Numerous studies have been reported on agerelated

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1 889 Effect of Age on Blood Pressure and Membrane-Dependent Vascular Responses in the Rat Edward E. Soltis Alterations in endothelium-dependent, sodium pump-mediated, and calcium-dependent responses of vascular smooth muscle were investigated in 5-7-, , and week-old male Sprague-Dawley rats. Age-dependent changes in systolic blood pressure were also determined. Although systolic blood pressure increased significantly with age, rats in all 3 age groups were considered normotensive. Initial studies on the passive force-response characteristics of strips of aortic and femoral arterial smooth muscle revealed that the level of passive force required for maximum active tension generation increased with increasing age. Subsequent studies were carried out using optimum passive force requirements. Endothelium-dependent relaxations of aortic smooth muscle induced by acetylcholine and the calcium ionophore A23187 decreased significantly with increasing age. An age-dependent decrease in the contractile response of aortic smooth muscle to ouabain and potassium-free physiological salt solution (PSS) was observed. Potassium relaxation of femoral smooth muscle following contraction to norepinephrine (NE) in a potassium-free PSS was also significantly attenuated with increasing age. No age-related alterations in calcium sensitivity (in the presence of 10~ 7 M NE) or calcium relaxation (membrane stabilization) of femoral arterial smooth muscle was seen. These results show that endothelium-dependent and sodium pump-mediated responses are reduced in vascular smooth muscle of the rat with increasing age. However, no changes in calcium-dependent responses are apparent. These observations are discussed in relation to the vascular changes observed in hypertension. (Circulation Research 1988;62: ) Numerous studies have been reported on agerelated changes in the morphological and functional characteristics of vascular smooth muscle. Decreases in the elasticity and distensibility of vascular tissues with increasing age have been associated primarily with alterations in vessel collagen and elastin. 1 ' 2 Increases in both the medial and intimal layers of the vessel also occur and appear to be due to several factors such as increased smooth 'muscle proliferation 3 and the increases in collagen and elastin. Studies on functional alterations in vascular smooth muscle have dealt primarily with changes in adrenergic responsiveness. Perhaps the most common rinding is a decrease in /3-adrenergic-mediated relaxation with increasing age. 4 " 6 Reports on the contractile response of vascular smooth muscle to norepinephrine (NE) (as well as other agonists), however, have been conflicting. An increase, 7 a decrease, 8 and no change 9 have been reported. Similar discrepancies concerning the role of calcium in altered vascular function with increasing age have also been reported. 810 An area of vascular tissue research that has received much attention in recent years is that concerned with alterations in the membrane characteristics of the From the Department of Physiology, University of Michigan, Ann Arbor, Mich. Supported by National Institutes of Health grant HL E.E.S. is the recipient of an Individual National Research Service Award (HL-06998). Address for correspondence: Edward E. Soltis, PhD, Campbell University, Department of Pharmaceutical Sciences, School of Pharmacy, Buies Creek, NC Received January 7, 1987; accepted July 10, vascular smooth muscle in hypertension. In particular, alterations in the sodium pump" 12 and the membrane stabilizing actions of calcium 1314 have been proposed as important in the functional changes that occur in the vasculature in hypertension. More recently, the endothelium has been suggested to have an important role in modulating vascular smooth muscle responsiveness to both vasodilator and vasoconstrictor agents It has been suggested that many of the vascular changes that occur in hypertension represent an accelerated form of the changes that occur in aging. However, no complete studies have been reported on age-related alterations in the sodium pump, calcium stabilization, or endothelium-dependent responses of vascular smooth muscle. The present study was performed to determine whether age-related changes occurred in these membrane-related functions in vascular smooth muscle from the rat and to determine their possible relation to changes in blood pressure with aging. The results show dramatic changes in both sodium pump-mediated and endothelium-dependent responses but no alterations in calcium-dependent membrane properties with increasing age. Materials and Methods Animals and General Procedures Male Sprague-Dawley rats (Charles Rivers, Portage, Mich.) 5-7,24-26, and weeks of age were used in this study. Animals were housed in stainless steel cages in a room illuminated from 0600 to 1800 hours and maintained at 26± 1 C. All animals received a standard rat chow diet (Ralston Purina, St. Louis, Mo.)

2 890 Circulation Research Vol 61, No 6, December 1987 and tap water ad libitum. Body weights and systolic blood pressure, measured using a standard tail-cuff technique in conscious animals, were recorded at the time of killing the animal. Both femoral arteries and the thoracic aorta were quickly removed from ether anesthetized rats and placed in a physiological salt solution (PSS) at room temperature. After the vessels were cleaned of fat and connective tissue, one helical strip from each femoral artery (1 x 10 mm) and two from the thoracic aorta (2x15 mm) were cut under a dissecting microscope. The strips were suspended in a muscle bath containing oxygenated (95% O 2-5% CO 2 ) PSS maintained at 37 C. The upper ends of the strips were connected to a force transducer (FT 03 Grass, Quincy, Mass.), and the responses recorded on a Grass Model 7 polygraph. At the end of each experiment, the strips were removed from the muscle bath, and the wet weight was determined. Composition of the PSS was as follows unless otherwise specified (in mm): NaCl 130, KC1 4.7, KH 2 PO , MgSO 4-7H 2 O 1.17, CaCl 2-2H 2 O 2.5, NaHCO , dextrose 5.5, CaNa, EDTA Passive Force-Response Studies Strips of femoral artery and aorta were placed under an initial passive force of 20 mg and 50 mg, respectively. At the initial passive force, the strips were equilibrated for 60 minutes, and afterwards, they were stimulated with 50 mm KC1, a dose that results in a maximal contraction. The contractions were allowed to reach a plateau, and active force generation was recorded. The tissues were rinsed with fresh PSS, and the contraction was allowed to return to the baseline passive force. Passive force was then increased to 40 mg in femoral arteries and 100 mg in aorta. The strips were allowed to stabilize at this new passive force and then were stimulated again with 50 mm KC1. The responses were recorded, and the tissues were rinsed with fresh PSS. This procedure was repeated at passive forces of 80, 160, 300, 500, and 750 mg in strips of femoral artery and at 250, 500, 1,000, 1,500, 2,000, and 2,500 mg in strips of aorta to determine the passive force at which maximum active tension (milligrams of active force divided by the wet weight of the strip) generation occurs in the vessels from the three age groups. Each individual strip was set at its own optimum passive force for the remainder of the experiment. Endothelium-Dependent Responses Strips of aorta were contracted with a dose of potassium chloride that resulted in a 50% of the maximal potassium chloride response (ED 50 ). This value was determined from a potassium chloride doseresponse curve generated after the passive forceresponse studies. Average ED 50 values are given in Table 1 for the three age groups. No differences in ED 50 values or maximum active tension were observed. The contractions to potassium chloride were allowed to plateau, and cumulative dose-response curves to either acetylcholine (ACh; 10 ~9 to 10 ~5 M) or the calcium ionophorea23187(10"'to 10~ 6 M) were generated and percent relaxation from the initial potassium chloride contraction recorded. The tissues were exposed to each dose of ACh and A23187 for 1 minute, a period that allowed for a maximal maintained response. Sodium Pump-Mediated Responses Aortic strips were exposed to either increasing concentrations of ouabain (10~ 8 to 10~ 3 M) or a potassium-free PSS (potassium chloride deleted and equimolar NaH 2 PO 4 substituted for KH 2 PO 4 ). Increases in force generation were recorded every 10 minutes for a period of 60 minutes in the potassium-free PSS. Tissues were exposed to each dose of ouabain for 10 minutes during which active force generation was recorded. This time frame allows for a maximum and steady-state contractile response to each dose of ouabain up to and including the 3 x 10" 4 M dose. Maximum force generation in response to 10" 3 M ouabain occurs after minutes of exposure. 17 However, the response to 10~ 3 M ouabain was recorded after 10 minutes to keep exposure time the same for each dose. The relaxation response to potassium addition following a contraction to NE in a potassium-free PSS was also examined. Strips of femoral artery were incubated in a potassium-free PSS for 15 minutes, and afterwards, the tissues were exposed to an ED 50 dose of NE and the contraction allowed to plateau. The ED 50 dose for NE was determined in the same manner as that for potassium chloride, and the values are listed in Table 1. No differences in ED 50 values or maximum active tension generation were observed. A dose-response curve to potassium chloride (0.1 to 3.0 mm) was then generated and the percent relaxation recorded. The strips remained in contact with each dose of potassium chloride for 1 minute, a time period that allowed for a maximal maintained response. Calcium-Dependent Responses Calcium sensitivity and the membrane stabilizing actions of calcium were determined in strips of femoral artery. For calcium sensitivity experiments, the tissues Table 1. ED 50 Values for Potassium Chloride Contraction in Aorta and NE Contraction in Femoral Artery Age group (weeks) KC1 (mm) NE(M) 5-7 (n = 6) (n = 6) 15.2± 1.3 (221 ±30) 14.8±1.1 (199±21) 16.1 ±0.8 (208±28) 3.2[±1.2]xl0" 8 (339±35) 1.8[±0.5]x 10-8 (281 ±27) 2.4[±0.9]xl0" 8 (291±23) Values are mean ± SEM. Values in parentheses represent maximum active tension (mg force/mg wet tissue wt). NE, norepinephrine.

3 Soltis Aging and Vascular Smooth Muscle Responsiveness 891 were exposed to a calcium-free EGTA (1.0 mm) PSS for 10 minutes. After 5 minutes, the tissues were stimulated with NE (10~ 7 M) to deplete membrane stores of calcium. After the 10-minute period, the tissues were rinsed with a calcium-free PSS, and NE (10~ 7 M) was added to the muscle bath (no contraction occurred). A calcium dose-response curve (0.01 to 2.5 mm) was then obtained and force generation recorded. Maximal maintained contractions to each dose were obtained after 2 minutes of exposure. Calcium stabilization of femoral arteries was determined by contracting the strips of femoral artery with an ED 50 dose of NE followed by a dose-response curve to calcium (4.5 to 20.5 mm) and recording the percent relaxation. The vessels were exposed to each concentration for a 1-minute period, which allowed for a maximal maintained response. Drugs Norepinephrine hydrochloride, acetylcholine chloride, and ouabain octahydrate were purchased from Sigma Chemical Co., St. Louis, Mo. Stock solutions were made in distilled water. The calcium ionophore A23187 was purchased from Calbiochem and dissolved in dimethylsulfoxide (DMSO). Dilutions were made in distilled water. The final concentration of DMSO in the muscle baths was no more than 0.1%. Preliminary experiments on exposure to DMSO alone revealed no effect on vascular smooth muscle responsiveness. As /3-adrenergic responsiveness has been shown to decrease with increasing age, propranolol hydrochloride (10~ 6 M, Sigma) was used to block /3-adrenergic stimulation by NE in all experiments to rule out any possible effect of this alteration on the responses investigated in this study. Statistics Threshold (ED, 0 ) and midrange (ED 50 ) values were determined after logit transformation of normalized dose-response curves. Data are expressed as mean ± SEM. One-way analysis of variance (ANOVA) followed by individual comparisons using the Newman-Keuls test were used to determine statistical significance. Significance was set at the/><0.05 level. Results Body weights, strip weights, and systolic blood pressures are presented in Table 2. Whereas a modest elevation in systolic blood pressure occurred in the and week-old rats compared with the 5-7-week-old rats, these animals remained normotensive (systolic blood pressure<140 mm Hg) through 1 year of age. Body weights and strip weights increased, significantly with increasing age. Passive Force-Response Studies Strips of femoral and aortic smooth muscle from each age group demonstrated the classic passive force-response (length-tension) characteristics (Figure 1A and IB). That is, with increasing passive force, active tension increased and reached a plateau. With increasing age, however, the optimum level of passive force required for maximum active tension generation increased in both femoral and aortic smooth muscle (Table 3). Active tension generation in response to a given passive force was also altered in both femoral and aortic smooth muscle with increasing age (Figure 1A and IB). At the lower end of the passive force-response curves (50-1,000 mg passive force in aorta and mg passive force in femoral artery), active tension generation decreased significantly with increasing age. As passive force was increased (1,500-2,500 mg passive force in aorta and mg passive force in femoral artery), active tension generation became similar in the three age groups, and no difference in maximum active tension was seen (Table 3). This is due to the observation that maximum active force (Table 3) increased significantly with increasing age. When the values for maximum active force are divided by the weight of the strips (which also increased with increasing age, Table 2), the response is normalized, and therefore, maximum active tension values are similar. Endothelium-Dependent Responses The endothelium-dependent relaxation responses to both ACh and A23187 were significantly attenuated in aortic smooth muscle with increasing age. No difference in the maximal relaxation response to ACh was seen. However, a significant decrease in the sensitivity was observed (Figure 2A). Both threshold (ED IO ) and midrange (ED 50 ) sensitivities were significantly decreased with increasing age (Table 4). Unlike the response to ACh, the maximal relaxation response to A23187 was significantly attenuated in the week-old rats (Figure 2B). As with the response to ACh, threshold and midrange sensitivities were decreased with increasing age (Table 4). Whereas aortic smooth muscle from 5-7- and week-old rats exhibited a contractile response to doses of A23187 greater than 10" 7 M, no such effect was seen in the week-old rats. Table 2. Body Weights, Strip Weights, and Systolic Blood Pressures Age group (weeks) 5-7 (n = 6) (n = 6) Body weight (g) 205 ± ±10* 589±14*t Aortic weight (mg) * *1 Femoral weight (mg) 1.06 ± ±0.12* 2.49±0.23*t Systolic blood pressure (mm Hg) 1.06 ±0 116±2* *t Values are mean± SEM. *Signiflcantly different from the 5-7-weeks-of-age group (p<0.05); tsignificantly different from the weeks-of-age group (p<0.05).

4 892 Circulation Research Vol 61, No 6, December Or B 5-7 WOA WOA WOA WOA UJ a > o <S o I I. J PASSIVE FORCE (mg) FIGURE 1. Passive force-response relations offemoral artery (Panel A) and aortic strips (PanelB) from rats 5-7, 24-26, and50-52 weeks of age (WOA). For statistical comparisons, values of passive force required for maximum active tension generation are listed in Table 3. Sodium Pump-Mediated Responses All three indicators of sodium pump-dependent responses (contractile responses to ouabain and potassium-free PSS in aorta and potassium relaxation in femoral artery) decreased with increasing age. The maximal contractile response to ouabain decreased with increasing age (Figure 3A and Table 5). While no difference in the response to ouabain at doses of 10~ 8 to 3 x 10" 5 M was seen between 5-7- and week-old rats, aorta from week-old rats exhibited a decreased response to all doses when compared with 5-7-week-old rats. The contractile response to potassium-free PSS was significantly decreased at all time points with increasing age (Figure 3B and Table 5). Potassium-relaxation experiments in femoral arteries revealed a similar pattern of sodium pump-dependent responsiveness as in the aorta. With increasing age, no change in the maximal relaxation response occurred. However, a significant decrease in the threshold and midrange sensitivities was observed (Figure O WOA' WOA^^ T J- 3C). In fact, at 0.1 and 0.3 mm KC1, femoral arteries from week-old rats exhibited a small contractile response. A similar response was seen at 0.1 mm KC1 in week-old rats. ED 10 and ED 50 values are presented in Table 5. Although femoral smooth muscle from each age group relaxed 100%, the concentration of potassium chloride at which this was achieved increased significantly with increasing age. The ED 100 values are presented in Table 5. Calcium-Dependent Responses Although calcium dose-response curves were generated up to 2.5 mm Ca, the data are expressed and graphed as the percent response to 1.6 mm Ca. This is due to the fact that the response at 2.5 mm Ca was no different than the response at 1.6 mm Ca. No difference in the calcium sensitivity of femoral smooth muscle in the presence of 10" 7 M NE was seen with increasing age (Figure 4A). Relaxation of femoral arterial smooth muscle induced by calcium (calcium -20 O t -40 < I : WOA' -100 u Ach (-log M) A23187 (-log M) FIGURE 2. Endothelium-dependent responses to acetylcholine (Ach) (Panel A) and the calcium ionophore A23187 (Panel B) in strips of aorta from rats 5-7, 24-26, and weeks of age (WOA). Statistical comparisons ofed l0 and ED!0 values for relaxation are listed in Table 4.

5 Soltis Aging and Vascular Smooth Muscle Responsiveness 893 Table 3. Values for Maximum Active Force, Maximum Active Tension, and the Optimum Passive Force for Maximum Active Tension Generation in Femoral and Aortic Smooth Muscle Age group (weeks) 5-7 (n = 6) (n = 6) A 204 ± ±19* 469±42*t Femoral B 197± ±27 C 292 ± ±73* 728±98*t A 467 ± ±33* 943±31*t Aorta B 165 ± ± ±25 C 1, ,722 ±158* 2,252 ±143*t A, Maximum active force; B, maximum active tension (mg force/tissue wt); C, optimum passive force (mg). Values are mean±sem. *Significantly different from 5-7-weeks-of-age group (p<0.05); tsignificantly different from weeks-of-age group (p<0.05). stabilization) was also unaltered with increasing age (Figure 4B). ED IO and ED 50 values for calcium sensitivity and calcium relaxation are listed in Table 6. Discussion While a number of studies on alterations in vascular reactivity with respect to aging have been reported, little information is available on changes in membrane functions that may be important in mediating or regulating these alterations in vascular smooth muscle responsiveness. In the present study, age-related alterations in sodium pump activity, membrane stabilizing actions of calcium, and endothelium-dependent responses in aortic and femoral arterial smooth muscle were investigated. Prior to these studies, passive force-response characteristics of the two types of vascular tissue were assessed to determine the level of passive force required for maximal active tension generation. As was expected, increasing the level of passive force placed on the strips resulted in an increase in active tension generation by both femoral and aortic smooth muscle in all age groups. The characteristics of the force-response curves, however, varied between the three age groups in both vascular tissues. With increasing age, a greater amount of passive force was _ JOC OO. * i 4».._. - " iob? WOA Ouabain (-log 500 o U or O > 200 u required to achieve maximum active tension generation. Another interesting observation is that at the lower levels of passive force the amount of active tension generated was lessened with increasing age. These observations most likely reflect a decreased elasticity and distensibility of the vascular tissues. That is, with increasing age, the amount of noncontractile tissue (collagen and elastin) increases. u As a result, a greater passive force (or stretch) is required to achieve a given level of active tension generation. Although with increasing age a greater passive force was required to achieve maximum active tension, maximum active tension itself was unaltered in the three age groups. This would suggest that when the strips are set at their optimal passive force the ability of the vascular smooth muscle to contract maximally is unaltered with increasing age. Maximum active force (milligrams) increased significantly with increasing age. However, since strip weight increased also, maximum active tension (force divided by tissue weight) became similar in the three age groups. This increase in maximum active force generation with increasing age may be due to a hyperplasia and/or hypertrophy of the vascular smooth muscle medial layer. 318 Decreases in the maximum active force and active tension generating ability of vascular smooth muscle./ ~ -H io JO I u eo -too O6 KCI ImM) FIGURE 3. Sodium pump-dependent contractile responses to ouabain (Panel A) and potassium-free PSS (Panel B) in strips of aorta and relaxation response to potassium addition (Panel C) in strips offemoral artery from rats 5-7, 24-26, and weeks of age (WOA). Statistical comparisons of maximum force generation in response tolo' 3 M ouabain and 60 minutes exposure to potassium-free PSS, as well as ED IO, ED^, ond ED m values for potassium relaxation, are given in Table 5.

6 894 Circulation Research Vol 61, No 6, December 1987 Table 4. ED 10 and ED 50 Values for Acetylcholine and A23187 Relaxation Responses in Aortic Smooth Muscle A Pg rn. T ACh(M) A23187,(M) (weeks) EDi 0 ED S0 ED,, ED,, 5-7 (n = 6) 5.2[±0.6]xl0" 9 3.1[±0.6]xl0~ 8, 2.9[±0.4]xl0" 9 1.4[±0.2]xl [± 1.2] x 10" 9 * 8.7[±1.9]X1O- 8 * 5.6[±0.5]xl0" 9 * 2.2[±O.2]X1O- 8 * (n = 6) 27.6[±4.5] x 10" 9 *t 22.0[±6.1]xl0-8 *t 14.2[±2.6] X 10" 9 *t 4.3[±0.3] x 10" 8 *t Values are mean±sem. *Significantly different from 5-7-weeks-of-age group (p<0.05); tsignificantly different from weeks-of-age group (p<0.05). have been reported to occur with increasing age The present study, however, shows no evidence for such an alteration. In support of this, a recent study by Duckies et al 9 also found no change in the maximum contractile ability of vascular smooth muscle in aging rats. It may be that in these previous studies the tissues were not set at their optimal passive force (or length) for maximal active force or tension generating capabilities. As mentioned previously, conflicting reports have appeared in the literature with respect to age-related alterations in vasoconstrictor responses to various agents and in particular to NE. In the present study, no differences in maximal active tension generation or sensitivity to NE in femoral arteries or potassium chloride in aorta were seen with increasing age. In a recent study, Duckies et al 9 also found no difference in the sensitivity or maximal contraction of femoral arteries to NE. Since they also performed passive force-response experiments to determine optimum conditions at which maximum active force generation occurs, the differences in NE responsiveness observed in other studies 78 may be the result of not performing appropriate passive force-response analysis on the vascular smooth muscle used at various ages. Because extracellular calcium plays a pivotal role in the excitation-contraction coupling process of vascular smooth muscle, the possibility of an altered calcium sensitivity of vascular smooth muscle occurring with increasing age was investigated. Results from previous studies have been conflicting on the importance of extracellular calcium in the altered vascular responsiveness seen with aging. Both Carrier et al 8 and Cohen and Berkowitz 10 observed a decreased sensitivity of rat aorta to NE with increasing age. However, whereas Carrier et al 8 demonstrated a decreased dependency of the NE response on extracellular calcium with increas- Table 5. Age group (weeks) 5_7 ( n = 6) («= 6) Indexes of Sodium Pump Activity A Ouabain (mg) 312± ±28* 5O±8*t B Potassium-free (mg) 484 ± * 241±13*t ing age, Cohen and Berkowitz 10 observed an increased dependency. That is, as extracellular calcium was decreased, the NE response was not affected in the study by Carrier et al 8 but was greatly diminished in the study by Cohen and Berkowitz. 10 In the present study, no indication of an altered vascular contractile responsiveness to calcium was observed. These data are supported by a recent study 20 demonstrating no change in 45 Ca uptake in rat aorta with increasing age. Also, no difference in high-affinity calcium-membrane binding sites was seen in aorta from 4- and 12-month-old rats. These binding sites have been suggested to have functional significance in that they appear to be associated with receptor-operated calcium channels. 20 In addition to its requirement for contraction, calcium also can cause relaxation of vascular smooth muscle, 21 and this action has been termed "membrane stabilization." 22 It has been suggested that this stabilizing action of calcium involves an alteration in the active and passive movement of major ions (including calcium) across the vascular smooth muscle membrane Conceivably, alterations in the ability of calcium to stabilize the membrane could result in an alteration in vascular smooth muscle responsiveness. 25 From the present study, it appears that the ability of calcium to stabilize the vascular smooth muscle membrane is not altered with increasing age. This is evident from the lack of an alteration in the ability of calcium to cause relaxation of already contracted vascular smooth muscle and is reflected also in the calciumsensitivity experiments. That is, one might expect the contractile response to calcium to be enhanced in the face of a reduction in its own stabilizing action. 23 The lack of an alteration in these calcium-dependent responses could account for the absence of any changes in NE and potassium chloride responsiveness. In recent years, several investigators have suggested ED ± ±0.04* 1.02±0.03*t C Potassium-relaxation ED ± * 1.67±0.06*t (mm) ED ± * 2.95±0.03*t A, Contractions (milligrams force) of aorta in response to 10~ 3 M ouabain; B, contractions (milligrams force) of femoral artery in response to 60 minutes exposure to potassium-free PSS; and C, ED 10, ED 50, and ED 1O o values for potassium-relaxation in femoral artery from 5-7, 24-26, and week-old rats. Values are mean±sem. *Significantly different from 5-7-weeks-of-age group (p<0.05); tsignificantly different from weeks-of-age group (p<0.05).

7 Soltis Aging and Vascular Smooth Muscle Responsiveness 895 Or ui 2 LJ 100 eo * 60 I X 40 2 o WOA 5-7 WOA WOA x L 5-7 WOA WOA WOA O Ca" (rnm) Ca** (mm) FIGURE 4. Calcium sensitivity (in the presence of 10~ 7 M norepinephrine (Panel A) and calcium relaxation (Panel B) of strips of femoral artery from rats 5-7, 24-26, and weeks of age (WOA). ED IO and ED S0 values are given in Table 6. that the endothelium of the vasculature plays a significant role in vasodilator and vasoconstrictor responses. l516 Although aging studies on morphologic changes in the endothelium have been reported, 2 *" 28 no such studies on functional alterations have appeared in the literature. A pronounced age-related decrease in the endothelium-dependent relaxation of aortic smooth muscle to ACh and A23187 was seen in the present study. Because previous studies have shown minimal changes in endothelial cell number and morphology with increasing age, 26 " 28 the present results would suggest a functional defect. A decrease in muscarinic receptor number and/or affinity for ACh could be one cause of the age-related decrease in endothelium-dependent relaxation. However, because A23187-induced relaxation was similarly decreased, the underlying defect is probably not receptor mediated but may involve the calciummobilization step of endothelium-derived relaxing factor (EDRF) release. This is not to say that other steps in the cascade process for the release of EDRF are not involved. Additionally, it may be quite possible that a simple mechanical barrier to EDRF is also the cause of the decreased response. It has been shown that with increasing age the subendothelium increases in thickness by as much as 5-10 times as a result of active migration of a number of different cell types into this layer. 28 This increased thickness could conceivably slow the diflfusion process of EDRF to the vascular smooth muscle cell following its release. Another possibility is a decrease in the activity of the vascular sodium pump. It has been shown that ouabain can significantly attenuate the relaxation response to ACh. 29 As was seen in the present study, measurements of sodium pump-mediated responses were decreased with increasing age. This decrease may mediate the endothelium-dependent relaxations seen with increasing age. A final possibility is a decreased sensitivity of the aortic smooth muscle to EDRF. The decreased endothelium-mediated response, however, is not likely to be due to a generalized defect in the ability of the aortic smooth muscle to relax. Although sensitivity was decreased, maximal relaxation in response to ACh was unaltered. In addition, no difference in calcium-induced relaxation was seen between the three age groups. Furthermore, previous studies'" have shown no age-related changes in the ability of vascular smooth muscle to relax in response to such agents as sodium nitrite and nitroglycerin. Taken together, these observations would suggest that the vascular smooth muscle retains its ability to relax with increasing age and any change in the response to a given agent is most likely the result of a specific alteration in the ability of the vascular smooth muscle to respond to that agent. A classic example would be the well-established observation of the age-related decrease in /3-adrenergic-mediated relaxation. * * Therefore, the decrease in endothelium-mediated relaxation is most likely a specific alteration and not a generalized defect in the ability of the aortic smooth muscle to relax. The contractile response to high doses of A23187 in 5-7- and week-old rats is probably due to increased levels of intracellular calcium in the aortic Table 6. ED 10 and ED 50 Values for Calcium Contractions and Calcium Relaxation in Rat Femoral Artery Calcium contraction (mm) Calcium relaxation (mm) Aee group (weeks) 5-7 (n = 6) (n = 6) Values are mean±sem. ED ± ± ED ± ± ±0.02 ED IO 3.01 ± ± ±0.26 ED ± ±0.21

8 896 Circulation Research Vol 61, No 6, December 1987 smooth muscle cell as a result of increased calcium influx or intracellular release. This lack of a contractile response in the week-old rats is puzzling, as no difference between the three age groups was seen in the calcium sensitivity or relaxation experiments. Other more complex factors such as an endothelial-derived contracting factor may be involved. Although endothelium-dependent relaxation responses were not performed in femoral arterial smooth muscle, similar age-related alterations would most likely be observed in this vessel when compared with the aorta. Quantitatively, the responses may not be the same, but qualitatively, the changes with age would be similar. Shirasaki et al 30 have obtained similar results using aorta and mesenteric artery and have suggested that the functional changes of the endothelial cells seen with aging are likely to be a generalized phenomenon of the vasculature and not specific for one vascular tissue. As with the endothelium, few reports on age-related alterations in the vascular sodium pump have appeared in the literature. Because the sodium pump plays an important role in regulating membrane potential in vascular smooth muscle and can have important effects on vascular responsiveness, it was of interest to determine whether any age-related alterations occurred in the sodium pump. A significant decrease in sodium pump-mediated responses was seen in the present study. Contractile responses to ouabain and potassiumfree PSS were significantly depressed, and'potassiuminduced relaxation was significantly attenuated with increasing age. Under the conditions of ouabain and potassium-free PSS in which the sodium pump is inhibited, intracellular sodium increases, and the vascular smooth muscle contracts due to increased calcium influx resulting from depolarization and/or decreased calcium efflux due to sodium/calcium exchange. 31 Potassium-induced relaxation following incubation in potassium-free PSS results from a hyperpolarization of the vascular smooth muscle due to stimulation of the sodium pump and transport of sodium out of the cell that had accumulated during the potassium-free period. 32 Therefore, each of these responses reflects the amount of sodium that had accumulated intracellularly. In each case, the accumulation of sodium appears to decrease with increasing age. In support of these findings, Cox et al 33 reported a decrease in intracellular sodium with increasing age in arterial smooth muscle from dogs. In a preliminary report, Moisey et al 34 also observed a decrease in intracellular sodium and sodium pump activity in tail artery from aging rats. Two possibilities are suggested for the differences observed in maximum active force generation induced by potassium-free and 10~ 3 M ouabain. The first possibility stems from the well-known fact that the sodium pump in rat vascular tissue is highly insensitive to inhibition by ouabain. This, of course, would result in a lower active force generation when compared with a contraction induced by a potassium-free solution. The second reason is due to a time effect. Tissues were exposed to potassium-free for a period of 60 minutes. Exposure to 10~ 3 M ouabain, however, was only for 10 minutes. Moreland et al 17 have shown that exposure of aortic smooth muscle to either potassium-free or 10" 3 M ouabain for the same time period (approximately 60 minutes) results in similar responses. Had the response to 10" 3 M ouabain been recorded for a longer time period (60 minutes) in each age group, the responses would have been similar to their respective responses in the 60-minute exposure to potassium-free. The absence of this data, however, does not detract from the significant finding in this study of the decreased responsiveness to ouabain and potassium-free in vascular smooth muscle from aging rats. As mentioned previously, the activity of the sodium pump may have an important role in endothelium-dependent relaxation responses. However, it is not known whether the reverse is true, i.e., whether or not the endothelium plays a role in contractile responses mediated by inhibition of the sodium pump. The endothelium is known to have effects on other contractile responses of vascular smooth muscle. However, whether this holds for sodium pump-mediated responses cannot be determined from the present study. As both endotheliumdependent and sodium pump-mediated responses were similarly affected with increasing age, a possible interaction of the two systems may exist in regulating each other's response. A recent review of vascular changes in hypertension and aging notes that most, if not all, of the alterations that occur in vascular smooth muscle in hypertension are actually an accelerated form of the changes that are seen in aging. 35 Alterations in sodium pump activity, endothelium-dependent relaxation, and calcium handling by the vascular smooth muscle have been shown to occur in hypertension, and it has been suggested that these changes can result in increased vascular smooth muscle responsiveness to NE Although sodium pump activity and endothelium-dependent relaxation were significantly altered in the present study, no changes in the vascular smooth muscle responsiveness to NE (or potassium chloride), as well as calcium, were seen. From these observations, it would appear that the alterations in vascular sodium pump and endothelium-dependent responses have little, if any, importance in causing changes in vascular responsiveness with aging. It may be quite possible, however, that the changes that occurred in these two responses were not significant enough to alter vascular smooth muscle responsiveness per se. It has been suggested that the underlying defect in hypertension is an altered calcium handling by the vascular smooth muscle that results in an increase in vascular resistance and elevation in blood pressure. 40 The absence of this defect in vascular smooth muscle in the aging animal may be the reason for the lack of a change in vascular responsiveness and subsequently the lack of an appreciable change in blood pressure observed in the present study. It appears, therefore, that the changes in the vascular smooth muscle membrane in hypertension are unique and not merely an acceleration of the changes seen in aging.

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Hypertension 1981;3(suppl II):II KEY WORDS age-related endothelium sodium pump calcium aorta femoral artery