Background and Purpose Aging is associated with a reduction in cerebral perfusion. Impaired vasodilatation in large brain arteries could be implicated. This study sought age-related changes in vasodilator responses to norepinephrine in rat basilar artery and investigated which aspects of norepinephrine’s action are responsible. To study the effect of aging per se, we used the rat, an animal with resistance to development of age-related pathologies.
Methods Vascular responses were studied in basilar arteries from young (3 to 4 months old) and old (20 to 22 months old) normotensive Sprague-Dawley rats with wire myography. Endothelial structure was assessed with confocal microscopy.
Results There was no age-related difference in blood pressure and in KCl or 5-hydroxytryptamine (5-HT) contractions. Relaxation to bradykinin or its absence predicted an intact or denuded endothelium, confirmed by confocal microscopy. Norepinephrine produced concentration-dependent relaxation that was significantly smaller in old rats, with or without endothelium. This response was significantly smaller in endothelium-denuded vessels, or after preincubation with NG-nitro-l-arginine methyl ester or propranolol, but not with rauwolscine.
Conclusions In old and young rats the vasodilator action of norepinephrine in basilar artery is dependent on β-adrenoceptors and nitric oxide. The impaired vasodilatation to norepinephrine found in the basilar artery from old rats might be caused by (1) a reduction in nitric oxide production and/or release or (2) β-adrenoceptor alteration at the endothelium and/or the vascular smooth muscle. This impairment of vasodilator function can be ascribed to the aging process per se and not to other age-related alterations, such as hypertension.
Aging is associated with an increased incidence of cerebrovascular disease. With age, there is an increase in cerebrovascular resistance, an upward shift in the lower limit of CBF autoregulation, and a reduction of hypercapnia-induced relaxation.1 These alterations contribute to the reduction in cerebral perfusion in the elderly, leading to a deterioration of mental and physical functions.2 3
Endothelial-mediated relaxations have been shown to be impaired in several vascular beds from aged animals, possibly because of a defect in the release and/or production of relaxing factors, mainly NO. NO plays a major role in the regulation of cerebral circulation4 and there is some evidence of an association between a defect in NO production and the aging process. Hypercapnia-induced relaxations, which are mediated by NO,4 5 have been shown to be decreased during aging,1 and there is also some evidence of an alteration in endothelium-dependent, but not endothelium-independent, relaxations in brain vessels. Reduced relaxation to acetylcholine, ADP, and BK has been described in the cerebral microvasculature.6 In large arteries, responses to thrombin but not other NO-mediated vasodilators, such as BK7 or calcium ionophore A23187,4 are affected by aging.
NO can be released by NE,8 as well as by a wide range of vasodilator agents and other constrictor amines, through stimulation of endothelial α2-adrenoceptors.9 10 In addition, NE might induce vasodilatation through endothelial11 12 or SMC β-adrenoceptors,13 and decreased β-adrenoceptor–mediated responses have been described in several vascular beds during aging.3 14 15
In brain vessels the picture is incomplete; it is not clear whether there is a general impairment in NO-mediated relaxations with aging, and the possibility of an alteration in β-adrenoceptor–mediated responses has not been fully explored. In addition, aging is usually accompanied by other cardiovascular alterations such as hypertension and/or atherosclerosis,3 also associated with an impairment in NO-mediated responses,4 making it difficult to separate the contribution of each disease.
The aim of this study was to determine the effect of aging on vasodilator responses to NE in the rat basilar artery. Endothelium-dependent and -independent mechanisms were evaluated, and the possible alteration of β-adrenoceptor–mediated relaxation, which is predominant in the basilar artery,12 was studied. We chose the rat as the experimental animal in order to be able to ascribe the vascular alterations to the aging process per se and not to age-related diseases, such as atherosclerosis or hypertension, because the rat has been shown to have a natural resistance to the development of age-related pathologies.16
Materials and Methods
Twenty-nine young (3 to 4 months old) and 29 old (20 to 22 months old) male Sprague-Dawley rats were used in the study. Nine old and 9 young rats were used for blood pressure measurements. The rats were anesthetized with sodium pentobarbital (60 mg/kg, IP). The arterial blood pressure was then measured using a Statham transducer (Hewlett-Packard recorder, model 7785A) connected to a cannula placed in the right carotid artery. Heart rate was derived from the arterial pulse. Blood pressure and heart rate values used for comparison between old and young animals were obtained after a 15-minute stabilization period. The rest of the animals were euthanatized by decapitation with a guillotine. The procedures followed were according to institutional guidelines. The brain was removed, it was placed in PSS, and the basilar artery was carefully dissected for wire myography.
Drugs and Solutions
All experiments were performed using PSS of the following composition (in mmol/L): NaCl 118.4, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, glucose 11.1, and the disodium salt of ethylenediamine tetraacetic acid (Na2EDTA) 0.023. The solution was bubbled with 95% O2-5% CO2 to give a pH of 7.4 at 37°C. The 125 mmol/L KCl solution was prepared by substitution of 125 mmol/L KCl for NaCl in PSS. Drugs used were NE bitartrate, BK, SNP, (−) isoproterenol (+) bitartrate salt, D-L propranolol HCl, L-NAME, 5-HT creatinine sulfate, and Rauw hydrochloride; all were purchased from Sigma Chemical Co. Stock solutions of NE were prepared in 0.023 mmol/L Na2EDTA. The rest of the drugs were dissolved in distilled water. The stock solutions were kept frozen and appropriate dilutions were made every day except for L-NAME, which was prepared daily.
Twenty rats from each age group were used for the functional study. Two segments of the basilar artery (2 mm length), one from an old and one from a young rat, were mounted simultaneously on a wire myograph (Myo-interface, model 410A, JP Trading). Briefly, two 40-μm tungsten wires were threaded through the lumen of the segment. One wire was attached to the stationary support driven by a micrometer, and the other was attached to an isometric force transducer and this in turn to a chart recorder (Linseis L6512B, Belmont Instruments) to measure force development. From each vessel, the resting tension–to–internal circumference ratio was determined and, with assumption of the Laplace relationship, the vessel was set to a normalized internal circumference of 0.91100, where 1100 is the internal circumference of the vessel under an effective transmural pressure of 100 mm Hg. After a 30-minute equilibration period, the vessels were exposed twice to 125 mmol/L KCl (osmotically corrected) to determine contractile function. The relaxant responses to agonists were determined in vessels precontracted with a submaximal concentration of 5-HT (0.1 μmol/L). To study the effects of blockers, they were previously incubated for 30 minutes, except for L-NAME, which was incubated for 60 minutes. Since preincubation with rauwolscine reduced the vasoconstriction to 0.1 μmol/L 5-HT, 0.3 μmol/L 5-HT was used to elicit the same level of contraction induced in the absence of the antagonist. Those vessels that did not contract to KCl or did not reach a stable contraction to 5-HT were discarded.
Test for Endothelial Function and Structure
The functional state of endothelium was tested at the beginning of the experiment by the ability of BK to elicit vasodilator responses. The vessels were precontracted with 0.1 μmol/L 5-HT and then relaxed with 3 μmol/L BK. A wide range of responses to BK was found in both age groups. In segments from young rats the responses ranged from 69% to 0% relaxation and in those from old rats from 42% to 0% relaxation. Segments that showed no detectable relaxation were considered endothelium denuded (5 from young and 7 from old rats). To select a comparable group of segments with functional endothelium in both age groups, we selected the 50% of vessels with the largest BK responses (9 from old and 9 from young rats). The state of the endothelial layer was further determined with an LSCM (Odyssey, Noran Instruments). At the end of the experiment the segments were fixed under tension with formalin 10% saline solution for 30 minutes. They were then stained for 30 minutes with the nuclear dye propidium iodide (10 μmol/L) and washed overnight in distilled water. Endothelial and SMC layers were observed with a ×40 oil immersion objective (NA 1.3, Nikon), using the argon-ion 529-nm line with a 550-nm long-pass barrier filter of the LSCM. Metamorph software (Universal Imaging Corporation) was used for image acquisition and analysis. The presence or absence of endothelium and the state of the SMC were then assessed by blind analysis of the images.
Statistics and Data Analysis
Vascular responses were calculated in active wall tension (mN/mm). Relaxations to drugs were calculated as percentage of previous contraction to 0.1 μmol/L or 0.3 μmol/L 5-HT. Sensitivities to drugs were determined in terms of pD2 value (negative logarithm of the concentration required to produce half-maximal response, pD2= –log10 EC50), and EC50 values were calculated by computer extrapolation from individual logarithm concentration-response curves. Results are expressed as mean±SEM and n denotes the number of animals used in each experiment. Student’s t tests were used in the statistical comparison of maximal responses and pD2 values. Deviations from the mean regarding the curves were statistically analyzed with two-factor ANOVA for unpaired experiments and two-factor repeated measures ANOVA for paired experiments.
Body weight was significantly increased in old rats. Systolic, diastolic, and mean arterial pressures were similar between old and young animals, whereas pulse pressure was significantly increased in old animals. Heart rate was significantly reduced in old rats (see Table⇓).
In the presence of functional endothelium, indicated by relaxation to BK and confocal microscopy (see below), contractile responses to a depolarizing solution of 125 mmol/L KCl were similar in young (3.92±0.27 mN/mm, n=9) and old (3.26±0.40 mN/mm, n=9) rats. KCl-induced contractile responses were also similar in vessels without functional endothelium in young (3.92±0.37 mN/mm, n=5) and old (3.09±0.07 mN/mm, n=7) rats.
The vasoconstriction induced by 0.1 μmol/L 5-HT was also similar at both age points in segments with endothelium (young, 1.75±0.25 mN/mm, n=9; old, 1.67±0.25 mN/mm, n=9) and without endothelium (young, 1.44±0.18 mN/mm, n=5; old, 1.45±0.22 mN/mm, n=7). The absence of endothelium made no difference within either age group.
In the presence of 1 μmol/L rauwolscine, the contractions induced by 0.1 μmol/L 5-HT were significantly reduced, in both young (control tissues, 1.60±0.25 mN/mm; +Rauw, 0.44±0.16 mN/mm, n=6, P<.01) and old (control tissues, 1.52±0.34 mN/mm; +Rauw, 0.48±0.19 mN/mm, n=6, P<.05) rats. Therefore, to induce a level of contraction similar to that obtained in the absence of the antagonist, 0.3 μmol/L 5-HT was needed (young, 1.73±0.23 mN/mm, n=6; old, 1.48±0.28 mN/mm, n=6).
Preincubation with propranolol or L-NAME did not affect 5-HT–induced vasoconstrictions (data not shown).
Endothelial Structure and Function
Blind LSCM analysis of the endothelial and SMC layers showed that arteries with the largest responses to 3 μmol/L BK had a well-preserved endothelial layer (Fig 1B⇓) and, therefore, were considered to have structurally intact endothelium. This was observed in both young (top image) and old (bottom image) rat basilar arteries. Vessels that did not relax to BK showed complete endothelial denudation or very few scattered endothelial cells (Fig 1C⇓). Vessels with intermediate responses to BK showed various degrees of endothelial denudation (data not shown). Endothelial denudation did not affect the SMC layer structure (Fig 1A⇓). This was also observed in both young (top images) and old (bottom images) rats. There was no difference in the structure of the nuclei between vessels from old and young rats, and endothelial and SMC nuclei both had similar shape, size, and normal orientation (Fig 1⇓).
Fig 2A⇓ shows the distribution of responses to 3 μmol/L BK in all the segments from young (n=20) and old (n=20) basilar arteries. This included intact and denuded segments, as well as those partially denuded, which were discarded. The responses to 3 μmol/L BK were significantly smaller in old rats. This difference was observed in segments with the largest responses to BK, which showed intact endothelium (young rats, 51.4±3.6, n=9; old rats, 31.3±2.3, n=9; P<.01), and when the segments with partial endothelial denudation were included (young rats, 36.2±4.1, n=15; old rats, 24.3±2.8, n=13; P<.05).
Concentration-Response Curves to BK and SNP
A further concentration-response curve to BK (0.1 nmol/L to 3 μmol/L) was performed on endothelium-intact segments. Maximum responses to BK were significantly smaller in old rats (Fig 2B⇑), whereas pD2 values were similar at both age points (young, 6.10±0.29, n=7; old, 5.99±0.07, n=7).
SNP elicited concentration-dependent vasodilator responses that reached almost 100% of the previous contraction to 5-HT and were similar in segments with or without endothelium (data not shown). There was no significant difference between young and old rats, either in the maximum response (Fig 2C⇑) or in the sensitivity (young pD2, 6.97±0.14, n=6; old pD2, 6.67±0.14, n=6).
Responses to NE and Isoproterenol
NE did not elicit vasoactive responses in basal conditions. When the vessel tone was raised with 0.1 μmol/L 5-HT, NE induced concentration-dependent relaxations, which were significantly smaller in vessels without endothelium in both young and old rats (Fig 3⇓).
Endothelium-intact segments from young rats reached a significantly greater maximum relaxation to NE (74.49±4.1%, n=9) when compared with old rats (31.6±3.9%, n=8, P<.01). In addition, endothelium-intact basilar arteries from young rats had significantly greater pD2 values than old animals (young, 6.13±0.12; old, 5.51±0.15; P<.01).
In endothelium-denuded segments from young rats, NE also elicited significantly greater maximum responses (25.24±2.4%, n=5) than those from old animals (6.17±2.25%, n=7, P<.01). Similar sensitivity to NE was observed in young (5.57±0.10) and old (5.36±0.16) rat basilar arteries in the absence of endothelium.
The effects of L-NAME, propranolol, and rauwolscine on NE- or isoproterenol-induced relaxations were analyzed in vessels with endothelium.
L-NAME (0.1 mmol/L) induced small sustained contractions that were similar in young (0.74±0.47 mN/mm, n=5) and old (0.82±0.59 mN/mm, n=5) rats. Preincubation with the NO-synthase inhibitor significantly reduced the maximum dilator responses to NE in both age groups (Fig 4A⇓ and 4B⇓). A decrease in sensitivity was also observed, in both young (NE, 6.08±0.20; NE+L-NAME, 4.75±0.02; n=5; P<.05) and old (NE, 5.14±0.24; NE+L-NAME, 4.69±0.001; n=5; P<.05) rats.
In segments with endothelium preincubated with 0.1 mmol/L L-NAME and precontracted with 0.1 μmol/L 5-HT, 30 μmol/L isoproterenol induced relaxation. The responses to the β-adrenoceptor agonist were significantly reduced in old animals (Fig 4C⇑).
In some segments from both groups of rats, 1 μmol/L propranolol induced small contractile responses in basal conditions (young, 0.36±0.10 mN/mm, n=3 of 6; old, 0.33±0.004 mN/mm, n=3 of 6). In the presence of the β-adrenoceptor antagonist the NE-induced relaxations were significantly reduced in both age groups (Fig 5⇓). In some segments the vasodilatations were transformed into contractions (Fig 5⇓).
Rauwolscine was without effect on the NE-induced relaxations. Neither the maximum responses (Fig 6⇓) nor the sensitivity (pD2) were modified in young (NE, 5.94±0.16; NE+Rauw, 5.67±0.14; n=7) or in old (NE, 5.42±0.10; NE+Rauw, 5.40±0.13; n=5) rats.
CBF is regulated by changing the cerebral arterial caliber, and cerebral arteries are expected to respond differently to chemical stimulation in young and aged animals.17 Our study shows that the vasodilator responses to NE and BK are reduced in old rats. Both responses require the participation of an intact endothelium and the ability of the tissue to synthesize NO. The age-related impairment in vasodilatation cannot be entirely attributed to endothelial malfunction, since there is some evidence for a deficit in SMC β-adrenoceptors and the dilatation that they mediate. There is a possibility of two sets of action for β-adrenoceptor activation in basilar artery: in SMCs and in endothelium. Changes in both with aging deserve further consideration.
There are some reports of rats becoming hypertensive with age.18 However, as previously described in rats from other strains—Ivanos,19 Wistar,20 and Fischer 4431 —the Sprague-Dawley rats used in this study remained normotensive at old age. Therefore, we can attribute the observed vascular alterations entirely to effects of aging rather than to hypertension, a risk factor related to aging and also associated with reduced vasodilator function. However, pulse pressure was significantly increased in old rats, as previously described in Wistar rats.20
Heart rate was significantly reduced in old Sprague-Dawley rats in our study. In anesthetized rats, resting heart rate has been reported to decrease during maturation and to decrease or to be unchanged by aging.21 The same phenomenon has also been reported in humans; young subjects have maximal heart rates of 180 to 200 beats/min, whereas heart rate is reduced to 140 to 160 beats/min around 70 years of age. This alteration has been partially attributed to the attenuation of tachycardic responses to β-adrenergic stimuli with aging.22
The present study found no differences in the contractions to 5-HT or KCl between old and young rats, suggesting no important changes in the contractile machinery during aging in the basilar artery. Alterations in responses to vasoconstrictor agents in old animals have been found in some vascular beds. However, this tendency is not uniform and it depends on species, vessel studied, and vasoconstrictor agent.3 In rabbit basilar arteries, no changes in the responses to NE have been found23 ; in dog cerebral arteries, contractions to NE are decreased, whereas KCl responses are increased during aging.17 It is important to take into account that some of the alterations in contractile responses observed with age could be caused by changes in the endothelium, which is an important modulator of vascular tone, and not related to a SMC dysfunction. Maximum contractile responses to 5-HT have been found to be markedly enhanced in canine basilar arteries after endothelial damage.24 In addition, Hajdu and coworkers25 showed that intra-atrial infusion of 5-HT reduced CBF in aged but not in adult rats, and suggested that an impaired endothelial function during aging was responsible for the augmented vasoconstrictor responses to the amine. In the rat basilar artery, we did not find differences in 5-HT contractions between segments with and without endothelium or between old and young rats. Our results with 5-HT might be explained by the low standard concentration used, which is needed for the study of the vasodilator function, and it is possible that differences in 5-HT responses could be observed at higher concentrations that induce maximal contractions.
The present work supports the hypothesis of an impairment of vasodilatation in the rat basilar artery during aging. This is likely to be at least partially caused by endothelial dysfunction. Vessels from old rats relaxed less to a maximum concentration of BK. BK-induced relaxations are endothelium dependent and mediated by NO in large cerebral arteries4 ; therefore, an impairment of NO production and/or release or access to the SMC could be responsible for the observed impairment in vasodilator function. A defect in guanylate cyclase activity or in further steps of the pathway, previously reported in mesenteric arteries from aged rats,26 is unlikely, because SNP, which vasodilates via a mechanism independent of NO release,27 elicited similar responses in basilar arteries from young and old rats. This result also demonstrates that SMC function was not impaired by aging in relation to induced relaxation.
In the absence of tone, NE did not elicit vasoactive responses in the basilar artery, and in preconstricted vessels, it induced concentration-dependent vasodilations. Only in the presence of the β-adrenoceptor antagonist propranolol did NE elicit a small vasoconstrictor effect. These results suggest a very limited α-adrenergic contractile response, as expected from an artery from the caudal region of the brain where β-adrenoceptor–mediated dilatation has been shown to predominate over α-adrenoceptor–mediated contraction.12 28 Propranolol practically blocked NE-induced relaxations, whereas rauwolscine produced no significant effect, in either the pD2 values or maximal responses. These results demonstrate that under the conditions used, the receptors involved in the vasodilator responses to NE in the basilar artery are mainly of the β-adrenoceptor subtype and rule out the possibility of an α2-adrenoceptor component, confirming the results from in vivo studies.29 Another study of wire myograph–mounted rat basilar artery, in which high KCl concentrations were used to induce tone, found a component of NE-induced vasodilatation that was resistant to propranolol and blocked by adding phentolamine.12
The reduction of contractile responses to 0.1 μmol/L 5-HT in the presence of rauwolscine can be explained by blockade of serotonergic receptors, mainly of the 5-HT1D subtype, by this α2-adrenoceptor antagonist, as has been previously reported.30
As previously shown by Hempelmann and Ziegler,12 NE-induced relaxations were greatly reduced in denuded segments from the basilar artery, suggesting that these responses are partially dependent on the presence of endothelium. The action of propranolol shows that the receptors implicated are likely β-adrenoceptors. There is evidence from autoradiographic studies of β-adrenoceptors associated with the endothelium11 and of their ability to release NO upon stimulation.31 NO is likely to be the mediator of the endothelial component of the NE-induced relaxations, because preincubation with L-NAME, an inhibitor of NO production, reduced the NE-induced responses. Our results differ from a previous in vivo study in rat basilar artery in which L-NAME did not have an effect on the NE-induced relaxations.29 The explanation for this difference could be the lower concentration of L-NAME used (10−5 mol/L) compared with ours (10−4 mol/L) and the shorter period of incubation (15 minutes versus 60 minutes), which might not have been able to block NO production completely. An antagonist effect of L-NAME on β-adrenoceptors, as previously described for muscarinic receptors,31 is not likely to occur, because the reduction of isoproterenol-mediated relaxations by L-NAME has been shown to be reversed by l-arginine14 and therefore caused by NO inhibition.
Our data fit the classic mechanism described for endothelium-derived NO-dependent relaxations. In addition to its role as an active vasodilator, NO may also be a permissive agent to SMC receptors in some systems, as described in rat middle cerebral arteries for α2-adrenoceptor–mediated relaxations.10 In this case, SMC α2-adrenoceptors are linked to a pertussis toxin–sensitive G protein, which upon activation reduces cytoplasmic calcium and allows for relaxation. The authors postulate that somewhere along the pathway between α2-adrenoceptor stimulation and calcium decrease, critical concentrations of cGMP produced by basal NO act permissively to allow the process to proceed. Thus, endothelial α2-adrenoceptors need not be involved. For endothelial β-adrenoceptors the classic mechanism has been suggested and it has been demonstrated that upon activation they stimulate NO release through a cAMP-dependent pathway.32 To the best of our knowledge there is no documented discussion of a permissive role of NO linked to SMC β-adrenoceptors. It is possible that the relaxations to NE observed in our study are mainly due to an action of NO, as postulated in the classic mechanism. However, a permissive role of NO linked to SMC β-adrenoceptors must also be taken into account as a possible explanation of our results. The likelihood of SMC β-adrenoceptors was emphasized by the large relaxation of isoproterenol even in the presence of L-NAME.
NE-induced relaxations were significantly reduced in the basilar artery from old rats. The effect of propranolol shows that these responses are mediated by β-adrenoceptors, suggesting an impairment of β-adrenoceptor vasodilator responses in the basilar artery during aging, as previously described in other vessels from different species.3 14 15 However, the question remains, Is the β-adrenoceptor deficit at the endothelial level, SMC level, or both? In old rats, the maximum responses to NE were decreased in segments with or without endothelium. Thus, if there is a change at a single site, this is more likely to be the SMC. It is nevertheless possible that both endothelial and SMC β-adrenoceptors might be affected in old rats. An endothelial alteration that involves a general impairment in receptor-induced production, diffusion, and/or release of NO is supported by the reduction in BK-mediated relaxations, also involving NO synthesis. Basal release of NO is probably normal, since L-NAME–induced contractions, which reflect the basal level of NO, were similar in old and young rats.
In old rats, NE-induced relaxations were also reduced in deendothelialized and L-NAME–treated segments, suggesting a defect at the level of the SMC β-adrenoceptors. To confirm this alteration, isoproterenol, a more specific β-adrenoceptor agonist, was used to induce relaxation with no countervailing α-adrenoceptor–mediated effects. The segments were pretreated with L-NAME and therefore only SMC β-adrenoceptor responses were studied. In these experimental conditions, isoproterenol-induced relaxations were reduced in arteries from old rats, suggesting a defect at the level of SMC β-adrenoceptors.
Speculations on the Mechanisms Involved and Implications
The present study shows an impairment of vasodilator mechanisms in the basilar artery from old rats, and it suggests the possibility of a defect not only at the level of the endothelium but also in SMC β-adrenoceptors. On the basis of previous studies and our own findings, several explanations can be suggested.
With respect to the decrease in endothelium-mediated responses in old rats, a first hypothesis could be a decrease in NO production in the basilar artery. Previous studies have shown that the age-related decrease in endothelium-dependent relaxation in rat carotid artery is not caused by an impaired release of relaxing factors.33 However, this possibility cannot be ruled out in the basilar artery.
A second possible explanation is the implication of free radicals. This is supported by several facts. First, the aging process appears to be associated with the generation of an excess of oxygen-derived free radicals caused by the reduction of natural antioxidant defenses.34 Second, free radicals have been shown to mediate the impairment of acetylcholine relaxations induced by elevation of pulse pressure.35 In the rats used in our study there was a significant increase in pulse pressure with aging, as previously shown in other rat strains20 and also in humans,21 22 in which there is an age-related increase in systolic blood pressure in particular. Therefore, it is possible that the generation of oxygen-derived free radicals by the increased pulse pressure and a reduction in the free-radical scavenging process during aging are responsible for NO inactivation.
A third explanation for the alteration of the endothelium-dependent vasodilator function in basilar arteries from old rats is physical abnormality of the endothelium. Elongation and thinning of endothelial cells have been described in aortas from 24-month-old Wistar rats.36 In addition, it has also been suggested that intimal thickening that occurs during aging37 could form a barrier between endothelium and media, leading to a decrease of the passage of vasoactive endothelial factors in the case of normal NO production. However, we ruled this possibility out because our results with LSCM showed no difference in the shape, orientation, or distribution of the endothelial cell nuclei between basilar arteries from young and old rats. In addition, we found a normal basal NO function, as shown by a similar contraction to L-NAME at both age points. This suggests that in the rat basilar artery, diffusion of endothelial substances to the media is not impaired. The aging process is also associated with degenerative processes of the arterial wall, such as atherosclerosis, mainly affecting large vessels. Pathological studies of the cerebral vasculature of humans indicate that atherosclerosis along the vessels of the circle of Willis is increased with age.2 However, this vascular alteration is less likely to occur in the rat, which has been shown to have a natural resistance to the development of atherosclerosis compared with other animals.16 No atheromatous degeneration has been found in 24-month-old rat aortas,36 a vessel usually affected. Therefore, the impairment of vasodilator function observed in rat basilar arteries is not likely to be attributable to this degenerative age-related process.
With respect to the mechanism responsible for SMC β-adrenoceptor dysfunction observed in the basilar artery from old rats, a possible hypothesis could be a reduction in the number and/or affinity of the SMC β-adrenoceptors with increasing age. Desensitization and internalization of β-adrenoceptors can occur because of elevations in catecholamine concentration,38 and an increase in endogenous catecholamines has been described during aging.39 Our own results in old Wistar Kyoto rats support this hypothesis. In a previous study with LSCM we have shown a significant reduction of the binding of β-adrenoceptor fluorescent ligand CGP 12177 in SMC layers from 1-year-old Wistar Kyoto rat basilar arteries when compared with those from 4-month-old rats. In addition, a correlation between the decrease in CGP 12177 binding and NE-induced relaxations was observed,40 suggesting a reduction in the number of the SMC β-adrenoceptors in the basilar artery with increasing age.
In summary, we have found diminished relaxations in the basilar artery from normotensive old rats. Reduced NO production or release, as well as alteration in the number and/or affinity of β-adrenoceptors, could be responsible for this reduction. Our results are also compatible with a permissive model of endothelial influence on SMC β-adrenoceptors and suggest that the basis of the vasodilator deficit may reside in the interaction between both systems. These vascular alterations can be ascribed to the aging process independent of other cardiovascular age-dependent diseases, such as hypertension and atherosclerosis, and might be related to the increase in pulse pressure. The impairment of vasodilator responses in the basilar artery, involving NO, an important regulator of CBF, might play a role in the altered cerebrovascular function associated with aging.
Selected Abbreviations and Acronyms
|CBF||=||cerebral blood flow|
|LSCM||=||laser scanning confocal microscope/microscopic/microscopy|
|L-NAME||=||NG-nitro-l-arginine methyl ester|
|PSS||=||physiological saline solution|
|SMC(s)||=||smooth muscle cell(s)|
This work was supported by grants from the Medical Research Council (Clinical Research Initiative in Heart Failure (PG 9307850) and the European Community BIOMED Program (BMHI-CT94-1375). We thank Dr Jesús Giraldo for his help with the statistical analysis.
- Received March 17, 1997.
- Revision received May 6, 1997.
- Accepted May 16, 1997.
- Copyright © 1997 by American Heart Association
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