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Effects of hyperthermia on plasma glycoprotein catabolism by the isolated perfused rat liver
03~-9629/s3$3.00+ O,GG 0 1983PergamonPressLtd
cbmp. Biochem Physioi. Vol. 75A. No. 3. pp. 391.--395,1983 Printed in Great Britain
EFFECTS OF ~YPERT~ER~~A ON PLASMA ~LYCUPR~TEIN CATABOLISM BY THE ISOLATED PERFUSED RAT LIVER JOSEPHL. SKIBBA, LAWRENCE P. MCKEAN
and JEFFREYL. WINKELHAKE
Departments of Surgery and Microbiology. The Medical College of Wisconsin, Milwaukee,
WI 53226, USA
Abstract-I. Chxrance and degradation of the glycoprotein, asiafofetuin (AF), by the isolated perfused rat liver at supranormal temperatures were investigated, 2. The half-life for disappearance of AF was similar at 37. 41. and 42’C. P > 0.05. 3. There was a significant difl’erencebetween the amount of hydrolysis of AF at 37, 41, and 42°C. P < 0.05. This indicates that there was significant retardation of lysosomal proteolysis or receptor cndocytosis by the hepatocyte at elevated temperatures.
INTRODUCTION Lysosome-dependent cell injury has been proposed as a causative factor leading to ceil death after hyperthermic exposure (Overgaard & Overgaard, 1972a.b; Overgaard, 1977). While hyperthermic treatment does not appear to affect endocytosis (Magun & Fennie,
1981: Magun, 1981; Weigel & Oka, 1981, 1982) heatof cells up to 42°C is associated with an increase in the number of lysosomes (Heine et nl., 1971X increased iysosomal enzyme activity in normal and malignant ceils (Barratt & Wills. 1979; Hume & Field. 1977; Hume et a!., 1978a,b; Keech & Wills. $979; Overgaard, 1976a,b; Overgaard (4i Nielsen, 1980; Overgaard & Overgaard, 1972a. 1972b; Overgaard & Paulsen, 1977) and increased membrane permeability of lysosomes (Hume et al., 1978a,b; Turano et al., 1970; VonArdenne, 1972). However, the degradation of internalized epidermai growth factor (EGF) by lysosomes was strongly inhibited in cultured Rat-2 cells at temperatures from 43 to 46°C (Magun & Fennie, 1981). Thus. the mechanism of hyperthermic cell killing is not well understood. With the exception of studies on mouse spleen (Barratt & Wills. 1979; Hume et al., 1978a,b), most investigations have focused on the effects of heat treatment on the lysosomes of cells in culture or of tumors. Very httle is known about the effects of hyperthermia on normal tissue metabolism. With the increased interest in hy~rtherrnic treatment of whole organs (such as the liver) for tumor therapy (Skibba et at., 1983; Storm et al., 1982), it is becoming important to understand the mechanisms of cell injury as a result of heat. One function of the liver is to specifically remove serum glycoproteins from the circulation when the penultimate galactose units of their carbohydrate moieties have been exposed by prior removal of terminal sialic acid residues (Ashwell & Morell, 1974: Ashwell & Harford, 1982). Asialoglycoproteins enter hepatocytes by receptor-mediated endocytosis with subsequent localization in lysosomes (LaBadie et ~1.. 197.5; Hubbard et RI., 1979). The peptide coming
ponent of the glycoprotein is rapidly hydrolyzed to free amino acids within Iysosomes (Dunn et al., 1979). To study this process, a highly glycosylated exogenous glycoprotein, fetuin (F). has served as an ideal probe for liver membrane and lysosome function. When asialofetuin (AF) is labeled with “‘1 or r311, the resulting hydrolyzed iodotyrosines are not accepted by tRNA and deiodination of this amino acid is rapid, thus reutilization of the radioactive iodine by the liver does not occur (Dunn et al., 1979). In the present study, the isolated perfused rat fiver model was used to evaluate normal hepatic functions of clearance and denudation of AF at supranormal temperatures. MATERIALS AND METHODS
Male Spra~u~~awle~ albino rats (King Rat, Oregon, WI) weighing 225-275g were fed laboratory chow ad lib and fasted 24-30 hr prior to liver perfusion.
391
Muteriuls Fetuin was purchased from Grand Island Biological Co. (Grand Island, NY). Na “‘1 and Nai3’I were obtained from New Engtand Nuclear Corp. (Boston, Mass). Fetuin was purchased from Sigma Chemical Co. (St, Louis, MO) and purified by column chromatography with Sephacryl S-200 prior to use. Na “*I and Na 13’1 were obtained from New England Nuclear Corp. (Boston, MA). Neuraminidase was from Rehring Diagnostics (Somerville, NJ) and was free of proteolytic activity. The technique for in siru perfusion of the liver in which accurate cont;oI of the liver temperature can be achieved was that described bv Collins & Skibba. 1980. The suraical procedure was carried out using ether anesthesia. Afte;administration of 250 units of heparin iv., the portal vein and thoracic segment of the inferior vena cava were cannulated and served as the inflow and outflow cannulas respectively. A liver flow rate of 12-15 mI/min was maintained with a hydrostatic pressure of I6cm. Thermocouple microprobes
JOSEPH L. SKIBBA er al.
39’
(Bailey Instrument Co., Saddle Brook, NY) were used to monitor and control the liver, cabinet, and perfusate temperatures. Perfusate and cabinet were heated to the desired temperature before initiation of the liver perfusion. The first 30 min of perfusion were allowed for temperature equilibration which was followed by a 120-min experimental period. The 150 ml recirculating perfusate consisted of whole rat blood diluted 1:Z with Krebs- Ringer bicarbonate solution, pH 7.4. The glycoproteins lZ51-F and 13’I-AF, 2O~lg each were added to the perfusate at the beginning of the 120 min experimental period. Prrpamtion
of ‘251-F and ‘3’1_AF
Fetuin was desialylated by treatment with neuraminidase for 24 hr at 37 ‘C in 50 mM acetate buffered saline, pH 5.5 with O.l”,, CaCI,. AF prepared in this manner had less than 0.5 mol NeuAc/mol AF. AF was separated from neuraminidase by column chromatography with Sephadex G-100 or Sephacryl S-200. The AF and F were radioiodinated using the iodine monochloride method as described by Winkelhake et (II., 1979 and dialyzed exhaustively against Krebs-Ringer bicarbonate. pH 7.4.
1N 9c 8C
7C 6C s E d 3 2 ? N % az
5C 4c
3c
20
10
Perfusate samples were removed at 2. 5, 10, 15. 30. 60, 90 and 120 min of perfusion. Perfusate plasma, 0.75 ml, was mixed with trichloroacetic acid to a final concentration of 109~. After centrifugation in a Beckman Microfuge, the acid soluble and acid precipi~blc fractions were counted. The “‘1 and t3t1 were measured in polystyrene test tubes (12 x 75 mm) on a Beckman Auto-Gamma Counter.
The significance of difference between means was determined by Student’s t-test or analysis of variance.
Time (minutes)
Fig. 1. Clearance of 13’I-AF from the perfusate by the liver 5 x 1O’cpm) and after a mixture of ‘311-AF (20,rg. ‘251-AF (2Oilg. 5 x 10’ cpm) was added. The first sample was taken at 2min. Uptake was measured as disappearance of acid precipitabie material expressed as the per cent of the acid precipitable material measured at 2 min. Four livers were perfused at each temperature. There was no significant difference with temperature.
RESULTS After addition of a mixture of “‘I-F, 2Opg and 1311-AF 20 hg to the perfusate, there was a similar rate of ‘disappearance of AF at 37, 41, and 42”‘, P > 0.05 (Fig. 1). The half-life for disappearance of the AF was consistent with the results of others (LaBadie et ul., 1975; Dunn et al., 1979). Figure 2 shows the rate of appearance of the TCA soluble t3’I from the i3’1-AF. There was a significant difference between the amount of hydrolysis of the i3iI-AF at 37, 41, and 42”, P < 0.05. This indicates that there was significant retardation of lysosomal proteolysis or receptor endocytosis by the hepatocyte at the elevated temperatures. The per cent of total radioactivity administered as 13i1 which remained in the liver after two hours of perfusion was calculated after counting a portion of liver. The results in Table 1 show there is a marked increase in total i3’1 retained in the liver at 41 and
42”C,
P
The clearance of iZ51-F was used as an internal control in these experiments. Data for Fig. 1 were considered good perfusions only if there was little or no clearance of “‘1-F (Fig. 3). However, at 41 and 42’C, the level of “‘1-F decreased by over 3O”j, from the initial value at 2 min. In addition, there was a significant increase in the acid soluble “‘1 in the perfusate from livers perfused at 41 and 42’C. This suggests that an alternate mechanism for clearance and proteolysis of glycoproteins may be operative at elevated temperatures. Only trace amounts of lz51 and i3iI appeared in the bile, which was collected during all perfusions.
At normothermic occurs immediately
temperature, endocytosis of AF upon binding by its receptor ,
1
...... 370 _-- 410 -42”
/
,
* S.E.M.
1 I
t
*.= 1___--I...’
*..* ....
I-
F&i--J2 ,/*
‘.’
MM
//” /’
rl
0
I
I
I
I
30
60
90
120
Time (minutes)
Fig. 2. Proteolysis of 13’I-AF was measured by appearance of acid soluble 13’1 in the perfusate expressed as per cent of the radioactivity measured in the first sample taken after 2 min. The data were obtained from the perfused livers of Fig. 1. The significance level with temperature was <0.05.
Glycoprotein catabolism in hyperthermic liver
(Dunn et al., 1980; To~leshaug et al., 1979) with accummulation in endocytic vesicles or phagosomes. Tolleshaug et ul. (1977) suggested that the pathway fromphagosome to lysasome was rate-limiting for ceIlular degradation of AF. Dunn et al. (1980) also identified fusion between pinocytic vesicles and lysosomes as the slowest metabolic event involved in the catabolism of AF by the liver. The processes involved in the metabolism of most asialoglycoproteins appear to be similar to those described for epidermal growth factor (EGF). Magun & Fennie (1981) found that heating to 45°C for 30min did not inhibit the ability of Rat-2 cells in culture to internalize bound EGF. However, hydrolysis of internalized EGF was inhibited at 43 to 46”C, but unaffected at 42°C (1981). The data presented here demonstrate that hepatocyte receptor-mediated cndocytosis of the glycoprotein, AF, was refractory to the effects of heat up to 42°C. However, while the uptake of AF by the perfused liver was unaffected by tem~ratures of 41 and 42”C, degradation of internalized AF was inhibited at these same temperatures (Fig. 2, Table 1). The accumulation of labeled AF within the liver at eievated temperature (Table 1) could have occurred on the cell surface or within the phagosomes and/or lysosomes. These findings are consistent with the findings in ceil cultures that degradation of EGF was inhibited at temperatures from 43 to 46°C. Our results are also similar to those found in the case of the heated mouse spleen where lysosomal response differed depending on whether the temperature was <42S”C or >42..5”C. Temperatures <425”C caused an increase in lysosomal enzyme activity, and > 425°C caused an increase in lysosomal membrane permeability and a decrease in enzyme activity (Hume et al., 1978a,b). Since internalization (endocytosis) is not apparently affected by heating cells to 42°C (Magun & Fennie, 198I ; Weigel & Oka, 1981, 1982), we feel this phase of catabolism can be discounted as the key site of hyperthermic damage to cells. If an increased permeability of the lysosomal membrane accounted for the inhibition of proteolysis of AF by the perfused liver, one would expect a gradual increase in the acid precipitable AF in the perfusate. This was not observed in the present study. Alternately, if the lesion occurs at the level of inhibition of lysosomal proteolytic activity, this could result from lack of sufficient ATP at hyperthermic temperatures for energy-requiring catabolism and/or the concomitant increase in lysosomal pH (Mego, 1979; Schneider, 1981). In fact, using the perfused rat liver. Skibba & Collins (1978) and Collins et al. (1980) found that a sharp decline in ATP-requiring biosyn-
!jp
.I.....
_*...410 - 42”
I30
f
S.E.M.
_J
60
”
Time (minutes)
Fig. 3. Disappearance of “‘1-F from the perfusate after addition of the sample as in Fig. 1 legend. The data were from the same perfused livers of Fig. 1 and expressed as per cent of the first sample taken at 2 min.
thetic functions occurred between 42 and 43°C suggesting a depression in hepatic ATP supply. Also consistent with the lack of sufficient ATP were the findings of Haveman (1980) in which lysosomes of murine mammary adenocarcinoma cells lost their capacity to accummulate acridine orange after hyperthermia. Here the author concluded that the ability of lysosomes to maintain a low internal pH was also destroyed by hyperthermia. An unaccounted for observation in our experiments was the occurrence of a small but significant amount of clearance and proteolysis of “‘1-F (the internal control) at 41 and 42°C (Figs 3 and 4). Initially it was thought that acceptable experiments would be those ,
/
I
I
.....*37” f S.E.M.
_-‘$lO
g E z
-
30
420 i
20 i-
Table I. Fraction of total ‘3’l-radioactivity remaining in the liver I20 min after the addition of 13’1-AF Temperature
y’, Total i 311
37 41 42
13.6 rt 1.0 50.3 + 12.1 71.2 + 12.7
Data from livers perfused in Figs 1 and 2. Significance level ~0.01, N = 4.
0
30
60
90
120
Time (minutes)
Fig. 4. Appearance of acid soluble *2sl and “‘1-F. The data were obtained from the perfused livers of Fig. 1 and expressed as in Fig. 2.
JOSEPH L. SKIBBAet ul.
3Y4
in which there was little or no disappearance of ““I-F. but this never occurred at elevated temperatures. Fetuin itself is resistant to heat coagulation in the presence of small amounts of salt (Spiro, 1960). Thus, denaturation and clearance by Kupffer cells would not explain this finding, and our further studies will probe the responsible mechanism(s). In conclusion. these initial studies demonstrate that in the perfused rat liver IysosomaI proteoiysis of the ~~si~~loglycoprotein. AF, is inhibited at 41 and 42,-C. While the process of endocytosis appears unaffected. several sites could be postulated to be the key step in this inhibition. For example, blockage may reside in the pathway of the transfer of endocytic vesicles to lysosomes, in the inhibition of fusion between the phagosomes and lysosomes, or in the lack of availability of ATP for maintenance of normal Iysosomal function. Continued use of giycoproteins such as AF as molecular probes should help resolve these possibilities and answer the question of whether the effect of heat on lysosomes is a primary one, or perhaps secondary to some other hyperthermia-induced change. Acktlo\rlrdge,,lenf,s--The authors are grateful to Bonnie Bates for her support and assistance in the preparation of thi$ manuscript.
REFERENCES ASHWI‘LI. G. & HARF~RII J. (1982) Carbohydrate-specific receptors of the liver. A. Ret:. ~i~~~~7f,~)z. 51, 53 I-554. ASHWELL G. & MOKELI. A. G. (1974) The role of surface carbohydrates in the hepatic recognition and transport of circulating glycoproteins. Adr. Enzpol. 41, 99-127. BARRAI”~ G. M. & WILLS E. D. (1979) The effect of hyp~rthernli~ and radiation on lysosomal enzyme activity of mouse mammary turnours. Eur. J. Ctmc,f,r IS. 243-250. COLLINS F. G., MITR~S F. A. & SKIBHA J. L. (1980) Effect of palmitate on hepatic biosynthetic functions at hyperthermic tcnlper~tures. ~~ef~7b~~is~t~ 29, 524 53 1. COLLIXS F. G. & SKI~BA J. L. (l9YOl Improved rn siru rat liver perfusion technique. J. Surg. RPS. 28, 65.--70. D~:N\ W. A.. HLJBBAKDA. L. & AK~NSOS JR N. N. (1980) Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes dul-ing heteropha8y of ‘*‘I-asialofetuin by the perfused rat liver. J. hiof. Cker~ 255, 5;71 5978. DLJ~N W. A.. LABADIE J. H. & ARC)NSON JR N. N. (1979) Inhibition of “‘I-asialofetuin catabolism by leupeptin in the perfused rat liver and in I+VCJ.J. hiol. C&m. 254, 4191 4196. HAVhMA\; J. (1980) The capacity of lysosomes of cultured mammalian cells to accumulate acrldine orange is destroyed by hyperthermia. Cell Tis.wc Rea. 213, 343-350. HEtx
U.. SVEKAK L., KONDRATICK J. &
BONAR R. A.
(1971) The behavior of hela S, cells under the influence of supranormal temperatures. .1. Clltrcutrtcct. Rrs. 34, 375 396. HOFEK. K. G. and MIVE~HI. N. F. (1980) Tumor cell sensitivity to hyperthermia as a function of extracellular and intracellular pH. 1. IIUI~ Catzcer inst. 65, Q-625. HtJtIt%AKt)A. L.. Wtt_so% G., ASHWELL G. & STUI(ENBROK H. (1979) An electron microscope autoradiographic
study of the carbohydrate recognition systems in rat liver. 1. Distribution of “’ I-ligands among the liver cell types. J. C
isolation and properties of a rabbit liver binding protein specific for asialogiycoproteins. J. hiol. Chrm. 249. 55365543. HUME S. P. & FIELD S. B. (1977) Acid phosphatasc activity following hyperthermia of mouse spieen and its implication in heat potentiation of x-ray damage. Ru~~j~r;~~~l Rrs. 12, 145-l 53. HLME S. P., ROIXRS M. A. &z FI~LU S. B. (1978a) Heat induced thermal resistance and its relationship to Iysosoma1 response. Inc. J. Rtdkr. Bid. 6, 503 5I 1. HLJME S. P., ROGERS M. A. & Fr~+_o S. 8. (1978b) TWO qualitatively different effects of hyperthermia on acid phosphatase staining in mouse spleen. dependent on the severity of the treatment. Int. J. Ruci~ut. Bid. 34, 40 l-409. KEE~H M. I,. & WILLS E. D. (19793 The effect of hyperthermia on activation of lysosomal enzymes in hela cells. Eur. J. Cllncrr 5, 1025 -1031. LABAIXF J. H.. C~IAPMAN K. P. & AKONSOK JR N. N. (1975) Glycoprotein catabolism in rat liver. Lysosomal diges-
tion of iodinated asialo-fetuin. ~i~~~l~~t?i. f. 152, 271-279. MAG~JN 9. E. (1981) Inhibition
and recovery of macromolecular synthesis. membrane transport, and lysosomal function following exposure of cultured cells to hyperthermia. Radicif. Rrs. 87. 657- 669. h’tAc;UN9. E, & FESNICC. W. (19X1) Effects of hyperthermia on binding. internalization and degradation of eipidermal growth factor. Ru(li(lr. Rrs. 86, 133 146. MEC~O J. L. (1979) The ATP-dependent proton pump in lysosome membranes. FEBS LLJIT.107, 113-l 16. OVFRGAARD J. (1976) InRuence of cxtracelltliar pH on the viability and morphology of tumor cells exposed to hyperthermia. J. nuf. Cuncer Inst. 56, 1243~125~~. OVERGAARI~J. (1976) Ultrastructure of a murine mammarv carcinoma exposed to hyperthermia i/i c,iro. Crilzrer Rex 36,9X3-987. OVERC;AARDJ. (1977) Effect of hyperthermia on malignant cells in riro. A review and a hypothesis. Crrrlc,er 39, 2637-2646. OVER~AAKD J. & NIELSEN 0. S. (1980) The role of tissue environmental factors on the kinetics and morpholo8~ of tumor cells exposed to hyperthermia. Ann. .V.Y. A<&. Sci. 335, 254278. OVFRGAARI> J. & POULS~N H. S. (1977) Effect of hypcrthcrmia and environmental acidity on the proteolytic activity in murine ascites tumor cells. J. 170I. Cartccr It7st. 58, 1159-1161. OVERGAARD K. & OVERC~ARD J. (1972a) Investigations on the possibility of a thermic tumour therapy I. Eur. J. Cancer 8, 65-78. OV~RC;AARI~K. & OVERGAARD J. (1972b) Investi~atiolls on the possibility of a thermic tumour therapy II. Eur. J. Cancer 8. 573-575. ScHNEIDCR D. L. (1981) ATP-dependent acidification 01 intact and disrupted lysosomes. Evidence for an ATPdriven proton pump. J. hioi. Clzm~ 256, 385% 3864. SKIHBA J. L., ALMAGRO U. A., CONI)ON R. E. & PETROFF JR R. J. (1983) A technique for isolation-perfusion of the canine liver with survival. J. Surq. Rrs. 34, 123-132. SKIBBA J. L. & COLLINS F. G. (1978) Effect of temperature on biochemical functions in the isolated perfused rat liver. J. Surg. Rex 24, 435- 441. Srr~o R. G. (1960) Studies on fetum, a glycoprotein of fetal serum. I. Isolation, chemical composition. and physicochemical properties. J. hio/. Chm. 235, 2860.2869. STORM F. K., KAISER L. R.. GOODNIGHTJ. E., HARRISOKW. H.. ELLIOTT R. S., GON~ESA. S. & MORT~K D. 1~. (1982) Thermochemotherapy for melanoma metastases in liver. Cancer 49, 1243- 1248. TOLLESHAUG T.. BEKC; T.. FIIOLIVH W. & NORLIM K. R. (1979) Intracellular localzation and degradation of asialoferuin in isolated rat hepatocytes. E~~~~bi~?~. hiqd7y.s. ilcrtr 585. 7 l-84.
Glycoprotein
catabolism
T~LLESHAUG H., BERG T., NILSSON M. & NORUM K. R. (1977) Uptake and degradation of ‘Z51-labeIIed asialofetuin by isolated hepatocytes. Biochim. hiophys. Acta 499, 73-84. TLIRANO C.. FERRARO A., STROM R., CAVALIERE R. & FANELLI A. R. (1970) The biochemical mechanism of selective heat sensitivity of cancer cells. III. Studies on lysosomes. Eur. J. Cancer 6, 67-72. VON ARDENNE M. (1972) Selective multiphase cancer therL~py: Conceptual aspects and experimental basis. Adu. Pharm. Chrmorkrr. 10, 33X-380. WEICZL P. H. & OKA J. A. (1982) Endocytosis and degra-
in hyperthermic
liver
395
dation mediated by the asialoglycoprotein receptor in isolated rat hepatocytes. J. biol. Chem. 257, 1201-1207. WEIGEL P. H. & OKA J. A. (1981) Temperature dependence of endocytosis mediated by the asialoglycoprotein receptor in isolated rat hepatocytes. J. hiol. Chem. 256, 2615-2617. WINKELHAKE J. L.. ELCOMBE B. M. & HODACH A. (1979) Immunological block to synthetic a-melanocyte-stimulating hormone: Melanocyte interaction by antibodies isolated from cell-column Immonoadsorbents. Cuw~r Rcs. 39. 3058 3064.
cbmp. Biochem Physioi. Vol. 75A. No. 3. pp. 391.--395,1983 Printed in Great Britain
EFFECTS OF ~YPERT~ER~~A ON PLASMA ~LYCUPR~TEIN CATABOLISM BY THE ISOLATED PERFUSED RAT LIVER JOSEPHL. SKIBBA, LAWRENCE P. MCKEAN
and JEFFREYL. WINKELHAKE
Departments of Surgery and Microbiology. The Medical College of Wisconsin, Milwaukee,
WI 53226, USA
Abstract-I. Chxrance and degradation of the glycoprotein, asiafofetuin (AF), by the isolated perfused rat liver at supranormal temperatures were investigated, 2. The half-life for disappearance of AF was similar at 37. 41. and 42’C. P > 0.05. 3. There was a significant difl’erencebetween the amount of hydrolysis of AF at 37, 41, and 42°C. P < 0.05. This indicates that there was significant retardation of lysosomal proteolysis or receptor cndocytosis by the hepatocyte at elevated temperatures.
INTRODUCTION Lysosome-dependent cell injury has been proposed as a causative factor leading to ceil death after hyperthermic exposure (Overgaard & Overgaard, 1972a.b; Overgaard, 1977). While hyperthermic treatment does not appear to affect endocytosis (Magun & Fennie,
1981: Magun, 1981; Weigel & Oka, 1981, 1982) heatof cells up to 42°C is associated with an increase in the number of lysosomes (Heine et nl., 1971X increased iysosomal enzyme activity in normal and malignant ceils (Barratt & Wills. 1979; Hume & Field. 1977; Hume et a!., 1978a,b; Keech & Wills. $979; Overgaard, 1976a,b; Overgaard (4i Nielsen, 1980; Overgaard & Overgaard, 1972a. 1972b; Overgaard & Paulsen, 1977) and increased membrane permeability of lysosomes (Hume et al., 1978a,b; Turano et al., 1970; VonArdenne, 1972). However, the degradation of internalized epidermai growth factor (EGF) by lysosomes was strongly inhibited in cultured Rat-2 cells at temperatures from 43 to 46°C (Magun & Fennie, 1981). Thus. the mechanism of hyperthermic cell killing is not well understood. With the exception of studies on mouse spleen (Barratt & Wills. 1979; Hume et al., 1978a,b), most investigations have focused on the effects of heat treatment on the lysosomes of cells in culture or of tumors. Very httle is known about the effects of hyperthermia on normal tissue metabolism. With the increased interest in hy~rtherrnic treatment of whole organs (such as the liver) for tumor therapy (Skibba et at., 1983; Storm et al., 1982), it is becoming important to understand the mechanisms of cell injury as a result of heat. One function of the liver is to specifically remove serum glycoproteins from the circulation when the penultimate galactose units of their carbohydrate moieties have been exposed by prior removal of terminal sialic acid residues (Ashwell & Morell, 1974: Ashwell & Harford, 1982). Asialoglycoproteins enter hepatocytes by receptor-mediated endocytosis with subsequent localization in lysosomes (LaBadie et ~1.. 197.5; Hubbard et RI., 1979). The peptide coming
ponent of the glycoprotein is rapidly hydrolyzed to free amino acids within Iysosomes (Dunn et al., 1979). To study this process, a highly glycosylated exogenous glycoprotein, fetuin (F). has served as an ideal probe for liver membrane and lysosome function. When asialofetuin (AF) is labeled with “‘1 or r311, the resulting hydrolyzed iodotyrosines are not accepted by tRNA and deiodination of this amino acid is rapid, thus reutilization of the radioactive iodine by the liver does not occur (Dunn et al., 1979). In the present study, the isolated perfused rat fiver model was used to evaluate normal hepatic functions of clearance and denudation of AF at supranormal temperatures. MATERIALS AND METHODS
Male Spra~u~~awle~ albino rats (King Rat, Oregon, WI) weighing 225-275g were fed laboratory chow ad lib and fasted 24-30 hr prior to liver perfusion.
391
Muteriuls Fetuin was purchased from Grand Island Biological Co. (Grand Island, NY). Na “‘1 and Nai3’I were obtained from New Engtand Nuclear Corp. (Boston, Mass). Fetuin was purchased from Sigma Chemical Co. (St, Louis, MO) and purified by column chromatography with Sephacryl S-200 prior to use. Na “*I and Na 13’1 were obtained from New England Nuclear Corp. (Boston, MA). Neuraminidase was from Rehring Diagnostics (Somerville, NJ) and was free of proteolytic activity. The technique for in siru perfusion of the liver in which accurate cont;oI of the liver temperature can be achieved was that described bv Collins & Skibba. 1980. The suraical procedure was carried out using ether anesthesia. Afte;administration of 250 units of heparin iv., the portal vein and thoracic segment of the inferior vena cava were cannulated and served as the inflow and outflow cannulas respectively. A liver flow rate of 12-15 mI/min was maintained with a hydrostatic pressure of I6cm. Thermocouple microprobes
JOSEPH L. SKIBBA er al.
39’
(Bailey Instrument Co., Saddle Brook, NY) were used to monitor and control the liver, cabinet, and perfusate temperatures. Perfusate and cabinet were heated to the desired temperature before initiation of the liver perfusion. The first 30 min of perfusion were allowed for temperature equilibration which was followed by a 120-min experimental period. The 150 ml recirculating perfusate consisted of whole rat blood diluted 1:Z with Krebs- Ringer bicarbonate solution, pH 7.4. The glycoproteins lZ51-F and 13’I-AF, 2O~lg each were added to the perfusate at the beginning of the 120 min experimental period. Prrpamtion
of ‘251-F and ‘3’1_AF
Fetuin was desialylated by treatment with neuraminidase for 24 hr at 37 ‘C in 50 mM acetate buffered saline, pH 5.5 with O.l”,, CaCI,. AF prepared in this manner had less than 0.5 mol NeuAc/mol AF. AF was separated from neuraminidase by column chromatography with Sephadex G-100 or Sephacryl S-200. The AF and F were radioiodinated using the iodine monochloride method as described by Winkelhake et (II., 1979 and dialyzed exhaustively against Krebs-Ringer bicarbonate. pH 7.4.
1N 9c 8C
7C 6C s E d 3 2 ? N % az
5C 4c
3c
20
10
Perfusate samples were removed at 2. 5, 10, 15. 30. 60, 90 and 120 min of perfusion. Perfusate plasma, 0.75 ml, was mixed with trichloroacetic acid to a final concentration of 109~. After centrifugation in a Beckman Microfuge, the acid soluble and acid precipi~blc fractions were counted. The “‘1 and t3t1 were measured in polystyrene test tubes (12 x 75 mm) on a Beckman Auto-Gamma Counter.
The significance of difference between means was determined by Student’s t-test or analysis of variance.
Time (minutes)
Fig. 1. Clearance of 13’I-AF from the perfusate by the liver 5 x 1O’cpm) and after a mixture of ‘311-AF (20,rg. ‘251-AF (2Oilg. 5 x 10’ cpm) was added. The first sample was taken at 2min. Uptake was measured as disappearance of acid precipitabie material expressed as the per cent of the acid precipitable material measured at 2 min. Four livers were perfused at each temperature. There was no significant difference with temperature.
RESULTS After addition of a mixture of “‘I-F, 2Opg and 1311-AF 20 hg to the perfusate, there was a similar rate of ‘disappearance of AF at 37, 41, and 42”‘, P > 0.05 (Fig. 1). The half-life for disappearance of the AF was consistent with the results of others (LaBadie et ul., 1975; Dunn et al., 1979). Figure 2 shows the rate of appearance of the TCA soluble t3’I from the i3’1-AF. There was a significant difference between the amount of hydrolysis of the i3iI-AF at 37, 41, and 42”, P < 0.05. This indicates that there was significant retardation of lysosomal proteolysis or receptor endocytosis by the hepatocyte at the elevated temperatures. The per cent of total radioactivity administered as 13i1 which remained in the liver after two hours of perfusion was calculated after counting a portion of liver. The results in Table 1 show there is a marked increase in total i3’1 retained in the liver at 41 and
42”C,
P
The clearance of iZ51-F was used as an internal control in these experiments. Data for Fig. 1 were considered good perfusions only if there was little or no clearance of “‘1-F (Fig. 3). However, at 41 and 42’C, the level of “‘1-F decreased by over 3O”j, from the initial value at 2 min. In addition, there was a significant increase in the acid soluble “‘1 in the perfusate from livers perfused at 41 and 42’C. This suggests that an alternate mechanism for clearance and proteolysis of glycoproteins may be operative at elevated temperatures. Only trace amounts of lz51 and i3iI appeared in the bile, which was collected during all perfusions.
At normothermic occurs immediately
temperature, endocytosis of AF upon binding by its receptor ,
1
...... 370 _-- 410 -42”
/
,
* S.E.M.
1 I
t
*.= 1___--I...’
*..* ....
I-
F&i--J2 ,/*
‘.’
MM
//” /’
rl
0
I
I
I
I
30
60
90
120
Time (minutes)
Fig. 2. Proteolysis of 13’I-AF was measured by appearance of acid soluble 13’1 in the perfusate expressed as per cent of the radioactivity measured in the first sample taken after 2 min. The data were obtained from the perfused livers of Fig. 1. The significance level with temperature was <0.05.
Glycoprotein catabolism in hyperthermic liver
(Dunn et al., 1980; To~leshaug et al., 1979) with accummulation in endocytic vesicles or phagosomes. Tolleshaug et ul. (1977) suggested that the pathway fromphagosome to lysasome was rate-limiting for ceIlular degradation of AF. Dunn et al. (1980) also identified fusion between pinocytic vesicles and lysosomes as the slowest metabolic event involved in the catabolism of AF by the liver. The processes involved in the metabolism of most asialoglycoproteins appear to be similar to those described for epidermal growth factor (EGF). Magun & Fennie (1981) found that heating to 45°C for 30min did not inhibit the ability of Rat-2 cells in culture to internalize bound EGF. However, hydrolysis of internalized EGF was inhibited at 43 to 46”C, but unaffected at 42°C (1981). The data presented here demonstrate that hepatocyte receptor-mediated cndocytosis of the glycoprotein, AF, was refractory to the effects of heat up to 42°C. However, while the uptake of AF by the perfused liver was unaffected by tem~ratures of 41 and 42”C, degradation of internalized AF was inhibited at these same temperatures (Fig. 2, Table 1). The accumulation of labeled AF within the liver at eievated temperature (Table 1) could have occurred on the cell surface or within the phagosomes and/or lysosomes. These findings are consistent with the findings in ceil cultures that degradation of EGF was inhibited at temperatures from 43 to 46°C. Our results are also similar to those found in the case of the heated mouse spleen where lysosomal response differed depending on whether the temperature was <42S”C or >42..5”C. Temperatures <425”C caused an increase in lysosomal enzyme activity, and > 425°C caused an increase in lysosomal membrane permeability and a decrease in enzyme activity (Hume et al., 1978a,b). Since internalization (endocytosis) is not apparently affected by heating cells to 42°C (Magun & Fennie, 198I ; Weigel & Oka, 1981, 1982), we feel this phase of catabolism can be discounted as the key site of hyperthermic damage to cells. If an increased permeability of the lysosomal membrane accounted for the inhibition of proteolysis of AF by the perfused liver, one would expect a gradual increase in the acid precipitable AF in the perfusate. This was not observed in the present study. Alternately, if the lesion occurs at the level of inhibition of lysosomal proteolytic activity, this could result from lack of sufficient ATP at hyperthermic temperatures for energy-requiring catabolism and/or the concomitant increase in lysosomal pH (Mego, 1979; Schneider, 1981). In fact, using the perfused rat liver. Skibba & Collins (1978) and Collins et al. (1980) found that a sharp decline in ATP-requiring biosyn-
!jp
.I.....
_*...410 - 42”
I30
f
S.E.M.
_J
60
”
Time (minutes)
Fig. 3. Disappearance of “‘1-F from the perfusate after addition of the sample as in Fig. 1 legend. The data were from the same perfused livers of Fig. 1 and expressed as per cent of the first sample taken at 2 min.
thetic functions occurred between 42 and 43°C suggesting a depression in hepatic ATP supply. Also consistent with the lack of sufficient ATP were the findings of Haveman (1980) in which lysosomes of murine mammary adenocarcinoma cells lost their capacity to accummulate acridine orange after hyperthermia. Here the author concluded that the ability of lysosomes to maintain a low internal pH was also destroyed by hyperthermia. An unaccounted for observation in our experiments was the occurrence of a small but significant amount of clearance and proteolysis of “‘1-F (the internal control) at 41 and 42°C (Figs 3 and 4). Initially it was thought that acceptable experiments would be those ,
/
I
I
.....*37” f S.E.M.
_-‘$lO
g E z
-
30
420 i
20 i-
Table I. Fraction of total ‘3’l-radioactivity remaining in the liver I20 min after the addition of 13’1-AF Temperature
y’, Total i 311
37 41 42
13.6 rt 1.0 50.3 + 12.1 71.2 + 12.7
Data from livers perfused in Figs 1 and 2. Significance level ~0.01, N = 4.
0
30
60
90
120
Time (minutes)
Fig. 4. Appearance of acid soluble *2sl and “‘1-F. The data were obtained from the perfused livers of Fig. 1 and expressed as in Fig. 2.
JOSEPH L. SKIBBAet ul.
3Y4
in which there was little or no disappearance of ““I-F. but this never occurred at elevated temperatures. Fetuin itself is resistant to heat coagulation in the presence of small amounts of salt (Spiro, 1960). Thus, denaturation and clearance by Kupffer cells would not explain this finding, and our further studies will probe the responsible mechanism(s). In conclusion. these initial studies demonstrate that in the perfused rat liver IysosomaI proteoiysis of the ~~si~~loglycoprotein. AF, is inhibited at 41 and 42,-C. While the process of endocytosis appears unaffected. several sites could be postulated to be the key step in this inhibition. For example, blockage may reside in the pathway of the transfer of endocytic vesicles to lysosomes, in the inhibition of fusion between the phagosomes and lysosomes, or in the lack of availability of ATP for maintenance of normal Iysosomal function. Continued use of giycoproteins such as AF as molecular probes should help resolve these possibilities and answer the question of whether the effect of heat on lysosomes is a primary one, or perhaps secondary to some other hyperthermia-induced change. Acktlo\rlrdge,,lenf,s--The authors are grateful to Bonnie Bates for her support and assistance in the preparation of thi$ manuscript.
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catabolism
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