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Endocrinology Vol. 141, No. 2 476-486
Copyright © 2000 by The Endocrine Society

ARTICLES

Breeding Stock-Specific Variation in Peptidylglycine {alpha}-Amidating Monooxygenase Messenger Ribonucleic Acid Splicing in Rat Pituitary1

Giuseppe D. Ciccotosto, Tracey A. Hand, Richard E. Mains and Betty A. Eipper

Departments of Neuroscience and Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Address all correspondence and requests for reprints to: Dr. Betty A. Eipper, Department of Neuroscience, WBSB 907, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205. E-mail: beipper{at}jhmi.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peptidylglycine {alpha}-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyl-terminal amidation of glycine-extended peptides in a two-step reaction involving a monooxygenase and a lyase. Several forms of PAM messenger RNA result from alternative splicing of the single copy PAM gene. The presence of alternately spliced exon A between the two enzymatic domains allows endoproteolytic cleavage to occur in selected tissues, generating soluble monooxygenase and membrane lyase from integral membrane PAM. While using an exon A antiserum, we made the unexpected observation that Charles River Sprague Dawley rats expressed forms of PAM containing exon A in their pituitaries, whereas Harlan Sprague Dawley rats did not. Forms of PAM containing exon A were expressed in the atrium and hypothalamus of both types of Sprague Dawley rat, although in different proportions. PAM transmembrane domain splicing also differed between rat breeders, and full-length PAM-1 was not prevalent in the anterior pituitary of either type of rat. Despite striking differences in PAM splicing, no differences in levels of monooxygenase or lyase activity were observed in tissue or serum samples. The splicing patterns of other alternatively spliced genes, pituitary adenylate cyclase-activating polypeptide receptor type 1 and cardiac troponin T, did not vary with rat breeder. Strain-specific variations in the splicing of transcripts such as PAM must be taken into account in analyzing the resultant proteins, and knowledge of these differences should identify variations with functional significance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PEPTIDYLGLYCINE {alpha}-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the carboxyl-terminal amidation of glycine-extended peptides in a two-step process (1). Peptidylglycine {alpha}-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction, and peptidyl-{alpha}-hydroxyglycine {alpha}-amidating lyase (PAL) catalyzes the second step. Several forms of PAM messenger RNA (mRNA) result from alternative splicing of the single copy rat PAM gene (2, 3, 4) (Fig. 1Go). PAM-1, the longest form, is composed of a signal and propeptide sequence followed by the PHM catalytic domain, a noncatalytic domain referred to as exon A (exon 16), the PAL catalytic domain, a transmembrane domain, and a carboxyl-terminal domain (1). Removal of exon A gives rise to PAM-2 and removal of exons A, Ba, and Bb gives rise to PAM-3. Splicing of the rat PAM gene is tissue specific and developmentally regulated (5, 6). In addition to alternative splicing, tissue-specific endoproteolytic cleavage of the various PAM proteins generates a wide variety of protein products (3, 6).



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  		Figure 1. PAM proteins and their cleaved products. PAM-1 is the largest PAM protein, and all amino acids are numbered as in rat PAM-1: PHM domain (36–392), PAL domain (498–831), and exon A (393–497). Cleavage within exon A occurs at Lys-Lys437, resulting in soluble PHM and membrane-bound PAL-CD. Exon B (832–917) contains alternatively spliced exons Ba (832–899) and Bb (900–917). PAM-1s, PAM-2s, and PALs are the products formed from cleavage between PAL and the transmembrane domain (TMD). The COOH-terminal domain extends from amino acid 891–976 and resides in the cytosol when part of PAM-1 or PAM-2. PAM-2 lacks the alternatively spliced exon A domain (dotted line), and PAM-3 lacks both the exon A and exon B domains. The apparent molecular masses of each PAM protein are listed. Cleavages are marked with the filled downward arrowhead.

 
In the rat, the presence of exon A allows endoproteolytic cleavage to occur in selected tissues, generating soluble PHM and membrane PAL from PAM-1; secretion of soluble PHM occurs along with secretion of peptide products (7). Lacking exon A, the bifunctional PAM-2 protein remains intact and membrane associated unless endoproteolytic cleavage separates PAL from the transmembrane domain. The functional significance of exon A is supported by the fact that transcripts with and without exon A have also been identified in human and Lymnaea (8, 9, 10). Although Lymnaea exon A shares only 8% sequence identity with rat exon A, it does have a Lys-Lys sequence that could constitute an endoproteolytic cleavage site (8). In Lymnaea, single cell PCR has been used to demonstrate that expression of PAM is limited to neurons producing amidated peptides, and that expression of PAM transcripts including exon A is further limited to a subset of these neurons. During rat development, the only PAM transcripts in the ependymal region of the spinal cord and the ventricular zone of the hippocampus lack exon A, again suggesting a functional role for this exon (11).

We used recombinant rat exon A to develop a rabbit polyclonal antiserum specific for forms of PAM that include this region. The polyclonal antiserum recognizes sites in both the PHM and PAL products generated from PAM-1 (12). In the process of using this antiserum, we made the unexpected observation that Sprague Dawley rats purchased from one breeder expressed forms of PAM containing exon A in their anterior pituitary and neurointermediate lobe, while Sprague Dawley rats obtained from a different breeder did not.

A number of reports describe substantial differences in experimental observations when using Sprague Dawley rats supplied by different breeders. Marked differences were observed in the projections from locus coeruleus neurons to the spinal cord when Sprague Dawley rats from different breeders were compared (13, 14). Similarly, differences in heart size (15), baseline renal function (16), level of induction of gallstone formation (17), medullary thick ascending limb adenylate cyclase response to arginine vasopressin (18), pituitary ACTH content and sensitivity of isolated adrenocortical cells to ACTH (19), and susceptibility to learned helplessness training (20) have been observed using rats from different breeders. These reports identify differences at the physiological and anatomical levels. In the present study we use RT-PCR and Western blot analysis to explore the differences in PAM expression at the molecular level.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of samples for Western blot analysis and measurement of enzyme activities
Adult male Sprague Dawley rats of similar age and weight (150–200 g) were obtained from two different breeders, Charles River Breeding Laboratories, Inc. (Wilmington, MA) and Harlan Sprague Dawley, Inc. (Frederick, MD). The rats were handled in accordance with guidelines of Johns Hopkins University animal care and use committee. The rats were killed with a guillotine; the anterior pituitary, neurointermediate pituitary (NIL), hypothalamus, and atrium were removed. Tissues were extracted in ice-cold 20 mM Na(N-Tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid)/10 mM mannitol, pH 7.4, and 1% Triton X-100, containing protease inhibitors (21). Homogenization was carried out with a Polytron (Brinkmann Instruments, Inc., Westbury, NY); homogenates were frozen and thawed three times and then centrifuged at 400 x g for 10 min at 4 C. The supernatants were removed and assayed for protein content using the bicinchoninic acid protein reagent kit (Pierce Chemical Co., Rockford, IL). Trunk blood was collected and allowed to clot before centrifugation; serum was kept frozen until analysis. Extracts (20 µg protein) were fractionated on either 10% or 12% polyacrylamide, 0.25% N,N'-methylene-bis-acrylamide/SDS gels, transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA), and visualized using the enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Arlington Heights, IL) (7, 22). Polyclonal rabbit antisera were raised against different regions of PAM: Ab JH1764 [rPAM-1-(37–382)] was used to detect PHM (7), Ab JH471 [rPAM-1-(464–864)] was used to detect PAL (7), Ab JH629 [rPAM-1-(409–497)]) was used to detect exon A (12), and Ab JH571 [rPAM-1-(898–976)] was used to detect the carboxyl-terminal domain of PAM (22). PHM and PAL enzyme assays were performed in duplicate on serial dilutions of tissue extracts and serum as previously described, using {alpha}-N-acetyl-Tyr-Val-Gly and {alpha}-N-acetyl-Tyr-Val-{alpha}-hydroxyglycine as substrates, respectively (23).

RNA extraction and PCR analysis
Tissue samples (anterior pituitary, hypothalamus, neurointermediate lobe, and atrium) from Sprague Dawley rats obtained from Charles River Breeding Laboratories, Inc. and Harlan Sprague Dawley, Inc., were divided into three groups, with three samples taken per group. Total RNA was isolated from the rat tissue samples using the RNA Stat-60 reagent (Tel-Test B, Friendswood, TX). RT PCR was used to evaluate the forms of endogenous PAM mRNA in the rat tissues. Total RNA (10–100 ng) was reverse transcribed using 0.5 µg oligo(deoxythymidine)12–18mer (Pharmacia Biotech) as primer in a 40-µl reaction volume containing reverse transcriptase buffer [50 mM Tris-HCl (pH 8.0), 50 mM KCl, 5 mM MgCl2, 5 mM dithiothreitol, and 50 µg/ml BSA], 0.5 µl RNasin (Promega Corp., Madison, WI), 0.5 mM deoxy-NTP mix (U.S. Biochemical Corp., Cleveland, OH), and 12.5 U avian myeloblastosis virus reverse transcriptase (Life Sciences Inc., St. Petersburg, FL) at 42 C for 60 min (RNAs were heat denatured at 65 C for 10 min before addition to reaction). Synthetic oligonucleotide primers used for PCR were as follows: sense primer across exon A of rPAM-1, S4 (1359–1375); antisense primer, A56 (1797–1812); sense primer across exon B, S64 (2637–2657); and antisense primers, A10 (3172–3188), A26 (3032–3048), and A29 (2894–2910). Controls included pituitary adenylate cyclase-activating polypeptide receptor type 1 (PACR1) and cardiac troponin T; the sense and antisense primers used for the PACR1 were PAC-S (1088–1107) and PAC-A (1498–1528), respectively; the sense and antisense primers used for troponin were TnT-S (68–88) and TnT-A (297–317), respectively. RT-PCRs were performed in a 50-µl volume (or a 100-µl volume for PACR1) consisting of 10 mM Tris-HCl (pH 9.0), 3 mM MgCl2, 0.01% (wt/vol) gelatin, 100 µM deoxy-NTP mix, 1 µM of each primer, varying amounts of complementary DNA (cDNA) or plasmid standard, and 2.5 U AmpliTaq DNA polymerase (Life Technologies, Inc., Gaithersburg, MD). Samples were overlaid with 2 drops of light mineral oil and subjected to 28 cycles of PCR in the Perkin-Elmer Corp./Cetus thermal cycler (Norwalk, CT). Cycling parameters were as follows: the initial denaturation step was at 95 C for 1 min; the repeat cycle consisted of annealing at 50 C for 1 min followed by extension at 72 C for 1 min and denaturation at 95 C for 1 min; the last extension time was for 10 min. After thermal cycling, the aqueous phase was treated with PCR Qiaquick purify (QIAGEN, Chatsworth, CA), resuspended in 10 µl sterile water, and then fractionated on a 2% agarose gel, a 1:1 mixture of agarose (Life Technologies, Inc., ultrapure), and NuSieve agarose (FMC Bioproducts, Rockland, ME) containing ethidium bromide in 0.5 M Tris, 0.45 M boric acid, and 5.0 mM Na2EDTA, pH 8.4 buffer. The PACR1 samples were subjected to 25 cycles of PCR using the following cycling parameters: the initial denaturation step was at 95 C for 5 min; the repeat cycle consisted of annealing at 57 C for 45 sec followed by extension at 72 C for 1 min and denaturation at 95 C for 45 sec; the last extension time was 10 min. PCR reaction sample (25 µl) was analyzed on a 3% agarose gel or purified using the Qiaquick purification kit. The purified samples were digested with AvaII and PvuII, and analyzed on a 3% agarose gel. Gels were prepared for Southern blot transfer by soaking for 10 min in 1.5 M NaCl and 0.5 N NaOH and then for 30 min in 1 M Tris-HCl (pH 8.0) and 1.5 M NaCl and transferred to Nytran (Schleicher & Schuell, Keene, NH) as previously described (24). The filters were hybridized with random primed PACR1 cDNA insert, washed (24), and exposed to film to verify the identity of the bands.

For Northern blot analysis, total RNA was fractionated on denaturing formaldehyde gels. POMC mRNA was visualized using a POMC probe [a 920-bp fragment was removed from the full-length mouse POMC cDNA (pMKSU16) by digestion with EcoRI and HindIII] (25). RNA loading was normalized using a probe for the ribosomal protein S26 (26).

RIA of POMC-related peptides
Rats were killed with a guillotine, and the anterior and neurointermediate pituitaries were extracted in 5 N acetic acid with protease inhibitors and then diluted 3-fold with water. Samples were split in half, and 2 mg/ml BSA was added to one lot; all samples were lyophilized. Samples not containing BSA were dissolved in RIA buffer and assayed for protein content using the bicinchoninic acid protein reagent kit (Pierce Chemical Co., Rockford, IL). Samples containing BSA were also dissolved in RIA buffer and used for RIAs. ACTH RIAs were performed using Ab Kathy (1:15,000), 125I-labeled ACTH-(1–39) (NEN Life Science Products, Boston, MA), and synthetic ACTH-(1–39) (Peninsula Laboratories, Inc., Belmont, CA) as the standard; this antibody only recognizes POMC products in which the COOH-terminal end of ACTH-(1–39) is exposed (21). Amidated joining peptide RIAs were performed using Ab Jamie (1:5,000), 125I-labeled Tyr-joining peptide-(12–18)-NH2, and synthetic amidated joining peptide (PNPSPAN-NH2) as the standard (Vega Biotechnologies, Tucson, AZ); this antibody requires the amidated COOH-terminus of joining peptide, but sees NH2-terminally extended peptides (27). Amidated {alpha}MSH RIAs were performed using Ab Wanda (1:40,000), [125I]Nle4,D-Phe7]{alpha}MSH (NEN Life Science Products), and synthetic amidated {alpha}MSH ({alpha}-N-acetyl-SYSMEHFRWGKPV-NH2) as standard; this antibody recognizes the amidated COOH-terminus of {alpha}MSH (28).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PAM protein expression differs in the pituitaries of Charles River and Harlan Sprague Dawley rats
In the course of using antisera to the exon A region of PAM-1 to characterize pituitary PAM, we made the surprising discovery that Sprague Dawley rats purchased from different breeders yielded very different patterns (Fig. 2Go). Although bands of about 120 and 46 kDa were readily apparent in anterior pituitaries from Charles River Sprague Dawley rats, no protein was detected in anterior pituitaries from Harlan Sprague Dawley rats. A dramatic difference was also observed when neurointermediate pituitaries were compared (Fig. 2Go). Endoproteolytic cleavage of PAM proceeds to a greater extent in the neurointermediate pituitary, and a 46-kDa product was the major exon A-containing PAM protein detected in Charles River Sprague Dawley rats; almost no exon A-containing PAM protein was detected in the neurointermediate pituitary of Sprague Dawley rats. In contrast, when extracts of hypothalamus and atrium were prepared, similar patterns were observed for rats from both breeders (Fig. 2Go). The tissue-specific cleavage of PAM-1 is readily apparent in comparing the PAM proteins detected in hypothalamus and atrium. Although intact PAM-1 is prevalent in atrium, cleaved 46-kDa PHM is the major product in hypothalamic extracts.



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  		Figure 2. Western blot analysis of PAM proteins containing exon A. Tissue extracts (20 µg total protein) prepared from anterior pituitary, neurointermediate pituitary, hypothalamus, and atrium of Charles River (C) and Harlan (H) Sprague Dawley rats were fractionated by SDS-PAGE and visualized using antiserum to exon A. Arrows indicate the locations of the molecular mass marker standards. For analysis with different PAM antisera, see Fig. 6Go. Membranes were stained with Coomassie brilliant blue to confirm equal protein loading of samples. Similar results were obtained with three sets of tissue extracts.

 
When aliquots of the same extracts were assayed for PHM and PAL enzymatic activities, a very different picture developed (Fig. 3Go). PHM and PAL enzymatic activities were readily detectable in the pituitaries of rats from both breeders, and the specific activities measured were not significantly different. Similarly, no differences in PHM and PAL enzymatic activities were seen for the neurointermediate pituitary, hypothalamus, and atrium when comparing Sprague Dawley rats from the two breeders (Fig. 3Go). Aliquots of serum were also assayed for PHM and PAL activity. Again, no significant differences were observed between Charles River and Sprague Dawley rats (Fig. 3Go). Serum samples were fractionated on a size exclusion column, and the profiles of PHM and PAL activities were determined (data not shown). Peaks of PHM and PAL activities were observed between the ovalbumin (43-kDa) and BSA (67-kDa) molecular mass standards, with identical elution profiles observed for the serum samples from Charles River and Sprague Dawley rats.



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  		Figure 3. PHM and PAL activities of tissue extracts and serum. PHM and PAL activities were determined in tissue extracts from the anterior pituitary (Ant Pit), neurointermediate pituitary (NI Pit), hypothalamus (Hypo), atrium, and serum of Charles River (C; filled bar) and Harlan (H; empty bar) Sprague Dawley rats. The data shown are the mean ± SEM from three separate analyses.

 
Alternative splicing across exon A differs in Charles River and Harlan Sprague Dawley rats
As our Western blots clearly demonstrated differences in the PAM proteins expressed in the pituitaries of Charles River and Sprague Dawley rats, we used RT-PCR to determine whether there were differences apparent at the RNA level. Primers spanning the exon A region of PAM-1 were employed; although transcripts that include exon A yield a 454-bp product, transcripts lacking exon A yield a 139-bp product (Fig. 4Go). RNA from the anterior pituitary of Charles River Sprague Dawley rats yielded similar amounts of both products. In contrast, RNA from the anterior pituitary of Sprague Dawley rats yielded only the smaller band derived from transcripts lacking exon A. A similar pattern was observed using cDNA from the neurointermediate pituitary; PAM transcripts containing exon A are prevalent in Sprague Dawley rats obtained from Charles River Breeding Laboratories, Inc., but not in Sprague Dawley rats obtained from Harlan Sprague Dawley, Inc.



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  		Figure 4. RT-PCR analysis of expression of PAM exon A in various tissues. A, Schematic of rPAM-1 and -2 cDNAs; the positions and orientations of the oligonucleotide primers used are indicated. B, Total RNA from the anterior pituitary (Ant Pit), neurointermediate pituitary (NI Pit), hypothalamus (Hypo), and atrium from Charles River (C) and Harlan (H) Sprague Dawley rats was reverse transcribed and amplified using the primers indicated. The positive control (+ve) consisted of products amplified from PAM-1 and PAM-2 plasmids; the negative control contained no cDNA (-ve). The PCR products were fractionated on agarose gels, and the ethidium bromide-stained gels were photographed. Similar results were obtained with two sets of RNA samples prepared from a separate set of rats from each supplier.

 
PAM transcripts that include exon A are readily detected in other tissues from Sprague Dawley rats. Hypothalamic and atrial cDNA prepared from both breeders of Sprague Dawley rats yielded PCR products that contained exon A and PCR products that lacked exon A (Fig. 4Go). Products including exon A were more prevalent in tissues prepared from Charles River rats than in tissues prepared from Harlan Sprague Dawley rats. Overall, the results obtained from the RT-PCR studies confirm the observations made at the protein levels (Fig. 2Go).

Alternative splicing across exons Ba and Bb differs in Charles River and Harlan Sprague Dawley rats
Having established that splicing of exon A differs in rats from the two breeders, we wanted to determine whether differences were also observed in splicing of cassette exons Ba and Bb (Figs. 1Go and 5AGo). To do this, three sets of diagnostic primer pairs were used (Fig. 5Go, A and B). As different primer sets may not be equally efficient, the absolute amount of product detected by the three sets of primer pairs cannot be compared. The products observed for anterior pituitary from Charles River and Harlan Sprague Dawley rats were again different (Fig. 5CGo). In Charles River Sprague Dawley rats, only two major splice variants were detected, and neither contained exon Bb; splice variants with and without exon Ba were detected. In Harlan Sprague Dawley rats, three major products were detected, including a form containing exon Bb (Fig. 5CGo, asterisk).



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  		Figure 5. RT-PCR analysis of expression of PAM exons Ba and Bb in various tissues. A, Schematic of rPAM-1 cDNA. The region surrounding exons Ba and Bb is magnified to show the oligonucleotide primer pairs used. B, Table summarizing the products expected from the primer pairs used. RT-PCR analysis of total RNA from the anterior pituitary (C) and neurointermediate pituitary, hypothalamus, or atrium (D) from Charles River (C) and Harlan (H) Sprague Dawley rats using the primer pairs indicated. The PAM-1 positive control (C; +ve) used all three primer pairs with the PAM-1 plasmid; the negative control (-ve) contained no cDNA. *, PCR products containing exon Bb. The PCR products were analyzed as described in Materials and Methods. Similar results were obtained with three sets of RNA samples prepared from a separate set of rats.

 
In contrast to the anterior pituitary, the neurointermediate pituitary, hypothalamus, and atrium displayed similar splicing profiles when comparing Charles River to Harlan Sprague Dawley rats (Fig. 5DGo). The neurointermediate pituitary contained two alternatively spliced forms, one containing both exons Ba and Bb and one containing only exon Ba. The hypothalamus and atrium have only a single variant containing both exons Ba and Bb (Fig. 5DGo). The splice variant that included exon Bb and lacked exon Ba was not detected in any of the tissue extracts examined.

PAM-1 is not a major transcript in the anterior pituitary of Sprague Dawley rats
We used antisera specific for PHM, PAL, and PAM-CD to relate the different splicing patterns detected for the PAM gene in Charles River and Harlan Sprague Dawley rats to different protein products (Fig. 6Go). We focused on anterior pituitary and atrium because the endoproteolytic cleavage of PAM is less extensive in these tissues, facilitating the correlation of PAM transcripts and PAM proteins. The PAM proteins encoded by the major splice variants in anterior pituitary of Charles River and Harlan Sprague Dawley rats are indicated in Fig. 6AGo. The lack of exon A or exon Bb in PAM transcripts from Harlan and Charles River Sprague Dawley rats, respectively, means that PAM-1 itself is not a major product in the rat anterior pituitary.



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  		Figure 6. Western blot analysis of PAM proteins using PHM, PAL, and PAM-CD antibodies. A, Table summarizes the PAM proteins encoded by the major splice variants detected in each tissue examined. Tissue extracts (20 µg total protein) prepared from anterior pituitary (B) and atrium (C) of Charles River (C) and Harlan (H) Sprague Dawley rats were fractionated by SDS-PAGE and visualized using antisera to PHM, PAL, and CD of PAM. Western blots were stained with Coomassie blue to confirm equal protein loading of samples. Similar results were obtained with three sets of tissue extracts.

 
The 118-kDa protein identified in the anterior pituitary of Charles River Sprague Dawley rats by antisera to exon A (Fig. 2Go), PHM, PAL, and CD (Fig. 6BGo) is PAM-1 that lacks exon Bb (Fig. 6AGo, PAM-1{Delta}Bb). The 110-kDa protein identified in Charles River Sprague Dawley rat anterior pituitary extracts by the exon A antibody (Fig. 2Go) is thought to correspond to a PAM-3 variant that includes exon A (Fig. 6AGo, PAM-3{Sigma}A). Cleavage of PAM-1{Delta}Bb or PAM-3{Sigma}A within exon A would yield similar 45-kDa PHM products, but different PAL products. The 65-kDa protein detected by antisera to exon A, PAL, and CD may be the fragment generated when this 110-kDa protein is cleaved to produce 45-kDa PHM (Figs. 2Go and 6AGo); further cleavage within the CD would generate the 60-kDa PAL protein detected by antisera to exon A and PAL. Harlan Sprague Dawley rat anterior pituitary contains no monofunctional PHM or PAL, confirming a crucial role for exon A in facilitating endoproteolytic cleavages separating the two catalytic domains. Similarly, the presence of exon A in PAM transcripts expressed in the neurointermediate pituitary of Charles River Sprague Dawley rats leads to accumulation of monofunctional PHM and PAL, which are absent from the neurointermediate pituitary of Harlan Sprague Dawley rats (data not shown).

PAM transcripts in the atria of Charles River and Harlan Sprague Dawley rats differ only in the increased prevalence of exon A-containing transcripts in Charles River rats; atrial PAM transcripts all contain exons Ba and Bb (Fig. 5DGo). Consistent with this, PAM proteins of 120 kDa predominate in the atria of Charles River Sprague Dawley rats, whereas PAM proteins of 105 kDa predominate in the atria of Harlan Sprague Dawley rats (Fig. 6CGo). Although a doublet of approximately 120-kDa PAM proteins is consistently detected in rats from both suppliers, this heterogeneity is thought to represent differences in glycosylation rather than the presence of splice variants (29). The 65-kDa protein detected by antisera to exon A, PAL, and CD represents the major cleavage product of PAM-1.

Alternative splicing of PACR1 in anterior pituitary and cardiac troponin T in atrium
The differences observed in the alternative splicing patterns of the PAM gene in Sprague Dawley rats purchased from different suppliers could be specific to the PAM gene or could generalize to other genes. PACR1 and cardiac troponin T are highly expressed in pituitary and atrium, respectively, and are known to undergo alternative splicing (30, 31, 32, 33, 34). To address this issue we compared the splicing patterns for these two genes in tissue from Sprague Dawley rats purchased from Charles River Breeding Laboratories, Inc. or Harlan Sprague Dawley, Inc. (Fig. 7Go).



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  		Figure 7. RT-PCR analysis of alternative splicing of PACR1 in anterior pituitary and cardiac troponin T in atrium. A, Schematic of PACR1 showing the alternatively spliced HIP and HOP exons and the six known isoforms of PACR1; diagnostic restriction sites are indicated. Total RNA from the anterior pituitary of Charles River (C) or Harlan (H) Sprague Dawley rats was subjected to RT-PCR using the primer pairs indicated (arrows). The PCR products were analyzed intact or after digestion with AvaII or PvuII and visualized by hybridization to PACR1 cDNA. B, Atrial cDNA amplified using the indicated primer pairs for cardiac troponin T (see Materials and Methods). The PCR products were fractionated on an ethidium-stained agarose gel. A mouse cardiac troponin T cDNA plasmid clone 6–4 was used as a positive control (+ve). The number of base pairs present in the PCR products derived from plasmid controls is indicated on the left. Similar results were obtained with two sets of RNA samples prepared from a separate pool of rats.

 
Alternative splicing of the PACR1 gene is tissue specific, with six isoforms identified (Fig. 7Go) (31, 35). To determine whether splicing of this gene differs in Charles River and Harlan Sprague Dawley rats, primer pairs that span the alternatively spliced HIP and HOP exons were used for RT-PCR (Fig. 7Go). Two PCR products were identified in the Sprague Dawley rats from both breeders, with no differences observed between rats from the two suppliers (Fig. 7Go, undigested, UD). The smaller band (307 bp) is the normal isoform of PACR1 and lacks both the HIP and HOP exons; the larger band (380–390 bp) includes the HOP1 exon, as it contained the restriction site for PvuII and BlpI (data not shown) and not that for AvaII (Fig. 7Go) QUOTE (32).

Cardiac troponin T undergoes alternative splicing in a developmentally regulated fashion; two isoforms are present in the fetal heart, and a single isoform is present in the adult heart (33, 36). To determine whether splicing of this gene differed in Charles River and Harlan Sprague Dawley rats, primer pairs that span the alternatively spliced exon 3–4 region were used for RT-PCR (Fig. 7Go). Two PCR products were identified in Sprague Dawley rats from both breeders, with no differences observed between rats from the two suppliers.

Expression of POMC differs in pituitaries of Charles River and Harlan Sprague Dawley rats
As POMC is highly expressed in the anterior and intermediate lobes of the pituitary, it was of interest to determine whether breeder-specific differences in POMC expression could be detected. Based on Northern blot analysis, levels of POMC mRNA were identical in the anterior and neurointermediate pituitaries of Sprague Dawley rats from both breeders (POMC mRNA to S26 mRNA ratios varied <10%; data not shown). Acid extracts of anterior and neurointermediate pituitaries were immunoassayed for ACTH, amidated joining peptide, and amidated {alpha}MSH. In the anterior pituitary, levels of ACTH and amidated joining peptide were indistinguishable in Charles River and Harlan Sprague Dawley rats (Fig. 8Go). Although the anterior pituitary contains very little amidated {alpha}MSH in comparison to ACTH (molar amounts are 50- to 100-fold lower), the amount of amidated {alpha}MSH in Charles River rat anterior pituitary was significantly less than the amount in Harlan rat anterior pituitary (P < 0.05; Fig. 8Go). When neurointermediate pituitary extracts were examined, breeder-specific differences were again observed. The amidated joining peptide content of Charles River Sprague Dawley rat neurointermediate pituitary was significantly greater than that in the Harlan rat (P < 0.05); neurointemediate pituitary levels of amidated {alpha}MSH were indistinguishable, eliminating intermediate lobe contamination as a source of the difference observed in the anterior pituitary.



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  		Figure 8. Immunoassay of POMC-derived peptides in pituitary extracts. The structures of POMC and the smaller peptides derived from it are shown; antibody specificities are indicated (# indicates specificity for the {alpha}-amide). Immunoactive ACTH, amidated joining peptide (JP-amide), and amidated {alpha}MSH ({alpha}MSH-amide) in extracts of anterior and neurointermediate pituitary from Charles River (C; filled bar) and Harlan (H; empty bar) Sprague Dawley rats were normalized to protein content. The data shown are the mean ± SEM from six separate analyses. The inset graph shows the data for {alpha}MSH-amide magnified. *, P < 0.05, by paired Student’s t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The anterior pituitary expresses many genes that undergo extensive alternative splicing, including the ATP-gated ion channel P2X2 (37), PACAP (38), and receptors for PACAP, vasoactive intestinal polypeptide, dopamine, and GH-releasing hormone (39, 40, 41, 42). The PAM gene is highly expressed in the pituitary and is also subject to extensive alternative splicing (2, 3, 4). We now show that the splicing pattern of the PAM gene differs dramatically between two breeders of Sprague Dawley rat. While PAM transcripts containing and lacking exon A were equally represented in the pituitaries of Charles River Sprague Dawley rats, no PAM transcripts containing exon A were detected in Harlan Sprague Dawley rats. The splicing patterns in the hypothalamus and atrium showed more subtle differences between rats from the two breeders.

Exon A serves no catalytic function in PAM (Fig. 1Go). Consistent with this, the catalytic activities of PHM and PAL were similar in pituitaries from the two breeders (Fig. 3Go). The presence of a Lys-Lys cleavage site within exon A allows the separation of soluble PHM from integral membrane PAL. Metabolic labeling experiments established that AtT-20 cells cleave about half of the newly synthesized PAM-1 between PHM and PAL within 3 h, whereas virtually no PAM-2 is cleaved in the same time period (43). Both PAM-1 and PAM-2 localize predominantly to the trans-Golgi network region of transfected hEK-293 and AtT-20 cells (7, 44). Furthermore, exon A is subject to other posttranslational processing modifications, including O-glycosylation and subsequent sulfation or sialylation of the O-linked oligosaccharide chains (45).

A potentially important function for exon A would be consistent with the finding of PAM transcripts with and without exon A in species from Lymnaea to humans (8, 10). Although poorly conserved, the exon A region of Lymnaea PAM has a potential endoproteolytic cleavage site (8). Unlike rat PAM-1, which has a single PHM domain, Lymnaea PAM has four PHM domains followed by an alternately spliced exon A region and PAL (8). Importantly, expression of forms of Lymnaea PAM with and without exon A is cell type specific. The lack of exon A in Xenopus laevis and Drosophila PAM suggests that exon A may have evolved later than the two enzymatic domains (9, 46). Although Xenopus laevis PAM lacks exon A, an different endoproteolytic cleavage site occurs between the PHM and PAL catalytic domains, thus enabling separation of soluble PHM from membrane PAL (9). In Drosophila, separate genes encode PHM and PAL, eliminating the need for an intervening cleavage site (46).

Splicing across exons Ba and Bb also differed in Harlan and Charles River Sprague Dawley rat anterior pituitary (3). Exon Ba encodes the transmembrane domain, and its presence or absence determines the membrane topology of PAM and its cellular localization (7, 47). Exon Bb encodes an 18-amino acid peptide [rPAM-1-(900–917)]; as PAM transcripts with exon Bb but lacking exon Ba were not identified in any tissue in either type of rat, when this 18-amino acid peptide is included, it is always situated adjacent to the cytosolic side of the transmembrane domain. Lys919 has been identified as a key site in the interaction of PAM-1 with cytosolic proteins such as P-CIP2 and kalirin, and the presence or absence of exon Bb could affect these interactions (48, 49).

Our data indicate that PAM-1 is not expressed in the anterior pituitary of Harlan or Charles River Sprague Dawley rats. Although PAM transcripts in the anterior pituitaries of Charles River Sprague Dawley rats include exon A, they lack exon Bb; an equivalent PAM transcript was identified in the initial cloning of bovine pituitary PAM (24) and in AtT-20 cells (GenBank no. AAB38364). The three major PAM splice variants identified in the anterior pituitary of Harlan rats all lack exon A. Based on RT-PCR and Western blot analysis, the anterior pituitaries of Charles River Sprague Dawley rats may express PAM proteins, which include exon A but lack both exons Ba and Bb (PAM-3/{Sigma}A). This soluble protein would be expected to undergo extensive endoproteolytic cleavage in exon A, and soluble PHM and PAL proteins of an appropriate size are detected with the exon A antibody. A similar form of PAM (clone 204) was identified in the anterior pituitary of Wistar rats (4) and Sprague Dawley rats (3).

Differences in PAM splicing were most dramatic in the anterior pituitary, but were not limited to this tissue. PAM-1 was more prevalent than PAM-2 in atria of adult Charles River rats and was less prevalent in atria of Sprague Dawley rats. In an earlier study using Sprague Dawley rats from Holtzman (Madison, WI), the prevalence of PAM-1 and PAM-2 mRNAs in the rat cardiac atrium and ventricle was found to vary dramatically during fetal and neonatal development (6). Although PAM-2 was the most abundant form during postnatal days 1–3, similar amounts of PAM-1 and PAM-2 were present on postnatal day 5, and PAM-1 was predominant from postnatal day 7 into adulthood (6).

Neither PACAP receptor 1 nor cardiac troponin T exhibited breeder-specific alternative splicing, suggesting that the difference between Charles River and Sprague Dawley rats is in the PAM gene rather than the splicing machinery. The exon A region of the PAM gene represents the site at which the PHM and PAL genes, which are expressed independently in Cnidarians (50) and Drosophila (46), were assembled into a single gene. In the rat, exon A is preceded by an 11.6-kb intron and followed by a more than 23-kb intron that includes an alternate polyadenylase addition site (51).

The pituitary gland consists of a complex mixture of cell types synthesizing and secreting different peptide hormones. We explored strain-specific differences in only one of these pituitary hormones, POMC. Differences in amidated {alpha}MSH content were observed in the anterior pituitary, and differences in amidated joining peptide content were observed in the neurointermediate pituitary. Strain-specific differences in the hypothalamic-pituitary-adrenal axis were previously noted in Sprague Dawley rats obtained from Holtzman and Taconic Farms, Inc. (Germantown, NY); both the pituitary content of ACTH and the sensitivity of isolated adrenocortical cells to ACTH stimulation differed (19). The differences we observed in amidated joining peptide and amidated {alpha}MSH levels were not associated with differences in POMC mRNA levels.

Thus, divergent experimental results obtained by different laboratories may be attributable simply to animals supplied by different breeders. The results from this study and the observations from several other reports demonstrating variability in experimental observations when comparing Sprague Dawley rats from different breeders (13, 14, 15, 16, 17, 18, 19, 20) clearly show that researchers should routinely report the strain and breeder of the animals used in their studies. For example, Petersen et al. found that Sprague Dawley rats from Charles River Breeding Laboratories, Inc., had significantly greater body weight, shorter lifespan, and a higher number of palpable masses and neoplasms than Sprague Dawley rats from Harlan Sprague Dawley, Inc. (52). Charles River reported decreased longevity in their outbred rat stocks and restructured its breeding system according to the International Genetic Standards (53). Historically, Sprague Dawley rats are direct descendants of an initial colony generated in the 1920s by Robert S. Dawley in which a hybrid hooded male and a female Wistar rat were crossed.2 Charles River obtained Sprague Dawley rats in 1950 from Sprague Dawley, Inc. (53). Perhaps the 40–50 yr of separate breeding colonies or the different types of selective rat breeding programs between the different suppliers has resulted in a multitude of differences, including the selective alternative splicing of the PAM gene and different steady state levels of selected POMC products.


   Acknowledgments
 
We thank Dr. Jian-Ping Jin (Department of Medical Biochemistry, Faculty of Medicine, University of Calgary, Calgary, Canada) for the mouse cardiac troponin T cDNA, and Dr. Victor May (Department of Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington, VT) for PACAP receptor-1 cDNA. We gratefully acknowledge Marie Bell for general laboratory assistance, and Lixian Jin for tissue culture.


   Footnotes
 
1 This work was supported by NIH Grant DK-32949 and Human Frontiers Science Program Organization Fellowship LT-647/97 (to G.D.C.). Back

2 http://www.harlan.com/us/index.htm. Back

Received August 4, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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R. El Meskini, R. E. Mains, and B. A. Eipper
Cell Type-Specific Metabolism of Peptidylglycine {alpha}-Amidating Monooxygenase in Anterior Pituitary
Endocrinology, August 1, 2000; 141(8): 3020 - 3034.
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