GUANYLIN AND UROGUANYLIN: A PROMISING NEXUS
IN INTESTINAL ELECTROLYTE AND FLUID HOMEOSTASIS

There are a group of proteins under the family guanylin peptides (GPs) that include guanylin (GN), uroguanylin (UGN), lymphoguanylin and renoguanylin (1). GN is a low molecular weight, heat-stable peptide first identified in rat jejunal extracts (2), while UGN was identified initially from opossum urine in the year 1993 (3). Both GN and UGN are secreted by the enteroendocrine cells of the intestinal mucosa. They are involved in the regulation of intestinal fluid homeostasis and maintenance of gut physiology. Salty meal increases the secretion of GN and UGN that inhibit sodium absorption followed by anion and water secretion. Later, they are absorbed into blood from the intestinal surface and transported to the kidney, resulting in hypernatremia and hypervolemia by promoting natriuresis, kaliuresis, and dieresis (4).

The intestine is the primary site for production of GPs. GN mostly occurs in the distal small intestine and colorectum, while UGN in the duodenum and proximal jejunum (5). Apart from these, the peptides are also found in the horses (6), mice (7), ells (8), opossum and bony fishes (9). However, the extraintestinal tissues like adrenal glands, reproductive organs, lungs, and pancreas can produce GPs that act through guanylate cyclase-C (GC-C). Interestingly, only the enterochromaffin cells of the digestive system produce GN and UGN (10, 11). The intestinal expression of UGN is also regulated by nutritional status and several hormones, including leptin, insulin and sexual hormones (12-14). In addition, dietary zinc appears to be a key regulator hor expression of GPs (15). The binding of GPs with receptors increases intracellular cGMP levels. Moreover, GPs can activate guanylate cyclase A and G-protein coupled receptors. The functions of GPs in the intestine and kidney are well established, but their roles in other tissues (liver, pancreas, lung, sweat glands) are still unclear. The intestinal GN and UGN bind to the cell membrane-bound GC-C and increase intracellular cyclic guanosine monophosphate (cGMP) levels in the enterocytes that activate the secretion of Cl– and HCO3– through the stimulation of cystic fibrosis transmembrane conductance regulator (CFTR) channels to maintain the salt balance in the intestinal lumen. On the other hand, GN and UGN decrease intestinal Na+ absorption by inhibiting the function of Na+/H+ exchanger, leading to retention and secretion of water from the intestine (16, 17). Moreover, UGN enhances salt and water loss from the kidney (18). GN and UGN are involved in the regulation of ion homeostasis and prevent hypernatremia after ingestion of salty foods. Although GN and UGN have a role in the prevention of hypernatremia after ingestion of salty foods, it has been hypothesized that low intestinal expression of proguanylin and prouroguanylin in murine models of obesity may contribute to an increase intestinal sodium absorption, thus favouring the development of obesity-associated hypertension (19). Deregulation of cGMP signaling is associated with several intestinal pathogeneses, viz. bowel transit disorders, inflammatory bowel disease, meconium ileus and colorectal cancer (20-26). Additionally, GN and UGN get associated with the development of extraintestinal diseases, including cystic fibrosis, asthma, kidney and heart diseases, obesity, and metabolic disorders (1). Like intestine, GN and UGN are also linked to water and electrolyte transport across the renal collecting ducts by GC-C independent mechanism, but phosphatase A2 and arachidonic acid dependent signaling pathway (27, 28). The present review has focused on the contribution of GN and UGN in intestinal physiology. Special emphasis has also been given to targeting the GC-C system for therapeutic purposes in order to prevent the intestinal disorders.

AN OVERVIEW OF THE STRUCTURE
OF GUANYLIN PEPTIDES

Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP)), and intestinal GN and UGN belong to the same class of natriuretic peptides. The genes of these natriuretic peptides are present in chromosome 1 in human and chromosome 4 in mouse, respectively (29). The genes of GN and UGN are Guca2a and Guca2b (30). Biochemically, GN and UGN are the class of peptide hormones that are synthesized as pre-pro-hormones (7, 31). The pre-pro form of GN contains 115 amino acids. Proteolytic cleavage produces pro-GN (94 amino acids) and mature GN (15 amino acids) (32). Similarly, human pre- pro-, and mature UGN comprise 112, 86 and 16 amino acids, respectively (33, 34). Hess et al. (35) reported that the circulating form of UGN carries 24 amino acids. On the other hand, heat-stable enterotoxin of Escherichia coli (STa; 19 amino acids) structurally resembles GN and UGN. Each of these peptides contains multiple disulfide bonds; GN and UGN contain two (between 4 – 12 and 7 – 15 cystein residues) while STa has three disulfide bonds (1, 36, 37). These disulfide bonds essentially control their activities (34, 38, 39).

N-terminal glutamate and aspartate of UGN act as pH sensor. Acidic pH of the upper intestine potentially increases the activity of UGN; it may due to the presence of two aspartate residues at the N-terminal end. Acidic pH influences the pKR (3.65) of an aspartate, leading to a conformational change in protein structure. This property improves the ligand-binding capacity of UGN. Experimental data reveal that pH 5.0 enhances the ligand-receptor affinity up to 100-fold compared to pH 8.0 (36, 40). However, GN does not contain aspartate residue at its N-terminal. The expression of GN occurs in the neutral pH or slightly alkaline environment of the lower intestine. These characteristics indicate the expression pattern of the GPs; UGN is expressed in the acidic environment of the duodenum, while GN expression happens to occur in the colonic area (5). GPs can be used for the treatment of gastrointestinal disorders. Synthetic GPs like linaclotide, plecanatide and dolcanatide have promising effects in the treatment of inflammation-mediated intestinal diorders (41, 42). Synthesis of these synthetic GPs is facing a problem due to the presence of multiple cysteine and disulfide bonds. The mutant form of GP contains less number of disulfide bonds, as the cysteine residue at the C-terminal is replaced by tyrosine residue and this mutant GP shows decreased level of binding-affinity and activation property of the GC-C (43).

A recent single-cell sequencing study has revealed that the genes encoding human proguanylin (GUCA2A) and prouroguanylin (GUCA2B) are mainly expressed in the BEST4 CFTR high-expressor (BCHE) cells of the small intestine (44). The genes of GN and UGN carry three exons and two introns (45, 46). The three exons of mature mRNA produce 115 and 112 amino acids for GN and UGN, respectively. Cleavage of the first 21 (GN) and 26 (UGN) amino acids from the N-terminal give pro-hormones. Finally, amino acid residues 101-115 (GN) and 97-112 (UGN) occur as short peptides (1). The amino acid sequences of GN and UGN of humans, murine, and porcine are almost similar. The second amino acid of GN of humans, mice, and pigs is glycine, aspargine and serine, respectively. However, the 13th amino acid of porcine is alanine instead of threonine in human and murine. The first (Asn→Gly) and last (Leu→Ser) differ in the sequences of human and porcine UGN (1).

The goblet cells, Paneth cells, colonic Paneth-like cells, tuft cells, epithelial cells and somatostatin secreting D-cells of the small intestine and colonic mucosa secrete GN (7, 30, 47-50). Intestinal enterochromaffin cells and intestinal epithelial cells express UGN (7, 11). Salt and intestinal pH are the primary regulators of the expression of GN and UGN in the intestine. Slightly acidic environment of the duodenum and proximal jejunum influences the release of UGN (40), while GN appears in neutral to slightly basic pH of the ileum and colorectal area (31, 40, 51). A high-salt diet increases the expression of GN and UGN (49, 52); while salt restriction reduces GN and UGN levels (47, 53). The expression of GN and UGN is Zn2+ dependent. The deficiency of Zn2+ promotes secretory diarrhea. Dietary zinc deficiency in rats increases UGN mRNA levels in both intestine and kidney that may relate to the fluid imbalance associated with this state (54, 55).

GUANYLATE CYCLASE-C DEPENDENT
SIGNALING PATHWAY

GN, UGN, and STa induce GC-C-mediated production of cGMP. The enzyme has been found in the intestine and colon of mammals, including humans. STa-specific GC-C is also present in extraintestinal tissues like the gall bladder, trachea, testis, kidney and adrenal gland (56). Cytoplasmic GC-C receptor is also expressed in the adipose tissue (13). The location and characteristics of GCs vary among the different tissues. Cytoplasmic GC is a soluble receptor, which is present in the skeletal muscle, platelets, lung, liver, kidney, heart and central nervous system (CNS). Its activation is mediated by nitric oxide (NO). Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) bind to GC-A, which is present in the smooth muscle, kidney, adrenal gland, heart and CNS. GC-B has a high affinity for C-type natriuretic peptides (CNP). GC-B is mostly present in fibroblasts, heart, CNS and others. But GC-C is restricted in the gastrointestinal tract (1). GC-D of the olfactory cells can also interact with GN and UGN (57). Thus, different GCs occur in various tissues of the body, but the relaxation of human umbilical arteries and veins does not fully GC-dependent. cAMP and cGMP pathways have partial effects. Avoidance of GC-dependent relaxation maintains the fetal circulation and prevents the shifting of blood to the umbilical vessels. These effects are essential to complete the term of the pregnancy. However, the contractility of the umbilical vessels can change at the last trimester of the pregnancy (58).

GC-C are homodimeric receptors that commonly occur on the apical brush borders of intestinal epithelial cells. They are distributed from the duodenum to the rectum. Each of these receptors has extracellular, transmembrane and cytosolic domains. The extracellular domain is present at the luminal face of the intestine and binds to the ligand. The cytosolic domain carries the kinase homology domain and catalytic domain (37, 59). The transmembrane domain bears close similarity with the structure of insulin-like growth factor (IGF) and epidermal growth factor (EGF) receptors (60). The kinase homology domain phosphorylates the receptor subunit for its activity, whereas the catalytic domain is involved in the synthesis of cGMP after cyclization from GTP. The C-terminal of each receptor interacts with the cytoskeleton proteins for its proper positioning (59). The catalytic activity of the GC-C is Mg2+-ATP dependent (61) and its activity becomes enhanced by protein kinase C (PKC) induced phosphorylation at its C-terminal (62).

MECHANISM OF ACTION
OF GUANYLIN RELATED PEPTIDES

Human GN and UGN are known as guanylate cyclase-activating peptide (GCAP)-1 and GCAP-2, respectively. GPs activate the membrane-bound GC-C at the apical side of the enterocytes. GC utilizes guanosine triphosphate (GTP) as a substrate for the synthesis of cGMP, which acts as a second messenger and stimulates the protein kinase G II (PKGII) (59, 63, 64). There are two isoforms of PKG, viz. PKGI and PKGII. In fact, PKGI is specific for the smooth muscle and regulates the contractility of the intestine (65). On the other hand, PKGII predominantly occurs in the intestinal epithelial cells that are involved in the regulation of luminal electrolytes and fluid secretion (66).

PKGs belong to the serine/threonine class of protein kinases, each comprising N-terminal, regulatory and catalytic domains. The N-terminal domain is responsible for homodimerization, autoinhibition, and subcellular localization. Two cGMP molecules bind to a specific site of the regulatory domain. The catalytic domain has two pockets for the binding of ATP and substrate (67). In the absence of cGMP, the N-terminal autoinhibitory site blocks the activity of the catalytic domain. The binding of cGMP at the regulatory domain makes a conformational change in the PKG that blocks the activity of the N-terminal inhibitory site and releases the catalytic domain for kinase activity (68).

A specific intracellular enzyme phosphodiesterase (PDE) degrades cyclic adenosine monophosphate (cAMP) and cGMP to terminate the cAMP/cGMP-mediated functions. There are several types of PDEs that are involved in the degradation of cGMP and cAMP to 5’GMP and 5’AMP, respectively. The activities of PDEs do not follow the uniform pattern. PDE-4,-7, and -8 specifically cleave cAMP, while PDE-5, -6, and -9 utilize cGMP as the substrate. A group of PDEs (PDE-1, -2, -3, -10, and -11) can hydrolyze both cAMP and cGMP (69, 70). Intestinal epithelial cells express PDE-1, -2, -3, -5, -9, and -10 (69, 71, 72). cGMP inhibits the activity of PDE 3 after binding to the regulatory site of the PDE (68). This inhibition indirectly increases the intracellular cAMP levels along with cGMP concentration. cAMP activates cAMP-dependent protein kinase A (PKA) for cellular functions. cGMP-induced PKGII and PKA activation regulates multiple physiological functions (37). The binding affinity of STa is much stronger than GN and UGN due to the presence of three disulfide bonds. The stronger binding capacity of STa increases the excess loss of electrolytes and water from the intestinal lumen leading to the onset of secretory diarrhea (73-76).

FUNCTIONS OF GUANYLIN AND UROGUANYLIN

Intestinal guanylin and uroguanylin are the endogenous paracrine hormones that primarily stimulate cell membranous GC-C receptor. They bring about intestinal fluid and ion homeostasis, associated with the maintenance of the intestinal barrier, inhibition of inflammation, visceral pain and regulation of tumourigenesis (Fig. 1) (5, 77-79).

Fig. 1. Schematic representation of guanylin and uroguanylin-induced and cGMP/PKGII-mediated multiple physiological functions in intestinal tract.
↑: Increase; ↓: Decrease; (–): Inhibition; IEC: intestinal epithelial cell; GN: guanylin; UGN: uroguanylin; STa: heat-stable enterotoxin of Escherichia coli; PKGII: protein kinase GII; PDE: phosphodiesterases; PKA: protein kinase A; TJ: tight junction; AJ: adherens junctions; MMP: matrix metalloproteinases; β-cat; beta cathenin; TCF: T-cell factor; PTEN: phosphase and tensin homolog; MLCK: myosin light chain kinase; Akt: protein kinase B; TNF-α: tumor necrosis factor-alpha; MRP4: multi-drug resistance protein 4; SP1: specificity protein 1.

Effect on intestinal electrolytes and fluid homeostasis

GN and UGN are involved in the regulation of pH balance and electrolytes transport in the intestinal lumen. Regulation of intestinal pH helps in the digestion and absorption of foods (80). The activation by GPs is pH-dependent, and low pH levels practically increase the intracellular cGMP concentration. The release of stomach content to the duodenum not only stimulates pancreatic bicarbonate secretion, but also increases UGN-induced cGMP accumulation in the duodenum. PKG II enhances cystic fibrosis transmembrane conductance regulator (CFTR) activity for the efflux of bicarbonate and Cl– into the intestinal lumen (68). However, GPs can control bicarbonate transport even in CFTR-deficient mice (81). PKG II also inhibits PDE 3 functions, resulting in an inhibition of cAMP degradation with concomitant activation of PKA, which in turn stimulates CFTR for the secretion of bicarbonate (82). CFTR is an ATP-gated anion channel. Slc26 family protein is another bicarbonate transporter. Both CFTR and Slc26 family transporter cooperatively maintain the bicarbonate secretion. There are 11 members in the Slc26 family; they can transport sulfate, iodide, formate, oxalate, hydroxyl ion, bicarbonate, and Cl– ions. In pancreas and digestive tract Slc26a3 (DRA), Slc26a6 (PAT-1, CFEX (Cl–/formate exchange)), and Slc26a9 are involved in the bicarbonate secretion (83). Each Slc26 polypeptide contains N-terminal cytoplasmic domains, 10–14 hydrophobic transmembrane spans and C-terminal cytoplasmic sulfate transporter anti-sigma factor antagonist (STAS) domains. The homo-oligomeric STAS domains regulate the cell surface expression of these proteins. They are also involved in the regulation of the CFTR (83). PKA-dependent phosphorylation of the regulatory domain of CFTR promotes its binding to the STAS domains of Slc26 transporters. This interaction synergistically activates the CFTR and the Slc26 transporters for bicarbonate secretion and Cl– absorption in the intestinal lumen (84). The binding of Slc26 transporters and CFTR are mediated by post-synaptic density 95/discs large/zona occludens 1 (PDZ) domains, which act as the protein-protein interaction motifs to make the functional state of many proteins, including CFTR and Slc26 (85).

On the contrary, PKGII inhibits the activity of sodium-hydrogen ion exchanger type 2/3 (NHE2/3) at the apical side of the duodenum. Inhibition of NHE2 lowers the H+ secretion into the intestinal lumen (68, 86). Thus, cGMP-dependent inhibition of NHE2/3 and CFTR-mediated increased bicarbonate secretion regulate the pH of the duodenum and thereby prevent hypernatremic effects of blood. These effects also hold the electrolytes and water into the intestinal lumen and restrict the rapid absorption of water as well as a sudden increase in blood volume (76, 87, 88). cGMP-mediated fluid secretion maintains the mucus hydration state and regulates intestinal pH; these effects prevent the colonization of the pathogenic bacteria over the surfaces of the intestinal cells (89, 90). Impairment in cGMP signaling causes the onset of diarrheal disease. Familial GC-C diarrhea syndrome (FGDS) is a common example in this context. Mutation in the catalytic domain of GC-C promotes the overproduction of cGMP in the presence of ligand, leading to the advancement of secretory diarrhea (20, 91, 92). It is evident that enterotoxigenic strains of E. coli can secret STa, which acts as a GC-C agonist and induces diarrhea (93).

Effects on epithelial cell proliferation

cGMP and PKGII restrict unwanted intestinal cell proliferation by arresting the cell cycle. In vitro studies have revealed that GC-C agonists like STa and UGN, 8-Br-cGMP, and the PDE inhibitor (zaprinast) can arrest the cell cycle (94). Alternatively, the inactivation of GC-C activity accelerates epithelial cell proliferation by promoting the activity of the G1/S checkpoint of the cell cycle (95). The progress of the cell cycle is influenced by pRb, CDK4, cyclinD1, β-catenin, while p27 downregulates the cell cycle (96). cGMP agonist or PDE inhibitor advances the activity of p21 and p27 to restrict the G1/S checkpoint (96-98). Moreover, PKG II increases the expression of p21 and p27 by recruiting the transcription factor specificity protein 1 (SP1) (98). Cell culture studies also reveal that inhibition of PDE5 and PDE10 elevates cGMP level (99, 100). Accumulation of cGMP directly inhibits the β-catenin/TCF (T-cell factor)-dependent proliferative activity (101). Kwon et al. (102) have reported that PKGII decreases β-catenin/TCF transcription by modulating the activity of c-Jun N-terminal kinase (JNK) and forkhead box O transcription factor 4 (FoxO4). PKGII can also suppress JNK release (103). cGMP modulates the activity of PTEN (phosphatase and tensin homolog) to inhibit the activity of Akt (protein kinase B) (96). Besides these, cGMP recruits calcium-sensing G-protein coupled receptor to the membrane, which functions as a cGMP-gated channel for the cellular entry of calcium ions. This effect opposes cell proliferation (104). Thus, cGMP exerts a complex regulatory mechanism over the cell cycle inducing and suppressing factors to control proliferation as well as renewed of the intestinal epithelial cells.

Effects on genetic stability and DNA repair system

cGMP signaling inhibits DNA double-strand breakage, abnormal mitotic spindle formation, chromosomal instability and to the contrary, cGMP pathway activates p53 and promotes DNA damage repair. Inappropriate function of GC-C induces DNA double-strand breakage and oxidative damage of DNA (105, 106). GC-C regulates p53 activity during DNA damage, which in turn triggers the DNA repair system to restore DNA integrity. Supplementation of GC-C agonist STa prevents radiation-induced gastrointestinal damage in mice (107). It has been suggested that cGMP signaling maintains genomic stability and metabolic activities, restricts reactive oxygen species generation and oxidative DNA damage in both in vivo and in vitro experiments (96). Deletion of GC-C expression-inducing intestinal transcription factor CDX2 advances the chromosomal aberrations by triggering the formation of the impaired anaphase-promoting complex (108, 109). Thus, cGMP prevents chromosomal instability, DNA damage, and abnormal mitosis leading to avoidance of abnormal cell growth and tumor formation in the intestinal wall.

Effects on intestinal barrier integrity and inflammatory responses

cGMP signaling regulates intestinal inflammatory responses and preserves intestinal barrier integrity. The disintegration of the intestinal barrier and chronic inflammation can cause the development of inflammatory bowel disease (IBD) irritable bowel syndrome (IBS), and sepsis; a mutation in the GC-C influences IBD (20, 21). Impaired GC-C function promotes the myosin light chain-mediated disassembly of tight junction that alters the intestinal permeability (110). The GC-C deficient (GC-C–/–) mice bring about decreases in the expression of junctional proteins like occludin, claudin-2, claudin-4 and JAMA, leading to the loss of intestinal barrier integrity. Similarly, inappropriate GC-C activity blocks the cGMP-mediated opposing effect on Akt, resulting in aberrant Akt signaling and the alteration of junctional protein expression (106). GC-C/cGMP signaling also influences the intestinal barrier maintenance by triggering the action of the transcription factor FoxO3a. Application of the GC-C agonist (8Br-cGMP) or the PDE5 inhibitor (vardenafil) in colitis mouse model inhibits Akt signaling, followed by activation of FoxO3a in order to enhance the intestinal barrier integrity (111).

Deletion of GC-C in mice alters the cytokine profile by inducing the expression of pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) (106, 110, 112). Moreover, in vitro studies reveal that colonocytes with GC-C–/– express a higher amount of TNF-α after induction of lipopolysaccharide (112). Administration of GC-C agonist plecanatide or PDE5 inhibitor sildenafil in the dextran sodium sulfate-induced intestinal inflammation study in mice showed a protective result against inflammatory responses by decreasing the recruitment of immune cells and the expression of pro-inflammatory cytokines (41, 113, 114). Thus, cGMP signaling essentially regulates the intestinal barrier integrity and prevents intestinal inflammatory responses to maintain the normal functions of the intestine.

Other effects

cGMP inhibits the expression of matrix metalloproteinases (115) and suppresses the epithelial-mesenchymal transition. Collectively, cGMP blocks cancer cell migration, invasion, and metastasis (115-117). Zuzga et al. (116) have reported that loss of cGMP increases the tumor cell migration. Thus, cGMP signaling inhibits matrix remodeling and cancer cell metastasis.

Although cGMP is an intracellular second messenger, it can also act as an extracellular signaling molecule and exerts paracrine effects. cGMP accumulates in the intracellular compartment and then releases into the extracellular environment through the membrane anion channel and multidrug resistance protein 4 (77, 118, 119). Extracellular cGMP interacts with the visceral nociceptive neurons and exhibits analgesic activity. Due to this property, cGMP can be used for the treatment of pain during IBD and other intestinal disorders (77, 118, 120).

GPs may regulate the food intake, obesity and development of colon carcinoma. In fact, GN and UGN exhibit their anti-obesity actions (121). UGN binds to its receptor GC-C in hypothalamic neurons containing anorexigenic peptide pro-opiomelanocortin (POMC), thereby decreasing appetite (122). Moreover, UGN increases energy expenditure through the activation of brown adipose tissue and browning in the white adipose tissue in murine models (123, 124). In humans, both GN and UGN activate lipolysis in visceral adipose tissue (13). GPs indirectly regulate lipolysis. They inhibit the PDE 3 in adipocytes and increase intracellular cAMP level that is the crucial factor for activation of adenosine monophosphate-activated protein kinase (AMPK) and lypolysis (125). Cell culture study of differentiated 3T3-l1 adipocytes reveals that 3-iodothyronamine induces phosphorylation of AMPK and nuclear localization of forkhead box O1 (FoxO1). At the downstream level these factors upregulate the expression of adipose triglyceride lipase, monoacylglycerol lipase and carnitine palmitoyltransferase 1, while downregulate the expression of acetyl-CoA carboxylase. Collectively, these enzymes enhance lipolytic action, mobilization of free fatty acids and glycerol, and increase the mitochondrial degradation of fatty acids (126). Importantly, circulating levels of prouroguanylin are decreased in children, adolescent and adult people with obesity (13, 127, 128, 129) as well as in experimental models of obesity (130), pointing to crucial role of UGN in obesity. Intestinal cell culture study exhibits the anti-proliferative effects of GPs. Proper expression of GPs protects colonic carcinogenesis (4). Moreover, dysfunction in GC-C/PKGII promotes colon cancer, and administration of GC-C agonists protects the progress of colorectal cancer (68, 131).

THERAPEUTIC IMPORTANCE OF GC-C ACTIVATORS

GPs or their analogs are becoming the attractive choice for the treatment of functional gastrointestinal disorders viz. chronic idiopathic constipation and IBD with or without constipation. The hyperactivity of GC-C in combating IBD is evident in humans and mice models (132). This property can be targeted for the treatment of constipation syndromes (5, 133). Therapeutic GC-C agonists linaclotide (134, 135) and plecanatide (136, 137) have got necessary clinical approval for the treatment of chronic idiopathic constipation and constipation-predominant IBD. GC-C agonist linaclotide also exerts a beneficial effect in whole body metabolism. Oral administration of linaclotide in mice with diet-induced obesity reduces body weight by activation of the sympathetic nervous system to trigger thermogenesis in brown adipose tissue, thereby stimulating energy expenditure (138). UGN analog (dolcanatide) has shown anti-inflammatory effects in the intestine of rodent models.

Linaclotide is a synthetic analog of E. coli enterotoxin STa, containing 14 amino acids. Structurally, it is slightly different from its parent compound. A single residue of leucine is substituted by tyrosine in its pharmacophore. Like STa, linaclotide has three disulfide bonds and acts as a GC-C agonist. Linaclotide is a hybrid design of STa, UGN, and GN. Its three disulfide bonds occur between CysI-CysIV (amino acids 1→6), CysII-CysV (amino acids 2→10), and CysIII-CysVI (amino acids 5→13) (139). The arrangement of these three disulfide bonds stabilizes three β-turns and holds the molecules into their active form. These structural features enhance the stability of the synthetic molecule compared to the endogenous peptides but the binding affinity to the GC-C remains the same (140, 141). Linaclotide is given orally to the patients during the clinical trial. There is a possibility of breakdown of a orally administered peptides into the gut due to the presence of protease enzymes, resulting in the limitation of therapeutic use for intestinal diseases. Linaclotide can tolerate gastric acidity and remains stable for up to 3 hours in the intestinal environment to elicit the proper response. It gives GC-C-active metabolite MM-419447 after cleavage of C-terminal Tyr, which drives its therapeutic activity (142, 143). Linaclotide shows the same affinity as STa for the GC-C and activates the cGMP-PKGII pathway (140, 142). It induces intestinal fluid and electrolyte secretion that makes a hydrated environment in the colorectal area and relieves the symptoms of constipation. This synthetic analog also exhibits analgesic properties and mitigates visceral pain (118, 142). The results of the clinical trials have revealed that linaclotide maintains spontaneous bowel movements and stool consistency that diminish abdominal discomfort (144).

Plecanatide is the synthetic analog of uroguanylin. Like uroguanylin, plecanatide also contains 16 amino acids but the aspartate in the third position from the N terminus is replaced by glutamate. Plecanatide is not absorbed systemically. This peptide shows similar therapeutic effects to linaclotide, but the activity starts from the duodenum in slightly acidic environments (145).

In summary, GN and UGN are the endogenous peptide hormones that trigger GC-C-cGMP signaling pathway for the regulation of intestinal fluid and electrolyte homeostasis. They are the prime regulators of CFTR channels and Na+/H+ exchangers, but can also modulate epithelial cell proliferation, DNA integrity, intestinal barrier function, inflammation, gut microbiome interaction and cell migration. Overstimulation of this pathway causes secretory diarrhea. However, improper activation promotes constipation, dysbiosis, chronic idiopathic constipation, IBD and colorectal cancer. Pharmacologically, they have several positive impacts to control intestinal disorders, including pain. As the GPs regulate the cell cycle, DNA instability, DNA repair, cell proliferation, extracellular matrix composition, cell junction, and cell invasiveness, they can be used as anti-tumor agents for the prevention of colorectal cancer. Many synthetic peptides (linaclotide, plecanatide, and dolcanatide) are developed as therapeutic agents. Linaclotide and plecanatide are now in the clinical trials for the treatment of functional gastrointestinal disorders and IBD. Thus, the integrative research on biochemical and physiological aspects of GN and UGN, and a more clinical trial will change the scenario of treatment pattern of many gastrointestinal diseases.

Authors’ contribution: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

Acknowledgements: The authors are grateful to the Principal of Midnapore College, Midnapore, West Bengal, India, and the Principal of Vidyasagar College, Kolkata, India for providing all kinds of facilities to prepare this manuscript.

Funding: No financial grant was available. This review article was self-supported by the authors.

Conflict of interests: None declared.

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