Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Thyroid Hormone Regulation of Peptidylglycine a-Amidating Monooxygenase Expression in Anterior Pituitary Gland L'Houcine Ouafik, Victor May, David W. Saffen, and Betty A. Eipper Department of Neuroscience Johns Hopkins University School of Medicine Baltimore, Maryland 21205 Department of Anatomy and Neurobiology University of Vermont College of Medicine (V.M.) Burlington, Vermont 05405 a large fraction of the anterior pituitary cell population upon surgical thyroidectomy. These results indicate that thyroid hormones are involved, either directly or indirectly, in regulating the expression of PAM in several cell types in the anterior pituitary gland. (Molecular Endocrinology 4: 1497-1505, 1990) Peptidylglycine a-amidating monooxygenase (PAM; EC 1.14.17.3) is a copper-, molecular oxygen-, and ascorbate-dependent enzyme which catalyzes the COOH-terminal amidation of bioactive peptides. Expression of PAM in the adult male rat anterior pituitary was evaluated after experimental manipulation of thyroid status. Levels of PAM mRNA increased 4- to 7-fold in animals made hypothyroid by treatment with 6-n-propyl-2-thiouracil or thyroidectomy and were not diminished below control levels in animals made hyperthyroid by treatment with T4. Treatment of thyroidectomized animals with T4 prevented the increase in PAM mRNA levels; similar doses of T4 returned serum TSH and anterior pituitary PAM mRNA to euthyroid values. Based on Northern blot analysis and amplification of fragments derived from rat PAM-1 by reverse transcription and the polymerase chain reaction, thyroid status did not affect the distribution of PAM mRNA among its various alternatively spliced forms. The specific activity of PAM in the anterior pituitary was increased slightly in both the soluble and particulate fractions from chemically hypothyroid rats; the majority of the PAM activity in the rat anterior pituitary was soluble, and increased secretion of enzyme may account for the lesser effect of chemical thyroidectomy on specific activity compared to mRNA levels. Western blot analysis demonstrated a 104-kDa PAM protein in particulate fractions prepared from control, PTUtreated, and T4-treated animals. The soluble fraction contained major PAM proteins of 95 and 75 kDa, and PTU treatment brought about an increase in the prevalence of the 75-kDa form of PAM protein. In situ hybridization studies using 35S-labeled fulllength RNA antisense transcripts of rat PAM-1 cDNA demonstrated an increase in levels of PAM mRNA in INTRODUCTION Peptidylglycine a-amidating monooxygenase (PAM; EC 1.14.17.3) is an essential posttranslational processing enzyme in the biosynthesis of a-amidated peptides (1, 2). PAM produces a-amidated peptide products from a variety of glycine-extended peptide substrates in a copper-, molecular oxygen-, and ascorbate-dependent manner. Complementary DNAs encoding PAM have been isolated from bovine intermediate pituitary, rat heart atrium, and frog skin libraries (3-6). The three species express mRNAs encoding PAM precursor proteins of approximately 100 kDa with an amino-terminal signal sequence, a large intragranular catalytic domain, a hydrophobic transmembrane domain, and a short cytoplasmic tail. PAM RNA transcripts undergo tissuespecific and developmentally regulated alternative splicing (4, 7). Soluble and membrane-associated PAM activities have been identified, and their distribution is tissue specific (8). Studies with purified PAM and PAM produced by AtT-20 mouse corticotropic tumor cells transfected with cDNA encoding bovine PAM indicate that a single enzyme can a-amidate a wide variety of amino acids (1, 2, 9, 10). Levels of amidation activity and PAM mRNA are regulated in a tissue-specific fashion in response to various drug treatments and endocrine manipulations both in vivo and in vitro (2, 11). 0888-8809/90/1497-1505$02.00/0 Molecular Endocrinology Copyright © 1990 by The Endocrine Society Although the major anterior pituitary hormones are not a-amidated, PAM levels in the anterior pituitary 1497 Vol4No. 10 MOL ENDO-1990 1498 gland are among the highest in rat tissues (12, 13). Several a-amidated peptides, including substance-P, neuropeptide-Y, and vasoactive intestinal peptide (VIP), have been identified in the rat anterior pituitary gland (14-16). Hypothyroidism has been shown to greatly increase the expression of each of these a-amidated peptides in the anterior pituitary gland (16-18). Previous studies demonstrated an effect of thyroidectomy on levels of PAM activity in serum and soluble PAM activity in the anterior pituitary (2). Although the effects of thyroid hormones on many cellular metabolic processes have been well documented (19, 20), little is known about the effects of thyroid hormones on peptide-processing enzymes. In the present study we have used several techniques to examine the effects of thyroid status on PAM expression in the anterior pituitary gland. Levels of PAM mRNA were evaluated using electrophoretic blot hybridization analysis, and changes in PAM mRNA forms were investigated using reverse transcription, followed by the polymerase chain reaction. Tissue levels of PAM activity were measured, and PAM protein forms were examined by Western blot analysis. In situ hybridization studies were conducted to determine whether changes in thyroid hormone status altered PAM mRNA levels in all or only a fraction of the total cell population of the anterior pituitary gland. RESULTS Regulation of PAM Expression by Thyroid Status In the first experimental paradigm, adult male rats were made hypothyroid by treatment with 6-n-propyl-2-thiouracil (PTU) or hyperthyroid by treatment with L-T4 (T4). The effectiveness of the treatment was verified by measurement of serum TSH levels (Fig. 1B). After treatment with T4, serum TSH levels were reduced compared to those in vehicle-injected control animals; similarly, serum TSH levels were elevated over control values in the PTU-treated hypothyroid rats. Anterior pituitary PAM expression in hyperthyroid and hypothyroid rats was assessed by Northern blot analysis to examine PAM mRNA levels and forms and by measurement of PAM activity. Total RNA prepared from the pituitaries of individual rats was subjected to Northern blot analysis, and PAM mRNA was visualized using a radiolabeled fragment derived from the 5' region of rat PAM-1 (rPAM-1) cDNA and capable of detecting all of the known forms of rat PAM mRNA (Fig. 1A). Pituitary PAM mRNA ranged from 3.6-3.8 kilobases in size, and the size distribution was unaltered by thyroid status. The PAM cDNA probe was removed from the blots, and the amount of ribosomal RNA present in each sample was determined by hybridization to a cDNA probe for ribosomal RNA (Fig. 1 A). The amount of PAM mRNA in each sample was then normalized to the amount of ribosomal RNA (Fig. 1B). Anterior pituitary PAM mRNA levels in the hypothyroid rats were increased 6.7 ± 0.6-fold over control values (n = 3; mean CONTROL PTU PAM 18S tf f iw^ * 0.8 •12.0 0.4 0.0 Con PTU Fig. 1. Effect of Thyroid Status on Expression of PAM mRNA in Anterior Pituitary A, Total RNA (10 ng) from pituitaries of individual euthyroid (control), hyperthyroid (T4), and hypothyroid (PTU) rats was fractionated on a denaturing 1 % agarose gel and transferred to Nytran. The blot was hybridized with a full-length rPAM-1 cDNA probe and exposed to x-ray film for 24 h at - 7 0 C with an intensifying screen. The blot was subsequently stripped and hybridized with the ribosomal RNA probe (18S). B, After densitization of the autoradiograms using computer-assisted densitometry and correction for nonlinearity of film grain density, levels of PAM mRNA were normalized to levels of ribosomal RNA on the same blot; this arbitrary ratio was used to express relative tissue PAM mRNA levels (•). Serum TSH levels (H) were assayed for each experimental animal, as described in Materials and Methods. Error bars indicate the SD. Stars indicate that the value is significantly different from the control (•, P < 0.05; • * , P < 0.005). Similar data were obtained in two additional independent experiments. ± SEM). There was no significant difference in anterior pituitary PAM mRNA levels between hyperthyroid and control animals. Since thyroid status had a dramatic effect on levels of PAM mRNA, the effect of thyroid status on anterior pituitary PAM specific activity was also assessed (Fig. 2). Treatment with PTU increased total PAM specific activity by approximately 60%, while treatment with T4 brought about a slight decrease in the specific activity of PAM in the anterior pituitary gland. The majority of the PAM activity (80-90%) was recovered in the soluble fraction, independent of thyroid status, and the specific activity of the soluble and particulate fractions mirrored the total specific activity (Fig. 2). The change in PAM specific activity after PTU treatment was in the same direction as the change in levels of PAM mRNA, but the magnitude of the change in activity was much smaller. This discrepancy could reflect increased secretion of PAM from the tissue or an alteration in the PAM protein present. Serum levels of PAM activity are in- Thyroid Hormone Regulation of PAM Expression 1499 A B T4 CON PTU T4 CON PTU —116 — 104 — — 97 — m — 66 — -i til •1 —104 . — 95 |— 84 — 75 —45- CONTROL PTU Fig. 2. Effect of Thyroid Status on PAM Specific Activity in Anterior Pituitary PAM specific activity in crude soluble (H) and paniculate (•) fractions prepared from two pooled anterior pituitary glands was measured as described in Materials and Methods. Total PAM specific activity (•) was calculated by taking into account the amount of protein in the two fractions. Data from two separate experiments (n = 4 for each treatment group in both experiments) were used to calculate the mean specific activity; each sample was assayed in duplicate. Error bars indicate the SEM. The asterisk indicates that the value is significantly different from the control (P < 0.05). creased in hypothyroidism (21), although the mechanism responsible for this increase has not been elucidated. To determine whether the forms of PAM protein present in the anterior pituitary were affected by thyroid status, equal amounts of protein prepared from the soluble and washed particulate fractions from control, PTU-treated, and T4-treated animals were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to Western blot analysis (Fig. 3). The antiserum used to visualize PAM proteins was raised to a synthetic peptide [bPAM-(561-579)] located in the intragranular domain of the bovine PAM precursor protein and was affinity purified before use. The major PAM protein in all of the anterior pituitary particulate fractions had a mass of 104 ± 2 kDa (Fig. 3A) and was also visualized by antiserum to a synthetic peptide [bPAM-(288-310)] located in the monooxygenase domain of the PAM precursor protein (data not shown). The qualitative pattern observed for the particulate fraction was independent of thyroid status. Analysis of the soluble fraction revealed the presence of multiple forms of PAM protein (Fig. 3B). In control and hyperthyroid animals, soluble PAM proteins of 95 and 75 kDa were predominant, with lesser amounts of 104- and 84-kDa PAM proteins. The same forms of PAM protein were present in PTU-treated animals, but the 75-kDa PAM protein was more prevalent. Consistent with the magnitude of the effect of thyroid status Fig. 3. Western Blot Analysis Two independent aliquots of particulate protein (5 ^g from T4-treated animals; 20 nQ from controls and PTU-treated animals; A) or soluble protein (50 ^g; B) prepared from the pooled pituitaries of two T4-treated, control (CON), or PTU-treated rats were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PAM proteins were visualized with affinity-purified antiserum to bPAM-(561 -579) and [125l]proteinA. The migration positions of the mol wt markers are indicated (center). The mol wt of PAM in the particulate fraction is shown on the left; the mol wt of the soluble PAM proteins are on the right. on PAM specific activity, the soluble protein from PTUtreated animals yielded a somewhat more intense signal than the same amount of protein from control or T4treated animals. Precise quantitative comparisons cannot yet be made, since the cross-reactivity of the antiserum with the various forms of PAM protein is not yet known. Proteins of the same mass were visualized with antisera to the monooxygenase domain of PAM (data not shown). Many of the PAM mRNAs that have been characterized encode precursor proteins that appear to undergo endoproteolytic cleavage to generate the major forms of PAM protein found in tissue extracts (2, 4, 8,13), and the 75-kDa PAM protein is thought to derive from larger precursor forms of PAM protein by endoproteolytic cleavage.1 Thus, the posttranslational processing of the PAM precursor in the pituitary appears to be sensitive to thyroid status. Thyroid Hormone Replacement Studies In the second experimental paradigm, the effect of thyroid hormone replacement on PAM expression in thyroidectomized rats was evaluated. Similar to hypothyroidism induced by PTU treatment, hypothyroidism caused by surgical thyroidectomy resulted in a 3.7 ± 1.1-fold (n = 2; mean ± range) increase in the level of PAM mRNA in the anterior pituitary gland. When the thyroidectomized animals were treated with increasing doses of T4, serum TSH levels declined from the elevated levels observed in hypothyroid animals to levels below those in euthyroid animals (Fig. 4B). When total RNA from the individual animals was fractionated on 1 Eipper, B. A., C. B.-R. Green, D. A. Stoffers, H. T. Keutmann, and L'H. Ouafik, manuscript in preparation. MOL ENDO-1990 1500 VoUNo. 10 A THYROXINE(«g) THX 0.25 r o 4 4 Ii 40 64 PAM THX 0.25 4.0 16 in the rat (Fig. 5, top) (4, 22). In the atrium, the major forms of PAM mRNA differ by the presence (rPAM-1, 4.2 kb) and absence (rPAM-2, 3.8 kb) of optional exon A (4); forms of PAM mRNA lacking all or part of optional exon B are found in the atrium (22), but are more prevalent in the pituitary (see Footnote 1). The forms of PAM mRNA in the pituitary are poorly resolved on denaturing agarose gels. To determine whether thyroid status altered the forms of PAM mRNA present in the anterior pituitary, cDNA was prepared by reverse transcription of RNA prepared from control, thyroidectomized, PTU-treated, and T4-treated animals. Pairs of oligonucleotides spanning the sequence of rPAM-1 were then used as primers in the polymerase chain reaction (Fig. 5). These studies were carried out to provide a comparison of the forms of PAM mRNA present; internal standards permitting correction for differing efficiencies of reverse transcription or amplifi- 64 fig T 4 Fig. 4. Effect of Thyroidectomy and T4 Replacement on Anterior Pituitary PAM mRNA Expression A, Total RNA (10 ng) from individual animals in each treatment group was subjected to Northern blot analysis, as described in Fig. 1A. The blot was hybridized first with the PAM cDNA probe and then stripped and hybridized with the ribosomal RNA probe. Similar results were observed in two independent experiments. B, Quantitative analysis of the blots shown in A was performed as described in Fig. 1B. Error bars indicate the range. Serum TSH levels in individual (n = 4) animals were measured as described; error bars indicate the SD. The serum TSH level in euthyroid animals was 1.4 ng/ml. denaturing agarose gels, treatment with increasing doses of T4 reduced PAM mRNA levels to control values (Fig. 4). As after treatment with PTU, no alteration in the size distribution of PAM mRNA could be discerned. When the levels of PAM mRNA were densitized and normalized to levels of ribosomal mRNA in each sample, the decline in anterior pituitary PAM mRNA expression was seen to occur over the same range of T4 that produced a decline in serum TSH to euthyroid levels (Fig. 4B). Thyroid hormone replacement decreased PAM mRNA levels maximally 4-fold from the elevated levels observed in hypothyroid animals; in agreement with the previous study, treatment with doses of T4 high enough to induce hyperthyroidism failed to reduce PAM mRNA levels below control values. As observed previously, levels of PAM activity in the serum of thyroidectomized rats were elevated over control levels (21). Effect of Thyroid Hormone Treatment on Expression of Different Alternatively Spliced Forms of PAM mRNA Multiple forms of PAM mRNA thought to arise via alternative splicing of a single gene have been identified AUG STOP 4 A B 94 -P771 ^_' To A rPAM-1 12 3 4 5/19 B — 1089 12 3 4 5 4/10 —1514 "1460 —1256 9/21 — 710 Fig. 5. Combined Reverse Transcriptase Polymerase Chain Reaction Demonstration of Alternatively Spliced Forms of PAM mRNA Schematic diagram of rPAM-1 cDNA shows the positions and orientations of oligonucleotide primers used for amplification. The initiation (AUG) and termination (STOP) codons as well as the positions of optional exons A and B (H) are shown. The PCR products obtained with the pairs of primers indicated on the left were fractionated on agarose gels, as described in Materials and Methods. The number of basepairs present in the amplified products derived from appropriate plasmid controls is indicated on the right. With primers 4/10 (which span optional exons A and B) the 1514-bp product corresponds to rPAM-2, and the 1256-bp product corresponds to rPAM-3 (22); the 1460-bp fragment derives from a form of PAM mRNA lacking part of optional exon B (see Footnote 1). Samples in each of the lanes are from 1) control, 2) T4-treated (40 »g T4/ day), 3) PTU-treated, and 4) thyroidectomized animals. Lane 5 in B is a zero DNA control. Thyroid Hormone Regulation of PAM Expression cation were not included in the samples, and the results do not provide a quantitative comparison of levels of PAM mRNA in different samples. Amplified fragments were fractionated on agarose gels and identified by comparison to fragments amplified from plasmids containing cDNA inserts corresponding to each of the various types of rPAM mRNA (Fig. 5). The first pair of oligonucleotides (5/19) spans the region from the 5' end of rPAM-1 to immediately before optional exon A; the second pair of oligonucleotides (4/10) spans optional exons A and B; the third pair (9/21) extends from the 3' end of optional exon B to immediately before the putative poly(A) addition signal. Based on amplification using these three sets of primers, thyroid status had no effect on the alternatively spliced forms of rPAM mRNA in the anterior pituitary; this observation is consistent with the results of Northern blot analysis, which failed to demonstrate an alteration in the mol wt pattern of PAM mRNAs after manipulation of thyroid status. In Situ Hybridization Studies 1501 of resolution, the increase in grain density appeared uniform across the entire anterior pituitary gland. The grain density diminished upon thyroid hormone replacement (Fig. 6, C and D). To determine the fraction of anterior pituitary cells exhibiting altered PAM expression in response to thyroid status, the tissue sections were exposed to photographic emulsion for 1 week and examined under brightfield microscopy (Fig. 7). In the control animals, cells expressing PAM mRNA were found scattered throughout the anterior pituitary gland. As observed previously, a subset of the cells was heavily labeled, but few cells were completely devoid of grains. This result is consistent with the immunocytochemical localization of high levels of PAM to a subset of gonadotropes, with moderate levels of PAM in corticotropes and lower levels in somatotropes and lactotropes (23). After thyroidectomy, PAM mRNA expression increased dramatically in a large percentage of the pituitary cells; the increase in PAM expression was clearly not limited to the population of anterior pituitary cells exhibiting The anterior pituitary gland is composed of several different cell types. To determine whether alterations in thyroid status affected PAM mRNA expression in a subset of the cell population, frozen anterior pituitary tissue from control rats, thyroidectomized rats, and thyroidectomized rats treated with T4 were prepared for in situ hybridization studies. Autoradiographic grains from the antisense PAM probe in the control anterior pituitary tissue represented basal PAM expression. In the thyroidectomized animals, the density of the autoradiographic grains increased significantly compared to that in controls (compare Fig. 6, A and B); at this level Fig. 6. Effect of Thyroid Hormone on Anterior Pituitary PAM mRNA Expression by in Situ Hybridization Anterior pituitary glands from control (A), thyroidectomized (B), and thyroidectomized rats given either 16 ^9 (C) or 4 ng (D) T4 replacement were separated from the neurointermediate lobe of the pituitary and embedded together, cryosectioned, and processed for in situ hybridization using 35S-labeled antisense PAM probe, as described in Materials and Methods. The sections were exposed to x-ray film for 3 days; the figure was printed directly from the autoradiogram. Heavy grain densities are represented by white areas. Fig. 7. High Resolution in Situ Hybridization for Anterior Pituitary PAM mRNA Cryosections from the study described in Fig. 6 were dipped in photographic emulsion for cellular resolution of autoradiographic grains. After a 1-week exposure period, the tissues were processed and stained with toluidine blue. Anterior pituitary glands from control (A) and thyroidectomized (B) rats were photographed in the brightfield mode. Scale bar = 50 ^m. MOL ENDO-1990 1502 high expression of PAM in the basal or euthyroid state. Grain density appeared to be increased in most of the cells of the anterior pituitary gland after thyroidectomy, with a subset of the cells exhibiting greatly enhanced grain densities compared to the majority of the cells. DISCUSSION These results demonstrate that PAM expression in the rat anterior pituitary is regulated by thyroid hormone. In the present study we showed that anterior pituitary PAM expression increased in hypothyroidism. Total PAM specific activity increased approximately 60% in PTU-treated rats, and PAM mRNA levels were elevated 4- and 7-fold in thyroidectomized and PTU-treated rats, respectively. The amount of PAM activity in pituitary extracts represents a balance among synthesis, storage, and secretion of the enzyme. Thus, the 7-fold increase in PAM mRNA levels observed in PTU-treated hypothyroid rats resulted in only a 60% increase in total PAM specific activity (Fig. 2). As observed previously (21), the anterior pituitary glands of surgically thyroidectomized rats exhibited a slight decrease in soluble (and total) PAM specific activity (data not shown) despite an increase in levels of PAM mRNA (Fig. 4). Similarly, elevated levels of preprohormone mRNA can be accompanied by reduced tissue levels of product peptide if secretion is stimulated sufficiently. Injection of hypothyroid animals with T4 restored PAM mRNA to control levels. By in situ hybridization, silver grains accumulated over the cytoplasm of groups of cells scattered throughout the anterior pituitary gland of the hypothyroid animals (Fig. 6, A and B). These results are interesting in light of the stimulatory effect of hypothyroidism on the production of many aamidated peptides synthesized in the anterior pituitary gland. Substance-P content in the anterior pituitary is markedly enhanced by hypothyroidism, restored to normal by T4 replacement, and reduced markedly below control levels in hyperthyroidism (16). Furthermore, the expression of preprotachykinin-A mRNA in the anterior pituitary gland is increased in hypothyroidism and suppressed below control values by T4 treatment (24). The effects of thyroidectomy on anterior pituitary neuropeptide-Y and VIP levels are strikingly similar to those described for substance-P (17,18). It will be of interest to use anterior pituitary tissue from hypothyroid rats as a model to study the colocalization of these peptides with PAM, since some of the peptides are present in very low abundance under normal physiological conditions. VIP cells, for example, are undetectable in normal rats, but increase dramatically in number after thyroidectomy (18,25). Coordinate regulation of expression of PAM and another a-amidated peptide, TRH, was previously observed during development of neonatal rat pancreas (26). Paracrine interactions mediated by some of these aamidated peptides may well play a role in the alterations Vol4No. 10 in pituitary function that occur in hypothyroidism and hyperthyroidism. Many anterior pituitary cell types, including somatotropes, lactotropes, and thyrotropes, are known to be responsive to thyroid hormones (2729). The function of the hypothalamic-pituitary-adrenal axis is also affected by thyroid status, although the fundamental mechanisms underlying this response are not clear (30, 31). In well studied examples, such as regulation of TSH (27), GH (32), PRL (28), S14 protein (33), malic enzyme (34), and a-myosin heavy chain (35), thyroid hormone has been shown to act mainly at the transcriptional level. It is not yet clear whether the effects of thyroid hormone on PAM expression in the anterior pituitary gland are direct or indirect. The control of the cytoplasmic level of PAM mRNA may involve changes in the rate of specific gene transcription, the rate of processing of the PAM primary transcript, or the stability of the nuclear or cytoplasmic PAM RNA sequences. Although our measurements reflect steady state levels of mRNA and do not allow us to distinguish changes in transcriptional rate from changes in RNA stability, these results indicate that any diminution in levels of thyroid hormone below euthyroid levels results in increased expression of PAM. Our current efforts are directed toward understanding the precise molecular mechanism for this regulation and the physiological significance of these widespread alterations in PAM expression in the anterior pituitary gland in hypothyroidism. MATERIALS AND METHODS Animals and Treatments Male Sprague-Dawley rats (150-200 g; Holtzman Laboratories, Madison, Wl) were maintained on a standard laboratory diet. In the first experimental paradigm, hyperthyroidism (n = 8) was induced by daily ip injection of T4 (15 ^g T4/100 g BW; Sigma Chemical Co., St. Louis, MO) for 15 days. Similarly, hypothyroidism (n = 8) was induced by daily ip injection of PTU (1 mg PTU/100 g BW; Sigma Chemical Co.). Both T4 and PTU were dissolved in 0.1 N NaOH and diluted to the appropriate concentration in normal saline solution. Control rats (n = 8) received daily ip injections of saline solution for the same period of time. The complete experiment was replicated independently three times. In the second experimental paradigm, male Sprague-Dawley rats (200 g) were thyroidectomized 6 weeks before thyroid hormone replacement. Each group of hypothyroid rats received daily ip injections of T4 (0.25, 1,4,16, 40, or 64 ng; n = 4 for each group) for 15 days. The experiment was replicated independently two times; control animals received sham injections of vehicle. At the end of each experiment, the animals were weighed, and trunk blood was collected for serum TSH measurements. Reagents for RIA forTSH/S were kindly provided by the NIDDK. Individual anterior pituitaries were rapidly removed for determination of PAM activity, preparation of total RNA, or in situ hybridization studies. Tissue Preparation and Amidation Assays Anterior pituitary tissue was homogenized in 20 mM NaTES, (A/-Tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid), 1503 Thyroid Hormone Regulation of PAM Expression pH 7.4, and 10 mM mannitol and separated into soluble and crude particulate fractions, as previously described (7, 8). Soluble fractions were assayed directly. The crude particulate fractions were solubilized by resuspension in the same buffer containing 1 % Triton X-100; after centrifugation for 60 min at 100,000 x g, the supernatants were used to measure solubilized membrane-associated PAM activity. Protein concentrations were determined using the bicinchoninic acid protein assay reagent (Pierce Chemical Co., Rockford, IL) and BSA as standard. Amidation assays were performed in duplicate, essentially as described previously (7, 13). Unless indicated otherwise, assays contained 20,000-25,000 cpm mono-[125l]D-Tyr-ValGly, 0.4 HM D-Tyr-Val-Gly, 400 HM ascorbate, 2 HM CUSO 4 , catalase (100 Mg/m|). and 2 fig protein in 120 mM NaTES buffer, pH 8.5. Reaction velocities are generally expressed as picomoles of product formed per ng protein/h (specific activity). The sum of the amount of PAM activity in the soluble and crude particulate fractions (taking into account the amount of protein in each fraction) represents total PAM activity per pituitary; normalization to total protein per pituitary yields total specific activity. The variation between duplicate samples was less than 5%. The reaction velocities reported are initial velocities, using a concentration of substrate about 10-fold below the Km of the enzyme for peptide substrate. In general, no more than 10% of the substrate was converted into product in the assay. Western Blot Analysis Samples were fractionated on slab gels containing 10% acrylamide and 0.25% A/.A/'-methylenebisacrylamide using the buffer system of Laemmli (36). Proteins were electrophoretically transferred to Immobilon membranes (Millipore Corp., Bedford, MA) in 25 mM Tris and 192 mM glycine, pH 8.3, containing 20% methanol (7). Mol wt was estimated by comparison with protein standards (Sigma Chemical Co.) fractionated in an adjacent lane. Immobilon strips were blocked with BSA, incubated with affinity-purified antiserum to bPAM[561 579] (Ab69, diluted 1:100), and processed essentially as described previously (7), except that [125l]protein-A (ICN, Costa Mesa, CA; 106 cpm/ml) was used to visualize the cross-reactive bands on autoradiographic film. An affinity-purified antiserum to bPAM[288-310] (Ab100, diluted 1:300) was used to confirm the results obtained with Ab69. RNA Isolation and Northern Blot Analysis Total RNA was prepared from individual anterior pituitaries using the acid guanidinium isothiocyanate-phenol-chloroform procedure (37). RNA was denatured and then electrophoresed on 1 % agarose gels containing 2.2 M formaldehyde, 20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA, pH 7.0, and transferred to Nytran (Schleicher and Schuell, Keene, NH) by capillary action in 20 x SSC (3.0 M NaCI and 0.3 M sodium citrate, pH 7.0). Filters were baked, prehybridized, hybridized, and washed as previously described (7). The 1.3-kb Pst\/ BamH\ fragment of rat PAM-1 cDNA (basepairs 351-1681) was labeled with [a-32P]dCTP to a specific activity of 109 cpm/ ng by random prime synthesis (Amersham Corp., Arlington Heights, IL) and used as probe (106 cpm/ml). Mol wt was estimated by comparison to a RNA ladder (Bethesda Research Laboratories, Gaithersburg, MD) fractionated in a parallel lane and stained with acridine orange or blotted and probed with labeled wild-type X-DNA. To correct for the actual amount of RNA applied to each lane, blots were stripped and hybridized to cDNA probes derived from frog ribosomal RNA (7, 13). For quantitation, autoradiograms were densitized using a LOATS RAS-1000 image analysis system (Amersham Corp.). Known amounts of cDNA probe were applied to a nitrocellulose membrane using a slot blot apparatus, and densitization of this autoradiogram provided a standard curve for converting integrated optical density into disintegrations per min. The amount of PAM mRNA (disintegrations per min) in each sample was then normalized to the amount of rRNA (disintegrations per min) in that sample and plotted as a ratio of PAM mRNA/ rRNA. Total RNA was also prepared from heart atria and apical ventricular tissues from control, PTU-treated, and T4-treated adult male rats and subjected to quantitative Northern blot analysis. Although preliminary data suggested effects of thyroid status on levels of PAM mRNA in the heart (38), neither PTU nor T4 treatment brought about a large change in levels of PAM mRNA in atrial or apical ventricular heart tissues. Thyroid hormones, however, do appear to affect PAM expression in primary neonatal heart atrial myocyte cultures.2 Combined Reverse Transcriptase Polymerase Chain Reaction Total RNA (5 ng) from control, hyperthyroid, and hypothyroid rats was reverse transcribed into cDNA using 1 ng oligo(dT)12. 18 (Pharmacia LKB Biotechnology, Piscataway, NJ) as primer in a 20-n\ reaction volume containing 50 mM Tris-HCI (pH 8.0), 50 mM KCI, 5 mM MgCI2, 5 /XM dithiothreitol, 50 ng/m\ BSA, 1.25 fi\ RNasin (Promega Corp., Madison, Wl), 0.5 mM each of four dNTPs, and 12 U AMV reverse transcriptase (Life Sciences, St. Petersburg, FL) at 42 C for 60 min. The synthetic oligonucleotide primers used in the polymerase chain reaction were all 17-mers. Primers yielding sense cDNA were (all basepair numbers are for rPAM-1) (4) no. 5 (366-382), no. 4 (1359-1375), and no. 9 (3124-3140). Primers yielding antisense cDNA were no. 19 (1455-1439), no. 10 (3188-3172), and no. 21 (3834-3816). Polymerase chain reactions were performed in a 50-^1 volume according to Cetus specifications: 10 mM Tris-HCI (pH 8.3; at 25 C), 50 mM KCI, 1.5 mM MgCI2, 0.01% gelatin, 200 MM each of four dNTPs, 1 HM each primer, cDNA derived from 50-250 ng total RNA, and 1.25 U Amplitaq DNA polymerase (Perkin Elmer Cetus, Norwalk, CT). Samples were overlayed with one drop of light mineral oil and subjected to 25 cycles in a MJ Research Thermal Cycler (MJ Research, Inc., Cambridge, MA). Cycling parameters were generally as follows. The initial denaturation step was performed at 94 C for 4 min; the repeat cycle consisted of annealing at 52 C for 1 min, followed by extention at 72 C for 3 min and denaturation at 94 C for 1 min. The last extension time was lengthened to 5 min. After thermal cycling, most of the oil was manually removed, and the remaining oil was extracted with chloroform. Samples were fractionated on agarose gels in 89 mM Tris, 89 mM boric acid, and 2.5 mM EDTA, pH 8.0. After staining with ethidium bromide, the gels were photographed. It should be emphasized that internal standards were not included during reverse transcription or amplification, and the amplified products can be compared only in a qualitative manner. In Situ Hybridization Anterior pituitaries were embedded in Tissue-Tek (Miles Laboratories, Elkhart, IN) and frozen in a dry ice-alcohol slurry (39). Cryosections (16 fim) of anterior pituitary glands from experimental and control animals were mounted on subbed glass slides and prepared for hybridization to RNA probes, as previously described (39). Radiolabeled riboprobes were prepared using uridine 5'-[a-35S-thio]triphosphate (New England Nuclear, Wilmington, DE) and T3 or T7 RNA polymerase (Promega Corp.) to synthesize, respectively, full-length RNA sense and antisense transcripts of rPAM-1 cDNA from plasmid Z6 (4). In situ hybridization was performed, as described by Segal and Shilo (40), with the following modifications. 35S-Labeled RNA probes were not denatured with alkali; prehybridization was usually omitted; hybridization was performed for 24 h in a moist chamber under unsealed silane-treated coverslips at 56 C, using 0.5-1 x 106 cpm probe for each section. Slides May, V., and K. M. Braas, unpublished observations. Vol4No. 10 MOL ENDO-1990 1504 were washed twice in 2 x SSC, incubated for 30 min at 30 C in 2 x SSC containing 10 ^g/ml RNase-A (Worthington Biochemicals, Freehold, NJ), washed twice for 30 min each time in 2 x SSC, and finally immersed in water for a few seconds. Sections were dehydrated by immersion in 100% ethanol, air dried, and exposed to Kodak film (Eastman Kodak, Rochester, NY) at 4 C for 3-7 days. For resolution at the cellular level, sections were exposed to Kodak autoradiography emulsion (NTB-2) for 1 week at room temperature, developed, and stained with toluidine blue. 10. 11. 12. Acknowledgments We wish to thank Doris A. Staffers for providing the rat PAM cDNA probes and teaching us the technique of RT-PCR, and Eileen Katsimpiris for preparing the affinity-purified antisera and performing the Western blot analyses. We also wish to thank Dick Mains and Karen Braas for their scientific support. Receipt of the reagents for the TSH0 RIA from the NIDDK and the National Hormone and Pituitary Program (University of Maryland School of Medicine) is gratefully acknowledged. 13. 14. 15. 16. Received June 11, 1990. Revision received July 18, 1990. Accepted July 18,1990. Address requests for reprints to: Dr. Betty A. Eipper, Department of Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205. This work was supported by Grants DK-32949 from NIH and DA-00098 from NIDA (to B.A.E.). 17. 18. REFERENCES 1. Bradbury AF, Smyth DG 1987 Biosynthesis of the Cterminal amide in peptide hormones. Biosci Rep 7:907916 2. Eipper BA, Mains RE 1988 Peptide a-amidation. Annu Rev Physiol 50:333-344 3. Eipper BA, Park LP, Dickerson IM, Keutmann HT, Thiele EA, Rodgriquez H, Schofield PR, Mains RE 1987 Structure of the precursor to an enzyme mediating COOHterminal amidation in peptide biosynthesis. Mol Endocrinol 1:777-790 4. Staffers DA, Green CB-R, Eipper BA 1989 Alternative splicing generates multiple forms of peptidylglycine aamidating monooxygenase in rat atrium. Proc Natl Acad Sci USA 86:735-739 5. Mizuno K, Ohsuye K, Wada Y, Fuchimura K, Tanaka S, Matsuo H 1987 Cloning and sequence of cDNA encoding a peptide C-terminal a-amidating enzyme from Xenopus laevis. Biochem Biophys Res Commun 148:546-552 6. Ohsuye K, Kitano K, Wada Y, Fuchimura K, Tanaka S, Mizuno K, Matsuo H 1988 Cloning of cDNA encoding a new peptide C-terminal a-amidating enzyme having a putative membrane spanning domain from Xenopus laevis skin. Biochem Biophys Res Commun 150:1275-1281 7. Ouafik L"H, May V, Keutmann HT, Eipper BA 1989 Developmental regulation of peptidylglycine a-amidating monooxygenase (PAM) in rat heart atrium and ventricle. J Biol Chem 264:5839-5845 8. May V, Cullen El, Braas KM, Eipper BA 1988 Membraneassociated forms of peptidylglycine a-amidating monooxygenase activity in rat pituitary. J Biol Chem 263:75507554 9. Perkins SN, Eipper BA, Mains RE 1990 Stable expression of full-length and truncated bovine peptidylgylcine a-ami- 19. 20. 21. 22. 23. 24. 25. 26. 27. dating monooxygenase complementary DNAs in cultured cells. Mol Endocrinol 4:132-139 Tamburini PP, Jones BN, Consalvo AP, Young SD, Lovato SJ, Gilligan JP, Wennogle LP, Erion M, Jeng AY 1988 Structure-activity relationships for glycine extended peptides and the a-amidating enzyme derived from medullary thyroid CA-77 cells. Arch Biochem Biophys 267:623-631 Thiele EA, Marek KL, Eipper BA 1989 Tissue-specific regulation of peptidyl-glycine a-amidating monooxygenase expression. Endocrinology 125:2279-2288 Sakata J, Mizuno K, Matsuo H 1986 Tissue distribution and characterization of peptide C-terminal a-amidating activity in rat. Biochem Biophys Res Commun 140:230236 Braas KM, Staffers DA, Eipper BA, May V 1989 Tissue specific expression of rat peptidylglycine a-amidating monooxygenase activity and mRNA. Mol Endocrinol 3:1387-1398 Arnaout MA, Garthwaite TL, Martinson DR, Hagen TC 1986 Vasoactive intestinal polypeptide is synthesized in anterior pituitary tissue. Endocrinology 119:2052-2057 DePalatis LR, Khorram O, Ho RH, Negro-Vilar A, McCann SM 1984 Partial characterization of immunoreactive substance P in the rat pituitary gland. Life Sci 34:225-235 Jones PM, Ghatei MA, Steel J, O'Halloran D, Gon G, Legon S, Burrin JM, Leonhardt U, Polak JM, Bloom SR 1989 Evidence for neuropeptide Y synthesis in the rat anterior pituitary and the influence of thyroid hormone status: comparison with vasoactive intestinal peptide, substance P, and neurotensin. Endocrinology 125:334341 Aronin N, Coslovsky R, Chase K 1988 Hypothyroidism increases substance P concentrations in the heterotopic anterior pituitary. Endocrinology 122:2911-2914 Lam KSL, Lechan RM, Minamitani N, Segerson TP, Reichlin S 1989 Vasoactive intestinal peptide in the anterior pituitary is increased in hypothyroidism. Endocrinology 124:1077-1084 Oppenheimer JH, Schwartz HL, Mariash CN, Kinlaw WB, Wong NCW, Freake HC 1987 Advances in our understanding of thyroid hormone action at the cellular level. Endocr Rev 8:288-308 Samuels HH, Forman BM, Horowitz ZD, Ye Z-S 1988 Regulation of gene expression by thyroid hormone. J Clin Invest 81:957-967 Mains RE, Myers AC, Eipper BA 1985 Hormonal, drug, and dietary factors affecting peptidyl glycine a-amidating monooxygenase activity in various tissues of the adult male rat. Endocrinology 116:2505-2515 Staffers DA, Eipper BA, 1989 Multiple forms of rat peptidylglycine a-amidating monooxygenase. In: Rotundo RL, Ahmad F, Bialy H, Black S, Brew K, Chaitin MH, Glaser L, Magleby KL, Neary JT, Van Brunt J, Whelan WJ (eds) ICSU Short Report 9, Advances in Gene Technology: Molecular Biology and Neuropharmacology. IRL Press, Oxford, p 120 May V, Ouafik L'H, Eipper BA, Braas KM 1990 Immunocytochemical and in situ hybridization studies of peptidylglycine a-amidating monooxygenase (PAM) in pituitary gland. Endocrinology 127:358-364 Jonassen JA, Mullikin-Kilpatrick D, McAdam A, Leeman SE 1987 Thyroid hormone status regulates preprotachykinin-A gene expression in male rat anterior pituitary. Endocrinology 121:1555-1561 Segerson TP, Lam KSL, Cacicedo L, Minamitani N, Fink JS, Lechan RM, Reichlin S 1989 Thyroid hormone regulates vasoactive intestinal peptide (VIP) mRNA levels in the rat anterior pituitary gland. Endocrinology 125:22212223 Ouafik L'H, Giraud P, Salers P, Dutour A, Castanas E, Boudouresque F, Oliver C1987 Evidence for high peptide a-amidating activity in the pancreas from neonatal rats. Proc Natl Acad Sci USA 84:261-264 Shupnik MA, Chin WW, Habener JF, Ridgway EC 1985 1505 Thyroid Hormone Regulation of PAM Expression 28. 29. 30. 31. 32. 33. 34. Transcriptional regulation of the thyrotropin subunit genes by thyroid hormone. J Biol Chem 260:2900-2903 Day RN, Maurer RA 1989 Thyroid hormone-responsive elements of the prolactin gene: evidence for both positive and negative regulation. Mol Endocrinol 3:931-938 Samuels MH, Wierman ME, Wang C, Ridgway EC 1989 The effect of altered thyroid status on pituitary hormone messenger ribonucleic acid concentrations in the rat. Endocrinology 124:2277-2282 Kamilaris TC, DeBold CR, Pavlou SN, Island DP, Hoursanidis A, Orth DN 1987 Effect of altered thyroid hormone levels on hypothalamic-pituitary-adrenal function. J Clin Endocrinol Metab 65:994-999 Sanchez-Franco F, Fernandez L, Fernandez G, Cacicedo L1989 Thyroid hormone action on ACTH secretion. Horm Metab Res 21:550-552 Ye Z-S, Forman BM, Aranda A, Pascula A, Park H-Y, Casanova J, Samuels HH 1988 Rat growth hormone gene expression. J Biol Chem 263:7821-7829 Jump DB 1989 Rapid induction of rat liver S14 gene transcription by thyroid hormone. J Biol Chem 264:46984703 Song M-KH, Dozin B, Grieco D, Rail JE, Nikodem VM 35. 36. 37. 38. 39. 40. 1988 Transcriptional activation and stabilization of malic enzyme mRNA precursor by thyroid hormone. J Biol Chem 263:17970-17974 Izumo S, Mahdavi V 1988 Thyroid hormone receptor a isoforms generated by alternative splicing differentially activate myosin HC gene transcription. Nature 334:539542 Laemmli UK 1970 Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (Lond) 227:680-685 Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal Biochem 162:156-159 May V, Ouafik L'H, Eipper BA, Thyroid hormone regulation of peptidylglycine a-amidating monooxygenase (PAM) expression. 71st Annual Meeting of The Endocrine Society, Seattle WA, 1989, p 90 (Abstract 272) Saffen DW, Cole AJ, Worley PF, Christy BA, Ryder K, Baraban JM 1988 Convulsant-induced increase in transcription factor messenger RNAs in rat brain. Proc Natl Acad Sci USA 85:7795-7799 Segal D, Shilo BZ 1986 Tissue localization of Drosophila melanogaster ras transcripts during development. Mol Cell Biol 6:2241-2248