Age-related changes in vascular responses: a review

Mechanisms of Ageing and Development

ELSEiVIER

and devebpment

79 (1995) 71-114

Review article

Age-related changes in vascular responses: a review Jestis Marin Departamento de Farmacologia y TerapPutica and Instituto Universitario de Investigacidn Gerontolhgica y Metabdlica, Facultad de Medicina, C/ Arzobispo Morcilio, 4, 28029-Madrid, Spain

Received 14 September 1994; revision received 30 December 1994; accepted 5 January 1995

Abstract

Normal aging is associated with different changes in the cardiovascular system that lead to an increase in pathological processes, such as hypertension, coronary artery disease, heart failure, and postural hypotension with enhancement of both morbidity and mortality. The vascular alterations consist of changes in the function and structure of the arteries, and increasing vascular stiffness, mainly when atherosclerosis is present, whose incidence is increased with age. The arteries accumulate lipids, collagen, and minerals. Cerebral perfusion may be reduced in the elderly, mainly regional cerebral blood flow, which leads to a deterioration of mental and physical functions. The degree of deterioration is increased when aging is associated with hypertension. Aging alters endothelial cells, which play an important role in vascular tone regulation. Such a process tends to reduce endothelium-dependent relaxations, and clearly reduces the vasodilation elicited by P-adrenoceptor agonists. The contractions induced by different agents, such as 5_hydroxytryptamine, histamine, high potassium and angiotensin are barely affected with aging, whereas those elicited by noradrenaline or endothelin are usually reduced. However, plasma noradrenaline levels are increased with age, mainly due to a reduction in the sensitivity of presynaptic n,-adrenoceptors and also of noradrenaline uptake. Sodium pump activity, that controls cellular ionic homeostasis, may be altered depending on animal

Abbreviations: NO, nitric oxide; NA, noradrenaline; ACh, acetylcholine; 5-HT, 5-hydroxytryptamine; WKY, Wistar-Kyoto rat; SHR, spontaneously hypertensive rat; EDRF, endothelium-derived relaxing factor; EDHF, endothelium-derived hyperpolarizing factor; EDCFs, endothelium-derived contracting factors; .L-NAME, NC-nitro-L-arginine methyl ester; MDA, malondialdehyde; PGI,, prostacyclin; VDCs, voltage-dependent calcium channels; ROCs, receptor-operated calcium channels; CM, calmodulin; MLC, myosin light chain; MLCK, myosin light chain kinase.

0047-6374/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0047-6374(94)01551-A

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J. Marin / Mech. Ageing Deu. 79 (1995) 71-114

species. Finally, vascular Ca2+ regulation appears to be altered and the extracellular Ca*+ dependence of contractile responses elicited by agonists is increased, which justifies the enhanced sensitivity to Ca2+ antagonists in senescence. Keywords:

Aging; Vasomotor responses; Nitric oxide; Endothelium;

Adrenoceptors;

Calcium

movement; Sodium pump; Sympathetic system

1. Introduction Aging, equivalent in this review to elderly, senescence, aged or advancing age, is a natural process that poses new social and sanitary problems in developed countries. Indeed, prolongation of human life span is a general phenomenon almost world-wide [l], and people with ages over 65 years constitute about 15% of developed country populations [Z]. Normal aging is associated with an impaired ability for adaptation to environmental changes [3]. Compliance and distensibility of the cardiovascular system diminish [l], as well as the cardiac output at supine rest [1,4]. Blood volume and total red cell mass show a clear, although modest, reduction with age [5,6] as does total volume of body fluids, which contributes to the relative deficiency of cardiovascular reflex regulation. This segment of population shows different pathological processes; those placed in the cardiovascular area, including hypertension, coronary artery disease, heart failure, and postural hypotension 171, are the most frequent. Age is usually associated with changes in the cardiovascular system, especially in the structure and function of the arteries [8,9]. Likewise, the risk of cardiovascular alterations is 2-5 times greater in elderly hypertensives than in normotensives of the same age [lo], and two times more compared with younger patients with the same arterial pressure [l I]. The cardiovascular alterations are responsible for 50% of morbidity and mortality in the elderly [12]. Both morbidity and mortality are dramatically increased when the aging process is associated with hypertension, which is a frequent pathology in this population [13]. In the elderly, cerebral perfusion is reduced, as a consequence of poor cerebral autoregulation associated with cerebral atherosclerosis, which alters both mental and physical functions; in addition, in patients with long-standing hypertension, the upper and lower limits of autoregulation tend to be shifted upwards [14], particularly in the elderly, so that even small decreases in blood pressure may induce significant reductions in cerebral blood flow [l]. Aging produces changes not only in vascular smooth muscle cells but also in endothelial cells [15]. The latter cells play a crucial role in vascular tone regulation [16,17]. Different studies show that aging reduces the relaxations elicited by some vasodilators that release nitric oxide (NO) from endothelial cells [18-201. The vasodilations elicited by p-adrenoceptor agonists in aging vessels from both human and rats are also reduced [7,21-231. However, the vasodilator responses induced by papaverine or nitrovasodilators in aged vessels are essentially unchanged [24,25].

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Vascular contractions induced by catecholamines, normally performed on isolated arterial strips from rats and sometimes from humans, are generally slightly reduce:d [1,26]. cr-Adrenoceptors play a leading role in the control of vascular resistance, which appears to be enhanced with increasing age [27]. This enhancement Iseems to be related to the increase in plasma levels of noradrenaline (NA) with age [1,28]. The small alteration of catecholamine response may be partly due to slight structural adaptive increases in wall-to-lumen ratio [29], that antagonizes the decline of strength. Studies concerning Ca’+ regulation in aged arteries point out some alterations in Ca2+ movements [30], as well as an increased sensitivity to Ca*+ antagonists [31], which may also justify the alterations of vascular responses mentioned above. Despite these studies in the last few years on the etiology and pathophysiological mechanisms involved in the vascular alterations with age, many aspects remain basically unknown, and there are a number of gaps in cardiovascular research on aging. The aim of the present review is to comment on the available knowledge concerning these vascular alterations, comprising changes in morphology, vascular reactivity, receptors, NA release, Ca2+ movements and blood pressure. 2. Morphological changes The vascular system is markedly altered with aging. Thus, the vessel walls enhance the content of lipids, collagen and minerals [2,32:33]. This enhancement reduces arterial distensibility [33,34] and increases vessel stiffness mainly if atherosclerosis is preserrt [2,34], which is frequent as the incidence of atherosclerosis increases with age [1,35]. The modifications in viscoelastic properties of the arterial wall that enhance vascular stiffness, participate in the increase in both systolic arterial pressure and blood pressure with age [2,36-391, and aIso the alterations in the response to vasoactive stimuli; these alterations are probably involved in the decrease in baroreceptor sensitivity [38]. However, carotid arterial compliance was found to be unchanged in older rats due to the significant increase in cross-sectional area [33]. The subendothelial layer thickens, by enhancing connective tissue content [40], and also the media of the large arteries [36] by increased calcification, protein content and lipid deposition, mainly around the internal elastic membrane [41,42]. These age-related changes tend to increase the above mentioned stiffness of the vessel wall and probably contribute to compensate for the loss of myocytes and to develop hypertrophy in the heart [43]. Aging also produces gradual morphological changes in veins and a decrease in their wall compliance [44], leading to a reduction of venous return [I]. The intraluminal blood flow may be less laminar, independent of atherosclerosis, because the endothelial cells become more heterogeneous in shape, size and axial orientation [40,42]. As previously commented, some changes at cardiac level appear with age, that lead to a reduction of myocardial cells as much in rats as in humans, although the existence of a certain degree of hypertrophy may compensate for this reduction [45,46].

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3. Endothelial

J. Marin / Mech. Ageing Dev. 79 (1995) 71-l 14

alterations

and NO synthesis

3.1. Involvement of endothelium in vascular responses

The obligatory role of endothelium in the vasodilation elicited by acetylcholine (ACh) was initially demonstrated by Furchgott and Zawadzki [47]. These authors also demonstrated the ability of endothelial cells to release a potent vasodilator factor, named endothelium-derived relaxing factor (EDRF), which modulates the vascular responses. This factor has been chemically identified as NO [48], that appears to originate from the terminal guanidino of L-arginine [49]. The EDRF (NO) or ‘endogenous nitrite’ could be released as NO, or more likely from a substance (RNO) containing NO from which it is easily liberated [16,50-521 (Fig. 1). The release of EDRF is a Ca2+-dependent p recess [17,53,54]. The fact that depolarization and ouabain inhibit EDRF-dependent vasorelaxation [55-571, indicates an involvement of Na + pump in the effect and/or the secretion of EDRF. NO

Fig. 1. Synthesis of NO from L-arginine and its stimulation by different agents that activate the corresponding receptors (P2, purinergic; M, muscarinic, c+ and /I-receptors) present on endothelial cells (EC). NO activates guanylate cyclase in the smooth muscle cell (SMC) increasing the intracellular levels of cGMP that produce vasodilation. Endothelium also synthesizes endothelium-derived contracting factors (EDCF, such as superoxide anion, 0, - , and endothelin-1, ET-l) that produce vasoconstriction: Isop., isoproterenol; NA, noradrenaline. For more details, see the text.

J. Marin 1 Mech. Ageing Dev. 79 (1995) 71-114

15

is a very diffusible compound that easily passes through cell membranes of smooth muscle cells, regulating their contractile activity [17,50,58,59]. The released EDRF has a short, but variable, half-life (6-50 s) [50]. This factor is a labile compound, which is easily inactivated by free oxygen radicals, and its inactivation is diminished by reduction of the 0, content of Krebs’ solution or by use of scavengers of superoxide anion (OZ.-) [50,60]. The fact that the half-lives of EDRIF and NO, under the same conditions, was similar (30 s) [48], supports the assumption that EDRF and NO were the same substance. Endothelium removal increases the vasoconstriction induced by different agents 150,611. This effect could be due to either an inhibitory effect exerted by EDRF on the contractile mechanisms of vascular smooth muscle cell or an EDRF release inhibition by these contractile agents. In any case, EDRF acpears to interfere with the contraction development. 3.2. Biosynthesis of NO ACh is not the only agent that causes the release of EDRF-NO; other agents are also able to elicit a release of this compound, such as 5-hydroxytryptamine (5-HT) or serotonin, thrombin, calcium ionophore A23187, histamine, substance P, ADP, ATP, arachidonic acid, changes in arterial pressure, electrical stimulation, etc. [16,17,48,50,58]. The endothelial NO synthesis is achieved by the constitutive NO synthase from the terminal guanidino of L-arginine [62,63]. This enzyme is Ca* + /calmodulin-dependent. Such a synthase is soluble and requires L-arginine, 0, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) as co-substrates [17,64,65]. They incorporate 0, into both NO and citrulline [66]. This synthase is a flavoprotein containing binding sites for flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) [64]. Such a NO synthase is competitively inhibited by NC-monomethyl-L-arginine and other L-arginine analogs [17,64]. Recently, it has been reported that vascular smooth muscle is also able to synthesize NO from L-arginine that contributes, with that formed by the endothelium, to the modulation of vascular tone [67-691. The NO synthase mediating the muscle-derived NO production may be of an inducible type [68]. These facts suggest that vascular smooth muscle not only responds to endothelial NO but has also the enzymatic machinery to generate NO from L-arginine directly. 3.3. 11’0 and the soluble guanylate cyclase The soluble guanylate cyclase is stimulated by EDRF-NO increasing the intracellular levels of 3’,5’-cyclic monophosphate (cGMP) in smooth muscle cells [50,58,70,71]. In addition, Ignarro [59] suggested that the endogenous receptor for NO is the heme moiety of this enzyme. This cGMP increase produces relaxation presumably by reduction of free Ca*+ in those cells [72,73]. NO also activates soluble guanylate cyclase of platelets, increasing their cGMP levels [74]. The mechanism involved in the platelet aggregation inhibition by NO is

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71-114

the same as the mechanism of vascular smooth muscle relaxation by NO, i.e. by reduction of intracellular free Ca* + concentration [59]. 3.4. Endo thelial changes It has already been commented that different agents can cause vasodilation by endothelial release of EDRF, and among them is histamine. Hence, changes in response to endothelium-dependent vasodilator agents, such as histamine or ACh may be due to alterations of endothelial cells or the corresponding receptors present in them. Histamine relaxes dog mesenteric arteries, partly due to endothelial release of prostacyclin (PGI,) J17,75-781. Endothelium-dependent relaxations to histamine are unchanged with age in rat aorta [79]. However, relaxations to histamine appear to decline during aging in the rat mesenteric artery [18] and beagle cerebral arteries

WI. In general, there is a tendency for the reduction with age of both the endothelium-dependent relaxation and endothelial cell ability to release NO [18-201, although the effect of age on endothelium-mediated responses varies with the species and vascular bed [19,81]. In contrast, vasodilations of aged vessels to papaverine or nitrovasodilators are essentially maintained [24,25]. The reduction of endothelium-dependent vasodilations with age could be attributed to a decrease in the synthesis of EDRF-NO and/or cGMP [82]. This reduction could be also due to an inhibition of the EDRF-NO access to smooth muscle cells by the thickening of endothelial and smooth muscle layers in aging. NG-Nitro-L-arginine methyl ester (L-NAME) produces an increase in basal tone of Wistar-Kyoto (WKY) rat aorta that is reduced with age; this increase is smaller in aortae from spontaneously hypertensive rat (SHR) compared with WKY of the same age [83], suggesting the existence of a tendency for the reduction of the basal release of NO in the hypertensive strain, as previously reported [84]. In addition, endothelial cells may also release endothelium-derived contracting factors (EDCFs) [77,85]. Among these factors endothelin is the most potent contractile agent synthesized by the endothelium [86,87] (Fig. l), and aging seems to reduce the responsiveness to endothelin, perhaps due to downregulation of the corresponding receptors [88]. On the other hand, Koga et al. [89] have observed, in vessels from WKY rat of advancing age (12-25 months), that ACh generates EDCFs, even in the absence of hypertension. This means that the reduction of endothelium-dependent relaxation may not be due to a reduction of EDRF-NO with advancing age, but to either an overproduction of the formation of EDCFs or to a hypersensitivity of smooth muscle cells to vasoconstrictor factors, masking the effect of vasodilator ones [89,90]. Another mechanism that may participate in endothelial cell deterioration is the generation of an excess of oxygen-derived free radicals due to a reduction of natural antioxidant defenses that appear to be associated with age [91]. Lipid peroxidation and peroxidation products, which seem to be increased with age, may have a role in the development of certain diseases [92] and also produce aging [93]. An indirect method of detecting the degree of lipid peroxidation is by measuring an end

J. Marin / Mech. Aping

Dev. 79 (1995) 71-I 14

II

product derived from hydroperoxides, such as malondialdehyde (MDA) [94]. This compound can alter the cellular membrane and DNA [94]. Experiments performed by ou.r group demonstrate that plasma MDA levels increase with age in healthy indivilduals [95] and Sprague-Dawley rats (unpublished results, Fig. 2). In vitro experiments with MDA concentrations similar to those found in plasma of these animals (about 1 PM) show the ability of this compound to reduce the relaxation elicite’d by ACh in rat tail artery; this effect is blocked by superoxide dismutase [96] (Fig. 2). Taking into account all these facts, one can suggest that the increase in MDA with aging may be involved in the morphological changes observed in the endothelium and in the inhibition of the endothelium-dependent relaxation with age. 4. Reactivity alterations 4.1. Changes in contractile responses Aging tends to show a modest reduction in maximum contractile responses induced by NA and other adrenoceptor agonists in isolated arterial preparations, mainly derived from rats [7,26,39,81,97-991. However, this tendency is not uniform as different results have been reported. For example, NA contractions are increased in isolated dog iliac, carotid, renal, and mesenteric arteries [loo], ear arteries from lamb and ewe [loll and rat aorta [102]. Yet, the isolated rabbit aorta [97] and basilar artery [ 1031 and rat femoral and carotid arteries [104] are unaffected, and NA contractions are reduced in the rat and guinea pig aorta [105-1071, rabbit aorta [108], rat tail artery 11091,dog [l lo] and monkey [l 1 l] mesenteric arteries and dog cerebral arteries [80]. This reduction in NA response can be due to a decrease in the sensitivity of the vessel [ 105,112,113], which seems to be produced by a decrease in cr,-adrenoceptor reserve [31], or a reduction of postsynaptic qadrenoceptor density, a:s reported in rat tail artery [114]. However, Benetos et al. [33] reported that aging (does not affect the affinity and density of rat carotid artery a,-adrenoceptors; this indicates that the origin of modifications in the contraction/relaxation mechanism with aging could be due to alterations in basal adrenergic tone or in the structure of the arterial wall [33]. It is necessary to point out that vascular a-adrenoceptors are of CI~-and cl,-subtypes [115,116]. The majority of aging studies do not distinguish between both subtypes, although normally the receptors involved are of q-subtype. However, in the human saphenous vein, adrenoceptor agonists induce contractions mediated mainly by qadrenoceptors [117]. A decrease with age of the inhibitory effect of yohimbine, an q-antagonist, on stimulation-induced contraction in this vein has been reported [117]. The contractile response to B-HT 933, an a,-agonist, increases with age in isolated subcutaneous human resistance arteries, whereas responses to NA, phenylephrine and perivascular nerve stimulation, mediated by or,-adrenoceptors, decrease, suggesting that the contractions mediated by q- and cr,-adrenoceptors are, respectively, increased and decreased with age in these arteries [39]. However, the function of postsynaptic a,-adrenoceptors in rat tail artery is not affected with age [114].

J. Marin / Mech. Ageing Dev. 79 (1995) 71-l 14

MDA plasma levels

1.0

zE

0.6

5 -5

0.4

0.0

24

5

30

AGE (months) 120

1

-----t-

Control (10) 1 pM MDA (10)

-Q-

60 60 ANOVA

NA 1clM

-6

-7

-6

-5

ACh (log M)

L

E 8

120-

-

Control (11) 150 UVml SOD

loo-

60 -

60ANOVA P=O.50

40 -

201 NA *clM

-6

-7

-6

ACh (log M)

-5

J. Marin / Mech. Ageing Dev. 79 (1995) 71-114

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Thle contraction caused by other drugs, such as 5-HT, histamine and angiotensin II, shows little or no changes with aging [2,7,19,50,81,98,118,119]. However, the contraction caused by 5-HT and the sensitivity to this amine was greater in coronary arteries from aged rats, which is associated with increased 5-HT,-receptor affinity and decreased receptor reserve [120]. Likewise, the contractile potency of histamine is reported to be decreased during maturation in dog cerebral arteries [80], and also that caused by 5-HT in the rat aorta [7]. However, the contraction caused by histamine increases with age in rabbit basilar arteries [121]. The maximum contractile responses caused by KC1 is usually scarcely modified [7,98,119,121], suggesting that the basic contractile machinery per se has become slightly altered with aging. This small alteration may be partly due to slight structural adaptive increases of wall-to-lumen ratio [29] that antagonizes the decline of strength. However, during maturation, an increase in the response to KC1 has been observed in dog cerebral [80] and coronary [122] arteries, and rabbit [ 1231 and rat [I 12,124] aorta. These alterations on contractile responses with age may be due to changes in endo thelial cells. Indeed, it has been reported that the endothelium modulates the contractions elicited by several agonists, such as NA and 5-HT [50,125,126], as endothelium removal increases contractile responses. This effect may be due to a reduction in stimulated influx of extracellular Ca2+ [125]. In the case of NA, this is due to that the activation of endothelial cr,-adrenoceptors by NA, or other a-agonists, can elicit EDRF release in some vessels [50,126]. However, this mechanism is not common for all arteries since endothelial a>-adrenoceptors do not exist in some arteries, for example in bovine coronary or human skin arteries [50]. Kaneko and Sunano [126] have also reported that endothelial a,-adrenoceptors also participate in the negative modulation of NA-induced contraction in rat aorta. The influence of age on the contractions modulated by endothelium has been scarcely studied. Among the agents studied concerning this subject is angiotensin II, whose contractions in rat aorta decrease while their endothelial modulation increases during the growth [ 1271. Likewise, the potent vasoconstrictions elicited by the agent of endothelial origin, endothelin [128], are reduced with age [87,129], probably due to a receptor desensitization [88]. In addition, endothelium-dependent contractile responses mediated by cholinergic muscarinic receptors are increased with aging in normotensive and hypertensive rats by the release of endothelial products formed through the cyclooxygenase pathway [89,90]. The different results reported on the contractile responses may be, in part, due to the expression of contractile results, since they are expressed in absolute terms, as a percentage of the maximum response elicited by KCl, tension produced per unit-cross sectional area or in relation to the maximum reached by the agonists. The

Fig. 2. Effect of age on plasma concentration of malondialdehyde (MDA) in WKY rats, and action of supero.uide dismutase (SOD, a scavenger of superoxide anion) in the inhibitory effect of MDA on the relaxations elicited by acetylcholine (ACh) in segments from tail artery precontracted with NA (1 ,uM). Results (mean + S.E.M., paired experiments) are expressed as a percentage of a previous tone with NA. *P < 0.05 ([96] and unpublished results). Number of segments used is shown in parentheses.

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latter is likely the most adequate [39]. In addition, other reasons for such distinct results may be due to species differences or to different experimental protocols. 4.2. Changes in vasodilator responses Endothelium-dependent relaxations [7,122] and those induced by P-adrenoceptor agonists, such as isoproterenol, are generally reduced in aged vessels from different animals, including human [7,21,8 1,98,130- 1341, even though conflicting results have been observed in this particular area [80,135], which will be commented on later. This reduction is not shared in the vasodilation caused by adenosine [136]. In contrast, the vasodilator effect of sodium nitroprusside, the reference agent, is usually similar in young and elderly normotensive human subjects [7,25]. However, the vasodilation caused by atria1 natriuretic factor is reduced without modification of the second messenger cGMP production [137]. Such results suggest age-related changes in the postreceptor cellular mechanisms. We comment below on the effects of aging on endothelium-dependent and endothelium-independent relaxations more extensively. 4.2.1. Endothelium -dependen t relaxation The ability of some vasodilators to produce relaxation mediated by the endothelial EDRF release has been reported [17,58,77]. The influence of age on endothelium-dependent relaxant responses varies with the species, vasodilator agent and vascular bed [7,19,81]. Thus, the endothelium-dependent relaxations induced by ACh, and related cholinergic muscarinic agonists, are reduced with age in different vascular preparations [7,25,110,121,122,138], although an increase [7,19,20,24] or no effect [7,19] have also been reported. In addition, ACh can produce relaxation by the release of endothelium-derived hyperpolarizing factor (EDHF), that hyperpolarizes smooth muscle cells [139,140]. In different arteries, there is a marked participation of EDHF on the relaxation elicited by ACh [16,140]. In rat mesenteric arteries, the hyperpolarization caused by ACh and its contribution to relaxation are markedly impaired in aged SHR and WKY rats, which explains, at least partially, the reduction in relaxation to ACh with age [141]. The endothelium-dependent relaxation caused by ATP and ACh in rat carotid artery is impaired in old and hypertensive rats, suggesting that aging and hypertension are risk factors that may augment the.disturbance of the cerebral circulation in pathologic conditions 1201. It is interesting to comment on the results reported by Hynes and Duckles [19] in some rat arteries. They found that the sensitivity of endothelium-mediated relaxation to cholinergic agonist methacholine, in aortic segments and perfused caudal arteries from Fischer rats, is increased with age. This effect occurs between the ages of 6 and 12 months and is maintained up to 27 months. No differences with age are observed in the perfused mesenteric bed. The relaxations of the perfused caudal artery to the calcium ionophore A23187 are not altered with age. These results suggest that the alteration of response to methacholine with age involves a change in the muscarinic receptor or the receptor-coupling mechanism.

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Histamine, an endothelium-dependent vasodilator, produces relaxations which decline during maturation and aging in the rat mesenteric artery [ 181.Histamine also induces relaxations directly related to age in mesenteric arteries from beagle, which are partly mediated by endothelial release of PGI, [I lo]; this release of PGI, appears to be lower in infant beagle mesenteric arteries than in those from older beagles [l IO]. The alteration of endothelium-dependent relaxations with aging may be influenced by tlhe mechanism involved in EDRF-NO release [19,142]. Thus, in pig coronary artery, NA produces endothelium-dependent relaxations by means of a,-adrenoceptor stimulation, which are reduced in older pigs (3-4 years) [142]. In contrast, endothelium-dependent vasodilations induced by other agents, such as substance P or bradykinin, are not modified with age [142]. Similarly, the endothelium-dependent relaxations induced by ATP in aortae from SHR and WKY rats [90] and those elicited by the calcium ionophore A231 87 in Fischer rat tail artery [19] are unaltered with age. However, relaxations elicited by ACh are reduced with advancing age [19,90]. The mechanism of EDRF-NO release caused by activation of qadrenoceptors by NA, or ACh stimulation of M, muscarinic receptors, involves G proteins. This mechanism is not implicated in the responses to substance P, bradykinin, ATP or calcium ionophore A23187. Such facts indicate that the NO release, in which G proteins are implicated, is more sensitive to the deleterious effect of aging [142]. Recently, we reported that low NA concentrations are able to induce contractions in aortic rings from SHR and WKY rats, whereas higher concentrations elicit relaxations [83]. These relaxations are reduced and abolished in rings from 3 and 6 month WKY rats, respectively. In rings from 5-week and 3-month SHR, the reduletion is similar to that obtained in 3- and 6-month WKY rats, respectively. This means that hypertension may resemble an ‘aged state’. This impairment appears to be d.ue to a dysfunction of endothelial /3_adrenoceptors leading to a reduced generation and/or release of NO. Indeed, NA-induced relaxation in both strains is blocked by the inhibitor of NO synthesis, L-NAME, and propranolol, but not modified by yohimbine or ouabain. These findings suggest that, in aortae from young WKY and prehypertensive SHR, NA induces vasodilations mediated by activation of endothelial j?-adrenoceptors and release of NO [83]. The precise mechanism by which vascular aging attenuates endothelium-dependent relaxation to NA is unknown. It has been suggested that the age-related lost of response to /?-receptor agonist may be due to an attenuated activation of CAMP-dependent protein kinase [143] or to a decrease in the function of the stimulatory GTP-binding protein [144]. NA-mediated relaxant responses are age-dependent in rat aorta, and this fact may explain the disparity of results between different authors concerning whether or not the vasodilation caused by a-adrenergic agonists is dependent on endothelium. In addition, these responses are impaired in hypertension. It is possible that desensitization of endothelial P-adrenoceptors in hypertension and aging is due to the existence of ebevated catecholamine levels in hypertensive and aged animals [7,145,146,147]. Isoproterenol also induces relaxations in these arteries that are abolished by propranolol and partially antagonized by endothelium removal and L-NAME, suggesting that both endothelial and smooth muscle /?-adrenoceptors are involved

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[83]. These results agree with previous reports in rat aorta, in which the relaxations induced by isoproterenol are inhibited by endothelial denudation [148]. The ability of isoproterenol to induce relaxant responses in vessels precontracted with NA has been reported [144,148,149]; these vasodilator responses have been thought to be mediated by smooth muscle /?-adrenoceptor activation of adenylate cyclase [150], and therefore are endothelium-independent. Other authors have demonstrated that methylene blue or hemoglobin, agents that interfere with NO action, inhibit the isoproterenol-induced relaxations [ 151,152]. These results indicate that endothelial P-adrenoceptors might play an important physiological function in the modulation of NA-induced tone, reducing the vascular contractions when the catecholamine concentration is elevated. 4.2.2. Endothelium-independent relax&ion Endothelium-independent vasodilations are also modified with age [7,20,22, 98,122,153]. The most studied agent has been the /?-adrenoceptor agonist isoproterenol. The relaxation caused by this agent is reduced during aging in the beagle mesenteric artery [l lo], rabbit basilar artery [ 1031, human saphenous [154] and dorsal hand [133] vein and rat pulmonary artery [132]. The reduction in P-adrenergic responses with advancing age [144] and hypertension is a very common phenomenon [54,155,156]. Such a fact could be attributed to fi-adrenoceptor desensitization due to the increase in endogenous catecholamines during aging [143], or to the enhanced sympathetic tone that has been observed in hypertension [54]. However, no modification of relaxant responses with aging in rat jugular vein and rat and rabbit portal vein has been also reported 171. Relaxations caused by papaverine in rat mesenteric artery [ 181and PGI, in beagle mesenteric arteries [I lo] do not change with age. However, the vasodilation caused by adenosine in the cremaster muscle microvasculature of Fischer 344 rats declines markedly with advancing age [157]. The relaxation to sodium nitroprusside or sodium nitrite decreases with aging in the rabbit aorta [123] but not in rat mesenteric arteries [22]. Nevertheless, Shirasaki et al. [158] observed no alteration on the vasorelaxation to sodium nitroprusside and sodium nitrite with hypertension and aging in de-endothelialized mesenteric artery and aorta from SHR and WKY, whereas a reduction in this effect has been reported in rat aorta [ 1591. In contrast, Shirasaki et al. [158] observed a lesser relaxation in intact vessels, probably due to the release of some endothelial vasoconstrictor factor in response to nitrovasodilators. 4.3. Alterations of M- and /3-adrenoceptors Vasoconstrictor responses mediated by a,-receptor seem to be relatively well maintained with aging [7,133], although an increase in the contractions mediated by a-adrenoceptors has been also reported [133,160]. However, the responses mediated by a,-adrenoceptors, present at pre- and postsynaptic sites, appear to be attenuated [7]. Since those present at presynaptic levels are involved in the negative modulation of NA release from adrenergic nerve terminals [7,161], their attenuation leads to a

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NA release enhancement per stimulus. This effect along with the fact that the uptalce of NA into these terminals is reduced accounts for, at least in part, the elevation of NA plasma levels usually observed with age [1,162]. The reduction of contractile responses mediated by postsynaptic qadrenoceptors, that predominate in veins [163], may participate in the attenuation of local vasoconstrictor tone. The sensitivity of cerebral a,-adrenoceptors seems to decline with age [7], indicating that the regulation of regional blood flow in the brain may be altered. In addition, /?-adrenoceptor-mediated responses in the cardiovascular system of human and animals seems to be usually reduced with aging [1,7,23,112,124,133,164]. The reduction of Q- and p-receptor-mediated responses with age in veins decreases the efficacy of the sympathetic system in the control of the venous system with advancing age, which is potentiated by increasing valvular incompetence [ 11.In addition, the attenuation of vasodilator p-tone may contribute to an increase in peripheral vascular resistance observed in elderly [38], to which the structural changes of the vessels and the facilitation of NA release, above described, may also contribute. The decline of responses caused by a-stimulation produces a reduction with age of the cardiac positive chronotropic and inotropic effects elicited by sympathetic activation, and a reduction in the renin secretion caused by sympathetic activation, whereas the vasodilation elicited by plasma adrenaline is attenuated [l]. A reduced positive effect of presynaptic p-receptors on NA release may exist, decreasing the transmitter release from perivascular adrenergic nerves with aging, that can be important in veins from aged individuals [163]. B-Blockers are usually contraindicated in the elderly due to the associated respiratory and peripheral vascular disease, and by the problems that resultant bradycardia may generate. Hypertension presents pathophysiological features in the elderly, such as a decreased cardiac output, reduced renal blood flow, low plasma levels of renin, and enhanced peripheral and renal vascular resistance in comparison with younger patients [5,165]. The effect of aging on a-adrenoceptor-binding sites has been studied in different tissues, and has yielded conflicting results. Thus, in maturation, the number of q-binding sites is increased [166] or reduced [167] in the rat heart, and unaltered [166] or enhanced [168] in the rat vas deferens. However, with aging, a decrease [ 169,170] or no alteration [167] in x-adrenoceptor number or affinity in rat ventricular myocardium has been reported. No alteration has been also observed in rabbit heart and spleen [171], and rat carotid artery [33]. In addition, in rat carotid artery, aging did not affect a,-adrenoceptor affinity and density [33]. The majority of binding studies indicate that the affinity of a-agonists for the binding sites does not change with aging or that this change is small [7]. Therefore, the changes in a-adrenoceptor agonist potency observed in reactivity experiments are probably due to a loss of receptors or to an alteration in the mechanisms beyond a-receptors activation [7]. There are conflicting results concerning if the reduction of the response to P-agonists is due to a decrease in cellular density of p-receptors and in the affinity for p-agonist binding [23], a decrease only in p-density without changing their

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affinity [33], or to other factors [1,7]. Different mechanisms may participate in cellular desensitization of /3-adrenergic stimulation with aging. These mechanisms operate at the level of the P-adrenergic receptors, G-protein, adenylate cyclase catalytic unit, and also at distal levels [7,23,172- 1751. All these mechanisms lead to the same result, i.e. an insufficient production of the second messenger adenosine 3’,5’-cyclic monophosphate (CAMP) following p-stimulation [1,7,28]. 5. Changes in Ca* + movements 5.1. Cellular calcium regulation Vascular smooth muscle cells are able to regulate Ca2+ metabolism, i.e. the maintenance of the Ca2+ gradient between the extracellular space (serum levels of ionized Ca2 + ) and the intracellular free Ca *+ . Indeed, the intracellular free Ca2 + concentration in the smooth muscle is about 0.1 PM in the basal state, i.e. 10 000 times lower than that present in the extracellular space, which is of mM order. When the cells are stimulated, the intracellular Ca2+ concentration increases, ranging from 1 to 10 PM [176-1781. The extracellular Ca2 + plays an important role in the contraction of vascular smooth muscle. There are different pathways for Ca2 + entry into the cell [176,17818 11: (1) through voltage-dependent Ca2 + channels (VDCs), or receptor-operated Ca2+ channels (ROCs), which are the most important pathways for Ca2+ influx; (2) through Na + channels, and (3) by a passive Ca* + leak, through leak channels, as a consequence of the favorable Ca2 + gradient, which can be blocked by La3 + but not by organic Ca2+ antagonists. Bevan et al. [182] have demonstrated the existence of other channels named stretch-operated Ca2 + channels. Ca2+ entry into smooth muscle cells, especially through VDCs, can be modified by Ca2+ entry modulators, i.e. by Ca2+ antagonists and Ca2 + agonists, which inhibit and facilitate Ca2 + influx, respectively [ 178,180- 1831. Dihydropyridines can be of antagonist type, such as nifedipine, or agonist, such as BAY K 8644, which is a nifedipine analog [ 183,184]. BAY K 8644 is able to produce direct contraction [185,186] or a moderated depolarization is needed with small K + concentrations (lo- 15 mM) to develop consistent contractile responses [ 184,185,187]. Elevated concentrations of intracellular Ca*+ are toxic for cells [188]. They are endowed with the following mechanisms to reduce toxic levels of Ca2 + and maintain Ca2 + homeostasis: (a) Ca2 + Mg* + -ATPase (Ca* + pump) of the plasma membrane, which produces Ca’ + extrusion in exchange for 2H + influx using the energy from ATP [179,189]. This ATPase of smooth muscle is calmodulin (CM)-dependent and the complex Caz + -CM stimulates the activity of this system [189,190]. In addition, the plasmalemmal Ca 2 + -ATPase has a high affinity but low capacity to pump Ca2 + out of the cell [ 190,191]. Such an exchange is coupled to the Na + , K + -ATPase, that produces an active extrusion of 3Na + and an accumulation of 2K + , thus maintaining the transmembrane Na + and K + gradients. (b) Na + -Ca2 + exchange system of plasmalemma (3Na + exchanged for 1Ca2 + on either side of the membrane), which has high capacity to produce Ca2+ efflux and low affinity [54,183,192].

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The intracellular free Ca* + increase produces the beginning of the contraction process. This consists of four Ca*+ ions binding to the corresponding binding sites of the CM. This process causes the formation of the Ca*+ -CM complex, which binds to the myosin light chain kinase (MLCK), forming the Ca2+ -CM-MLCK complex, the active form of this enzyme. This complex catalyzes phosphorylation of the 20 OOO-Dalight chain subunit of myosin (MLC), which stimulates actin-myosin interaction, causing smooth muscle contraction [2]. When intracellular Ca*+ concentrations are lower than 1 ,uM, the Ca*+ -CM-MLCK complex is dissociated [193]. and MLCK is transformed into its inactive form. Furthermore, other mechanisms by which Ca2+ regulates smooth muscle contraction have been suggested. Among them, the most studied is that mediated by the protein caldesmon, a Ca’ + -CM-binding protein, which regulates actin-myosin interactions [ 1941, increasing or reducing these interactions in the presence or absence of the Ca*+-CM complex [ 194,195]. 5.2. Alteration of Ca2+ movements and e#ect of Ca”

entry modulators

Sin.ce Ca* + has an imp ortant function on vascular tone, which can be altered with age, one can assume that age may alter the Ca*+ movements and the action of Ca*+ entry modulators. The impaired regulation of cellular Ca*+ movements seems a rather generalized manifestation of the elderly. The apparent incapacity of aged cells to respond, in many cases, to certain neurotransmitters and related stimuli may be, at least in part, reversed by manipulation of Ca*+ movements [3]. Cassie et al. [30] suggest that age increases the sensitivity to Ca* + in rat mesenteric arteries, and Pedersen [ 1961 observed a significant increment in Ca* + -dependency with age in the thoracic aortae from WKY rats. In addition, the potencies for K+ (whose responses are dependent on extracellular Ca* + ) and CaCl, (in K + -depolarized aortae), but not the maximum contraction, are reduced in aortae from older rats [197]. Such a reduction is less pronounced in pulmonary artery from aged rats [197]. This suggests age-related changes in the resting membrane potential or in VDCs, as well as that the Ca*+ sensitivity is different depending on the vessel and the vasoconstrictor agent. Furthermore, it has been reported that Ca2+ placed in intracellular stores has special importance in the contraction induced by NA in older rats [198]. It is necessary to comment that NA uses both intracellular and extracellular Ca* + for contraction, and depending on the type of artery, one or other predominates. NA essentially acts on smooth muscle a,-adrenoceptors of rat aorta.; NA-cr-adrenoceptors interaction produces mobilization of intracellular Ca* + , and an opening of plasma membrane Ca2 + channels, permitting extracellular Ca*+ entry; this Ca2+ entry is involved in the tonic response, and is inhibited by Ca*+ entry blockers, such as dihydropyridines [ 199,200]. In rat aortic rings contracted with NA, the vasodepressor effect of the Ca2+ antagonist diltiazem [3 I,1 191 or felodipine [ 1191 is greater in aged than in young rats, ;and a similar effect is observed by reducing extracellular Ca* + [31]. In our laboratory, we observed that the relaxant effects of nifedipine in these arteries precontracted with K + is reduced with age (unpublished results, Fig. 3). However,

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a,b,c

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Nifedipine (log M) Fig. 3. Effect of age on the relaxation elicited by nifedipine in WKY aortic rings precontracted with 50 mM K+ Results (mean k S.E.M.) are expressed as a percentage of a previous tone with 50 mM K + Number of segments used is shown in parentheses. Analysis of variance (ANOVA) among different ages: a = P < 0.05, vs. 5 weeks; b = P < 0.05. vs. 3 months; c = P < 0.05, vs. 6 months (unpublished results).

the rat pulmonary artery, none of both above age-related changes are observed [31], and in rabbit aorta, a reduction of the sensitivity to verapamil with age has been reported [123]. The increase in the depressor effect of diltiazem seems to be due to a reduction in a-adrenoceptor reserve in this artery [31], because the affinity of these receptors for NA is not changed with age [112]. In contrast, the responses to other agents, such as 5-HT and IS+ are not reduced by diltiazem [119], suggesting that its inhibitory effect depends on the contractile agent. Another study with diltiazem shows that this agent causes a nonparallel shift of the concentrationresponse curve to NA and 5-HT in rat aortic preparation, associated with a reduction in maximum responses [201]. The maximum contraction reduction caused by this Caz+ antagonist decreases with age from 3 to 10 weeks, but increases from 10 to 40 weeks; on the other hand, the maximum contraction to 5-HT was unaltered. An increased relationship between the change in diltiazem inhibition and a-receptor reserve was observed, which may be involved in the effect of this Ca2+ antagonist [201]. Cassie et al. [30] describe that the sensitivity of diltiazem to inhibit K+ inducedcontraction was greater in mesenteric arteries of WKY than SHR from adults rats, suggesting a lower Ca2+ influx dependency through Ca2+ channels, or an alteration of these channels, in SHR vessels. The increase in the sensitivity to Ca2+ antagonists with age justifies the use of these drugs in senescence [8,202]. The in

J. Marin / Mech. Ageing Dev. 79 (1995) 71-114

300,

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+

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Fig. 4. Effect of age and partial depolarization with 15 mM K + on the concentration-response curve to BAY KL88644 in WKY rat aortic segments. Results (mean k S.E.M.) are expressed as a percentage of the previous contraction induced by 75 mM K+. Number of segments used is shown in parentheses. Analysis of variance (ANOVA) has been achievedbetweenthe responsesobtainedin segments subjected to basal condition and partially depolarized [206].

different sensitivities to Ca2 + antagonists may be due to age-related modifications in the number or affinity of binding sites for these drugs. Thus, a reduction in the affinity of Ca2+ antagonists for their binding sites on Ca* + channels [31] and an enhancement of these sites for dihydropyridines and the affinity constant [203] has been reported; the latter findings would explain their greater effect in older animals. Few studies have analyzed the influence of age on the contractile responses elicited by the Ca2+ agonist, BAY K 8644. Wanstall and O’Donnell [197] report that the responses caused by this agonist are reduced in aorta from older rats partially depolarized with low K+ concentrations (6 mM). Such an effect suggests that tlhe resting membrane potential may be more negative in aged vessels, which could explain the increased potency of Ca2 + antagonists, such as diltiazem. In cardiac muscle, conflicting results have been obtained, since a decrease [204] or increase [205] in the response to BAY K 8644 have been described. In our labora.tory, we observed that age does not increase the contractile response to BAY K 8644 in WKY rat aortic segments either in basal conditions or partially depolarized with 15 mM K + , although the responses obtained in the latter case were greater than that obtained in basal situations [206] (Fig. 4). 5.3. Role of endothelium on the eflect of Ca2+ entry modulators The ability of endothelial cells to synthesize mediators of vasodilator and vasoconstrictor responses is well established [16,50,207]. The release of endothelial factors requires an increase in intracellular levels of free Ca*+ [16,50]. The actions of Ca’ + entry modulators on EDRF release are not clear up to now, and the same occurs with the type of Ca2+ channels present in these cells [16,208]. The effect of Ca*+ antagonists on endothelium-mediated responses varies, depending on the type of the vessel, the vasoconstrictor agent and the Ca2+

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antagonist used. Thus, Rubanyi et al. [209] observe that the presence or absence of endothelium does not modify the vasodilator responses elicited by different Ca2+ antagonists in dog femoral artery contracted with prostaglandin F,,. Nevertheless, Miller and Stoclet [210] find that the relaxant effect of the Ca2+ antagonist flunarizine is greater in rat aorta with endothelium precontracted with prostaglandin F,, or phenylephrine. Vanhoutte [21 l] reported that the vasodilation elicited by nisoldipine in dog coronary artery contracted with a-adrenoceptor agonists was greater when the endothelium was present. In our laboratory, we observed that the relaxation caused by nifedipine in rat aortic segments precontracted with 50 mM K+ is greater in the absence of endothelium, and this effect is essentially unaffected with age (unpublished results, Fig. 5). Likewise, in SHR and WKY rat aortic segments in basal situation or partially depolarized with 15 mM K+, respectively, we observed that the endothelium positively modulates the contractile response to BAY K 8644; this effect is maintained with age in aortae from normotensive and hypertensive rats [206] (Figs. 6,7). This endothelial influence may be due to the release of an endothelial contractile factor(s) by BAY K 8644 [212]. However, other authors have observed that the vasoconstrictor effects of BAY K 8644 are negatively modulated by the endothelium, probably due to endothelial release of a vasodilator factor [213-2151. 6. Sodium pump activity Na+, K + -ATPase or Na + pump is situated in the plasma membrane of practically all eukaryotic cells, including vascular smooth muscle cells, whose function is to maintain the transmembranous ion balance needed to regulate membrane potential, i.e. it plays an essential role in cell survival [54,216-2181. It is also thought to be critically implicated in functions, such as cellular growth and differentiation and contraction of vascular smooth muscle, by virtue of its role in the maintenance of the cellular ionic milieu [54,216,217,219]. The pump functions in a cyclic manner, transporting 2 mol of K+ intracellularly for every 3 mol of Na+ moved extracellularly, and the energy for this transport is provided by the free energy of hydrolysis of one ATP mol by cycle [217] (Fig. 8). The Na + pump molecule consists of CI-and p-subunits [220,221]; the transmembrane g-subunit is responsible for most of the activities and has an intracellular ATP hydrolytic site and extracellular digitalis glycoside-binding site [221-2251 (Fig. 8). At least, it has been described as three isoforms of the cc-subunit (oL,,a, and a3) and two of the p-subunit (p, and j3J [223-2251; each isoform of the a-subunit has a different sensitivity to digitalis glycoside [226-2281. Hence, differences in the sensitivity to digitalis among species, for example the scarce sensitivity to the

Fig. 5. Effect of endothelium removal (E - ) on the concentration-response curve to nifedipine in aortic segments from WKY rats of different ages precontracted with 50 mM K + Results (mean + S.E.M.) are expressed as a percentage of a previous tone with 50 mM K + Number of segments used is shown in parentheses (unpublished results).

J. Marin / Mrch. Ageing Dev. 7Y (1995) 71-114

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J. Marin 1 Mech. Ageing Dev. 79 (1995) ?1- II4

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sodium pump inhibitor ouabain of rat blood vessels [229,230], may be related to respective isoform expressed [224,228]. In addition, the isoform expressed in a tissue may be altered in pathologic processes, such as hypertension [231] and probably also with age (see below). Experiments achieved by our group show age-related increases in the sensitivity to th.e digitalis, ouabain, in WKY rat aorta, which is negatively modulated by endothelium up to 6 months and positively at 18 months [232,233]. This increase was lmore rapidly reached in aortae from SHR, but in this case, the endothelium positively modulates the ouabain response up to 12 months, and the contraction obtained at 18 months was reduced and not affected by endothelium [233] (Fig. 9). Such results suggest that hypertension produces a faster aging of endothelium, as the response obtained in segments from WKY rats at 18 months was similar to that observed at 3-6 months in SHR segments. Likewise, they suggest that the endothelium modulates the sodium pump and therefore, the intracellular ionic concentration. The increase in sensitivity could be due to an age-related increase in the expression of high affinity isoforms (Q and CIJ of the sodium pump for the digitalis. Indeed, changes in the expression of the a-subunit of the sodium pump with age have been reported [223,234-2361. A known indirect measure of Na+ pump activity is by measuring the magnitude of the relaxation elicited by low K+ concentrations, since this effect is blocked by ouabain. In this sense, it has been reported that the relaxant response elicited by low KC (5 mM) in mesenteric arteries from beagle does not change with age, indicating that Na + pump activity is unaltered [ 1lo]. Similar experiments were achieved in cerebral arteries from beagles, which show that Nat pump activity is also not modified with aging [SO]. However, an age-related increase in this activity was observed in rabbit basilar arteries [103]. Taking these results together suggests that aging may alter vascular Nat pump activllty, and that this alteration depends on the vessels and animal species. 7. Changes in the sympathetic system Aging appears to produce changes in the structure of neurons and nerves [237,238], and on adrenergic neurotransmission that produces modifications on catecholamine plasma level, cardiovascular content of these amines (which is an index of adrenergic innervation), their neuronal uptake and presynaptic q-adrenoceptors [1,7], i.e. there is certain loss of adrenergic control of the vascular system. It is interesting to note that the NA release from adrenergic nerve endings by nerve stimulation is negatively regulated by a feedback mechanism mediated by presynaptic qadrenoceptors whereby NA modulates its own release [ 161,239,240].

Fig. 6. Effect of endothelium removal (E - ) on the contraction-response curve to BAY K 8644 in aortic segments from WKY rats of different ages partially depolarized with I5 mM K + Results (mean k S.E.M.) are expressed as a percentage of the previous contraction induced by 75 mM K + Number of segments used is shown in parentheses [206].

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SHR (BASAL)

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Et+)(6) EC)(16)

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Et+) (9) Et-1 (7) ANOVA

Fig. 7. Effect of endothelium removal (E - ) on the contraction-response curve to BAY K 8644 in aortic segments from SHR of different ages in basal situation. Results (mean f S.E.M.) are expressed as a percentage of the previous contraction induced by 75 mM K + Number of segments used is shown in parentheses [206].

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93

Na?, K+-ATPase Extracellular

[K’],=4 mM [Na+],= 140 mM [Cl; H&O; ] g, etc. = 150 mM

~150

mM

Resting membrane potential about -57 mV Fig. 8. Structure of Na+, K + -ATPase (ouabain) binding site and the intracellular are also another two sites for binding intracellular space, respectively. For more

(containing x- and b-subunits) with extracellular digitalis hydrolytic site for ATP, both placed in the a-subunit. There and exchanging 2K + /3Na+ from the extracellular and details, see the text.

In addition, 20% of the released NA diffuses away, and the remainder is taken up by nerve adrenergic varicosities [ 1,7,161,239] (Fig. 10). 7.1. .4lterations in the structure of the nerves Aging seems to produce modifications in different nerves, including adrenergic ones. Thus, alterations in their structure, reduction in the number of neurons and loss of nerve fibers have been reported [237,238,241,242]. A reduction with aging of: innervation of rat plexus [243], catecholamines in human sympathetic ganglia [244], guinea pig myenteric neurons [245], and neuron number of dorsal motor nucleus of the vagus and nucleus ambiguus of mouse [246] has been observed. In addition, as aging produces changes in postsynaptic structures, these changes may be responsible for the loss of nerve fibers [242].

J. Marin 1 Mech. Ageing Dev. 79 (1995) 71-114

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2500

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J. Marin / Mech. Ageing Dev. 79 (1995) 71-I 14

Presynaptic

Aging: J. Uptake of NA $ a2 presynap. and p posts. activity 1 Innervation [ -LNA] ‘T’Plasma [NA]: TA.P. 1 NA metabolism Fig. IO. Mechanisms and receptors involved in the release and uptake of NA at presynaptic level and the effect of NA at postsynaptic level. The effect of aging on these mechanisms is also indicated. MAO, monc,amino oxidase that metabolizes the NA; R, receptor. For more details, see the text.

At rat cardiovascular level, it has been reported that the catecholamine content of heart [247-2491 and certain arteries [109,237,250-2521 decreases with age, whereas in rat mesenteric and renal arteries and veins, it is unaltered [251]. In rat tail artery, the NA content is increased [253], unaltered [251] or decreased [109]. In general, aging seems to produce a decline in adrenergic innervation in different tissues. These alterations induced by aging modify the neurotransmitter content in the nerve terminals and the transmitter release by nerve impulse, as reported in rat atria [254] and heart [255]. However, the capacity of perivascular adrenergic terminals for taking up [3H]NA does not seem to be modified with age [104]. In contrast, an enhancement of both catecholamine synthesis by the sympathetic nervous system in older rats, and the activity of tyrosine hydroxylase in superior cervical ganglion, which is involved in NA synthesis, have been reported [256]. This enhancement could be to compensate for the reduction in catecholamine content.

Fig. 9. Effect of endothelium removal (E - ) on the contraction elicited by 1 mM ouabain in aortic segments from SHR and WKY rats of different ages. Results (mean k S.E.M.) are expressed as a percentage of the previous contraction induced by 75 mM K + . Number of the segments used is shown in parentheses. *P < 0.05; **P < 0.01 with regard to segments with endothelium; +P < 0.05; ++P < 0.831with regard to segments from rats of 5 weeks [233].

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7.2. Presynaptic a,-adrenoceptors

It was previously commented that presynaptic cr,-adrenoceptors negatively regulate the neuronal release of NA. This indicates that an increase or decrease in the sensitivity of these receptors may result in a decrease or increase in NA release, respectively. A reduction in the sensitivity of presynaptic a,-adrenoceptors with aging has been described in different tissues, such as rat isolated heart [257], pithed

*Ooo 1 *A-_ 1500 1

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Fig. 1I. Effect of age on the [‘H]NA release induced by electrical field stimulation (200 mA, 0.3 ms, 4 Hz for 1 min) from rat mesenteric arteries from SHR and WKY of different ages preincubated with this tritiated amine. The arteries are subjected to five successive electrical stimulation periods [Sl-S5] separated by an interval of 30 min. Only the means are indicated to simplify the figure. Results (mean k S.E.M.) of electrical stimulation-induced tritium release are expressed in dpm/mg of tissue. The values obtained at 5 weeks were significant (ANOVA) with regard to those found at the rest of ages in SHR strain, but only at 3 months in WKY strain; n = 5-9 [263].

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Yohimbine (log M) Fig. 12. Effect of age on the [7H]NA release induced by electrical field stimulation (200 mA, 0.3 ms, 4 Hz for I min) from rat mesenteric arteries from SHR and WKY of different ages preincubated with this tritiated amine and exposed to different concentrations of phentolamine and yohimbine and to five succe:ssive electrical stimulation periods [Sl-S5] separated by an interval of 30 min. Resuits (mean f. S.E.hl.) of electrical stimulation-induced tritium release are by the ratios Sx/Sl (SX = S2, S3, S4 and S5); *P c: 0.05, with respect to 5 weeks, n = 6-14 [263].

rat heart [258], rat vas deferens [259], rat cerebral cortex [260] and rat tail artery [253,261,262], but not in human saphenous vein [259]. In our laboratory, we observed that [‘H]NA release from rat mesenteric arteries induced by electrical stimulation decreases with maturation in SHR, whereas it is practically unaltered in the same arteries of WKY rats [263] (Fig. 11). The reduction in the sensitivity of presynaptic a,-adrenoceptors with aging may compensate for the decrease on [3H]-NA release in rat mesenteric arteries [263] (Fig. 12), as the NA release would be facilitated. The decreased responsiveness of presynaptic cr,-adrenoceptors, and the resultant enhancement in the NA release, may explain the increased plasma NA levels in elderly, and consequently the blood pressure increase with age [253].

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7.3. Neuronal uptake

The effect of aging on the NA uptake into adrenergic nerve endings is not clear, because an increase or a decrease in neuronal uptake has been reported [7], although there is some evidence suggesting that it may decline with age, at least in some tissues [7,254,258]. A reduction in the density of adrenergic nerves, previously discussed, or an enhancement of the distance between the nerve terminals and the cardiovascular adrenoceptors [264], probably due to the morphological alterations produced by aging, may be the reason for the reduced neuronal NA uptake. A reduced NA uptake results in an enhanced rate of spillover and a reduced rate of clearance from the plasma [265]. The increased plasma levels of NA in the elderly may be explained by a reduced neuronal uptake [I ,7]. 7.4. Plasma catecholamine concentrations The plasma NA levels in humans originate from adrenergic synapse spillover [162,266], mainly from the vessels of skeletal muscle [l], but also from lungs, heart, and kidneys [ 162,267]. Aging usually produces an increase in human plasma levels of NA both at rest [7,145,268,269], and in response to physiological stress [145,270,271]. Adrenaline released from the adrenal medulla may be higher in young than old individuals [l]. In addition, the effect of age on adrenaline plasma levels is not clear, because both an increase [271] or no change [145,268] have been reported. NA spillover into the urine and elsewhere tends to be lower in young than in older subjects [162,272]. In the rat, plasma levels of both NA and adrenaline have been reported to be increased with age [273]. The elevated plasma NA concentration in elderly subjects may be due to [1,146,162,274] (Fig. 10): (1) an enhancement of transmitter release per impulse, probably due to a reduction in presynaptic a,-receptor activity (previously discussed), although an attenuated influence of stimulatory presynaptic P-receptors would tend to antagonize such a mechanism; (2) a reduced transmitter uptake, with more NA remaining in the synaptic gap - most experimental findings support this view [162,266,274], and (3) a reduced turnover rate of circulating plasma NA [275,276], especially when regional blood flow levels and renal excretion are low. All these facts suggest that the average of sympathetic discharge is increased more in older than in younger subjects, and that the mechanisms involved are compensatory sympathetic adjustments to antagonize the reduction of effector responses in aging. 8. Age-related changes in blood pressure and in different vascular beds 8.1. Blood pressure changes

The incidence of hypertension increases in the elderly (30-65% of this segment of the population), and isolated systolic hypertension is frequent (about 18% of those over 80 years) [10,11,277]. The older hypertensives have twofold or more risk of

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cardiovascular complications than younger hypertensives [2,11], and an elevated incidence of several associated diseases [2]. The older normotensive subjects possess low renin and low serum calcium, and the latter finding correlates with the development of hypertension [27X]. The alterations in viscoelastic properties with the associated increase in vascular stiffness participate in the increase in both systolic arterial pressure and blood pressure with age [2,36-391. The enhancement in resting vascular resistance with age also contributes to this increase [l]. Hypertensi’on generally appears to increase cardiovascular aging [20,24]; this process is reversed to some degree by antihypertensive treatment [279]. In elderly subjects, there is usually an increase in plasma NA concentration (discussed earlier) [1,146,162] due to a decrease in presynaptic a,-receptor activity and transmitter uptake [162,266,274], associated with a reduced turnover rate of circulating plasma NA [275,276] (Fig. 10). In addition, in humans, a marked reduction in the baroreflex effects on vascular beds frequently participates in the blood pressure changes with age [1,280,281]. The baroreflex, mediated by baro- and volume-receptors, functions by means of efferent hormonal links, mediated by P-adrenergic stimulation of renin release [282], and subsequent angiotensin formation and aldosterone release [l]. In the elderly, there is also a certain reduction in blood volume that probably contributes to deficiencies in cardiovascular reflex regulation associated with the treatment with diuretic drugs, changes in body position, accidental fluid losses, etc. [l]. 8.2. Cerebrovascular bed In aging, there is normally a small blood flow reduction per unit tissue weight, which is associated with a reduced cerebral metabolism, probably due to age-dependent losses of neurons [l]. It should be noted that local blood flow in the brain is coupled with metabolic demand [283], probably by both chemical and neural mechanisms [284]. Th.e structure and mechanics of brain vessels appear to be altered with aging, which predisposes to stroke [285]. The ratio collagen/elastin is enhanced in rat internal carotid artery and decreases the distensibility [32]. A similar phenomenon occu’rs in human vertebral and basilar arteries [286]. Recently, it has been reported that, during aging, rat cerebral arterioles undergo atrophy, and a reduction of distensibility as well as of distensible elements, such as elastin and smooth muscle [287]. Cerebral microvessels from aging rats show ultrastructural changes due to degeneration processes or to perivascular deposition of collagen fibrils, proteins debris, etc. Chronic treatment with nifedipine (16-30 months) strongly reduces aberrant perivascular deposits of fibrillar and basement membrane material without modification of degeneration process of pericytes [288,289]. By means of positron emission tomographic images of regional cerebral blood (rCBF) achieved in 30 normal volunteers, an age-related decrease in adjusted rCBF in thee cingulate, parahippocampal, superior temporal, medial frontal, and posterior parietal cortices bilaterally, and in the left posterior prefrontal cortices was demonstrated. Such reductions suggest a regionally specific loss of cerebral function with

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age. The affected areas were all limbic, or associated, cortices. Therefore, these decreases may constitute the cerebral substrate of the cognitive changes that occur during normal aging [290]. Another study performed in healthy volunteers showed that the regional metabolic rate of oxygen decreases with aging, especially in the left caudate region, although both CBF and oxygen extraction fraction were variable and less age-dependent [291]. These authors conclude that regional metabolic rate may be reflecting healthy brain aging more properly. The autoregulation of CBF flow may be less rapid and marked in the elderly [I]. Furthermore, if the aged subjects present proximal arterial obstructions, which are compensated by distal autoregulatory dilatation, the autoregulation is greatly decreased [ 11. There are numerous pathological problems in the elderly, among them is reduced cerebral perfusion, which leads to a decrease in both mental and physical functions. This decrease is essentially due to the poor cerebral autoregulation associated with cerebral atherosclerosis; in addition, in hypertensive patients, the upper and lower limits of autoregulation tend to be shifted upward [14], essentially in senescence, in which small reductions in blood pressure may induce significant falls of cerebral blood flow [l]. The influence of age on isolated cerebral vessel reactivity has also been studied. Thus, cerebral arteries from beagles of different ages were exposed to the contractile agents NA, 5-HT, histamine and angiotensin II, and also to nicotine and electrical stimulation that evoke relaxant responses [80]. The results suggest that the sensitivity or the quantity of a,-adrenergic and H,-histaminergic receptors is high in the cerebral arteries of infant beagles and declines with age; the affinity of serotonergic receptors also decreases with age, whereas the biosynthesis of PGI, in the vascular wall appears to increase, maintaining Na+ pump activity [80]. Similar experiments performed in helical strips of basilar arteries from rabbits show that responses to contractile agents and Na + pump activity are increased with the age. Nevertheless, the relaxation to isoproterenol is reduced, and unaffected those elicited by adenosine and prostaglandin E, [103]. Analogous experiments achieved in cerebral arterial strips from monkeys of different ages, suggest that adrenergic nerves innervating immature monkey cerebral arteries contribute to the regulation of vascular tone predominantly over nonadrenergic, noncholinergic vasodilator nerves, whereas the vasodilator nerves play a major role in the mature monkey arteries. a,-Adrenoceptor subtype appears to be mainly involved in the NA-induced contraction of young monkey cerebral arteries, as it is in adult monkey arteries [l 111. Human basilar arteries show a reduction with aging of the endothelium-dependent relaxation induced by thrombin, but the concentration needed to reach 50% maximum relaxation is unaffected, and the maximum relaxation in response to bradykinin, calcium ionophore A23187, and sodium nitroprusside is also unaffected [292]. In addition, cerebral arterioles of aged rats show a reduced relaxation to ACh, ADP and bradykinin, whereas the vasodilations in response to nitroglycerin are similar in adults and aged rats [153]. Thus, dilator responses of cerebral arterioles to agonists that may release EDRF are altered in aged compared with adult rats. Impaired vasodilation in aged rats does not appear to be related to

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production of a cyclooxygenase constrictor substance [153]. Recently, it has been reported that the endothelium-dependent relaxation induced by ACh is reduced with age in female but not in male rabbits [121]. 8.3. Pulmonary vascular bed The pulmonary vessels have a thin wall, as corresponds to the low pressure of pulmonary circulation, tolerate marked hydrostatic pressure differences between apex and base, and possess important elastic properties [l]. Aging produces changes in the compliance of the pulmonary vasculature, as occurs in systemic vasculature, with functional consequences [l]. 8.4. Coronary vascular bed Humans coronary arteries are usually afflicted by atherosclerotic disturbances, which alter the coronary circulation, producing an increase in coronary resistance that compensates for the reduction in nutritional needs of the elderly myocardium

VI. To,da et al. [293] observed, in helical strips of beagle coronary arteries precontracted with prostaglandin Fz,, that NA, adrenaline, electrical stimulation and tyramine produce contractions at low drug concentrations or low electrical stimulation frequencies, and relaxations at higher concentrations and frequencies, which are mediated by a,-adrenoceptors and a-adrenoceptors, respectively. They also observed that the contractions mediated by a,-adrenoceptors decrease with age, whereas P-adrenoceptor-mediated relaxations increase with age. They found that the functions of a,-adrenoceptors appear to be in the order of large > medium > small-size coronary arteries from beagles of different ages, and that B-receptor funct:ions are in reverse order. This indicates that the use of P-blockers could be dangerous in older individuals due to the reduction of the vasodilator /?-tone in coronary circulation, especially in some angina1 patients. In human coronary arteries, the endothelium-dependent dilation evoked by ACh may be decreased with aging [294]. In addition, in isolated pig coronary arteries, NA causes endothelium-dependent relaxation in the presence of propranolol [142]. The maximum relaxation was significantly greater in the young than in the aged group, which is inhibited by yohimbine. However, the relaxations elicited by substance P, bradykinin, and nitroglycerin are not significantly different between the young and the aged group. Thus, vascular aging may affect the sympathetic regulation of the coronary arterial tone by attenuation of endothelium-dependent relaxation to catecholamines via cr,-adrenoceptors [ 1421. Acknowledgments This work was supported by Grants from D.G.I.C.Y.T. (FAR91-0205 and PM9;!-0037) and Bayer Espaiia, and it is ascribed to the Biomed Project No. BM Hl-CT94-1375 of E.E.C.

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