n innervate both pial and intracerebral vessels 14,28. in support of this is the fact that all adrenergic fibers disappear after superior cervical gan-glionectomy 9.36. A l s o , it has been shown that adrenergic fibers originating in the locus coeruleus may innervate capillaries 2

4 . H o w e v e r , it is questionable that

adre-nergic fibers originating in the brain stem truly innervate cerebral vessels, since in many cases the nerve fibers are separated from vessels by a thick basement m e m b r a n e2 8

. W i t h catecholamine fluorescence techniques the density and distri-bution of adrenergic fibers have been shown to be v e r y similar in pial arteries of several animals and of humans H /?—, ax— and a2- a d r e n e r g i c receptors have been

identified and characterized in experiments using pharmacological models 9,14.55. in the peripheral vessels generally a receptors mediante vasoconstriction while ¡3 receptors mediate vasodilation. T h i s appears to be also true in the brain vasculature 5,54,55. In the cat, stimulation of the cervical sympathetic nerves produces pial arteriolar vasoconstriction, as well as a mild decrease in C B F 2

8 . T h e magnitude of C B F

decrease caused by sympathetic stimulation depends on the species s t u d i e d7

. A l s o , no significant constrictor response w a s detectable in smaller arterioles ( < 1 0 0 u m ) 6 i . T h e physiological significance of the pial arteries' constriction by sympathetic nerve stimulation is not c l e a r7

respon-siveness of different vessels in one species and in vessels from other species 14.17. For example, norepinephrine-induced contraction of human and monkey cerebral arteries are mediated by ax adrenoreceptors, while in d o g s this effect appears to be

mediated by <*2 adrenoreceptors 55. Cerebral blood vessels responses to sympathetic

nerve stimulation are relatively weak, clearly attributable to the v e r y l o w responsi-veness of their a-adrenergic receptors to norepinephrine 28-55. T h i s has led some investigators to suggest that cerebrovascular a-adrenoreceptors are different from those found in peripheral arteries 8.14. Stimulation of /?-adrenergic receptors causes cerebrovascular dilation. Evidence derived from a l a r g e variety of vascular preparations seems to exclude any dependence of this ^-adrenergic dilation on the endothelium 54,55. Of interest, however, is the fact that lack of endothelial cells potentiates the v a s o -constriction effect of adrenergic agents. It is possible that viable endothelial cells could constantly release a specific factor acting on the adrenergic receptor-mediated mechanism of constriction 54. T h i s finding may be of considerable importance in understanding pathophysiological phenomena involving alterations of endothelial func-tion, whether by mechanical lesion 28 or chemical lesion 50. T h i s endothelial role in the modulation of adrenergic cerebral vasoconstriction is likely to be related to development of cerebral vasospasm following traumatic brain injury or subarachnoid hemorrhage 26,47. A l s o , it appears to be related to the vasoparalysis that follows such pathological states 31,50. Although the lack of E D R F may not be the only cause of the abnormal vascular behavior in these pathological states, it seems likely that the endothelial cells may play an active role in modulating cerebrovascular responses to adrenergic stimuli 54.

Cholinergic innervation — Pial arteries are supplied with a well developed

plexus of nonsympathetic cholinergic nerves. T h e close association of their endings with the smooth muscle cells at the surface of the media layer fulfills accepted ultrastructural criteria for a functioning neurovascular relationship 9,28. T h e presence of muscarine receptors in intracerebral microvessels has been more controversial. It is believed that the cholinergic innervation of the intracerebral vessels, in the case of small arterioles and capillaries, is of intracerebral origin since there is no peri-vascular space at this peri-vascular level. T h i s system w o u l d parallel the adrenergic system arising from the locus coeruleusis. Although there is no direct evidence that such a system exists 14,28) indirect evidence supports the presence of a cholinergic

control of the cerebral vasculature. For example, administration of acetylcholine intravascularly dilates pial arterioles and increases cerebral blood flow. Carbachol applied locally dilates pial arteries 50. T h e autoregulatory cerebral vasodilation following a decrease in systemic arterial blood pressure is blocked by atropine 36. T h e currently observed presence of muscarinic binding sites and choline acetyltrans-ferase ( C h A T ) activity in brain capillary fractions also suggests that there may be a cholinergic innervation of brain capillaries 13,15,51. H o w e v e r , this C h A T activity could be due to contamination by brain tissue instead of C h A T activity in the actual vessels' w a l l . Thus, the C h A T data alone is not sufficient to support the idea of cholinergic control of intracerebral arterioles and c a p i l l a r i e s1 4

. Parasympatho-mimetic compounds produce either a relaxation or a contraction of the cerebral vasculature. T h e relaxation occurs at l o w doses, and the response is inhibited in a competitive manner by atropine- T h e contraction occurs with high doses and appears to be mediated by muscarinic receptors 9. W h e n cholinergic fibers are activated by transmural electrical stimulation of cerebral arteries from cats, they induce significant vasodilation 7,28. T h i s vasodilation is not blocked by atropine 36. T h u s , besides its direct postjunctional vasodilatory action on muscarinic receptors in the vascular smooth muscle, cholinergic innervation can also promote pial vasodilation indirectly, through an inhibition of the norepinephrine release v i a the nicotinic receptors present on the perivascular sympathetic f i b e r s9

. Recently, in vitro studies have shown that Ach-induced relaxation of l a r g e cerebral arteries of cats and rabbits is dependent upon intact endothelium 35,54} suggesting that E D R F may play a role on the

of collagenase-treated cerebral vessel preparation strongly suggests that muscarinic receptors are located in the membrane of capillary endothelial cells. M o r e muscarinic binding sites w e r e found in microvessel fractions than in capillaries, suggesting that in the former preparation, [3/ / ] Q N B probably binds to smooth muscle cells as well

as to the endothelium13. T n u s , Ach- induced relaxation of l a r g e cerebral vessels may be not dependent only upon E D R F . T h e presence of A c h receptors in the endothelial cells membrane suggests that E D R F play a role in the intracerebral vessel tonus control and may participate in the capillary blood flow regulation. Lesion of the endothelium of pial microvessels in vivo48 by exposure of the vessels to filtered light from a mercury lamp, in presence of intravascular sodium fluorescein suppressed the Ach dependent relaxation. M o r e o v e r , after endothelial lesion the microvessels constricted in presence of A c h . T h e s e findings support the importance of E D R F on the control of cerebrosvascular circulation in vivo so.

Peptidergic innervation — Research on the innervation of the cerebral blood

vessels have disclosed peptidecontaining nerve fibers running along the pial blood vessels ( e . g . , substance P , V I P , pancreatic polypeptide, gastrin-releasing polypeptide, neurotensin, serotonin, neuropeptide Y , and somatostatin)37.38. T h e origin of these fibers is still unknown. T h e y may be derived from the parasympathetic ganglions around the brain, but the question remains unresolved. Other peptides such as cholecystokinin and proopiomelanocortin related peptides have had their action tested in the pial arterioles. T h e s e studies demonstrated that such peptides are not important in cerebrovascular control when applied in physiological concentrations 38,56. M o r e is known about the action of V I P and substance P on cerebral vasculature. V I P - c o n t a i n i n g fibers reveal a spiral pattern similar to the muscle cell pattern. VIP-immunoreactive nerve terminals are present primarily in the inner layer of the adventitia. T h e intraventricular and intra-arterial injection of V I P is followed by increased C B F . In addition, topical application of V I P to the cerebral arterioles and veins causes vasodilation 10.58. T h e location of the V I P terminal suggest a direct action of V I P on the muscle itself. T h e dependence on endothelium for VIP-induced dilation awaits testing. Substance P-containing fibers have a meshwork pattern in the cerebral blood vessels. Ultrastructural observation has shown that substance P immunoreactivity terminal boutons are present in the outer layer of the adventitia apart from the smooth muscle cells. T o p i c a l application of substance P to the pial arteriole and vein causes vasodilation 10. Intracerebral injection of substance P leads to an increase of the local C B F io. T h u s substance P , as in the systemic circulation, also causes vasodilation in the cerebral vasculature. T h i s effect may v e r y well be endotheliums dependent as is the case in other vascular beds 19.43.

E D R F A N D T H E C E R E B R O V A S C U L A R C H E M I C A L R E G U L A T I O N

T h e neurogenic control of cerebral circulation is not sufficient to explain physiological changes in C B F that take place under particular circumstances, such as variations in arterial blood pressure, changes in arterial PC02 and arterial P 02,

and responses of the cerebral vasculature to oscillations in the brain metabolic status. A s w e described above, the sympathetic vasoconstriction is rather weak. Furthermore, a generalized sympathetic or parasympathetic discharge w o u l d not control regional cerebral blood f l o w in areas of increased metabolism. T h u s , in addition to the factors that externally regulate cerebral circulation, and in addition to the neurogenic control, the cerebral vessels themselves possess the ability to regulate their diameter. In this section w e will review these intrinsic control mechanisms influencing cerebral blood flow and their possible relationship with the endothelium dependent relaxing factor.

Arterial PC02 and P02 cerebrovascular regulation — Arterial hypercapnia

dilates cerebral blood vessels 50, increases C B F , and l o w e r s cerebrovascular resistance ( C V R ^2 7

. Arterial hvpocapnia causes reverse changes in these variables. T h e effect of C02 is dependent on changes in hydrogen ion concentration of the extracellular

fluid in the vicinity of the cerebral blood vessels l . Molecular C02 and the

the increase in C B F during hypercapnia w a s severely reduced f o l l o w i n g administration of indomethacin 47. Several P G (PGI2, PGE2, PGG2 and PGD2) and their precursor,

arachidonic acid, dilate pial arterioles when applied topically. In addtion, l a r g e cerebral vessels synthesize P Q n.12,23. H o w e v e r , the participation of P G in response to C02 seems unlikely, since f o l l o w i n g the administration of cyclooxygenase, a severe

inhibitor of the vasodilator effect of arachidonic acid, the responses of pial vessels to arterial hypercapnia, hypocapnia, and hypoxia are not altered 2.28. T h e absence of significant involvement of changes in P G synthesis in the responses to C02 or to

hypoxia do not exclude the possibility that P G may be important mediators of other physiological or of abnormal responses in the brain circulation 58. T h e r e is evidence from experimental studies trat in pathological states such as mechanical brain injury and arterial hypertension, arachidonate is metabolized via the c y c l o o x y g a n a s e or lipoxyganase pathways. Its metabolism by either pathway generates a powerful free o x y g e n radical which closely resembles the free hydroxyl radical in its reactivity !8,29. T h e action of this free radical on cerebral arterioles causes their vasodilator response to topical acetylcholine to be converted to vasoconstriction 25. T h e vasodilation to topical acetylcholine is restored by topical application of superoxide dismutase and catalase. T h e s e results show that superoxide and other radicals generated in pathological conditions interfere with acetylcholineinduced endothelium dependent v a s o d i -lation, probably because they destroy the endothelium derived relaxant factor 59. Although it is known that following the mentioned pathological states lesions exist in the cerebral arteriole endothelium 28,59,60, that responsiveness to changes in

PC0

is impaired, and that the Ach-induced vasodilation is converted to vasoconstriction, there is no direct evidence of causal relationship 31, Further in vivo studies using the l i g h t / d y e model of microvascular endothelial denudation 48,50, with topical phar-macological manipulation and variation of systemic parameters may g i v e insights as to the role of E D R F in cerebrovascular chemical regulation. Arterial hypoxia dilates pial arterioles, increases C B F , and l o w e r s C V R2

? . T h e s e effects appear to be related to changes in local metabolism, since they are reverted by local application of o x y g e -nated fluorocarbons 28. Hypoxia-induced vasodilation is also dependent on the artery's previous tonus and the magnitude of the change in P 02 43,44.

Metabolic cerebrovascular regulation — T h e r e is a strong relationship between

the level of functional activity and metabolic rate of the brain on the one hand, and global or regional C B F on the other 27,34. Physiological activation of specific areas of the cortex by various types of sensory stimulation, or in association with motor activity, leads to an increased blood f l o w to the activated areas. T h e s e areas also present increased metabolism 34. it is generally believed that the relationship between C B F and metabolism is dependent on the production of vasodilator meta-bolites by the neural cells. C B F , in turn, is modulated by these metabolite levels in the perivasular space. T h e r e are a l a r g e number of candidates that may serve as mediators of metabolic flow regulation. A t present, adenosine, hydrogen ions and potassium ions appear to be the most promising candidates. Adenosine is a strong dilator of pial vessels when applied in the perivascular space. Brain adenosine con-centration increases under conditions of arterial hypoxia, ischemia, or increased metabolic activity of the brain 28,45,46. Although dependence on endothelium of the adenosine cerebrovascular relaxation has not been tested, data from other vascular beds suggest that it may not be E D R F mediated 43,44.

Cerebrovascular auto regulation — T w o basic mechanisms have been proposed

to explain autoregulation in the brain: the myogenic mechanism, which holds that cerebral vessels are responsive to changes in transmural pressure, and the metabolic mechanism, the basic premise of which is that changes in cerebral vascular caliber are the result of alterations in the concentration of vasodilator metabolites. T h e s e , in turn, are induced by alterations in blood f l o w secondary to the changes in pressure. T h e second theory is more likely 28. Adenosine concentration in the extracellular brain tissue has been demonstrated to rise quickly during hypotension 28. Adjustment of pial arteriole diameter during variations in arterial blood pressure is not followed by changes in extracellular H+ and K+ activity 57. Chemical regulation and autoregu-lation of C B F are not dependent on a and /3-adrenoceptors 5. Thus, adenosine, known to be an endothelium independent vasorelaxant in several vascular beds 43,44, j s a

strong candidate for mediating of vasodilation during pressure autoregulation 45,46. It would also be of interest to test whether or not the viscosity autoregulation 42 j s

C O N C L U S I O N S

A t this point in time there is no direct evidence that E D R F plays a vital role in cerebrovascular regulation. H o w e v e r , indirect evidence suggests that E D R F may be involved in the modulation of cerebral sympathetic vasocontriction and parasym-pathetic vasodilation. T h e r e are no experiments that have tested the known chemical regulators of the cerebrovascular circulation in models of denuded endothelim cerebral vessels. Findings in experimental models of p a t h o l o g i c a l entities such as mechanical head injury, arterial hypertension and subarachnoid hemorrhage, known to have cerebrovascular endothelium lesions, s u g g e s t that E D R F serves an important function in regulating cerebral circulation under such circumstances. In vitro studies of isolated l a r g e cerebral vessels and cultured cerebral microvessels 6, complemented with in vivo studies of l i g h t / d y e denuded pial arteries, m a y clarify the r o l e of endothelium dependent r e l a x i n g factor in the regulation of cerebral circulation.

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