0013-7227/79/1041-0226$02.00 Endocrinology Copyright © 1979 by The Endocrine Society

Vol. 104, No. 1

Printed in U.SA.

Studies on Rat Pituitary Homografts. I. In Vitro Biosynthesis and Release of Growth Hormone and Prolactin A. ZANINI, G. GIANNATTASIO, P. DE CAMILLI, A. E. PANERAI, E. E. MULLER, AND J. MELDOLESI C.N.R. Center of Cytopharmacology and Department of Pharmacology, University of Milan, 20129 Milan, and (E.E.M.) Institute of Pharmacology and Pharmacognosy, University of Cagliari, 09100 Cagliari, Italy

proximately 87, 91, and 93%, respectively. These results might be due primarily to a decrease in the number of somatotrophs and/or in their secretory activity, with relatively minor changes in GH intracellular transport, and turnover. In contrast, a clearcut fall in in vitro turnover was detected for PRL, as shown by the fact that decreases in biosynthesis and release per mg tissue protein of this hormone (approximately -95% and -99%, respectively) by far exceed the decrease in the tissue concentration (-74%). These data indicate that the in vitro secretory activity of mammotrophs is greatly reduced in the grafts with respect to the normotopic glands. Thus, the high secretory activity previously reported in hypophysectomized rats bearing pituitary grafts should be attributed to the lack of the inhibitory control of the central nervous system rather than to an increase in secretory capacity under nonrestrained conditions. (Endocrinology 104: 226, 1979)

ABSTRACT. Homologous anterior pituitaries grafted under the kidney capsule in hypophysectomized rats were studied 30 days after transplantation. Some cells maintained the ultrastructural features peculiar to the various cell types of normotopic glands, while the others were characterized by few, small, dense granules, spherical or polymorphic, located peripherally in the cytoplasm. This picture might be due to a functional adaptation which occurs in pituitary cells still producing different hormones, once removed from central nervous system control. The major change in polypeptide hormone composition of graft homogenates relative to normotopic pituitaries is the fall in GH and PRL concentration. The in vitro incorporation of L[3H]leucine into the two hormones and the release of radioactive GH and PRL from L-[3H]leucine-prelabeled tissue fragments are also greatly decreased. The decrease in concentration, in vitro biosynthesis, and release of GH per mg tissue protein are ap-

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VER the last few years it has become increasingly clear that the responsiveness of pituitary somatotrophs and mammotrophs to secretagogues is critically dependent on the influence of environmental stimuli. In particular, it is now known that injection of TRH elicits a marked increase of PRL release in many animal species (1-6), whereas the release of GH is unaffected (7) or transiently modified only when large doses are used (8, 9). In most studies carried out on rats, the release of neither hormone was considerably increased by in vitro exposure of pituitary slices to TRH (3, 10-12), even if under these conditions the neurohormone is able to overcome the inhibition of PRL secretion brought about by dopamine (11). At variance with these results is the finding that in hypophysectomized (hypox) rats bearing a pituitary graft under the kidney capsule the injection of TRH is a potent stimulus not only for PRL but also for GH release (8, 13). The GH responsiveness of pitui-

tary grafts to TRH was found to appear within a few days after the transplantation and to increase thereafter, reaching a maximum after about 30 days (8). These findings indicate that pituitary somatotrophs, once removed from the direct control of the central nervous system (CNS), might undergo a functional adaptation involving the expression of specific features that are hardly detected in the normotopic glands. These observations are of interest also because abnormal sensitivity of GH-producing cells to TRH has been described in man in a variety of pathological conditions, such as acromegaly (14, 15), mental depression (16), anorexia nervosa (17), and liver disease (18). Hence, knowledge of the cellular events occurring in grafted pituitaries might be of relevance also for the understanding of at least some aspects of the physiopathology of the human disorders alluded to above. The study of the secretory activity of 30-day-old pituitary grafts (8) has been further expanded. In the present paper we will report: 1) a detailed investigation of the in vitro synthesis and release of proteins by the ectopic glands; and 2) the morphological characterization of the

Received October 14, 1977. Address requests for reprints to: Dr. J. Meldolesi, Department of Pharmacology, University of Milano, Via Vanvitelli 32, 20129 Milano, Italy. 226

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GH AND PRL IN PITUITARY GRAFTS

grafts used in the study. In the following article the profound effects of TRH, given in vivo as well as in vitro, on the secretory activity of the GH- and PRL-producing cells of the grafts will be reported (19). Materials and Methods Hypox female Sprague-Dawley rats (110-140 g BW) and intact female littermates (which served as pituitary donors or as controls) were obtained through the courtesy of the Carlo Erba Drug Co. (Milan, Italy). After hypophysectomy (10-15 days) the rats received a pituitary transplant under the kidney capsule according to a procedure previously described (8). After transplantation, animals were weighed every other day; only animals that showed a constant body weight increase (1-2 g/day) throughout this period were used. Incubation procedures Thirty days after transplantation, hypox-transplanted rats were killed, and the ectopic pituitary glands were removed and quickly transferred to ice-cold, oxygenated Krebs-Ringer bicarbonate solution (pH 7.4), supplemented with glucose and with a complete set of amino acids (KRB) (20). The fragments of glandular tissue were trimmed free of the remnants of the kidney capsule, pooled, and then incubated as specified below. In separate experiments, pituitary glands obtained from intact female rats were cut into four to six slices, which were pooled and then incubated in vitro. Incubations were carried out at 37 C in 10-ml Ehrlenmeyer flasks containing 1 ml oxygenated KRB, using a shaking bath operated at ~60 cycles/min. Two different protocols were used. In the experiments aimed to investigate hormone biosynthesis the pituitary fragments (10-20 mg wet weight per ml of medium) were pulse-labeled for 15 min at 37 C in KRB containing L-[3H]leucine (30 jiCi/ml; SA, 1 mCi/jumol) after a 10-min preincubation at 4 C in the same medium. The fragments were then carefully rinsed with ice-cold, nonradioactive KRB and homogenized immediately thereafter in glass-distilled water (300 /il for either 6 ectopic or 3 normotopic glands). In the experiments aimed to investigate the release of radioactive hormones, the pituitary fragments were labeled as described above, but for 90 min. After this period they were rinsed in KRB containing an excess (2 HIM) of unlabeled L-leucine (chasemedium) and then ireincubated in the latter for a total of 60 min, with changes of medium occurring after 10 and 20 min (chase incubation). In a few experiments, the pituitary fragments were incubated in nonradioactive KRB, and the release of GH and PRL was measured by RIA. At the end of the incubation, pituitary tissue fragments were collected and homogenized in glass-distilled water (300 ju,l for either 12 ectopic or 5 normotopic glands), while the chase media were centrifuged at high speed (105,000 x g, for 60 min in a Spinco type 40 rotor) to remove collagen fibers and any cell debris. Only the supernatants of these centrifugations were used for the analyses, while the pellets [which contained virtually no trichloroacetic acid (TCA)-insoluble radioactivity] were discarded. For further details on the incubation procedures see Ref. 20. Analytical procedures Protein assay was carried out according to Lowry et al. (21). Radioactive proteins; were precipitated with 10% TCA, washed

227

twice with 5% TCA, dissolved in Packard Instagel, and counted in a Intertechnique SL 30 liquid scintillation spectrometer. The counts were corrected for background, and correction for quenching was made by external standardization. GH and PRL were purified from homogenates and media by Na dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE), as previously described (22). Before electrophoresis, the proteins of the media were concentrated by TCA precipitation (final TCA concentration, 5%), followed by centrifugation. To assure a quantitative recovery and to facilitate the identification of the hormone bands, a mixture of bovine serum albumin, rat GH (rGH), and rat PRL (rPRL) were added as carrier. Samples of tissue and media, buffered at pH 6.7, were dissolved with 1% SDS in the presence of 2.5% /?-mercaptoethanol, then heated for 2 min in boiling water and finally applied to the gels within the next 10 min. Electrophoresis was carried out with 0.1% SDS-Tris-glycine buffer, pH 8.9, in a discontinuous system composed of a spacer gel (4% polyacrylamide in 50 mM Trisphosphate buffer, pH 6.7) and a running gel (10% polyacrylamide in 375 mM Tris-HCl buffer, pH 8.9). Both gels contained 0.1% SDS. After electrophoresis, the gels were fixed with TCA and stained with Coomassie brilliant blue (22). Stained gels were analyzed quantitatively in a Joice and Loebl MK 11 microdensitometer; reference calibration curves for rGH and rPRL were constructed by running known amounts of hormone standards under identical conditions, as previously described (22). Stained gels were sliced into segments 1.5 mm thick. The latter were dried, dissolved in 0.2 ml 30% H2O2, mixed with 10 ml Instagel, and counted (20). RIA determinations of GH and PRL in grafts and media were performed according to the methods of Schalch and Reichlin (23) and Niswender et al. (24), respectively, using materials supplied by the NIAMDD, Bethesda, MD. Results were expressed in terms of the NIH standards GH-RP-1 and PRL-RP-1 (potency of 1.5 and 11.0 IU/mg, respectively). Sensitivity was 0.1 ng for both assays. Biochemical data were analyzed statistically by the Student's t test. The level of significance was chosen as P < 0.05. Light and electron microscopy Small tissue cubes (0.5-1 mm thick) from ectopic and normotopic pituitaries were trimmed with a razor blade and fixed for 2 h at room temperature in 2.5% glutaraldehyde buffered at pH 7.4 with 0.12 M cacodylate buffer containing 1% sucrose. After washing with the buffer-sucrose solution, the specimens were postfixed with 1% OsO4 in veronal acetate buffer (0.056 M), pH 7.4, for 2 h at 4 C, then stained in block with 0.5% uranyl acetate in the same buffer (pH 6.2), and embedded in Epon 812. Sections 1 /xm thick were stained with toluidine blue and examined by light microscopy. Thin sections were cut with a diamond knife in Reichert or LKB ultramicrotomes, doubly stained with uranyl acetate and lead citrate, and examined in a Philips EM 200 or EM 300 electron microscope. Materials L-[3H]Leucine was purchased from the Radiochemical Centre Ltd., Amersham, England. rGH and rPRL standards (NIAMDD-RP1) were the kind gift of Dr. A. D. Parlow, Harbor General Hospital, Torrance, CA.

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ZANINI ET AL. Results

Morphological study One month after transplantation, the size of the grafted pituitaries is greatly reduced. Their volume is of the order of -1.80 mm3 (-2.5 X -1.2 X -0.6 mm) and their average weight is approximately 2.0 mg. Examination by light microscopy (Fig. 1) revealed that the center of the grafts (—5% of the total volume) is occupied by fibrous tissue surrounded by well preserved glandular tissue. In the latter, most cells are characterized by a large cytoplasm; nuclei are clear and usually contain a discrete nucleolus. Smaller cells with dense nuclei, sometimes displaying a wheel-like arrangement of the chromatin, are also seen. Most of these cells are located within capillary lumena and in pericapillary spaces, while others are intermixed with the large cells. In different grafts, their contribution varies from -5-20% of the total cell population. Electron microscopy of the peripheral areas revealed that the large cells are true parenchymal elements (Figs. 2-6). In their overall architecture they resemble normal pituitary cells, since they contain secretory granules, a well developed roughsurfaced endoplasmic reticulum, and a large Golgi complex, often organized into numerous parallel stacks. However, only a few elements maintain structural features typical of the various cell types of the normotopic gland. In many others, the granules [usually spherical, dense, and small (100-200 m/x in diameter)] are much reduced in number and often aligned at the extreme periphery of the cytoplasm (Figs. 3-6). Larger granules, sometimes irregular in shape, are also frequently observed. Some cells exhibit large lysosomes, multivesicular bodies, and residual bodies, containing dense myelin figures. The latter organelles might be the final result of increased autophagy. In contrast,

Endo • 1979 Vol 104 • No 1

images attributable to crinophagy (25) were never observed. The small cells with dense nuclei are identified as small lymphocytes (Fig. 5). Some of these are arranged to form isolated clusters; others are intermingled with the parenchymal cells. Plasma cells (Fig. 6), macrophages, and fibroblasts are also encountered. Polypeptide composition and in vitro protein synthesis Figure 7 compares the distribution of polypeptides and their radioactivity in SDS gels of homogenates prepared from ectopic and normotopic pituitary tissue fragments, labeled in vitro for 15 min with L-[3H]leucine. It is clear from the densitometric tracings that at this level of resolution no gross qualitative differences in polypeptide composition between the two homogenates are detectable. In contrast, considerable quantitative differences are readily apparent. These differences are relatively minor in the gel regions where polypeptides larger than a mol wt of 30,000 are separated (slices 1-40); in fact, in comparison with their normotopic counterparts, the heterotopic homogenates show a decrease in the high molecular weight region (especially at the level of the peak labeled with a double arrow) and a marked increase of the peak at a molecular weight of ~ 50,000 (single arrow). In the low molecular weight region of the gel (slices 40-60), a massive reduction of GH and PRL is clearly evident in the ectopic glands. As reported in Table 1, the concentration of the two hormones, measured by quantitative microdensitometry, amounts to only 13.3% and 26% of the values found in the normal glands, respectively. The

FIG. 1. Low power light micrograph of a toluidine blue-stained section through the whole thickness of a 30-day-old pituitary graft. Approximately half of the section is shown. Fibrous tissue is localized at the center as well as at the surface, whereas the rest of the graft is accounted for primarily by large parenchymal cells of variable size and density and by blood capillaries. Small cells with dense nuclei (sometimes showing a wheel-like arrangement of the chromatin) are also present. They are concentrated in capillary lumena and pericapillary spaces (inset; X100 and X450).

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229

FIG. 2. This low power electron micrograph illustrates the structural heterogeneity of the parenchymal cells of 30-day-old grafts. With respect to the situation observed in the normotopic gland (not shown in figures), the number and average size of secretory granules is greatly diminished in most cells. Moreover a marked heterogeneity also exists in relation to granule shape. For instance, large polymorphic granules, similar to the typical PRL granules of normal mammotrophs, often coexist with numerous, small, spherical granules (for example in the cells labeled C1-C4). In several cells the small spherical granules are aligned at the cytoplasmic periphery. Lysosomes and residual bodies are indicated by arrows and arrowheads (X4.500).

drastic decrease of GH and PRL in the grafts was also demonstrated by RIA. The values obtained by this method are even lower than those found by microdensitometry (~6 and ~12.4% of that of normal glands, respectively; not shown in tables). The in vitro incorporation of L-[3H]leucine into the proteins of ectopic pituitaries is reduced to approximately one third of that observed in normotopic glands (Table 2). The analysis of the distribution of labeled peptides in SDS gels (Fig. 7) revealed that this reduction is not homogeneous. In particular, the radioactivity recovered in the intermediate region of the gel, where the peptides ranging in molecular weight between ~60,000-30,000 are known to migrate, was either unchanged (slices 16-24) or even slightly increased (slices 25-40). The same can be said for the low molecular weight peptides running close to the gel front (slices 50-60). A totally different pattern was found both in the high molecular weight region (in which the labeling is considerably reduced) and in the GH and PRL bands. The two large radioactivity peaks which in the gels of the normotopic glands coincide with these two bands, appear to be nearly absent in the grafts. In quantitative terms (Table 2), the contribution of the two hormones, which in the normal pituitary accounts for over 34% of the radioactivity incorporated into protein

in vitro, is decreased to approximately 7%. On a total tissue protein basis, the grafts synthesize GH and PRL at rates which are only 8.9% and 4.8% of those present in normotopic glands, respectively. Also the specific radioactivity of the hormones is decreased in the grafts: to a moderate degree in the case of GH, to less than 24% in the case of PRL. In vitro protein release The in vitro release of protein (studied by monitoring the accumulation in the chase medium of pituitary proteins labeled by a prior incubation of tissue fragments in the presence of L-[3H]leucine) was found to be strikingly different in normotopic and ectopic glands. During chase incubation normal glands (Fig. 8B) release a considerable amount of their protein-incorporated radioactivity. The major component was found to be PRL, which by itself accounts for over 40% of the total protein radioactivity released. The contribution of GH is of the order of 15%. In contrast, the release of radioactive protein by the ectopic glands (Fig. 8A) is very low and the contribution to this of the two hormones is minimal. Most of the TCAinsoluble radioactivity discharged by these glands was found to be accounted for by two polypeptides with

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Endo i 1979 Vol 104 , N o l

FIG. 3. The field includes several parenchymal cells characterized by a various complement of secretory granules. The three cells at the right top of the figure (C1-C3) contain granules of irregular shape, mostly of large size, in Ci (which resembles the PRL cells of normotopic pituitary) and of small size in C2. The other cells of the figure contain mostly dense, small, spherical granules, preferentially aligned at the cell periphery. The same localization is seen in C2. MV, Multivesicular bodies; L, lysosomes (X8000).

approximately 55,000 and 25,000 mol wt. Labeled polypeptides with analogous migration rates in SDS gels were identified also among the proteins released by normotopic glands (arrowheads in Fig. 8B); however, their relative contribution is much smaller than that of GH and PRL. In Fig. 9, the in vitro release of GH and PRL from normotopic and ectopic glands is plotted against time and expressed as the percentage of the total GH and PRL radioactivity present in the tissue plus media at the end of the incubation. With respect to normotopic glands, the percentage of release of GH in ectopic glands is slightly decreased. However, the difference is not statistically significant. In contrast, for PRL the decrease is clearcut (—80% to -85%). The data reported in Fig. 9 were also calculated on a tissue protein basis. When expressed this way, the values for radioactive GH released into the medium at the different timepoints of the chase incubation were approximately 7% of the values found for normotopic glands, while those for PRL were around 1% (not shown in Tables). Moreover, in a few cases, the release of GH and PRL was estimated also by RIA. In these experiments, the amounts of hormone released in 20 min from pituitary fragments, which had been preincubated for 90 min, were the following: GH, 2.85 and 0.16 jug; PRL, 3.84

and 0.058 /xg/mg tissue protein in normotopic and ectopic pituitaries, respectively. These RIA results are in good agreement with those obtained by the radiochemical procedure and, therefore, confirm that GH and PRL release is greatly reduced in the grafts incubated in vitro.1 Discussion Pituitary glands grafted under the kidney capsule of hypophysectomized rats are a classical experimental model which has been extensively used in endocrinology mainly for differentiating the direct responses of the gland from those mediated through the CNS. On the other hand, it has recently become clear that pituitary glands devoid of CNS influences acquire peculiarities which are hardly detectable in the normotopic gland (8, 26). In this and in the companion article (19), we report studies on the morphology and in vitro synthesis and release of GH and PRL under basal conditions and after 1 Since the RIA data are expected to be more severely affected by hormone leakage (defined as the fraction of the tissue hormone store released unspecifically from damaged cells during the time periods of incubation) than data obtained by the radiochemical procedure (see Discussion), it would appear that leakage from the grafts was smaller than that from the normotopic glands. This was to be expected, since the pituitary grafts were very small and thin and were incubated intact, whereas the larger normotopic glands were sliced before incubation.

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231

FIG. 4. Of the four parenchymal cells shown in the field, one (Ci) contains large, polymorphic granules typical of PRL cells of normotopic pituitaries, while the others are characterized by small, spherical granules, mostly located at the cytoplasmic periphery. Note the large and elaborated Golgi complex in C2 and C3 (GC). MV, Multivesicular bodies; L, lysosomes; $ , grazing section of the nuclear periphery and envelope (X 10,500).

TRH stimulation of ectopic pituitaries 30 days after transplantation. To our knowledge, the in vitro approach has never been applied before to the study of pituitary grafts. Previous studies of the ultrastructure of rat pituitary grafts agree that within a few days after transplantation, most parenchymal cells become chromophobic as a consequence of a drastic decrease in their secretory granules (27-31). In contrast, disagreement remains as to the classification of these chromophobic cells among the cell types present in the normal pituitary. Based on the presence of large and polymorphic mature secretory granules, of immature granules concentrated in the Golgi area, and of elaborate arrays of rough endoplasmic reticulum cisternae, many of these cells were identified as mammotrophs by some authors (27, 30, 31). Others, in contrast, have suggested that after transplantation, GH and PRL cells might transform into corticotrophs, since many cells in the grafts exhibit small dense secretory granules aligned at the extreme periphery of the cytoplasm (29) (a pattern which in the normotopic rat pituitary is typical of ACTH-producing cells; see Ref. 32). In the present study we have confirmed that only a minority of the parenchymal cells present in 30-day-old

grafts retain the morphology of the various cell types present in the normotopic pituitary, whereas most of the others appear morphologically very similar. The latter are characterized by a low number of secretory granules, which are in general dense, small, spherical, and often peripherally located. However, many of these cells also contain a variable proportion of large polymorphic granules. In our opinion, the disappearance of the typical, differentiated morphology of pituitary cells does not necessarily indicate the prevalence of one single cell type. In this respect it should be emphasized that the identification of the different cell types present in the normal pituitary is based mainly on the size, shape, density, number, and topological distribution of the secretory granules (32, 33) and that these features can change in pituitary cells depending on a variety of factors (e.g. the degree of stimulation; see Refs. 33 and 34 for reviews). We believe therefore that the identification of the parenchymal cells present in the grafts cannot be made with certainty by conventional electron microscopy but requires specific immunocytochemical techniques (35, 36) which so far have not been used to investigate the problem. The ultrastructural modifications observed in the

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Endo • 1979 Vol 104 • No 1

FIGS. 5 and 6. Parenchymal cells similar to those shown in Figs. 2-4 are seen in close apposition to either a small lymphocyte (LC; Fig. 5), characterized by the condensed arrangement of chromatin, or to a plasma cell (PC; Fig. 6), recognizable by the highly developed rough-surfaced endoplasmic reticulum (which contains a dense fibrillar material and is heavily covered with polysomes) and Golgi complex (GC). Lysosomes in parenchymal cells are indicated by arrows, MV, Multivesicular body. The extracellular space is occupied by a filamentous material (#; X8200 and X7900). TABLE 1. GH and PRL content (jug/mg total tissue protein) of ectopic and normotopic pituitary glands

Ectopic glands Normotopic glands

GH

PRL

12.09 ± 0.68 90.60 ± 6.33

7.35 ± 0.52 28.30 ± 1.83

Results are the average of four experiments ± SE.

grafts might be the expression of an adaptative process due to the interruption of the trophic influence of the CNS. In this respect, it is of interest that in the rat fetus, in which the CNS control of the pituitary gland is not fully developed (see Ref. 37), the gland has an overall appearance similar to that observed in our pituitary grafts (38). A variable number of immunocompetent cells (lymphocytes and plasma cells) as well as macrophages was found in ectopic pituitaries. In spite of this, we believe that our biochemical data remain basically valid; in fact, in the grafts 1) most cells are morphologically well preserved parenchymal cells, 2) the protein composition of the homogenate is modified only quantitatively, and 3) GH and PRL cells exhibit the ability of hormone synthesis and release, which is greatly stimulated by exposure to TRH (see Ref. 19). In addition, it should be emphasized that in this work the in vitro release of GH and PRL was studied primarily by determining the distribution in the tissue fragments and incubation fluids of the radioactivity incorporated into the two hormones during

pulse labeling with L-[3H]leucine. As discussed in previous reports on the pituitary (20) as well as on other secretory systems (39), this method has the advantage over simple hormone assays in that it eliminates, to a considerable extent, the artifactual contribution of the cells already damaged at the beginning of the experiment, as well as that of free organelles and cell debris, which have little or no GH- and PRL-synthesizing capacity. Most of our biochemical studies were carried out by high resolution SDS-PAGE. As discussed previously (22), this technique seems adequate to yield detailed information on the overall protein composition of the pituitary gland. Moreover, stained gels can be used to estimate quantitatively the tissue content as well as the radioactivity of GH and PRL, because the two hormones are separated into two large, well identified peaks, which contain only very small amounts of contaminants (22). In contrast, no information can be obtained on the other pituitary hormones because they are not resolved from the other proteins of the homogenate. In view of their size, the subunits of both gonadotropins and of TSH, as well as ACTH, are expected to migrate at or close to the front of our SDS gels. The content, synthesis, and release of GH and PRL were found to be greatly affected in the ectopic pituitaries when compared to their normotopic counterparts. In agreement with previous observations (40, 41), we found that the tissue concentration of the two hormones is

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GH AND PRL IN PITUITARY GRAFTS

TABLE 2. TCA-insoluble and GH and PRL radioactivity of ectopic and normotopic pituitary glands pulse-labeled with L-[3H]leucine for 15 min TCA-insoluble radioactivity (dpm/mg total protein)

GH

dpm/mg Total protein

dpm/mg GH

dpm/mg Total protein

dpm/mg PRL

5,700 (5,500-5,900) 63,800 (48,900-78,700)

417,300 (402,900-431,700) 730,700 (595,100-866,300)

5,300 (5,200-5,400) 108,600 (89,300-127,900)

712,300 (601,300-823,300) 3,146,800 (2,835,800-3,457,800)

155,900 (126,400-185,400)° 502,000 (501,500-502,500)

Ectopic glands Normotopic glands

PRL

Results are the averages of two experiments. " Ranges. PRL

o

PRL

JGH I

30 J 20. 10.

ili

3.5. 3.0. 2.5. 2.0 1.5. N PRL

1.0. 10

20

30

40 50

gel slices

60

+

10

20

30

40

gel slices

50

60

+

FlG. 7. Distribution of protein and protein-bound radioactivity in SDS polyacrylamide gels of homogenates obtained from ectopic (A) and normotopic (B) pituitary glands pulse-labeled with L-[3H]leucine for 15 min. The positions of GH and PRL are labeled. The single and double thick arrows point to two other regions of the electrophoretic pattern in which major differences between ectopic and normotopic pituitary homogenates are detectable (see text).

lowered (to approximately 1/7 and 1/4, respectively). This finding appears consistent with our morphological observations, since in the normotopic pituitary at least 80% of GH and PRL is stored in secretory granules (22), which are greatly decreased in the grafts. The fall of the total hormone content per gland is even larger than the decrease in tissue concentration, since the volume of 30day-old grafts is considerably smaller than that of normotopic glands. Moreover, the in vitro synthesis and release of both hormones were dramatically decreased (see Table 2 and Figs. 8 and 9). In particular, while GH and PRL account for over half of the radioactive proteins discharged in vitro by prelabeled normotopic glands, the major components released from the grafts migrate in SDS gels in two peaks corresponding to peptides with approximately 55,000 and 25,000 mol wt. In our opinion, the possibility that these peaks represent precursors of GH and PRL or anomalous aggregates of finished hormone molecules is unlikely for the following reasons. 1) In the case of GH

0.5.

s*J

!\\T

10 20 30 i,0 50 60

,A 10 20 30 40

50 60

gel slices + gel slices + FIG. 8. Distribution in SDS polyacrylamide gels of protein radioactivity, released into the medium from ectopic (A) and normotopic (B) pituitary glands pulse-labeled with L-[3H]leucine for 90 min, during 10 min of chase incubation.

and PRL the existence of prohormones has been excluded (42). In contrast, pre-GH and pre-PRL have been described (43-45). However, prehormones, which are characterized by an additional peptide covalently linked at the N-terminus, are not real precursors. They are synthesized in heterologous cell-free systems but never found in intact pituitary cells because the additional peptides are cleaved by specific proteolysis during growth of the nascent chains (45). 2) Boiling in SDS in the presence of reducing agents (as done in the present work) produces a complete denaturation of the proteins with dissociation of all noncovalent bonds. Thus, the SDS-PAGE patterns refer to denaturated polypeptides, not to native proteins or protein aggregates (46). 3) The results obtained by PAGE separation of discharged radioactive GH and PRL were confirmed by RIAs carried out in separate experiments. The two peaks separated in the SDS gels could be accounted for by secretory peptides, as yet unidentified, discharged also by the rat normotopic pituitary. Alternatively, they could be due not to hormone but to heavy and light chains of immunoglobulins secreted

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ZANINI ET AL.

234

GH

PRL ING

a>

40.

40.

30.

30.

c o

S o

1120

20.

10.

10.

10 20

60

10 20

60

min in chase FIG. 9. In vitro release of pulse-labeled GH and PRL. Tissue fragments [from ectopic (EG) and normotopic (NG) glands] were pulse-labeled in vitro with L-[3H]leucine for 90 min and then reincubated in chase medium for a total of 60 min, with changes of medium occurring after 10 and 20 min. Data are expressed as the percentage of total (tissue plus media) GH and PRL radioactivity recovered in the media at the time indicated. Results are averages of 2-3 experiments ± SE. Statistically significant differences between ectopic and normotopic pituitaries are labeled by asterisks.

at a very high rate by immunocompetent cells (47). The latter interpretation would be consistent with the size of the two peptides as well as with their relative proportion. The behavior of GH and PRL cells of the pituitary grafts was found to be quite different with respect to the in vitro synthesis and release of the two hormones. In somatotrophs, the specific radioactivity after in vitro labeling (Table 2) and the in vitro intracellular turnover of the hormone (which can be derived from the data on hormone release reported in Fig. 9) are both reduced with respect to the normal gland, but only by approximately 40% and 25%, respectively. When the rates of in vitro biosynthesis and release are calculated per mg tissue protein, values of approximately —91% and -93% with respect to the normotopic glands are obtained. These values are not far from that observed for the tissue concentration of GH (—87%). Thus, the fall of GH secretory activity occurring in the grafts does not imply drastic changes in the balance between biosynthesis, storage, and release of the hormone and might be due primarily to a proportional decrease in somatotrophs with respect to the other cell types and/or a decrease of the secretory activity of each single somatotroph. This latter mechanism is also suggested by our morphological studies, in which most of the pituitary cells were found to be poor in secretory granules. In contrast, the rates of in vitro biosynthesis and release of PRL were reduced in the grafts to a much larger extent than the tissue concentration of the hor-

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mone. When calculated per mg tissue protein, the observed decreases were of the order of —95%, —99%, and 74%, respectively. Since the duration of the pulse incubation with L-[3H]leucine used in our experiments (90 min) exceeds the intracellular transport time of PRL in normal mammotrophs {i.e. the time needed for newly synthesized hormone molecules to become stored in intracellular granules and therefore available for discharge ~45 min; see Ref. 32), we do not know the rate at which the latter function is carried out in the grafted cells. However, because our release experiments (Fig. 9) indicate that, during chase incubation, newly synthesized PRL is discharged at an approximately constant rate from both normotopic and ectopic glands, there is no doubt that the time of intracellular transport in the grafted cells cannot be longer than the pulse incubation time (i.e. 90 min). Thus, the large disparity between the decrease in release and tissue concentration of PRL, documented by the present experiments in grafted mamotrophs, cannot be accounted for by possible changes in the rate of intracellular transport and should be attributed to a drastic decrease in the intracellular turnover of the hormone. The findings on PRL synthesis and turnover might appear surprising in view of numerous studies which have shown that in the hypox rat, pituitary grafts are able to sustain a considerable PRL secretion, as revealed by high plasma levels of the hormone as well as by the occurrence of PRL-dependent processes. These findings have been also confirmed by some of us in graft-bearing rats comparable to those used in the present investigation (8). Since, as already mentioned, the total PRL content of the gland is much lower in grafts than in normotopic glands, these results strongly suggest that in vivo, the hormone might turn over faster in ectopic than in normotopic cells. In fact, a 2-fold increase in in vivo PRL turnover in grafts was reported previously by Nicoll and Swearingen (40). The striking difference between the in vitro and in vivo behavior of ectopic and normotopic mammotrophs is probably due to their different regulation. In this respect, it should be emphasized that normotopic PRL cells function in vivo under the control of the CNS. The overall result of this control is a strong inhibition of their secretory activity. Therefore, when normotopic glands are removed and incubated in vitro, PRL cells undergo a potent activation (11, 48). When a pituitary gland is transplanted under the kidney capsule, activation of mammotrophs analogously occurs, as revealed by the large increase in the plasma concentration of PRL in transplanted animals (8). However, this activation is not permanent. In fact, the secretory activity of PRL cells decreases progressively within 2 weeks to reach a relatively low level, which nevertheless remains higher than

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GH AND PRL IN PITUITARY GRAFTS the CNS-inhibited activity level of their normotopic counterparts. When ectopic glands are placed in vitro, no derepression of their mammotrophs occurs. Our present observation that the release of PRL from the grafts incubated in vitro is carried out at a relatively low rate therefore appears quite reasonable. In conclusion, our results strongly indicate that the high in vivo PRL secretory activity of pituitary grafts is not due to the expansion of the functional potential of the mammotrophic cells but results primarily from the lack of the inhibitory control of the CNS. In addition, transplanted cells might become more sensitive to secretagogues, a possibility that is considered in detail in the companion article (19). Acknowledgments The photographic assistance of Mr. F. Crippa and P. Tinelli is gratefully acknowledged. The authors thank Drs. D. Cocchi and V. Locatelli for carrying out the RIAs of GH and PRL.

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