Lysosome-Mediated Apoptosis is Associated with Cathepsin D-Specific Processing of Bid at Phe24, Trp48, and Phe183

Bax-mediated permeabilization of the outer mitochondrial membrane and release of apoptogenic factors into the cytosol are key events that occur during apoptosis. Likewise, apoptosis is associated with permeabilization of the lysosomal membrane and release of lysosomal cathepsins into the cytosol. This report identifies proteolytically active cathepsin D as an important component of apoptotic signaling following lysosomal membrane permeabilization in fibroblasts. Lysosome-mediated cell death is associated with degradation of Bax sequestering 14-3-3 proteins, cleavage of the Bax activator Bid, and translocation of Bax to mitochondria, all of which were cathepsin D-dependent. Processing of Bid could be reproduced by enforced lysosomal membrane permeabilization, using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride (MSDH). We identified three cathepsin D-specific cleavage sites in Bid, Phe24, Trp48, and Phe183. Cathepsin D-cleaved Bid induced Bax-mediated release of cytochrome c from purified mitochondria, indicating that the fragments generated are functionally active. Moreover, apoptosis was associated with cytosolic acidification, thereby providing a more favorable environment for the cathepsin D-mediated cleavage of Bid. Our study suggests that cytosolic cathepsin D triggers Bax-mediated cytochrome c release by proteolytic activation of Bid.     

Regionally selective changes in brain lysosomes occur in the transition from young adulthood to middle age in rats

The possibility that brain aging in rats exhibits regional variations of the type found in humans was studied using lysosomal chemistry as a marker. Age-related (two vs 12months; male Sprague-Dawley) differences in cathepsin D immunostaining were pronounced in the superficial layers of entorhinal cortex and in hippocampal field CA1, but not in neocortex and field CA3. Three changes were recorded: an increase in the intraneuronal area occupied by labeled lysosomes; clumping of immunopositive material within neurons; more intense cytoplasmic staining. Western blot analyses indicated that the increases involved the active forms of cathepsin D rather than their proenzyme. Shrinkage of cathepsin-D-positive neuronal cell bodies was observed in entorhinal cortex but not in neocortical sampling zones. Age-related lysosomal changes as seen with cathepsin B immunocytochemistry were considerably more subtle than those obtained with cathepsin D antibodies. In contrast, a set of glial and/or vascular elements located in a distal dendritic field of the middle-aged hippocampus was much more immunoreactive for cathepsin B than cathepsin D. The areas exhibiting sizeable changes in the present study are reported to be particularly vulnerable to aging in humans.The results thus suggest that aspects of brain aging common to mammals help shape neurosenescence patterns in humans.     

Accumulation of inactive cathepsin D in old rats

From the 2nd to the 29th month of life seven different rat organs (liver, heart, kidney, lung, spleen, skeletal muscle, intestinal mucosa) show a continuous increase in the concentration of protein reacting with rabbit antiserum against rat liver cathepsin D. However, the activity of aspartate proteinases, probably mainly cathepsin D, does not show the same increase. This result is interpreted as accumulation of enzymatically inactive but immunologically reactive cathepsin D. This phenomenon, which is well known for some cytosolic enzymes in senescent animals, is first reported to occur for a lysosomal proteinase as well.     

Microinjection of cathepsin d induces caspase-dependent apoptosis in fibroblasts

Recent reports have indicated that enzymes such as cathepsins D and B are translocated from lysosomal compartments to the cytosol early during apoptosis. We have previously noted that a translocation of cathepsins D and B occur before cytochrome c release and caspase activation in cardiomyocytes and human fibroblasts during oxidative stress-induced apoptosis. In the present report, we use a microinjection technique to investigate if cytosolic location of the cathepsins D and B are important for induction of apoptosis. We found that microinjection of cathepsin D into the cytosol of human fibroblasts caused apoptosis, which was detected as changes in distribution of cytochrome c, cell shrinkage, activation of caspases, chromatin condensation, and formation of pycnotic nuclei. No apoptosis was, however, induced by microinjection of cathepsin B. Moreover, apoptosis was prevented in fibroblasts pretreated with a caspase-3-like inhibitor, and also when microinjected with cathepsin D mixed with the cathepsin D inhibitor, pepstatin A. These results show that cytosolic cathepsin D can act as a proapoptotic mediator upstream of cytochrome c release and caspase activation in human fibroblasts.     

The Role of Cathepsin D in the Pathogenesis of Tuberculosis: A Histochemical Study Employing Unlabeled Antibodies and the Peroxidase-Antiperoxidase Complex

Cathepsin D was visualized in free pulmonary alveolar macrophages (AM), in oil-induced peritoneal macrophages (MN) and in rabbit pulmonary and dermal BCG lesions with unlabeled antibodies and the peroxidase-antiperoxidase (PAP) complex. Large amounts of cathepsin D were present in AM and lower amounts in MN. In the lung this enzyme was richest in the alveolar macrophages that accumulated around the BCG lesions. In the dermal lesions, cathepsin D was in highest concentration in macrophages at the border of the necrotic (liquefying) centers. It was also found in high concentration in keratinizing cells of the dermal epithelium and hair follicles. It did not, however, increase appreciably in many of the activated macrophages that stained intensely for the lysosomal enzyme β-galactosidase. In fact, many epithelioid cells with high β-galactosidase activity contained no visible cathepsin D. This proteinase does not, therefore, seem to be primarily involved in the lymphocyte-mediated macrophage activation associated with acquired cellular resistance to tubercle bacilli. It is probably more involved with cell autolysis, with the digestion of ingested necrotic debris and, in all likelihood, with the process of liquefaction, the most adverse event in the pathogenesis of tuberculosis in man.     

Protection of hypoxic cytotoxicity by glucocorticoid in the liver

Induction of hypoxia for 2.5 h in perfused cat livers resulted in a 10-fold increase in cathepsin D and a 15-fold increase in lactic dehydrogenase (LDH) activities in the perfusate and a 42% depression of the clearance rate of particles by the reticuloendothelial system (RES). Addition of 10^–3 M methylprednisolone (MP) to the perfusate only slightly retarded the release of LDH, but significantly (P<0.05) inhibited cathepsin D release by 90% at 150 min. Liver flow did not change during the perfusion period when MP was added to control or hypoxic livers. Similarly, MP did not significantly alter oxygen consumption in perfused livers. However, MP protected against the reduction in carbon clearance during hypoxia without inducing blockade of colloidal carbon clearance. Moreover, opsonization of carbon with autologous plasma did not improve the clearance of colloidal carbon above that observed in nonopsonized experiments. Thus, pharmacologic doses of a synthetic glucocorticoid protected RES cells and release of lysosomal hydrolases during severe hypoxia in isolated perfused cat livers probably by stabilization of cellular and intracellular (lysosomal) membranes.Supported in part by grant HL-17745 from the National Heart and Lung Institute of the NIH.     

Aktivitätsverhalten lysosomaler proteinabbauender Enzyme der Rattenleber bei der Galaktosamin-Hepatitis: Kathepsin D,Kathepsin A und saure Carboxypeptidase

Zusammenfassung In der vorliegenden Arbeit wird über das Verhalten der lysosomalen Enzyme Kathepsin D, Kathepsin A und saure Carboxypeptidase bei einer akut und einer subakut verlaufenden Galaktosamin-Hepatitis der Ratte berichtet. Es werden Rattenlebern nach der Methode von de Duve durch Zentrifugation in ein Lysosomen-Sediment und einen Lysosomen-Überstand fraktioniert. Ein Teil der Leber wird zur Bestimmung der Gesamtaktivität der oben genannten Enzyme homogenisiert. Bei der akuten Hepatitis nehmen nach 25 Std die Enzymaktivitäten in der Lysosomen-Sediment-Fraktion ab, in der Lysosomen-Überstands-Fraktion zu. Die Aktivität dieser Enzyme pro g Leberfrischgewicht nimmt bis auf die von Kathepsin A signifikant ab. Bei der subakuten Galaktosamin-Hepatitis ist nach 8 Tagen ein signifikanter Anstieg der 3 Enzymaktivitäten in der Lysosomen-Überstandsfraktion nachweisbar; er ist im Vergleich zur akuten Schädigung weniger ausgeprägt. Die Aktivitäten von Kathepsin D und A pro g Leberfrischgewicht nehmen zu; diese Zunahme im Verlauf der subakuten Hepatitis könnte Ausdruck einer Regenerationsleistung der Leber sein.Unter der Wirkung von Galaktosamin werden die Lysosomen instabiler.

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Distribution and activity of lysosomal protein catabolicenzymes of rat liver in galactosamine hepatitis: Cathepsin D, cathepsin A and acid carboxypeptidase

Summary Changes of activity and distribution in rat liver of the lysosomal enzymes cathepsin D, cathepsin A, and acid carboxypeptidase are described as a result of D-galactosamine action. Hepatitis was provoked by acute and by prolonged administration of D-galactosamine. A lysosomal pellet and a lysosomal supernatant fraction were prepared from rat liver by centrifugation according to de Duve. Total activity of the respective enzymes mentioned above was assayed in the liver homogenate. A loss of enzyme activities in the lysosomal pellet and an increase in the supernatant fraction was observed after 25 hours in acute hepatitis following D-galactosamine administration. Total activities of cathepsin D and acid carboxypeptidase per gram liver wet weight were significantly decreased. Administration of D-galactosamine over 8 days resulted in a significant increase of the three enzyme activities in the lysosomal supernatant fraction; as compared to the acute hepatitis, however, this increase was less marked. Total activities of cathepsin D and A per gram liver wet weight increased during prolonged D-galactosamine administration; this is interpreted as a result of liver regeneration.D-Galactosamine action in liver leads to more labile lysosomes.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

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Die Ergebnisse wurden zum großen Teil im Rahmen der Inaugural-Dissertation von Gunther Wangemann gewonnen.     

Lysosomal cathepsin B plays an important role in antigen processing, while cathepsin D is involved in degradation of the invariant chain inovalbumin-immunized mice

We previously reported that CA074, a specific inhibitor of cathepsin B, modulates specific immune responses from the T helper 2 (Th2) type to Th1 type in BALB/c mice infected with Leishmania major. In the present study, we found that a similar type of immune deviation was also induced in mice immunized with ovalbumin (OVA). However, treatment of mice with pepstatin A, a specific cathepsin D inhibitor, suppressed the OVA-specific proliferation of lymphocytes and blocked the development of both Th1 and Th2 cellular responses. These inhibitors did not appear to have any direct influence in vitro on functions of naive lymphocytes. OVA antigen (47 000 MW) was digested mainly into 40 000 MW protein in vitro by lysosomal proteases from naive BALB/c mice, and its digestion was markedly inhibited by the addition of CA074, but not by addition of pepstatin A, during incubation. However, pepstatin A strongly suppressed the degradation of the major histocompatibility complex class II-associated invariant chain (Ii) molecule in vivo and in vitro. Thus, cathepsin B appears to process antigens directed to preferential activation of Th2 cells, while cathepsin D may be responsible for the degradation of Ii, the processing of which is essential in initiating the antigen-specific activation of Th1 and Th2 CD4+ T cells. These lysosomal proteases may have different functions in regulating immune responses.     

Neuronal and microglial cathepsins in aging and age-related diseases

It has been long believed that cathepsins compensate for each other because of their overlapping substrate specificities. However, there is increasing evidence that disturbance of the normal balance of their enzymatic activities is the first insult in brain aging and age-related diseases. The imbalance of cathepsins may further cause age-related neuropathological changes such as accumulation of autophagic vacuoles and the formation of ceroid-lipofuscin leading to neuronal dysfunction and damage. Leakage of cathepsins due to the fragility of lysosomal membranes during aging also contributes to neurodegeneration. Furthermore, the deficiency of cathepsin D has been recently revealed to provoke a novel type of lysosomal storage disease associated with massive neurodegeneration. In these animals, microglia are activated to initiate inflammatory and cytotoxic responses by binding and phagocytosis of storage neurons. Activated microglia also release some members of cathepsins to induce neuronal death by degrading extracellular matrix proteins. Thus the microglial activation possibly through sensing neuronal storage may also be an important causative factor for neurodegeneration in lysosomal storage diseases and age-related diseases such as Alzheimer's disease. This review describes the pathological roles of neuronal and microglial cathepsins in brain aging and age-related diseases.     

Activities of lysosomal enzymes and levels of serum amyloid A (SAA) in blood plasma of hamsters during casein induction of AA-amyloidosis

Casein-induced amyloidosis in hamsters was found to be of the AA-type, as shown by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis of the major low-molecular weight component of the amyloid fibrils. Levels of serum amyloid A (SAA) and the activities of cathepsin D, beta-N-glucosaminidase, serine esterase, lactate dehydrogenase (LDH) and gamma glutamyl transpeptidase (GGT) were measured in the blood plasma during induction of amyloidosis. During the pre-amyloid phase an increase was observed in all these parameters. During the deposition of amyloid, an increase was observed in the activities of the lysosomal enzymes cathepsin D and beta-N-glucosaminidase, which was significantly correlated with amyloid deposition. Serine esterase activities did not show any relationship to amyloid deposition. LDH and GGT activities were normal in the amyloid phase. SAA levels were lower during amyloid deposition than during the pre-amyloid phase. These findings indicate that a specific release of lysosomal contents from mononuclear phagocytic cells is involved in the pathogenesis of AA-amyloidosis. Amyloid deposition may be the result of: (i) extrusion of intralysosomal protein AA or pre-amyloid, followed by extracellular formation of amyloid fibrils; (ii) secretion of lysosomal enzymes, followed by extracellular cleavage of SAA and subsequent aggregation of protein AA with other components.     

Activities of lysosomal enzymes and levels of serum amyloid A (SAA) in blood plasma of hamsters during casein induction of AA-amyloidosis.

Casein-induced amyloidosis in hamsters was found to be of the AA-type, as shown by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and amino acid analysis of the major low-molecular weight component of the amyloid fibrils. Levels of serum amyloid A (SAA) and the activities of cathepsin D, beta-N-glucosaminidase, serine esterase, lactate dehydrogenase (LDH) and gamma glutamyl transpeptidase (GGT) were measured in the blood plasma during induction of amyloidosis. During the pre-amyloid phase an increase was observed in all these parameters. During the deposition of amyloid, an increase was observed in the activities of the lysosomal enzymes cathepsin D and beta-N-glucosaminidase, which was significantly correlated with amyloid deposition. Serine esterase activities did not show any relationship to amyloid deposition. LDH and GGT activities were normal in the amyloid phase. SAA levels were lower during amyloid deposition than during the pre-amyloid phase. These findings indicate that a specific release of lysosomal contents from mononuclear phagocytic cells is involved in the pathogenesis of AA-amyloidosis. Amyloid deposition may be the result of: (i) extrusion of intralysosomal protein AA or pre-amyloid, followed by extracellular formation of amyloid fibrils; (ii) secretion of lysosomal enzymes, followed by extracellular cleavage of SAA and subsequent aggregation of protein AA with other components.     

Degradation of gonococcal outer membrane proteins by human neutrophil lysosomal proteases.

Interest in the molecular mechanisms of leukocyte bactericidal activity led us to study the effects of human neutrophil lysosomal proteases on the outer membrane (OM) proteins of Neisseria gonorrhoeae. A protease fraction containing cathepsin G and elastase activity was partially purified by gel filtration chromatography of acetate extracts of purified neutrophil granules. OM was obtained from gonococci by French press-Sarkosyl or by LiCl2 extraction. The principal (protein I) and opacity-associated (proteins II) OM proteins of N. gonorrhoeae were hydrolyzed by lysosomal proteases; proteins II were more susceptible to hydrolysis than protein I. Treatment of whole gonococci, with subsequent purification of OM, or direct treatment of purified OM led to identical hydrolysis of OM proteins by lysosomal proteases as indicated by sodium dodecyl sulfate-polyacrylamide gel patterns. Similarly, hydrolysis of purified OM proteins was identical whether OM was treated with unfractionated granule extract or with the partially purified lysosomal proteases, indicating that the observed hydrolysis by unfractionated lysosomal contents was due solely to the lysosomal protease fraction. Hydrolysis of OM proteins was dependent upon the concentration of proteases, time, and temperature. Hydrolysis of proteins II was observed with as little as 1 microgram of proteases per ml for 1 h at 37 degrees C. OM incubated alone or with heat-inactivated proteases showed no hydrolytic activity. The addition of 25 mM Na+, K+, Mg2+, or Ca2+ to incubation mixtures containing proteases and OM did not alter hydrolytic activity as indicated by sodium dodecyl sulfate-polyacrylamide gel patterns.     

Progestin increases cathepsin D synthesis in uterine luminal epithelial cells

Early in blastocyst implantation, cells of the uterine luminal epithelium deteriorate and die in response to the presence of the blastocyst. Destruction of the epithelial cells appears to depend on control of the autophagic activity and enzyme content of lysosomes in these cells. Concentrations of the lysosomal proteinase, cathepsin D, have been identified in luminal epithelial cells, and these studies examined changes in epithelial cathepsin D activity and their hormonal control during early pseudopregnancy in the rat. Cathepsin D activity in luminal epithelial cells increases during early pseudopregnancy to maximal levels at the time of sensitivity to deciduogenic stimuli. Rates of cathepsin D synthesis in luminal epithelial cells also increase during early pseudopregnancy, but neither enzyme activity nor rates of synthesis increase in stromal-myometrial tissues. In ovariectomized rats, progestins rather than estradiol increase cathepsin D activity and rates of synthesis in luminal epithelial cells. These studies suggest that cell death in the luminal epithelium during blastocyst implantation may depend in part on the accumulation of lysosomal cathepsin D in these cells in response to progesterone secretion during early pregnancy.     

Lysosome is a primary organelle in B cell receptor-mediated apoptosis: an indispensable role of Syk in lysosomal function

To investigate the mechanism of B cell receptor (BCR)-mediated apoptosis, we utilized immature B cell lines, DT40 and WEHI-231. In both cell lines, BCR-crosslinking caused the increase in lysosomal pH with early apoptotic changes characterized by chromatin condensation and phosphatidylserine exposure. This increase was detected in c-Abl-deficient DT40 cells but not in Syk-deficient cells, which corresponded to the fact that the former cells but not the latter revealed BCR-induced apoptosis. In contrast, BCR-crosslinking caused no apparent change in mitochondrial transmembrane potential. Therefore, the lysosomal change might be a primary event in BCR-induced apoptosis in DT40 cells. The increased activity of cathepsin B and apoptosis-preventing effect of a cathepsin inhibitor suggested a significant role of lysosomal enzymes in this apoptosis. By microscopic studies, lysosomes of wild-type DT40 cells fused to BCR-carrying endosomes became enlarged and accumulated one another. In contrast, these changes of lysosomal dynamics did not occur in Syk-deficient cells but transfer of wild-type Syk restored the lysosomal changes and apoptosis. These results demonstrated that the lysosomal change accompanied with the activation of lysosomal enzymes is a primary step in BCR-crosslinking-mediated apoptosis and Syk is responsible for this step through the fusion of BCR-carrying endosomes to lysosomes.     

Age-dependent change in activities of lysosomal enzymes in rat brain

The age-dependent change in activities of seven lysosomal enzymes (cathepsin D, β-glucuronidase, acid phosphatase, acid/alkaline DNases and acid/alkaline RNases) was studied in four brain regions (cerebrum, hippocampus, pons and cerebellum) of Wistar rats. The activity of cathepsin D was significantly increased with aging in the four regions. The age-dependent change in activities of acid and alkaline DNases showed the characteristic regional difference, and the ratio of acid to alkaline DNases was increased with aging in all regions. Acid RNase showed the lowest activity in 18-month-old rats, and alkaline RNase activity was decreased with aging. The activity of β-glucuronidase was higher in 2-month-old rats in all of the regions studied. Acid phophatase showed no significant age-dependent change except in pons. The study demonstrated that all of the lysosomal enzyme activities do not change in parallel with aging, and that the age-dependent change showed the characteristic regional difference.     

Changes of skeletal muscle proteases activities during a chronic low-frequency stimulation period

Modifications of muscular contractile patterns by chronic low-frequency stimulation induce structural, physiological, and biochemical transformations in fast-twitch fibers that cause them to act like slow-twitch muscle. During this transformation many changes in protein pattern appear and the proteolytic system may be involved in those changes. The activities of cathepsin L, B, H, D, the level of cystatin, as well as the calpain activity in rabbit fast-twitch muscle have been compared with those of slow-twitch muscle. The results show that fast-twitch muscle has lower cathepsin activities and higher calpain activities than slow-twitch muscle. Chronic low-frequency stimulation was applied for 24 days to fast-twitch muscles and changes in proteases and protease inhibitors (cystatin and calpastatin) were studied. After 7-14 days of stimulation, lysosomal cathepsin L, B, and D and cytoplasm calpain and proteosome activities increased several-fold. Involvement of the phagocyte cells in the protein fiber turnover was minimal. Although the turnover of contractile proteins during muscle electrostimulation takes place in synchrony with changes in the muscle proteolytic system, the stimulation period used did not attain the total transformation from fast- to slow-twitch muscle proteolytic pattern.     

Lysosomal alterations in hypoxic and reoxygenated hearts. II. Immunohistochemical and biochemical changes in cathepsin D.

Sublethal hypoxic injury in rat and rabbit hearts was accompanied by a biochemical redistribution of cathepsin D activity from the particulate to the supernatant fraction of the tissue homogenate, which was partially reversible on reoxygenation. Immunofluorescent staining for cathepsin D failed to reveal major anatomic release of the acid hydrolase until necrosis was present, suggesting that the earlier biochemical redistribution was primarily a result of increased lysosomal fragility during homogenization, with significant intracellular diffusion of the enzyme occurring only as irreversible damage took place. Hypoxia produced enlargement of both cathepsin-D-staining lysosomes and nonstaining vacuoles, as well as their aggregation. These changes were intensified during reoxygenation and recovery of reversibly damaged hearts, suggesting a possible role for the lysosomal-vacuolar apparatus in myocytic repair following hypoxic injury.     

Loss of CB1 receptors leads to decreased cathepsin D levels and accelerated lipofuscin accumulation in the hippocampus

Early onset of age-related changes in the brain of cannabinoid 1 receptor knockout (Cnr1^−/−) mice suggests that cannabinoid 1 (CB1) receptor activity significantly influences the progression of brain aging. In the present study we show that lack of CB1 receptors leads to a significant increase in lipofuscin accumulation and a reduced expression and activity of cathepsin D, lysosomal protease implicated in the degradation of damaged macromolecules, in the hippocampus of 12-month-old mice. The impaired clearance of damaged macromolecules due to the low cathepsin D levels and not enhanced oxidative stress may be responsible for the lipofuscin accumulation because macromolecule oxidation levels were comparable between the genotypes within the same age group. The altered levels of autophagy markers p62 and LC3-II suggest that autophagy is upregulated in CB1 knockout mice. Increased autophagic flux in the absence of CB1 receptors is probably a compensatory mechanism to partially counteract decreased lysosomal degradation capacity. Together, these results suggest that CB1 receptor activity affects lysosomal activity, degradation of damaged macromolecules and thus it may influence the course and onset of brain aging.     

Characterization of liver lysosomal enzyme activity in hepatocytes, Kupffer and endothelial cells during aging: effect of dietary restriction

The specific activity of 4 lysosomal enzymes was studied in homogenate, hepatocytes, Kupffer and endothelial cells isolated from the livers of female Sprague-Dawley rats aged 3.5, 12 and 24 months. Cells were obtained by enzymatic digestion and centrifugal elutriation. Cell viability was not affected by age or diet. In hepatocytes, the activities of all enzymes (acid phosphatase, beta-galactosidase, arylsulfatase B and cathepsin D) increased with age in rats fed ad libitum (A) but were not altered significantly by dietary restriction. The activities of all enzymes except acid phosphatase were systematically higher at 3.5 months of age in Kupffer and endothelial cells than in hepatocytes. Acid phosphatase, arylsulfatase B and cathepsin D activities increased with age in both Kupffer and endothelial cells. Beta galactosidase was decreased significantly with age in Kupffer cells but was elevated in endothelial cells. Rats exposed to dietary restriction (R) showed higher activities of beta-galactosidase, arylsulfatase B and cathepsin D when compared to corresponding A animals with the exception of the younger age group. No clear cut pattern was observed in acid phosphatase activity. Thus, the activities of liver lysosomal enzymes increase with age but the pattern of change differs with respect to enzyme and cell populations. The heightened enzyme activity in Kupffer and endothelial cells from R rats may reflect a more efficient phagocytic capacity in these animals.     

Studies of prostatic cytosol protein kinases of the aging ACI rat

Changes in prostatic cytosolic cyclic AMP (cAMP)-dependent and independent protein kinases associated with aging in ACI rats were determined. A decrease in prostatic weight and tissue protein content (but not DNA) with age was noted. The cytosolic cAMP-dependent protein kinase was present predominantly as the type II isozyme. Total content of this enzyme per prostate declined by 37% in 12-month-old rats but remained relatively stable thereafter. However, the activity ratio (+cAMP/-cAMP) increased with aging, indicating an elevation in the amount of the holoenzyme or a decrease in the amount of the free catalytic subunit. Concomitantly the specific activity (activity/unit protein in the cytosol) of this enzyme was found to be increased; this is most likely due to the loss of bulk proteins in the cytosol, and may indicate a slower loss of this enzyme protein in relation to other cytosolic proteins. By comparison, the cAMP-independent protein kinase demonstrated a marked and progressive decline in amount as well as in specific activity with aging, e.g. 80-90% decline at 12 months of age. The above changes in these enzymes did not appear to be due to low circulating androgen in the animals, since they were not reversed on administration of 3.0 mg of testosterone propionate per rat daily for four consecutive days. It is therefore suggested that the above-described changes in prostatic cytosolic protein kinases are primarily associated with cellular senescence in this organ.