317

and Cellular Endocrinology, 13 (1979) 317-332 o Elsevier/North-Holland Scientific Pubishers, Ltd.

Molecular

SOME BIOCHEMICAL CHARACTERISTICS PITUITARY GLAND *

OF HORMONE-SECRETING

TUMORS AND OF THE HOST’S ANTERIOR

PITUITARY

Ivan NAGY ** and Robert M. MACLEOD Division of Endocrinology, Department of Medicine, cine, Charlottesville, VA 22908 (U.S.A.) Received

7 November

1978; accepted

University of Virginia, School of Medi-

29 December

1978

The activity of some glycolytic, oxidative, and degradative enzymes was studied in transplanted rat hormone-secreting pituitary tumors MtTW15 and 7315a and in the host pituitary gland. The elevated serum-hormone concentrations produced by 7315a tumor decreased the size of the host’s pituitary gland, its hormone content, and G6P-DH, LDH, PK, and ICDH, but produced no changes in MDH, acid phosphatase, cathepsin-D, and LYSAR enzyme activities (mU/mg tissue). LDH and PK activities were greater in unit weight of pituitary tumors than in pituitary glands. Although more G6P-DH was found in MtTWlS tumor than in normal pituitary tissue, less of the enzyme was detected in 7315a pituitary tumor. It is concluded that elevated serum pituitary hormones selectively decrease hormone production and the activity of some enzymes in the pituitary gland, presumably through a feedback mechanism. Keywords:

pituitary;

pituitary

tumor;

prolactin;

enzymes;

metabolism.

High levels of circulating hormones secreted by implanted pituitary tumors decrease the host’s anterior pituitary weight and suppress polypeptide hormone synthesis and release (MacLeod et al., 1966; Chen et al., 1967; MacLeod and Abad, 1968; MacLeod et al., 1969). In earlier reports we documented anterior pituitary RNA metabolism decrease and the alteration of carbonic anhydrase in the rat pituitary gland (Kimura and MacLeod, 1975a,b). Pituitary polyribosomes from rats bearing hormone-secreting pituitary tumors show less efficient protein synthesis in vitro than those from normal rats (Reagen et al., 1974). The present study was designed to determine whether elevated serum-hormone levels originating from transplanted pituitary tumors alter the activity of some gly-

* This study Institute. ** On leave (Hungary).

was supported from

by USPHS Research

the Central

Laboratory

Grant

of the

CA-O7535-15

Paul

Helm

from the National

Pediatric

Hospital,

Cancer

Budapest

318

1. Nagy, R.M. MacLeod

colytic and citric acid cycle enzymes in the host’s pituitary gland. Close associations between hormonal function and enzyme activity have been presented, but no experimental evidence exists comparing the glycolytic and oxidative metabolic activity present in hormone-secreting pituitary tumors with those in the anterior pituitary gland (Anda et al., 19’76a,b; Field et al., 1960, Hoch-Ligeti et al., 1967; Krass et al., 1971; Luine et al., 1974, 1975;Nagy et al., 1978a,b,c). Morphological studies of the anterior pituitary gland demonstrate clearly the role of lysosomes in intracellular polypeptide translocation (Farquhar, 1971; Farquhar et al., 1975, Smith and Farquhar, 1966). It has been assumed that lysosomal and cytoplasmatic peptidases and proteinases have a role in degrading endogenous pituitary hormones and in regulating pituitary amino acid homeostasis (Adams and Smith, 19.51; Ellis and Nuenkc, 1967; Farquhar, 1971; LaBella and Brown, 1959; McDonald et al., 1968; Farquhar et al., 1975; Meyer and Clifton, 1956; Pickup and Hope, 1971; Smith and Farquhar, 1966; Vanha-Pertula, 1969). In the present study we also employed a lysosomal marker enzyme and determined peptidase activity in an attempt to determine differences in biochemical characteristics of normal pituitary tissue and tumors.

MATERIAL AND METHODS Preparation of tissue homogenates. We inoculated mature female Buffalo rats with pituitary tumor 73 15a and Wistar-Furth females with MtTW 15 tumor as previously described (MacIeod et al., 1969). Tumor-bearing animals and corresponding controls were kept under normal laboratory conditions. When the tumors reached approximately 2 X 4 cm (at about 8-9 weeks), the rats were decapitated and blood collected for prolactin radioimmunoassay. The anterior pituitary gland and the tumor were removed and weighed. Using a glass homogenator fitted with a glass pestle, we homogenized the tissue using ice-cold 0.5 M t~etanolam~ne buffer, pH 7.5, 12 mglml. The homogenate was twice sonicated at 30-40 W for 15 sec. Dehydrogenase and protein determinations were performed on whole, sonicated tissue homogenates and on supernatant fractions from sonicated homogenates centrifuged at 10 800 X g for 20 min. For phosphatase, peptidase, and proteinase determinations, we homogenized pooled tissue in 0.1% TRITON X-100 containing buffer, and analyzed the 10 800 X g supemat~t of those samples. These latter analyses were also performed on the pituitary glands of normal mature female and male Sprague-Dawley rats. One group of these animals received a single subcutaneous injection of 100 pg polyestradiol phosphate (Estradurin, Ayerst Laboratory Inc., New York, U.S.A.) and sacrificed 72 h later. B~~ch~rni~alanalyses. We used kinetic measurerllents at 25’C based on the NAD NADH and NADP/NADPH transformations (Bergmeyer and Gawehn, 1974) to determine the activity of the following dehydrogenases: D-glucose-6-phosphate:

Hormone-secreting pituitary tumors

319

NADP oxidoreductase, EC 1 .l .I .49 (G6P-DH); L-1actate:NAD oxidoreductase, EC 1 .l .1.27 (LDH); ATP:pyruvate 20phosphotransferase, EC 2.7.1.40 (PK); threoD,-isocitrate:NADP oxidoreductase, EC 1 .I .42 (ICDH); and L-malate:NAD oxidoreductase, EC 1 .l .1.37 (MDH). We determined orthophoshoric monoester phosphohydrolase, EC 3.1.3.2 (acid phosphatase) by two-point method (Bergmeyer and Gawehn, 1974), using 4-nitrophenylphosphate with and without tartrate inhibition as substrate after 30 min incubation at 25°C. After 4 h incubation at 37°C we measured cathepsin-D (EC 3.4.23.5) hemoglobin-splitting activity (Pisarev and Altschuler, 1973). Kinetic measurements of L-leucyl-arylamidase EC 3.4.11.2 (LEUAR) and of L-lysine-arylamidase EC 3.4.11.2 (LYSAR) were performed at 2S°C, using L,-leucine-p-nitroanilide and L-lysine-p-nitroanihde as substrate (Ellis and Perry, 1968). The 7315a tumor tissue was soft and easy to homogenize; biochemical analyses could be performed without difficulty in the crude tissue homogenates (0.3-3.0 mg tissue/ml buffer; 40-200 ~1 sample in 1.0-l .2 ml assay volume). MtTWlS tumors had more connective tissue, and the completion of its disintegration was uncertain. Therefore analyses were performed on the 10 800 Xg supernatant. In one experiment we compared enzyme activities in centrifuged and noncentrifuged preparations. Our observations revealed negligible loss in G6P-DH, LDH, PK, ICDH, and MDH activity after centrifugation. Therefore data on those substances from the two types of extracts are comparable. However, we did observe 28.9% loss of total acid phosphatase activity and 16.5% loss of protein concentration in the centrifuged preparation. Rat-serum prolactin was measured by double antibody radioimmunoassay using materials and protocols supplied by the NIAMDD Rat Pituitary Hormone distribution program, Bethesda, MD. The results are presented in terms of NIAMDD rat prolactin RP-3. Coomassie Brillian Blue G-250 was used for protein determinations (Bradford, 1976). We received some biochemical and chemical compounds as gifts from Boehringer Mannheim Company (Mannheim, G.F.R.) and purchased the remainder from Sigma Chemical Company (St. Louis, U.S.A.). All results are expressed as mean f SEM, with statistical analysis by analysis of variance.

RESULTS Transplantation of these pituitary tumors into specific hosts caused remarkable changes in serum-hormone concentrations and the size of the pituitary gland. The data in Table 1 show that pituitary tumors 73 15a and MtTW15 produce extremely high serum-prolactin concentrations. As previously reported, tumor-bearing rats had significantly smaller pituitary glands than did control animals (Chen et al., 1967; MacLeod et al., 1966; MacLeod and Abad, 1968; MacLeod et al., 1969). The ante-

320

I. Nagy, R.M. MacLeod

Table 1 Body, tumor, and anterior pituitary weight and serum prolactin concentration (Mean f SEM) Groups

Number of animals

9. Sprague-Dawley control female 10. Sprague-Dawley control male 11. Sprague-Dawley estradiol male

Tumor weight (9)

Anterior pituitary weight (mg)

256 + 4

1. Buffalo control female 2. Buffalo 7315a female 3. Buffalo control female 4. Buffalo 7315a female 5. Wistar-Furth control female 6. Wistar-Furth MtTWl5 female 7. Wistar-Furth control female 8. Wistar-Furth MtlWlS female

Body weight (g)

297 * 17

10.86 + 0.58 67.9

t 3.9

228 2 8 253 + 4

5.78 f 0.15 a)

of the animals

Serum prolactin (ng/ml)

123 +

49

10 368 f 2540 a)

9.78 +z0.36 28.6

f 3.1

6.96 k 0.58 a)

7

216 + 6

I

354 k 14

8

207 + 9

12

306 f 11

30

223 f

2

8.02 + 0.28

30

239 f

2

6.23 + 0.23

30

230 + 2

12.93 c 0.77 16.3

5 2.7

10.22 ? 0.21 a) 11.05 f 0.39

5.19 + 1.2

8.23 k 0.26 a)

33k

28

13 988 + 2659 a) 31k

29

6 523 f 1368 a)

8.49 f 0.17 a)

a) P < 0.01 versus corresponding control. Each treatment group comprised 6-30 rats. Replicate analyses were performed on 3-10 subgroups each containing pooled pituitary glands from l-3 rats.

rior pituitary bearing

gland protein content and concentration were significantly less in rats the 73 1.5a tumors than in rats without the tumors (Table 2). MtTW 15 hosts

had the same pituitary gland protein concentration as control animals, but significantly lower total gland protein content. The activities of several enzymes in pituitary gland and tumor preparations were measured. G6P-DH activity in control Buffalo anterior pituitary glands was more than double that in Wistar-Furth controls (Fig. 1). G6P-DH activity decreased approximately 70% in the pituitary glands of rats bearing the 73 15a tumor and 50% in the pituitary glands of rats bearing the MtTW15 tumor. We found consistently that Wistar-Furth control pituitary glands contain significantly less G6P-DH activity (mU/mg tissue) than MtTW15 tumors, whereas Buffalo control pituitary glands con-

Hormone-secreting pituitary tumors Table 2 Protein in tumors

and anterior

Groups

1. Buffalo control female 2. Buffalo 7315a female 3. Buffalo control female 4. Buffalo 7315a female 5. Wistar-Furth control female 6. Wistar-Furth MtTWlS female 7. Wistar-Furth control female 8. Wistar-Furth MtTW15 female 9. Sprague-Dawley control female 10. Sprague-Dawley control male 11. Sprague-Dawley estradiol male

pituitary

Treatment of the homogenate

321

(Mean + SEM) Tumor &mg

pg/mg

sonicated sonicated Triton centrifuged Triton centrifuged sonicated centrifuged sonicated centrifuged Triton centrifuged Triton centrifuged Triton centrifuged Triton centrifuged Triton centrifuged

Anterior

72.9 f 4.9

75.7 f 4.3

100.5

pituitary

tissue

f 4.4

tissue

&gland

114.7 * 3.9

1248.1

77.6 5 6.8 b) 118.6

f 0.7

107.6

t 1.5 b)

478.5

f 82.7 c 51.5 b)

__

83.5 c 5.2

1069.7

79.3 + 6.7

810.3

i 68.8 + 68.9 a)

105.7 + 2.3 93.2 k 3.4

100.2

i- 2.4

125.9

f 1.3

127.0 -c 1.4 130.8

a) P < 0.05, b) P < 0.01 versus corresponding controls. Each treatment group comprised 6-30 rats. Replicate analyses groups each containing pooled pituitary glands from l-3 rats.

f 1.8

were performed

-

on 3-10

sub-

tamed more activity than did the 73 15a tumors. Table 3 compares changes in G6P-DH, LDH, and PK activity (mU/mg protein and total gland activity) in the pituitary glands and pituitary tumors. The data once again show significantly decreased G6P-DH activity in the pituitary glands of tumor-bearing rats. LDH concentration and total activity in MtTW15 host pituitary glands showed significant decrease compared with the glands of non-tumor animals. As one would expect, LDH concentration was greater (Fig. 2) in the pituitary tumor itself than in the pituitary gland. We observed no change in LDH activity (mU/mg tissue) in 7315a host pituitary glands, but total gland LDH activity was greatly decreased by the hormones secreted by the tumor. The PK-activity (mU/mg tissue) was not significantly changed in the pituitary gland of the host (Fig. 3).

I. Nagy, R.M. MacLeod

322

‘w T

mu/w

1

x-

-xx-

r

-

-

iP GROUPS:

1

BUFfAL

CONTROL FEMALE

AP

AP 2 731% FEMALE

,5

1

WIST+R-FURTH

CONTROL FEMALE

Fig 1. Glucosed-phosphate dehydrogenase activity anterior pituitary gland; TU, pituitary tumor.

(mU/mg

TU 6

MtTW IS FEMALE

wet tissue) in pituitary

tissues. AP,

The elevated serum hormones caused an increase in PK activity (mU/mg protein) in MtTWlS host pituitary glands, while the host’s total gland PK was unchanged (Table 3). PK activity in the MtTWlS and 7315a tumors was almost identical, and considerably higher than normal pituitary gland activity. Table 4 summarizes the data on ICDH and MDH activity. ICDH and MDH activity (mU/mg tissue) in the pituitary glands was the same in both control groups. ICDH activity was decreased in all host pituitary glands, but the suppression was greater in the pituitary ‘glands of animals bearing MtTWlS tumors than in 7315a hosts. The same holds for total gland MDH activity. However host-gland MDH activity (mU/mg tissue) was not affected by the tumor hormones. Table 5 gives data on pituitary total acid phosphatase and tartrate-inhibited acid phosphatase activity. Hormones secreted by the tumors had no observable effect on pituitary gland phosphatase activity. Acid phosphatase activity was unaffected in the pituitary glands and MtTW15 tumor, but was significantly decreased in 7315a tumor. Estradiol was without effect on phosphatase activity.

33.9 * 9.0

88.8 r 6.0

56.9 f 3.0

31.4 f 3.8

90.3 * 4.0

Gland

Tumor

Gland

Gland

Tumor

IAP-2AP 2TU-2AP 5AP-6AP 5AP-6TU 6AP-6TU

84.4 c 8.4

Gland

I. Non-tumor control (B) 2.7315a tumor-bearing (B) 2.7315a tumor-bearing (B) 5. Non-tumor control (W/F) 6. MtTWl5 tumor-bearing CWIF) 6. MtTWlS tumor-bearing (W/F)