Journals of Gerontology Series A: Biological Sciences and Medical Sciences Large Type Edition
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Agirregoitia, N.
Right arrow Articles by Casis, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Agirregoitia, N.
Right arrow Articles by Casis, L.
The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 58:B792-B797 (2003)
© 2003 The Gerontological Society of America

Effect of Aging on Rat Tissue Peptidase Activities

Naiara Agirregoitia, Javier Gil, Fátima Ruiz, Jon Irazusta and Luis Casis

Department of Physiology, Medical School, University of the Basque Country, Bizkaia, Spain.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
The process of aging is known to involve alterations in the activity of peptidases and proteases. However, the precise changes in the activity of many peptidases in aged tissues have not yet been fully characterized, and both decreases and increases in both peptidase activity and peptide levels have been reported to occur during the aging process. In the present study, we measured the activity of several peptidases in selected tissues (brain cortex, brain stem, liver, kidney, heart, and lung) of the young adult (3 months old) and aged (18 months old and 22 months old) rat. The activities of prolyl endopeptidase, pyroglutamyl peptidase I, puromycin sensitive aminopeptidase, and aminopeptidase N were assayed using ß-naphthylamine aminoacidic derivatives as substrates. The activity of the soluble fractions of prolyl endopeptidase was found to be reduced in the lungs of aged animals, while reduced activity of soluble pyroglutamyl peptidase I and also aminopeptidase N was measured in the aged kidney and heart, respectively. In contrast, increased activity of particulate prolyl endopeptidase was measured in the brain stem of older animals. Since most of these changes can be correlated with known alterations in the levels of peptides controlled by each enzyme, the results of the present study indicate that the studied peptidases may play an important role in regulating tissue peptide levels during aging.

MANY proteases or peptidases have been reported to play a role in the process of aging (1). Indeed, it has been proposed that reduced intracellular protein catabolism may be one of the hallmarks of aging (2). In contrast, other studies have reported that many proteases, which play essential roles in regulating peptide metabolism, are up-regulated in aged tissues (1,3). Thus, it is presently unclear how changes in the levels of peptides and in the activity of peptidases in different tissues contribute to the aging process.

Enkephalins and thyrotropin-releasing hormone (TRH) are peptides that have been implicated in the aging processes (4,5). Their action in the central nervous system is well characterized. TRH levels are reduced in the brain of aged rats, compared with young adult animals (6). This decrease can be prevented if rats are treated with inhibitors of the enzymes that degrade the tripeptide (7). In addition, decreased levels of met-enkephalin have been reported in the striatum (8). On the other hand, increases in the levels of opioid peptides are thought to have important functional implications for the senescent heart (4,9). Since aging has been associated with both decreased and increased levels of certain peptides, it is clearly important to characterize the activity of the associated peptidases in aging tissue.

Due to their short chain length, the tripeptide TRH (pGlu-His-Pro-NH2) and the pentapeptides Met- and Leu-enkephalin (Tyr-Gly-Gly-Phe-Met or -Leu) are especially susceptible to the action of peptidases. Indeed, the degradation pathways associated with these peptidases are better understood than those associated with the metabolism of other longer peptides (10,11). Three enzymes are known to be involved in the primary degradation of TRH: prolyl endopeptidase, which is responsible of the deamidation of TRH (12), and pyroglutamyl peptidase (PGP) I (13) and II (14), which remove the N-terminal pyroglutamyl. Prolyl endopeptidase and PGP I are widely distributed in most tissues and body fluids (15–17). These two enzymes are mainly cytosolic, but membrane-associated forms of both enzymes have also been described (18,19). In contrast, PGP II is a very specific enzyme whose expression is restricted mainly to brain synaptosomes (20).

Puromycin-sensitive aminopeptidase and aminopeptidase N are known to participate in the degradation of members of the enkephalin family of opioid peptides (11). Puromycin-sensitive aminopeptidase (PSA) is mainly soluble, although a membrane form has been described in the brain (21). In contrast, aminopeptidase N is a membrane-bound, particulate enzyme (22). Nevertheless, both enzymes are widely distributed in most tissues (22,23).

Prolyl endopeptidase, which is capable of degrading TRH in addition to other proline-containing peptides such as vasopressin and substance P (SP), may play a role in aging processes since its activity has been reported to be associated with neuronal degeneration (24). Moreover, intracerebroventricular administration of inhibitors of prolyl endopeptidase has been reported to improve the functioning of TRHergic neurons, which are deteriorated in the aged rat (6). Alzheimer's disease, which can be considered as a form of precocious aging, has been proposed by some researchers to be a proteolytic disorder (25). In keeping with this idea, postmortem brain tissue from patients with Alzheimer's disease presented altered prolyl endopeptidase activity in comparison with aged-matched tissue from normal individuals. However, these differences involved both decreased (26) and increased (27) enzymatic activity and, consequently, the role of peptidases in aging remains unclear. In other degenerative illnesses, such as amyotrophic lateral sclerosis, the levels of activity of prolyl endopeptidase and PGP I were found to be altered (28).

It is presently clear that peptidases participate in the aging of the central nervous system. However, the role played by these enzymes in the aging of other tissues is more uncertain. In the present work, we examined age-related alterations in the activity of four ubiquitous peptidases involved in TRH or enkephalin hydrolysis in selected structures (brain cortex, brain stem, liver, kidney, heart, and lung) of young adult and old rats. Our results indicate that a number of these peptidases may participate in regulating the levels of important peptides during the process of aging.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Animals
Three-month-old (n = 14), 18-month-old (n = 8), and 22-month-old (n = 7) adult male Sprague-Dawley rats housed under controlled environmental conditions (22°C and a 12-h light–dark cycle) with free access to food and water were used in this study. Experiments were carried out in accordance with the European Community Council Directive. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Preparation of Soluble and SolubilizedParticulate Fractions
Dissected structures (brain frontal cortex, brain stem, liver, kidney, heart, and lung) were homogenized in 10 mM Tris-HCl buffer, pH 7.4, for 2 minutes at 800 rpm using a Heidolph PZR 50 Selecta homogenizer (Schwabach, Germany) and ultracentrifuged in a Centrikon T-2070 Kontron Instruments (Milan, Italy) apparatus at 100,000 g for 35 minutes. The resulting supernatants were used to measure soluble enzyme activities and protein concentrations. To avoid contamination with soluble enzymes, the resulting pellets were washed 3 times by suspension in 10 mM Tris-HCl buffer, pH 7.4. Subsequently, pellets were homogenized in 4M NaCl solution and then centrifuged to release loosely bound proteins from the membrane. The resulting pellet was then homogenized in 10 mM Tris-HCl buffer, pH 7.4, plus 0.1% Triton X-100 (Sigma Chemical Co., St. Louis, MO) for 30 seconds at 800 rpm and ultracentrifuged at 100,000 g for 35 minutes. The supernatants thus obtained were used to determine the solubilized particulate enzyme activities and protein concentrations. All steps were carried out at 4°C.

Enzyme Assays
Prolyl endopeptidase activity was fluorimetrically measured using Z-Gly-Pro-ß-naphthylamide as a substrate (29). pGlu-ß-naphthylamide was the substrate used to measure pyroglutamyl peptidase I (PGP I) (18), using DTT (dithithreitol), which activates this enzyme while it inhibits the activity of PGP II (10). Aminopeptidase N and puromycin-sensitive aminopeptidase (PSA) activities were measured in triplicate using Ala-ß-naphthylamide as a substrate following the method described by Alba and colleagues (30). Enzymatic activity was detected in the absence and presence of 20 µM puromycin, which is known to completely inhibit puromycin-sensitive aminopeptidase (31). PSA activity was calculated by subtracting activity measured in the presence of puromycin from activity measured without the inhibitor. These assays are based on the fluorescence of ß-naphthylamine generated from the hydrolysis of the substrate by the enzyme. Reactions were initiated by adding 10 µl of sample to the assay mixture. After 30 minutes incubation at 37°C, 1 ml of 0.1 M sodium acetate buffer (pH 4.2) was added to the mixture to terminate the reaction. The released ß-naphthylamine was determined by measuring the fluorescent intensity at 412 nm with excitation at 345 nm. Tubes without samples were used to determine background fluorescence. Relative fluorescence was converted into picomoles of product using a standard curve, constructed with increasing concentrations of ß-naphthylamine.

Protein concentration was measured in triplicate by the method described by Bradford (32). The results were recorded as units of peptidase (UP) per milligram of protein. One unit of peptidase activity is the amount of enzyme that releases 1 picomole of ß-naphthylamine per minute.

Statistical Analyses
Data were analyzed statistically using SPSS version 10 software (SPSS, Inc., Chicago, IL). One-way analysis of variance (ANOVA) was performed to detect differences in each parameter during aging. When differences were detected, ANOVA was followed by Scheffé multiple comparisons test. Statistically significant differences were considered at p <.05.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
The activity of soluble prolyl endopeptidase from lung was found to decrease significantly with aging (ANOVA test, p <.05) (Figure 1A). Thus the activity in 22-month-old rats (1384 ± 149 UP/mg protein) was found to be significantly lower (Scheffé test, p <.05) than in 3-month-old animals (1818 ± 86 UP/mg protein). In contrast, the activity of particulate prolyl endopeptidase (Figure 1B) was higher (ANOVA test, p <.05; Scheffé test, p <.05) in the brain stem of both in 18-month-old animals (570 ± 84 UP/mg protein) and in 22-month-old animals (570 ± 22 UP/mg protein), than in 3-month-old rats (405 ± 29 UP/mg protein). No significant changes in prolyl endopeptidase activity were observed in other structures (brain cortex, liver, kidney, and heart).



View larger version (37K):
[in this window]
[in a new window]
  		Figure 1. Prolyl endopeptidase activities in soluble (sol) (A) and particulate (par) (B) homogenates of the brain cortex (BC), brain stem (BS), liver (Li), kidney (Ky), heart (He), and lung (Lu) from 3-month-old rats, 18-month-old rats, and 22-month-old rats. Values are expressed as mean ± SEM (standard error of mean) units of peptidase (UP) per milligram of protein (prot). Statistically significant differences are represented as follows: # p <.05, comparing 3-month-old rats and 18-month-old-rats; * p <.05, comparing 3-month-old rats and 22-month-old rats

 
Similar measurements of PGP I activity in the same tissues were performed (Figure 2). Soluble activity (Figure 2A) decreased with aging in the kidney (ANOVA test, p <.005), being lower in 18-month-old animals (2847 ± 361 UP/mg protein; Scheffé test, p <.05) and 22-month-old animals (2209 ± 353 UP/mg protein; Scheffé test, p <.005) than in their younger counterparts (3904 ± 222 UP/mg protein). In the lungs, decreased PGP I activity was found to be associated with aging (ANOVA test, p <.05). However, the Scheffé test did not detect statistically significant differences comparing the different groups of animals. Alterations in the activity of the particulate enzyme were not found to be statistically significant in the studied age groups (Figure 2B).



View larger version (27K):
[in this window]
[in a new window]
  		Figure 2. Pyroglutamyl peptidase I activities in soluble (sol) (A) and particulate (par) (B) homogenates of the brain cortex, brain stem, liver, kidney, heart, and lung from 3-month-old rats, 18-month-old rats, and 22-month-old rats. Values are given as mean ± SEM (standard error of mean). Statistically significant differences are represented as follows: # p <.05, comparing 3-month-old rats and 18-month-old rats; ** p <.005, comparing 3-month-old rats and 22-month-old rats. BC = brain cortex; BS = brain stem; Li = liver; Ky = kidney; He = heart; Lu = lung

 
Finally, we studied the activities of aminopeptidase N and PSA in the same tissues of young and old adult rats (Figure 3). Aminopeptidase N activity (Figure 3B) was found to be decreased (Scheffé test, p <.05) in heart tissue in 22-month-old rats (1818 ± 259 UP/mg protein) in comparison with 3-month-old rats (2918 ± 259 UP/mg protein). In contrast, significant changes in the activity of PSA were not observed with aging (Figure 3A).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 3. Puromycin-sensitive aminopeptidase (A) and aminopeptidase N (B) activities in homogenates of the brain cortex, brain stem, kidney, liver, heart, and lung from 3-month-old rats, 18-month-old rats, and 22-month-old rats. Values are given as mean ± SEM (standard error of mean). Statistically significant differences are represented as follows: * p <.05, comparing 3-month-old rats and 22-month-old rats. BC = brain cortex; BS = brain stem; Li = liver; Ky = kidney; He = heart; Lu = lung

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
In the present study, we found decreased activity of soluble peptidases in various tissues from aged rats. Thus, the levels of activity of soluble prolyl endopeptidase and PGP I were found to be significantly reduced in aged rat lung and kidney tissue, respectively. However, the observed decreases were found to be restricted to only 2 out of 6 assayed structures. Consequently, our study does not support the idea of a general age-related up-regulation or down-regulation of peptidases, at least in the case of the studied enzymes.

Oxidative stress may contribute to the proteolytic alterations that occur as the result of aging (33). Interestingly, prolyl endopeptidase is especially sensitive to oxidative stress (34) and changes in redox potential have been reported to regulate this enzyme (35). Since the lung is frequently exposed to increased oxidative stress (36) and has been reported to be very susceptible to oxygen-induced effects (37), the observed decrease in lung prolyl endopeptidase activity may well be due to increased oxidative stress in the aging lung. Indeed, increases in the levels of oxidatively modified protein in the cell have been associated with aging (38). It would be expected that reduced levels of activity of prolyl endopeptidase and also PGP I in the aged lung would lead to an increase in the levels of peptides, which are susceptible to inactivation by these enzymes. In keeping with this idea, increased levels of immunoreactivity of SP, peptide degraded by prolyl endopeptidase, were reported in aged lungs (39). Moreover, administration of prolyl endopeptidase inhibitors led to increased SP immunoreactivity in the brain (40), suggesting that SP levels could be controlled by prolyl endopeptidase activity. Increased levels of TRH would also be expected in the aging lung, although this peptide has been reported to be involved in lung maturation (41) rather than senescence.

Interestingly, increased prolyl endopeptidase activity has been reported in primary lung tumors (42). This opposite regulation of prolyl endopeptidase in senescent versus tumor lung tissue raises the possibility that deregulated prolyl endopeptidase activity may contribute to lung tumorigenesis, since it is thought that replicative senescence evolved to protect higher eukaryotes, particularly mammals, from developing cancer (43).

In the aged kidney, we detected reduced activity of soluble PGP I. This alteration is consistent with the finding that the levels of activity of renal brush-border enzymes were reduced in aged rats (44). Interestingly, the TRH tripeptide, which can be degraded by PGP I, can act centrally (45) and peripherally (46) as a hypertensive agent, because antisense or antiserum inhibition of the tripeptide reduces arterial blood pressure in spontaneously hypertensive rats. The present results indicate that the decrease in the activity of enzymes capable of degrading TRH in the kidney of aged rats could be relevant to age-related hypertension. Further studies will be required to clarify this potentially important issue.

The activity of particulate peptidases was also found to be modified with aging, in particular, in the brain and heart. In contrast to the situation in the aging lung in which levels of SP have been reported to be up-regulated, in the brain, a loss of SP immunoreactivity was observed with age. Our finding that particulate prolyl endopeptidase activity increased in the brain stem of aged rats may account for reduced levels of peptides in the aging brain stem. In contrast, the absence of changes in prolyl endopeptidase activity in the brain cortex does not correlate with other findings, such as the report that TRH levels were reduced in the brain cortex of aged rats, compared with young adult animals (6). In addition, prolyl endopeptidase inhibitors have been reported to restore arginine-vasopressin and TRH-like immunoreactivity, as well as that of SP in certain brain areas of aged animals (5,40). However, in these cases, reduced peptide synthesis or processing rather than increased levels of brain endopeptidase may underlie the reduced levels of TRH or SP, which has been reported by others.

Met-enkephalin has been described as an opioid growth factor in the heart with autocrine and/or paracrine activity (47). Aminopeptidase N is one of the principal enzymes involved in enkephalin degradation. We observed that its activity significantly decreased in the heart with senescence, being lower in 22-month-old rats in comparison with 3-month-old animals. This is consistent with the finding that tissue enkephalin levels are higher in 22–24-month-old rats than in younger counterparts (4,9). Interestingly, the levels of proenkephalin mRNA have been reported to reach a maximum in 18-month-old rats, decreasing afterwards (9). Thus, the levels of tissue enkephalins do not strictly correlate with the relative abundance of the corresponding mRNA, suggesting that the decrease in aminopeptidase N activity, which we observed between 18 and 22 months of age, may account for the higher levels of enkephalins in 22-month-old animals, when levels of the corresponding mRNAs have begun to decline. Since opioid receptor stimulation can negatively modulate several characteristics of cardiac myocyte contraction (48), our findings of reduced aminopeptidase activity could have important clinical implications for the aging heart.

In summary, we have detected in a variety of tissues, age-related changes in the activity of several peptidases that are known to participate in the metabolism of TRH and enkephalin. Our findings suggest that these peptidases play an important role in controlling tissue peptide levels during aging. The present results also indicate that aging is not associated with a general up-regulation or down-regulation of the activity of the studied peptidases, but rather that such alterations occur in a region- and peptidase-specific fashion.


   Acknowledgments
 
This work has been supported by grants from the Basque Government (PI-1998-26), the University of the Basque Country (00081.327-EA-7984/2000), and the Gangoiti-Barrera Foundations. N. Agirregoitia and F. Ruiz are recipients of grants from the Spanish Ministerio de Educación Cultura y Deporte. We thank Raquel Villares for her helpful technical assistance as well as the Academic Consulting and Translating Services.

Address correspondence to Jon Irazusta, PhD, Department of Physiology, Medical School, University of the Basque Country, P.O. Box 699 Bilbao, Bizkaia, Spain. E-mail: ofpirasj{at}lg.ehu.es


   Footnotes
 
Decision Editor: James R. Smith, PhD

Received February 3, 2003

Accepted May 22, 2003


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Jiang CH, Tsien JZ, Schulz PG, Hu YH. The effects of aging on gene expression in the hypothalamus and cortex of mice. Proc Natl Acad Sci U S A.. 2001;98:1930-1934.[Abstract/Free Full Text]
  2. Hayashi T, Goto S. Age related changes in the 20S and 26S proteasome activities in the liver of male F344 rats. Mech Ageing Dev.. 1998;102:55-66.[Medline]
  3. Keppler D, Walter R, Perez C, Sierra F. Increased expression of mature cathepsin B in aging rat liver. Cell Tissue Res.. 2000;302:181-188.[Medline]
  4. Caffrey JL, Boluyt MO, Younes A, et al. Aging, cardiac proenkephalin mRNA and enkephalin peptides in the Fisher 344 rat. J Mol Cell Cardiol.. 1994;26:701-711.[Medline]
  5. Miyamoto M, Hirai K, Heya T, Nagaoka A. Effect of a sustained release formulation of thyrotropin-releasing hormone on behavioural abnormalities in senescence-accelerated mice. Eur J Pharmacol.. 1994;271:357-266.[Medline]
  6. Shinoda M, Okayamiya K, Toide K. Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on thyrotropin-releasing hormone-like immunoreactivity in the cerebral cortex and the hippocampus of aged rats. Jpn J Pharmacol.. 1995;69:273-276.[Medline]
  7. Toide K, Fujiwara T, Iwamoto Y, Shinoda M, Okamiya K, Kato T. Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on neuropeptide metabolism in the rat brain. N-S Arch Pharmacol.. 1996;353:355-362.
  8. Zoli M, Ferraguti F, Toffano G, Fuxe K, Agnati LF. Neurochemical alterations but not nerve cell loss in aged rat neostriatum. J Chem Neuroanat.. 1993;6:131-145.[Medline]
  9. Boluyt MO, Younes A, Caffrey JL, et al. Age-associated increase in rat cardiac opioid production. Am J Physiol.. 1993;265:H212-H218.
  10. O'Cuinn G, O'Connor B, Elmore M. Degradation of thyrotropin-releasing hormone and luteinizing hormone by enzymes in brain tissue. J Neurochem.. 1990;54:1-13.[Medline]
  11. Hersh LB. Degradation of enkephalins. The search for an enkephalinase. Mol Cell Biochem.. 1982;47:35-43.[Medline]
  12. Cunningham PM, O'Connor C. Proline specific peptidases. Biochim Biophys Acta.. 1997;1343:160-186.[Medline]
  13. Cummings PM, O'Connor C. Bovine brain pyroglutamyl aminopeptidase (type-1): purification and characterisation of a neuropeptide-inactivate peptidase. Int J Biochem Cell Biol.. 1996;28:883-893.[Medline]
  14. Wilk S, Wilk EK. Rabbit brain pyroglutamate aminopeptidase II, a membrane-bound TRH degrading activity: purification and specificity studies. Ann NY Acad Sci.. 1989;553:556-558.
  15. Fuse Y, Polk DH, Lam RW, Reviczy AL, Fischer DA. Distribution and ontogeny of thyrotropin releasing hormone degrading enzymes in rats. Am J Physiol.. 1990;259:E787-E791.
  16. Irazusta J, Larrinaga G, González-Maeso J, Gil J, Meana JJ, Casis L. Distribution of prolyl endopeptidase activities in rat and human brain. Neurochem Int.. 2002;40:337-345.[Medline]
  17. Goossens F, DeMeester I, Vanhoof G, Scharpe S. Distribution of prolyl oligopeptidase in human peripheral tissues and body fluids. Eur J Clin Chem Clin Biochem.. 1996;34:17-22.[Medline]
  18. Alba F, Arenas C, López MA. Comparison of soluble and membrane-bound pyroglutamyl peptidase I activities in rat brain tissues in the presence of detergents. Neuropeptides.. 1995;29:103-107.[Medline]
  19. O'Leary RM, O'Connor B. Identification and localisation of a synaptosomal membrane prolyl endopeptidase from bovine brain. Eur J Biochem.. 1995;227:277-283.[Medline]
  20. Friedman TC, Wilk S. Delineation of a particulate thyrotropin-releasing hormone-degrading enzyme in rat brain by use of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase. J Neurochem. 46:1231–1239.
  21. Dyer SH, Slaughter CA, Orth K, Moomaw CR, Hersh LB. Comparison of the soluble and membrane-bound forms of the puromycin-sensitive enkephalin degrading aminopeptidases from rat. J Neurochem.. 1990;54:547-554.[Medline]
  22. Gros C, Giros B, Schwartz JC. Identification of aminopeptidase as enkephalin-inactivating enzyme in rat cerebral membranes. Biochemistry.. 1985;24:2179-2185.[Medline]
  23. Fernández D, Valdivia A, Irazusta J, Ochoa C, Casis L. Peptidase activities in human semen. Peptides.. 2002;23:461-468.[Medline]
  24. Laitinen KSM, van-Groen T, Tanila H, Venalainen J, Mannisto PT, Alafuzoff IT. Brain prolyl oligopeptidase activity is associated with neuronal damage rather than beta-amyloid accumulation. NeuroReport.. 2001;12:3309-3312.[Medline]
  25. Saido TC. Alzheimer disease as proteolytic disorders. Anabolism and catabolism of beta-amyloid. Neurobiol Aging.. 1998,;19:S69-S75.[Medline]
  26. Mantle D, Falkous G, Ishiura S, Blanchard PJ, Perry EK. Comparison of praline endopeptidase activity in brain tissue from normal cases and cases with Alzheimer's disease, Lewy body dementia, Parkinson's disease and Huntington's disease. Clin Chim Acta.. 1996;249:129-139.[Medline]
  27. Aoyagi T, Wada T, Nagai M, et al. Deficiency of kallikrein-like enzyme activities in cerebral tissue with Alzheimer's disease. Experientia.. 1990;46:94-97.[Medline]
  28. Shaw PJ, Ince PG, Falkous G, Mantle D. Cytoplasmic, lysosomal and matrix protease activities in spinal cord tissue from amyotrophic lateral sclerosis (ALS) and control patients. J Neurol Sci.. 1996;139:71-75.[Medline]
  29. Yoshimoto T, Ogita K, Walter R, Koida M, Tsuru D. Synthesis of a new fluorogenic substrate and distribution of the endopeptidase in rat tissues and body fluids of man. Biochim Biophys Acta.. 1979;568:184-192.
  30. Alba F, Iribar C, Ramírez M, Arenas C. A fluorimetric method for the determination of brain aminopeptidases. Arch Neurobiol.. 1989;52:169-173.
  31. Giros B, Gros C, Solhone B, Schwartz JC. Characterization of aminopeptidases responsible for inactivating endogenous (Met5)enkephalin in brain slices using peptidase inhibitors and anti-aminopeptidase M antibodies. Mol Pharmacol.. 1986;29:281-287.[Abstract]
  32. Bradford MM. A rapid and sensitive method for the quantification of microgram of protein utilizing the principle of protein-dye binding. Anal Biochem.. 1976;72:248-254.[Medline]
  33. Keller JN, Hanni KB, Markesbery WR. Possible involvement of proteasome inhibition in aging: implications for oxidative stress. Mech Ageing Dev.. 2000;113:61-70.[Medline]
  34. Terwel D, Bothmer J, Wolf E, Meng F, Jolles J. Affected enzymes activities in Alzheimer's disease are sensitive to antemortem hypoxia. J Neurol Sci.. 1998;161:47-58.[Medline]
  35. Tsukahara T, Ishiura S, Sugita H. Regulation of prolyl endopeptidase activity by the intracellular redox state. J Biol Chem.. 1990;265:21448-21453.[Abstract/Free Full Text]
  36. Ahotupa M, Mantyla E, Peltola V, Puntala A, Toivonen H. Pro-oxidant effects of normobaric hyperoxia in rat tissues. Acta Physiol Scand.. 1992;145:151-157.[Medline]
  37. Takahashi A, Aoshiba K, Nagai A. Apoptosis of wound fibroblasts induced by oxidative stress. Exp Lung Res.. 2002;28:275-284.[Medline]
  38. Conconi M, Friguet B. Proteasome inactivation upon aging and on oxidation-effect of HSP 90. Mol Biol Rep.. 1997;24:45-50.[Medline]
  39. Amenta F, Spillantini MG, Cavalotti C, Geppeti P. Increase in substance P levels in the lung of the old rats. Arch Gerontol Geriatric.. 1988;7:215-219.
  40. Toide K, Fujiwara T, Okamiya K, Iwamoto Y, Kato T. Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on prolyl endopeptidase activity and substance P- and arginine-vasopressin-like immunoreactivity in the brains of aged rats. J Neurochem.. 1995;65:234-240.[Medline]
  41. Ansari MA, Demello DE, Polk DH, Devaskar UP. Thyrotropin-releasing hormone accelerates fetal mouse lung ultrastructural maturation via stimulation of extra thyroidal pathway. Pediatr Res.. 1997;42:709-714.[Medline]
  42. Sedo A, Krepela E, Kasafirek E. Dipeptidyl peptidase IV, prolyl endopeptidase and cathepsin B activities in primary human lung tumors and lung parenchyma. J Cancer Res Clin Oncol.. 1991;117:249-253.[Medline]
  43. Campisi J. Cancer, aging and cellular senescence. In Vivo.. 2000;14:183-188.[Medline]
  44. Nakano M, Tauchi H. Age-related alteration of renal brush border leucine aminopeptidase in rat kidney cortex. Mech Ageing Dev.. 1998;45:51-58.
  45. Garcia SI, Alvarez AL, Porto PI, Garfunkel VM, Finkielman S, Pirola CJ. Antisense inhibition of thyrotropin releasing hormone reduces arterial blood pressure in spontaneously hypertensive rats. Hypertension.. 2001;37:365-370.[Abstract/Free Full Text]
  46. Goldstein DJ, Ropchak TJ, Kelly GB, Keiser HR. TRH reverses the effect of captopril in the renal hypertensive rat. Eur J Pharmacol.. 1983;93:293-294.[Medline]
  47. McLaughlin PJ, Wu Y. Opioid gene expression in developing and adult rat heart. Dev Dynam.. 1998;211:153-163.[Medline]
  48. Ventura C, Spurgeon H, Lakatta EG, Guarnieri C, Capogrossi MC. Kappa and delta opioid receptor stimulation affects cardiac myocyte function and Ca2+ release from and intracellular pool in myocytes and neurons. Circ Res.. 1992;70:66-81.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. J. Trask, D. B. Averill, D. Ganten, M. C. Chappell, and C. M. Ferrario
Primary role of angiotensin-converting enzyme-2 in cardiac production of angiotensin-(1-7) in transgenic Ren-2 hypertensive rats
Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H3019 - H3024.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Agirregoitia, N.
Right arrow Articles by Casis, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Agirregoitia, N.
Right arrow Articles by Casis, L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
All GSA journals The Gerontologist
Journals of Gerontology Series B: Psychological Sciences and Social Sciences