Renal effects of serine-7 analog of lymphoguanylin in ex vivo rat kidney

Manasses C. Fonteles1, Stephen L. Carrithers2, Helena S. A. Monteiro1, Andre F. Carvalho1, Gustavo R. Coelho1, Richard N. Greenberg2, and Leonard R. Forte3

1 Clinical Research Unit of Federal University of Ceara and Ceara State University, 60434 Fortaleza-CE, Brazil; 2 Department of Internal Medicine, Division of Infectious Disease, University of Kentucky and Lexington Veterans Affairs Medical Center, Lexington, Kentucky 40506; and 3 Department of Pharmacology, School of Medicine, Missouri University and Truman Memorial Veterans Affairs Hospital, Columbia, Missouri 65212


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Guanylin and uroguanylin compose a family of natriuretic, diuretic, and kaliuretic peptides that bind to and activate apical membrane receptor guanylyl cyclase signaling molecules in renal and intestinal epithelia. Recently, a complementary DNA encoding an additional member of the guanylin family of cGMP-regulating peptides was isolated from lymphoid tissues of the opossum and was termed lymphoguanylin (LGN). A peptide analog of opossum LGN was synthesized containing a single disulfide bond with the internal cysteine-7 replaced by a serine residue (LGNCys7right-arrow Ser7). The biological activity of LGNSer was tested by using a cGMP bioassay with cultured T84 (human intestinal) cells and opossum kidney (OK) cells. LGNSer has potencies and efficacies for activation of cGMP production in the intestinal and kidney cell lines that are 100- and 1,000-fold higher than LGN, respectively. In the isolated perfused rat kidney, LGNSer stimulated a maximal increase in fractional Na+ excretion from 24.8 ± 3.0 to 36.3 ± 3.3% 60 min after administration and enhanced urine flow from 0.15 ± 0.01 to 0.24 ± 0.01 ml · g-1 · min-1. LGNSer (0.69 µM) also increased fractional K+ excretion from 27.3 ± 2.3 to 38.0 ± 3.0% and fractional Cl- excretion from 26.1 ± 0.8 to 43.5 ± 1.9. A ninefold increase in the urinary excretion of cGMP from 1.00 ± 0.04 to 9.28 ± 1.14 pmol/ml was elicited by LGNSer, whereas cAMP levels were not changed on peptide administration. These findings demonstrate that LGNSer, which contains a single disulfide bond like native LGN, activates guanylyl cyclase-C (GC-C) receptors in T84 and OK cells and may be very helpful in studying the physiological importance of activation of GC-C in vivo. LGNSer also exhibits full activity in the isolated perfused kidney equivalent to that observed previously with opossum uroguanylin, suggesting a physiological role for LGN in renal function. Thus the single amino acid substitution enhances the activity and potency of LGN.

uroguanylin; guanylin; Escherichia coli heat-stable enterotoxin; guanylyl cyclase-C; kidney; cyclic guanosine 5'-monophosphate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GUANYLIN AND UROGUANYLIN ARE small cysteine-rich peptides that were initially isolated from rat jejunum and opossum urine, respectively (5, 20). Both of these peptides share strong structural similarity with heat-stable toxin (STa) peptides secreted by the enterotoxigenic strains of Escherichia coli that cause secretory diarrhea in humans and other animals (12). Guanylin and uroguanylin are produced at high levels in the intestinal mucosa, where they are secreted into the intestinal lumen and into the circulation. However, these peptides have been found in other tissues, including kidney, adrenal medulla, lymphoid tissues, myocardium, brain, and the airway epithelium (6, 8, 12, 38). These peptides bind to and activate apical membrane guanylyl cyclases (GCs) that are expressed in the intestine, kidney, and other epithelia (9). Guanylin and uroguanylin markedly stimulate intestinal chloride and bicarbonate secretion in the intestine via activation of cGMP accumulation in target cells located throughout the intestinal tract (19, 22). Guanylin, uroguanylin, and STa also increase urinary sodium, chloride, potassium, and water excretion in vivo in mice and in the ex vivo perfused rat kidney (2, 11, 18, 29).

Identification of both membrane receptor GCs and a cGMP signaling pathway for STa in the opossum kidney and cultured opossum kidney (OK) cells provided the first evidence for renal mechanisms involving intestinal cGMP-regulating peptides. Fonteles and coworkers (29) then demonstrated that STa induces natriuresis, diuresis, and kaliuresis in the isolated perfused rat kidney. Guanylin and uroguanylin were shown to possess similar natriuretic, diuretic, and kaliuretic activities compared with STa in intestinal and specific renal model systems (2, 11, 25, 29). Guanylin and/or uroguanylin may act physiologically as "intestinal natriuretic hormones" in an endocrine axis linking the intestine to the kidney (15, 18). Such an intestine-kidney signaling mechanism may contribute to a rapid and sustained increase in the urinary excretion of NaCl after a high-salt meal (1, 11, 15, 27).

Recently, a third member of the endogenous guanylin peptide family of cGMP-regulating hormones was identified by molecular cloning of cDNAs encoding the polypeptide precursor preprolymphoguanylin (13). Lymphoguanylin (LGN) is widely expressed in many lymphoid tissues of opossums, but the mRNA for this peptide is most abundant in the kidney and heart. This finding suggests that LGN may participate in a paracrine or autocrine intrarenal cGMP signaling mechanism that could influence Na+ and Cl- transport within the kidney (13). Like guanylin and uroguanylin, the bioactive form of LGN is found as a 15-residue peptide located at the COOH terminus of a larger precursor molecule (5, 13, 20). The amino acid sequence of LGN shares 80% identity with opossum uroguanylin but only 40% identity with guanylin. However, LGN has unique structural features that make it a distinct member of this family of peptides. Native LGN has three cysteine residues, and thus only one intramolecular disulfide bond can be formed in this peptide. In contrast, all of the guanylin and uroguanylin peptides identified thus far contain two intramolecular disulfide bonds, which were previously thought to be required for full biological activity of these peptides (Fig. 1). In a previous study, synthetic LGN was prepared containing three cysteine residues as derived from the cDNA structure of preprolymphoguanylin (13). This peptide was considerably less potent and less efficacious than either uroguanylin or guanylin when tested in either OK or T84 intestinal cell cGMP accumulation bioassays. Thus we prepared an analog of LGN containing only two cysteines, which facilitated the formation of a single disulfide bond and proper folding of this peptide (Fig. 1). We hypothesized that this analog, LGNCys7right-arrow Ser7, would increase intracellular cGMP in T84 and OK epithelial cells and cause a significant natriuretic response in the isolated perfused rat kidney. In the present study, we demonstrate that LGNCys7right-arrow Ser7 has full potency and efficacy in the stimulation of cGMP accumulation in both T84 and OK cells compared with the pharmacological properties of uroguanylin. In the isolated perfused rat kidney, LGNSer markedly increases the urinary excretion of Na+, K+, Cl-, and cGMP equivalent to the renal responses that were previously reported for uroguanylin and STa in this organ system (11).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1.   Primary structures of the active forms of lymphoguanylinSer; opossum lymphoguanylin; uroguanylin, and guanylin from opossum, human, and rat; and the Eschericha coli heat-stable enterotoxin (STa). The cysteine residues involved in the intramolecular disulfide bonds are underlined (single-letter code was used to represent the amino acid sequence of each peptide). Lines connecting the cysteine residues within each of these peptides denote the intramolecular disulfide bonds for each peptide group.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Synthesis of LGNSer. A modified form of opossum LGN (LGNCys7right-arrow Ser7; QEECELSINMACTGY) and native opossum uroguanylin were synthesized by solid-phase synthesis and purified by C18 reverse-phase HPLC as described in detail elsewhere (1, 5, 20).

Perfused rat kidney protocol. Adult Wistar-Kyoto rats of either sex, weighing 250-300 g, were anesthetized with pentobarbital sodium (50 mg/kg body wt ip). Before each experiment, the animals were fasted for 24 h with free water ad libitum. The perfusate solution consisted of a modified Krebs-Henseleit solution (MKHS) with the following composition (in mM): 147 Na+, 5 K+, 2.5 Ca2+, 2.0 Mg2+, 110 Cl-, 25 HCO3-, 1 SO42-, and 1 PO43-. Six grams of bovine serum albumin were added to 100 ml of MKHS after a previous dialysis for 48 h at 4-6°C against a volume 10 times larger. Immediately before the beginning of each perfusion protocol, 100 mg of glucose, 50 mg of urea, and 50 mg of inulin (Sigma, St. Louis, MO) were added to the MKHS in a final volume of 100 ml. The pH was then adjusted to 7.4, and the solution was placed in the perfusion line.

The isolated perfused rat kidney assay was performed according to methods previously described by Bowman, as modified by Fonteles et al. (10, 11, 29) by employing a Silastic membrane oxygenator into the perfusion line (PO2 = 450-500 Torr). The renal artery of the right kidney of each animal was cannulated via the superior mesenteric artery without flow interruption to avoid ischemic damage and placed into the perfusion line. The animals were then killed by exsanguination. A period of 15-20 min was allowed for blood washout. Before the infusion of the test peptide, a 30-min internal control period was permitted.

Three groups of isolated perfused rat kidneys were tested: 1) a control group in which the kidneys were perfused only with the MKHS described above; 2) another group, in which LGNSer (0.23 µM) was added as a bolus to the perfusate reservoir after a 30-min internal control period; and 3) a group in which LGNSer was placed in the perfusate reservoir at a final concentration of 0.69 µM after a 30-min internal control period. Functional observations were made for 120 min for each of these groups.

The perfusion pressure was determined at the tip of a stainless steel cannula and was allowed to fluctuate under experimental conditions but was carefully kept at 120-140 mmHg during the 30-min internal control period of each group by adjusting the flow rate of the peristaltic pump. Samples of both urine and perfusate were collected at 10-min intervals for biochemical analysis (see below). The glomerular filtration rate (GFR), the fractional excretions of Na+ (%ENa+), K+ (%EK+), and Cl- (%ECl-), osmolar clearance (Cosm), and the fractional tubular reabsorptions of Na+ (%RNa+), K+ (%RK+), and Cl- (%RCl-) were determined by conventional clearance formulas as described in detail elsewhere (10, 31).

Biochemical analyses. The perfusate and urine aliquots (50 µl) obtained were analyzed for Na+ and K+ by flame photometry, and Cl- was determined by spectrophotometry. Inulin was determined according to the method described by Wasler and coworkers (37) as modified by Fonteles et al. (10, 11). Osmolality was determined by freezing-point depression (Needham Heights, MA).

Urinary cGMP and cAMP measurements. Immediately after each experiment, the tubes containing urine samples were reweighed for determination of urine volume. cGMP and cAMP levels in urine were determined by a cGMP enzymeimmunoassay (EIA; Amersham Pharmacia Biotech, Piscataway, NJ) or a cAMP EIA (Amersham Pharmacia Biotech), respectively. Briefly, an aliquot from the urine samples was diluted 1:10 with a sodium acetate assay buffer in a final volume of 150 µl, followed by acetylation with alpha -amino-N/tetrethylammonium (1:2, vol/vol). Then, 100 µl of cGMP- or cAMP-rabbit antiserum were added to each well of a microtiter plate coated with anti-rabbit IgG antibody. Fifty microliters of each acetylated urine sample were then added to each well, and the plate was incubated for 2 h at 4°C. The cGMP- or cAMP-peroxidase conjugate (100 µl) was then placed in each well, and the plate was incubated at 4°C for 1 h. Each well was washed four times with PBS with 0.05% Tween 20, and 200 µl of 1,1'-trimethylene-bis(4-formylpyridinium bromide) dioxime substrate were placed into each well and incubated for 30 min at 25°C. Then, 100 µl of a 1 M sulfuric acid solution were added to each well. After 10 min at RT, the absorbance at 450 nm was measured by spectrophotometry (model 2550, EIA Reader, Bio-Rad Laboratories). The cGMP and cAMP concentrations of each urine sample were determined in duplicate or triplicate by comparison with known standards.

Cell culture. T84 cells were cultured in DMEM/Ham's F-12 medium (1:1) supplemented with 5% fetal bovine serum, 60 mg of penicillin, and 100 mg of streptomycin/ml as described in detail elsewhere (3, 5, 13, 20). OK (OK-epsilon ) cells were cultured in DMEM and Ham's F-12 medium (1:1) supplemented with 5% fetal bovine serum as previously described (13, 16).

cGMP accumulation bioassay. T84 and OK cells were cultured in 24-well plastic dishes, and cGMP levels were measured in control and agonist-stimulated cells by RIA (1, 5, 13, 20). Synthetic peptides were diluted in 200 µl DMEM containing 20 mM HEPES and 1 mM of IBMX at pH 7.4. Cells were washed with DMEM before addition of 200 µl DMEM containing either agonist peptides or vehicle to T84 or OK cells, followed by incubation at 37°C for 40 min. After incubation, the medium was aspirated, the cells were washed with PBS, and 0.2 ml of 3.3% perchloric acid was added to each well to extract cGMP. The cell extract was neutralized and used for measurement of cGMP by RIA as previously described (5, 20). cAMP levels were not measured from these cellular extracts because neither uroguanylin nor STa increases this cyclic nucleotide in these cells (1, 5, 16).

Data analyses. For the experiments with perfused kidneys, the data (means ± SE) were averaged in triplicate between 30-min intervals. Statistical analyses were carried out by one-way ANOVA and by Student's t-test when applicable. There were at least four to five perfused-kidney experiments for each data point.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The synthetic LGNSer peptide containing a single disulfide bond was tested for biological activity by using both OK and T84 cell cGMP accumulation bioassays (Fig. 2). LGNSer stimulates cGMP production with apparent potencies and efficacies that are equal to those observed for opossum uroguanylin in these cGMP bioassays with kidney and intestinal cell lines. Thus LGNSer appears to be a full agonist for the receptor GC signaling molecules that are expressed in these cultured cells (for human T84 cells, GC-C; for OK cells, OK-GC), even though this peptide contains only one intramolecular disulfide bond.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 2.   Activation of receptors on T84 and opossum kidney (OK) cells. T84 cells (A) or OK cells (B) were exposed to vehicle or specific concentrations of uroguanylin (UGN) or lymphoguanylinSer (LGNSer) for 40 min at 37°C. Intracellular cGMP was measured by enzyme-linked immunoassay as described in MATERIALS AND METHODS. These data represent the average of 2 different determinations.

LGNSer (0.23 µM) induced a significant natriuretic response in the isolated perfused kidney, reflected as both an increase in %ENa+ from 24.7 ± 3.0 to 36.3 ± 3.3% at 90 min (P <=  0.05; Fig. 3) and a decrease in %RNa+ (Table 1). A prominent and sustained diuresis was also observed at this concentration of LGNSer, with urine flow increasing from 0.15 ± 0.01 to 0.24 ± 0.01 ml · g-1 · min-1 (P < 0.05; Fig. 4). The changes in %EK+ and %RK+ elicited by 0.23 µM LGNSer were not statistically significant (Tables 1 and 2). At this concentration of LGN, no changes occurred in either %ECl- or %RCl- (Tables 1 and 2). The urinary excretion of cGMP increased significantly (from 1.02 ± 0.04 to 8.84 ± 0.82 pmol/ml, P <=  0.05) at 60 min after treatment with LGNSer (Fig. 5, 90-min time point). The increase in urinary cGMP excretion paralleled the increase in Na+ excretion and urine flow. No significant changes were observed in either GFR or perfusion pressure (Table 2). In addition, urinary cAMP levels in the rat kidneys treated with LGNSer did not vary from those in the control kidneys. Also, by the end of the experiment, the cAMP levels were virtually undetectable in both the control and LGNSer-treated rat kidneys.


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 3.   Fractional sodium excretion (%ENa+) changes induced by LGNSer in rat kidneys perfused with a modified Krebs-Henseleit solution containing albumin (6 g/100 ml). Values are means ± SE. *P <=  0.05 (ANOVA and Student's t-test), compared with the 30-min internal control.


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Data from control and LGNSer-treated rat kidneys perfused with a modified Krebs-Henseleit solution containing albumin (6 g/100 ml)



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 4.   Urine flow changes (UF; ml/g/min) induced by LGNSer in the isolated perfused rat kidney. *P <=  0.05, compared with the 30-min internal control.


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Data from control and LGNSer-treated rat kidneys perfused with a modified Krebs-Henseleit solution containing albumin 6 g%



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   Urinary recovery of cGMP (pmol/ml; A) and cAMP (fmol/ml; B) in control and LGNSer-treated rat kidneys. *P <=  0.05, compared with 30-min internal control.

At the higher concentration of LGNSer (0.69 µM), we found a significant increase in the %RNa+ (Fig. 3) and a corresponding decrease in %RNa+ from 77.1 ± 1.0 to 64.0 ± 0.7% by 90 min (P <=  0.05; Table 1). LGNSer also induced a diuresis, with urine flow increasing from 0.19 ± 0.02 to 0.33 ± 0.02 ml · g-1 · min-1 by 120 min (P <=  0.05) (Fig. 4). At this concentration of LGNSer, we also observed a significant kaliuresis, with an increase in %EK+ (Table 2) and a decrease in %RK+ from 72.6 ± 2.3 to 62.0 ± 2.9% (P <=  0.05). A significant increase in %ECl- from 26.1 ± 0.8 to 37.9 ± 1.9% (P <=  0.05) and a decrease in %RCl- were also observed (Tables 1 and 2). An increase in perfusion pressure with a higher LGNSer concentration was observed (Table 2).

LGNSer elicited striking increases in the urinary excretion of cGMP at 0.69 µM, with cGMP increasing from the control level of 1.00 ± 0.04 to 9.28 ± 1.14 pmol cGMP/ml by 90 min after peptide administration (P <=  0.05) (Fig. 5). There were no significant changes in GFR (Table 2), osmolar clearance (data not shown), and cAMP (Fig. 5) at the concentrations of LGNSer that were tested in these experiments. Thus LGNSer elicits marked increases in urinary cGMP excretion in association with substantial increases in the excretion of Na+, K+, and water, probably through activation of receptor GC signaling molecules located on the surface of target cells in the renal tubules.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A synthetic form of LGN containing two cysteine residues and a single disulfide bond has been prepared (LGNSer) and found to be equipotent compared with native opossum uroguanylin in both the OK and T84 cell cGMP bioassays. In marked contrast, we previously found that synthetic LGN prepared with three cysteine residues in its native form had markedly reduced potency compared with guanylin or uroguanylin in these bioassays (12). This finding is consistent with the hypothesis that a fully active guanylin-like peptide that contains only one intramolecular disulfide bond can activate the cognate guanylin receptors of kidney and intestine. Therefore, the secondary structure and three-dimensional conformation of this LGN analog must be similar to those previously elucidated for guanylin and uroguanylin containing two disulfide bonds (12). The advantages of preparing a guanylin peptide with a single disulfide bond are that simple air-oxidation steps can be used to elicit folding of this peptide into an active conformation. In contrast, we previously used a more complex method of directed disulfide bond formation to ensure that the intramolecular disulfides that are formed in the guanylin peptides are those connecting the first to third and second to fourth cysteine residues in the linear peptide chain (Fig. 1) (5, 20). If directed folding in a stepwise manner is not used for the guanylin and uroguanylin peptides, little biologically active peptide is formed because the subsequent disulfide bond formation results in an inactive disulfide isomer of the guanylin peptides (24). The synthesis of LGNSer containing a single disulfide bond will facilitate the preparation of a fully active guanylin-family peptide to further investigate the physiological roles of this novel class of cGMP-regulating peptides.

The present study characterizes the renal effects of a new member of the guanylin peptide family. LGNSer has natriuretic, diuretic, and kaliuretic activities in the isolated perfused rat kidney. The renal effects of LGNSer are surprisingly quite similar to those previously described for uroguanylin (11). At the concentrations of uroguanylin and LGNSer that have been tested, both peptides elicit prominent natriuretic responses in the isolated perfused kidney (11). It has also been demonstrated that E. coli STa, the molecular mimic of guanylin and uroguanylin, has natriuretic, diuretic, and kaliuretic activities in the isolated perfused rat kidney (29).

Receptors for guanylin/uroguanylin/STa have been shown to be localized to both medullary and cortical segments in the rat nephron after 125I-labeled guanylin was intravenously injected (17). Also, autoradiography employing 125I-labeled STa to opossum kidney tissue sections demonstrated binding sites for STa receptors (16, 25). Carrithers et al. (3) report the presence of GC-C receptors along distinct regions of the rat nephron. This suggests that receptors for the guanylin family of peptides may exist on cells located in different segments of the nephron, thus influencing tubular function (12). In the opossum, the guanylin/uroguanylin/LGN/STa receptor is known as OK-GC, which is the renal isoform of mammalian intestinal GC-C found in mice, rats, and humans (26, 30). OK-GC is present in OK cells, and GC-C is present in T84 cells (14, 25). On ligand binding in intestinal or kidney epithelial cells, these receptors increase the intracellular content of cGMP. We have demonstrated in this study that LGNSer stimulates cGMP accumulation in both the OK and T84 cells with potencies and efficacies that are comparable to that of native opossum uroguanylin. Furthermore, LGNSer is over 100 times more potent than native LGN in T84 cells and OK cells, respectively (13). These results suggest that LGNSer can activate either OK-GC or GC-C.

The fact that urinary cGMP excretion is increased ninefold 60 min after LGNSer administration in the isolated perfused rat kidney suggests that this cyclic nucleotide may mediate the natriuretic effects of LGN. Furthermore, the natriuresis and the urinary cGMP accumulation are consistent with the hormone effect in the kidney preparation. On the other hand, cAMP levels do not increase in the urine fractions (Fig. 5). The basal levels observed in the control and treatment periods are likely to be remnants of vascular cAMP in this system. Additional studies have supported the fact that 8-bromoadenosine 3',5'-cyclic monophosphate causes natriuresis (29). These effects were also observed with STa, which is the bacterial mimic peptide of the endogenous family of guanylin peptides. Thus the increases in urinary Na+, K+, and Cl- on LGNSer treatment are consistent with a cGMP-dependent mechanism.

In the intestine, it has been shown that STa and guanylin specifically signal through cGMP-dependent protein kinase II (cGK-II; 33-36), causing Cl- secretion from native epithelial cells. However, guanylin/uroguanylin/LGN target cells may also increase transepithelial Cl- and HCO3- secretion by interacting with and stimulating the enzymatic activities of cAMP-dependent protein kinase A-II (PKA-II; 12, 15). Disabling the gene encoding cGK-II results in a loss of fluid secretion responses to STa and guanylin in vivo and a marked decrease in anion secretion in vitro (33). Short-circuit current responses to STa/guanylin in vitro were partly retained in the intestine of cGK-II(-/-) mice, indicating that alternative pathways exist for cGMP regulation by the guanylin peptides. It was previously shown in T84 cells that STa/guanylin-induced cGMP activates Cl- secretion by cross-activation of PKA-II rather than by activation of cGK-II (12, 15). A third cGMP signaling mechanism may be through cGMP binding to specific sites on cGMP-dependent phosphodiesterases, leading to decreased cAMP hydrolysis. Thus high levels of cGMP would indirectly increase intracellular cAMP, which, in turn, activates PKA-II (7). The renal mechanism(s) for the family of guanylin-like peptides is unknown and has not been investigated. Also, there is no evidence to demonstrate that LGN signals through cGK-II in the kidney. Presently, we are testing specific inhibitors to the cGK-II and PKA pathways to determine the downstream signaling pathway(s) for guanylin-like peptide (and LGN) action in the kidney.

Because LGN was identified in a metatherian (marsupial) mammal (Didelphis virginiana), it is possible that that the LGN gene could have arisen by gene duplication after the evolutionary divergence of eutherian (placental) and metatherian mammals occurred (23). An ancestral uroguanylin-like gene could be the evolutionary precursor for both uroguanylin and LGN genes of the opossum. Because a homolog of LGN has not yet been identified in representative eutherian mammals, a physiological role in the body for LGN can only be postulated for the opossum at the present time (13).

In conclusion, an analog of LGN containing a single disulfide bond increased cGMP in OK and T84 (human intestinal) cells. This analog, LGNSer, is more potent than native LGN and equipotent to uroguanylin in both of these in vitro cell models. Treatment of the ex vivo perfused kidneys of rats with LGNSer resulted in substantial increases in the urinary excretion of Na+, K+, water, and cGMP. In the opossum, LGN may participate in an intrarenal paracrine signaling pathway that influences kidney function via the second-messenger cGMP and influence sodium transport in the kidney. Furthermore, LGNSer, which is more easily synthesized than LGN, uroguanylin, or guanylin, can be employed to elucidate the renal mechanisms as well as the specific nephron sites of action for the guanylin family of peptides.


    ACKNOWLEDGEMENTS

We gratefully acknowledge Jose Amadeus for animal care, Silvia F. Franca for expert technical assistance, and Domingos Barreto for biochemical determinations.


    FOOTNOTES

The present research was supported by a grant form the Conselho Nacional de Pesquisas, Brazil (to M. C. Fonteles). L. R. Forte, R. N. Greenberg, and S. L. Carrithers were supported by the Medical Research Service, Department of Veterans Affairs, Washington, DC.

Part of this work was published as an abstract for Experimental Biology '99, Washington, DC, April 27-21, 1999.

Address for reprint requests and other correspondence: M. C. Fonteles, Clinical Research Unit, CP 3229 UPC-UFC/UECe, 60434 Fortaleza, Ceara, Brazil (E-mail: reitor{at}uece.br).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 March 2000; accepted in final form 11 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Carrithers, SL, Eber S, Forte LR, and Greenberg RN. Increased urinary excretion of uroguanylin in patients with congestive heart failure. Am J Physiol Heart Circ Physiol 278: H538-H547, 2000[Abstract/Free Full Text].

2.   Carrithers, SL, Hill MJ, Johnson BR, O'Hara SM, Jackson BA, Ott CE, Lorenz J, Mann EA, Giannella RA, Forte LR, and Greenberg RN. Renal effects of guanylin and uroguanylin in vivo. Braz J Med Biol Res 32: 1337-1344, 1999[ISI][Medline].

3.   Carrithers, SL, Taylor B, Cai WY, Johnson BR, Ott CE, Greenberg RN, and Jackson BA. Guanylyl cyclase-C receptor mRNA distribution along the rat nephron. Regul Pept 24: 65-74, 2000.

4.   Coulsor, R. Metabolism and excretion of exogenous adenosine-3',5'-monophosphate and guanosine-3',5'-monophosphate. Studies in the isolated perfused rat kidney and in the intact rat. J Biol Chem 251: 4958-4967, 1976[Abstract].

5.   Currie, MG, Fok KF, Kato J, Moore RJ, Hamra FK, Duffin KL, and Smith CE. Guanylin: an endogenous activator of intestinal guanylate cyclase. Proc Natl Acad Sci USA 88: 947-951, 1992.

6.   De Sauvage, FJ, Keshav S, Kuang WJ, Gillet N, Henzel W, and Goeddel DV. Precursor structure, expression and tissue distribution of human guanylin. Proc Natl Acad Sci USA 89: 9089-9093, 1992[Abstract].

7.   Dousa, TP. Cyclic-3',5'-nucleotide phosphodiesterase isozymes in cell biology and pathophysiology of the kidney. Kidney Int 55: 29-62, 1999[ISI][Medline].

8.   Fan, X, Hamra FK, Freeman RH, Eber SL, Krause WJ, Lim RW, Pace VM, Currie MG, and Forte LR. Uroguanylin: cloning of preprouroguanylin cDNA, mRNA expression in the intestine and heart and isolation of uroguanylin and prouroguanylin from plasma. Biochem Biophys Res Commun 219: 457-462, 1996[ISI][Medline].

9.   Fan, X, Wang Y, London RM, Eber SL, Krause WJ, Freeman RH, and Forte LR. Signaling pathways for guanylin and uroguanylin in the digestive, renal, central nervous, reproductive and lymphoid systems. Endocrinology 138: 4636-4648, 1997[Abstract/Free Full Text].

10.   Fonteles, MC, Cohen JJ, Black AJ, and Wertheim SJ. Support of kidney function by long-chain fatty acids derived from renal tissue. Am J Physiol Renal Fluid Electrolyte Physiol 244: F235-F246, 1983[ISI][Medline].

11.   Fonteles, MC, Greenberg RN, Monteiro HSA, Currie MG, and Forte LR. Natriuretic and kaliuretic activities of guanylin and uroguanylin in the isolated perfused rat kidney. Am J Physiol Renal Physiol 275: F191-F197, 1998[Abstract/Free Full Text].

12.   Forte, LR. Guanylin regulatory peptides: structures, biological activities mediated by cyclic GMP and pathobiology. Regul Pept 81: 25-39, 1999[ISI][Medline].

13.   Forte, LR, Eber SL, Fan X, London RM, Wang Y, Rowland LM, Chin DT, Freeman RH, and Krause WJ. Lymphoguanylin: cloning and characterization of a unique member of the guanylin peptide family. Endocrinology 140: 1800-1806, 1999[Abstract/Free Full Text].

14.   Forte, LR, Eber SL, Turner JT, Freeman RH, Fok KF, and Currie MG. Guanylin stimulation of Cl- secretion in human intestinal T84 cells via cyclic guanosine monophosphate. J Clin Invest 91: 2423-2428, 1993[ISI][Medline].

15.   Forte, LR, Fan X, and Hamra FK. Salt and water homeostasis: uroguanylin is a circulating peptide hormone with natriuretic activity. Am J Kidney Dis 28: 296-304, 1996[ISI][Medline].

16.   Forte, LR, Krause WJ, and Freeman RH. Receptors and cGMP signaling mechanism for E. coli enterotoxin in opossum kidney. Am J Physiol Renal Fluid Electrolyte Physiol 255: F1040-F1046, 1988[Abstract/Free Full Text].

17.   Furuya, S, Naruse S, Ando E, Nokihara K, and Hayakawa T. Effects and distribution of intravenously injected 125I-guanylin in rat kidney examined by high-resolution light microscopic radioautography. Anat Embryol (Berl) 196: 185-193, 1997[ISI][Medline].

18.   Greenberg, RN, Hill M, Craytzer J, Krause WJ, Eber SL, Hamra FK, and Forte LR. Comparison of effects of uroguanylin, guanylin, Eschericha coli heat-stable STa in mouse intestine and kidney: evidence that uroguanylin is an intestinal natriuretic hormone. J Invest Med 45: 276-282, 1997[ISI][Medline].

19.   Guba, M, Kuhn M, Forssmann WG, Classen M, Gregor M, and Seidler U. Guanylin strongly stimulates rat duodenal HCO3- secretion: proposed mechanisms and comparison with other secretagogues. Gastroenterology 111: 1558-1568, 1996[ISI][Medline].

20.   Hamra, FK, Forte LR, Eber SL, Pidhorodjeckyj NV, Krause WJ, Freeman RH, Chin DT, Tompkins JA, K, Fok F, Smith CE, Smith KL, Siegel NR, and Currie MG. Uroguanylin: structure and activity of a second endogenous peptide that stimulates intestinal guanylate cyclase. Proc Natl Acad Sci USA 90: 10464-10468, 1993[Abstract].

21.   Heuze-Joubert, I, Mennecier P, Simonet S, Laubie M, and Verbeuren TJ. Effect of vasodilators, including nitric oxide, on the release of cGMP and cAMP in the isolated perfused rat kidney. Eur J Pharmacol 220: 161-171, 1992[ISI][Medline].

22.   Ieda, H, Naruse S, Kintagawa M, Ishiguro H, and Hayakawa T. Effects of guanylin and uroguanylin on rat jejunal fluid and electrolyte transport: comparison with heat-stable enterotoxin. Regul Pept 79: 165-171, 1999[ISI][Medline].

23.   Janke, A, Xu X, and Amarson U. The complete mitochondrial genome of walaroo (Macropus robustus) and the phylogenetic relationship among Monotremata, Marsupiala, and Eutheria. Proc Natl Acad Sci USA 94: 1276-1281, 1997[Abstract/Free Full Text].

24.   Klodt, J, Kuhn M, Marx UC, Martin S, Rosch P, Forsmmann WG, and Andermann K. Synthesis, biological activity, and isomerism of guanylate-cyclase C-activating peptides guanylin and uroguanylin. J Pept Res 50: 222-230, 1997[ISI][Medline].

25.   Krause, WJ, Freeman RH, and Forte LR. Autoradiographic demonstration of specific binding sites for E. coli enterotoxin in various epithelia of the North American opossum. Cell Tissue Res 260: 387-394, 1990[ISI][Medline].

26.   Krause, WJ, London RM, Freeman RH, and Forte LR. The guanylin and uroguanylin peptide hormones and their receptors. Acta Anat 160: 213-231, 1997[ISI][Medline].

27.   Lennane, RJ, Peart WS, Carey RM, and Shaw J. Comparison of natriuresis after oral and intravenous sodium loading in sodium-depleted rabbits: evidence for a gastrointestinal or portal monitor of sodium intake. Clin Sci Mol Med 49: 433-436, 1975[ISI][Medline].

28.   Li, Z, Perkins AG, Peters MF, Campa MJ, and Goy MF. Purification, cDNA sequence and tissue distribution of rat uroguanylin. Regul Pept 68: 45-56, 1997[ISI][Medline].

29.   Lima, AA, Monteiro HSA, and Fonteles MC. The effects of Eschericha coli heat-stable enterotoxin in renal sodium tubular transport. Pharmacol Toxicol 70: 163-167, 1992[ISI][Medline].

30.   London, RM, Eber SL, Visweswariah SS, Krause WJ, and Forte LR. Structure and activity of OK-GC: a kidney receptor activated by guanylin peptides. Am J Physiol Renal Physiol 276: F882-F891, 1999[Abstract/Free Full Text].

31.   Martinez-Maldonato, M, and Opava-Stitzer S. Free water clearance during saline, manitol, glucose and urea diuresis in rats. J Physiol (Lond) 280: 487-497, 1978[ISI][Medline].

32.   Miyazato, M, Nakazato M, Matsukara S, Kangawa K, and Matsuo H. Uroguanylin gene expression in the alimentary tract and extra-intestinal tissues. FEBS Lett 398: 170-174, 1996[ISI][Medline].

33.   Pfeifer, A, Aszodi A, Seidler U, Ruth P, Hoffman F, and Fassler R. Intestinal secretory defects and dwarfism in mice lacking cGMP-dependent protein kinase II. Science 274: 2082-2086, 1996[Abstract/Free Full Text].

34.   Vaandrager, AB, Bot AGM, and DeJonge HR. Guanosine 3',5'-cyclic monophosphate-dependent protein kinase II mediates heat-stable enterotoxin-provoked chloride secretion in rat intestine. Gastroenterology 112: 437-443, 1997[ISI][Medline].

35.   Vaandrager, AB, Edixhoven M, Bot AGM, Kroos MA, Jarchau T, Lohmann S, Genieser HG, and DeJonge HR. Endogenous type II cGMP-dependent protein kinase exists as a dimer in membranes and can be functionally distinguished from the type I isoforms. J Biol Chem 272: 11816-11823, 1997[Abstract/Free Full Text].

36.   Vaandrager, AB, Smolenski A, Tilly BC, Houtsmuller AB, Ehlert EME, Bot AGM, Edixhoven M, Boomaars WEM, Lohmann SM, and DeJonge HR. Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl- channel activation. Proc Natl Acad Sci USA 95: 1466-1471, 1998[Abstract/Free Full Text].

37.   Wasler, M, Davidosn DG, and Orloff J. The renal clearance of alkali-stable inulin. J Clin Invest 34: 1520-1523, 1955[ISI].

38.   Whitaker, TL, Witte DP, Scott MC, and Cohen MB. Uroguanylin and guanylin: distinct but ovelapping patterns of messenger RNA expression in mouse intestine. Gastroenterology 113: 1000-1006, 1997[ISI][Medline].

39.   Wiegand, RC, Kato J, Huang MD, Fok KF, Kachur JF, and Currie MG. Human guanylin: cDNA isolation, structure and activity. FEBS Lett 311: 150-154, 1992[ISI][Medline].


Am J Physiol Renal Fluid Electrolyte Physiol 280(2):F207-F213




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (3)
Google Scholar
Articles by Fonteles, M. C.
Articles by Forte, L. R.
Articles citing this Article
PubMed
PubMed Citation
Articles by Fonteles, M. C.
Articles by Forte, L. R.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online