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Contractile and Metabolic Properties of Longitudinal Smooth Muscle from Rat Urinary Bladder and the Effects of Aging
0022-534 7 /93/ 1502- 0529$03. 00/0 THE JOURNAL O F UROL O G Y Copyright © 19 9 3 by AMERICAN UROLO GICAL A S S OCIATI O N , !NC .
Voi. 150, 529-536,
Printed
1993
U. S. A.
CONTRACTILE AND METABOLIC PROPERTIES OF LONGITUDINAL SMOOTH MUSCLE FROM RAT URINARY BLADDER AND THE EFFECTS OF AGING DAVID D . MUNRO* AND IGOR R. WENDT Department of Physiology, Monash University, Clayton, Victoria, Australia
ABSTRACT Longitudinal smooth muscle strips taken from the urinary bladders of Sprague-Dawley rats, aged approximately 6 months and 24 months, were examined under a variety of conditions. Force development in response to electrical field stimulation and to cumulative addition of Ca2+ in the continual presence of 64 mM. KCl was the same in both adult and aged preparations. In response to cumulative additions of carbachol, however, it was observed that there was a shift to the right of the dose-response curve and a decrease in maximal force in the aged muscle strips. The maximal velocity of shortening was significantly lower in muscles from aged animals than in those from young adult animals. The metabolic tension cost during isometric contraction was, however, the same in both groups suggesting that the decline in Vus is largely due to factors not influencing energetic cost. The aged muscles also exhibited a lower basal metabolic rate and a reduced contribution of aerobic glycolysis to total metabolic energy production during both quiescence and stimulation under normoxic conditions than did muscles from young adult animals. They were, however, able to increase their rate of lactate production to the same levels as young adult muscles when stimulated under anoxic conditions. KEY WORDS: bladder; aging; muscle, smooth; metabolism Alterations in the normal voiding pattern of the urinary bladder are a common problem in the elderly population. Uri nary incontinence is a frequently reported dysfunction with major medical, p sychological, social and economic implica tions. 1 In addition, a reduction in urinary flow rate, increased frequency of micturition, incomplete voiding and reduced blad der capacity are often reported to accompany aging. 2-4 Smooth muscle is an integral component of the lower urinary tract and, ultimately, normal voiding is dependent upon the ability of the bladder musculature to respond appropriately to stimulation. It is possible, therefore, that any changes or impairment in the operative contractile or metabolic capacity of the smooth mus cle of the bladder may be reflected in voiding function. Results from previous studie s of the effect of increasing age on rat urinary bladder function have been disparate. In isolated whole bladder preparations, an increase in the maximum con tractile response to acetylcholine was found to occur in bladders taken from aged rats, compared with those taken from young adults. 5 No age-related alterations were observed in response to phenylephrine or isoprenaline. In a subsequent study, how ever, the age-related response to acetylcholine was apparent only in the bladder base, while being completely absent in the bladder body. 6 In contrast, it has been observed that the ability of the whole isolated rat bladder preparation to contract and relax in the presence of autonomic agonists, bethanechol, phen ylephrine and isoprenaline did not change with age. 7 In s mooth muscle, as in skeletal muscle, the immediate source of chemical energy is adenosine triphosphate (ATP ) . The bio chemical p athways for ATP synthesis have been identified in smooth muscle with the enzymes of the glycolytic pathway, the tricarboxylic acid cycle and the respiratory chain contributing to ATP production. Smooth muscles in general, however, have relatively low total phosphagen reserves, 8 and this necessitates a close coupling of metabolic restorative processes to high energy phosphate usage if contractile activity is to be sustained for substantial periods of time. 9 • 1 0 An unusual feature of the
Accepted for publication February 8, 1993. * Requests for reprints: Department of Physiology, Monash Univer sity, Clayton, 3168, Victoria, Australia. 529
metabolism of smooth muscle is the substantial contribution of glycolysis to total metabolic energy production. Isometrically contracting smooth muscle of rabbit urinary bladder, under fully oxygenated conditions, can derive as much as 3 5 % of the total energy from glycolysis. 1 1 · 1 2 In oxygenated vascular smooth muscle preparations, glycolysis may also provide as much as 30% of the total energy required during contraction, 1 3 · 14 whereas contributions of 10 to 20% may be present in intestinal 15 and uterine 16 smooth muscle. It has been suggested that this reflects a functional compartmentation of metabolism in smooth muscle with aerobic glycolysis preferentially provid ing metabolic support for membrane-associated processes, while oxidative metabolism is more strongly correlated with the contractile process. 1 0 The present study was undertaken to determine whether there were any changes in the contractile or metabolic proper ties of urinary bladder smooth muscle associated with aging. Aspects of the relationship between energy metabolism and contractile activity were investigated under both aerobic and anaerobic conditions. Energy turnover was assessed from meas urements of the rates of oxygen consumption and lactate pro duction to allow the relative contributions of oxidative and glycolytic metabolism to be distinguished. MATERIALS AND METHO D S
The urinary bladder was removed fr o m either young adult ( 140 ± 10 days, n = 19) or aged ( 790 ± 84 days, n = 14) male Sprague-Dawley rats that had been killed by cervical disloca tion. The bladder was opened by a longitudinal incision, emptied of urine and rinsed in Ca2+ -free Krebs-Henseleit solution. The bladder was transferred to a dissecting dish where it was pinned out, mucosal side up, and submerged in oxygenated Ca2+ -free Krebs -Henseleit solution. A strip of the outer longi tudinal smooth muscle layer was carefully dissected free from the bladder body wall, tied at both ends with 5 - zero noncapillary silk and transferred to the appropriate apparatus for measurement of shortening velocity or force production, concomitantly with either oxygen consumption or lactate production . The muscle strips used in this study had an average length of 13.8
530
BLADDER S M O OTH MUSCLE AND EFFECTS O F AGING
± 0.5 mm. and weighed on average 5.2 ± 0.4 mg.; the average cross-sectional area was 0.59 ± 0.06 mm. 2 (as estimated by the ratio of muscle mass to length and assuming a muscle density of 1 mg./mm. 3 ) . In all cases, the muscle was mounted i n the appropriate experimental chamber in Ca2+ - free Krebs-Henseleit solution and left to undergo stress relaxation under a load of 1 gm. for 30 minutes before the commencement of any measurements. The Krebs-Henseleit solution had the following composition (in mM. ) : 118 NaCl, 4.75 KCl, 1 . 18 MgS04 , 1 . 18 KH2 P0 4 , 24.8 NaHC0 3 , 2 .5 CaCb and 10 glucose. The Ca2+ -free solution was prepared by simply omitting the CaCb. A "High KCl" solution was prepared by partial substitution (64 mM.) of NaCl by KCL The solutions were continuously aerated with either 95% Or 5% CO 2 or 95% Nr5% CO2 and had a pH of 7.36 at 27C, the temperature at which all the experiments were performed. All contractions in this study were isometric, with the muscle length set to be optimal for force development. In all experi ments the muscle was held vertically in the appropriate cham ber with the lower tie rigidly fixed and the upper tie connected, via a light stainless steel rod, to a lever system equipped with a force transducer. The total compliance of the force measuring system, including the ties and the connecting rod, was 0.4 mm./ N. Electrical field stimulation was achieved via platinum elec trodes brought into contact with the muscle. Contractions were initiated for 45 seconds using 10 volt square pulses of 1 milli second duration at frequencies ranging from 0.2 to 10 Hz. Dose response curves were also obtained for carbachol (Sigma, St. Louis, Missouri) , 1 x 10-s M. to 1 X 10-e M., and for the cumulative addition of Ca 2+ , 0.1 mM. to 5.0 mM., in the presence of 64 mM. KCL Measurement of oxygen consumption. Oxygen consumption (J02 ) was measured in a flow-through type system utilizing a Clark oxygen probe (Yellow Springs Instruments, YSI 4004) . Solution of a constant oxygen tension (680 mm. Hg) is drawn past the muscle, which is housed in a narrow stainless steel chamber (volume 0.4 ml.) , and then past the oxygen probe situated downstream from the muscle chamber. In this flow through system, the output of the electrode is constant in the absence of the tissue or when the rate of oxygen consumption by the tissue is constant. Oxygen consumption is determined from the difference betwee n oxygen content of the solution entering the muscle chamber and that leaving the muscle chamber, together with knowledge of the flow rate of solution past the muscle. The oxygen content of the solution entering the muscle chamber (that is, zero oxygen consumption refer ence) can be sampled by the same oxygen probe and was repeatedly checked during the course of each experiment. The muscle itself was mounted on a specially constructed stainless steel holder equipped with a grid of platinum stimu lating electrodes. This holder was then inserted into the muscle chamber, and the entire assembly was immersed in a large capacity temperature regulated water bath. Measurement of lactate production. A fluorometric assay was used to determine lactate production (JLA c). During these ex periments the muscles were mounted on the same holder used in the oxygen consumption measuring experiment, but were placed in small glass vials containing 2 ml. Krebs-Henseleit solution aerated with either 95% Or5 % CO 2 or 95% N2 -5% CO2 • Following completion of the experimental protocol, the muscle was removed from the vial and the solution was stored at -40C to await subsequent assay. The sampling time for determination of lactate production was 30 minutes. Such a time period was used for determination both of basal lactate production and lactate produced during contractures. Since only lactate appearing in the solution was assayed, the possi bility exists that lactate accumulation in the tissue could lead to an underestimate of the true total lactate production. In all cases, however, basal samples were collected before and after sampling periods of contractions. The levels before and after
were similar, suggesting that all lactate produced in association with the contractions had effluxed into the bath within the sampling period. Measurement of shortening velocity. Muscle strips were mounted on a Cambridge Technology 300H ergometer system (Cambridge, Massachusetts) which can measure both length and tension changes of the muscle simultaneously. The muscle was attached to the ergometer arm by means of a fine tungsten alloy wire and was stimulated via platinum ring electrodes. Unloaded shortening velocity (Vus ) was estimated by use of the slack test. The slack test involves applying a variable rapid step shortening to the muscle while it is undergoing isometric contraction. Such a procedure introduces slack into the cou pling between the muscle and the ergometer arm, and the muscle may then shorten under effectively zero load until the slack is taken up. A measurement of the time taken from the imposition of the shortening to the onset of tension redevel opment may then be made. This procedure was repeated at various step length magnitudes on each muscle examined. The amplitudes of the steps were plotted against the time required to redevelop tension following shortening. Such a plot gave a linear relation in which the slope of the relationship repre sented unloaded shortening velocity and the intercept denoted the length step required to fully discharge the series elastic component of the muscle strips. Although various length steps were imposed on the muscle, it was always shortened to the same final length. The final length achieved was within the range 0.80 to 0.85 L0 where L0 is the optimal length for force development. The length steps used to estimate Vus were taken from the range 0.10 to 0.30 L0 Five length steps from within this range were presented in random order to the muscles, and the response was stored on computer for later analysis. The influence of time was established by determining Vus at 5, 60 and 300 seconds after the initiation of isometric contraction, and these different contraction durations were also presented to the muscle in random order. Statistics. All data are presented as means ± the standard error of the mean. Differences between means have been as sessed using the Student's t test applied in paired or unpaired fashion as appropriate. ,
•
RESULTS
Isolated whole urinary bladders taken from the aged animals weighed on average 204 ± 17 mg. (n = 14) and were significantly (p <0.05) heavier than those taken from the young adults, 139 ± 10 mg. (n = 19) . There was also a corresponding rise in body weight of the aged animals, thus indicating that bladder weight may simply have increased in line with body weight. Therefore, the ratio of bladder weight to body weight was determined for each animal, the results being, on average, 0.037 ± 0.004 mg./ gm. and 0.027 ± 0.002 mg./gm. for the aged and young adult animals, respectively. Analysis of these results demonstrated that the ratio was significantly higher for the aged animals (p < 0.05 ) . Such a result shows that the bladder of the aged animal has increased in size by a greater proportion than the overall body weight, a finding indicative of some hypertrophy of the bladder. Isometric force generation. The contractile responses initiated by electrical field stimulation for both muscle groups are given in figure 1. The values shown are peak force production and the force level at the conclusion of the 45-second stimulus regimen. Both age groups exhibited identical force profiles to electrical field stimulation reaching the same peak force levels, then declined in force throughout the duration of the stimulus to the same end force values. The ability to respond to cumu lative additions of Ca2+ in the continual presence of 64 mM. KCl was also the same for both muscle groups (fig. 2). The aged muscles could produce comparable force to those of young adults at any Ca2 + concentration and also could produce the same maximal force level. In response to cumulative additions
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BLADDER S M O OTH JV! U S CLE AND EFFECTS O F AGING 5
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FIG. 2. Force development by young adult (closed circles, n = 7) and aged (open circles, n = 8) muscle preparations in response to cumulative addition of Ca2+ in presence of 64 mM. KC!, (values are means ± SE).
of carbachol, however, two main differences were associated with the aged preparations (fig. 3). First, there was a significant decrease in the maximum force levels produced in response to carbachol (p <0.05), and second there was a shift to the right of the dose-response curve. Interestingly, in addition to the aged muscle strips producing significantly lower maximal force levels in response to carbachol, the time taken to reach peak force was significantly longer compared with that taken by the young adult strips. As shown in figure 4, there was no difference between the time to peak force of the two muscle groups when stimulated with 64 mM. KCl (2.5 mM. Ca2+ ) . However, when
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FIG. 4. Time t o peak fo rce values fo r young adult (hatched bars, n = 6) and aged (open bars, n =6 7) muscle preparations stimulated by either 64 mM. KC! or 1 X 10� M. carbachol in presence of 2.5 mM. Ca'+ . *Denotes value significantly different from corresponding young adult muscle value (p <0.05).
challenged with carbachol the aged muscles took significantly longer to reach peak force (p < 0.05 ) . Unloaded shortening velocity and series elastic component.
Figure 5 represents the Vu, values estimated from the slack test for both young adult and aged muscle strips at 5, 60 and 300 seconds after the initiation of contraction. The values of esti mated Vu, for rat urinary bladder smooth muscle strips are in the range of previously reported values for many other smooth muscle preparations, 10 including rabbit urinary bladder. 1 7 It is evident, however, that Vu, is lower in the aged muscle strips. This difference between the aged and young adult muscles was statistically significant at each of the three times examined (p <0.05 for 5, 60 and 300 seconds). The data presented in figure
532
BLADDER SMOOTH MUSCLE AND EFFECTS OF AGING
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Fm. 5. Unloaded shortening velocity (Vu,) of young adult (closed circles, n = 10) and aged (open circles, n = 6) muscle strips plotted against time at release. Muscles were subjected to electrical field stimulation (10 Hz) and rapidly released to slack length through varying length steps at 5, 60 and 300 seconds after the commencement of stimulation. Unloaded shortening velocity values of aged muscle strips are significantly lower than young adult values at each of three times of release (p < 0.05). Note also decline in Vu, with increasing duration of contraction in both muscle groups.
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consumption rate. When 2.5 mM. Ca2+ was added to the solu tion, however, the muscles developed spontaneous contractile activity with a corresponding rise of oxygen consumption above the basal rate. Both young adult and aged muscles exhibited such spontaneous contractile behavior and, in order to inves tigate any differences between the two, the peak force produced and the frequency of the spontaneous contractions were deter mined for each muscle. Although the frequency of the sponta neous peaks was the same for the young adult and the aged muscles (1.13 ± 0.23 per minute and 1 . 1 7 ± 0.20 per minute, respectively) the peak force produced during spontaneous con tractions by the young adult muscles in Ca2+ -containing solu tion was significantly (p <0.05) greater than that produced by the aged muscles (70 ± 5, and 52 ± 6 mN./mm. 2 respectively) . Measurements of oxygen consumption and lactate produc tion were made on unstimulated muscles in Ca2+ free and Ca2+ containing solution (fig. 7). In Ca2+ free solution, although both muscle groups were quiescent, the aged muscles had a signifi cantly lower oxygen consumption rate (p <0.05). Measurements of lactate production during the same condition also show a significantly lower rate of lactate production associated with the aged muscles (p <0.05). Following the addition of 2.5 mM. Ca2+ to the solution bathing the muscle, both muscle groups
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5 m in 2.5 m M Ca 2 + FIG. 6. Original records of force (lower trace) and oxygen consump tion ( upper trace) of strip of longitudinal smooth muscle of rat urinary bladder (young adult). Downward arrow above oxygen trace indicates point at which muscle was introduced into recording chamber. Muscle was initially in Ca2 + -free solution, and 2.5 mM. Ca2+ was added at point indicated by upward arrow. This induced spontaneous contractile ac tivity with corresponding increase in oxygen consumption. Zero oxygen consumption is represented by long-dashed line, while short-dashed line indicates level of basal oxygen consumption of quiescent muscle. Dashed line in lower panel represents passive force level. 5 also show clear evidence for a decline in Vus over time of contraction. A slowing of V u, associated with increasing dura tion of contraction up to 300 seconds was apparent in both the young adult and aged muscle preparations, with Vus at 300 seconds being reduced to 46 % and 42% of its value at 5 seconds in the young adult and aged muscles respectively. The series elastic component exhibited no evidence of a systematic alteration with increasing duration of contraction within either age group (p >0.05). The young adult muscle strips had a mean series elastic component of 0.063 ± 0.003L0 and the aged a mean of 0.083 ± 0.004 Lo . Between group statistical analysis revealed that the series elastic component was not significantly different in the aged muscle strips (p > 0.05). Force and energy expenditure under basal conditions. An original record of oxygen consumption and force production is shown in figure 6. As is illustrated, muscle preparations were quiescent in Ca2+ -free solution with a constant basal oxygen
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2 . 5 m M ca2+ ca2+ free FIG. 7. Rates of oxygen consumption (J02 ) and lactate production (JLAc) for young adult (hatched bars, n = 10 for J02 , n = 7 for J LAc) and aged (open bars, n = 8 for Jo22+, n = 6 for JLAc) muscle strips in absence and presence of 2.5 mM. Ca . *Denotes value significantly different from corresponding young adult value (p < 0.05).
BLADDER S M O OTH M U S C L E A N D EFFECTS
subsequently developed spontaneous force. Associated with this was a corresponding rise in both oxygen consumption and lactate production above the quiescent basal levels. The aged muscles, however, again had significantly lower oxygen con sumption and lactate production rates when compared with those of the young adults (p <0.05). Relation between force production and oxygen consumption. In both the young adult and aged preparations there was a linear correlation between steady state force production and oxygen consumption rate. This is clearly indicated in figure 8, where force production has been varied by stimulating the muscles with 64 mM. KCl in the presence of graded concentra tions of Ca2 +. Under this stimulation protocol, the muscles produced graded and sustained steady levels of fo rce that facil itated the quantitation of steady state J02 and force. Whilst the data shown in figure 8 indicate that the aged muscles consumed slightly less oxygen than the young adult muscles at equivalent levels of force, it should be borne in mind that the Jo2 values represent total oxygen consumption and include the basal component. In fact the intercept values in figure 8 represent the basal J02 in Ca2+-free 64 mM. KCl solution. When the basal oxygen consumption is subtracted, it is clear that there is no difference in suprabasal J02 at any given force level between the young adult and aged muscles. There were no significant differences between the regression lines fitted to the pooled data for each age group as shown in figure 8 (p <0.05). It was more difficult to establish steady state force levels with carbachol stimulation, since carbachol generally caused an initial rapid fo rce development followed by a decay in force during which phasic contractions developed. Nevertheless, analysis of the average fo rce maintained during this latter phase revealed that it was linearly correlated with the average J02 over this period, and again there were no differences between the young adult and aged preparations in the relation between steady state J 02 and force under carbachol stimulation. Aerobic force and lactate production. Aerobic lactate produc tion rates increased from the unstimulated basal conditions in response to contractions induced by both high KCl and car bachol (table 1). In both conditions , the young adult muscles had significantly higher aerobic lactate production rates (p < 0.05). Although significantly more force was produced by the young adults in response to carbachol, no such difference in
AGING
force was apparent '.¥hen stimulated by KCL Thus it would appear that the lower lactate production rate is not attributable to a diminished force response. Effect of anoxia on force and lactate production. Table l also illustrates the effect of anoxia on force production and lactate production rate. Even in a fully oxygenated environment, there is some loss of force over the 30-minute recording period. Such loss is described by the percentage of peak force being produced by the muscle strip at the completion of 30 minutes. It is quite clear, however, that when the muscles are placed in an anoxic environment, this loss of force is significantly greater in both muscles, and fo r both stimulating conditions (p <0.05), al though it does appear that fo rce is considerably better main tained when stimulated by carbachol than when stimulated by high KCl under both aerobic and anaerobic conditions. Stimulation in an anaerobic environment produced a signif icant rise in the rate of lactate production fo r both muscle groups in response to both stimuli (p <0.05). Under these circumstances the rates of lactate production achieved by the aged muscles were the same as those for the young adult muscles (p >0.05). Since JLAc during stimulation under aerobic condi tions was lower in the aged muscles, the increase from the aerobic to the anaerobic level was proportionally greater in the aged muscles. Rate of A TP synthesis. The rate of ATP synthesis (JATP) was calculated from the rates of oxygen consumption and lactate production as: JATP = 6.42 X Jo2 + 1.25 X JLAc, 9 • 10' 18 Table 2 shows the results of these JATP calculations for young adult and aged muscles under the various conditions. In the Ca2+ free condition, JATP was considerably lower in the aged muscle, The percentage of ATP supplied aerobic glycolysis was also lower than the value for the young adult muscles. With the addition of Ca2 + to the solution and the subsequent development of spontaneous contractile activity, JATP rose from the quiescent levels fo r both muscle groups. The percentage of ATP being supplied by aerobic glycolysis did not change, however, from the quiescent values. During high KCl and carbachol stimula tion there was a considerable rise in JATP of both muscle groups and an increase in the fractional contribution of aerobic gly colysis to total metabolism. The young adult muscles exhibited higher levels of total JATP and a higher JLAc component than the aged muscles under both stimulating conditions. DISCUSSION
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The ability of longitudinal smooth muscle strips from rat urinary bladder to produce force in response to electrical field stimulation or high KCl appears to be unaltered with aging. However, there is some diminution in the contractile response to carbachol in the aged muscles. This is demonstrated decreased maximal force levels, a shift to the right of the dose response curve and an increase in the time taken to reach peak force values. Previous studies have either reported an age related increase in responsiveness to muscarinic stimulation, or no change in contractile response compared with young adult animals. 5- 7 The preparations used in those studies, however, have been isolated whole bladders, examined either as intact bladders or spirally cut bladder strips. When comparing results obtained with longitudinal smooth muscle preparations, as in the present investigation, with those obtained with whole blad ders, one should bear in mind the nature of the experimental muscle preparation. Due to the complex composition of the whole bladder preparation, direct comparison to responses of isolated longitudinal smooth muscle can not be confidently made. A clear difference in the contractile properties of the young and aged preparations was observed in terms of the unloaded shortening velocity (Vus ) . The significantly lower V us in the aged smooth muscle strips may indicate that these muscles have an intrinsically lower rate of crossbridge cycling than muscles from young adult animals. This could occur if there
534
BLADDER S M O OTH MUSCLE AND EFFECTS O F AGING
TABLE 1.
Force production (mN./mm.2) and lactate production (µmol./gm./min. _ ,) of longitudinal smooth muscle of rat urinary bladder under aerobic and anaerobic conditions Young adult muscles (n = 7) Aerobic
Aged muscles (n = 7)
Anaerobic
Aerobic
64 mM. KC! (2. 5 mM. Ca J
Anaerobic
2+
Peak Force End Force Lactate production
140 ± 71 ± (48.9% ± 0.30 ±
10 12 6.4) 0.07
118 ± 29 ± (23.3% ± 0.60 ±
Carbaclwl (1
Peak Force End Force
13t 4t 2.6)tO% 0.15 X
145 ± 1 1 54 ± 7 (39.1 % ± 6.8) 0.15 ± 0.04*
100 ± 13t 15 ± St (14.4% ± 2.0)t 0.59 ± 0.14
1 0-• M.)
150 ± 19 119 ± lOt 109 ± 13 88 ± 14t 85 ± 14 51 ± 7t 65 ± 9 36 ± 7t (60.8% ± 5.5) (42.8% ± 5.5)t (62.3% ± 13.7) (41.3% ± 2.l)t Lactate Production 0.34 ± 0.o7 0.58 ± 0.14 0.14 ± 0.02* 0.44 ± 0.04 Values are means ± SE t Significantly different from corresponding aerobic values in same muscle group (p < 0.05, independent t-test). * Significantly different from corresponding value of young adult muscles (p<0.05, independent t-test). End force values represent the force level being produced by the muscle at the completion of the 30-min measuring period. Percentage that end constitutes of peak force is shown in parentheses. TABLE 2.
Calculated rate of ATP synthesis and estimated contribution of oxidative metabolism and glycolysis to total metabolism for young adult and aged longitudinal muscle of the rat urinary bladder during unstimulated and stimulated conditions Young adult muscles
Aged muscles
JATP µmol./gm./
JATP µmol./gm./ % from % from % from % from min- 1 min- 1 Jo, JLAC J o, JLAC Ca2+ -free Ringer 1.40 91.1% 8.9% 1.03 96.3 % 3.7% 2.5 mM Ca2+ Ringer 2.47 92.3% 7.7% 1.77 95.6% 4.4% 64 mM. KCl 3 .04 87.8% 12.2% 2.49 92.0% 8.0% 3.52 Carbachol (1 x 10-• M.) 88.0% 12.0% 2.96 94.0% 6.0% JATP represents the estimated rate of ATP usage as calculated: JATP = J02 (6.42) + JLAc (1.25). Percentage values are also estimated from the equation given.
was some transition in the isoform of myosin with aging as has been reported to occur with aging in cardiac muscle. 19 At the present time, however, there is no evidence to suggest any such effects of aging on the properties of smooth muscle myosin. Arner et al. 20 reported a reduced Vmax in hypertrophied rat portal vein, which they ascribed to a lower rate of crossbridge turnover considered to be due to alterations in either the activation mechanisms or the intrinsic properties of the con tractile system itself. Alternatively the lower Vu, could be accounted for by an increased internal load in the aged prepa rations. This increase could possibly result if there were struc tural alterations in the aged tissues. An increase in the relative amount of intermediate filaments has been observed in hyper trophic smooth muscle 21 ; however, the possibility of such alter ations occurring in aged smooth muscle remains unclear. Unloaded shortening velocity of both the young adult and aged muscle strips declined with increasing duration of con traction. The values of Vu, after 300 seconds of contraction were approximately 46% and 42% of those after 5 seconds of contraction in the young adult and aged muscles, respectively. A similar decline in Vu, with increasing duration of contraction has been observed in several vascular22 • 23 and visceraP 0• 24 smooth muscles. Previous studies have linked the decline in Vu, with the development of the "latch state" during contraction of smooth muscle. 22 • 2 5 The present observations of a decline in Vu, over time in rat urinary bladder smooth muscle are in general agreement with such previous studies and, if interpreted within the framework of the latch hypothesis, would indicate that the latch phenomenon continues to be expressed in aged smooth muscle preparations. The aged bladder preparations exhibited a reduced basal metabolic rate compared with the young adult muscles when quiescent in Ca2+ -free solution. This was manifest in both significantly lower oxygen consumption and lactate production rates (and, hence, calculated JATP; see figure 7 and table 2). Energy turnover of quiescent muscle reflects the energy require ments of processes involved in maintaining cellular homeosta sis. The increased bladder weight-to-body weight ratio in the aged animals indicates that the aged bladder is hypertrophied.
If this is associated with an increase in cell size, then the depressed basal metabolism may be related to the greater size of the aged bladder smooth muscle cells. These larger cells would have a lower surface-to-volume ratio; hence ionic ho meostasis might be more easily maintained, requiring a lower level of energy expenditure, as has been suggested to occur in hypertrophied cardiac muscle. 26 In apparent contrast, however, it has recently been reported that bladder strips hypertrophied as a result of outlet obstruction did not exhibit any detectable change in the basal Jo2 or J LAc as compared with control bladder strips. 18 The hypertrophied bladders examined in that study were, however, obtained from young adult rats that had been subjected to severe outlet obstruction for a relatively brief time (10 days) . The depressed basal metabolism observed in the present study may be largely attributable to age-related factors unrelated to hypertrophy, or if it is in fact due to hypertrophy, may reflect the different nature and time course of the hyper trophic stimulus in the naturally aging animal. A further factor that may contribute to the lower basal metabolism in the aged muscle is a reduced rate of protein turnover. Decreases in the rates of protein synthesis and degradation have been reported to be associated with aging in cardiac muscle. 19 The increase in JATP from quiescent to spontaneously con tracting preparations was proportionally the same for both muscle groups, indicating that under these conditions about 40% of the total energy flux could be ascribed to the contractile activity as such. Although proportionally the increases were the same, the total calculated rate of JATP remained consider ably lower in the aged muscles in comparison to the young adults. The lower J ATP in the aged muscles is probably reflective of the significantly lower average peak force level produced during spontaneous contractile activity by these preparations, together with the underlying lower basal JATP, as determined in the Ca2+ free state. Muscle strips from both the young adult and aged bladders derived a portion of their total energy needs from aerobic glycolysis; however, the proportional contribution of aerobic glycolysis to total energy production was less in the aged preparations. In the quiescent state, aerobic glycolysis contrib-
BLADDER S M OOTH MUSCLE AND EFFECTS OF AGING uted approximately 8.5 % of total ATP generation in the young adult muscles and 4% in the aged muscles while, in the stimu lated state, the contributions of aerobic glycolysis to total metabolism were approximately 1 2 % and 6 to 8% in the young adult and aged muscles, respectively. The values for the young adult preparations are in good agreement with those reported by Arner et aL, 1 8 also for rat urinary bladder. A substantial contribution of aerobic glycolysis to total metabolism appears to be a feature common to most smooth muscles, with contri butions of 5 to 1 5 % under quiescent conditions and 10 to 35% under stimulated conditions, having been reported for a variety of vascular and visceral smooth muscles. 9 · 1 0· 1 1 • 1 5 • 16• 27 The lower aerobic lactate production rates observed in the aged muscles do not appear to be due to a limitation in the ability of the aged muscles to generate ATP via glycolysis since these preparations are able to increase their rate of lactate production to the same levels as young adult muscles when stimulated by high KC! or carbachol under anoxic conditions. It has been proposed that aerobic glycolysis may play an important role in preferentially providing ATP for membrane associated processes, such as the Na+ - K + pump, in smooth muscle. 1 0 In this context, it is interesting to speculate that the reduced J LAc observed in the aged preparations may represent a lower energy requirement of such membrane-associated proc esses. Again this may be related to the apparent hypertrophy of the aged bladder and the possible increase in cell size, which would result in a decrease in the amount of cell membrane relative to cell volume. The rate of oxygen consumption was observed to correlate linearly with isometric force in both the young adult and aged muscles. This correlation was most clearly indicated when the muscles were stimulated with 64 mM. KCl in the presence of varying concentrations of extracellular Ca2+ (fig. 8). A linear relation between J 02 and force has previously been observed in a variety of smooth muscle preparations. 9- 1 1 • 28 There was no difference in the relation between suprabasal J 02 and force between the young adult and aged muscles. This indicates that the energetic cost of maintaining a given force level, as derived from oxidative metabolism, is the same in the two muscle groups. Even when the lower J LAc component of the aged muscles is taken into account, the metabolic tension cost (su prabasal J ATP per unit tension) is not significantly different between the young adult and aged muscles. Lactate production was only measured during maximal contractions in the present study and not during the graded contractions; however, it has been shown that J LAc varies linearly with tension in rabbit urinary bladder smooth muscle. 1 1 The unchanged metabolic tension cost in the aged muscles suggests that the cross-bridge turnover rate is not altered with aging. This would in turn indicate that the lower V u, of the aged preparations is perhaps more likely to be the result of some phenomenon such as an increase in internal load rather than some change in the intrin sic properties of the contractile system itself. Under anaerobic conditions force production was reduced in both the young adult and aged muscles. The extent of the reduction in force was similar in the two groups when stimu lated with carbachol; however, with high KC! stimulation the reduction in force was slightly greater in the aged muscles. Both the young adult and aged muscles appeared to be less able to sustain force under anoxic conditions when stimulated with high KC! rather than carbachoL The rate of lactate production during stimulation under anoxic conditions increased to similar levels in both groups; however, as J LA C under aerobic conditions was less in the aged muscles, the proportionate increase was in fact greater in these muscles. J LAc during stimulation under anaerobic conditions was approximately 2-fold and 4-fold higher than during stimulation under aerobic conditions in the young adult and aged muscles, respectively. When calculated as J A TP (J LAc x 1.25), these rates of lactate production under anaerobic conditions represent only approx-
535
imately 20 to 25% of the total JATP observed with the same stimulation under aerobic conditions, where both oxidative metabolism and aerobic glycolysis can contribute to ATP pro duction. Since the anaerobic force levels remain, on average, at >50% of the aerobic levels over the 30-minute stimulation period, this implies that the metabolic tension cost is substan tially less during contractions in an anoxic environment. Arner et aL 1 8 reported a similar finding and suggested that anoxic contractions for both control and hypertrophic detrusor muscle are associated with a considerably lower cross-bridge turnover rate compared with normoxic contractions. Results obtained in our laboratory (Munro and Wendt, unpublished observations) provide data demonstrating that Vus is significantly decreased during anoxic contractions, suggesting that a reduced cross bridge cycling rate may account, at least in part, for the de creased tension cost during anoxia. However, a full understand ing of the mechanisms that permit continued function with an apparently greatly reduced metabolic cost under anoxic condi tions is yet to be gained. In conclusion, aging was found not to affect the force-gen erating capacity of longitudinal smooth muscle of the rat uri nary bladder, although there was a decrease in muscarinic responsiveness. The maximal velocity of shortening was signif icantly longer in muscles from aged animals than in those from young adult animals. The metabolic tension cost during iso metric contraction was, however, the same in both groups, suggesting that the decline in Vu s is due largely to factors not influencing energetic cost. The aged muscles also exhibited a lower basal metabolic rate and a reduced contribution of aerobic glycolysis to total metabolic energy production during both quiescence and stimulation under normoxic conditions than did muscles from young adult animals. They were, however, able to increase their rate of lactate production to the same levels as young adult muscles when stimulated under anoxic conditions. REFERENCES
L Campbell, A. J., Reinken, J. and McCosh, L.: Incontinence in the elderly: prevalence and prognosis. Age Ageing, 1 4 : 65, 1985. 2. Andersen, J, T., Jacobsen, 0., Worm-Petersen, J. and Hald, T.: Bladder function in healthy elderly males. Scand. J. UroL Ne phroL, 1 2 : 123, 1978. 3. Castleden, C. M., Duffin, H. M. and Asher, M. J.: Clinical and urodynamic studies in 100 elderly incontinent patients. Br. Med. J., 282: 1 103, 1981. 4. Drach, G. W., Layton, T. N. and Binard, W. J.: Male peak urinary flow rate: relations to volume voided and age. J. UroL, 1 2 2 : 210, 1979, 5. Kolta, M. G,, Wallace, L. J. and Gerald, M. C.: Age-related changes in sensitivity of rat urinary bladder to autonomic agents. Mech. Ageing Dev., 2 7 : 183, 1984. 6. Ordway, G. A., Esbenshade, T. A., Kolta, M. G,, Gerald, IVL C, and Wallace, L. J.: Effect of age on cholinergic muscarinic respon siveness and receptors in the rat urinary bladder. J. UroL, 136: 492, 1986, 7. Chun, A, L., Wallace, L. J., Gerald, M. C., Wein, A. J. and Levin, R M.: Effects of age on urinary bladder function in the male rat. J. UroL, 1 4 1 : 1 70, 1989. 8. Butler, T. M. and Davies, R. E . : High-energy phosphates in smooth muscle. In: Handbook of Physiology-The Cardiovascular System IL Edited by D. F. Bohr, A. P. Somlyo and H. V. Sparks, Jr. Bethesda: American Physiological Society, pp. 237-252, 1980. 9. Paul, R J.: Chemical energetics of vascular smooth muscle. In: Handbook of Physiology-The Cardiovascular System IL Edited by D. F. Bohr, A. P. Somlyo, and H. V. Sparks, Jr. Bethesda: American Physiological Society, pp. 201-235, 1980. 10. Paul, R J.: Smooth muscle mechanochemical energy conversion: relations between metabolism and contractility. In: Physiology of the Gastrointestinal Tract. Edited by L. R Johnson. New York: Raven Press, 2nd edn. , pp. 483-506, 1987. l L Wendt, L R and Gibbs, C. L.: Energy expenditure of longitudinal smooth muscle of rabbit urinary bladder. Am. J. PhysioL, 252: C88, 1987.
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12. Wendt, I. R.: Effects of substrate and hypoxia on smooth muscle metabolism and contraction. Am. J. Physiol., 256: C719, 1989. 13. Gliick, E. and Paul, R. J.: The aerobic metabolism of porcine carotid artery and its relationship to isometric force: energy cost of isometric contraction. Pfliigers Arch., 370: 9, 1977. 14. Hellstrand, P., Johansson, B. and Norberg, K.: Mechanical, elec trical, and biochemical effects of hypoxia and substrate removal on spontaneously active vascular smooth muscle. Acta Physiol. Scand., 100: 69, 1977. 15. Casteels, R. and Wuytack, F.: Aerobic and anaerobic metabolism in smooth muscle cells of taenia coli in relation to active ion transport. J. Physiol. (Lond) ., 250: 203, 1975. 16. Kroeger, E. A.: Effect of ionic environment on oxygen uptake and lactate production of myometrium. Am. J. Physiol., 230: 158, 1976. 17. Uvelius, B.: Shortening velocity, active force and homogeneity of contraction during electrically evoked twitches in smooth muscle from rabbit urinary bladder. Acta Physiol. Scand., 1 06: 481, 1979. 18. Arner, A., Malmqvist, U. and Uvelius, B.: Metabolism and force in hypertrophied smooth muscle from rat urinary bladder. Am. J. Physiol., 258: C923, 1990. 19. Lakatta, E. G.: Cardiac muscle changes in senescence. Ann. Rev. Physiol., 49: 519, 1987. 20. Arner, A., Malmqvist, U. and Uvelius, B.: Effects of Ca2+ on force-
21. 22. 23. 24. 25. 26.
27. 28.
velocity characteristics of normal and hypertrophic smooth mus cle of the rat portal vein. Acta Physiol. Scand., 124: 525, 1985. Berner, P. F., Somlyo, A. V. and Somlyo, A. P.: Hypertrophy induced increase in intermediate filaments in vascular smooth muscle. J. Cell. Biol., 88: 96, 1981. Dillon, P. F., Aksoy, M. 0., Driska, S. P. and Murphy, R. A.: Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science, 2 1 1 : 495, 1981. Krisanda, J. M. and Paul, R. J.: Energetics of isometric contraction in porcine carotid artery. Am. J. Physiol., 246: C510, 1984. Walker, J. S., Wendt, I. R. and Gibbs, C. L.: Heat production of rat anococcygeus muscle during isometric contraction. Am. J. Physiol., 255: C536, 1988. Hai, C-H. and Murphy, R. A.: Regulation of shortening velocity by cross-bridge phosphorylation in smooth muscle. Am. J. Physiol., 255: C86, 1988. Gibbs, C. L., Wendt, I. R., Kotsanas, G., Young, I. R. and Woolley, G.: Mechanical, energetic, and biochemical changes in long-term pressure overload of rabbit heart. Am. J. Physiol., 259: H849, 1990. Davidheiser, S., Joseph, J. and Davies, R. E.: Separation of aerobic glycolysis from oxidative metabolism and contractility in rat anococcygeus muscle. Am. J. Physiol., 247: C335, 1984. Paul, R. J. and Peterson, J. R.: Relation between length, isometric force, and 0 2 consumption rate in vascular smooth muscle. Am. J. Physiol., 228: 915, 1975.
Voi. 150, 529-536,
Printed
1993
U. S. A.
CONTRACTILE AND METABOLIC PROPERTIES OF LONGITUDINAL SMOOTH MUSCLE FROM RAT URINARY BLADDER AND THE EFFECTS OF AGING DAVID D . MUNRO* AND IGOR R. WENDT Department of Physiology, Monash University, Clayton, Victoria, Australia
ABSTRACT Longitudinal smooth muscle strips taken from the urinary bladders of Sprague-Dawley rats, aged approximately 6 months and 24 months, were examined under a variety of conditions. Force development in response to electrical field stimulation and to cumulative addition of Ca2+ in the continual presence of 64 mM. KCl was the same in both adult and aged preparations. In response to cumulative additions of carbachol, however, it was observed that there was a shift to the right of the dose-response curve and a decrease in maximal force in the aged muscle strips. The maximal velocity of shortening was significantly lower in muscles from aged animals than in those from young adult animals. The metabolic tension cost during isometric contraction was, however, the same in both groups suggesting that the decline in Vus is largely due to factors not influencing energetic cost. The aged muscles also exhibited a lower basal metabolic rate and a reduced contribution of aerobic glycolysis to total metabolic energy production during both quiescence and stimulation under normoxic conditions than did muscles from young adult animals. They were, however, able to increase their rate of lactate production to the same levels as young adult muscles when stimulated under anoxic conditions. KEY WORDS: bladder; aging; muscle, smooth; metabolism Alterations in the normal voiding pattern of the urinary bladder are a common problem in the elderly population. Uri nary incontinence is a frequently reported dysfunction with major medical, p sychological, social and economic implica tions. 1 In addition, a reduction in urinary flow rate, increased frequency of micturition, incomplete voiding and reduced blad der capacity are often reported to accompany aging. 2-4 Smooth muscle is an integral component of the lower urinary tract and, ultimately, normal voiding is dependent upon the ability of the bladder musculature to respond appropriately to stimulation. It is possible, therefore, that any changes or impairment in the operative contractile or metabolic capacity of the smooth mus cle of the bladder may be reflected in voiding function. Results from previous studie s of the effect of increasing age on rat urinary bladder function have been disparate. In isolated whole bladder preparations, an increase in the maximum con tractile response to acetylcholine was found to occur in bladders taken from aged rats, compared with those taken from young adults. 5 No age-related alterations were observed in response to phenylephrine or isoprenaline. In a subsequent study, how ever, the age-related response to acetylcholine was apparent only in the bladder base, while being completely absent in the bladder body. 6 In contrast, it has been observed that the ability of the whole isolated rat bladder preparation to contract and relax in the presence of autonomic agonists, bethanechol, phen ylephrine and isoprenaline did not change with age. 7 In s mooth muscle, as in skeletal muscle, the immediate source of chemical energy is adenosine triphosphate (ATP ) . The bio chemical p athways for ATP synthesis have been identified in smooth muscle with the enzymes of the glycolytic pathway, the tricarboxylic acid cycle and the respiratory chain contributing to ATP production. Smooth muscles in general, however, have relatively low total phosphagen reserves, 8 and this necessitates a close coupling of metabolic restorative processes to high energy phosphate usage if contractile activity is to be sustained for substantial periods of time. 9 • 1 0 An unusual feature of the
Accepted for publication February 8, 1993. * Requests for reprints: Department of Physiology, Monash Univer sity, Clayton, 3168, Victoria, Australia. 529
metabolism of smooth muscle is the substantial contribution of glycolysis to total metabolic energy production. Isometrically contracting smooth muscle of rabbit urinary bladder, under fully oxygenated conditions, can derive as much as 3 5 % of the total energy from glycolysis. 1 1 · 1 2 In oxygenated vascular smooth muscle preparations, glycolysis may also provide as much as 30% of the total energy required during contraction, 1 3 · 14 whereas contributions of 10 to 20% may be present in intestinal 15 and uterine 16 smooth muscle. It has been suggested that this reflects a functional compartmentation of metabolism in smooth muscle with aerobic glycolysis preferentially provid ing metabolic support for membrane-associated processes, while oxidative metabolism is more strongly correlated with the contractile process. 1 0 The present study was undertaken to determine whether there were any changes in the contractile or metabolic proper ties of urinary bladder smooth muscle associated with aging. Aspects of the relationship between energy metabolism and contractile activity were investigated under both aerobic and anaerobic conditions. Energy turnover was assessed from meas urements of the rates of oxygen consumption and lactate pro duction to allow the relative contributions of oxidative and glycolytic metabolism to be distinguished. MATERIALS AND METHO D S
The urinary bladder was removed fr o m either young adult ( 140 ± 10 days, n = 19) or aged ( 790 ± 84 days, n = 14) male Sprague-Dawley rats that had been killed by cervical disloca tion. The bladder was opened by a longitudinal incision, emptied of urine and rinsed in Ca2+ -free Krebs-Henseleit solution. The bladder was transferred to a dissecting dish where it was pinned out, mucosal side up, and submerged in oxygenated Ca2+ -free Krebs -Henseleit solution. A strip of the outer longi tudinal smooth muscle layer was carefully dissected free from the bladder body wall, tied at both ends with 5 - zero noncapillary silk and transferred to the appropriate apparatus for measurement of shortening velocity or force production, concomitantly with either oxygen consumption or lactate production . The muscle strips used in this study had an average length of 13.8
530
BLADDER S M O OTH MUSCLE AND EFFECTS O F AGING
± 0.5 mm. and weighed on average 5.2 ± 0.4 mg.; the average cross-sectional area was 0.59 ± 0.06 mm. 2 (as estimated by the ratio of muscle mass to length and assuming a muscle density of 1 mg./mm. 3 ) . In all cases, the muscle was mounted i n the appropriate experimental chamber in Ca2+ - free Krebs-Henseleit solution and left to undergo stress relaxation under a load of 1 gm. for 30 minutes before the commencement of any measurements. The Krebs-Henseleit solution had the following composition (in mM. ) : 118 NaCl, 4.75 KCl, 1 . 18 MgS04 , 1 . 18 KH2 P0 4 , 24.8 NaHC0 3 , 2 .5 CaCb and 10 glucose. The Ca2+ -free solution was prepared by simply omitting the CaCb. A "High KCl" solution was prepared by partial substitution (64 mM.) of NaCl by KCL The solutions were continuously aerated with either 95% Or 5% CO 2 or 95% Nr5% CO2 and had a pH of 7.36 at 27C, the temperature at which all the experiments were performed. All contractions in this study were isometric, with the muscle length set to be optimal for force development. In all experi ments the muscle was held vertically in the appropriate cham ber with the lower tie rigidly fixed and the upper tie connected, via a light stainless steel rod, to a lever system equipped with a force transducer. The total compliance of the force measuring system, including the ties and the connecting rod, was 0.4 mm./ N. Electrical field stimulation was achieved via platinum elec trodes brought into contact with the muscle. Contractions were initiated for 45 seconds using 10 volt square pulses of 1 milli second duration at frequencies ranging from 0.2 to 10 Hz. Dose response curves were also obtained for carbachol (Sigma, St. Louis, Missouri) , 1 x 10-s M. to 1 X 10-e M., and for the cumulative addition of Ca 2+ , 0.1 mM. to 5.0 mM., in the presence of 64 mM. KCL Measurement of oxygen consumption. Oxygen consumption (J02 ) was measured in a flow-through type system utilizing a Clark oxygen probe (Yellow Springs Instruments, YSI 4004) . Solution of a constant oxygen tension (680 mm. Hg) is drawn past the muscle, which is housed in a narrow stainless steel chamber (volume 0.4 ml.) , and then past the oxygen probe situated downstream from the muscle chamber. In this flow through system, the output of the electrode is constant in the absence of the tissue or when the rate of oxygen consumption by the tissue is constant. Oxygen consumption is determined from the difference betwee n oxygen content of the solution entering the muscle chamber and that leaving the muscle chamber, together with knowledge of the flow rate of solution past the muscle. The oxygen content of the solution entering the muscle chamber (that is, zero oxygen consumption refer ence) can be sampled by the same oxygen probe and was repeatedly checked during the course of each experiment. The muscle itself was mounted on a specially constructed stainless steel holder equipped with a grid of platinum stimu lating electrodes. This holder was then inserted into the muscle chamber, and the entire assembly was immersed in a large capacity temperature regulated water bath. Measurement of lactate production. A fluorometric assay was used to determine lactate production (JLA c). During these ex periments the muscles were mounted on the same holder used in the oxygen consumption measuring experiment, but were placed in small glass vials containing 2 ml. Krebs-Henseleit solution aerated with either 95% Or5 % CO 2 or 95% N2 -5% CO2 • Following completion of the experimental protocol, the muscle was removed from the vial and the solution was stored at -40C to await subsequent assay. The sampling time for determination of lactate production was 30 minutes. Such a time period was used for determination both of basal lactate production and lactate produced during contractures. Since only lactate appearing in the solution was assayed, the possi bility exists that lactate accumulation in the tissue could lead to an underestimate of the true total lactate production. In all cases, however, basal samples were collected before and after sampling periods of contractions. The levels before and after
were similar, suggesting that all lactate produced in association with the contractions had effluxed into the bath within the sampling period. Measurement of shortening velocity. Muscle strips were mounted on a Cambridge Technology 300H ergometer system (Cambridge, Massachusetts) which can measure both length and tension changes of the muscle simultaneously. The muscle was attached to the ergometer arm by means of a fine tungsten alloy wire and was stimulated via platinum ring electrodes. Unloaded shortening velocity (Vus ) was estimated by use of the slack test. The slack test involves applying a variable rapid step shortening to the muscle while it is undergoing isometric contraction. Such a procedure introduces slack into the cou pling between the muscle and the ergometer arm, and the muscle may then shorten under effectively zero load until the slack is taken up. A measurement of the time taken from the imposition of the shortening to the onset of tension redevel opment may then be made. This procedure was repeated at various step length magnitudes on each muscle examined. The amplitudes of the steps were plotted against the time required to redevelop tension following shortening. Such a plot gave a linear relation in which the slope of the relationship repre sented unloaded shortening velocity and the intercept denoted the length step required to fully discharge the series elastic component of the muscle strips. Although various length steps were imposed on the muscle, it was always shortened to the same final length. The final length achieved was within the range 0.80 to 0.85 L0 where L0 is the optimal length for force development. The length steps used to estimate Vus were taken from the range 0.10 to 0.30 L0 Five length steps from within this range were presented in random order to the muscles, and the response was stored on computer for later analysis. The influence of time was established by determining Vus at 5, 60 and 300 seconds after the initiation of isometric contraction, and these different contraction durations were also presented to the muscle in random order. Statistics. All data are presented as means ± the standard error of the mean. Differences between means have been as sessed using the Student's t test applied in paired or unpaired fashion as appropriate. ,
•
RESULTS
Isolated whole urinary bladders taken from the aged animals weighed on average 204 ± 17 mg. (n = 14) and were significantly (p <0.05) heavier than those taken from the young adults, 139 ± 10 mg. (n = 19) . There was also a corresponding rise in body weight of the aged animals, thus indicating that bladder weight may simply have increased in line with body weight. Therefore, the ratio of bladder weight to body weight was determined for each animal, the results being, on average, 0.037 ± 0.004 mg./ gm. and 0.027 ± 0.002 mg./gm. for the aged and young adult animals, respectively. Analysis of these results demonstrated that the ratio was significantly higher for the aged animals (p < 0.05 ) . Such a result shows that the bladder of the aged animal has increased in size by a greater proportion than the overall body weight, a finding indicative of some hypertrophy of the bladder. Isometric force generation. The contractile responses initiated by electrical field stimulation for both muscle groups are given in figure 1. The values shown are peak force production and the force level at the conclusion of the 45-second stimulus regimen. Both age groups exhibited identical force profiles to electrical field stimulation reaching the same peak force levels, then declined in force throughout the duration of the stimulus to the same end force values. The ability to respond to cumu lative additions of Ca2+ in the continual presence of 64 mM. KCl was also the same for both muscle groups (fig. 2). The aged muscles could produce comparable force to those of young adults at any Ca2 + concentration and also could produce the same maximal force level. In response to cumulative additions
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BLADDER S M O OTH JV! U S CLE AND EFFECTS O F AGING 5
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of carbachol, however, two main differences were associated with the aged preparations (fig. 3). First, there was a significant decrease in the maximum force levels produced in response to carbachol (p <0.05), and second there was a shift to the right of the dose-response curve. Interestingly, in addition to the aged muscle strips producing significantly lower maximal force levels in response to carbachol, the time taken to reach peak force was significantly longer compared with that taken by the young adult strips. As shown in figure 4, there was no difference between the time to peak force of the two muscle groups when stimulated with 64 mM. KCl (2.5 mM. Ca2+ ) . However, when
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challenged with carbachol the aged muscles took significantly longer to reach peak force (p < 0.05 ) . Unloaded shortening velocity and series elastic component.
Figure 5 represents the Vu, values estimated from the slack test for both young adult and aged muscle strips at 5, 60 and 300 seconds after the initiation of contraction. The values of esti mated Vu, for rat urinary bladder smooth muscle strips are in the range of previously reported values for many other smooth muscle preparations, 10 including rabbit urinary bladder. 1 7 It is evident, however, that Vu, is lower in the aged muscle strips. This difference between the aged and young adult muscles was statistically significant at each of the three times examined (p <0.05 for 5, 60 and 300 seconds). The data presented in figure
532
BLADDER SMOOTH MUSCLE AND EFFECTS OF AGING
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consumption rate. When 2.5 mM. Ca2+ was added to the solu tion, however, the muscles developed spontaneous contractile activity with a corresponding rise of oxygen consumption above the basal rate. Both young adult and aged muscles exhibited such spontaneous contractile behavior and, in order to inves tigate any differences between the two, the peak force produced and the frequency of the spontaneous contractions were deter mined for each muscle. Although the frequency of the sponta neous peaks was the same for the young adult and the aged muscles (1.13 ± 0.23 per minute and 1 . 1 7 ± 0.20 per minute, respectively) the peak force produced during spontaneous con tractions by the young adult muscles in Ca2+ -containing solu tion was significantly (p <0.05) greater than that produced by the aged muscles (70 ± 5, and 52 ± 6 mN./mm. 2 respectively) . Measurements of oxygen consumption and lactate produc tion were made on unstimulated muscles in Ca2+ free and Ca2+ containing solution (fig. 7). In Ca2+ free solution, although both muscle groups were quiescent, the aged muscles had a signifi cantly lower oxygen consumption rate (p <0.05). Measurements of lactate production during the same condition also show a significantly lower rate of lactate production associated with the aged muscles (p <0.05). Following the addition of 2.5 mM. Ca2+ to the solution bathing the muscle, both muscle groups
-
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5 m in 2.5 m M Ca 2 + FIG. 6. Original records of force (lower trace) and oxygen consump tion ( upper trace) of strip of longitudinal smooth muscle of rat urinary bladder (young adult). Downward arrow above oxygen trace indicates point at which muscle was introduced into recording chamber. Muscle was initially in Ca2 + -free solution, and 2.5 mM. Ca2+ was added at point indicated by upward arrow. This induced spontaneous contractile ac tivity with corresponding increase in oxygen consumption. Zero oxygen consumption is represented by long-dashed line, while short-dashed line indicates level of basal oxygen consumption of quiescent muscle. Dashed line in lower panel represents passive force level. 5 also show clear evidence for a decline in Vus over time of contraction. A slowing of V u, associated with increasing dura tion of contraction up to 300 seconds was apparent in both the young adult and aged muscle preparations, with Vus at 300 seconds being reduced to 46 % and 42% of its value at 5 seconds in the young adult and aged muscles respectively. The series elastic component exhibited no evidence of a systematic alteration with increasing duration of contraction within either age group (p >0.05). The young adult muscle strips had a mean series elastic component of 0.063 ± 0.003L0 and the aged a mean of 0.083 ± 0.004 Lo . Between group statistical analysis revealed that the series elastic component was not significantly different in the aged muscle strips (p > 0.05). Force and energy expenditure under basal conditions. An original record of oxygen consumption and force production is shown in figure 6. As is illustrated, muscle preparations were quiescent in Ca2+ -free solution with a constant basal oxygen
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2 . 5 m M ca2+ ca2+ free FIG. 7. Rates of oxygen consumption (J02 ) and lactate production (JLAc) for young adult (hatched bars, n = 10 for J02 , n = 7 for J LAc) and aged (open bars, n = 8 for Jo22+, n = 6 for JLAc) muscle strips in absence and presence of 2.5 mM. Ca . *Denotes value significantly different from corresponding young adult value (p < 0.05).
BLADDER S M O OTH M U S C L E A N D EFFECTS
subsequently developed spontaneous force. Associated with this was a corresponding rise in both oxygen consumption and lactate production above the quiescent basal levels. The aged muscles, however, again had significantly lower oxygen con sumption and lactate production rates when compared with those of the young adults (p <0.05). Relation between force production and oxygen consumption. In both the young adult and aged preparations there was a linear correlation between steady state force production and oxygen consumption rate. This is clearly indicated in figure 8, where force production has been varied by stimulating the muscles with 64 mM. KCl in the presence of graded concentra tions of Ca2 +. Under this stimulation protocol, the muscles produced graded and sustained steady levels of fo rce that facil itated the quantitation of steady state J02 and force. Whilst the data shown in figure 8 indicate that the aged muscles consumed slightly less oxygen than the young adult muscles at equivalent levels of force, it should be borne in mind that the Jo2 values represent total oxygen consumption and include the basal component. In fact the intercept values in figure 8 represent the basal J02 in Ca2+-free 64 mM. KCl solution. When the basal oxygen consumption is subtracted, it is clear that there is no difference in suprabasal J02 at any given force level between the young adult and aged muscles. There were no significant differences between the regression lines fitted to the pooled data for each age group as shown in figure 8 (p <0.05). It was more difficult to establish steady state force levels with carbachol stimulation, since carbachol generally caused an initial rapid fo rce development followed by a decay in force during which phasic contractions developed. Nevertheless, analysis of the average fo rce maintained during this latter phase revealed that it was linearly correlated with the average J02 over this period, and again there were no differences between the young adult and aged preparations in the relation between steady state J 02 and force under carbachol stimulation. Aerobic force and lactate production. Aerobic lactate produc tion rates increased from the unstimulated basal conditions in response to contractions induced by both high KCl and car bachol (table 1). In both conditions , the young adult muscles had significantly higher aerobic lactate production rates (p < 0.05). Although significantly more force was produced by the young adults in response to carbachol, no such difference in
AGING
force was apparent '.¥hen stimulated by KCL Thus it would appear that the lower lactate production rate is not attributable to a diminished force response. Effect of anoxia on force and lactate production. Table l also illustrates the effect of anoxia on force production and lactate production rate. Even in a fully oxygenated environment, there is some loss of force over the 30-minute recording period. Such loss is described by the percentage of peak force being produced by the muscle strip at the completion of 30 minutes. It is quite clear, however, that when the muscles are placed in an anoxic environment, this loss of force is significantly greater in both muscles, and fo r both stimulating conditions (p <0.05), al though it does appear that fo rce is considerably better main tained when stimulated by carbachol than when stimulated by high KCl under both aerobic and anaerobic conditions. Stimulation in an anaerobic environment produced a signif icant rise in the rate of lactate production fo r both muscle groups in response to both stimuli (p <0.05). Under these circumstances the rates of lactate production achieved by the aged muscles were the same as those for the young adult muscles (p >0.05). Since JLAc during stimulation under aerobic condi tions was lower in the aged muscles, the increase from the aerobic to the anaerobic level was proportionally greater in the aged muscles. Rate of A TP synthesis. The rate of ATP synthesis (JATP) was calculated from the rates of oxygen consumption and lactate production as: JATP = 6.42 X Jo2 + 1.25 X JLAc, 9 • 10' 18 Table 2 shows the results of these JATP calculations for young adult and aged muscles under the various conditions. In the Ca2+ free condition, JATP was considerably lower in the aged muscle, The percentage of ATP supplied aerobic glycolysis was also lower than the value for the young adult muscles. With the addition of Ca2 + to the solution and the subsequent development of spontaneous contractile activity, JATP rose from the quiescent levels fo r both muscle groups. The percentage of ATP being supplied by aerobic glycolysis did not change, however, from the quiescent values. During high KCl and carbachol stimula tion there was a considerable rise in JATP of both muscle groups and an increase in the fractional contribution of aerobic gly colysis to total metabolism. The young adult muscles exhibited higher levels of total JATP and a higher JLAc component than the aged muscles under both stimulating conditions. DISCUSSION
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FIG. 8. Relation between steady state force production and rate of oxygen consumption following cumulative addition of Ca2+ in presence of 64 mM. KC!, to young adult (closed circles, n = 7) and aged (open circles, n = 8) muscle preparations (values are means ± SE) . In both age groups oxygen consumption correlates linearly with force (r = 0.992, young adults; r = 0.989, aged muscles) .
The ability of longitudinal smooth muscle strips from rat urinary bladder to produce force in response to electrical field stimulation or high KCl appears to be unaltered with aging. However, there is some diminution in the contractile response to carbachol in the aged muscles. This is demonstrated decreased maximal force levels, a shift to the right of the dose response curve and an increase in the time taken to reach peak force values. Previous studies have either reported an age related increase in responsiveness to muscarinic stimulation, or no change in contractile response compared with young adult animals. 5- 7 The preparations used in those studies, however, have been isolated whole bladders, examined either as intact bladders or spirally cut bladder strips. When comparing results obtained with longitudinal smooth muscle preparations, as in the present investigation, with those obtained with whole blad ders, one should bear in mind the nature of the experimental muscle preparation. Due to the complex composition of the whole bladder preparation, direct comparison to responses of isolated longitudinal smooth muscle can not be confidently made. A clear difference in the contractile properties of the young and aged preparations was observed in terms of the unloaded shortening velocity (Vus ) . The significantly lower V us in the aged smooth muscle strips may indicate that these muscles have an intrinsically lower rate of crossbridge cycling than muscles from young adult animals. This could occur if there
534
BLADDER S M O OTH MUSCLE AND EFFECTS O F AGING
TABLE 1.
Force production (mN./mm.2) and lactate production (µmol./gm./min. _ ,) of longitudinal smooth muscle of rat urinary bladder under aerobic and anaerobic conditions Young adult muscles (n = 7) Aerobic
Aged muscles (n = 7)
Anaerobic
Aerobic
64 mM. KC! (2. 5 mM. Ca J
Anaerobic
2+
Peak Force End Force Lactate production
140 ± 71 ± (48.9% ± 0.30 ±
10 12 6.4) 0.07
118 ± 29 ± (23.3% ± 0.60 ±
Carbaclwl (1
Peak Force End Force
13t 4t 2.6)tO% 0.15 X
145 ± 1 1 54 ± 7 (39.1 % ± 6.8) 0.15 ± 0.04*
100 ± 13t 15 ± St (14.4% ± 2.0)t 0.59 ± 0.14
1 0-• M.)
150 ± 19 119 ± lOt 109 ± 13 88 ± 14t 85 ± 14 51 ± 7t 65 ± 9 36 ± 7t (60.8% ± 5.5) (42.8% ± 5.5)t (62.3% ± 13.7) (41.3% ± 2.l)t Lactate Production 0.34 ± 0.o7 0.58 ± 0.14 0.14 ± 0.02* 0.44 ± 0.04 Values are means ± SE t Significantly different from corresponding aerobic values in same muscle group (p < 0.05, independent t-test). * Significantly different from corresponding value of young adult muscles (p<0.05, independent t-test). End force values represent the force level being produced by the muscle at the completion of the 30-min measuring period. Percentage that end constitutes of peak force is shown in parentheses. TABLE 2.
Calculated rate of ATP synthesis and estimated contribution of oxidative metabolism and glycolysis to total metabolism for young adult and aged longitudinal muscle of the rat urinary bladder during unstimulated and stimulated conditions Young adult muscles
Aged muscles
JATP µmol./gm./
JATP µmol./gm./ % from % from % from % from min- 1 min- 1 Jo, JLAC J o, JLAC Ca2+ -free Ringer 1.40 91.1% 8.9% 1.03 96.3 % 3.7% 2.5 mM Ca2+ Ringer 2.47 92.3% 7.7% 1.77 95.6% 4.4% 64 mM. KCl 3 .04 87.8% 12.2% 2.49 92.0% 8.0% 3.52 Carbachol (1 x 10-• M.) 88.0% 12.0% 2.96 94.0% 6.0% JATP represents the estimated rate of ATP usage as calculated: JATP = J02 (6.42) + JLAc (1.25). Percentage values are also estimated from the equation given.
was some transition in the isoform of myosin with aging as has been reported to occur with aging in cardiac muscle. 19 At the present time, however, there is no evidence to suggest any such effects of aging on the properties of smooth muscle myosin. Arner et al. 20 reported a reduced Vmax in hypertrophied rat portal vein, which they ascribed to a lower rate of crossbridge turnover considered to be due to alterations in either the activation mechanisms or the intrinsic properties of the con tractile system itself. Alternatively the lower Vu, could be accounted for by an increased internal load in the aged prepa rations. This increase could possibly result if there were struc tural alterations in the aged tissues. An increase in the relative amount of intermediate filaments has been observed in hyper trophic smooth muscle 21 ; however, the possibility of such alter ations occurring in aged smooth muscle remains unclear. Unloaded shortening velocity of both the young adult and aged muscle strips declined with increasing duration of con traction. The values of Vu, after 300 seconds of contraction were approximately 46% and 42% of those after 5 seconds of contraction in the young adult and aged muscles, respectively. A similar decline in Vu, with increasing duration of contraction has been observed in several vascular22 • 23 and visceraP 0• 24 smooth muscles. Previous studies have linked the decline in Vu, with the development of the "latch state" during contraction of smooth muscle. 22 • 2 5 The present observations of a decline in Vu, over time in rat urinary bladder smooth muscle are in general agreement with such previous studies and, if interpreted within the framework of the latch hypothesis, would indicate that the latch phenomenon continues to be expressed in aged smooth muscle preparations. The aged bladder preparations exhibited a reduced basal metabolic rate compared with the young adult muscles when quiescent in Ca2+ -free solution. This was manifest in both significantly lower oxygen consumption and lactate production rates (and, hence, calculated JATP; see figure 7 and table 2). Energy turnover of quiescent muscle reflects the energy require ments of processes involved in maintaining cellular homeosta sis. The increased bladder weight-to-body weight ratio in the aged animals indicates that the aged bladder is hypertrophied.
If this is associated with an increase in cell size, then the depressed basal metabolism may be related to the greater size of the aged bladder smooth muscle cells. These larger cells would have a lower surface-to-volume ratio; hence ionic ho meostasis might be more easily maintained, requiring a lower level of energy expenditure, as has been suggested to occur in hypertrophied cardiac muscle. 26 In apparent contrast, however, it has recently been reported that bladder strips hypertrophied as a result of outlet obstruction did not exhibit any detectable change in the basal Jo2 or J LAc as compared with control bladder strips. 18 The hypertrophied bladders examined in that study were, however, obtained from young adult rats that had been subjected to severe outlet obstruction for a relatively brief time (10 days) . The depressed basal metabolism observed in the present study may be largely attributable to age-related factors unrelated to hypertrophy, or if it is in fact due to hypertrophy, may reflect the different nature and time course of the hyper trophic stimulus in the naturally aging animal. A further factor that may contribute to the lower basal metabolism in the aged muscle is a reduced rate of protein turnover. Decreases in the rates of protein synthesis and degradation have been reported to be associated with aging in cardiac muscle. 19 The increase in JATP from quiescent to spontaneously con tracting preparations was proportionally the same for both muscle groups, indicating that under these conditions about 40% of the total energy flux could be ascribed to the contractile activity as such. Although proportionally the increases were the same, the total calculated rate of JATP remained consider ably lower in the aged muscles in comparison to the young adults. The lower J ATP in the aged muscles is probably reflective of the significantly lower average peak force level produced during spontaneous contractile activity by these preparations, together with the underlying lower basal JATP, as determined in the Ca2+ free state. Muscle strips from both the young adult and aged bladders derived a portion of their total energy needs from aerobic glycolysis; however, the proportional contribution of aerobic glycolysis to total energy production was less in the aged preparations. In the quiescent state, aerobic glycolysis contrib-
BLADDER S M OOTH MUSCLE AND EFFECTS OF AGING uted approximately 8.5 % of total ATP generation in the young adult muscles and 4% in the aged muscles while, in the stimu lated state, the contributions of aerobic glycolysis to total metabolism were approximately 1 2 % and 6 to 8% in the young adult and aged muscles, respectively. The values for the young adult preparations are in good agreement with those reported by Arner et aL, 1 8 also for rat urinary bladder. A substantial contribution of aerobic glycolysis to total metabolism appears to be a feature common to most smooth muscles, with contri butions of 5 to 1 5 % under quiescent conditions and 10 to 35% under stimulated conditions, having been reported for a variety of vascular and visceral smooth muscles. 9 · 1 0· 1 1 • 1 5 • 16• 27 The lower aerobic lactate production rates observed in the aged muscles do not appear to be due to a limitation in the ability of the aged muscles to generate ATP via glycolysis since these preparations are able to increase their rate of lactate production to the same levels as young adult muscles when stimulated by high KC! or carbachol under anoxic conditions. It has been proposed that aerobic glycolysis may play an important role in preferentially providing ATP for membrane associated processes, such as the Na+ - K + pump, in smooth muscle. 1 0 In this context, it is interesting to speculate that the reduced J LAc observed in the aged preparations may represent a lower energy requirement of such membrane-associated proc esses. Again this may be related to the apparent hypertrophy of the aged bladder and the possible increase in cell size, which would result in a decrease in the amount of cell membrane relative to cell volume. The rate of oxygen consumption was observed to correlate linearly with isometric force in both the young adult and aged muscles. This correlation was most clearly indicated when the muscles were stimulated with 64 mM. KCl in the presence of varying concentrations of extracellular Ca2+ (fig. 8). A linear relation between J 02 and force has previously been observed in a variety of smooth muscle preparations. 9- 1 1 • 28 There was no difference in the relation between suprabasal J 02 and force between the young adult and aged muscles. This indicates that the energetic cost of maintaining a given force level, as derived from oxidative metabolism, is the same in the two muscle groups. Even when the lower J LAc component of the aged muscles is taken into account, the metabolic tension cost (su prabasal J ATP per unit tension) is not significantly different between the young adult and aged muscles. Lactate production was only measured during maximal contractions in the present study and not during the graded contractions; however, it has been shown that J LAc varies linearly with tension in rabbit urinary bladder smooth muscle. 1 1 The unchanged metabolic tension cost in the aged muscles suggests that the cross-bridge turnover rate is not altered with aging. This would in turn indicate that the lower V u, of the aged preparations is perhaps more likely to be the result of some phenomenon such as an increase in internal load rather than some change in the intrin sic properties of the contractile system itself. Under anaerobic conditions force production was reduced in both the young adult and aged muscles. The extent of the reduction in force was similar in the two groups when stimu lated with carbachol; however, with high KC! stimulation the reduction in force was slightly greater in the aged muscles. Both the young adult and aged muscles appeared to be less able to sustain force under anoxic conditions when stimulated with high KC! rather than carbachoL The rate of lactate production during stimulation under anoxic conditions increased to similar levels in both groups; however, as J LA C under aerobic conditions was less in the aged muscles, the proportionate increase was in fact greater in these muscles. J LAc during stimulation under anaerobic conditions was approximately 2-fold and 4-fold higher than during stimulation under aerobic conditions in the young adult and aged muscles, respectively. When calculated as J A TP (J LAc x 1.25), these rates of lactate production under anaerobic conditions represent only approx-
535
imately 20 to 25% of the total JATP observed with the same stimulation under aerobic conditions, where both oxidative metabolism and aerobic glycolysis can contribute to ATP pro duction. Since the anaerobic force levels remain, on average, at >50% of the aerobic levels over the 30-minute stimulation period, this implies that the metabolic tension cost is substan tially less during contractions in an anoxic environment. Arner et aL 1 8 reported a similar finding and suggested that anoxic contractions for both control and hypertrophic detrusor muscle are associated with a considerably lower cross-bridge turnover rate compared with normoxic contractions. Results obtained in our laboratory (Munro and Wendt, unpublished observations) provide data demonstrating that Vus is significantly decreased during anoxic contractions, suggesting that a reduced cross bridge cycling rate may account, at least in part, for the de creased tension cost during anoxia. However, a full understand ing of the mechanisms that permit continued function with an apparently greatly reduced metabolic cost under anoxic condi tions is yet to be gained. In conclusion, aging was found not to affect the force-gen erating capacity of longitudinal smooth muscle of the rat uri nary bladder, although there was a decrease in muscarinic responsiveness. The maximal velocity of shortening was signif icantly longer in muscles from aged animals than in those from young adult animals. The metabolic tension cost during iso metric contraction was, however, the same in both groups, suggesting that the decline in Vu s is due largely to factors not influencing energetic cost. The aged muscles also exhibited a lower basal metabolic rate and a reduced contribution of aerobic glycolysis to total metabolic energy production during both quiescence and stimulation under normoxic conditions than did muscles from young adult animals. They were, however, able to increase their rate of lactate production to the same levels as young adult muscles when stimulated under anoxic conditions. REFERENCES
L Campbell, A. J., Reinken, J. and McCosh, L.: Incontinence in the elderly: prevalence and prognosis. Age Ageing, 1 4 : 65, 1985. 2. Andersen, J, T., Jacobsen, 0., Worm-Petersen, J. and Hald, T.: Bladder function in healthy elderly males. Scand. J. UroL Ne phroL, 1 2 : 123, 1978. 3. Castleden, C. M., Duffin, H. M. and Asher, M. J.: Clinical and urodynamic studies in 100 elderly incontinent patients. Br. Med. J., 282: 1 103, 1981. 4. Drach, G. W., Layton, T. N. and Binard, W. J.: Male peak urinary flow rate: relations to volume voided and age. J. UroL, 1 2 2 : 210, 1979, 5. Kolta, M. G,, Wallace, L. J. and Gerald, M. C.: Age-related changes in sensitivity of rat urinary bladder to autonomic agents. Mech. Ageing Dev., 2 7 : 183, 1984. 6. Ordway, G. A., Esbenshade, T. A., Kolta, M. G,, Gerald, IVL C, and Wallace, L. J.: Effect of age on cholinergic muscarinic respon siveness and receptors in the rat urinary bladder. J. UroL, 136: 492, 1986, 7. Chun, A, L., Wallace, L. J., Gerald, M. C., Wein, A. J. and Levin, R M.: Effects of age on urinary bladder function in the male rat. J. UroL, 1 4 1 : 1 70, 1989. 8. Butler, T. M. and Davies, R. E . : High-energy phosphates in smooth muscle. In: Handbook of Physiology-The Cardiovascular System IL Edited by D. F. Bohr, A. P. Somlyo and H. V. Sparks, Jr. Bethesda: American Physiological Society, pp. 237-252, 1980. 9. Paul, R J.: Chemical energetics of vascular smooth muscle. In: Handbook of Physiology-The Cardiovascular System IL Edited by D. F. Bohr, A. P. Somlyo, and H. V. Sparks, Jr. Bethesda: American Physiological Society, pp. 201-235, 1980. 10. Paul, R J.: Smooth muscle mechanochemical energy conversion: relations between metabolism and contractility. In: Physiology of the Gastrointestinal Tract. Edited by L. R Johnson. New York: Raven Press, 2nd edn. , pp. 483-506, 1987. l L Wendt, L R and Gibbs, C. L.: Energy expenditure of longitudinal smooth muscle of rabbit urinary bladder. Am. J. PhysioL, 252: C88, 1987.
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BLADDER SMOOTH MUSCLE AND EFFECTS OF AGING
12. Wendt, I. R.: Effects of substrate and hypoxia on smooth muscle metabolism and contraction. Am. J. Physiol., 256: C719, 1989. 13. Gliick, E. and Paul, R. J.: The aerobic metabolism of porcine carotid artery and its relationship to isometric force: energy cost of isometric contraction. Pfliigers Arch., 370: 9, 1977. 14. Hellstrand, P., Johansson, B. and Norberg, K.: Mechanical, elec trical, and biochemical effects of hypoxia and substrate removal on spontaneously active vascular smooth muscle. Acta Physiol. Scand., 100: 69, 1977. 15. Casteels, R. and Wuytack, F.: Aerobic and anaerobic metabolism in smooth muscle cells of taenia coli in relation to active ion transport. J. Physiol. (Lond) ., 250: 203, 1975. 16. Kroeger, E. A.: Effect of ionic environment on oxygen uptake and lactate production of myometrium. Am. J. Physiol., 230: 158, 1976. 17. Uvelius, B.: Shortening velocity, active force and homogeneity of contraction during electrically evoked twitches in smooth muscle from rabbit urinary bladder. Acta Physiol. Scand., 1 06: 481, 1979. 18. Arner, A., Malmqvist, U. and Uvelius, B.: Metabolism and force in hypertrophied smooth muscle from rat urinary bladder. Am. J. Physiol., 258: C923, 1990. 19. Lakatta, E. G.: Cardiac muscle changes in senescence. Ann. Rev. Physiol., 49: 519, 1987. 20. Arner, A., Malmqvist, U. and Uvelius, B.: Effects of Ca2+ on force-
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