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Adrenal neuropeptide Y mRNA but not preproenkephalin mRNA induction by stress is impaired by aging in Fischer 344 rats
Mechanisms of Ageing and Development 101 (1998) 233 – 243
Adrenal neuropeptide Y mRNA but not preproenkephalin mRNA induction by stress is impaired by aging in Fischer 344 rats Jeffrey H. Silverstein a,c,d,e,*, Joseph Beasley c, Tooru M. Mizuno b,f, Evan London a, Charles V. Mobbs b,e,f a Anesthesia Section, Veterans Affairs Medical Center, Bronx, NY 10468, USA Pathology and Diagnostic Laboratory Ser6ice, Veterans Affairs Medical Center, Bronx, NY 10468, USA c Department of Anesthesiology, Mount Sinai School of Medicine, 1 Gusta6e L. Le6y Place, New York, NY 10029 -6574, USA d Department of Surgery, Mount Sinai School of Medicine, 1 Gusta6e L. Le6y Place, New York, NY 10029 -6574, USA e Department of Geriatrics and Adult De6elopment, Mount Sinai School of Medicine, 1 Gusta6e L. Le6y Place, New York, NY 10029 -6574, USA f Fishberg Center for Neurobiology, Mount Sinai School of Medicine, 1 Gusta6e L. Le6y Place, New York, NY 10029 -6574, USA b
Received 27 August 1997; received in revised form 27 November 1997; accepted 2 December 1997
Abstract Relatively few molecular markers of stress have been studied in aged individuals. Interactions of age and stress on adrenal neuropeptide Y (NPY) and preproenkephalin (ppENK) expression have not been reported. The purpose of these studies was to characterize the adrenal NPY and ppENK responses to stress using a common stressor, physical restraint for 2 h, in Fischer 344 rats at 7, 16 and 23 months of age. Northern blot techniques were used to evaluate induction by stress of adrenal NPY mRNA and adrenal ppENK mRNA. Two humoral responses to stress, serum glucose and corticosterone, were measured to corroborate
* Corresponding author. Tel.: +1 212 2416426; fax: + 1 212 8763906; e-mail: Jeff – [email protected] 0047-6374/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 4 7 - 6 3 7 4 ( 9 7 ) 0 0 1 7 7 - 2
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that a stress response occurred. We observed that the induction by stress of adrenal NPY mRNA is impaired with age but the stress-induced elevation of adrenal ppENK mRNA, blood glucose, and corticosterone show no evidence of age-related impairments. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neuropeptide Y; Enkephalins; Stress, mechanical; Glucose; Adrenal; Aging
1. Introduction A variety of physiologic and behavioral systems are activated by stress. Alterations of the ability to respond to stressors, involving either excessive or inadequate responses in either duration or magnitude, have been speculated to lead to disease (Selye, 1976). Numerous investigators have proposed that responses to stress change with age (Sapolsky et al., 1983; Brodish and Odio, 1989; Blake et al., 1991; de Kloet, 1992). The basal activity of two major stress systems, the sympathetic nervous system (Ziegler et al., 1976) and the hypothalamic–pituitary–adrenal (HPA) system (Landfield and Eldridge, 1994), may increase with age in some systems, and the HPA response to stress may effectively increase with age, due to impaired negative feedback (Sapolsky et al., 1986a). The one molecular stress response which has been examined, the induction of hsp 70 mRNA, has been reported to be attenuated with aging response to restraint stress (Blake et al., 1991; Udelsman et al., 1993) and mild heat shock (Heydari et al., 1993; Wu et al., 1993). Neuropeptide Y (NPY) is a vasoconstrictor peptide which is abundantly present in peripheral noradrenergic nerves, the brain, adrenal medulla, cardiac neurons and the myenteric plexus of the gut. Recent work suggests an important role for NPY in the stress response (Zukowska-Grojec, 1995) and particularly its induction in the rat adrenal gland following stress (Hiremagalur et al., 1994). The ability of opioid peptides to inhibit the release of various neurotransmitters supports their possible function as neuromodulators. Proenkephalin was first discovered in bovine adrenal cortex where enkephalin biosynthesis was elucidated (Lewis et al., 1980). Preproenkephalin (ppENK) mRNA has been shown to be induced by restraint stress (Dowds et al., 1994). In the present study, the effect of age on these two molecular responses to restraint stress was evaluated. Corticosterone and glucose were also evaluated to ensure that the individuals were in fact responding to the stress paradigm.
2. Materials and methods
2.1. Animals Fisher 344 male rats were purchased at ages 6, 15 and 22 months of age from NIA/Harlan Sprague Dawley. Care and use were in accord with the guidelines of
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the Mount Sinai School of Medicine and the Bronx Veterans Affairs Medical Center. The animals were individually housed (10¦ × 8¦× 7¦ with wire mesh floors suspended on a Lab Products rack; Lab Products, Rochelle Park, NJ) and maintained in a 12-h light, 12-h dark cycle (lights on at 07:00 h) with ad libitum access to food (laboratory autoclavable rodent diet 5010, Purina) and water. Prior to arrival, the rats were fed NIH 31 diet (Harlan Tekald Laboratory Diet — sterilized) which differs from our rat chow in that the Pruina chow has 23.0% crude protein versus 18.4% for NIH 31. The animals are housed for approximately 1 month in this facility prior to experimentation. Sentinel animals were sacrificed for monitoring for murine virus antibodies (sendai, PVM, SDA/ RCV, KRV, H-1, GD-7, Reo-3, LCMV, MAD, Ectro, CARB, RCMV, Hant, Mycoplasma pulmonis, E. cunicuili, B. piliformis) following arrival and this process was carried out at least every 6 months. Beginning at 09:00, animals were restrained for 120 min, a time chosen based on the expected peak for NPY mRNA and ppENK mRNA (Nankova et al., 1996). Restraint consisted of immobilization of all four extremities by padded cords designed to avoid injury to the limbs. The head was not restrained. Rats were sacrificed at the end of the stress period, by exposure to carbon dioxide for : 1.5 min following which the animals were decapitated. In all cases, rats were sacrificed between 11:00 and 13:00 h. Control animals were removed from their home cage and immediately sacrificed. The trunk blood was collected into EDTA coated tubes on ice. Blood glucose was measured immediately after decapitation by a One-Touch II glucose meter (Lifescan, Milpitas, CA) using a fresh drop of trunk blood. The glucose meter was calibrated daily. Animals were examined for macroscopic pathology. Two animals were removed from the 23-month-old group (one each from the restraint and control groups) due to pituitary tumors. No renal or other intraabdominal tumors were noted. Both adrenals were removed, cleaned of extraneous fat, and immediately frozen for subsequent analysis. Adrenal glands for each animal were maintained separately for the entire experiment. Plasma corticosterone was measured by radioimmunoassay (corticosterone — 125I RIA kit, ICN Biomedicals, Costa Mesa, CA).
2.2. RNA extraction and Northern blot analysis Total RNA from : 50 mg of each individual adrenal gland was extracted in RNAzol B (Tel-Test, Friendswood, TX) and chloroform, and precipitated by isopropanol. Each pellet was resuspended in 25 ml of diethylpyrocarbonate (DEPC) treated water. The quantity and integrity of the isolated RNA was determined by both UV absorbance spectroscopy (260 nm/280 nm) and agarose electrophoresis, visualizing the intact ribosomal RNA bands with ethidium bromide. Ten micrograms of total RNA was denatured by incubating with glyoxal and dimethyl sulfoxide for 1 h at 50°C, loaded into 1.5% agarose gel, then separated by electrophoresis at 80 V for 1.5 h. RNA was transferred to Immobilon S® (Millipore) by capillary elution in 20× standard saline citrate (SSC)
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(1× SSC is 0.15 M NaCl/15 mM sodium citrate, pH 7.0). After being washed with 6 × SSC, the membrane was baked at 80°C for 1 h and exposed to the UV crosslinker (300× 100 mJ/cm2) to fix RNA. The membrane was pre-hybridized in a solution containing 25 mM NaPO4 (pH 7.0), 4 × SSC, 5 mM EDTA, 5 × Denhardt’s solution, 0.5% (vol/vol) SDS, 50% (vol/vol) deionized formamide, and denatured salmon sperm DNA at 100 mg/ml (5 Prime3 Prime, Boulder, CO) at 42°C overnight. Hybridization was done in the same buffer containing 10% (wt/vol) dextran sulfate and 32P-labeled NPY or (ppENK) gene probe at 1.6 mCi/ml at 42°C overnight. The membrane was washed twice in 1 × SSC, 0.1% SDS for 20 min at room temperature, followed by two washes in 0.1 × SSC/ 0.1% SDS for 15 min at room temperature. Subsequently, the membrane was washed in 0.1× SSC, 0.1% SDS for 3 h at 55°C and then exposed to autoradiography film (DuPont NEN, Wilmington, DE) between intensifying screens for 1–48 h at − 70°C. (To monitor RNA loading, the membrane was reprobed and hybridized with 32P-labeled probe encoding 18S ribosomal RNA.) The total integrated densities of hybridization signals were determined by computerized densitometric scanning (MCID, St. Catherine’s, ON).
2.3. Template production and probe labeling The NPY probe was prepared by amplifying a source DNA fragment (kindly provided by S. Sabol) using PCR with N-terminal primer: 5%-CTAGGTAACAAACGAATGGGG-3% and C-terminal primer: 5%CCACATGGAAGGGTCTTCAAG-3% (Higuchi et al. 1988a). A ppENK mRNA probe was prepared by labeling a PvuII-generated fragment from rat preproenkephalin cDNA (kindly provided by R. Howells) using amplified primer extension with C-terminal primer: 5%-TGCTCGTGCTGTCTTCATCATTG-3% (Howells, 1986). To make a single-stranded labeled probe, a mixture of 1× PCR Buffer; 1.5 mM MgCl2, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dGTP (Gene Amp RNA PCR Core Kit, Perkin Elmer, Foster City, CA), 50 pmol C% terminal NPY primer or 50 pmol C% terminal ppENK primer, 50 ng NPY or 50 ng ppENK template, 500 mCi [32P]dCTP (DuPont NEN, Wilmington, DE), and 25 units of Taq polymerase (Gene Amp RNA PCR Core Kit, Perkin Elmer, Boulder, CO), in a total volume of 50 ml, was amplified in a thermocycler (Thermolyne, Temp-Tronic, Dubuque, IA). The amplification condition was programmed for 70 cycles of: 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s. Labeled probes were purified by G-50 spin columns (5 Prime – 3 Prime, Boulder, CO) and stored at − 70°C until use.
2.4. Statistical analysis Statistical analysis was performed using JMP software (Version 2, SAS, Cary, NC); analysis of variance (ANOVA) was calculated using the general linear model. ANOVA was followed by the Tukey–Kramer honestly significant difference pair-wise comparison, unless otherwise specified.
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3. Results Adrenal NPY mRNA per microgram RNA was significantly influenced by stress (PB0.05, ANOVA; Fig. 1). Although by ANOVA the main effect of age was not significant, post-hoc comparisons indicated that the effect of stress was attenuated during age. In 7-month-old rats, stress significantly induced NPY mRNA by 143% (PB0.05, Tukey) and in 16-month old rats, stress significantly induced NPY mRNA by 118% (P B0.05, Tukey). In contrast, the effect of stress on NPY mRNA in 23-month-old rats was not significant (34%; P\ 0.05, Tukey). To further assess the source of the differential effects of stress at different ages, the effect of age was analyzed separately in control and stressed rats. As analyzed by ANOVA in control rats, NPY mRNA increased significantly with age (65% increase from 7 to 23 months, P B0.05, Tukey). However, the effect of age on NPY mRNA in stressed rats was not significant (in contrast to the significant 65% increase exhibited in non-stressed rats, NPY mRNA actually decreased by a non-significant 9% from 7 to 23 months in stressed rats). Like NPY mRNA, adrenal ppENK mRNA was significantly increased by stress (P B 0.05, ANOVA; Fig. 2), with no main effect of age. However, in contrast to NPY mRNA, post-hoc comparisons did not indicate that the effect of stress on ppENK mRNA was attenuated with age. In 7-, 16- and 23-month-old rats, stress significantly induced ppENK mRNA by 93%, 84%, and 122%, respectively (at each age, P B0.05, Tukey). Furthermore, analysis of ppENK mRNA in control and stressed rats separately did not indicate an effect of age (a decrease by 12% in 23vs. 7-month-old non-stressed rats, and no change in stressed rats, P\ 0.05, Tukey).
Fig. 1. Northern blot analysis of NPY mRNA expression in the adrenal glands of 7-, 16- and 23-month-old rats exposed to stress as compared to controls. Data are expressed as the mean 9S.E.M. of individual analysis (n= 6) for each group. Densitometry is expressed as optical density.
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Fig. 2. Northern blot analysis of preproenkephalin mRNA expression in the adrenal glands of 7-, 16and 23-month-old rats exposed to stress as compared to controls. Data are expressed as the mean 9 S.E.M. of individual analysis (n= 6) for each group. Densitometry is expressed as optical density.
Like ppENK mRNA, blood glucose was significantly increased by stress (PB 0.05, ANOVA), with no evidence of an effect of age (Fig. 3). In 7-, 16- and 23-month-old rats, stress significantly induced blood glucose by 53%, 73%, and
Fig. 3. Glucose levels from trunk blood immediately following stress in 7-, 16- and 23-month-old rats as compared to controls (data are mean 9S.E.M., n = 6, for all groups).
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Fig. 4. Corticosterone levels immediately following stress in 7-, 16- and 23-month-old rats as compared to controls (data are mean 9 S.E.M., n =6, for all groups).
82%, respectively (at each age, PB0.05, Tukey). Furthermore, analysis of blood glucose in control and stressed rats separately did not indicate an effect of age (a decrease by 12% in 23- vs. 7-month-old non-stressed, and an increase of 4% in 7vs. 23-month-old stressed rats, respectively, P\ 0.05, Tukey). Plasma corticosterone was significantly increased by stress (PB 0.05, ANOVA), but, in contrast to other parameters, ANOVA also indicated a significant effect of age (P B 0.05, ANOVA) (Fig. 4). In 7-, 16- and 23-month-old rats, stress significantly increased plasma corticosterone by 928%, 270%, and 665%, respectively (at each age, P B 0.05, Tukey). Furthermore, plasma corticosterone was increased by 174% in 23- vs. 7-month-old stressed rats (PB 0.05, Tukey).
4. Discussion The purpose of this study was to assess the effect of age on the response to stress of adrenal NPY and ppENK mRNA. Few studies have demonstrated impaired responses to stress with aging. Indeed, some evidence supports the concept that the basal activity of two major stress systems, the sympathetic nervous system (Ziegler et al., 1976) and the HPA system (Landfield and Eldridge, 1994), increase with age in some systems, and that the HPA response to stress may effectively increase with age, due to impaired negative feedback (Sapolsky et al., 1986b). On the other hand, the one molecular response which has been examined, the induction of hsp 70 mRNA, has been reported to be attenuated with age in response to restraint stress (Blake et al., 1991; Udelsman et al., 1993) and mild heat shock (Heydari et al., 1993; Wu et al., 1993). In the present study, responses of NPY and ppENK to
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stress were differentially affected by aging. The induction of adrenal NPY mRNA by stress was impaired with age (Fig. 1), whereas the stress-induced elevation of blood glucose (Fig. 3) and adrenal ppENK mRNA (Fig. 2) showed no evidence of age-related impairments. The failure to observe a statistically significant elevation of adrenal NPY mRNA in the oldest rats might have several explanations. First, an effect of stress may be present in the older rats, but masked by increased variance with age. However, two observations argue against this hypothesis. The standard errors for NPY mRNA in both control and stress-induced rats were similar at all three ages (Fig. 1). Furthermore, the magnitude of induction in the oldest rats (34%) was substantially lower than in younger rats (143%), with an intermediate magnitude of induction (118%) in the middle-aged rats. A second explanation might be that the baseline of expression of NPY mRNA increases with age, i.e. the main effect of age in control rats (an age-related increase which is significant by analysis of variance) is in the opposite direction to the effect of age in stressed rats. Thus a ceiling effect might prevent further induction in the oldest rats, artificially decreasing the magnitude of induction. If there were such a ceiling effect, the NPY mRNA levels in the oldest rats should cluster toward the high end of the values exhibited by younger rats; however, the data from the oldest rats did not exhibit such behavior. For example, taking the median value of NPY mRNA in the young rats as a criterion, 50% of young rats met or exceeded this median value (by definition), and 66% of middleaged rats exceeded this value, but none of the older rats exceeded this value. Therefore there was no evidence that a ceiling effect artificially reduced the magnitude of the stress effect in older rats. Nevertheless, it is possible that if the baseline level of NPY mRNA did not increase with age, the effect of stress in the older rats would have been significant, although decreased in magnitude compared to young rats. For example, a t-test comparison of the old stressed rats, compared to the young control rats (an invalid comparison made for exploratory purposes only) indicated a significant difference (PB 0.05, t-test). Therefore we hypothesize that the lack of a statistically significant stress effect on adrenal NPY mRNA expression in 23-month-old rats is due to two factors: an increased baseline expression, and a decreased (but possibly non-zero) magnitude of induction by stress. The cause of the increased baseline and decreased induction remains to be elucidated by future experiments. Ultimately, the relevance of this response has to be related to an increase in and measurable effect of the NPY peptide. In this regard, our data are consistent with a reported increase in adrenal NPY immunoreactivity in aging up to 18 months reported by Higuchi et al. (Higuchi et al., 1988b). Interestingly, in their experiments, serum NPY was not similarly increased. Although we conclude that the induction of NPY mRNA by stress is attenuated with age, this loss of responsivity to stress is clearly not general. Elevations of enkephalins are a potentially interesting stress response in that enkephalins are associated with the development of antinociception (Calcagnetti et al., 1992) and may be associated with an alteration of anesthetic requirements following stress (Silverstein and Beasley, 1995). Adrenal ppENK mRNA has been reported to exhibit increased expression following stress (DeCristofaro et al., 1993). In the
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current study induction of adrenal ppENK mRNA by stress was not impaired with age. The response of plasma glucose, which is probably mediated by the sympathetic nervous system (Miles et al., 1991), and glucocorticoids, which have been studied extensively in both stress and aging (Sabatino et al., 1991; de Kloet, 1992), were assessed to be sure that the animals were indeed responding to stress. Although basal activity of the sympathetic nervous system has been reported to increase with age (Cizza et al., 1995) there was no effect of age on basal plasma glucose or on the response of plasma glucose to stress, in agreement with previous studies (Odio and Brodish, 1990). It should be noted that baseline levels of glucose were relatively low compared to some published values of glucose taken from the tail vein of well fed rats. This may reflect the fact that our values were taken from trunk blood, which may sometimes exhibit slightly lower values than tail vein blood (C. Mobbs, unpublished data). Corticosterone levels in both non-stressed and stressed individuals were significantly higher in the older rats than in the younger rats. The age-related increase in blood corticosterone in the non-stressed individuals, which parallels the age-related increase in adrenal NPY mRNA, is consistent with previous reports (Hess and Riegle, 1970; Tang and Phillips, 1978). Early reports suggested that corticosterone regulation is altered with age in the absence of stress (Britton et al., 1975). Our studies also agree with previous studies that the induction of glucocorticoids by restraint stress in rats is not attenuated in aging rats. Indeed, although the present study does not address the question of total responsiveness of the glucocorticoid system since only one time point after stress was assessed, previous investigators have reported enhanced corticosterone secretion after stress in aging rats (Sabatino et al., 1991), in contrast to the impaired induction of adrenal NPY mRNA (Fig. 2) and hsp 70 mRNA (Blake et al., 1991). While our data are consistent with a differential age effect on stress responses measured in this study, there are many areas which remain to be investigated. The current study examines a single time point. A full time course examination of each of these responses is necessary. Furthermore, the current study provides no insight into the mechanism or physiologic significance of the described result. For example, while the regulation of NPY mRNA changes with age, this may be a result of transcriptional alterations in adrenal cells, an alteration in the signaling mechanism or the result of counterregulatory phenomena. These issues remain to be investigated. While much remains to be elucidated concerning NPY and ppENK in the aging stress response, the reported glucose and corticosterone levels represent simple evaluations of stress responses which have been extensively studied, particularly in aging (Sabatino et al., 1991). The current study does not evaluate adrenal secretory capacity by time integration in aging and does not address the issue of free versus bound hormone concentrations since glucose and corticosterone were used only to ensure that impaired molecular responses to stress could be interpreted in the context of clear evidence that the animals were responding to stress at a physiological level. NPY mRNA was assessed as previous studies have shown that adrenal NPY responds to restraint stress (Hiremagalur et al., 1994) and represents a physiologi-
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cally important component of the sympathetic response to stress (Zukowska-Grojec, 1995). NPY is co-localized with norepinephrine and secreted from the adrenal in response to stress (Zukowska-Grojec and Wahlestedt, 1993; Zukowska-Grojec, 1995). NPY appears to enhance the activity or effectiveness of the sympathetic nervous system (Westfall et al., 1987). A recent report described an increase in adrenal NPY mRNA levels from 1 to 8.25 months of age (Higuchi et al., 1991). Although induction of NPY mRNA by stress during aging has not been previously examined, impaired induction of NPY by stress in aging individuals could conceivably pre-dispose to physiologic derangements such as hypotension, as has been reported during septic shock (Arnalich et al., 1994). ppENK mRNA has also been shown to be induced by stress (Dowds et al., 1994), but the effect of age has not been previously examined.
Acknowledgements This work was partially supported by a Brookdale National Fellowship to J.H. Silverstein.
References Arnalich, F., Sanchez, J.F., Martinez, M., Jimenez, M., Lopez, J., Vazquez, J.J., Hernanz, A., 1994. Changes in plasma concentrations of vasoactive neuropeptides in patients with sepsis and septic shock. Life Sci. 56, 75–81. Blake, M.J., Udelsman, R., Feulner, G.J., Norton, D.D., Holbrook, N.J., 1991. Stress-induced heat shock protein 70 expression in adrenal cortex: an adrenocorticotropic hormone-sensitive, age-dependent response. Proc. Natl. Acad. Sci. USA 88, 9873 – 9877. Britton, G.W., Rotenberg, S., Freeman, C., Britton, V.J., Karoly, K., Ceci, L., Klug, T.L., Lacko, A.G., Adelman, R.C., 1975. Regulation of corticosterone levels and liver enzyme activity in aging rats. Adv. Exp. Med. Biol. 61, 209–228. Brodish, A., Odio, M., 1989. Age-dependent effects of chronic stress on ACTH and corticosterone responses to an acute novel stress. Neuroendocrinology 49, 496 – 501. Calcagnetti, D.J., Stafinsky, J.L., Crisp, T., 1992. A single restraint stress exposure potentiates analgesia induced by intrathecally administered DAGO. Brain Res. 592, 305 – 309. Cizza, G., Pacak, K., Kvetnansky, R., Palkovits, M., Goldstein, D.S., Brady, L.S., Fukuhara, K., Bergamini, E., Kopin, I.J., Blackman, M.R., Chrousos, G.P., Gold, 1995. Decreased stress responsivity of central and peripheral catecholaminergic systems in aged 334/N Fischer rats. J. Clin. Invest. 95, 1217–1224. de Kloet, E.R., 1992. Corticosteroids, stress, and aging (Review). Ann. New York Acad. Sci. 663, 357 – 371. DeCristofaro, J.D., Weisinger, G., La Gamma, E.F., 1993. Cholinergic regulation of rat preproenkephalin RNA in the adrenal medulla. Mol. Brain Res. 18, 133 – 140. Dowds, T., Nankova, B., Hiremagalur, B., Kvetnansky, R., Sabban, E.L., 1994. Effect of immobilization stress on expression of mRNAs for adrenomedullary proenkephalin and neuropeptide Y in normal and hypophysectomized rats. Soc. Neurosci. 20, 374 Abstract. Hess, G.D., Riegle, G.D., 1970. Adrenocortical responsiveness to stress and ACTH in aging rats. J. Gerontol. 25, 354–358. Heydari, A.R., Wu, B., Takahashi, R., Strong, R., Richardson, A., 1993. Expression of heat shock protein 70 is altered by age and diet at the level of transcription. Mol. Cell Biol. 13, 2909 – 2918.
J.H. Sil6erstein et al. / Mechanisms of Ageing and De6elopment 101 (1998) 233–243
243
Higuchi, H., Yang, H.Y., Sabol, S.L., 1988a. Rat neuropeptide Y precursor gene expression. mRNA structure, tissue distribution, and regulation by glucocorticoids, cyclic AMP, and phorbol ester. J. Biol. Chem. 263, 6288–6295. Higuchi, H., Yang, H.Y., Costa, E., 1988b. Age-related bidirectional changes in neuropeptide Y peptides in rat adrenal glands, brain, and blood. J. Neurochem. 50, 1879 – 1886. Higuchi, H., Yokokawa, K., Iwasa, A., Yoshida, H., Miki, N., 1991. Age-dependent increase in neuropeptide Y gene expression in rat adrenal gland and specific brain areas. J. Neurochem. 57, 1840–1847. Hiremagalur, B., Kvetnansky, R., Nankova, B., Fleischer, J., Geertman, R., Fukuhara, K., Viskupic, E., Sabban, E.L., 1994. Stress elicits trans-synaptic activation of adrenal neuropeptide Y gene expression. Mol. Brain Res. 27, 138–144. Howells, R.D., 1986. Proenkephalin biosynthesis in the rat. NIDA Res. Monogr. 70, 43 – 65. Landfield, P.W., Eldridge, J.C., 1994. Evolving aspects of the glucocorticoid hypothesis of brain aging: Hormonal modulation of neuronal calcium homeostasis. Neurobiol. Aging 15, 579 – 588. Lewis, R.V., Stern, A.S., Kimura, S., Rossier, J., Stein, S., Udenfriend, S., 1980. An about 50,000-dalton protein in adrenal medulla: a common precursor of [Met]- and [Leu]enkephalin. Science 208, 1459–1461. Miles, P.D.G., Yamatani, K., Lickley, H.L.A., Vranic, M., 1991. Mechanism of glucoregulatory responses to stress and their deficiency in diabetes. Proc. Natl. Acad. Sci. USA 88, 1296 – 1300. Nankova, B., Hiremagalur, B., Menezes, A., Zeman, R., Sabban, E., 1996. Promoter elements and second messenger pathways involved in transcriptional activation of tyrosine hydroxylase by ionomycin. Brain Res. Mol. Brain Res., 35, 164–172. Odio, M.R., Brodish, A., 1990. Glucoregulatory responses of adult and aged rats after exposure to chronic stress. Exp. Gerontol. 25, 159–172. Sabatino, F., Masoro, E.J., McMahan, C.A., Kuhn, R.W., 1991. Assessment of the role of the glucocorticoid system in aging processes and in the action of food restriction. J. Gerontol. 46, B171–179. Sapolsky, R.M., Krey, L.C., McEwen, B.S., 1983. The adrenocortical stress-response in the aged male rat: impairment of recovery from stress. Exp. Gerontol. 18, 55 – 64. Sapolsky, R.M., Krey, L.C., McEwen, B.S., 1986a. The adrenocortical axis in the aged rat: impaired sensitivity to both fast and delayed feedback inhibition. Neurobiol. Aging 7, 331 – 335. Sapolsky, R.M., Krey, L.C., McEwen, B.S., 1986b. The neuroendocrinology of stress and aging: the glucocorticoid cascade hypothesis (Review). Endocrine Rev. 7, 284 – 301. Selye, H., 1976. Diseases of adaptation. In: Anonymous Stress in Health and Disease. Butterworth, Boston, pp. 725–895. Silverstein, J.H., Beasley, J., 1995. Effects of age on stress alteration of anesthestic requirements. Neurosci. Abstr. 21, 281.9 (Abstract). Tang, F., Phillips, J.G., 1978. Some age-related changes in pituitary-adrenal function in the male laboratory rat. J. Gerontol. 33, 377–382. Udelsman, R., Blake, M.J., Stagg, C.A., Li, D.G., Putney, D.J., Holbrook, N.J., 1993. Vascular heat shock protein expression in response to stress. Endocrine and autonomic regulation of this age-dependent response. J. Clin. Invest. 91, 465–473. Westfall, T.C., Carpentier, S., Chen, X., Beinfeld, M.C., Naes, L., Meldrum, M.J., 1987. Prejunctional and postjunctional effects of neuropeptide Y at the noradrenergic neuroeffector junction of the perfused mesenteric arterial bed of the rat. J. Cardiovasc. Pharmacol. 10, 716 – 722. Wu, B., Gu, M.J., Heydari, A.R., Richardson, A., 1993. The effect of age on the synthesis of two heat shock proteins in the hsp70 family. J. Gerontol. 48, B50 – 56. Ziegler, M.G., Lake, C.R., Kopin, I.J., 1976. Plasma noradrenaline increases with age. Nature 261, 333–335. Zukowska-Grojec, Z., 1995. Neuropeptide Y. A novel sympathetic stress hormone and more (Review). Ann. New York Acad. Sci. 771, 219– 233. Zukowska-Grojec, Z., Wahlestedt, C., 1993. Origin and actions of neuropeptide Y in the cardiovascular system. In: Colmers, W.M., Wahlestedt, C. (Eds.), The Biology of Neuropeptide Y and Related Peptides. Humana Press, Totowa, NJ, pp. 315 – 388.
Adrenal neuropeptide Y mRNA but not preproenkephalin mRNA induction by stress is impaired by aging in Fischer 344 rats Jeffrey H. Silverstein a,c,d,e,*, Joseph Beasley c, Tooru M. Mizuno b,f, Evan London a, Charles V. Mobbs b,e,f a Anesthesia Section, Veterans Affairs Medical Center, Bronx, NY 10468, USA Pathology and Diagnostic Laboratory Ser6ice, Veterans Affairs Medical Center, Bronx, NY 10468, USA c Department of Anesthesiology, Mount Sinai School of Medicine, 1 Gusta6e L. Le6y Place, New York, NY 10029 -6574, USA d Department of Surgery, Mount Sinai School of Medicine, 1 Gusta6e L. Le6y Place, New York, NY 10029 -6574, USA e Department of Geriatrics and Adult De6elopment, Mount Sinai School of Medicine, 1 Gusta6e L. Le6y Place, New York, NY 10029 -6574, USA f Fishberg Center for Neurobiology, Mount Sinai School of Medicine, 1 Gusta6e L. Le6y Place, New York, NY 10029 -6574, USA b
Received 27 August 1997; received in revised form 27 November 1997; accepted 2 December 1997
Abstract Relatively few molecular markers of stress have been studied in aged individuals. Interactions of age and stress on adrenal neuropeptide Y (NPY) and preproenkephalin (ppENK) expression have not been reported. The purpose of these studies was to characterize the adrenal NPY and ppENK responses to stress using a common stressor, physical restraint for 2 h, in Fischer 344 rats at 7, 16 and 23 months of age. Northern blot techniques were used to evaluate induction by stress of adrenal NPY mRNA and adrenal ppENK mRNA. Two humoral responses to stress, serum glucose and corticosterone, were measured to corroborate
* Corresponding author. Tel.: +1 212 2416426; fax: + 1 212 8763906; e-mail: Jeff – [email protected] 0047-6374/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 0 0 4 7 - 6 3 7 4 ( 9 7 ) 0 0 1 7 7 - 2
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that a stress response occurred. We observed that the induction by stress of adrenal NPY mRNA is impaired with age but the stress-induced elevation of adrenal ppENK mRNA, blood glucose, and corticosterone show no evidence of age-related impairments. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neuropeptide Y; Enkephalins; Stress, mechanical; Glucose; Adrenal; Aging
1. Introduction A variety of physiologic and behavioral systems are activated by stress. Alterations of the ability to respond to stressors, involving either excessive or inadequate responses in either duration or magnitude, have been speculated to lead to disease (Selye, 1976). Numerous investigators have proposed that responses to stress change with age (Sapolsky et al., 1983; Brodish and Odio, 1989; Blake et al., 1991; de Kloet, 1992). The basal activity of two major stress systems, the sympathetic nervous system (Ziegler et al., 1976) and the hypothalamic–pituitary–adrenal (HPA) system (Landfield and Eldridge, 1994), may increase with age in some systems, and the HPA response to stress may effectively increase with age, due to impaired negative feedback (Sapolsky et al., 1986a). The one molecular stress response which has been examined, the induction of hsp 70 mRNA, has been reported to be attenuated with aging response to restraint stress (Blake et al., 1991; Udelsman et al., 1993) and mild heat shock (Heydari et al., 1993; Wu et al., 1993). Neuropeptide Y (NPY) is a vasoconstrictor peptide which is abundantly present in peripheral noradrenergic nerves, the brain, adrenal medulla, cardiac neurons and the myenteric plexus of the gut. Recent work suggests an important role for NPY in the stress response (Zukowska-Grojec, 1995) and particularly its induction in the rat adrenal gland following stress (Hiremagalur et al., 1994). The ability of opioid peptides to inhibit the release of various neurotransmitters supports their possible function as neuromodulators. Proenkephalin was first discovered in bovine adrenal cortex where enkephalin biosynthesis was elucidated (Lewis et al., 1980). Preproenkephalin (ppENK) mRNA has been shown to be induced by restraint stress (Dowds et al., 1994). In the present study, the effect of age on these two molecular responses to restraint stress was evaluated. Corticosterone and glucose were also evaluated to ensure that the individuals were in fact responding to the stress paradigm.
2. Materials and methods
2.1. Animals Fisher 344 male rats were purchased at ages 6, 15 and 22 months of age from NIA/Harlan Sprague Dawley. Care and use were in accord with the guidelines of
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the Mount Sinai School of Medicine and the Bronx Veterans Affairs Medical Center. The animals were individually housed (10¦ × 8¦× 7¦ with wire mesh floors suspended on a Lab Products rack; Lab Products, Rochelle Park, NJ) and maintained in a 12-h light, 12-h dark cycle (lights on at 07:00 h) with ad libitum access to food (laboratory autoclavable rodent diet 5010, Purina) and water. Prior to arrival, the rats were fed NIH 31 diet (Harlan Tekald Laboratory Diet — sterilized) which differs from our rat chow in that the Pruina chow has 23.0% crude protein versus 18.4% for NIH 31. The animals are housed for approximately 1 month in this facility prior to experimentation. Sentinel animals were sacrificed for monitoring for murine virus antibodies (sendai, PVM, SDA/ RCV, KRV, H-1, GD-7, Reo-3, LCMV, MAD, Ectro, CARB, RCMV, Hant, Mycoplasma pulmonis, E. cunicuili, B. piliformis) following arrival and this process was carried out at least every 6 months. Beginning at 09:00, animals were restrained for 120 min, a time chosen based on the expected peak for NPY mRNA and ppENK mRNA (Nankova et al., 1996). Restraint consisted of immobilization of all four extremities by padded cords designed to avoid injury to the limbs. The head was not restrained. Rats were sacrificed at the end of the stress period, by exposure to carbon dioxide for : 1.5 min following which the animals were decapitated. In all cases, rats were sacrificed between 11:00 and 13:00 h. Control animals were removed from their home cage and immediately sacrificed. The trunk blood was collected into EDTA coated tubes on ice. Blood glucose was measured immediately after decapitation by a One-Touch II glucose meter (Lifescan, Milpitas, CA) using a fresh drop of trunk blood. The glucose meter was calibrated daily. Animals were examined for macroscopic pathology. Two animals were removed from the 23-month-old group (one each from the restraint and control groups) due to pituitary tumors. No renal or other intraabdominal tumors were noted. Both adrenals were removed, cleaned of extraneous fat, and immediately frozen for subsequent analysis. Adrenal glands for each animal were maintained separately for the entire experiment. Plasma corticosterone was measured by radioimmunoassay (corticosterone — 125I RIA kit, ICN Biomedicals, Costa Mesa, CA).
2.2. RNA extraction and Northern blot analysis Total RNA from : 50 mg of each individual adrenal gland was extracted in RNAzol B (Tel-Test, Friendswood, TX) and chloroform, and precipitated by isopropanol. Each pellet was resuspended in 25 ml of diethylpyrocarbonate (DEPC) treated water. The quantity and integrity of the isolated RNA was determined by both UV absorbance spectroscopy (260 nm/280 nm) and agarose electrophoresis, visualizing the intact ribosomal RNA bands with ethidium bromide. Ten micrograms of total RNA was denatured by incubating with glyoxal and dimethyl sulfoxide for 1 h at 50°C, loaded into 1.5% agarose gel, then separated by electrophoresis at 80 V for 1.5 h. RNA was transferred to Immobilon S® (Millipore) by capillary elution in 20× standard saline citrate (SSC)
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(1× SSC is 0.15 M NaCl/15 mM sodium citrate, pH 7.0). After being washed with 6 × SSC, the membrane was baked at 80°C for 1 h and exposed to the UV crosslinker (300× 100 mJ/cm2) to fix RNA. The membrane was pre-hybridized in a solution containing 25 mM NaPO4 (pH 7.0), 4 × SSC, 5 mM EDTA, 5 × Denhardt’s solution, 0.5% (vol/vol) SDS, 50% (vol/vol) deionized formamide, and denatured salmon sperm DNA at 100 mg/ml (5 Prime3 Prime, Boulder, CO) at 42°C overnight. Hybridization was done in the same buffer containing 10% (wt/vol) dextran sulfate and 32P-labeled NPY or (ppENK) gene probe at 1.6 mCi/ml at 42°C overnight. The membrane was washed twice in 1 × SSC, 0.1% SDS for 20 min at room temperature, followed by two washes in 0.1 × SSC/ 0.1% SDS for 15 min at room temperature. Subsequently, the membrane was washed in 0.1× SSC, 0.1% SDS for 3 h at 55°C and then exposed to autoradiography film (DuPont NEN, Wilmington, DE) between intensifying screens for 1–48 h at − 70°C. (To monitor RNA loading, the membrane was reprobed and hybridized with 32P-labeled probe encoding 18S ribosomal RNA.) The total integrated densities of hybridization signals were determined by computerized densitometric scanning (MCID, St. Catherine’s, ON).
2.3. Template production and probe labeling The NPY probe was prepared by amplifying a source DNA fragment (kindly provided by S. Sabol) using PCR with N-terminal primer: 5%-CTAGGTAACAAACGAATGGGG-3% and C-terminal primer: 5%CCACATGGAAGGGTCTTCAAG-3% (Higuchi et al. 1988a). A ppENK mRNA probe was prepared by labeling a PvuII-generated fragment from rat preproenkephalin cDNA (kindly provided by R. Howells) using amplified primer extension with C-terminal primer: 5%-TGCTCGTGCTGTCTTCATCATTG-3% (Howells, 1986). To make a single-stranded labeled probe, a mixture of 1× PCR Buffer; 1.5 mM MgCl2, 0.2 mM dATP, 0.2 mM dTTP, 0.2 mM dGTP (Gene Amp RNA PCR Core Kit, Perkin Elmer, Foster City, CA), 50 pmol C% terminal NPY primer or 50 pmol C% terminal ppENK primer, 50 ng NPY or 50 ng ppENK template, 500 mCi [32P]dCTP (DuPont NEN, Wilmington, DE), and 25 units of Taq polymerase (Gene Amp RNA PCR Core Kit, Perkin Elmer, Boulder, CO), in a total volume of 50 ml, was amplified in a thermocycler (Thermolyne, Temp-Tronic, Dubuque, IA). The amplification condition was programmed for 70 cycles of: 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s. Labeled probes were purified by G-50 spin columns (5 Prime – 3 Prime, Boulder, CO) and stored at − 70°C until use.
2.4. Statistical analysis Statistical analysis was performed using JMP software (Version 2, SAS, Cary, NC); analysis of variance (ANOVA) was calculated using the general linear model. ANOVA was followed by the Tukey–Kramer honestly significant difference pair-wise comparison, unless otherwise specified.
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3. Results Adrenal NPY mRNA per microgram RNA was significantly influenced by stress (PB0.05, ANOVA; Fig. 1). Although by ANOVA the main effect of age was not significant, post-hoc comparisons indicated that the effect of stress was attenuated during age. In 7-month-old rats, stress significantly induced NPY mRNA by 143% (PB0.05, Tukey) and in 16-month old rats, stress significantly induced NPY mRNA by 118% (P B0.05, Tukey). In contrast, the effect of stress on NPY mRNA in 23-month-old rats was not significant (34%; P\ 0.05, Tukey). To further assess the source of the differential effects of stress at different ages, the effect of age was analyzed separately in control and stressed rats. As analyzed by ANOVA in control rats, NPY mRNA increased significantly with age (65% increase from 7 to 23 months, P B0.05, Tukey). However, the effect of age on NPY mRNA in stressed rats was not significant (in contrast to the significant 65% increase exhibited in non-stressed rats, NPY mRNA actually decreased by a non-significant 9% from 7 to 23 months in stressed rats). Like NPY mRNA, adrenal ppENK mRNA was significantly increased by stress (P B 0.05, ANOVA; Fig. 2), with no main effect of age. However, in contrast to NPY mRNA, post-hoc comparisons did not indicate that the effect of stress on ppENK mRNA was attenuated with age. In 7-, 16- and 23-month-old rats, stress significantly induced ppENK mRNA by 93%, 84%, and 122%, respectively (at each age, P B0.05, Tukey). Furthermore, analysis of ppENK mRNA in control and stressed rats separately did not indicate an effect of age (a decrease by 12% in 23vs. 7-month-old non-stressed rats, and no change in stressed rats, P\ 0.05, Tukey).
Fig. 1. Northern blot analysis of NPY mRNA expression in the adrenal glands of 7-, 16- and 23-month-old rats exposed to stress as compared to controls. Data are expressed as the mean 9S.E.M. of individual analysis (n= 6) for each group. Densitometry is expressed as optical density.
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Fig. 2. Northern blot analysis of preproenkephalin mRNA expression in the adrenal glands of 7-, 16and 23-month-old rats exposed to stress as compared to controls. Data are expressed as the mean 9 S.E.M. of individual analysis (n= 6) for each group. Densitometry is expressed as optical density.
Like ppENK mRNA, blood glucose was significantly increased by stress (PB 0.05, ANOVA), with no evidence of an effect of age (Fig. 3). In 7-, 16- and 23-month-old rats, stress significantly induced blood glucose by 53%, 73%, and
Fig. 3. Glucose levels from trunk blood immediately following stress in 7-, 16- and 23-month-old rats as compared to controls (data are mean 9S.E.M., n = 6, for all groups).
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Fig. 4. Corticosterone levels immediately following stress in 7-, 16- and 23-month-old rats as compared to controls (data are mean 9 S.E.M., n =6, for all groups).
82%, respectively (at each age, PB0.05, Tukey). Furthermore, analysis of blood glucose in control and stressed rats separately did not indicate an effect of age (a decrease by 12% in 23- vs. 7-month-old non-stressed, and an increase of 4% in 7vs. 23-month-old stressed rats, respectively, P\ 0.05, Tukey). Plasma corticosterone was significantly increased by stress (PB 0.05, ANOVA), but, in contrast to other parameters, ANOVA also indicated a significant effect of age (P B 0.05, ANOVA) (Fig. 4). In 7-, 16- and 23-month-old rats, stress significantly increased plasma corticosterone by 928%, 270%, and 665%, respectively (at each age, P B 0.05, Tukey). Furthermore, plasma corticosterone was increased by 174% in 23- vs. 7-month-old stressed rats (PB 0.05, Tukey).
4. Discussion The purpose of this study was to assess the effect of age on the response to stress of adrenal NPY and ppENK mRNA. Few studies have demonstrated impaired responses to stress with aging. Indeed, some evidence supports the concept that the basal activity of two major stress systems, the sympathetic nervous system (Ziegler et al., 1976) and the HPA system (Landfield and Eldridge, 1994), increase with age in some systems, and that the HPA response to stress may effectively increase with age, due to impaired negative feedback (Sapolsky et al., 1986b). On the other hand, the one molecular response which has been examined, the induction of hsp 70 mRNA, has been reported to be attenuated with age in response to restraint stress (Blake et al., 1991; Udelsman et al., 1993) and mild heat shock (Heydari et al., 1993; Wu et al., 1993). In the present study, responses of NPY and ppENK to
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stress were differentially affected by aging. The induction of adrenal NPY mRNA by stress was impaired with age (Fig. 1), whereas the stress-induced elevation of blood glucose (Fig. 3) and adrenal ppENK mRNA (Fig. 2) showed no evidence of age-related impairments. The failure to observe a statistically significant elevation of adrenal NPY mRNA in the oldest rats might have several explanations. First, an effect of stress may be present in the older rats, but masked by increased variance with age. However, two observations argue against this hypothesis. The standard errors for NPY mRNA in both control and stress-induced rats were similar at all three ages (Fig. 1). Furthermore, the magnitude of induction in the oldest rats (34%) was substantially lower than in younger rats (143%), with an intermediate magnitude of induction (118%) in the middle-aged rats. A second explanation might be that the baseline of expression of NPY mRNA increases with age, i.e. the main effect of age in control rats (an age-related increase which is significant by analysis of variance) is in the opposite direction to the effect of age in stressed rats. Thus a ceiling effect might prevent further induction in the oldest rats, artificially decreasing the magnitude of induction. If there were such a ceiling effect, the NPY mRNA levels in the oldest rats should cluster toward the high end of the values exhibited by younger rats; however, the data from the oldest rats did not exhibit such behavior. For example, taking the median value of NPY mRNA in the young rats as a criterion, 50% of young rats met or exceeded this median value (by definition), and 66% of middleaged rats exceeded this value, but none of the older rats exceeded this value. Therefore there was no evidence that a ceiling effect artificially reduced the magnitude of the stress effect in older rats. Nevertheless, it is possible that if the baseline level of NPY mRNA did not increase with age, the effect of stress in the older rats would have been significant, although decreased in magnitude compared to young rats. For example, a t-test comparison of the old stressed rats, compared to the young control rats (an invalid comparison made for exploratory purposes only) indicated a significant difference (PB 0.05, t-test). Therefore we hypothesize that the lack of a statistically significant stress effect on adrenal NPY mRNA expression in 23-month-old rats is due to two factors: an increased baseline expression, and a decreased (but possibly non-zero) magnitude of induction by stress. The cause of the increased baseline and decreased induction remains to be elucidated by future experiments. Ultimately, the relevance of this response has to be related to an increase in and measurable effect of the NPY peptide. In this regard, our data are consistent with a reported increase in adrenal NPY immunoreactivity in aging up to 18 months reported by Higuchi et al. (Higuchi et al., 1988b). Interestingly, in their experiments, serum NPY was not similarly increased. Although we conclude that the induction of NPY mRNA by stress is attenuated with age, this loss of responsivity to stress is clearly not general. Elevations of enkephalins are a potentially interesting stress response in that enkephalins are associated with the development of antinociception (Calcagnetti et al., 1992) and may be associated with an alteration of anesthetic requirements following stress (Silverstein and Beasley, 1995). Adrenal ppENK mRNA has been reported to exhibit increased expression following stress (DeCristofaro et al., 1993). In the
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current study induction of adrenal ppENK mRNA by stress was not impaired with age. The response of plasma glucose, which is probably mediated by the sympathetic nervous system (Miles et al., 1991), and glucocorticoids, which have been studied extensively in both stress and aging (Sabatino et al., 1991; de Kloet, 1992), were assessed to be sure that the animals were indeed responding to stress. Although basal activity of the sympathetic nervous system has been reported to increase with age (Cizza et al., 1995) there was no effect of age on basal plasma glucose or on the response of plasma glucose to stress, in agreement with previous studies (Odio and Brodish, 1990). It should be noted that baseline levels of glucose were relatively low compared to some published values of glucose taken from the tail vein of well fed rats. This may reflect the fact that our values were taken from trunk blood, which may sometimes exhibit slightly lower values than tail vein blood (C. Mobbs, unpublished data). Corticosterone levels in both non-stressed and stressed individuals were significantly higher in the older rats than in the younger rats. The age-related increase in blood corticosterone in the non-stressed individuals, which parallels the age-related increase in adrenal NPY mRNA, is consistent with previous reports (Hess and Riegle, 1970; Tang and Phillips, 1978). Early reports suggested that corticosterone regulation is altered with age in the absence of stress (Britton et al., 1975). Our studies also agree with previous studies that the induction of glucocorticoids by restraint stress in rats is not attenuated in aging rats. Indeed, although the present study does not address the question of total responsiveness of the glucocorticoid system since only one time point after stress was assessed, previous investigators have reported enhanced corticosterone secretion after stress in aging rats (Sabatino et al., 1991), in contrast to the impaired induction of adrenal NPY mRNA (Fig. 2) and hsp 70 mRNA (Blake et al., 1991). While our data are consistent with a differential age effect on stress responses measured in this study, there are many areas which remain to be investigated. The current study examines a single time point. A full time course examination of each of these responses is necessary. Furthermore, the current study provides no insight into the mechanism or physiologic significance of the described result. For example, while the regulation of NPY mRNA changes with age, this may be a result of transcriptional alterations in adrenal cells, an alteration in the signaling mechanism or the result of counterregulatory phenomena. These issues remain to be investigated. While much remains to be elucidated concerning NPY and ppENK in the aging stress response, the reported glucose and corticosterone levels represent simple evaluations of stress responses which have been extensively studied, particularly in aging (Sabatino et al., 1991). The current study does not evaluate adrenal secretory capacity by time integration in aging and does not address the issue of free versus bound hormone concentrations since glucose and corticosterone were used only to ensure that impaired molecular responses to stress could be interpreted in the context of clear evidence that the animals were responding to stress at a physiological level. NPY mRNA was assessed as previous studies have shown that adrenal NPY responds to restraint stress (Hiremagalur et al., 1994) and represents a physiologi-
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cally important component of the sympathetic response to stress (Zukowska-Grojec, 1995). NPY is co-localized with norepinephrine and secreted from the adrenal in response to stress (Zukowska-Grojec and Wahlestedt, 1993; Zukowska-Grojec, 1995). NPY appears to enhance the activity or effectiveness of the sympathetic nervous system (Westfall et al., 1987). A recent report described an increase in adrenal NPY mRNA levels from 1 to 8.25 months of age (Higuchi et al., 1991). Although induction of NPY mRNA by stress during aging has not been previously examined, impaired induction of NPY by stress in aging individuals could conceivably pre-dispose to physiologic derangements such as hypotension, as has been reported during septic shock (Arnalich et al., 1994). ppENK mRNA has also been shown to be induced by stress (Dowds et al., 1994), but the effect of age has not been previously examined.
Acknowledgements This work was partially supported by a Brookdale National Fellowship to J.H. Silverstein.
References Arnalich, F., Sanchez, J.F., Martinez, M., Jimenez, M., Lopez, J., Vazquez, J.J., Hernanz, A., 1994. Changes in plasma concentrations of vasoactive neuropeptides in patients with sepsis and septic shock. Life Sci. 56, 75–81. Blake, M.J., Udelsman, R., Feulner, G.J., Norton, D.D., Holbrook, N.J., 1991. Stress-induced heat shock protein 70 expression in adrenal cortex: an adrenocorticotropic hormone-sensitive, age-dependent response. Proc. Natl. Acad. Sci. USA 88, 9873 – 9877. Britton, G.W., Rotenberg, S., Freeman, C., Britton, V.J., Karoly, K., Ceci, L., Klug, T.L., Lacko, A.G., Adelman, R.C., 1975. Regulation of corticosterone levels and liver enzyme activity in aging rats. Adv. Exp. Med. Biol. 61, 209–228. Brodish, A., Odio, M., 1989. Age-dependent effects of chronic stress on ACTH and corticosterone responses to an acute novel stress. Neuroendocrinology 49, 496 – 501. Calcagnetti, D.J., Stafinsky, J.L., Crisp, T., 1992. A single restraint stress exposure potentiates analgesia induced by intrathecally administered DAGO. Brain Res. 592, 305 – 309. Cizza, G., Pacak, K., Kvetnansky, R., Palkovits, M., Goldstein, D.S., Brady, L.S., Fukuhara, K., Bergamini, E., Kopin, I.J., Blackman, M.R., Chrousos, G.P., Gold, 1995. Decreased stress responsivity of central and peripheral catecholaminergic systems in aged 334/N Fischer rats. J. Clin. Invest. 95, 1217–1224. de Kloet, E.R., 1992. Corticosteroids, stress, and aging (Review). Ann. New York Acad. Sci. 663, 357 – 371. DeCristofaro, J.D., Weisinger, G., La Gamma, E.F., 1993. Cholinergic regulation of rat preproenkephalin RNA in the adrenal medulla. Mol. Brain Res. 18, 133 – 140. Dowds, T., Nankova, B., Hiremagalur, B., Kvetnansky, R., Sabban, E.L., 1994. Effect of immobilization stress on expression of mRNAs for adrenomedullary proenkephalin and neuropeptide Y in normal and hypophysectomized rats. Soc. Neurosci. 20, 374 Abstract. Hess, G.D., Riegle, G.D., 1970. Adrenocortical responsiveness to stress and ACTH in aging rats. J. Gerontol. 25, 354–358. Heydari, A.R., Wu, B., Takahashi, R., Strong, R., Richardson, A., 1993. Expression of heat shock protein 70 is altered by age and diet at the level of transcription. Mol. Cell Biol. 13, 2909 – 2918.
J.H. Sil6erstein et al. / Mechanisms of Ageing and De6elopment 101 (1998) 233–243
243
Higuchi, H., Yang, H.Y., Sabol, S.L., 1988a. Rat neuropeptide Y precursor gene expression. mRNA structure, tissue distribution, and regulation by glucocorticoids, cyclic AMP, and phorbol ester. J. Biol. Chem. 263, 6288–6295. Higuchi, H., Yang, H.Y., Costa, E., 1988b. Age-related bidirectional changes in neuropeptide Y peptides in rat adrenal glands, brain, and blood. J. Neurochem. 50, 1879 – 1886. Higuchi, H., Yokokawa, K., Iwasa, A., Yoshida, H., Miki, N., 1991. Age-dependent increase in neuropeptide Y gene expression in rat adrenal gland and specific brain areas. J. Neurochem. 57, 1840–1847. Hiremagalur, B., Kvetnansky, R., Nankova, B., Fleischer, J., Geertman, R., Fukuhara, K., Viskupic, E., Sabban, E.L., 1994. Stress elicits trans-synaptic activation of adrenal neuropeptide Y gene expression. Mol. Brain Res. 27, 138–144. Howells, R.D., 1986. Proenkephalin biosynthesis in the rat. NIDA Res. Monogr. 70, 43 – 65. Landfield, P.W., Eldridge, J.C., 1994. Evolving aspects of the glucocorticoid hypothesis of brain aging: Hormonal modulation of neuronal calcium homeostasis. Neurobiol. Aging 15, 579 – 588. Lewis, R.V., Stern, A.S., Kimura, S., Rossier, J., Stein, S., Udenfriend, S., 1980. An about 50,000-dalton protein in adrenal medulla: a common precursor of [Met]- and [Leu]enkephalin. Science 208, 1459–1461. Miles, P.D.G., Yamatani, K., Lickley, H.L.A., Vranic, M., 1991. Mechanism of glucoregulatory responses to stress and their deficiency in diabetes. Proc. Natl. Acad. Sci. USA 88, 1296 – 1300. Nankova, B., Hiremagalur, B., Menezes, A., Zeman, R., Sabban, E., 1996. Promoter elements and second messenger pathways involved in transcriptional activation of tyrosine hydroxylase by ionomycin. Brain Res. Mol. Brain Res., 35, 164–172. Odio, M.R., Brodish, A., 1990. Glucoregulatory responses of adult and aged rats after exposure to chronic stress. Exp. Gerontol. 25, 159–172. Sabatino, F., Masoro, E.J., McMahan, C.A., Kuhn, R.W., 1991. Assessment of the role of the glucocorticoid system in aging processes and in the action of food restriction. J. Gerontol. 46, B171–179. Sapolsky, R.M., Krey, L.C., McEwen, B.S., 1983. The adrenocortical stress-response in the aged male rat: impairment of recovery from stress. Exp. Gerontol. 18, 55 – 64. Sapolsky, R.M., Krey, L.C., McEwen, B.S., 1986a. The adrenocortical axis in the aged rat: impaired sensitivity to both fast and delayed feedback inhibition. Neurobiol. Aging 7, 331 – 335. Sapolsky, R.M., Krey, L.C., McEwen, B.S., 1986b. The neuroendocrinology of stress and aging: the glucocorticoid cascade hypothesis (Review). Endocrine Rev. 7, 284 – 301. Selye, H., 1976. Diseases of adaptation. In: Anonymous Stress in Health and Disease. Butterworth, Boston, pp. 725–895. Silverstein, J.H., Beasley, J., 1995. Effects of age on stress alteration of anesthestic requirements. Neurosci. Abstr. 21, 281.9 (Abstract). Tang, F., Phillips, J.G., 1978. Some age-related changes in pituitary-adrenal function in the male laboratory rat. J. Gerontol. 33, 377–382. Udelsman, R., Blake, M.J., Stagg, C.A., Li, D.G., Putney, D.J., Holbrook, N.J., 1993. Vascular heat shock protein expression in response to stress. Endocrine and autonomic regulation of this age-dependent response. J. Clin. Invest. 91, 465–473. Westfall, T.C., Carpentier, S., Chen, X., Beinfeld, M.C., Naes, L., Meldrum, M.J., 1987. Prejunctional and postjunctional effects of neuropeptide Y at the noradrenergic neuroeffector junction of the perfused mesenteric arterial bed of the rat. J. Cardiovasc. Pharmacol. 10, 716 – 722. Wu, B., Gu, M.J., Heydari, A.R., Richardson, A., 1993. The effect of age on the synthesis of two heat shock proteins in the hsp70 family. J. Gerontol. 48, B50 – 56. Ziegler, M.G., Lake, C.R., Kopin, I.J., 1976. Plasma noradrenaline increases with age. Nature 261, 333–335. Zukowska-Grojec, Z., 1995. Neuropeptide Y. A novel sympathetic stress hormone and more (Review). Ann. New York Acad. Sci. 771, 219– 233. Zukowska-Grojec, Z., Wahlestedt, C., 1993. Origin and actions of neuropeptide Y in the cardiovascular system. In: Colmers, W.M., Wahlestedt, C. (Eds.), The Biology of Neuropeptide Y and Related Peptides. Humana Press, Totowa, NJ, pp. 315 – 388.