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Cloning and mRNA expression of guanylin, uroguanylin, and guanylyl cyclase C in the Spinifex hopping mouse, Notomys alexis
GENERAL AND COMPARATIVE
ENDOCRINOLOGY General and Comparative Endocrinology 132 (2003) 171–179 www.elsevier.com/locate/ygcen
Communication in Genomics and Proteomics
Cloning and mRNA expression of guanylin, uroguanylin, and guanylyl cyclase C in the Spinifex hopping mouse, Notomys alexis John A. Donald* and Ray C. Bartolo School of Biological and Chemical Sciences, Deakin University, Geelong, Victoria 3217, Australia Accepted 21 January 2003
Abstract Guanylin and uroguanylin are peptides that activate guanylyl cyclase C (GC-C) receptors in the intestine and kidney, which causes an increase in the excretion of salt and water. The Spinifex hopping mouse, Notomys alexis, is a desert rodent that can survive for extended periods without free access to water and it was hypothesised that to conserve water, the expression of guanylin, uroguanylin, and GC-C would be down-regulated to reduce the excretion of water in urine and faeces. Accordingly, this study examined the expression of guanylin, uroguanylin, and GC-C mRNA in Notomys under normal (access to water) and water-deprived conditions. Initially, guanylin and uroguanylin cDNAs encoding the full open reading frame were cloned and sequenced. A PCR analysis showed guanylin and uroguanylin mRNA expression in the small intestine, caecum, proximal and distal colon, heart, and kidney. In addition, a partial GC-C cDNA was obtained and GC-C mRNA expression was demonstrated in the proximal and distal colon, but not the kidney. Subsequently, a semi-quantitative PCR method showed that water deprivation in Notomys caused a significant increase in guanylin and uroguanylin mRNA expression in the distal colon, and in guanylin and GC-C mRNA expression in the proximal colon. No significant difference in guanylin and uroguanylin mRNA expression was observed in the kidney. The results of this study indicate that there is, in fact, an up-regulation of the colonic guanylin system in Notomys after 7 days of water deprivation. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Guanylin; Uroguanylin; Guanylyl cyclase C; Colon; Water deprivation; Notomys alexis
1. Introduction Studies into the activation of particulate guanylyl cyclase (GC) receptors by heat stable enterotoxins secreted by bacteria, led to the belief that an endogenous ligand that activated guanylyl cyclase existed in the intestine (Guarino et al., 1987). Subsequently, Currie et al. (1992) performed cGMP bioassay experiments using intestinal extracts, which led to the discovery of a 15 amino acid peptide that was named guanylin. Following the discovery of guanylin, similar experimental approaches revealed a factor in opossum urine that stimulated cGMP production in T84 cells (Hamra et al., 1993). This factor was identified as a 15 amino acid peptide that was related to guanylin and was called uroguanylin (Hamra et al., 1993). Following the cDNA * Corresponding author. Fax: +61-52-272022. E-mail address: [email protected] (J.A. Donald).
cloning of guanylin and uroguanylin, hybridisation analysis led to the discovery of an mRNA species in the spleen that was later identified as a third peptide, which was called lymphoguanylin (Forte et al., 1999). Guanylin, uroguanylin, and lymphoguanylin have been termed the guanylin family of peptides (called here guanylin-related peptides). Guanylin and uroguanylin mRNAs are highly expressed in the intestinal mucosa, but there are differences in expression levels along the length of the intestine (Forte, 1999; Hamra et al., 1996). In rats, the highest levels of guanylin expression occur in the distal small intestine and large intestine, whereas uroguanylin expression is highest in the proximal small intestine (Forte, 1999; Hamra et al., 1996; Nakazato et al., 1998). Guanylin and uroguanylin are also expressed in the kidney (Fan et al., 1997; Nakazato et al., 1998) and uroguanylin is present in the heart (Fan et al., 1996). Both peptides occur in the plasma, which indicates they
0016-6480/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0016-6480(03)00082-0
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act as both endocrine and paracrine regulators of biological function (Date et al., 1999; Fan et al., 1996). The guanylin-related peptides increase the intracellular level of cGMP by binding to and activating a particulate GC receptor called GC-C, which is related to the GC receptors of the natriuretic peptide receptor family (Forte and Currie, 1995; London et al., 1999; Vaandrager, 2002). Accordingly, GC-C receptors are located in the intestinal epithelium and renal tubules, the two primary targets of guanylin-related peptides (Carrithers et al., 2000; Li and Goy, 1993). In the intestine, activation of GC-C stimulates the secretion of Cl into the intestinal lumen and inhibits the reabsorption of sodium and water (Forte and Currie, 1995). This results in the loss of salt and water via the intestine and is the genesis of diarrhoea mediated by bacterial heat stable enterotoxins (Forte and Currie, 1995). In the kidney, activation of GC-C mediates diuresis, natriuresis, and kaliuresis; uroguanylin is considered the dominant regulator of ion transport in the kidney (Fan et al., 1997; Fonteles et al., 1998; Forte et al., 2000a). The combined actions of guanylin-related peptides in the intestine and kidney has led to the idea that these peptides form a link between the intestine and kidney to assist in the regulation of sodium (and water) balance in the body (Forte et al., 2000a). Many desert animals can survive without free drinking water by a number of adaptations that enable them to stay in water balance (Degen, 1997). In particular, the kidney and colon are modified to maximise the reabsorption of water, and thereby reduce water loss in the urine and faeces (Degen, 1997). Since guanylinrelated peptides act to increase water and salt excretion it is interesting to consider their role in animals that are minimising the excretion of water. However, there is no data on the biology of guanylin-related peptides in desert animals. The aim of the present study was to clone guanylin, uroguanylin, and GC-C (partial) cDNAs from the Spinifex hopping mouse, Notomys alexis (called Notomys here), a desert-dwelling rodent that occurs in central and western Australia. Subsequently, the expression of guanylin, uroguanylin, and GC-C mRNA was determined in the colon and kidney of Notomys after 7 days of water deprivation and compared to that in control animals with free access to water.
water ad libitum and were fed mixed bird seed and fresh apple. 2.1. Amplification, cloning, and sequencing of guanylin, uroguanylin, and partial GC-C cDNAs Total RNA was isolated using Trizol (Invitrogen), which utilises the single step phenol/guanidine isothiocyanate method (Chomczynski and Sacchi, 1987). The RNA concentration was determined by spectrophotometry at 260 nm. First strand cDNA was synthesised from total RNA using Superscript II (Invitrogen) as per the manufacturerÕs protocol. Kidney cDNA was used for the cloning of guanylin and uroguanylin, and colon cDNA was used to obtain a partial clone of the GC-C. The guanylin, uroguanylin, and GC-C primers were designed in regions that showed conservation between mouse and rat sequences that were obtained from GenBank (National Centre for Biotechnology, NCBI); the guanylin and uroguanylin primers spanned three intron–exon boundaries of the mouse genes (Sciaky et al., 1994; Whitaker et al., 1997). The primer sequences were as follows: guanylin forward 50 -gctgcattgcatactgcta cc-30 , guanylin reverse 50 -cttccacatgggctgagag-30 ; uroguanylin forward 50 -cagaggtgtgagctgggaag-30 , uroguanylin reverse 50 -tatgggcagggtaggctgtg-30 ; GC-C forward 50 gccttggacatcctcagcttc-30 , GC-C reverse 50 -ttg gtccttca tcccagtcag-30 . All PCRs were performed as follows: 1 PCR buffer, 0.2 mM dNTPs, 0.8 lM forward and reverse primers, 2.0 U Taq DNA polymerase (Invitrogen), 2.5 mM MgCl2 , and 2 ll of the cDNA reaction. Amplification for each primer set was performed as follows: cycle 1, 94 °C for 300 s, 60 °C for 60 s, and 72 °C for 60 s; cycles 2–34, 94 °C for 60 s, 60 °C for 60 s, and 72 °C for 60 s; cycle 35, 94 °C for 60 s, 60 °C for 60 s, and 72 °C for 300 s. PCR products of the expected size were purified and cloned into a pCR 2.1 vector, which was then transformed into competent Escherichia coli TOP10F0 cells. Cloned cDNAs were sequenced on an Applied Biosystems automated sequencer (Australian Genome Research Facility, Queensland). The Basic Local Alignment Search Tool (BLAST) program at NCBI was used to search GenBank for identity. Alignments were carried out on the nucleotide and deduced protein sequences using ClustalW (http://www.ebi.ac.uk/ clustalw/). 2.2. Water deprivation experiment
2. Materials and methods Spinifex hopping mice, Notomys, (25–30 g) were obtained from a breeding colony in the Deakin University Animal House, Geelong. Under normal conditions, animals were housed in rat boxes containing straw and sawdust for bedding, at a constant temperature of 21 °C and a 12:12 h light–dark cycle. The animals received
The experiment consisted of a control group, in which Notomys received water ad libitum, and an experimental group, in which the mice were subjected to 7 days without free access to water; this was called water deprivation (WD). One week prior to the commencement of WD, mature Notomys were chosen and randomly separated into a control group (n ¼ 8) and a WD
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group (n ¼ 8). All mice were ear-tagged to enable identification. The animals were housed in their groups in sand-filled glass aquaria (W 100 H 40 L 50 cm) allowing room for communal sleeping burrows. Each group was fed 20 g of millet seed daily; millet seed has a high water content and is low in protein (nitrogen) (Murray and Dickman, 1994). The hopping mice were weighed daily. At the end of the WD period, both control and experimental Notomys were anaesthetised by halothane inhalation followed by cervical dislocation. Following sacrifice, blood samples were obtained via cardiac puncture using a 1 ml syringe coated with EDTA (10 mg/ml) and the haematocrit was determined using an Ames haematocrit centrifuge. The heart, kidneys, small intestine, caecum, and proximal and distal colon were removed and frozen in liquid N2 and stored until the RNA was extracted. 2.3. Analysis of guanylin, uroguanylin, and GC-C mRNA expression RT-PCR was used to determine the mRNA expression of guanylin, uroguanylin and GC-C mRNA in tissues from control and water-deprived Notomys. New guanylin (forward only) and uroguanylin primers were used as follows: guanylin forward 50 -accatgaatgcctgtgtgc-3; uroguanylin forward 50 -actatgtcaggaagccaactgtg-30 , uroguanylin reverse 50 -cacagctcacattcgtcggtg-30 . Total RNA isolation and cDNA synthesis were performed as described above. For the analysis of mRNA expression in different tissues, 2 lg of total RNA from each tissue was used. The PCR was performed for 22 cycles for GC-C and 23 cycles for guanylin and uroguanylin, which was in the linear ranges of all cDNAs used in the expression study. Amplification was performed using an initial cycle of 94 °C for 300 s, 60 °C for 60 s, and 72 °C for 60 s. Cycles 2– 21/22 consisted of 94 °C for 60 s, 60 °C for 60 s, and 72 °C for 60 s, and the parameters of cycle 22/23 were 94 °C for 60 s, 60 °C for 60 s, and 72 °C for 300 s. To quantify the level of mRNA expression between control and water-deprived Notomys, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control. It was assumed that the levels of GAPDH expression were constant in control and water-deprived Notomys (Sturzenbaum and Kille, 2001). The GAPDH primers were forward 50 -gaag gtcggtgtgaacggatttg-30 , and reverse 50 -ttactccttggaggccatgtagg-30 ; these primers generated a 999 base pair product. In preliminary experiments, the linear amplification range of each cDNA was determined by running multiplex PCR for a varying number of cycles between 17 and 35. The mRNA expression analysis was performed at a cycle number at the mid-point of the linear amplification curve. Initially, the linear range was established for amplification of each individual PCR product. The linear range was also determined in the various tissues used in
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the mRNA expression experiments, and both control and experimental samples to ensure that there was no variation between tissues, individual animals or sample groups. In the distal colon and kidney, guanylin, uroguanylin, and GAPDH primers were included in the multiplex reaction. However, in the proximal colon, the expression of guanylin and GAPDH and uroguanylin and GAPDH was determined separately. The expression of GC-C mRNA was determined in a multiplex PCR with GAPDH primers. PCR were assembled as described above, except that 1 ll of the cDNA reaction was used. For quantification of the PCR products, the reactions were spiked with 2.5 lCi of ½a-32 PdCTP. As the radiolabelled dCTP is randomly incorporated into the PCR products, the level of radiation emitted by a PCR product is directly proportional to the amount of the PCR product. The amplified PCR products were separated by electrophoresis on a 1.5% agarose gel in TBE running buffer at 100 V. The gel was incubated in 0.5 lg/ml of ethidium bromide and visualised on an UV light box and the relevant bands were excised using a scalpel blade and placed in microcentrifuge tubes. The amount of isotope ½a-32 P incorporated into the PCR products was measured by placing the tubes in vials, and counting in a scintillation counter (Tri-Carb 2000CA Liquid Scintillation Counter, Packard). Quantification of the mRNA expression was determined by normalising the levels of guanylin, uroguanylin, and GCC amplification against that of GAPDH. The values from control animals were adjusted to represent 100%, and the level of expression of guanylin, uroguanylin and GC-C in water-deprived Notomys was calculated as a percentage of the control. 2.4. Data analysis All data are represented as means one standard error (SE). To test the difference between control and experimental groups, a StudentÕs t test were performed using the SPSS 10 statistical package. A p value 60:05 was considered as statistically significant. 2.5. Materials ½a-32 PdCTP (3000 Ci/mmol) was purchased from Amersham Pharmacia Biotech. All other chemicals were either reagent or molecular grade and were purchased from Sigma or Invitrogen. 3. Results 3.1. Cloning and sequencing of guanylin, uroguanylin, and GC-C cDNAs A cDNA of 410 base pairs was amplified using the guanylin primers. Analysis of the nucleotide sequence showed highest homology to that of mouse and rat
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guanylin. The Notomys guanylin cDNA contained an open reading frame of 345 nucleotides encoding preproguanylin (Fig. 1A). The nucleotide sequence of Notomys guanylin showed 91 and 92% identity to that of mouse and rat, respectively. The deduced amino acid sequence of Notomys preproguanylin was 94% homol-
ogous to rat and mouse; the mature guanylin peptide was identical (Fig. 1B). A cDNA of 416 base pairs was amplified using the uroguanylin primers. Analysis of the nucleotide sequence showed highest homology to that of mouse and rat uroguanylin. The Notomys uroguanylin cDNA contained an open reading frame of 321 nucle-
Fig. 1. (A) Nucleotide and deduced amino acid sequences of a preproguanylin cDNA from Notomys. Numbers to the right indicate the base pair and amino acid number, respectively. The underlined sequences are the primer sites and the asterisk represents the stop codon. The mature guanylin peptide is indicated by the dashed underline. (B) Alignment of the deduced amino acid sequence of Notomys preproguanylin with other guanylin sequences. The alignment was performed using ClustalW. The asterisks indicate conserved amino acids between Notomys, mouse (AAB05758) and rat (AAA41300). The vertical lines indicated conserved amino acids between Notomys, mouse, rat, human (XP_046404), opossum (Fan et al., 1997), and zebrafish.
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otides encoding preprouroguanylin (Fig. 2A). The nucleotide sequence of Notomys uroguanylin showed 91 and 90% identity to that of mouse and rat, respectively. The deduced amino acid sequence of Notomys preprouroguanylin was 91% homologous to rat and mouse; the mature uroguanylin peptide was identical (Fig. 2B). A cDNA of 327 base pairs was amplified using the GCC primers (Fig. 3A). Analysis of the nucleotide sequence showed highest homology to that of mouse GC-C. The deduced amino acid of the partial GC-C cDNA was identical to that of the same region of mouse GC-C (Fig. 3B).
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3.2. Tissue expression of guanylin, uroguanylin, and GCC mRNA A multiplex PCR was performed to detect the expression of guanylin and uroguanylin mRNA in the heart, kidney, small intestine, caecum, and proximal and distal colon of Notomys. The caecum and proximal and distal colon had a greater expression of guanylin than uroguanylin, but in the small intestine, uroguanylin mRNA expression was greater than that of guanylin. The heart and kidney had lower levels of guanylin and uroguanylin mRNA expression (Fig. 4A). In contrast to
Fig. 2. (A) Nucleotide and deduced amino acid sequences of a preprouroguanylin cDNA from Notomys. Numbers to the right indicate the base pair and amino acid number, respectively. The underlined sequences are the primer sites and the asterisk represents the stop codon. The mature uroguanylin peptide is indicated by the dashed underline. (B) Alignment of the deduced amino acid sequence of Notomys preprouroguanylin with other uroguanylin sequences. The alignment was performed using ClustalW. The asterisks indicate conserved amino acids between Notomys, mouse (AAB82750) and rat (AAB18331). The vertical lines indicated conserved amino acids between Notomys, mouse, rat, human (1588627), opossum (AAB00553), and eel (CAC35449).
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Fig. 3. (A) Nucleotide and deduced amino acid sequences of a partial GC-C cDNA from Notomys. (B) Alignment of the deduced amino acid sequence of a partal Notomys GC-C with that of mouse (XM_132928). The alignment was performed using ClustalW. The asterisks indicate conserved amino acids between Notomys and mouse.
Fig. 4. (A) Image of a PCR amplification showing the expression of guanylin (391 base pairs) and uroguanylin (298 base pairs) mRNA in heart (1), kidney (2), small intestine (3), caecum (4), proximal colon (5), and distal colon (6) from Notomys. The left lane is a marker lane and the 500 base pair marker is boxed. The two bands below the 500 base pair band are 400 and 300 base pairs, respectively. (B) Image showing PCR amplification of a 327 base pair GC-C mRNA in the same tissues as (A). Lane assignment and markers as for (A).
guanylin and uroguanylin, no expression of GC-C mRNA was observed in the heart and kidney, but it was found in each of the regions of the gut (Fig. 4B). 3.3. Effect of water deprivation on guanylin, uroguanylin and GC-C mRNA expression Notomys subjected to WD steadily lost weight until the seventh day when weight had decreased by 22:3 1:7%; control animals maintained a stable weight. There was no significant difference in haematocrit between control (52:0 1:02%) and water-deprived
(50:2 1:72%) Notomys. The expression of guanylin, uroguanylin, and GC-C mRNA was compared between control and water-deprived Notomys. In the distal colon, there was a significant increase in the expression of guanylin and uroguanylin mRNA; the increase in guanylin mRNA expression was greater than that of uroguanylin (Fig. 5A). However, there was no difference in GC-C mRNA expression in the distal colon between control and water-deprived Notomys. In the proximal colon, there was a significant increase in guanylin and GC-C mRNA expression but not in uroguanylin mRNA expression (Fig. 5B). In the kidney, no significant difference in guanylin and uroguanylin mRNA expression was found (Fig. 5C).
4. Discussion Guanylin and uroguanylin are peptides that regulate intestinal and renal transepithelial fluid and electrolyte transport by activating guanylyl cyclase C receptors and generating cGMP (Forte, 1999). The binding of guanylin and uroguanylin to GC-C leads to the secretion of chloride and a parallel secretion of sodium and water; in the gut and kidney this results in the loss of salt and water from the body (Forte and Currie, 1995; Joo et al., 1998). In the gut, one of the targets for guanylinrelated peptides is the colon because of its role in modifying the salt and water content of the faeces prior to excretion. High levels of guanylin and GC-C mRNA expression occur in this tissue (Forte, 1999), and it has
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Fig. 5. Histograms showing the relative level of mRNA expression of guanylin, uroguanylin, and GC-C (colon only) in the distal colon (A), proximal colon (B), and kidney (C) of control () and 7 day waterdeprived (j) Notomys. The guanylin, uroguanylin or GC-C to GAPDH ratios for the control mice were set at 100%. Asterisks (*) indicate statistical significance ðp 6 0:05Þ from control values. Significant increases in guanylin and uroguanylin mRNA expression were found in the distal colon, but only guanylin and GC-C mRNA expression was significantly increased in the proximal colon.
been shown that this expression is sensitive to the level of salt intake (Carrithers et al., 2002; Li et al., 1996). In addition, the guanylin-related peptides (primarily uroguanylin) stimulate GC-C in the kidney to increase salt and water excretion (Forte et al., 2000a,b). In desert rodents such as Notomys, the reabsorption of water from the colon and kidney is one of the key adaptations for reducing water loss in arid environments (Degen,
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1997). It was, therefore, predicted that the expression of guanylin, uroguanylin and GC-C mRNA would be down-regulated in the colon and kidney during WD in Notomys. In order to study the expression of guanylin and uroguanylin mRNA in Notomys, cDNAs encoding the prepropeptides were amplified and cloned. Not surprisingly, the nucleotide and deduced amino acid sequences of Notomys guanylin and uroguanylin cDNAs showed very high homology to mammalian sequences; the mature peptides were identical to those of mouse and rat. Rodent guanylin differs by only one residue from human guanylin and three residues from opossum guanylin (Forte et al., 2000b). In contrast, rodent uroguanylin differs by three amino acid residues from human and opossum uroguanylin; human uroguanylin also has a C-terminal leucine residue that is only present in eel (Comrie et al., 2001; Forte et al., 2000b). The expression of guanylin and uroguanylin mRNA was demonstrated in a range of tissues from Notomys, using multiplex RT-PCR. Both guanylin and uroguanylin mRNA expression were found in the proximal and distal colon, caecum, small intestine, kidney, and heart, as has been found in mouse and rat. The expression of guanylin mRNA was notably higher than uroguanylin in the colon, which is consistent with previous studies that have shown a more important role for guanylin than uroguanylin in this tissue (Forte, 1999). The expression of uroguanylin mRNA in the heart has been described previously, and it has been proposed that uroguanylin may be released into the circulation in a similar manner to atrial natriuretic peptide (Fan et al., 1996). A partial cDNA encoding GC-C was cloned from the colon of Notomys, and the deduced amino acid sequence was identical to mouse, showing it was GC-C. The expression of GC-C mRNA was found in the small intestine, caecum, and the proximal and distal colon, but not in the heart and kidney. The absence of GC-C mRNA expression in the kidney was very surprising since GC-C expression has been clearly demonstrated in the rat kidney (Carrithers et al., 2000), and guanylin and uroguanylin can affect renal function (Forte et al., 2000a). Given that both guanylin and uroguanylin are expressed in the kidney of Notomys, further analysis on the presence or absence of GC-C proteins in the kidney is required. Following molecular cloning of guanylin, uroguanylin, and GC-C, the expression of their respective mRNAs was determined in normal and water-deprived Notomys, in order to examine how water restriction effects the level of mRNA expression. Notomys can survive long periods of WD, and maintain plasma volume and osmolarity at similar levels to Notomys with free access to water (Heimeier et al., 2002; MacMillen and Lee, 1969; Weaver et al., 1994). A previous study found
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that this homeostasis occurs without any long-term upregulation of vasopressin and the renin–angiotensin system (Weaver et al., 1994). However, the role of regulatory molecules that generate cGMP has not been extensively investigated in desert rodents. In the present study, a 7 day WD experiment was performed since this represents the acute phase of the adaptation to WD. In response to WD, there was an average decrease in body weight of 22.3% at day 7, compared to control animals, which is mainly due to a reduction in fat stores. A similar change in body weight was found in previous WD studies with Notomys (Heimeier et al., 2002; MacMillen and Lee, 1969; Weaver et al., 1994). If the WD period is extended beyond 7 days, the loss in body weight stabilises and the animals actually gain weight by 14 days of WD (Heimeier et al., 2002; MacMillen and Lee, 1969; Weaver et al., 1994). In this study, there was no significant difference in haematocrit between control and water-deprived animals, which suggests that ECF dehydration did not occur in water-deprived Notomys. The expression of guanylin mRNA in the proximal and distal colon significantly increased in response to WD; in addition uroguanylin mRNA expression was increased in the distal colon. An increase in mRNA expression suggests that there is also an increase in the production of guanylin and uroguanylin peptides in the colon. This result was surprising since one of the functions of the colonic epithelium is to reduce water loss in the faeces to prevent dehydration (Forte and Currie, 1995); an increase in the synthesis of guanylin and uroguanylin peptides in the colon of water-deprived Notomys would appear detrimental to this function. It is, therefore, interesting to speculate why there is an upregulation in the expression of guanylin and uroguanylin in the colon during WD. Previous studies have shown that the expression of guanylin and uroguanylin in the intestine of rats is affected by oral salt intake. For example, low salt intake results in a down-regulation of guanylin expression in the distal colon (Carrithers et al., 2002; Li et al., 1996) but high salt intake had no effect in the colon (Carrithers et al., 2002). Furthermore, salt loading increased guanylin secretion in the rat small intestine (Kita et al., 1999). Therefore, it is possible that there are differences in the amount of salt in the colon between control and water-deprived Notomys that could modulate the expression of guanylin and uroguanylin mRNA. As with guanylin and uroguanylin, a downregulation of GC-C mRNA expression was not observed following WD. Although guanylin and uroguanylin mRNA expression increased markedly in the distal colon a parallel increase in GC-C expression was not observed. In contrast, GC-C mRNA expression was significantly higher in the proximal colon in which a small increase in guanylin mRNA expression was observed. Thus, it appears that there is differential regu-
lation of GC-C mRNA transcription in the proximal and distal segments of the colon during WD. Following the discovery of guanylin and uroguanylin, it was shown that GC-C are present in the kidney and that the peptides stimulate the excretion of water and salt via cGMP (Forte et al., 2000a,b). Guanylin is less potent than uroguanylin in eliciting diuresis and natriuresis in perfused rat kidneys, as guanylin is inactivated in the renal tubules (Fonteles et al., 1998; Forte et al., 2000b). It was thus proposed that uroguanylin is part of an endocrine axis linking the intestine and the kidney that is regulated by dietary salt (Forte et al., 2000a,b). However, recent studies have shown that guanylin and uroguanylin are expressed in the kidney (Fan et al., 1997), which indicates the peptides may act in a paracrine/autocrine fashion. The distribution patterns of the peptides vary since guanylin mRNA is most highly expressed in the collecting ducts of mouse whereas uroguanylin expression is highest in the proximal tubules (Potthast et al., 2001). Both mRNA species were expressed in the kidney of Notomys, but the anatomical location was not determined. It was predicted a priori that the renal guanylin and uroguanylin mRNA expression would be down-regulated during WD to reduce the level of cGMP-mediated excretion, if indeed GC-C protein are present in the kidney of Notomys (see above). However, the renal expression of guanylin and uroguanylin mRNAs was not affected by WD as compared to control Notomys. Interestingly, this result is consistent with data from a WD experiment in laboratory mice in which no effect on renal expression was found (Potthast et al., 2001). In contrast, a salt load in the drinking water mediated an increase in renal uroguanylin mRNA expression but not guanylin expression (Potthast et al., 2001). The absence of any upregulation of uroguanylin mRNA expression during WD in Notomys may be due to the fact that plasma osmolarity is insignificantly changed, and therefore, there is no signal that leads to changes in uroguanylin mRNA transcription.
References Carrithers, S.L., Taylor, B., Cai, W.Y., Johnson, B.R., Ott, C.E., Greenberg, R.N., Jackson, B.A., 2000. Guanylyl cyclase-C receptor mRNA distribution along the rat nephron. Regul. Pept. 95, 65–74. Carrithers, S.L., Jackson, B.A., Cai, W.Y., Greenberg, R.N., Ott, C.E., 2002. Site specific effects of dietary salt intake on guanylin and uroguanylin mRNA expression in rat intestine. Regul. Pept. 107, 87–95. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162, 156–159. Comrie, M.M., Cutler, C.P., Cramb, G., 2001. Cloning and expression of guanylin from the European eel (Anguilla anguilla). Biochem. Biophys. Res. Commun. 281, 1078–1085.
J.A. Donald, R.C. Bartolo / General and Comparative Endocrinology 132 (2003) 171–179 Currie, M.G., Fok, K.F., Kato, J., Moore, R.J., Hamra, F.K., Duffin, K.L., Smith, C.E., 1992. Guanylin: an endogenous activator of intestinal guanylate cyclase. Proc. Natl. Acad. Sci. USA 89, 947–951. Date, Y., Nakazato, M., Yamaguchi, H., Kangawa, K., Kinoshita, Y., Chiba, T., Ueta, Y., Yamashita, H., Matsukura, S., 1999. Enterochromaffin-like cells, a cellular source of uroguanylin in rat stomach. Endocrinology 140, 2398–2404. Degen, A., 1997. Ecophysiology of Small Desert Mammals. SpringerVerlag, Germany. Fan, X., Hamra, F.K., Freeman, R.H., Eber, S.L., Krause, W.J., Lim, R.W., Pace, V.M., Currie, M.G., Forte, L.R., 1996. Uroguanylin: cloning of preproguanylin, mRNA expression in the intestine and heart and isolation of uroguanylin and prouroguanylin from plasma. Biochem. Biophys. Res. Commun. 219, 457–462. Fan, X., Wang, Y., London, R.M., Eber, S.L., Krause, W.J., Freeman, R.H., Forte, L.R., 1997. Signaling pathways for guanylin and uroguanylin in the digestive, renal, central nervous, reproductive, and lymphoid systems. Endocrinology 138, 4636–4648. Fonteles, M.C., Greenberg, R.N., Monteiro, H.S., Currie, M.G., Forte, L.R., 1998. Natriuretic and kaliuretic activities of guanylin and uroguanylin in the isolated perfused rat kidney. Am. J. Physiol. 275, F191–F197. Forte, L.R., 1999. Guanylin regulatory peptides: structures, biological activities mediated by cyclic GMP and pathobiology. Regul. Pept. 81, 25–39. Forte, L.R., Currie, M.G., 1995. Guanylin: a peptide regulator of epithelial transport. FASEB J. 9, 643–650. Forte, L.R., Eber, S.L., Fan, X., London, R.M., Wang, Y., Rowland, L.M., Chin, D.T., Freeman, R.H., Krause, W.J., 1999. Lymphoguanylin: cloning and characterization of a unique member of the guanylin peptide family. Endocrinology 140, 1800–1806. Forte, L.R., London, R.M., Freeman, R.H., Krause, W.J., 2000a. Guanylin peptides: renal actions mediated by cyclic GMP. Am. J. Physiol. 278, F180–F191. Forte, L.R., London, R.M., Krause, W.J., Freeman, R.H., 2000b. Mechanisms of guanylin action via cyclic GMP in the kidney. Annu. Rev. Physiol. 62, 673–695. Guarino, A., Cohen, M.B., Thompson, M., Dharmasathphorn, K., Giannella, R., 1987. T84 cell receptor binding and guanylyl cyclase activation by Escherichia coli heat-stable toxin. Am. J. Physiol. 253, G775–G780. Hamra, F.K., Fan, X., Krause, W.J., Freeman, R.H., Chin, D.T., Smith, C.E., Currie, M.G., Forte, L.R., 1996. Prouroguanylin and proguanylin: purification from colon, structure, modulation of bioactivity by proteases. Endocrinology 137, 257–265. Hamra, F.K., Forte, L.R., Eber, S.L., Pidhorodeckyj, N.V., Krause, W.J., Freeman, R.H., Chin, D.T., Tompkins, J.A., Fok, K.F., Smith, C.E., Duffin, K.L., Siegel, N.R., Currie, M.G., 1993. Uroguanylin: structure and activity of a second endogenous peptide that stimulates intestinal guanylate cyclase. Proc. Natl. Acad. Sci. USA 90, 10464–10468. Heimeier, R.A., Davis, B.J., Donald, J.A., 2002. The effect of water deprivation on the expression of atrial natriuretic peptide and its receptors in the spinifex hopping mouse, Notomys alexis. Comp. Biochem. Physiol. Part A, Molecular and Integrative Physiology 132 (4), 893–903.
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Joo, N.S., London, R.M., Kim, H.D., Forte, L.R., Clarke, L.L., 1998. Regulation of intestinal Cl and HCO 3 secretion by uroguanylin. Am. J. Physiol. 274, G633–G644. Kita, T., Kitamura, K., Eto, T., 1999. Marked increase of guanylin secretion in response to salt loading in the rat small intestine. Am. J. Physiol. 277, G960–G966. Li, Z.P., Goy, M.F., 1993. Peptide-regulated guanylate cyclase pathways in rat colon—in situ localisation of GC-A, GC-C and guanylin messenger RNA. Am. J. Physiol. 265, G394–G402. Li, Z., Knowles, J.W., Goyeau, D., Prabhakar, S., Short, D.B., Perkins, A.G., Goy, M.F., 1996. Low salt intake down-regulates the guanylin signaling pathway in rat distal colon. Gastroenterology 111, 1714–1721. London, R.M., Eber, S.L., Visweswariah, S.S., Krause, W.J., Forte, L.R., 1999. Structure and activity of OK-GC: a kidney receptor guanylate cyclase activated by guanylin peptides. Am J. Physiol. Renal. Physiol. 276, F882–F891. MacMillen, R.E., Lee, A.K., 1969. Water metabolism of Australian hopping mice. Comp. Biochem. Physiol. 28, 493–514. Murray, B.R., Dickman, C.R., 1994. Food preference and seed selection in two species of Australian desert rodents. Wildlife Res. 21, 647–655. Nakazato, M., Yamaguchi, H., Date, Y., Miyazato, M., Kangawa, K., Goy, M.F., Chino, N., Matsukura, S., 1998. Tissue distribution, cellular source, and structural analysis of rat immunoreactive uroguanylin. Endocrinology 139, 5247–5254. Potthast, R., Ehler, E., Scheving, L.A., Sindic, A., Schlatter, E., Kuhn, M., 2001. High salt intake increases uroguanylin expression in mouse kidney. Endocrinology 142, 3087–3097. Sciaky, D., Kosiba, J.L., Cohen, M.B., 1994. Genomic sequence of the murine guanylin gene. Genomics 24, 583–587. Sturzenbaum, S.R., Kille, P., 2001. Control genes in quantitative molecular biological techniques: the variability of invariance. Comp. Biol. Physiol. B 130, 281–289. Vaandrager, A.B., 2002. Structure and function of the heat-stable enterotoxin receptor/guanylyl cyclase C. Mol. Cell. Biochem. 230, 73–83. Weaver, D., Walker, L., Alcorn, D., Skinner, S., 1994. The contributions of renin and vasopressin to the adaptation of the Australian Spinifex hopping mouse (Notomys alexis) to free water deprivation. Comp. Biochem. Physiol. 108, 107–116. Whitaker, T.L., Steinbrecher, K.A., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Cohen, M.B., 1997. The uroguanylin gene (Guca1b) is linked to guanylin (Guca2) on mouse chromosome 4. Genomics 45, 348–354.
Further reading Nakazato, M., 2001. Guanylin family: new intestinal peptides regulating electrolyte and water homeostasis. J. Gastroenterol. 36, 219–225. Wiegand, R.C., Kato, J., Currie, M.G., 1992. Rat guanylin cDNA: characterisation of the precursor of an endogenous activator of the intestinal guanylate cyclase. Biochem. Biophys. Res. Commun. 185, 812–817.
ENDOCRINOLOGY General and Comparative Endocrinology 132 (2003) 171–179 www.elsevier.com/locate/ygcen
Communication in Genomics and Proteomics
Cloning and mRNA expression of guanylin, uroguanylin, and guanylyl cyclase C in the Spinifex hopping mouse, Notomys alexis John A. Donald* and Ray C. Bartolo School of Biological and Chemical Sciences, Deakin University, Geelong, Victoria 3217, Australia Accepted 21 January 2003
Abstract Guanylin and uroguanylin are peptides that activate guanylyl cyclase C (GC-C) receptors in the intestine and kidney, which causes an increase in the excretion of salt and water. The Spinifex hopping mouse, Notomys alexis, is a desert rodent that can survive for extended periods without free access to water and it was hypothesised that to conserve water, the expression of guanylin, uroguanylin, and GC-C would be down-regulated to reduce the excretion of water in urine and faeces. Accordingly, this study examined the expression of guanylin, uroguanylin, and GC-C mRNA in Notomys under normal (access to water) and water-deprived conditions. Initially, guanylin and uroguanylin cDNAs encoding the full open reading frame were cloned and sequenced. A PCR analysis showed guanylin and uroguanylin mRNA expression in the small intestine, caecum, proximal and distal colon, heart, and kidney. In addition, a partial GC-C cDNA was obtained and GC-C mRNA expression was demonstrated in the proximal and distal colon, but not the kidney. Subsequently, a semi-quantitative PCR method showed that water deprivation in Notomys caused a significant increase in guanylin and uroguanylin mRNA expression in the distal colon, and in guanylin and GC-C mRNA expression in the proximal colon. No significant difference in guanylin and uroguanylin mRNA expression was observed in the kidney. The results of this study indicate that there is, in fact, an up-regulation of the colonic guanylin system in Notomys after 7 days of water deprivation. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Guanylin; Uroguanylin; Guanylyl cyclase C; Colon; Water deprivation; Notomys alexis
1. Introduction Studies into the activation of particulate guanylyl cyclase (GC) receptors by heat stable enterotoxins secreted by bacteria, led to the belief that an endogenous ligand that activated guanylyl cyclase existed in the intestine (Guarino et al., 1987). Subsequently, Currie et al. (1992) performed cGMP bioassay experiments using intestinal extracts, which led to the discovery of a 15 amino acid peptide that was named guanylin. Following the discovery of guanylin, similar experimental approaches revealed a factor in opossum urine that stimulated cGMP production in T84 cells (Hamra et al., 1993). This factor was identified as a 15 amino acid peptide that was related to guanylin and was called uroguanylin (Hamra et al., 1993). Following the cDNA * Corresponding author. Fax: +61-52-272022. E-mail address: [email protected] (J.A. Donald).
cloning of guanylin and uroguanylin, hybridisation analysis led to the discovery of an mRNA species in the spleen that was later identified as a third peptide, which was called lymphoguanylin (Forte et al., 1999). Guanylin, uroguanylin, and lymphoguanylin have been termed the guanylin family of peptides (called here guanylin-related peptides). Guanylin and uroguanylin mRNAs are highly expressed in the intestinal mucosa, but there are differences in expression levels along the length of the intestine (Forte, 1999; Hamra et al., 1996). In rats, the highest levels of guanylin expression occur in the distal small intestine and large intestine, whereas uroguanylin expression is highest in the proximal small intestine (Forte, 1999; Hamra et al., 1996; Nakazato et al., 1998). Guanylin and uroguanylin are also expressed in the kidney (Fan et al., 1997; Nakazato et al., 1998) and uroguanylin is present in the heart (Fan et al., 1996). Both peptides occur in the plasma, which indicates they
0016-6480/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0016-6480(03)00082-0
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act as both endocrine and paracrine regulators of biological function (Date et al., 1999; Fan et al., 1996). The guanylin-related peptides increase the intracellular level of cGMP by binding to and activating a particulate GC receptor called GC-C, which is related to the GC receptors of the natriuretic peptide receptor family (Forte and Currie, 1995; London et al., 1999; Vaandrager, 2002). Accordingly, GC-C receptors are located in the intestinal epithelium and renal tubules, the two primary targets of guanylin-related peptides (Carrithers et al., 2000; Li and Goy, 1993). In the intestine, activation of GC-C stimulates the secretion of Cl into the intestinal lumen and inhibits the reabsorption of sodium and water (Forte and Currie, 1995). This results in the loss of salt and water via the intestine and is the genesis of diarrhoea mediated by bacterial heat stable enterotoxins (Forte and Currie, 1995). In the kidney, activation of GC-C mediates diuresis, natriuresis, and kaliuresis; uroguanylin is considered the dominant regulator of ion transport in the kidney (Fan et al., 1997; Fonteles et al., 1998; Forte et al., 2000a). The combined actions of guanylin-related peptides in the intestine and kidney has led to the idea that these peptides form a link between the intestine and kidney to assist in the regulation of sodium (and water) balance in the body (Forte et al., 2000a). Many desert animals can survive without free drinking water by a number of adaptations that enable them to stay in water balance (Degen, 1997). In particular, the kidney and colon are modified to maximise the reabsorption of water, and thereby reduce water loss in the urine and faeces (Degen, 1997). Since guanylinrelated peptides act to increase water and salt excretion it is interesting to consider their role in animals that are minimising the excretion of water. However, there is no data on the biology of guanylin-related peptides in desert animals. The aim of the present study was to clone guanylin, uroguanylin, and GC-C (partial) cDNAs from the Spinifex hopping mouse, Notomys alexis (called Notomys here), a desert-dwelling rodent that occurs in central and western Australia. Subsequently, the expression of guanylin, uroguanylin, and GC-C mRNA was determined in the colon and kidney of Notomys after 7 days of water deprivation and compared to that in control animals with free access to water.
water ad libitum and were fed mixed bird seed and fresh apple. 2.1. Amplification, cloning, and sequencing of guanylin, uroguanylin, and partial GC-C cDNAs Total RNA was isolated using Trizol (Invitrogen), which utilises the single step phenol/guanidine isothiocyanate method (Chomczynski and Sacchi, 1987). The RNA concentration was determined by spectrophotometry at 260 nm. First strand cDNA was synthesised from total RNA using Superscript II (Invitrogen) as per the manufacturerÕs protocol. Kidney cDNA was used for the cloning of guanylin and uroguanylin, and colon cDNA was used to obtain a partial clone of the GC-C. The guanylin, uroguanylin, and GC-C primers were designed in regions that showed conservation between mouse and rat sequences that were obtained from GenBank (National Centre for Biotechnology, NCBI); the guanylin and uroguanylin primers spanned three intron–exon boundaries of the mouse genes (Sciaky et al., 1994; Whitaker et al., 1997). The primer sequences were as follows: guanylin forward 50 -gctgcattgcatactgcta cc-30 , guanylin reverse 50 -cttccacatgggctgagag-30 ; uroguanylin forward 50 -cagaggtgtgagctgggaag-30 , uroguanylin reverse 50 -tatgggcagggtaggctgtg-30 ; GC-C forward 50 gccttggacatcctcagcttc-30 , GC-C reverse 50 -ttg gtccttca tcccagtcag-30 . All PCRs were performed as follows: 1 PCR buffer, 0.2 mM dNTPs, 0.8 lM forward and reverse primers, 2.0 U Taq DNA polymerase (Invitrogen), 2.5 mM MgCl2 , and 2 ll of the cDNA reaction. Amplification for each primer set was performed as follows: cycle 1, 94 °C for 300 s, 60 °C for 60 s, and 72 °C for 60 s; cycles 2–34, 94 °C for 60 s, 60 °C for 60 s, and 72 °C for 60 s; cycle 35, 94 °C for 60 s, 60 °C for 60 s, and 72 °C for 300 s. PCR products of the expected size were purified and cloned into a pCR 2.1 vector, which was then transformed into competent Escherichia coli TOP10F0 cells. Cloned cDNAs were sequenced on an Applied Biosystems automated sequencer (Australian Genome Research Facility, Queensland). The Basic Local Alignment Search Tool (BLAST) program at NCBI was used to search GenBank for identity. Alignments were carried out on the nucleotide and deduced protein sequences using ClustalW (http://www.ebi.ac.uk/ clustalw/). 2.2. Water deprivation experiment
2. Materials and methods Spinifex hopping mice, Notomys, (25–30 g) were obtained from a breeding colony in the Deakin University Animal House, Geelong. Under normal conditions, animals were housed in rat boxes containing straw and sawdust for bedding, at a constant temperature of 21 °C and a 12:12 h light–dark cycle. The animals received
The experiment consisted of a control group, in which Notomys received water ad libitum, and an experimental group, in which the mice were subjected to 7 days without free access to water; this was called water deprivation (WD). One week prior to the commencement of WD, mature Notomys were chosen and randomly separated into a control group (n ¼ 8) and a WD
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group (n ¼ 8). All mice were ear-tagged to enable identification. The animals were housed in their groups in sand-filled glass aquaria (W 100 H 40 L 50 cm) allowing room for communal sleeping burrows. Each group was fed 20 g of millet seed daily; millet seed has a high water content and is low in protein (nitrogen) (Murray and Dickman, 1994). The hopping mice were weighed daily. At the end of the WD period, both control and experimental Notomys were anaesthetised by halothane inhalation followed by cervical dislocation. Following sacrifice, blood samples were obtained via cardiac puncture using a 1 ml syringe coated with EDTA (10 mg/ml) and the haematocrit was determined using an Ames haematocrit centrifuge. The heart, kidneys, small intestine, caecum, and proximal and distal colon were removed and frozen in liquid N2 and stored until the RNA was extracted. 2.3. Analysis of guanylin, uroguanylin, and GC-C mRNA expression RT-PCR was used to determine the mRNA expression of guanylin, uroguanylin and GC-C mRNA in tissues from control and water-deprived Notomys. New guanylin (forward only) and uroguanylin primers were used as follows: guanylin forward 50 -accatgaatgcctgtgtgc-3; uroguanylin forward 50 -actatgtcaggaagccaactgtg-30 , uroguanylin reverse 50 -cacagctcacattcgtcggtg-30 . Total RNA isolation and cDNA synthesis were performed as described above. For the analysis of mRNA expression in different tissues, 2 lg of total RNA from each tissue was used. The PCR was performed for 22 cycles for GC-C and 23 cycles for guanylin and uroguanylin, which was in the linear ranges of all cDNAs used in the expression study. Amplification was performed using an initial cycle of 94 °C for 300 s, 60 °C for 60 s, and 72 °C for 60 s. Cycles 2– 21/22 consisted of 94 °C for 60 s, 60 °C for 60 s, and 72 °C for 60 s, and the parameters of cycle 22/23 were 94 °C for 60 s, 60 °C for 60 s, and 72 °C for 300 s. To quantify the level of mRNA expression between control and water-deprived Notomys, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control. It was assumed that the levels of GAPDH expression were constant in control and water-deprived Notomys (Sturzenbaum and Kille, 2001). The GAPDH primers were forward 50 -gaag gtcggtgtgaacggatttg-30 , and reverse 50 -ttactccttggaggccatgtagg-30 ; these primers generated a 999 base pair product. In preliminary experiments, the linear amplification range of each cDNA was determined by running multiplex PCR for a varying number of cycles between 17 and 35. The mRNA expression analysis was performed at a cycle number at the mid-point of the linear amplification curve. Initially, the linear range was established for amplification of each individual PCR product. The linear range was also determined in the various tissues used in
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the mRNA expression experiments, and both control and experimental samples to ensure that there was no variation between tissues, individual animals or sample groups. In the distal colon and kidney, guanylin, uroguanylin, and GAPDH primers were included in the multiplex reaction. However, in the proximal colon, the expression of guanylin and GAPDH and uroguanylin and GAPDH was determined separately. The expression of GC-C mRNA was determined in a multiplex PCR with GAPDH primers. PCR were assembled as described above, except that 1 ll of the cDNA reaction was used. For quantification of the PCR products, the reactions were spiked with 2.5 lCi of ½a-32 PdCTP. As the radiolabelled dCTP is randomly incorporated into the PCR products, the level of radiation emitted by a PCR product is directly proportional to the amount of the PCR product. The amplified PCR products were separated by electrophoresis on a 1.5% agarose gel in TBE running buffer at 100 V. The gel was incubated in 0.5 lg/ml of ethidium bromide and visualised on an UV light box and the relevant bands were excised using a scalpel blade and placed in microcentrifuge tubes. The amount of isotope ½a-32 P incorporated into the PCR products was measured by placing the tubes in vials, and counting in a scintillation counter (Tri-Carb 2000CA Liquid Scintillation Counter, Packard). Quantification of the mRNA expression was determined by normalising the levels of guanylin, uroguanylin, and GCC amplification against that of GAPDH. The values from control animals were adjusted to represent 100%, and the level of expression of guanylin, uroguanylin and GC-C in water-deprived Notomys was calculated as a percentage of the control. 2.4. Data analysis All data are represented as means one standard error (SE). To test the difference between control and experimental groups, a StudentÕs t test were performed using the SPSS 10 statistical package. A p value 60:05 was considered as statistically significant. 2.5. Materials ½a-32 PdCTP (3000 Ci/mmol) was purchased from Amersham Pharmacia Biotech. All other chemicals were either reagent or molecular grade and were purchased from Sigma or Invitrogen. 3. Results 3.1. Cloning and sequencing of guanylin, uroguanylin, and GC-C cDNAs A cDNA of 410 base pairs was amplified using the guanylin primers. Analysis of the nucleotide sequence showed highest homology to that of mouse and rat
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guanylin. The Notomys guanylin cDNA contained an open reading frame of 345 nucleotides encoding preproguanylin (Fig. 1A). The nucleotide sequence of Notomys guanylin showed 91 and 92% identity to that of mouse and rat, respectively. The deduced amino acid sequence of Notomys preproguanylin was 94% homol-
ogous to rat and mouse; the mature guanylin peptide was identical (Fig. 1B). A cDNA of 416 base pairs was amplified using the uroguanylin primers. Analysis of the nucleotide sequence showed highest homology to that of mouse and rat uroguanylin. The Notomys uroguanylin cDNA contained an open reading frame of 321 nucle-
Fig. 1. (A) Nucleotide and deduced amino acid sequences of a preproguanylin cDNA from Notomys. Numbers to the right indicate the base pair and amino acid number, respectively. The underlined sequences are the primer sites and the asterisk represents the stop codon. The mature guanylin peptide is indicated by the dashed underline. (B) Alignment of the deduced amino acid sequence of Notomys preproguanylin with other guanylin sequences. The alignment was performed using ClustalW. The asterisks indicate conserved amino acids between Notomys, mouse (AAB05758) and rat (AAA41300). The vertical lines indicated conserved amino acids between Notomys, mouse, rat, human (XP_046404), opossum (Fan et al., 1997), and zebrafish.
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otides encoding preprouroguanylin (Fig. 2A). The nucleotide sequence of Notomys uroguanylin showed 91 and 90% identity to that of mouse and rat, respectively. The deduced amino acid sequence of Notomys preprouroguanylin was 91% homologous to rat and mouse; the mature uroguanylin peptide was identical (Fig. 2B). A cDNA of 327 base pairs was amplified using the GCC primers (Fig. 3A). Analysis of the nucleotide sequence showed highest homology to that of mouse GC-C. The deduced amino acid of the partial GC-C cDNA was identical to that of the same region of mouse GC-C (Fig. 3B).
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3.2. Tissue expression of guanylin, uroguanylin, and GCC mRNA A multiplex PCR was performed to detect the expression of guanylin and uroguanylin mRNA in the heart, kidney, small intestine, caecum, and proximal and distal colon of Notomys. The caecum and proximal and distal colon had a greater expression of guanylin than uroguanylin, but in the small intestine, uroguanylin mRNA expression was greater than that of guanylin. The heart and kidney had lower levels of guanylin and uroguanylin mRNA expression (Fig. 4A). In contrast to
Fig. 2. (A) Nucleotide and deduced amino acid sequences of a preprouroguanylin cDNA from Notomys. Numbers to the right indicate the base pair and amino acid number, respectively. The underlined sequences are the primer sites and the asterisk represents the stop codon. The mature uroguanylin peptide is indicated by the dashed underline. (B) Alignment of the deduced amino acid sequence of Notomys preprouroguanylin with other uroguanylin sequences. The alignment was performed using ClustalW. The asterisks indicate conserved amino acids between Notomys, mouse (AAB82750) and rat (AAB18331). The vertical lines indicated conserved amino acids between Notomys, mouse, rat, human (1588627), opossum (AAB00553), and eel (CAC35449).
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Fig. 3. (A) Nucleotide and deduced amino acid sequences of a partial GC-C cDNA from Notomys. (B) Alignment of the deduced amino acid sequence of a partal Notomys GC-C with that of mouse (XM_132928). The alignment was performed using ClustalW. The asterisks indicate conserved amino acids between Notomys and mouse.
Fig. 4. (A) Image of a PCR amplification showing the expression of guanylin (391 base pairs) and uroguanylin (298 base pairs) mRNA in heart (1), kidney (2), small intestine (3), caecum (4), proximal colon (5), and distal colon (6) from Notomys. The left lane is a marker lane and the 500 base pair marker is boxed. The two bands below the 500 base pair band are 400 and 300 base pairs, respectively. (B) Image showing PCR amplification of a 327 base pair GC-C mRNA in the same tissues as (A). Lane assignment and markers as for (A).
guanylin and uroguanylin, no expression of GC-C mRNA was observed in the heart and kidney, but it was found in each of the regions of the gut (Fig. 4B). 3.3. Effect of water deprivation on guanylin, uroguanylin and GC-C mRNA expression Notomys subjected to WD steadily lost weight until the seventh day when weight had decreased by 22:3 1:7%; control animals maintained a stable weight. There was no significant difference in haematocrit between control (52:0 1:02%) and water-deprived
(50:2 1:72%) Notomys. The expression of guanylin, uroguanylin, and GC-C mRNA was compared between control and water-deprived Notomys. In the distal colon, there was a significant increase in the expression of guanylin and uroguanylin mRNA; the increase in guanylin mRNA expression was greater than that of uroguanylin (Fig. 5A). However, there was no difference in GC-C mRNA expression in the distal colon between control and water-deprived Notomys. In the proximal colon, there was a significant increase in guanylin and GC-C mRNA expression but not in uroguanylin mRNA expression (Fig. 5B). In the kidney, no significant difference in guanylin and uroguanylin mRNA expression was found (Fig. 5C).
4. Discussion Guanylin and uroguanylin are peptides that regulate intestinal and renal transepithelial fluid and electrolyte transport by activating guanylyl cyclase C receptors and generating cGMP (Forte, 1999). The binding of guanylin and uroguanylin to GC-C leads to the secretion of chloride and a parallel secretion of sodium and water; in the gut and kidney this results in the loss of salt and water from the body (Forte and Currie, 1995; Joo et al., 1998). In the gut, one of the targets for guanylinrelated peptides is the colon because of its role in modifying the salt and water content of the faeces prior to excretion. High levels of guanylin and GC-C mRNA expression occur in this tissue (Forte, 1999), and it has
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Fig. 5. Histograms showing the relative level of mRNA expression of guanylin, uroguanylin, and GC-C (colon only) in the distal colon (A), proximal colon (B), and kidney (C) of control () and 7 day waterdeprived (j) Notomys. The guanylin, uroguanylin or GC-C to GAPDH ratios for the control mice were set at 100%. Asterisks (*) indicate statistical significance ðp 6 0:05Þ from control values. Significant increases in guanylin and uroguanylin mRNA expression were found in the distal colon, but only guanylin and GC-C mRNA expression was significantly increased in the proximal colon.
been shown that this expression is sensitive to the level of salt intake (Carrithers et al., 2002; Li et al., 1996). In addition, the guanylin-related peptides (primarily uroguanylin) stimulate GC-C in the kidney to increase salt and water excretion (Forte et al., 2000a,b). In desert rodents such as Notomys, the reabsorption of water from the colon and kidney is one of the key adaptations for reducing water loss in arid environments (Degen,
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1997). It was, therefore, predicted that the expression of guanylin, uroguanylin and GC-C mRNA would be down-regulated in the colon and kidney during WD in Notomys. In order to study the expression of guanylin and uroguanylin mRNA in Notomys, cDNAs encoding the prepropeptides were amplified and cloned. Not surprisingly, the nucleotide and deduced amino acid sequences of Notomys guanylin and uroguanylin cDNAs showed very high homology to mammalian sequences; the mature peptides were identical to those of mouse and rat. Rodent guanylin differs by only one residue from human guanylin and three residues from opossum guanylin (Forte et al., 2000b). In contrast, rodent uroguanylin differs by three amino acid residues from human and opossum uroguanylin; human uroguanylin also has a C-terminal leucine residue that is only present in eel (Comrie et al., 2001; Forte et al., 2000b). The expression of guanylin and uroguanylin mRNA was demonstrated in a range of tissues from Notomys, using multiplex RT-PCR. Both guanylin and uroguanylin mRNA expression were found in the proximal and distal colon, caecum, small intestine, kidney, and heart, as has been found in mouse and rat. The expression of guanylin mRNA was notably higher than uroguanylin in the colon, which is consistent with previous studies that have shown a more important role for guanylin than uroguanylin in this tissue (Forte, 1999). The expression of uroguanylin mRNA in the heart has been described previously, and it has been proposed that uroguanylin may be released into the circulation in a similar manner to atrial natriuretic peptide (Fan et al., 1996). A partial cDNA encoding GC-C was cloned from the colon of Notomys, and the deduced amino acid sequence was identical to mouse, showing it was GC-C. The expression of GC-C mRNA was found in the small intestine, caecum, and the proximal and distal colon, but not in the heart and kidney. The absence of GC-C mRNA expression in the kidney was very surprising since GC-C expression has been clearly demonstrated in the rat kidney (Carrithers et al., 2000), and guanylin and uroguanylin can affect renal function (Forte et al., 2000a). Given that both guanylin and uroguanylin are expressed in the kidney of Notomys, further analysis on the presence or absence of GC-C proteins in the kidney is required. Following molecular cloning of guanylin, uroguanylin, and GC-C, the expression of their respective mRNAs was determined in normal and water-deprived Notomys, in order to examine how water restriction effects the level of mRNA expression. Notomys can survive long periods of WD, and maintain plasma volume and osmolarity at similar levels to Notomys with free access to water (Heimeier et al., 2002; MacMillen and Lee, 1969; Weaver et al., 1994). A previous study found
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that this homeostasis occurs without any long-term upregulation of vasopressin and the renin–angiotensin system (Weaver et al., 1994). However, the role of regulatory molecules that generate cGMP has not been extensively investigated in desert rodents. In the present study, a 7 day WD experiment was performed since this represents the acute phase of the adaptation to WD. In response to WD, there was an average decrease in body weight of 22.3% at day 7, compared to control animals, which is mainly due to a reduction in fat stores. A similar change in body weight was found in previous WD studies with Notomys (Heimeier et al., 2002; MacMillen and Lee, 1969; Weaver et al., 1994). If the WD period is extended beyond 7 days, the loss in body weight stabilises and the animals actually gain weight by 14 days of WD (Heimeier et al., 2002; MacMillen and Lee, 1969; Weaver et al., 1994). In this study, there was no significant difference in haematocrit between control and water-deprived animals, which suggests that ECF dehydration did not occur in water-deprived Notomys. The expression of guanylin mRNA in the proximal and distal colon significantly increased in response to WD; in addition uroguanylin mRNA expression was increased in the distal colon. An increase in mRNA expression suggests that there is also an increase in the production of guanylin and uroguanylin peptides in the colon. This result was surprising since one of the functions of the colonic epithelium is to reduce water loss in the faeces to prevent dehydration (Forte and Currie, 1995); an increase in the synthesis of guanylin and uroguanylin peptides in the colon of water-deprived Notomys would appear detrimental to this function. It is, therefore, interesting to speculate why there is an upregulation in the expression of guanylin and uroguanylin in the colon during WD. Previous studies have shown that the expression of guanylin and uroguanylin in the intestine of rats is affected by oral salt intake. For example, low salt intake results in a down-regulation of guanylin expression in the distal colon (Carrithers et al., 2002; Li et al., 1996) but high salt intake had no effect in the colon (Carrithers et al., 2002). Furthermore, salt loading increased guanylin secretion in the rat small intestine (Kita et al., 1999). Therefore, it is possible that there are differences in the amount of salt in the colon between control and water-deprived Notomys that could modulate the expression of guanylin and uroguanylin mRNA. As with guanylin and uroguanylin, a downregulation of GC-C mRNA expression was not observed following WD. Although guanylin and uroguanylin mRNA expression increased markedly in the distal colon a parallel increase in GC-C expression was not observed. In contrast, GC-C mRNA expression was significantly higher in the proximal colon in which a small increase in guanylin mRNA expression was observed. Thus, it appears that there is differential regu-
lation of GC-C mRNA transcription in the proximal and distal segments of the colon during WD. Following the discovery of guanylin and uroguanylin, it was shown that GC-C are present in the kidney and that the peptides stimulate the excretion of water and salt via cGMP (Forte et al., 2000a,b). Guanylin is less potent than uroguanylin in eliciting diuresis and natriuresis in perfused rat kidneys, as guanylin is inactivated in the renal tubules (Fonteles et al., 1998; Forte et al., 2000b). It was thus proposed that uroguanylin is part of an endocrine axis linking the intestine and the kidney that is regulated by dietary salt (Forte et al., 2000a,b). However, recent studies have shown that guanylin and uroguanylin are expressed in the kidney (Fan et al., 1997), which indicates the peptides may act in a paracrine/autocrine fashion. The distribution patterns of the peptides vary since guanylin mRNA is most highly expressed in the collecting ducts of mouse whereas uroguanylin expression is highest in the proximal tubules (Potthast et al., 2001). Both mRNA species were expressed in the kidney of Notomys, but the anatomical location was not determined. It was predicted a priori that the renal guanylin and uroguanylin mRNA expression would be down-regulated during WD to reduce the level of cGMP-mediated excretion, if indeed GC-C protein are present in the kidney of Notomys (see above). However, the renal expression of guanylin and uroguanylin mRNAs was not affected by WD as compared to control Notomys. Interestingly, this result is consistent with data from a WD experiment in laboratory mice in which no effect on renal expression was found (Potthast et al., 2001). In contrast, a salt load in the drinking water mediated an increase in renal uroguanylin mRNA expression but not guanylin expression (Potthast et al., 2001). The absence of any upregulation of uroguanylin mRNA expression during WD in Notomys may be due to the fact that plasma osmolarity is insignificantly changed, and therefore, there is no signal that leads to changes in uroguanylin mRNA transcription.
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Further reading Nakazato, M., 2001. Guanylin family: new intestinal peptides regulating electrolyte and water homeostasis. J. Gastroenterol. 36, 219–225. Wiegand, R.C., Kato, J., Currie, M.G., 1992. Rat guanylin cDNA: characterisation of the precursor of an endogenous activator of the intestinal guanylate cyclase. Biochem. Biophys. Res. Commun. 185, 812–817.