bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of 11β-hydroxysteroid dehydrogenase enzymes in the rat kidney by estradiol
Authors: Gomez-Sanchez Elise P. , Ganjam Venkataseshu , Chen Yuan Jian , Liu Ying , Zhou Ming Yi , Toroslu Cigdem , Romero Damian G. , Hughson Michael D. , de Rodriguez Angela , Gomez-Sanchez Celso E.
Published: March 2015

The 11β-hydroxysteroid dehydrogenase (11βHSD) type 1 (11βHSD1) enzyme is an NADP+-dependent oxidoreductase, usually reductase, of major glucocorticoids. The NAD+-dependent type 2 (11βHSD2) enzyme is an oxidase that inactivates cortisol and corticosterone, conferring extrinsic specificity of the mineralocorticoid receptor for aldosterone. We reported that addition of a reducing agent to renal homogenates results in the monomerization of 11βHSD2 dimers and a significant increase in NAD+-dependent corticosterone conversion. Estrogenic effects on expression, dimerization, and activity of the kidney 11βHSD1 and -2 enzymes are described herein. Renal 11βHSD1 mRNA and protein expressions were decreased to very low levels by estradiol (E2) treatment of both intact and castrated male rats; testosterone had no effect. NADP+-dependent enzymatic activity of renal homogenates from E2-treated rats measured under nonreducing conditions was less than that of homogenates from intact animals. Addition of 10 mM DTT to aliquots from these same homogenates abrogated the difference in NADP+-dependent activity between E2-treated and control rats. In contrast, 11βHSD2 mRNA and protein expressions were significantly increased by E2treatment. There was a marked increase in the number of juxtamedullary proximal tubules stained by the antibody against 11βHSD2 after the administration of E2. Notwithstanding, neither the total corticosterone and 11-dehydrocorticosterone excreted in the urine nor their ratio differed between E2- and vehicle-treated rats. NAD+-dependent enzymatic activity in the absence or presence of a reducing agent demonstrated that the increase in 11βHSD2 protein was not associated with an increase in in vitro activity unless the dimers were reduced to monomers.
α-Melanocyte-stimulating hormone is a novel regulator of bone
Authors: Cornish Jillian , Callon Karen E. , Mountjoy Kathleen G. , Bava Usha , Lin Jian-Ming , Myers Damian E. , Naot Dorit , Reid Ian R.
Published: March 2015

α-Melanocyte-stimulating hormone (α-MSH), a 13-amino acid peptide produced in the brain and pituitary gland, is a regulator of appetite and body weight, and its production is regulated by leptin, a factor that affects bone mass when administered centrally. α-MSH acts via melanocortin receptors. Humans deficient in melanocortin receptor 4 (MC4-R) have increased bone mass, and MC4-R has been identified in an osteoblast-like cell line. Thus α-MSH may act directly on the skeleton, a question addressed by the present studies. In primary cultures of osteoblasts and chondrocytes, α-MSH dose dependently (≥10−9M) stimulated cell proliferation. In bone marrow cultures, α-MSH (>10−9M) stimulated osteoclastogenesis. Systemic administration of α-MSH to mice (20 injections of 4.5 μg/day) decreased the trabecular bone volume in the proximal tibiae from 19.5 ± 1.8 to 15.2 ± 1.4% ( P = 0.03) and reduced trabecular number ( P = 0.001). Radiographic indexes of trabecular bone, assessed by phase-contrast X-ray imaging, confirmed the bone loss. It is concluded that α-MSH acts directly on bone, increasing bone turnover, and, when administered systemically, it decreases bone volume. The latter result may also be contributed to by α-MSH effects elsewhere, such as the adipocyte, pancreatic β-cell, or central nervous system.
Somatotropic axis in hypocretin-deficient narcoleptic humans: altered circadian distribution of GH-secretory events
Authors: Overeem Sebastiaan , Kok Simon W. , Lammers Gert Jan , Vein Alla A. , Frölich Marijke , Meinders Arend E. , Roelfsema Ferdinand , Pijl Hanno
Published: March 2015

Narcolepsy is a sleep disorder caused by impaired hypocretin (orexin) neurotransmission. Growth hormone (GH) secretion may be altered in narcolepsy for various reasons. Slow-wave sleep episodes, which are closely associated with GH-secretory events, are more randomly dispersed over 24 h in narcoleptics. Furthermore, hypocretins may inhibit pituitary GH release. We assessed the function of the somatotropic axis in narcolepsy by deconvolving 24-h (10-min sampling interval) plasma GH concentration profiles in seven hypocretin-deficient narcoleptic patients and in seven healthy controls matched for age, sex, and body weight. Both basal and pulsatile GH secretion rate and secretagogue-induced GH release were similar in patients and controls. However, narcoleptics secreted ∼50% of their total production during the daytime, whereas controls secreted only 25% during the day. Also, the GH output pattern of narcoleptics was significantly less regular. We propose that hypocretin deficiency disrupts the circadian distribution of hypothalamic GH-releasing hormone release in narcoleptic patients to simultaneously cause daytime GH release and promote their propensity to fall asleep during the day.
Elevated plasma nonesterified fatty acids are associated with deterioration of acute insulin response in IGT but not NGT
Authors: Stefan Norbert , Stumvoll Michael , Bogardus Clifton , Tataranni P. Antonio
Published: March 2015

High concentrations of nonesterified fatty acids (NEFA) are a risk factor for developing type 2 diabetes in Pima Indians. In vitro and in vivo, chronic elevation of NEFA decreases glucose-stimulated insulin secretion. We hypothesized that high fasting plasma NEFA would increase the risk of type 2 diabetes by inducing a worsening of glucose-stimulated insulin secretion in Pima Indians. To test this hypothesis, fasting plasma NEFA concentrations, body composition, insulin action (M), acute insulin response (AIR, 25-g IVGTT), and glucose tolerance (75-g OGTT) were measured in 151 Pima Indians [107 normal glucose tolerant (NGT), 44 impaired glucose tolerant (IGT)] at the initial visit. These subjects, participants in ongoing studies of the pathogenesis of obesity and type 2 diabetes, had follow-up measurements of body composition, glucose tolerance, M, and AIR. In NGT individuals, cross-sectionally, high fasting plasma NEFA concentrations at the initial visit were negatively associated with AIR after adjustment for age, sex, percent body fat, and M ( P = 0.03). Longitudinally, high fasting plasma NEFA concentrations at the initial visit were not associated with change in AIR. In individuals with IGT, cross-sectionally , high fasting plasma NEFA concentrations at the initial visit were not associated with AIR. Longitudinally, high fasting plasma NEFA concentrations at the initial visit were associated with a decrease in AIR before ( P< 0.0001) and after adjustment for sex, age at follow-up, time of follow-up, change in percent body fat and insulin sensitivity, and AIR at the initial visit ( P = 0.0006). In conclusion, findings in people with NGT indicate that fasting plasma NEFA concentrations are not a primary etiologic factor for β-cell failure. However, in subjects who have progressed to a state of IGT, chronically elevated NEFA seem to have a deleterious effect on insulin-secretory capacity.
The melanocortin system
Authors: Gantz Ira , Fong Tung M.
Published: March 2015

The melanocortin system consists of melanocortin peptides derived from the proopiomelanocortin gene, five melanocortin receptors, two endogenous antagonists, and two ancillary proteins. This review provides an abbreviated account of the basic biochemistry, pharmacology, and physiology of the melanocortin system and highlights progress made in four areas. In particular, recent pharmacological and genetic studies have affirmed the role of melanocortins in pigmentation, inflammation, energy homeostasis, and sexual function. Development of selective agonists and antagonists is expected to further facilitate the investigation of these complex physiological functions and provide an experimental basis for new pharmacotherapies.
Fat accumulation in the rat during early pregnancy is modulated by enhanced insulin responsiveness
Authors: Ramos M. P. , Crespo-Solans M. D. , del Campo S. , Cacho J. , Herrera E.
Published: March 2015

Insulin sensitivity has been implicated in the variation of fat accumulation in early gestation by as-yet-unknown mechanisms. In the present study, we analyzed the insulin sensitivity of lipolysis and lipogenesis in lumbar adipocytes from rats at 0, 7, 14, and 20 days of gestation. In adipocytes of 7-day pregnant rats, we found a twofold decrease in both β-agonist (isoproterenol and BRL-37344)-stimulated lipolysis and β3-adrenoceptor protein but not in lipolysis initiated by forskolin or isobutylmethylxanthine, suggesting a modification of the lipolytic pathway at the receptor level. Whereas adipocytes from 7-day pregnant rats showed a twofold increase in fatty acid synthesis from glucose, those from 20-day pregnant animals displayed a decreased lipogenic activity. Insulin responsiveness of the lipolytic and lipogenic pathways was analyzed by dose-response experiments, giving evidence for the involvement of improved insulin responsiveness in the enhanced lipogenic and reduced lipolytic activities of adipocytes in early pregnancy. In contrast, insulin resistance is responsible for lower antilipolytic and lipogenic actions of insulin in late pregnant animals. In conclusion, the present study shows that enhanced adipose tissue insulin responsiveness during early pregnancy contributes to maternal fat accumulation, whereas decreased insulin responsiveness during late gestation modulates fat breakdown.
Direct assessment of muscle glycogen storage after mixed meals in normal and type 2 diabetic subjects
Authors: Carey P. E. , Halliday J. , Snaar J. E. M. , Morris P. G. , Taylor R.
Published: March 2015

To understand the day-to-day pathophysiology of impaired muscle glycogen storage in type 2 diabetes, glycogen concentrations were measured before and after the consumption of sequential mixed meals (breakfast: 190.5 g carbohydrate, 41.0 g fat, 28.8 g protein, 1,253 kcal; lunch: 203.3 g carbohydrate, 48.1 g fat, 44.0 g protein, 1,497.5 kcal) by use of natural abundance 13C magnetic resonance spectroscopy. Subjects with diet-controlled type 2 diabetes ( n = 9) and age- and body mass index-matched nondiabetic controls ( n = 9) were studied. Mean fasting gastrocnemius glycogen concentration was significantly lower in the diabetic group (57.1 ± 3.6 vs. 68.9 ± 4.1 mmol/l; P < 0.05). After the first meal, mean glycogen concentration in the control group rose significantly from basal (97.1 ± 7.0 mmol/l at 240 min; P = 0.005). After the second meal, the high level of muscle glycogen concentration in the control group was maintained, with a further rise to 108.0 ± 11.6 mmol/l by 480 min. In the diabetic group, the postprandial rise was markedly lower than that of the control group (65.9 ± 5.2 mmol/l at 240 min, P < 0.005, and 70.8 ± 6.7 mmol/l at 480 min, P = 0.01) despite considerably greater serum insulin levels (752.0 ± 109.0 vs. 372.3 ± 78.2 pmol/l at 300 min, P = 0.013). This was associated with a significantly greater postprandial hyperglycemia (10.8 ± 1.3 vs. 5.3 ± 0.2 mmol/l at 240 min, P < 0.005). Basal muscle glycogen concentration correlated inversely with fasting blood glucose ( r = −0.55, P < 0.02) and fasting serum insulin ( r = −0.57, P < 0.02). The increment in muscle glycogen correlated with initial increment in serum insulin only in the control group ( r = 0.87, P< 0.002). This study quantitates for the first time the subnormal basal muscle glycogen concentration and the inadequate glycogen storage after meals in type 2 diabetes.
On-line analysis of gap junctions reveals more efficient electrical than dye coupling between islet cells
Authors: Quesada Ivan , Fuentes Esther , Andreu Etelvina , Meda Paolo , Nadal Angel , Soria Bernat
Published: March 2015

Pancreatic β-cells constitute a well-communicating multicellular network that permits a coordinated and synchronized signal transmission within the islet of Langerhans that is necessary for proper insulin release. Gap junctions are the molecular keys that mediate functional cellular connections, which are responsible for electrical and metabolic coupling in the majority of cell types. Although the role of gap junctions in β-cell electrical coupling is well documented, metabolic communication is still a matter of discussion. Here, we have addressed this issue by use of a fluorescence recovery after photobleaching (FRAP) approach. This technique has been validated as a reliable and noninvasive approach to monitor functional gap junctions in real time. We show that control pancreatic islet cells did not exchange a gap junction-permeant molecule in either clustered cells or intact islets of Langerhans under conditions that allowed cell-to-cell exchange of current-carrying ions. Conversely, we have detected that the same probe was extensively transferred between islet cells of transgenic mice expressing connexin 32 (Cx32) that have enhanced junctional coupling properties. The results indicate that the electrical coupling of native islet cells is more extensive than dye communication. Dye-coupling domains in islet cells appear more restricted than previously inferred with other methods.
Insulin modulates PC-1 processing and recruitment in cultured human cells
Authors: Menzaghi C. , Di Paola R. , Baj G. , Funaro A. , Arnulfo A. , Ercolino T. , Surico N. , Malavasi F. , Trischitta V.
Published: March 2015

We evaluated whether insulin signaling modulates plasma cell glycoprotein (PC-1) plasma membrane recruitment, posttranslational processing, and gene expression in human cultured cell lines. Insulin induced a fourfold increase ( P < 0.01) of membrane PC-1 expression by rapid and sensitive mechanism(s). This effect was reduced ( P < 0.05–0.01) by inhibition of phosphatidylinositol 3-kinase (200 nmol/l wortmannin) and S6 kinase (50 nmol/l rapamycin) activities and intracellular trafficking (50 μmol/l monensin) and was not accompanied by PC-1 gene expression changes. Moreover, at Western blot, insulin elicited the appearance, in both plasma membrane and cytosol, of a PC-1-related 146-kDa band (in addition to bands of 163, 117, 106, and 97 kDa observed also in absence of insulin) that was sensitive to endoglycosidase H. Finally, inhibition of PC-1 translocation to plasma membrane, by wortmannin pretreatment, increases insulin-stimulated receptor autophosphorylation. Our data indicate that insulin stimulates PC-1 posttranslational processing and translocation to the plasma membrane, which in turn impairs insulin receptor signaling. Bidirectional cross talk between insulin and PC-1, therefore, takes place, which may be part of the hormone self-desensitization mechanism.
Inhibition of IFN-γ-induced STAT1 activation by 15- deoxy-Δ12,14-prostaglandin J2
Authors: Weber Sarah M. , Scarim Anna L. , Corbett John A.
Published: March 2015

The inhibitory actions of 15-deoxy-Δ12,14-prostaglandin J2(PGJ2) on inflammatory gene expression have been attributed to the ability of this prostaglandin to inhibit the activation of NF-κB. In this study, we have identified an additional signaling pathway sensitive to inhibition by PGJ2. We show that PGJ2inhibits interferon (IFN)-γ-stimulated phosphorylation and DNA-binding activity of STAT1. The inhibitory actions on STAT1 phosphorylation are first apparent after a 1- to 2-h incubation and are maximal after a 6-h incubation with PGJ2, and they correlate with the expression of heat shock protein (HSP)70 in islets. In previous studies, we have correlated the inhibitory actions of PGJ2on inducible nitric oxide synthase (iNOS) expression and NF-κB activation in response to IL-1 with the increased expression of HSP70. Using overexpression and antisense depletion, we provide evidence that HSP70 does not mediate the inhibitory actions of PGJ2on IL-1-induced NF-κB or IFN-γ-induced STAT1 activation or cytokine-stimulated iNOS expression by β-cells. Last, we show that the inhibitory actions of a short 6-h pulse with PGJ2on IL-1 plus IFN-γ-stimulated iNOS expression and NO production by β-cells are persistent for extended periods (≤48 h). These findings suggest that PGJ2inhibits multiple cytokine-signaling pathways (IL-1 and IFN-γ), that the inhibitory actions are persistent for extended periods, and that increased HSP70 expression correlates with, but does not appear to mediate, the inhibitory actions of PGJ2on IL-1 and IFN-γ signaling in β-cells.
Glutamine cycling in isolated working rat heart
Authors: Cohen David M. , Guthrie Patrick H. , Gao Xiaolian , Sakai Ryosei , Taegtmeyer Heinrich
Published: March 2015

To what extent does glutamine turnover keep pace with oxidative metabolism in the rat heart? To address this question, the following groups of substrates were presented to the isolated, working rat heart: 1) glucose (5 mM), insulin (40 μU/ml), and [2-13C]acetate (5 mM; high workload, n = 5); 2) pyruvate (2.5 mM) and [2-13C]acetate (5 mM; normal workload, n = 5); or 3) propionate (1 mM) and [2-13C]acetate (2.5 mM; normal workload, n = 3). In a subset of these experiments, the exchange of glutamate and glutamine was quantified by separation with ion exchange chromatography and analysis by GC-MS. There was an apparent equilibration of mass isotopomers of glutamate and glutamine after 50 min of perfusion, although the extent of equilibration was not determined. The fractional enrichment in glutamine was 31% of the enrichment of glutamate with the three different perfusates. From high-resolution nuclear magnetic resonance spectra, we found a ratio of glutamine to glutamate content of 94.1, 53.4, and 96.9%, respectively, for each experimental group. In experiments for which l-[1-13C]glutamine (5 mM) was included in the perfusate of group 2, [1-13C]glutamine was detected in the heart, but transfer of13C from glutamine to glutamate was not detected ( n = 4). We conclude that, in the perfused working heart, production of glutamine by amidation of glutamate takes place and can be detected, whereas the reverse process, generation of glutamate from glutamine, remains undetected.
Glucose metabolism and glutamate analog acutely alkalinize pH of insulin secretory vesicles of pancreatic β-cells
Authors: Eto Kazuhiro , Yamashita Tokuyuki , Hirose Kenzo , Tsubamoto Yoshiharu , Ainscow Edward K. , Rutter Guy A. , Kimura Satoshi , Noda Mitsuhiko , Iino Masamitsu , Kadowaki Takashi
Published: March 2015

We studied acute changes of secretory vesicle pH in pancreatic β-cells with a fluorescent pH indicator, lysosensor green DND-189. Fluorescence was decreased by 0.66 ± 0.10% at 149 ± 16 s with 22.2 mM glucose stimulation, indicating that vesicular pH was alkalinized by ∼0.016 unit. Glucose-responsive pH increase was observed when cytosolic Ca2+influx was blocked but disappeared when an inhibitor of glycolysis or mitochondrial ATP synthase was present. Glutamate dimethyl ester (GME), a plasma membrane-permeable analog of glutamate, potentiated glucose-stimulated insulin secretion at 5 mM without changing cellular ATP content or cytosolic Ca2+concentration ([Ca2+]). Application of GME at basal glucose concentration decreased DND-189 fluorescence by 0.83 ± 0.19% at 38 ± 2 s. These results indicated that the acutely alkalinizing effect of glucose on β-cell secretory vesicle pH was dependent on glucose metabolism but independent of modulations of cytosolic [Ca2+]. Moreover, glutamate derived from glucose may be one of the mediators of this alkalinizing effect of glucose, which may have potential relevance to the alteration of secretory function by glutamate.
Sexually dimorphic effects of maternal alcohol intake and adrenalectomy on left ventricular hypertrophy in rat offspring
Authors: Wilcoxon Jennifer Slone , Schwartz Jeff , Aird Fraser , Redei Eva E.
Published: March 2015

In humans, low birth weight and increased placental weight can be associated with cardiovascular disease in adulthood. Low birth weight and increased placental size are known to occur after fetal alcohol exposure or prenatal glucocorticoid administration. Thus the effects of removing the alcohol-induced increase in maternal corticosterone by maternal adrenalectomy on predictors of cardiovascular disease in adulthood were examined in rats. Alcohol exposure of dams during the last 2 wk of gestation resulted in significantly decreased fetal weight and increased placental weight on gestational day 21. Adult female, but not male, offspring of alcohol-consuming mothers exhibited left ventricular hypertrophy. Placental 11β-hydroxysteroid dehydrogenase-2 (11β-HSD-2) mRNA levels, measured by Northern blot, were decreased in females but not males. Adrenalectomy of alcohol-consuming dams reversed the increase in placental weight and the decrease in female placental 11β-HSD-2 expression and eliminated the left ventricular hypertrophy of adult female offspring. These data suggest that alcohol-induced changes in placental 11β-HSD-2 mRNA levels and left ventricular weight are coupled in female offspring only and depend on maternal adrenal status.
Interaction of free fatty acids and epinephrine in regulating hepatic glucose production in conscious dogs
Authors: Chu Chang An , Galassetti Pietro , Igawa Kayano , Sindelar Dana K. , Neal Doss W. , Burish Mark , Cherrington Alan D.
Published: March 2015

To determine the effects of an increase in lipolysis on the glycogenolytic effect of epinephrine (EPI), the catecholamine was infused portally into 18-h-fasted conscious dogs maintained on a pancreatic clamp in the presence [portal (Po)-EPI+FFA, n = 6] and absence (Po-EPI+SAL, n = 6) of peripheral Intralipid infusion. Control groups with high glucose (70% increase) and free fatty acid (FFA; 200% increase; HG+FFA, n = 6) and high glucose alone (HG+SAL, n = 6) were also included. Hepatic sinusoidal EPI levels were elevated (Δ568 ± 77 and Δ527 ± 37 pg/ml, respectively) in Po-EPI+SAL and EPI+FFA but remained basal in HG+FFA and HG+SAL. Arterial plasma FFA increased from 613 ± 73 to 1,633 ± 101 and 746 ± 112 to 1,898 ± 237 μmol/l in Po-EPI+FFA and HG+FFA but did not change in EPI+SAL or HG+SAL. Net hepatic glycogenolysis increased from 1.5 ± 0.3 to 3.1 ± 0.4 mg · kg−1· min−1( P < 0.05) by 30 min in response to portal EPI but did not rise (1.8 ± 0.2 to 2.1 ± 0.3 mg · kg−1· min−1) in response to Po-EPI+FFA. Net hepatic glycogenolysis decreased from 1.7 ± 0.2 to 0.9 ± 0.2 and 1.6 ± 0.2 to 0.7 ± 0.2 mg · kg−1· min−1by 30 min in HG+FFA and HG+SAL. Hepatic gluconeogenic flux to glucose 6-phosphate increased from 0.6 ± 0.1 to 1.2 ± 0.1 mg · kg−1· min−1( P < 0.05; by 3 h) and 0.7 ± 0.1 to 1.6 ± 0.1 mg · kg−1· min−1( P < 0.05; at 90 min) in HG+FFA and Po-EPI+FFA. The gluconeogenic parameters remained unchanged in the Po-EPI+SAL and HG+SAL groups. In conclusion, increased FFA markedly changed the mechanism by which EPI stimulated hepatic glucose production, suggesting that its overall lipolytic effect may be important in determining its effect on the liver.
High glucose-mediated effects on endothelial cell proliferation occur via p38 MAP kinase
Authors: McGinn S. , Saad S. , Poronnik P. , Pollock C. A.
Published: March 2015

The mitogen-activated protein (MAP) kinases contribute to altered cell growth and function in a variety of disease states. However, their role in the endothelial complications of diabetes mellitus remains unclear. Human endothelial cells were exposed for 72 h to 5 mM (control) or 25 mM (high) glucose or 5 mM glucose plus 20 mM mannitol (osmotic control). The roles of p38 and p42/44 MAP kinases in the high glucose-induced growth effects were determined by assessment of phosphorylated MAP kinases and their downstream activators by Western blot and by pharmacological inhibition of these MAP kinases. Results were expressed as a percentage (means ± SE) of control. High glucose increased the activity of total and phosphorylated p38 MAP kinase ( P < 0.001) and p42/44 MAP kinase ( P < 0.001). Coexposure of p38 MAP kinase blocker with high glucose reversed the antiproliferative but not the hypertrophic effects associated with high-glucose conditions. Transforming growth factor (TGF)-β1 increased the levels of phosphorylated p38 MAP kinase, and p38 MAP kinase blockade reversed the antiproliferative effects of this cytokine. The high glucose-induced increase in phosphorylated p38 MAP kinase was reversed in the presence of TGF-β1 neutralizing antibody. Although hyperosmolarity also induced antiproliferation ( P < 0.0001) and cell hypertrophy ( P < 0.05), there was no change in p38 activity, and therefore inhibition of p38 MAP kinase had no influence on these growth responses. Blockade of p42/44 MAP kinase had no effect on the changes in endothelial cell growth induced by either high glucose or hyperosmolarity. High glucose increased p42/44 and p38 MAP kinase activity in human endothelial cells, but only p38 MAP kinase mediated the antiproliferative growth response through the effects of autocrine TGF-β1. High glucose-induced endothelial cell hypertrophy was independent of activation of the MAP kinases studied. In addition, these effects were independent of any increase in osmolarity associated with high-glucose exposure.
Unique regulation of anion/HCO3- exchangers by constitutive nitric oxide in rabbit small intestine
Authors: Coon Steven , Sundaram Uma
Published: March 2015

In the rabbit small intestine, there are three functionally different brush-border membrane (BBM) anion/HCO3- exchangers: 1) Cl/HCO3- exchange on the BBM of villus cells responsible for coupled NaCl absorption; 2) Cl/HCO3- exchange on the BBM of crypt cells possibly involved in HCO3- secretion; and 3) short-chain fatty acid (SCFA)/HCO3- exchange on the BBM of villus cells, which facilitates SCFA absorption. Although constitutive nitric oxide (cNO) has been postulated to alter many gastrointestinal tract functions, how cNO may specifically alter these three transporters is unknown. Inhibition of cNO synthase with NG-nitro-l-arginine methyl ester (l-NAME) 1) did not affect villus cell BBM Cl/HCO3 change, 2) stimulated crypt cell BBM Cl/HCO3- exchange, and 3) inhibited villus cell BBM SCFA/HCO3- exchange. d-NAME, an inactive analog of l-NAME, and l- N6-(1-iminoethyl)lysine, a more selective inhibitor of inducible NO, did not affect these transport processes. Kinetic studies demonstrated that 1) the mechanism of inhibition of crypt cell BBM Cl/HCO3- exchange is secondary to a decrease in the maximal rate of uptake of Cl, without an alteration in the affinity of the transporter for Cl, and 2) the mechanism of stimulation of villus cell BBM SCFA/HCO3- exchange is secondary to an increase in the affinity of the transporter for SCFA without an alteration in the maximal rate of uptake of SCFA. These results indicate that cNO uniquely regulates the three BBM anion/HCO3- transporters in the rabbit small intestine.
Na+/H+exchanger blockade inhibits enterocyte inflammatory response and protects against colitis
Authors: Németh Zoltán H. , Deitch Edwin A. , Szabó Csaba , Mabley Jon G. , Pacher Pál , Fekete Zoltán , Hauser Carl J. , Haskó György
Published: March 2015

Na+/H+exchangers (NHEs) are integral transmembrane proteins found in all mammalian cells. There is substantial evidence indicating that NHEs regulate inflammatory processes. Because intestinal epithelial cells express a variety of NHEs, we tested the possibility that NHEs are also involved in regulation of the epithelial cell inflammatory response. In addition, since the epithelial inflammatory response is an important contributor to mucosal inflammation in inflammatory bowel disease (IBD), we examined the role of NHEs in the modulation of disease activity in a mouse model of IBD. In human gut epithelial cells, NHE inhibition using a variety of agents, including amiloride, 5-( N-methyl- N-isobutyl)amiloride, 5-( N-ethyl- N-isopropyl)- amiloride, harmaline, clonidine, and cimetidine, suppressed interleukin-8 (IL-8) production. The inhibitory effect of NHE inhibition on IL-8 was associated with a decrease in IL-8 mRNA accumulation. NHE inhibition suppressed both activation of the p42/p44 mitogen-activated protein kinase and nuclear factor-κB. Finally, NHE inhibition ameliorated the course of IBD in dextran sulfate-treated mice. Our data demonstrate that inhibition of NHEs may be an approach worthy of pursuing for the treatment of IBD.
II. Modulating leukocyte recruitment to splanchnic organs to reduce inflammation
Authors: Bonder Claudine S. , Kubes Paul
Published: March 2015

A hallmark feature of intestinal inflammation is the recruitment and extravasation of numerous cell types from the blood to the afflicted site. Much of what we know about the mechanisms of leukocyte recruitment to splanchnic organs comes from an extensive series of studies on neutrophils in the mesenteric microvasculature. In this themes article, we highlight the important findings from these experiments but also emphasize some of the limitations. In fact, there is a growing body of evidence that neutrophil recruitment may be quite different in the mesentery than in other splanchnic organs. For example, the molecular mechanisms underlying neutrophil recruitment into the liver are quite different than the mesentery and are dependent on the type of inflammatory disease. We also discuss the effect of modulating leukocyte recruitment to splanchnic organs in chronic inflammation and emphasize that the approaches that have been successful in acute inflammation may be less effective in such conditions as inflammatory bowel disease (IBD). One obvious reason for this observation is the growing body of evidence to suggest that the initiation and maintenance of IBD is, in part, due to dysregulated or inappropriately activated populations of infiltrating T lymphocyte subsets. Therefore, we also discuss some interesting new approaches to limiting lymphocyte recruitment into the inflamed intestine either by targeting T helper (Th)1 vs. Th2 lymphocytes or perhaps by allowing the recruitment of regulatory T cells. Inhibiting specific adhesion molecules or specific chemokine receptors may work in this regard.
Decrease in activity of smooth muscle L-type Ca2+ channels and its reversal by NF-κB inhibitors in Crohn's colitis model
Authors: Kinoshita Kazuya , Sato Koichi , Hori Masatoshi , Ozaki Hiroshi , Karaki Hideaki
Published: March 2015

We investigated the mechanisms of dysmotility of the colonic circular muscle of the Crohn's disease rat model. Contractions induced by KCl, carbachol, and Bay K 8644 were decreased in circular smooth muscles isolated from 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat colon. However, the absolute force and Ca2+ sensitivity of contractile proteins were not affected as assessed in α-toxin permeabilized smooth muscle. The current density of the L-type Ca2+ channel in circular smooth muscle cells was significantly decreased in the TNBS-treated colonic cells. However, expressions of the L-type Ca2+ channel mRNA and protein did not differ between control and TNBS-treated preparations. Pretreatment with the NF-κB inhibitors pyrrolidinedithiocarbamate and sulfasalazine partially recovered the decreased contractility and current density of the L-type Ca2+ channel by TNBS treatment. These results suggest that the decrease in the contraction of circular smooth muscle isolated from TNBS-induced colitis rat colon, which may be related to gut dysmotility in Crohn's disease, is attributable to the decreased activity of the L-type Ca2+ channel. The dysfunction of the L-type Ca2+ channel may be mediated by NF-κB-dependent pathways.
RhoA stimulates IEC-6 cell proliferation by increasing polyamine-dependent Cdk2 activity
Authors: Guo Huazhang , Ray Ramesh M. , Johnson Leonard R.
Published: March 2015

Although RhoA plays an important role in cell proliferation and in Ras transformation in fibroblasts and mammary epithelial cells, its role in intestinal epithelial cells (IEC) is unknown. In a previous study (Ray RM, Zimmerman BJ, McCormack SA, Patel TB, and Johnson LR. Am J Physiol Cell Physiol 276: C684–C691, 1999), we showed that polyamine depletion [dl-α-difluoromethylornithine (DFMO) treatment] strongly inhibits the proliferation of IEC. In this report, we examined the effect of RhoA on IEC-6 cell proliferation and whether polyamine depletion inhibits cell proliferation in the presence of constitutively active RhoA. Constitutively active RhoA and vector-transfected IEC-6 cell lines were grown in the presence or absence of DFMO, which causes polyamine depletion by inhibiting ornithine decarboxylase, the first rate-limiting step in polyamine synthesis. Constitutively active RhoA significantly increased the rate of cell proliferation. These cells also lost contact inhibition and formed conspicuous foci when they were fully confluent. Decreased p21Waf1/Cip1 expression and increased cyclin-dependent kinase (Cdk2) mRNA levels and activity accompanied the increased proliferation. The inhibition of p21Waf1/Cip1 was independent of p53. There was no activation of the Ras-Raf-MEK-ERK pathway in the RhoA-transfected cell line. Polyamine depletion totally prevented the effect of activated RhoA on IEC-6 cell proliferation, focus formation, and Cdk2 expression. The stability of mRNA and protein for Cdk2 and p21Waf1/Cip1 in V14-RhoA cells was not significantly different from that of vector-transfected cells. In conclusion, RhoA activation decreased p21Waf1/Cip1 expression and increased basal and serum-induced ornithine decarboxylase activity, Cdk2 expression, Cdk2 protein, and Cdk2 activity, leading to the stimulation of IEC proliferation and transformation. Polyamine depletion totally prevented RhoA's effect on proliferation by decreasing Cdk2 expression and activity.
The ERK pathway involves positive and negative regulations of HT-29 colorectal cancer cell growth by extracellular zinc
Authors: Park Ki-Sook , Lee Nam-Gu , Lee Ki-Hoo , Seo Jeong Taeg , Choi Kang-Yell
Published: March 2015

Dietary zinc is an important trace element in the body and is related to both cell proliferation and growth arrest. A recent study found that extracellular zinc-sensing receptors trigger intracellular signal transduction in HT-29 human colorectal cancer cells. However, the signaling mechanism causing this growth regulation by extracellular zinc is not clearly understood. At 10- and 100-μM levels of ZnCl2treatment, HT-29 cell growth and proliferation increased and decreased, respectively, in a minimally serum-starved medium (MSSM). A lack of significant increase in intracellular zinc levels after zinc treatment suggested that this differential growth regulation of HT-29 cells by extracellular zinc is acquired by receptor-mediated signal transduction. Moreover, this zinc-induced growth regulation was differentially affected by PD-98059, suggesting the involvement of the ERK pathway. Transient ERK activation and subsequent cyclin D1 induction were observed on adding 10 μM ZnCl2in MSSM in the presence of cell proliferation. On the other hand, prolonged ERK activity was observed with a subsequent increase of cyclin D1 and p21Cip/WAF1on adding 100 μM ZnCl2in MSSM, and this was associated with nonproliferation. Moreover, this ERK activation and cyclin D1 and p21Cip/WAF1induction were abolished by PD-98059 pretreatment. The differential regulations of cell growth, ERK activities, and cyclin D1 and p21Cip/WAF1inductions were also observed in serum-enriched medium containing higher zinc concentrations. Therefore, differential cell cycle regulator induction occurs by a common ERK pathway in the differential growth regulation of HT-29 cells by extracellular zinc.
Inhibitory interactions between 5-HT3 and P2X channels in submucosal neurons
Authors: Barajas-López Carlos , Montaño Luis M. , Espinosa-Luna Rosa
Published: March 2015

Inhibitory interactions between 5-HT subtype 3 (5-HT3) and P2X receptors were characterized using whole cell recording techniques. Currents induced by 5-HT ( I 5-HT) and ATP ( I ATP) were blocked by tropisetron (or ondansetron) and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, respectively. Currents induced by 5-HT + ATP ( I 5-HT+ATP) were only as large as the current induced by the most effective transmitter, revealing current occlusion. Occlusion was observed at membrane potentials of −60 and 0 mV (for inward currents), but it was not present at +40 mV (for outward currents). Kinetic and pharmacological properties of I 5-HT+ATP indicate that they are carried through 5-HT3 and P2X channels. Current occlusion occurred as fast as activation of I 5-HT and I ATP, was still present in the absence of Ca2+ or Mg2+, after adding staurosporine, genistein, K-252a, or N-ethylmaleimide to the pipette solution, after substituting ATP with ∝,β-methylene ATP or GTP with GTP-γ-S in the pipette, and was observed at 35°C, 23°C, and 8°C. These results are in agreement with a model that considers that 5-HT3 and P2X channels are in functional clusters and that these channels might directly inhibit each other.
Anti-inflammatory effects of angiotensin II AT1 receptor antagonism prevent stress-induced gastric injury
Authors: Bregonzio Claudia , Armando Ines , Ando Hiromichi , Jezova Miroslava , Baiardi Gustavo , Saavedra Juan M.
Published: March 2015

Stress reduces gastric blood flow and produces acute gastric mucosal lesions. We studied the role of angiotensin II in gastric blood flow and gastric ulceration during stress. Spontaneously hypertensive rats were pretreated for 14 days with the AT1 receptor antagonist candesartan before cold-restraint stress. AT1 receptors were localized in the endothelium of arteries in the gastric mucosa and in all gastric layers. AT1 blockade increased gastric blood flow by 40–50%, prevented gastric ulcer formation by 70–80% after cold-restraint stress, reduced the increase in adrenomedullary epinephrine and tyrosine hydroxylase mRNA without preventing the stress-induced increase in adrenal corticosterone, decreased the stress-induced expression of TNF-α and that of the adhesion protein ICAM-1 in arterial endothelium, decreased the neutrophil infiltration in the gastric mucosa, and decreased the gastric content of PGE2. AT1 receptor blockers prevent stress-induced ulcerations by a combination of gastric blood flow protection, decreased sympathoadrenal activation, and anti-inflammatory effects (with reduction in TNF-α and ICAM-1 expression leading to reduced neutrophil infiltration) while maintaining the protective glucocorticoid effects and PGE2 release. Angiotensin II has a crucial role, through stimulation of AT1 receptors, in the production and progression of stress-induced gastric injury, and AT1 receptor antagonists could be of therapeutic benefit.
Inosine reduces inflammation and improves survival in a murine model of colitis
Authors: Mabley J. G. , Pacher P. , Liaudet L. , Soriano F. G. , Haskó G. , Marton A. , Szabó C. , Salzman A. L.
Published: March 2015

Inosine, a naturally occurring purine formed from the breakdown of adenosine, has recently been shown to exert powerful anti-inflammatory effects both in vivo and in vitro. This study evaluated inosine as a potential therapy for colitis. Colitis was induced in mice by the administration of dextran sulfate sodium (DSS). Oral treatment with inosine was begun either before the onset of colitis or as a posttreatment once colitis was established. Evaluation of colon damage and inflammation was determined grossly (body wt, rectal bleeding), histologically, and biochemically (colon levels of MPO, MDA, and cytokines). DSS-induced colitis significantly increased inflammatory cell infiltration into the colon. DSS-induced colitis also increased colon levels of lipid peroxidation, cytokines, and chemokines. Inosine protected the colon from DSS-induced inflammatory cell infiltration and lipid peroxidation. Inosine also partially reduced these parameters in an experimental model of established colitis. Thus inosine treatment may be a potential therapy in colitis.
Physiological regulation and NO-dependent inhibition of migrating myoelectric complex in the rat small bowel by OXA
Authors: Ehrström M. , Näslund E. , Ma J. , Kirchgessner A. L. , Hellström P. M.
Published: March 2015

Orexin A (OXA)-positive neurons are found in the lateral hypothalamic area and the enteric nervous system. The aim of this study was to investigate the mechanism of OXA action on small bowel motility. Electrodes were implanted in the serosa of the rat small intestine for recordings of myoelectric activity during infusion of saline or OXA in naive rats, vagotomized rats, rats pretreated with guanethidine (3 mg/kg) or Nω-nitro-l-arginine (l-NNA; 1 mg/kg). Naive rats were given a bolus of the orexin receptor-1 (OX1R) antagonist (SB-334867-A; 10 mg/kg), and the effect of both OXA and SB-334867-A on fasting motility was studied. Double-label immunocytochemistry with primary antibodies against OXA, neuronal nitric oxide synthase (nNOS), and OX1R was performed. OXA induced a dose-dependent prolongation of the cycle length of the migrating myoelectric complex (MMC) and, in the higher doses, replaced the activity fronts with an irregular spiking pattern. Vagotomy or pretreatment with guanethidine failed to prevent the response to OXA. The OXA-induced effect on the MMC cycle length was completely inhibited by pretreatment with l-NNA ( P < 0.05), as did SB-334867-A. The OX1R antagonist shortened the MMC cycle length from 14.1 (12.0–23.5) to 11.0 (9.5–14.7) min ( P < 0.05) during control and treatment periods, respectively. Colocalization of OXA and nNOS was observed in myenteric neurons of the duodenum and nerve fibers in the circular muscle. Our results indicate that OXA inhibition of the MMC involves the OX1R and that activation of a l-arginine/NO pathway possibly originating from OX1R/nNOS-containing neurons in the myenteric plexus may mediate this effect. Endogenous OXA may have a physiological role in regulating the MMC.
Mechanisms of airway protection during retching, vomiting, and swallowing
Authors: Lang Ivan M. , Dana Nicole , Medda Bidyut K. , Shaker Reza
Published: March 2015

We investigated the mechanisms of airway protection and bolus transport during retching and vomiting by recording responses of the pharyngeal, laryngeal, and hyoid muscles and comparing them with responses during swallowing and responses of the gastrointestinal tract. Five dogs were chronically instrumented with electrodes on the striated muscles and strain gauges on smooth muscles. Retching and vomiting were stimulated by apomorphine (5–10 ug/kg iv). During retching, the hyoid and thyroid descending and laryngeal abductor muscles were activated; between retches, the hyoid, thyroid, and pharyngeal elevating, and laryngeal adductor muscles were activated. Vomiting always occurred during the ascending phase of retching and consisted of three sequential phases of hyoid and pharyngeal muscle activation culminating in simultaneous activation of all recorded elevating and descending laryngeal, hyoid, and pharyngeal muscles. Retrograde activation of esophagus and pharyngeal muscles occurred during the later phases, and laryngeal adductor was maximally activated in all phases of the vomit. During swallowing, the laryngeal adductor activation was followed immediately by brief activation of the laryngeal abductor. We concluded that retching functions to mix gastric contents with refluxed intestinal secretions and to impart an orad momentum to the bolus before vomiting. During retches, the airway is protected by glottal closure, and between retches, it is protected by ascent of the larynx and closure of the upper esophageal sphincter. The airway is protected by maximum glottal closure during vomiting. During swallowing, the airway is protected by laryngeal elevation and glottal closure followed by brief opening of the glottis, which may release subglottal pressure expelling material from the laryngeal vestibule.
Flow fields generated by peristaltic reflex in isolated guinea pig ileum: impact of contraction depth and shoulders
Authors: Jeffrey Brian , Udaykumar Holavanahalli S. , Schulze Konrad S.
Published: March 2015

The guinea pig ileum responds to distension with characteristic wall movements, luminal pressure gradients, and outflow (the peristaltic reflex). To date, little is known about whether the peristaltic reflex generates flow events other than laminar flow. Here we used a numerical method to solve for the flow generated by moving walls to assess occlusive contractions ( case 1), nonocclusive contractions ( case 2), and contractions with steep shoulders ( case 3) for which visual parameters of wall movements are published. We found that all three contraction cases produced pressure differentials across the coapting segment, downstream and reverse flow, and vortical flow patterns that redistributed particles and mixed liquids. Contractions generated pressures and shear stresses, particularly along the moving section of the wall. The nonocclusive contraction was much less effective than the occlusive contraction with the steep shoulders; the occlusive contraction with flat shoulders had an intermediate effect. Our analysis shows that even peristaltic contractions produce not only laminar flow but also many flow events likely to promote digestion and absorption. The visual patterns of contractions impact the patterns of luminal flow, and precise definition of wall movements is critical to quantify the fluid mechanical consequences of intestinal contractions.
A neurokinin 1 receptor antagonist reduces an ongoing ileal pouch inflammation and the response to a subsequent inflammatory stimulus
Authors: Stucchi Arthur F. , Shebani Khaled O. , Leeman Susan E. , Wang Chi-Chung , Reed Karen L. , Fruin A. Brent , Gower Adam C. , McClung James P. , Andry Christopher D. , O'Brien Michael J. , Pothoulakis Charalabos , Becker James M.
Published: March 2015

Ileal pouch-anal anastomosis (IPAA) is an excellent surgical option for patients with chronic ulcerative colitis (CUC) requiring colectomy; however, persistent episodes of ileal pouch inflammation, or pouchitis, may result in debilitating postoperative complications. Because considerable evidence implicates substance P (SP) as an inflammatory mediator of CUC, we investigated whether SP participates in the pathophysiology of pouchitis. With the use of a rat model of IPAA that we developed, we showed that ileal pouch MPO levels and neurokinin 1 receptor (NK-1R) protein expression by Western blot analysis were significantly elevated 28 days after IPAA surgery. In situ hybridization and immunohistochemistry showed that the increase in NK-1R protein expression was localized to the lamina propria and epithelia of pouch ileum. The intraperitoneal administration of the NK-1R antagonist (NK-1RA) CJ-12,255 for 4 days, starting on day 28, was effective in reducing MPO levels. Starting on day 28, animals with IPAA were given 5% dextran sulfate sodium (DSS) in their drinking water for 4 days, which caused histological and physical signs of clinical pouchitis concomitant with significant increases in ileal pouch MPO concentrations as well as NK-1R protein expression by Western blot analysis. In situ hybridization and immunohistochemistry showed that the increase in NK-1R protein expression was especially evident in crypt epithelia of pouch ileum. When the NK-1RA was administered 1 day before starting DSS and continued for the duration of DSS administration, the physical signs of clinical pouchitis and the rise in MPO were prevented. These data implicate SP in the pathophysiology of pouchitis and suggest that NK-1RA may be of therapeutic value in the management of clinical pouchitis.