the effects of purified cathepsin d infusions in intact animals

The Effects of Purified Cathepsin D Infusions in Intact Animals

Mark S. Mason, MD, Charlottesville, Virginia

Stephen L. Wangensteen, MD,’ Charlottesville, Virginia

Lysosomes, first described by de Duve et al [I] in 1955, are a class of subcellular organelles that contain a variety of hydrolytic enzymes with acid pH optima. These organelles normally function as the cyto- plasmic vacuolar digestive system and provide for degradation of endogenous and exogenous substances. Under physiologic conditions, lysosomal enzymatic action is controlled by a single limiting lipoprotein membrane. This membrane functionally isolates lysosomes from the surrounding cytoplasm and prevents autolysis [2]. A number of pathophys- iologic states have been described in which normal lysosomal membrane integrity is lost. Hypoxia and acidosis, as occur in circulatory shock, have been shown to cause labilization of lysosomal membranes and release of lysosomal acid hydrolases [3-51. It has been postulated that in shock, activation of these hydrolytic enzymes initiates cellular autolysis, cell death, and local tissue degeneration. After their in- troduction into the systemic circulation, they may cause adverse effects to distant tissues, thus leading to propagation of the shock syndrome and a state of irreversibility.

A large body of suggestive evidence implicates lysosomal hydrolytic enzymes in the pathogenesis of shock. Multiple investigations have demonstrated increased levels of circulating lysosomal enzymes in a variety of shock states and these levels have been noted to reflect both the severity and duration of shock [6-161. Histologic and histochemical studies have revealed lysosomal disruption and enzyme release from various tissues during shock [7,11,17], and infusions of exogenous heterogeneous lysosomal extracts in intact animals have been found to produce circulatory disturbances [18,19]. Finally, these enzymes may cause microcirculatory changes, including stasis of blood flow, altered capillary permeability, and leukocyte capillary adhesion and immigration during shock [20].

From the Department of Surgery, University of Virginia Medical Center, Charlottesville, Virginia. This work was supported by a contract from the US Army Medical Research and Development Command, #DADA 17-73- c-3099.

Present address and reprint requests: Stephen L. Wangensteen. MD, Department of Surgery, University of Arizona Health Sciences Center, Tucson, Arizona 85724.

Of the numerous lysosomal enzymes, acid phosphatase, beta-glucuronidase, and the acid proteases have received the greatest attention. The acid proteases and in particular the cathepsins have been implicated as the agents most responsible for deleterious effects. A significant relationship between serum catheptic activity and mortality has been demonstrated ih hemorrhagic shock [21]. Further- more, protease inhibitors, particularly those that oppose lysosomal cathepsins, have been shown to afford protection in experimental shock states [22,23]. Quantitatively, cathepsin D has been found to be one of the major acid proteases, and it is be- lieved to have a distribution limited only to lysosomes [24,25]. Recent investigations in our own laboratory demonstrated a 47 per cent increase in survival in rabbits subjected to hemorrhagic shock after pre- treatment with cathepsin D antiserum [26]. Al- though the evidence is indirect, this suggests that removal of cathepsin D by formation of antigen antibody complexes prevents the deleterious effects of this potent acid protease in shock.

In recent years, methods have been devised for the isolation and purification of cathepsin D. It is the purpose of the present study to (1) adopt a method for obtaining highly purified cathepsin D and (2) directly study the effects of infusion of large amounts of this enzyme in the intact animal.

Material and Methods

Cdthepsin. D Purification. The method of Barrett [27], with modifications, was used for cathepsin D isolation. Commercially obtained young rabbit liver (2.0 kg) was

homogenized in 4.0 1 of 1 per cent saline solution containing 2 per cent l-butanol. The pH of the homogenate was adjusted to 7.0 by the addition of 2 M tris base HCl buffer. The homogenate was then centrifuged at 1,500 X g for 30 minutes at 15%. The resultant precipitate was discarded and the supernatant was adjusted to pH 3.6 and incubated for 12 hours at 37%. The supetnate was cooled to 0°C and 0.9 volumes of anhydrous acetone were added. The preparation was centrifuged at 1500 X g for 15 minutes at 0°C the precipitate was discarded, and a second volume of acetone equal to the first was added to the supernate. The subsequent precipitate was collected by vacuum filtration. The filtrate was discarded and the precipitate redispersed

278 The American Journal of Surgery

Cathepsin D Infusion

in a minimum volume of 50 mm ethylenediaminetetra- acetic acid (EDTA) trisodium salt in a buffer solution at pH 8.0. The preparation was dialyzed over a 24 hour period against two changes of the EDTA tris buffer solution and then redialyzed for 24 hours against a 2 mm tris HCl buffer solution of pH 9.0 until the pH of the preparation was greater than 7.5. The preparation was then filtered as be- fore, the filtrate preserved, and the precipitate discarded. Approximately 300 cc of enzyme preparation was available at this stage. The preparation was applied to a column and those samples with high (> 20 units/ml) activity were pooled and dialyzed for 12 hours. The dialysate was applied to another column and the column eluates with high cathepsin D activity were pooled and stored at O’C.

Biochemicat Determinations. Plasma cathepsin D activity was assayed using a combination of the methods of Barrett [27] and Anson [28]. The assay is based on the determination of acid-soluble peptides released from hemoglobin substrate by enzyme sample. Cathepsin D spe- cific activity was expressed as milliequivalents of tyrosine X lo-” produced per milliliter sample per milligram protein per hour at 37°C. Relative activity was defined as milliequivalents tyrosine X 10e4 produced per milliliter sample per hour at 37°C.

Protein was determined by the method of Lowry et al 1291 for the purification preparations and by the biuret technic for plasma samples. 1 Na+ 1 and 1 K+ 1 were determined by the flame spectrophotometer. 1 Cl-1 was analyzed by a chlorimeter. Other biochemical parameters were determined by Biomedical Laboratories, Inc, Richmond, Virginia.

Intact Animal Preparation. New Zealand white rabbits, weighing between 3 and 3.5 kg, were anesthetized by the administration of acepromazine (0.3 mg intravenously) and sodium pentobarbital(20 mg/kg intravenously). The left carotid artery was cannulated with a 14 F polyethylene catheter. Mean arterial blood pressure (MABP) and heart rate (HR) were monitored using a Statham P-23 Db pressure transducer and recorded on a Hewlett-Packard 8805 B carrier amplifier. A rectal probe connected to a tele- thermometer was inserted for monitoring core body temperature. Initial MABP, HR, and temperature were recorded and a 3 cc blood sample was obtained for cathepsin D assay. The purified cathepsin D preparation was given intravenously during a 5 minute interval. The volume for infusion was calculated, on the basis of animal weight, to give serum cathepsin D levels equal to or greater than those found in severe hemorrhagic shock. During the ensuing 2 hour experimental period, MABP, HR, and temperature were recorded and 3 cc blood samples were obtained for cathepsin D assay at 15 minute intervals. To maintain fluid balance 3 cc of heparinized saline solution was infused after each blood sampling. At the termination of the experiment, the arterial cannula was removed, the animal returned to its cage, and its condition observed at 8,16, and 24 hours after infusion.

Statistical Analysis. All reported values, unless other- wise indicated, are expressed as the mean f the standard error of the mean (SEM). Analysis for statistical signifi-

TABLE I Procedural Analysis of Cathepsin D Isolation

Relative Activity Yield

(units/ml) (%)

Homogenate 109.3 f 13.9 100 Dialyzed extract 59.3 f 5.2 55.6 f 4.6 DEAE cellulose 51.4 f 4.6 46.6 f 4.6 CM cellulose 44.9 f 4.9 43.0 f 4.3

Note: The results are mean values based on several isolation preparations.

cance was made by the paired t test with probability values less than 0.05 considered statistically significant.

Results

Cathepsin D Isolation and Purification. Using a

total of 16 kg rabbit liver, 794 ml of purified enzyme extract containing a mean 44.9 f 4.9 enzyme units/ml was produced. (Table I.) High yields averaging 43 per cent of the initial homogenate activity were obtained. Overall, the procedure gave an approximate 490-fold

increase in purity. Purification, particularly after diethylaminoethanol (DEAE) cellulose column

chromatography was less than expected and the final

preparations averaged 109.6 f 21.9 enzyme units/mg protein with a mean 0.56 f 0.13 mg/ml total protein. Assay for enzymes other than cathepsin D were

performed on the purified enzyme extract. Acid phosphatase and beta-glucuronidase activities were not detectable in any of the final preparations. Other liver enzymes were present in small quantities. (Table II.) As expected, sodium concentrations were

high, but other electrolytes were absent with the exception of trace amounts of calcium. Cholesterol was found to be the other nonprotein contaminant

most consistently present.

TABLE II Biochemical Analysis of Cathepsin D Preparations

Cathepsin D 44.9 f 4.9 U/ml @-glucuronidase 0 f 0 U/ml Acid phosphatase OfOUlml Alkaline phosphatase Lactic dehydrogenase SGOT Gamma glutamyl Transpeptidase Sodium Potassium Chloride Calcium

5.5 f 3.3 miu/ml 6.2 f 6.2 miu/ml 4.2 f 2.1 miu/ml

0.7 f 0.3 miu/ml 192 f 6 mEq/l

Of 0 mEq/l 0 f 0 mEq/l

0.6 f 0.3 mg/lOO ml Glucose Cholesterol

5.5 f 2.2 mg/lOO ml 13.0 f 1.5 mg/lOO ml

Triglycerides 0 l 0 mg/iOO ml Total bilirubin 0.02 f 0.02 mg/lOO ml

Note: The results are mean values based on several isolation preparations.

Volume 134, August 1977 279

Mason and Wangensteen

TABLE III Physlologlc Responses to Cathepsln D lnfuslons

Mean Arterial Interval Blood Pressure Heart Rate Temperature (min) (mm Hg) (beatdmin) (“C)

Preinfusion 0 88.4 f 2.5 239 f 14 38.1 f 0.1 Postinfusion 15 91.7 f 3.0 222 f 14 37.9 f 0.1

30 87.7 f 3.4 218 f 10 38.1 f 0.3 60 87.9 f 3.9 221 f 15 37.7 f 0.1 l 90 86.9 f 3.4 240 f 10 37.6 f 0.1’

120 86.4 f 3.5 251 f9 37.6 f 0.2’

l p <0.005.

Physiologic Studies. Normal plasma cathepsin D was measured in fifty-eight healthy New Zealand white rabbits and found to be 1.7 f 0.2 units/mg protein (0.9 f 0.1 units/ml). Following infusion of the purified cathepsin D preparation in seven normal rabbits, enzyme activity increased dramatically. At 15 minutes cathepsin D activity was observed to increase 467 per cent to 38.2 f 4.4 units/mg protein. (Figure 1.) Over the following 105 minutes a gradual but progressive decrease in plasma cathepsin activity occurred, probably as a result of clearance of the enzyme by the reticuloendothelial system. However, at 2 hours after infusion, plasma cathepsin D remained significantly elevated over initial levels (p <0.005) at 20.1 f 3.95 enzyme units/mg protein, which is in the range observed in shock states [13].

Table III summarizes the hemodynamic and temperature responses to the infusion of purified cathepsin D. No statistically significant change was noted in either MABP or HR during the entire 2 hour experiment. A significant decline in temperature (p CO.005) was observed at 60 minutes after infusion and persisted for the remaining experimental period. NO animal died after infusion and all animals were in good health 24 hours after infusion.

Comments

Previous studies have demonstrated multiple deleterious effects after the infusion of lysosomal preparations into experimental animals. Gazzaniga and O’Connor [18] reported the occurrence of hypotension, altered blood coagulation, granulocyto- penia, bowel mucosal hemorrhage, mesenteric in- farction, and hepatic congestion in intact animals after the infusion of autologous kidney lysosomal enzyme extracts. Glenn et al [19] observed hypotension with decreased cardiac output and superior mesenteric artery blood flow after infusion of lysosomal extracts into normal dogs. Animals subjected to major reticuloendothelial exclusion by splenectomy and portacaval shunt demonstrated profound circulatory effects which correlated with high plasma cathepsin D levels following infusion of lysosomal

280

preparations. Despite these findings, several problems are in-

herent in previous infusion studies. Foremost is that previously infused extracts contained innumerable enzymes derived from multiple subcellular sources and included mitochondria, endoplasmic reticulum, and microsomes, as welI as lysosomes. Furthermore, the preparations contained large quantities of nonenzymatic proteinaceous contaminants of cellular and intercellular origin. It is, therefore, difficult to ascribe the observed results directly to lysosomal enzymatic action or to products of their action. The present study deleted these extraneous factors so that the effects of a single lysosomal enzymatic species-cathepsin D-could be observed.

The method of Barrett [27] for the isolation and purification of cathepsin D was found to be repro- ducible and gave quantities of purified enzymes which were sufficient for physiologic study. Although Barrett [27] reported higher increases in purification (870 vs 490), our final preparations were sufficiently low in total protein content and contaminants. The major criticism of the procedure emphasized by Smith and Turk [30] is that it is time-consuming and may allow proteolysis of cathepsin D by other enzymes present in the system during isolation.

Our investigations failed to demonstrate any hemodynamic effects of purified cathepsin D infusions in normal rabbits. MABP and HR remained stable throughout the experiment. A significant depression in core body temperature was observed in all of the animals infused with cathepsin D. No other untoward effects were observed, and all animals survived the experiment.

Fredlund et al [12] emphasized that plasma lysosomal enzyme disappearance was uniphasic and exponential regardless of the dose of infusates. Our experiments confirmed this finding for cathepsin D in the intact rabbit. There was no discernible sepa- ration into distinct phases of elimination and the enzyme followed a gradual rate of disappearance along an arithmetic curve. (Figure 1.) When plotted on a semilogarithmic scale, the disappearance rate was nearly linear, (Figure 2.) If the disappearance

The Ametlcan Journal ol Surgery

Cathepsin D Infusion

0 15 30 45 60 75 90 105 120 I , I # , I I ,

O 15 30 45 60 75 90 105 120

Time (min) Time (min)

Figure 1. Plasma CathePsk 0 aCtMtit3S after a single infu- FIgwe 2. SemWogarWhmk p/at of plasma cathepsk D dis- sion of a purWied enzyme extract. The dWference between appearance afler a single InksIon of a purWied enzyme pre- and posthfuskn enzyme activities is statktkaWy sig- extract. Plot is akng a least squares Wne of best f/t. (Cor- nWkant (p X0.001). relation coeniclent = 0.98).

rate is extrapolated, the half life of cathepsin D in the circulation is approximately 90 minutes.

The reticuloendothelial system has been shown to be in large part responsible for the clearance of circulating lysosomal enzymes. Lentz and Smith [IO] demonstrated reduced levels of plasma acid phosphatase and beta-glucuronidase during hypotension in animals pretreated with reticuloendothelial stimulating agents. Zymosan, a complete cell wall extract and known stimulant of reticuloendothelial function, was found to give the smallest increases in plasma lysosomal hydrolase activity during shock. Surgically accomplished, reticuloendothelial exclusion prolongs half life of the enzyme and results in greater increases in plasma levels during infusion studies. Fredlund et al [12] documented prolonged hydrolase disappearance following portacaval shunt. In one case, reestablished hepatic circulation resulted in the redevelopment of a normal elimination rate. Glenn et al [19] subjected dogs to splenectomy, as well as portacaval shunt prior to the infusion of un- purified lysosomal preparations. This resulted in a 170 per cent increase in plasma cathepsin D levels over those of intact, infused animals and the enzyme was found to be elevated over prolonged periods. In the present series, plasma cathepsin D increases were so massive, (467 per cent) that reticuloendothelial exclusion was deemed unnecessary. Even at 2 hours following infusion, enzyme levels were markedly increased (240 per cent). Indeed, Glenn et al [19] did

not achieve greater initial increases (400 per cent) and at 2 hours levels had fallen below those in our series. Presumably, infusion of a highly purified and concentrated enzyme preparation overcomes the ability of the reticuloendothelial system to clear the enzyme normally. This is substantiated by the fact that we found the half life of cathepsin D to be 90 minutes as compared to less than 60 minutes in the series in intact animals of Glenn et al [19].

It is apparent that the administration of large amounts of cathepsin D in intact animals does not cause the cardiovascular disturbances seen in shock states. The enzyme or products of its action do not have direct vasculotoxic or vasoactive effects which are apparent in the intact animal. However, it is possible that detrimental effects of the enzyme are confined to the intracellular or intercellular level. The liberation of lysosomal enzymes has been shown to result in digestion of cell membranes and struc- tural alterations in cell peripheries [31]. The appearance of cathepsin D in shock plasma might then represent a phenomenon secondary to cellular damage. Moreover, as Bitensky, Chagen, and Cun- ningham [32] have suggested, deleterious effects might not result from direct enzymatic action but rather from the inability of lysosomes to function in detoxification reactions. The subsequent accumu- lation of toxic substances might then be the damag- ing factor. In either case, infusions of exogenous enzymes would have little effect in normal animals.

voluln. 184, Augusl lo77 201

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Mason and Wangensteen

Cathepsin D is known to have a distinctly acid pH optimum 1331. At normal blood pH the enzyme may express only minimal activity and it may well depend on the development of metabolic derangements, particularly acidosis, prior to its activation. This is an equally likely explanation for the failure of cathepsin D infusions to affect alterations in intact experimental animals. The difficulties inherent in experiments with lysosomal enzyme infusions in animals made acidotic have been emphasized [12]. Preparatory infusions of acidifying solutions have

been found to cause profound metabolic and hemodynamic changes which complicate the interpreta- tion of changes secondary to lysosomal enzyme infusions. However, further infusion studies in animals subjected to various degrees of shock may help to delineate the role of cathepsin D in the pathogenesis of circulatory shock.

9. Dumont AE, Weissmann G: Lymphatic transport of &glucuronidase during hemorrhagic shock. Nature 201: 1231, 1964.

10. Lentz PE, Smith JJ: Effect of reticuloendothelial stimulation on plasma acid hydrolase activity in hemorrhaaic shock. Proc $0~ Exp Biol &ed 124: 1243,. 1967. -

11. Sutherland NG, Bounous G, Gurd FN: Role of intestinal mucosal lysosomal enzymes in the pathogenesis of shock. J Trauma 8: 350, 1968.

12. Fredlund PE, Ockerman PA, Vang JO: Plasma activities of acid hydrolases in experimental oligemic shock in the pig. Am J Surg 124: 300. 1972.

13. Courtice FC, Adams EP, Shannon AD, Bishop DM: Acid hydrolases in the rabbit in hemorrhagic shock. 0 J Exp Physiol 59: 31, 1974.

14. Glenn TM, Lefer AM: Role of lysosomes in the pathogenesis of splanchnic ischemic shock in cats. Circ Res 27: 783, 1970.

15. Alho AV, Motsay GT, Lillehei RC: Lysosomal enzymes in endotoxin shock. JAMA 212: 869, 1970.

16. Warren HS, Ferguson WW, Wangensteen SL: Lysosomal enzyme release in canine endotoxin shock treated with methylprednisolone. Am Surg 39: 608, 1973.

17. Kerr JFR: A histochemical study of hypertrophy and ischemic injury of rat liver with special reference to changes in lysosomes. J P&hoi Bacterial 90: 4 19, 1965.

18. Gazzaniga AB, O’Conner NE: Effects of intravenous infusion of autologous kidney lysosomal enzymes in the dog. Ann Surg 172: 804, 1970.

Summary

The method of Barrett, with modifications, was used for the isolation of rabbit liver cathepsin D. Infusions of this purified preparation into intact animals resulted in marked increases in plasma cathepsin D activity but failed to reproduce the cardiovascular disturbances seen in shock states. A significant depression in core body temperature was observed in all of the animals infused with cathepsin D. However, no other untoward effects were observed. The plasma disappearance of cathepsin D was found to be uniphasic and exponential.

References

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27. Barrett AJ: Lysosomal acid proteinase of rabbit liver. Biochem J 104: 601, 1967.

28. Anson ML: The estimation of cathepsin with hemoglobin and the partial purification of cathepsin. J Gen Physiol20: 565, 1936.

29. Lowry OH, Rosebrough NT, Farr LA, Randall RJ: Protein mea- surement with the folin phenol reagent. J Biol Chem 193: 265, 1951.

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the effects of purified cathepsin d infusions in intact animals

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