Urinary angiotensin-converting enzyme activity in type 2 diabetes mellitus: its relationship to diabetic nephropathy

Urinary angiotensin-converting enzyme (ACE) and urinaryN-acetyl--d-glucosaminidase (NAG) were measured after a 5-year interval in 38 non-azotemic type 2 diabetic patients. Of these patients at baseline, 16 had nil nephropathy, 15 had incipient nephropathy, and 7 had overt nephropathy. During the follow-up, 6 and 1 of the 16 patients with nil nephropathy developed incipient and overt nephropathy, respectively. Four of the 15 patients with incipient nephropathy progressed to overt nephropathy. The 7 patients with overt nephropathy continued to have overt nephropathy, with slight azotemia in one. Urinary ACE and NAG levels were normal at baseline and showed no significant elevations at follow-up in the patients with nil nephropathy, no significant changes at baseline and modest elevations at follow-up in the patients with incipient nephropathy, and high at baseline and marked elevations at follow-up in the patients with overt nephropathy. In all patients, urinary ACE during the follow-up was positively correlated with urinary albumin or NAG, but not with glomerular filtration rate. Urinary ACE may be of poor prognostic value for the follow-up of diabetic patients, which is at variance with urinary albumin.     

Genetic epistasis of adiponectin and PPARgamma2 genotypes in modulation of insulin sensitivity: a family-based association study

Aims/hypothesis Genetic interactions in modulating the phenotypes of a complex trait, such as insulin sensitivity, were usually taken for granted. However, this has not been commonly shown. Previous studies have suggested that both PPAR2 and adiponectin genes could influence insulin sensitivity. Therefore it is likely that they could modulate insulin sensitivity through gene to gene interactions.Methods We genotyped 1793 subjects of Chinese and Japanese descendents from 601 hypertensive families recruited in Sapphire study for a T94G in the adiponectin gene exon 2 and the PPAR2 Pro12Ala polymorphisms. Serum insulin concentrations and insulin resistance index (HOMA_IR) were used as the markers of insulin sensitivity.Results We found that the T allele of adiponectin gene was associated with a higher Ins60 and higher area under curve of insulin (AUCi) in OGTT utilizing all subjects in a mixed model that corrected for family effects. Important interactions between adiponectin and PPAR2 genotypes were found in fasting insulin concentrations (Ins0), insulin concentrations at 2-h (Ins120) in OGTT and insulin resistance index (HOMA_IR). The main effects of the PPAR2 genotypes were in the plasma glucose concentrations in OGTT. In contrast, the main effects of adiponectin genotypes were in every insulin variable, including Ins0, Ins60, Ins120, AUCi and HOMA_IR. The subjects carrying the adiponectin G allele and the PPAR2 Ala12 allele seemed to be more insulin sensitive.Conclusion/interpretation These results showed that adiponectin is a genetic factor associated with insulin sensitivity. Interactions with PPAR2 genotypes modified this association.Abbreviations PPAR peroxisome proliferator-activated receptor - HOMA homeotasis model assessment - T2DM Type 2 diabetes mellitus     

Labeling of pancreatic glycogen by d-[U-14C]glucose in hyperglycemic rats

Under conditions of sustained hyperglycemia, glycogen accumulates in pancreatic islets, but not so in acinar pancreatic cells. We investigated whether advantage could be taken of such a situation in the perspective of the noninvasive imaging of the endocrine pancreas. Control rats or animals injected with streptozotocin (STZ) were infused with solutions of d-glucose mixed with a tracer amount of d-[U-¹⁴C]glucose, and the radio-active glycogen content of both liver and pancreas was then measured. After 48 h of infusion, the radio-active glycogen content of the pancreas was 30 times lower in STZ rats than in control animals, coinciding with a 50 times lower insulin content. In the control rats, a sizable labeling of pancreatic glycogen was also recorded when d-[U-¹⁴C]glucose was infused for only the last 4 h of unlabeled d-glucose infusion; such a labeling was not decreased when the animals were further infused for 1 h with only the unlabeled hexose. Moreover, a pronounced difference in the pancreatic gland and blood radioactive content of control rats was still observed when the hyperglycemic animals were killed only 40 min after the iv injection of d-[U-¹⁴C]glucose. In STZ rats transplanted with islets and later infused with d-[U-¹⁴C]glucose, the total radioactive content and radioactive glycogen content were both much higher in the transplanted islets than in the pancreatic gland. These results allow one to define the conditions under which the administration of either 2-deoxy-2-[¹⁸F]fluoro-d-glucose or ¹¹C-labeled d-glucose could conceivably be used to favor the selective labeling of the endocrine, as distinct from exocrine, pancreas.     

[dl-1,2-Bis(2-hydroxyphenyl)ethylenediamine]dichloroplatinum(II), a new compound for the therapy of ovarian cancer

Summary The synthesis of diastereoisomeric [1,2-bis (2-hydroxyphenyl)ethylenediamine]dichloroplatinum(II) complexes,dl-3-PtCl₂ andmeso-3-PtCl₂, and their evaluation on the hormone-independent, human MDA-MB231 breast cancer cell line, on the cisplatin-sensitive and -resistant L1210 leukemia cell line, on the cisplatinresistant human NIH:OVCAR 3 ovarian cancer cell line, on the P-388 leukemia of the mouse and on the cisplatinsensitive and -resistant Ehrlich ascites tumor of the mouse are described. On all tumor modelsdl-3-PtCl₂ produces a marked inhibitory effect. The diastereoisomermeso-3-PtCl₂ is less active and more toxic. It is striking thatdl-3-PtCl₂ leads to a pronounced inhibition of all cisplatin-resistant tumors. At non-toxic concentrationsdl-3-PtCl₂ produces cytocidal effects on the NIH:OV-CAR 3 cell line. Thereforedl-3-PtCl₂ is of interest for further evaluation for the therapy of ovarian cancer.     

Potentiation and prolongation of the insulinotropic action of glucagon-like peptide 1 by methyl pyruvate or dimethyl ester of L-glutamic acid in a type 2 diabetes animal model

Methyl pyruvate and the dimethyl ester of l-glutamic acid were administered intravenously, as a primed constant infusion (1.0–2.0 μmol followed by 0.5–1.0 μmol/min, both expressed per gram of body wt), in adult rats that had been injected with streptozotocin during the neonatal period. Each ester augmented plasma insulin concentration and potentiated and/or prolonged the insulinotropic action of glucagon-like peptide 1 (GLP-1) injected intravenously (5 pmol/g of body wt) at min 5 of the test. It is proposed, therefore, that suitable nonglucidic nutrients, susceptible to bypassing the site-specific defects of d-glucose transport and metabolism responsible for the preferential impairment of the B-cell secretory response to d-glucose in non-insulin-dependent diabetes, could be used to optimize the insulinotropic action of GLP-1.     

<Emphasis Type="Italic">N</Emphasis>-Acetyl-<Emphasis Type="SmallCaps">l</Emphasis>-Glutamine,A Liquid-Stable Source of Glutamine,Partially Prevents Changes in Body Weight and on Intestinal Immunity Induced by Protein Energy Malnutrition in Pigs

The goal of this study was to evaluate the preventive effect of free glutamine versus N-acetyl-l-glutamine, a liquid-stable source of glutamine, on gut damage induced by protein energy malnutrition in pigs. Healthy pigs (n=6) were fed a liquid formula for 30 days. Three subgroups of malnourished pigs (n=6) received daily 20% of the food intake recorded in control group, supplemented with calcium caseinate, glutamine, or N-acetyl-l-glutamine. Body weight was recorded, and small intestinal samples were evaluated for biochemical and immunologic parameters. Suppression in body weight gain was significantly lower in pigs fed with N-acetyl-l-glutamine than in the rest of malnourished pigs. Total number of lymphocytes, CD21⁺ B cells and CD4⁺ T cells in ileal Peyer patches were not significantly different in malnourished pigs fed with N-acetyl-l-glutamine and in healthy pigs. In conclusion, N-acetyl-l-glutamine has a moderate protective effect, partially preventing changes induced by protein energy malnutrition.     

Ascorbic acid absorption in Crohn's disease

Total body pool and intestinal absorption of ascorbic acid were studied in 12 patients undergoing operation for Crohn's disease (six with fistulae and six without) and in six control patients undergoing operation for reasons other than Crohn's disease.l-[caxboxyl-¹⁴C]Ascorbic acid, 0.19–0.40 megabecquerels (MBq), was given orally. After a period of equilibration, the labeled ascorbic acid was flushed out of the patient's body tissues using large doses of unlabeled ascorbic acid. Intestinal absorption of ascorbic acid, assessed from the total cumulative urinary¹⁴C recovery, was found to be similar in patients with fistulizing Crohn's disease (73.9±8.45%), those without fistulas (72.8±11.53%), and in controls (80.3±8.11%). Total body pools of ascorbic acid, calculated using the plasma¹⁴C decay curves, were similar in patients with Crohn's disease with fistulas (17.1±5.91 mg/kg), patients without fistulas (9.6±3.58 mg/kg), and in controls (13.3±4.28 mg/kg). The results indicate that ascorbic acid absorption is normal in patients with both fistulizing and nonfistulizing Crohn's disease. The results suggest that routine supplements of vitamin C are not necessary unless oral ascorbic acid intake is low.The authors thank Hoffmann La Roche for performing the ascorbic acid analyses. The study was supported by grants from the Sheila Dawber Fund, the North Western Regional Health Authority and Hoffmann La Roche.     

Correlation of GLUT-1 Overexpression, Tumor Size, and Depth of Invasion with 18F-2-fluoro-2-deoxy-d-glucose Uptake by Positron Emission Tomography in Colorectal Cancer

We investigated the wide variability of 18F-2-fluoro-2-deoxy-d-glucose (FDG) uptake, semiquantified as standardized uptake value (SUV), in positron emission tomography (PET) scanning, in 20 patients with colorectal cancer (CRC), including 1 with synchronous hepatic metastasis. The sensitivity of PET in CRC diagnosis was 100%, with a mean SUV of 8.0 (3.1–11.9). Tumor size and depth of invasion were associated with higher SUVs (P=.0004, .042, respectively). Strong glucose transporter-1 (GLUT-1) expression had significantly positive correlation with the SUV (r=.619, P=.003). GLUT-1 expression revealed positive staining in 17 (85%) of the 20 primary lesions. The central part of the tumor, thought to be relatively hypoxic, had stronger GLUT-1 expression and a higher SUV than the periphery, in both the primary tumor and hepatic metastatic foci. Our data suggest that the SUVs of FDG uptake in PET may be a noninvasive biomarker for advanced CRC, indicative of a large hypoxic tumor with deep invasion.     

Combination therapy with interferon-alpha(2b), ribavirin,and amantadine in chronic hepatitis C nonresponders to interferon and ribavirin

Standard therapies for the treatment of hepatitis C are ineffective in almost 50% of patients. Amantadine is an antiviral agent that may have activity against hepatitis C virus. In this pilot study, we evaluated the efficacy of a combination of interferon, ribavirin, and amantadine in patients with chronic hepatitis C who had previously failed 6–12 months of treatment with interferon and ribavirin. In this prospective open-label study, 23 patients were treated with a combination of interferon-_2b 3 million units subcutaneously three times per week, ribavirin 1000–1200 mg daily, and amantadine 100 mg twice daily for 6–12 months. Treatment was discontinued at 6 months if the patients had detectable HCV RNA by PCR. All patients were followed for 6 months after the completion of treatment. At the end of treatment, the biochemical response was 47% and the virological response was 30%. However, the rate of sustained virological response was only 13% (3/23). There were no unexpected side effects with triple therapy. In conclusion, triple therapy with interferon, ribavirin and amantadine resulted in a low sustained viral clearance in chronic hepatitis C patients who had previously failed interferon and ribavirin combination therapy.     

Haplotypes of PPARGC1A are associated with glucose tolerance, body mass index and insulin sensitivity in offspring of patients with type 2 diabetes

Aims/hypothesis Decreased expression of the peroxisomal proliferator activated receptor gamma coactivator 1 alpha gene (PPARGC1A) is found in patients with type 2 diabetes, and variants in this gene have been linked with type 2 diabetes. Therefore, we investigated the effects of single nucleotide polymorphisms in PPARGC1A on body composition and glucose tolerance and on insulin sensitivity and secretion.Methods Non-diabetic offspring (n=156, age 34.9±0.5 years [mean±SEM], BMI 26.2±0.4 kg/m²) underwent an OGTT and an IVGTT and the hyperinsulinaemic-euglycaemic clamp. The promoter and coding regions of PPARGC1A were sequenced.Results Two haplotype blocks in PPARGC1A were observed, one in the promoter region (G-1774A, A-1679G, T-1422C, A-1278G, C-543A) and one in the coding region and 3 regions (Thr394Thr, Asp475Asp, Gly482Ser, Thr528Thr, Thr612Met, G+2381A). The coding region haplotype carrying the rare allele in codons 482 and 528 was associated with elevated glucose levels in an OGTT (p=0.024, adjusted for age, sex and BMI) and a haplotype carrying the rare alleles in codons 394 and 475 was associated with low BMI (p=0.033), high rates of whole-body glucose uptake (p=0.045) and low glucose levels in the OGTT (p=0.037).Conclusions/interpretation We conclude that PPARGC1A is likely to contribute to the risk of diabetes in offspring of patients with type 2 diabetes.J. Pihlajamäki and M. Kinnunen contributed equally to this study     

Cytotoxic effect of a fusion protein from transforming growth factora andPseudomonas exotoxin on rat and human bladder carcinoma cells in vitro

A protein formed by fusion of transforming growth factor withPseudomonas exotoxin (TGF-PE40) has been shown to have the ability to kill or inhibit the growth of several carcinoma cell lines. This study was designed to evaluate thein vitro cytotoxic effects of TGF-PE40 on rat and human bladder carcinoma cell lines with different biological potential, and normal rat urothelial cells. The rat cell lines used were D44c, LMC19, and MYU3L, which were established in our laboratory. Human cell lines used were RT4, T24, and 253J. As a normal control, we used the first-passage culture of normal rat bladder urothelium (RU-P1). We examined the number and affinity of epidermal growth factor receptors (EGFR) in these cells, the ability of TGF-PE40 to bind EGFR, and the cytotoxic effect of TGF-PE40 and PE40. Rat cell lines, D44c, LMC19, and MYU3L (EGFR=4.9×10³–11.4×10³/cell) had ED₅₀ values (the concentration of TGF-PE40 needed to reduce the viable cell population by 50%) of 180 pM, 540 pM and 6000 pM respectively; forc ₁ (the concentration required to achieve complete inhibition of growth under continuous serum stimulation) TGF-PE40 concentrations of 10⁴ pM, 10⁴ pM and higher than 10⁴ pM respectively were required. Human cell lines, RT4, T24, and 253J (EGFR=32×10³–126×10³/cell) had ED₅₀ values of 20 pM, 66 pM, and 330 pM respectively and T24 showedc ₁ values of 10³ pM. RU-P1 (EGFR =92.6×10³/cell) had the highest ED₅₀ value of 8000 pM. These data indicate that the susceptibility to TGF-PE40 does not always depend on the number of EGFR, that cells having a relatively small number of EGFR respond well to TGF-PE40, and that normal urothelial cells are more resistant to TGF-PE40 than are cancer cells. The differential effect of TGF-PE40 on normal and neoplastic cells provides a rational basis for its use in vivo to control tumor growth.Supported by National Institutes of Health Grant CA14649     

Differences in the regional distribution and response to ischaemia of adenosine-regulating enzymes in the heart

Summary The activities of the adenosine-generating enzyme 5-nucleotidase and the adenosine-degrading enzyme adenosine deaminase were determined for four regions of rat hearts prior to and following 10–60 min of ischaemia. Whereas adenosine deaminase was uniformly active throughout the heart, 5-nucleotidase was twice as active in atrial than in ventricular myocardium, and more active in the right than in the left ventricles in normoxic tissues. In isolated heart preparations normoxic perfusion decreased adenosine deaminase and increased 5-nucleotidase activity compared to levelsin vivo. Global ischaemia for 10 min elevated adenosine deaminase activity but had no effect on 5-nucleotidase activity. However, 30 min of ischaemia decreased 5-nucleotidase activity by 50% in all regions of the heart. These changed levels were not altered by 10 min of reperfusion. The fall in 5-nucleotidase activity with ischaemia occurred only in the 90% of this enzyme which is membrane-bound. The reasons for the marked differences in distribution and responses to ischaemia of these two enzymes have yet to be elucidated but metabolic inhibitors seem unlikely to be involved.     

Positron emission tomography imaging of adenoviral-mediated transgene expression in liver cancer patients

BACKGROUND & AIMS: In gene-therapy protocols, imaging of gene expression is needed to evaluate the transduction efficiency of the vector, its tissue distribution, and the duration of transgene expression and to assess the feasibility of repeated vector administration. METHODS: We have used positron emission tomography with a fluorine-18-labeled penciclovir analogue to monitor thymidine kinase gene expression after intratumoral injection of a first-generation recombinant adenovirus in patients with hepatocellular carcinoma. Patients were enrolled in a pilot clinical trial and treated with escalating doses of the vector. Two days after adenovirus inoculation, transgene expression was evaluated during the first hours after administration of the radiotracer both on the treated lesion and on a whole-body basis. RESULTS: Transgene expression in the tumor was dependent on the injected dose of the adenovirus and was detectable in all patients who received > or = 10(12) viral particles. However, when the study was repeated 9 days after vector injection, no expression could be observed. It is interesting to note that no specific expression of the transgene could be detected in distant organs or in the surrounding cirrhotic tissue in any of the cases studied. CONCLUSIONS: Our findings show the real possibility of imaging transgene expression in humans by using viral vectors. We show that hepatocarcinoma is a permissive tumor for adenoviral infection and that the nontumoral cirrhotic liver is spared from transduction when the vector is administered by intratumoral injection. These results show that positron emission tomography imaging may help in the design of gene-therapy strategies and in the clinical assessment of new-generation vectors.     

Interleukin 15 is a potent stimulant of intraepithelial lymphocytes

Background & Aims: This study examined the effects of interleukin (IL)-15 on intraepithelial lymphocytes (IELs) because they resemble memory cells that react to IL-15 and are located next to epithelial cells that produce IL-15. Methods: Proliferative responses were measured by [³H]thymidine uptake; interferon (IFN)-γ production by enzyme-linked immunosorbent assay; and cytotoxicity production by lysis of ⁵¹Cr-labeled HT-29 cells. Results: The proliferative response of IELs was much greater with IL-15 than with equivalent amounts of IL-2 or IL-7 (P < 0.001); the same level of blastogenesis was induced by 10³-fold less IL-15 than IL-2. Production of IFN-γ was also highest when IELs were stimulated with IL-15. IELs lysed more ⁵¹Cr-labeled HT-29 cells when cultured for 72 hours with IL-15 (48% ± 3% at 25:1 effector-to-target ratio) than with IL-2 (27% ± 3%) or IL-7 (12% ± 2%) (P < 0.0001). Similarly, limiting dilution analysis revealed a greater frequency of cytotoxic precursors in IELs that were stimulated by IL-15 rather than IL-2: 1/467 vs. 1/1900. But IL-15 did not alter the number of natural killer cells, as determined by quantitating CD16 and CD56 by immunofluorescence. Rather, it increased serine esterase content in IELs. Conclusions: IL-15 is the most potent of the known cytokines for IELs, inducing the highest levels of proliferation, IFN-γ production, and cytotoxicity.GASTROENTEROLOGY 1998;115:1439-1445     

Selective glutathione depletion of mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor

Background & Aims: Tumor necrosis factor (TNF)-α induces cell injury by generating oxidative stress from mitochondria. The purpose of this study was to determine the effect of ethanol on the sensitization of hepatocytes to TNF-α. Methods: Cultured hepatocytes from ethanol-fed (ethanol hepatocytes) or pair-fed (control hepatocytes) rats were exposed to TNF-α, and the extent of oxidative stress, gene expression, and viability were evaluated. Results: Ethanol hepatocytes, which develop a selective deficiency of mitochondrial glutathione (mGSH), showed marked susceptibility to TNF-α. The susceptibility to TNF-α, manifested as necrosis rather than apoptosis, was accompanied by a progressive increase in hydrogen peroxide that correlated inversely with cell survival. Nuclear factor κB activation by TNF-α was significantly greater in ethanol hepatocytes than in control hepatocytes, an effect paralleled by the expression of cytokine-induced neutrophil chemoattractant. Similar sensitization of normal hepatocytes to TNF-α was obtained by depleting the mitochondrial pool of GSH with 3-hydroxyl-4-pentenoate. Restoration of mGSH by S-adenosyl-L-methionine or by GSH–ethyl ester prevented the increased susceptibility of ethanol hepatocytes to TNF-α. Conclusions: These results indicate that mGSH controls the fate of hepatocytes in response to TNF-α. Its depletion caused by alcohol consumption amplifies the power of TNF-α to generate reactive oxygen species, compromising mitochondrial and cellular functions that culminate in cell death.GASTROENTEROLOGY 1998;115:1541-1551     

T75M-KCNJ2 mutation causing Andersen-Tawil syndrome enhances inward rectification by changing Mg2+ sensitivity

Andersen-Tawil syndrome (ATS) is a multisystem inherited disease exhibiting periodic paralysis, cardiac arrhythmias, and dysmorphic features. In this study, we characterized the KCNJ2 channels with an ATS mutation (T75M) which is associated with cardiac phenotypes of bi-directional ventricular tachycardia, syncope, and QT(c) prolongation. Confocal imaging of GFP-KCNJ2 fusion proteins showed that the T75M mutation impaired membrane localization of the channel protein, which was restored by co-expression of WT channels with T75M channels. Whole-cell patch-clamp experiments in CHO-K1 cells showed that the T75M mutation produced a loss-of-function of the channel. When both WT and the T75M were co-expressed, the T75M mutation showed dominant-negative effects on inward rectifier K+ current densities, with prominent suppression of outward currents at potentials between 0 mV and +80 mV over the E(K). Inside-out patch experiments in HEK293T cells revealed that co-expression of WT and the T75M channels enhanced voltage-dependent block of the channels by internal Mg2+, resulting in enhanced inward rectification at potentials 50 mV more positive than the E(K). We suggest that the T75M mutation causes dominant-negative suppression of the co-expressed WT KCNJ2 channels. In addition, the T75M mutation caused alteration of gating kinetics of the mutated KCNJ2 channels, i.e., increased sensitivity to intracellular Mg2+ and resultant enhancement of inward rectification. The data presented suggest that the mutation may influence clinical features, but it does not directly show this.     

Control of cardiac myofilament activation and PKC-betaII signaling through the actin capping protein, CapZ

Actin capping protein (CapZ) anchors the barbed ends of sarcomeric actin to the Z-disc. Myofilaments from transgenic mice (TG-CapZ) expressing a reduced amount of CapZ demonstrate altered function and protein kinase C (PKC) signaling [Pyle WG, Hart MC, Cooper JA, Sumandea MP, de Tombe PP, and Solaro RJ., Circ. Res. 90 (2002) 1299-306]. The aims of the current study were to determine the direct effects of CapZ on myofilament function and on PKC signaling to the myofilaments. Our studies compared mechanical properties of single myocytes from TG-CapZ mouse hearts to wild-type myocytes from which CapZ was extracted using PIP(2). We found that myofilaments from CapZ-deficient transgenic myocardium exhibited increased Ca(2+) sensitivity and maximum isometric tension. The extraction of CapZ from wild-type myofilaments replicated the increase in maximum isometric tension, but had no effect on myofilament Ca(2+) sensitivity. Immunoblot analysis revealed that the extraction of CapZ was associated with a reduction in myofilament-associated PKC-beta(II) and that CapZ-deficient transgenic myofilaments also lacked PKC-beta(II). Treatment of wild-type myofilaments with recombinant PKC-beta(II) reduced myofilament Ca(2+) sensitivity, whereas this effect was attenuated in myofilaments from TG-CapZ mice. Our results indicate that cardiac CapZ directly controls maximum isometric tension generation, and establish CapZ as an important component in anchoring PKC-beta(II) at the myofilaments, and for mediating the effects of PKC-beta(II) on myofilament function.     

Autocrine/paracrine regulation of the growth of the biliary tree by the neuroendocrine hormone serotonin

BACKGROUND & AIMS: The biliary tree is the target of cholangiopathies that are chronic cholestatic liver diseases characterized by loss of proliferative response and enhanced apoptosis of cholangiocytes, the epithelial cells lining the biliary tree. The endogenous factors that regulate cholangiocyte proliferation are poorly understood. Therefore, we studied the role of the neuroendocrine hormone serotonin as a modulator of cholangiocyte proliferation. METHODS: The presence of the serotonin 1A and 1B receptors on cholangiocytes was evaluated. We then tested whether the activation of such receptors by the administration of the selective agonists modifies cholangiocyte proliferation and functional activity both in vivo and in vitro. In addition, the intracellular signal mediating the serotonin receptor action in cholangiocytes was characterized. We studied the expression and secretion of serotonin by cholangiocytes and the effects of the neutralization of the secreted hormone on the growth of the biliary tree. RESULTS: Cholangiocytes express the serotonin 1A and 1B receptors. Their activation markedly inhibits the growth and choleretic activity of the biliary tree in the bile duct-ligated rat, a model of chronic cholestasis. Such changes are mediated by enhanced d -myo-inositol 1,4,5-triphosphate/Ca 2+ /protein kinase C signaling and the consequent inhibition of the adenosine 3',5'-cyclic monophosphate/protein kinase A/Src/extracellular signal-regulated kinase 1/2 cascade. Cholangiocytes secrete serotonin, the blockage of which enhances cholangiocyte proliferation in the course of cholestasis. CONCLUSIONS: We observed the existence of an autocrine loop based on serotonin that limits the growth of the biliary tree in the course of chronic cholestasis. Our novel findings might open new approaches for the management of cholangiopathies.     

Revisiting the evolution of gonadotropin-releasing hormones and their receptors in vertebrates: secrets hidden in genomes

Gonadotropin-releasing hormone (GnRH) and its G protein-coupled receptor, GnRHR, play a pivotal role in the control of reproduction in vertebrates. To date, many GnRH and GnRHR genes have been identified in a large variety of vertebrate species using conventional biochemical and molecular biological tools in combination with bioinformatic tools. Phylogenetic approaches, primarily based on amino acid sequence identity, make it possible to classify these multiple GnRHs and GnRHRs into several lineages. Four vertebrate GnRH lineages GnRH1, GnRH2, GnRH3, and GnRH4 (for lamprey) are well established. Four vertebrate GnRHR lineages have also been proposed—three for nonmammalian GnRHRs and mammalian GnRHR2 as well as one for mammalian GnRHR1. However, these phylogenetic analyses cannot fully explain the evolutionary origins of each lineage and the relationships among the lineages. Rapid and vast accumulation of genome sequence information for many vertebrate species, together with advances in bioinformatic tools, has allowed large-scale genome comparison to explore the origin and relationship of gene families of interest. The present review discusses the evolutionary mechanism of vertebrate GnRHs and GnRHRs based on extensive genome comparison. In this article, we focus only on vertebrate genomes because of the difficulty in comparing invertebrate and vertebrate genomes due to their marked divergence.