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Uptake, localization, and retention of gonadotropin-releasing hormone and gonadotropin-releasing hormone analogs in rat gonadotrophs
Molecular and Cellular Endocrinology,
0 Elsevier/North-Holland
19 (1980) 101-l 12 Scientific Publishers, Ltd.
101
UPTAKE, LOCALIZATION, AND RETENTION OF GONADOTROPIN-RELEASING HORMONE AND GONADOTROPIN-RELEASING HORMONE ANALOGS IN RAT GONADOTROPHS * T.M. DUELLO ** and T.M. NETT Department of Physiology and Biophysics, College of Vetennary Medicine and Biomedical Sciences, Colorado State University, Fort CoNins, CO 80523 (U.S.A.)
Received 1 October 1979; accepted 27 March 1980
Uptake and retention of GnRH by pituitary was compared to that of GnRH-ethylamide (GnRH-EA) and D-Ala6 -GnRH-ethylamide (D-Ala6 -GnRH-EA) to determine if differences in these parameters might partially account for the increased biopotency of these superactive analogs. Ovariectomized estrogen/progesterone-treated rats were given an intracarotid injection of either 1251-labeledGnRH, GnRH-EA, or -D-Ala6-GnRH-EA. Maximum uptake occurred at 2 mm for GnRH (8000 cpm/pit), 5 min for GnRH-EA (10 000 cpm/pit), and 45 mm for D-Ala6 -GnRH-EA (24 000 cpm/pit). Thereafter pituitary content decreased out to 1, 2, and 4 h, resp. Specific uptake of all 3 peptides was shown autoradiographically to be restricted only to immunostained gonadotrophs with the silver grains being localized intracellularly at all time points studied. Serum concentrations of LH increased in response to all 3 peptides in proportion to their uptake and did not begin to decrease until after maximum uptake of each peptide had occurred. Thus, the extent of uptake and length of retention of these peptides in the pituitary correlate well with their relative biopotencies, D-Ala6-GnRH-EA > GnRH-EA > GnRH. Keywords:
GnRH; analogs; internalization;
LH.
Following elucidation of the amino acid sequence of gonadotropin-releasing hormone, or GnRH (Burgus et al., 1971; Matsuo et al., 1971) concerted efforts were directed at determining which amino acids were necessary for biological activity. While the entire molecule was shown to be involved in binding to receptor, the histidy1 and tryptophanyl residues in positions 2 and 3 appeared to directly affect biopotency. Simple substitutions, or deletions, at these positions significantly decreased or abolished gonadotropin-releasing activity (Coy et al., 1973; Monahan et al., 1973). In contrast, D-amino substitutions at position 6 and propylamide or ethylamide substitutions at position 10 resulted in molecules with enhanced abil-
* This work was supported by NIH Grants HD07841 and HD07031. ** To whom reprint requests should be sent.
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ities to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (Fujino et al., 1972, 1974; Monahan et al., 1973). Reeves et al. (1977) postulated that the enhanced biological activity of these analogs might reflect differences in their half-lives in the systemic circulation, longer binding to receptors, or increased binding due to a preferred molecular conformation. When 2 superactive analogs, both with substitutions at position 6 and 10, were compared to GnRH, no differences in their half-lives in serum were observed (Reeves et al., 1977) but both analogs were taken up to greater extents and retained in the pituitary for longer periods of time than the parent molecule. While differences in these parameters may account for differences in biopotency, additional explanations are possible. For example, Koch et al. (1977) showed that enzymic preparations from rat hypothalamus and anterior pituitary, known to preferentially cleave GnRH at the Gly6Leu’ position, were less effective in degrading analogs with substitutions at positions 6 and/or 7. Thus, they suggested that the rate of degradation of GnRH and GnRH analogs may be a physiological determinant of biopotency. In an effort to examine more closely the importance of uptake and retention in the regulation of the release of LH by GnRH, GnRH was compared to desGlyNH:‘-GnRH-ethylamide (GnRH-EA) and D-Ala6, des-GlyNH~“-GnRI-ethylamide (D-Ala6-G&H-EA). The biopotency of these peptides is 3-fold and . lo-30-fold greater than that of GnRH in a variety of different biological assays (cf. review by Schally, 1978).
MATERIALS
AND METHODS
Adult Sprague-Dawley rats, ovariectomized for 2-3 weeks, were each given a subcutaneous injection of 50 ng of estradiol benzoate and 25 mg of progesterone in safflower oil. 36 h later rats were anesthesized with Ketamine (12 mg/lOO g wt.)/ Acepromazine (0.25 mg/l.OO g wt.) after which the carotid artery was exposed and isolated. 3 groups of rats, a minimum of 56 rats per group, were given intracarotid injections of 17 pmoles of 1 of 3 ‘251-labeled peptides: GnRH (Parke Davis, Inc.), GnRH-EA (Abbott Laboratories), or D-Ala6-GnRH-EA (Dr. M. Fujino, Osaka, Japan). Radioiodination of hormones was performed as previously described (Wagner et al., 1979). Monoiodinated peptide was separated from diiodinated peptide on a 0.6 X 25 cm column of QAE Sephadex (Q-25-120; Sigma Chemical Co.). Essentially no free iodide elutes from this column (Wagner et al., 1979). The pattern of elution of unlabeled GnRH was determined by radioimmunoassay (Nett and Adams, 1977) and found to be less than 2% of the peak of monoiodinated peptide. All preparations were used within 24 h of radioiodination and were of identical specific activity (1574 Ci/mmole). Following the injection, the carotid was ligated to prevent excessive bleeding. The rats were decapitated and exsangumated at specific intervals thereafter at which time pituitary, thyroid, adrenal, liver, kidney, uterus, brain, and spleen tissue were removed, weighed, and the radioactivity present in
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each tissue was quantified using a Nuclear Chicago Automatic gamma spectrometer. Concentrations of LH in serum were determined by the radioimmunoassay method of Niswender et al. (1969). The ratio of tissue cpm/mg to serum cpm/pl (T/S) were used to express uptake in each tissue with significant uptake defined as a TS > 1. Tissue specificity of uptake was determined by measuring the ability of an excess of unlabeled peptide to block uptake of the labeled peptide. If uptake was specifically blocked the T/S should approximate 1. 8-10 rats in each group were given jugular injections of either a 200- or lOOO-fold excess of D-Ala6-GnRH-EA 1, 10 or 60 min prior to the injection of 1 of the 3 labeled peptides. D-Ala6GnRH-EA was used to block all 3 peptides since it had been shown to be retained by the pituitary longer than GnRH (Reeves et al., 1977). Therefore, we reasoned that it should be most effective in blocking the binding of the labeled peptides. In addition, it has been shown that D-Ala6-GnRH-EA (Crowder et al., 1980) dissociates from sheep pituitary homogenates approx. 7 times more slowly than GnRH (Wagner et al., 1979). To further examine the specificity of uptake by the pituitary, each of 6 rats were given an intracarotid injection of 17 pmoles of 1 of 2 1251labeled fragments of GnRH containing amino acids l-6 or amino acids 5-10. Uptake of each was determined as for the other ‘251-labeled peptides. Cellular specificity was assessed using a combined immunocytochemical-autoradiographic technique to determine whether the labeled peptides were taken up only by gonadotrophs. Pituitaries from rats in groups receiving 12’I-GnRH or -D-Ala6-GnRH-EA injections were cut into pieces approx. 1 mm3 and fixed for 24 h at 4°C in a 1% solution of glutaraldehyde in 0.1 M cacodylate buffer containing 5% sucrose (PH 7.3). The tissues were then washed in buffer, dehydrated in a graded series of ethanol, and embedded in Epon 812. Loss of radioactivity in each step was monitored. 1-I-(sections were cut, mounted on glass slides, and stored overnight in an oven at 60°C. The sections were then etched, dehydrated and bleached as described by Baskin et al. (1979) and incubated in Tris-buffered saline containing 0.25% gelatin for a minimum of 10 min. The immunoglobulin-enzyme bridge method of Mason et al. (1969), as modified by Sternberger et al. (1970) was used to immunostain gonadotrophs. The procedure included the following steps: (1) initial incubation of sections with rabbit antiserum to ovine LH (R440, provided by Dr. G.D. Niswender) at 1 : 10 000 at 4°C for 24 h; (2) incubation with sheep antirabbit y-globulin produced in this laboratory and used at 1 : 100 for 10 min; (3) incubation with horseradish peroxidase-anti-peroxidase (PAP, Cappel Laboratories) at 1 : 100 for 10 min; and (4) reaction of the peroxidase with a solution of 3,3’diaminobenzidine tetrahydrochloride (0.3 mg/ml; Sigma Chemical Co.) and Hz02 (0.05%) in a 0.05 M Tris buffer (pH 7.6) for l-5 min. Specificity controls entailed the substitution of normal rabbit serum (1 : 10 000) for the antiserum and incubation of the anti-LH serum with 1 pg/ml of ovine LH (NIH-oLHS19) for 48 h at 4’C. A physiologic control entailed ascertaining whether immunostaining was restricted to the castration cells seen in the pituitaries of additional rats ovariectomized for 5 or more weeks. Following incubation with the substrate solution,
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slides were rinsed in water, blotted dry, coated with Kodak NTB3 Nuclear Track Emulsion by the dip method, and stored in light proof dessicated boxes at 4’C for l-4 days. At the end of the exposure time, autoradiograms were developed in freshly prepared Kodak D-19 developer for 45 set and fixed in Kodak fixer for 4 min. The emulsion was scraped from the back of the slide with a razor blade whereupon the slides were air dried, coversripped, and viewed in a Zeiss microscope.
RESULTS at various Pituitary uptake of r2’I-GnRH, -GnRH-EA, and -D-Ala6-GnRH-EA intervals is shown in Fig. 1 (bars). Both analogs were taken up to greater extents and over longer periods of time than was GnRH. While pituitary content of GnRH began to decrease approx. 2 min after the iqection, pituitary content of GnRHEA and D-Alar’-GnRH-EA did not decrease until 5 and 45 min, resp. At these times pituitary content of the labeled analogs were 125 and 300% of that of GnRH
TIME (min) Fig. 1. Uptake by the pituitary of radiolabeled GnRH and analogs and the resultant release of LH. Rats were given intracarotid injections of 1 of the 3 radiolabeled peptides and the pituitaries removed at specified intervals. Pituitary content of radiolabeled peptide is indicated by the bars (left axis), while the LH released in response to each treatment is indicated by the stippled area (right axis).
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at 2 min. Injection of twice as much ‘251-D-Ala6-GnRH-EA did not result in greater uptake by the pituitary gland indicating that the concentration of peptide was not limiting uptake. While GnRH was retained in the pituitary for approx. 60 min, GnRH-EA and D-Ala6-GnRH-EA were retained much longer with the T/S remaining above 1 for 2 and 4 h, resp. All 3 radioiodinated preparations were biologically active as evidenced by the LH released in response to the injections (Fig. 1, stippled area), the magnitude of which could not be attributed to the 2% contamination with unlabeled hormone (unpublished data). Peak release of LH in response to GnRH and GnRH-EA was seen at 5min postinjection and did not return to basal concentrations for approx. 1 and 2 h, resp. However, at 5-min postinjection serum concentrations of LH due to GnRH-EA were 2.7 times greater than after injection of GnRH. At 45 min, peak serum concentrations of LH due to D-Ala6-GnRH-EA were 6.4 times greater than at 5 min after the injection of GnRH and had not returned to baseline by 4 h. When a 200-fold excess of unlabeled D-Ala6-GnRH-EA was given via the jugular vein 1 min prior to the intracarotid injection of labeled peptide, uptake of both GnRI-EA and D-Ala6-GnRH-EA by the pituitary was consistently blocked when the pituitaries were removed at time points longer than 10 min, i.e., the T/S approximated 1 (Table 1). This 1-min pretreatment failed to block uptake of GnRH by the pituitary as did a lo-min pretreatment, a 1-min pretreatment with a lOOOfold excess, or injection of both labeled and unlabeled peptide simultaneously. GnRH uptake was blocked, however, when the pretreatment entailed injection of a 200-fold excess of unlabeled peptide which was allowed to circulate for 60 min prior to the injection of the labeled peptide. This treatment presumably saturated receptors for GnRH as uptake could not be blocked further by pretreating with a 200-fold excess 60 min and again 1 min prior to the injection of the labeled peptide. The T/S for adrenal, uterus, brain, and spleen were consistently less than 1, for
Table 1 Uptake of radiolabeled peptides by pituitary of rats receiving no pretreatment of a pretreatment with unlabeled D-Ala6-GnRH-EA. Uptake of l2 5 I-GnRH could not be blocked by the 1-min pretreatment with unlabeled D-Ala6-GnRH-EA. Uptake of 12sIGnRH-EA and -DAla6-GnRH-EA could be blocked by this pretreatment at time points after 10 mm T/S t [ S.E.] Treatment
Time (min)
Treated
Cold-blocked
GnRH GnRH-EA D-Ala6 -GnRH-EA
2 20 60
1.3 f 0.2 3.4 f 0.4 11.2 f 0.9
1.3 f 0.2 1.3 f 0.2 1.6 +z0.7
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liver slightly greater than 1, and for thyroid and kidney they were often in excess of 10, indicating a greater degree of uptake of the peptide or a breakdown product of the peptide. Since none of these values were affected by any of the pretreatment regimens, specific uptake was restricted to pituitary tissue only. During the processing of pituitaries lo-SO% of the radioactivity was lost in the fixative and the first buffer wash. This was due to the fact that the concentration of labeled peptide was in excess of the dose necessary to occupy available receptors for GnRH on a single passage through the pituitary. This excess was necessary to insure that low serum concentrations of radiolabeled peptide would not limit further uptake on recirculation. However, that radiolabeled peptide retained in the pituitary throughout processing represented specific uptake as a combined immunocytochemical-autoradiographic method showed silver grains (i.e., labeled peptides) were restricted only to those cells which immunostained for LH. The immunocytochemical localization (Fig. 2a) was considered specific as: (1) no staining occurred when normal rabbit serum was substituted for the antiserum; (2) no staining occurred when the antiserum was absorbed with 1 fig/ml of LH (Fig. 2b); and (3) staining was restricted to the castration cells in the pituitaries of rats which had
Specific uptake of GnRN by rat gonadotrophs
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Fig, 2. (a) Immunostaining of gonadotrophs by antiserum to LH. @I The immunostaining was prevented when the antiserum was absorbed with 1 p&n1 LH. The astesisks indicate landmarks common to both fields (X670).
been avariectomized for 5 or mare weeks. At I, 2 and 5 min after injection of either “‘I-GnRH or -n-Ala6-GnRH-EA, a few cells which immunostained were shown autoradio~a~hica~y to be heaviiy labeled (Fig. 3a), though grains over vascular and ~nters~tia1 tissue in these areas were often abundant. However, if the labeled D-Ala’-GnRH-EA was allowed to circulate for 60 min, immunostained cells throughout the pituitary became uniformly labeled and vascular and interstitial labeling tended to decrease (Fig. 3b). At all times studied silver grains were localized only over the cytoplasm of the i~u~ost~e~ cells and were never observed over nucfei or circumscribing the cell at the plasma membrane. This localization was not affected by prior immunostaining of the tissue. The cellular uptake was considered spccitic since silver grains were not found over immunostained gonadotrophs in rats where uptake was blocked by the pretreatment with the unlabeled D-A&-G&II-EA (Fig_ 3~). Since GnRH has a half-life of approx. 7 min in the systemic circulation (Redding
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Specific uptake of GnRH by rat gonadotrophs’
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and Schally, 1973), the possibility existed that what was binding to gonadotrophs and being localized were radiolabeled fragments of the peptides. However, when fragments of GnRH containing amino acids l-6 or 5-10 were radiolabeled and injected, only nonspecific radioactivity remained associated with the pituitary 2 min after the injection and the T/S was always less than 1.
DISCUSSION The extent of uptake and length of retention of r2’I-GnRH, G&I-I--EA and -D-Ala6-GnRH-EA in the pituitary correlates well with the ability of each peptide to release LH as shown here and as reported previously (Reeves et al., 1977). GnRH-EA, which has the position-10 substitution only, was taken up to a greater extent, was retained longer, and caused greater release of LH than GnRH, though not to the extent of D-Ala6-GnRI-EA, which has both the position-6 and -10 substitutions. This greater uptake of both superactive analogs is reflected in their higher T/S. While the T/S for GnRH uptake by pituitary is only slightly greater than 1, the ratios for aII 3 peptides are probably deceptively low at intervals of 5 min or less since serum cpm/fi are artifactually high in the exsanguinated blood. This is probably due to the fact that the radioactivity was not equilibrated with the entire fluid volume of the rat due to ligature of the carotid artery through which the labeled peptide was injected. Regardless of whether the T/S is low at time points under 5 min, it would nonetheless have been expected that this value would decrease if uptake were indeed blocked by the unlabeled peptide. This, however, was not the case. The factors responsible for the inability to consistently block uptake at time points earlier than 10 min are no doubt the same as those responsible for the inability to block uptake of GnRH at all It was assumed that the majority of the binding would take place during the first passage through the pituitary, the peptide then becoming diluted in the entire systemic circulation. However, just as the labeled GnRI--EA and the D-Ala6-G&I-I-EA required repeated passage for maximal uptake, the unlabeled peptide as well apparently required more than 1 min for receptor saturation throughout the pituitary to occur. Only when the unlabeled D-Ala6-GnRH-EA, present in a 200-fold excess, was allowed to recirculate with the labeled G&H-EA or D-Ala6-GnRI-EA, could it effectively compete for receptors at time points
Fig. 3. (a) An immunostained gonadotroph in the pituitary of a rat treated with ’ 2s IGnRH for 2 min. Note that it is heavily labeled with silver grains (X1050). (b) Labeling was not as heavy, though more uniform throughout the pituitary, in gonadotrophs of rats treated with ’ 25I-DAla6-GnRH-EA for 60 min. Note the hypertrophy and vacuolation of these castration cells in this rat ovariectomized for 6 weeks (X830). (c) When rats were pretreated with a 200-fold excess of unlabeled D-Ala6-GnRH-EA, uptake of the radiolabeled peptides was blocked. As a result silver grains were not localized over gonadotrophs in these rats (X830).
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after 10 min. The cytologic findings support this reasoning. While only a fraction of gonadotrophs were labeled at 1, 2 and 5 min, uniform labeling was seen after 60 min of recirculation. At this time the I-min pretreatment could be shown autoradiographically to have blocked uptake of GnRH-EA and D-Ala6-GnRH-EA. In the case of GnRH, longer periods of recirculation of unlabeled D-Ala6-GnRH-EA and labeled GnRH did not aid in the blocking of uptake of labeled GnRH as peak uptake of this peptide occurred within 2 min after injection. Uptake was blocked when 60 min was allowed for the recirculation and uniform distribution of unlabeled peptide, which saturated receptors and blocked binding of the radiolabeled GnRH administered thereafter. As noted the T/S in thyroid, liver and kidney are high, though specific binding could not be demonstrated. As suggested by Reeves et al. (1977) the thyroid is no doubt concentrating the iodine, not the peptides, while the liver and kidney are major sites of degradation and excretion. Hopkins and Gregory (1977) demonstrated binding of GnRH to the plasma membrane of gonadotrophs using a ferritin-GnRH conjugate. Plasma-membrane labeling was also seen in association with other secretory cell types, somatotrophs and thyrotrophs in particular. Only in gondadotrophs were lysosome-like structures found labeled with the ferritin-GnRH conjugate. In the study reported here silver grains were never found associated with any cell types except those immunostained for LHand then only with the interior of the cell, not in association exclusively with the periphery. If labeled peptide was bound to the smooth surface of the cell, it would be expected that as the 1251-disintegrated, electrons would be given off in a random fashion and silver grains would be found randomly distributed in and around the cell. As stated, silver grains were not found circumscribing gonadotrophs or over the plasma membrane in these 1-p sections, suggesting that the labeled peptide was indeed intracellular. The possibility exists that plasma-membrane bound counts were washed off during the washing and dehydration of the tissue. As there is no way of determining how much of the radiolabeled peptide which washed out was ever specifically bound, we chose to express uptake as total cpm/pituitary. While nonspecific counts may have been greater at early time points, this did not seem to distort the results as increased uptake over time could be demonstrated. While the use of frozen sections would have circumvented the loss of radioactivity by the washing and dehydration, the resolution of the autoradiographic localization would be much less in a 5-p section as compared to a 1-n section and a valid comparison could not be made. The fact that both analogs were taken up to greater degrees than GnRH offers one possible explanation for their increased biopotencies. This greater uptake is probably not due to differences in the half-life in the circulation as Reeves et al. (1977) showed that disappearance rates for GnRH and D-Ala6-G&H--EA did not differ significantly. Nor would it appear to be due to a difference in their binding affinities as the initial rates of uptake for all 3 peptides were similar, in agreement with work reported by Heber and Ode11 (1978). A third explanation for the
Specific uptake of GnRH by rat gonadotrophs
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increased uptake of the analogs remains to be considered, namely, the possibility that the increased uptake is due to binding of the analogs to a greater number of receptors. The initial binding of peptide to the receptor for GnRH immediately following injection may serve to stimulate the release of LH, resulting in the exocytosis of secretory granules. If receptors for GnRH are present in the limiting membrane of secretory granules as shown immunocytochemically by Sternberger and Petrali (1975), release of the contents of the granules and incorporation of the granule membrane into the plasma membrane would result in insertion of new receptors for GnRH into the plasma membrane, the presence of which could explain the increased uptake of analogs over time, as well as the inability to block uptake of labeled peptides at short time intervals. If new receptors were inserted via granules as LH was released, receptor numbers could theoretically be maintained or increase over the period during which LH was released. New receptors inserted in this manner would then be available to bind more labeled peptide, uptake increasing concomitantly’with the release of LH. As GnRH-EA and D-Ala6-GnRH-EA released more LH, more receptors would be available for more binding and greater uptake would result. Uptake would decrease either when the supply of new receptors was exhausted or when the concentration of peptide in the serum decreased. Such a phenomena may account for the self-priming effect of GnRH when consecutive doses are administered (Fink et al., 1976). Along this same line, if a pretreatment with unlabeled peptide served to release LH via exocytosis of granules and receptor numbers increased, those first new receptors would be occupied predominantly by the labeled peptide injected 1 min thereafter. If these rats were sacrificed 2 min later, the majority of receptors would be occupied by labeled peptide and it would not be possible to demonstrate a statistical difference in cpm/ pituitary or T/S between these animals and those treated for 2 min with labeled peptide only. However, if the labeled and unlabeled peptide were allowed to equilibrate and recirculate for a longer period, the unlabeled peptide could thereby effectively compete for receptors at later time points, preventing the binding of labeled peptide. This would then explain the inability to block uptake of all peptides at early time points. This explanation is not to negate that the longer retention times for the analogs shown here and the slower rates of degradation for analogs with position-6 substitutions as shown by Koch et al. (1977) might also play a part in the ability of the analogs to release significantly greater amounts of LH. It is intriguing to speculate as to the function the internalization of GnRH might play. If the receptor is internalized as well, it could be a means whereby the plasma membrane is ridded of occupied receptors. The receptor itself might have an intracellular action or it may be simply degraded or recycled. Farquhar (1978) has shown that pituitary plasma membrane may be recycled via the Golgi apparatus, so possibly receptors are recycled as well. Regardless of the fate of the receptor, the internalization of the GnRH could possibly serve one or several purposes, such as the stimulation of the release of a readily releasable pool of LH, regulation of the transfer of newly synthesized LH to a readily releasable pool, or regulation of the
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glycosylation of the LH as postulated by Liu and Jackson (1977). Since Hopkins and Gregory (1977) localized the ferritin-GnRH conjugate in lysosome-like structures, it may be that the GnRH is internalized only to be degraded. However, unconjugated ferritin can be localized in lysosomes as well (Farquhar, 1978), so it is difficult to assess whether this is the normal route for GnRH metabolism following internalization. Ultrastructural autoradiographic studies are in progress to determine what organelles the 1251-GnRH becomes associated with upon entry into gonadotrophs.
REFERENCES Baskin, D.G., Erlandsen, S.L., and Parsons, J.A. (1979) J. Histochem. Cytochem. 27,867-872. Burgus, R., Butcher, M., Ling, N., Monahan, M., Rivier, J., Fellows, R., Amoss, M., Blackwell, R., Vale, W., and Guillemin, R. (1971) C.R. Acad. Sci. (Paris) 272,1611-1613. Coy, D.H., Vilchez-Martinez, J.A., Coy, E.J., Arimura, A., and Schally, A.V. (1973) J. Clin. Endocrinol. Metab. 37, 331-333. Crowder, M.E., Moss, G.E., and Nett, T.M. (1980), submitted. Farquhar, M.G. (1978) J. Cell Biol. 77, R35-R42. Fink, G., Chiappa, S.A., and Aiyer, M.S. (1976) J. Endocrinol. 69, 359-372. Fujino, M., Kobayashi, S., Obayashi, M., Shinagawa, S., Fukuda, T., Kitada, C., Nakagama, R., Yamakazi, I., White, W.R., and Rippel, R.H. (1972) Biochem. Biophys. Res. Commun. 49, 863-869. Fujino, M., Fukuda, T., Shinagawa, S., Kobayashi, S., Yamazaki, I., Nakayama, R., Seely, J.H., White, W.R., and Rippel, R.H. (1974) Biochem. Biophys. Res. Commun. 60,406-413. Heber, D., and Odell, W.D. (1978) Biochem. Biophys. Res. Commun. 82,67-73. Hopkins, CR., and Gregory, H. (1977) J. Cell Biol. 75,528-540. Koch, Y., Baram, T., Hazum, E., and Fridkin, M. (1977) Biochem. Biophys. Res. Commun. 74, 488-491. Liu, T., and Jackson, G.L. (1977) Endocrinology 100, 1294-1302. Mason, T.E., Phifer, R.F., Spicer, S.S., Swallow, R.A., and Dreskin, R.B. (1969) J. Histochem. Cytochem. 92,231-238. Matsuo, H.Y., Baba, Y., Nair, R.M.G., Arimura, A., and Schally, A.V. (1971) Biochem. Biophys. Res. Commun. 43,1334-1339. Monahan, M.W., Am&s, MS., Anderson, H.A., and Vale, W. (1973) Biochemistry 12, 46164620. Nett, T.M., and Adams, T.E. (1977) Endocrinology 101,1135-1144. Niswender, G.D., Reichert Jr., L.E., Midgley Jr., A.R., and Nalbandov, A.V. (1969) Endocrinology 84,1166-l 173. Redding, T.W., and Schally, A.V. (1973) Life Sci. 12,23-32. Reeves, J.J., Tarnavsky, G.K., Becker, S.R., Coy, D., and Schally, A.V. (1977) Endocrinology 101,540-547. Schally, A.V. (1978) Science 202, 18-28. Sternberger, L.A., and Petrali, J.P. (1975) Cell Tiss. Res. 162, 141-176. Sternberger, L.A., Hardy Jr., P.H., Cuculis, J.J., and Meyer, H.G. (1970) J. Histochem. Cytothem. 18,315-333. Wagner, T.O.F., Adams, T.E., and Nett, T.M. (1979) Biol. Reprod. 20, 140-149.
0 Elsevier/North-Holland
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UPTAKE, LOCALIZATION, AND RETENTION OF GONADOTROPIN-RELEASING HORMONE AND GONADOTROPIN-RELEASING HORMONE ANALOGS IN RAT GONADOTROPHS * T.M. DUELLO ** and T.M. NETT Department of Physiology and Biophysics, College of Vetennary Medicine and Biomedical Sciences, Colorado State University, Fort CoNins, CO 80523 (U.S.A.)
Received 1 October 1979; accepted 27 March 1980
Uptake and retention of GnRH by pituitary was compared to that of GnRH-ethylamide (GnRH-EA) and D-Ala6 -GnRH-ethylamide (D-Ala6 -GnRH-EA) to determine if differences in these parameters might partially account for the increased biopotency of these superactive analogs. Ovariectomized estrogen/progesterone-treated rats were given an intracarotid injection of either 1251-labeledGnRH, GnRH-EA, or -D-Ala6-GnRH-EA. Maximum uptake occurred at 2 mm for GnRH (8000 cpm/pit), 5 min for GnRH-EA (10 000 cpm/pit), and 45 mm for D-Ala6 -GnRH-EA (24 000 cpm/pit). Thereafter pituitary content decreased out to 1, 2, and 4 h, resp. Specific uptake of all 3 peptides was shown autoradiographically to be restricted only to immunostained gonadotrophs with the silver grains being localized intracellularly at all time points studied. Serum concentrations of LH increased in response to all 3 peptides in proportion to their uptake and did not begin to decrease until after maximum uptake of each peptide had occurred. Thus, the extent of uptake and length of retention of these peptides in the pituitary correlate well with their relative biopotencies, D-Ala6-GnRH-EA > GnRH-EA > GnRH. Keywords:
GnRH; analogs; internalization;
LH.
Following elucidation of the amino acid sequence of gonadotropin-releasing hormone, or GnRH (Burgus et al., 1971; Matsuo et al., 1971) concerted efforts were directed at determining which amino acids were necessary for biological activity. While the entire molecule was shown to be involved in binding to receptor, the histidy1 and tryptophanyl residues in positions 2 and 3 appeared to directly affect biopotency. Simple substitutions, or deletions, at these positions significantly decreased or abolished gonadotropin-releasing activity (Coy et al., 1973; Monahan et al., 1973). In contrast, D-amino substitutions at position 6 and propylamide or ethylamide substitutions at position 10 resulted in molecules with enhanced abil-
* This work was supported by NIH Grants HD07841 and HD07031. ** To whom reprint requests should be sent.
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ities to release luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (Fujino et al., 1972, 1974; Monahan et al., 1973). Reeves et al. (1977) postulated that the enhanced biological activity of these analogs might reflect differences in their half-lives in the systemic circulation, longer binding to receptors, or increased binding due to a preferred molecular conformation. When 2 superactive analogs, both with substitutions at position 6 and 10, were compared to GnRH, no differences in their half-lives in serum were observed (Reeves et al., 1977) but both analogs were taken up to greater extents and retained in the pituitary for longer periods of time than the parent molecule. While differences in these parameters may account for differences in biopotency, additional explanations are possible. For example, Koch et al. (1977) showed that enzymic preparations from rat hypothalamus and anterior pituitary, known to preferentially cleave GnRH at the Gly6Leu’ position, were less effective in degrading analogs with substitutions at positions 6 and/or 7. Thus, they suggested that the rate of degradation of GnRH and GnRH analogs may be a physiological determinant of biopotency. In an effort to examine more closely the importance of uptake and retention in the regulation of the release of LH by GnRH, GnRH was compared to desGlyNH:‘-GnRH-ethylamide (GnRH-EA) and D-Ala6, des-GlyNH~“-GnRI-ethylamide (D-Ala6-G&H-EA). The biopotency of these peptides is 3-fold and . lo-30-fold greater than that of GnRH in a variety of different biological assays (cf. review by Schally, 1978).
MATERIALS
AND METHODS
Adult Sprague-Dawley rats, ovariectomized for 2-3 weeks, were each given a subcutaneous injection of 50 ng of estradiol benzoate and 25 mg of progesterone in safflower oil. 36 h later rats were anesthesized with Ketamine (12 mg/lOO g wt.)/ Acepromazine (0.25 mg/l.OO g wt.) after which the carotid artery was exposed and isolated. 3 groups of rats, a minimum of 56 rats per group, were given intracarotid injections of 17 pmoles of 1 of 3 ‘251-labeled peptides: GnRH (Parke Davis, Inc.), GnRH-EA (Abbott Laboratories), or D-Ala6-GnRH-EA (Dr. M. Fujino, Osaka, Japan). Radioiodination of hormones was performed as previously described (Wagner et al., 1979). Monoiodinated peptide was separated from diiodinated peptide on a 0.6 X 25 cm column of QAE Sephadex (Q-25-120; Sigma Chemical Co.). Essentially no free iodide elutes from this column (Wagner et al., 1979). The pattern of elution of unlabeled GnRH was determined by radioimmunoassay (Nett and Adams, 1977) and found to be less than 2% of the peak of monoiodinated peptide. All preparations were used within 24 h of radioiodination and were of identical specific activity (1574 Ci/mmole). Following the injection, the carotid was ligated to prevent excessive bleeding. The rats were decapitated and exsangumated at specific intervals thereafter at which time pituitary, thyroid, adrenal, liver, kidney, uterus, brain, and spleen tissue were removed, weighed, and the radioactivity present in
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each tissue was quantified using a Nuclear Chicago Automatic gamma spectrometer. Concentrations of LH in serum were determined by the radioimmunoassay method of Niswender et al. (1969). The ratio of tissue cpm/mg to serum cpm/pl (T/S) were used to express uptake in each tissue with significant uptake defined as a TS > 1. Tissue specificity of uptake was determined by measuring the ability of an excess of unlabeled peptide to block uptake of the labeled peptide. If uptake was specifically blocked the T/S should approximate 1. 8-10 rats in each group were given jugular injections of either a 200- or lOOO-fold excess of D-Ala6-GnRH-EA 1, 10 or 60 min prior to the injection of 1 of the 3 labeled peptides. D-Ala6GnRH-EA was used to block all 3 peptides since it had been shown to be retained by the pituitary longer than GnRH (Reeves et al., 1977). Therefore, we reasoned that it should be most effective in blocking the binding of the labeled peptides. In addition, it has been shown that D-Ala6-GnRH-EA (Crowder et al., 1980) dissociates from sheep pituitary homogenates approx. 7 times more slowly than GnRH (Wagner et al., 1979). To further examine the specificity of uptake by the pituitary, each of 6 rats were given an intracarotid injection of 17 pmoles of 1 of 2 1251labeled fragments of GnRH containing amino acids l-6 or amino acids 5-10. Uptake of each was determined as for the other ‘251-labeled peptides. Cellular specificity was assessed using a combined immunocytochemical-autoradiographic technique to determine whether the labeled peptides were taken up only by gonadotrophs. Pituitaries from rats in groups receiving 12’I-GnRH or -D-Ala6-GnRH-EA injections were cut into pieces approx. 1 mm3 and fixed for 24 h at 4°C in a 1% solution of glutaraldehyde in 0.1 M cacodylate buffer containing 5% sucrose (PH 7.3). The tissues were then washed in buffer, dehydrated in a graded series of ethanol, and embedded in Epon 812. Loss of radioactivity in each step was monitored. 1-I-(sections were cut, mounted on glass slides, and stored overnight in an oven at 60°C. The sections were then etched, dehydrated and bleached as described by Baskin et al. (1979) and incubated in Tris-buffered saline containing 0.25% gelatin for a minimum of 10 min. The immunoglobulin-enzyme bridge method of Mason et al. (1969), as modified by Sternberger et al. (1970) was used to immunostain gonadotrophs. The procedure included the following steps: (1) initial incubation of sections with rabbit antiserum to ovine LH (R440, provided by Dr. G.D. Niswender) at 1 : 10 000 at 4°C for 24 h; (2) incubation with sheep antirabbit y-globulin produced in this laboratory and used at 1 : 100 for 10 min; (3) incubation with horseradish peroxidase-anti-peroxidase (PAP, Cappel Laboratories) at 1 : 100 for 10 min; and (4) reaction of the peroxidase with a solution of 3,3’diaminobenzidine tetrahydrochloride (0.3 mg/ml; Sigma Chemical Co.) and Hz02 (0.05%) in a 0.05 M Tris buffer (pH 7.6) for l-5 min. Specificity controls entailed the substitution of normal rabbit serum (1 : 10 000) for the antiserum and incubation of the anti-LH serum with 1 pg/ml of ovine LH (NIH-oLHS19) for 48 h at 4’C. A physiologic control entailed ascertaining whether immunostaining was restricted to the castration cells seen in the pituitaries of additional rats ovariectomized for 5 or more weeks. Following incubation with the substrate solution,
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slides were rinsed in water, blotted dry, coated with Kodak NTB3 Nuclear Track Emulsion by the dip method, and stored in light proof dessicated boxes at 4’C for l-4 days. At the end of the exposure time, autoradiograms were developed in freshly prepared Kodak D-19 developer for 45 set and fixed in Kodak fixer for 4 min. The emulsion was scraped from the back of the slide with a razor blade whereupon the slides were air dried, coversripped, and viewed in a Zeiss microscope.
RESULTS at various Pituitary uptake of r2’I-GnRH, -GnRH-EA, and -D-Ala6-GnRH-EA intervals is shown in Fig. 1 (bars). Both analogs were taken up to greater extents and over longer periods of time than was GnRH. While pituitary content of GnRH began to decrease approx. 2 min after the iqection, pituitary content of GnRHEA and D-Alar’-GnRH-EA did not decrease until 5 and 45 min, resp. At these times pituitary content of the labeled analogs were 125 and 300% of that of GnRH
TIME (min) Fig. 1. Uptake by the pituitary of radiolabeled GnRH and analogs and the resultant release of LH. Rats were given intracarotid injections of 1 of the 3 radiolabeled peptides and the pituitaries removed at specified intervals. Pituitary content of radiolabeled peptide is indicated by the bars (left axis), while the LH released in response to each treatment is indicated by the stippled area (right axis).
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at 2 min. Injection of twice as much ‘251-D-Ala6-GnRH-EA did not result in greater uptake by the pituitary gland indicating that the concentration of peptide was not limiting uptake. While GnRH was retained in the pituitary for approx. 60 min, GnRH-EA and D-Ala6-GnRH-EA were retained much longer with the T/S remaining above 1 for 2 and 4 h, resp. All 3 radioiodinated preparations were biologically active as evidenced by the LH released in response to the injections (Fig. 1, stippled area), the magnitude of which could not be attributed to the 2% contamination with unlabeled hormone (unpublished data). Peak release of LH in response to GnRH and GnRH-EA was seen at 5min postinjection and did not return to basal concentrations for approx. 1 and 2 h, resp. However, at 5-min postinjection serum concentrations of LH due to GnRH-EA were 2.7 times greater than after injection of GnRH. At 45 min, peak serum concentrations of LH due to D-Ala6-GnRH-EA were 6.4 times greater than at 5 min after the injection of GnRH and had not returned to baseline by 4 h. When a 200-fold excess of unlabeled D-Ala6-GnRH-EA was given via the jugular vein 1 min prior to the intracarotid injection of labeled peptide, uptake of both GnRI-EA and D-Ala6-GnRH-EA by the pituitary was consistently blocked when the pituitaries were removed at time points longer than 10 min, i.e., the T/S approximated 1 (Table 1). This 1-min pretreatment failed to block uptake of GnRH by the pituitary as did a lo-min pretreatment, a 1-min pretreatment with a lOOOfold excess, or injection of both labeled and unlabeled peptide simultaneously. GnRH uptake was blocked, however, when the pretreatment entailed injection of a 200-fold excess of unlabeled peptide which was allowed to circulate for 60 min prior to the injection of the labeled peptide. This treatment presumably saturated receptors for GnRH as uptake could not be blocked further by pretreating with a 200-fold excess 60 min and again 1 min prior to the injection of the labeled peptide. The T/S for adrenal, uterus, brain, and spleen were consistently less than 1, for
Table 1 Uptake of radiolabeled peptides by pituitary of rats receiving no pretreatment of a pretreatment with unlabeled D-Ala6-GnRH-EA. Uptake of l2 5 I-GnRH could not be blocked by the 1-min pretreatment with unlabeled D-Ala6-GnRH-EA. Uptake of 12sIGnRH-EA and -DAla6-GnRH-EA could be blocked by this pretreatment at time points after 10 mm T/S t [ S.E.] Treatment
Time (min)
Treated
Cold-blocked
GnRH GnRH-EA D-Ala6 -GnRH-EA
2 20 60
1.3 f 0.2 3.4 f 0.4 11.2 f 0.9
1.3 f 0.2 1.3 f 0.2 1.6 +z0.7
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liver slightly greater than 1, and for thyroid and kidney they were often in excess of 10, indicating a greater degree of uptake of the peptide or a breakdown product of the peptide. Since none of these values were affected by any of the pretreatment regimens, specific uptake was restricted to pituitary tissue only. During the processing of pituitaries lo-SO% of the radioactivity was lost in the fixative and the first buffer wash. This was due to the fact that the concentration of labeled peptide was in excess of the dose necessary to occupy available receptors for GnRH on a single passage through the pituitary. This excess was necessary to insure that low serum concentrations of radiolabeled peptide would not limit further uptake on recirculation. However, that radiolabeled peptide retained in the pituitary throughout processing represented specific uptake as a combined immunocytochemical-autoradiographic method showed silver grains (i.e., labeled peptides) were restricted only to those cells which immunostained for LH. The immunocytochemical localization (Fig. 2a) was considered specific as: (1) no staining occurred when normal rabbit serum was substituted for the antiserum; (2) no staining occurred when the antiserum was absorbed with 1 fig/ml of LH (Fig. 2b); and (3) staining was restricted to the castration cells in the pituitaries of rats which had
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Fig, 2. (a) Immunostaining of gonadotrophs by antiserum to LH. @I The immunostaining was prevented when the antiserum was absorbed with 1 p&n1 LH. The astesisks indicate landmarks common to both fields (X670).
been avariectomized for 5 or mare weeks. At I, 2 and 5 min after injection of either “‘I-GnRH or -n-Ala6-GnRH-EA, a few cells which immunostained were shown autoradio~a~hica~y to be heaviiy labeled (Fig. 3a), though grains over vascular and ~nters~tia1 tissue in these areas were often abundant. However, if the labeled D-Ala’-GnRH-EA was allowed to circulate for 60 min, immunostained cells throughout the pituitary became uniformly labeled and vascular and interstitial labeling tended to decrease (Fig. 3b). At all times studied silver grains were localized only over the cytoplasm of the i~u~ost~e~ cells and were never observed over nucfei or circumscribing the cell at the plasma membrane. This localization was not affected by prior immunostaining of the tissue. The cellular uptake was considered spccitic since silver grains were not found over immunostained gonadotrophs in rats where uptake was blocked by the pretreatment with the unlabeled D-A&-G&II-EA (Fig_ 3~). Since GnRH has a half-life of approx. 7 min in the systemic circulation (Redding
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and Schally, 1973), the possibility existed that what was binding to gonadotrophs and being localized were radiolabeled fragments of the peptides. However, when fragments of GnRH containing amino acids l-6 or 5-10 were radiolabeled and injected, only nonspecific radioactivity remained associated with the pituitary 2 min after the injection and the T/S was always less than 1.
DISCUSSION The extent of uptake and length of retention of r2’I-GnRH, G&I-I--EA and -D-Ala6-GnRH-EA in the pituitary correlates well with the ability of each peptide to release LH as shown here and as reported previously (Reeves et al., 1977). GnRH-EA, which has the position-10 substitution only, was taken up to a greater extent, was retained longer, and caused greater release of LH than GnRH, though not to the extent of D-Ala6-GnRI-EA, which has both the position-6 and -10 substitutions. This greater uptake of both superactive analogs is reflected in their higher T/S. While the T/S for GnRH uptake by pituitary is only slightly greater than 1, the ratios for aII 3 peptides are probably deceptively low at intervals of 5 min or less since serum cpm/fi are artifactually high in the exsanguinated blood. This is probably due to the fact that the radioactivity was not equilibrated with the entire fluid volume of the rat due to ligature of the carotid artery through which the labeled peptide was injected. Regardless of whether the T/S is low at time points under 5 min, it would nonetheless have been expected that this value would decrease if uptake were indeed blocked by the unlabeled peptide. This, however, was not the case. The factors responsible for the inability to consistently block uptake at time points earlier than 10 min are no doubt the same as those responsible for the inability to block uptake of GnRH at all It was assumed that the majority of the binding would take place during the first passage through the pituitary, the peptide then becoming diluted in the entire systemic circulation. However, just as the labeled GnRI--EA and the D-Ala6-G&I-I-EA required repeated passage for maximal uptake, the unlabeled peptide as well apparently required more than 1 min for receptor saturation throughout the pituitary to occur. Only when the unlabeled D-Ala6-GnRH-EA, present in a 200-fold excess, was allowed to recirculate with the labeled G&H-EA or D-Ala6-GnRI-EA, could it effectively compete for receptors at time points
Fig. 3. (a) An immunostained gonadotroph in the pituitary of a rat treated with ’ 2s IGnRH for 2 min. Note that it is heavily labeled with silver grains (X1050). (b) Labeling was not as heavy, though more uniform throughout the pituitary, in gonadotrophs of rats treated with ’ 25I-DAla6-GnRH-EA for 60 min. Note the hypertrophy and vacuolation of these castration cells in this rat ovariectomized for 6 weeks (X830). (c) When rats were pretreated with a 200-fold excess of unlabeled D-Ala6-GnRH-EA, uptake of the radiolabeled peptides was blocked. As a result silver grains were not localized over gonadotrophs in these rats (X830).
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after 10 min. The cytologic findings support this reasoning. While only a fraction of gonadotrophs were labeled at 1, 2 and 5 min, uniform labeling was seen after 60 min of recirculation. At this time the I-min pretreatment could be shown autoradiographically to have blocked uptake of GnRH-EA and D-Ala6-GnRH-EA. In the case of GnRH, longer periods of recirculation of unlabeled D-Ala6-GnRH-EA and labeled GnRH did not aid in the blocking of uptake of labeled GnRH as peak uptake of this peptide occurred within 2 min after injection. Uptake was blocked when 60 min was allowed for the recirculation and uniform distribution of unlabeled peptide, which saturated receptors and blocked binding of the radiolabeled GnRH administered thereafter. As noted the T/S in thyroid, liver and kidney are high, though specific binding could not be demonstrated. As suggested by Reeves et al. (1977) the thyroid is no doubt concentrating the iodine, not the peptides, while the liver and kidney are major sites of degradation and excretion. Hopkins and Gregory (1977) demonstrated binding of GnRH to the plasma membrane of gonadotrophs using a ferritin-GnRH conjugate. Plasma-membrane labeling was also seen in association with other secretory cell types, somatotrophs and thyrotrophs in particular. Only in gondadotrophs were lysosome-like structures found labeled with the ferritin-GnRH conjugate. In the study reported here silver grains were never found associated with any cell types except those immunostained for LHand then only with the interior of the cell, not in association exclusively with the periphery. If labeled peptide was bound to the smooth surface of the cell, it would be expected that as the 1251-disintegrated, electrons would be given off in a random fashion and silver grains would be found randomly distributed in and around the cell. As stated, silver grains were not found circumscribing gonadotrophs or over the plasma membrane in these 1-p sections, suggesting that the labeled peptide was indeed intracellular. The possibility exists that plasma-membrane bound counts were washed off during the washing and dehydration of the tissue. As there is no way of determining how much of the radiolabeled peptide which washed out was ever specifically bound, we chose to express uptake as total cpm/pituitary. While nonspecific counts may have been greater at early time points, this did not seem to distort the results as increased uptake over time could be demonstrated. While the use of frozen sections would have circumvented the loss of radioactivity by the washing and dehydration, the resolution of the autoradiographic localization would be much less in a 5-p section as compared to a 1-n section and a valid comparison could not be made. The fact that both analogs were taken up to greater degrees than GnRH offers one possible explanation for their increased biopotencies. This greater uptake is probably not due to differences in the half-life in the circulation as Reeves et al. (1977) showed that disappearance rates for GnRH and D-Ala6-G&H--EA did not differ significantly. Nor would it appear to be due to a difference in their binding affinities as the initial rates of uptake for all 3 peptides were similar, in agreement with work reported by Heber and Ode11 (1978). A third explanation for the
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increased uptake of the analogs remains to be considered, namely, the possibility that the increased uptake is due to binding of the analogs to a greater number of receptors. The initial binding of peptide to the receptor for GnRH immediately following injection may serve to stimulate the release of LH, resulting in the exocytosis of secretory granules. If receptors for GnRH are present in the limiting membrane of secretory granules as shown immunocytochemically by Sternberger and Petrali (1975), release of the contents of the granules and incorporation of the granule membrane into the plasma membrane would result in insertion of new receptors for GnRH into the plasma membrane, the presence of which could explain the increased uptake of analogs over time, as well as the inability to block uptake of labeled peptides at short time intervals. If new receptors were inserted via granules as LH was released, receptor numbers could theoretically be maintained or increase over the period during which LH was released. New receptors inserted in this manner would then be available to bind more labeled peptide, uptake increasing concomitantly’with the release of LH. As GnRH-EA and D-Ala6-GnRH-EA released more LH, more receptors would be available for more binding and greater uptake would result. Uptake would decrease either when the supply of new receptors was exhausted or when the concentration of peptide in the serum decreased. Such a phenomena may account for the self-priming effect of GnRH when consecutive doses are administered (Fink et al., 1976). Along this same line, if a pretreatment with unlabeled peptide served to release LH via exocytosis of granules and receptor numbers increased, those first new receptors would be occupied predominantly by the labeled peptide injected 1 min thereafter. If these rats were sacrificed 2 min later, the majority of receptors would be occupied by labeled peptide and it would not be possible to demonstrate a statistical difference in cpm/ pituitary or T/S between these animals and those treated for 2 min with labeled peptide only. However, if the labeled and unlabeled peptide were allowed to equilibrate and recirculate for a longer period, the unlabeled peptide could thereby effectively compete for receptors at later time points, preventing the binding of labeled peptide. This would then explain the inability to block uptake of all peptides at early time points. This explanation is not to negate that the longer retention times for the analogs shown here and the slower rates of degradation for analogs with position-6 substitutions as shown by Koch et al. (1977) might also play a part in the ability of the analogs to release significantly greater amounts of LH. It is intriguing to speculate as to the function the internalization of GnRH might play. If the receptor is internalized as well, it could be a means whereby the plasma membrane is ridded of occupied receptors. The receptor itself might have an intracellular action or it may be simply degraded or recycled. Farquhar (1978) has shown that pituitary plasma membrane may be recycled via the Golgi apparatus, so possibly receptors are recycled as well. Regardless of the fate of the receptor, the internalization of the GnRH could possibly serve one or several purposes, such as the stimulation of the release of a readily releasable pool of LH, regulation of the transfer of newly synthesized LH to a readily releasable pool, or regulation of the
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glycosylation of the LH as postulated by Liu and Jackson (1977). Since Hopkins and Gregory (1977) localized the ferritin-GnRH conjugate in lysosome-like structures, it may be that the GnRH is internalized only to be degraded. However, unconjugated ferritin can be localized in lysosomes as well (Farquhar, 1978), so it is difficult to assess whether this is the normal route for GnRH metabolism following internalization. Ultrastructural autoradiographic studies are in progress to determine what organelles the 1251-GnRH becomes associated with upon entry into gonadotrophs.
REFERENCES Baskin, D.G., Erlandsen, S.L., and Parsons, J.A. (1979) J. Histochem. Cytochem. 27,867-872. Burgus, R., Butcher, M., Ling, N., Monahan, M., Rivier, J., Fellows, R., Amoss, M., Blackwell, R., Vale, W., and Guillemin, R. (1971) C.R. Acad. Sci. (Paris) 272,1611-1613. Coy, D.H., Vilchez-Martinez, J.A., Coy, E.J., Arimura, A., and Schally, A.V. (1973) J. Clin. Endocrinol. Metab. 37, 331-333. Crowder, M.E., Moss, G.E., and Nett, T.M. (1980), submitted. Farquhar, M.G. (1978) J. Cell Biol. 77, R35-R42. Fink, G., Chiappa, S.A., and Aiyer, M.S. (1976) J. Endocrinol. 69, 359-372. Fujino, M., Kobayashi, S., Obayashi, M., Shinagawa, S., Fukuda, T., Kitada, C., Nakagama, R., Yamakazi, I., White, W.R., and Rippel, R.H. (1972) Biochem. Biophys. Res. Commun. 49, 863-869. Fujino, M., Fukuda, T., Shinagawa, S., Kobayashi, S., Yamazaki, I., Nakayama, R., Seely, J.H., White, W.R., and Rippel, R.H. (1974) Biochem. Biophys. Res. Commun. 60,406-413. Heber, D., and Odell, W.D. (1978) Biochem. Biophys. Res. Commun. 82,67-73. Hopkins, CR., and Gregory, H. (1977) J. Cell Biol. 75,528-540. Koch, Y., Baram, T., Hazum, E., and Fridkin, M. (1977) Biochem. Biophys. Res. Commun. 74, 488-491. Liu, T., and Jackson, G.L. (1977) Endocrinology 100, 1294-1302. Mason, T.E., Phifer, R.F., Spicer, S.S., Swallow, R.A., and Dreskin, R.B. (1969) J. Histochem. Cytochem. 92,231-238. Matsuo, H.Y., Baba, Y., Nair, R.M.G., Arimura, A., and Schally, A.V. (1971) Biochem. Biophys. Res. Commun. 43,1334-1339. Monahan, M.W., Am&s, MS., Anderson, H.A., and Vale, W. (1973) Biochemistry 12, 46164620. Nett, T.M., and Adams, T.E. (1977) Endocrinology 101,1135-1144. Niswender, G.D., Reichert Jr., L.E., Midgley Jr., A.R., and Nalbandov, A.V. (1969) Endocrinology 84,1166-l 173. Redding, T.W., and Schally, A.V. (1973) Life Sci. 12,23-32. Reeves, J.J., Tarnavsky, G.K., Becker, S.R., Coy, D., and Schally, A.V. (1977) Endocrinology 101,540-547. Schally, A.V. (1978) Science 202, 18-28. Sternberger, L.A., and Petrali, J.P. (1975) Cell Tiss. Res. 162, 141-176. Sternberger, L.A., Hardy Jr., P.H., Cuculis, J.J., and Meyer, H.G. (1970) J. Histochem. Cytothem. 18,315-333. Wagner, T.O.F., Adams, T.E., and Nett, T.M. (1979) Biol. Reprod. 20, 140-149.