There is considerable evidence from animal and human research that adrenergic control of cardiovascular function declines with age (1). Several studies reported decreased cardiac norepinephrine (NE) content per gram heart weight (2,3) and increased adrenergic nerve terminal degeneration in old rats (4). Within the cardiac adrenergic neuroeffector junction, presynaptic release of NE (5-9) and postsynaptic β1-adrenergic receptor-mediated responses to adrenergic stimulation (10-15) decline with age. Although there seems to be general agreement about these age-related changes within the cardiac adrenergic neuroeffector junction, there are conflicting reports on the influence of age on neuronal NE transport capacity in the heart. Kreider et al. (15), by using the isolated heart preparation, reported that cocaine, an neuronal NE uptake blocker, enhanced the heart rate response to NE in 12- and 24-month-old rat hearts but not in 6-month-old rats. Similar findings were reported by Daly et al. (16), who showed that cocaine increased the quantity of NE overflow in 12- and 24-month-old but not 6-month-old rats during nerve stimulation of the isolated heart. These studies suggest that aging increases the neuronal NE uptake capacity of the heart. However, other investigators reported that cocaine augments cardiac NE release and the pressor and tachycardia responses to NE in young rats but not old rats (17,18). Reduced NE uptake capacity also was reported in the hearts of elderly men (19).
The activity of NE released by adrenergic nerves is terminated for the most part by an active neuronal uptake of the neurotransmitter by the nerve terminal (20). Neuronal uptake is an adenosine triphosphate (ATP)-dependent process that relies on the Na+ gradient to drive the transport of NE into the nerve. In the rat heart, only ∼9% of released NE diffuses out of the synapse and spills over into the blood (20). Therefore age-related changes in neuronal uptake of NE can markedly alter synaptic NE concentration, which determines the response of the end organ to NE. The purpose of this study was to characterize neuronal NE uptake in cardiac synaptosomes prepared from young and old male F344 rat hearts. We determined both the maximal rate of NE transport (Vmax) and transporter affinity for NE as reflected by the Km. In addition, the sensitivity of cardiac synaptosomal NE uptake to inhibition by desmethylimipramine (DMI), a selective neuronal NE-uptake blocker, was assessed.
The effects of dietary restriction (DR) on cardiac synaptosomal NE uptake also was examined because previous studies suggested that part of the beneficial action of DR in extending life span may be related to changes in cardiac adrenergic nerve terminal function (21,22).
MATERIALS AND METHODS
Ad libitum (AL) and dietary restricted (DR) male F344 rats were obtained from the National Center for Toxicological Research (NCTR), where they were maintained under barrier conditions. The AL animals were fed on a pasteurized rodent diet and autoclaved water adjusted to pH 3. Starting at weaning, the DR animals were given 60% of the daily caloric intake consumed by AL rats of the same age. In our animal facility, the DR diet was maintained as provided by the NCTR. On arrival in our animal facility, the rats were free of infectious disease, as verified by serologic assay, and were housed one rat per cage under barrier conditions in standard filtered cages, in a temperature-regulated environment (21 ± 1°C) with a 12-h light/dark cycle. The rats were used 1 to 3 weeks after arrival in our facility. The animal facility is AALAC approved with licensed staff veterinarian.
Preparation of cardiac synaptosomes
Synaptosomes were prepared from rat hearts by methods described in our earlier publications (7-9,21,23). Each animal was killed by decapitation. The heart was cleaned in ice-cold 0.32 M sucrose, blotted dry, and then weighed. The heart was minced in ice-cold 0.32 M sucrose containing 1 mM EGTA and then digested for 40 min at 37°C in HEPES buffered saline (HBS) containing 12 units of collagenase (class II, Worthington Biochemical Corp., Freehold, NJ, U.S.A.) per milligram of tissue. After rinsing the tissue with buffer to remove the collagenase, the digested tissue was homogenized in 0.32 M sucrose by using a Teflon/glass homogenizer. Cellular debris was pelleted by centrifugation at 650 g for 10 min. The supernatant was centrifuged at 21,000 g for 20 min, and the resulting pellet (P2) was resuspended in cold buffer. The P2 pellet contains the cardiac synaptosomes (23).
Measurement of neuronal NE uptake
The capacity to accumulate NE was determined in cardiac synaptosomes according to the method of Aloyo et al. (23), with some modifications. Duplicate aliquots of 100 μg of synaptosomal protein in 100 μl of HBS were incubated at 37°C for 10 min before starting the uptake reactions by adding [3H]NE at concentrations of 50, 100, 200, and 400 nM. The reactions continued for 10 min. An additional set of duplicate aliquots containing 1 μM desmethylimipramine (DMI), a specific neuronal NE-uptake blocker, also were incubated with each [3H]NE concentration. At the end of 10 min, each aliquot was vacuum filtered through a Whatman GF/B filter (Whatman International Ltd., Maidstone, England) and rinsed 3 times with 3 ml of HBS. The filters were placed in scintillation fluid, vortexed, and allow to sit overnight. Each sample was counted in a Beckman scintillation counter. DMI-sensitive NE uptake was determined by subtracting the average DPM of aliquots containing DMI from the average DPM from aliquots without DMI and then converting DPM to pmoles of NE (see Fig. 1A).
The Vmax and Km for NE-uptake activity were determined by plotting the reciprocals of the DMI-sensitive NE uptake (pmol/min/mg protein) and the [3H]NE concentration (nM), a transformation known as Lineweaver-Burk plots (24). After this transformation, the data form a straight line, the Vmax is equal to 1/y-intercept, and the Km is equal to the slope × Vmax (see Fig. 1B).
Duplicate aliquots of 100 μg of synaptosomal protein in 100 μl of HBS were incubated with 1 nM to 1 μM DMI at 37°C for 10 min before starting the uptake reactions by adding [3H]NE at 300 nM. The reactions continued for 10 min and were stopped as described earlier. Preliminary experiments (data not shown) and prior studies (23) showed that maximal inhibition of NE uptake in cardiac synaptosomes occurs at 1 μM of DMI in all ages and treatment groups. Data from the concentration-response curves for DMI were used to calculate a median inhibitory concentration (IC50) for each individual animal by using the sigmoidal dose-response equation with variable slope contained in the GraphPad PRISM data-analysis software package. This equation also calculates a Hill slope.
Endogenous NE content of cardiac synaptosome preparation
Triplicate samples of 50 μl of the P2 were stored at −70°C and later analyzed for endogenous NE content by using alumina extraction and high-performance liquid chromatography/electrochemical detection methods (16). Endogenous NE content is an index of the number of synaptosomes in each preparation.
Endogenous NE content of whole hearts
Whole hearts were flash frozen in liquid N2. The frozen hearts were thawed and minced in 5 ml/g of 1N acetic acid and 0.02N HCl, and then homogenized by using a polytron at setting 8 for 1 min. The homogenate was centrifuged for 30 min at 20,000 g, and the supernatant removed and placed on ice. The pellet was resuspended in 5 ml/g of 1N acetic acid and 0.02N HCl, rehomogenized, and centrifuged for 30 min at 20,000 g. The second supernatant was combined with the first, and 150 μl of the supernatant was analyzed for endogenous NE content by using alumina extraction and high-performance liquid chromatography/electrochemical detection methods (16).
The data are presented as mean ± standard error of the mean (SEM). A two-factor analysis of variance (ANOVA) was used to determine the effect of age and diet on Vmax, Km, NE content, and IC50 for DMI and NE content. Interaction between age and diet was included in the model. Further ANOVA and the Fisher PLSD (protected least significant difference) mean separation tests were used to distinguish significant age effects within diet groups and significant diet effects within age groups. Significance for the ANOVA and Fisher PLSD was set at p < 0.05. The means of the Hill slopes for each experimental group were analyzed with a one sample t test (p < 0.05) to determine whether the Hill slopes were significantly different from 1.
Synaptosomal NE uptake
Vmax and Km for NE transport are presented in Table 1. When expressed per mg protein, the Vmax for NE transport declined significantly in AL rats between 12 and 24 months, but there were no age-related changes in DR rats. Moreover, Vmax for transport was significantly higher in DR rats as compared with AL rats. When expressed per ng NE in the cardiac synaptosome preparation (P2), the Vmax for NE transport in AL rats was significantly less at 12 and 24 months compared with that at 6 months. In DR rats, Vmax for NE transport was significantly less at 24 months compared with that at 6 and 12 months. No differences between AL and DR rats were noted when the data were expressed in terms of ng NE. The differences in Vmax between AL and DR rats when expressed per mg protein are probably the result of an increased yield of synaptosomes in the DR rats, as suggested by the significantly higher endogenous NE content in the P2 fraction of DR rats (see Table 1).
The Km for NE uptake was not different between age or diet groups.
Figure 2 shows the concentration-response curves for DMI inhibition of NE transport into cardiac synaptosomes. The sensitivity to the inhibition by DMI, as indicated by the IC50 values, did not change with age or diet (Table 2). The means of the Hill slopes for each age and diet group were not significantly different from 1.
Endogenous NE content of the heart
The total NE content of the whole heart did not change with age or diet (Table 3). However, when expressed per gram heart weight, there was a significant decline in NE content in 24-month-old AL rats compared with younger AL rats, but no change with age in DR rats. When expressed per gram heart weight, DR rats had significantly greater NE content than AL rats at all ages. The differences in NE content per gram heart weight probably reflect the increase in heart size with age in AL rats and the smaller size of the DR hearts.
We showed that a brief period of collagenase digestion before fractionation of the heart with a Teflon/glass homogenizer produces viable cardiac synaptosomes. In this preparation, NE accumulation is attenuated by DMI, at 2°C, in buffer in which choline replaces sodium, and in hypoosmotic buffer, but is insensitive to metanephrine, a nonneuronal NE-uptake blocker (23). These findings are characteristic of intact synaptosomes (25) and indicate that the site of NE uptake is the synaptosome. Therefore cardiac synaptosomes are ideal for investigating cardiac neuronal uptake in aged animals.
The results of this study indicate that the capacity to transport NE into cardiac synaptosomes declines with age in the male F344 rat. These results are in agreement with those of Docherty and Hyland (17) and Borton and Docherty (18) but disagree with those of Krieder et al. (15) and Daly et al. (16). Buchholz and Duckles (26) reported no change in NE uptake with age in the rat tail artery and concluded that the amount of NE taken up is proportional to the amount of NE released. The major difference between these studies and our study is the use of cardiac synaptosomes to measure NE uptake capacity directly, whereas the other studies measured changes in NE release after exposure to a neuronal-uptake blocker such as cocaine. Daly et al. (16) showed that in the presence of cocaine, no increases in NE spillover occurred in young rat hearts, whereas in older rat hearts, NE spillover was increased. Buchholz and Duckles (26) proposed that the response to cocaine in young rat hearts may be masked by the activity of presynaptic α2-adrenergic receptors, which inhibit NE release. Presynaptic α2-adrenergic receptor activity was reported to decline with age in the rat heart (27). As the concentration of NE in the cardiac synaptic clef increases in the presence of cocaine, the increased stimulation of presynaptic α2-adrenergic receptors in the young hearts may cancel the actions of cocaine, resulting in a net lack of change in NE spillover. The reduction in presynaptic α2-adrenergic receptor-mediated inhibition of NE release in old rat hearts may yield a net apparent increase in NE spillover in the presence of cocaine. At the concentrations that block NE uptake, cocaine's local anesthetic effects (28) also may limit NE release; therefore complete blockade of NE uptake by cocaine may also result in greatly reduced NE release. Indeed, because in the rat, only one tenth of NE released into the synaptic cleft spills over into the circulation (20), if complete blockade had taken place, NE spillover should have been 10 times the noncocaine levels. For these reasons, indirect estimates of NE-uptake capacity by using cocaine may be complicated by factors that influence NE release.
Our results for Km and Vmax at 6 months are similar to those reported for cardiac synaptosomes by Aloyo et al. (23; 430 nM and 1.10 pmol/min/mg protein) and Dumont and Lemaire (29; 385 nM and 0.6 pmol/min/mg protein). The Km values obtained in our study are also in accord with those previously reported by Iverson (30) in the isolated rat heart and by Slotkin et al. (31) in rat brain synaptosomes. Vmax is dependent on the number of NE-transporter sites present in the synaptosome, and therefore differences in yield of synaptosomes per mg protein between the age groups may affect Vmax. The apparent increase in Vmax in DR rats compared with AL rats seems to be the result of a greater yield of synaptosomes per mg protein because the differences disappear after correcting Vmax for ng NE per mg of protein. Using the NE content of the synaptosome preparation to correct for differences in synaptosome yield assumes that the content of NE per synaptosome is the same between young and old rats and between AL and DR rats. This may be a safe assumption because total NE content per heart did not change with age in our study. Other investigators also reported that total NE content per heart in the rat does not change with age (32-34), but NE/g heart weight declines significantly with age (2,3,34). In DR rats, however, the total NE content per heart is similar to that in AL rats, but the weight of the heart is reduced by ≥25%, suggesting that DR does not alter the number of cardiac adrenergic nerves per heart or that the content of NE per nerve is increased by DR. If the latter is true, then our use of endogenous NE to correct for synaptosome yield may under estimate the Vmax in DR rats. Kim et al. (22) also reported that 6-month-old male DR F344 rats had higher endogenous NE per gram heart weight, higher endogenous NE content in the P2 fraction, and higher Vmax per mg protein compared with 6-month-old AL rats. Our study indicates that DR delays the age-related reduction in NE uptake capacity seen in AL rats.
Our results agree with those of Esler et al. (19) in the aging human heart. In this study, an increase in cardiac NE spillover was attributed to a reduced neuronal uptake of NE because the transcardiac extraction of plasma radiolabeled NE was lower in the older subjects. Our results, as well as those of Esler et al. (19), suggested that the number of NE transporters may decline with age. To our knowledge, the effect of age on the number of NE transporter sites in the heart has not been measured. No change in affinity of the transporter for NE and no change in the sensitivity of DMI argue against an age-related change in transporter binding of NE, because DMI competes with NE for the same binding sites on the NE transporter (35). Because NE uptake is a Na+-dependent process (35), age-related changes in the binding of this ion may alter NE uptake. Other possible mechanisms for a decrease in NE-uptake capacity with age may be reduced Na+ concentration gradient due to defects in the Na+/K+ ATPase pump and changes in membrane structure that interfere with movement of the transporter across the membrane. NE-transporter function may be influenced by age-related differences in the local concentration of adenosine, histamine (36), and ATP (37). Adenosine and histamine have been shown to reduce NE uptake in the heart during ischemia (36), and ATP enhances NE uptake into PC12 cells (37). Studies measuring DMI-binding sites in the rat heart showed that clonidine treatment, which reduces NE release by activating α2-adrenergic receptors, reduces DMI-binding sites, and amphetamine treatment, which increases NE release, increases DMI binding (38). These findings suggest that the age-related decline in cardiac NE release noted in several previous studies in the rat (5-9,16,21) may directly influence NE uptake and result in reduced NE uptake with age.
In conclusion, our studies suggest that neuronal NE-uptake capacity declines in the aging heart. At present there is no clear explanation for this decline.
Acknowledgment: This study was supported by a grant from the National Institutes of Health (AG 11060).
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