Brazilian Journal of Medical and Biological Research (1999) 32: 1329-1336 Guanylin peptides and cGMP 1329 ISSN 0100-879X Guanylin peptides: cyclic GMP signaling mechanisms L.R. Forte1,2, 1Harry S. Truman Veterans’ Hospital, Departments of R.H. Freeman3, 2Pharmacology, 3Physiology and 4Pathology and Anatomical Sciences, W.J. Krause4 and School of Medicine, Missouri University, Columbia, MO, USA R.M. London1,2 Abstract Correspondence Guanylate cyclases (GC) serve in two different signaling pathways Key words L.R. Forte involving cytosolic and membrane enzymes. Membrane GCs are · Kidney Department of Pharmacology receptors for guanylin and atriopeptin peptides, two families of cGMP- · Intestine School of Medicine · regulating peptides. Three subclasses of guanylin peptides contain one Guanylate cyclase Missouri University · Chloride secretion intramolecular disulfide (lymphoguanylin), two disulfides (guanylin M-515 Medical Sciences Building · Sodium excretion Columbia, MO 65212 and uroguanylin) and three disulfides (E. coli stable toxin, ST). The - USA peptides activate membrane receptor-GCs and regulate intestinal Cl - Fax: +1-573-884-4558 and HCO3 secretion via cGMP in target enterocytes. Uroguanylin and E-mail: [email protected] ST also elicit diuretic and natriuretic responses in the kidney. GC-C is an intestinal receptor-GC for guanylin and uroguanylin, but GC-C Presented at the Meeting “NO Brazil, Basic and Clinical may not be involved in renal cGMP pathways. A novel receptor-GC Aspects of Nitric Oxide”, expressed in the opossum kidney (OK-GC) has been identified by Foz do Iguaçu, PR, Brazil, molecular cloning. OK-GC cDNAs encode receptor-GCs in renal March 10-13, 1999. tubules that are activated by guanylins. Lymphoguanylin is highly expressed in the kidney and heart where it may influence cGMP pathways. Guanylin and uroguanylin are highly expressed in intestinal mucosa to regulate intestinal salt and water transport via paracrine Received May 28, 1999 actions on GC-C. Uroguanylin and guanylin are also secreted from Accepted June 22, 1999 intestinal mucosa into plasma where uroguanylin serves as an intesti- nal natriuretic hormone to influence body Na+ homeostasis by endo- crine mechanisms. Thus, guanylin peptides control salt and water transport in the kidney and intestine mediated by cGMP via membrane receptors with intrinsic guanylate cyclase activity. Introduction as biologically active, 13, 14 and 15 amino acid peptides using the T84 cell cGMP bio- Guanylin, uroguanylin and lymphogua- assay (2). Lymphoguanylin was recently iden- nylin are heat-stable peptides that regulate tified using a PCR-based homology cloning the enzymatic activity of cell-surface guany- strategy to isolate cDNAs from opossum late cyclase signaling molecules. Guanylin spleen and other lymphoid tissues, which was the first endogenous peptide identified encode a 109 amino acid precursor that is and was isolated as a 15 amino acid peptide most similar in its primary structure to pre- from rat intestine that stimulates cGMP pro- prouroguanylin (3). Guanylin and uroguany- duction in T84 intestinal cells (1). Urogua- lin have two intramolecular disulfide bonds, nylin was next isolated from opossum urine but lymphoguanylin has only one disulfide Braz J Med Biol Res 32(11) 1999 1330 L.R. Forte et al. bond. A synthetic form of lymphoguanylin protein (11,12). CFTR and GC-receptors are also activates guanylate cyclase (GC)-recep- localized together in apical plasma mem- tors in human T84 intestinal and opossum branes of target enterocytes. The first GC- kidney (OK) cells. The family of guanylin receptor for guanylin family peptides was regulatory peptides share similarities both in identified at the molecular level (i.e., GC-C) primary structures and biological activities by molecular cloning of cDNAs encoding an with the heat-stable enterotoxin (stable toxin, intestinal membrane protein that binds 125I- ST) peptides secreted by strains of enteric ST with high affinity and is activated by ST microorganisms that cause a watery form of (13). Transgenic suckling mice with dis- diarrhea similar to that seen in cholera (4). abled GC-C genes have a marked reduction Thus, bacteria-derived ST peptides are mo- in the intestinal fluid secretion response to E. lecular mimics of guanylin peptides acting to coli ST, indicating that GC-C is responsible stimulate fluid and electrolyte secretion into for a major component of the intestinal ac- the lumen of the intestine by activation of tions of guanylin peptides to enhance fluid native guanylin receptors located on the api- secretion (14,15). However, about 10% of cal surfaces of enterocytes lining the intes- specific 125I-ST binding to receptor sites on tine. Biologically active peptides in the intestinal membranes still remain functional guanylin family of proteins are found at the in GC-C-knock out (GC-C-KO) animals (15). COOH-termini of longer precursor polypep- This suggests that an additional gene or mul- tides that are secreted as biologically inac- tiple genes encoding GC-receptors for tive prohormones or protoxins (5-8). Pro- guanylin agonists exist in the mouse ge- teolytic enzymes serve as converting en- nome. zymes to activate the proguanylins or proSTs, Bacterial STs were the first peptides but these important enzymes have not been shown to activate GC-receptor signaling identified thus far. molecules in the intestine, thus causing se- cretory diarrhea (4). Moreover, the intestinal Physiological actions of guanylin GC targets for ST peptides in the intestine peptides were considered unique and not existing outside the intestinal epithelium until we Physiological actions of the endogenous discovered that E. coli ST also stimulates guanylin peptides include the regulation of GC-receptors found on the surface of OK intestinal fluid secretion during digestion, and potoroo kidney (PtK-2) cell lines (Fig- and neutralization of HCl in the duodenum ure 1; Ref. 16). It can be seen from one of our and of organic acids derived from enteric original experiments that these two kidney bacteria in the large intestine (9,10). Guany- cell lines have remarkable cGMP responses lins and STs stimulate the electrogenic se- to E. coli ST, but only small cGMP re- cretion of both chloride and bicarbonate an- sponses to atriopeptin-A and no detectable ions, which provides the physiological driv- responses to the nitric oxide donor, sodium ing force to accomplish fluid secretion into nitroprusside. The additional demonstration the intestinal lumen. Control of these intesti- that kidney cortex has specific GC-receptors nal functions by guanylin peptides is medi- that are activated by ST implies that this ated via intracellular cGMP through activa- class of cell surface GC-receptors plays a tion of cGMP-dependent protein kinase II much broader physiological role in the body (cG-kinase II) and/or cAMP-dependent pro- than was previously recognized (17-19). It tein kinase II (cA-kinase II) with subsequent should be emphasized that these findings phosphorylation of the cystic fibrosis trans- were made well before the first guanylin membrane conductance regulator (CFTR) peptides were isolated (1,2). Putative ST Braz J Med Biol Res 32(11) 1999 Guanylin peptides and cGMP 1331 receptors were also found in other epithelia nylin are also found in the circulation and it of the opossum in addition to the receptors is likely that the gastrointestinal (GI) tract is localized to brush border membranes (BBM) a main source of the plasma peptides (7,33, of epithelial cells lining the intestinal tract 34). Secretion of uroguanylin from GI mu- and within renal tubules (16-19). The exist- cosa into the plasma in response to oral NaCl ence of renal, hepatic, airway and testicular may explain the prolonged increase in uri- ST receptors predicted that endogenous nary sodium excretion that occurs following ST-like peptides exist to regulate the activity a high salt meal (26,27). of the extra-intestinal as well as intestinal GC-receptors. These seminal experiments Identification of a kidney led directly to the subsequent isolation of GC-receptor for uroguanylin guanylin and uroguanylin peptides from in- testine and urine, respectively (1,2). Both We sought to elucidate the primary struc- guanylin and uroguanylin are produced in ture of a membrane GC expressed in cul- the intestine, but uroguanylin is the major tured OK cells and in the opossum kidney bioactive peptide found in urine, which con- because the prior discovery of this renal GC- tains either no guanylin or very small a- receptor was a stimulus that ultimately led to mounts of the peptide (2,20-22). Uroguany- the isolation of guanylin and uroguanylin lin and ST stimulate the enzymatic activity (1,2,16-19). A PCR-based cloning strategy of renal tubular GC-receptors and increase was used to isolate 3762-bp cDNAs from the urinary excretion of sodium, potassium RNA/cDNAs expressed in OK cells and and water in both the perfused rat kidney ex opossum kidney cortex (35,36). Transfec- vivo and the mouse in vivo (16-19,23-25). tion and expression of the OK-guanylate Guanylin is less potent in the stimulation of cyclase (OK-GC) cDNA into COS and urinary Na+ and water excretion compared HEK293 cells produces a cell surface GC- to either uroguanylin or ST, but guanylin receptor of ~160 kDa size that is activated by does have marked kaliuretic activity in the uroguanylin, guanylin and E. coli ST pep- perfused rat kidney (24). Thus, uroguanylin tides. OK-GC cDNA contains an open read- has biological activity consistent with a pep- ing frame encoding a 1049 residue mature tide hormone that influences renal function by regulating the urinary excretion of so- 70000 13000 dium chloride as a physiological mechanism OK PtK-2 12000 that contributes to the maintenance of Na+ 60000 11000 balance in the body. A natriuretic peptide 50000 10000 cells) cells) such as uroguanylin was predicted to exist in 6 6 9000 the digestive system for release into the blood- 40000 8000 stream for the purpose of stimulating the 30000 7000 urinary excretion of NaCl following a salty 20000 6000 meal (26,27).