Novel sites for expression of an Escherichia coli heat-stable enterotoxin receptor in the developing
rat
D. WAYNE LANEY, JR., ELIZABETH A. MANN, STEVEN C. DELLON, DOUGLAS R. PERKINS, RALPH A. GIANNELLA, AND MITCHELL B. COHEN Division of Pediatric Gastroenterology and Nutrition, Children’s Hospital Medical Center, Division of Digestive Diseases, University of Cincinnati College of Medicine, and Veterans Affairs Medical Center, Cincinnati, Ohio 45220 Laney, D. Wayne, Jr., Elizabeth A. Mann, Steven C. Dellon, Douglas R. Perkins, Ralph A. Giannella, and Mitchell B. Cohen. Novel sites for expression of an Escherichia coli heat-stable enterotoxin receptor in the developing rat. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G816G821, 1992.~Escherichia coli heat-stable enterotoxin (STa) mediates diarrhea1 disease by binding to and activating an intestinal transmembrane receptor, guanylate cyclase C (GC-C). To test the hypotheses that there was 1) increased perinatal expression of GC-C in rat intestine and 2) GC-C expression and STa binding in extraintestinal tissues of immature rat, we prepared whole cell membranes and total RNA from jejunum, ileum, colon, liver, kidney, heart, lung, brain, testis, and placenta of rats ranging in age from 12 days gestation to adult. Northern analysis demonstrated the presence of a unique 3.8-kb mRNA transcript at all ages in the jejunum, ileum, colon, and, to a lesser degree, in the testis. GC-C was also detected by Northern analysis in liver (from gestational age 18 days through 14 days postnatal) and in placenta. Steady-state mRNA encoding GC-C was not detected by Northern analysis in the other organs examined. GC-C-specific mRNA expression was greatest in the perinatal period in the jejunum, ileum, and liver. Specific binding of l”‘I-labeled STa was found in each of the tissue membranes in which GC-C mRNA was present; binding was not present in those tissues that had no detectable GC-C mRNA. The existence of GC-C in extraintestinal organs in the rat, and the developmental changes in GC-C expression support our hypothesis that GC-C, apart from its role as an STa receptor in mediating diarrhea1 disease, also serves as a receptor for an endogenous ligand. diarrhea1 disease; guanylate cyclase; guanosine 3’,5’-cyclic monophosphate; guanylate cyclase C; bacterial toxins; ontogeny; guanylin
Escherichia coli are an important cause of diarrhea1 disease, especially in infants and children in developing countries (1). The toxins elaborated by these organisms include a family of heat-stable peptide toxins (STa) that bind to specific receptors on the intestinal brush-border membrane (BBM), activate guanylate cyclase, and stimulate net fluid secretion (10). A pattern of increased STa receptor density occurs in the BBM of pig and human intestine in the perinatal period (6, 14, 21) and in the immature rat (7). Schulz and co-workers (26) have described a cDNA encoding a novel member of the guanylate cyclase family of transmembrane proteins, denoted guanylate cyclase C (GC-C), which serves as a receptor for STa in rat intestine. This protein has an extracellular domain that includes the STa binding region and an intracellular do-
ENTEROTOXIGENIC
main that is the site of guanylate cyclase activity. Using Northern analysis, these investigators (26) were unable to demonstrate the existence of GC-C mRNA in any extraintestinal organ of the adult rat, confirming previous studies showing that STa-stimulated guanylate cyclase activation was limited to the intestine of the adult rat (23). The STa receptor has been shown to be present outside the intestine in the epithelial cells of the kidney and testis of the adult North American opossum (12). Subsequent surveys of adult opossum tissues, using in vitro autoradiographic techniques, demonstrated STa binding in the liver, gallbladder, and trachea as well (16). The presence of this toxin receptor in extraintestinal sites has not yet been explained but suggests the existence of an endogenous ligand(s). A peptide with 40% homology to STa has recently been isolated from rat jejunum (9). This peptide, termed guanylin, was shown to activate intestinal guanylate cyclase in the adult and fetal intestine and to displace STa binding to cultured T8* intestinal epithelial cells. Thus guanylin may represent an endogenous ligand. Based on the observations that the STa receptor exists in greater numbers in immature animals and that the STa receptor is expressed in extraintestinal organs of the opossum, we hypothesized that this protein is also expressed in extraintestinal tissues in the immature rat. Using Northern analysis, we have demonstrated expression of GC-C in the rat liver and placenta in the perinatal period and in low levels in the testis throughout development. We have also shown that membranes prepared from both the intestine and these extraintestinal organs bind STa. Maximal expression of both GC-C mRNA and 125I-STa binding activity in the rat intestine and liver occurred in the perinatal period. The existence of GC-C in extraintestinal organs in the rat and the developmental changes in GC-C expression provide additional evidence for the role of an endogenous ligand for the STa receptor. MATERIALS
AND METHODS
Membrane preparation. Sprague-Dawley rats were obtained from Zivic-Miller (Zelienople, PA) and allowed access to standard rat food and water. Animals were studied at ages ranging from 1 day to adult (~250 g). Timed pregnant females were used to provide fetal animals, gestational age (G) G12-G21. Animals 51 day of age were killed by decapitation; animals >l
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day of age were killed by injection with pentobarbital sodium. Jejunum, ileum, colon, liver, kidney, heart, lung, brain, testis, and placenta were harvested, rinsed of blood in ice-cold saline, and immediately frozen intact in liquid nitrogen. Whole cell membranes were prepared by combining frozen tissue samples, finely crushed with a mortar and pestle, with 2 mM tris(hydroxymethyl)aminomethane (Tris)/50 mM mannito1 buffer, pH 7.1, in a ratio of 1:30 (wt/vol). This mixture was then homogenized using a Tissumizer (Tekmar, Cincinnati, OH). Samples were centrifuged at 500 g for 3 min to remove connective tissue fragments. The remaining supernatants were centrifuged at 30,000 g for 10 min at 4°C and the resulting pellets were resuspended in phosphate-buffered saline [(in mM) 140 NaCl, 1.5 KH,PO,, 8 Na,HPO,, pH 7.41 and frozen for binding studies. Protein content was assayed with the use of a commercially available assay (Bio-Rad, Richmond, CA). STa binding assays. STa was prepared from E. coli strain 18D (28), labeled with 12”I-Na by the lactoperoxidase technique, and separated from unlabeled STa by high-pressure liquid chromatography as previously described (29). Binding assays were performed by incubating 25 pg of whole cell membranes with 50,000 counts per minute (cpm; -50 PM) of 12”1-STa for 90 min at 37°C in 500 ~1 of 100 mM sodium acetate buffer, pH 4.8, as previously described (6). Nonspecific binding was determined in the presence of 1 PM unlabeled STa, and the results were expressed as counts per minute specifically bound per 25 pg membrane protein. The apparent affinity constant of binding (K,) was determined from analysis of competitive displacement of radiolabeled STa with the use of the computer program LIGAND (22) as previously described (7). Polymerase chain reaction amplification of GC-C, preparation of cDNA probe, and Northern and slot blot analyses. A 2-pg aliquot of total RNA isolated from adult rat jejunum was used in a reverse transcriptase reaction containing 20 pm01 of an antisense primer 5-GCTCCGATCCGTTCTTGTAA-3 from the published rat GC-C cDNA sequence (26) and 600 U Moloney murine leukemia virus reverse transcriptase (BRL, Gaithersburg, MD). The resulting cDNA was then used as a template for a polymerase chain reaction in the presence of (in mM) 10 Tris. HCl, pH 8.3, 50 KCl, 2.5 MgCl,, 0.2 of each nucleotide, as well as 30 pmol of each rat GC-C primer, antisense and sense (5-AACCCACGCTGATGTTCTGC-3), and 2 U of AmpliTaq Polymerase (Perkin Elmer Cetus, Norwalk, CT). The product of this reaction, a fragment encompassing nucleotides 54-602 (548 base pairs) of the rat GC-C cDNA, was gel purified and cloned into Bluescript (Stratagene, La Jolla, CA) by blunt end ligation. The identity of the recombinant clone (p5GCC) was confirmed by sequencing (25) with T7 DNA polymerase (Pharmacia, Piscataway, NJ). Aliquots of tissue obtained from the same animals from which membranes were prepared were used to isolate total RNA by acid guanidine isothiocyanate-phenol-chloroform extraction (4). For Northern analysis, 20 pg RNA samples were denatured with glyoxal, fractionated by electrophoresis in a 1% agarose gel, and transferred to a nylon membrane (Magna NT, MSI, Westboro, MA) by capillary action. Samples were cross-linked to the membrane with the use of ultraviolet light and hybridized (5) with the 5 CC-C cDNA fragment, which had been labeled with [:32P]deoxycytidine triphosphate by random primer extension (Megaprime, Amersham, Arlington Heights, IL). Blots were washed under high stringency conditions (0.1 X standard sodium citrate, 65°C) and then subjected to autoradiography using Kodak XAR-5 film (Eastman Kodak, Rochester, NY). The films were exposed from 2 to 10 days at -80°C using 2 Lightning Plus intensifying screens (New England Nuclear, Boston, MA). Slot blot analysis was performed using lo-Mg aliquots of total RNA denatured with formaldehyde and bound to a nylon membrane using the Minifold II Slot-Blotter (Schleicher and
RECEPTOR
G817
EXPRESSION
Schuell, Keene, NH) according to the manufacturer’s protocols. Cross-linking and hybridization were performed in the manner used for Northern analysis. Comparison of relative mRNA abundance was accomplished by hybridizing the membranes with a :“P-labeled 18s ribosomal RNA probe (18) and quantitating the autoradiographic signals by densitometry (LKB, Gaithersburg, MD). Data presentation and analysis. For binding studies, each point was determined in duplicate, and the experiments repeated in triplicate. For slot blot analysis, a minimum of three autoradiographic signals was evaluated for each data point. Data from both slot blot analysis and i2”I-STa binding studies were evaluated using the unpaired Student’s t test. All data are means + SE. RESULTS
Northern analysis. To examine the expression of GC-C in the rat, total RNA was isolated from ileum, jejunum, colon, liver, heart, lung, kidney, brain, testis, and placenta. Northern analysis of total RNA prepared from whole rat intestine (G18 and G20) and from rat jejunum (1 day through adult) demonstrated the presence of a 3.8-kb transcript at all ages examined. GC-C expression was also present at all ages in the ileum and colon (data not shown). As shown in Figure 1, total RNA from 1 day rat liver and intestine, 18 days testis, and G19 placenta also hybridized with the 5 GC-C fragment. A single 3%kb transcript was present by Northern analysis of RNA from liver (G18 through 14 days), testis (14 days through E 3
Fig. 1. Northern analysis of RNA from immature rat organs. Total RNA (20 pg/lane) isolated from multiple organs of immature rat, was hybridized under stringent conditions with 548-bp 5 cDNA fragment of guanylate cyclase C (GC-C) as described in METHODS. A single 3.8-kb transcript is present in 1 day rat jejunum, ileum, colon, and liver (lanes 1-4), 18 day testis (lane 5), as well as in G19 placenta (lane 9). No transcript is present in lanes 6-8 (kidney, brain, heart). 28s and 18s ribosomal RNA markers are indicated on right. Lanes 1 and 2 are overexposed in this film to demonstrate faint signals in other organs. Bottom panel, same blot rehybridized with a :=P-labeled oligonucleotide complementary to 18s ribosomal RNA to demonstrate equal loading of RNA.
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G818
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adult), and placenta (G16-G21) (data not shown). No hybridization was detectable with RNA from kidney, brain, lung, and heart from animals at GE?, GZO, 1 day, 7 days, 14 days, 16 days, 18 days, 20 days, 22 days, 28 days, and adult. Slot blot analyses and 1251-S7’abinding. To quantitate the relative amounts of GC-C specific mRNA present within each organ at various age points, we performed
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slot blot analyses. Binding of 12”I-STa to whole cell membranes prepared from the same organs was measured as an indication of GC-C protein expression. Figure 2 illustrates the results of slot blot analysis and I”“1-STa binding for fetal intestine (Fig. 2A) and separately for postnatal jejunum (Fig. 2B), ileum (Fig. 2C), and colon (Fig. 20). In fetal intestine (Fig. 2A), GC-C mRNA expression and STa binding were similar to what was observed in the adult jejunum but rapidly increased to a maximal level at postnatal day 1 (Fig. 2B). In the jejunum (Fig. 2B), the relative abundance of GC-C mRNA in the 1 day rat was four times greater than in the adult rat (4.05 t 0.30 vs. 1.00 t 0.17, respectively; P < 0.001). There was a progressive decrease of mRNA levels with maturation. A similar pattern was seen for jejunal STa binding activity in the jejunum, although STa binding in the 1 day rat was only twofold greater than that in the adult (15,253 t 1,343 vs. 8,503 t 815 cpm ‘““I-STa specifically bound/25 ,ug protein, respectively;
P = 0.006).
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RECEPTOR
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(days)
Fig. ‘2. Slot blot analysis and l”Y-labeled heat-stable toxin (STa) specific binding in rat fetal intestine (A) and in postnatal jejunum (B), ileum (C), and colon (D). STa receptor mRNA (0) was quantitated by densitometric analysis of slot blots hybridized with 548-bp 5’ cDNA fragment of CC-C and normalized by rehybridization with 18s oligonucleotide probe. Values plotted are mRNA abundance compared with binding (0) is presented as counts adult levels (means + SE). l”‘I-STa per minute (cpm) specifically bound per 25 ,ug membrane protein (means t SE). Nonspecific binding in fetal intestine, postnatal jejunum, and postnatal ileum ranged from 1.2 to 5.2% of total cpm bound. In colon, nonspecific binding ranged from 2.2 to 10.2% of total cpm bound.
In the rat ileum (Fig. 2C), a twofold elevation in GC-C mRNA abundance was noted in 1 day vs. adult rats (1.94 t 0.04 vs. 1.00 t 0.15, respectively; P < 0.001). STa binding activity increased from 1 day to 2 days, reaching a level 2.5 times greater than adult ileum (17,341 t 1,098 vs. 7,044 t 289 cpm 12”I-STa specifically bound/25 pg protein, respectively; P < 0.001). Both steady-state mRNA levels and STa binding activity appeared to increase at 18 days during the weaning period. After day 18, STa binding activity decreased to adult levels, whereas GC-C mRNA levels remained essentially unchanged. In the colon (Fig. 2D), GC-C mRNA levels remained stable without significant variation during postnatal development. However, STa receptor binding activity was 2.5fold greater in l-day colon compared with the adult colon (8,666 t 998 vs. 3,211 t 217 cpm l”jI-STa specifically bound/25 pg protein, respectively; P = 0.003). Comparison of GC-C mRNA abundance (densitometric absorbance of GC-C hybridization normalized by comparison to densitometric absorbance of 18s hybridization) among the jejunum, ileum, and colon revealed a change in the relative amounts in each organ with maturation. In the l-day rat, GC-C mRNA abundance was greatest in the ileum (0.914 t 0.02), followed by the jejunum (0.700 t 0.07), and the colon (0.350 t 0.08). In the adult, mRNA abundance was greatest in the ileum and colon (0.470 t 0.15 and 0.424 t 0.10, respectively), with levels in the jejunum substantially lower (0.173 t 0.14). These maturation-related changes in the relative amounts of GC-C mRNA present in the intestine suggest that each organ has a specific pattern of differential regulation. Comparison of 1251-STabinding in the jejunum, ileum, and colon (Fig. 2) also demonstrated changes in relative amounts with maturation. In the l-day rat intestine, the greatest amount of 12”I-STa binding was found in the jejunum, followed by the ileum, and the colon. In the adult, binding was similar in the jejunum and ileum with less binding present in the colon. These changes in relative amounts of binding did not directly correlate with changes in GC-C mRNA levels. In the liver (Fig. 3A), GC-C mRNA was detected from G16 to adult by slot blot analysis. It was highest at G19 where it was present in amounts approximately sevenfold
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CO,51 HEAT-STABLE
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EXPRESSION
1251-STaspecifically bound/25 pg protein, respectively; P < 0.001). Binding activity was slightly decreased at 1 day and then fell between 1 and 7 days to adult levels. -8 In the rat testis (Fig. 3B), GC-C mRNA expression at 7 -7 2 the earliest time point examined (7 days), was 2.4fold s greater than the amount present in adult testis (P = 0.01). 5 E 5 Steady-state mRNA levels showed a gradual decrease to 1251-STabinding in the testis was present fi ii theadultlevel. at all agestested. Binding was maximal between 7 and 14 days and then decreased by 16 days to adult levels. -2 g; .Similar slot blot analysis and binding studies were done T-i -1 5 using rat placental tissue (Fig. 3C). GC-C mRNA was cr. \ present throughout late gestation (G16-G21), as was 1251STa binding; earlier time points were not examined. No change in the pattern of GC-C expression (mRNA or 5 I 1251-STa binding) in the placenta was observed. 52 To characterize the kinetics of STa binding in rat liver, 2 Q, a:: we determined the ability of increasing concentrations of E : 55 STa to competitively displace radiolabeled STa from g 2 membranes prepared from liver tissue of l-day rats and k+ [r ILo compared this with the binding kinetics of 1 day jejunum m (Fig. 4). Unlabeled STa specifically inhibited the binding Z.? of labeled STa to jejunal membranes in a dose-dependent manner, similar to that observed in the l-day liver (K, = 1.87 t 0.22 x log vs. 1.21 t 0.32 X 10’ l/mol, respectively). Thus, by two criteria, hybridization to same size GC-C transcript under stringent conditions in both liver and intestine and analysis of STa binding kinetics, the STa receptors present in liver and intestine appear to be 5 identical. 2 DISCUSSION !.O4: 2” Us .5 E:
2
RECEPTOR
6001
We surveyed a variety of extraintestinal organs in the rat and found 1251-STabinding and a 3%kb GC-C transcript in the liver, testis, and placenta. This transcript is the same size as that which was shown to encode both the binding and the guanylate cyclase domains of GC-C in the adult rat small intestine (26). In the liver, GC-C mRNA expression is greatest in the
I
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I G18 Age
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(days)
Fig. 3. Slot blot analysis and lY-STa specific binding in rat liver (A), testis (B), and placenta (C). STa receptor mRNA (0) and 12”I-STa specific binding (0) are shown as described in the legend to Fig. 2. In liver and testis, mRNA is expressed as relative abundance compared with adult levels. In placenta, relative abundance is determined in cornparison to G21 levels. Data are means t SE. Nonspecific binding in immature liver (11 day) ranged from 8.9 to 19.7% of total cpm bound. At age 27 days, nonspecific binding was 51.5 to 80.7% of total cpm bound. In testis, nonspecific binding was observed to be 15.5 to 71.3% of total cpm bound, with percentage of nonspecific binding increasing with older-aged animals. Nonspecific binding in placenta was ~16% of total cpm bound.
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greater than in adult liver (7.3 t 1.2 vs. 1.0 t 0.29, respectively; P = 0.002). By 1 day, the amount of mRNA decreased to one-half the level present at G19 and continued to decreaseto the adult level. 1251-STabinding was present at all ages in the liver. Binding was maximal at the G19 time point where it was more than fivefold greater than the adult level (1,941 t 31 vs. 368 t 61 cpm
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Fig. 4. Competitive inhibition of ‘*“I-STa binding by unlabeled STa: 1 day rat jejunum (0) vs. 1 day rat liver (0). Increasing concentrations of unlabeled STa were incubated with a constant amount of 12.51-STa for 90 min at 37°C. Specific binding of 12”1-STa to jejunum and liver membranes is expressed as fraction of 12,51-STa bound in absence of added cold toxin (B/B,). Data points are means + SE.
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perinatal period and decreases with maturation. Similarly, the concentrations of numerous hepatic enzymes exhibit changes in levels of expression with development (13). The perinatal development of the liver is associated with marked changes in the hematopoietic component, circulation patterns, rates of bile flow, and protein synthesis; however, it is not known if these alterations influence the expression of GC-C. Furthermore, it is not known which of the numerous cell populations that make up the liver tissue are responsible for the observed GC-C expression and 12sI-STa binding. Although both binding and mRNA expression are maximal at GI9, the relative rate of decrease between 1 and 7 days is greater for 1251STa binding than for GC-C mRNA expression, suggesting that steady-state mRNA levels are only one of several factors regulating GC-C expression. We observed low levels of l”‘I-STa binding with a high nonspecific component in adult liver and detected no GC-C signal by Northern analysis of RNA from adult liver. However, with the use of the more sensitive and quantitative slot blot analysis, low levels of GC-C mRNA were detected. We also observed GC-C mRNA expression and STa binding in the testis and in the placenta. In the placenta, GC-C mRNA expression and STa binding were constant at the time points evaluated (G16-GZ). GC-C mRNA expression in the rat testis decreased -2.4fold from age 7 days to adult while binding exhibited a more complex pattern. The range of binding observed (-200-900 cpm specifically bound) was substantially less than that observed in the intestine but was similar to that observed in the liver and placenta. This may be due to the presence of multiple cell types in these organs, only one of which bears the STa receptor. Future studies with cell-specific RNA and in situ hybridization will further characterize the expression of GC-C in each of these extraintestinal tissues. We found no evidence of GC-C mRNA in rat brain or heart at any age point. Surprisingly, we also found no evidence of GC-C expression in the kidney, an organ with structural and enzymatic homology to the intestine and from which an endogenous ligand for GC-C has recently been isolated (9). Because an STa receptor has been documented in the opossum kidney (l2), our inability to detect GC-C in rat kidney may indicate low levels of expression undetectable by Northern analysis or a species-specific pattern of GC-C distribution. The presence of GC-C in multiple tissues is analogous to the distribution of other members of the guanylate cyclase family in multiple tissues. mRNA encoding for GC-A, a receptor for both atria1 natriuretic peptide and brain natriuretic peptide, is present in heart and brain tissue of multiple species (19, 30) and has also been described in the kidney, adrenal gland, terminal ileum, and adipose tissue of humans (3, 17). Similarly, GC-B, which is thought to be the primary receptor for a central nervous system natriuretic peptide, is present not only in the brain, but in human placenta, pituitary gland, and terminal ileum as well (2, 27). An increased density of STa receptors is present in the intestine of several species of immature animals (6, 14, 2 I). However, the relationship between the maturational changes in GC-C mRNA expression and STa binding (GC-C function) has not previouslv been explored. In the
RECEPTOR
EXPRESSION
jejunum, GC-C mRNA expression and STa binding exhibit concurrent maximal levels in the first days of life and then decrease in a parallel fashion. These similar changes suggestthat GC-C expression is under transcriptional control in the jejunum during the perinatal period. At the time of weaning (14-21 days), an increase in GC-C protein expression occurs without a significant increase in GC-C mRNA expression. This suggeststhat translational or posttranslational events modulate GC-C expression at weaning. Similarly, after weaning (22 days), STa binding decreases by approximately one-third, whereas GC-C expression remains relatively constant through adulthood. In the ileum, GC-C mRNA expression and 12”1-STa binding also decrease with maturation. However, unlike the pattern in the jejunum, the decrease in both parameters is of similar magnitude (-2-fold). At present, only one receptor for STa has been identified. It is possible, however, that there are other receptors in addition to GC-C. We cannot exclude the possibility that the binding we observed is due to these other receptors. The presence of another STa binding specieswould provide an alternative explanation for the discordance between our observed 12”I-STa binding and GC-C mRNA levels. Other brush-border membrane proteins present in the small intestine are known to exhibit changes in expression or activity that correspond to changes in development. These changes may be grouped into two patterns. The first pattern is demonstrated by lactase and neur aminidase, which are expressed maximally at birth. Levels of these enzymes decrease during the weaning period in rats, reaching very low or nearly absent “adult” levels by the end of the third postnatal week (11,15). The second pattern is exemplified by digestive enzymes including amylase, maltase, sucrase, and intestinal aminooligopeptidase. These enzymes are present in minimal levels in the perinatal period and then increase at the time of weaning to achieve levels in the range of those measured in the adult (8, 15, 24). The ontogeny of GC-C expression in the small intestine clearly differs from both of these patterns in that expression is maximal in the perinatal period but not absent in the adult. At the time of weaning, the serum levels of glucocorticoids and thyroxine increase and are thought to be partly responsible for the elevation in disaccharidases, which occur during that period (15). Glucocorticoids have also been shown to be involved in the decrease in lactase levels that occurs in the weaning period (15). The relationship of these and other hormones changes to changes in GC-C levels is not yet known. In contrast to the small intestine, GC-C mRNA levels exhibit little change with development in the colon. STa binding, however, exhibits a 2.5fold decline from 1 day to adult levels, suggesting posttranscriptional regulatory mechanisms. In addition, unlike the pattern observed in the small intestine, binding in the colon decreasesto near adult levels before the time of weaning (14 days). Previous studies in our laboratory using different techniques, have also shown STa binding and STa-mediated guanylate cyclase stimulation in colonocytes (20). Although we have demonstrated 1251-STabinding and GC-C mRNA expression to be present in extraintestinal sites in the rat, we have not determined whether the
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GC-C present is activated by STa. Furthermore, we have not determined whether extraintestinal GC-C is linked to chloride secretion as has been demonstrated in the intestine (10). Our studies demonstrate the existence of GC-C mRNA and GC-C protein at all ages examined in the jejunum, ileum, and colon. The amount of GC-C protein expressed in these organs, as measured by STa binding, is greater than levels measured at equivalent agesin extraintestinal sites. Both the developmental pattern and the higher level of GC-C expression in the intestine suggest that GC-C has an endogenous function in the intestine as well. Because GC-C mediates STa-induced diarrhea1 disease by alterations in epithelial cell ion flux, the endogenous function of GC-C could also be to regulate epithelial cell ion flux in response to an endogenous ligand. Recently, guanylin, a 15 amino acid peptide with 40% homology to STa has been identified as the first putative endogenous ligand for the STa receptor (9). The physiological functions of guanylin in the intestine and in extraintestinal sites of GC-C expression as well as the existence of other endogenous ligands for the STa receptor are currently under investigation. The authors thank Dr. William F. Balistreri for helpful suggestions regarding this article. This work was presented in part at Digestive Disease Week, American Gastroenterological Association, lo-13 May 1992, in San Francisco, CA, and published in abstract form (Gastroenterology 102: A562, 1992). This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Health Grant DK-01908, an American Gastroenterological Association/Glaxo Industry Award, Veterans Affairs Research Grant 539-3108-01, and by a Bristol-Myers Perinatal Research Center Award. Address for reprint requests: M. B. Cohen, Div.of Pediatric Gastroenterology and Nutrition, Children’s Hospital Medical Center, 3250 Elland Ave., Cincinnati, OH, 45229. Received
28 May
1992; accepted
in final
form
13 August
1992.
REFERENCES 1. Black, R. E., K. H. Brown, S. Becker, A. R. M. Abdul Alim, and I. Huq. Longitudinal studies of infectious diseases and physical growth of children in rural Bangladesh. II. Incidence of diarrhea and association with known pathogens. Am. J. Epidemiol. 115: 315-324, 1982. 2. Chang, M. S., D. G. Lowe, M. Lewis, R. Hellmiss, E. Chen, and D. V. Goeddel. Differential activation by atria1 and brain natriuretic peptides of two receptor guanylate cyclases. Nature Lond. 341: 68-72, 1989. 3. Chinkers, M., and D. L. Garbers. The protein kinase domain of the ANP receptor is required for signaling. Science Wash. DC 245: 1392-1394, 1989. 4. Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987. 5. Church, G. M., and W. Gilbert. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81: 1991-1995, 1984. 6. Cohen, M. B., A. Guarino, R. Shukla, and R. A. Giannella. Age-related differences in receptors for Escherichia coli heat-stable enterotoxin in the small and large intestine of children. Gastroenterology 94: 367-373, 1988. 7. Cohen, M. B., M. S. Moyer, M. Luttrell, and R. A. Giannella. The immature rat small intestine exhibits an increased sensitivity and response to Escherichia coli heat-stable enterotoxin. Pediatr. Res. 20: 555-560, 1986. 8. Cohen, M. L., N. A. Santiago, J.-S. Zhu, and G. M. Gray. Differential regulation of intestinal amino-oligopeptidase gene expression in neonatal and adult rats. Am. J. PhysioZ. 261 (Gastrointest. Liver Physiol. 24): G866-G871, 1991. 9. Currie, M. G., K. F. Fok, J. Kato, R. J. Moore, F. K. Hamura. K. L. Duffin. and C. E. Smith. Guanvlin: an endog-
RECEPTOR
EXPRESSION
G821
of intestinal guanylate cyclase. Proc. Natl. Acad. 1992. 10. DeJonge, H. R. The mechanism of action of Escherichia coli heat-stable toxin. Biochem. Sot. Trans. 12: 180-184, 1984. 11. Dickson, J. J., and M. Messer. Intestinal neuraminidase activity of suckling rats and other mammals: relationship to the sialic acid content of milk. Biochem. J. 170: 407-413, 1978. 12. Forte, L. R., W. J. Krause, and R. H. Freeman. Escherichia coZi enterotoxin receptors: localization in opossum kidney, intestine, and testis. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F874-F881, 1989. 13. Greengard, Olga. Enzyme differentiation of human liver: comparison with the rat model. Pediatr. Res. 11: 669-676, 1977. 14. Guarino, A., M. B. Cohen, and R. A. Giannella. Small and large intestinal guanylate cyclase activity in children: effect of age and stimulation by Escherichia coli heat-stable enterotoxin. Pediatr. Res. 21: 551-555, 1987. 15. Henning, S. J. Biochemistry of intestinal development. Environ. Health Perspect. 33: 9-16, 1979. 16. Krause, W. J., R. H. Freeman, and L. R. Forte. Autoradiographic demonstration of specific binding sites for E. coZi enterotoxin in various epithelia of the North American opossum. Cell Tissue Res. 260: 387-394, 1990. 17. Lowe, D. G., M. S. Chang, R. Hellmiss, E. Chen, S. Singh, and D. L. Garbers. Human atria1 natriuretic peptide receptor defines a new paradigm for second messenger signal transduction. EMBO J. 8: 1377-1384, 1989. 18. Mann, E. A., and J. B. Lingrel. Developmental and tissuespecific expression of rat T-kininogen. Biochem. Biophys. Res. Commun. 174: 417-423, 1991. 19. Mantyh, C. R., L. Kruger, N. C. Brecha, and P. W. Mantyh. Localization of specific binding sites for atria1 natriuretic factor in peripheral tissues of the guinea pig, rat, and human. Hypertension Dallas 8: 712-721, 1986. 20. Mezoff, A. G., R. A. Giannella, M. N. Eade, and M. B. Cohen. Escherichia coli enterotoxin (ST,) binds to receptors, stimulates guanyl cyclase, and impairs absorption in rat colon. Gastroenterology 102: 816-822, 1992. 21 Mezoff, A. G., N. J. Jensen, and M. B. Cohen. Mechanisms of increased susceptibility of immature and weaned pigs to Escherichia coli heat-stable enterotoxin. Pediatr. Res. 29: 424-428, 1990. 22 Munson, P. J., and D. Rodbard. LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107: 220-239, 1980. 23 Rao, M. C., S. Guandalini, P. L. Smith, and M. Field. Mode of action of heat-stable E. coli enterotoxin: tissue and subcellular specificities and role of cyclic GMP. Biochem. Biophys. Acta 632: 660-672, 1980. 24 Rubino, A., F. Zimbalatti, and S. Auricchio. Intestinal disaccharidase activities in adult and suckling rats. Biochim. Biophys. Acta 92: 305-311, 1964. 25. Sanger, F., S. Nicklen, and A. R. Coulson. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467, 1977. S., C. K. Green, P. S. T. Yuen, and D. L. Garbers. 26. Schulz, Guanylyl cyclase is a heat-stable enterotoxin receptor. CeZL 63: 941-948, 1990. 27. Schulz, S., S. Singh, R. A. Bellet, G. Singh, D. J. Tubb, H. Chin, and D. L. Garbers. The primary structure of a plasma membrane guanylate cyclase demonstrates diversity within this new receptor family. Cell 58: 1155-1162, 1989. S. J., S. E. Asher, and R. A. Giannella. Purification 28. Staples, and characterization of heat-stable enterotoxin produced by a strain of Escherichia coli pathogenic for man. J. BioZ. Chem. 255: 4716-4721, 1980. M. R., M. Luttrell, G. Overmann, and R. A. 29. Thompson, Giannella. Biological and immunological characteristics of 12FiI-4 Tyr and -18 Tyr Escherichia coli heat-stable enterotoxin species purified by high- performance liquid chromatography. Anal. Biothem. 148: 26-36, 1985. J. N., A. Augustine, D. V. Goeddel, and D. G. 30. Wilcox, Lowe. Differential regional expression of three natriuretic peptide receptor genes within primate tissue. Mol. CeZl.BioL. 11: 34543462, 1991. enous
activator
Sci. USA 89: 947-951,
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rat
D. WAYNE LANEY, JR., ELIZABETH A. MANN, STEVEN C. DELLON, DOUGLAS R. PERKINS, RALPH A. GIANNELLA, AND MITCHELL B. COHEN Division of Pediatric Gastroenterology and Nutrition, Children’s Hospital Medical Center, Division of Digestive Diseases, University of Cincinnati College of Medicine, and Veterans Affairs Medical Center, Cincinnati, Ohio 45220 Laney, D. Wayne, Jr., Elizabeth A. Mann, Steven C. Dellon, Douglas R. Perkins, Ralph A. Giannella, and Mitchell B. Cohen. Novel sites for expression of an Escherichia coli heat-stable enterotoxin receptor in the developing rat. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G816G821, 1992.~Escherichia coli heat-stable enterotoxin (STa) mediates diarrhea1 disease by binding to and activating an intestinal transmembrane receptor, guanylate cyclase C (GC-C). To test the hypotheses that there was 1) increased perinatal expression of GC-C in rat intestine and 2) GC-C expression and STa binding in extraintestinal tissues of immature rat, we prepared whole cell membranes and total RNA from jejunum, ileum, colon, liver, kidney, heart, lung, brain, testis, and placenta of rats ranging in age from 12 days gestation to adult. Northern analysis demonstrated the presence of a unique 3.8-kb mRNA transcript at all ages in the jejunum, ileum, colon, and, to a lesser degree, in the testis. GC-C was also detected by Northern analysis in liver (from gestational age 18 days through 14 days postnatal) and in placenta. Steady-state mRNA encoding GC-C was not detected by Northern analysis in the other organs examined. GC-C-specific mRNA expression was greatest in the perinatal period in the jejunum, ileum, and liver. Specific binding of l”‘I-labeled STa was found in each of the tissue membranes in which GC-C mRNA was present; binding was not present in those tissues that had no detectable GC-C mRNA. The existence of GC-C in extraintestinal organs in the rat, and the developmental changes in GC-C expression support our hypothesis that GC-C, apart from its role as an STa receptor in mediating diarrhea1 disease, also serves as a receptor for an endogenous ligand. diarrhea1 disease; guanylate cyclase; guanosine 3’,5’-cyclic monophosphate; guanylate cyclase C; bacterial toxins; ontogeny; guanylin
Escherichia coli are an important cause of diarrhea1 disease, especially in infants and children in developing countries (1). The toxins elaborated by these organisms include a family of heat-stable peptide toxins (STa) that bind to specific receptors on the intestinal brush-border membrane (BBM), activate guanylate cyclase, and stimulate net fluid secretion (10). A pattern of increased STa receptor density occurs in the BBM of pig and human intestine in the perinatal period (6, 14, 21) and in the immature rat (7). Schulz and co-workers (26) have described a cDNA encoding a novel member of the guanylate cyclase family of transmembrane proteins, denoted guanylate cyclase C (GC-C), which serves as a receptor for STa in rat intestine. This protein has an extracellular domain that includes the STa binding region and an intracellular do-
ENTEROTOXIGENIC
main that is the site of guanylate cyclase activity. Using Northern analysis, these investigators (26) were unable to demonstrate the existence of GC-C mRNA in any extraintestinal organ of the adult rat, confirming previous studies showing that STa-stimulated guanylate cyclase activation was limited to the intestine of the adult rat (23). The STa receptor has been shown to be present outside the intestine in the epithelial cells of the kidney and testis of the adult North American opossum (12). Subsequent surveys of adult opossum tissues, using in vitro autoradiographic techniques, demonstrated STa binding in the liver, gallbladder, and trachea as well (16). The presence of this toxin receptor in extraintestinal sites has not yet been explained but suggests the existence of an endogenous ligand(s). A peptide with 40% homology to STa has recently been isolated from rat jejunum (9). This peptide, termed guanylin, was shown to activate intestinal guanylate cyclase in the adult and fetal intestine and to displace STa binding to cultured T8* intestinal epithelial cells. Thus guanylin may represent an endogenous ligand. Based on the observations that the STa receptor exists in greater numbers in immature animals and that the STa receptor is expressed in extraintestinal organs of the opossum, we hypothesized that this protein is also expressed in extraintestinal tissues in the immature rat. Using Northern analysis, we have demonstrated expression of GC-C in the rat liver and placenta in the perinatal period and in low levels in the testis throughout development. We have also shown that membranes prepared from both the intestine and these extraintestinal organs bind STa. Maximal expression of both GC-C mRNA and 125I-STa binding activity in the rat intestine and liver occurred in the perinatal period. The existence of GC-C in extraintestinal organs in the rat and the developmental changes in GC-C expression provide additional evidence for the role of an endogenous ligand for the STa receptor. MATERIALS
AND METHODS
Membrane preparation. Sprague-Dawley rats were obtained from Zivic-Miller (Zelienople, PA) and allowed access to standard rat food and water. Animals were studied at ages ranging from 1 day to adult (~250 g). Timed pregnant females were used to provide fetal animals, gestational age (G) G12-G21. Animals 51 day of age were killed by decapitation; animals >l
G816 Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 16, 2019.
ESCHERICHIA
COLI HEAT-STABLE
day of age were killed by injection with pentobarbital sodium. Jejunum, ileum, colon, liver, kidney, heart, lung, brain, testis, and placenta were harvested, rinsed of blood in ice-cold saline, and immediately frozen intact in liquid nitrogen. Whole cell membranes were prepared by combining frozen tissue samples, finely crushed with a mortar and pestle, with 2 mM tris(hydroxymethyl)aminomethane (Tris)/50 mM mannito1 buffer, pH 7.1, in a ratio of 1:30 (wt/vol). This mixture was then homogenized using a Tissumizer (Tekmar, Cincinnati, OH). Samples were centrifuged at 500 g for 3 min to remove connective tissue fragments. The remaining supernatants were centrifuged at 30,000 g for 10 min at 4°C and the resulting pellets were resuspended in phosphate-buffered saline [(in mM) 140 NaCl, 1.5 KH,PO,, 8 Na,HPO,, pH 7.41 and frozen for binding studies. Protein content was assayed with the use of a commercially available assay (Bio-Rad, Richmond, CA). STa binding assays. STa was prepared from E. coli strain 18D (28), labeled with 12”I-Na by the lactoperoxidase technique, and separated from unlabeled STa by high-pressure liquid chromatography as previously described (29). Binding assays were performed by incubating 25 pg of whole cell membranes with 50,000 counts per minute (cpm; -50 PM) of 12”1-STa for 90 min at 37°C in 500 ~1 of 100 mM sodium acetate buffer, pH 4.8, as previously described (6). Nonspecific binding was determined in the presence of 1 PM unlabeled STa, and the results were expressed as counts per minute specifically bound per 25 pg membrane protein. The apparent affinity constant of binding (K,) was determined from analysis of competitive displacement of radiolabeled STa with the use of the computer program LIGAND (22) as previously described (7). Polymerase chain reaction amplification of GC-C, preparation of cDNA probe, and Northern and slot blot analyses. A 2-pg aliquot of total RNA isolated from adult rat jejunum was used in a reverse transcriptase reaction containing 20 pm01 of an antisense primer 5-GCTCCGATCCGTTCTTGTAA-3 from the published rat GC-C cDNA sequence (26) and 600 U Moloney murine leukemia virus reverse transcriptase (BRL, Gaithersburg, MD). The resulting cDNA was then used as a template for a polymerase chain reaction in the presence of (in mM) 10 Tris. HCl, pH 8.3, 50 KCl, 2.5 MgCl,, 0.2 of each nucleotide, as well as 30 pmol of each rat GC-C primer, antisense and sense (5-AACCCACGCTGATGTTCTGC-3), and 2 U of AmpliTaq Polymerase (Perkin Elmer Cetus, Norwalk, CT). The product of this reaction, a fragment encompassing nucleotides 54-602 (548 base pairs) of the rat GC-C cDNA, was gel purified and cloned into Bluescript (Stratagene, La Jolla, CA) by blunt end ligation. The identity of the recombinant clone (p5GCC) was confirmed by sequencing (25) with T7 DNA polymerase (Pharmacia, Piscataway, NJ). Aliquots of tissue obtained from the same animals from which membranes were prepared were used to isolate total RNA by acid guanidine isothiocyanate-phenol-chloroform extraction (4). For Northern analysis, 20 pg RNA samples were denatured with glyoxal, fractionated by electrophoresis in a 1% agarose gel, and transferred to a nylon membrane (Magna NT, MSI, Westboro, MA) by capillary action. Samples were cross-linked to the membrane with the use of ultraviolet light and hybridized (5) with the 5 CC-C cDNA fragment, which had been labeled with [:32P]deoxycytidine triphosphate by random primer extension (Megaprime, Amersham, Arlington Heights, IL). Blots were washed under high stringency conditions (0.1 X standard sodium citrate, 65°C) and then subjected to autoradiography using Kodak XAR-5 film (Eastman Kodak, Rochester, NY). The films were exposed from 2 to 10 days at -80°C using 2 Lightning Plus intensifying screens (New England Nuclear, Boston, MA). Slot blot analysis was performed using lo-Mg aliquots of total RNA denatured with formaldehyde and bound to a nylon membrane using the Minifold II Slot-Blotter (Schleicher and
RECEPTOR
G817
EXPRESSION
Schuell, Keene, NH) according to the manufacturer’s protocols. Cross-linking and hybridization were performed in the manner used for Northern analysis. Comparison of relative mRNA abundance was accomplished by hybridizing the membranes with a :“P-labeled 18s ribosomal RNA probe (18) and quantitating the autoradiographic signals by densitometry (LKB, Gaithersburg, MD). Data presentation and analysis. For binding studies, each point was determined in duplicate, and the experiments repeated in triplicate. For slot blot analysis, a minimum of three autoradiographic signals was evaluated for each data point. Data from both slot blot analysis and i2”I-STa binding studies were evaluated using the unpaired Student’s t test. All data are means + SE. RESULTS
Northern analysis. To examine the expression of GC-C in the rat, total RNA was isolated from ileum, jejunum, colon, liver, heart, lung, kidney, brain, testis, and placenta. Northern analysis of total RNA prepared from whole rat intestine (G18 and G20) and from rat jejunum (1 day through adult) demonstrated the presence of a 3.8-kb transcript at all ages examined. GC-C expression was also present at all ages in the ileum and colon (data not shown). As shown in Figure 1, total RNA from 1 day rat liver and intestine, 18 days testis, and G19 placenta also hybridized with the 5 GC-C fragment. A single 3%kb transcript was present by Northern analysis of RNA from liver (G18 through 14 days), testis (14 days through E 3
Fig. 1. Northern analysis of RNA from immature rat organs. Total RNA (20 pg/lane) isolated from multiple organs of immature rat, was hybridized under stringent conditions with 548-bp 5 cDNA fragment of guanylate cyclase C (GC-C) as described in METHODS. A single 3.8-kb transcript is present in 1 day rat jejunum, ileum, colon, and liver (lanes 1-4), 18 day testis (lane 5), as well as in G19 placenta (lane 9). No transcript is present in lanes 6-8 (kidney, brain, heart). 28s and 18s ribosomal RNA markers are indicated on right. Lanes 1 and 2 are overexposed in this film to demonstrate faint signals in other organs. Bottom panel, same blot rehybridized with a :=P-labeled oligonucleotide complementary to 18s ribosomal RNA to demonstrate equal loading of RNA.
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G818
ESCHERICHIA
COLI
HEAT-STABLE
adult), and placenta (G16-G21) (data not shown). No hybridization was detectable with RNA from kidney, brain, lung, and heart from animals at GE?, GZO, 1 day, 7 days, 14 days, 16 days, 18 days, 20 days, 22 days, 28 days, and adult. Slot blot analyses and 1251-S7’abinding. To quantitate the relative amounts of GC-C specific mRNA present within each organ at various age points, we performed
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slot blot analyses. Binding of 12”I-STa to whole cell membranes prepared from the same organs was measured as an indication of GC-C protein expression. Figure 2 illustrates the results of slot blot analysis and I”“1-STa binding for fetal intestine (Fig. 2A) and separately for postnatal jejunum (Fig. 2B), ileum (Fig. 2C), and colon (Fig. 20). In fetal intestine (Fig. 2A), GC-C mRNA expression and STa binding were similar to what was observed in the adult jejunum but rapidly increased to a maximal level at postnatal day 1 (Fig. 2B). In the jejunum (Fig. 2B), the relative abundance of GC-C mRNA in the 1 day rat was four times greater than in the adult rat (4.05 t 0.30 vs. 1.00 t 0.17, respectively; P < 0.001). There was a progressive decrease of mRNA levels with maturation. A similar pattern was seen for jejunal STa binding activity in the jejunum, although STa binding in the 1 day rat was only twofold greater than that in the adult (15,253 t 1,343 vs. 8,503 t 815 cpm ‘““I-STa specifically bound/25 ,ug protein, respectively;
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Fig. ‘2. Slot blot analysis and l”Y-labeled heat-stable toxin (STa) specific binding in rat fetal intestine (A) and in postnatal jejunum (B), ileum (C), and colon (D). STa receptor mRNA (0) was quantitated by densitometric analysis of slot blots hybridized with 548-bp 5’ cDNA fragment of CC-C and normalized by rehybridization with 18s oligonucleotide probe. Values plotted are mRNA abundance compared with binding (0) is presented as counts adult levels (means + SE). l”‘I-STa per minute (cpm) specifically bound per 25 ,ug membrane protein (means t SE). Nonspecific binding in fetal intestine, postnatal jejunum, and postnatal ileum ranged from 1.2 to 5.2% of total cpm bound. In colon, nonspecific binding ranged from 2.2 to 10.2% of total cpm bound.
In the rat ileum (Fig. 2C), a twofold elevation in GC-C mRNA abundance was noted in 1 day vs. adult rats (1.94 t 0.04 vs. 1.00 t 0.15, respectively; P < 0.001). STa binding activity increased from 1 day to 2 days, reaching a level 2.5 times greater than adult ileum (17,341 t 1,098 vs. 7,044 t 289 cpm 12”I-STa specifically bound/25 pg protein, respectively; P < 0.001). Both steady-state mRNA levels and STa binding activity appeared to increase at 18 days during the weaning period. After day 18, STa binding activity decreased to adult levels, whereas GC-C mRNA levels remained essentially unchanged. In the colon (Fig. 2D), GC-C mRNA levels remained stable without significant variation during postnatal development. However, STa receptor binding activity was 2.5fold greater in l-day colon compared with the adult colon (8,666 t 998 vs. 3,211 t 217 cpm l”jI-STa specifically bound/25 pg protein, respectively; P = 0.003). Comparison of GC-C mRNA abundance (densitometric absorbance of GC-C hybridization normalized by comparison to densitometric absorbance of 18s hybridization) among the jejunum, ileum, and colon revealed a change in the relative amounts in each organ with maturation. In the l-day rat, GC-C mRNA abundance was greatest in the ileum (0.914 t 0.02), followed by the jejunum (0.700 t 0.07), and the colon (0.350 t 0.08). In the adult, mRNA abundance was greatest in the ileum and colon (0.470 t 0.15 and 0.424 t 0.10, respectively), with levels in the jejunum substantially lower (0.173 t 0.14). These maturation-related changes in the relative amounts of GC-C mRNA present in the intestine suggest that each organ has a specific pattern of differential regulation. Comparison of 1251-STabinding in the jejunum, ileum, and colon (Fig. 2) also demonstrated changes in relative amounts with maturation. In the l-day rat intestine, the greatest amount of 12”I-STa binding was found in the jejunum, followed by the ileum, and the colon. In the adult, binding was similar in the jejunum and ileum with less binding present in the colon. These changes in relative amounts of binding did not directly correlate with changes in GC-C mRNA levels. In the liver (Fig. 3A), GC-C mRNA was detected from G16 to adult by slot blot analysis. It was highest at G19 where it was present in amounts approximately sevenfold
Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 16, 2019.
ESCHERICHIA
CO,51 HEAT-STABLE
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1251-STaspecifically bound/25 pg protein, respectively; P < 0.001). Binding activity was slightly decreased at 1 day and then fell between 1 and 7 days to adult levels. -8 In the rat testis (Fig. 3B), GC-C mRNA expression at 7 -7 2 the earliest time point examined (7 days), was 2.4fold s greater than the amount present in adult testis (P = 0.01). 5 E 5 Steady-state mRNA levels showed a gradual decrease to 1251-STabinding in the testis was present fi ii theadultlevel. at all agestested. Binding was maximal between 7 and 14 days and then decreased by 16 days to adult levels. -2 g; .Similar slot blot analysis and binding studies were done T-i -1 5 using rat placental tissue (Fig. 3C). GC-C mRNA was cr. \ present throughout late gestation (G16-G21), as was 1251STa binding; earlier time points were not examined. No change in the pattern of GC-C expression (mRNA or 5 I 1251-STa binding) in the placenta was observed. 52 To characterize the kinetics of STa binding in rat liver, 2 Q, a:: we determined the ability of increasing concentrations of E : 55 STa to competitively displace radiolabeled STa from g 2 membranes prepared from liver tissue of l-day rats and k+ [r ILo compared this with the binding kinetics of 1 day jejunum m (Fig. 4). Unlabeled STa specifically inhibited the binding Z.? of labeled STa to jejunal membranes in a dose-dependent manner, similar to that observed in the l-day liver (K, = 1.87 t 0.22 x log vs. 1.21 t 0.32 X 10’ l/mol, respectively). Thus, by two criteria, hybridization to same size GC-C transcript under stringent conditions in both liver and intestine and analysis of STa binding kinetics, the STa receptors present in liver and intestine appear to be 5 identical. 2 DISCUSSION !.O4: 2” Us .5 E:
2
RECEPTOR
6001
We surveyed a variety of extraintestinal organs in the rat and found 1251-STabinding and a 3%kb GC-C transcript in the liver, testis, and placenta. This transcript is the same size as that which was shown to encode both the binding and the guanylate cyclase domains of GC-C in the adult rat small intestine (26). In the liver, GC-C mRNA expression is greatest in the
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Fig. 3. Slot blot analysis and lY-STa specific binding in rat liver (A), testis (B), and placenta (C). STa receptor mRNA (0) and 12”I-STa specific binding (0) are shown as described in the legend to Fig. 2. In liver and testis, mRNA is expressed as relative abundance compared with adult levels. In placenta, relative abundance is determined in cornparison to G21 levels. Data are means t SE. Nonspecific binding in immature liver (11 day) ranged from 8.9 to 19.7% of total cpm bound. At age 27 days, nonspecific binding was 51.5 to 80.7% of total cpm bound. In testis, nonspecific binding was observed to be 15.5 to 71.3% of total cpm bound, with percentage of nonspecific binding increasing with older-aged animals. Nonspecific binding in placenta was ~16% of total cpm bound.
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greater than in adult liver (7.3 t 1.2 vs. 1.0 t 0.29, respectively; P = 0.002). By 1 day, the amount of mRNA decreased to one-half the level present at G19 and continued to decreaseto the adult level. 1251-STabinding was present at all ages in the liver. Binding was maximal at the G19 time point where it was more than fivefold greater than the adult level (1,941 t 31 vs. 368 t 61 cpm
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Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (132.174.254.157) on January 16, 2019.
G820
ESCHERICHIA
COLI HEAT-STABLE
perinatal period and decreases with maturation. Similarly, the concentrations of numerous hepatic enzymes exhibit changes in levels of expression with development (13). The perinatal development of the liver is associated with marked changes in the hematopoietic component, circulation patterns, rates of bile flow, and protein synthesis; however, it is not known if these alterations influence the expression of GC-C. Furthermore, it is not known which of the numerous cell populations that make up the liver tissue are responsible for the observed GC-C expression and 12sI-STa binding. Although both binding and mRNA expression are maximal at GI9, the relative rate of decrease between 1 and 7 days is greater for 1251STa binding than for GC-C mRNA expression, suggesting that steady-state mRNA levels are only one of several factors regulating GC-C expression. We observed low levels of l”‘I-STa binding with a high nonspecific component in adult liver and detected no GC-C signal by Northern analysis of RNA from adult liver. However, with the use of the more sensitive and quantitative slot blot analysis, low levels of GC-C mRNA were detected. We also observed GC-C mRNA expression and STa binding in the testis and in the placenta. In the placenta, GC-C mRNA expression and STa binding were constant at the time points evaluated (G16-GZ). GC-C mRNA expression in the rat testis decreased -2.4fold from age 7 days to adult while binding exhibited a more complex pattern. The range of binding observed (-200-900 cpm specifically bound) was substantially less than that observed in the intestine but was similar to that observed in the liver and placenta. This may be due to the presence of multiple cell types in these organs, only one of which bears the STa receptor. Future studies with cell-specific RNA and in situ hybridization will further characterize the expression of GC-C in each of these extraintestinal tissues. We found no evidence of GC-C mRNA in rat brain or heart at any age point. Surprisingly, we also found no evidence of GC-C expression in the kidney, an organ with structural and enzymatic homology to the intestine and from which an endogenous ligand for GC-C has recently been isolated (9). Because an STa receptor has been documented in the opossum kidney (l2), our inability to detect GC-C in rat kidney may indicate low levels of expression undetectable by Northern analysis or a species-specific pattern of GC-C distribution. The presence of GC-C in multiple tissues is analogous to the distribution of other members of the guanylate cyclase family in multiple tissues. mRNA encoding for GC-A, a receptor for both atria1 natriuretic peptide and brain natriuretic peptide, is present in heart and brain tissue of multiple species (19, 30) and has also been described in the kidney, adrenal gland, terminal ileum, and adipose tissue of humans (3, 17). Similarly, GC-B, which is thought to be the primary receptor for a central nervous system natriuretic peptide, is present not only in the brain, but in human placenta, pituitary gland, and terminal ileum as well (2, 27). An increased density of STa receptors is present in the intestine of several species of immature animals (6, 14, 2 I). However, the relationship between the maturational changes in GC-C mRNA expression and STa binding (GC-C function) has not previouslv been explored. In the
RECEPTOR
EXPRESSION
jejunum, GC-C mRNA expression and STa binding exhibit concurrent maximal levels in the first days of life and then decrease in a parallel fashion. These similar changes suggestthat GC-C expression is under transcriptional control in the jejunum during the perinatal period. At the time of weaning (14-21 days), an increase in GC-C protein expression occurs without a significant increase in GC-C mRNA expression. This suggeststhat translational or posttranslational events modulate GC-C expression at weaning. Similarly, after weaning (22 days), STa binding decreases by approximately one-third, whereas GC-C expression remains relatively constant through adulthood. In the ileum, GC-C mRNA expression and 12”1-STa binding also decrease with maturation. However, unlike the pattern in the jejunum, the decrease in both parameters is of similar magnitude (-2-fold). At present, only one receptor for STa has been identified. It is possible, however, that there are other receptors in addition to GC-C. We cannot exclude the possibility that the binding we observed is due to these other receptors. The presence of another STa binding specieswould provide an alternative explanation for the discordance between our observed 12”I-STa binding and GC-C mRNA levels. Other brush-border membrane proteins present in the small intestine are known to exhibit changes in expression or activity that correspond to changes in development. These changes may be grouped into two patterns. The first pattern is demonstrated by lactase and neur aminidase, which are expressed maximally at birth. Levels of these enzymes decrease during the weaning period in rats, reaching very low or nearly absent “adult” levels by the end of the third postnatal week (11,15). The second pattern is exemplified by digestive enzymes including amylase, maltase, sucrase, and intestinal aminooligopeptidase. These enzymes are present in minimal levels in the perinatal period and then increase at the time of weaning to achieve levels in the range of those measured in the adult (8, 15, 24). The ontogeny of GC-C expression in the small intestine clearly differs from both of these patterns in that expression is maximal in the perinatal period but not absent in the adult. At the time of weaning, the serum levels of glucocorticoids and thyroxine increase and are thought to be partly responsible for the elevation in disaccharidases, which occur during that period (15). Glucocorticoids have also been shown to be involved in the decrease in lactase levels that occurs in the weaning period (15). The relationship of these and other hormones changes to changes in GC-C levels is not yet known. In contrast to the small intestine, GC-C mRNA levels exhibit little change with development in the colon. STa binding, however, exhibits a 2.5fold decline from 1 day to adult levels, suggesting posttranscriptional regulatory mechanisms. In addition, unlike the pattern observed in the small intestine, binding in the colon decreasesto near adult levels before the time of weaning (14 days). Previous studies in our laboratory using different techniques, have also shown STa binding and STa-mediated guanylate cyclase stimulation in colonocytes (20). Although we have demonstrated 1251-STabinding and GC-C mRNA expression to be present in extraintestinal sites in the rat, we have not determined whether the
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ESCHERICHIA
COLI
HEAT-STABLE
GC-C present is activated by STa. Furthermore, we have not determined whether extraintestinal GC-C is linked to chloride secretion as has been demonstrated in the intestine (10). Our studies demonstrate the existence of GC-C mRNA and GC-C protein at all ages examined in the jejunum, ileum, and colon. The amount of GC-C protein expressed in these organs, as measured by STa binding, is greater than levels measured at equivalent agesin extraintestinal sites. Both the developmental pattern and the higher level of GC-C expression in the intestine suggest that GC-C has an endogenous function in the intestine as well. Because GC-C mediates STa-induced diarrhea1 disease by alterations in epithelial cell ion flux, the endogenous function of GC-C could also be to regulate epithelial cell ion flux in response to an endogenous ligand. Recently, guanylin, a 15 amino acid peptide with 40% homology to STa has been identified as the first putative endogenous ligand for the STa receptor (9). The physiological functions of guanylin in the intestine and in extraintestinal sites of GC-C expression as well as the existence of other endogenous ligands for the STa receptor are currently under investigation. The authors thank Dr. William F. Balistreri for helpful suggestions regarding this article. This work was presented in part at Digestive Disease Week, American Gastroenterological Association, lo-13 May 1992, in San Francisco, CA, and published in abstract form (Gastroenterology 102: A562, 1992). This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Health Grant DK-01908, an American Gastroenterological Association/Glaxo Industry Award, Veterans Affairs Research Grant 539-3108-01, and by a Bristol-Myers Perinatal Research Center Award. Address for reprint requests: M. B. Cohen, Div.of Pediatric Gastroenterology and Nutrition, Children’s Hospital Medical Center, 3250 Elland Ave., Cincinnati, OH, 45229. Received
28 May
1992; accepted
in final
form
13 August
1992.
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Sci. USA 89: 947-951,
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