Fibroblast growth factor-21 regulates PPARγ activity and the antidiabetic actions of thiazolidinediones.

Fibroblast growth factor-21 (FGF21) is a circulating hepatokine that beneficially affects carbohydrate and lipid metabolism. Here, we report that FGF21 is also an inducible, fed-state autocrine factor in adipose tissue that functions in a feed-forward loop to regulate the activity of peroxisome proliferator-activated receptor γ (PPARγ), a master transcriptional regulator of adipogenesis. FGF21 knockout (KO) mice display defects in PPARγ signaling including decreased body fat and attenuation of PPARγ-dependent gene expression. Moreover, FGF21-KO mice are refractory to both the beneficial insulin-sensitizing effects and the detrimental weight gain and edema side effects of the PPARγ agonist rosiglitazone. This loss of function in FGF21-KO mice is coincident with a marked increase in the sumoylation of PPARγ, which reduces its transcriptional activity. Adding back FGF21 prevents sumoylation and restores PPARγ activity. Collectively, these results reveal FGF21 as a key mediator of the physiologic and pharmacologic actions of PPARγ.

Molecular Basis of Bile Acid-FXR-FGF15/19 Signaling Axis.

Bile acids (BAs) are a group of amphiphilic molecules consisting of a rigid steroid core attached to a hydroxyl group with a varying number, position, and orientation, and a hydrophilic side chain. While BAs act as detergents to solubilize lipophilic nutrients in the small intestine during digestion and absorption, they also act as hormones. Farnesoid X receptor (FXR) is a nuclear receptor that forms a heterodimer with retinoid X receptor α (RXRα), is activated by BAs in the enterohepatic circulation reabsorbed via transporters in the ileum and the colon, and plays a critical role in regulating gene expression involved in cholesterol, BA, and lipid metabolism in the liver. The FXR/RXRα heterodimer also exists in the distal ileum and regulates production of fibroblast growth factor (FGF) 15/FGF19, a hormone traveling via the enterohepatic circulation that activates hepatic FGF receptor 4 (FGFR4)-ß-klotho receptor complex and regulates gene expression involved in cholesterol, BA, and lipid metabolism, as well as those regulating cell proliferation. Agonists for FXR and analogs for FGF15/19 are currently recognized as a promising therapeutic target for metabolic syndrome and cholestatic diseases.

PPARγ-K107 SUMOylation regulates insulin sensitivity but not adiposity in mice.

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is a master regulator of adipocyte differentiation and is the target for the insulin-sensitizing thiazolidinedione (TZD) drugs used to treat type 2 diabetes. In cell-based in vitro studies, the transcriptional activity of PPARγ is inhibited by covalent attachment of small ubiquitin-related modifier (SUMOylation) at K107 in its N terminus. However, whether this posttranslational modification is relevant in vivo remains unclear. Here, using mice homozygous for a mutation (K107R) that prevents SUMOylation at this position, we demonstrate that PPARγ is SUMOylated at K107 in white adipose tissue. We further show that in the context of diet-induced obesity PPARγ-K107R-mutant mice have enhanced insulin sensitivity without the corresponding increase in adiposity that typically accompanies PPARγ activation by TZDs. Accordingly, the PPARγ-K107R mutation was weaker than TZD treatment in stimulating adipocyte differentiation in vitro. Moreover, we found that both the basal and TZD-dependent transcriptomes of inguinal and epididymal white adipose tissue depots were markedly altered in the K107R-mutant mice. We conclude that PPARγ SUMOylation at K107 is physiologically relevant and may serve as a pharmacologic target for uncoupling PPARγ's beneficial insulin-sensitizing effect from its adverse effect of weight gain.

Hepatic uptake of conjugated bile acids is mediated by both sodium taurocholate cotransporting polypeptide and organic anion transporting polypeptides and modulated by intestinal sensing of plasma bile acid levels in mice.

The Na+ -taurocholate cotransporting polypeptide (NTCP/SLC10A1) is believed to be pivotal for hepatic uptake of conjugated bile acids. However, plasma bile acid levels are normal in a subset of NTCP knockout mice and in mice treated with myrcludex B, a specific NTCP inhibitor. Here, we elucidated which transport proteins mediate the hepatic uptake of conjugated bile acids and demonstrated intestinal sensing of elevated bile acid levels in plasma in mice. Mice or healthy volunteers were treated with myrcludex B. Hepatic bile acid uptake kinetics were determined in wild-type (WT), organic anion transporting polypeptide (OATP) knockout mice (lacking Slco1a/1b isoforms), and human OATP1B1-transgenic mice. Effects of fibroblast growth factor 19 (FGF19) on hepatic transporter mRNA levels were assessed in rat hepatoma cells and in mice by peptide injection or adeno-associated virus-mediated overexpression. NTCP inhibition using myrcludex B had only moderate effects on bile acid kinetics in WT mice, but completely inhibited active transport of conjugated bile acid species in OATP knockout mice. Cholesterol 7α-hydroxylase Cyp7a1 expression was strongly down-regulated upon prolonged inhibition of hepatic uptake of conjugated bile acids. Fgf15 (mouse counterpart of FGF19) expression was induced in hypercholanemic OATP and NTCP knockout mice, as well as in myrcludex B-treated cholestatic mice, whereas plasma FGF19 was not induced in humans treated with myrcludex B. Fgf15/FGF19 expression was induced in polarized human enterocyte-models and mouse organoids by basolateral incubation with a high concentration (1 mM) of conjugated bile acids. CONCLUSION: NTCP and OATPs contribute to hepatic uptake of conjugated bile acids in mice, whereas the predominant uptake in humans is NTCP mediated. Enterocytes sense highly elevated levels of (conjugated) bile acids in the systemic circulation to induce FGF15/19, which modulates hepatic bile acid synthesis and uptake. (Hepatology 2017;66:1631-1643).

Genomic and expression analysis of canine calcitonin receptor-stimulating peptides and calcitonin/calcitonin gene-related peptide.

Calcitonin receptor-stimulating peptides (CRSPs) are new members of the calcitonin/calcitonin gene-related peptide (CT/CGRP) family identified in pigs, dogs and other domestic animals, and CRSP-1 is an active ligand for the CT receptor (CT-R). We recently sequenced porcine CRSP genes (Crsps) and found similarity with the CT/CGRP gene (Ct/Cgrp) in sequence and genomic organization. In this study, we identified five Crsps, Crsp-1 to Crsp-5, in dogs. Crsp-1 has five exons with an exon-intron organization identical to that of porcine Crsp-1 or Crsp-2, while Crsp-2 and Crsp-3 have additional CT-2- and CT-3-coding exons like Ct/Cgrp. Crsp-2 was renamed as Ct-2/Crsp-2 because both CRSP-2 and CT-2 mRNAs were tissue-specifically expressed. Crsp-4 and Crsp-5 are presumably generated by retrotransposition. We postulate that Crsps were generated from the gene duplication of Ct/Cgrp, and gained their diversity during mammalian evolution. Among the canine CTs and CRSPs, CRSP-1, CT-1 and CT-2 are active ligands for the CT-R, but CRSP-2 and others are inactive. Canine CRSP-1 and CT-2 are expressed in the central and peripheral systems, while CT-1 is localized in the thyroid gland. These findings indicate that dogs can be used for an experimental model as analysing the physiological roles of the CT/CGRP/CRSP family.

A novel member of the calcitonin gene-related peptide family, calcitonin receptor-stimulating peptide, inhibits the formation and activity of osteoclasts.

We isolated a novel peptide, calcitonin receptor-stimulating peptide-1 (CRSP-1), from porcine brain and found that the administration of this peptide into rats induced a transient decrease in plasma calcium concentration. Therefore, we investigated the effects of CRSP-1 on osteoclastogenesis. Osteoclast-like cells were formed from spleen cells or bone marrow cells by a combination of the receptor activator of nuclear factor-kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). CRSP-1 dose-dependently inhibited the formation of multinucleated osteoclast-like cells, and a calcitonin receptor inhibitor antagonized in part the inhibition of osteoclast formation by CRSP-1. Furthermore, CRSP-1 destroyed the actin ring that is a typical index of osteoclast resorption activity; it contributed to this action via the signaling pathway of protein kinase A. Our findings indicate that CRSP-1 inhibits osteoclastogenesis by inhibiting the formation and activity of multinucleated osteoclasts. The inhibitory effects of CRSP-1 on osteoclast metabolism were similar in degree to those of porcine calcitonin. CRSP-1 might provide a clue to the development of tools useful in the prevention and treatment of osteoporosis.

Membrane guanylyl cyclase receptors: an update.

Recent studies have demonstrated key roles for several membrane guanylyl cyclase receptors in the regulation of cell hyperplasia, hypertrophy, migration and extracellular matrix production, all of which having an impact on clinically relevant diseases, including tissue remodeling after injury. Additionally, cell differentiation, and even tumor progression, can be profoundly influenced by one or more of these receptors. Some of these receptors also mediate important communication between the heart and intestine, and the kidney to regulate blood volume and Na+ balance.

Central effects of calcitonin receptor-stimulating peptide-1 on energy homeostasis in rats.

The CT-R [calcitonin (CT) receptor] is expressed in the central nervous system and is involved in the regulation of food intake, thermogenesis, and behaviors. CT-R-stimulating peptide-1 (CRSP-1), a potent ligand for the CT-R, was recently isolated from the porcine brain. In this study, we first confirmed that porcine CRSP-1 (pCRSP-1) enhanced the cAMP production in COS-7 cells expressing recombinant rat CT-R, and then we examined the central effects of pCRSP-1 on feeding and energy homeostasis in rats. Intracerebroventricular (icv) administration of pCRSP-1 to free-feeding rats suppressed food intake in a dose-dependent manner. Chronic icv infusion of pCRSP-1 suppressed body weight gain over the infusion period. Furthermore, icv administration of pCRSP-1 increased body temperature and decreased locomotor activity. The central effects of pCRSP-1 were more potent than those of porcine CT in rats. In contrast, ip administration of pCRSP-1 did not elicit any anorectic or catabolic effects. Administration icv of pCRSP-1 also induced mild dyskinesia of the lower extremities and decreased gastric acid output. Fos expression induced by icv administration of pCRSP-1 was detected in the neurons of the paraventricular nucleus, dorsomedial hypothalamic nucleus, arcuate nucleus, locus coeruleus, and nucleus of solitary tract, areas that are known to regulate feeding and energy homeostasis. Administration icv of pCRSP-1 increased plasma concentrations of ACTH and corticosterone, implying that the hypothalamic-pituitary-adrenocortical axis might be involved in catabolic effects of pCRSP-1. These results suggest that CRSP-1 can function as a ligand for the CT-R and may act as a catabolic signaling molecule in the central nervous system.

Isolation and characterization of a glycine-extended form of calcitonin receptor-stimulating peptide-1: another biologically active form of calcitonin receptor-stimulating peptide-1.

In this study, we isolated a peptide eliciting a potent stimulatory effect on cAMP production in LLC-PK(1) cells from acid extracts of porcine brain. By structural analysis, this peptide was determined to be a C-terminal glycine-extended form of calcitonin receptor-stimulating peptide-1 (CRSP-1-Gly). Synthetic CRSP-1-Gly enhanced the cAMP production in COS-7 cells expressing calcitonin (CT) receptor as strongly as CRSP-1. Measurement of immunoreactive (IR) CRSP-1-Gly by radioimmunoassay using the specific antisera against CRSP-1-Gly showed that a relatively high level (>1pmol/g wet weight) of IR-CRSP-1-Gly was detected in the midbrain, hypothalamus, anterior and posterior lobes of pituitary, and thyroid gland, and the ratio of IR-CRSP-1-Gly to total IR-CRSP-1 varies from 0.02 to 0.35 in each tissue. These results suggest that CRSP-1-Gly is actually present in the tissues as one of major endogenous molecular forms of CRSP-1, and can regulate the cells expressing the CT receptor both in the central nervous system and peripheral tissues in a manner similar to that of CRSP-1. IR-CRSP-2 and IR-CRSP-3 are also present in the brain and other tissues, but their tissue concentrations are 33% on average and less than 3% that of total IR-CRSP-1, respectively.

Detection of FGF15 in plasma by stable isotope standards and capture by anti-peptide antibodies and targeted mass spectrometry.

Fibroblast growth factor 15 (FGF15) has been proposed as a postprandial hormone that signals from intestine to liver to regulate bile acid and carbohydrate homeostasis. However, detecting FGF15 in blood using conventional techniques has proven difficult. Here, we describe a stable isotope standards and capture by anti-peptide antibodies (SISCAPA) assay that combines immuno-enrichment with selected reaction monitoring (SRM) mass spectrometry to overcome this issue. Using this assay, we show that FGF15 circulates in plasma in an FXR and circadian rhythm-dependent manner at concentrations that activate its receptor. Consistent with the proposed endocrine role for FGF15 in liver, mice lacking hepatocyte expression of the obligate FGF15 co-receptor, ß-Klotho, have increased bile acid synthesis and reduced glycogen storage despite having supraphysiological plasma FGF15 concentrations. Collectively, these data demonstrate that FGF15 functions as a hormone and highlight the utility of SISCAPA-SRM as a sensitive assay for detecting low-abundance proteins in plasma.

Specificity of porcine calcitonin receptor and calcitonin receptor-like receptor in the presence of receptor-activity-modifying proteins.

Adrenomedullin (AM), calcitonin gene-related peptide (CGRP), amylin (AMY) and calcitonin (CT) are members of the CGRP/CT superfamily of peptides. Among them, AM and CGRP are reported to share a core receptor, the calcitonin receptor-like receptor (CRLR), and the specificity of the CRLR is determined by the expression levels of receptor-activity-modifying proteins (RAMPs). In the case of AMY, co-expression of the calcitonin receptor (CTR) and RAMPs was recently reported to form its specific receptor. However, detailed analysis of the receptor specificity of the CRLR and CTR in the presence of RAMPs has so far been performed mainly in the human system. Thus, we cloned cDNAs encoding porcine CRLR, RAMP1, RAMP2 and RAMP3 precursors from a porcine lung and hypothalamus cDNA library, and determined their sequences. Then, porcine RAMPs, CRLR and CTR were expressed in COS-7 or porcine vascular smooth muscle cells, and the resulting receptor complexes were analyzed by the cyclic adenosine 3,5-monophosphate (cAMP) production assay. The specificity of CRLR was clearly determined by the expression of RAMPs; RAMP1 converted CRLR to CGRP receptor, while RAMP2 and RAMP3 converted it to AM receptor, but the affinity of CTR for AMY was not increased by the expression of any known RAMPs. In contrast to previous findings, porcine CTR and RAMP did not appear to form an AMY receptor having sufficient affinity and specificity for the physiological interaction.

Structure and biological properties of three calcitonin receptor-stimulating peptides, novel members of the calcitonin gene-related peptide family.

In this review, we describe the structure and biological properties of calcitonin receptor-stimulating peptide-1 (CRSP-1), CRSP-2 and CRSP-3, the novel members of the CGRP family. CRSP-1, which has been identified in the pig, cow, dog, and horse, is a specific ligand for the calcitonin (CT) receptor, and porcine CRSP-1 elicits a 100-fold greater effect on a recombinant porcine CT receptor than porcine CT, although this peptide has high structural similarity with CGRP. CRSP-1 is expressed and synthesized mainly in the central nervous system (CNS), pituitary and thyroid gland. In an in vivo experiment, bolus administration of CRSP-1 into rats reduced the plasma calcium concentration, but did not alter blood pressure, indicating its action as a CT receptor agonist in the peripheral circulation. In the CNS, CRSP-1 is also deduced to be an endogenous agonist for the CT receptor. CRSP-2 has been identified in the pig and dog, and CRSP-3 has been identified only in the pig. They are expressed and synthesized mainly in the CNS and thyroid gland. However, their endogenous molecular forms, receptors, and biological activity remain unidentified.

Novel fish-derived adrenomedullin in mammals: structure and possible function.

Adrenomedullin (AM) has been recognized as a member of the calcitonin (CT)/CT gene-related peptide (CGRP) family. However, an independent AM family consisting of five paralogous peptides exists in teleost fish. Among them, the peptide named AM1 is an ortholog of mammalian AM as determined by the linkage analysis of orthologous genes and the presence of proAM N-terminal 20 peptide (PAMP)-like sequence in the prosegment. Since the peptides named AM2 and 3 are distinct from other members with respect to the precursor sequence, tissue distribution of the transcripts, and exon-intron organization, we searched for their mammalian orthologs from genome databases, which resulted in an identification of AM2 in human, rat, and mouse. AM2 was expressed abundantly in the submaxillary gland, kidney, and some vascular and digestive tissues of mice. AM2 injected in vivo induced potent cardiovascular and renal effects in mice. In the heart and kidney of mice, AM2 was localized in endothelial cells of the coronary vessels and in glomeruli and vasa recta, respectively. AM2 increased cAMP accumulation in cells expressing human CT receptor-like receptor (CRLR) and one of receptor activity-modifying proteins (RAMPs), but it was no more potent than CGRP and AM. AM2 was also less potent than CT in cells expressing CT receptor and RAMP. There remains a possibility that a new AM2-specific receptor or an additional RAMP that enables CRLR to be an AM2-specific receptor, exists in mammals.

Mutations in Tyr808 reveal a potential auto-inhibitory mechanism of guanylate cyclase-B regulation.

In this study, Tyr808 in GC-B (guanylate cyclase-B), a receptor of the CNP (C-type natriuretic peptide), has been shown to be a critical regulator of GC-B activity. In searching for phosphorylation sites that could account for suppression of GC-B activity by S1P (sphingosine-1-phosphate), mutations were introduced into several candidate serine/threonine and tyrosine residues. Although no novel phosphorylation sites that influenced the suppression of GC-B were identified, experiments revealed that mutations in Tyr808 markedly enhanced GC-B activity. CNP-stimulated activities of the Y808F and Y808A mutants were greater than 30-fold and 70-fold higher, respectively, than that of WT (wild-type) GC-B. The Y808E and Y808S mutants were constitutively active, expressing 270-fold higher activity without CNP stimulation than WT GC-B. Those mutations also influenced the sensitivity of GC-B to a variety of inhibitors, including S1P, Na3VO4 and PMA. Y808A, Y808E and Y808S mutations markedly weakened S1P- and Na3VO4-dependent suppression of GC-B activity, whereas Y808E and Y808S mutations rather elevated cGMP production. Tyr808 is conserved in all membrane-bound GCs and located in the niche domain showing sequence similarity to a partial fragment of the HNOBA (haem nitric oxide binding associated) domain, which is found in soluble GC and in bacterial haem-binding kinases. This finding provides new insight into the activation mechanism of GCs.

The starvation hormone, fibroblast growth factor-21, extends lifespan in mice.

Fibroblast growth factor-21 (FGF21) is a hormone secreted by the liver during fasting that elicits diverse aspects of the adaptive starvation response. Among its effects, FGF21 induces hepatic fatty acid oxidation and ketogenesis, increases insulin sensitivity, blocks somatic growth and causes bone loss. Here we show that transgenic overexpression of FGF21 markedly extends lifespan in mice without reducing food intake or affecting markers of NAD+ metabolism or AMP kinase and mTOR signaling. Transcriptomic analysis suggests that FGF21 acts primarily by blunting the growth hormone/insulin-like growth factor-1 signaling pathway in liver. These findings raise the possibility that FGF21 can be used to extend lifespan in other species.DOI:http://dx.doi.org/10.7554/eLife.00065.001.

CNP/GC-B system: a new regulator of adipogenesis.

Adipogenesis is regulated by a wide variety of compounds. An adipogenic cocktail containing insulin (INS), dexamethasone (DEX) and 3-isobutyl-1-methyl xanthine (IBMX) is routinely used to induce adipogenesis in 3T3-L1 preadipocytes, but the biochemical actions in adipogenesis of IBMX, a non-specific phosphodiesterase inhibitor, are not completely understood. In this study we show that C-type natriuretic peptide (CNP) is an endogenous adipogenesis regulator which can largely replace the function of IBMX. In 3T3-L1 preadipocytes, CNP potently elevated cGMP production through guanylyl cyclase-B (GC-B). Lipid droplets were evident in these cells upon stimulation with CNP for 12 days in the presence of INS and DEX, and their adiposity, evaluated by Oil Red O, was significantly higher than in cells stimulated with INS and DEX only. Membrane-permeable cGMP analogue also enhanced adiposity when cells were cultured together with INS and DEX, and KT5823, a non-specific cGMP-dependent kinase (cGK) inhibitor, suppressed the stimulatory effect of IBMX on adipogenesis, revealing that IBMX-stimulated adipogenesis is mediated through cGK. The enhancement of adiposity elicited by CNP was accompanied by increased mRNA levels of adipocyte-specific genes including those encoding peroxisome proliferator-activated receptor gamma and glucose transporter 4. Interestingly, the mRNA level of CNP itself was markedly enhanced in 3T3-L1 cells upon stimulation with INS, DEX and IBMX, reaching a maximum at 8h incubation with the cocktail. These observations suggest that the CNP/GC-B system participates in regulation of adipogenesis, particularly at an early stage in the process.

Calcitonin receptor-stimulating peptide: Its evolutionary and functional relationship with calcitonin/calcitonin gene-related peptide based on gene structure.

This review focuses on the evolutionary and functional relationship of calcitonin receptor-stimulating peptide (CRSP) with calcitonin (CT)/calcitonin gene-related peptide (CGRP) in mammals. CRSP shows high sequence identity with CGRP, but distinct biological properties. CRSP genes (CRSPs) have been identified in mammals such as pigs and dogs of the Laurasiatheria, but not in primates and rodents of the Euarchontoglires or in non-placental mammals. CRSPs have genomic organizations highly similar to those of CT/CGRP genes (CT/CGRPs), which are located along with CGRPs in a locus between CYP2R1 and INSC, while the other members of the CGRP superfamily, adrenomedullin and amylin, show genomic organizations and locations distinct from CT, CGRP, and CRSP. Thus, we categorized these three peptides into the CT/CGRP/CRSP family. Non-placental mammals having one and placental mammals having multiple CT/CGRP/CRSP family genes suggests that multiplicity of CT/CGRP started at an early stage of mammalian evolution. In the placental mammals, Laurasiatheria generally possesses multiple CRSPs and only one CT/CGRP, while Euarchontoglires possesses CT/CGRP and CGRPbeta but no CRSP, indicating an increase in the diversity and multiplicity of this family of genes in mammalian evolution. Phylogenetic analysis suggests that some CRSPs have been generated very recently in mammalian evolution. Taken together, the increase in the number and complexity of the CT/CGRP/CRSP family genes may have due to evolutionary pressure to facilitate adaptation during mammalian evolution. In this regard, it is important to elucidate the physiological roles of CT, CGRP and CRSP from the viewpoint of the CT/CGRP/CRSP family even in Euarchontoglires.

Identification and biological activity of ovine and caprine calcitonin receptor-stimulating peptides 1 and 2.

We have recently reported the isolation of three new members of the calcitonin (CT) gene-related peptide family of peptides, the CT receptor (CT-R)-stimulating peptides (CRSPs). We now report the sequencing and characterization of ovine/caprine CRSP-1 and caprine CRSP-2. Mature ovine and caprine CRSP-1 are identical and have strong structural homology to CRSP-1s identified to date from other species. As with other CRSP-1s, ovine/caprine CRSP-1 binds to and activates the CT-R but not the CT-like receptor (CL-R) in combination with the receptor activity-modifying proteins (RAMPs). By contrast, caprine CRSP-2 does not activate any of these receptor-RAMP complexes. Intravenous infusions of ovine CRSP-1 to normal conscious sheep induced dose-dependent reduction in plasma total Ca levels (P=0.02) and corrected Ca levels (P=0.017) associated with increases in plasma cAMP (P=0.002). CRSP-1 reduced both plasma amino-terminal pro-C-type natriuretic peptide levels (P=0.006) and plasma renin activity (P=0.028). There were no significant effects observed on hemodynamic or renal indices measured. In conclusion, we have sequenced ovine/caprine CRSP-1 and caprine CRSP-2 precursors. This newly identified CRSP-1 has been shown to share the structural and biological features of CRSP-1s known to date. In vivo studies confirm that ovine CRSP-1 reduces plasma Ca levels in sheep, presumably via a cAMP-mediated mechanism. By contrast, caprine CRSP-2 did not stimulate any combination of CT-R, CL-R, and RAMPs. Accession numbers of cDNA determined in this study are caprine CRSP-1, AB364646; caprine CRSP-2, AB364647; and ovine CRSP-1, AB364648.

Calcitonin receptor-stimulating peptide-1 regulates ion transport and growth of renal epithelial cell line LLC-PK1.

Calcitonin receptor-stimulating peptide-1 (CRSP-1) is a peptide recently identified from porcine brain by monitoring the cAMP production through an endogenous calcitonin (CT) receptor in the renal epithelial cell line LLC-PK(1). Here we investigated the effects of CRSP-1 on the ion transport and growth of LLC-PK(1) cells. CRSP-1 inhibited the growth of LLC-PK(1) cells with a higher potency than porcine CT. CRSP-1 enhanced the uptake of (22)Na(+) into LLC-PK(1) cells more strongly than did CT and slightly reduced the (45)Ca(2+) uptake. The enhancement of the (22)Na(+) uptake was abolished by 5-(N-ethyl-N-isopropyl) amiloride, a strong Na(+)/H(+) exchanger (NHE) inhibitor for NHE1, even at a concentration of 1x10(-8)M, although other ion transporter inhibitors did not affect the (22)Na(+) uptake. These results indicate that CRSP-1 enhances the (22)Na(+) uptake by the specific activation of NHE1. Taken together, CRSP-1 is considered to be a new regulator for the urinary ion excretion and renal epithelial cell growth.